WO2022212845A2

WO2022212845A2 - Pd-1- and ox40l-based chimeric proteins

Info

Publication number: WO2022212845A2
Application number: PCT/US2022/023054
Authority: WO
Inventors: Arundathy PANDITE; Fatima RANGWALA; Thomas LAMPKIN; Taylor Schreiber; George FROMM; Suresh DE SILVA
Original assignee: Shattuck Labs, Inc.
Priority date: 2021-04-01
Filing date: 2022-04-01
Publication date: 2022-10-06
Also published as: WO2022212845A3

Abstract

The present technology relates, inter alia, to methods of treating cancer with chimeric proteins comprising an extracellular domain of human programmed cell death protein 1 (PD-1) and an extracellular domain of human 0X40 Ligand (OX40L), including doses and regimens.

Description

PD-1- and 0X4QL-BASED CHIMERIC PROTEINS

TECHNICAL FIELD

The present technology relates to, inter alia, compositions and methods, including chimeric proteins that find use in the treatment of disease, such as immunotherapies for cancer comprising doses, dosing regimens that including diphasic dosing or dosing regimens comprising three cycles.

PRIORITY

This application claims the benefit of, and priority to, U.S. Provisional Application Nos. 63/169,336, filed April 1 , 2021 ; 63/229,245, filed August 4, 2021 ; 63/231 ,581 , filed August 10, 2021 ; and 63/278,565, filed November 12, 2021 , the contents of each of which are hereby incorporated by reference in their entireties.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The contents of the text file named “SHK-047PC_Sequence Listing_ST25”, which was created on March 31, 2022 and is 37,135 bytes in size, are hereby incorporated herein by reference in their entireties.

BACKGROUND

The field of cancer immunotherapy has grown tremendously over the past several years. This has been largely driven by the clinical efficacy of antibodies targeting the family of checkpoint molecules (e.g., CTLA- 4 and PD-1/L1) in many tumor types. However, despite this success, clinical response to these agents as monotherapy occurs in a minority of patients (10-45% in various solid tumors), and these therapies are hindered by side effects.

Discovery of the proper dose and regimen of such agents is crucial to efficacious treatment of cancers. Developing novel treatment strategies, including dosing and regimens, remains a formidable task given the complexity of the human immune system, the high cost, and the potential for toxicity which may result from such interventions.

SUMMARY

In various aspects, the present technology provides for compositions and methods that are useful for cancer immunotherapy. For instance, the present technology, in part, relates to doses and treatment regimens of specific chimeric proteins that simultaneously block immune inhibitory signals and stimulate immune activating signals. Importantly, inter alia, the present technology provides for improved chimeric proteins that can maintain a stable and reproducible multimeric state. Accordingly, the present compositions and methods overcome various deficiencies in producing bi-specific agents. Using this approach, combination immunotherapy can be achieved by a single chimeric protein, having superior preclinical activity compared to the separate administration of two individual antibodies against each of the identical targets. Further, the present technology allows for treatment of human cancer patients with amounts of the present chimeric proteins to yield successful therapy. The present technology relates to chimeric proteins comprising an extracellular domain of human programmed cell death protein 1 (PD-1) and an extracellular domain of human 0X40 ligand (OX40L). PD-1 is Type I transmembrane protein, which binds, at least, PD-L1 and PD-L2 on the surface of human tumor cells; this binding blocks an inhibitory signal produced by the tumor cell, or other cells in the tumor microenvironment. Thus, the PD-1 end of a chimeric protein disrupts, blocks, reduces, inhibits and/or sequesters the transmission of immune inhibitory signals, e.g., originating from a cancer cell that is attempting to avoid its detection and/or destruction. OX40L is a Type II transmembrane that binds an OX40L receptor {e.g., 0X40) on the surface of primary peripheral blood mononuclear cells (PBMCs), as well as tissue- resident antigen presenting cells; this binding provides immune stimulatory properties upon anti-cancer immune cells. Thus, the OX40L end of a chimeric protein enhances, increases, and/or stimulates the transmission of an immune stimulatory signal to the 0X40 expressing immune cell. Together, chimeric proteins of the present technology are capable of treating cancer via two distinct mechanisms.

In a chimeric protein of the present technology, the extracellular domain of human PD-1 (a Type I transmembrane protein) is located at the chimeric protein’s amino terminus (see, by way of non-limiting example, FIG. 1A, left protein), whereas the extracellular domain of human OX40L (a Type II transmembrane protein), is located at the chimeric protein’s carboxy terminus (see, by way of non-limiting example, FIG. 1 A, right protein). The extracellular domain of PD-1 contains the functional domains that are responsible for interacting with other binding partners (either ligands or receptors) in the extracellular environment (see, FIG. 1B, left protein) and the extracellular domain of OX40L contains the functional domains that are responsible for interacting with other binding partners (either ligands or receptors) in the extracellular environment (see, FIG. 1B, right protein).

An aspect of the present technology is a method for treating a cancer in a human subject. The method comprising a step of administering to the human subject a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, in which (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1 ), (b) is a linker adjoining the first and second domains, wherein the linker comprises at least one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L). See, by way of non-limiting examples, FIG. 1C and FIG. 1D. See, also, FIG. 3A.

In embodiments, the dose of the chimeric protein administered is at least 0.0001 mg/kg, e.g., between about 0.0001 mg/kg and about 50.0 mg/kg. The chimeric protein may be administered at an initial dose {e.g., at one of about 0.0001, about 0.001, about 0.003, about 0.01, about 0.03, about 0.1, about 0.3, about 1, about

2, about 3, about 4, about 6, about 8, about 10, about 12, about 15, about 18, about 21, about 24, about 25, about 27, about 30, about 33, about 35, about 36, about 40, about 42, about 45, about 48, and about 50 mg/kg) and the chimeric protein is administered in one or more subsequent administrations (e.g., at one or more of about 0.0001, about 0.001, about 0.003, about 0.01, about 0.03, about 0.1, about 0.3, about 1, about 2, about

3, about 4, about 6, about 8, about 10, about 12, about 15, about 18, about 21, about 24, about 25, about 27, about 30, about 33, about 35 , about 36, about 40, about 42, about 45, about 48, and about 50 mg/kg mg/kg). In embodiments, the initial dose is less than the dose for at least one of the subsequent administrations (e.g. each of the subsequent administrations) or the initial dose is the same as the dose for at least one of the subsequent administrations (e.g., each of the subsequent administrations). In embodiments, the starting dose and/or the subsequent doses is the maximum tolerated dose or less than the maximum tolerated dose. In embodiments, the chimeric protein is administered at least about one time a month, e.g., at least about two times a month, at least about three times a month, and at least about four times a month. In embodiments, the chimeric protein is first administered once a week for three weeks and the chimeric protein is then administered about once every three weeks or once every four weeks; alternately, the chimeric protein is first administered once a week for three weeks and the chimeric protein is then administered about two times per month, e.g., once a week for three weeks and the chimeric protein is then administered about once every two weeks.

In embodiments, the cancer is selected from melanoma, non-small cell lung cancer (squamous, adeno, or adeno-squamous), urothelial cancer, renal cell cancer, squamous cell cervical cancer, gastric or gastroesophageal junction adenocarcinoma, squamous cell carcinoma of the anus, squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin, Hodgkin’s disease, diffuse large B cell lymphoma, and microsatellite instability high or mismatch repair deficient solid tumors, excluding CNS tumors. In embodiments, the cancer comprises an advanced solid tumor (local and/or metastatic) or advanced lymphoma. In some aspects, the present technology relates to biphasic or a three-cycle regimen for treating cancer that maximizes potential clinical efficacy, while avoiding substantial side effects. In a biphasic dosing regimen, the first phase is intended to increase the binding of target receptors (PD-L1 and 0X40) in tumor by the chimeric proteins disclosed herein to therapeutically relevant levels. The second phase is intended to maintain the binding of target receptors (PD-L1 and 0X40) in tumor by the chimeric proteins disclosed herein at therapeutically relevant levels.

Accordingly, in one aspect, the present technology relates to a method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein:

(a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1),

(b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L), wherein the step of administering comprises biphasic dosing. In some embodiments, the first phase, and the second phase each independently comprise a dosing frequency of from about twice a week to about once every two months.

In one aspect, the present technology relates to a method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L). In a three cycle dosing regimen, the first cycle is intended to increase the binding of target receptors (PD-L1 and 0X40) in tumor by the PD-1- Fc-OX40L chimeric proteins disclosed herein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61). The second cycle is intended to modulate the binding of target receptors (PD-L1 and 0X40) in tumor by the chimeric proteins disclosed herein to therapeutically relevant levels. The third phase is intended to maintain the binding of target receptors (PD-L1 and 0X40) in tumor by the chimeric proteins disclosed herein at therapeutically relevant levels. Accordingly, the dosing frequency of the first cycle, the dosing frequency of the second cycle and the dosing frequency of the third cycle may be same or different. Therefore, in some embodiments, the first cycle, the second cycle and the third cycle each independently comprise a dosing frequency of from about twice a week to about once every two months. In some embodiments, the dosing frequency of the first cycle, the second cycle and the third cycle are each independently selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. Additionally, or alternatively, in some embodiments, the first cycle, the second cycle and the third cycle each independently last from about two days to about 12 months. In some embodiments, the first cycle, the second cycle and the third cycle independently lasts from about two weeks to about 2 months. In some embodiments, the first cycle lasts from about two weeks to about 2 months; the second cycle lasts from about 2 weeks to about 12 months and the third cycle lasts from about 2 weeks to about 6 months.

Additionally, or alternatively, in some embodiments, the effective amount for the first cycle, the second cycle and the third cycle each independently comprise about 0.01 mg/kg to about 10 mg/ml. In some embodiments, the effective amount for the first cycle, the second cycle and the third cycle each independently selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg, and any range including and/or in between any two of the preceding values. The effective amount for the first cycle, the second cycle and the third cycle may be the same or different. In some embodiments, the chimeric proteins disclosed herein is the human PD-1-Fc- OX40L chimeric protein.

In one aspect, the present disclosure relates to a method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L) with a dosing regimen selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the dosing regimen is about every week to about every 2 weeks, about every 10 days to about every 3 weeks, or about every 2 weeks to about every 4 weeks.

In one aspect, the present disclosure relates to a method for inducing lymphocyte expansion in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein:

(b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L). In some embodiments, the human subject is dosed with a dosing regimen selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the dosing regimen is about every week to about every 2 weeks, about every 10 days to about every 3 weeks, or about every 2 weeks to about every 4 weeks. In some embodiments, the initial dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg. In some embodiments, the dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg. In embodiments, the dose is administered with a once a week or a once every two weeks schedule. In embodiments, the method further comprising administration of a priming dose to the subject.

In one aspect, the present disclosure relates to a method for inducing lymphocyte margination in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein:

(b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L). In some embodiments, the human subject is dosed with a dosing regimen selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the dosing regimen is about every week to about every 2 weeks, about every 10 days to about every 3 weeks, or about every 2 weeks to about every 4 weeks. In some embodiments, the initial dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about

30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg. In some embodiments, the dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about

48 mg/kg, and about 50 mg/kg. In embodiments, the dose is administered with a once a week or a once every two weeks schedule. In embodiments, the method further comprising administration of a priming dose to the subject.

In one aspect, the present disclosure relates to a method of evaluating the efficacy of a cancer treatment in a subject in need thereof comprising, the method comprising the steps of: administering a dose of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L), wherein the dose of from about 0.03 mg/kg to about 50 mg/kg; obtaining a biological sample from the subject; performing an assay on the biological sample to determine level and/or activity of a cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFa; and continuing dosing if the subject has an increase in the level and/or activity of at least one cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFa.

In one aspect, the present disclosure relates to a method of selecting a subject for treatment with a therapy for cancer, the method comprising the steps of: administering a dose of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L), wherein the dose of from about 0.03 mg/kg to about 50 mg/kg; obtaining a biological sample from the subject; performing an assay on the biological sample to determine level and/or activity of a cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFa; and selecting the subject for treatment with the therapy for cancer if the subject has an increase in the level and/or activity of at least one cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFo.

In some embodiments of any of the aspects disclosed herein, the cancer is selected from melanoma, nonsmall cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), squamous cell carcinoma of the skin (skin-SCC), urothelial cancer, cervical cancer, gastric cancer, gastric or gastro- esophageal junction adenocarcinoma, renal cell carcinoma (RCC), squamous cell carcinoma (SCCA), squamous cell carcinoma of the anus, Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor, with the proviso that the microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor is not a CNS tumor. In any of the embodiments disclosed herein, the number of proliferating CD4 central and/orthe number effector memory T cells in the subject may increase compared to the number of proliferating CD4 central and/or the number effector memory T cells in the subject before administration of the chimeric protein, or compared to the number of proliferating CD4 central and/or the number effector memory T cells in a subject that was not administered the chimeric protein, and/or compared to a control. In any of the embodiments disclosed herein, the treatment produces at least one therapeutic effect chosen from a reduction in size of a tumor, reduction in number of metastatic lesions over time, complete response, partial response, and stable disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A to FIG. 1D show schematic illustrations of Type I transmembrane proteins (FIG. 1A and FIG. 1B, left proteins) and Type II transmembrane proteins (FIG. 1A and FIG. 1B, right proteins). A Type I transmembrane protein and a Type II transmembrane protein may be engineered such that their transmembrane and intracellular domains are omitted and the transmembrane proteins’ extracellular domains are adjoined using a linker sequence to generate a single chimeric protein. As shown in FIG. 1C and FIG. 1D, the extracellular domain of a Type I transmembrane protein, e.g., PD-1, and the extracellular domain of a Type II transmembrane protein, e.g., OX40L, are combined into a single chimeric protein. FIG. 1C depicts the linkage of the Type I transmembrane protein and the Type II transmembrane protein by omission of the transmembrane and intracellular domains of each protein, and where the liberated extracellular domains from each protein have been adjoined by a linker sequence. The extracellular domains in this depiction may include the entire amino acid sequence of the Type I protein {e.g., PD-1) and/or Type II protein {e.g., OX40L) which is typically localized outside the cell membrane, or any portion thereof which retains binding to the intended receptor or ligand. Moreover, the chimeric protein comprises sufficient overall flexibility and/or physical distance between domains such that a first extracellular domain (shown at the left end of the chimeric protein in FIG. 1C and FIG. 1D) is sterically capable of binding its receptor/ligand and/or a second extracellular domain (shown at the right end of the chimeric protein in FIG. 1C and FIG. 1D) is sterically capable of binding its receptor/ligand. FIG. 1D depicts adjoined extracellular domains in a linear chimeric protein wherein each extracellular domain of the chimeric protein is facing “outward”.

FIG. 2 shows immune inhibitory and immune stimulatory signaling that is relevant to the present technology (from Mahoney, Nature Reviews Drug Discovery 2015: 14;561 -585), the entire contents of which are hereby incorporated by reference.

FIG. 3A shows, without wishing to be bound by theory, an in silico predicted structure of a monomeric PD-1- Fc-OX40L chimeric protein. FIG. 3B shows, without wishing to be bound by theory, four potential configurations of PD-1-Fc-OX40L chimeric proteins. FIG. 3C shows visualization by electron microscopy of SL-279252 hexamers (top two images) and SL-279252 trimers (bottom two images). FIG. 3D shows nonlimiting potential N- and O-glycosylation sites of SL-279252 of some embodiments. The potential N glycosylation sites of some embodiments are in bold and underlined, and the potential 0 glycosylation sites of some embodiments have a gray box around the specific glycosylated amino acid. FIG. 3E shows an exemplary nucleotide sequence encoding precursor and mature SL-279252 (SEQ ID NO: 60). Leader sequence is indicated by a boldface-underlined font. Spaces are placed between codons and translation and with numbers per line are shown at the end of the line.

FIG. 4A shows that tumor cells may express PD-L1 on the cell surface, which can bind to PD-1 expressed by a T cell (FIG. 4B). This interaction suppresses activation of T cells. A chimeric protein comprising the extracellular domain of PD-1 , adjoined to the extracellular domain of OX40L may bind to PD-L1 on the surface of a tumor cell, preventing binding to PD-1 on the surface of a T cell (FIG. 4C). The chimeric protein may then “dangle” from the surface of the tumor cell, and the OX40L portion of the chimeric protein may then bind to 0X40 expressed on the surface of the T cell. This would result in replacement of an inhibitory PD-L1 signal with a co-stimulatory OX40L signal to enhance the anti-tumor activity of T cells.

FIG. 5A and FIG. 5B depict, without wishing to be bound by theory, the mechanism of action of the PD-1-Fc- OX40L chimeric protein (SL-279252). FIG. 5A compares mechanisms of action between an anti-OX40 antibody (as an example) and the SL-279252 chimeric protein. FIG. 5B compares mechanisms of action between a checkpoint inhibitor (using an anti-PD-1 antibody such as pembrolizumab (KEYTRUDA) or as an example) and the SL-279252 chimeric protein.

FIG. 6A and FIG. 6B show in vivo efficacy of the murine PD-1-Fc-OX40L in an MC38 tumor model.

FIG. 7A shows the evolution of in vivo tumor size after CT26 tumor inoculation for each group of mice described in the figure. FIG. 7B and FIG. 7C show the overall survival percentage, and statistics, of mice and tumor rejection through forty days after tumor inoculation. In FIG. 7B, the different treatment conditions are identified as: untreated: “a”, “e”, and “h”; aPD-L1 (10F.9G2): “b”; aPD-1 (RMP1-14): “c”; aOX40 (0X86): “d”; PD-L1/OX40: T; aPD-1 (RMP1-14)/OX40: “g”; PD-1-Fc-OX40L (100 mg x 2): “i”; PD-1-Fc-OX40L (150 mg x 2): “j”; and PD-1-Fc-OX40L (300 mg x 2): “k”.

FIG. 8A shows Western blot analysis of SL-279252 performed by probing purified chimeric protein with human anti-PD-1, anti-Fc, and anti-OX40L, under non-reducing and reducing conditions, and ± the deglycosylase PNGase F. FIG. 8B shows a functional ELISA using capture with recombinant hPD-L1 followed by detection with recombinant hOX40-His and then anti-His-HRP. HVEM-His served as a negative control. FIG. 8C shows a functional PD-L1 blocking assay for testing the ability of SL-279252 to outcompete PD-1 -Biotin for binding to plate-bound PD-L1 in an ELISA format. Avidin-HRP was used for signal detection. (ELISA= enzyme-linked immunosorbent assay; HRP = horse radish peroxidase; HVEM-his = herpes virus entry mediator- histidine; PNGase = Peptide N-glycosidase).

FIG. 9A to FIG. 9C are flow cytometry binding analyses showing binding of SL-279252 to cells expressing, respectively, PD-L1 (FIG. 9A), PD-L2 (FIG. 9B), or 0X40 (FIG. 9C). Each figure shows a titration curve for increasing concentrations of the chimeric protein.

FIG. 10A is a schematic of a tumor/T cell co-culture functional assay. CD3+ human T cells stimulated for 48 hours with suboptimal levels of CD3/CD28/IL-2, were plated on mitomycin-c treated PD-L1 i_o (PC3) and PD- L1 high (HCC827) tumor cells ± SL-279252, for an additional 3-5 days (days 5-7 of the entire time-course). ‘FP’ stands for fusion protein and refers to the PD-1-Fc-OX40L ARC (SL-279252). FIG. 10B shows that on day six of the assay, SL-279252 induced higher levels of secreted IL2 in PC3 cells (FIG. 10B, left bundle) than in HCC827 cells (FIG. 10B, right bundle). FIG. 10C shows a flow cytometry analysis of cells taken from the tumor/T cell co-culture functional assay outlined in FIG. 10A. The left-most bars indicate the proportion of CD4+ or CD8+ cells expressing Ki67 (as an indicator of proliferation) in the absence of tumor cells. The second from left bars indicates the proportion of CD4+ or CD8+ cells expressing Ki67 in the presence of tumor cells but without SL-279252, whereas the third-from left and right-most bars indicate the proportion of CD4+ or CD8+ cells expressing Ki67 in the presence of 500 ng or 5 g of SL-279252, respectively. On days five (top) and seven (bottom) of the assay, floating T cells were collected and subjected to extra- and intracellular flow cytometry in order to assess proliferation (Ki67) and markers of T cell activation (IFNy & TNFa).

FIG. 11 shows that SL-279252 stimulates IL-2 secretion from human peripheral blood mononuclear cells (PBMCs) only when the TCR on a T cell is engaged with MHC Class II on APCs. Ten distinct healthy donor PBMC samples were assessed across a wide concentration range of SL-279252 (from 0.10 pM to 200 nM), with either 50 or 100 ng/mL of staphylococcus enterotoxin B (SEB). The IL-2 baseline corrected mean replicate data was fit to a standard E_max model with variable slope as shown allowing for the calculation of an individual ECso for SL-279252 for each donor. The geometric mean (95% Cl) for the ECso values of SL- 279252 following SEB superantigen stimulation of ten human donors at 50 and 100 ng/mL were similar at 0.4866 (0.2684 to 0.8823) and 0.5903 (0.3689 to 0.9446) nM, respectively.

FIG. 12A shows the workflow of a SEB superantigen assay in which total primary human PBMCs were harvested and treated with Staphylococcal enterotoxin B ± SL-279252 and benchmark controls. Culture supernatants were collected 3 days later and assessed for secreted levels of IL-2 by ELISA. FIG. 12B shows results from the SEB superantigen assay.

FIG. 13 shows an inverse relationship between the logarithm of dose and the logarithm of clearance for cynomolgus monkeys administered SL-279252.

FIG. 14 shows that SL-279252 localizes to the immune synapse and coordinates tumor cell killing. At 90 min, the SL-279252 chimeric protein (red label) is distributed across the cell membrane. T-cells are labelled in green and tumor cells are labelled in yellow with tumor nuclei in blue. The SL-279252 chimeric protein then redistributes to the immune synapse between the T cell and the tumor cell (135 min). At 180 min, apoptosis ofthe tumor cell is indicated by cleavage of caspase 3/7 (green). At 225 min, the T cells disengage with tumor cell death and apoptotic membrane blebs visualized at 360 min.

FIG. 15 shows a schematic of the design of the Phase 1 clinical trial of SL-279252. The Phase 1 clinical trial is a first in human, open label, multi-center, dose escalation and dose expansion study in subjects with advanced solid tumors or lymphomas. The primary objective of this study is to evaluate the safety, tolerability of SL-279252. The secondary objective of this study is to evaluate the recommended phase 2 dose (RP2D), pharmacokinetic (PK), anti-tumor activity and pharmacodynamic effects of SL-279252. The exploratory objective of this study is to evaluate the pharmacodynamic (PD) markers in blood and tumor. The study design consisted of Dose Escalation Cohorts and Dose Expansion Cohorts, shown on the left and right hand side, respectively. The dose levels (DL) DL1 through DL10 used in this study were: DL1 is a 0 0001 mg/kg dose; DL2 is a 0001 mg/kg dose; DL3 is a 0003 mg/kg dose; DL4 is a 0.01 mg/kg dose; DL5 is a 0.03 mg/kg dose; DL6 is a 0.1 mg/kg dose; DL7 is a 0.3 mg/kg dose; DL8 is a 1 mg/kg dose; DL9 is a 3 mg/kg dose; and DL10 is a 10 mg/kg. The abbreviations used include: D = Day; q28d = Every 28 days; and DLT = dose limiting toxicity.

FIG. 16 shows simulation of likely human concentrations of a human PD-1-Fc-OX40L chimeric protein (SL- 279252) by dose (mg/kg).

FIG. 17 shows the comparison of parameters associated with efficacy of SL-279252 in mouse, monkey and man.

FIG. 18Ato FIG. 18L are graphs illustrating elevations observed in subjects treated with SL-279252. Shown are the dose-dependent elevations serum levels of IFNy (FIG. 18A), IFNa (FIG. 18B), IL-27 (FIG. 18C), CCL2 (FIG. 18D), CCL3 (FIG. 18E), CCL4 (FIG. 18F), IL-2 (FIG. 18G), TNFa (FIG. 18H), IL-18 (FIG. 181), IL-15 (FIG. 18J) and IL-6 (FIG. 18K) before and after administration of the indicated doses of SL-279252 are shown. FIG. 18L contains graphs illustrating the elevations of serum levels of IL-6 and IL-10 in subjects treated with SL-279252.

FIG. 19Ato FIG. 19F are graphs illustrating the relation between the serum cytokines levels and the observed Cmax values at different does of SL-279252. The levels of IFNy (FIG. 19A), IL-2 (FIG. 19B), IFNa (FIG. 19C), IL-27 (FIG. 19D), CCL2 (FIG. 19E), and IL-6 (FIG. 19F) were plotted as a function of the observed Cmax values at the indicated doses of SL-279252. These data illustrate that dose-dependent serum cytokine elevations observed in subjects treated with SL-279252.

FIG. 20A and FIG. 20B show the comparison of the pharmacokinetics (PK) of SL-279252 at Cycle 1 Day 1 (C1 D1) and Cycle 1 Day 15 (C1 D15). FIG. 20A shows the pharmacokinetics (PK) of SL-279252 at Cycle 1 Day 1 (C1 D1) following the administration of the indicated dose levels (DL) of SL-279252. FIG. 20A shows the pharmacokinetics (PK) of SL-279252 at Cycle 1 Day 15 (C1 D15) following the administration of the indicated dose levels (DL) of SL-279252. Serum concentrations of SL-279252 at the indicated time postdosing are plotted. As shown, at dose level 6 (DL6; 1 mg/kg dose), SL-279252 was detectable until 96 hours following post-dosing.

FIG. 21 shows a graph illustrating the lack of complement activation in humans following the dosing of SL- 279252. Predose, 1 hour post-dose and 24 hours post dose plasma levels of the terminal complement complex (sC5b9) at Cycle 1 Day 1 (C1D1), Cycle 1 Day 15 (C1D15) and Cycle 2 Day 1 (C2D1) are shown. Shown are the data for individual subjects receiving dose level DL1 (00001 mg/kg), DL2 (0001 mg/kg), DL3 (0003 mg/kg), DL4 (0.01 mg/kg), DL5 (0.03 mg/kg), DL6 (0.1 mg/kg). The dotted line shows the normal range of sC5b9 for healthy adults.

FIG.22A to FIG.22E show graphs of lymphocyte counts in subjects dosed with indicated doses of over time up to Cycle 2 Day 16 (C2D16, Day 42).

FIG. 23A to FIG. 23B show graphs of lymphocyte counts change post dose in comparison to predose for each subject by dose level at Cycle 1 Day 1 (C1D1; FIG. 23A), and Cycle 2 Day 1 (C2D1; FIG. 23B). For FIG.23B, post-dose sample not drawn for subject in DL2; DL4 - subject withdrew due to progressive disease; DL7 - 3 subjects had not reached C2D1 at the time of this experiment. Dose level DL1 was 00001 mg/kg; DL2 was 0001 mg/kg; DL3 was 0003 mg/kg; DL4 was 0.01 mg/kg; DL5 was 0.03 mg/kg; DL6 was 0.1 mg/kg; DL7 was 0.3 mg/kg; and DL8 was 1 mg/kg.

FIG.24A to FIG.24D show the pharmacokinetics (PK) in the indicated subjects 0101 (receiving 00001 mg/kg SL-279252), 0201 (receiving 0 001 mg/kg SL-279252), 0102 (receiving 0 003 mg/kg SL-279252), 0301 (receiving 0003 mg/kg SL-279252), 0302 (receiving 001 mg/kg SL-279252), 0103 (receiving 003 mg/kg SL- 279252), 0203 (receiving 003 mg/kg SL-279252), 0106 (receiving 01 mg/kg SL-279252), 0107 (receiving 0 1 mg/kg SL-279252) , and 0204 (receiving 01 mg/kg SL-279252) at Cycle 1 Day 1 (C1D1; FIG. 24A, FIG. 24C and FIG.24D) or Cycle 1 Day 15 (C1D15; FIG.24B).

FIG.25 shows the summary of pharmacokinetics (PK) endpoints SL-279252 at Cycle 1 Day 1 (C1 D1), Cycle 1 Day 15 (C1 D15), and Cycle 2 Day 1 for subjects 0101 (receiving 00001 mg/kg SL-279252), 0201 (receiving 0001 mg/kg SL-279252), 0102 (receiving 0003 mg/kg SL-279252), 0301 (receiving 0003 mg/kg SL-279252), 0302 (receiving 001 mg/kg SL-279252), 0103 (receiving 003 mg/kg SL-279252), 0203 (receiving 003 mg/kg SL-279252), 0106 (receiving 0 1 mg/kg SL-279252), 0107 (receiving 0 1 mg/kg SL-279252), and 0204 (receiving 01 mg/kg SL-279252).

FIG. 26 shows the receptor occupancy at Cycle 1 Day 1 (C1D1) for all patients that received doses DL1 (0 0001 mg/kg dose), DL2 (0001 mg/kg dose), DL3 (0003 mg/kg dose), DL4 (0.01 mg/kg dose), DL5 (0.03 mg/kg dose), DL6 (0.1 mg/kg dose), DL7 (0.3 mg/kg dose), DL8 (1 mg/kg dose), and DL9 (3 mg/kg dose).

FIG. 27 shows a graph showing the fraction of CD4- X40+ and SL-279252 (ARC)-lymphocyte in all CD3+ cells in peripheral blood as a function of time following the administration of SL-279252 for several patients treated at 0.3, 1, or 3 mg/kg doses. These data illustrate the margination of CD4OX40+ cells in patients treated with SL-279252.

FIG. 28 shows a graph comparing the anti-tumor activity of the murine PD-1-Fc-OX40L in mouse CT26 mouse colon carcinoma allograft model in comparison with checkpoint blocking antibodies alone, costimulator agonist antibodies alone, or a combination of checkpoint blocking and co-stimulator agonist antibodies. Mice were inoculated with CT26 cells. On day 5 and on day 7, the mice were treated with murine PD-1-Fc-OX40L chimeric protein, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-OX40 antibody, combination of the anti-PD-L1 antibody and the anti-OX40 antibody, and combination of the anti-PD-1 antibody and the anti-OX40 antibody. Tumor volumes were plotted as a function of time. Mice that rejected the tumors were counted. Some mice were re-challenged with CT26 cells and tumor rejection was reevaluated. The primary column in the panels below represents the number of mice that rejected the primary tumor. The re-challenge column in the panels below represents the number of mice that rejected the primary tumor and were also capable of rejecting a second tumor challenge without repeat treatment, demonstrating a durable adaptive immune response.

FIG. 29 shows the histochemical analysis of lungs of from untreated and SL-279252-treated monkeys illustrating the migration of lymphocytes. Cynomolgus monkeys were administered 5 consecutive weekly doses of 40 mg/kg SL-279252. Illustrative lung section from a control and SL-279252 treated animal are shown.

FIG. 30 shows the lymphocyte expansion from pre-dose to day 15 in non-human primates. Cynomolgus monkeys were treated on Day 1 and 8 with the indicated dose of SL-279252 or a vehicle control. Total lymphocyte counts were obtained prior to the dose on Day 1, and then again on Day 15. These data demonstrate a dose-dependent expansion in the total number of circulating lymphocytes in cynomolgus macaques following SL-279252 infusion. Each data point indicates an individual animal.

FIG. 31 shows the post-dose lymphocyte margination from day 15 to day 16. Cynomolgus monkeys were treated with SL-279252 on Day 1, 8 and 15 at the indicated dose. Pre- and post-dose lymphocyte counts were obtained on day 15 prior to the third dose, and on day 16 approximately 24 hours after the third dose. The number of peripheral blood lymphocytes was observed to decrease in a dose-dependent manner following the Day 15 dose, and is plotted above as the (100 - ((# of lymphocytes on Day 16) / (# of lymphocytes on Day 15) x 100). Each data point indicates an individual animal. FIG. 32 shows the dose escalation per keyboard design (n = 43). This dosing schedule allowed an accelerated titration with n > 1 subjects per cohort until grade 2 (G2) toxicity was observed or dose level 6 (DL6) was reached. Subjects received intravenous (IV) administration of SL-279252 on Schedule 1 or Schedule 2 until clinical disease progression, unacceptable toxicity or withdrawal of consent took place.

FIG. 33 is a plot showing percentage change in target tumor size from baseline. The dose levels and response are shown using letters and numbers, respectively. Arrows in place of dose level bars indicate continued treatment.

FIG. 34 shows the duration that the patients have stayed on study treatment. DLBCL = diffuse large B cell lymphoma; Esoph Adeno = esophageal adenocarcinoma; GEJ = gastroesophageal junction; MSI-H = microsatellite instability high; NSCLC = non-small cell lung cancer; RCC = renal cell carcinoma; RECIL - response evaluation criteria for lymphomas; SCC = squamous cell carcinoma; SCCAC = squamous cell carcinoma of the anal canal; SCCC = squamous cell carcinoma of the cervix; SCCHN = squamous cell carcinoma of head and neck; SNSCLC = squamous non-small cell lung cancer; SCC skin = squamous cell carcinoma of skin.

FIG. 35A and FIG. 35B show the pharmacokinetics (PK) of SL-279252 at Cycle 1 Day 1 (FIG. 35A), Cycle 1 Day 15 (FIG. 35B) at the indicated doses.

FIG. 36 shows the maximal 0X40 receptor engagement (RE) by dose. Flow cytometry panels were designed to evaluate 0X40 CD4 T cell receptor engagement on subject peripheral blood samples (N=43). Maximal 0X40 receptor engagement at the indicated time point is shown.

FIG. 37 demonstrates that binding of SL-279252 to 0X40+ CD4 T cells is durable. Maximal 0X40 receptor engagement (RE) at Cycle 1 Day 1 (C1 D1 pre) and Cycle 1 Day 15 (C1 D15 pre) are compared at the indicated doses.

FIG. 38 shows the changes in CD8+T cells as a function of dose of SL-279252. Ki67 CD8 central memory T cells (left panel), Ki67 CD8 effector memory T cells (middle panel), and CD8 central memory T cells (right panel) are shown.

FIG. 39A to FIG. 39C show the increased CD8 T cell infiltration in on-treatment biopsies in response to SL- 279252 treatment. FIG. 39A show the CD8 cells in tumor biopsies before and after treatment. FIG. 39B show the CD8⁺ granzyme B⁺ cells in tumor biopsies before and after treatment. FIG. 39C show the CD8⁺ Ki67⁺ cells in tumor biopsies before and after treatment. FIG. 40 shows the increase in CD8/GZMB/Nkp46 in MSI-H CRC Subject dosed at 3 mg/kg.

FIG. 41 A and FIG. 41 B show the dose-dependent binding and margination of CD4- )X40+T cells at Day 1 (FIG. 41 A) or Day 29 (FIG. 41 B) at increasing doses of SL-279252.

DETAILED DESCRIPTION

The present technology is based, in part, on the discovery that chimeric proteins can be engineered from the extracellular, or effector, regions of human programmed cell death protein 1 (PD-1) and human 0X40 Ligand (OX40L). Further, the present technology is based, in part, on the discovery of certain doses of such agents for anti-cancer safety and efficacy in human patients. The present technology is based, in part, on the observation of a gradual increase in Cmax with doses subsequent to first dose of fusion proteins of the present technology, and further improved Cmax with further doses. Accordingly, in some aspects, the present technology relates to biphasic or a three-cycle regimen for treating cancer that maximizes potential clinical efficacy, while avoiding substantial side effects. These, PD-1- and OX40L-based chimeric proteins can simultaneously block immune inhibitory signals and stimulate immune activating signals, at least in the treatment of cancer. The present technology is also based, in part, on the observation of a gradual increase in Cmax with doses subsequent to first dose of fusion proteins of the present technology, and further improved Cmax with further doses. Accordingly, in some aspects, the present technology relates to biphasic or a three-cycle regimen for treating cancer. In a biphasic dosing regimen, the first phase is intended to increase the binding of target receptors (PD-L1 and 0X40) in tumor by the chimeric proteins disclosed herein to therapeutically relevant levels. The second phase is intended to maintain the binding of target receptors (PD-L1 and 0X40) in tumor by the chimeric proteins disclosed herein at therapeutically relevant levels.

The present chimeric proteins provide advantages including, without limitation, ease of use and ease of production. This is because two distinct immunotherapy agents are combined into a single product which may allow for a single manufacturing process instead of two independent manufacturing processes. In addition, administration of a single agent instead of two separate agents allows for easier administration and greater patient compliance. Further, in contrast to, for example, monoclonal antibodies, which are large multimeric proteins containing numerous disulfide bonds and post-translational modifications such as glycosylation, the present chimeric proteins are easier and more cost effective to manufacture. Furthermore, while individual immunotherapy agents may or may not exert therapeutic effects in the place, at the same time, a single agent instead of two separate agents ensures their concerted action at the same microenvironment at the same time. Importantly, since a chimeric protein of the present technology (via binding of the extracellular domain of PD- 1 to its receptor/ligand on a cancer cell) disrupts, blocks, reduces, inhibits, and/or sequesters the transmission of immune inhibitory signals, e.g., originating from a cancer cell that is attempting to avoid its detection and/or destruction, and ( ia binding of OX40L to its receptor) enhances, increases, and/or stimulates the transmission of an immune stimulatory signal to an anti-cancer immune cell, it can provide an anti-tumor effect by two distinct pathways; this dual-action is more likely to provide any anti-tumor effect in a patient and/or to provide an enhanced anti-tumor effect in a patient. Furthermore, since such chimeric proteins can act via two distinct pathways, they can be efficacious, at least, in patients who respond poorly to treatments that target one of the two pathways. Thus, a patient who is a poor responder to treatments acting via one of the two pathway, can receive a therapeutic benefit by targeting the other pathway.

Chimeric Proteins

The chimeric proteins of the present technology comprise an extracellular domain of PD-1 and an extracellular domain of OX40L which together can simultaneously block immune inhibitory signals and stimulate immune activating signals.

Aspects of the present technology provide a chimeric protein comprising a general structure of: N terminus - (a) - (b) - (c) - C terminus, where (a) is a first domain comprising an extracellular domain of PD-1, (b) is a linker adjoining the first domain and the second domain, e.g., the linker comprising at least one cysteine residue capable of forming a disulfide bond and/or comprising a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of OX40L; wherein the linker connects the first domain and the second domain.

In some embodiments, the first domain comprises substantially all of the extracellular domain of PD-1. In some embodiments, the first domain is capable of binding a PD-1 ligand. In some embodiments, the first domain is capable of binding a PD-1 ligand (e.g. PD-L1 and PD-L2) expressed on cancer cell surface. In some embodiments, the first domain is capable of inhibiting the binding of a PD-1 ligand (e.g. PD-L1 and PD- L2) to the PD-1 protein located on T cell surface. In some embodiments, the first domain is capable of inhibiting an immunosuppressive signal. In some embodiments, the first domain is capable of blocking the suppression of the activation of the T cell, which is caused by binding a PD-1 ligand expressed on cancel cell surface to PD-1 protein located on T cell surface. In some embodiments, the second domain is capable of binding an OX40L receptor. In some embodiments, the second domain comprises substantially all of the extracellular domain of OX40L. In some embodiments, the second domain is capable of activating an immune stimulatory signal.

In some embodiments, the chimeric protein is a recombinant fusion protein, e.g., a single polypeptide having the extracellular domains disclosed herein. For example, in embodiments, the chimeric protein is translated as a single unit in a prokaryotic cell, a eukaryotic cell, or a cell-free expression system.

In some embodiments, the present chimeric protein is producible in a mammalian host cell as a secretable and fully functional single polypeptide chain.

In some embodiments, chimeric protein refers to a recombinant protein of multiple polypeptides, e.g., multiple extracellular domains disclosed herein, that are combined ( via covalent or non-covalent bonding) to yield a single unit, e.g., in vitro {e.g., with one or more synthetic linkers disclosed herein).

In some embodiments, the chimeric protein is chemically synthesized as one polypeptide or each domain is chemically synthesized separately and then combined. In some embodiments, a portion of the chimeric protein is translated and a portion is chemically synthesized.

In some embodiments, an extracellular domain refers to a portion of a transmembrane protein which is capable of interacting with the extracellular environment. In some embodiments, an extracellular domain refers to a portion of a transmembrane protein which is sufficient for binding to a ligand or receptor and is effective in transmitting a signal to a cell. In some embodiments, an extracellular domain is the entire amino acid sequence of a transmembrane protein which is normally present at the exterior of a cell or of the cell membrane. In some embodiments, an extracellular domain is that portion of an amino acid sequence of a transmembrane protein which is external of a cell or of the cell membrane and is needed for signal transduction and/or ligand binding as may be assayed using methods know in the art (e.g., in vitro ligand binding and/or cellular activation assays).

Transmembrane proteins typically consist of an extracellular domain, one or a series of transmembrane domains, and an intracellular domain. Without wishing to be bound by theory, the extracellular domain of a transmembrane protein is responsible for interacting with a soluble receptor or ligand or membrane-bound receptor or ligand [i.e., a membrane of an adjacent cell). Without wishing to be bound by theory, the transmembrane domain(s) is responsible for localizing the transmembrane protein to the plasma membrane. Without wishing to be bound by theory, the intracellular domain of a transmembrane protein is responsible for coordinating interactions with cellular signaling molecules to coordinate intracellular responses with the extracellular environment (or visa-versa).

There are generally two types of single-pass transmembrane proteins: Type I transmembrane proteins which have an extracellular amino terminus and an intracellular carboxy terminus (see, FIG. 1A, left protein) and Type II transmembrane proteins which have an extracellular carboxy terminus and an intracellular amino terminus (see, FIG. 1A, right protein). Type I and Type II transmembrane proteins can be either receptors or ligands. For Type I transmembrane proteins (e.g., PD-1), the amino terminus of the protein faces outside the cell, and therefore contains the functional domains that are responsible for interacting with other binding partners (either ligands or receptors) in the extracellular environment (see, FIG. 1B, left protein). For Type II transmembrane proteins (e.g., OX40L), the carboxy terminus of the protein faces outside the cell, and therefore contains the functional domains that are responsible for interacting with other binding partners (either ligands or receptors) in the extracellular environment (see, FIG. 1B, right protein). Thus, these two types of transmembrane proteins have opposite orientations to each other relative to the cell membrane.

Chimeric proteins of the present technology comprise an extracellular domain of PD-1 and an extracellular domain of OX40L. Thus, a chimeric protein of the present technology comprises, at least, a first domain comprising the extracellular domain of PD-1 , which is connected - directly or via a linker- to a second domain comprising the extracellular domain of OX40L. As illustrated in FIG. 1C and FIG. 1D, when the domains are linked in an amino-terminal to carboxy-terminal orientation, the first domain is located on the “left”’ side of the chimeric protein and is “outward facing” and the second domain is located on “right” side of the chimeric protein and is “outward facing”.

Other configurations of first and second domains are envisioned, e.g., the first domain is outward facing and the second domain is inward facing, the first domain is inward facing and the second domain is outward facing, and the first and second domains are both inward facing. When both domains are “inward facing”, the chimeric protein would have an amino-terminal to carboxy-terminal configuration comprising an extracellular domain of OX40L, a linker, and an extracellular domain of PD-1. In such configurations, it may be necessary for the chimeric protein to include extra “slack”, as described elsewhere herein, to permit binding domains of the chimeric protein to one or both of its receptors/ligands.

Constructs could be produced by cloning of the nucleic acids encoding the three fragments (the extracellular domain of PD-1 , followed by a linker sequence, followed by the extracellular domain of OX40L) into a vector (plasmid, viral or other) wherein the amino terminus of the complete sequence corresponded to the ‘left’ side of the molecule containing the extracellular domain of PD-1 and the carboxy terminus of the complete sequence corresponded to the ‘right’ side of the molecule containing the extracellular domain of OX40L. In some embodiments of chimeric proteins having one of the other configurations, as described above, a construct would comprise three nucleic acids such that the translated chimeric protein produced would have the desired configuration, e.g., a dual inward-facing chimeric protein. Accordingly, in some embodiments, the present chimeric proteins are engineered as such.

Programmed cell death protein 1, or PD-1, is a cell surface protein present on T cells and other white blood cells. It binds to two ligands, PD-L1 and PD-L2, which can be expressed by tumor cells as well as other immune cells in the tumor microenvironment. When PD-L1 binds to PD-1 , the resulting PD-1 signaling limits the capacity of T cells to kill tumor cells. Anti-PD-1 antibodies disrupt binding of PD-1 to PD-L1 to restore baseline tumor cell-killing activity of T cells. While anti-PD-1/PD-L1 antibodies have achieved significant clinical and commercial success, a majority of patients with cancer do not benefit from this class of therapy, as evidenced by a response rate of 35% or less in patients with melanoma, NSCLC, bladder cancer, HNSCC, and other cancers. A limitation of anti-PD-1/PD-L1 antibodies is their inability to provide a signal that directly amplifies the ability of T cells to kill tumor cells. Achieving this enhanced tumor-killing effect necessitates the introduction of a distinct mechanism to complement checkpoint blockade. One such approach is the stimulation of costimulatory receptors. Most current approaches attempt to simultaneously exploit both pathways by co-administering anti-PD-1/PD-L1 antibodies with costimulatory receptor agonists. However, these attempts have not been successful in clinical trials, which may be is due to the structural mismatch between existing bivalent antibodies and the trimeric TNF receptor superfamily.

PD-L1 and PD-L2 can be expressed by tumor cells (and also other immune cells in the tumor microenvironment) and bind to PD-1 receptor expressed by tumor infiltrating lymphocytes (FIG. 5B, left panel). When this occurs, PD-1 signaling in lymphocytes, including T cells, limits the capacity of those T cells to kill tumor cells. Inhibition of the PD-1 receptor on lymphocytes by PD-1 (or PD-L1) blocking antibodies (including pembrolizumab (KEYTRUDA)and nivulomab (OPDIVO)) limits PD-L1 binding to PD-1, and thus maintains the baseline capacity of T cells to kill tumor cells (FIG. 5B, middle panel). While other programs sought to block PD-1 and activate 0X40 signaling by administering multiple therapeutics, SL-279252 seeks to do so within a single therapeutic. Importantly, unlike the bivalent structure of existing antibodies, the hexameric structure of SL-279252 is designed to effectively trimerize and directly activate 0X40 receptors. In contrast, FIG. 5B shows the proposed mechanism of action of SL-279252, without bound by theory. Without bound by theory, SL-279252 functions both to block PD-L1 binding to PD-1 (analogous to the activity of PD-1/L1 blocking antibodies) and also stimulates 0X40 receptors on T cells, leading to increased activation and tumor cell killing capacity. As disclosed herein, in preclinical studies, SL-279252 was found to be a highly potent stimulator of an adaptive immune response, and also demonstrated greater anti-tumor activity than anti-PD-1 antibodies or 0X40- agonist antibodies, either alone or in combination.

PD-1-FC-OX40L Chimeric Protein

In some embodiments, the chimeric protein is capable of contemporaneously binding the human PD-1 ligand and the human OX40L receptor, wherein the PD-1 ligand is PD-L1 or PD-L2 and the OX40L receptor is 0X40.

The chimeric protein has a general structure of: N terminus - (a) - (b) - (c) - C terminus, in which (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L). In some embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond.

Chimeric proteins of the present technology have a first domain which is sterically capable of binding its ligand/receptor and/or a second domain which is sterically capable of binding its ligand/receptor. This means that there is sufficient overall flexibility in the chimeric protein and/or physical distance between an extracellular domain (or portion thereof) and the rest of the chimeric protein such that the ligand/receptor binding domain of the extracellular domain is not sterically hindered from binding its ligand/receptor. This flexibility and/or physical distance (which is herein referred to as “slack”) may be normally present in the extracellular domain(s), normally present in the linker, and/or normally present in the chimeric protein (as a whole). Alternately, or additionally, the chimeric protein may be modified by including one or more additional amino acid sequences (e.g., the joining linkers described below) or synthetic linkers (e.g., a polyethylene glycol (PEG) linker) which provide additional slack needed to avoid steric hindrance.

In some embodiments, the chimeric proteins of the present technology comprise variants of the extracellular domain of PD-1. As examples, the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of PD-1 , e.g., human PD-1.

In some embodiments, the extracellular domain of PD-1 has the following amino acid sequence:

LDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQP

GQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH

PSPSPRPAGQFQ (SEQ ID NO: 57).

In some embodiments, a chimeric protein comprises a variant of the extracellular domain of PD-1. As examples, the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 57.

In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 57. One of ordinary skill may select variants of the known amino acid sequence of PD-1 by consulting the literature, e.g. Zhang et al. “Structural and Functional Analysis of the Costimulatory Receptor Programmed Death-1” Immunity. Mar; 20(3):337-47 (2004); Lin et al. “The PD-1/PD-L1 complex resembles the antigenbinding Fv domains of antibodies and T cell receptors”, Proc Natl Acad Sci U SA. 105(8):3011 -6 (2008); Zak et al. “Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1”, St cture. 23(12):2341 -2348 (2015); and Cheng ef al. “Structure and Interactions of the Human Programmed Cell Death 1 Receptor”, JBiol Chem. 288(17): 11771 -85 (2013), each of which is incorporated by reference in its entirety.

In some embodiments, the chimeric proteins of the present technology comprise variants of the extracellular domain of OX40L. As examples, the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of OX40L, e.g., human OX40L.

In some embodiments, the extracellular domain of OX40L has the following amino acid sequence:

QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHY

QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL

(SEC ID NO: 58).

In some embodiments, a chimeric protein comprises a variant of the extracellular domain of OX40L. As examples, the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 58.

In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 58.

One of ordinary skill may select variants of the known amino acid sequence of OX40L by consulting the literature, e.g. Croft, et al, "The Significance of 0X40 and OX40L to T cell Biology and Immune Disease," Immunol Rev., 229(1): 173-191 (2009); and Baum, et al., "Molecular characterization of murine and human 0X40/0X40 ligand systems: identification of a human 0X40 ligand as the HTLV-1 -regulated protein gp34," The EMBO Journal, 13(77): 3992-4001 (1994), each of which is incorporated by reference in its entirety.

In some embodiments, the linker of a chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker of a chimeric protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker of a chimeric protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker of a chimeric protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker of a chimeric protein comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, a chimeric protein of the present technology comprises: (1) a first domain comprising the amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, a chimeric protein of the present technology comprises: (1) a first domain comprising the amino acid sequence that is at least 95% identical to SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence that is at least 95% identical to SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, a chimeric protein of the present technology comprises: (1) a first domain comprising the amino acid sequence that is at least 97% identical to SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence that is at least 97% identical to SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is that is at least 97% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, a chimeric protein of the present technology comprises: (1) a first domain comprising the amino acid sequence that is at least 98% identical to SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence that is at least 98% identical to SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, a chimeric protein of the present technology comprises: (1) a first domain comprising the amino acid sequence that is at least 99% identical to SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence that is at least 99% identical to SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, a chimeric protein of the present technology comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, a chimeric protein of the present technology comprises: (1) a first domain comprising the amino acid sequence identical to SEQ ID NO: 57, (b) a second domain comprises the amino acid sequence that is to SEQ ID NO: 58, and (c) a linker comprises an amino acid sequence that is that is identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, a PD-1-Fc-OX40L chimeric protein of the present technology has the following amino acid sequence:

LDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPG QDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPS

PSPRPAGQFQSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVWDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKIEGRMDQVSHRYPRIQSIKVQFTEYKKEK GFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL

TYKDKVYLN DNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 59).

In some embodiments, the chimeric protein of the present technology comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 potential N glycosylation sites (exemplary potential N-glycosylation sites present in SEQ ID NO: 59 are shown in bold above; see also FIG. 3D). In some embodiments, the chimeric protein of the present technology comprises at least two potential N glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least four potential N glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least six potential N glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least eight potential N glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least ten potential N glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least 1 , 2, 3, 4, 5, 6, 7, or 8 potential 0 glycosylation sites (exemplary potential O-glycosylation sites present in SEQ ID NO: 59 are shown in bold underlined font above). In some embodiments, the chimeric protein of the present technology comprises at least two potential 0 glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least four potential 0 glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least six potential 0 glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least eight potential 0 glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least two potential N glycosylation sites, and at least two potential 0 glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least four potential N glycosylation sites, and at least four potential 0 glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least six potential N glycosylation sites, and at least six potential 0 glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least eight potential N glycosylation sites, and at least eight potential 0 glycosylation sites. In some embodiments, the chimeric protein of the present technology comprises at least ten potential N glycosylation sites, and at least eight potential 0 glycosylation sites. In some embodiments, the chimeric protein expressed in Chinese Hamster Ovary (CHO) cells is glycosylated.

In some embodiments, the chimeric protein of the present technology comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 potential N glycosylation sites.

There are eleven cysteines present in the SL-279252 chimeric protein. In some embodiments, the SL-279252 chimeric protein has no disulfide bonds. In some embodiments, the SL-279252 chimeric protein has no disulfide bond between C30=C69. In some embodiments, the SL-279252 chimeric protein has at least one, or at least two, or at least 3, or at least 4, or at least 5 or six disulfide bonds. In some embodiments, the SL- 279252 chimeric protein has at least one, or at least two interchain disulfide bonds. In some embodiments, the SL-279252 chimeric protein has at least one, or at least two, or at least 3 intrachain disulfide bonds. In some embodiments, the SL-279252 chimeric protein has a C150=C150 interchain disulfide bond. In some embodiments, the SL-279252 chimeric protein has a C153=C153 interchain disulfide bond. In some embodiments, the SL-279252 chimeric protein has a C185=C245 disulfide bond. In some embodiments, the SL-279252 chimeric protein has a C291 =C349 disulfide bond. In some embodiments, the SL-279252 chimeric protein has a C424=C508 disulfide bond.

In some embodiments, the molecular formula of the SL-279252 chimeric protein, predicted from the amino acid sequence, is C2562H3973N695O782S18. The predicted molecular weight for the monomeric chimeric protein of SEQ ID NO: 59 is 57.6 kDa. The predicted molecular weight for the glycosylated monomeric chimeric protein of SEQ ID NO: 59 is 136 kDa.

In some embodiments, the PD-1-Fc-OX40L chimeric protein of the present technology is encoded by the following nucleotide sequence (leader sequence is shown by a bold-underlined font):

ATGGAATGGTCTTGGGTCTTTCTGTTCTTTCTTTCTGTGACAACCGGCGTGCACTCCCTCGAC

T CGCCGGACCGGCCCT GGAACCCT CCAACTTT CT CACCCGCCCT GCT GGT CGT GACCGAGG GAGACAACGCGACTTT CACCT GTT CGTT CT CCAACACCT CCGAAT CCTT CGT GCT GAACT GGT ACCGGATGTCCCCTAGCAATCAGACCGATAAGCTGGCCGCTTTTCCGGAGGATAGGAGCCAG CCGGGCCAAGATTGCCGCTT CCGCGT GACT CAGCT GCCGAACGGCAGAGACTT CCACAT GT C CGT CGT GCGGGCGCGCCGGAACGACT CCGGAACCT AT CT CT GCGGAGCCATTT CCCT GGCC CCGAAGGCACAGAT CAAGGAGT CGCT GAGAGCT GAGCT GAGGGT CACCGAAAGACGGGCCG AGGT CCCT ACCGCCCACCCCT CCCCCT CCCCT CGCCCGGCCGGCCAGTT CCAGAGCAAAT A CGGGCCGCCCT GCCCT CCCT GCCCGGCGCCGGAGTT CCT GGGCGGTCCT AGCGT GTT CCTG TT CCCACCCAAGCCCAAGGAT CAGCTT AT GAT CT CCCGGACCCCCGAAGT GACCT GT GT GGT GGT GGACGT GT CCCAAGAGGACCCT GAAGT GCAGTT CAATT GGT ACGT GGAT GGCGT GGAAG T GCACAACGCCAAGACTAAGCCT CGCGAAGAACAGTT CAACAGCACCT ACAGAGT GGTGTCC GT GTT GACCGT GCT GCACCAAGACT GGCTGT CGGGCAAAGAGT ACAAGT GCAAGGT GTCCTC CAAGGGCCT CCCGT CGT CAAT CGAAAAGACCATT AGCAACGCAACCGGACAGCCCCGAGAAC CACAGGT CT ACACCTT GCCGCCAAGCCAGGAGGAAAT GACT AAGAACCAGGT GT CCCT GACT T GCCT CGT GAAGGGATTTT ACCCT AGCGACAT CGCCGT GGAAT GGGAGT CCAACGG ACAGCC TGAAAACAACTATAAGACTACGCCCCCCGTGCTGGATTCCGACGGAAGCTTCTTCCTGTACTC CCGGCT CACCGT GGAT AAGT CACGGT GGCAGGAAGGGAACGT GTT CT CAT GCTCGGT CCT GC ACGAAGCCCTT CAT AACCACT ACACGCAGAAAT CGCT GAGCCT GT CCCT GGGGAAGAT CGAG GGAAGGAT GGACCAAGT GT CGCAT CGGT ACCCACGGATT CAGT CCAT CAAGGT GCAGTT CAC CGAGT ACAAGAAGGAGAAGGGTTT CATT CT GACT AGCCAGAAAGAGGACGAGAT CAT GAAGG T CCAGAACAATT CCGT GAT CAT CAACT GT GACGGATT CT ACCT GATT AGCCT GAAGGGTTACTT CT CACAAGAAGT GAACATTT CACT GCATT ACCAGAAGGACGAGGAGCCGCT GTT CCAATT GAA GAAGGT CCGCT CCGT GAACT CCCT GAT GGT CGCGT CACT CACTT ACAAGGACAAGGT CT ACC T GAACGT GACCACCGACAAT ACCT CCCT CGACGATTT CCACGT GAACGGCGGAGAACTT AT C CT CAT CCACCAAAACCCGGGGGAGTT CTGCGTGCT CT AG (SEQ ID NO: 60)

In some embodiments, the SEQ ID NO: 60 encodes for a precursor of the PD-1-Fc-OX40L chimeric protein of the present technology having following amino acid sequence (leader sequence is shown by an italic font, the extracellular domain of PD-1 is shown by an underlined font, the extracellular domain of OX40L is indicated by a bold-face font, and an Fc domain is shown in a bold-underlined font):

MEl/l/StWFLFFLSVTTGUHSLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSESFVLNWYR

MSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQ

IKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQ LMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWL

SGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL SLGKIEGRMDQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKG YFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILI HQNPGEFCVL (SEQ NO: 61).

The chimeric protein of SEQ ID NO: 59 or SEQ ID NO: 61 (also referred to herein as SL-279252) is a recombinant fusion glycoprotein comprising the extracellular domain of human PD-1 (PDCD1, CD279), a central domain including the hinge-CH2-CH3 region from human immunoglobulin constant gamma 4 (IGHG4, lgG4), and the extracellular domain of human OX40L (TNFSF4, CD252). The linear configuration of SL- 279252 is PD-1-FC-OX40L The tertiary structure of SL-279252, predicted by RaptorX, and without wishing to be bound by theory, is shown in FIG. 3A.

The dual-sided nature of the chimeric proteins disclosed herein, such as the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 or SEQ ID NO: 61), is designed to intercept one of the key immunosuppressive pathways within the tumor microenvironment (TME): the PD-1 - programmed cell death ligand 1 /programmed cell death-ligand 2 axis (PD-L1/PD-L2).

Tumor cells may express PD-L1 (or PD-L2) on their cell surface, which can bind to PD-1 expressed by a T cell to suppress activation of the T cell (FIG. 4A and FIG. 4B). Thus, the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 or SEQ ID NO: 61) can bind to PD-L1 and PD-L2 expressed on the surface of tumor and antigen presenting cells, with the PD-1 domain of the PD-1-Fc-OX40L chimeric proteins disclosed herein intended to provide competitive inhibition of PD-L1 , and to replace the PD-L1 inhibitory signal with functionally trimerized/hexamerized OX40L, resulting in an incoming T cell experiencing co-stimulation via engagement through its 0X40 receptor instead of suppression through PD-1 interactions. In other words, because the extracellular domains (ECDs) of PD-1 and OX40L are physically linked to one another and localized to the TME, tumor infiltrating T cells will receive co-stimulation at the same time they recognize a tumor antigen via its T cell receptor (TCR). Importantly, because the ECDs of PD-1 and OX40L are physically linked to one another, and localized to the TME, tumor infiltrating T cells will receive costimulation at the same time they recognize a tumor antigen via the T cell receptor. Together, these would result in replacement of an inhibitory PD-L1 signal with a co-stimulatory OX40L signal to enhance the anti-tumor activity of T cells. The three constituent components of the chimeric proteins disclosed herein, including the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 or SEQ ID NO: 61), have unique attributes that facilitate dimerization or oligomerization. The extracellular domain of PD-1 normally exists as a monomer and is not known to form higher-order homomeric complexes. The central Fc domain contains cysteine residues that are capable of disulfide bonding to form a dimeric structure. In some embodiments, the chimeric proteins disclosed herein, including the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59), contains an S228P mutation in the hinge region of the Fc domain to prevent Fab arm exchange. The OX40L domain is known to form homotrimeric complexes, which are stabilized through noncovalent, electrostatic interactions. Although the chimeric proteins disclosed herein, including the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61), are expressed as a continuous monomeric protein by production cell lines, the resulting monomeric proteins self-assemble into higher-order species based on these disulfide and charge-based interactions of OX40L (creating a trimer) and the combined influence of these attractive forces, resulting in a hexamer (dimer of trimers). The majority (>80%) of the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) comprises the hexamer and trimer forms, which have similar activity. Importantly, because the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61), are comprised of hexamers and trimers, they stimulate 0X40 signaling in the absence of cross-linking by Fc receptors or any other cross-linking agent. The predicted tertiary structures of the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) as a monomer and in various oligomeric states, based on disulfide (Fc) and charge-based (OX40L) interactions are illustrated in FIG. 3B. FIG. 3C shows visualization by electron microscopy of the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) hexamers (top two images) and the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) trimers (bottom two images).

It is noteworthy that, unlike monoclonal antibodies, Fc receptor cross-linking is not required for functional activity of the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61). As shown in FIG. 5A (top left panel), a pair of anti-OX40 antibodies bind two different 0X40 receptors and each Fab portion binds with high affinity. Flowever, 0X40 receptors cannot be trimerized by two antibodies in the absence of cross-linking; therefore, no signaling occurs. As shown in FIG. 5A (top right panel), two antibodies are cross- linked by FcR (Fc Receptors; expressed by an accessory cell) leading to 0X40 trimer formation; of course, the T cell pictured must also recognizing a tumor antigen, via its T cell Receptor (TCR) in order for 0X40 signaling to be permissive. As shown in FIG. 5A (bottom left panel), if antibody doses are too high, then both FcR and 0X40 can become saturated which prevents cross-linking. This may explain why the activity of certain agonist antibodies is paradoxically reduced as the dose level increases or when they are combined with other antibodies that compete for Fc receptor binding. In contrast, as shown in FIG. 5A (bottom right panel), the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) forms trimers/hexamers and activates OX 40 without the need for cross-linking.

As shown in FIG. 5B (left panel), PD-L1 and PD-L2 can be expressed by tumor cells (and also other immune cells in the tumor microenvironment) and bind to PD-1 receptor expressed by tumor infiltrating lymphocytes (left panel). When this occurs, PD-1 signaling in lymphocytes, including T cells, limits the capacity of those T cells to kill tumor cells. As shown in FIG.5B (middle panel), the inhibition of the PD-1 receptor on lymphocytes by PD-1 (or PD-L1) blocking antibodies (including pembrolizumab (KEYTRUDA)and nivulomab (OPDIVO)) can limit PD-L1 binding to PD-1, and thus maintaining the baseline capacity of T cells to kill tumor cells (middle panel). On the other hand, as shown in FIG. 5B (right panel), the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) can simultaneously function both to block PD-L1 binding to PD-1 (analogous to the activity of PD-1/L1 blocking antibodies) and also to stimulate 0X40 receptors on T cells, leading to increased activation and tumor cell killing capacity.

In some embodiments, a chimeric protein comprises a variant of the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61). As examples, the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 99.2%, or at least about 99.4%, or at least about 99.6%, or at least about 99.8% sequence identity with SEQ ID NO: 59 OR SEQ ID NO: 61.

In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 59 OR SEQ ID NO: 61.

In some embodiments, the first domain is capable of binding a PD-1 ligand.

In some embodiments, the first domain comprises substantially all of the extracellular domain of PD-1. In some embodiments, the second domain is capable of binding an OX40L receptor.

In some embodiments, the second domain comprises substantially all of the extracellular domain of OX40L

In some embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from lgG4, e.g., human lgG4.

In some embodiments, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ

ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, the first domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain of a chimeric protein comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 57.

In some embodiments, the second domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

58. In some embodiments, the second domain of a chimeric protein comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 58.

In some embodiments, the (a) the first domain comprises the amino acid sequence of SEQ ID NO: 57, (b) the second domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) the linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, the chimeric protein further comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5 and/or SEQ ID NO: 7. In some embodiments, the chimeric protein further comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5 and/or SEQ ID NO: 7. In some embodiments, the chimeric protein further comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5 and/or SEQ ID NO: 7. In some embodiments, the chimeric protein further comprises the amino acid sequence of SEQ ID NO: 5 and/or SEQ ID NO: 7.

In some embodiments, the chimeric protein comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 59 OR SEQ ID NO: 61 , e.g., at least about 98% identical to SEQ ID NO: 59 OR SEQ ID NO: 61, at least about 99% identical to SEQ ID NO: 59 OR SEQ ID NO: 61, at least about 99.2% identical to SEQ ID NO: 59 OR SEQ ID NO: 61 , at least about 99.4% identical to SEQ ID NO: 59 OR SEQ ID NO: 61 , at least about 99.6% identical to SEQ ID NO: 59 OR SEQ ID NO: 61, or at least about 99.8% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 59 OR SEQ ID NO: 61.

In any herein-disclosed aspect and embodiment, the chimeric protein may comprise an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences disclosed herein. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

In some embodiments, the substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, /V-formylmethionine b-alanine, GABA and d-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general).

Mutations may also be made to the nucleotide sequences of the chimeric proteins by reference to the genetic code, including taking into account codon degeneracy.

In some embodiments, a chimeric protein is capable of binding human ligand(s)/receptor(s).

In some embodiments, each extracellular domain (or variant thereof) of the chimeric protein binds to its cognate receptor or ligand with a KD of about 1 nM to about 5 nM, for example, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM. In some embodiments, the chimeric protein binds to a cognate receptor or ligand with a KD of about 5 nM to about 15 nM, for example, about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM, about 9.5 nM, about 10 nM, about 10.5 nM, about 11 nM, about 11.5 nM, about 12 nM, about 12.5 nM, about 13 nM, about 13.5 nM, about 14 nM, about 14.5 nM, or about 15 nM.

In some embodiments, each extracellular domain (or variant thereof) of the chimeric protein binds to its cognate receptor or ligand with a KD of less than about 1 mM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 150 nM, about 130 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, or about 5 nM, or about 1 nM (as measured, for example, by surface plasmon resonance or biolayer interferometry).

In some embodiments, the chimeric protein binds to human PD-L1 or PD-L2 with a KD of about 1 nM to about 5 nM, for example, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM. In some embodiments, the chimeric protein binds to human PD-L1 with a KD of less than about 3 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (as measured, for example, by surface plasmon resonance or biolayer interferometry). In some embodiments, the chimeric protein binds to human PD-L2 with a KD of less than about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (as measured, for example, by surface plasmon resonance or biolayer interferometry).

In some embodiments, the chimeric protein binds to human 0X40 with a KD of less than about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (as measured, for example, by surface plasmon resonance or biolayer interferometry).

As used herein, a variant of an extracellular domain is capable of binding the receptor/ligand of a native extracellular domain. For example, a variant may include one or more mutations in an extracellular domain which do not affect its binding affinity to its receptor/ligand; alternately, the one or more mutations in an extracellular domain may improve binding affinity for the receptor/ligand; or the one or more mutations in an extracellular domain may reduce binding affinity for the receptor/ligand, yet not eliminate binding altogether. In some embodiments, the one or more mutations are located outside the binding pocket where the extracellular domain interacts with its receptor/ligand. In some embodiments, the one or more mutations are located inside the binding pocket where the extracellular domain interacts with its receptor/ligand, as long as the mutations do not eliminate binding altogether. Based on the skilled artisan’s knowledge and the knowledge in the art regarding receptor-ligand binding, s/he would know which mutations would permit binding and which would eliminate binding.

In some embodiments, the chimeric protein exhibits enhanced stability and protein half-life.

A chimeric protein of the present technology may comprise more than two extracellular domains. For example, the chimeric protein may comprise three, four, five, six, seven, eight, nine, ten, or more extracellular domains. A second extracellular domain may be separated from a third extracellular domain via a linker, as disclosed herein. Alternately, a second extracellular domain may be directly linked (e.g., via a peptide bond) to a third extracellular domain. In some embodiments, a chimeric protein includes extracellular domains that are directly linked and extracellular domains that are indirectly linked via a linker, as disclosed herein.

Linkers

In some embodiments, the chimeric protein comprises a linker. In some embodiments, the linker comprising at least one cysteine residue capable of forming a disulfide bond. The at least one cysteine residue is capable of forming a disulfide bond between a pair (or more) of chimeric proteins. Without wishing to be bound by theory, such disulfide bond forming is responsible for maintaining a useful multimeric state of chimeric proteins. This allows for efficient production of the chimeric proteins; it allows for desired activity in vitro and in vivo.

In a chimeric protein of the present technology, the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence.

In some embodiments, the linker is derived from naturally-occurring multi-domain proteins or is an empirical linker as described, for example, in Chichili etai., Protein Sci. 22(2): 153-167 (2013); Chen etai., Adv Drug DelivRev. 65(10): 1357-1369 (2013), the entire contents of which are hereby incorporated by reference. In some embodiments, the linker may be designed using linker designing databases and computer programs such as those described in Chen et al., Adv Drug Deliv Rev. 65(10): 1357-1369 (2013); and Crasto et al., Protein Eng. 13(5):309-312 (2000), the entire contents of which are hereby incorporated by reference.

In some embodiments, the linker comprises a polypeptide. In some embodiments, the polypeptide is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long. For example, the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11 , about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.

In some embodiments, the linker is flexible.

In some embodiments, the linker is rigid.

In some embodiments, the linker is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines).

In some embodiments, the linker comprises a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1, and lgA2)). The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of lgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. lgG2 has a shorter hinge than lgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of lgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the lgG2 molecule. lgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the lgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In lgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in lgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of lgG4 is shorter than that of lgG1 and its flexibility is intermediate between that of lgG1 and lgG2. The flexibility of the hinge regions reportedly decreases in the order lgG3>lgG1>lgG4>lgG2. In some embodiments, the linker may be derived from human lgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.

According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., Immunological Reviews 130:87 (1992). The upper hinge region includes amino acids from the carboxyl end of Cm to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id. The core hinge region of wild-type human lgG1 contains the sequence CPPC (SEQ ID NO: 24) which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. In some embodiments, the present linker comprises, one, or two, or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1 and lgA2)). The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, lgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin. In some embodiments, the linker of the present technology comprises one or more glycosylation sites.

In some embodiments, the linker comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1 and lgA2)).

In a chimeric protein of the present technology, the linker comprises a hinge-CH2-CH3 Fc domain derived from lgG4. In some embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human lgG4. In some embodiments, the linker comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3 (e.g., at least 95% identical to the amino acid sequence of SEQ ID NO: 2.). In some embodiments, the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 4-50 (or a variant thereof). In some embodiments, the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 4-50 (or a variant thereof); wherein one joining linker is N terminal to the hinge-CH2-CH3 Fc domain and another joining linker is C terminal to the hinge-CH2-CH3 Fc domain.

In some embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human lgG1 antibody. In some embodiments, the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn). In some embodiments, the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present chimeric proteins.

In some embodiments, the Fc domain in a linker contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311, 416, 428, 433, or 434 (in accordance with Kabat numbering, as in as in Kabat, etal., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference), or equivalents thereof. In some embodiments, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In some embodiments, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine. In some embodiments, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In some embodiments, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In some embodiments, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In some embodiments, the amino acid substitution at amino acid residue 309 is a substitution with proline. In some embodiments, the amino acid substitution at amino acid residue 311 is a substitution with serine. In some embodiments, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine. In some embodiments, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In some embodiments, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In some embodiments, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine. In some embodiments, the amino acid substitution at amino acid residue 416 is a substitution with serine. In some embodiments, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In some embodiments, the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine. In some embodiments, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine.

In some embodiments, the Fc domain linker (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference). In some embodiments, the IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation. In some embodiments, the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation. In some embodiments, the IgG constant region includes an YTE and KFH mutation in combination.

In some embodiments, the linker comprises an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference). Illustrative mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In some embodiments, the IgG constant region comprises a M428L/N434S mutation or LS mutation. In some embodiments, the IgG constant region comprises a T250Q/M428L mutation or QL mutation. In some embodiments, the IgG constant region comprises an N434A mutation. In some embodiments, the IgG constant region comprises a T307A/E380A/N434A mutation or AAA mutation. In some embodiments, the IgG constant region comprises an I253A/H310A/H435A mutation or IHH mutation. In some embodiments, the IgG constant region comprises a H433K/N434F mutation. In some embodiments, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination.

Additional exemplary mutations in the IgG constant region are described, for example, in Robbie, et al., Antimicrobial Agents and Chemotherapy 57(12):6147-6153 (2013); Dall’Acqua et al, Journal Biol Chem 281 (33):23514-24 (2006); Dall’Acqua et al., Journal of Immunology 169:5171-80 (2002); Ko et al. Nature 514:642-645 (2014); Grevys et al. Journal of Immunology 194(11 ):5497-508 (2015); and U.S. Patent No. 7,083,784, the entire contents of which are hereby incorporated by reference.

An illustrative Fc stabilizing mutant is S228P. Illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311 S and the present linkers may comprise 1 , or 2, or 3, or 4, or 5 of these mutants.

In some embodiments, the chimeric protein binds to FcRn with high affinity. In some embodiments, the chimeric protein may bind to FcRn with a KD of about 1 nM to about 80 nM. For example, the chimeric protein may bind to FcRn with a KD of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, or about 80 nM. In some embodiments, the chimeric protein may bind to FcRn with a KD of about 9 nM. In some embodiments, the chimeric protein does not substantially bind to other Fc receptors {i.e. other than FcRn) with effector function.

In some embodiments, the Fc domain in a linker has the amino acid sequence of SEQ ID NO: 1 (see Table 1, below), or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto. In some embodiments, mutations are made to SEQ ID NO: 1 to increase stability and/or half-life. For instance, in embodiments, the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 2 (see Table 1, below), or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto. For instance, in embodiments, the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 3 (see Table 1, below), or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto.

Further, one or more joining linkers may be employed to connect an Fc domain in a linker (e.g., one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto) and the extracellular domains. For example, any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or variants thereof may connect an extracellular domain as disclosed herein and an Fc domain in a linker as disclosed herein. Optionally, any one of SEQ ID NOs: 4 to 50, or variants thereof are located between an extracellular domain as disclosed herein and an Fc domain as disclosed herein.

In some embodiments, the present chimeric proteins may comprise variants of the joining linkers disclosed in Table 1, below. For instance, a linker may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 4 to 50.

In some embodiments, the first and second joining linkers may be different or they may be the same.

Without wishing to be bound by theory, including a linker comprising at least a part of an Fc domain in a chimeric protein, helps avoid formation of insoluble and, likely, non-functional protein concatemers and/or aggregates. This is in part due to the presence of cysteines in the Fc domain which are capable of forming disulfide bonds between chimeric proteins.

In some embodiments, a chimeric protein may comprise one or more joining linkers, as disclosed herein, and lack a Fc domain linker, as disclosed herein.

In some embodiments, the first and/or second joining linkers are independently selected from the amino acid sequences of SEQ ID NOs: 4 to 50 and are provided in Table 1 below:

Table 1: Illustrative linkers (Fc domain linkers and joining linkers)

n some embodiments, the joining linker substantially comprises glycine and serine residues {e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines). For example, in embodiments, the joining linker is (Gly4Ser)_n, where n is from about 1 to about 8, e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO: 25 to SEQ ID NO: 9, respectively). In some embodiments, the joining linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 33). Additional illustrative joining linkers include, but are not limited to, linkers having the sequence LE, (EAAAK)_n (n=1-3) (SEQ ID NO: 36 to SEQ ID NO: 38), A(EAAAK)_nA (n = 2-5) (SEQ ID NO: 39 to SEQ ID NO: 42), A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 43), PAPAP (SEQ ID NO: 44), KESGSVSSEQLAQFRSLD (SEQ ID NO: 45), GSAGSAAGSGEF (SEQ ID NO: 46), and (XP)_n, with X designating any amino acid, e.g., Ala, Lys, or Glu. In some embodiments, the joining linker is GGS. In some embodiments, a joining linker has the sequence (Gly)_n where n is any number from 1 to 100, for example: (Gly)s (SEQ ID NO: 34) and (Gly)e (SEQ ID NO: 35).

In some embodiments, the joining linker is one or more of GGGSE (SEQ ID NO: 47), GSESG (SEQ ID NO: 48), GSEGS (SEQ ID NO: 49), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 50), and a joining linker of randomly placed G, S, and E every 4 amino acid intervals. In some embodiments, the chimeric protein comprises a joining linker comprising the amino acid sequence of SEQ ID NO: 5 and/or SEQ ID NO: 7.

In some embodiments, where a chimeric protein comprises an extracellular domain (ECD) of PD-1, one joining linker preceding an Fc domain, a second joining linker following the Fc domain, and an ECD of OX40L, the chimeric protein may comprise the following structure: ECD of human PD-1 - Joining Linker 1 - Fc Domain - Joining Linker 2 - ECD of human OX40L

The combination of a first joining linker, an Fc Domain linker, and a second joining linker is referend to herein as a “modular linker”. In some embodiments, a chimeric protein comprises a modular linker as shown in

Table 2:

TABLE 2: Illustrative modular linkers

n some embodiments, the present chimeric proteins may comprise variants of the modular linkers disclosed in Table 2, above. For instance, a linker may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 51 to 56.

In some embodiments, the linker may be flexible, including without limitation highly flexible. In some embodiments, the linker may be rigid, including without limitation a rigid alpha helix. Characteristics of illustrative joining linkers is shown below in Table 3: TABLE 3: Characteristics of illustrative joining linkers

In some embodiments, the linker may be functional. For example, without limitation, the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the present chimeric protein. In another example, the linker may function to target the chimeric protein to a particular cell type or location.

In some embodiments, a chimeric protein comprises only one joining linkers.

In some embodiments, a chimeric protein lacks joining linkers.

In some embodiments, the linker is a synthetic linker such as polyethylene glycol (PEG).

In some embodiments, a chimeric protein has a first domain which is sterically capable of binding its ligand/receptor and/or the second domain which is sterically capable of binding its ligand/receptor. Thus, there is enough overall flexibility in the chimeric protein and/or physical distance between an extracellular domain (or portion thereof) and the rest of the chimeric protein such that the ligand/receptor binding domain of the extracellular domain is not sterically hindered from binding its ligand/receptor. This flexibility and/or physical distance (which is referred to as “slack”) may be normally present in the extracellular domain(s), normally present in the linker, and/or normally present in the chimeric protein (as a whole). Alternately, or additionally, an amino acid sequence (for example) may be added to one or more extracellular domains and/or to the linker to provide the slack needed to avoid steric hindrance. Any amino acid sequence that provides slack may be added. In some embodiments, the added amino acid sequence comprises the sequence (Gly)_n where n is any number from 1 to 100. Additional examples of addable amino acid sequence include the joining linkers described in Table 1 and Table 3. In some embodiments, a polyethylene glycol (PEG) linker may be added between an extracellular domain and a linker to provide the slack needed to avoid steric hindrance. Such PEG linkers are well known in the art.

In some embodiments, a chimeric protein of the present technology comprises the extracellular domain of human PD-1 (or a variant thereof), a linker, and the extracellular domain of human OX40L (or a variant thereof). In some embodiments, the linker comprises a hinge-CH2-CH3 Fc domain, e.g., from an lgG1 or from lgG4, including human lgG1 or lgG4. Thus, in embodiments, a chimeric protein of the present technology comprises the extracellular domain of human PD-1 (or a variant thereof), linker comprising a hinge-CH2-CH3 Fc domain, and the extracellular domain of human OX40L (or a variant thereof). Such a chimeric protein may be referred to herein as “hPD-1-Fc-OX40L” or “SL-279252”.

Diseases , Methods of Treatment, and Mechanisms of Action

The chimeric proteins disclosed herein, including the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 OR SEQ ID NO: 61), finds use in methods for treating both advanced solid tumors and advanced lymphomas. These tumor types include: melanoma, non-small cell lung cancer (squamous, adeno, adeno-squamous), urothelial cancer, renal cell cancer, squamous cell cervical cancer, gastric or gastro-esophageal junction adenocarcinoma, squamous cell carcinoma of the anus, squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin, and microsatellite instability high or mismatch repair deficient solid tumors excluding central nervous system (CNS) tumors. Other tumors of interest include Hodgkin’s lymphoma (HL) and diffuse large B cell lymphoma.

In some embodiments, the cancer comprises an advanced solid tumor (local and/or metastatic).

In some embodiments, the cancer comprises an advanced lymphoma.

Aspects of the present technology provide methods of treating cancer. The methods comprise a step of administering to a subject in need thereof an effective amount of a chimeric protein, e.g., in a pharmaceutical composition, as disclosed herein.

It is often desirable to enhance immune stimulatory signal transmission to boost an immune response, for instance to enhance a patient’s anti-tumor immune response.

In some embodiments, the chimeric protein of the present technology comprises an extracellular domain of human PD-1, which disrupts, blocks, reduces, inhibits, and/or sequesters the transmission of immune inhibitory signals, e.g., originating from a cancer cell that is attempting to avoid its detection and/or destruction, and an extracellular domain of human OX40L, which enhances, increases, and/or stimulates the transmission of an immune stimulatory signal to an anti-cancer immune cell. Thus, the simultaneous binding of the extracellular domain of PD-1 to its ligand/receptor and the binding of the extracellular domain of OX40L with its receptor will prevent the transmission of an immunosuppressive signal from the cancer cell and will have stimulate immune activity in an immune system cell. In other words, chimeric proteins of the present technology are capable of treating cancer via two distinct mechanisms.

In some embodiments, the present technology pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors. As disclosed elsewhere herein, the treatment of cancer involves, in embodiments, modulating the immune system with the present chimeric proteins to favor of increasing or activating immune stimulatory signals. In some embodiments, the method reduces the amount or activity of regulatory T cells (Tregs) as compared to untreated subjects or subjects treated with antibodies directed to PD-1, OX40L, and/or their respective ligands or receptors. In some embodiments, the method increases priming of effector T cells in draining lymph nodes of the subject as compared to untreated subjects or subjects treated with antibodies directed to PD-1, OX40L, and/or their respective ligands or receptors. In some embodiments, the method causes an overall decrease in immunosuppressive cells and a shift toward a more inflammatory tumor environment as compared to untreated subjects or subjects treated with antibodies directed to the PD-1, OX40L, and/or their respective ligands or receptors.

In some embodiments, the present chimeric proteins are capable of, or can be used in methods comprising, modulating the amplitude of an immune response, e.g. modulating the level of effector output. In some embodiments, e.g. when used for the treatment of cancer, the present chimeric proteins alter the extent of immune stimulation as compared to immune inhibition to increase the amplitude of a T cell response, including, without limitation, stimulating increased levels of cytokine production, proliferation or target killing potential. In some embodiments, the patient’s T cells are activated and/or stimulated by the chimeric protein, with the activated T cells being capable of dividing and/or secreting cytokines.

Cancers or tumors refer to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system {e.g., virus infected cells). The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor. In contrast, the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part. The metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis. The cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body. The cancer may be due to a process such as lymphatic or hematogenous spread. The cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor. The cancer may be this new tumor, which may be a metastatic (or secondary) tumor.

The cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor. The cells of the tumor may be like those in the original tumor. As an example, if a breast cancer or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells. The tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.

The cancer may have an origin from any tissue. The cancer may originate from melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively. The cancer may also be a hematological malignancy, which may be leukemia or lymphoma. The cancer may invade a tissue such as liver, lung, bladder, or intestinal.

In some embodiments, the chimeric protein is used to treat a subject that has a treatment-refractory cancer.

I n some embodiments, the chimeric protein is used to treat a subject that is refractory to one or more immune- modulating agents. For example, in embodiments, the chimeric protein is used to treat a subject that presents no response to treatment, or whose disease progresses, after 12 weeks or so of treatment. For instance, in embodiments, the subject is refractory to one or more PD-1 and/or PD-L1 and/or PD-L2 agents, including, for example, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab (TECENTRIQ, GENENTECH), and/or MPDL3280A (ROCHE)-refractory patients. For instance, in embodiments, the subject is refractory to an anti-CTLA-4 agent, e.g., ipilimumab (YERVOY)- refractory patients {e.g., melanoma patients). Accordingly, in embodiments the present technology provides methods of cancer treatment that rescue patients that are non-responsive to various therapies, including monotherapy of one or more immune-modulating agents.

In some embodiments, the present technology provides chimeric proteins which target a cell or tissue within the tumor microenvironment. In some embodiments, the cell or tissue within the tumor microenvironment expresses one or more targets or binding partners of the chimeric protein. The tumor microenvironment refers to the cellular milieu, including cells, secreted proteins, physiological small molecules, and blood vessels in which the tumor exists. In some embodiments, the cells or tissue within the tumor microenvironment are one or more of: tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor. In some embodiments, the present chimeric protein targets a cancer cell. In some embodiments, the cancer cell expresses one or more of targets or binding partners of the chimeric protein.

The activation of regulatory T cells is critically influenced by costimulatory and co-inhibitory signals. Two major families of costimulatory molecules include the B7 and the tumor necrosis factor (TNF) families. These molecules bind to receptors on T cells belonging to the CD28 or TNF receptor families, respectively. Many well-defined co-inhibitors and their receptors belong to the B7 and CD28 families.

In some embodiments, an immune stimulatory signal refers to a signal that enhances an immune response. For example, in the context of oncology, such signals may enhance antitumor immunity. For instance, without limitation, immune stimulatory signal may be identified by directly stimulating proliferation, cytokine production, killing activity, or phagocytic activity of leukocytes. For example, a chimeric protein may directly stimulate the proliferation and cytokine production of individual T cell subsets. Another example includes direct stimulation of an immune inhibitory cell with through a receptor that inhibits the activity of such an immune suppressor cell. This would include, for example, stimulation of CD4+FoxP3+ regulatory T cells, which would reduce the ability of those regulatory T cells to suppress the proliferation of conventional CD4+ or CD8+ T cells. In another example, this would include stimulation of 0X40 on the surface of an antigen presenting cell, causing activation of antigen presenting cells including enhanced ability of those cells to present antigen in the context of appropriate native costimulatory molecules, including those in the B7 or TNF superfamily. In another example, the chimeric protein causes activation of the lymphoid cell and/or production of pro-inflammatory cytokines or chemokines to further stimulate an immune response, optionally within a tumor.

In some embodiments, the present chimeric proteins are capable of, or find use in methods involving, enhancing, restoring, promoting and/or stimulating immune modulation. In some embodiments, the present chimeric proteins described herein, restore, promote and/or stimulate the activity or activation of one or more immune cells against tumor cells including, but not limited to: T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g. M1 macrophages), B cells, and dendritic cells. In some embodiments, the present chimeric proteins enhance, restore, promote and/or stimulate the activity and/or activation of T cells, including, by way of a non-limiting example, activating and/or stimulating one or more T- cell intrinsic signals, including a pro-survival signal; an autocrine or paracrine growth signal; a p38 MAPK-, ERK-, STAT-, JAK-, AKT- or PI3K-mediated signal; an anti-apoptotic signal; and/or a signal promoting and/or necessary for one or more of: pro-inflammatory cytokine production or T cell migration or T cell tumor infiltration.

In some embodiments, the present chimeric proteins are capable of, or find use in methods involving, causing an increase of one or more of T cells (including without limitation cytotoxic T lymphocytes, T helper cells, natural killer T (NKT) cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, and macrophages {e.g., one or more of M1 and M2) into a tumor or the tumor microenvironment. In some embodiments, the chimeric protein enhances recognition of tumor antigens by CD8+ T cells, particularly those T cells that have infiltrated into the tumor microenvironment. In some embodiments, the present chimeric protein induces CD19 expression and/or increases the number of CD19 positive cells (e.g., CD19 positive B cells). In some embodiments, the present chimeric protein induces IL-15Ra expression and/or increases the number of IL-15Ra positive cells (e.g., IL-15Ra positive dendritic cells).

In some embodiments, the present chimeric proteins are capable of, or find use in methods involving, inhibiting and/or causing a decrease in immunosuppressive cells (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs)), and particularly within the tumor and/or tumor microenvironment (TME). In some embodiments, the present therapies may alter the ratio of M1 versus M2 macrophages in the tumor site and/or TME to favor M1 macrophages.

In some embodiments, the present chimeric proteins are able to increase the serum levels of various cytokines including, but not limited to, one or more of IFNy, TNFa, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL- 17A, IL-17F, and IL-22. In some embodiments, the present chimeric proteins are capable of enhancing IL- 2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, TNFa or IFNy in the serum of a treated subject. In some embodiments, administration of the present chimeric protein is capable of enhancing TNFa secretion. In a specific embodiment, administration of the present chimeric protein is capable of enhancing superantigen mediated TNFa secretion by leukocytes. Detection of such a cytokine response may provide a method to determine the optimal dosing regimen for the indicated chimeric protein. In a chimeric protein of the present technology, the chimeric protein is capable of increasing or preventing a decrease in a sub-population of CD4+ and/or CD8+ T cells.

In a chimeric protein of the present technology, the chimeric protein is capable of enhancing tumor killing activity by T cells.

In some embodiments, the chimeric protein activates the human subject’s T cells when bound by the OX40L domain of the chimeric protein and (a) one or more tumor cells are prevented from transmitting an immunosuppressive signal when bound by the first domain of the chimeric protein, (b) a quantifiable cytokine response in the peripheral blood of the subject is achieved, and/or (c) tumor growth is reduced in the subject in need thereof as compared to a subject treated with 0X40 agonist antibodies and/or PD-L1 blocking antibodies.

In some embodiments, the present chimeric proteins inhibit, block and/or reduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; or stimulate, induce, and/or increase cell death of a pro-tumor T cell. T cell exhaustion is a state of T cell dysfunction characterized by progressive loss of proliferative and effector functions, culminating in clonal deletion. Accordingly, a pro-tumor T cell refers to a state of T cell dysfunction that arises during many chronic infections, inflammatory diseases, and cancer. This dysfunction is defined by poor proliferative and/or effector functions, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Illustrative pro-tumor T cells include, but are not limited to, Tregs, CD4+ and/or CD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2 cells and Th17 cells. Checkpoint inhibitory receptors refer to receptors expressed on immune cells that prevent or inhibit uncontrolled immune responses. In contrast, an anti-tumor CD8+ and/or CD4+ T cell refers to T cells that can mount an immune response to a tumor.

In some embodiments, the present chimeric proteins are capable of, and can be used in methods comprising, increasing a ratio of effector T cells to regulatory T cells. Illustrative effector T cells include ICOS⁺ effector T cells; cytotoxic T cells (e.g., ab TCR, CD3⁺, CD8⁺, CD45RO⁺); CD4⁺ effector T cells (e.g., ab TCR, CD3⁺, CD4⁺, CCR7⁺, CD62Lhi, IL7R/CD127⁺); CD8⁺ effector T cells (e.g., ctf TCR, CD3⁺, CD8⁺, CCR7⁺, CD62Lhi, IL-7R/CD127⁺); effector memory T cells (e.g., CD62Llow, CD44⁺, TCR, CD3⁺, IL 7R/CD127⁺, IL-15R⁺, CCR7low); central memory T cells (e.g., CCR7⁺, CD62L⁺, CD27⁺; or CCR7hi, CD44⁺, CD62Lhi, TCR, CD3⁺, IL-7R/CD127⁺, IL-15R⁺); CD62L⁺ effector T cells; CD8⁺ effector memory T cells (TEM) including early effector memory T cells (CD27⁺ CD62L-) and late effector memory T cells (CD27- CD62L-) (TemE and TemL, respectively); CD127(⁺)CD25(low/-) effectorT cells; CD127(-)CD25(-) effectorT cells; CD8⁺stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca(⁺)); TH1 effector T-cells (e.g., CXCR3⁺, CXCR6⁺ and CCR5⁺; or o TCR, CD3⁺, CD4⁺, IL-12R⁺, IFNyR⁺, CXCR3⁺), TH2 effector T cells (e.g., CCR3⁺, CCR4⁺ and CCR8⁺; or ab TCR, CD3⁺, CD4⁺, IL-4R⁺, IL-33R⁺, CCR4⁺, IL-17RB⁺, CRTH2⁺); TH9 effector T cells (e.g., ab TCR, CD3⁺, CD4⁺); TH17 effector T cells (e.g., ab TCR, CD3⁺, CD4⁺, IL-23R⁺, CCR6⁺, IL-1 R⁺); CD4⁺CD45RO⁺CCR7⁺ effectorT cells, CD4⁺CD45RO⁺CCR7(-) effectorT cells; and effectorT cells secreting IL-2, IL-4 and/or IFN-g. Illustrative regulatory T cells include ICOS⁺ regulatory T cells, CD4⁺CD25⁺FOXP3⁺ regulatory T cells, CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25- regulatory T cells, CD4⁺CD25high regulatory T cells, TIM-3⁺PD-1⁺ regulatory T cells, lymphocyte activation gene-3 (LAG-3)⁺ regulatory T cells, CTLA- 4/CD152⁺ regulatory T cells, neuropilin-1 (Nrp-1 )⁺ regulatory T cells, CCR4⁺CCR8⁺ regulatory T cells, CD62L (L-selectin)⁺ regulatory T cells, CD45RBIow regulatory T cells, CD127low regulatory T cells, LRRC32/GARP⁺ regulatory T cells, CD39⁺ regulatory T cells, GITR⁺ regulatory T cells, LAP⁺ regulatory T cells, 1 B11⁺ regulatory T cells, BTLA⁺ regulatory T cells, type 1 regulatory T cells (Tr1 cells), T helper type 3 (Th3) cells, regulatory cell of natural killer T cell phenotype (NKTregs), CD8⁺ regulatory T cells, CD8⁺CD28- regulatory T cells and/or regulatory T-cells secreting IL-10, IL-35, TGF-b, TNF-a, Galectin-1, IFN-y and/or MCP1.

In some embodiments, the chimeric protein of the invention causes an increase in effector T cells (e.g., CD4 CD25- T cells).

In some embodiments, the chimeric protein causes a decrease in regulatory T cells (e.g., 004- ^025+ T cells).

In some embodiments, the chimeric protein generates a memory response which may, e.g., be capable of preventing relapse or protecting the animal from a recurrence and/or preventing, or reducing the likelihood of, metastasis. Thus, an animal treated with the chimeric protein is later able to attack tumor cells and/or prevent development of tumors when rechallenged after an initial treatment with the chimeric protein. Accordingly, a chimeric protein of the present technology stimulates both active tumor destruction and also immune recognition of tumor antigens, which are essential in programming a memory response capable of preventing relapse.

In some embodiments, the chimeric protein is capable of causing activation of antigen presenting cells. In some embodiments, the chimeric protein is capable enhancing the ability of antigen presenting cells to present antigen. In some embodiments, the present chimeric proteins are capable of, and can be used in methods comprising, transiently stimulating effector T cells for longer than about 12 hours, about 24 hours, about 48 hours, about 72 hours or about 96 hours or about 1 week or about 2 weeks. In some embodiments, the transient stimulation of effector T cells occurs substantially in a patient’s bloodstream or in a particular tissue/location including lymphoid tissues such as for example, the bone marrow, lymph-node, spleen, thymus, mucosa- associated lymphoid tissue (MALT), non-lymphoid tissues, or in the tumor microenvironment.

In a chimeric protein of the present technology, the present chimeric protein unexpectedly provides binding of the extracellular domain components to their respective binding partners with slow off rates (Kd or K₀ff). In some embodiments, this provides an unexpectedly long interaction of the receptor to ligand and vice versa. Such an effect allows for a longer positive signal effect, e.g., increase in or activation of immune stimulatory signals. For example, the present chimeric protein, e.g., via the long off rate binding allows sufficient signal transmission to provide immune cell proliferation, allow for anti-tumor attack, allows sufficient signal transmission to provide release of stimulatory signals, e.g., cytokines.

In a chimeric protein of the present technology, the chimeric protein is capable of forming a stable synapse between cells. The stable synapse of cells promoted by the chimeric proteins {e.g., between cells bearing negative signals) provides spatial orientation to favor tumor reduction - such as positioning the T cells to attack tumor cells and/or sterically preventing the tumor cell from delivering negative signals, including negative signals beyond those masked by the chimeric protein of the invention. In some embodiments, this provides longer on-target {e.g., intratumoral) half-life ( ) as compared to serum ti/2 of the chimeric proteins. Such properties could have the combined advantage of reducing off-target toxicities associated with systemic distribution of the chimeric proteins. In embodiments, the chimeric protein has a serum half-life (ti/2) of more than 6 hr, or more than 8 hr, or more than 12 hr, or more than 14 hr, or more than 18 hr, or more than 20 hr, or more than 22 hr, or more than 24 hr.

In some embodiments, the chimeric protein is capable of providing a sustained immunomodulatory effect.

The present chimeric proteins provide synergistic therapeutic effects {e.g., anti-tumor effects) as it allows for improved site-specific interplay of two immunotherapy agents. In some embodiments, the present chimeric proteins provide the potential for reducing off-site and/or systemic toxicity.

In some embodiments, the present chimeric protein exhibit enhanced safety profiles. In embodiment, the present chimeric protein exhibit reduced toxicity profiles. For example, administration of the present chimeric proteins may result in reduced side effects such as one or more of diarrhea, inflammation {e.g., of the gut), or weight loss, which occur following administration of antibodies directed to the ligand(s)/receptor(s) targeted by the extracellular domains of the present chimeric proteins. In some embodiments, the present chimeric protein provides improved safety, as compared to antibodies directed to the ligand(s)/receptor(s) targeted by the extracellular domains of the present chimeric proteins, yet, without sacrificing efficacy.

In some embodiments, the present chimeric proteins provide reduced side-effects, e.g., Gl complications, relative to current immunotherapies, e.g., antibodies directed to ligand(s)/receptor(s) targeted by the extracellular domains of the present chimeric proteins. Illustrative Gl complications include abdominal pain, appetite loss, autoimmune effects, constipation, cramping, dehydration, diarrhea, eating problems, fatigue, flatulence, fluid in the abdomen or ascites, gastrointestinal (Gl) dysbiosis, Gl mucositis, inflammatory bowel disease, irritable bowel syndrome (IBS-D and IBS-C), nausea, pain, stool or urine changes, ulcerative colitis, vomiting, weight gain from retaining fluid, and/or weakness.

In any of the embodiments disclosed herein, the number of proliferating CD4 central and/orthe number effector memory T cells in the subject may increase compared to the number of proliferating CD4 central and/or the number effector memory T cells in the subject before administration of the chimeric protein, or compared to the number of proliferating CD4 central and/or the number effector memory T cells in a subject that was not administered the chimeric protein, and/or compared to a control. In any of the embodiments disclosed herein, the treatment produces at least one therapeutic effect chosen from a reduction in size of a tumor, reduction in number of metastatic lesions over time, complete response, partial response, and stable disease.

Pharmaceutical composition

Aspects of the present technology include a pharmaceutical composition comprising a therapeutically effective amount of a chimeric protein as disclosed herein.

Any chimeric protein disclosed herein may be used in a pharmaceutical composition.

In some embodiments, a chimeric protein disclosed herein is provided as a sterile frozen solution in a vial or as a sterile liquid solution in a vial. A drug product comprising a chimeric protein disclosed herein comprises a sterile-filtered, formulated chimeric protein disclosed herein solution filled into a 10 mL single use glass vial stoppered with a FLUROTEC® rubber stopper and sealed with an aluminum flip off seal. In some embodiments, a chimeric protein disclosed herein is formulated at between about 10mg/mL to about 30 mg/mL, e.g., about 20 mg/mL in between about 30 mM to about 70 mM L-histidine, e.g., about 50 mM L- histidine and between about 125 mM and about 400 mM sucrose, e.g., about 250 mM sucrose in water for injection. In some embodiments, each vial contains about 1 mL of drug product or about 20 mg of a chimeric protein disclosed herein.

The chimeric proteins disclosed herein, including the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 OR SEQ ID NO: 61), can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically-acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

In some embodiments, the compositions disclosed herein are in the form of a pharmaceutically acceptable salt.

Further, any chimeric protein disclosed herein can be administered to a subject as a component of a composition, e.g., pharmaceutical composition, that comprises a pharmaceutically acceptable carrier or vehicle. Such pharmaceutical compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration. Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In some embodiments, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent disclosed herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent disclosed herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

In some embodiments, the compositions, e.g., pharmaceutical compositions, disclosed herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like). In some embodiments, the chimeric proteins may by conjugated and/or fused with another agent to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In some embodiments, the chimeric proteins may be fused or conjugated with one or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, and the like. In some embodiments, each of the individual chimeric proteins is fused to one or more of the agents described in Strohl, BioD gs 29(4):215—239 (2015), the entire contents of which are hereby incorporated by reference.

The present technology includes the disclosed chimeric protein in various formulations of pharmaceutical composition. Any chimeric protein disclosed herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. DNA or RNA constructs encoding the protein sequences may also be used. In some embodiments, the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.

Where necessary, the pharmaceutical compositions comprising the chimeric protein (can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.

The pharmaceutical compositions comprising the chimeric protein of the present technology may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the pharmaceutical compositions are prepared by uniformly and intimately bringing therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art)

In some embodiments, any chimeric protein disclosed herein is formulated in accordance with routine procedures as a pharmaceutical composition adapted for a mode of administration disclosed herein. Administration , Dosing , and Treatment Regimens

In some embodiments, a chimeric protein disclosed herein is presented as a sterile frozen solution at a concentration of about 20 mg/mL and a total volume of about 1 mL, optionally in a 10 mL glass vial. In some embodiments, a chimeric protein disclosed herein is administered by intravenous (IV) infusion following dilution with normal saline. Starting dose, dose escalation schema and dose schedules of certain embodiments are presented below.

In some embodiments, the dose of the chimeric protein administered is at least 0.0001 mg/kg, e.g., between about 0.0001 mg/kg and about 6.0 mg/kg.

In some embodiments, the chimeric protein is administered at an initial dose (e.g., one of about 0.0001, about 0.001, about 0.003, about 0.01 , about 0.03, about 0.1, about 0.3, about 1 , about 2, about 3, about 4, about 6, about 8, about 10, about 12, about 15, about 18, about 21, about 24, about 25, about 27, about 30, about 33, about 35, and about 36 mg/kg) and the chimeric protein is administered in one or more subsequent administrations. In some embodiments, the one or more subsequent administrations has a dose of one or more of about 0.0001, about 0.001 , about 0.003, about 0.01, about 0.03, about 0.1, about0.3, about 1 , about 2, about 3, about 4, about 6, about 8, about 10, about 12, about 15, about 18, about 21, about 24, about 25, about 27, about 30, about 33, about 35, and about 36 mg/kg. In some embodiments, subjects are administered one or more priming doses (e.g., one of about 0.0001, about 0.001, about 0.003, about 0.01, about 0.03, about 0.1, about 0.3, about 1, about 2, about 3, about 4, about 6, about 8, about 10, about 12, about 15, about 18, about 21, about 24, about 25, about 27, about 30, about 33, about 35, and about 36 mg/kg) before actual initial or maintenance dosing.

In embodiments, the starting dose and/or the subsequent doses is the maximum tolerated dose or less than the maximum tolerated dose.

In some embodiments, the dose escalates between one or more subsequent dose in log increments, e.g., 0.0001 mg/kg to 0.001 mg/kg, 0.001 mg/kg to 0.01 mg/kg, and 0.01 mg/kg to 0.1 mg/kg.

In some embodiments, the dose escalates between one or more subsequent dose in about half log increments, e.g., 0.001 mg/kg to 0.003 mg/kg and 0.003 mg/kg to 0.01 mg/kg.

In some embodiments, the initial dose is less than the dose for at least one of the subsequent administrations., e.g., each of the subsequent administrations. In some embodiments, the initial dose is the same as the dose for at least one of the subsequent administrations, e.g., each of the subsequent administrations.

In some embodiments, the chimeric protein is administered at least about one time a week.

In some embodiments, the chimeric protein is administered at least about one time a month.

In some embodiments, the chimeric protein is administered at least about two times a month.

In some embodiments, the chimeric protein is administered at least about three times a month.

In some embodiments, the chimeric protein is first administered once a week for three weeks and the chimeric protein is then administered about once every three weeks or once every four weeks.

In some embodiments, the chimeric protein is first administered once a week for three weeks and the chimeric protein is then administered about two times per month. For example, the chimeric protein is first administered once a week for three weeks and the chimeric protein is then administered about once every two weeks.

In some embodiments, the chimeric protein is administered at least about four times a month. For example, the chimeric protein is administered about once a week.

A chimeric protein may be administered intravenously by intravenous infusion or bolus injection into the bloodstream. In some embodiments, subjects are administered one or more priming doses (e.g., one of about 0.0001, about 0.001, about 0.003, about 0.01, about 0.03, about 0.1, about 0.3, about 1, about 2, about 3, about 4, about 6, about 8, about 10, about 12, about 15, about 18, about 21, about 24, about 25, about 27, about 30, about 33, about 35, and about 36 mg/kg) before actual initial or maintenance dosing.

In some embodiments, the present chimeric protein allows for a dual effect that provides less side effects than are seen in conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ). For example, the present chimeric proteins reduce or prevent commonly observed immune-related adverse events that affect various tissues and organs including the skin, the gastrointestinal tract, the kidneys, peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and the endocrine system; such as hypophysitis, colitis, hepatitis, pneumonitis, rash, and rheumatic disease.

Dosage forms suitable for intravenous administration include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (i e.g ., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

The dosage of any chimeric protein disclosed herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject’s general health, and the administering physician’s discretion.

The data disclosed herein demonstrate a gradual increase in Cmax with doses subsequent to initial doses. Furthermore, fourth dose (second cycle, first dose) showed a distinct Cmax compared to the third dose (first cycle, third dose). Without being bound by theory, the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) shows a latency in Cmax. Accordingly, in one aspect, the present disclosure relates to a biphasic or a triphasic dosing of fusion proteins of the present technology, disclosed herein in the methods of treatment disclosed herein. In a biphasic dosing regimen, the first phase is intended to increase the binding of target receptors (PD-L1 and 0X40) in tumor by the fusion proteins of the present technology to therapeutically relevant levels. The second phase is intended to maintain the binding of target receptors (PD- L1 and 0X40) in tumor by the fusion proteins of the present technology at therapeutically relevant levels. Accordingly, the doses and regimen of the chimeric proteins disclosed herein (e.g. the PD-1-Fc-OX40L chimeric protein such as SEQ ID NO: 59 OR SEQ ID NO: 61) are useful in the methods of treatment disclosed herein. As demonstrated herein, the doses, and/or dosing frequency of the fusion proteins of the present technology may be different during the first and the second phase.

The results disclosed herein also support a three-cycle dosing regimen since the Cmax at C2D1 was higher than the Cmax at C1 D15. In a three cycle dosing regimen, the first cycle is intended to increase the binding of target receptors (PD-L1 and 0X40) in tumor by the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 OR SEQ ID NO: 61). The second cycle is intended to modulate the binding of target receptors (PD-L1 and 0X40) in tumor by the chimeric protein disclosed herein to therapeutically relevant levels. The third phase is intended to maintain the binding of target receptors (PD-L1 and 0X40) in tumor by the chimeric protein disclosed herein at therapeutically relevant levels. Accordingly, the doses and regimen of the chimeric proteins disclosed herein are useful in the methods of treatment disclosed herein. As demonstrated herein, the doses, and/or dosing frequency of the chimeric proteins disclosed herein may be different during the first cycle, the second cycle and the third cycle.

In one aspect, the present technology relates to a method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L), wherein the step of administering comprises biphasic dosing. In some embodiments, the first phase, and the second phase each independently comprise a dosing frequency of from about twice a week to about once every two months. In some embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond.

In one aspect, the present disclosure relates to a method for inducing lymphocyte margination in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L). In some embodiments, the human subject is dosed with a dosing regimen selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the dosing regimen is about every week to about every 2 weeks, about every 10 days to about every 3 weeks, or about every 2 weeks to about every 4 weeks. In some embodiments, the initial dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg. In some embodiments, the dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg. In embodiments, the dose is administered with a once a week or a once every two weeks schedule. In embodiments, the method further comprising administration of a priming dose to the subject.

In some embodiments, the initial dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg. In some embodiments, the maintenance dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg. In embodiments, the dose is administered with a once a week or a once every two weeks schedule. In embodiments, the method further comprising administration of a priming dose to the subject.

In some embodiments, the dose is administered with a weekly (once every week) or a biweekly (once every two weeks) regimen. In some embodiments, the dose is administered with a once weekly dosing regimen. In some embodiments, the dose is administered with a once every 7 days dosing regimen. In some embodiments, the dose is administered with a once every two weeks dosing regimen.

In some embodiments, the dosing frequency of the first phase, and the dosing frequency of the second phase are the same. In other embodiments, the dosing frequency of the first phase, and the dosing frequency of the second phase are different.

In some embodiments, the dosing frequency of the first phase is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In some embodiments, the dosing frequency of the first phase is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months.

In some embodiments, the dosing frequency of the second phase is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In some embodiments, the dosing frequency of the second phase is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months.

In some embodiments, the dosing frequency of the first phase is selected from from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks; and the frequency of the second phase is selected from from about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks.

Additionally, or alternatively, in some embodiments, the first phase, and the second phase each independently last from about two days to about 12 months. In some embodiments, the first phase lasts from about two weeks to about 2 months; and the second phase lasts from about 2 weeks to about 12 months. In some embodiments, the first phase lasts from about two weeks to about 1 month; and the second phase lasts from about 2 weeks to about 12 months. In some embodiments, the first phase lasts from about two weeks to about 1 month; and the second phase lasts from about 4 weeks to about 12 months.

Additionally, or alternatively, in some embodiments, the effective amount for the first phase, the second phase and the third phase each independently comprise about 0.01 mg/kg to about 10 mg/ml. In some embodiments, the effective amount for the first phase, the second phase and the third phase each independently selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg, and any range including and/or in between any two of the preceding values. In some embodiments, the effective amount for the first phase, the second phase and the third phase each independently selected from from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, and about 10 mg/kg to about 50 mg/kg. In some embodiments, the effective amount for the first phase, the second phase and the third phase are same. In some embodiments, the effective amount for the first phase, the second phase and the third phase are different. In some embodiments, the effective amount for the first phase is greater than the effective amount for the second phase. In some embodiments, the effective amount for the first phase is from about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg; and the effective amount for the second phase is from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg.

In some embodiments, the chimeric proteins disclosed herein is the human PD-1 -Fc-OX40L chimeric protein. In one aspect, the present technology relates to a method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L), wherein the step of administration comprises a first cycle, a second cycle and a third cycle. In some embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. In some embodiments, the first cycle, the second cycle and the third cycle each independently comprise a dosing frequency of from about twice a week to about once every two months. In some embodiments, the dosing frequency of the first cycle, the dosing frequency of the second cycle and the dosing frequency of the third cycle are the same. In some embodiments, the dosing frequency of the first cycle, the dosing frequency of the second cycle and the dosing frequency of the third cycle are different. In some embodiments, the dosing frequency of the first cycle is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months.

In some embodiments, the dosing frequency of the first cycle is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the dosing frequency of the second cycle is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In some embodiments, the dosing frequency of the second cycle is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the dosing frequency of the third cycle is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In some embodiments, the dosing frequency of the third cycle is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the dosing frequency of the first cycle is selected from from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks; and the frequency of the second cycle is selected from from about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks.

Additionally, or alternatively, in some embodiments, the first cycle, the second cycle and the third cycle each independently last from about two days to about 12 months. In some embodiments, the first cycle lasts from about two weeks to about 2 months; and the second cycle lasts from about 2 weeks to about 12 months. In some embodiments, the first cycle lasts from about two weeks to about 2 months; the second cycle lasts from about 2 weeks to about 12 months and the third cycle lasts from about 2 weeks to about 6 months.

Additionally, or alternatively, in some embodiments, the effective amount for the first cycle, the second cycle and the third cycle each independently comprise about 0.01 mg/kg to about 10 mg/ml. In some embodiments, the effective amount for the first cycle, the second cycle and the third cycle each independently selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg, and any range including and/or in between any two of the preceding values. In some embodiments, the effective amount for the first cycle, the second cycle and the third cycle each independently selected from from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, and about 10 mg/kg to about 50 mg/kg.

In some embodiments, the effective amount for the first cycle, the second cycle and the third cycle are same. In other embodiments, the effective amount for the first cycle, the second cycle and the third cycle are different. In some embodiments, the effective amount for the first cycle is greater than the effective amount for the second cycle. I n other embodiments, the effective amount for the first cycle is lesser than the effective amount for the second cycle. In yet other embodiments, the effective amount for the first cycle and the effective amount for the second cycle are the same.

In some embodiments, the effective amount for the first cycle is from about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg; and the effective amount for the second cycle is from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg.

In some embodiments, the chimeric proteins disclosed herein is the human PD-1 -Fc-OX40L chimeric protein.

In one aspect, the present disclosure relates to a method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L) with a dosing regimen, wherein the dosing regimen comprises dosing with a frequency in the range of about every three days to about every 2 months. In some embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. In some embodiments, the dosing regimen is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In some embodiments, the dosing regimen is selected from about every week, about every 10 days, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In some embodiments, the dosing regimen is about every 2 weeks, about every 3 weeks, or about every 4 weeks.

In one aspect, the present disclosure relates to a method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L) with a dosing regimen selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. In some embodiments, the dosing regimen is about every week to about every 2 weeks, about every 10 days to about every 3 weeks, or about every 2 weeks to about every 4 weeks.

In some embodiments of any of the aspects disclosed herein, the first domain is capable of binding a PD-1 ligand. In some embodiments, the first domain comprises substantially all of the extracellular domain of PD- 1. In some embodiments, the second domain is capable of binding an OX40L receptor. In some embodiments, the second domain comprises substantially all of the extracellular domain of OX40L. In some embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from lgG4, e.g., human lgG4. In some embodiments, the linker comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the first domain comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the first domain comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, (a) the first domain comprises the amino acid sequence of SEQ ID NO: 57, (b) the second domain comprises the amino acid sequence of SEQ ID NO: 58, and (c) the linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, the chimeric protein further comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7. In some embodiments, the chimeric protein further comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 59 OR SEQ ID NO: 61.

Additionally or alternatively, in some embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99.2% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99.4% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99.6% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99.8% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the human subject has received, been tolerant to, or is ineligible for standard therapy and/or the cancer has no approved therapy considered to be standard of care.

In some embodiments of any of the aspects disclosed herein, the human subject is not receiving a concurrent chemotherapy, immunotherapy, biologic or hormonal therapy.

In one aspect, the present disclosure relates to a chimeric protein for use in the method of any of the embodiments disclosed herein.

In one aspect, the present technology relates to a chimeric protein comprising an amino acid sequence that is at least about 98% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the chimeric protein comprises an amino acid sequence that is identical to SEQ ID NO: 59 OR SEQ ID NO: 61.

The dosing frequency of the first phase, and the dosing frequency of the second phase may be same or different. In some embodiments, the dosing frequency of the first phase and the dosing frequency of the second phase are each independently selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months. In some embodiments, the dosing frequency of the first phase is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months. In some embodiments, the first phase, and the second phase each independently last from about two days to about 12 months. For example, in some embodiments, the first phase lasts from about two weeks to about

2 months; and the second phase lasts from about 2 weeks to about 12 months. In some embodiments, the first phase lasts from about two weeks to about 1 month; and the second phase lasts from about 2 weeks to about 12 months. In some embodiments, the first phase lasts from about two weeks to about 1 month; and the second phase lasts from about 4 weeks to about 12 months.

The effective amount for the first phase, the second phase and the third phase may be same or different. In some embodiments, the effective amount for the first phase, the second phase and the third phase each independently comprise about 0.01 mg/kg to about 10 mg/ml. In some embodiments, the effective amount for the first phase is from about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg; and the effective amount for the second phase is from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about

3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg. In some embodiments, the chimeric proteins disclosed herein is the human PD-1-Fc-OX40L chimeric protein.

In some embodiments, the human PD-1-Fc-OX40L chimeric protein is capable of providing a sustained immunomodulatory effect.

In some embodiments, the linker comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the linker comprises hinge-CH2-CH3 Fc domain derived from IgG. In some embodiments, the linker comprises hinge-CH2-CH3 Fc domain derived from an IgG selected from lgG1 and lgG4. In some embodiments, the linker comprises hinge-CH2-CH3 Fc domain derived from human lgG1 or human lgG4. In some embodiments, the linker comprises hinge-CH2-CH3 Fc domain derived from lgG4. In some embodiments, the hinge-CH2-CH3 Fc domain is derived from human lgG4.

Additionally, or alternatively, in some embodiments, the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 57.

Additionally, or alternatively, in some embodiments, the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 58. Additionally, or alternatively, in some embodiments, the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the human PD-1-Fc- OX40L chimeric protein comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 59 OR SEQ ID NO: 61. In some embodiments, the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is identical to SEQ ID NO: 59 OR SEQ ID NO: 61.

Methods of Selecting a Subject for Treatment and Evaluating the Efficacy of Cancer Treatment

In one aspect, the present disclosure relates to a method of selecting a subject for treatment with a therapy for a cancer, the method comprising the steps of: (i) administering a dose of a chimeric protein, wherein the dose of from about 0.03 mg/kg to 50 mg/kg; wherein the chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1 ), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L); (ii) obtaining a biological sample from the subject; (iii) performing an assay on the biological sample to determine level and/or activity of a cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFa; and (iv) selecting the subject for treatment with the therapy for cancer if the subject has an increase in the level and/or activity of at least one cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNo, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFo.

In one aspect, the present disclosure relates to a method for evaluating the efficacy of a cancer treatment in a subject in need thereof, wherein the subject is suffering from cancer, the method comprising the steps of: (i) administering a dose of a chimeric protein, wherein the dose of from about 0.03 mg/kg to 50 mg/kg; wherein the chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L); (ii) obtaining a biological sample from the subject; (iii) performing an assay on the biological sample to determine a level and/or activity of a cytokine selected from IFNy, IFNa, IL-27, CCL2, CCL3, CCL4, IL-2, TNFa and, and IL- 18; and (iv) continuing administration of the chimeric protein if the subject has an increase in the level and/or activity of at least one cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFa.

In some embodiments, the increase is calculated in comparison to the level and/or activity of the cytokine in another biological sample in the subject prior to administering the dose of the chimeric protein to the subject. In some embodiments, the increase is calculated in comparison to a level and/or activity of the cytokine in another biological sample from a different subject that has not been administered the dose of the chimeric protein. In some embodiments, the increase is calculated in comparison to a level and/or activity of the cytokine in a negative control. In some embodiments, the negative control is devoid of the cytokine. In some embodiments, the negative control contains the levels of the cytokine found in individuals that are not undergoing an inflammatory response. In some embodiments, the increase occurs by a factor of at least about 0.1 x, about 0.2*, about 0.3*, about 0.4*, about 0.5*, about 0.6*, about 0.7*, about 0.8*, about 0.9*, about 1 x, about 1.1 *, about 1.2^c, about 1.3^c, about 1.4^c, about 1.5^c, about 1.6^c, about 1.7^c, about 1.8^c, about 1.9x, about 2^c, about 2.1 x, about 2.2^c, about 2.3x, about 2.4^c, about 2.5x, about 2.6x, about 2.7^c, about 2.8x, about 2.9x, about 3x, about 3.1 x, about 3.2^c, about 3.3x, about 3.4^c, about 3.5x, about 3.6x, about 3.7x, about 3.8x, about 3.9x, about 4^c, about 4.1 x, about 4.2^c, about 4.3x, about 4.4^c, about 4.5x, about 4.6x, about 4.7^c, about 4.8x, about 4.9x, about 5x, about 5.1 x, about 5.2^c, about 5.3x, about 5.4^c, about 5.5x, about 5.6x, about 5.7^c, about 5.8x, about 5.9x, about 6x, about 6.1 x, about 6.2^c, about 6.3x, about 6.4x, about 6.5x, about 6.6x, about 6.7^c, about 6.8x, about 6.9x, about 7^c, about 7.1 x, about 7.2^c, about 7.3x, about 7.4^c, about 7.5x, about 7.6x, about 7.7^c, about 7.8x, about 7.9x, about 8x, about 8.1 x, about 8.2x, about 8.3*, about 8.4*, about 8.5*, about 8.6*, about 8.7*, about 8.8*, about 8.9*, about 9*, about 9.1 x, about 9.2^c, about 9.3x, about 9.4^c, about 9.5x, about 9.6x, about 9.7^c, about 9.8x, about 9.9x, or about 10x compared to the negative control.

In some embodiments, the increase is calculated in comparison to a level and/or activity of the cytokine in a positive control. In some embodiments, the positive control comprises the cytokine. In some embodiments, the positive control comprises the levels of the cytokine found in individuals that are undergoing an inflammatory response.

Additionally, or alternatively, in some embodiments, the subject has a decrease in the level and/or activity of at least one cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFa. In some embodiments, the decrease is calculated in comparison to the level and/or activity of the cytokine in another biological sample in the subject prior to administering the dose of the chimeric protein to the subject. In some embodiments, the decrease is calculated in comparison to a level and/or activity of the cytokine in another biological sample from a different subject that has not been administered the dose of the chimeric protein. In some embodiments, the decrease is calculated in comparison to a level and/or activity of the cytokine in a negative control. In some embodiments, the negative control is devoid of the cytokine. In some embodiments, the negative control contains the levels of the cytokine found in individuals that are not undergoing an inflammatory response. In some embodiments, the decrease occurs by a factor of at least about 0.1 x, about 0.2^c, about 0.3^c, about 0.4^c, about 0.5^c, about 0.6^c, about 0.7^c, about 0.8^c, about 0.9^c, about 1 x, about 1.1 ^c, about 1.2^c, about 1.3^c, about 1.4^c, about 1.5^c, about 1.6^c, about 1.7^c, about 1.8^c, about 1.9x, about 2^c, about 2.1 x, about 2.2^c, about 2.3x, about 2.4^c, about 2.5x, about 2.6x, about 2.7^c, about 2.8x, about 2.9x, about 3x, about 3.1 x, about 3.2^c, about 3.3x, about 3.4^c, about 3.5x, about 3.6x, about 3.7x, about 3.8x, about 3.9x, about 4^c, about 4.1 x, about 4.2^c, about 4.3x, about 4.4^c, about 4.5x, about 4.6x, about 4.7^c, about 4.8x, about 4.9x, about 5x, about 5.1 x, about 5.2^c, about 5.3x, about 5.4^c, about 5.5x, about 5.6x, about 5.7^c, about 5.8x, about 5.9x, about 6x, about 6.1 x, about 6.2^c, about 6.3x, about 6.4x, about 6.5x, about 6.6x, about 6.7^c, about 6.8x, about 6.9x, about 7^c, about 7.1 x, about 7.2^c, about 7.3x, about 7.4^c, about 7.5x, about 7.6x, about 7.7^c, about 7.8x, about 7.9x, about 8x, about 8.1 x, about 8.2x, about 8.3x, about 8.4^c, about 8.5x, about 8.6x, about 8.7^c, about 8.8x, about 8.9x, about 9x, about 9.1 x, about 9.2^c, about 9.3x, about 9.4^c, about 9.5x, about 9.6x, about 9.7^c, about 9.8x, about 9.9x, or about 10x compared to the negative control. In some embodiments, the decrease is calculated in comparison to a level and/or activity of the cytokine in a positive control. In some embodiments, the positive control comprises the cytokine. In some embodiments, the positive control comprises the levels of the cytokine found in individuals that are undergoing an inflammatory response.

In some embodiments of any of the aspects disclosed herein, the cancer is selected from melanoma, non- small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), squamous cell carcinoma of the skin (skin-SCC), urothelial cancer, cervical cancer, gastric cancer, gastric or gastroesophageal junction adenocarcinoma, renal cell carcinoma (RCC), squamous cell carcinoma (SCCA), squamous cell carcinoma of the anus, Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor, with the proviso that the microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor is not a CNS tumor.

In some embodiments, the biological sample is a body fluid, a sample of separated cells, a sample from a tissue or an organ, or a sample of wash/rinse fluid obtained from an outer or inner body surface of a subject. In some embodiments, the biological sample is a body fluid selected from blood, plasma, serum, lacrimal fluid, tears, bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy sample, cell-containing body fluid, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, washing or lavage such as a ductal lavage or broncheoalveolar lavage, aspirate, scraping, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluids, secretions, and/or excretions, and/or cells therefrom. In some embodiments, the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen. In some embodiments, the biological sample is a tumor sample derived from a tumor selected from melanoma, nonsmall cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), squamous cell carcinoma of the skin (skin-SCC), urothelial cancer, cervical cancer, gastric cancer, gastric or gastro- esophageal junction adenocarcinoma, renal cell carcinoma (RCC), squamous cell carcinoma (SCCA), squamous cell carcinoma of the anus, Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor, with the proviso that the microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor is not a CNS tumor. In some embodiments, the biological sample is obtained by a well-known technique including, but not limited to scrapes, swabs or biopsies. In some embodiments, the biological sample is obtained by needle biopsy. In some embodiments, the biological sample is obtained by use of brushes, (cotton) swabs, spatula, rinse/wash fluids, punch biopsy devices, puncture of cavities with needles or surgical instrumentation. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or include cells from an individual from whom the biological sample is obtained. In some embodiments, a biological sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, the biological sample is obtained by methods selected from the group consisting of biopsy {e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid {e.g., blood, lymph, feces etc.), etc. In some embodiments, the biological sample is originates from a tumor, blood, liver, the urogenital tract, the oral cavity, the upper aerodigestive tract the epidermis, or anal canal. It is to be understood that the biological sample may be further processed in order to carry out the method of the present technology. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.

In some embodiments, the level and/or activity of the cytokine is measured by one or more of RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS).

In some embodiments, the level and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds to one or more of the cytokines. In some embodiments, the agent that specifically binds to one or more of the cytokines is an antibody or fragment thereof. In some embodiments, the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or fragment thereof.

In some embodiments, the level and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds to one or more of the nucleic acids. In some embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

In some embodiments, the evaluating comprises anyone of diagnosis, prognosis, and response to treatment. In some embodiments, the evaluating informs classifying the subject into a high or low risk group. In some embodiments, the high risk classification comprises a high level of cancer aggressiveness, wherein the aggressiveness is characterizable by one or more of a high tumor grade, low overall survival, high probability of metastasis, and the presence of a tumor marker indicative of aggressiveness. In some embodiments, the low risk classification comprises a low level of cancer aggressiveness, wherein the aggressiveness is characterizable by one or more of a low tumor grade, high overall survival, low probability of metastasis, and the absence and/or reduction of a tumor marker indicative of aggressiveness. In some embodiments, the low risk or high risk classification is indicative of withholding of neoadjuvant therapy. In some embodiments, the low risk or high risk classification is indicative of withholding of adjuvant therapy. In some embodiments, the low risk or high risk classification is indicative of continuing of the administration of the chimeric protein. In some embodiments, the low risk or high risk classification is indicative of withholding of the administration of the chimeric protein.

In some embodiments, the evaluating is predictive of a positive response to and/or benefit from the administration of the chimeric protein. In some embodiments, the evaluating is predictive of a negative or neutral response to and/or benefit from the administration of the chimeric protein. In some embodiments, the evaluating informs continuing the administration or withholding of the administration of the chimeric protein. In some embodiments, the evaluating informs continuing of the administration of the chimeric protein. In some embodiments, the evaluating informs changing the dose of the chimeric protein. In some embodiments, the evaluating informs increasing the dose of the chimeric protein. In some embodiments, the evaluating informs decreasing the dose of the chimeric protein. In some embodiments, the evaluating informs changing the regimen of administration of the chimeric protein. In some embodiments, the evaluating informs increasing the frequency of administration of the chimeric protein.

In some embodiments, the evaluating informs administration of neoadjuvant therapy. In some embodiments, the evaluating informs administration of adjuvant therapy. In some embodiments, the evaluating informs withholding of neoadjuvant therapy. In some embodiments, the evaluating informs changing of neoadjuvant therapy. In some embodiments, the evaluating informs changing of adjuvant therapy. In some embodiments, the evaluating informs withholding of adjuvant therapy.

In some embodiments, the evaluating is predictive of a positive response to and/or benefit from neoadjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from neoadjuvant chemotherapy. In some embodiments, the evaluating is predictive of a positive response to and/or benefit from adjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from adjuvant chemotherapy. In some embodiments, the evaluating is predictive of a negative or neutral response to and/or benefit from neoadjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from neoadjuvant chemotherapy. In some embodiments, the evaluating is predictive of a negative or neutral response to and/or benefit from adjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from adjuvant chemotherapy.

In some embodiments, the evaluating informs decreasing the frequency of administration of the chimeric protein. In some embodiments,

In some embodiments, the neoadjuvant therapy and/or adjuvant therapy is checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an agent that targets one of TIM-3, BTLA, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPa, ICOS, CD172a, and TMIGD2.

In one aspect, the present disclosure relates to a method of selecting a subject for treatment with a therapy for a cancer, the method comprising obtaining a tumor sample from the subject, and evaluating the level and/or activity of a PD-1 ligand in the tumor sample, and if PD-1 ligand-positive tumor cells are detected, administering a chimeric protein, at a dose of from about 0.03 mg/kg to about 36 mg/kg; wherein the chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L). In embodiments, the level and/or activity of a PD-1 ligand is detected by contacting the tumor sample or a cell free extract thereof with an anti- PD-1 ligand antibody, or a binding fragment thereof. In embodiments, the level and/or activity of a PD-1 ligand is detected by contacting the tumor sample or a cell free extract thereof with PD-1 protein or a PD-1 ligandbinding fragment thereof. In embodiments, the level and/or activity of a PD-1 ligand is detected by contacting the tumor sample or a cell free extract thereof with a nucleic acid specific to a PD-1 ligand. In embodiments, the PD-1 ligand is PD-L1 or PD-L2.

In embodiments, the level and/or activity of the PD-1 ligand is measured by one or more of RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS).

In embodiments, the level and/or activity of the PD-1 ligand is measured by contacting the sample with an agent that specifically binds to one or more of the PD-1 ligands. In some embodiments, the agent that specifically binds to one or more of the PD-1 ligands is an antibody or fragment thereof. In some embodiments, the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or fragment thereof. In embodiments, the level and/or activity of the PD-1 ligand is measured by contacting the sample with an agent that specifically binds to one or more of the nucleic acids. In some embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

Subjects and/or Animals

In some embodiments, the subject and/or animal is a human. In some embodiments, the human is a pediatric human. In some embodiments, the human is an adult human. In some embodiments, the human is a geriatric human. In some embodiments, the human may be referred to as a patient.

In some embodiments, the human subject has received, been tolerant to, or is ineligible for standard therapy and/or the cancer has no approved therapy considered to be standard of care. Standard of care is the treatment that is accepted by medical experts as a proper treatment for a certain type of cancer and that is widely used by healthcare professionals, and also called best practice, standard medical care, and standard therapy. For example, radical surgery has been reported to be the standard of care for fit stage I non-small cell lung cancer (NSCLC) patients. Zarogoulidis et al, J Thorac Dis. 5(Suppl 4): S389-S396 (2013). In the curative setting, high-dose cisplatin concurrent with radiotherapy has been reported to be the standard of care, either as primary treatment or after surgery for head and neck squamous cell carcinoma (HNSCC). Oosting and Haddard, Front Oncol. 9: 815 (2019). Some cancers have no standard of care either because no treatment is accepted by medical experts as a proper treatment, or no treatment exists.

In embodiments, the human subject has a tumor that expresses a PD-1 ligand. In embodiments, the human subject has a tumor that is known to express a PD-1 ligand. In embodiments, the human subject is known to have PD-L1 positive tumor. In embodiments, the human subject is known to have PD-L2 positive tumor. In embodiments, that subject is previously treated is being treated with an anti-PD-1 or anti-PD-L1 agent. In embodiments, that subject is previously treated is being treated with an anti-PD-1 or anti-PD-L1 agent selected from nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab (TECENTRIQ, GENENTECH), and/or MPDL3280A (ROCHE). In embodiments, that subject is refractory to an anti-PD-1 or anti-PD-L1 agent.

In some embodiments, the human subject is ineligible for standard therapy may be exclusion criteria such as blood count, organ function, co-morbid conditions {e.g. heart disease, such as individuals with baseline abnormal electrocardiogram readings, uncontrolled diabetes, kidney disease, liver disease), women who are or may become pregnant, prior cancer treatments, exposure to certain medications, demographics, disease characteristics, overall illness burden, prior cancer history, and physiological reserve.

In some embodiments, the human subject has received more than two prior checkpoint inhibitor-containing treatment regimens, e.g., as a monotherapy or as a combination immunotherapy.

In some embodiments, the human subject is refractory to a prior checkpoint inhibitor therapy. For example, the subject is experiencing or has experienced disease progression within three months of treatment initiation of the prior checkpoint inhibitor therapy.

In some embodiments, the human subject has a life expectancy of greater than 12 weeks.

In some embodiments, the human subject has a measurable disease by iRECIST (solid tumors) or RECIL 2017 (lymphoma).

In some embodiments, the human subject is not receiving a concurrent chemotherapy, immunotherapy, biologic or hormonal therapy. However, the human subject may be receiving concurrent hormonal therapy for non-cancer related conditions is acceptable.

In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.

Aspects of the present technology include use of a chimeric protein as disclosed herein in the manufacture of a medicament, e.g., a medicament for treatment of cancer.

Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein

In some embodiments, the chimeric proteins disclosed herein {e.g. a recombinant, chimeric glycoprotein comprising the extracellular domain of human PD-1, a central domain from the human immunoglobulin constant gamma 4 (lgG4), and the extracellular domain of human OX40L, i.e., hPD-1-Fc-OX40L) binds to its cognate target molecules PD-L1, PD-L2, and 0X40 with nanomolar affinity in a dose-dependent manner, both individually and simultaneously. The chimeric proteins disclosed herein displayed slower dissociation kinetics when bound to PD-L1, PD-L2, and 0X40 compared to its interactions with control molecules suggesting that the fusion of PD-1 and OX40L via an Fc domain increases receptor-occupancy time, a beneficial characteristic in a tumor microenvironment.

Binding of the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) to 0X40 was shown to increase NFKB signaling and increase secretion of IL2 from CD3+ T cells in the presence of tumor cells expressing high levels of PD-L1. It also was found to stimulate expression of Ki67 (an intracellular marker for cell proliferation) in CD4+ and CD8+ T cells and increase expression of IFNy and TNFa in human CD8+ T cells.

In a staphylococcus enterotoxin B (SEB) assay, the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 OR SEQ ID NO: 61 )stimulated higher cytokine production from human PBMCs than its components alone or in combination, suggesting that the physical tethering of the PD-1 and OX40L domains by the Fc fragment provides a greater IL2 response than either ligand/receptor separately. The geometric mean for the ECso values of the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) following SEB super antigen stimulation, 50 ng/mL and 100 ng/mL, were 0.4866 nM and 0.5903 nM, respectively; however, because SEB is capable of activating a large proportion of TCRs present in a PBMC sample, these values likely over-estimate the minimal concentration at which an additional immune stimulating agent (such as the PD-1-Fc-OX40L chimeric protein {e.g. SEQ ID NO: 59 OR SEQ ID NO: 61)) may enhance immune responses in patients. As tumor-antigen specific immune responses in human cancer patients comprise only a small proportion of the overall T cell-mediated immune response repertoire, it is likely that the estimated ECso values for the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) from the SEB assay provide a conservative estimate for the dose level at which similar responses could be seen in human cancer patients. In similar experiments with PBMCs from cynomolgus monkeys, the species utilized in the below-disclosed non-human primate studies, IL-2 secretion was observed at similar concentrations of the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) to that of the human PBMCs. In comparison to commercial antibodies, the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) also induced higher expression of IL2, further supporting the expectation that the bispecific actions of the PD-1-Fc- OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) will improve upon the success rate of immune checkpoint or 0X40 single-targeted therapies currently available in the clinical setting. In summary, the PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) selectively and specifically binds to its intended targets of PD-L1 and 0X40 with high affinity. The PD-1-Fc-OX40L chimeric protein (e.g. SEQ ID NO: 59 OR SEQ ID NO: 61) has exhibited functional activity associated with the binding of both targets in a variety of in vitro assays including in vitro anti-tumor models. In vivo anti-tumor activity of a murine version of the protein was demonstrated in mouse tumor models. Minimal cross-reactivity with nonspecific targets was observed in human tissues. The minimum anticipated biological effect level (MABEL) based on the ECso of the SEB super antigen stimulation assay was determined to be 0.587 nM or 33.8 ng/mL.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

The examples herein are provided to illustrate advantages and benefits of the present technology and to further assist a person of ordinary skill in the art with preparing or using the chimeric proteins of the present technology. The examples herein are also presented in order to more fully illustrate the preferred aspects of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects or embodiments of the present technology described above. The variations, aspects or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.

Example 1: Murine PD-1-Fc-OX40L Provides In Vivo Anti-Tumor Activity

SL-279252 (PD-1-Fc-OX40L; SEQ ID NO: 59) is a recombinant, chimeric hexameric, bi-functional fusion protein comprising the extracellular domains of human programmed cell death 1 (PD-1 ; also known as PDCD1 ; or CD279) and tumor necrosis factor ligand superfamily member 4 (TNFSF4; also known as 0X40 ligand; OX40L; or CD252), linked by a central Fc domain from the human immunoglobulin lgG4. SL-279252 simultaneously binds to both tumor necrosis factor receptor superfamily member 4 (TNFRSF4; 0X40) and PD-L1 and/or PD-L2 expressed on cancer cells. Specifically, SL-279252 comprises the extracellular domain (ECD) of PD-1 (70 pM affinity to PD-L1) linked to the ECD of OX40L (324 pM affinity to 0X40) through an Fc linker protein. Stimulation of 0X40 promotes cytokine production and induces proliferation of memory and effector T lymphocytes against tumor cells, while PD-1 binding disrupts PD-1 signaling and restores immune function through the activation of T cells. In this example, the murine mPD-1-Fc-OX40L) was studied head-to-head with mouse anti-PD-1 antibodies, mouse anti-PD-L1 antibodies, and mouse anti-OX40 antibodies both alone and in combination and with respect to tumor control and rejection rates.

In vivo tumor efficacy experiments were performed in mice, using the murine equivalent of SL-279252 (mPD- 1-FC-OX40L). Prior to performing these in vivo studies, analogous, in vitro assays were performed to characterize the structure and function of mPD-1-Fc-OX40L in a similar manner to the SL-279252 (as described below in Example 2; data not shown). Murine PD-1 -Fc-OX40L was produced, purified, and shown to bind PD-L1/L2 and 0X40 simultaneously and with high affinity. Murine PD-1-Fc-OX40L exhibited functional activity in vitro, and significantly outperformed PD-1/L1 blockade as a monotherapy, outperformed 0X40 agonist monotherapy, and outperformed the combination of antibody-mediated PD-1/L1 blockade with 0X40 agonist therapy in a variety of in vitro functional assays.

The anti-tumor activity mPD-1-Fc-OX40L was then assessed in vivo by implanting murine colon tumors, comprising MC38 or CT26 tumor cells, into C57BL/6 and BALB/c mice, respectively, and then treating the mice with anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-OX40 antibodies, a combination of the two antibodies, or mPD-1-Fc-OX40L Both of these tumor models, MC38 and CT26, are known to respond to antibody-mediated PD-1 blockade.

Mice were inoculated subcutaneously on their rear flank with 5x10⁵ MC38 cells on day 0. On days five and seven and once the tumors established the mice were treated with two doses by IP injection and were about 4-6 mm in diameter; each dose consisting of 100 g of either mPD-1 -Fc-OX40L or the indicated antibodies, either alone or in combination. In FIG. 6A, each line represents the tumor growth from an individual mouse. On day forty, surviving mice were re-challenged with a secondary tumor consisting of 5x10⁵ cells on the opposing rear flank. Five new untreated mice were also inoculated on day forty as a control for tumor rechallenge growth. Rejection of primary and secondary tumors is shown in FIG. 6A.

Mice harboring MC38 tumors that were administered mPD-1 -Fc-OX40L had higher rates of tumor rejection (80%) than either antibody given as monotherapy (anti-OX40: 60% and anti-PD-L1 : 20%) and produced similar rates of rejection to the antibody combination treatment group (100%) (FIG. 6A). To assess the durability of an immune response without re-treatment, mice that rejected the initial tumor were re-challenged with the parental MC38 tumor on the opposite flank on day forty. Of the five mice initially cured with the antibody combination, only one of five (20%) rejected the tumor re-challenge. In the mPD-1 -Fc-OX40L group, two of four (50%) mice rejected the tumor challenge suggesting, without wishing to be bound by theory, that an initial memory response was generated against the tumor, which led to protection against a subsequent tumor challenge (FIG. 6A). Kaplan-Meier curves, generated for each treatment group to assess overall survival, show that control of tumor growth correlated well with overall survival sixty-five days after initial tumor inoculation (FIG. 6B).

FIG. 7A to FIG.7C show results from in vivo tumor studies demonstrating that the mPD-1 -Fc-OX40L chimeric protein has significant anti-tumor activity in a CT26 tumor re-challenge model. Here, BALB/c mice were implanted subcutaneously on their rear flank with 5x10⁵ CT26 colon cancer cells on day zero, and then treated with two doses by IP injection on days five and seven once the tumors established and were about 4-6 mm in diameter; the doses consisted of 100 g for each antibody dose and either 100 g, 150 g, or 300 g for each dose of the mPD-1 -Fc-OX40L chimeric protein. In FIG. 7A, each line represents the tumor growth from an individual mouse. The first dotted line (about day eighteen) represents the average time when all untreated mice reached tumor burden. On day thirty, surviving mice were re-challenged with a secondary tumor consisting of 3x10⁵ cells on the opposing rear flank. Thirteen new untreated mice were also inoculated on day forty as a control for tumor re-challenge growth. All mice treated with the mPD-1 -Fc-OX40L chimeric protein that survived until the day thirty challenge, were combined when plotting the re-challenge tumor growth curves. Kaplan-Meier curves were generated for each treatment group and plotted to compare sample groups (FIG. 7B).

In the CT26 tumor model, the higher doses (150 g x2 and 300 g *2) were shown to outperform the lower dose group (100 g *2). Even in the low-dose group, tumor control was significantly better for mPD-1 -Fc- OX40L than for a monotherapy antibody treatment to PD-1 (RMP1-14), to PD-L1 (10F.9G2), or to 0X40 (0X86); the tumor control for the mPD-1 -Fc-OX40L was roughly equivalent to the antibody combinations (FIG. 7A). A dose-dependent increase in overall survival and tumor rejection was observed when mPD-1 - Fc-OX40L was administered at either 150 g or 300 g per injection (FIG. 7B). Taken together, the results from the in vivo assay indicates that mPD-1 -Fc-OX40L is highly functional and efficacious at controlling tumors in mice. The data suggest, inter alia, that the incorporation of PD-1 and OX40L into the chimeric protein’s structure may provide mechanistic benefits and the potential for anti-tumor efficacy in humans. Importantly, the mPD-1 -Fc-OX40L chimeric protein was effectively able to kill tumor cells and/or reduce tumor growth when re-challenged (which illustrates a cancer relapse). Thus, the mPD-1 -Fc-OX40L chimeric protein appears to generate a memory response which may be capable of preventing relapse. FIG. 7B and FIG. 7C show the overall survival percentage, and statistics, of mice and tumor rejection through forty days after tumor inoculation. Dosages used in the in vivo mouse studies are briefly described in Table 4:

The above data clearly demonstrate, inter alia, that the anti-tumor activity of the murine equivalent of SL- 279252 (mPD-1-Fc-OX40L) in established murine tumors was significantly superior to either PD-1 blocking, 0X40 agonist, or combination antibody therapy. Thus, mPD-1-Fc-OX40L in vivo has significant functional activity in treating cancer. Accordingly, the PD-1-Fc-OX40L chimeric fusion protein is useful in the methods of treatment of cancer disclosed herein.

Example 2: Characterization of the Human PD-1-Fc-OX40L Chimeric Protein (SL-279252)

The human PD-1-Fc-OX40L chimeric protein (SL-279252) was expressed in Chinese Hamster Ovary (CHO) cells, purified and characterized. As shown in FIG. 3D, there are up to 10 potential N glycosylation sites and / or up to 8 potential O glycosylation sites in the amino acid sequence of SL-279252. The SL-279252 expressed in CHO cells was found to have post-translational modifications. The post translations modifications, including glycosylation that were detected in SL-279252 expressed and purified from CHO cells is shown in the Table below:

The N-glycan Content (% of Total) for SL-279252 Reference Standard NB6690p20 is shown in the Table below:

It is noted that while a bulk preparation showed the existence of the above modifications, it is possible that individual molecules have a range of modifications that differ from some other molecules. It is also noted that post-translational modifications (e.g., glycosylation) may change when the manufacturing process is changed, or scaled up or down.

There are 11 cysteines present in the sequence with up to 5 potentially capable of forming disulfide bond pairs. The following disulfide bonds were identified:

C30=C69 Not detected C150=C150 Interchain

C153=C153 Interchain

C185=C245

C291 =C349

C424=C508 Accordingly, the predicted molecular weight for the monomeric chimeric protein of SEQ ID NO: 59 (SL- 279252) is 57.6 kDa. The predicted molecular weight for the glycosylated monomeric chimeric protein of SEQ ID NO: 59 is 136 kDa.

An in vitro binding affinity assessment of a human PD-1 -Fc-OX40L chimeric protein (SL-279252) to recombinant human programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), and 0X40 was performed using surface plasmon resonance (SPR) analysis. Additionally, given that SL-279252 contains an internal Fc domain comprising the hinge-CH2-CH3 domain of human lgG4, binding was also assessed to recombinant human Fcg receptors: R1A, RUB, RIIIB and the neonatal Fc receptor (FcRn). To determine the on-rates (Ka), off-rates (Kd) and binding affinities (KD) of SL-279252 to its intended binding targets, histidine-tagged versions of the human recombinant proteins were immobilized to a Ni-sulfate activated ProteOn sensor chip. Control analytes for binding to their respective partners and human lgG1 were used as positive controls for binding to Fcg receptors and FcRn.

The results of the SPR assays show that SL-279252 binds to human PD-L1, PD-L2, and 0X40 with high affinity and with a slow off-rate, the latter of which indicates a longer on-target resident time. This could be of benefit within the tumor microenvironment where PD-L1 and PD-L2 are known to be expressed. Additionally, SL-279252 did not bind to recombinant FcgRIA (Table 5) or FcgRIIB and FcgRIIIB receptors {data not shown) but binds to FcRn receptor as intended by design (Table 5). These features are important as they suggest, inter alia, that Fc effector functions (such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity) are unlikely to be triggered by the chimeric protein once in a human. The interaction of SL-279252 to the neonatal Fc receptor is beneficial since it could potentially lead to a longer half-life of the chimeric protein in serum, due to recycling of SL-279252-bound FcRn back to a cell’s surface.

Table 5: Surface plasmon resonance binding results for SL-279252

E = exponent of 10 (x10^E); ND = not detected

The biochemical characterization and in vitro functional activity of SL-279252 (human PD-1-Fc-OX40L) was determined by several methods.

Structural characterizations were performed to confirm that all three domains of the human PD-1-Fc-OX40L (SL-279252) are intact and recognizable in a protein-detection assay.

Western blot analysis was performed on purified fusion protein which were probed with human anti-PD-1, anti-Fc, or anti-OX40L (FIG. 8A). SL-279252 was detected by all three antibodies and when the chimeric protein was run under reducing conditions, it migrated at approximately 75 kDa. Roughly 50% of the non- reduced protein ran as a dimer, which was a potential advantage, given the known in vivo oligomerization associated with OX40L signaling and function. The predicted molecular weight for SL-279252 is about 57.6 kDa. However, the reduced fraction of SL-279252 was detected at a higher molecular weight, which, without wishing to be bound by theory, was believed to be due to glycosylation. This was verified by treating SL- 279252 with a protein deglycosylase, PNGase F. Following deglycosylation, the reduced fraction of SL- 279252 migrated exactly at the predicted molecular weight of about 57.6 kDa. This provided evidence that SL-279252 is modified through glycosylation, which is known to have essential roles in the proper folding and stability of proteins, and in cell-to-cell adhesion (Dalziel M, Dwek RA. Science 2014; Maverakis E, Lebrilla CB. JAutoimmun. 2015). Together, these structural characterizations revealed a glycosylated protein that forms a dimer under non-reducing conditions, i.e. by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

ELISA (enzyme-linked immunosorbent assay) assays were used to demonstrate the binding affinity of the different domains of SL-279252 to their respective binding partners. More specifically, to determine whether SL-279252 retained binding to its dual targets (PD-L1 and 0X40), functional ELISA assays were developed to quantify and demonstrate simultaneous binding of SL-279252’s PD-1 extracellular domain to recombinant PD-L1 and SL-279252’s OX40L extracellular domain to recombinant 0X40. See, FIG. 8B, top right image. FIG. 8B, bottom left image shows a dual-binding ELISA assay demonstrating the ability of SL-279252 to bind and engage both targets (PD-L1 and OX40-His) simultaneously. Increasing concentrations of SL-279252 were incubated with a fixed amount of plate-bound recombinant human PD-L1 protein. Thereafter, recombinant OX40-His protein or a control His-tagged protein (HVEM-His) was incubated with the complex and binding was detected via an HRP-conjugated anti-His antibody. The results clearly show that SL-279252 binds to PD-L1 and 0X40 simultaneously and with high specificity.

Additionally, an ELISA-based blocking/competition assay was performed to demonstrate that SL-279252 could out-compete biotinylated human PD-1 for binding to plate-bound, recombinant human PD-L1. In this assay, recombinant human PD-L1 was coated on high-binding ELISA plates. Horseradish peroxidase (HRP) signal was produced using detection with biotinylated human PD-1, followed by contact with an avidin-HRP. See, FIG. 8C, right image. As shown in FIG. 8C, left image, in the negative control (a chimeric protein that does not contain a PD-L1 binding domain) the signal for biotinylated PD-1 was not disrupted (FIG. 8C, top curve/Square symbols). Notably, SL-279252 blocked biotinylated PD-1 binding to PD-L1 (thereby decreasing HRP signal), in a concentration-dependent manner (FIG. 8C, bottom curve/circle symbols). The results of this assay demonstrated inter alia, that hPD-1-Fc-OX40L (SL-279252) strongly competes with biotinylated PD-1 for binding to recombinant human PD-L1 and with an effective concentration at 50% of maximal response (ECso) of 6.68 nM.

Collectively, these studies demonstrated: (1) both domains of SL-279252 bind their respective targets with high affinity and (2) SL-279252 is capable of binding both targets simultaneously in a dose-dependent manner.

SL-279252 (hPD-1-Fc-OX40L) contains both the extracellular domain of PD-1, and the extracellular domain of 0X40 ligand (OX40L). The PD-1 domain of SL-279252 functions as a decoy receptor and provides competitive inhibition of PD-L1 and PD-L2. In addition, the OX40L domain of the chimeric protein directly binds to and activates 0X40 on the surface of immune cells. To confirm functionality SL-279252, the ability of the chimeric protein to bind mammalian cells which over-expressed human PD-L1, PD-L2, and 0X40 was assessed. Here, mammalian cell lines (Jurkat and CHO-K1) were genetically engineered to express full- length versions of human PD-L1, PD-L2, and 0X40. An untreated control and nine test concentrations of SL-279252 (0.0032, 0.0064, 0.16, 0.4, 1.0, 2.5, 5.0, 10, and 20 pg/mL) were evaluated, and with SL-279252’s binding to cells detected using a flow cytometry. Minimal background binding of SL-279252 was determined in parental CHO-K1 or Jurkat cells that did not express the target ligands and receptor (FIG. 9A to FIG. 9C). Mean fluorescence intensity (MFI) values were plotted against the logio nM test concentrations of the chimeric protein for each of the cell lines and the ECso of binding was calculated using the logio(agonist) versus response - variable slope (four parameters) fit. These results indicate, inter alia, that SL-279252 binds to human PD-L1, PD-L2, and 0X40 expressed on cells in a dose-dependent manner with ECso values of 27 nM, 47 nM, and 6 nM, respectively (FIG. 9A to FIG. 9C).

The functional activity of SL-279252 was determined by three separate in vitro functional assays including: (1) an NFkB-luciferase reporter system, (2) a tumor/T cell co-culture cytokine release assay, and (3) an SEB superantigen assay for determining cytokine release in comparison to other human antibody therapeutics.

An OX40-dependent NFkB-luciferase reporter assay was performed using Jurkat cells; this assay evidenced that SL-279252 stimulates a signaling cascade downstream of 0X40. Consistently, addition of SL-279252 was shown to stimulate the NFkB-luciferase reporter in a concentration-dependent manner. Importantly, Fc receptors were absent in this assay. Thus, the data indicates that Fc receptor cross-linking is not required for functional activity of SL-279252.

A cytokine release, tumor co-culture assay was performed to assess the ability of SL-279252 to stimulate anti-tumor T cell activity in inducing the expression of IL-2 in T cells. This assay is illustrated in FIG. 10A.

A cytokine release, tumor co-culture assay was performed to assess the ability of SL-279252 to stimulate anti-tumor T cell activity in inducing the expression of IL-2 in T cells. This assay is illustrated in FIG. 10A. As shown in FIG. 10A„ primary human CD3+T cells (isolated from human peripheral blood leukocytes) were sub-optimally stimulated with CD3/CD28 T cell activator beads (to upregulate PD-1 and 0X40), before co- culture with either a PD-L1 i_ow prostate cancer cell (human PC3) or a PD-L1 high lung adenocarcinoma cell (human HCC827), in the presence or absence of SL-279252 (500 ng and 5 g concentrations). Thus, this assay allowed assessment of the effector function and proliferation of T cells using IL2 secretion and a flow cytometry-based immune assessment. Results of the assay showed that IL-2 secretion from CD3+T cells (measured by ELISA) was reduced in the presence of tumor cells expressing high levels of PD-L1 ; however, IL-2 secretion could be restored with the addition SL-279252 (FIG. 10B). SL-279252 induced higher levels of secreted IL2 in PC3 cells (FIG. 10B, left bundle) than in HCC827 cells (FIG. 10B, right bundle). As human T cells produced significantly more IL-2 when co-cultured with the PC3 cell line than with the HCC827 cell line, this suggests that the quantity of PD-L1 inhibited IL-2 production. Further analysis of the human T cells from this assay by intracellular flow cytometry demonstrated that SL-279252 stimulated increased expression of Ki67, an intracellular marker for cell proliferation, in both CD4+ and CD8+ T cells, and that SL-279252 increased expression of interferon gamma (IFNy) and tumor necrosis factor alpha (TNFa) in human CD8+T cells (FIG. 10C).

Another assay to characterize the functional activity of the SL-279252 chimeric protein is the superantigen cytokine release assay. In this assay, increasing concentrations of staphylococcus enterotoxin B (SEB) were used to activate human peripheral blood leukocytes in the presence of various test agents. The SEB cytokine release assay is a highly sensitive method that provides a readout on T cell receptor (TCR)-dependent T cell activation following SEB-mediated ligation of the TCR with MHC Class II molecules on antigen presenting cells (APCs). The SEB assay was used to determine whether SL-279252 stimulated cytokine release downstream of TCR ligation in either human peripheral blood mononuclear cells (PBMCs; FIG. 11) or in cynomolgus macaque PBMCs {data not shown). PBMCs were cultured with multiple concentrations of SEB, in the presence of a human IgG control or a dose titration of SL-279252; from 0.10 pM to 200 nM for human PBMCs (from a total of ten donors) or from 5 pM to 50 nM for cynomolgus PBMCs (from total of three monkeys). After three days in culture, IL-2 secretion was assessed from supernatants by ELISA. Following SEB-mediated TCR/MHC Class II ligation, SL-279252 induced potent dose-dependent stimulation of IL-2; which was consistent and reproducible across ten distinct, healthy human donor PBMC samples and three distinct, healthy cynomolgus monkey PBMC samples. This cytokine-inducing activity was shown to be SEB (TCR/MHC II) dependent. When SEB was removed, SL-279252 (either soluble or plate-bound) was unable to stimulate cytokine secretion (IL-2, IFNy, or TNFa) from these unstimulated PBMCs [data not shown). Taken together, these results provide direct evidence for one of the proposed mechanisms of action for SL- 279252 in which the chimeric protein stimulates IL-2 secretion from human PBMCs only when the TCR on a T cell is engaged with MHC Class II on APCs, here using the superantigen SEB.

The SEB assay was also performed with commercially-available single-sided fusion proteins, including PD- 1-Fc, Fc-OX40L, or the combination of single-sided fusion proteins. The steps in the assay are illustrated in FIG. 12A. SL-279252 stimulated higher cytokine production from human PBMCs than either fusion protein component alone or in combination, suggesting, without wishing to be bound by theory, that the physical tethering of both checkpoint-blocking and immune-stimulating signals (in the hPD-1-Fc-OX40L) provides a greater IL-2 response than either signal alone. The relative potency of SL-279252 was compared to sequence equivalents of several human antibody therapeutics in human leukocytes which were incubated with increasing concentrations of the super antigen, SEB in the presence of pembrolizumab (aPD-1), nivolumab (aPD-1), tavolixizumab (aOX40), the combination of pembrolizumab/tavolixizumab, the combination of nivolumab /tavolixizumab, or SL-279252 (FIG. 12B). In this assay, SL-279252 outperformed the three clinical-stage antibodies (i.e., pembrolizumab, nivolumab, and tavolixizumab) which target the same signaling factors as SL-279252 when given as monotherapies or in combination in the SEB assay.

Taken together, these data indicate that the two extracellular domains of hPD-1-Fc-OX40L (SL-279252) maintain functionality within the context of the chimeric protein; thus allowing for engagement of cognate ligands and receptor. The Fc domain within SL-279252 provides sufficient flexibility and distance between the PD-1 and OX40L extracellular domains to allow proper engagement with their binding targets that are expressed on a cell’s surface. In addition, SL-279252 demonstrates functional activity in vitro. Finally, the data suggest that the bispecific actions of SL-279252 may have advantages when compared to targeting of PD-1 and 0X40 via a single-targeted antibody therapy.

Example 3: Characterization of the Human PD-1-Fc-OX40L Chimeric Protein (SL-279252) in Monkeys

SL-279252 (hPD-1-Fc-OX40L) was evaluated in cynomolgus monkeys to help identify characteristics relevant to SL-279252 administration in humans. The cynomolgus monkey was selected for use in the nonclinical studies, including safety, tolerability, pharmokinetic, and toxicology studies, at least, because SL- 279252 was found to be cross-reactive with simian 0X40 and PD-L1 in functional ELISA assays but not cross-reactive with murine or canine 0X40 and PD-L1. These studies included a Dose Range Finding (DRF) study and Good Laboratory Practice (GLP) study with doses ranging from 0.03 to 100 mg/kg and in various dosing schedules, including weekly dosing. As shown in FIG. 13, an inverse relationship between the logarithm of dose and the logarithm of clearance for cynomolgus monkeys administered SL-279252 was observed.

Table 6 presents a list of pharmacokinetic studies and toxicokinetic performed with SL-279252 in the cynomolgus monkey.

Table 6

Importantly, no mortality occurred in monkeys administered SL-279252 at less than or equal to 10 mg/kg. Laboratory changes observed in these monkeys included a positive Anti-Drug Antibody (ADA) response, increased complement split products, increased cytokines, increased fibrinogen, and/or decreased albumin. These findings were indicative of a potential immune/inflammatory response which were not associated with clinical signs. In summary, no severe clinical signs or symptoms of toxicity and no adverse macroscopic or microscopic findings were observed in monkeys administered weekly doses of less than or equal to 10 mg/kg/dose.

Example 4: In Silico , In Vitro , and Ex Vivo Characterization of Human PD-1-Fc-OX40L Chimeric Proteins

A proprietary in silico technology, iTope™, was used to determine if SL-279252 contained peptide sequences that would bind human MHC class II alleles. iTope™ models the binding between amino acid side chains of a peptide and specific binding pockets within the binding grooves of thirty-four human MHC class II alleles. The T Cell Epitope Database™ (TCED™) database served as the source for known T cell epitopes previously identified by EpiScreen™ T cell epitope mapping assays of antibody variable regions. When iTope™ and TCED™ analyses were applied, no promiscuous high or moderate affinity MHC class II binding sequences were identified in the SL-279252 sequence and no matches to previously identified T cell epitopes in the TCED™ were found. These findings suggest that SL-279252 would be considered of low risk for immunogenicity in humans based on its sequence.

In general, protein therapeutics that induce >10% positive responses in the EpiScreen™ T cell proliferation assay are associated with an increased risk of immunogenicity in the clinic. To assess immunogenicity risk for SL-279252, PBMCs were collected from a cohort of fifty healthy human donors to test in the EpiScreen™ assay. This cohort size well-represents the number and frequency for human leukocyte antigen-antigen-D related (HLA-DR) and HLA-DQ allotypes expressed in European/North American populations and in human populations elsewhere. For each donor, SL-279252 (1.0 mg/mL), a reproducibility control (cells incubated with 0.3 mM keyhole limpet hemocyanin [KLH]), a clinical benchmark control (cells incubated with 5 mM exenatide), and a culture medium-only control were included, and T cell proliferation and IL-2 secretion were used to measure T cell activation. Cultures were incubated for a total of eight days with the test agents. On days five, six, seven, and eight, aliquots of cells were processed and pulsed with 0.75 mq [³H]-Thymidine for a further eighteen hours before harvesting on filter mats. Counts per minute were determined by scintillation counting. PBMC from the same cohort donors were also analyzed in an IL-2 ELISpot assay following an eight-day incubation. For proliferation and IL-2 ELISpot assays, an empirical threshold stimulation index >1.9 has been established as the threshold for a positive response. Donors that were positive at least a single time point during the time course were deemed as positive. SL-279252 induced a positive response at a frequency of 8%, suggesting a low risk for immunogenicity in humans as shown in Table 7.

Table 7: Magnitude of positive T cell proliferation responses

The following studies were designed to assess the potential for SL-279252 to directly activate donor PBMCs in the absence of any T cell receptor activating, or other activating agents. In vitro cytokine release assays using healthy donor PBMCs treated with a dose titration of SL-279252 were performed. SL-279252 was assessed using both soluble and plate-bound conditions. End points were assessed using ELISA by measuring PBMC activation through the secretion of the cytokines IL-2, IFNy, and TNFa. No detectable changes in IL-2, IFNy, or TNFa were observed in human PBMC supernatants after co-incubation with dose titrations of SL-279252, in the soluble or plate-bound formats. As anticipated, the SEB positive control stimulated the production/secretion of all three cytokines in both formats tested. These findings indicate that SL-279252 is incapable of activating PBMCs in the absence of either TCR/MHC Class II ligation, cognate antigen recognition, high-level expression of the 0X40 receptor, or a combination of these factors. These studies suggest a decreased risk of off-target/non-specific immune activation in human patients treated with SL-279252.

In vitro assays were performed to determine whether SL-279252 directly activated human and/or cynomolgus complement proteins. The results indicate that SL-279252 can activate human and cynomolgus complement proteins in vitro at concentrations above 15.3 pg/mL. Finally, a comparison between SL-279252 peak serum concentrations from MPI studies indicates that the in vitro assays provide conservative estimates for serum concentrations of SL-279252 at which complement activation is likely to occur.

A study was performed to evaluate potential tissue cross-reactivity of SL-279252 in cryosectioned human tissues. SL-279252 conjugated to 5-(6)-carboxyfl uorescei n (1.0 and 10.0 pg/mL) was used to stain cryosectioned human tissue obtained from thirty-seven normal tissues from at least three separate donors. Buffer without primary antibody was used as an assay control.

The reactivity was consistent with expected expression of PD-L1 and/or 0X40. However, there was unexpected reactivity in epithelial cells in the gastrointestinal tract (cytoplasm, variably present at both concentrations of test article), kidney (cytoplasm, high concentration of test article), salivary gland (membrane and cytoplasm, high concentration of test article), ureter (cytoplasm, both concentrations of test article), and cervix (cytoplasm, high concentration of test article only), endothelial cells within the placenta (membrane and cytoplasm, high concentration of test article only), and in the extracellular matrix of fibrous stroma in multiple locations (high concentration of test article only). Negative results were obtained for the remaining tissues.

The biologic relevance of intracellular reactivity alone is low, as the SL-279252 chimeric protein is unlikely to have access to the cytoplasmic compartment in vivo. In addition, the unexpected reactivity observed in the tissues noted above was only observed at high concentrations of SL-279252 and likely reflects background staining in the assay. A plasma cell membrane microarray assay (Retrogenix, Inc.) was used to determine the specific cell surface binding partners of SL-279252. The primary screening step assessed SL-279252 binding against fixed human HEK293 cells, individually expressing 4,575 human proteins. Most of the proteins used in the microarray primary screen were cell-surface membrane proteins. Binding to target-expressing and untransfected cells by SL-279252 and other Fc-fusion proteins was assessed using an AlexaFluor647-labelled anti-human IgG Fc detection antibody followed by fluorescence imaging. All identified hits in the primary screen underwent confirmation and specificity testing. Identified binding partners were re-expressed and probed with SL-279252 individually in the presence of positive or negative controls to determine reproducibility of binding to each test protein. This approach confirmed specific binding of SL-279252 to its intended targets, PD-L1, PD-L2, and 0X40. The only off-target interaction that was noted was binding to galectin 1 which is routinely seen with other Fc-fusion proteins.

Table 8, below, provides a summary comparison of data across various studies, including the SEB Assay, the observed cynomolgus monkey exposure, and the simulated human exposure values derived using the PK model. The simulated human Cmax and AUC(0-24h) geometric means, including those for the recommended starting dose of 0.0001 mg/kg, are shown. The Cmax for the starting dose of 0.0001 mg/kg is approximately one tenth of the human SEB Assay ECso geometric mean value of 33.8 ng/mL.

Table 8: Summary Comparison of SEB Assay ECso, Observed Cynomolgus Monkey

Exposure and Simulated Human Exposure

The predicted Cmax and the proportion of simulated subjects that exceed the 2.5% Cl of the SEB Assay EC50 is presented in Table 9. None of the simulated concentrations at this dose exceed the 2.5% Cl of the EC50.

Table 9: SEB EC50 and predicted Cmax in humans

The geometric mean for the EC50 values of SL-279252 following SEB superantigen stimulation, 50 ng/mL and 100 ng/mL, were 0.4866 nM and 0.5903 nM, respectively. However, because SEB is capable of activating a large proportion of TCRs present in a PBMC sample, it is predicted to overestimate the minimal concentration at which an additional immune stimulating agent (such as SL-279252) may enhance immune responses in patients. As tumor-antigen specific immune responses in cancer patients comprise only a small proportion of the overall T cell-mediated immune response repertoire, it is likely that the estimated EC50 values for SL-279252 from the SEB assay provide a conservative estimate for the dose level at which similar responses could be seen in cancer patients. For these reasons the EC50 of the SEB assay was chosen to select a starting dose based on the minimum anticipated biological effect level of SL-279252.

The SL-279252 chimeric protein was designed to have a bi-functional mechanism of action in which it can contemporaneously bind to a PD-L1/PD-L2 on one cell and to an 0X40 on a second cell. As support for this bi-functional mechanism of action, multiple in vitro cell tethering assays were performed. First, PD-1- Fc-OX40L was incubated with two different cell types, either over-expressing human PD-L1 or 0X40, with each independently labelled with different fluorophores. Using flow cytometry, co-localization of these two cell populations, and a shift in forward/side scatter (indicating a larger size of the two tethered cells than would be observed with a single cell) was observed only when those cells were cultured with hPD-1-Fc- OX40L (SL-279252), and not the separate administration of both single-side FP controls (PD-1-Fc or Fc- OX40L). Second, to assess the ability of SL-279252 to physically “tether” tumor cells with T cells and stimulate potent cytotoxic activity, time-lapse immunofluorescent live cell imaging was used. The PD-L1 high human non-small cell lung cancer cell line NCI-H2023, was co-cultured with human CD3+ T cells that were stimulated for two days with suboptimal CD3, CD28, and IL-2 (FIG. 14). Prior to co-culture, the tumor cells were labeled with a cell tracker and nuclear stain and pre-treated with 150 nM of SL-279252 that was fluorescently-labelled with an Alexa Fluor 647 fluorochrome. As T cells migrated in close proximity to tumor cells, the density of detectable SL-279252 increased significantly, suggesting that the hPD-1-Fc-OX40L chimeric protein is able to coat PD-L1 on the tumor cell surface, and then functionally cluster 0X40 receptors on the T cell at the physical interface between an interacting T cell and tumor cell. This increase in SL-279252 co-localization is soon followed by a burst of apoptotic activity in the tumor cells {e.g., cleaved caspase 3/7). These data provide evidence that the role of SL-279252 appears bi-functional and in turn exerts potent tumor-killing activity through simultaneous interaction with two different cells.

Example 5: In Vivo Administration of Human PD-1-Fc-OX40L Chimeric Proteins

SL-279252 is a recombinant, chimeric glycoprotein comprising the extracellular domain of human PD-1, a central domain from the human immunoglobulin constant gamma 4 (lgG4), and the extracellular domain of human OX40L

FIG. 15 shows a schematic of the design of the Phase 1 clinical trial of SL-279252, which is a first in human, open label, multi-center, dose escalation and dose expansion study in subjects with advanced solid tumors or lymphomas. As shown in FIG. 15, the primary objective of this study is to evaluate the safety, tolerability of SL-279252. The secondary objective of this study is to evaluate the recommended phase 2 dose (RP2D), pharmacokinetic (PK), anti-tumor activity and pharmacodynamic effects of SL-279252 (FIG. 15). The exploratory objective of this study is to evaluate the pharmacodynamic (PD) markers in blood and tumor (FIG. 15).

A Phase 1 dose-escalation and dose-expansion trial of SL-279252 in patients with advanced solid tumors and lymphoma is currently on-going. The primary objective of the Phase 1 trial is to assess the safety and tolerability of SL-279252. The secondary objectives include evaluation of the pharmacokinetic and pharmacodynamic profiles as well as the anti-tumor activity of SL-279252. Anti-tumor response according to immune Response Evaluation Criteria in Solid Tumors or Response Evaluation Criteria in Lymphoma 2017 is being evaluated. These are standard, widely accepted criteria to evaluate tumor response in oncology clinical trials. An RP2D and schedule will be identified for SL-279252 following the completion of the Phase 1 trial.

Patients with relapsed, advanced, or metastatic solid tumors or lymphoma who have received standard of care therapies, including anti-PD-1/PD-L1 antibodies, are eligible to enroll in the trial. As shown in FIG. 15, in the dose-escalation portion of this study, up to six patients were treated at each of ten dose levels ranging from 0.0001 mg/kg to 6 mg/kg. Patient samples were evaluated to determine the pharmacokinetic profile, receptor occupancy on PD-L1 and 0X40 peripheral blood immune phenotyping, changes in immune cell infiltration in tumor biopsies, and evidence for elevation in multiple serum cytokines.

Following completion of the dose-escalation portion of the study, a dose and schedule will be selected for evaluation in up to two expansion cohorts. We anticipate treating a total of approximately 80 patients in the dose- escalation and dose-expansion portions of this clinical trial. An overview of the Phase 1 trial design evaluating SL-279252 in patients with advanced solid tumors and lymphoma is below:

Blood was collected prior to and following SL-279252 administration for serial assessment of PK and relevant biomarkers (e.g., cytokines, complement, immunophenotyping, etc.). Tumor tissue (archival tissue and predose, on-treatment and at progression fresh tumor biopsies) will be obtained from subjects.

The SL-279252 chimeric protein was administered to human subjects with advanced solid tumors (local and/or metastatic) and lymphomas. Cancer types were selected from melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), squamous cell carcinoma of the skin (skin- SCC), urothelial cancer, cervical cancer, gastric cancer, gastric or gastro-esophageal junction adenocarcinoma, renal cell carcinoma (RCC), squamous cell carcinoma of the anus (SCCA), Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), microsatellite instability-low (MSI-L) or mismatch repair deficient (MMRD) solid tumors (excluding CNS tumors) (FIG. 15). Preferably, the human subjects had received, been tolerant to, and/or are otherwise ineligible for standard therapy and/or the cancer has no approved therapy considered to be standard of care. A pharmaceutical composition comprising SL-279252 was prepared at 20 mg/mL and supplied as a frozen liquid. Following thawing, SL-279252 was diluted in sterile normal saline (0.9%). Each IV infusion will take approximately five to sixty minutes depending on dose, with shorter infusion times at lower doses.

As shown in FIG. 15, the study design consisted of Dose Escalation Cohorts and Dose Expansion Cohorts. The safety and tolerability evaluations of SL-279252 were begun on Schedule 1 of the dose escalation phase. The dose levels (DL) DL1 through DL10 used in this study were: DL1 is a 00001 mg/kg dose; DL2 is a 0 001 mg/kg dose; DL3 is a 0 003 mg/kg dose; DL4 is a 0.01 mg/kg dose; DL5 is a 0.03 mg/kg dose; DL6 is a 0.1 mg/kg dose; DL7 is a 0.3 mg/kg dose; DL8 is a 1 mg/kg dose; DL9 is a 3 mg/kg dose; and DL10 is a 10 mg/kg. Dose escalation was carried out per Keyboard Design and up to 2 schedules may be evaluated. Schedule 1 comprised administering SL-279252 on days one, eight, and fifteen of a twenty-eight-day cycle and once every two weeks thereafter on days one and fifteen in twenty-eight-day cycles, followed by dosing every 28 days (q28d). The second schedule (Schedule 2) comprised once-weekly doses of SL-279252 on days one, eight, fifteen, and twenty-two of a twenty-eight-day cycle.

Without wishing to be bound by theory, due to the large molecular weight of SL-279252, especially as trimeric and hexameric complexes, SL-279252 was expected to have limited distribution from the central compartment following IV infusion. Additionally, it was expected that SL-279252 is metabolized via endocytosis and protein degradation pathways as is typical for glycoproteins. Also, since SL-279252 binds with high affinity to receptors located on particular types of blood cells, it is expected that that specific receptor binding is the primary clearance mechanism for the removal of SL-279252 from the serum compartment. FIG. 16 shows simulations of likely human concentrations of SL-279252 by dose (mg/kg). Gray lines and shaded areas represent median and 95% Prediction Interval for a single dose of 0.0001 to 4 mg/kg SL- 279252 administered to humans. Doses and corresponding Cmax median and 95% Cl are displayed along the top of each graph. Blue lines and shaded areas are geometric mean and 95% Cl of the EC50 for the SEB Assay (33.8 ng/mL). Red dotted lines are the lower limit of quantitation for the current bioanalytical assay (25 ng/mL). Assay development is ongoing to increase sensitivity.

As shown in FIG. 17, no PD effects observed in mice and monkeys at the doses of <1 mg/kg, and <0.1 mg/kg, respectively, and no PD effects expected in man at the doses of <0.1 mg/kg. Antigen-specific T cell proliferation observed in mice at the doses of >1 mg/kg (FIG. 17). Serum cytokine elevations were observed at the doses of >2 mg/kg in mice, >1 mg/kg in monkeys, and were anticipated in humans at the doses of >0.1 mg/kg (FIG. 17). Further, as shown in FIG. 17, tumor was efficacy observed in mice at the doses of >4 mg/kg. Likewise, lymphocyte proliferation and margination to tissues was observed in monkeys at the doses of >1 mg/kg. Therefore, lymphocyte proliferation and tumor efficacy expected in man at the doses of >1 mg/kg (FIG. 17). Finally, whereas no toxicities observed in mice at the doses up to 40 mg/kg, infusion reactions were observed in monkeys at the doses of >10 mg/kg. Therefore, as shown in FIG. 17, the expected maximum administered dose in humans was estimated to be 10 mg/kg.

Example 6: Dose-Dependent Induction of Serum Cytokines by SL-279252

To understand the physiological impact of SL-279252 in human subjects, levels of in CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-10, IL-18, IL-15, IL-27, and TNFa were studied following the administration of SL- 279252 in comparison to baseline. As shown in FIG. 18A, the levels of IFNy were induced at doses of >0.03 mg/kg compared to the baseline. Similarly, the levels of IFNa (FIG. 18B), IL-27 (FIG. 18C), (CCL2 (FIG. 18D), CCL3 (FIG. 18E), CCL4 (FIG. 18F), IL-2 (FIG. 18G), TNFa (FIG. 18H), IL-18 (FIG. 181), IL-15 (FIG. 18J) and IL-6 (FIG. 18K) were also increased at doses of >0.03 mg/kg compared to the baseline or lower doses of SL-279252. FIG. 18L contains graphs illustrating the elevations of serum levels of IL-6 and IL-10 in subjects treated with SL-279252. The induction of all observed cytokines was generally dose-dependent, although some outliers were observed. The profile of cytokines were consistent with cell-mediated immunity. Dose dependent on-target cytokines response indicating that there are no neutralizing antibodies. Moreover, consistent concentration: time profiles with repeat dosing indicated that no ADA-drug complexes were formed. Accordingly, these data did not show any safety concerns. As shown below, pharmacodynamic effects will be observed at doses predicted by the PK/PD modeling.

To understand whether the observed induction of the cytokines correlated with the maximal serum concentration of SL-279252 (Cmax), serum cytokines levels were plotted as a function of the observed Cmax values at different does of SL-279252. As shown in FIG. 19A to FIG. 19F a linear relation between was found between the serum cytokines levels and the observed Cmax values at different does of SL-279252. Specifically, he levels of IFNy (FIG. 19A), IL-2 (FIG. 19B), IFNa (FIG. 19C), IL-27 (FIG. 19D), CCL2 (FIG. 19E), and IL-6 (FIG. 19F) increased with increasing Cmax values at different administered does of SL- 279252. These data illustrate that the observed serum cytokine elevations observed in subjects treated with SL-279252 was dependent on Cmax.

Therefore, these results demonstrate that the doses and regimen of SL-279252, the PD-1-Fc-OX40L chimeric fusion protein, used herein causes induction of pro-inflammatory cytokine production, which in turn causes T cell migration and/or T cell tumor infiltration, and immune/inflammatory response. Accordingly, the doses and regimen of SL-279252 disclosed herein are useful in the methods of treatment disclosed herein.

Example 7; Pharmacokinetics of SL-279252

To understand the stability of SL-279252 in serum, blood was drawn at various times following the administration of SL-279252 Cycle 1 Day 1 (C1 D1) and assayed for SL-279252. As shown in FIG. 20A and FIG. 24A, the serum concentration as well as the time for which detectable SL-279252 persisted in blood increased with increasing doses of SL-279252. The highest serum concentration of SL-279252 (Cmax) as well as the total drug exposure across time, as measured by the area under the curve (AUC) showed dose- dependent increases in exposure as shown in FIG. 25. SL-279252 was detectable until 96 hours following post-dosing at dose level 6 (DL6; 0.1 mg/kg dose) (FIG. 20A, FIG. 24A, FIG. 24C, and FIG. 24D). At DL6, SL-279252 was detectable at low concentrations at -168 hours (data not shown). These results suggest, inter alia, that SL-279252 remains detectable in humans much longer than seen in monkeys. Its half-life is therefore, comparable to that of Enbrel®, which has a 3 day half-life.

To understand the stability of SL-279252 during subsequent administrations, blood SL-279252 levels were compared at Cycle 1 Day 1 (C1 D1), Cycle 1 Day 15 (C1 D15) and Cycle 2 Day 1 (C2D1). Dose-dependent increases in total exposure to SL-279252 at ), Cycle 1 Day 15 (C1 D15) were evidenced by both Cmax and AUC at C1 D15 and C2D1, similar to C1 D1 (See FIG. 20B and FIG. 24B). The Cmax and AUC values are shown in FIG.25. Most other subjects showed higher Cmax at C1 D15 and/or C2D1 than at C1 D1. In several cases, a durable increase a progressive increase in Cmax was observed at C2D1 and C1 D15 compared to C1 D1 respectively, and C2D1 compared to C1 D15 at dose level 6 (DL6, 0.1 mg/kg). See, e.g. subjects 0107, 0302, 0203. Without being bound by theory, these results suggest, without wishing to be bound by theory, saturation of target receptors (PD-L1 and 0X40) in the peripheral compartment leading to a gradual increase in Cmax. These data also suggest lack of increased clearance due to humoral response or the like following subsequent dosing. Since the schedule of administration was Day 1, Day 8 and Day 15 in the first cycle, followed by a once every 2 weeks administration in the second cycle (FIG. 15), these results suggest inter alia, durable occupancy of target receptors (PD-L1 and 0X40) in the peripheral compartment even after lack of administration of the drug for a week (at C1 D15) or two weeks (at C2D1 ).

Therefore, these data demonstrate that initial doses of SL-279252 saturate the target receptors (PD-L1 and 0X40) in tumor, leading to a gradual increase in Cmax with doses subsequent to first dose. Furthermore, fourth dose (second cycle, first dose) showed a distinct Cmax compared to the third dose (first cycle, third dose). Without being bound by theory, accordingly, these data demonstrate that the immunological effects due to SL-279252 exhibits a latency.

Accordingly, these results provide a scientific basis for a biphasic or a triphasic dosing of SL-279252 which may be used in the methods of treatment disclosed herein.

To explore the observed gradual increase in Cmax further, the pharmacokinetics results from various subjects were compared. The data were from the following subjects at Cycle 1 Day 1 (C1D1) and Cycle 1 Day 15 were compared: (C1 D15)0101 (receiving 0 0001 mg/kg SL-279252), 0201 (receiving 0 001 mg/kg SL- 279252), 0102 (receiving 0003 mg/kg SL-279252), 0301 (receiving 0003 mg/kg SL-279252), 0302 (receiving 001 mg/kg SL-279252), 0103 (receiving 003 mg/kg SL-279252), 0203 (receiving 003 mg/kg SL-279252), 0106 (receiving 01 mg/kg SL-279252), 0107 (receiving 01 mg/kg SL-279252), and 0204 (receiving 01 mg/kg SL-279252). As shown in FIG. 24A and FIG. 24B, which plot the serum concentration of SL-279252 on a logarithmic scale, the concentrations SL-279252 decline occurred in a biphasic fashion. These data further showed that the observed Cmax closely matching model-predicted Cmax. Half-life is ~4 hours at 0.003 mg/kg in humans, which (increased with dose) this was higher than the observed half-life of 0.79 hr at 0.1 mg/kg in NHP.

Therefore, these results demonstrate, inter alia, that the doses and regimen of SL-279252, the PD-1-Fc- OX40L chimeric fusion protein, used herein has a longer half-life in humans compared to monkeys. Moreover, these results suggest that dose level 6 (DL6, 0.1 mg/kg) lead to extended saturation of target receptors (PD- L1 and 0X40) in the peripheral compartment, and longer duration of effect.

Example 8: Lack of Complement Activation in Humans Following the Dosing of SL-279252

Since complement activation, in association with anti-drug antibody responses, was observed in NHP, to understand whether SL-279252 induces complement in humans, the terminal complement complex (sC5b9) was measured predose, 1 hour post-dose and 24 hours post dose at Cycle 1 Day 1 (C1D1), Cycle 1 Day 15 (C1 D15) and Cycle 2 Day 1 (C2D1 ). Levels of sC5b9 were plotted from subjects 0101 and 0102 that received the dose of 00001 mg/kg, 0201 that received the dose of 0001 mg/kg, and 0301 , and 0302 that received the dose of 0003 mg/kg. As shown in FIG. 21, the levels of the terminal complement complex (sC5b9) were within the normal range following the dosing of SL-279252. No complement activation was observed in humans, including patients receiving SL-279252 Q2W for >7 months (data not shown). Example 9: Lymphocyte Profiles Following the Dosing of SL-279252

To understand whether the observed stability of SL-279252 and induction of proinflammatory cytokines by SL-279252, lymphocyte counts were measured in subjects dosed with various doses of over time. As shown in FIG. 22A, small changes in lymphocyte counts, if any, were observed even after 44 days in the subjects treated with dose levels DL1 (0 0001 mg/kg dose), DL2 (0 001 mg/kg dose), and DL3 (0 003 mg/kg dose) (FIG.22B). Similarly, the subjects treated with dose levels DL4 (0.01 mg/kg dose) and DL5 (0.03 mg/kg dose) also exhibited minor changes in lymphocyte counts, if any, during 44 days of treatment. Likewise, the subjects treated with dose levels DL6 (0.1 mg/kg dose; FIG. 22C), DL7 (0.3 mg/kg dose; FIG. 22D) also exhibited slight changes in lymphocyte counts, if any, until day 44 of treatment. FIG. 23E, the changes in lymphocyte counts were small at the doses of 0.3 mg/kg or lower. As shown in FIG. 22A, small changes in lymphocyte counts, if any, were observed even after 16 days in the subjects treated with dose levels DL8 (1 mg/kg dose).

The changes lymphocyte counts post dose in comparison to predose for each subject by dose level were measured and plotted. As shown in FIG. 23A, the post dose changes in lymphocyte counts at Cycle 1 Day 1 (C1 D1 ) in comparison to pre-dose levels were small. Similarly, the post dose changes in lymphocyte counts at Cycle 1 Day 1 (C2D1) in comparison to pre-dose levels were small (FIG. 23B).

Example 10: Receptor Occupancy by SL-279252

To understand occupancy of the 0X40 by the OX40L domain of SL-279252, the extent of reduction of CD4+ 0X40+ lymphocytes in peripheral blood was enumerated as a function of SL-279252 dose. Specifically, the percent reduction of CD4+ 0X40+ lymphocytes was determined at Cycle 1 Day 1 (C1 D1 ) for all patients that received doses DL1 (00001 mg/kg dose), DL2 (0001 mg/kg dose), DL3 (0003 mg/kg dose), DL4 (0.01 mg/kg dose), DL5 (0.03 mg/kg dose), DL6 (0.1 mg/kg dose), DL7 (0.3 mg/kg dose), DL8 (1 mg/kg dose), and DL9 (3 mg/kg dose). As shown in FIG. 26, a dose-dependent and saturable decrease in reduction of CD4+ 0X40+ lymphocytes was observed. The data were also more consistent between replicates and between patients within a given cohort. Further, these data demonstrate a good 0X40 receptor occupancy (about 75%) at doses 1 mg/kg and higher.

These results indicate, inter alia, that SL-279252 causes a dose-dependent and saturable occupancy of the 0X40 by the OX40L domain of SL-279252. Furthermore, these data demonstrate therapeutic effects of SL- 279252. Example 11: Lymphocyte Margination bySL-279252

Next, the fate of the CD4+ 0X40+ lymphocytes upon the treatment with SL-279252 was explored. Briefly, subjects were dosed with SL-279252 at 0.3, 1, or 3 mg/kg doses. Peripheral blood was drawn predose and at 4 hr and 24 hr post dose of Cycle 1 Dose 1 (C1 D1). The fraction of CD4+ 0X40+ SL-279252 (ARC)- cells of total CD3+ cells was measured and plotted as a function of time following the administration of SL-279252. As shown in FIG. 27, the fraction of CD4- )X40+ and SL-279252 (ARC)-lymphocyte decreased within two hours of dosing. Therefore, all of the 0X40+ cells in blood are leaving the blood in about 2 h.

These data illustrate the margination of CD4+OX40+ cells in patients treated with SL-279252.

Example 12: Lymphocyte Margination bySL-279252

The fate of the CD4+ 0X40+ lymphocytes upon the treatment with SL-279252 was further explored in cynomolgus monkeys. Cynomolgus monkeys were administered 5 consecutive weekly doses of 40 mg/kg SL-279252. Lymphocytes were stained in various tissue sections by immunohistochemistry and visualized microscopically. FIG. 29 shows the illustrative histochemical analysis of lungs of from untreated and SL- 279252-treated cynomolgus monkeys. As shown in FIG. 29, the lung sections from SL-279252-treated cynomolgus monkeys showed higher levels of CD4+ 0X40+ lymphocytes compared to the lung sections from untreated cynomolgus monkeys. Similarly, the cynomolgus monkeys treated with SL-279252 were observed to have dose-dependent migration of lymphocytes to the liver, gastrointestinal tract and lungs (data not shown). These data illustrate that the lymphocytes migrated to liver, gastrointestinal tract and lungs.

These data demonstrate that SL-279252 induced dose-dependent migration of lymphocytes to the liver, gastrointestinal tract and lungs. Collectively, these data provide evidence of on-target biology driven by 0X40, and these effects were accompanied by distinct changes in multiple serum cytokines.

Example 13: Lymphocyte Expansion and Margination Induced by SL-279252 in Non-Human Primates

Next, the numbers of lymphocytes following the treatment with SL-279252 was explored in cynomolgus monkeys. As discussed above, repeated dosing of SL-279252 was evaluated in non-human primate (NHP) dose-range finding and GLP toxicity studies. In these studies, SL-279252 was evaluated over a course of 5 once-weekly doses, across a dose range of 0.1-100 mg/kg. Cynomolgus monkeys were treated on Day 1 and 8 with the indicated dose of SL-279252 or a vehicle control. Total lymphocyte counts were obtained prior to the dose on Day 1, and then again on Day 15. FIG. 30 shows the lymphocyte expansion from pre-dose to day 15 in non-human primates. Each data point indicates an individual animal. Next, the lymphocyte margination was further explored following the treatment with SL-279252 in cynomolgus monkeys. Cynomolgus monkeys were treated with SL-279252 on Day 1 , 8 and 15 at the indicated dose. Pre- and post-dose lymphocyte counts were obtained on day 15 prior to the third dose, and on day 16 approximately 24 hours after the third dose. The number of peripheral blood lymphocytes was observed to decrease in a dose-dependent manner following the Day 15 dose, and is plotted in FIG. 31 as the (100 - ((# of lymphocytes on Day 16) / (# of lymphocytes on Day 15) x 100). Each data point indicates an individual animal. These data further illustrate shows the post-dose lymphocyte margination from day 15 to day 16.

These data demonstrate a dose-dependent expansion in the total number of circulating lymphocytes in cynomolgus macaques following SL-279252 infusion. Taken together with the cytokine data shown above, these data indicate that SL-279252 was a potent immune stimulator in NHP. A dose-dependent expansion was observed in the total number of lymphocytes in NHP, post-dose partitioning of lymphocytes into specific tissue sites, and elevations in multiple serum cytokines.

Example 14: Anti-tumor activity of Mouse Surrogate of SL-279252

A murine surrogate of SL-279252, mouse PD-1-Fc-OX40L, was produced so that the activity of mouse PD- 1-Fc-OX40L could be compared to both PD-1 blocking and 0X40 agonist antibodies. See: Fromm et al. Agonist redirected checkpoint, PD-1-Fc-OX40L, for cancer immunotherapy Journal for ImmunoTherapy of Cancer 6:149 (2018).

Mice were inoculated with CT26 cells. On day 5 and on day 7, the mice were treated with murine PD-1-Fc- OX40L chimeric protein, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-OX40 antibody, combination of the anti-PD-L1 antibody and the anti-OX40 antibody, and combination of the anti-PD-1 antibody and the anti-OX40 antibody. Tumor volumes were plotted as a function of time. Mice that rejected the tumors were counted. Some mice were re-challenged with CT26 cells and tumor rejection was reevaluated. FIG.28 shown a graph comparing the anti-tumor activity of the murine PD-1-Fc-OX40L in mouse CT26 mouse colon carcinoma allograft model in comparison with checkpoint blocking antibodies alone, co-stimulator agonist antibodies alone, or a combination of checkpoint blocking and co-stimulator agonist antibodies. The primary column in FIG. 28 shows the number of mice that rejected the primary tumor. The re-challenge column in the panels FIG. 28 shows the number of mice that rejected the primary tumor and were also capable of rejecting a second tumor challenge without repeat treatment, demonstrating a durable adaptive immune response. As shown in FIG. 28, the murine PD-1-Fc-OX40L showed more potent antitumor activity in in mouse CT26 mouse colon carcinoma allograft model compared with checkpoint blocking antibodies alone, co-stimulator agonist antibodies alone, or a combination of checkpoint blocking and co-stimulator agonist antibodies. Interestingly, the murine PD-1-Fc-OX40L showed more potent antitumor activity than anti-PD-1 antibody, anti- 0X40 antibody or a combination thereof.

These data demonstrate the antitumor activity of SL-279252, illustrating that SL-279252 is useful in treating cancer.

Example 15: Dosing in Human Clinical Trial of SL-279252

A total of 29 patients have received treatment with SL-279252 up to a dose of 6 mg/kg in the dose-escalation portion of the Phase 1 trial. Overall, SL-279252 has been observed to be well tolerated. Treatment-related adverse events, including immune-related events, have been reported in some patients, but there have not been any dose-limiting toxicities as of September 9, 2020, as shown in Table 10. A maximum tolerated dose has not been reached. Preliminary pharmacokinetic activity has been evaluated in 22 patients treated across a dose range of 0.0001 to 3 mg/kg. Exposure of SL-279252 as determined by the maximum peak drug concentration, or Cmax, and the area under the curve, or AUC, increased with dose escalation in a linear fashion. The pharmacokinetic profile consists of a distribution phase and an elimination phase. We believe this distribution phase indicated rapid binding to the target receptors. Following repeat dosing, a consistent Cmax and AUC was observed without evidence of accelerated drug clearance. The volume of distribution of drug indicated that SL-279252 distributed beyond the circulatory compartment into tissues.

Adverse events, or AEs, were classified according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE— version 5.0). As of September 9, 2020, treatment-related AEs have been reported in 13 patients. In 12 patients, these AEs have been Grade 1 or 2 in severity. One patient experienced a Grade 3 treatment-related AEs. No treatment-related Grade 4 or 5 adverse events, treatment- related serious adverse events or dose limiting toxicities have been reported.

Summary of Treatment-Related Adverse Events and Series Adverse Events

Table 10: Overall summary for Adverse Events (total number of patients = 29)

Number of patients

Any Adverse Events: 25

Grade 5 AEs 0

Grade 4 AEs 0 Grade 3 AEs 7

Dose Limiting Toxicities 0

Serious Adverse Events 7

Treatment-related SAE 0

T reatment-related Adverse Events 13

Grade 5 AE 0

Grade 4 AE 0

Grade 3 AE¹ 1

Grade 2 AE² 4

Grade 1 AE³ 9

¹Grade 3 treatment-related AE: neutropenia (transient, self-limiting)

²Grade 2 treatment-related AE: rash maculo-papular (n=2), rash (n=1), pruritus (n=1), neutrophil count decreased (n=1), and infusion reaction (n=1).

³Grade 1 treatment-related AE: night sweats (n=2), constipation (n=2), rash maculo-papular (n=1), asthenia (n=1), decreased appetite (n=1), diarrhea (n=1), dizziness (n=1), fatigue (n=1), headache (n=1), hyperkalemia (n=1), hypothyroidism (n=1), localized edema (n=1), neuropathy peripheral (n=1), pyrexia (n=1), vertigo (n=1).

Preliminary pharmacodynamic activity has also been evaluated in 22 patients treated across a dose-range of 0.0001 to 3 mg/kg. Post-dose receptor occupancy on OX40-positive lymphocytes was observed in a dose- dependent fashion, and the total number of OX40-positive cells in the blood declined rapidly post-infusion of SL- 279252. The post-infusion decreases in OX40-positive lymphocytes provides evidence of on-target biology. In NHP, similar post-infusion decreases in lymphocytes were associated with migration of lymphocytes into tissues.

These results demonstrate, inter alia, that SL- 279252 is well tolerated with a minimal toxic dose (MTD) of more than 6 mg/kg (the maximal dose examined herein). Further, these results indicate that a weekly (once every week or every 7 days), or a biweekly (once every two weeks) dosing may be appropriate for treatment. A total of 43 patients were treated in the initially planned dose-escalation cohorts, beginning from the first dose level of 0.0001 mg/kg through 6.0 mg/kg, and 2 dosing schedules were evaluated. Any dose-limiting toxicities were not observed through the highest dose level. In a heavily pre-treated, predominantly PD-1/L1 experienced patient population the following responses were observed: 1 durable partial response, an additional unconfirmed partial response, and additional patients with tumor shrinkage at the higher dose levels of SL-279252 as monotherapy. This activity is in line with the response rates reported in a heterogeneous population of checkpoint refractory patients who have been re-treated with approved PD-1 antibody therapies, and overall disease control rates in the 25-40% range. Dose dependent 0X40 receptor engagement of 0X40 expressing T cells was also observed, and a primary pharmacodynamic effect showed a rapid egress of these target cells from the circulation, which has not been reported for prior 0X40 agonist antibodies.

Example 16: Phase 1 Dose Escalation and Dose Expansion Study in Subjects with Advanced Solid Tumors or Lymphomas

Methods:

The first-in-human, Phase 1 dose escalation study is evaluating SL-279252 as monotherapy in patients with advanced solid tumors or lymphomas. Objectives include evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose (RP2D), pharmacokinetic (PK) parameters, pharmacodynamic (PD) effects, and anti-tumor activity per Immune Response Evaluation Criteria in Solid Tumors (iRECIST).

Results:

43 patients were enrolled and dosed intravenously with SL-279252 (median age 64 years; 56% male; median [range] of 3 [0-5] prior systemic therapies for metastatic disease): 30 patients were treated on schedule 1 (day 1, 8, 15, 29, then every 2 weeks) from dose level 0.0001-6 mg/kg, and 13 patients were treated on schedule 2 (weekly) from dose level 0.3-3 mg/kg. 58% of patients were PD-1/PD-L1 inhibitor experienced, and most tumors lacked PD-L1 expression. Common (>15%) treatment emergent adverse events (AEs) of any grade (G) were constipation in 11 (26%) patients, back pain in 8 (19%) patients, anemia in 7 (16%) patients and decreased appetite in 7 (16%) patients. Infusion-related reactions (G1/2) were noted in 3 (7%) patients. G3 treatment-related AEs (TRAEs) were neutropenia (2%) and hypercalcemia (2%); no G4/5 TRAEs or DLTs occurred. SL-279252 Cmax and AUC increased linearly up to 3mg/kg, and greater than proportional increase in AUC was observed at 6 mg/kg. The preliminary T½ was ~23 hours. Dose-dependent receptor occupancy on Oϋ4- )C40+ T cells persisted for >7 days and these cells rapidly marginated from the peripheral blood post infusion. Increases in the number of proliferating central and/or effector memory T cells were seen in some patients at doses of >1 mg/kg. Analysis of paired tumor biopsies is ongoing. Best response was 1 durable confirmed partial response (iPR, in a patient suffering from ocular melanoma, 4 prior systemic regimens) in a patient who remained on treatment for >1 year, and stable disease (iSD) in 12 patients (1 unconfirmed iPR). iSD for > 24 weeks occurred in 6/12 patients.

Conclusions:

SL-279252 is well-tolerated in patients with refractory solid tumors with no maximum tolerated dose (MTD) reached. OX40-dependent PD effects and durable anti-tumor activity was observed. Trends for PK/PD effects at >1 mg/kg suggest dose exploration in PD-L1 expressing cancers is warranted beyond 6 mg/kg.

Example 17: Additional Dose Escalation Cohorts in the SL-279252 Phase 1 Clinical Trial

The PK profile of SL-279252 is being exhaustively characterized through the 6 mg/kg dose level, and the data suggest the potential for further escalation in pharmacodynamic activity at doses beyond 6 mg/kg. Because emerging data indicated that additional dose levels would enable a more complete assessment of PK, PD and anti-tumor activity, dose escalation will be through at least two additional dose levels, of 12 mg/kg and 24 mg/kg. Patients with known PD-L1 positive tumors will also be enrolled and clinical response rates will be observed in that population.

It is anticipated that 0X40 activation will increase the response rates beyond what is expected of PD-L1 inhibition in PD-1 experienced patients. It is anticipated that anti-PD-1 therapy experienced patients or the patients known PD-L1 positive tumors will show evidence of clinical efficacy that is better than that of anti- PD-1 therapy.

Example 18: The Progress of the SL-279252 Phase 1 Clinical Trial

The study design is shown in FIG. 32 (see also FIG. 15). This design involved an accelerated titration with n > 1 subjects per cohort until grade 2 (G2) toxicity was observed or dose level 6 (DL6) was reached. Subjects received intravenous (IV) administration of SL-279252 on Schedule 1 or Schedule 2 until clinical disease progression, unacceptable toxicity or withdrawal of consent took place. Schedule 2 was further explored at a dose range where immunologic activity was being observed. 43 subjects received IV SL-279252 (median age = 64; age range of 31-82). The subjects had median of 3 prior systemic therapies (range: 0-5) for metastatic disease. 56% of the subjects were male. A total of 25 subjects (58%) were checkpoint inhibitor (CPI) experienced. Tumor characteristics are shown in the Table 11: Table 11 : Tumor Characteristics (All Treated Subjects); Total (N = 43)

^* Endometrial, upper gastrointestinal track, and ileum and appendix; † mucosal melanoma of the vulvar (n=1) and cutaneous melanoma of the toe (=1)

As shown in Table 11, the most common tumor types were ocular melanoma (n=7; 16%), NSCLC adenocarcinoma (n=6; 14%), gastric adenocarcinoma (n=5; 12%), RCC (n=4; 9%) and HNSCC (n=4; 9%).

Table 12 summarizes all adverse events.

Table 12: All Causality Adverse events (AEs) in >10% of Subjects

No DLTs have been observed with SL-279252 on either schedule at doses ranging from 0.0001 mg/kg to 6.0 mg/kg. 40 subjects (93%) experienced an adverse event (AE) on treatment (Table 12). 19 subjects (44%) had treatment-related AEs (TRAEs). The most common reported TRAEs, occurring in > 5% of subjects, were maculo-papular rash (n=4; 9%), infusion related reaction (IRR; n=3; 7%), asthenia, constipation, decreased appetite, fatigue, hypothyroidism, night sweats and pruritis (remainder were n=2; 5%). The only G3 TRAEs were neutropenia (2%) and hypercalcemia (2%); each occurred in 1 subject. No G4/5 TRAEs occurred. Infusion related reactions (IRRs) occurred in 3 subjects (7%) dosed at 1.0 mg/kg (n=2) or 3.0 mg/kg (n=1); 6 events were G2 in severity and 1 event was G1 in severity. IRRs were manageable and did not prevent completion of IV dosing or lead to discontinuation of SL-279252. Most subjects (81%) discontinued SL- 279252 due to radiological or clinical progression; 2 subjects discontinued due to AEs unrelated to SL- 279252, 2 subjects withdrew consent, and 1 subject died due to cholecystitis and renal failure that were deemed to be unrelated to SL-279252 per investigator attribution.

Responses were classified using Response Evaluation Criteria in Solid Tumours for immune-based therapeutics (iRECIST). Tumor size was tracked and plotted as percentage change in target tumor size from baseline (FIG. 33). As shown in FIG. 33, the best response was 1 durable confirmed partial response (iPR; ocular melanoma, 4 prior systemic regimens, CPI-experienced) in a subject who remained on treatment for >1 yr. Stable disease (iSD) was observed in 12 patients (with 1 unconfirmed iPR in a CPI-experienced subject with cutaneous melanoma). iSD for > 24 weeks occurred in 5/12 subjects. Among these 5 subjects, 4 subjects had received prior CPI therapy targeting PD-1, PD-L1 or CTLA-4.

FIG. 34 shows the duration that the patients have stayed on study treatment. As shown in FIG. 34, median number of doses administered to patients was 7 (range 2-32). Median duration the patients remained on study treatment was 1.9 months (range 0.5-15.4; FIG. 34).

Pharmacokinetics (PK) profiles are well-characterized for doses > 0.003 mg/kg (FIG.20A and FIG.20B; FIG. 24A to FIG. 24D; FIG. 25; and FIG. 35A and FIG. 35B). FIG. 35A shows the updated pharmacokinetics (PK) of SL-279252 at Cycle 1 Day 1 for different doses. FIG. 35B shows the updated pharmacokinetics (PK) of SL-279252 at Cycle 1 Day 15 for different doses. As shown in FIG. 35A and FIG. 35B, SL-279252 Cmax and AUC increased linearly up to 3.0 mg/kg. A greater than proportional increase in AUC was observed at 6.0 mg/kg (FIG. 35A and FIG. 35B). The preliminary half-life (T½) was approximately 23 hours. For both schedules, within subject exposure (Cmax and AUC₂₄) was similar on Day 1, 15, and 29 (FIG. 35A and FIG. 35B), indicating no accumulation or time-dependent changes in PK. A trend towards non-linear increase in exposure was apparent for AUC and reflected by a decrease in CL with increasing dose (FIG. 35A and FIG. 35B). Preliminary observations, without wishing to be bound by theory, suggest saturation of target mediated drug disposition (FIG. 35A and FIG. 35B).

High background interference in samples from healthy volunteers and cancer patients was detected during anti-drug antibody (ADA) assay development presumed to be due to circulating anti-PD-1 therapeutic antibodies while in some cases the potential source of the interference has not been identified. Study subjects have demonstrated a high rate of positive tests at baseline prior to any treatment with SL-279252. Delayed infusion related reaction (IRR) (n=3) and/or accelerated clearance (n=2) was observed in 4/43 subjects. These events may indicate clinically relevant, treatment-emergent ADA. Efforts to develop validated assays to assess ADA confirmation and neutralization are continued. To evaluate receptor engagement, flow cytometry panels were designed to evaluate 0X40 CD4 T cell receptor engagement on subject peripheral blood samples (N=43). Maximum 0X40 receptor engagement across all SL- 279252 doses and schedules occurring during cycle 1 was plotted (FIG. 36). As shown in FIG. 36, maximal 0X40 RE was noted at doses > 1 mg/kg. Low level of PD-L1 expression on peripheral leukocytes precluded determination of PD-L1 receptor occupancy. Further, the dose-dependent binding and margination of CD4- )X40+T cells at Day 1 (FIG. 41 A) or Day 29 (FIG. 41 B) was observed as doses of SL-279252 were increased.

Flow cytometry analysis of T cells was in peripheral blood prior to dosing at Cycle 1 Day 1 (C1 D1 pre) and Cycle 1 Day 15 (C1 D15 pre). At baseline (Cycle 1 Day 1 (C1 D1 pre)) subjects had low median levels of (background) ARC staining on 0X40+ CD4 T cells (FIG. 37). As shown in FIG. 37, the background signal increased prior to administration of the third infusion (C1 D15 Pre). These results suggest, inter alia, that repeated SL-279252 dosing leads to durable binding for at least 7 days.

The Ki67 CD8 central memory T cells, Ki67 CD8 effector memory T cells, and CD8 central memory T cells were measured following dosing at increasing levels. The changes in CD8 central memory T cells (FIG. 38, left panel), Ki67 CD8 effector memory T cells (FIG. 38, middle panel), and CD8 central memory T cells (FIG. 38, right panel) as a function of dose of SL-279252 were plotted. These data showed increases in the number of proliferating and total CD8 central memory and effector memory T cells were seen at doses of > 1.0 mg/kg in some subjects (FIG. 38). No significant trends of change in cytokines or chemokines were observed following IV doses of SL-279252.

CD8 T cell infiltration in on-treatment biopsies was measured in pre-treatment biopsy samples and while on- treatment FIG. 39A to FIG. 39C. The on-treatment biopsy timepoint was at week 3 in the first treatment cycle between days 16-23. Tumor CD8+( FIG. 39A), CD8- 3ranzyme B(GZMB)+ (FIG. 39B) and CD8+Ki67+ (FIG. 39C) density were plotted for pre and on-treatment biopsies. As shown in FIG. 39A to FIG. 39C, an increased infiltration of CD8 T cells in tumors was observed in many samples. NK cells, CD8+ cells and CD8- 3ranzyme B(GZMB)+ cells were also observed in tumor biopsy samples by immunohistochemistry. As shown in FIG. 40, an increase in CD8/GZMB/Nkp46 was observed in MSI-H CRC Subject dosed at 3 mg/kg.

In summary, these results suggest, inter alia, that SL-279252 was well-tolerated in heavily-pretreated subjects with refractory solid tumors with no MTD reached. SL-279252 has exhibited antitumor activity in predominantly CPI-experienced subjects dosed on Schedule 1 (D1, D8, D15, then q2 weeks in 28d cycles). 1 confirmed iPR at 1.0 mg/kg, iSD as best response occurred in 12 subjects (5/12 subjects had iSD for >24 weeks; and 1 unconfirmed iPR at 6 mg/kg). SL-279252 exhibited linear PK at doses up to 3.0 mg/kg, and a greater than proportional increase in AUC was observed at 6.0 mg/kg suggesting, without wishing to be bound by theory, potential receptor saturation. The preliminary half-life (T½) was approximately 23 hours. Dose-dependent 0X40 receptor engagement on CD4OX40+T cells and OX40-dependent PD effects have been observed in subjects dosed with SL-279252 on Schedule 1. Trends for PK/PD effects and durable antitumor activity at doses of SL-279252 > 1.0 mg/kg suggests dose exploration in PD-L1 expressing cancers was warranted beyond 6.0 mg/kg. Dose escalation of SL-279252 will be continued at 12.0 mg/kg to fully characterize PK, PD, and antitumor activity.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present technology is not entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

EQUIVALENTS

While the invention has been disclosed in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments disclosed specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

CLAIMS What is claimed is:

1 . A method for treating a cancer in a human subject comprising a step of administering to the human subject a chimeric protein having a general structure of:

N terminus - (a) - (b) - (c) - C terminus, wherein:

(b) is a linker adjoining the first and second domains, wherein the linker comprises at least one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH2-CH3 Fc domain, and

(c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L), wherein the dose of the chimeric protein administered is at least about 0.0001 mg/kg.

2. The method of claim 1 , wherein the dose of the chimeric protein administered is between about 0.0001 mg/kg and about 50.0 mg/kg.

3. The method of claim 1 or claim 2, wherein the dose selected from about 1 mg/kg, about 3 mg/kg, about 6 mg/kg, or about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about

25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about

42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg.

4. The method of claim 1, wherein the chimeric protein is administered at an initial dose and one or more subsequent administrations.

5. The method of claim 4, wherein the initial dose is one of about 0.0001 mg/kg, about 0.001 mg/kg, about 0.003 mg/kg, about 0.01 mg/kg, about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 .0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, about 6.0 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, or about 50 mg/kg.

6. The method of claim 4 or claim 5, wherein the one or more subsequent administrations has a dose of one or more of about 0.0001 mg/kg, about 0.001 mg/kg, about 0.003 mg/kg, about 0.01 mg/kg, about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, about 6.0 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about

27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about

45 mg/kg, about 48 mg/kg, or about 50 mg/kg.

7. The method of any one of claims 4 to 6, wherein the initial dose is less than the dose for at least one of the subsequent administrations.

8. The method of claim 7, wherein the initial dose is less than the dose for each of the subsequent administrations.

9. The method of any one of claims 4 to 6, wherein the initial dose is the same as the dose for at least one of the subsequent administrations.

10. The method of claim 9, wherein the initial dose is the same as the dose for each of the subsequent administrations.

11. The method of any one of claims 1 to 10, wherein the chimeric protein is administered at least about one time a month.

12. The method of any one of claims 1 to 11 , wherein the chimeric protein is administered at least about two times a month.

13. The method of any one of claims 1 to 12, wherein the chimeric protein is administered at least about three times a month.

14. The method of claim 13, wherein the chimeric protein is first administered once a week for three weeks and the chimeric protein is then administered about once every three weeks or once every four weeks.

15. The method of claim 13, wherein the chimeric protein is first administered once a week for three weeks and the chimeric protein is then administered about two times per month.

16. The method of claim 15, wherein the chimeric protein is first administered once a week for three weeks and the chimeric protein is then administered about once every two weeks.

17. The method of any one of claims 1 to 13, wherein the chimeric protein is administered at least about four times a month.

18. The method of claim 14, wherein the chimeric protein is administered about once a week.

19. The method of any one of claims 1 to 18, wherein the cancer comprises an advanced solid tumor (local and/or metastatic) or a lymphoma.

20. The method of any one of claims 1 to 19, wherein the cancer is selected from melanoma, non-small cell lung cancer (squamous, adeno, or adeno-squamous), urothelial cancer, renal cell cancer, squamous cell cervical cancer, gastric or gastro-esophageal junction adenocarcinoma, squamous cell carcinoma of the anus, squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin, Hodgkin's disease, diffuse large B cell lymphoma, and microsatellite instability high or mismatch repair deficient solid tumors, excluding CNS tumors.

21. The method of any one of claims 1 to 20, wherein the first domain is capable of binding a PD-1 ligand.

22. The method of any one of claims 1 to 21, wherein the first domain comprises substantially all of the extracellular domain of PD-1.

23. The method of any one of claims 1 to 22, wherein the second domain is capable of binding an OX40L receptor.

24. The method of any one of claims 1 to 23, wherein the second domain comprises substantially all of the extracellular domain of OX40L.

25. The method of any one of claims 1 to 24, wherein the linker comprises a hinge-CH2-CH3 Fc domain derived from lgG4, e.g., human lgG4.

26. The method of any one of claims 1 to 25, wherein the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

27. The method of any one of claims 1 to 26, wherein the first domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 57.

28. The method of any one of claims 1 to 27, wherein the second domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58.

29. The method of any one of claims 1 to 28, wherein

(a) the first domain comprises the amino acid sequence of SEQ ID NO: 57,

(b) the second domain comprises the amino acid sequence of SEQ ID NO: 58, and

(c) the linker comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

30. The method of any one of claims 1 to 29, wherein the chimeric protein further comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

31. The method of any one of claims 1 to 30, wherein the chimeric protein further comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7.

32. The method of any one of claims 1 to 29, wherein the chimeric protein comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

33. The method of claim 32, wherein the chimeric protein comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

34. The method of claim 33 wherein the chimeric protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

35. The method of claim 34, wherein the chimeric protein comprises an amino acid sequence that is at least about 99.2% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

36. The method of claim 35, wherein the chimeric protein comprises an amino acid sequence that is at least about 99.4% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

37. The method of claim 36, wherein the chimeric protein comprises an amino acid sequence that is at least about 99.6% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

38. The method of claim 37, wherein the chimeric protein comprises an amino acid sequence that is at least about 99.8% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

39. The method of claim 38, wherein the chimeric protein comprises the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61.

40. The method of any one of claims 1 to 38, wherein the human subject has received, been tolerant to, or is ineligible for standard therapy and/or the cancer has no approved therapy considered to be standard of care.

41. The method of any one of claims 1 to 39, wherein the human subject is not receiving a concurrent chemotherapy, immunotherapy, biologic or hormonal therapy.

42. A chimeric protein for use in the method of any one of claims 1 to 40.

43. A chimeric protein comprising an amino acid sequence that is at least about 98% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

44. The chimeric protein of claim 42, wherein the chimeric protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

45. The chimeric protein of claim 43, wherein the chimeric protein comprises an amino acid sequence that is identical to SEQ ID NO: 59 or SEQ ID NO: 61.

46. A method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of:

N terminus - (a) - (b) - (c) - C terminus, wherein:

(b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and

(c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L); wherein the step of administering comprises a biphasic dosing.

47. The method of claim 46, wherein the first phase, and the second phase each independently comprise a dosing frequency of from about twice a week to about once every two months.

48. The method of claim 46 or claim 47, wherein the dosing frequency of the first phase, and the dosing frequency of the second phase are the same.

49. The method of claim 46 or claim 47, wherein the dosing frequency of the first phase, and the dosing frequency of the second phase are different.

50. The method of any one of claims 46-49, wherein the dosing frequency of the first phase is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months.

51 . The method of any one of claims 46-50, wherein the dosing frequency of the first phase is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months.

52. The method of any one of claims 46-51, wherein the dosing frequency of the second phase is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months.

53. The method of any one of claims 46-52, wherein the dosing frequency of the second phase is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months.

54. The method of any one of claims 46-53, wherein the dosing frequency of the second phase is selected from from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks; and the frequency of the second phase is selected from from about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks.

55. The method of any one of claims 46-54, wherein the first phase, and the second phase each independently last from about two days to about 12 months.

56. The method of any one of claims 46-55, wherein the first phase lasts from about two weeks to about 2 months; and the second phase lasts from about 2 weeks to about 12 months.

57. The method of any one of claims 46-56, wherein the first phase lasts from about two weeks to about 1 month; and the second phase lasts from about 2 weeks to about 12 months.

58. The method of any one of claims 46-57, wherein the first phase lasts from about two weeks to about 1 month; and the second phase lasts from about 4 weeks to about 12 months.

59. The method of any one of claims 46-58, wherein the effective amount for the first phase, the second phase and the third phase each independently comprise about 0.01 mg/kg to about 10 mg/ml.

60. The method of any one of claims 46-59, wherein the effective amount for the first phase, the second phase and the third phase each independently selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg, and any range including and/or in between any two of the preceding values.

61 . The method of any one of claims 46-60, wherein the effective amount for the first phase, the second phase and the third phase each independently selected from from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, and about 10 mg/kg to about 50 mg/kg.

62. The method of any one of claims 46-61, wherein the effective amount for the first phase, the second phase and the third phase are same.

63. The method of any one of claims 46-62, wherein the effective amount for the first phase, the second phase and the third phase are different.

64. The method of any one of claims 46-63, wherein the effective amount for the first phase is greater than the effective amount for the second phase.

65. The method of any one of claims 46-64, wherein the effective amount for the first phase is from about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg; and the effective amount for the second phase is from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg.

66. The method of any one of claims 46-65, wherein the human PD-1-Fc-OX40L chimeric protein is capable of providing a sustained immunomodulatory effect.

67. The method of any one of claims 46-66, wherein the linker comprises hinge-CH2-CH3 Fc domain derived from lgG4.

68. The method of claim 67, wherein the hinge-CH2-CH3 Fc domain is derived from human lgG4.

69. The method of claim 67, wherein the linker comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

70. The method of claim 67, wherein the linker comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

71. The method of any one of claims 46-70, wherein the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57.

72. The method of claim 71, wherein the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 57.

73. The method of any one of claims 46-66, wherein the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58.

74. The method of claim 73, wherein the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 58.

75. The method of any one of claims 46-74, wherein the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

76. The method of any one of claims 46-75, wherein the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

77. The method of any one of claims 46-76, wherein the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

78. The method of any one of claims 46-77, wherein the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

79. A method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein:

(c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L); wherein the step of administering comprises a first cycle, a second cycle and/or a third cycle.

80. The method of claim 79, wherein the first cycle, the second cycle and the third cycle each independently comprise a dosing frequency of from about twice a week to about once every two months.

81 . The method of claim 79 or claim 80, wherein the dosing frequency of the first cycle, the dosing frequency of the second cycle and the dosing frequency of the third cycle are the same.

82. The method of claim 79 or claim 80, wherein the dosing frequency of the first cycle, the dosing frequency of the second cycle and the dosing frequency of the third cycle are different.

83. The method of any one of claims 79 to 82, wherein the dosing frequency of the first cycle is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months.

84. The method of any one of claims 79 to 83, wherein the dosing frequency of the first cycle is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months.

85. The method of any one of claims 79 to 84, wherein the dosing frequency of the second cycle is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months.

86. The method of any one of claims 79 to 85, wherein the dosing frequency of the second cycle is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months.

87. The method of any one of claims 79 to 86, wherein the dosing frequency of the third cycle is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months.

88. The method of any one of claims 79 to 87, wherein the dosing frequency of the third cycle is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months.

89. The method of any one of claims 79 to 88, wherein the dosing frequency of the first cycle is selected from from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks; and the frequency of the second cycle is selected from from about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks.

90. The method of any one of claims 79 to 89, wherein the first cycle, the second cycle and the third cycle each independently last from about two days to about 12 months.

91. The method of any one of claims 79 to 90, wherein the first cycle lasts from about two weeks to about 2 months; and the second cycle lasts from about 2 weeks to about 12 months.

92. The method of any one of claims 79 to 91, wherein the first cycle lasts from about two weeks to about 2 months; the second cycle lasts from about 2 weeks to about 12 months and the third cycle lasts from about 2 weeks to about 6 months.

93. The method of any one of claims 79 to 92, wherein the effective amount for the first cycle, the second cycle and the third cycle each independently comprise about 0.01 mg/kg to about 10 mg/ml.

94. The method of any one of claims 79 to 93, wherein the effective amount for the first cycle, the second cycle and the third cycle each independently selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg, and any range including and/or in between any two of the preceding values.

95. The method of any one of claims 79 to 94, wherein the effective amount for the first cycle, the second cycle and the third cycle each independently selected from from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, and about 10 mg/kg to about 50 mg/kg.

96. The method of any one of claims 79 to 95, wherein the effective amount for the first cycle, the second cycle and the third cycle are same.

97. The method of any one of claims 79 to 96, wherein the effective amount for the first cycle, the second cycle and the third cycle are different.

98. The method of any one of claims 79 to 97, wherein the effective amount for the first cycle is greater than the effective amount for the second cycle.

99. The method of any one of claims 79 to 98, wherein the effective amount for the first cycle is from about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg; and the effective amount for the second cycle is from about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 1 mg/kg to about 10 mg/kg, about 3 mg/kg to about 30 mg/kg, or about 10 mg/kg to about 50 mg/kg.

100. The method of any one of claims 79-99, wherein the human PD-1-Fc-OX40L chimeric protein is capable of providing a sustained immunomodulatory effect.

101. The method of any one of claims 79-100, wherein the linker comprises hinge-CH2-CH3 Fc domain derived from lgG4.

102. The method of claim 101, wherein the hinge-CH2-CH3 Fc domain is derived from human lgG4.

103. The method of claim 101, wherein the linker comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

104. The method of claim 103, wherein the linker comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

105. The method of any one of claims 79-104, wherein the extracellular domain of human programmed cell death protein 1 (PD-1) comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57.

106. The method of claim 105, wherein the extracellular domain of human programmed cell death protein 1 (PD- 1) comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 57.

107. The method of any one of claims 79-106, wherein the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58.

108. The method of claim 107, wherein the extracellular domain of human 0X40 ligand (OX40L) comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 58.

109. The method of any one of claims 79-108, wherein the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

110. The method of any one of claims 79-109, wherein the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

111. The method of any one of claims 79-110, wherein the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

112. The method of any one of claims 79-111, wherein the human PD-1-Fc-OX40L chimeric protein comprises an amino acid sequence that is identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

113. A method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of:

N terminus - (a) - (b) - (c) - C terminus, wherein:

(c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L); wherein the dosing regimen comprises dosing with a frequency in the range of about every three days to about every 2 months.

114. The method of claim 113, wherein the dosing regimen is selected from about every three days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months.

115. The method of claim 113 or claim 114, wherein the dosing regimen is selected from about every week, about every 10 days, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about 7 seven weeks, about every 8 weeks and about every 2 months.

116. The method of any one of claims 113 to 115, wherein the dosing regimen is about every 2 weeks, about every 3 weeks, or about every 4 weeks.

117. A method for treating a cancer in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge- CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L) with a dosing regimen selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and , about every 6 weeks to about every 2 months.

118. The method of claim 117, wherein the dosing regimen is about every week to about every 2 weeks, about every 10 days to about every 3 weeks, or about every 2 weeks to about every 4 weeks.

119. The method of any one of claims 46 to 118, wherein the cancer is selected from melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), squamous cell carcinoma of the skin (skin- SCC), urothelial cancer, cervical cancer, gastric cancer, gastric or gastro-esophageal junction adenocarcinoma, renal cell carcinoma (RCC), squamous cell carcinoma (SCCA), squamous cell carcinoma of the anus, Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor, with the proviso that the microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor is not a CNS tumor.

120. The method of any one of claims 46 to 119, wherein the first domain is capable of binding a PD-1 ligand.

121. The method of any one of claims 46 to 120, wherein the first domain comprises substantially all of the extracellular domain of PD-1.

122. The method of any one of claims 46 to 121, wherein the second domain is capable of binding an OX40L receptor.

123. The method of any one of claims 46 to 122, wherein the second domain comprises substantially all of the extracellular domain of OX40L.

124. The method of any one of claims 46 to 123, wherein the linker comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

125. The method of any one of claims 46 to 124, wherein the first domain comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57.

126. The method of any one of claims 46 to 125, wherein the second domain comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58.

127. The method of any one of claims 46 to 80, wherein

(a) the first domain comprises the amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 57,

(b) the second domain comprises the amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 58, and

(c) the linker comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

128. The method of any one of claims 46 to 127, wherein the chimeric protein further comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

129. The method of any one of claims 46 to 128, wherein the chimeric protein further comprises the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7.

130. The method of any one of claims 46 to 129, wherein the chimeric protein comprises an amino acid sequence that is at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 59 or SEQ ID NO: 61 .

131 . The method of claim 130, wherein the chimeric protein comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

132. The method of claim 131 , wherein the chimeric protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

133. The method of claim 132, wherein the chimeric protein comprises an amino acid sequence that is at least about 99.2% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

134. The method of claim 133, wherein the chimeric protein comprises an amino acid sequence that is at least about 99.4% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

135. The method of claim 134, wherein the chimeric protein comprises an amino acid sequence that is at least about 99.6% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

136. The method of claim 135, wherein the chimeric protein comprises an amino acid sequence that is at least about 99.8% identical to SEQ ID NO: 59 or SEQ ID NO: 61.

137. The method of claim 136, wherein the chimeric protein comprises the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61.

138. The method of any one of claims 46 to 137, wherein the human subject has received, been tolerant to, or is ineligible for standard therapy and/or the cancer has no approved therapy considered to be standard of care.

139. The method of any one of claims 46 to 138, wherein the human subject is not receiving a concurrent chemotherapy, immunotherapy, biologic or hormonal therapy.

140. A method for evaluating the efficacy of cancer treatment in a subject in need thereof, wherein the subject is suffering from a cancer, the method comprising the steps of:

(i) administering a dose of a chimeric protein, wherein the dose of from about 0.03 mg/kg to 50 mg/kg; wherein the chimeric protein having a general structure of:

N terminus - (a) - (b) - (c) - C terminus, wherein:

(b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge- CH2-CH3 Fc domain, and

(c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L);

(ii) obtaining a biological sample from the subject;

(iii) performing an assay on the biological sample to determine level and/or activity of a cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNo, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFo; and

(iv) continuing administration of the chimeric protein if the subject has an increase in the level and/or activity of at least one cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFa.

141. A method of selecting a subject for treatment with a therapy for a cancer, the method comprising the steps of:

N terminus - (a) - (b) - (c) - C terminus, wherein:

(ii) obtaining a biological sample from the subject; (iii) performing an assay on the biological sample to determine level and/or activity of a cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNo, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFo; and

(iv) selecting the subject for treatment with the therapy for cancer if the subject has an increase in the level and/or activity of at least one cytokine selected from CCL2, CCL3, CCL4, IFNy, IFNa, IL-2, IL-6, IL-18, IL-15, IL-27, and TNFo.

142. The method of claim 140 or claim 141, wherein the cancer is selected from melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), squamous cell carcinoma of the skin (skin-SCC), urothelial cancer, cervical cancer, gastric cancer, gastric or gastro-esophageal junction adenocarcinoma, renal cell carcinoma (RCC), squamous cell carcinoma (SCCA), squamous cell carcinoma of the anus, Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor, with the proviso that the microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor.

143. The method of any one of claims 140-142, wherein the biological sample is a body fluid selected from blood, plasma, serum, lacrimal fluid, tears, bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy sample, cell- containing body fluid, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, washing or lavage such as a ductal lavage or broncheoalveolar lavage, aspirate, scraping, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluids, secretions, and/or excretions, and/or cells therefrom.

144. The method of any one of claims 140-143, wherein the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen.

145. The method of any one of claims 140-144, wherein the biological sample is a tumor sample derived from a tumor selected from melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), squamous cell carcinoma of the skin (skin-SCC), urothelial cancer, cervical cancer, gastric cancer, gastric or gastro-esophageal junction adenocarcinoma, renal cell carcinoma (RCC), squamous cell carcinoma (SCCA), squamous cell carcinoma of the anus, Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor, with the proviso that the microsatellite instability-low (MSI-L) and mismatch repair deficient (MMRD) solid tumor.

146. The method of any one of claims 140-145, wherein the biological sample is obtained by a technique selected from scrapes, swabs, and biopsy.

147. The method of any one of claims 140-146, wherein the biological sample is obtained by use of brushes, (cotton) swabs, spatula, rinse/wash fluids, punch biopsy devices, puncture of cavities with needles or surgical instrumentation.

148. The method of any one of claims 140-147, wherein the level and/or activity of the cytokine is measured by RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof.

149. The method of any one of claims 140-148, wherein the cytokine is measured by contacting the sample with an agent that specifically binds to one or more of the cytokines.

150. The method of claim 149, wherein the agent that specifically binds to one or more of the cytokines is an antibody or fragment thereof.

151. The method of claim 150, wherein the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or fragment thereof.

152. The method of any one of claims 140-151, wherein the level and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds to one or more of the nucleic acids.

153. The method of claim 152, wherein the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

154. The method of any one of claims 140 or 142-153, wherein the evaluating comprises diagnosis, prognosis, or response to treatment.

155. The method of any one of claims 140 or 142-154, wherein the evaluating informs classifying the subject into a high or low risk group.

156. The method of claim 155, wherein the high risk classification comprises a high level of cancer aggressiveness, wherein the aggressiveness is characterizable by one or more of a high tumor grade, low overall survival, high probability of metastasis, and the presence of a tumor marker indicative of aggressiveness.

157. The method of claim 155 or claim 156, wherein the low risk classification comprises a low level of cancer aggressiveness, wherein the aggressiveness is characterizable by one or more of a low tumor grade, high overall survival, low probability of metastasis, and the absence and/or reduction of a tumor marker indicative of aggressiveness.

158. The method of any one of claims 155 to 157, wherein the low risk or high risk classification is indicative of withholding of neoadjuvant therapy.

159. The method of any one of claims 155 to 158, wherein the low risk or high risk classification is indicative of withholding of adjuvant therapy.

160. The method of any one of claims 155 to 159, wherein the low risk or high risk classification is indicative of continuing of the administration of the chimeric protein.

161. The method of any one of claims 155 to 160, wherein In some embodiments, the low risk or high risk classification is indicative of withholding of the administration of the chimeric protein.

162. The method of any one of claims 140 or 142-161, wherein the evaluating is predictive of a positive response to and/or benefit from the administration of the chimeric protein.

163. The method of any one of claims 140 or 142-161, wherein the evaluating is predictive of a negative or neutral response to and/or benefit from the administration of the chimeric protein.

164. The method of any one of claims 140 or 142-163, wherein the evaluating informs continuing the administration or withholding of the administration of the chimeric protein.

165. The method of claim 164, wherein the evaluating informs continuing of the administration of the chimeric protein.

166. The method of claim 164 or claim 165, wherein the evaluating informs changing the dose of the chimeric protein.

167. The method of any one of claims 164 to 166, wherein the evaluating informs increasing the dose of the chimeric protein.

168. The method of any one of claims 164 to 167, wherein the evaluating informs decreasing the dose of the chimeric protein.

169. The method of any one of claims 164 to 168, wherein the evaluating informs changing the regimen of administration of the chimeric protein.

170. The method of any one of claims 164 to 169, wherein the evaluating informs increasing the frequency of administration of the chimeric protein.

171. The method of any one of claims 140 or 142 to 170, wherein the evaluating informs administration of neoadjuvant therapy.

172. The method of any one of claims 140 or 142 to 171 , wherein the evaluating informs withholding of neoadjuvant therapy.

173. The method of any one of claims 140 or 142 to 172, wherein the evaluating informs administration of adjuvant therapy.

174. The method of any one of claims 140 or 142 to 173, wherein the evaluating informs changing of neoadjuvant therapy.

175. The method of any one of claims 140 or 142 to 173, wherein the evaluating informs changing of adjuvant therapy.

176. The method of any one of claims 140 or 142 to 175, wherein the evaluating informs withholding of adjuvant therapy.

177. The method of any one of claims 140 or 142 to 176, wherein the evaluating is predictive of a positive response to and/or benefit from neoadjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from neoadjuvant chemotherapy.

178. The method of any one of claims 140 or 142 to 176, wherein the evaluating is predictive of a negative or neutral response to and/or benefit from neoadjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from neoadjuvant chemotherapy.

179. The method of any one of claims 140 or 142 to 178, wherein the evaluating is predictive of a positive response to and/or benefit from adjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from adjuvant chemotherapy.

180. The method of any one of claims 140 or 142 to 178, wherein the evaluating is predictive of a negative or neutral response to and/or benefit from adjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from adjuvant chemotherapy.

181. The method of any one of claims 158 to 180, wherein the neoadjuvant therapy and/or adjuvant therapy is a chemotherapeutic agent.

182. The method of any one of claims 158 to 180, wherein the neoadjuvant therapy and/or adjuvant therapy is a cytotoxic agent.

183. The method of any one of claims 158 to 180, wherein the neoadjuvant therapy and/or adjuvant therapy is checkpoint inhibitor.

184. A method for inducing lymphocyte expansion in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L).

185. A method for inducing lymphocyte margination in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L).

186. A method for inducing lymphocyte margination in a human subject in need thereof the method comprising a step of administering to the human subject an effective amount of a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a first domain comprising an extracellular domain of human programmed cell death protein 1 (PD-1), (b) is a linker adjoining the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human 0X40 ligand (OX40L).

187. The method of any one of claim 1-139 or 184-186, wherein the initial dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg.

188. The method of any one of claim 1-139 or 184-187, wherein the maintenance dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg.

189. The method of any one of claim 1-139 or 184-188, wherein the dose is selected from about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, about 21 mg/kg, about 24 mg/kg, about 25 mg/kg, about 27 mg/kg, about 30 mg/kg, about 33 mg/kg, about 35 mg/kg, about 36 mg/kg, about 40 mg/kg, about 42 mg/kg, about 45 mg/kg, about 48 mg/kg, and about 50 mg/kg.

190. The method of any one of claim 1-139 or 184-186, wherein the dose is administered with a once a week or a once every two weeks schedule.

191. The method of any one of claim 1-139 or 184-190, further comprising administration of a priming dose to the subject.

192. The method of any one of claim 1- 139 or 184-191, wherein the number of proliferating CD4 central and/or the number effector memory T cells in the subject increases compared to the number of proliferating CD4 central and/or the number effector memory T cells in the subject before administration of the chimeric protein, or compared to the number of proliferating CD4 central and/or the number effector memory T cells in a subject that was not administered the chimeric protein, and/or compared to a control.

193. The method of any one of claim 1-139 or 184-192, wherein the treatment produces at least one therapeutic effect chosen from a reduction in size of a tumor, reduction in number of metastatic lesions over time, complete response, partial response, and stable disease.