CN118510812A

CN118510812A - Chimeric proteins based on SIRP1A and CD40L

Info

Publication number: CN118510812A
Application number: CN202280087887.5A
Authority: CN
Inventors: A·潘迪特; F·朗瓦拉; T·兰普金; T·施赖伯; G·弗罗姆; S·达西瓦
Original assignee: Shattuck Labs Inc
Current assignee: Shattuck Labs Inc
Priority date: 2021-11-12
Filing date: 2022-11-11
Publication date: 2024-08-16

Abstract

The present disclosure relates in part to compositions and methods, including chimeric proteins, useful in immunotherapy for treating diseases, such as cancer.

Description

Chimeric proteins based on SIRP1A and CD40L

Technical Field

The present technology relates in particular to compositions and methods (including chimeric proteins) useful in immunotherapy, including dosages, dosing regimens including bi-phasic administration, or dosing regimens including three cycles, for treating diseases such as cancer.

Priority

The application claims U.S. provisional application No. 63/278,567 filed on 11/12 of 2021; and U.S. provisional application No. 63/371,083, filed on 8/11 of 2022, the contents of each of which are hereby incorporated by reference in their entirety.

Sequence listing

The present application contains a sequence table that is submitted via EFS-Web in XML format. The content of the XML copy named "SHK-055PC_116981-5055_sequence_Listing" was created at month 9 of 2022, 11, and is 82,547 bytes in size, and the content of which is incorporated herein by reference in its entirety.

Background

Over the past several years, there has been tremendous development in the area of cancer immunotherapy. This is driven in large part by the clinical efficacy of antibodies targeting the checkpoint family of molecules (e.g., CTLA-4 and PD-1/L1) in many tumor types. However, despite this success, clinical responses to these agents as monotherapy occur in a small number of patients (10% -45% of various solid tumors), and these therapies are hampered by side effects.

Rational dose selection and optimization of dosing regimens are of clinical importance and are a prerequisite to enhancing patient compliance and achieving maximum clinical benefit. Development of dosing regimens typically relies on pharmacokinetic/pharmacodynamic studies performed in animal models. However, since immunotherapy does not exert a direct antiproliferative activity on cancer cells, but is expected to exploit tumor immunity, some animal studies are generally performed using murine substitutes, and thus pharmacokinetic/pharmacodynamic studies cannot be performed. In addition, physicians can vary the dosage regimen of immunotherapy depending on the immunogenicity of the tumor, the stage of the disease, and the patient's physical condition. Thus, there is a need to develop new dosing strategies and protocols.

Disclosure of Invention

Thus, in various aspects, the present disclosure provides compositions and methods useful for developing strategies for developing dosages and dosing regimens for cancer immunotherapy. The present disclosure is based in part on the following findings: cd80+ cells, cd8+ cells, granzyme b+ cells, cd68+ cells, ki67+ cells and PD-l1+ immune cells in the Tumor Microenvironment (TME) are increased following administration of the sirpa-Fc-CD 40L chimeric protein; b cells and/or cd40+ cells collected from the peripheral blood margin (marginate) within hours after administration and returned to the peripheral blood within days; and the level of certain serum cytokines increases within hours after administration and decreases within days after administration of the sirpa-Fc-CD 40L chimeric protein.

One aspect of the present disclosure is a method for treating cancer in a human subject. In embodiments, the method comprises: (i) Administering to a human subject a first dose of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background level of cells in a pre-dose biological sample obtained from a subject prior to administration of a first dose has been measured, wherein the marker is selected from one or more of cd80+ cells, cd8+ cells, granzyme b+ cells, cd68+ cells, ki67+ cells, and PD-l1+ immune cells. In embodiments, the method further comprises administering a second dose of the chimeric protein to the human subject if the post-administration level of the cell is greater than the background level of the cell. In embodiments, the second dose is administered at least about 48 hours after the first dose is administered. In embodiments, the biological sample is a biopsy sample or a surgical specimen. In embodiments, the biological sample is a tumor biopsy or a tumor surgical specimen. In embodiments, the level of cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting (IN CELL WESTERN), immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

One aspect of the present disclosure is a method for treating cancer in a human subject. In embodiments, the method comprises: (i) Administering to a human subject a first dose of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background level and/or activity of B cells and/or cd40+ cells in a first biological sample obtained from a subject prior to administration of a first dose has been measured. In embodiments, the level and/or activity of B cells and/or cd40+ cells in a second biological sample obtained from the subject after administration of the first dose has been measured N hours after administration. In an embodiment, N is a number between 1 and 24. In embodiments, the level and/or activity of the B cells and/or cd40+ cells N hours after administration is less than the background level and/or activity of the B cells and/or cd40+ cells. In embodiments, the post-administration M-day level and/or activity of B cells and/or cd40+ cells in a third biological sample obtained from the subject after administration of the first dose has been measured. In an embodiment, M is a number between 1 and 28. In embodiments, the method further comprises (ii) administering a second dose of the chimeric protein to the human subject if the level and/or activity of the B cells and/or cd40+ cells M days after administration is at least about 50% greater than the level and/or activity of the B cells and/or cd40+ cells N hours after administration. In embodiments, the second dose is administered at least about 48 hours after the first dose is administered. In embodiments, the first biological sample, the second biological sample, and the third biological sample are blood. In embodiments, the level and/or activity of B cells and/or cd40+ cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof.

One aspect of the present disclosure is a method for treating cancer in a human subject. In embodiments, the method comprises: (i) Administering to a human subject a first dose of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background amount and/or activity of a cytokine selected from the group consisting of CCL2, CXCL9, CXCL10, ifnα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC in a first biological sample obtained from a subject prior to administration of a first dose has been measured. In embodiments, the amount and/or activity of the cytokine in the second biological sample obtained from the subject N hours after administration of the first dose has been measured. In an embodiment, N is a number between 1 and 24. In embodiments, the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine. In embodiments, the amount and/or activity of the cytokine in a third biological sample obtained from the subject M days after administration of the first dose has been measured. In an embodiment, M is a number between 1 and 28. In embodiments, the method further comprises (ii) administering a second dose of the chimeric protein to the human subject if the amount and/or activity of the cytokine M days after administration is at least about 30% less than the amount and/or activity of the cytokine N hours after administration. In embodiments, the second dose is administered at least about 48 hours after the first dose is administered. In embodiments, the first biological sample, the second biological sample, and the third biological sample are blood. In embodiments, the amount and/or activity of the cytokine is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof.

One aspect of the present disclosure is a method for assessing the efficacy of a cancer treatment in a subject in need thereof, wherein the subject has cancer. In embodiments, the method comprises: (i) Obtaining a biological sample obtained from a subject who has received a first dose of chimeric protein. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background level and/or activity of cells in a biological sample obtained from a subject prior to administration of a first dose has been measured. In embodiments, the cell is selected from one or more of a cd80+ cell, a cd8+ cell, a granzyme b+ cell, a cd68+ cell, a ki67+ cell, and a PD-l1+ immune cell. In embodiments, the method further comprises (ii) determining post-administration levels and/or activity of cells in the biological sample. In embodiments, the method further comprises (iii) determining that the chimeric protein is effective if the post-administration level and/or activity of the cell is greater than the background level and/or activity of the cell. In embodiments, the biological sample is a tumor biopsy or a tumor surgical specimen. In embodiments, the level and/or activity of the cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof.

One aspect of the disclosure is a method of selecting a subject for treatment with a therapy for cancer. In embodiments, the method comprises: (i) Obtaining a biological sample obtained from a subject who has received a first dose of chimeric protein. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background level and/or activity of cells in a biological sample obtained from a subject prior to administration of a first dose has been measured. In embodiments, the cell is selected from one or more of a cd80+ cell, a cd8+ cell, a granzyme b+ cell, a cd68+ cell, a ki67+ cell, and a PD-l1+ immune cell. In embodiments, the method further comprises (ii) determining post-administration levels and/or activity of cells in the biological sample. In embodiments, the method further comprises (iii) selecting the subject for treatment with the chimeric protein if the post-administration level and/or activity of the cell is greater than the background level and/or activity of the cell. In embodiments, the biological sample is a tumor biopsy or a tumor surgical specimen. In embodiments, the level and/or activity of the cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof.

One aspect of the present disclosure is a method for assessing the efficacy of a cancer treatment in a subject in need thereof, wherein the subject has cancer. In embodiments, the method comprises: (i) A first biological sample obtained from a subject that has received a first dose of chimeric protein is obtained. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the method further comprises determining the background level and/or activity of B cells and/or cd40+ cells in the first biological sample. In embodiments, the method further comprises (iii) obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24. In embodiments, the method further comprises determining the level and/or activity of B cells and/or cd40+ cells in the second biological sample N hours after administration. In embodiments, the method further comprises (iv) obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises determining the level and/or activity of B cells and/or cd40+ cells in the third biological sample M days after administration. In embodiments, the method further comprises (v) determining that the chimeric protein is effective if the level and/or activity of the B cell and/or the cd40+ cell at N hours after administration is less than the background level and/or activity of the B cell and/or the cd40+ cell and/or if the level and/or activity of the B cell and/or the cd40+ cell at M days after administration is at least about 50% greater than the level and/or activity of the B cell and/or the cd40+ cell at N hours after administration. In embodiments, the first biological sample, the second biological sample, and the third biological sample are blood. In embodiments, the level and/or activity of B cells and/or cd40+ cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof.

One aspect of the disclosure is a method of selecting a subject for treatment with a therapy for cancer. In embodiments, the method comprises: (i) A first biological sample obtained from a subject that has received a first dose of chimeric protein is obtained. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the method further comprises determining the background level and/or activity of B cells and/or cd40+ cells in the first biological sample. In embodiments, the method further comprises (iii) obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24. In embodiments, the method further comprises determining the level and/or activity of B cells and/or cd40+ cells in the second biological sample N hours after administration. In embodiments, the method further comprises (iv) obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises determining the level and/or activity of B cells and/or cd40+ cells in the third biological sample M days after administration. In embodiments, the method further comprises (v) selecting the subject for treatment with the chimeric protein if the N hour post-administration level and/or activity of the B cell and/or cd40+ cell is less than the background level and/or activity of the B cell and/or cd40+ cell and/or if the M day post-administration level and/or activity of the B cell and/or cd40+ cell is at least about 50% greater than the N hour post-administration level and/or activity of the B cell and/or cd40+ cell. In embodiments, the first biological sample, the second biological sample, and the third biological sample are blood. In embodiments, the level and/or activity of B cells and/or cd40+ cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof.

One aspect of the present disclosure is a method for assessing the efficacy of a cancer treatment in a subject in need thereof, wherein the subject has cancer. In embodiments, the method comprises: (i) A first biological sample obtained from a subject that has received a first dose of chimeric protein is obtained. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the method further comprises (ii) determining the background amount and/or activity of the cytokine in the first biological sample. In embodiments, the cytokine is selected from the group consisting of CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1 β, MIP-1 α, and MDC. In embodiments, the method further comprises (iii) obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24. In embodiments, the method further comprises determining the amount and/or activity of the cytokine in the second biological sample N hours after administration. In embodiments, the method further comprises (iv) obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises determining the amount and/or activity of the cytokine in the third biological sample M days after administration. In embodiments, the method further comprises (v) determining that the chimeric protein is effective if the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine and/or if the amount and/or activity of the cytokine M days after administration is at least about 50% less than the amount and/or activity of the cytokine N hours after administration. In embodiments, the first biological sample, the second biological sample, and the third biological sample are blood. In embodiments, the amount and/or activity of the cytokine is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof.

One aspect of the disclosure is a method of selecting a subject for treatment with a therapy for cancer. In embodiments, the method comprises: (i) A first biological sample obtained from a subject that has received a first dose of chimeric protein is obtained. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the method further comprises (ii) determining the background amount and/or activity of the cytokine in the first biological sample. In embodiments, the cytokine is selected from the group consisting of CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1 β, MIP-1 α, and MDC. In embodiments, the method further comprises (iii) obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24. In embodiments, the method further comprises determining the amount and/or activity of the cytokine in the second biological sample N hours after administration. In embodiments, the method further comprises (iv) obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises determining the amount and/or activity of the cytokine in the third biological sample M days after administration. In embodiments, the method further comprises (v) selecting the subject for treatment with the chimeric protein if the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine and/or if the amount and/or activity of the cytokine M days after administration is at least about 50% less than the amount and/or activity of the cytokine N hours after administration. In embodiments, the first biological sample, the second biological sample, and the third biological sample are blood. In embodiments, the amount and/or activity of the cytokine is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof.

In embodiments, the first domain is capable of binding to a CD172a (sirpa) ligand. In embodiments, the first domain comprises substantially all of the extracellular domain of CD172a (sirpa). In embodiments, the first domain comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID NO. 57.

In embodiments, the second domain is capable of binding to a CD40 receptor. In embodiments, the second domain comprises substantially all of the extracellular domain of CD 40L. In embodiments, the second domain comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID NO. 58.

In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG 4. In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from human IgG 4. In embodiments, the linker comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO. 3.

In 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 about 90%, 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% identical to the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3. In embodiments, the chimeric protein further comprises the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 7. In an embodiment, the chimeric protein further comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7.

In embodiments, the chimeric protein comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61.

In embodiments, the first dose of chimeric protein is in the range of about 0.03mg/kg to 10mg/kg. In embodiments, the first dose is about 0.003, or about 0.01, or about 0.03, or about 0.1, or about 0.3, or about 1, or about 2, or about 3, or about 4, or about 6, or about 8, or about 10mg/kg.

In embodiments, the methods of any aspect disclosed herein further comprise administering a second dose of the chimeric protein. In embodiments, the second dose is administered at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 14 days, or at least about 21 days, or at least about 28 days after administration of the first dose. In embodiments, the second dose of chimeric protein is in the range of about 0.03mg/kg to 10mg/kg. In embodiments, the second dose is about 0.003, or about 0.01, or about 0.03, or about 0.1, or about 0.3, or about 1, or about 2, or about 3, or about 4, or about 6, or about 8, or about 10mg/kg.

Drawings

The patent or application document includes at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the patent office upon request and payment of the necessary fee.

FIG. 1 shows a graphical representation of a SIRPalpha-Fc-CD 40L chimeric protein (SL-172154). SL-172154 (SIRPalpha-Fc-CD 40L) is a hexameric bifunctional fusion protein consisting of SIRPalpha (binding affinity to CD47 is 0.628 nM) linked to CD40L (binding affinity to CD40 is 4.74 nM) via an Fc linker protein.

Fig. 2 shows the dose escalation (n=15) designed according to the modified toxicity probability (mTPI-2). The planned dose escalation is performed in half-log increments. At least 3 subjects were included at sequential Dose Levels (DL) and evaluated for Dose Limiting Toxicity (DLT) during the first treatment period. The subjects received Intravenous (IV) administration of SL-172154 according to schedule 1 or schedule 2 until disease progression, unacceptable toxicity occurred, or consent was withdrawn.

Figure 3 shows tumor response and treatment duration. iUPD = unidentified progressive disease (iRECIST) NE = non-evaluable; PD = progressive disease; SD = stable disease

Figures 4A to 4D show reproducible increases in serum cytokines following repeated administration of SL-172154. Changes in CCL2 (MCP-1) (fig. 4A), CCL4 (MIP-1β) (fig. 4B), CCL3 (MIP-1α) (fig. 4C), and CCL22 (MDC) (fig. 4D) levels are shown.

FIGS. 5A and 5B show dose-dependent and reproducible increases in serum IL-12. Fig. 5A shows that the subject level of interleukin 12 (IL-12), a TH1 pro-inflammatory response mediator, over time represents the periodic effector cytokine response observed in the study subjects. Fig. 5B shows the median response (horizontal bars) at the first infusion, initially shown as dose-dependent.

FIGS. 6A and 6B show that SL-172154 preferentially binds CD47 on leukocytes rather than erythrocytes. Fig. 6A shows CD47 Receptor Occupancy (RO) as assessed by Fluorescence Activated Cell Sorting (FACS) analysis of both Red Blood Cells (RBCs) and White Blood Cells (WBCs) using whole blood. One hour after infusion on cycle 1 day 1 (C1D 1), the median CD47 RO on leukocytes (horizontal bars) was about 80%. Fig. 6B shows that CD47 RO on RBC is <5% for all dose levels.

Figures 7A-7C show SL-172154 stimulation of dose-dependent B cell edge set and activation. Fig. 7A shows that median frequency of edge set (marginating) cells increases in a dose-dependent manner (horizontal bars). Receptor engagement was about 100% at all dose levels (data not shown). Fig. 7B shows that the median B cell frequency was restored to the pre-infusion level by the next infusion, maintaining the cycle pattern of egress and return for each infusion cycle. Figure 7C shows that returning B cells showed an increase in co-stimulatory marker CD86 as well as maturation marker CD95, indicating that SL-172154 can induce phenotypic changes.

FIGS. 8A and 8B show different distributions of TNF alpha and interleukin-6 (IL-6) relative to CD40 mAb. FIG. 8A shows the induction of TNFα at different doses of CP-870,893 (left panel) or SL-172154 (right panel). FIG. 8B shows induction of IL-6 at different doses of CP-870,893 (left panel) or SL-172154 (right panel). CP-870,893 data is from Vonderheide et al, J Clin Oncol 25:876-883 (2007).

FIGS. 9A and 9B show that SL-172154 induces an innate immune response in the Tumor Microenvironment (TME). FIG. 9A shows immunohistochemical analysis of biopsy samples from patient A before and after administration of SL-172154. Monocytes were detected by CD68 (a protein highly expressed by cells in the monocyte lineage) staining. FIG. 9B shows upregulation of the activation markers CD40 and MHC class II in TME of tumor biopsy samples after treatment with SL-172154 as compared to pre-treatment biopsy samples.

FIGS. 10A and 10B show that SL-172154 induces an adaptive immune response in the Tumor Microenvironment (TME). FIG. 10A shows CD8+ cells, granzyme B+ cells, CD68+ cells and Ki67+ cells in biopsy samples from patient A before and after administration of SL-172154. Cd8+ cells, granzyme b+ cells, cd68+ cells and ki67+ cells were increased in post-treatment biopsy samples compared to pre-treatment biopsy samples. Fig. 10B is a graph comparing Tumor Proportion Score (TPS) and Combined Positive Score (CPS).

FIG. 11 shows a planned clinical development strategy for SL-172154. Such strategies include assays of SL-172154 monotherapy in ovarian cancer, combination therapy of SL-172154+ liposomal doxorubicin in ovarian cancer, combination therapy of SL-172154+ azacytidine + valnemulin in AML, combination therapy of SL-172154+ azacytidine in HR-MDS, and combination therapy of SL-172154+ azacytidine in TP53 mutant AML.

FIG. 12 shows an increase in CD80+ cell abundance and/or CD80 expression in tumors following administration of SL-172154.

Detailed Description

The present disclosure is based in part on the following findings: after administration of sirpa-Fc-CD 40L chimeric proteins, which are well tolerated without evidence of DLT or anemia, thrombocytopenia, liver dysfunction, cytokine release syndrome or pneumonia, cd80+ cells, cd8+ cells, granzyme b+ cells, cd68+ cells, ki67+ cells and PD-l1+ immune cells in the Tumor Microenvironment (TME) are increased. The present disclosure is also based in part on the following findings: b cells and/or CD40 ⁺ cells collect from the peripheral blood and the levels of congenital and adaptive serum cytokines such as CCL2, CXCL9, CXCL10, ifnα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC increase within hours after administration of the sirpa-Fc-CD 40L chimeric protein, and B cells and/or CD40 ⁺ cells return to the peripheral blood, and the levels of congenital and adaptive serum cytokines such as CCL2, CXCL9, CXCL10, ifnα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC decrease within about one day after administration of the sirpa-Fc-CD 40L chimeric protein. In some embodiments, the level of B cells and/or CD40 ⁺ cells and the level of congenital and adaptive serum cytokines such as CCL2, CXCL9, CXCL10, ifnα, IL15, IL23, IL-12, MCP-1, MIP-1 β, MIP-1 a, and MDC are near background levels during one or two days of administration of the sirpa-Fc-CD 40L chimeric protein.

Importantly, since the chimeric proteins of the present disclosure disrupt, block, reduce, inhibit and/or sequester (via binding of the extracellular domain of CD172a (sirpa) to its receptor/ligand on cancer cells), e.g., the transmission of immunosuppressive signals from cancer cells that seek to avoid phagocytosis and/or destruction thereof and enhance, increase and/or stimulate the transmission of immunostimulatory signals to anti-cancer immune cells (via binding of CD40L to its receptor), it can provide anti-tumor effects by two different pathways; this dual effect is more likely to provide any anti-tumor effect in the patient and/or to provide an enhanced anti-tumor effect in the patient. Furthermore, because such chimeric proteins can function via two different pathways, they can be effective, at least in patients who respond poorly to therapies targeting one of the two pathways. Thus, patients with poor response to treatment acting via one of the two pathways may obtain therapeutic benefit by targeting the other pathway.

Chimeric proteins

The chimeric proteins of the present disclosure comprise the extracellular domain of CD172a (sirpa) and the extracellular domain of CD40L, which together can block an immunosuppressive signal and stimulate an immune activation signal simultaneously.

Aspects of the present disclosure provide chimeric proteins comprising the following general structure: an N-terminus- (a) - (b) - (C) -C-terminus, wherein (a) is a first domain comprising an extracellular domain of CD172a (sirpa), (b) is a linker adjoining the first domain and the second domain, e.g., a linker comprising at least one cysteine residue capable of forming a disulfide bond and/or comprising a hinge-CH 2-CH3 Fc domain, and (C) is a second domain comprising an extracellular domain of CD 40L; wherein the linker connects the first domain and the second domain.

In embodiments, the first domain comprises substantially all of the extracellular domain of CD172a (sirpa). In embodiments, the first domain is capable of binding to a CD172a (sirpa) ligand. In embodiments, the first domain is capable of binding to a CD172a (sirpa) ligand (e.g., CD 47) expressed on the surface of a cancer cell. In embodiments, the first domain is capable of inhibiting the binding of a CD172a (sirpa) ligand (e.g., CD 47) to CD172a (sirpa) proteins located on bone marrow cells and hematopoietic stem cells and neurons. In embodiments, the first domain is capable of inhibiting an immunosuppressive signal. In embodiments, the first domain is capable of inhibiting an immunosuppressive signal. In embodiments, the first domain is capable of inhibiting macrophage checkpoints or "do not eat me" signals. In embodiments, therapies using SIRPa-Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61) stimulate macrophages to phagocytose tumor cells and effectively present tumor antigens of the phagocytosed tumor cells to T cells.

In embodiments, the second domain is capable of binding to a CD40 receptor. In embodiments, the second domain comprises substantially all of the extracellular domain of CD 40L. In embodiments, the second domain is capable of activating an immunostimulatory signal.

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

In embodiments, the chimeric proteins of the invention may be produced in mammalian host cells as a single polypeptide chain that is secreted and fully functional.

In embodiments, a chimeric protein refers to a plurality of recombinant proteins having a plurality of polypeptides, e.g., a plurality of extracellular domains disclosed herein, combined (via covalent or non-covalent bonding) to produce a single unit, e.g., in vitro (e.g., having one or more synthetic linkers disclosed herein).

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

In embodiments, an extracellular domain refers to a portion of a transmembrane protein capable of interacting with the extracellular environment. In embodiments, an extracellular domain refers to a portion of a transmembrane protein that is sufficient to bind to a ligand or receptor and efficiently transmit a signal to a cell. In embodiments, the extracellular domain is the entire amino acid sequence of a transmembrane protein that is normally present outside of a cell or cell membrane. In embodiments, the extracellular domain is a portion of the amino acid sequence of a transmembrane protein that is external to the cell or cell membrane and is required for signal transduction and/or ligand binding, as may be determined using methods known in the art (e.g., in vitro ligand binding and/or cell 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 the interaction with a soluble receptor or ligand or membrane-bound receptor or ligand (i.e., the membrane of an adjacent cell). Without wishing to be bound by theory, one or more transmembrane domains are responsible for localizing transmembrane proteins to the plasma membrane. Without wishing to be bound by theory, the intracellular domain of transmembrane proteins is responsible for coordinating interactions with cell signaling molecules to coordinate intracellular reactions with the extracellular environment (and vice versa).

There are generally two types of single pass transmembrane proteins: type I transmembrane proteins having an extracellular amino terminus and an intracellular carboxy terminus and type II transmembrane proteins having an extracellular carboxy terminus and an intracellular amino terminus. Type I and type II transmembrane proteins may be receptors or ligands. For a type I transmembrane protein (e.g., CD172a (sirpa)), the amino terminus of the protein faces the outside of the cell and thus contains a functional domain responsible for interaction with other binding partners (ligands or receptors) in the extracellular environment. For type II transmembrane proteins (e.g., CD 40L), the carboxy-terminus of the protein faces the outside of the cell and thus contains a functional domain responsible for interaction with other binding partners (ligands or receptors) in the extracellular environment. Thus, the two types of transmembrane proteins have opposite orientations relative to the cell membrane.

Describing CD47 as "do not eat me" signal in a broad range of cancers motivates the search for a combination of "eat me" signals that can enhance anti-tumor immunity in the case of CD47 blockade. WILLINGHAM et al ,The CD47-signal regulatory protein alpha(SIRPa)interaction is a therapeutic target for human solid tumors.Proc Natl Acad Sci U S A 109:6662-6667(2012);Jaiswal et al .,CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis.Cell 138:271-285(2009);Weiskopf et al ,Engineered SIRPalpha variants as immunotherapeutic adjuvants to anticancer antibodies.Science 341:88-91(2013).CD47 blocking and preclinical combinations of ADCP competent antibodies (including rituximab and trastuzumab) can enhance tumor phagocytosis. Kauder et al ,ALX148 blocks CD47 and enhances innate and adaptive antitumor immunity with a favorable safety profile.PLoS One 13:e0201832(2018);Chao et al ,Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma.Cell 142:699-713(2010);Chao et al ,Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47.Sci Transl Med 2:63ra94(2010);Advani et al ,CD47 Blockade by Hu5F9-G4 and rituximab in non-Hodgkin's lymphoma.N Engl J Med379:1711-1721(2018);Zhao et al ,CD47-signal regulatory protein-alpha(SIRPalpha)interactions form a barrier for antibody-mediated tumor cell destruction.Proc Natl Acad Sci U S A 108:18342-18347(2011). at least 50% of patients with recurrent or refractory diffuse large B cell lymphoma or follicular lymphoma treated with a combination of Hu5F9-G4 (a humanized IgG4 isotype CD47 blocking mAb) and rituximab showed an objective response. Advani et al ,CD47 Blockade by Hu5F9-G4 and rituximab in non-Hodgkin's lymphoma.N Engl J Med 379:1711-1721(2018).CD47 block boost immune ignoring antigen presentation in tumors (Tseng et al ,Anti-CD47antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response.Proc Natl Acad Sci U S A 110:11103-11108(2013)), but only sporadic clinical responses were observed when using CD 47/sirpa blocking therapeutics as monotherapy or in combination with PD-1/L1 blocking antibodies.

Disruption of CD47 binding to sirpa has become a promising immunotherapeutic strategy for advanced cancers by enhancing antibody-dependent cell phagocytosis (ADCP) of the targeted antibodies. Preclinical CD 47/sirpa blockade induces anti-tumor activity by increasing phagocytosis of tumor cells by macrophages and enhancing cross-presentation of tumor antigens by dendritic cells to cd8+ T cells; both processes are enhanced by CD40 signaling. A novel double sided fusion protein is produced herein that incorporates the extracellular domains of sirpa and CD40L linked by a central Fc domain, referred to as sirpa-Fc-CD 40L. As shown herein, sirpa-Fc-CD 40L chimeric proteins bind CD47 and CD40 with high affinity and activate CD40 signaling without Fc receptor cross-linking. No evidence of hemolysis, hemagglutination or thrombocytopenia was observed in vitro or in cynomolgus monkeys. Furthermore, as shown herein, sirpa-Fc-CD 40L chimeric proteins perform better than CD47 blocking and CD40 agonist antibodies in murine CT26 tumor models and act synergistically with immune checkpoint blocking of PD-1 and CTLA 4. sirpa-Fc-CD 40L activates type I interferon responses in macrophages and enhances the activity of ADCP competent targeting antibodies in vitro and in vivo. These data indicate that while CD 47/sirpa inhibition can enhance tumor cell phagocytosis, activation of CD 40-mediated type I interferon responses provides a bridge between macrophage and T cell-mediated immunity, thereby significantly enhancing persistent tumor control and rejection.

The chimeric proteins of the present disclosure comprise the extracellular domain of CD172a (sirpa) and the extracellular domain of CD 40L. Thus, the chimeric proteins of the present disclosure comprise at least a first domain comprising an extracellular domain of CD172a (sirpa) linked directly or through a linker to a second domain comprising an extracellular domain of CD 40L. When the domains are linked in an amino-terminal to carboxy-terminal orientation, the first domain is on the "left" side of the chimeric protein and faces "outward", and the second domain is on the "right" side of the chimeric protein and faces "outward".

Other configurations of the first domain and the second domain are contemplated, e.g., the first domain faces outward and the second domain faces inward, the first domain faces inward and the second domain faces outward, and both the first domain and the second domain face inward. When both domains are "inward facing", the chimeric protein will have an amino-to-carboxy-terminal configuration comprising the extracellular domain of CD40L, a linker, and the extracellular domain of CD172a (sirpa). In such configurations, the chimeric protein may have to comprise additional "slack" to allow the domain of the chimeric protein to bind to one or both of its receptors/ligands, as described elsewhere herein.

Constructs can be produced by cloning nucleic acids encoding three fragments (extracellular domain of CD172a (sirpa), followed by a linker sequence, then extracellular domain of CD 40L) into a vector (plasmid, virus or otherwise), wherein the amino-terminus of the complete sequence corresponds to the "left" of a molecule containing extracellular domain of CD172a (sirpa) and the carboxy-terminus of the complete sequence corresponds to the "right" of a molecule containing extracellular domain of CD 40L. In some embodiments of chimeric proteins having one of the other configurations described above, the construct will comprise three nucleic acids such that the translated chimeric protein produced will have the desired configuration, e.g., a dual inward facing chimeric protein. Thus, in embodiments, the chimeric proteins of the invention are so engineered.

CD172a (SIRPalpha) -Fc-CD40L chimeric proteins

In embodiments, the chimeric protein is capable of binding to both a human CD172a (sirpa) ligand and a human CD40 receptor, wherein the CD172a (sirpa) ligand is CD47 and the CD40L receptor is CD40.

The chimeric proteins have the following general structure: the N-terminus- (a) - (b) - (C) -C-terminus, wherein (a) is a first domain comprising the extracellular domain of human signal-regulating protein a (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (C) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond.

The chimeric proteins of the present disclosure have a first domain capable of spatially binding to their ligand/receptor and/or a second domain capable of spatially binding to their ligand/receptor. This means that there is sufficient overall flexibility in the chimeric protein and/or there is a physical distance between the extracellular domain (or portion thereof) and the remainder of the chimeric protein such that the ligand/receptor binding domain of the extracellular domain binds its ligand/receptor in space unimpeded. Such flexibility and/or physical distance (referred to herein as "relaxation") may typically be present in one or more extracellular domains, typically in a linker, and/or typically in a chimeric protein (as a whole). Alternatively or additionally, the chimeric proteins may be modified by inclusion of one or more additional amino acid sequences (e.g., a splice linker as described below) or synthetic linkers (e.g., a polyethylene glycol (PEG) linker), which provide additional relaxation needed to avoid steric hindrance.

In embodiments, the chimeric proteins of the present disclosure comprise a variant of the extracellular domain of CD172a (sirpa). As an example, a 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 to a known amino acid sequence of CD172a (sirpa), for example, human CD172a (sirpa).

In embodiments, the extracellular domain of CD172a (sirpa) has the following amino acid sequence:

EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY(SEQ ID NO:57).

In embodiments, the chimeric protein comprises a variant of the extracellular domain of CD172a (sirpa). As an example, a 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 to SEQ ID NO 57.

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

The skilled artisan can select variants of known amino acid sequences of CD172a (sirpa) by reference, for example HATHERLEY et al ,"Paired receptor specificity explained by structures of signal regulatory proteins alone and complexed with CD47."Mol Cell 31:266-277(2008);Hatherley et al ,"The Structure of the Macrophage Signal Regulatory Protein Alpha(Sirpalpha)Inhibitory Receptor Reveals a Binding Face Reminiscent of that Used by T Cell Receptors."J Biol Chem 282:14567(2007);Hatherley et al ,"Structure of Signal-Regulatory Protein Alpha:A Link to Antigen Receptor Evolution."J Biol Chem 284:26613(2009);Hatherley et al ,"Polymorphisms in the Human Inhibitory Signal-Regulatory Protein Alpha Do not Affect Binding to its Ligand Cd47."J Biol Chem 289:10024(2014);Ring et al ,"Anti-SIRP alpha antibody immunotherapy enhances neutrophil and macrophage antitumor activity."Proc Natl Acad Sci U S A 114:E10578-E10585(2017),, each of which is incorporated by reference in its entirety.

In embodiments, the chimeric proteins of the present disclosure comprise a variant of the extracellular domain of CD 40L. As an example, a 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 to a known amino acid sequence of CD40L, e.g.

In embodiments, the extracellular domain of CD40L has the following amino acid sequence:

HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL(SEQ ID NO:58).

In embodiments, the chimeric protein comprises a variant of the extracellular domain of CD 40L. As an example, a 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 to SEQ ID NO 58.

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

The skilled artisan can select variants of known amino acid sequences of CD40L by reference, for example, karpisas et al ,"2A crystal structure of an extracellular fragment of human CD40 ligand."Structure 3:1031-1039(1995);Karpusas et al ,"Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody."Structure 9:321-329(2001);Silvian et al ,"Small Molecule Inhibition of the TNF Family Cytokine CD40 Ligand through a Subunit Fracture Mechanism."ACS Chem Biol 6:636-647(2011);An et al ,"Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation."J Biol Chem 286:11226-11235(2011);Karnell et al ,"A CD40L-targeting protein reduces autoantibodies and improves disease activity in patients with autoimmunity."Sci Transl Med 11(2019),, each of which is incorporated by reference in its entirety.

In embodiments, the linker of 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 the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3. In embodiments, the linker of the 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 embodiments, the linker of the 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 embodiments, the linker of the 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 embodiments, the linker of the 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 embodiments, the linker of the chimeric protein comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

In embodiments, the chimeric proteins of the present disclosure comprise: (1) A first domain comprising an 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 comprising an 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 comprising 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 embodiments, the chimeric proteins of the present disclosure comprise: (1) A first domain comprising an amino acid sequence at least 95% identical to SEQ ID No. 57; (b) A second domain comprising an amino acid sequence at least 95% identical to SEQ ID No. 58; and (c) a linker comprising 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 embodiments, the chimeric proteins of the present disclosure comprise: (1) A first domain comprising an amino acid sequence at least 97% identical to SEQ ID No. 57; (b) A second domain comprising an amino acid sequence at least 97% identical to SEQ ID No. 58; and (c) a linker comprising 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 embodiments, the chimeric proteins of the present disclosure comprise: (1) A first domain comprising an amino acid sequence at least 98% identical to SEQ ID No. 57; (b) A second domain comprising an amino acid sequence at least 98% identical to SEQ ID No. 58; And (c) a linker comprising 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 embodiments, the chimeric proteins of the present disclosure comprise: (1) A first domain comprising an amino acid sequence at least 99% identical to SEQ ID No. 57; (b) A second domain comprising an amino acid sequence at least 99% identical to SEQ ID No. 58; and (c) a linker comprising 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 embodiments, the chimeric proteins of the present disclosure comprise: (1) A first domain comprising the amino acid sequence of SEQ ID NO. 57; (b) A second domain comprising the amino acid sequence of SEQ ID NO. 58; and (c) a linker comprising 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 embodiments, the chimeric proteins of the present disclosure comprise: (1) A first domain comprising the same amino acid sequence as SEQ ID No. 57; (b) A second domain comprising the same amino acid sequence as SEQ ID No. 58; and (c) a linker comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

In embodiments, the CD172a (sirpa) -Fc-CD40L chimeric proteins of the present disclosure have the following amino acid sequences (the extracellular domain of CD172a (sirpa) is shown in bold font, the extracellular domain of CD40L is underlined, the Fc domain is shown in italics):

The 792 amino acid sequence (excluding the leader sequence) of the CD172a (SIRPalpha) -Fc-CD40L chimeric protein (SL-172154) is shown above. The CD172a (SIRPalpha) -Fc-CD40L chimeric protein exists in an oligomeric form. There are 17 cysteines in the amino acid sequence, of which there may be 8 disulfide pairs. Glycosylation of both N and O linkages has been demonstrated.

In embodiments, the chimeric proteins of the present disclosure comprise at least 1,2, 3,4, 5, 6, 7, 8, 9, or 10 potential N-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 2 potential N-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 4 potential N-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 6 potential N-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 8 potential N-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 10 potential N-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 1,2, 3,4, 5, 6, 7, or 8 potential O glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 2 potential O glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 4 potential O glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 6 potential O glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 8 potential O glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 2 potential N-glycosylation sites and at least 2 potential O-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 4 potential N-glycosylation sites and at least 4 potential O-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 6 potential N-glycosylation sites and at least 6 potential O-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 8 potential N-glycosylation sites and at least 8 potential O-glycosylation sites. In embodiments, the chimeric proteins of the present disclosure comprise at least 10 potential N-glycosylation sites and at least 8 potential O-glycosylation sites. In embodiments, the chimeric protein expressed in Chinese Hamster Ovary (CHO) cells is glycosylated.

17 Cysteines were present in the SL-172154 chimeric protein. In some embodiments, the SL-172154 chimeric protein has no disulfide bonds. In some embodiments, the SL-172154 chimeric protein has at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 disulfide bonds. In some embodiments, the SL-172154 chimeric protein has at least 1 or at least 2 interchain disulfide bonds. In some embodiments, the SL-172154 chimeric protein has at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8 intrachain disulfide bonds. In some embodiments, the SL-172154 chimeric protein has a c350=c350 interchain disulfide bond. In some embodiments, the SL-172154 chimeric protein has c353=c353 interchain disulfide bonds. In some embodiments, the SL-172154 chimeric protein has c153=c153 interchain disulfide bonds. In some embodiments, the SL-172154 chimeric protein has a c25=c91 disulfide bond. In some embodiments, the SL-172154 chimeric protein has c140=c198 disulfide bonds. In some embodiments, the SL-172154 chimeric protein has a c243=c301 disulfide bond. In some embodiments, the SL-172154 chimeric protein has c385=c445 disulfide bonds. In some embodiments, the SL-172154 chimeric protein has a c491=c549 disulfide bond. In some embodiments, the SL-172154 chimeric protein has a c603=c615 disulfide bond. In some embodiments, the SL-172154 chimeric protein has c709=c725 disulfide bonds. In some embodiments, the SL-172154 chimeric protein has c140=c243=c709/C725 hybrid disulfide bonds. In some embodiments, the SL-172154 chimeric protein has a C615 (chain 1) =c 615 (chain 2) hybrid disulfide bond.

In some embodiments, the CD172a (sirpa) -Fc-CD40L chimeric proteins of the disclosure are encoded by the following nucleotide sequences (the leader sequences are shown in bold-underlined font):

In some embodiments, SEQ ID NO:60 encodes a precursor (leader sequence shown in italics) of a CD172a (SIRPalpha) -Fc-CD40L chimeric protein of the present disclosure having the amino acid sequence:

The chimeric protein of SEQ ID NO. 59 (also referred to herein as SL-172154) is a recombinant fusion glycoprotein comprising the extracellular domain of human CD172a (SIRPalpha) (PDCD 1, CD272 a), the central domain comprising the hinge-CH 2-CH3 region from human immunoglobulin constant gamma 4 (inhibitory receptor SHPS-1, igG 4), and the extracellular domain of human CD40L (TNFSF 5, TRAP, CD 154). The linear configuration of SL-172154 is CD172a (SIRPalpha) -Fc-CD40L.

The predicted molecular weight of the monomeric chimeric protein of SEQ ID NO. 59 is 88.1kDa. The predicted molecular weight of the glycosylated monomeric chimeric protein of SEQ ID NO. 59 is about 115kDa.

The bilateral nature of chimeric proteins disclosed herein, such as a CD172a (SIRPalpha) -Fc-CD40L chimeric protein (e.g., SEQ ID NO:59 or SEQ ID NO: 61), is intended to intercept one of the key immunosuppressive pathways within the Tumor Microenvironment (TME): CD172a (sirpa) -CD47 macrophage checkpoint.

Tumor cells can express CD47 on their cell surface, and CD47 can bind to CD172a (sirpa) expressed by macrophages to inhibit phagocytosis of tumor cells. Thus, a CD172a (SIRPalpha) -Fc-CD40L chimeric protein (e.g., SEQ ID NO:59 or SEQ ID NO: 61) can bind to CD47 expressed on the tumor surface, wherein the CD172a (SIRPalpha) domain of the CD172a (SIRPalpha) -Fc-CD40L chimeric proteins disclosed herein is intended to provide competitive inhibition of CD47, and replace the CD47 inhibition signal with a functional trimerized/hexameric CD40L, resulting in the entry of T cells undergoing co-stimulation through engagement of their CD40 receptor, rather than inhibition by CD172a (SIRPalpha) interaction. In other words, since the extracellular domains (ECDs) of CD172a (sirpa) and CD40L are physically linked to each other and localized to TME, tumor infiltrating T cells will receive co-stimulation while recognizing tumor antigens through their T Cell Receptor (TCR). Importantly, since the ECDs of CD172a (sirpa) and CD40L are physically linked to each other and localized to TME, tumor infiltrating T cells will receive co-stimulation while recognizing tumor antigens through the T Cell Receptor (TCR). Together, these will result in replacement of the inhibitory CD47 signal by the co-stimulatory CD40L signal, thereby enhancing the anti-tumor activity of T cells.

The three components of the chimeric proteins disclosed herein, including the CD172a (SIRPalpha) -Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61), have unique properties that promote dimerization or oligomerization. The extracellular domain of CD172a (sirpa) is usually in monomeric form and it is not clear whether it will form higher order homologous complexes. The central Fc domain contains cysteine residues that are capable of disulfide bonding to form a dimeric structure. In embodiments, the chimeric proteins disclosed herein, including CD172a (SIRPalpha) -Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61) contain an S228P mutation in the hinge region of the Fc domain to prevent Fab arm exchange. It is known that the CD40L domain can form homotrimeric complexes that are stabilized by non-covalent electrostatic interactions. Although the chimeric proteins disclosed herein, including CD172a (SIRPalpha) -Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61), are expressed by the production cell line as continuous monomeric proteins, the resulting monomeric proteins self-assemble into higher order species based on these disulfide bonds of CD40L and on the combined effects of charge-based interactions (producing trimers) and these attractive forces, thereby producing hexamers (dimers of trimers). Most (> 80%) of the CD172a (SIRPalpha) -Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61) comprise hexamer and trimer forms, which have similar activity. Importantly, since the CD172a (SIRPalpha) -Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61) consist of hexamers and trimers, they stimulate CD40 signaling without cross-linking of the Fc receptor or any other cross-linking agent. Based on disulfide (Fc) and charge-based (CD 40L) interactions, The predicted tertiary structure of the CD172a (SIRPalpha) -Fc-CD40L chimeric protein (e.g., SEQ ID NO:59 or SEQ ID NO: 61) in monomeric and various oligomeric states shows visualization of the hexamer (top two images) and the trimer (bottom two images) of the CD172a (SIRPalpha) -Fc-CD40L chimeric protein (e.g., SEQ ID NO:59 or SEQ ID NO: 61) by electron microscopy. thus, a CD172a (SIRPalpha) -Fc-CD40L chimeric protein (e.g., SEQ ID NO:59 or SEQ ID NO: 61) forms a trimer/hexamer and activates CD40 without cross-linking. Notably, unlike monoclonal antibodies, fc receptor crosslinking is not necessary for the functional activity of CD172a (SIRPalpha) -Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61).

In embodiments, the chimeric protein comprises a variant of a CD172a (SIRPalpha) -Fc-CD40L chimeric protein (e.g., SEQ ID NO:59 or SEQ ID NO: 61). As an example, a 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 6%, or at least about 99.8% identity to SEQ ID No. 61.

In embodiments, the first domain of 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 the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61. In embodiments, the first domain of the 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 embodiments, the first domain of the 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 embodiments, the first domain of the 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 embodiments, the first domain of the 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 embodiments, the first domain of the chimeric protein comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61.

In embodiments, the first domain is capable of binding to a CD172a (sirpa) ligand.

In embodiments, the first domain comprises substantially all of the extracellular domain of CD172a (sirpa).

In embodiments, the second domain is capable of binding to a CD40 receptor.

In embodiments, the second domain comprises substantially all of the extracellular domain of CD 40L.

In embodiments, the linker comprises a hinge-CH 2-CH3Fc domain derived from IgG4, e.g., human IgG 4.

In 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 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 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 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 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 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 embodiments, the linker comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

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

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

In 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 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 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 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 embodiments, the chimeric protein further comprises the amino acid sequence of SEQ ID NO. 5 and/or SEQ ID NO. 7.

In 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 embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61.

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

In embodiments, the amino acid mutation is an amino acid substitution, and may include conservative substitutions and/or non-conservative substitutions. "conservative substitutions" may be made, for example, based on the 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 divided into the following six standard amino acid groups: (1) hydrophobicity: met, ala, val, leu, ile; (2) neutral hydrophilicity: cys, ser, thr; asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe. As used herein, a "conservative substitution" is defined as the exchange of an amino acid for another amino acid listed in the same set of six standard amino acid sets shown above. For example, asp is exchanged by Glu to retain a negative charge in the polypeptide so modified. In addition, glycine and proline may be substituted for each other based on their ability to disrupt the alpha-helix. As used herein, a "non-conservative substitution" is defined as the exchange of an amino acid by another amino acid listed in a different one of the six standard amino acid groups (1) to (6) shown above.

In embodiments, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminocaproic acid, aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocysteine, sulfoalanine, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methylaminoacid, C alpha-methylaminoacid, nalpha-methylaminoacid, and general amino acid analogs.

Reference may also be made to the genetic code, including the mutation of the nucleotide sequence of the chimeric protein taking into account the degeneracy of the codons.

In embodiments, the chimeric protein is capable of binding to one or more human ligands/receptors.

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

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

In embodiments, the chimeric protein binds to human CD47 with a K _D of about 1nM to about 5nM, e.g., about 1nM, about 1.5nM, about 2nM, about 2.5nM, about 3nM, about 3.5nM, about 4nM, about 4.5nM, or about 5 nM. In embodiments, the chimeric protein binds to human CD47 at K _D (e.g., as measured by surface plasmon resonance or biolayer interferometry) of less than about 3nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 90pM, about 80pM, about 70pM, about 60pM, about 55pM, about 50pM, about 45pM, about 40pM, about 35pM, about 30pM, about 25pM, about 20pM, about 15pM, or about 10pM, or about 1 pM.

In embodiments, the chimeric protein binds to human CD40 at a K _D (e.g., as measured by surface plasmon resonance or bio-layer interferometry) of less than about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 90pM, about 80pM, about 70pM, about 60pM, about 55pM, about 50pM, about 45pM, about 40pM, about 35pM, about 30pM, about 25pM, about 20pM, about 15pM, or about 10pM, or about 1 pM.

As used herein, a variant of an extracellular domain is capable of binding to a receptor/ligand of a native extracellular domain. For example, a variant may comprise one or more mutations in the extracellular domain that do not affect its binding affinity for its receptor/ligand; or one or more mutations in the extracellular domain may improve binding affinity for the receptor/ligand; or one or more mutations in the extracellular domain may reduce the binding affinity for the receptor/ligand, but not completely eliminate the binding. In embodiments, one or more mutations are located outside the binding pocket, wherein the extracellular domain interacts with its receptor/ligand. In embodiments, one or more mutations are located inside the binding pocket, wherein the extracellular domain interacts with its receptor/ligand, provided that the mutation does not completely eliminate binding. Based on the knowledge of the skilled artisan and the knowledge in the art about receptor-ligand binding, he/she will know which mutations will allow binding and which mutations will eliminate binding.

In embodiments, the chimeric proteins exhibit enhanced stability and protein half-life.

The chimeric proteins of the present disclosure may comprise more than two extracellular domains. For example, a chimeric protein may comprise three, four, five, six, seven, eight, nine, ten, or more extracellular domains. As disclosed herein, the second extracellular domain can be separated from the third extracellular domain via a linker. Or the second extracellular domain may be directly linked (e.g., via a peptide bond) to the third extracellular domain. In embodiments, the chimeric proteins comprise directly linked extracellular domains and extracellular domains linked indirectly via a linker, as disclosed herein.

Joint

In embodiments, the chimeric protein comprises a linker.

In embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. 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, this disulfide bond formation is responsible for maintaining the useful multimeric state of the chimeric protein. This allows for efficient production of the chimeric protein; it allows for the desired activity in vitro and in vivo.

In the chimeric proteins of the present disclosure, the linker is a polypeptide selected from the group consisting of a flexible amino acid sequence, an IgG hinge region, or an antibody sequence.

In embodiments, the linker is derived from a naturally occurring multidomain Protein, or is a Protein sci.22 (2): 153-167 (2013) as, for example, in Chichili et al; an empirical joint as described in Chen et al, adv Drug Deliv Rev.65 (10): 1357-1369 (2013), the entire contents of which are hereby incorporated by reference. In some embodiments, the joints may be designed using a joint design database and computer program such as those described in the following documents: 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 embodiments, the linker comprises a polypeptide. In 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 length of 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.

In embodiments, the joint is flexible.

In embodiments, the joint is rigid.

In embodiments, the linker comprises substantially 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% glycine and serine).

In embodiments, the linker comprises a hinge region of an antibody (e.g., igG, igA, igD and IgE, including subclasses (e.g., igG1, igG2, igG3, and IgG4, and IgA1 and IgA 2)). The hinge region seen in IgG, igA, igD and IgE class antibodies acts as a flexible spacer, thereby allowing the Fab portion to move freely in space. In contrast to the constant region, the hinge domain is structurally diverse, differing in both sequence and length in immunoglobulins and subclasses. For example, hinge regions in the IgG subclass vary in length and flexibility. The hinge region of IgG1 comprises amino acids 216-231 and, because it is free and flexible, the Fab fragment can rotate about its axis of symmetry and move within a sphere centered on the first of the two heavy chain inter-disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacking glycine residues is relatively short and contains a rigid polyproline duplex, stabilized by an additional inter-heavy chain disulfide bridge. These properties limit the flexibility of IgG2 molecules. IgG3 differs from the other subclasses by its unique extended hinge region (about four times the length of the IgG1 hinge) which contains 62 amino acids (comprising 21 prolines and 11 cysteines) forming an inflexible polyproline duplex. In IgG3, the Fab fragment is relatively far from the Fc fragment, giving the molecule greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight than the other subclasses. The hinge region of IgG4 is shorter than IgG1 and its flexibility is between that of IgG1 and IgG2. The flexibility of the hinge region is reported to decrease in the following order: igG3> IgG1> IgG4> IgG2. In embodiments, the linker may be derived from human IgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.

According to crystallographic studies, the immunoglobulin hinge region can be functionally further subdivided into three regions: an upper hinge region, a core region, and a lower hinge region. See Shin et al Immunological Reviews 130:87 (1992). The upper hinge region includes amino acids from the carboxy terminus of C _H1 to the first residue in the hinge that restricts movement (typically the first cysteine residue that forms an interchain disulfide bond between two heavy chains). The length of the upper hinge region is related to the segment flexibility of the antibody. The core hinge region contains an inter-heavy chain disulfide bond, and the lower hinge region engages the amino terminus of the C _H2 domain and contains the residues in C _H2. As above. The core hinge region of wild-type human IgG1 contains the sequence CPPC (SEQ ID NO: 24) which, when dimerized by disulfide bond formation, produces a cyclic octapeptide which is thought to act as a pivot, thereby imparting flexibility. In embodiments, the linker of the invention comprises one, two, or three of the upper hinge, core, and lower hinge regions of any antibodies (e.g., igG, igA, igD and IgE, including subclasses (e.g., igG1, igG2, igG3, and IgG4, and IgA1 and IgA 2)). The hinge region may also contain one or more glycosylation sites, including many structurally different types of sites for carbohydrate attachment. For example, igA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, thereby conferring resistance to intestinal proteases on the hinge region polypeptide, and is considered to be an advantageous property of secretory immunoglobulins. In embodiments, the linker of the present disclosure comprises one or more glycosylation sites.

In embodiments, the linker comprises an Fc domain of an antibody (e.g., igG, igA, igD and IgE, including subclasses (e.g., igG1, igG2, igG3, and IgG4, and IgA1 and IgA 2)).

In the chimeric proteins of the disclosure, the linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG 4. In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from human IgG 4. In 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 NOs 1 to 3. In 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 NOs 1 to 3 (e.g., at least 95% identical to the amino acid sequence of SEQ ID NO: 2). In embodiments, the linker comprises one or more adaptor linkers, such adaptor linkers are independently selected from SEQ ID NOs 4-50 (or variants thereof). In embodiments, the linker comprises two or more splice linkers, each splice linker being independently selected from the group consisting of SEQ ID NOs 4-50 (or variants thereof); one of the linker is N-terminal to the hinge-CH 2-CH3 Fc domain and the other linker is C-terminal to the hinge-CH 2-CH3 Fc domain.

In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from a human IgG1 antibody. In embodiments, the Fc domain exhibits increased affinity and enhanced binding to neonatal Fc receptor (FcRn). In embodiments, the Fc domain comprises one or more mutations that increase affinity for FcRn and enhance binding to FcRn. Without wishing to be bound by theory, it is believed that the increased affinity for and increased binding to FcRn increases the in vivo half-life of the chimeric proteins of the invention.

In embodiments, the Fc domain in the linker comprises one or more amino acid substitutions at amino acid residues 250, 252, 254, 256, 308, 309, 311, 416, 428, 433, or 434 (according to Kabat numbering, e.g., kabat, et al, sequences of Proteins of Immunological Interest, 5 th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, md. (1991), expressly incorporated herein by reference), or an equivalent thereof. In embodiments, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In embodiments, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan, or threonine. In embodiments, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In embodiments, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In embodiments, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In embodiments, the amino acid substitution at amino acid residue 309 is a substitution with proline. In embodiments, the amino acid substitution at amino acid residue 311 is a substitution with serine. In 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 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 embodiments, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In embodiments, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine, or asparagine. In embodiments, the amino acid substitution at amino acid residue 416 is a substitution with serine. In embodiments, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In embodiments, the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine. In embodiments, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine or tyrosine.

In embodiments, the Fc domain linker (e.g., comprising an IgG constant region) comprises one or more mutations, such as substitutions at amino acid residues 252, 254, 256, 433, 434, or 436 (according to Kabat numbering, e.g., kabat, et al, sequences of Proteins of Immunological Interest, 5 th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, md. (1991), expressly incorporated herein by reference). In embodiments, the IgG constant regions comprise a triple M252Y/S254T/T256E mutation or a YTE mutation. In embodiments, the IgG constant region comprises a triple H433K/N434F/Y436H mutation or a KFH mutation. In embodiments, the IgG constant region comprises a combination of YTE and KFH mutations.

In embodiments, the linker comprises an IgG constant region comprising one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (according to Kabat numbering, e.g., kabat, et al, sequences of Proteins of Immunological Interest, 5 th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, md. (1991), expressly incorporated herein by reference). Exemplary mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N F, N S and H435A. In embodiments, the IgG constant region comprises an M428L/N434S mutation or an LS mutation. In embodiments, the IgG constant region comprises a T250Q/M428L mutation or a QL mutation. In embodiments, the IgG constant region comprises the N434A mutation. In embodiments, the IgG constant region comprises a T307A/E380A/N434A mutation or an AAA mutation. In embodiments, the IgG constant region comprises the I253A/H310A/H435A mutation or IHH mutation. In embodiments, the IgG constant region comprises the H433K/N434F mutation. In embodiments, the IgG constant region comprises the combined M252Y/S254T/T256E and H433K/N434F mutations.

Additional exemplary mutations in IgG constant regions are described, for example, in Robbie et al, antimicrobial AGENTS AND Chemotherapy (12): 6147-6153 (2013); dall' Acqua et al Journal Biol Chem (33): 23514-24 (2006); dall' Acqua et al Journal of Immunology 169:5171-80 (2002); ko et al Nature514:642-645 (2014); grevys et al Journal of Immunology (11): 5497-508 (2015); and U.S. patent number 7,083,784, the entire contents of which are hereby incorporated by reference.

An illustrative Fc-stabilized mutant is S228P. Illustrative mutants that extend Fc half-life are T250Q, M428L, V T, L P309P and Q311S, and the linker of the invention may comprise 1, or 2, or 3, or 4, or 5 of these mutants.

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

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

In addition, one or more splice junctions can be used to connect the Fc domain (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% identical thereto) and the extracellular domain in the junction. For example, any 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 link the extracellular domain as disclosed herein and the Fc domain in the linker as disclosed herein. Optionally, any one of SEQ ID NOs 4 to 50 or variants thereof is located between the extracellular domain as disclosed herein and the Fc domain as disclosed herein.

In embodiments, the chimeric proteins of the invention may comprise variants of the splice junctions disclosed in table 1 below. For example, 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 to the amino acid sequence of any of SEQ ID NO.

In embodiments, the first and second splice joints may be different or they may be the same.

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

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

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

Table 1: illustrative linkers (Fc domain linker and linker)

In embodiments, the junction joint consists essentially 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% glycine and serine). For example, in embodiments, the splice joint is (Gly ₄Ser)_n, where n is about 1 to about 8, such as 1,2, 3,4, 5, 6, 7, or 8 (SEQ ID NO:25 to SEQ ID NO:9, respectively) in embodiments, the splice joint sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 33) additional illustrative splice joints include, but are not limited to, those having the sequence LE, (EAAAK) _n (n=1-3) (SEQ ID NO:36 to SEQ ID NO: 38), A (EAAAK) _n A (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, where X represents any amino acid, such as Ala, lys, or Glu. In embodiments, the splice joint is GGS. In embodiments, the splice joint has the sequence (Gly) _n, where n is any number from 1 to 100, such as (Gly) ₈ (SEQ ID NO: 34) and (Gly) ₆ (SEQ ID NO: 35).

In embodiments, the splice junctions are one or more of GGGSE(SEQ ID NO:47)、GSESG(SEQ ID NO:48)、GSEGS(SEQ ID NO:49)、GEGGSGEGSSGEGSSSE GGGSEGGGSEGGGSEGGS(SEQ ID NO:50) and splice junctions placed randomly G, S and E every 4 amino acid intervals.

In embodiments, the chimeric protein comprises a junction linker comprising the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 7.

In embodiments wherein the chimeric protein comprises the extracellular domain (ECD) of CD172a (sirpa), one engagement linker before the Fc domain, a second engagement linker after the Fc domain, and the ECD of CD40L, the chimeric protein may comprise the following structure:

ECD-linker 1-Fc domain of human CD172a (SIRPalpha) -linker 2-ECD of human CD40L

The combination of the first binding linker, fc domain linker, and second binding linker is referred to herein as a "modular linker". In embodiments, the chimeric protein comprises a modular linker as shown in table 2:

table 2: illustrative Modular connector

In embodiments, the chimeric proteins of the invention may comprise variants of the modular linkers disclosed in table 2 above. For example, 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 to the amino acid sequence of any of SEQ ID NO.

In embodiments, the joint may be flexible, including but not limited to highly flexible. In embodiments, the linker may be rigid, including but not limited to a rigid alpha helix. The features of an illustrative splice joint are shown in table 3 below:

table 3: features of illustrative joint

In embodiments, the linker may be functional. For example, but not limited to, the linker may function to increase folding and/or stability, increase expression, improve pharmacokinetics, and/or improve the biological activity of the chimeric proteins of the invention. In another example, the linker can function to target the chimeric protein to a particular cell type or location.

In embodiments, the chimeric protein comprises only one junction linker.

In embodiments, the chimeric protein lacks a splice junction.

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

In embodiments, the chimeric protein has a first domain capable of spatially binding its ligand/receptor and/or a second domain capable of spatially binding its ligand/receptor. Thus, there is sufficient overall flexibility in the chimeric protein and/or there is a physical distance between the extracellular domain (or portion thereof) and the remainder of the chimeric protein such that the ligand/receptor binding domain of the extracellular domain binds its ligand/receptor in space unimpeded. Such flexibility and/or physical distance (which is referred to as "relaxation") may typically be present in one or more extracellular domains, typically in a linker, and/or typically in a chimeric protein (as a whole). Alternatively or additionally, amino acid sequences (for example) may be added to one or more extracellular domains and/or linkers to provide the necessary relaxation to avoid steric hindrance. Any amino acid sequence that provides relaxation may be added. In embodiments, the added amino acid sequence comprises the sequence (Gly) _n, where n is any number from 1 to 100. Additional examples of amino acid sequences that may be added include the splice junctions described in tables 1 and 3. In embodiments, a polyethylene glycol (PEG) linker may be added between the extracellular domain and the linker to provide the relaxation required to avoid steric hindrance. Such PEG linkers are well known in the art.

In embodiments, the chimeric proteins of the present disclosure comprise the extracellular domain of human CD172a (sirpa) (or variant thereof), a linker, and the extracellular domain of human CD40L (or variant thereof). In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain, e.g., from IgG1 or from IgG4 (including human IgG1 or IgG 4). Thus, in embodiments, the chimeric proteins of the present disclosure comprise the extracellular domain of human CD172a (sirpa) (or variant thereof), a linker comprising a hinge-CH 2-CH3 Fc domain, and the extracellular domain of human CD40L (or variant thereof). Such chimeric proteins may be referred to herein as "hCD172a (sirpa) -Fc-CD40L" or "SL-172154".

Disease, treatment method and mechanism of action

One aspect of the present disclosure is a method for treating cancer in a human subject. In embodiments, the method comprises: (i) Administering to a human subject a first dose of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background level and/or activity of cells in a biological sample obtained from a subject prior to administration of a first dose has been measured, wherein the marker is selected from one or more of cd80+ cells, cd8+ cells, granzyme b+ cells, cd68+ cells, ki67+ cells, and PD-l1+ immune cells. In embodiments, the method further comprises (ii) administering a second dose of the chimeric protein to the human subject if the post-administration level and/or activity of the cell is greater than the background level and/or activity of the cell. In embodiments, the second dose is administered at least about 48 hours after the first dose is administered.

In embodiments, the biological sample is selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph fluid, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid or lavage, aspirate, scrapings, bone marrow specimens, biopsy specimens, and surgical specimens. In embodiments, the biological sample is a biopsy sample or a surgical specimen. In embodiments, the biological sample is a tumor biopsy or a tumor surgical specimen. In embodiments, the tumor biopsy is derived from a tumor selected from the group consisting of ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous Squamous Cell Carcinoma (CSCC), and head and neck Squamous Cell Carcinoma (SCCHN).

In embodiments, the level and/or activity of the cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof. In embodiments, the level and/or activity of a cell is measured by contacting the sample with an agent that specifically binds to one or more molecules selected from the group consisting of CD80, CD8, granzyme B, CD, ki67, and PD-L1. In embodiments, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In embodiments, the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof.

In embodiments, the level and/or activity of a cell is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding one or more of CD80, CD8, granzyme B, CD, ki67, and PD-L1. In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

One aspect of the present disclosure is a method for treating cancer in a human subject. In embodiments, the method comprises: (i) Administering to a human subject a first dose of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background level and/or activity of B cells and/or cd40+ cells in a first biological sample obtained from a subject prior to administration of a first dose has been measured. In embodiments, the level and/or activity of B cells and/or cd40+ cells in a second biological sample obtained from the subject after administration of the first dose has been measured N hours after administration. In an embodiment, N is a number between 1 and 24. In embodiments, the level and/or activity of the B cells and/or cd40+ cells N hours after administration is less than the background level and/or activity of the B cells and/or cd40+ cells. In embodiments, the post-administration M-day level and/or activity of B cells and/or cd40+ cells in a third biological sample obtained from the subject after administration of the first dose has been measured. In an embodiment, M is a number between 1 and 28. In embodiments, the method further comprises (ii) administering a second dose of the chimeric protein to the human subject if the level and/or activity of the B cells and/or cd40+ cells M days after administration is at least about 50% greater than the level and/or activity of the B cells and/or cd40+ cells N hours after administration. In embodiments, the second dose is administered at least about 48 hours after the first dose is administered.

In embodiments, N is less than 12, or less than 8, or less than 6, or less than 4, or less than 3, or less than 2, or less than 1. In embodiments, M is less than 21, or less than 14, or less than 12, or less than 10, or less than 8, or less than 6, or less than 4, or less than 2. In embodiments, N is less than 12, or less than 8, or less than 6, or less than 4, or less than 3, or less than 2, or less than 1; and M is less than 21, or less than 14, or less than 12, or less than 10, or less than 8, or less than 6, or less than 4, or less than 2.

In embodiments, the level and/or activity of the B cells and/or cd40+ cells N hours after administration is greater than the background level and/or activity of the B cells and/or cd40+ cells. In embodiments, the level and/or activity of the B cells and/or cd40+ cells N hours after administration is less than the background level and/or activity of the B cells and/or cd40+ cells. In embodiments, the level and/or activity of the B cells and/or cd40+ cells N hours after administration is within about 10%, or about 20%, or about 30%, or about 40% of the background level and/or activity of the B cells and/or cd40+ cells.

In embodiments, the first biological sample, the second biological sample, and the third biological sample are independently selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, fecal matter, lymph fluid, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid or lavage, aspirate, scraping, bone marrow specimen, biopsy specimen, and surgical specimen. In embodiments, the first biological sample, the second biological sample, and the third biological sample are blood.

In embodiments, the level and/or activity of B cells and/or cd40+ cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof. In embodiments, the level and/or activity of B cells and/or cd40+ cells is measured by contacting the sample with an agent that specifically binds to CD40 and/or a B cell marker. In embodiments, the B cell marker is selected from the group consisting of CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD, CD95, igM, igD, and CD40. In embodiments, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In embodiments, the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof. In embodiments, the level and/or activity of B cells and/or cd40+ cells is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding CD40 and/or B cell markers. In embodiments, the B cell marker is selected from the group consisting of CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD, CD95, igM, igD, and CD40. In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

One aspect of the present disclosure is a method for treating cancer in a human subject. In embodiments, the method comprises: (i) Administering to a human subject a first dose of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background amount and/or activity of a cytokine selected from the group consisting of CCL2, CXCL9, CXCL10, ifnα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC in a first biological sample obtained from a subject prior to administration of a first dose has been measured. In embodiments, the amount and/or activity of the cytokine in the second biological sample obtained from the subject N hours after administration of the first dose has been measured. In an embodiment, N is a number between 1 and 24. In embodiments, the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine. In embodiments, the amount and/or activity of a cytokine in a third biological sample obtained from a subject after administration of a first dose has been measured M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises (ii) administering a second dose of the chimeric protein to the human subject if the amount and/or activity of the cytokine M days after administration is at least about 30% less than the amount and/or activity of the cytokine N hours after administration. In embodiments, the second dose is administered at least about 48 hours after the first dose is administered.

One aspect of the present disclosure is a method for treating cancer in a human subject. In embodiments, the method comprises: (i) Administering to a human subject a first dose of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background level of cells in a pre-dose biological sample obtained from a subject prior to administration of a first dose has been measured, wherein the marker is selected from one or more of cd80+ cells, cd8+ cells, granzyme b+ cells, cd68+ cells, ki67+ cells, and PD-l1+ immune cells. In embodiments, the method further comprises administering a second dose of the chimeric protein to the human subject if the post-administration level of the cell is greater than the background level of the cell. In embodiments, the second dose is administered at least about 48 hours after the first dose is administered.

In embodiments, the biological sample is selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph fluid, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid or lavage, aspirate, scrapings, bone marrow specimens, biopsy specimens, and surgical specimens. In embodiments, the biological sample is a biopsy sample or a surgical specimen. In embodiments, the biological sample is a tumor biopsy or a tumor surgical specimen. In embodiments, the tumor biopsy sample or tumor surgical specimen is derived from a tumor selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous Squamous Cell Carcinoma (CSCC), and head and neck Squamous Cell Carcinoma (SCCHN).

In embodiments, the level and/or activity of the cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof. In embodiments, the level and/or activity of a cell is measured by contacting the sample with an agent that specifically binds to one or more molecules selected from the group consisting of CD80, CD8, granzyme B, CD, ki67, and PD-L1. In embodiments, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In embodiments, the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof. In embodiments, the level and/or activity of a cell is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding one or more of CD80, CD8, granzyme B, CD, ki67, and PD-L1. In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

One aspect of the present disclosure is a method for treating cancer in a human subject. In embodiments, the method comprises: (i) Administering to a human subject a first dose of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the background amount and/or activity of a cytokine selected from the group consisting of CCL2, CXCL9, CXCL10, ifnα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC in a first biological sample obtained from a subject prior to administration of a first dose has been measured. In embodiments, the amount and/or activity of the cytokine in the second biological sample obtained from the subject N hours after administration of the first dose has been measured. In an embodiment, N is a number between 1 and 24. In embodiments, the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine. In embodiments, the amount and/or activity of the cytokine in a third biological sample obtained from the subject M days after administration of the first dose has been measured. In an embodiment, M is a number between 1 and 28. In embodiments, the method further comprises (ii) administering a second dose of the chimeric protein to the human subject if the amount and/or activity of the cytokine M days after administration is at least about 30% less than the amount and/or activity of the cytokine N hours after administration. In embodiments, the second dose is administered at least about 48 hours after the first dose is administered.

In embodiments, the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine. In embodiments, the amount and/or activity of the cytokine N hours after administration is less than the background amount and/or activity of the cytokine. In embodiments, the amount and/or activity of the cytokine N hours after administration is within about 10%, or about 20%, or about 30%, or about 40% of the background amount and/or activity of the cytokine.

In embodiments, the amount and/or activity of the cytokine is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or combinations thereof. In embodiments, the amount and/or activity of a cytokine is measured by contacting the sample with an agent that specifically binds to the cytokine. In embodiments, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In embodiments, the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof. In embodiments, the amount and/or activity of a cytokine is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding the cytokine. In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

The chimeric proteins disclosed herein, including CD172a (SIRPalpha) -Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61), are useful in methods of treating both advanced solid tumors and advanced lymphomas. These tumor types include: melanoma, non-small cell lung cancer (squamous, glandular, adeno-squamous), urothelial cancer, renal cell carcinoma, cervical squamous cell carcinoma, gastric adenocarcinoma or gastro-esophageal junction adenocarcinoma, anal squamous cell carcinoma, head and neck squamous cell carcinoma, skin squamous cell carcinoma, and high microsatellite instability or mismatch repair deficient solid tumors (excluding Central Nervous System (CNS) tumors). Other tumors of interest include Hodgkin's Lymphoma (HL), diffuse large B-cell lymphoma, acute Myeloid Leukemia (AML), and high-risk myelodysplastic syndrome (HR-MDS).

In embodiments, the cancer comprises advanced solid tumors (localized and/or metastatic). In embodiments, the human subject has cancer, wherein the cancer treated is characterized by macrophages in the tumor microenvironment and/or tumor cells in the tumor that are cd47+ cells. In embodiments, administration of the sirpa-Fc-CD 40L chimeric protein blocks the "do not eat me" signal and/or stimulates the "eat me" signal of tumor cells. In embodiments, therapies using SIRP alpha-Fc-CD 40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61) stimulate macrophages to phagocytose tumor cells and effectively present tumor antigens of the phagocytosed tumor cells to T cells.

In embodiments, the cancer is a solid cancer. In embodiments, the cancer is a solid tumor. In embodiments, the cancer is a metastatic cancer. In embodiments, the cancer is a hematologic cancer. In embodiments, the cancer expresses CD47.

In embodiments, the cancer comprises advanced lymphoma. In embodiments, the cancer comprises Acute Myeloid Leukemia (AML). In embodiments, the cancer comprises p53 mutant AML. In embodiments, the cancer comprises high-risk myelodysplastic syndrome (HR-MDS).

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

It is often desirable to enhance immunostimulatory signaling to enhance an immune response, for example, to enhance an anti-tumor immune response in a patient.

In embodiments, the chimeric proteins of the present disclosure comprise an extracellular domain of human CD172a (sirpa) that disrupts, blocks, reduces, inhibits and/or sequesters the transmission of immunosuppressive signals, e.g., derived from cancer cells that attempt to avoid detection and/or disruption thereof; and the extracellular domain of human CD40L that enhances, increases and/or stimulates the delivery of immunostimulatory signals to anti-cancer immune cells. Thus, the simultaneous binding of the extracellular domain of CD172a (sirpa) to its ligand/receptor and the binding of the extracellular domain of CD40L to its receptor will prevent the transmission of immunosuppressive signals from cancer cells and will have stimulatory immune activity in immune system cells. In other words, the chimeric proteins of the present disclosure are capable of treating cancer by two different mechanisms.

In embodiments, the present disclosure relates to cancers and/or tumors; for example, the treatment or prevention of cancer and/or tumors. As disclosed elsewhere herein, in embodiments, the treatment of cancer involves modulating the immune system with the chimeric proteins of the invention to facilitate increasing or activating the immunostimulatory signal. In embodiments, the method reduces the amount or activity of regulatory T cells (tregs) compared to untreated subjects or subjects treated with antibodies to CD172a (sirpa), CD40L, and/or their respective ligands or receptors. In embodiments, the method increases priming of effector T cells in a draining lymph node of a subject as compared to an untreated subject or a subject treated with antibodies to CD172a (sirpa), CD40L, and/or their respective ligands or receptors. In embodiments, the method results in an overall reduction of immunosuppressive cells and a shift to a more inflammatory tumor environment compared to untreated subjects or subjects treated with antibodies to CD172a (sirpa), CD40L, and/or their respective ligands or receptors.

In embodiments, the chimeric proteins of the invention are capable of modulating the magnitude of an immune response (e.g., modulating the level of effector output) or are useful in methods comprising modulating the magnitude of an immune response (e.g., modulating the level of effector output). In embodiments, for example when used in the treatment of cancer, the chimeric proteins of the invention alter the degree of immune stimulation compared to immunosuppression to increase the magnitude of the T cell response, including but not limited to stimulating increased levels of cytokine production, proliferation or target killing potential. In embodiments, the T cells of the patient are activated and/or stimulated by the chimeric protein, wherein the activated T cells are capable of dividing and/or secreting cytokines.

Cancer or tumor refers to uncontrolled cell growth and/or abnormally increased cell survival and/or inhibition of apoptosis, which interfere with normal function of body organs and systems. Including benign and malignant cancers, polyps, hyperplasia, dormant state tumors, or micrometastases. In addition, cells having abnormal proliferation that are not impeded by the immune system (e.g., virus-infected cells) are included. The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be a region of cancer cells at a clinically detectable site of origin, and may be a primary tumor. In contrast, metastatic cancer can be the spread of a disease from one organ or part to another non-adjacent organ or part. Metastatic cancers can be caused by cancer cells that have the ability to penetrate and infiltrate surrounding normal tissue in a localized area, forming a new tumor, which can be localized metastasis. Cancer cells may also be caused by cancer cells that have the ability to penetrate lymphatic and/or blood vessel walls, after which the cancer cells can circulate through the blood stream (and thus become tumor cells in the circulation) to other sites and tissues in the body. Cancers may arise from processes such as lymphatic or blood borne disseminated. Cancer can also be caused by tumor cells that reside at another site, re-penetrate the blood vessel or wall, continue to multiply, and eventually form another clinically detectable tumor. The cancer may be such a new tumor, which may be a metastatic (or secondary) tumor.

Cancers may be caused by metastatic tumor cells, which may be secondary or metastatic tumors. The cells of the tumor may be similar to the cells in the original tumor. As an example, if breast or colon cancer metastasizes to the liver, a secondary tumor, while present in the liver, consists of abnormal breast or colon cells rather than abnormal hepatocytes. Thus, the tumor in the liver may be metastatic breast cancer or metastatic colon cancer, rather than liver cancer.

The cancer may originate from any tissue. Cancers may originate from melanoma, colon, breast or prostate; and thus may consist of cells that are initially skin, colon, breast or prostate tissue, respectively. The cancer may also be a hematological malignancy, which may be leukemia or lymphoma. Cancers can invade tissues such as the liver, lung, bladder, or intestine.

In embodiments, the chimeric proteins are used to treat a subject with refractory cancer. In embodiments, the chimeric proteins are used to treat subjects refractory to one or more immunomodulatory agents. For example, in embodiments, the chimeric protein is used to treat a subject that does not respond to treatment or its disease progression after about 12 weeks of treatment. For example, in embodiments, the subject is refractory to one or more CD172a (sirpa) and/or CD47 agents, including, for example, mo Luoli mab (5F 9), hu5F9-G4, CC-90002, ti-061, SRF231, TT I-621, TTI-622, or ALX148 refractory patients. For example, in embodiments, the subject is refractory to an anti-CTLA-4 agent, such as an ipilimumab (YERVOY) refractory patient (e.g., a melanoma patient). Thus, in embodiments, the present disclosure provides methods of treating cancer that rescue patients who are unresponsive to various therapies, including monotherapy with one or more immunomodulators.

In embodiments, the present disclosure provides chimeric proteins that target cells or tissues within a tumor microenvironment. In embodiments, cells or tissues within the tumor microenvironment express one or more targets or binding partners of the chimeric protein. Tumor microenvironment refers to the cellular environment, including the cells in which the tumor is present, secreted proteins, physiological small molecules, and blood vessels. In embodiments, the cells or tissue within the tumor microenvironment are one or more of the following: tumor blood vessels; tumor infiltrating lymphocytes; fibroblast reticulocytes; endothelial Progenitor Cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; a component of extracellular matrix (ECM); dendritic cells; an antigen presenting cell; t cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to the tumor. In embodiments, the chimeric proteins of the invention target cancer cells. In embodiments, the cancer cell expresses one or more targets or binding partners of the chimeric protein.

Activation of regulatory T cells is severely affected by co-stimulatory and co-inhibitory signals. Two major families of costimulatory molecules include the B7 and Tumor Necrosis Factor (TNF) families. These molecules bind to receptors belonging to the CD28 or TNF receptor family on T cells, respectively. Many well-defined co-inhibitors and their receptors belong to the B7 and CD28 families.

In embodiments, an immunostimulatory signal refers to a signal that enhances an immune response. For example, in the context of oncology, such signals may enhance anti-tumor immunity. For example, but not limited to, an immunostimulatory signal may be identified by directly stimulating proliferation, cytokine production, killing activity, or phagocytic activity of leukocytes. For example, the chimeric proteins may directly stimulate proliferation and cytokine production by a subset of individual T cells. Another example includes directly stimulating immunosuppressive cells through receptors that inhibit the activity of such immunosuppressive cells. For example, this would include stimulating cd4+foxp3+ regulatory T cells, which would reduce the ability of those regulatory T cells to suppress proliferation of conventional cd4+ or cd8+ T cells. In another example, this would include stimulating CD40 on the surface of antigen presenting cells, thereby causing activation of antigen presenting cells, including enhancement of the ability of those cells to present antigen in the context of appropriate natural costimulatory molecules, including those in the B7 or TNF superfamily. In another example, the chimeric protein causes activation of lymphoid cells and/or production of pro-inflammatory cytokines or chemokines, thereby further stimulating an immune response, optionally within a tumor.

In embodiments, the chimeric proteins of the invention are capable of enhancing, restoring, promoting and/or stimulating immune modulation, or are suitable for use in methods involving enhancing, restoring, promoting and/or stimulating immune modulation. In embodiments, the chimeric proteins of the invention 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 embodiments, the chimeric proteins of the invention enhance, restore, promote and/or stimulate the activity and/or activation of T cells, including (as non-limiting examples) activating and/or stimulating one or more T cell endogenous signals, including pro-survival signals; an autocrine or paracrine growth signal; p38 MAPK-, ERK-, STAT-, JAK-, AKT-, or PI 3K-mediated signaling; an anti-apoptotic signal; and/or facilitate one or more of the following and/or signals necessary for one or more of the following: pro-inflammatory cytokine production or T cell migration or T cell tumor infiltration.

In embodiments, the chimeric proteins of the invention are capable of causing an increase in one or more of T cells (including but not limited to 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 tumor microenvironment, or are suitable for use in methods involving causing an increase in one or more of T cells (including but not limited to 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 tumor microenvironment. In embodiments, the chimeric proteins enhance the recognition of tumor antigens by cd8+ T cells, particularly those T cells that have penetrated into the tumor microenvironment. In embodiments, the chimeric proteins of the invention induce CD19 expression and/or increase the number of CD19 positive cells (e.g., CD19 positive B cells). In embodiments, the chimeric proteins of the invention induce IL-15Rα expression and/or increase the number of αIL-15R positive cells (e.g., IL-15Rα positive dendritic cells).

In embodiments, the chimeric proteins of the invention are capable of inhibiting and/or eliciting immunosuppressive cells (e.g., bone Marrow Derived Suppressor Cells (MDSCs), regulatory T cells (tregs), tumor-associated neutrophils (TAN), M2 macrophages, and tumor-associated macrophages (TAMs)), and particularly decrease within a tumor and/or Tumor Microenvironment (TME), or are useful in methods involving inhibiting and/or eliciting immunosuppressive cells (e.g., bone Marrow Derived Suppressor Cells (MDSCs), regulatory T cells (tregs), tumor-associated neutrophils (TAN), M2 macrophages, and tumor-associated macrophages (TAMs)), and particularly decrease within a tumor and/or Tumor Microenvironment (TME). In embodiments, the therapies of the invention may alter the ratio of M1 to M2 macrophages in the tumor site and/or TME to favor M1 macrophages. In embodiments, the sirpa-Fc-CD 40L chimeric protein inhibits/reduces/eliminates "do not eat me" signaling on tumor cells by Sipr a/CD 47. In embodiments, the sirpa-Fc-CD 40L chimeric protein renders the tumor more susceptible to attack by the immune system of the subject. In embodiments, the sirpa-Fc-CD 40L chimeric protein renders the tumor more susceptible to attack by the innate immune system of the subject. In embodiments, the sirpa-Fc-CD 40L chimeric protein renders the tumor more susceptible to the subject's adaptive immune system attack. In embodiments, the sirpa-Fc-CD 40L chimeric protein can inhibit/reduce/eliminate binding of tumor over-expressed CD47 to sirpa expressed by phagocytes, thereby allowing phagocytic removal of cancer cells and/or immunogenic processing of tumor antigens by macrophages and/or dendritic cells. In embodiments, administration of the sirpa-Fc-CD 40L chimeric protein blocks the "do not eat me" signal and/or stimulates the "eat me" signal of tumor cells. In embodiments, therapies using SIRP alpha-Fc-CD 40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61) stimulate macrophages to phagocytose tumor cells and effectively present tumor antigens of the phagocytosed tumor cells to T cells.

In embodiments, the chimeric proteins of the invention are capable of increasing serum levels of various cytokines including, but not limited to, one or more of the following: IFNγ, TNFα, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, IL-17F, and IL-22. In embodiments, the chimeric proteins of the invention are capable of enhancing IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, or IFNγ in the serum of a treated subject. In embodiments, the chimeric proteins of the invention do not increase serum levels of certain cytokines. In embodiments, the chimeric proteins of the invention do not increase serum levels of IL-6 and/or TNFα. In embodiments, the chimeric proteins of the invention do not increase serum levels of IL-6 and/or TNF alpha in the serum of a treated subject. In embodiments, the chimeric proteins of the invention do not increase the serum level of IL-6 and/or tnfα in the serum of the treated subject, but increase the levels of other cytokines including, but not limited to CCL2, IL-8, and CXCL9 in the serum of the treated subject. Detection of such cytokine responses may provide a means for determining the optimal dosing regimen for the indicated chimeric proteins.

In the chimeric proteins of the present disclosure, the chimeric proteins are capable of increasing or preventing the decrease in cd4+ and/or cd8+ T cell subsets.

In the chimeric proteins of the present disclosure, the chimeric proteins are capable of enhancing tumor killing activity of T cells.

In embodiments, the chimeric protein activates T cells of a human subject when bound by the CD40L 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 a subject in need thereof as compared to a subject treated with a CD40 agonist antibody and/or a CD47 blocking antibody.

In embodiments, the chimeric proteins of the invention inhibit, block and/or reduce cell death of anti-tumor cd8+ and/or cd4+ T cells; or to stimulate, induce and/or increase cell death of neoplastic T cells. T cell depletion is a state of T cell dysfunction characterized by proliferation and gradual loss of effector function, ultimately leading to clonal loss. Thus, a pro-neoplastic T cell refers to a state of T cell dysfunction that occurs during many chronic infections, inflammatory diseases, and cancers. Such dysfunction is defined by poor proliferation and/or effector function, sustained expression of inhibitory receptors, and transcriptional states that differ from those of functional effector or memory T cells. Depletion can prevent optimal control of infection and tumors. Illustrative pro-tumor T cells include, but are not limited to, treg, cd4+ and/or cd8+ T cells, th2 cells and Th17 cells that express one or more checkpoint inhibitory receptors. Checkpoint inhibitory receptors refer to receptors expressed on immune cells that can prevent or inhibit uncontrolled immune responses. In contrast, anti-tumor cd8+ and/or cd4+ T cells refer to T cells that can mount an immune response against a tumor.

In embodiments, the chimeric proteins of the invention are capable of increasing the ratio of effector T cells to regulatory T cells and are useful in methods comprising increasing the ratio of effector T cells to regulatory T cells. Illustrative effector T cells include ICOS ⁺ effector T cells; cytotoxic T cells (e.g., αβ TCR, CD3 ⁺、CD8⁺、CD45RO⁺);CD4⁺ effector T cells (e.g., αβ TCR, CD3 ⁺、CD4⁺、CCR7⁺、CD62Lhi、IL^-7R/CD127⁺);CD8⁺ effector T cells (e.g., αβ TCR, CD3 ⁺、CD8⁺、CCR7⁺、CD62Lhi、IL^-7R/CD127⁺); Effector memory T cells (e.g., CD62L low, CD44 ⁺、TCR、CD3⁺、IL^-7R/CD127⁺、IL-15R⁺, CCR7 low); center 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 (CD 27 ⁺CD62L^-) and late effector memory T cells (CD 27 ^-CD62L^-) (TemE and TemL, respectively); CD127 (⁺) CD25 (low/-) effector T cells; CD127 (^-)CD25(^-) effector T 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 αβ TCR, CD3 ⁺、CD4⁺、IL-12R⁺、IFNγR⁺、CXCR3⁺), TH2 effector T cells (e.g., CCR3 ⁺、CCR4⁺ and CCR8 ⁺; Or αβTCR、CD3⁺、CD4⁺、IL-4R⁺、IL-33R⁺、CCR4⁺、IL-17RB⁺、CRTH2⁺);TH9 effector T cells (e.g., αβ TCR, CD3 ⁺、CD4⁺); TH17 effector T cells (e.g., ,αβTCR、CD3⁺、CD4⁺、IL-23R⁺、CCR6⁺、IL-1R⁺);CD4⁺CD45RO⁺CCR7⁺ effector T cells, CD4 ⁺CD45RO⁺CCR7(^-) effector T cells; and effector T cells that secrete IL-2, IL-4 and/or IFN-gamma. 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 ⁺ CD25 highly regulatory T cells, TIM-3 ⁺CD172a(SIRPα)⁺ regulatory T cells, lymphocyte activating 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, CD45RB low regulatory T cells, CD127 low regulatory T cells, LRRC32/GARP ⁺ regulatory T cells, CD39 ⁺ regulatory T cells, GITR ⁺ regulatory T cells, LAP ⁺ regulatory T cells, 1B11 ⁺ regulatory T cells, BTLA ⁺ regulatory T cells, type 1 regulatory T cells (Tr 1 cells), T helper 3 (Th 3) cells, regulatory cells of natural killer T cell phenotype (NKTreg), CD8 ⁺ regulatory T cells, CD8 ⁺CD28^- regulatory T cells and/or regulatory T cells that secrete IL-10, IL-35, TGF-beta, TNF-alpha, galectin-1, IFN-gamma and/or MCP 1.

In embodiments, the chimeric proteins of the invention cause an increase in effector T cells (e.g., cd4+cd25-T cells).

In embodiments, the chimeric protein causes a decrease in regulatory T cells (e.g., cd4+cd25+ T cells).

In embodiments, the chimeric proteins produce a memory response that may, for example, be capable of preventing relapse or protecting an animal from relapse and/or preventing metastasis, or reducing the likelihood of metastasis. Thus, animals treated with the chimeric proteins are able to attack tumor cells and/or prevent tumor progression later on re-challenge after initial treatment with the chimeric proteins. Thus, the chimeric proteins of the present disclosure stimulate active tumor destruction and also stimulate immune recognition of tumor antigens, which is essential in programming memory responses that can prevent recurrence.

In embodiments, the chimeric protein is capable of causing activation of antigen presenting cells. In embodiments, the chimeric protein is capable of enhancing the ability of antigen presenting cells to present antigen.

In embodiments, the chimeric proteins of the invention are capable of transiently stimulating effector T cells for more 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, and are useful in methods comprising stimulating effector T cells for more 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 embodiments, transient stimulation of effector T cells occurs substantially in the patient's blood stream or in specific tissues/sites (including lymphoid tissues such as, for example, bone marrow, lymph nodes, spleen, thymus, mucosa-associated lymphoid tissue (MALT), non-lymphoid tissues) or in the tumor microenvironment.

In the chimeric proteins of the present disclosure, the chimeric proteins of the present invention unexpectedly provide for the binding of an extracellular domain component to its corresponding binding partner with a slow off-rate (Kd or K _off). In embodiments, this provides unexpectedly long interactions of the receptor with the ligand, and vice versa. This effect allows for longer positive signaling effects, such as an increase or activation of the immunostimulatory signal. For example, the chimeric proteins of the invention (e.g., by long off-rate binding) allow sufficient signaling to provide immune cell proliferation, allow anti-tumor attack, allow sufficient signaling to provide release of stimulatory signals (e.g., cytokines).

In the chimeric proteins of the present disclosure, the chimeric proteins are capable of forming stable synapses between cells. The stable synapses of the cells promoted by the chimeric proteins (e.g., between cells with negative signals) provide spatial orientation to facilitate tumor reduction-such as locating T cells to attack tumor cells and/or spatially preventing tumor cells from transmitting negative signals, including negative signals other than those masked by the chimeric proteins of the invention. In embodiments, this provides a longer mid-target (e.g., intratumoral) half-life (t _1/2) as compared to serum t _1/2 of the chimeric protein. Such properties may have the combined advantage of reducing off-target toxicity associated with systemic distribution of the chimeric protein.

In embodiments, the chimeric protein is capable of providing sustained immunomodulatory effects.

The chimeric proteins of the invention provide synergistic therapeutic effects (e.g., anti-tumor effects) because it allows for improved site-specific interaction of two immunotherapeutic agents. In embodiments, the chimeric proteins of the invention provide the potential to reduce ectopic and/or systemic toxicity.

In embodiments, the chimeric proteins of the invention exhibit enhanced safety features. In embodiments, the chimeric proteins of the invention exhibit reduced toxicity profiles. For example, administration of the chimeric proteins of the invention may produce reduced side effects, such as one or more of diarrhea, inflammation (e.g., intestinal inflammation), or weight loss, that occur after administration of antibodies to one or more ligands/receptors targeted by the extracellular domain of the chimeric proteins of the invention. In embodiments, the chimeric proteins of the invention provide improved safety, but without sacrificing efficacy, compared to antibodies directed against one or more ligands/receptors targeted by the extracellular domain of the chimeric proteins of the invention.

In embodiments, the chimeric proteins of the invention provide reduced side effects, such as G I complications, relative to current immunotherapy, such as antibodies directed against one or more ligands/receptors targeted by the extracellular domain of the chimeric proteins of the invention. Illustrative G I complications include abdominal pain, anorexia, autoimmune effects, constipation, cramps, dehydration, diarrhea, eating problems, fatigue, flatulence, abdominal dropsy or ascites, gastrointestinal (G I) dysbiosis, G I mucositis, inflammatory bowel disease, irritable bowel syndrome (ibs-D and ibs-C), nausea, pain, stool or urine changes, ulcerative colitis, vomiting, weight gain due to effusion and/or weakness.

Pharmaceutical composition

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

Any of the chimeric proteins disclosed herein can be used in a pharmaceutical composition.

In embodiments, the chimeric proteins disclosed herein are provided in the form of a sterile frozen solution in a vial or a sterile liquid solution in a vial. Pharmaceutical products comprising the chimeric proteins disclosed herein include sterile filtered formulated chimeric protein solutions disclosed herein, which are filled to useThe rubber stopper was plugged into a 10mL disposable glass vial sealed with an aluminum flip seal. In embodiments, the chimeric proteins disclosed herein are formulated in about 30mM to about 70mM L-histidine (e.g., about 50mM L-histidine) and about 125mM to about 400mM sucrose (e.g., about 250mM sucrose) in water for injection at about 10mg/mL to about 30mg/mL, e.g., about 20 mg/mL. In embodiments, each vial contains about 1mL of the pharmaceutical product or about 20mg of the chimeric protein disclosed herein.

The chimeric proteins disclosed herein, including the CD172a (SIRPalpha) -Fc-CD40L chimeric proteins (SEQ ID NO:59 or SEQ ID NO: 61) may have sufficiently basic functional groups that can react with mineral or organic acids, or carboxyl groups that can react with mineral or organic bases, to form pharmaceutically acceptable salts. As is well known in the art, pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids. Such salts include, for example, those described in Journal of Pharmaceutical Science,66,2-19 (1977) and The Handbook of Pharmaceutical Salts; pharmaceutically acceptable salts listed in Properties, selection, and use.p.h.stahl and c.g.weruth (editions), verlag, zurich (Switzerland), 2002, which are hereby incorporated by reference in their entirety.

In embodiments, the compositions disclosed herein are in the form of pharmaceutically acceptable salts.

Furthermore, any of the chimeric proteins disclosed herein can be administered to a subject as a composition, e.g., a component of a pharmaceutical composition, comprising a pharmaceutically acceptable carrier or vehicle. Such pharmaceutical compositions may optionally comprise a suitable amount of a pharmaceutically acceptable excipient in order to provide a form for proper administration. The pharmaceutical excipients may 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 may be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. In embodiments, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the agents disclosed herein are administered intravenously. Saline and dextrose in water and glycerol solutions can also be employed as liquid excipients, particularly 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 of the agents disclosed herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

In embodiments, a composition disclosed herein, e.g., a pharmaceutical composition, is resuspended in saline buffer (including but not limited to TBS, PBS, etc.).

In embodiments, the chimeric proteins may extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties by conjugation and/or fusion with another agent. In embodiments, the chimeric protein can be fused or conjugated to 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 an embodiment, each of the individual chimeric proteins is fused to one or more agents described in Stroh l, bioDrugs (4): 215-239 (2015), the entire contents of which are hereby incorporated by reference.

The present disclosure includes the disclosed chimeric proteins in various formulations of pharmaceutical compositions. Any of the chimeric proteins disclosed herein can take the form of a solution, suspension, emulsion, drop, tablet, pill, pellet, capsule, liquid-containing capsule, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, or any other suitable form for use. DNA or RNA constructs encoding protein sequences may also be used. In embodiments, the composition is in the form of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences1447-1676 (Al fonso r. Gennaro et al, 19 th edition 1995), which is incorporated herein by reference.

If desired, the pharmaceutical composition comprising the chimeric protein may further comprise a solubilizing agent. In addition, the agent may be delivered using suitable vehicles or delivery devices known in the art. The composition for administration may optionally contain a local anesthetic, such as, for example, lidocaine, to reduce pain at the injection site.

Pharmaceutical compositions comprising the chimeric proteins of the present disclosure may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of combining the therapeutic agent with a carrier composed of one or more additional ingredients. Generally, the pharmaceutical compositions are prepared by uniformly and intimately bringing into association the therapeutic agent with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into dosage forms (e.g., wet or dry granulation, powder blends, etc., and then tableting using conventional methods known in the art) of the desired formulation.

In embodiments, any chimeric protein disclosed herein is formulated according to conventional procedures into a pharmaceutical composition suitable for the mode of administration disclosed herein.

Administration, dosing and treatment regimens

One aspect of the present disclosure is a method for assessing the efficacy of a cancer treatment in a subject in need thereof, wherein the subject has cancer. In embodiments, the method comprises: (i) A first biological sample obtained from a subject that has received a first dose of chimeric protein is obtained. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the method further comprises determining the background level and/or activity of B cells and/or cd40+ cells in the first biological sample. In embodiments, the method further comprises (iii) obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24. In embodiments, the method further comprises determining the level and/or activity of B cells and/or cd40+ cells in the second biological sample N hours after administration. In embodiments, the method further comprises (iv) obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises determining the level and/or activity of B cells and/or cd40+ cells in the third biological sample M days after administration. In embodiments, the method further comprises (v) determining that the chimeric protein is effective if the level and/or activity of the B cell and/or the cd40+ cell at N hours after administration is less than the background level and/or activity of the B cell and/or the cd40+ cell and/or if the level and/or activity of the B cell and/or the cd40+ cell at M days after administration is at least about 50% greater than the level and/or activity of the B cell and/or the cd40+ cell at N hours after administration.

One aspect of the disclosure is a method of selecting a subject for treatment with a therapy for cancer. In embodiments, the method comprises: (i) A first biological sample obtained from a subject that has received a first dose of chimeric protein is obtained. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the method further comprises determining the background level and/or activity of B cells and/or cd40+ cells in the first biological sample. In embodiments, the method further comprises (iii) obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24. In embodiments, the method further comprises determining the level and/or activity of B cells and/or cd40+ cells in the second biological sample N hours after administration. In embodiments, the method further comprises (iv) obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises determining the level and/or activity of B cells and/or cd40+ cells in the third biological sample M days after administration. In embodiments, the method further comprises (v) selecting the subject for treatment with the chimeric protein if the N hour post-administration level and/or activity of the B cell and/or cd40+ cell is less than the background level and/or activity of the B cell and/or cd40+ cell and/or if the M day post-administration level and/or activity of the B cell and/or cd40+ cell is at least about 50% greater than the N hour post-administration level and/or activity of the B cell and/or cd40+ cell.

One aspect of the present disclosure is a method for assessing the efficacy of a cancer treatment in a subject in need thereof, wherein the subject has cancer. In embodiments, the method comprises: (i) A first biological sample obtained from a subject that has received a first dose of chimeric protein is obtained. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the method further comprises (ii) determining the background amount and/or activity of the cytokine in the first biological sample. In embodiments, the cytokine is selected from the group consisting of CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1 β, MIP-1 α, and MDC. In embodiments, the method further comprises (iii) obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24. In embodiments, the method further comprises determining the amount and/or activity of the cytokine in the second biological sample N hours after administration. In embodiments, the method further comprises (iv) obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises determining the amount and/or activity of the cytokine in the third biological sample M days after administration. In embodiments, the method further comprises (v) determining that the chimeric protein is effective if the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine and/or if the amount and/or activity of the cytokine M days after administration is at least about 50% less than the amount and/or activity of the cytokine N hours after administration.

One aspect of the disclosure is a method of selecting a subject for treatment with a therapy for cancer. In embodiments, the method comprises: (i) A first biological sample obtained from a subject that has received a first dose of chimeric protein is obtained. In embodiments, the chimeric protein has the following general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L). In embodiments, the method further comprises (ii) determining the background amount and/or activity of the cytokine in the first biological sample. In embodiments, the cytokine is selected from the group consisting of CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1 β, MIP-1 α, and MDC. In embodiments, the method further comprises (iii) obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24. In embodiments, the method further comprises determining the amount and/or activity of the cytokine in the second biological sample N hours after administration. In embodiments, the method further comprises (iv) obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28. In embodiments, the method further comprises determining the amount and/or activity of the cytokine in the third biological sample M days after administration. In embodiments, the method further comprises (v) selecting the subject for treatment with the chimeric protein if the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine and/or if the amount and/or activity of the cytokine M days after administration is at least about 50% less than the amount and/or activity of the cytokine N hours after administration.

In embodiments, the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine. In embodiments, the amount and/or activity of the cytokine N hours after administration is less than the background amount and/or activity of the cytokine. In embodiments, the amount and/or activity of the cytokine N hours after administration is within about 10%, or about 20%, or about 30%, or about 40% of the background amount and/or activity of the cytokine. In embodiments, the first biological sample, the second biological sample, and the third biological sample are independently selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, fecal matter, lymph fluid, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid or lavage, aspirate, scraping, bone marrow specimen, biopsy specimen, and surgical specimen. In embodiments, the first biological sample, the second biological sample, and the third biological sample are blood.

In embodiments, the chimeric protein comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61. In embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61. In embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99.2% identical to the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61. In embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99.4% identical to the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61. In embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99.6% identical to the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61. In embodiments, the chimeric protein comprises an amino acid sequence that is at least about 99.8% identical to the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61. In embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61.

In embodiments, the human subject has or is suspected of having an advanced solid tumor or lymphoma. In embodiments, the human subject has or is suspected of having a cancer selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous Squamous Cell Carcinoma (CSCC), and head and neck Squamous Cell Carcinoma (SCCHN). In embodiments, the human subject has failed one or more platinum-based therapies. In embodiments, the human subject is not suitable for receiving platinum therapy. In embodiments, the human subject does not receive concurrent chemotherapy, immunotherapy, biological or hormonal therapy, and/or wherein the human subject has received standard therapy, is resistant to standard therapy, or is not suitable to receive standard therapy, and/or the cancer is not considered an approved therapy for standard care.

In embodiments, the chimeric proteins disclosed herein are present in the form of a sterile frozen solution at a concentration of about 20mg/mL and a total volume of about 1mL, optionally in a 10mL glass vial. In embodiments, the chimeric proteins disclosed herein are administered by Intravenous (IV) infusion after dilution with physiological saline. The starting dose, dose escalation regimen and dose schedule of certain embodiments are presented below.

In embodiments, the dose of chimeric protein administered is at least 0.0001mg/kg, for example between about 0.0001mg/kg and about 10mg/kg. In embodiments, the dosage of chimeric protein administered is at least about 0.3mg/kg, for example at least about 0.3mg/kg, or about 1.0mg/kg, or about 2mg/kg, or about 3, about 4mg/kg, or about 6mg/kg, or about 8mg/kg, or about 10mg/kg. In embodiments, the chimeric protein is administered at a dose of at least about 1mg/kg, for example at least about 1.0mg/kg, or about 2mg/kg, or about 3, about 4mg/kg, or about 6mg/kg, or about 8mg/kg, or about 10mg/kg. In embodiments, the dosage of sirpa-Fc-CD 40L chimeric protein is not limited by anemia or other cytopenia effects, and therefore is higher than allowed compared to certain other therapeutic agents (e.g., anti-CD 47 antibodies or sirpa Fc fusion proteins). Furthermore, in embodiments, low dose priming is not required.

In embodiments, the administration is intravenous administration. In embodiments, the administration is intratumoral administration. In embodiments, administration is by injection. In embodiments, administration is by infusion. In embodiments, administration is by intravenous infusion. In embodiments, administration is by intratumoral injection.

In embodiments, the chimeric protein is administered at an initial dose (e.g., about 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, or about 10 mg/kg) and the chimeric protein is administered in one or more subsequent administrations. In embodiments, one or more subsequent administrations have a dosage of 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 and about 10 mg/kg.

In embodiments, the initial dose and/or subsequent doses are maximum tolerated doses or less than maximum tolerated doses.

In embodiments, the dose is incremented in logarithmic increments between one or more subsequent doses, e.g., 0.0001mg/kg to 0.001mg/kg, 0.001mg/kg to 0.01mg/kg, and 0.01mg/kg to 0.1mg/kg.

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

In embodiments, one or more platinum-based therapies of a human subject have failed and are optionally unsuitable for receiving platinum therapy. In embodiments, the human subject does not receive concurrent chemotherapy, immunotherapy, biological or hormonal therapy, and/or wherein the human subject has received standard therapy, is resistant to standard therapy, or is not suitable to receive standard therapy, and/or the cancer is not considered an approved therapy for standard care.

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

In embodiments, the initial dose is the same as at least one dose in a subsequent administration, e.g., each dose in a subsequent administration.

In embodiments, the chimeric protein is administered at least about once a month.

In embodiments, the chimeric protein is administered at least about twice per month.

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

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

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

In embodiments, the chimeric protein is administered at least about four times per month. For example, the chimeric protein is administered about once a week. In embodiments, the chimeric protein is administered once a week (once every seven days). In embodiments, the chimeric protein is administered once every two weeks.

In embodiments, administration of the sirpa-Fc-CD 40L chimeric protein does not cause anemia or another cytopenia in the patient. In embodiments, administration does not cause lysis of RBCs. In embodiments, administration of a sirpa-Fc-CD 40L chimeric protein is less likely to cause anemia or another cytopenia than, for example, an anti-CD 47 Ab. In embodiments, the dosage of sirpa-Fc-CD 40L chimeric protein is not limited by anemia or other cytopenia effects, and therefore is higher than allowed compared to certain other therapeutic agents (e.g., anti-CD 47 antibodies or sirpa Fc fusion proteins). Furthermore, in embodiments, low dose priming is not required.

Another advantage provided by SIRPalpha-Fc-CD 40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO: 61) is that, despite targeting, they do not cause anemia or another cytopenia in the patient. This is because, although the CD 47/sirpa interaction plays a key role in RBC lysis, as shown herein, sirpa-Fc-CD 40L chimeric proteins do not cause RBC lysis. Thus, the methods of the invention are less likely to cause anemia or another cytopenia than, for example, anti-CD 47 abs.

The chimeric proteins may be administered intravenously into the blood by intravenous infusion or bolus injection. For patients with advanced ovarian cancer, fallopian tube cancer, and primary peritoneal cancer, the chimeric protein may be administered intravenously by intravenous infusion.

The chimeric proteins may be administered by intratumoral injection. In embodiments, the therapeutic dose for intratumoral administration is equal to or less than the therapeutic dose of intravenous infusion. In embodiments, the therapeutic dose for intratumoral administration is equal to the therapeutic dose of intravenous infusion. In embodiments, the therapeutic dose for intratumoral administration is less than the therapeutic dose of intravenous infusion. In embodiments, for patients with advanced or metastatic CSCC and HNSCC, therapeutic doses for intratumoral administration.

In embodiments, the chimeric proteins of the invention allow for dual effects that provide fewer side effects than those observed with conventional immunotherapy (e.g., treatment with one or more of OPD IVO, KEYTRUDA, YERVOY, and TECENTRIQ). For example, the chimeric proteins of the invention reduce or prevent immune-related adverse events that are commonly observed that affect various tissues and organs, including the skin, gastrointestinal tract, kidneys, peripheral and central nervous systems, liver, lymph nodes, eyes, pancreas, and endocrine systems; such as pituitary inflammation, colitis, hepatitis, pneumonia, rash and rheumatism.

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 a sterile solid composition (e.g., a lyophilized composition) which may be dissolved or suspended in a sterile injection medium at the point of use. They may contain, for example, suspending or dispersing agents known in the art.

The dosage and dosing regimen of any of the chimeric proteins disclosed herein can depend on various parameters, including, but not limited to, the disease being treated, the general health of the subject, and the discretion of the administering physician.

In one aspect, the present disclosure relates to a method for treating cancer in a human subject in need thereof, the method comprising the step of administering to the human subject an effective amount of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein a (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L), wherein the administering step comprises two-stage administration. In embodiments, the first and second phases each independently comprise a dosing frequency of about twice weekly to about once every two months. In embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. In embodiments, the chimeric protein exhibits a linear dose response within the following dose ranges: such as from about 0.3mg/kg to about 3mg/kg, or from about 0.5mg/kg to about 3mg/kg, or from about 0.7mg/kg to about 3mg/kg, or from about 1mg/kg to about 3mg/kg, or from about 1.5mg/kg to about 3mg/kg, or from about 2mg/kg to about 3mg/kg, or from about 2.5mg/kg to about 3mg/kg, or from about 0.3mg/kg to about 2.5mg/kg, or from about 0.3mg/kg to about 2mg/kg, or from about 0.3mg/kg to about 1.5mg/kg, or from about 0.3mg/kg to about 1.0mg/kg, or from about 0.3mg/kg to about 0.5mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped dose response.

In embodiments, the frequency of administration in the first phase is the same as the frequency of administration in the second phase. In other embodiments, the frequency of administration of the first phase is different from the frequency of administration of the second phase.

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

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

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

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

Additionally or alternatively, in embodiments, the effective amounts of the first, second, and third stages each independently comprise about 0.01mg/kg to about 10mg/ml. In embodiments, the effective amounts of the first, second, and third phases are each independently selected from about 0.03mg/kg, about 0.1mg/kg, about 0.3mg/kg, about 1mg/kg, about 3mg/kg, about 10mg/kg, and include any two of the foregoing values and/or any range between any two of the foregoing values. In embodiments, the effective amounts of the first, second, and third phases are each independently selected from about 0.01mg/kg to about 0.1mg/kg, about 0.03mg/kg to about 0.3mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.3mg/kg to about 3mg/kg, and about 1mg/kg to about 10mg/kg. In embodiments, the effective amounts of the first, second, and third stages are the same. In embodiments, the effective amounts of the first, second, and third stages are different. In embodiments, the effective amount of the first stage is greater than the effective amount of the second stage. In embodiments, the effective amount of the first stage is about 0.03mg/kg to about 0.3mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.3mg/kg to about 3mg/kg, or about 1mg/kg to about 10mg/kg; and the effective amount of the second stage is about 0.01mg/kg to about 0.1mg/kg, about 0.03mg/kg to about 0.3mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.3mg/kg to about 3mg/kg, or about 1mg/kg to about 10mg/kg.

In embodiments, the chimeric proteins disclosed herein are human CD172a (sirpa) -Fc-CD40L chimeric proteins.

In one aspect, the present disclosure relates to a method for treating cancer in a human subject in need thereof, the method comprising the step of administering to the human subject an effective amount of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L), wherein the administering step comprises a first cycle, a second cycle, and a third cycle. In embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. In embodiments, the first period, the second period, and the third period each independently comprise a dosing frequency of about twice weekly to about once every two months. In 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 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 embodiments, the dosing frequency of the first period is selected from about once every three days, about twice a week, about once every 10 days, about twice a 3 week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once a month, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, and about once every 2 months. In embodiments, the chimeric protein exhibits a linear dose response within the following dose ranges: such as from about 0.3mg/kg to about 3mg/kg, or from about 0.5mg/kg to about 3mg/kg, or from about 0.7mg/kg to about 3mg/kg, or from about 1mg/kg to about 3mg/kg, or from about 1.5mg/kg to about 3mg/kg, or from about 2mg/kg to about 3mg/kg, or from about 2.5mg/kg to about 3mg/kg, or from about 0.3mg/kg to about 2.5mg/kg, or from about 0.3mg/kg to about 2mg/kg, or from about 0.3mg/kg to about 1.5mg/kg, or from about 0.3mg/kg to about 1.0mg/kg, or from about 0.3mg/kg to about 0.5mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped dose response.

In embodiments, the dosing frequency of the first period is selected from about once every 3 days to about once every 10 days, about once every week to about once every 2 weeks, about once every 10 days to about once every 3 weeks, about once every 2 weeks to about once every 4 weeks, about once every 3 weeks to about once every 5 weeks, about once every 4 weeks to about once every 6 weeks, about once every 5 weeks to about once every 7 weeks, about once every 6 weeks to about once every 8 weeks, and about once every 6 weeks to about once every 2 months. In embodiments, the dosing frequency of the second period is selected from about once every three days, about twice a week, about once every 10 days, about twice a3 week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once a month, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, and about once every 2 months. In embodiments, the dosing frequency of the second period is selected from about once every 3 days to about once every 10 days, about once every week to about once every 2 weeks, about once every 10 days to about once every 3 weeks, about once every 2 weeks to about once every 4 weeks, about once every 3 weeks to about once every 5 weeks, about once every 4 weeks to about once every 6 weeks, about once every 5 weeks to about once every 7 weeks, about once every 6 weeks to about once every 8 weeks, and about once every 6 weeks to about once every 2 months. In embodiments, the dosing frequency of the third period is selected from about once every three days, about twice a week, about once every 10 days, about twice a3 week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once a month, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, and about once every 2 months. In embodiments, the dosing frequency of the third period is selected from about once every 3 days to about once every 10 days, about once every week to about once every 2 weeks, about once every 10 days to about once every 3 weeks, about once every 2 weeks to about once every 4 weeks, about once every 3 weeks to about once every 5 weeks, about once every 4 weeks to about once every 6 weeks, about once every 5 weeks to about once every 7 weeks, about once every 6 weeks to about once every 8 weeks, and about once every 6 weeks to about once every 2 months. In embodiments, the dosing frequency of the first cycle is selected from about once every 3 days to about once every 10 days, about once every week to about once every 2 weeks, about once every 10 days to about once every 3 weeks; and the frequency of the second period is selected from about once per week to about once every 2 weeks, about once every 10 days to about once every 3 weeks, about once every 2 weeks to about once every 4 weeks, about once every 3 weeks to about once every 5 weeks, about once every 4 weeks to about once every 6 weeks.

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

Additionally or alternatively, in embodiments, the effective amounts of the first period, the second period, and the third period each independently comprise about 0.01mg/kg to about 10mg/ml. In embodiments, the effective amounts of the first, second, and third periods are each independently selected from about 0.03mg/kg, about 0.1mg/kg, about 0.3mg/kg, about 1mg/kg, about 3mg/kg, about 10mg/kg, and include any two of the foregoing values and/or any range between any two of the foregoing values. In embodiments, the effective amounts of the first, second, and third cycles are each independently selected from about 0.01mg/kg to about 0.1mg/kg, about 0.03mg/kg to about 0.3mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.3mg/kg to about 3mg/kg, and about 1mg/kg to about 10mg/kg.

In embodiments, the effective amounts of the first period, the second period, and the third period are the same. In other embodiments, the effective amounts of the first period, the second period, and the third period are different. In embodiments, the effective amount of the first period is greater than the effective amount of the second period. In other embodiments, the effective amount of the first period is less than the effective amount of the second period. In other embodiments, the effective amount of the first period is the same as the effective amount of the second period.

In embodiments, the effective amount of the first period is about 0.03mg/kg to about 0.3mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.3mg/kg to about 3mg/kg, or about 1mg/kg to about 10mg/kg; and the effective amount of the second period is about 0.01mg/kg to about 0.1mg/kg, about 0.03mg/kg to about 0.3mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.3mg/kg to about 3mg/kg, or about 1mg/kg to about 10mg/kg.

In one aspect, the present disclosure relates to a method for treating cancer in a human subject in need thereof, the method comprising the step of administering to the human subject an effective amount of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein a (CD 172a (sirpa)), (b) is a linker contiguous with the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L), wherein the dosing regimen comprises dosing at a frequency ranging from about once every three days to about once every 2 months. In embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. In embodiments, the dosing regimen is selected from about once every three days, about twice a week, about once every 10 days, about twice a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once a month, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, and about once every 2 months. In embodiments, the dosing regimen is selected from about once every week, about once every 10 days, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every month, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, and about once every 2 months. In embodiments, the dosing regimen is about once every 2 weeks, about once every 3 weeks, or about once every 4 weeks.

In one aspect, the present disclosure relates to a method for treating cancer in a human subject in need thereof, the method comprising the step of administering to the human subject an effective amount of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising an extracellular domain of human signal-regulating protein a (CD 172a (sirpa)), and (b) is a linker contiguous with the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising an extracellular domain of human CD40 ligand (CD 40L), wherein the dosing regimen is selected from about once every 3 days to about once every 10 days, about once every week to about once every 2 weeks, about once every 10 days to about once every 3 weeks, about once every 2 weeks to about once every 4 weeks, about once every 3 weeks to about once every 5 weeks, about once every 4 weeks to about once every 6 weeks, about once every 5 weeks to about once every 7 weeks, about once every 6 weeks to about once every 8 weeks, and about once every 6 weeks to about every 2 months. In embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. In embodiments, the dosing regimen is from about once per week to about once every 2 weeks, from about once every 10 days to about once every 3 weeks, or from about once every 2 weeks to about once every 4 weeks. In embodiments, the chimeric protein exhibits a linear dose response within the following dose ranges: such as from about 0.3mg/kg to about 3mg/kg, or from about 0.5mg/kg to about 3mg/kg, or from about 0.7mg/kg to about 3mg/kg, or from about 1mg/kg to about 3mg/kg, or from about 1.5mg/kg to about 3mg/kg, or from about 2mg/kg to about 3mg/kg, or from about 2.5mg/kg to about 3mg/kg, or from about 0.3mg/kg to about 2.5mg/kg, or from about 0.3mg/kg to about 2mg/kg, or from about 0.3mg/kg to about 1.5mg/kg, or from about 0.3mg/kg to about 1.0mg/kg, or from about 0.3mg/kg to about 0.5mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped dose response.

In some embodiments of any aspect disclosed herein, the first domain is capable of binding to a CD172a (sirpa) ligand. In embodiments, the first domain comprises substantially all of the extracellular domain of CD172a (sirpa). In embodiments, the second domain is capable of binding to a CD40 receptor. In embodiments, the second domain comprises substantially all of the extracellular domain of CD 40L. In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG4, e.g., human IgG 4. In 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 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 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 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 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 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 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 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 an embodiment, the first domain comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO. 57.

In some embodiments of any aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect disclosed herein, the second domain comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO. 58. In 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 embodiments, the chimeric protein further comprises the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 7. In an embodiment, the chimeric protein further comprises the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7. In 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 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 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 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 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 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 embodiments, the chimeric protein comprises the same amino acid sequence as SEQ ID NO 59 or SEQ ID NO 61. In 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 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 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 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 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 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 embodiments, the chimeric protein comprises the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 61. In embodiments, the human subject has received standard therapy, is resistant to standard therapy, or is unsuitable for receiving standard therapy, and/or the cancer is not considered an approved therapy for standard care.

In one aspect, the present disclosure relates to a method of promoting lymphocyte migration from peripheral blood into secondary lymphoid organs (e.g., lymph nodes and spleen) in a human subject in need thereof, the method comprising the step of administering to the human subject an effective amount of a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD 40L).

In some embodiments of any aspect disclosed herein, the human subject is not undergoing concurrent chemotherapy, immunotherapy, biological or hormonal therapy.

In one aspect, the disclosure relates to chimeric proteins for use in the methods of any of the embodiments disclosed herein.

In one aspect, the disclosure relates to chimeric proteins comprising an amino acid sequence that is at least about 98% identical to SEQ ID NO 59 or SEQ ID NO 61. In 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 embodiments, the chimeric protein comprises the same amino acid sequence as SEQ ID NO 59 or SEQ ID NO 61.

The frequency of administration in the first stage and the frequency of administration in the second stage may be the same or different. In embodiments, the dosing frequency of the first stage and the dosing frequency of the second stage are each independently selected from about every three days, about twice a week, about once a week, about every 10 days, about twice a week, about every 2 weeks, about once a week 3, about once a week 4, about once a month, about once a week 5, about once a week 6, about once a week 7, about once a week 8, and about once a month 2. In embodiments, the dosing frequency of the first stage is selected from about once every 3 days to about once every 10 days, about once every week to about once every 2 weeks, about once every 10 days to about once every 3 weeks, about once every 2 weeks to about once every 4 weeks, about once every 3 weeks to about once every 5 weeks, about once every 4 weeks to about once every 6 weeks, about once every 5 weeks to about once every 7 weeks, about once every 6 weeks to about once every 8 weeks, and about once every 6 weeks to about once every 2 months.

In embodiments, the first stage and the second stage each independently last from about two days to about 12 months. For example, in embodiments, the first stage lasts from about 2 weeks to about 2 months; and the second phase lasts from about 2 weeks to about 12 months. In embodiments, the first stage lasts from about 2 weeks to about 1 month; and the second phase lasts from about 2 weeks to about 12 months. In embodiments, the first stage lasts from about 2 weeks to about 1 month; and the second phase lasts from about 4 weeks to about 12 months.

The effective amounts of the first stage, the second stage, and the third stage may be the same or different. In embodiments, the effective amounts of the first, second, and third stages each independently comprise about 0.01mg/kg to about 10mg/ml. In embodiments, the effective amount of the first stage is about 0.03mg/kg to about 0.3mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.3mg/kg to about 3mg/kg, or about 1mg/kg to about 10mg/kg; and the effective amount of the second stage is about 0.01mg/kg to about 0.1mg/kg, about 0.03mg/kg to about 0.3mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.3mg/kg to about 3mg/kg, or about 1mg/kg to about 10mg/kg. In embodiments, the chimeric proteins disclosed herein are human CD172a (sirpa) -Fc-CD40L chimeric proteins.

In embodiments, the human CD172a (sirpa) -Fc-CD40L chimeric protein is capable of providing sustained immunomodulatory effects.

In 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 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 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 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 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 embodiments, the linker comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG. In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG selected from IgG1 and IgG4. In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG1 or human IgG4. In embodiments, the linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG4. In embodiments, the hinge-CH 2-CH3 Fc domain is derived from human IgG4.

Additionally or alternatively, in embodiments, the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)) 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 an embodiment, the extracellular domain of human signal-regulating protein α (CD 172a (SIRPalpha)) comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO 57. In an embodiment, the extracellular domain of human signal-regulating protein α (CD 172a (SIRPalpha)) comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO 57. In an embodiment, the extracellular domain of human signal-regulating protein α (CD 172a (SIRPalpha)) comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO 57. In an embodiment, the extracellular domain of human signal-regulating protein α (CD 172a (SIRPalpha)) comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO 57. In an embodiment, the extracellular domain of human signal-regulating protein α (CD 172a (SIRPalpha)) comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 57.

Additionally or alternatively, in embodiments, the extracellular domain of human CD40 ligand (CD 40L) 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 an embodiment, the extracellular domain of human CD40 ligand (CD 40L) comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 58. In an embodiment, the extracellular domain of human CD40 ligand (CD 40L) comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO. 58. In an embodiment, the extracellular domain of human CD40 ligand (CD 40L) comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO. 58. In an embodiment, the extracellular domain of human CD40 ligand (CD 40L) comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO. 58. In an embodiment, the extracellular domain of human CD40 ligand (CD 40L) comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO. 58.

Additionally or alternatively, in embodiments, the human CD172a (SIRPalpha) -Fc-CD40L 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 embodiments, the human CD172a (SIRPalpha) -Fc-CD40L chimeric protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO 59 or SEQ ID NO 61. In embodiments, the human CD172a (SIRPalpha) -Fc-CD40L chimeric protein comprises an amino acid sequence that is at least 96% identical to SEQ ID NO 59 or SEQ ID NO 61. In embodiments, the human CD172a (SIRPalpha) -Fc-CD40L chimeric protein comprises an amino acid sequence that is at least 97% identical to SEQ ID NO 59 or SEQ ID NO 61. In embodiments, the human CD172a (SIRPalpha) -Fc-CD40L chimeric protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO 59 or SEQ ID NO 61. In embodiments, the human CD172a (SIRPalpha) -Fc-CD40L chimeric protein comprises an amino acid sequence that is at least 99% identical to SEQ ID NO 59 or SEQ ID NO 61. In embodiments, the human CD172a (SIRPalpha) -Fc-CD40L chimeric protein comprises the same amino acid sequence as SEQ ID NO 59 or SEQ ID NO 61.

In one aspect, the present disclosure relates to a method for treating cancer in a human subject, the method comprising: (i) Administering to the human subject a chimeric protein having the general structure: n-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises at least one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of a human CD40 ligand (CD 40L); and (ii) administering a second therapeutic agent. In embodiments, the chimeric protein is administered at a dose of between about 0.0001mg/kg and about 10mg/kg. In embodiments, the dosage of chimeric protein administered is at least about 0.3mg/kg, for example at least about 0.3mg/kg, or about 1.0mg/kg, or about 2mg/kg, or about 3, about 4mg/kg, or about 6mg/kg, or about 8mg/kg, or about 10mg/kg. In embodiments, the chimeric protein is administered at a dose of at least about 1mg/kg, for example at least about 1.0mg/kg, or about 2mg/kg, or about 3, about 4mg/kg, or about 6mg/kg, or about 8mg/kg, or about 10mg/kg. In embodiments, the chimeric protein exhibits a linear dose response within the following dose ranges: such as from about 0.3mg/kg to about 3mg/kg, or from about 0.5mg/kg to about 3mg/kg, or from about 0.7mg/kg to about 3mg/kg, or from about 1mg/kg to about 3mg/kg, or from about 1.5mg/kg to about 3mg/kg, or from about 2mg/kg to about 3mg/kg, or from about 2.5mg/kg to about 3mg/kg, or from about 0.3mg/kg to about 2.5mg/kg, or from about 0.3mg/kg to about 2mg/kg, or from about 0.3mg/kg to about 1.5mg/kg, or from about 0.3mg/kg to about 1.0mg/kg, or from about 0.3mg/kg to about 0.5mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped dose response.

In embodiments, administration of the chimeric protein results in a CD47 Receptor Occupancy (RO) on the white blood cells of at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% as compared to RO of a second subject and/or an external control prior to administration of the chimeric protein, without administration of the chimeric protein. In embodiments, administration of the chimeric protein results in a CD47 Receptor Occupancy (RO) on B cells of at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% as compared to RO of a second subject and/or an external control prior to administration of the chimeric protein, without administration of the chimeric protein. In embodiments, administration of the chimeric protein results in an increase in the amount or activity of one or more of IL-12, MCP-1, MIP-1β, MIP-1α, and MDC% as compared to RO of a second subject prior to administration of the chimeric protein, without administration of the chimeric protein, and/or an external control.

In one aspect, the present disclosure relates to a method for treating cancer in a human subject, the method comprising administering to a subject in need thereof: a chimeric protein having the general structure of N-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises at least one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of a human CD40 ligand (CD 40L); wherein: the subject is undergoing or has undergone treatment with a second therapeutic agent. In embodiments, the chimeric protein is administered at a dose of between about 0.0001mg/kg and about 10mg/kg. In embodiments, the dosage of chimeric protein administered is at least about 0.3mg/kg, for example at least about 0.3mg/kg, or about 1.0mg/kg, or about 2mg/kg, or about 3, about 4mg/kg, or about 6mg/kg, or about 8mg/kg, or about 10mg/kg. In embodiments, the chimeric protein is administered at a dose of at least about 1mg/kg, for example at least about 1.0mg/kg, or about 2mg/kg, or about 3, about 4mg/kg, or about 6mg/kg, or about 8mg/kg, or about 10mg/kg. In embodiments, the chimeric protein exhibits a linear dose response within the following dose ranges: such as from about 0.3mg/kg to about 3mg/kg, or from about 0.5mg/kg to about 3mg/kg, or from about 0.7mg/kg to about 3mg/kg, or from about 1mg/kg to about 3mg/kg, or from about 1.5mg/kg to about 3mg/kg, or from about 2mg/kg to about 3mg/kg, or from about 2.5mg/kg to about 3mg/kg, or from about 0.3mg/kg to about 2.5mg/kg, or from about 0.3mg/kg to about 2mg/kg, or from about 0.3mg/kg to about 1.5mg/kg, or from about 0.3mg/kg to about 1.0mg/kg, or from about 0.3mg/kg to about 0.5mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped dose response.

In one aspect, the present disclosure relates to a method for treating cancer in a human subject, the method comprising administering to a subject in need thereof a second anti-cancer therapeutic agent, wherein the subject is undergoing or has undergone treatment with a chimeric protein having the general structure of N-terminal- (a) - (b) - (C) -C-terminal, wherein: (a) is a first domain comprising the extracellular domain of human signal-regulating protein α (CD 172a (sirpa)), (b) is a linker adjoining the first domain and the second domain, wherein the linker comprises at least one cysteine residue capable of forming a disulfide bond and/or comprises a hinge-CH 2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of a human CD40 ligand (CD 40L). In embodiments, the chimeric protein is administered at a dose of between about 0.0001mg/kg and about 10 mg/kg. In embodiments, the chimeric protein exhibits a linear dose response within the following dose ranges: such as from about 0.3mg/kg to about 3mg/kg, or from about 0.5mg/kg to about 3mg/kg, or from about 0.7mg/kg to about 3mg/kg, or from about 1mg/kg to about 3mg/kg, or from about 1.5mg/kg to about 3mg/kg, or from about 2mg/kg to about 3mg/kg, or from about 2.5mg/kg to about 3mg/kg, or from about 0.3mg/kg to about 2.5mg/kg, or from about 0.3mg/kg to about 2mg/kg, or from about 0.3mg/kg to about 1.5mg/kg, or from about 0.3mg/kg to about 1.0mg/kg, or from about 0.3mg/kg to about 0.5mg/kg. In embodiments, the chimeric protein does not exhibit a bell-shaped dose response.

In embodiments, the chimeric protein is administered prior to the second therapeutic agent. In embodiments, the second therapeutic agent is administered prior to the chimeric protein. In embodiments, the second therapeutic agent and the chimeric protein are administered substantially together.

In embodiments, the second therapeutic agent is selected from the group consisting of antibodies and chemotherapeutic agents. In embodiments, the antibody is capable of Antibody Dependent Cellular Cytotoxicity (ADCC). In embodiments, the antibody is selected from the group consisting of cetuximab, rituximab, obrituximab, hul4.18k322a, hul 3F8, denotuximab, and trastuzumab. In embodiments, the antibody is capable of antibody-dependent cellular phagocytosis (ADCP). In embodiments, the antibody is selected from cetuximab, darimumab, rituximab, and trastuzumab. In embodiments, the antibody is capable of binding to a molecule selected from the group consisting of carcinoembryonic antigen (CEA), EGFR, HER-2, epithelial cell adhesion molecule (EpCAM) and human epithelial mucin-1, CD20, CD30, CD38, CD40 and CD 52. In embodiments, the antibody is capable of binding EGFR. In embodiments, the antibody is selected from Mab a13, AMG595, cetuximab (Erbitux, C225), panitumumab (ABX-EGF, vectibinx), dituximab (ABT 806), dituximab, ma Foduo statin, degree Li Getuo Mab (MEHD 7945A, RG 7597), furyimumab (Sym 004), GC1118, infliximab (GA 201), matuzumab (EMD 72000), nesuximab (Portrazza), nituzumab (h-R3), anitumumab (vectibinx, ABX-EGF), zafiuzumab, humMR1, and totuuximab. In embodiments, the antibody is cetuximab.

In embodiments, the chemotherapeutic agent is an anthracycline. In embodiments, the anthracycline is selected from doxorubicin, daunorubicin, epirubicin, and idarubicin, and pharmaceutically acceptable salts, acids, or derivatives thereof. In embodiments, the chemotherapeutic agent is doxorubicin.

In embodiments, the dose of chimeric protein administered is at least about 0.0001mg/kg, for example between about 0.0001mg/kg and about 10.0 mg/kg. The chimeric protein may be administered in 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, or about 10.0 mg/kg), and the chimeric protein is administered in one or more subsequent administrations (e.g., 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, and about 10 mg/kg). In embodiments, the dosage of chimeric protein administered is at least about 0.3mg/kg, for example at least about 0.3mg/kg, or about 1.0mg/kg, or about 2mg/kg, or about 3, about 4mg/kg, or about 6mg/kg, or about 8mg/kg, or about 10mg/kg. In embodiments, the chimeric protein is administered at a dose of at least about 1mg/kg, for example at least about 1.0mg/kg, or about 2mg/kg, or about 3, about 4mg/kg, or about 6mg/kg, or about 8mg/kg, or about 10mg/kg. In embodiments, the initial dose is less than the dose of at least one of the subsequent administrations (e.g., the dose of each of the subsequent administrations), or the initial dose is the same as the dose of at least one of the subsequent administrations (e.g., the dose of each of the subsequent administrations). In embodiments, the initial dose and/or subsequent doses are maximum tolerated doses or less than maximum tolerated doses. In embodiments, the chimeric protein is administered at least about once a month, for example at least about twice a month, at least about three times a month, and at least about four times a month. In embodiments, the chimeric protein is administered first once a week for three weeks, then about once every three weeks or once every four weeks; or the chimeric protein may be administered first once a week for three weeks, then about twice a month (e.g., once a week) for three weeks, then about once every two weeks.

In embodiments, the cancer comprises advanced solid tumors (localized and/or metastatic) or lymphomas. In embodiments, the cancer is selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous Squamous Cell Carcinoma (CSCC), and head and neck Squamous Cell Carcinoma (SCCHN). In embodiments, the cancer comprises advanced solid tumors (localized and/or metastatic) or advanced lymphomas.

Examples

The examples herein are provided to illustrate the advantages and benefits of the present technology and to further assist one of ordinary skill in the art in preparing or using chimeric proteins of the present technology. The embodiments herein are also provided 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 technology, as defined by the appended claims. The examples may include or incorporate any of the variations, aspects or implementations of the techniques of the invention described above. The variations, aspects, or embodiments described above may further each include or incorporate any or all of the other variations, aspects, or embodiments of the technology.

Example 1: phase 1 clinical trial of SIRP alpha-Fc-CD 40L chimeric protein (SL-172154)

This first phase 1 dose escalation study of humans is currently evaluating SL-172154 as monotherapy in subjects with platinum-resistant ovarian cancer. The main goal of the study was to assess security; the maximum tolerated dose or maximum administered dose of SL-172154 was identified. The secondary objective of the study was to identify doses and schedules (i.e. recommended phase 2 dose [ RP2D ]), characterize PK and immunogenicity, and evaluate the anti-tumor activity of solid tumors according to recistv 1.1. The exploratory goals are: receptor occupancy of sirpa and CD40 on PBMCs was assessed and Pharmacodynamic (PD) effects in blood and tumors were studied.

The planned dose escalation was performed in half-log increments (fig. 1). At least 3 subjects were included at sequential Dose Levels (DL) and evaluated for Dose Limiting Toxicity (DLT) during the first treatment period. The subjects received Intravenous (IV) administration of SL-172154 according to schedule 1 or schedule 2 until disease progression, unacceptable toxicity occurred, or consent was withdrawn. Currently 10mg/kg are recruited.

The main inclusion criteria are:

locally advanced or metastatic ovarian cancer, primary peritoneal cancer or fallopian tube cancer.

Is refractory to one or more existing therapies and is not suitable for receiving platinum therapy. Subjects with homologous recombination-deficient positive disease must have previously received PARPi, whether or not bevacizumab is combined.

Age 18 years or older

ECOG behavior state 0 or 1

Measurable disease according to RECIST v1.1

The main exclusion criteria were:

primary platinum refractory as defined by progression during prior platinum therapy or within 1 month

Previous treatment with anti-CD 47 or anti-sirpa targeting agents or CD40 agonists.

Recorded medical history with autoimmune disease or active pneumonia

Concurrent use of systemic corticosteroids or other immunosuppressive drugs

Subject characteristics are shown in table 4:

TABLE 4 tumor characterization

15 Subjects with a median age of 67 years (range 33-79 years), who had previously received median 5-line systemic therapy (range 2-7), were treated with IV SL-172154 at 4 dose levels according to the 2 schedules. Of the 15 subjects 13 were at baseline ECOGPS at 1.6 subjects were treated with 0.1mg/kg (n=3) and 0.3mg/kg (n=3) according to schedule 1 (days 1, 8, 15, 29, once every 2 weeks). 9 subjects were treated at 0.3mg/kg (n=3), 1.0mg/kg (n=3) and 3.0mg/kg (n=3) according to schedule 2 (once a week). As shown in table 1, most subjects had primary ovarian cancer (n=9; 60%), FIGO stage IV (n=9; 60%), high grade disease (n=11; 73%), and serous cancer histology (n=11; 73%). Adverse events are listed in table 5.

Table 5. The most common AE in all treated subjects

No DLT was observed at doses ranging from 0.1mg/kg to 3.0mg/kg according to SL-172154 for either schedule. 15 subjects (100%) experienced Adverse Events (AE) at the time of treatment. The most common AEs (all causal relationships) occurring in > 3 subjects included fatigue, infusion-related reactions (IRR), nausea, diarrhea, loss of appetite, dehydration and itching (table 2). 14 subjects (93%) had treatment-related AE (TRAE). The most common TRAEs occurring in ≡2 subjects include IRR (n=8; 53%), fatigue (n=7; 47%), nausea (n=4; 27%), loss of appetite (n=3; 20%), chills (n=2; 13%), diarrhea (n=2; 13%) and dyspnea (n=2, 13%). No G3 TRAE was reported. 8 subjects (53%) had infusion-related responses (IRR) when dosed at 0.3mg/kg (n=3), 1.0mg/kg (n=2) and 3.0mg/kg (n=3); 1 IRR event was G3 and was considered secondary to iron infusion, 14 IRR severity was G2, and 2 IRR severity was G1.IRR may be controlled by precursor administration without preventing intravenous administration from being completed or causing SL-172154 to be disabled. 13 subjects (87%) had been deactivated with SL-172154;12 were discontinued due to radiological or clinical progression and 1 subject opted to stop treatment.

Figure 3 shows tumor response and treatment duration. Efficacy of 14 post-baseline scans the best response of the subjects can be assessed:

–SD，n＝4

–PD，n＝9

ne, n=1 (subject has SD, but does not meet the SD minimum time interval specified by the regimen)

1 Subject (3 mg/kg) did not reach disease assessment in the first treatment at week 8

Pharmacokinetics:

Maximum concentration (C _max) and area under the curve, extrapolation of time 0 to infinity (AUC _inf) increases disproportionately with increasing dose.

Clearance decreases with increasing dose.

PK presented biphasic at 1.0 and 3.0mg/kg dose.

At the doses studied, the elimination phase of the curve has not been fully characterized.

Figures 4A to 4D show reproducible increases in serum cytokines following repeated administration of SL-172154. Blood was collected from the subjects at the indicated times to prepare plasma. Cytokine levels were determined using the multiplex ECLELISA method and the selected dose level of cytokine was shown. The pro-inflammatory chemokines CCL2 (MCP-1) (fig. 4A), CCL4 (MIP-1β) (fig. 4B), CCL3 (MIP-1α) (fig. 4C) and CCL22 (MDC) (fig. 4D) exhibited dose-dependent increases in plasma levels in all post-infusion measurements. The duration and magnitude of these increases are equal throughout all infusion time intervals.

Figures 7A-7C show SL-172154 stimulation of dose-dependent B cell edge set and activation. B cells represent a large number of circulating immune cells that express high levels of CD 40. Fluorescence Activated Cell Sorting (FACS) sets were designed to query CD40 receptor occupancy, activation and maturation status. In C1D 1, almost all (about 80%) of the cd40+ B cells were pooled or withdrawn from circulation within one hour after infusion. Figure 7A shows that median frequency of edge set cells increases in a dose-dependent manner (horizontal bars). Receptor engagement was about 100% at all dose levels (data not shown). Fig. 7B shows that the median B cell frequency was restored to the pre-infusion level by the next infusion, maintaining the cycle pattern of egress and return for each infusion cycle. Figure 7C shows that returning B cells showed an increase in co-stimulatory marker CD86 as well as maturation marker CD95, indicating that SL-172154 can induce phenotypic changes. Similarly, cd14+ monocytes collect or exit the cycle within one hour after infusion; the pre-infusion level is restored by the time of the next infusion, maintaining the outbound and inbound cycle patterns for each infusion cycle. The observed edge set pattern was driven mainly by cd86+ classical and non-classical monocytes (data not shown).

FIGS. 8A and 8B show different distributions of TNF alpha and interleukin-6 (IL-6) relative to CD40 mAb. FIG. 8A shows the induction of TNFα at different doses of CP-870,893 (left panel) or SL-172154 (right panel). FIG. 8B shows induction of IL-6 at different doses of CP-870,893 (left panel) or SL-172154 (right panel). CP-870,893 data is from Vonderheide et al JClin Oncol 25:876-883 (2007). These results demonstrate, inter alia, that SL-172154 does not induce TNFα and interleukin-6 (IL-6) relative to CD40 mAb. Dose Limiting Toxicity (DLT) is due to Cytokine Release Syndrome (CRS), which limits dose escalation of CD40 agonist mAb. Interestingly, no significant increase in TNFα and IL-6 was observed in the case of SL-172154. Thus, the SL-172154 current dose is administered at 10 times the CP-870,893 dose.

FIGS. 10A and 10B show that SL-172154 induces an adaptive immune response in the Tumor Microenvironment (TME). FIG. 9A shows CD8+ cells, granzyme B+ cells, CD68+ cells and Ki67+ cells in biopsy samples from patient A before and after administration of SL-172154. Cd8+ cells, granzyme b+ cells, cd68+ cells and ki67+ cells were increased in post-treatment biopsy samples compared to pre-treatment biopsy samples. Fig. 9B is a graph comparing Tumor Proportion Score (TPS) and Combined Positive Score (CPS). The induction of PD-L1 on immune cells is a result of cd8+ T cell activation.

These results indicate that SL-172154 is well tolerated with no evidence of DLT or anemia, thrombocytopenia, liver dysfunction, cytokine release syndrome or pneumonia. The dose was continued to be increased by 10 mg/kg. Preliminary PK parameters for SL-172154 indicate drug treatment mediated by receptor binding targets. At the dose studied, a high receptor occupancy of SL-172154 was observed on CD47+ leukocytes with minimal binding to RBC. SL-172154 binding to CD40+ B cells and monocytes resulted in rapid activation and marginality following infusion. Periodic increases in congenital and adaptive serum cytokines are consistent with CD40 receptor engagement and activation. IL-6 or TNF alpha did not increase, nor was there evidence of bell-shaped dose response. SL-172154 was well tolerated at doses saturating both CD40 and CD47, with evidence that mid-target PD activity had not reached a plateau, warranting further dose escalation. These results further demonstrate, without being bound by theory, that SL-172154 induces both innate and adaptive immune responses in the Tumor Microenvironment (TME).

Example 2: increased CD80 expression in tumors following administration of SIRPalpha-Fc-CD 40L chimeric protein (SL-172154)

SL-172154 is intratumorally administered to ovarian cancer patients. Tumor biopsy samples were obtained from the patient before and after SL-172154 administration. Biopsy samples were analyzed for CD80 expression in tumors. As shown in FIG. 12, the samples obtained after administration of SL-172154 showed an increase in the abundance of CD80+ cells and/or CD80 expression in tumors after administration of SL-172154, as compared to the biopsy samples obtained prior to administration of SL-172154.

Incorporated by reference

All patents and publications mentioned herein are hereby incorporated by reference in their entirety.

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 disclosure is not entitled to antedate such disclosure by virtue of prior application.

As used herein, all headings are for organization only and are not intended to limit the disclosure in any way. The contents of any individual portion may be equally applicable to all portions.

Equivalent scheme

While the application has been disclosed in connection with specific embodiments thereof, it will be understood that the application is capable of additional modifications and is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application 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, many equivalents to the specific embodiments disclosed herein. Such equivalents are intended to be encompassed by the scope of the appended claims.

Claims

1. A method for treating cancer in a human subject, the method comprising:

(i) Administering to the human subject a first dose of a chimeric protein having the general structure:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

(a) Is a first domain comprising the extracellular domain of human signal-regulating protein alpha (CD 172a (SIRPa)),

(B) Is a linker linking the first domain and the second domain, wherein the linker comprises a hinge-CH 2-CH3 Fc domain, and

(C) Is a second domain comprising the extracellular domain of the human CD40 ligand (CD 40L),

Wherein the background level of cells in a pre-dose biological sample obtained from the subject prior to administration of the first dose has been measured, wherein the cells are selected from one or more of cd80+ cells, cd8+ cells, granzyme b+ cells, cd68+ cells, ki67+ cells, and PD-l1+ immune cells;

(ii) Administering a second dose of said chimeric protein to said human subject if the post-administration level of said cells is greater than said background level of said cells,

Wherein the second dose is administered at least about 48 hours after administration of the first dose.

2. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid or lavage, aspirate, swabs, bone marrow specimens, biopsy specimens, and surgical specimens.

3. The method of claim 2, wherein the biological sample is a biopsy sample or a surgical specimen, optionally wherein the biological sample is a tumor biopsy sample or a tumor surgical specimen.

4. The method of claim 3, wherein the tumor biopsy sample or the tumor surgical specimen is derived from a tumor selected from the group consisting of ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous Squamous Cell Carcinoma (CSCC), and head and neck Squamous Cell Carcinoma (SCCHN).

5. The method of any one of claims 1 to 4, wherein the level of the cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

6. The method of claim 5, wherein the level of the cells is measured by contacting the sample with an agent that specifically binds to one or more of CD80, CD8, granzyme B, CD, ki67, and PD-L1.

7. The method of claim 6, wherein the agent that specifically binds to the one or more molecules is an antibody or fragment thereof.

8. The method of claim 7, wherein the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof.

9. The method of claim 5, wherein the level of the cells is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding one or more of CD80, CD8, granzyme B, CD, ki67, and PD-L1.

10. The method of claim 9, wherein the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

11. A method for treating cancer in a human subject, the method comprising:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

Wherein the background level and/or activity of B cells and/or cd40+ cells in a first biological sample obtained from the subject prior to administration of the first dose has been measured;

Wherein the post-dose N hours level and/or activity of B cells and/or cd40+ cells in a second biological sample obtained from the subject after administration of the first dose has been measured, wherein N is a number between 1 and 24, wherein the post-dose N hours level and/or activity of B cells and/or cd40+ cells is less than the background level and/or activity of B cells and/or cd40+ cells;

Wherein the post-administration M-day level and/or activity of B cells and/or cd40+ cells in a third biological sample obtained from the subject after administration of the first dose has been measured, wherein M is a number between 1 and 28; and

(Ii) Administering a second dose of said chimeric protein to said human subject if said post-administration M day level and/or activity of B cells and/or CD40+ cells is at least about 50% greater than said post-administration N hour level and/or activity of B cells and/or CD40+ cells,

12. The method of claim 11, wherein:

N is less than 12, or less than 8, or less than 6, or less than 4, or less than 3, or less than 2, or less than 1; and/or

M is less than 21, or less than 14, or less than 12, or less than 10, or less than 8, or less than 6, or less than 4, or less than 2.

13. The method of claim 11, wherein:

n is less than 12, or less than 8, or less than 6, or less than 4, or less than 3, or less than 2, or less than 1; and

14. The method of any one of claims 11 to 13, wherein the post-administration N hours level and/or activity of B cells and/or cd40+ cells is greater than the background level and/or activity of B cells and/or cd40+ cells.

15. The method of any one of claims 11 to 13, wherein the level and/or activity of B cells and/or cd40+ cells N hours after the administration is less than the background level and/or activity of B cells and/or cd40+ cells.

16. The method of any one of claims 11 to 13, wherein the post-administration N hours level and/or activity of B cells and/or cd40+ cells is within about 10%, or about 20%, or about 30%, or about 40% of the background level and/or activity of B cells and/or cd40+ cells.

17. The method of claim 11, wherein the first biological sample, the second biological sample, and the third biological sample are independently selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue, or fine needle biopsy samples, cell-containing bodily fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, rinse or lavage, aspirate, scrapings, bone marrow specimens, biopsy specimens, and surgical specimens.

18. The method of claim 17, wherein the first biological sample, the second biological sample, and the third biological sample are blood.

19. The method of any one of claims 11 to 18, wherein the level and/or activity of B cells and/or cd40+ cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or a combination thereof.

20. The method of claim 19, wherein the level and/or activity of B cells and/or cd40+ cells is measured by contacting the sample with an agent that specifically binds to CD40 and/or a B cell marker.

21. The method of claim 20, wherein the B cell marker is selected from the group consisting of CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD, CD95, igM, igD, and CD40.

22. The method of claim 20 or claim 21, wherein the agent that specifically binds to the one or more molecules is an antibody or fragment thereof.

23. The method of claim 22, wherein the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof.

24. The method of claim 19, wherein the level and/or activity of B cells and/or cd40+ cells is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding CD40 and/or a B cell marker.

25. The method of claim 24, wherein the B cell marker is selected from the group consisting of CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD, CD95, igM, igD, and CD40.

26. The method of claim 24 or claim 25, wherein the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

27. A method for treating cancer in a human subject, the method comprising:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

Wherein the background amount and/or activity of a cytokine selected from the group consisting of CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1 β, MIP-1 α and MDC in a first biological sample obtained from the subject prior to administration of the first dose has been measured, and

Wherein the amount and/or activity of the cytokine in a second biological sample obtained from the subject after administration of the first dose has been measured N hours after administration, wherein N is a number between 1 and 24, wherein the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine;

Wherein the amount and/or activity of the cytokine in a third biological sample obtained from the subject after administration of the first dose has been measured M days after administration, wherein M is a number between 1 and 28; and

(Ii) Administering a second dose of said chimeric protein to said human subject if said amount and/or activity of said cytokine is at least about 30% less than said amount and/or activity of said cytokine N hours after administration,

28. The method of claim 27, wherein:

29. The method of claim 27, wherein:

30. The method of any one of claims 27 to 29, wherein the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine.

31. The method of any one of claims 27 to 29, wherein the amount and/or activity of the cytokine N hours after administration is less than the background amount and/or activity of the cytokine.

32. The method of any one of claims 27 to 29, wherein the amount and/or activity of the cytokine N hours after administration is within about 10%, or about 20%, or about 30%, or about 40% of the background amount and/or activity of the cytokine.

33. The method of claim 27, wherein the first biological sample, the second biological sample, and the third biological sample are independently selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue, or fine needle biopsy samples, cell-containing bodily fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, rinse or lavage, aspirate, scrapings, bone marrow specimens, biopsy specimens, and surgical specimens.

34. The method of claim 33, wherein the first biological sample, the second biological sample, and the third biological sample are blood.

35. The method of any one of claims 27 to 34, wherein the amount and/or activity of the cytokine is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

36. The method of claim 35, wherein the amount and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds to the cytokine.

37. The method of claim 36, wherein the agent that specifically binds to the one or more molecules is an antibody or fragment thereof, optionally wherein the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or fragment thereof.

38. The method of claim 35, wherein the amount and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding the cytokine.

39. The method of claim 38, wherein the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

40. The method of any one of claims 1 to 39, wherein the second dose is administered at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 14 days, or at least about 21 days, or at least about 28 days after administration of the first dose.

41. A method for assessing the efficacy of a cancer treatment in a subject in need thereof, wherein the subject has cancer, the method comprising:

(i) Obtaining a biological sample from the subject that has received a first dose of chimeric protein;

Wherein the chimeric protein has the general structure:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

(C) A second domain comprising the extracellular domain of a human CD40 ligand (CD 40L);

Wherein the background level and/or activity of cells in a biological sample obtained from the subject prior to administration of the first dose has been measured, wherein the cells are selected from one or more of cd80+ cells, cd8+ cells, granzyme b+ cells, cd68+ cells, ki67+ cells and PD-l1+ immune cells;

(ii) Determining the post-administration level and/or activity of said cells in said biological sample; and

(Iii) If the post-administration level and/or activity of the cell is greater than the background level and/or activity of the cell, the chimeric protein is determined to be effective.

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

Wherein the chimeric protein has the general structure:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

(Iii) If the post-administration level and/or activity of the cell is greater than the background level and/or activity of the cell, the subject is selected for treatment with the chimeric protein.

43. The method of claim 41 or claim 42, wherein the biological sample is selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, fecal matter, lymph fluid, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid or lavage, aspirate, scraping, bone marrow specimen, biopsy specimen, and surgical specimen.

44. The method of claim 43, wherein the biological sample is a biopsy or surgical specimen.

45. The method of claim 44, wherein the biological sample is a tumor biopsy or a tumor surgical specimen.

46. The method of claim 45, wherein the tumor biopsy or the tumor surgical specimen is derived from a tumor selected from the group consisting of ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous Squamous Cell Carcinoma (CSCC), and head and neck Squamous Cell Carcinoma (SCCHN).

47. The method of any one of claims 41-46, wherein the level of the cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

48. The method of claim 47, wherein the level of the cells is measured by contacting the sample with an agent that specifically binds to one or more molecules selected from the group consisting of CD80, CD8, granzyme B, CD, ki 67, and PD-L1.

49. The method of claim 48, wherein the agent that specifically binds to the one or more molecules is an antibody or fragment thereof.

50. The method of claim 49, wherein the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof.

51. The method of claim 47, wherein the level of the cells is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding one or more of CD80, CD8, granzyme B, CD, ki 67, and PD-L1.

52. The method of claim 51, wherein the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

53. A method for assessing the efficacy of a cancer treatment in a subject in need thereof, wherein the subject has cancer, the method comprising:

(i) Obtaining a first biological sample obtained from the subject that has received a first dose of chimeric protein;

Wherein the chimeric protein has the general structure:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

(ii) Determining the background level and/or activity of B cells and/or cd40+ cells in the first biological sample;

(iii) Obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24, and determining the level and/or activity of B cells and/or cd40+ cells in the second biological sample N hours after administration;

(iv) Obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28, and determining the post-administration M-day level and/or activity of B cells and/or cd40+ cells in the third biological sample; and

(V) The chimeric protein is determined to be effective if the post-administration N-hour level and/or activity of B cells and/or cd40+ cells is less than the background level and/or activity of B cells and/or cd40+ cells and/or if the post-administration M-day level and/or activity of B cells and/or cd40+ cells is at least about 50% greater than the post-administration N-hour level and/or activity of B cells and/or cd40+ cells.

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

(i) Obtaining a biological sample obtained from the subject that has received the first dose of chimeric protein;

Wherein the chimeric protein has the general structure:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

(V) Selecting the subject for treatment with the chimeric protein if the post-dose N-hour level and/or activity of B cells and/or cd40+ cells is less than the background level and/or activity of B cells and/or cd40+ cells and/or if the post-dose M-day level and/or activity of B cells and/or cd40+ cells is at least about 50% greater than the post-dose N-hour level and/or activity of B cells and/or cd40+ cells.

55. The method of claim 53 or claim 54, wherein:

56. The method of claim 53 or claim 54, wherein:

57. The method of any one of claims 53-56, wherein the level and/or activity of B cells and/or cd40+ cells N hours after said administration is greater than the background level and/or activity of B cells and/or cd40+ cells.

58. The method of any one of claims 53-56, wherein the level and/or activity of B cells and/or cd40+ cells N hours after said administration is less than the background level and/or activity of B cells and/or cd40+ cells.

59. The method of any one of claims 53-56, wherein the level and/or activity of B cells and/or cd40+ cells N hours after said administration is within about 10%, or about 20%, or about 30%, or about 40% of the background level and/or activity of B cells and/or cd40+ cells.

60. The method of claim 53 or claim 54, wherein the first biological sample, the second biological sample, and the third biological sample are independently selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid or lavage, aspirate, scrape, bone marrow specimen, biopsy specimen, and surgical specimen.

61. The method of claim 60, wherein the first biological sample, the second biological sample, and the third biological sample are blood.

62. The method of any one of claims 53 to 61, wherein the level and/or activity of B cells and/or cd40+ cells is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS) or a combination thereof.

63. The method of claim 62, wherein the level and/or activity of B cells and/or cd40+ cells is measured by contacting the sample with an agent that specifically binds to CD40 and/or a B cell marker.

64. The method of claim 63, wherein the B cell marker is selected from the group consisting of CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD, CD95, igM, igD, and CD40.

65. The method of claim 63 or claim 64, wherein the agent that specifically binds to the one or more molecules is an antibody or fragment thereof.

66. The method of claim 65, wherein the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof.

67. The method of claim 62, wherein the level and/or activity of B cells and/or cd40+ cells is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding CD40 and/or a B cell marker.

68. The method of claim 67, wherein the B cell marker is selected from the group consisting of CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD, CD95, igM, igD, and CD40.

69. The method of claim 67 or claim 68, wherein said agent that specifically binds to one or more of said nucleic acids is a nucleic acid primer or probe.

70. A method for assessing the efficacy of a cancer treatment in a subject in need thereof, wherein the subject has cancer, the method comprising:

Wherein the chimeric protein has the general structure:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

(ii) Determining the background amount and/or activity of a cytokine selected from the group consisting of CCL2, CXCL9, CXCL10, ifnα, IL15, IL23, IL-12, MCP-1, MIP-1 β, MIP-1 α, and MDC in the first biological sample;

(iii) Obtaining a second biological sample obtained from the subject N hours after administration, wherein N is a number between 1 and 24, and determining the amount and/or activity of the cytokine in the second biological sample N hours after administration;

(iv) Obtaining a third biological sample obtained from the subject M days after administration, wherein M is a number between 1 and 28, and determining the amount and/or activity of the cytokine in the third biological sample M days after administration; and

(V) The chimeric protein is determined to be effective if the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine and/or if the amount and/or activity of the cytokine M days after administration is at least about 50% less than the amount and/or activity of the cytokine N hours after administration.

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

Wherein the chimeric protein has the general structure:

n-terminal- (a) - (b) - (C) -C-terminal,

Wherein:

(V) Selecting the subject for treatment with the chimeric protein if the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine and/or if the amount and/or activity of the cytokine M days after administration is at least about 50% less than the amount and/or activity of the cytokine N hours after administration.

72. The method of claim 70 or claim 71, wherein:

73. The method of claim 70 or claim 71, wherein:

74. The method of any one of claims 70-73, wherein the amount and/or activity of the cytokine N hours after administration is greater than the background amount and/or activity of the cytokine.

75. The method of any one of claims 70-73, wherein the amount and/or activity of the cytokine N hours after administration is less than the background amount and/or activity of the cytokine.

76. The method of any one of claims 70-73, wherein the amount and/or activity of the cytokine N hours after administration is within about 10%, or about 20%, or about 30%, or about 40% of the background amount and/or activity of the cytokine.

77. The method of claim 70 or claim 71, wherein the first biological sample, the second biological sample, and the third biological sample are independently selected from the group consisting of blood, plasma, serum, blood cells, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid or lavage, aspirate, scrape, bone marrow specimen, biopsy specimen, and surgical specimen.

78. The method of claim 77, wherein said first biological sample, said second biological sample, and said third biological sample are blood.

79. The method of any one of claims 70-78, wherein the amount and/or activity of the cytokine is measured by RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

80. The method of claim 79, wherein the amount and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds to the cytokine.

81. The method of claim 80, wherein the agent that specifically binds to the one or more molecules is an antibody or fragment thereof.

82. The method of claim 81, wherein the antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, or a fragment thereof.

83. The method of claim 79, wherein the amount and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding the cytokine.

84. The method of claim 83, wherein the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

85. The method of any one of claims 1-84, wherein the first dose of the chimeric protein is in the range of about 0.03mg/kg to 10 mg/kg.

86. The method of claim 85, wherein the first dose is about 0.003, or about 0.01, or about 0.03, or about 0.1, or about 0.3, or about 1, or about 2, or about 3, or about 4, or about 6, or about 8, or about 10mg/kg.

87. The method of any one of claims 41-86, further comprising administering a second dose of the chimeric protein.

88. The method of claim 87, wherein the second dose is administered at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 14 days, or at least about 21 days, or at least about 28 days after administration of the first dose.

89. The method of claim 87 or claim 88, wherein the second dose of the chimeric protein is in the range of about 0.03mg/kg to 10 mg/kg.

90. The method of any one of claims 87 to 89, wherein the second dose is about 0.003, or about 0.01, or about 0.03, or about 0.1, or about 0.3, or about 1, or about 2, or about 3, or about 4, or about 6, or about 8, or about 10mg/kg.

91. The method of any one of claims 1-90, wherein the first domain is capable of binding a CD172a (sirpa) ligand.

92. The method of any one of claims 1-91, wherein the first domain comprises substantially all of the extracellular domain of CD172a (sirpa).

93. The method of any one of claims 1-92, wherein the second domain is capable of binding to a CD40 receptor.

94. The method of any one of claims 1-93, wherein the second domain comprises substantially all of the extracellular domain of CD 40L.

95. The method of any one of claims 1-94, wherein the linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG 4.

96. The method of claim 95, wherein the linker comprises a hinge-CH 2-CH3 Fc domain derived from human IgG 4.

97. The method of any one of claims 1 to 96, wherein the linker comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID No. 1, SEQ ID No. 2, or SEQ ID No. 3.

98. The method of any one of claims 1 to 97, wherein the first domain comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID NO: 57.

99. The method of any one of claims 1 to 98, wherein the second domain comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID No. 58.

100. The method of any one of claims 1 to 99, 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 about 90%, 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% identical to the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3.

101. The method of any one of claims 1 to 100, wherein the chimeric protein further comprises the amino acid sequence of SEQ ID No. 5 or SEQ ID No. 7.

102. The method of any one of claims 1-101, wherein the chimeric protein further comprises the amino acid sequences of SEQ ID No. 5 and SEQ ID No. 7.

103. The method of any one of claims 1 to 102, wherein the chimeric protein comprises an amino acid sequence that is at least about 90%, 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% identical to the amino acid sequence of SEQ ID No. 59 or SEQ ID No. 61.

104. The method of claim 103, wherein the chimeric protein comprises an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID No. 59 or SEQ ID No. 61.

105. The method of claim 103 or claim 104, wherein the chimeric protein comprises 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8 amino acid mutations relative to the amino acid sequence of SEQ ID No. 59 or SEQ ID No. 61.

106. The method of any one of claims 1-105, wherein the human subject has or is suspected of having an advanced solid tumor or lymphoma.

107. The method of any one of claims 1-106, wherein the human subject has or is suspected of having a cancer selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous Squamous Cell Carcinoma (CSCC), and head and neck Squamous Cell Carcinoma (SCCHN).

108. The method of any one of claims 1-107, wherein the human subject has failed one or more platinum-based therapies.

109. The method of claim 108, wherein the human subject is not suitable for receiving further platinum therapy.

110. The method of any one of claims 1-108, wherein the human subject is not suitable for receiving platinum therapy.

111. The method of any one of claims 1-100, wherein the human subject has not received concurrent chemotherapy, immunotherapy, biological or hormonal therapy, and/or wherein the human subject has received standard therapy, is resistant to standard therapy, or is not suitable to receive standard therapy, and/or the cancer has not been considered as an approved therapy for standard care.