KR20240118207A - SIRP1A- and CD40L-based chimeric proteins - Google Patents

Abstract

The present disclosure relates, in part, to compositions and methods comprising chimeric proteins that find use in the treatment of disease, such as immunotherapy for cancer.

Description

SIRP1A- and CD40L-based chimeric proteins

Technology field

The present technology particularly relates to chimeric proteins that find use in the treatment of vaginal diseases, including dosing regimens comprising dosing regimens comprising dosing, biphasic dosing or three cycles, such as immunotherapy for cancer. It relates to compositions and methods.

preference

This application relates to U.S. Provisional Patent Application No. 63/278,567, filed on November 12, 2021; and U.S. Provisional Patent Application No. 63/371,083, filed on August 11, 2022, the contents of each of which are incorporated herein by reference in their entirety.

Sequence Listing

This application includes a sequence listing submitted in XML format via EFS-Web. The content of the XML copy file name "SHK-055PC_116981-5055_Sequence_Listing" was created on November 9, 2022, has a capacity of 82,547 bytes, and the content is incorporated herein by reference in its entirety.

The field of cancer immunotherapy has grown tremendously over the past few years. This has largely been driven by the clinical efficacy of antibodies targeting families of checkpoint molecules (eg, CTLA-4 and PD-1/L1) in multiple tumor types. However, despite this success, clinical responses to these agents as monotherapy occur in a small number of patients (10 to 45% in a variety of solid tumors), and these therapies are hampered by side effects.

Rational dose selection and optimization of dosing regimen are clinically important and are prerequisites for improving patient compliance and achieving maximum clinical benefit. Development of dosing regimens typically follows pharmacokinetic/pharmacodynamic studies performed in animal models. However, because immunotherapies do not exert direct antiproliferative activity on cancer cells, but are instead expected to exploit tumor immunity, murine surrogates are typically used in some animal studies, making pharmacokinetic/pharmacodynamic studies unavailable. . Additionally, doctors may vary the dosage regimen of immunotherapy based on the immunogenicity of the tumor, disease stage, and physical condition of the patient. Therefore, novel strategies for developing dosing and therapy are needed.

Accordingly, in various aspects, the present disclosure provides compositions and methods useful for developing strategies for developing doses and dosing regimens for cancer immunotherapy. The present disclosure provides that CD80+ cells, CD8+ cells, granzyme B+ cells, CD68+ cells, Ki67+ cells and PD-L1+ immune cells are increased in the tumor microenvironment (TME) following administration of SIRPα-Fc-CD40L chimeric protein; B cells and/or CD40+ cells margin from the peripheral blood within hours of administration and return to the peripheral blood within a few days; It is based in part on the finding that levels of certain serum cytokines are increased within hours of dosing and decreased within a few days following administration of the SIRPα-Fc-CD40L chimeric protein.

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background level of cells is measured in a pre-dose biological sample obtained from the subject prior to administration of the first dose, and the markers include CD80+ cells, CD8+ cells, granzyme B+ cells, CD68+ cells, Ki67+ cells, and PD-L1+ immune cells. is selected from one or more of: In an embodiment, the method further comprises administering a second dose of the chimeric protein to the human subject when the post-administration level of the cells is greater than the background level of the cells. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose. In an embodiment, the biological sample is a biopsy sample or surgical specimen. In an embodiment, the biological sample is a tumor biopsy sample or tumor surgical specimen. In embodiments, cellular levels are measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof.

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background level and/or activity of B cells and/or CD40+ cells is measured in a first biological sample obtained from the subject prior to administration of the first dose. In an embodiment, the level and/or activity of B cells and/or CD40+ cells N hours after administration is measured in a second biological sample obtained from the subject following administration of the first dose. In embodiments, N is a number from 1 to 24. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at M days post-administration is measured in a third biological sample obtained from the subject following administration of the first dose. In embodiments, M is a number from 1 to 28. In an embodiment, the method comprises: (ii) the level and/or activity of B cells and/or CD40+ cells at M days post-administration is at least about and administering to the human subject a second dose of the chimeric protein when 50% higher. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose. In an embodiment, the first biological sample, the second biological sample and the third biological sample are blood. In an embodiment, the level and/or activity of B cells and/or CD40+ cells is measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or It is measured by a combination of these.

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background amount and/or activity of a cytokine selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α and MDC is determined by administration of the first dose. It was measured in a first biological sample previously obtained from the subject. In an embodiment, the amount and/or activity of the cytokine N hours after administration is measured in a second biological sample obtained from the subject after administration of the first dose. In embodiments, N is a number from 1 to 24. In an embodiment, the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine. In an embodiment, the amount and/or activity of the cytokine at M days post-administration is measured in a third biological sample obtained from the subject after administration of the first dose. In embodiments, M is a number from 1 to 28. In an embodiment, the method comprises (ii) preparation of the chimeric protein when the amount and/or activity of the cytokine at M days after administration is at least about 30% lower than the amount and/or activity of the cytokine at N hours after administration. It further comprises administering 2 doses to the human subject. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose. In an embodiment, 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 determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. It is measured.

Aspects of the disclosure are methods of assessing the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject is suffering from cancer. In an embodiment, the method comprises (i) obtaining a biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the background level and/or activity of cells is measured in a biological sample obtained from the subject prior to administration of the first dose. In an embodiment, 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. In an embodiment, the method further comprises (ii) determining post-administration levels and activity of cells in the biological sample. In an embodiment, the method further comprises the step of (iii) determining that the chimeric protein is efficacious if the level and/or activity of the cells after administration is greater than the background level and/or activity of the cells. In an embodiment, the biological sample is a tumor biopsy sample or tumor surgical specimen. In embodiments, cellular levels and/or activity are measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or a combination thereof. .

An aspect of the disclosure is a method of selecting a subject for treatment with therapy for cancer. In an embodiment, the method comprises (i) obtaining a biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the background level and/or activity of cells is measured in a biological sample obtained from the subject prior to administration of the first dose. In an embodiment, 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. In an embodiment, the method further comprises (ii) determining post-administration levels and activity of cells in the biological sample. In an embodiment, the method further comprises the step of (iii) selecting the subject for treatment with the chimeric protein if the level and/or activity of the cells after administration is greater than the background level and/or activity of the cells. In an embodiment, the biological sample is a tumor biopsy sample or tumor surgical specimen. In embodiments, cellular levels and/or activity are measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or a combination thereof. .

Aspects of the disclosure are methods of assessing the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject is suffering from cancer. In an embodiment, the method comprises (i) obtaining a first biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the method further comprises determining the background level and/or activity of B cells and/or CD40+ cells in the first biological sample. In an embodiment, the method further comprises the step of (iii) obtaining a second biological sample obtained from the subject at N hours after administration, where N is 1 to 24. In an embodiment, the method further comprises determining the level and/or activity of B cells and/or CD40+ cells in the second biological sample at N hours after administration. In an embodiment, the method further comprises (iv) obtaining a third biological sample obtained from the subject on M days after administration, wherein M is 1 to 28. In an embodiment, the method further comprises determining the level and/or activity of B cells and/or CD40+ cells in a third biological sample at day M after administration. In an embodiment, the method comprises: (v) the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells, and/or A chimeric protein is efficacious if the level and/or activity of B cells and/or CD40+ cells at M days after administration is at least about 50% higher than the level and/or activity of B cells and/or CD40+ cells at N hours after administration. It further includes the step of determining that there is. In an embodiment, the first biological sample, the second biological sample and the third biological sample are blood. In an embodiment, the level and/or activity of B cells and/or CD40+ cells is measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or It is measured by a combination of these.

An aspect of the disclosure is a method of selecting a subject for treatment with therapy for cancer. In an embodiment, the method comprises (i) obtaining a first biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the method further comprises determining the background level and/or activity of B cells and/or CD40+ cells in the first biological sample. In an embodiment, the method further comprises the step of (iii) obtaining a second biological sample obtained from the subject at N hours after administration, where N is 1 to 24. In an embodiment, the method further comprises determining the level and/or activity of B cells and/or CD40+ cells in the second biological sample at N hours after administration. In an embodiment, the method further comprises (iv) obtaining a third biological sample obtained from the subject on M days after administration, wherein M is 1 to 28. In an embodiment, the method further comprises determining the level and/or activity of B cells and/or CD40+ cells in a third biological sample at day M after administration. In an embodiment, the method comprises (v) the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells, and/or Treatment with a chimeric protein, if the level and/or activity of B cells and/or CD40+ cells at M days after administration is at least about 50% higher than the level and/or activity of B cells and/or CD40+ cells at N hours after administration. It further includes the step of selecting an object. In an embodiment, the first biological sample, the second biological sample and the third biological sample are blood. In an embodiment, the level and/or activity of B cells and/or CD40+ cells is measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or It is measured by a combination of these.

Aspects of the disclosure are methods of assessing the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject is suffering from cancer. In an embodiment, the method comprises (i) obtaining a first biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the method further comprises the step of (ii) determining the background amount and/or activity of the cytokine in the first biological sample. In an embodiment, the cytokine is selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC. In an embodiment, the method further comprises the step of (iii) obtaining a second biological sample obtained from the subject at N hours after administration, where N is 1 to 24. In an embodiment, the method further comprises determining the amount and/or activity of the cytokine in the second biological sample at N hours after administration. In an embodiment, the method further comprises (iv) obtaining a third biological sample obtained from the subject on M days after administration, wherein M is 1 to 28. In an embodiment, the method further comprises determining the amount and/or activity of the cytokine in a third biological sample at M days after administration. In an embodiment, the method comprises: (v) the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine; and/or the amount and/or activity of the cytokine at M days after administration and /or determining that the chimeric protein is efficacious if the activity is at least about 50% less than the amount and/or activity of the cytokine at N hours after administration. In an embodiment, 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 determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. It is measured.

An aspect of the disclosure is a method of selecting a subject for treatment with therapy for cancer. In an embodiment, the method comprises (i) obtaining a first biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the method further comprises the step of (ii) determining the background amount and/or activity of the cytokine in the first biological sample. In an embodiment, the cytokine is selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC. In an embodiment, the method further comprises the step of (iii) obtaining a second biological sample obtained from the subject at N hours after administration, where N is 1 to 24. In an embodiment, the method further comprises determining the amount and/or activity of the cytokine in the second biological sample at N hours after administration. In an embodiment, the method further comprises (iv) obtaining a third biological sample obtained from the subject on M days after administration, wherein M is 1 to 28. In an embodiment, the method further comprises determining the amount and/or activity of the cytokine in a third biological sample at M days after administration. In an embodiment, the method comprises: (v) the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine; and/or the amount of the cytokine at M days after administration. and/or if the activity is at least about 50% less than the amount and/or activity of the cytokine at N hours after administration, selecting the subject for treatment with the chimeric protein. In an embodiment, 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 determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. It is measured.

In an embodiment, the first domain is capable of binding CD172a (SIRPα) ligand. In an embodiment, the first domain comprises substantially all of the extracellular domain of CD172a (SIRPα). In an embodiment, the first domain 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. Contains amino acid sequences.

In an embodiment, the second domain is capable of binding the CD40 receptor. In an embodiment, the second domain comprises substantially all of the extracellular domain of CD40L. In an embodiment, the second domain 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. Contains amino acid sequences.

In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4. In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from human IgG4. In an embodiment, the linker 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%, with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. or comprises amino acid sequences that are at least about 99% identical.

In an embodiment, (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 SEQ ID NO: 1, SEQ ID NO: 2 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: 3. In an embodiment, 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 an embodiment, the chimeric protein 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 the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61. Contains 99% identical amino acid sequences.

In an embodiment, the first dose of chimeric protein ranges from about 0.03 mg/kg to 10 mg/kg. In an embodiment, 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 10 mg/kg.

In an embodiment, the method of any aspect disclosed herein further comprises administering a second dose of the chimeric protein. In an embodiment, 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 It is administered over 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. In an embodiment, the second dose of chimeric protein ranges from about 0.03 mg/kg to 10 mg/kg. In an embodiment, 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 10 mg/kg.

The patent or patent application includes at least one drawing executed in color. Copies of such patents or patent application publications with color drawings will be provided by the Office upon request and payment of the necessary fee.
Figure 1 shows a diagrammatic representation of the SIRPα-Fc-CD40L chimeric protein (SL-172154). SL-172154 (SIRPα-Fc-CD40L) is a hexamer consisting of SIRPα (binding affinity for CD47 is 0.628 nM) linked to CD40L (binding affinity for CD40 is 4.74 nM) via an Fc linker protein. It is a soluble fusion protein.
Figure 2 is Modified toxicity probability (mTPI-2) indicates dose escalation (N=15) per design. The planned capacity increase is in 1/2 log increments. At least three subjects were enrolled at sequential dose levels (DL) and assessed for dose limiting toxicity (DLT) in the first cycle of treatment. Subjects will receive intravenous (IV) administration of SL-172154 on Schedule 1 or Schedule 2 until disease progression, unacceptable toxicity, or withdrawal of consent.
Figure 3 shows tumor response and duration of treatment. iUPD = Unidentified progressive disease (iRECIST) NE = Not evaluable; PD = progressive disease; SD = stable disease
Figures 4A - 4D show reproducible increases in serum cytokines following repeated administration of SL-172154. Shows changes in CCL2(MCP-1) ( Figure 4A ), CCL4(MIP-1β) ( Figure 4B ), CCL3(MIP-1α) ( Figure 4C ), and CCL22(MDC) ( Figure 4D ) levels.
Figures 5A and 5B show dose-dependent and reproducible increases in serum IL-12. Figure 5A shows that subject levels interleukin 12 (IL-12), a mediator of the TH1 pro-inflammatory response over time, is a hallmark of the cyclic effector cytokine response observed in study subjects. Figure 5B shows the median response (horizontal bars) at the first injection and appears preliminary to be dose dependent.
Figures 6A and 6B demonstrate that SL-172154 preferentially binds to CD47 on leukocytes but not RBCs. Figure 6A shows CD47 receptor occupancy (RO) assessed by fluorescence activated cell sorting (FACS) analysis using whole blood for both red blood cells (RBCs) and white blood cells (WBCs). One hour after infusion on Cycle 1 Day 1 (C1D1), the median CD47 RO (horizontal bar) for leukocytes is approximately 80%. Figure 6B shows that CD47 RO on RBC is less than 5% for all dose levels.
Figures 7A - 7C demonstrate that SL-172154 stimulates dose-dependent B cell marginalization and activation. Figure 7A shows that the median frequency of marginal cells increases in a dose-dependent manner (horizontal bars). Receptor engagement is approximately 100% at all dose levels (data not shown). Figure 7B shows that median B cell frequency returns to pre-infusion levels by the next infusion and returns to each infusion cycle, maintaining an egress cycle pattern. Figure 7C shows that reverting B cells show an increase in the costimulatory marker CD86 as well as the maturation marker CD95, suggesting that SL-172154 can induce phenotypic changes.
Figures 8A and 8B show distinct profiles of TNFα and interleukin-6 (IL-6) for CD40 mAb. Figure 8A shows the induction of TNFα at various doses of CP-870,893 (left panel) or SL-172154 (right panel). Figure 8B shows the induction of IL-6 at various doses of CP-870,893 (left panel) or SL-172154 (right panel). CP-870,893 data is derived from Vonderheide et al ., J Clin Oncol 25:876-883 (2007).
Figures 9A and 9B demonstrate that SL-172154 induces an innate immune response in the tumor microenvironment (TME). Figure 9A shows immunohistochemical analysis of a biopsy sample from Patient A before and after administration of SL-172154. Monocytes were detected by staining for CD68 (a protein highly expressed by cells of the monocyte lineage). Figure 9B demonstrates upregulation of CD40 and MHC class II, activation markers of the TME, in tumor biopsy samples following treatment with SL-172154 compared to pre-treatment biopsy samples.
Figures 10A and 10B demonstrate that SL-172154 induces an adaptive immune response in the tumor microenvironment (TME). Figure 10A shows CD8+ cells, granzyme B+ cells, CD68+ cells, and Ki67+ cells in a biopsy sample from Patient A before and after administration of SL-172154. CD8+ cells, granzyme B+ cells, CD68+ cells and Ki67+ cells are increased in post-treatment biopsy samples compared to pre-treatment biopsy samples. Figure 10B is a plot comparing tumor proportion score (TPS) and combined positive score (CPS).
Figure 11 depicts the planned clinical development strategy for SL-172154. The strategies are SL-172154 monotherapy in ovarian cancer, SL-172154 + liposomal doxorubicin combination in ovarian cancer, SL-172154 + azacitidine + venetoclax in AML, and SL-172154 + aza in HR-MDS. Includes trials of combination therapy with cytidine and SL-172154 plus azacytidine in TP53 mutant AML.
Figure 12 shows increased abundance of CD80+ cells and/or CD80 expression following administration of SL-172154.

The present disclosure provides that CD80+ cells, CD8+ cells, granzyme B+ cells, CD68+ cells, Ki67+ cells, and PD-L1+ immune cells are increased in the tumor microenvironment (TME) following administration of SIRPα-Fc-CD40L chimeric protein, which results in DLT or It is based in part on the finding that it is well tolerated, with no evidence of anemia, thrombocytopenia, liver dysfunction, cytokine release syndrome, or pneumonitis. The present disclosure also provides that B cells and/or CD40 ⁺ cells migrate from the peripheral blood and release innate and adaptive serum cytokines, such as CCL2, CXCL9, CXCL10, IFNα, within a few hours after administration of the SIRPα-Fc-CD40L chimeric protein. The finding of increased levels of IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α and MDC, innate and adaptive serum cytokines, as well as the return of B cells and/or CD40 ⁺ cells to the peripheral blood. , e.g., CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α and MDC levels are reduced within approximately 1 day of administration of the SIRPα-Fc-CD40L chimeric protein. It is based in part on In some embodiments, B cell and/or CD40 ⁺ cell levels as well as innate and adaptive serum cytokines, such as CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP Levels of -1α and MDC approach background levels within 1 or 2 days of the SIRPα-Fc-CD40L chimeric protein.

Importantly, the chimeric proteins of the present disclosure (e.g., through binding of the extracellular domain of CD172a (SIRPα) to receptors/ligands on cancer cells) may target cancer cells in an attempt to avoid phagocytosis and/or destruction. destroy, block, reduce, inhibit and/or sequester the transmission of immunoinhibitory signals derived from, enhance, increase and/or (through binding of CD40L to its receptor) the transmission of immunostimulatory signals to anti-cancer immune cells. Because it stimulates, it provides antitumor effects by two distinct pathways; This dual-action is more likely to provide any anti-tumor effect in the patient and/or provide an enhanced anti-tumor effect in the patient. Furthermore, because these chimeric proteins can act through two separate pathways, they may be efficacious, at least in patients who respond poorly to treatments targeting one of the two pathways. Therefore, patients who are poor responders to treatments acting through one of the two pathways may benefit therapeutically from targeting the other pathway.

chimeric protein

The chimeric protein of the present disclosure comprises the extracellular domain of CD172a (SIRPα) and the extracellular domain of CD40L, which together can simultaneously block immune inhibitory signals and stimulate immune activation signals.

Aspects of the disclosure provide a chimeric protein comprising the general structure of the N terminus - (a) - (b) - (c) - C terminus, wherein (a) comprises the extracellular domain of CD172a (SIRPα). a first domain, and (b) comprises a linker adjacent the first domain and the second domain, e.g., at least one cysteine residue capable of forming a disulfide bond and/or comprising a hinge-CH2-CH3 Fc domain. is a linker, and (c) is a second domain comprising the extracellular domain of CD40L; A linker connects the first domain and the second domain.

In an embodiment, the first domain comprises substantially all of the extracellular domain of CD172a (SIRPα). In an embodiment, the first domain is capable of binding CD172a (SIRPα) ligand. In an embodiment, the first domain can bind CD172a (SIRPα) ligand (e.g., CD47) expressed on the surface of cancer cells. In embodiments, the first domain can inhibit binding of a CD172a(SIRPα) ligand (e.g., CD47) to CD172a(SIRPα) protein located on myeloid and hematopoietic stem cells and neurons. In embodiments, the first domain can inhibit immunosuppressive signals. In embodiments, the first domain can inhibit immunosuppressive signals. In embodiments, the first domain may inhibit macrophage checkpoint or “do not eat me” signaling. In an embodiment, therapy with a SIRPα-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) stimulates macrophages to phagocytose tumor cells and render tumor antigens of the phagocytic tumor cells to T cells. Present effectively.

In an embodiment, the second domain is capable of binding the CD40 receptor. In an embodiment, the second domain comprises substantially all of the extracellular domain of CD40L. In embodiments, the second domain can activate an immune stimulatory signal.

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

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

In an embodiment, the chimeric protein is multiple polypeptides that are combined (via covalent or non-covalent linkages) to yield a single unit, e.g., in vitro (e.g., by one or more synthetic linkers disclosed herein), For example, it refers to a recombinant protein of multiple extracellular domains disclosed herein.

In an embodiment, the chimeric protein can be chemically synthesized as one polypeptide or each domain can be chemically synthesized separately and then combined. In an embodiment, part of the chimeric protein is translated and part is chemically synthesized.

In an embodiment, an extracellular domain refers to the portion of a transmembrane protein that can interact with the extracellular environment. In an embodiment, an extracellular domain refers to a portion of a transmembrane protein that is sufficient to bind a ligand or receptor and is effective in transmitting a signal to the cell. In an embodiment, the extracellular domain is the entire amino acid sequence of a transmembrane protein that normally resides outside the cell or outside the cell membrane. In an embodiment, the extracellular domain is external to the cell or cell membrane and is involved in signal transduction and/or ligand binding that can be analyzed using methods known in the art (e.g., in vitro ligand binding and/or cell activation assays). It is the corresponding part of the amino acid sequence of the transmembrane protein required for.

Transmembrane proteins typically consist of an extracellular domain, one or a series of transmembrane domains, and an intracellular domain. Without being bound by theory, the extracellular domain of a transmembrane protein results in an interaction with a soluble receptor or ligand or a membrane-bound receptor or ligand (i.e., the membrane of an adjacent cell). Without being bound by theory, the transmembrane domain(s) serve to localize the transmembrane protein to the plasma membrane. Without being bound by theory, the intracellular domains of transmembrane proteins serve to coordinate interactions with cell signaling molecules to coordinate intracellular responses with the extracellular environment (and vice versa).

There are generally two types of single-pass transmembrane proteins: type I transmembrane proteins, which have an extracellular amino terminus and an intracellular carboxy terminus, and type II transmembrane proteins, which have an extracellular carboxy terminus and an intracellular amino terminus. Type I and II transmembrane proteins can be either receptors or ligands. For type I transmembrane proteins (e.g., CD172a (SIRPα)), the amino terminus of the protein faces extracellularly and is therefore a functional domain responsible for interacting with other binding partners (ligands or receptors) in the extracellular environment. Includes. Type II transmembrane proteins (e.g. In the case of CD40L), the carboxy terminus of the protein faces outside the cell and therefore contains a functional domain that is responsible for interacting with other binding partners (ligands or receptors) in the extracellular environment. Therefore, these two types of transmembrane proteins have opposite orientations with respect to the cell membrane.

The description of CD47 as a “don't eat me” signal in a wide range of cancers stimulated the exploration of what combinations of “eat me” signals could enhance antitumor immunity in the setting 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 USA 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)]. A preclinical combination of CD47 blocking and ADCP-competent antibodies, including rituximab and trastuzumab, enhances 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 Med 379:1711-1721 (2018); Zhao et al ., CD47-signal regulatory protein-alpha (SIRPalpha) interactions form a barrier for antibody-mediated tumor cell destruction. Proc Natl Acad Sci USA 108:18342-18347 (2011)]. At least 50% of patients with relapsed or refractory diffuse large B-cell lymphoma or follicular lymphoma treated with Hu5F9-G4, humanized, IgG4 isotype, CD47-blocking mAb in combination with rituximab demonstrate 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 blockade improves antigen presentation in immune-neglected tumors (Tseng et al ., Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response. Proc Natl Acad Sci USA 110 :11103-11108 (2013)), only sporadic clinical responses were observed when using CD47/SIRPα blocking treatments as monotherapy or in combination with PD-1/L1-blocking antibodies.

Disrupting the binding of CD47 to SIRPα has emerged as a promising immunotherapeutic strategy for advanced cancer by enhancing antibody-dependent cellular phagocytosis (ADCP) of targeted antibodies. Preclinically, CD47/SIRPα blockade induces antitumor activity by increasing phagocytosis of tumor cells by macrophages and enhancing cross-presentation of tumor antigens to CD8+ T cells by dendritic cells; Both of these processes are enhanced by CD40 signaling. A novel, bilateral fusion protein was generated incorporating the extracellular domains of SIRPα and CD40L adjacent to the central Fc domain, also referred to herein as SIRPα-Fc-CD40L. As shown herein, the SIRPα-Fc-CD40L chimeric protein bound CD47 and CD40 with high affinity and activated CD40 signaling in the absence of Fc receptor replacement binding. No evidence of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or in cynomolgus macaques. Additionally, as shown herein, the SIRPα-Fc-CD40L chimeric protein outperformed CD47 blockade and CD40 enteric antibody in the murine CT26 tumor model and was upregulated by immune checkpoint blockade of PD-1 and CTLA4. SIRPα-Fc-CD40L activated type I interferon responses in macrophages and enhanced the activity of ADCP-competent targeting antibodies both in vitro and in vivo. These data demonstrate that CD47/SIRPα inhibition was able to enhance tumor cell phagocytosis, whereas CD40-mediated activation of type I interferon responses significantly enhanced sustainable tumor control and rejection of macrophage- and T-cell-mediated immunity. It was explained that it provided a bridge between the two.

The chimeric protein of the present disclosure comprises the extracellular domain of CD172a (SIRPα) and the extracellular domain of CD40L. Accordingly, the chimeric protein used in the present disclosure has at least a second domain comprising the extracellular domain of CD40L - directly or It comprises a first domain comprising the extracellular domain of CD172a (SIRPα), which is connected - via a linker. When the domains are joined in amino-terminal to carboxy-terminal orientation, the first domain is located on the "left" side of the chimeric protein, "facing out", and the second domain is located on the "right" side of the chimeric protein. and “turns outward.”

Alternative configurations of the first domain and the second domain are contemplated, for example, the first domain facing outward and the second domain facing inward, the first domain facing inward and the second domain facing outward, Both the first domain and the second domain face inward. When both domains are "faced inward", the chimeric protein will have an amino-terminus to carboxy-terminus configuration comprising CD40L, the linker, and the extracellular domain of CD172a (SIRPα). In this configuration, the chimeric protein may be required to contain additional "slack" as described elsewhere herein to enable the domain of the chimeric protein to bind to one or both of its receptors/ligands. there is.

The construct can be generated by cloning the nucleic acid encoding these three fragments (the extracellular domain of CD172a (SIRPα), followed by the linker sequence, then the extracellular domain of CD40L) into a vector (plasmid, virus, or other). , the amino terminus of the complete sequence corresponds to the 'left' side of the molecule containing the extracellular domain of CD172a (SIRPα), and the carboxy terminus of the complete sequence corresponds to the 'right' side of the molecule containing the extracellular domain of CD40L. do. In some embodiments, of the chimeric proteins having one of the different configurations as described above, the construct is comprised of three nucleic acids such that the resulting translated chimeric protein has the desired configuration, e.g., a dual inward-facing chimeric protein. will include Accordingly, in an embodiment, the chimeric protein is engineered as such.

CD172a(SIRPα)-Fc-CD40L chimeric protein

In an embodiment, the chimeric protein is capable of simultaneously binding a human CD172a(SIRPα) ligand and a human CD40 receptor, wherein the CD172a(SIRPα) ligand is CD47 and the CD40L receptor is CD40.

The chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) is a first protein comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second linker comprising the extracellular domain of human CD40 ligand (CD40L). It's a domain. In an embodiment, the linker includes at least one cysteine residue capable of forming a disulfide bond.

The chimeric protein of the present disclosure has a first domain capable of sterically binding its ligand/receptor and/or a second domain capable of sterically binding its ligand/receptor. This is due to the overall flexibility of the chimeric protein and/or the physical cohesion between the extracellular domain (or part thereof) and the rest of the chimeric protein such that the ligand/receptor binding domain of the extracellular domain is not sterically hindered from binding to its ligand/receptor. This means that the distance is sufficient. Early flexibility and/or physical distance (referred to herein as “slack”) may be normally present in the extracellular domain(s),/or be normally present in the linker, and/or be normally present in the chimeric protein (as a whole). there is. Alternatively, or additionally, the chimeric protein may contain one or more additional amino acid sequences (e.g., binding linkers described below) or synthetic linkers that provide the additional slack necessary to avoid steric hindrance (e.g., polyethylene glycol (PEG) ) can be modified by including a linker).

In an embodiment, the chimeric protein of the present disclosure comprises a variant of the extracellular domain of CD172a (SIRPα). By way of example, the variant may be at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about, the known amino acid sequence of CD172a(SIRPα), e.g., human CD172a(SIRPα). 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.

In an embodiment, the extracellular domain of CD172a (SIRPα) has the following amino acid sequence:

In an embodiment, the chimeric protein comprises a variant of the extracellular domain of CD172a (SIRPα). For example, the variant is 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. You can have it.

In an embodiment, 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.

Those skilled in the art will see, 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.” Variants of the known amino acid sequence of CD172a (SIRPα) can be selected by referring to Proc Natl Acad Sci USA 114: E10578-E10585 (2017), each of which is incorporated by reference in its entirety.

In an embodiment, the chimeric protein of the present disclosure comprises a variant of the extracellular domain of CD40L. By way of example, the variant may be 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, the known amino acid sequence of CD40L, e.g., human CD40L. 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 may have at least about 99% sequence identity.

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

In an embodiment, the chimeric protein comprises a variant of the extracellular domain of CD40L. By way of example, the variant is 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. You can have it.

In an embodiment, 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.

Those skilled in the art will appreciate the literature, e.g. Karpusas et al., “ 2 A 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 herein by reference in its entirety.

In an embodiment, the linker of the chimeric protein has 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. Includes. In an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, the chimeric protein of the disclosure comprises (1) a first protein 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; domain, (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) SEQ ID NO: 1, and 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: 2 or SEQ ID NO: 3.

In an embodiment, the chimeric protein of the disclosure comprises (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 domain, 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 an embodiment, the chimeric protein of the disclosure comprises (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 domain, 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 an embodiment, the chimeric protein of the disclosure comprises (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 domain, 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 an embodiment, the chimeric protein of the disclosure comprises (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 domain, 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 an embodiment, the chimeric protein of the disclosure comprises (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) SEQ ID NO: 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 an embodiment, the chimeric protein of the disclosure comprises (1) a first domain comprising an amino acid sequence identical to SEQ ID NO: 57, (b) a second domain comprising an amino acid sequence identical to SEQ ID NO: 58, and (c) a sequence It includes a linker containing 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 an embodiment, the CD172a(SIRPα)-Fc-CD40L chimeric protein of the present disclosure has the following amino acid sequence (the extracellular domain of CD172a(SIRPα) is shown in bold, the extracellular domain of CD40L is underlined, Fc domains are shown in italics):

The 792 amino acid sequence (not including the leader sequence) of the CD172a(SIRPα)-Fc-CD40L chimeric protein (SL-172154) is shown above. The CD172a(SIRPα)-Fc-CD40L chimeric protein exists in an oligomeric profile. There are 17 cysteines in the amino acid sequence, with 8 possible disulfide pairs. Both N- and O-linked glycosylation were identified.

In an embodiment, the chimeric protein of the disclosure comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 potential N glycosylation sites. In an embodiment, the chimeric protein of the present disclosure includes at least two potential N glycosylation sites. In an embodiment, the chimeric protein of the present disclosure includes at least four potential N glycosylation sites. In an embodiment, the chimeric protein of the present disclosure includes at least 6 potential N glycosylation sites. In an embodiment, the chimeric protein of the present disclosure comprises at least 8 potential N glycosylation sites. In an embodiment, the chimeric protein of the present disclosure comprises at least 10 potential N glycosylation sites. In an embodiment, the chimeric protein of the present disclosure includes at least 1, 2, 3, 4, 5, 6, 7, or 8 potential O glycosylation sites. In an embodiment, the chimeric protein of the present disclosure includes at least two potential O glycosylation sites. In an embodiment, the chimeric protein of the present disclosure includes at least four potential O glycosylation sites. In an embodiment, the chimeric protein of the present disclosure comprises at least 6 potential O glycosylation sites. In an embodiment, the chimeric protein of the present disclosure comprises at least 8 potential O glycosylation sites. In an embodiment, the chimeric protein of the disclosure comprises at least two potential N glycosylation sites, and at least two potential O glycosylation sites. In an embodiment, the chimeric protein of the present disclosure comprises at least four potential N glycosylation sites, and at least four potential O glycosylation sites. In an embodiment, the chimeric protein of the present disclosure comprises at least 6 potential N glycosylation sites, and at least 6 potential O glycosylation sites. In an embodiment, the chimeric protein of the disclosure comprises at least 8 potential N glycosylation sites, and at least 8 potential O glycosylation sites. In an embodiment, the chimeric protein of the present disclosure comprises at least 10 potential N glycosylation sites, and at least 8 potential O glycosylation sites. In an embodiment, the chimeric protein expressed in Chinese hamster ovary (CHO) cells is glycosylated.

There are 17 cysteines in the SL-172154 chimeric protein. In some embodiments, the SL-172154 chimeric protein is free of disulfide bonds. In some embodiments, the SL-172154 chimeric protein is 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 It has 9, or at least 10 disulfide bonds. In some embodiments, the SL-172154 chimeric protein has at least one or at least two 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 interchain disulfides. has a bond. In some embodiments, the SL-172154 chimeric protein has C350=C350 interchain disulfide bonds. In some embodiments, the SL-172154 chimeric protein has a C353=C353 interchain disulfide bond. 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 a C140 = C198 disulfide bond. In some embodiments, the SL-172154 chimeric protein has a C243 = C301 disulfide bond. In some embodiments, the SL-172154 chimeric protein has a C385 = C445 disulfide bond. 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 a C709 = C725 disulfide bond. In some embodiments, the SL-172154 chimeric protein has C140 = C243 = C709/C725 scrambled disulfide bonds. In some embodiments, the SL-172154 chimeric protein has a C615 (Chain 1) = C615 (Chain 2) scrambled disulfide bond.

In some embodiments, the CD172a(SIRPα)-Fc-CD40L chimeric protein of the present disclosure is encoded by the following nucleotide sequence (leader sequence shown in bold-underlined pond):

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

The chimeric protein of SEQ ID NO: 59 (also referred to herein as SL-172154) includes the extracellular domain of human CD172a (SIRPα) (PDCD1, CD272a), the hinge of human immunoglobulin constant gamma 4 (inhibitory receptor SHPS-1, IgG4) It is a recombinant fusion glycoprotein containing the central domain containing the -CH2-CH3 region and the extracellular domain of human CD40L (TNFSF5, TRAP, CD154). The linear configuration of SL-172154 is CD172a(SIRPα)-Fc-CD40L.

The predicted molecular weight for the monomeric chimeric protein of SEQ ID NO:59 is 88.1 kDa. The predicted molecular mass for the glycosylated monomeric chimeric protein of SEQ ID NO:59 is approximately 115 kDa.

The double-faced nature of the chimeric proteins disclosed herein, such as the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61), allows them to inhibit one of the important immunosuppressive pathways within the tumor microenvironment (TME). CD172a (SIRPα) - designed to block the CD47 macrophage checkpoint.

Tumor cells can express CD47 on the cell surface, which can bind to CD172a (SIRPα) expressed by macrophages, inhibiting phagocytosis of tumor cells. Accordingly, the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) can bind CD47 expressed on the tumor surface, and the CD172a(SIRPα)-Fc-CD40L disclosed herein The CD172a(SIRPα) domain of the chimeric protein provides competitive inhibition of CD47 and replaces the CD47 inhibitory signal with functionally trimerized/hexamerized CD40L, via its CD40 receptor instead of inhibition via CD172a(SIRPα) interaction. It is intended to generate incoming T cells that experience costimulation through engagement. In other words, because the extracellular domains (ECD) of CD172a (SIRPα) and CD40L are physically connected to each other and localized to the TME, tumor-infiltrating T cells simultaneously recognize tumor antigens through their T cell receptors (TCRs). It will accept empty stimulation. Importantly, because the ECDs of CD172a (SIRPα) and CD40L are physically linked to each other and localized to the TME, tumor-infiltrating T cells will receive co-stimulation simultaneously with their recognition of tumor antigens via T cell receptors. Taken together, these will cause the inhibitory CD47 signal to be replaced by the costimulatory CD40L signal, thereby enhancing the antitumor activity of T cells.

The three components of the chimeric proteins disclosed herein, including the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61), have unique properties that promote dimerization or oligomerization. . The extracellular domain of CD172a (SIRPα) usually exists as a monomer and is not known to form higher-order homomeric complexes. The central Fc domain contains cysteine residues that can disulfide bond to form a dimeric structure. In an embodiment, a chimeric protein disclosed herein comprising a CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) comprises an S228P mutation in the hinge region of the Fc domain to modify the Fab arm. prevent exchange. The CD40L domain is known to form a homotrimeric complex that is stabilized through non-covalent, electrostatic interactions. The chimeric proteins disclosed herein, including the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61), are expressed as continuous monomeric proteins by the production cell line, but the resulting monomeric proteins have this Based on the charge-based interactions of cargo and CD40L (generating trimers) and the combined influence of these attractions, higher order species are assembled, generating hexamers (dimers of trimers). The majority (>80%) of the CD172a(SIRPα)-Fc-CD40L chimeric proteins (e.g., SEQ ID NO:59 or SEQ ID NO:61) contain hexameric and trimeric forms with similar activities. Importantly, because the CD172a(SIRPα)-Fc-CD40L chimeric proteins (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) consist of hexamers and trimers, they exhibit the absence of cross-linking by Fc receptors or any other cross-linking agent. stimulates CD40 signaling. Predicted 3 of the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) as a monomer and in various oligomeric states based on disulfide (Fc) and charge-based (CD40L) interactions The secondary structures are the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO:59 or SEQ ID NO:61) hexamer (top two images) and the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., Shows visualization by electron microscopy of the SEQ ID NO:59 or SEQ ID NO:61) trimer (bottom two images). Accordingly, the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) forms a trimer/hexamer and activates CD40 without the need for cross-linking. It is noteworthy that, unlike monoclonal antibodies, Fc receptor cross-linking does not require the functional activity of the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61).

In an embodiment, the chimeric protein comprises a variant of the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61). By way of example, the variant is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least About 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74 %, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least About 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% %, or at least about 99.2%, or at least about 99.4%, or at least about 99.6%, or at least about 99.8%.

In an embodiment, 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. do. 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:59 or SEQ ID NO:61. 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:59 or SEQ ID NO:61. 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:59 or SEQ ID NO:61. 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:59 or SEQ ID NO:61. 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:59 or SEQ ID NO:61.

In an embodiment, the first domain is capable of binding CD172a (SIRPα) ligand.

In an embodiment, the first domain comprises substantially all of the extracellular domain of CD172a (SIRPα).

In an embodiment, the second domain is capable of binding the CD40 receptor.

In an embodiment, the second domain comprises substantially all of the extracellular domain of CD40L.

In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4, eg, human IgG4.

In an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, (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 is SEQ ID NO: 1 or SEQ ID NO: 2. or an amino acid sequence that is at least 95% identical to SEQ ID NO:3.

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

In an embodiment, the chimeric protein 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, or at least about identical to SEQ ID NO: 59 or SEQ ID NO: 61. 99% identical, or 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, or at least about 99.6% identical to SEQ ID NO: 59 or SEQ ID NO: 61, or or an amino acid sequence that is at least about 99.8% identical to SEQ ID NO:59 or SEQ ID NO:61. In an embodiment, 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, a chimeric protein may comprise an amino acid sequence with one or more amino acid mutations relative to any of the proteins disclosed herein. In embodiments, one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

In embodiments, amino acid mutations are amino acid substitutions and may include conservative and/or non-conservative substitutions. “Conservative substitutions” may be made, for example, based on similarity in polarity, charge, capacity, solubility, hydrophobicity, hydrophilicity and/or amphiphilic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) Acid: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) aromatics: Trp, Tyr, Phe. As used herein, “conservative substitution” is defined as exchanging an amino acid for another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in this modified polypeptide. Additionally, glycine and proline can be substituted for each other based on their ability to disrupt the α-helix. As used herein, “non-conservative substitution” is defined as exchanging an amino acid for another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

In embodiments, substitutions can also be made with non-classical amino acids (e.g., generally selenocysteine, pyrrolysine, N -formylmethionine β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA) , D-isomers of common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-aminohexane Acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t -butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids and amino acid analogs).

Mutations can also be made in the nucleotide sequence of a chimeric protein with reference to the genetic code, including taking codon degeneracy into account.

In an embodiment, the chimeric protein is capable of binding human ligand(s)/receptor(s).

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

In an embodiment, each extracellular domain (or variant thereof) of the chimeric protein has about 1 μM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, 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 30n M, Binds to its cognate receptor or ligand with a K _D of less than about 25 nM, about 20 nM, about 15 nM, about 10 nM, or about 5 nM, or about 1 nM.

In embodiments, the chimeric protein has a K of about 1 nM to about 5 nM, e.g., about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM. _D binds to human CD47. In an embodiment, the chimeric protein has a molecular weight of about 3 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 55 pM, about 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or Binds to human CD47 with a K _D of about 10 pM, or less than about 1 pM.

In an embodiment, the chimeric protein has a molecular weight (e.g., as measured by surface plasmon resonance or biolayer interferometry) of about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 55 pM, about 50 pM, about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about Binds to human CD40 with a K _D of less than 1 pM.

As used herein, variants of the extracellular domain can bind receptors/ligands of the native extracellular domain. For example, a variant may contain one or more mutations in the extracellular domain that do not affect the binding affinity for its receptor/ligand; Alternatively, one or more mutations in the extracellular domain may improve binding affinity for the receptor/ligand; Alternatively, one or more mutations in the extracellular domain may reduce binding affinity for the receptor/ligand, but do not completely eliminate binding. In an embodiment, one or more mutations are located outside the binding pocket where the extracellular domain interacts with its receptor/ligand. In an embodiment, one or more mutations are located within the binding pocket where the extracellular domain interacts with its receptor/ligand, unless the mutation completely abolishes binding. Based on the knowledge of those skilled in the art and knowledge of receptor-ligand binding, it will be appreciated that mutations enable binding and eliminate binding.

In an embodiment, the chimeric protein exhibits improved stability and protein half-life.

Chimeric proteins of the present disclosure may comprise more than two extracellular domains. For example, a chimeric protein may contain 3, 4, 5, 6, 7, 8, 9, 10 or more extracellular domains. The second extracellular domain can be separated from the third extracellular domain via a linker as disclosed herein. Alternatively, the second extracellular domain may be linked directly (e.g., via a peptide bond) to the third extracellular domain. In an embodiment, the chimeric protein comprises an extracellular domain that is directly linked and an extracellular domain that is indirectly linked through a linker, as disclosed herein.

linker

In an embodiment, the chimeric protein includes a linker.

In an embodiment, the linker includes at least one cysteine residue capable of forming a disulfide bond. At least one cysteine residue may form a disulfide bond between one (or more) pair of chimeric proteins. Without being bound by theory, this disulfide bond formation results in maintaining the useful multimeric state of the chimeric protein. This allows efficient production of chimeric proteins; Enables desired activity in vitro and in vivo .

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

In an embodiment, the linker is derived from a naturally occurring multi-domain protein or as described, for example, in Chichili et al. , Protein Sci. 22(2):153-167 (2013); Chen et al. , Adv Drug Deliv Rev. 65(10):1357-1369 (2013), the entire contents of which are incorporated herein by reference. In an embodiment, the linker is as described in Chen et al. , Adv Drug Deliv Rev . 65(10):1357-1369 (2013); and Crasto et al. , Protein Eng . 13(5):309-312 (2000)], the entire contents of which are incorporated herein by reference.

In an embodiment, the linker comprises a polypeptide. In an embodiment, the polypeptide is 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 It is 150 amino acids long, or less than about 100 amino acids long. For example, the linker is 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 It may be less than 5, about 4, about 3, or about 2 amino acids long.

In an embodiment, the linker is flexible.

In an embodiment, the linker is rigid.

In an embodiment, the linker contains 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 an embodiment, the linker comprises the hinge region of an antibody (e.g., of the IgG, IgA, IgD, and IgE subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)) . The hinge region found in IgG, IgA, IgD and IgE class antibodies acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant region, the hinge domain is structurally diverse, varying in both sequence and length across immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies across IgG subclasses. The hinge region of IgG1 encompasses amino acids 216 to 231, and because it is freely flexible, the Fab fragments can rotate about their axis of symmetry and move within a sphere centered on the first of the two interheavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1 with 12 amino acid residues and 4 disulfide bridges. The hinge region of IgG2 is devoid of glycine residues and contains a relatively short, rigid poly-proline helix stabilized by extra heavy chain disulfide bridges. These properties limit the flexibility of the IgG2 molecule. IgG3 differs from other subclasses by a unique extended hinge region (approximately four times the length of the IgG1 hinge) containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. forms. In IgG3, the Fab fragment is located relatively far away from the Fc fragment, giving the molecule greater flexibility. The extended hinge of IgG3 also results in a higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge region reportedly decreases in the following order: IgG3>IgG1>IgG4>IgG2. In an embodiment, 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 further divided into three regions: upper hinge region, core region and lower hinge region. See Shin et al. , Immunological Reviews 130:87 (1992)]. The upper hinge region includes amino acids from the carboxyl end of C _H1 to the first residue of the hinge, which limits movement, and generally to the first cysteine residue, which forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the flexibility of the antibody segment. The core hinge region contains an interheavy chain disulfide bridge, and the lower hinge region binds the amino-terminal end of the C _H2 domain and contains residues at C _H2 . See 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, results in a cyclic octapeptide that is believed to act as a pivot, thus conferring flexibility. In an embodiment, the present linker links the upper hinge region, core region, and subclasses of any antibody (e.g., IgG, IgA, IgD, and IgE (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2) includes one or two or three of the lower hinge regions. The hinge region may also contain one or more glycosylation sites, including multiple structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within the 17 amino acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, which is considered an advantageous property for secretory immunoglobulins. In an embodiment, the present disclosure includes one or more glycosylation sites.

In an embodiment, the linker comprises the Fc domain of an antibody (e.g., of the IgG, IgA, IgD, and IgE subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)) .

In the chimeric proteins of the present disclosure, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4. In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from human IgG4. In an embodiment, 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 of SEQ ID NO: 1 through SEQ ID NO: 3. In an embodiment, the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any of SEQ ID NO: 1 through SEQ ID NO: 3 (e.g., at least 95% identical to the amino acid sequence of SEQ ID NO: 2). In an embodiment, the linker comprises one or more binding linkers, wherein such binding linkers are independently selected from SEQ ID NOs: 4-50 (or variants thereof). In an embodiment, the linker comprises two or more binding linkers, each binding linker being independently selected from SEQ ID NOs: 4-50 (or variants thereof); One binding linker is N-terminal to the hinge-CH2-CH3 Fc domain and the other binding linker is C-terminal to the hinge-CH2-CH3 Fc domain.

In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human IgG1 antibody. In an embodiment, the Fc domain exhibits increased affinity and improved binding to the neonatal Fc receptor (FcRn). In an embodiment, the Fc domain comprises one or more mutations that increase affinity and enhance binding to FcRn. Without being bound by theory, it is believed that the increased affinity and improved binding to FcRn increases the in vivo half-life of the chimeric protein.

In an embodiment, the Fc domain in the linker comprises amino acid residues 250, 252, 254, 256, 308, 309, 311, 416, 428, 433, or 434 (Kabat, et al. , expressly incorporated herein by reference). Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), or their equivalents. In an embodiment, the amino acid substitution at amino acid residue 250 is with glutamine. In an embodiment, the amino acid substitution at amino acid residue 252 is with tyrosine, phenylalanine, tryptophan, or threonine. In an embodiment, the amino acid substitution at amino acid residue 254 is with threonine. In an embodiment, the amino acid substitution at amino acid residue 256 is with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In an embodiment, the amino acid substitution at amino acid residue 308 is with threonine. In an embodiment, the amino acid substitution at amino acid residue 309 is with proline. In an embodiment, the amino acid substitution at amino acid residue 311 is with serine. In an embodiment, the amino acid substitution at amino acid residue 385 is with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine, or glycine. In an embodiment, the amino acid substitution at amino acid residue 386 is with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In an embodiment, the amino acid substitution at amino acid residue 387 is with arginine, proline, histidine, serine, threonine, or alanine. In an embodiment, the amino acid substitution at amino acid residue 389 is with proline, serine, or asparagine. In an embodiment, the amino acid substitution at amino acid residue 416 is with serine. In an embodiment, the amino acid substitution at amino acid residue 428 is with leucine. In an embodiment, the amino acid substitution at amino acid residue 433 is with arginine, serine, isoleucine, proline, or glutamine. In an embodiment, the amino acid substitution at amino acid residue 434 is with histidine, phenylalanine, or tyrosine.

In an embodiment, the Fc domain linker (e.g., comprising an IgG constant region) comprises one or more mutations, such as a substitution at amino acid residues 252, 254, 256, 433, 434, or 436 (as explicitly incorporated herein by reference) According to Kabat numbering as in Kabat, et al. , Sequences of Proteins of Immunological Interest, 5th Ed., National Institutes of Health, Bethesda, Md. In an embodiment, the IgG constant region comprises triple M252Y/S254T/T256E mutations or YTE mutations. In an embodiment, the IgG constant region comprises triple H433K/N434F/Y436H mutations or KFH mutations. In an embodiment, the IgG constant region comprises YTE and KFH mutations in combination.

In an embodiment, 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 (see references expressly incorporated herein by reference) [According to Kabat numbering as in Kabat, et al ., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Exemplary mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S and H435A. In an embodiment, the IgG constant region comprises the M428L/N434S mutation or the LS mutation. In an embodiment, the IgG constant region comprises a T250Q/M428L mutation or a QL mutation. In an embodiment, the IgG constant region comprises the N434A mutation. In an embodiment, the IgG constant region comprises the T307A/E380A/N434A mutation or the AAA mutation. In an embodiment, the IgG constant region comprises the I253A/H310A/H435A mutation or the IHH mutation. In an embodiment, the IgG constant region comprises the H433K/N434F mutation. In an embodiment, the IgG constant region comprises a combination of M252Y/S254T/T256E and H433K/N434F mutations.

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

An exemplary Fc stabilizing mutant is S228P. Exemplary Fc half-life extending mutants are T250Q, M428L, V308T, L309P and Q311S, and the linker may contain 1, or 2, or 3, or 4, or 5 of these mutants.

In an embodiment, the chimeric protein binds FcRn with high affinity. In an embodiment, the chimeric protein is capable of binding FcRn with a K _D of about 1 nM to about 80 nM. For example, the chimeric protein has about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 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, Alternatively, it can bind to FcRn with a K _D of about 80nM. In an embodiment, the chimeric protein is capable of binding FcRn with a K _D of about 9 nM. In an embodiment, the chimeric protein does not substantially bind to other Fc receptors that have effector functions (i.e., other than FcRn).

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

Additionally, one or more binding linkers may bind an Fc domain (e.g., or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3) and an extracellular domain. You can. For example, any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or variants thereof may comprise an extracellular domain as disclosed herein. It can be connected to the Fc domain of a linker as shown. Optionally, any one of SEQ ID NOs: 4-50, or a variant thereof, is located between the extracellular domain as disclosed herein and the Fc domain as disclosed herein.

In embodiments, the chimeric protein may comprise a variant of the connecting linker disclosed in Table 1 below. For example, the linker is 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 may have at least about 99% sequence identity.

In embodiments, the first and second binding linkers can be different or they can be the same.

Without being bound by theory, including a linker comprising at least a portion of the Fc domain in the chimeric protein avoids the formation of insoluble and, possibly, non-functional protein concatemers and/or aggregates. do. This is, in part, due to the presence of cysteines in the Fc domain that can form disulfide bonds between chimeric proteins.

In an embodiment, the chimeric protein may comprise one or more linking linkers as disclosed herein and may lack an Fc domain linker as disclosed herein.

In an embodiment, the first and/or second connecting linker is independently selected from the amino acid sequences of SEQ ID NOs: 4-50 and are provided in Table 1 below:

In an embodiment, the binding linker comprises glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycine and serine). For example, in an embodiment, the binding linker is (Gly ₄ Ser) _n , where n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (each as SEQ ID NO: 25 to SEQ ID NO: 9). In an embodiment, the binding linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 33). Additional exemplary linking linkers include the sequence LE; (EAAAK) _n (n = 1 to 3) (SEQ ID NO: 36 to SEQ ID NO: 38), A (EAAAK) _n A (n = 2 to 5) (SEQ ID NO: 39 to SEQ ID NO: 42), A (EAAAK) ₄ ALEA (EAAAK) ₄ A (SEQ ID NO: 43), PAPAP (SEQ ID NO: 44), KESGSVSSEQLAQFRSLD (SEQ ID NO: 45), GSAGSAAGSGEF (SEQ ID NO: 46) and (XP) linkers with _n , but are not limited to represents any amino acid, such as Ala, Lys or Glu. In an embodiment, the binding linker is GGS. In an embodiment, the binding linker has the sequence (Gly) _n , where n is any number from 1 to 100, for example (Gly) ₈ (SEQ ID NO: 34) and (Gly) ₆ (SEQ ID NO: 35) )am.

In an embodiment, the binding linker is GGGSE (SEQ ID NO: 47), GSESG (SEQ ID NO: 48), GSEGS (SEQ ID NO: 49), GEGGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 50), and G, S, and E randomly positioned every four amino acids. One or more binding linkers.

In an embodiment, the chimeric protein comprises a connecting linker comprising the amino acid sequence of SEQ ID NO:5 and/or SEQ ID NO:7.

In an embodiment, where the chimeric protein comprises an extracellular domain (ECD) of CD172a (SIRPα), one connecting linker before the Fc domain, a second connecting linker after the Fc domain, and the ECD of CD40L, the chimeric protein has the following: May contain structures:

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

The combination of a first connecting linker, an Fc domain linker, and a second connecting linker is referred to herein as a “modular linker”. In an embodiment, the chimeric protein comprises a modular linker as shown in Table 2 :

In an embodiment, the chimeric protein may comprise a variant of the modular linker disclosed in Table 2 above. For example, the linker is 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 may have at least about 99% sequence identity.

In embodiments, the linker may be flexible, including but not limited to a high degree of flexibility. In embodiments, the linker may be rigid, including but not limited to a rigid alpha helix. Characteristics of exemplary connecting linkers are shown in Table 3 below:

In embodiments, the linker may be functional. For example, but not limited to, linkers may improve folding and/or stability, improve expression, improve pharmacokinetics, and/or improve bioactivity of the chimeric protein. It can do what it says. In another example, a linker may serve to target the chimeric protein to a specific cell type or location.

In an embodiment, the chimeric protein includes only one connecting linker.

In an embodiment, the chimeric protein lacks a connecting linker.

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

In an embodiment, the chimeric protein has a first domain capable of sterically binding its ligand/receptor and/or a second domain capable of sterically binding its ligand/receptor. Accordingly, the overall flexibility of the chimeric protein and/or the gap between the extracellular domain (or portion thereof) and the rest of the chimeric protein such that the ligand/receptor binding domain of the extracellular domain is not sterically hindered from binding to its ligand/receptor. Physical distance is sufficient. Early flexibility and/or physical distance (referred to as “slack”) may be normally present in the extracellular domain(s),/or be normally present in the linker, and/or be normally present in the chimeric protein (as a whole). Alternatively, or additionally, amino acid sequences (for example) may be added to one or more extracellular domains and/or to the linker to provide the necessary slack to avoid steric hindrance. Any amino acid sequence that provides slack can be added. In an embodiment, 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 can be added include the connecting linkers listed in Tables 1 and 3 . In an embodiment, a polyethylene glycol (PEG) linker may be added between the extracellular domain and the linker to provide the necessary slack to avoid steric hindrance. Such PEG linkers are well known in the art.

In an embodiment, the chimeric protein of the present disclosure comprises an extracellular domain of human CD172a (SIRPα) (or a variant thereof), a linker, and an extracellular domain of human CD40L (or a variant thereof). In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain from, e.g., an IgG1 or IgG4, including a human IgG1 or IgG4. Accordingly, in an embodiment, the chimeric protein of the present disclosure comprises an extracellular domain of human CD172a(SIRPα) (or a variant thereof), a linker comprising a hinge-CH2-CH3 Fc domain, and an extracellular domain of human CD40L (or a variant thereof). Includes domain. This chimeric protein may be referred to herein as “hCD172a(SIRPα)-Fc-CD40L” or “SL-172154”.

Diabetes mellitus, treatment methods and mechanism of action

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background level and/or activity of cells is measured in a pre-dose biological sample obtained from the subject prior to administration of the first dose, and the markers include CD80+ cells, CD8+ cells, granzyme B+ cells, CD68+ cells, Ki67+ cells, and PD -Selected from one or more of L1+ immune cells. In an embodiment, the method further comprises the step of (ii) administering a second dose of the chimeric protein to the human subject when the post-administration level and/or activity of the cells is greater than the background level and/or activity of the cells. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose.

In an embodiment, the biological sample is blood, plasma, serum, blood cells, lacrimal fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid. , pleural fluid, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, irrigations or washes, aspirates, scrapings, bone marrow specimens, biopsy specimens and surgical specimens. In an embodiment, the biological sample is a biopsy sample or surgical specimen. In an embodiment, the biological sample is a tumor biopsy sample or tumor surgical specimen. In an embodiment, the tumor biopsy sample derived from the tumor is selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous squamous cell carcinoma (CSCC), and squamous cell carcinoma of the head and neck (SCCHN).

In embodiments, cellular levels and/or activity are measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or a combination thereof. . In an embodiment, the level and/or activity of the cells is measured by contacting the sample with an agent that specifically binds to one or more molecules selected from CD80, CD8, granzyme B, CD68, Ki67, and PD-L1. In an embodiment, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In an embodiment, the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof.

In an embodiment, the level and/or activity 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, CD68, Ki67, and PD-L1. . In an embodiment, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background level and/or activity of B cells and/or CD40+ cells is measured in a first biological sample obtained from the subject prior to administration of the first dose. In an embodiment, the level and/or activity of B cells and/or CD40+ cells N hours after administration is measured in a second biological sample obtained from the subject following administration of the first dose. In embodiments, N is a number from 1 to 24. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at M days post-administration is measured in a third biological sample obtained from the subject following administration of the first dose. In embodiments, M is a number from 1 to 28. In an embodiment, the method comprises: (ii) the level and/or activity of B cells and/or CD40+ cells at M days post-administration is at least about and administering to the human subject a second dose of the chimeric protein when 50% higher. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose.

In an embodiment, 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 an embodiment, 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 an embodiment, 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; 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 an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is greater than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is about 10%, or about 20%, or about 30% of the background level and/or activity of B cells and/or CD40+ cells. %, or within about 40%.

In an embodiment, the first biological sample, the second biological sample, and the third biological sample are blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing Nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, excretions, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, scrapings, bone marrow specimens, biopsy specimens. and independently selected from surgical specimens. In an embodiment, the first biological sample, the second biological sample and the third biological sample are blood.

In an embodiment, the level and/or activity of B cells and/or CD40+ cells is measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or It is measured by a combination of these. In an embodiment, the level and/or activity of B cells and/or CD40+ cells are measured by contacting the sample with an agent that specifically binds to CD40 and/or B-cell markers. In an embodiment, the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. In an embodiment, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In an embodiment, the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. In an embodiment, the level and/or activity of B cells and/or CD40+ cells are 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 an embodiment, the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. In an embodiment, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background amount and/or activity of a cytokine selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α and MDC is determined by administration of the first dose. It was measured in a first biological sample previously obtained from the subject. In an embodiment, the amount and/or activity of the cytokine N hours after administration is measured in a second biological sample obtained from the subject after administration of the first dose. In embodiments, N is a number from 1 to 24. In an embodiment, the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine. In an embodiment, the amount and/or activity of the cytokine at M days after administration is measured in a third biological sample obtained from the subject after administration of the first dose, where M is a number from 1 to 28. In an embodiment, the method comprises (ii) preparation of the chimeric protein when the amount and/or activity of the cytokine at M days after administration is at least about 30% lower than the amount and/or activity of the cytokine at N hours after administration. It further comprises administering 2 doses to the human subject. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose.

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background level of cells is measured in a pre-dose biological sample obtained from the subject prior to administration of the first dose, and the markers include CD80+ cells, CD8+ cells, granzyme B+ cells, CD68+ cells, Ki67+ cells, and PD-L1+ immune cells. is selected from one or more of: In an embodiment, the method further comprises administering a second dose of the chimeric protein to the human subject when the post-administration level of the cells is greater than the background level of the cells. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose.

In an embodiment, the biological sample is blood, plasma, serum, blood cells, lacrimal fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid. , pleural fluid, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, irrigations or washes, aspirates, scrapings, bone marrow specimens, biopsy specimens and surgical specimens. In an embodiment, the biological sample is a biopsy sample or surgical specimen. In an embodiment, the biological sample is a tumor biopsy sample or tumor surgical specimen. In an embodiment, the tumor biopsy sample or tumor surgical specimen derived from the tumor is selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous squamous cell carcinoma (CSCC), and squamous cell carcinoma of the head and neck (SCCHN).

In embodiments, cellular levels and/or activity are measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or a combination thereof. . In an embodiment, the level and/or activity of cells is measured by contacting the sample with an agent that specifically binds to one or more molecules selected from CD80, CD8, granzyme B, CD68, Ki67, and PD-L1. In an embodiment, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In an embodiment, the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. In an embodiment, the level and/or activity 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, CD68, Ki67, and PD-L1. . In an embodiment, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background level and/or activity of B cells and/or CD40+ cells is measured in a first biological sample obtained from the subject prior to administration of the first dose. In an embodiment, the level and/or activity of B cells and/or CD40+ cells N hours after administration is measured in a second biological sample obtained from the subject following administration of the first dose. In embodiments, N is a number from 1 to 24. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at M days post-administration is measured in a third biological sample obtained from the subject following administration of the first dose. In embodiments, M is a number from 1 to 28. In an embodiment, the method comprises: (ii) the level and/or activity of B cells and/or CD40+ cells at M days after administration is at least about about less than the level and/or activity of B cells and/or CD40+ cells at N hours after administration; and administering to the human subject a second dose of the chimeric protein when 50% higher. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose.

In an embodiment, 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 an embodiment, 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 an embodiment, 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; 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 an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is greater than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is about 10%, or about 20%, or about 30% of the background level and/or activity of B cells and/or CD40+ cells. %, or within about 40%.

In an embodiment, the first biological sample, the second biological sample, and the third biological sample are blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing Nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, excretions, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, scrapings, bone marrow specimens, biopsy specimens. and independently selected from surgical specimens. In an embodiment, the first biological sample, the second biological sample and the third biological sample are blood.

In an embodiment, the level and/or activity of B cells and/or CD40+ cells is measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or It is measured by a combination of these. In an embodiment, the level and/or activity of B cells and/or CD40+ cells are measured by contacting the sample with an agent that specifically binds to CD40 and/or B-cell markers. In an embodiment, the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. In an embodiment, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In an embodiment, the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. In an embodiment, the level and/or activity of B cells and/or CD40+ cells are 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 an embodiment, the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. In an embodiment, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

Aspects of the disclosure are methods of treating cancer in a human subject. In an embodiment, the method comprises administering to a human subject a first dose of a chimeric protein having the general structure of (i) N terminus - (a) - (b) - (c) - C terminus, a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)), and (b) is a linker adjacent to the first and second domains, wherein the linker is a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the background amount and/or activity of a cytokine selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α and MDC is determined by administration of the first dose. It was measured in a first biological sample previously obtained from the subject. In an embodiment, the amount and/or activity of the cytokine N hours after administration is measured in a second biological sample obtained from the subject after administration of the first dose. In embodiments, N is a number from 1 to 24. In an embodiment, the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine. In an embodiment, the amount and/or activity of the cytokine at M days post-administration is measured in a third biological sample obtained from the subject after administration of the first dose. In embodiments, M is a number from 1 to 28. In an embodiment, the method comprises (ii) preparation of the chimeric protein when the amount and/or activity of the cytokine at M days after administration is at least about 30% lower than the amount and/or activity of the cytokine at N hours after administration. It further comprises administering 2 doses to the human subject. In an embodiment, the second dose is administered at least about 48 hours after administration of the first dose.

In an embodiment, 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 an embodiment, 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 an embodiment, 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; 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 an embodiment, the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine. In an embodiment, the amount and/or activity of the cytokine at 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 at 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 an embodiment, the first biological sample, the second biological sample, and the third biological sample are blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing Nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, excretions, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, scrapings, bone marrow specimens, biopsy specimens. and independently selected from surgical specimens. In an embodiment, 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 determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. It is measured. In an embodiment, the amount and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds the cytokine. In an embodiment, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In an embodiment, the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. In an embodiment, 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. In an embodiment, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

Chimeric proteins disclosed herein, including the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61), find use in methods of treating both advanced solid tumors and advanced lymphomas. do. These tumor types include melanoma, non-small cell lung cancer (squamous, adenocarcinoma, adenocarcinoma-squamous cell), urothelial carcinoma, renal cell carcinoma, squamous cell cervical cancer, adenocarcinoma of the stomach or gastro-oesophageal junction, squamous cell carcinoma of the anus, and squamous cell of the head and neck. Includes high or mismatch repair deficient cell carcinoma, squamous cell carcinoma of the skin, and solid tumors excluding microsatellite instability high or 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 an embodiment, the cancer comprises an advanced solid tumor (local and/or metastatic). In an embodiment, the human subject has cancer, and the cancer being treated is characterized by having macrophages in the tumor microenvironment and/or having tumor cells that are CD47+ cells in the tumor. In an embodiment, administration of the SIRPα-Fc-CD40L chimeric protein blocks “don’t eat me” signaling and/or stimulates “eat me” signaling in tumor cells. In an embodiment, therapy with a SIRPα-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) stimulates macrophages to phagocytose tumor cells and render tumor antigens of the phagocytic tumor cells to T cells. Present effectively.

In an embodiment, the cancer is a solid tumor. In an embodiment, the cancer is a solid tumor. In an embodiment, the cancer is metastatic cancer. In an embodiment, the cancer is a hematological cancer. In an embodiment, the cancer expresses CD47.

In an embodiment, the cancer comprises advanced lymphoma. In an embodiment, the cancer comprises acute myeloid leukemia (AML). In an embodiment, the cancer comprises p53 mutant AML. In an embodiment, the cancer comprises high risk myelodysplastic syndrome (HR-MDS).

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

It is often desirable to enhance immunostimulatory signaling to augment the immune response, for example, to enhance the patient's anti-tumor immune response.

In an embodiment, the chimeric proteins of the present disclosure disrupt, block, reduce, inhibit, and/or sequester the transmission of immune inhibitory signals, e.g., derived from cancer cells attempting to evade detection and/or destruction. The extracellular domain of human CD172a (SIRPα), and the extracellular domain of human CD40L, which enhances, increases and/or stimulates the transduction of immunostimulatory signals to anti-cancer immune cells. Therefore, simultaneous binding of the extracellular domain of CD172a (SIRPα) to its ligand/receptor and binding of the extracellular domain of CD40L to its receptor will prevent the transmission of immunosuppressive signals from cancer cells and stimulate immune activity in immune system cells. will be. In other words, the chimeric proteins of the present disclosure can treat cancer through two distinct mechanisms.

In embodiments, the present disclosure relates to cancer and/or tumors; For example, it relates to the treatment or prevention of cancer and/or tumors. As disclosed elsewhere herein, cancer treatment, in embodiments, involves modulating the immune system with the chimeric proteins of the invention to favor increased or activated immune stimulatory signals. In embodiments, the method reduces the amount or activity of regulatory T cells (Treg) as compared to an untreated subject or a subject treated with an antibody associated with CD172a (SIRPα), CD40L, and/or their respective ligands or receptors. In embodiments, the method increases priming of effector T cells in the draining lymph nodes of a subject compared to an untreated subject or a subject treated with an antibody associated with CD172a (SIRPα), CD40L, and/or their respective ligands or receptors. In embodiments, the method comprises an overall reduction in immunosuppressive cells and migration to a more inflammatory tumor environment when compared to an untreated subject or a subject treated with antibodies associated with CD172a (SIRPα), CD40L, and/or their respective ligands or receptors. causes

In embodiments, the chimeric proteins can be used in methods that can or involve modulating the magnitude of an immune response, e.g., modulating the level of effector output. In embodiments, for example, when used to treat cancer, the present chimeric protein alters the degree of immune stimulation compared to immunosuppression to increase the amplitude of T cell responses, which may increase the likelihood of cytokine production, proliferation, or target killing. These include, but are not limited to, those that stimulate increased levels. In an embodiment, the patient's T cells are activated and/or stimulated by the chimeric protein, and the activated T cells are capable of dividing and/or secreting cytokines.

Cancer or tumor refers to the uncontrolled growth of cells and/or abnormally increased cell survival and/or inhibition of apoptosis that interferes with the normal functioning of the body's organs and systems. These include benign and malignant cancers, polyps, hyperplasia, as well as occult tumors or micrometastases. Also included are cells with abnormal proliferation that are not interfered with by the immune system (e.g., virally infected cells). The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be an area of cancer cells at the original site that becomes clinically detectable, or may be a primary tumor. In contrast, metastatic cancer may be the spread of disease from one organ or part to another non-adjacent organ or part. Metastatic cancer can be caused by cancer cells gaining the ability to invade and infiltrate normal tissue surrounding a localized area to form a new tumor, which may be a local metastasis. Cancer can also be caused by cancer cells acquiring the ability to penetrate lymphatic and/or blood vessel walls, and then cancer cells can circulate through the bloodstream to other areas and tissues in the body (thereby becoming a circulating tumor). become a cell). Cancer may be due to processes such as lymphatic or hematogenous spread. Cancer can also be caused by tumor cells that lodge in other areas, re-infiltrate through blood vessels or walls, and continue to proliferate, eventually forming another clinically detectable tumor. The cancer may be this new tumor, which may be a metastatic (or secondary) tumor.

Cancer may be caused by metastasized tumor cells, which may be secondary or metastatic tumors. The tumor cells will be identical to those of the original tumor. For example, if breast or colon cancer spreads to the liver, the secondary tumor, while present in the liver, consists of abnormal breast or colon cells rather than abnormal liver cells. Therefore, the tumor in the liver may be metastatic breast cancer or metastatic colon cancer rather than liver cancer.

Cancer can originate from any tissue. The cancer may originate from melanoma, colon, breast or prostate, and therefore may be composed of cells that are originally skin, colon, breast or prostate, respectively. Cancer can also be a hematological malignancy, which can be leukemia or lymphoma. Cancer can invade tissues such as the liver, lungs, bladder, or intestines.

In an embodiment, the chimeric protein is used to treat a subject with treatment-refractory cancer. In an embodiment, the chimeric protein is used to treat a subject who is refractory to one or more immuno-modulators. For example, in an embodiment, the chimeric protein is used to treat a subject who has no response to treatment after about 12 weeks of treatment, or a subject whose disease progresses. For example, in an embodiment, the subject is refractory to one or more CD172a (SIRPα) and/or CD47 agents, e.g., magrolimab (5F9), Hu5F9-G4, CC-90002, Ti-061, SRF231 , including patients refractory to TTI-621, TTI-622, or ALX148. For example, in an embodiment, the subject is a patient refractory to an anti-CTLA-4 agent, e.g., ipilimumab (YERVOY)-refractory patient (e.g., a melanoma patient). Accordingly, in embodiments, the present disclosure provides methods of treating cancer that rescue patients who are unresponsive to various therapies, including monotherapy of one or more immuno-modulatory agents.

In an embodiment, the present disclosure provides chimeric proteins that target cells or tissues within the tumor microenvironment. In an embodiment, cells or tissues within the tumor microenvironment express one or more targets or binding partners of the chimeric protein. The tumor microenvironment refers to the cellular environment that includes cells, secreted proteins, physiological small molecules, and blood vessels in which the tumor resides. In embodiments, cells or tissues within the tumor microenvironment include tumor blood vessels; Tumor-infiltrating lymphocytes; fibroblast fibroblast reticulocyte; vascular endothelial progenitor cells (EPC); cancer-related fibroblasts; Peripheral cells; other stromal cells; Components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cell; regulatory T cells; macrophages; neutrophils; and one or more of other immune cells located proximal to the tumor. In an embodiment, the chimeric protein targets cancer cells. In an embodiment, the cancer cell expresses one or more of the target or binding partner of the chimeric protein.

Activation of regulatory T cells is critically influenced 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 on T cells belonging to the CD28 or TNF receptor families, respectively. A number of well-defined co-inhibitors and their receptors belong to the B7 and CD28 families.

In an embodiment, an immunostimulatory signal refers to a signal that enhances an immune response. For example, in the context of oncology, these signals may enhance anti-tumor immunity. For example, without limitation, immunostimulatory signals can be identified by directly stimulating proliferation, cytokine production, killing activity, or phagocytosis of white blood cells. For example, chimeric proteins can directly stimulate proliferation and cytokine production of individual T cell subsets. Other examples include direct stimulation of immunosuppressive cells through receptors that inhibit the activity of these immunosuppressive cells. This includes, for example, stimulation of CD4+FoxP3+ regulatory T cells, which reduces the ability of those regulatory T cells to inhibit proliferation of conventional CD4+ or CD8+ T cells. In another example, this includes stimulation of CD40 on the surface of an antigen-presenting cell, enhancing the ability of that cell to present antigen in the context of an appropriate natural costimulatory molecule, including molecules of the B7 or TNF superfamily. causes the activation of In another example, the chimeric protein selectively further stimulates an immune response within the tumor by causing activation of lymphoid cells and/or production of pro-inflammatory cytokines or chemokines.

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

In an embodiment, the chimeric protein is a T cell (including but not limited to cytotoxic T lymphocytes, T helper cells, natural killer T (NKT) cells), B cells, natural killer cells within a tumor or tumor microenvironment. Use in a method that can cause or involves causing an increase in one or more of (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes and macrophages (e.g., one or more of M1 and M2) discover In an embodiment, the chimeric protein enhances recognition of tumor antigens by CD8+ T cells, particularly those T cells infiltrating the tumor microenvironment. In an embodiment, the chimeric protein induces CD19 expression and/or CD19 positive cells (e.g., increases the number of CD19 positive B cells). In an embodiment, the chimeric protein induces IL-15Rα expression and/or increases the number of IL-15Rα positive cells (e.g., IL-15Rα positive dendritic cells).

In an embodiment, the chimeric protein may be particularly useful for suppressing immunosuppressive cells (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor-associated neutrophils (TANs) within the tumor and/or tumor microenvironment (TME). ), M2 macrophages and tumor-associated macrophages (TAMs)) and/or find use in a method involving inhibiting and/or causing a reduction. In embodiments, the therapy may alter the ratio of M1 to M2 macrophages in the tumor site and/or the TME to favor M1 macrophages. In an embodiment, the SIRPα-Fc-CD40L chimeric protein inhibits/reduces/eliminates the transmission of the “don’t eat me” signal to tumor cells through Sipr1a/CD47. In an embodiment, the SIRPα-Fc-CD40L chimeric protein makes tumors more likely to be attacked by the subject's immune system. In an embodiment, the SIRPα-Fc-CD40L chimeric protein renders tumors more likely to be attacked by the subject's innate immune system. In an embodiment, the SIRPα-Fc-CD40L chimeric protein renders tumors more likely to be attacked by the subject's adaptive immune system. In an embodiment, the SIRPα-Fc-CD40L chimeric protein inhibits/reduces/eliminates the binding of tumor-overexpressed CD47 to phagocyte-expressed SIRPα to achieve phagocytic clearance of cancer cells and/or immunity to tumor antigens by macrophages and/or dendritic cells. Original processing may be permitted. In an embodiment, administration of the SIRPα-Fc-CD40L chimeric protein blocks “don’t eat me” signaling and/or stimulates “eat me” signaling in tumor cells. In an embodiment, therapy with a SIRPα-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) stimulates macrophages to phagocytose tumor cells and render tumor antigens of the phagocytic tumor cells to T cells. Present effectively.

In an embodiment, the chimeric protein comprises one or more of: IFNγ, TNFα, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, IL-17F, and IL-22. It can increase serum levels of various cytokines, including but not limited to. In embodiments, the chimeric protein may enhance IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, or IFNγ in the serum of the treated subject. In an embodiment, the chimeric protein does not increase serum levels of a particular cytokine. In an embodiment, the chimeric protein does not increase serum levels of IL-6 and/or TNFα. In an embodiment, the chimeric protein does not increase serum levels of IL-6 and/or TNFα in the serum of the treated subject. In an embodiment, the chimeric protein does not increase serum levels of IL-6 and/or TNFα in the serum of the treated subject, but includes, but is not limited to, CCL2, IL-8, and CXCL9 in the serum of the treated subject. Increases levels of other cytokines. Detection of these cytokine responses may provide a way to determine the optimal dosing regimen for the indicated chimeric protein.

In the chimeric proteins of the present disclosure, the chimeric proteins can increase or prevent decline in subpopulations of CD4+ and/or CD8+ T cells.

In the chimeric proteins of the present disclosure, the chimeric proteins can enhance tumor killing activity by T cells.

In an embodiment, 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 transmit an immunosuppressive signal when bound by the first domain of the chimeric protein. (b) a quantifiable cytokine response is achieved in the subject's peripheral blood, and/or (c) tumor growth is reduced compared to subjects treated with a CD40 agonist antibody and/or a CD47 blocking antibody. It is reduced in subjects that need it.

In an embodiment, the chimeric protein inhibits, blocks and/or reduces cell death of anti-tumor CD8+ and/or CD4+ T cells; or stimulate, induce and/or increase cell death of pre-neoplastic T cells. T cell exhaustion is a state of T cell dysfunction characterized by progressive loss of proliferative and effector functions, ending in clonal exhaustion. Thus, preneoplastic T cells refer to a state of T cell dysfunction that occurs during a number of chronic infectious inflammatory diseases and cancer. This dysfunction is defined by poor proliferative and/or effector function, and persistent expression and transcriptional status of inhibitory receptors independent of functional effector or memory T cells. Depletion prevents optimal control of infections and tumors. Exemplary pre-neoplastic T cells include, but are not limited to, Tregs, CD4+ and/or CD8+ T cells that express one or more checkpoint inhibitory receptors, Th2 cells, and Th17 cells. Checkpoint inhibitory receptors refer to receptors expressed on immune cells that prevent or inhibit uncontrolled immune responses. In contrast, anti-tumor CD8+ and/or CD4+ T cells refer to T cells that can initiate an immune response against a tumor.

In embodiments, the chimeric protein can increase the ratio of effector T cells to regulatory T cells and can be used in methods comprising the same. Exemplary 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., CD62Llow, CD44 ⁺ , TCR, CD3 ⁺ , IL ^- 7R/CD127 ⁺ , IL-15R ⁺ , CCR7low); Central memory T cells (e.g. , CCR7 ⁺ , CD62L ⁺ , CD27 ⁺ ; or CCR7hi, CD44 ⁺ , CD62Lhi, TCR, CD3 ⁺ , IL-7R/CD127 ⁺ , IL-15R ⁺ ); CD62L ⁺ effector T cells; CD8 ⁺ effector memory T cells (TEM), including early effector memory T cells (CD27 ⁺ CD62L ^- ) and late effector memory T cells (CD27 ^- CD62L ^- ) (TemE and TemL, respectively); CD127( ⁺ )CD25(low/-) 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 αβ TCR, 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., αβ TCR, CD3 ⁺ , CD4 ⁺ , IL-23R ⁺ , CCR6 ⁺ , IL-1R ⁺ ); CD4 ⁺ CD45RO ⁺ CCR7 ⁺ effector T cells, CD4 ⁺ CD45RO ⁺ CCR7( ^- ) effector T cells; and effector T cells secreting IL-2, IL-4 and/or IFN-γ. Exemplary regulatory T cells include ICOS ⁺ regulatory T cells, CD4 ⁺ CD25 ⁺ FOXP3 ⁺ regulatory T cells, CD4 ⁺ CD25 ⁺ regulatory T cells, CD4 ⁺ CD25 ^- regulatory T cells, CD4 ⁺ CD25high regulatory T cells, TIM-3 ⁺ CD172a (SIRPα) ⁺ regulatory T cells, lymphocyte activation gene-3 (LAG-3) ⁺ regulatory T cells, CTLA-4/CD152 ⁺ regulatory T cells, neuropilin-1 (Nrp-1) ⁺ regulatory T cells, CCR4 ⁺ CCR8 ⁺ regulatory T cells, CD62L (L-selectin) ⁺ regulatory T cells, CD45RBlow regulatory T cells, CD127low 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 (Tr1 cells), T helper type 3 (Th3) cells, regulatory cells of the natural killer T cell phenotype (NKTregs), CD8 ⁺ regulatory T cells , CD8 ⁺ CD28 ^- regulatory T cells and/or regulatory T-cells secreting IL-10, IL-35, TGF-β, TNF-α, galectin-1, IFN-γ and/or MCP1.

In an embodiment, the chimeric protein of the invention results in an increase in effector T cells (e.g., CD4+CD25- T cells).

In an embodiment, the chimeric protein causes a decrease in regulatory T cells (e.g., CD4+CD25+ T cells).

In an embodiment, the chimeric protein generates a memory response that, for example, prevents relapse or protects the animal from relapse and/or prevents metastasis or reduces the likelihood of metastasis. Accordingly, animals treated with the chimeric protein are subsequently able to attack tumor cells and/or prevent the development of tumors when rechallenged following initial treatment with the chimeric protein. Accordingly, the chimeric proteins of the present disclosure stimulate both active tumor destruction and also immune recognition of tumor antigens, which are essential for programming memory responses that can prevent relapse.

In embodiments, the chimeric protein can cause activation of an antigen presenting cell. In embodiments, the chimeric protein can enhance the ability of an antigen presenting cell to target the antigen of interest.

In embodiments, the chimeric protein is capable of transiently stimulating effector T cells for at least about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours, or about 1 week, or about 2 weeks; , can be used in a method including this. In an embodiment, the transient stimulation of effector T cells is substantially in the patient's bloodstream or in a specific cell comprising lymphoid tissue, such as bone marrow, lymph nodes, spleen, thymus, mucosa-associated lymphoid tissue (MALT), non-lymphoma tissue. Occurs in the tissue/site or in the tumor microenvironment.

In the chimeric proteins of the present disclosure, the chimeric proteins unexpectedly provide binding of extracellular domain components to their respective binding partners at a slow off rate (K _d or K _off ). In embodiments, this provides unexpectedly long interactions of the receptor to the ligand and vice versa. This effect allows for longer positive signaling effects, such as increased or activated immune stimulatory signals. For example, the present chimeric protein may enable sufficient signaling, e.g., through long off-rate binding, to provide immune cell proliferation, enable anti-tumor attack, and enable sufficient signaling to provide Stimulating signals, such as release of cytokines, are provided.

In the chimeric proteins of the present disclosure, the chimeric proteins are capable of forming stable synapses between cells. Stable synapses of cells facilitated by a chimeric protein (e.g., between cells carrying a negative signal) position T cells to attack tumor cells and/or target cells beyond those masked by the chimeric protein of the invention. It provides spatial orientation advantageous for tumor reduction, such as sterically preventing tumor cells from transmitting negative signals, including negative signals. In an embodiment, this provides a longer on-target (e.g., intratumoral) half-life (t _1/2 ) compared to the serum t _1/2 of the chimeric protein. These properties could have the combined advantage of reducing off-target toxicity associated with systemic distribution of the chimeric protein.

In embodiments, the chimeric protein may provide a sustained immunomodulatory effect.

This chimeric protein provides a synergistic therapeutic effect (e.g., antitumor effect) because it allows for improved site-specific interaction of the two immunotherapy agents. In an embodiment, the present chimeric protein provides the potential for reducing off-site and/or systemic toxicity.

In an embodiment, the chimeric protein exhibits an improved safety profile. In an embodiment, the chimeric protein exhibits a reduced toxicity profile. For example, administration of the chimeric protein may cause diarrhea, inflammation (e.g., intestinal), or weight loss that occurs following administration of antibodies directed to the ligand(s)/receptor(s) targeted by the extracellular domain of the chimeric protein. May result in reduced side effects, such as one or more of the following: In an embodiment, the chimeric protein also provides improved safety compared to antibodies directed to the ligand(s)/receptor(s) targeted by the extracellular domain of the chimeric protein without sacrificing efficacy.

In an embodiment, the subject chimeric protein has reduced side effects against current immunotherapies, e.g., antibodies directed to the ligand(s)/receptor(s) targeted by the extracellular domain of the subject chimeric protein, e.g. Provides GI complications. Exemplary GI complications include abdominal pain, loss of appetite, autoimmune effects, constipation, cramping, dehydration, diarrhea, eating problems, fatigue, flatulence, intra-abdominal fluid or ascites, gastrointestinal (GI) dysbiosis, GI mucositis, and inflammatory bowel disease. , irritable bowel syndrome (IBS-D and IBS-C), nausea, pain, stool or urine changes, ulcerative colitis, vomiting, weight gain from fluid retention, and/or weakness.

pharmaceutical composition

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

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

In an embodiment, the chimeric proteins disclosed herein are provided as a sterile frozen solution in a vial or as a sterile liquid solution in a vial. The drug product comprising the chimeric protein disclosed herein is a sterile-filtered, formulated chimeric protein solution disclosed herein filled in 10 mL single-use glass vials capped with Flurotec® rubber stoppers and sealed with aluminum flip off seals. Includes. In an embodiment, the chimeric protein disclosed herein is administered at about 10 mg/ml to about 30 mg/ml, e.g., about 20 mg/ml at about 30mM to about 70mM L-histidine, e.g., about 20 mg/ml in water for injection. Formulated with 50mM L-histidine and about 125mM to about 400mM sucrose, for example about 250mM sucrose. In an embodiment, each vial contains about 1 mL of drug product, or about 20 mg of the chimeric protein disclosed herein.

Chimeric proteins disclosed herein, including the CD172a(SIRPα)-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61), contain a sufficiently basic functional group capable of reacting with anhydrous or organic acid or an inorganic or organic base. It has a carboxyl group that can react with and can form a pharmaceutically acceptable salt. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids, as is well known in the art. Such salts are described, for example, in Journal of Pharmaceutical Science , 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use . PH Stahl and CG Wermuth (eds.), Verlag, Zurich (Switzerland) 2002].

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

Additionally, any of the chimeric proteins disclosed herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle, e.g., a pharmaceutical composition. Such pharmaceutical compositions may optionally contain suitable amounts of pharmaceutically acceptable excipients to provide a form for appropriate administration. Pharmaceutical excipients may be oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., and liquids such as water. Pharmaceutical excipients may be, for example, saline solution, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, auxiliaries, stabilizers, thickeners, lubricants and colorants may be used. In an embodiment, the pharmaceutically acceptable excipient when administered to a subject is sterile. Water is a useful excipient when any of the formulations disclosed herein are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, Includes ethanol, etc. Any of the formulations disclosed herein may, if desired, also include minor amounts of wetting or emulsifying agents or pH buffering agents.

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

In embodiments, the chimeric protein may be conjugated and/or fused with other agents to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In an embodiment, the chimeric protein is PEG, It may be fused or conjugated to one or more of GLK, CTP, transferrin, etc. In an embodiment, each of the individual chimeric proteins is fused to one or more of the agents described in Strohl, BioDrugs 29(4):215-239 (2015), which is incorporated herein by reference in its entirety.

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

If desired, the pharmaceutical composition comprising the chimeric protein (may also comprise a solubilizing agent. Additionally, the formulation may be delivered by a suitable vehicle or delivery device as is known in the art. The composition for administration may be Optionally, a local anesthetic, for example lignocaine, may be included to reduce pain at the injection site.

Pharmaceutical compositions comprising the chimeric proteins of the present disclosure can conveniently be provided in unit dosage form and can be prepared by any method well known in the pharmaceutical art. These methods generally include combining the therapeutic agent with a carrier that constitutes one or more accessory ingredients. Typically, a pharmaceutical composition is uniformly and intimately combined with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, the product is formed into the desired dosage form (e.g., wet or dry granules, powder formulations). etc., are then manufactured by forming them into dosage forms (tablets) using conventional methods known in the art.

In embodiments, any of the chimeric proteins disclosed herein are formulated according to routine procedures as pharmaceutical compositions suitable for the modes of administration disclosed herein.

Administration, Dosing and Treatment Regimen

Aspects of the disclosure are methods of assessing the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject is suffering from cancer. In an embodiment, the method comprises (i) obtaining a biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the background level and/or activity of cells is measured in a biological sample obtained from the subject prior to administration of the first dose. In an embodiment, 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. In an embodiment, the method further comprises (ii) determining post-administration levels and activity of cells in the biological sample. In an embodiment, the method further comprises the step of (iii) determining that the chimeric protein is efficacious if the level and/or activity of the cells after administration is greater than the background level and/or activity of the cells. In an embodiment, the biological sample is a tumor biopsy sample or tumor surgical specimen. In embodiments, cellular levels and/or activity are measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or a combination thereof. .

An aspect of the disclosure is a method of selecting a subject for treatment with therapy for cancer. In an embodiment, the method comprises (i) obtaining a biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the background level and/or activity of cells is measured in a biological sample obtained from the subject prior to administration of the first dose. In an embodiment, 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. In an embodiment, the method further comprises (ii) determining post-administration levels and activity of cells in the biological sample. In an embodiment, the method further comprises the step of (iii) selecting the subject for treatment with the chimeric protein if the level and/or activity of the cells after administration is greater than the background level and/or activity of the cells. In an embodiment, the biological sample is a tumor biopsy sample or tumor surgical specimen. In embodiments, cellular levels and/or activity are measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or a combination thereof. .

In an embodiment, the biological sample is blood, plasma, serum, blood cells, lacrimal fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid. , pleural fluid, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, irrigations or washes, aspirates, scrapings, bone marrow specimens, biopsy specimens and surgical specimens. In an embodiment, the biological sample is a biopsy sample or surgical specimen. In an embodiment, the biological sample is a tumor biopsy sample or tumor surgical specimen. In an embodiment, the tumor biopsy sample or tumor surgical specimen derived from the tumor is selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous squamous cell carcinoma (CSCC), and squamous cell carcinoma of the head and neck (SCCHN).

In embodiments, cellular levels and/or activity are measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or a combination thereof. . In an embodiment, the level and/or activity of cells is measured by contacting the sample with an agent that specifically binds to one or more molecules selected from CD80, CD8, granzyme B, CD68, Ki67, and PD-L1. In an embodiment, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In an embodiment, the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. In an embodiment, the level and/or activity 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, CD68, Ki67, and PD-L1. . In an embodiment, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

Aspects of the disclosure are methods of assessing the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject is suffering from cancer. In an embodiment, the method comprises (i) obtaining a first biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the method further comprises determining the background level and/or activity of B cells and/or CD40+ cells in the first biological sample. In an embodiment, the method further comprises the step of (iii) obtaining a second biological sample obtained from the subject at N hours after administration, where N is 1 to 24. In an embodiment, the method further comprises determining the level and/or activity of B cells and/or CD40+ cells in the second biological sample at N hours after administration. In an embodiment, the method further comprises (iv) obtaining a third biological sample obtained from the subject on M days after administration, wherein M is 1 to 28. In an embodiment, the method further comprises determining the level and/or activity of B cells and/or CD40+ cells in a third biological sample at day M after administration. In an embodiment, the method comprises: (v) the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells, and/or A chimeric protein is efficacious if the level and/or activity of B cells and/or CD40+ cells at M days after administration is at least about 50% higher than the level and/or activity of B cells and/or CD40+ cells at N hours after administration. It further includes the step of determining that there is.

An aspect of the disclosure is a method of selecting a subject for treatment with therapy for cancer. In an embodiment, the method comprises (i) obtaining a first biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the method further comprises determining the background level and/or activity of B cells and/or CD40+ cells in the first biological sample. In an embodiment, the method further comprises the step of (iii) obtaining a second biological sample obtained from the subject at N hours after administration, where N is 1 to 24. In an embodiment, the method further comprises determining the level and/or activity of B cells and/or CD40+ cells in the second biological sample at N hours after administration. In an embodiment, the method further comprises (iv) obtaining a third biological sample obtained from the subject on M days after administration, wherein M is 1 to 28. In an embodiment, the method further comprises determining the level and/or activity of B cells and/or CD40+ cells in a third biological sample at day M after administration. In an embodiment, the method comprises: (v) the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells, and/or Treatment with a chimeric protein, if the level and/or activity of B cells and/or CD40+ cells at M days after administration is at least about 50% higher than the level and/or activity of B cells and/or CD40+ cells at N hours after administration. It further includes the step of selecting an object.

In an embodiment, 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 an embodiment, 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 an embodiment, 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; 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 an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is greater than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than the background level and/or activity of B cells and/or CD40+ cells. In an embodiment, the level and/or activity of B cells and/or CD40+ cells at N hours after administration is about 10%, or about 20%, or about 30% of the background level and/or activity of B cells and/or CD40+ cells. %, or within about 40%.

In an embodiment, the first biological sample, the second biological sample, and the third biological sample are blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing Nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, excretions, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, scrapings, bone marrow specimens, biopsy specimens. and independently selected from surgical specimens. In an embodiment, the first biological sample, the second biological sample and the third biological sample are blood.

In an embodiment, the level and/or activity of B cells and/or CD40+ cells is measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or It is measured by a combination of these. In an embodiment, the level and/or activity of B cells and/or CD40+ cells are measured by contacting the sample with an agent that specifically binds to CD40 and/or B-cell markers. In an embodiment, the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. In an embodiment, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In an embodiment, the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. In an embodiment, the level and/or activity of B cells and/or CD40+ cells are 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 an embodiment, the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. In an embodiment, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

Aspects of the disclosure are methods of assessing the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject is suffering from cancer. In an embodiment, the method comprises (i) obtaining a first biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the method further comprises the step of (ii) determining the background amount and/or activity of the cytokine in the first biological sample. In an embodiment, the cytokine is selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC. In an embodiment, the method further comprises the step of (iii) obtaining a second biological sample obtained from the subject at N hours after administration, where N is 1 to 24. In an embodiment, the method further comprises determining the amount and/or activity of the cytokine in the second biological sample at N hours after administration. In an embodiment, the method further comprises (iv) obtaining a third biological sample obtained from the subject on M days after administration, wherein M is 1 to 28. In an embodiment, the method further comprises determining the amount and/or activity of the cytokine in a third biological sample at M days after administration. In an embodiment, the method comprises: (v) the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine; and/or the amount and/or activity of the cytokine at M days after administration and /or determining that the chimeric protein is efficacious if the activity is at least about 50% less than the amount and/or activity of the cytokine at N hours after administration.

An aspect of the disclosure is a method of selecting a subject for treatment with therapy for cancer. In an embodiment, the method comprises (i) obtaining a first biological sample obtained from a subject who has received a first dose of the chimeric protein. In an embodiment, the chimeric protein has the general structure of N terminus - (a) - (b) - (c) - C terminus, where (a) represents the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first and second domains, wherein the linker includes a hinge-CH2-CH3 Fc domain, and (c) is an extracellular domain of human CD40 ligand (CD40L). It is a second domain containing. In an embodiment, the method further comprises the step of (ii) determining the background amount and/or activity of the cytokine in the first biological sample. In an embodiment, the cytokine is selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α, and MDC. In an embodiment, the method further comprises the step of (iii) obtaining a second biological sample obtained from the subject at N hours after administration, where N is 1 to 24. In an embodiment, the method further comprises determining the amount and/or activity of the cytokine in the second biological sample at N hours after administration. In an embodiment, the method further comprises (iv) obtaining a third biological sample obtained from the subject on M days after administration, wherein M is 1 to 28. In an embodiment, the method further comprises determining the amount and/or activity of the cytokine in a third biological sample at M days after administration. In an embodiment, the method comprises: (v) the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine; and/or the amount of the cytokine at M days after administration. and/or if the activity is at least about 50% less than the amount and/or activity of the cytokine at N hours after administration, selecting the subject for treatment with the chimeric protein.

In an embodiment, 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 an embodiment, 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 an embodiment, 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; 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 an embodiment, the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine. In an embodiment, the amount and/or activity of the cytokine at 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 at 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 an embodiment, the first biological sample, the second biological sample, and the third biological sample are blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing Nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, excretions, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, scrapings, bone marrow specimens, biopsy specimens. and independently selected from surgical specimens. In an embodiment, 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 determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. It is measured. In an embodiment, the amount and/or activity of the cytokine is measured by contacting the sample with an agent that specifically binds the cytokine. In an embodiment, the agent that specifically binds to one or more molecules is an antibody or fragment thereof. In an embodiment, the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. In an embodiment, 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. In an embodiment, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

In an embodiment, the first dose of chimeric protein ranges from about 0.03 mg/kg to 10 mg/kg. In an embodiment, 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 10 mg/kg.

In an embodiment, the method of any aspect disclosed herein further comprises administering a second dose of the chimeric protein. In an embodiment, 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 It is administered over 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. In an embodiment, the second dose of chimeric protein ranges from about 0.03 mg/kg to 10 mg/kg. In an embodiment, 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 10 mg/kg.

In an embodiment, the method of any aspect disclosed herein further comprises administering a second dose of the chimeric protein. In an embodiment, 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 It is administered over 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. In an embodiment, the second dose of chimeric protein ranges from about 0.03 mg/kg to 10 mg/kg. In an embodiment, 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 10 mg/kg.

In an embodiment, the first domain is capable of binding CD172a (SIRPα) ligand. In an embodiment, the first domain comprises substantially all of the extracellular domain of CD172a (SIRPα). In an embodiment, the first domain 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. Includes amino acid sequence.

In an embodiment, the second domain is capable of binding the CD40 receptor. In an embodiment, the second domain comprises substantially all of the extracellular domain of CD40L. In an embodiment, the second domain 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. Includes amino acid sequence.

In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4. In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from human IgG4. In an embodiment, the linker 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%, with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. or comprises amino acid sequences that are at least about 99% identical.

In an embodiment, (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 SEQ ID NO: 1, SEQ ID NO: 2 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: 3. In an embodiment, 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 an embodiment, the chimeric protein 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 the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61. Contains 99% identical amino acid sequences. In an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61.

In an embodiment, the human subject has or is suspected of having an advanced solid tumor or lymphoma. In an embodiment, the human subject has, or is suspected of having, a cancer selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, squamous cell carcinoma of the skin (CSCC), and squamous cell carcinoma of the head and neck (SCCHN). In an embodiment, the human subject has failed one or more platinum-based therapies. In an embodiment, the human subject is ineligible for platinum therapy. In an embodiment, the human subject is not receiving concurrent chemotherapy, immunotherapy, biologic, or hormonal therapy, and/or the human subject has received standard therapy, is tolerant of, or is ineligible for standard therapy, and/or the cancer is subject to standard therapy. There are no approved therapies considered curative.

In an embodiment, the chimeric proteins disclosed herein are provided as a sterile frozen solution at a concentration of about 20 mg/ml and a total volume of about 1 ml, optionally in 10 ml glass vials. In an embodiment, the chimeric proteins disclosed herein are administered by intravenous (IV) infusion following dilution with normal saline. Starting doses, dose escalation plans, and dosing schedules for certain embodiments are presented below.

In an embodiment, the dose of chimeric protein administered is at least 0.0001 mg/kg, such as about 0.0001 mg/kg to about 10 mg/kg. In an embodiment, the dose of chimeric protein administered is at least about 0.3 mg/kg, such as at least about 0.3 mg/kg, or about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4 mg. /kg, or about 6 mg/kg, or about 8 mg/kg, or about 10 mg/kg. In an embodiment, the dose of chimeric protein administered is at least about 1 mg/kg, such as at least about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4 mg/kg, or about 6 mg. /kg, or about 8 mg/kg, or about 10 mg/kg. In embodiments, the dose of the SIRPα-Fc-CD40L chimeric protein is not limited by anemia or other cytopenic effects and is therefore higher compared to certain other therapeutic agents (e.g., anti-CD47 antibodies or SIRPalphaFc fusion proteins). Capacity is acceptable. Additionally, in embodiments, low dose priming is not necessary.

In an embodiment, administration is intravenous. In an embodiment, administration is intratumoral. In an embodiment, administration is by injection. In an embodiment, administration is by injection. In an embodiment, administration is performed by intravenous infusion. In an embodiment, administration is performed by intratumoral injection.

In an embodiment, the approximate chimeric protein is administered at an initial dose (e.g., 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 doses. In embodiments, about one or more subsequent administrations are administered at 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 It has one or more doses of about 10 mg/kg.

In embodiments, the starting dose and/or subsequent doses are at or below the maximum tolerated dose.

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

In an embodiment, the dose is increased in about 1/2 log increments between one or more subsequent doses, e.g., from 0.001 mg/kg to 0.003 mg/kg and from 0.003 mg/kg to 0.01 mg/kg.

In an embodiment, the human subject has failed one or more platinum-based therapies and is optionally ineligible for platinum therapy. In an embodiment, the human subject is not receiving concurrent chemotherapy, immunotherapy, biologic, or hormonal therapy, and/or the human subject has received standard therapy, is tolerant of, or is ineligible for standard therapy, and/or the cancer is subject to standard therapy. There are no approved therapies considered curative.

In an embodiment, the initial dose is less than at least one of the subsequent administrations, e.g., less than the dose for each subsequent administration.

In an embodiment, the initial dose is equal to at least one of the subsequent administrations, e.g., the dose for each subsequent administration.

In an embodiment, the chimeric protein is administered at least about once per month.

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

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

In an embodiment, the chimeric protein is first administered once weekly for three weeks, and then the chimeric protein is administered about once every three weeks or once every four weeks.

In an embodiment, the chimeric protein is first administered once weekly for three weeks, and then the chimeric protein is administered about twice per month. For example, the chimeric protein is initially administered once a week for three weeks, and then the chimeric protein is administered approximately once every two weeks.

In an embodiment, the chimeric protein is administered at least about 4 times per month. For example, the chimeric protein is administered approximately once a week. In an embodiment, the chimeric protein is administered once per week (once every 7 days). In an embodiment, the chimeric protein is administered once every two weeks.

In an embodiment, administration of the SIRPα-Fc-CD40L chimeric protein does not cause anemia or other cytopenias in the patient. In an embodiment, administration does not cause lysis of RBCs. In an embodiment, administration of a SIRPα-Fc-CD40L chimeric protein is less likely to cause anemia or other cytopenias than, for example, an anti-CD47 Ab. In embodiments, the dose of the SIRPα-Fc-CD40L chimeric protein is not limited by anemia or other cytopenic effects and is therefore higher compared to certain other therapeutic agents (e.g., anti-CD47 antibodies or SIRPalphaFc fusion proteins). Capacity is acceptable. Additionally, in embodiments, low dose priming is not necessary.

Another advantage offered by the SIRPα-Fc-CD40L chimeric protein (e.g., SEQ ID NO: 59 or SEQ ID NO: 61) is that it does not cause anemia or other cytopenias in the patient despite targeting. This is because, as shown herein, the CD47/SIRPα interaction plays an important role in the lysis of RBCs, but the SIRPα-Fc-CD40L chimeric protein does not cause lysis of RBCs. Therefore, the method is less likely to cause anemia or other cytopenias than, for example, anti-CD47 Abs.

Chimeric proteins can be administered intravenously into the bloodstream by intravenous infusion or bolus injection. The chimeric protein can be administered intravenously by intravenous infusion to patients suffering from advanced ovarian cancer, fallopian tube cancer, and primary peritoneal cancer.

Chimeric proteins can be administered by intratumoral injection. In embodiments, the therapeutic dose for intratumoral administration is equal to or less than the therapeutic dose for intravenous infusion. In an embodiment, the therapeutic dose for intratumoral administration is the same as the therapeutic dose for intravenous infusion. In an embodiment, the therapeutic dose for intratumoral administration is less than the therapeutic dose for intravenous infusion. In an embodiment, the therapeutic dose for intratumoral administration to a patient suffering from advanced or metastatic CSCC and HNSCC.

In embodiments, the present chimeric protein allows for a dual effect providing fewer side effects than seen with conventional immunotherapy (e.g., treatment with one or more of OPDIVO, KEYTRUDA, YERVOY and TECENTRIQ). For example, the chimeric protein may be involved in a variety of commonly observed immune-related adverse reactions affecting various tissues and organs, including the skin, gastrointestinal tract, kidneys, peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and endocrine system. For example, it reduces or prevents hypophysitis, colitis, hepatitis, pneumonia, rash and rheumatic diseases.

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

The dosage, as well as the dosing schedule, of any of the chimeric proteins disclosed herein may depend on a variety of 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 disclosure relates to a method of treating cancer in a human subject in need thereof, the method comprising the general structure of the N terminus - (a) - (b) - (c) - C terminus. administering to a human subject an effective amount of the chimeric protein, wherein (a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a(SIRPα)), and (b) is the first and A linker adjacent to the second domain, wherein the linker includes a hinge-CH2-CH3 Fc domain, (c) is a second domain including the extracellular domain of human CD40 ligand (CD40L), and the administering step is phase 2 dosing. Includes. In an embodiment, the first phase and the second phase each independently comprise a dosing frequency of about twice a week to about once every two months. In an embodiment, the linker includes at least one cysteine residue capable of forming a disulfide bond. In an embodiment, the chimeric protein is, for example, from about 0.3 mg/kg to about 3 mg/kg, or from about 0.5 mg/kg to about 3 mg/kg, or from about 0.7 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 3 mg/kg, or about 2.5 mg/kg to about 3 mg/kg. kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about 0.3 mg/kg to about 2 mg/kg, or about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 It exhibits a linear dose response in a dose range of mg/kg, or about 0.3 mg/kg to about 0.5 mg/kg. In an embodiment, the chimeric protein does not exhibit a bell-shaped dose response.

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

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

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

In embodiments, the dosing frequency of the first phase is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks; The frequency of the second phase can be from about every 1 week to about every 2 weeks, from about every 10 days to about every 3 weeks, from about every 2 weeks to about every 4 weeks, from about every 3 weeks to about every 5 weeks, from about every 4 weeks to about Selected every 6 weeks.

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

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

In an embodiment, the chimeric protein disclosed herein is a human CD172a(SIRPα)-Fc-CD40L chimeric protein.

In one aspect, the disclosure relates to a method of treating cancer in a human subject in need thereof, the method comprising the general structure of the N terminus - (a) - (b) - (c) - C terminus. administering to a human subject an effective amount of the chimeric protein, wherein (a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a(SIRPα)), and (b) is the first and a linker adjacent to the second domain, wherein the linker comprises a hinge-CH2-CH3 Fc domain, (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L), and the administering step is the first cycle. , including the second cycle and the third cycle. In an embodiment, the linker includes at least one cysteine residue capable of forming a disulfide bond. In an embodiment, the first cycle, the second cycle, and the third cycle each independently comprise a dosing frequency of about 2 times per week to about 1 time every 2 months. In an embodiment, 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 an embodiment, 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 an embodiment, the dosing frequency in the first cycle is about every 3 days, about twice a week, about every week, about every 10 days, about 2 times every 3 weeks, about every 2 weeks, about every 3 weeks, about Every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, and about every 2 months. In an embodiment, the chimeric protein is, for example, from about 0.3 mg/kg to about 3 mg/kg, or from about 0.5 mg/kg to about 3 mg/kg, or from about 0.7 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 3 mg/kg, or about 2.5 mg/kg to about 3 mg/kg. kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about 0.3 mg/kg to about 2 mg/kg, or about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 It exhibits a linear dose response in a dose range of mg/kg, or about 0.3 mg/kg to about 0.5 mg/kg. In an embodiment, the chimeric protein does not exhibit a longitudinal dose response.

In embodiments, the dosing frequency in the first cycle is from about every 3 days to about every 10 days, from about every week to about every 2 weeks, from about every 10 days to about every 3 weeks, from about every 2 weeks to about every 4 weeks. , about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and about every 6 weeks to about every 2 months. is selected from In an embodiment, the dosing frequency in the second cycle is about every 3 days, about twice a week, about every week, about every 10 days, about 2 times every 3 weeks, about every 2 weeks, about every 3 weeks, about Every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, and about every 2 months. In embodiments, the dosing frequency in the second cycle is from about every 3 days to about every 10 days, from about every week to about every 2 weeks, from about every 10 days to about every 3 weeks, from about every 2 weeks to about every 4 weeks. , about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and about every 6 weeks to about every 2 months. is selected from In an embodiment, the dosing frequency in the third cycle is about every 3 days, about twice a week, about every week, about every 10 days, about 2 times every 3 weeks, about every 2 weeks, about every 3 weeks, about Every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, and about every 2 months. In an embodiment, the dosing frequency in the third cycle is from about every 3 days to about every 10 days, from about every week to about every 2 weeks, from about every 10 days to about every 3 weeks, from about every 2 weeks to about every 4 weeks. , about every 3 weeks to about every 5 weeks, about every 4 weeks to about every 6 weeks, about every 5 weeks to about every 7 weeks, about every 6 weeks to about every 8 weeks, and about every 6 weeks to about every 2 months. is selected from In an embodiment, the dosing frequency in the first cycle is selected from about every 3 days to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks; The frequency of the second cycle can be from about every 1 week to about every 2 weeks, from about every 10 days to about every 3 weeks, from about every 2 weeks to about every 4 weeks, from about every 3 weeks to about every 5 weeks, from about every 4 weeks to about every 4 weeks. Selections are made approximately every 6 weeks.

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

Additionally or alternatively, in embodiments, the effective amount for the first cycle, second cycle, and third cycle each independently comprises about 0.01 mg/kg to about 10 mg/ml. In embodiments, the effective amount for the first cycle, second cycle and third cycle is about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 10 mg/kg, and any two of the foregoing values and/or any range between the two, each independently selected. In embodiments, the effective amount for the first cycle, second cycle, and third cycle is about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about are each independently selected from 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, and about 1 mg/kg to about 10 mg/kg.

In an embodiment, the effective amounts for the first cycle, second cycle, and third cycle are the same. In other embodiments, the effective amounts for the first, second and third cycles are different. In an embodiment, the effective amount for the first cycle is greater than the effective amount for the second cycle. In another embodiment, the effective amount for the first cycle is less than the effective amount for the second cycle. In another embodiment, the effective amount for the first cycle and the effective amount for the second cycle are the same.

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

In an embodiment, the chimeric protein disclosed herein is a human CD172a(SIRPα)-Fc-CD40L chimeric protein.

In one aspect, the disclosure relates to a method of treating cancer in a human subject in need thereof, the method comprising the general structure of the N terminus - (a) - (b) - (c) - C terminus. Administering to a human subject an effective amount of the chimeric protein in a dosage regimen, wherein (a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a(SIRPα)), and (b) is: a linker adjacent the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L), the dosing regimen being approximately It involves dosing at a frequency ranging from every three days to about two months. In an embodiment, the linker includes at least one cysteine residue capable of forming a disulfide bond. In embodiments, the dosing regimen is about every 3 days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, about every 2 weeks, about every 3 weeks, about every 4 weeks, selected from approximately every month, approximately every 5 weeks, approximately every 6 weeks, approximately every 7 weeks, approximately every 8 weeks, and approximately every 2 months. In embodiments, the dosage regimen is about every week, about every 10 days, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about every 7 weeks. , about every 8 weeks and about every 2 months. In embodiments, the dosage regimen is about every 2 weeks, about every 3 weeks, or about every 4 weeks.

In one aspect, the disclosure relates to a method of treating cancer in a human subject in need thereof, the method comprising the general structure of the N terminus - (a) - (b) - (c) - C terminus. administering to a human subject an effective amount of the chimeric protein, wherein (a) is a first domain comprising the extracellular domain of human signal regulatory protein α (CD172a(SIRPα)), and (b) is the first and a linker adjacent to the second domain, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and (c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L), the dosing regimen being approximately every 3 days. to about every 10 days, about every week to about every 2 weeks, about every 10 days to about every 3 weeks, about every 2 weeks to about every 4 weeks, about every 3 weeks to about every 5 weeks, about every 4 weeks. to about every 6 weeks, from about every 5 weeks to about every 7 weeks, from about every 6 weeks to about every 8 weeks, and from about every 6 weeks to about every 2 months. In an embodiment, the linker includes at least one cysteine residue capable of forming a disulfide bond. In embodiments, the dosage regimen is from about every week to about every 2 weeks, from about every 10 days to about every 3 weeks, or from about every 2 weeks to about every 4 weeks. In an embodiment, the chimeric protein is, for example, from about 0.3 mg/kg to about 3 mg/kg, or from about 0.5 mg/kg to about 3 mg/kg, or from about 0.7 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 3 mg/kg, or about 2.5 mg/kg to about 3 mg/kg. kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about 0.3 mg/kg to about 2 mg/kg, or about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 It exhibits a linear dose response in a dose range of mg/kg, or about 0.3 mg/kg to about 0.5 mg/kg. In an embodiment, the chimeric protein does not exhibit a longitudinal dose response.

In some embodiments of any of the aspects disclosed herein, the first domain is capable of binding CD172a (SIRPα) ligand. In an embodiment, the first domain comprises substantially all of the extracellular domain of CD172a (SIRPα). In an embodiment, the second domain is capable of binding the CD40 receptor. In an embodiment, the second domain comprises substantially all of the extracellular domain of CD40L. In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4, eg, human IgG4. In an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 has 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 Includes. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:58. In some embodiments of any of the aspects disclosed herein, the second domain comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO:58. In an embodiment, (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 is SEQ ID NO: 1 or SEQ ID NO: 2. or an amino acid sequence that is at least 95% identical to SEQ ID NO:3.

In an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, the chimeric protein comprises an amino acid sequence identical to SEQ ID NO:59 or SEQ ID NO:61. In an embodiment, 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 an embodiment, 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, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61. In an embodiment, the human subject is receiving, tolerating, or is ineligible for standard therapy and/or the cancer has no approved therapy considered standard of care.

In one aspect, the disclosure relates to a method for promoting the migration of lymphocytes from peripheral blood to secondary lymphoid organs (e.g., lymph nodes) and the spleen in a human subject in need thereof, comprising: comprising administering to a human subject an effective amount of a chimeric protein having the general structure of N terminus - (a) - (b) - (c) - C terminus, wherein (a) is human signal regulatory protein α (CD172a ( a first domain comprising the extracellular domain of SIRPα)), (b) a linker adjacent to the first and second domains, the linker comprising a hinge-CH2-CH3 Fc domain, and (c) a human CD40 ligand. The second domain includes the extracellular domain of (CD40L).

In some embodiments of any of the aspects disclosed herein, the human subject is not receiving concurrent chemotherapy, immunotherapy, biotherapy, 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 a chimeric protein comprising an amino acid sequence that is at least 98% identical to SEQ ID NO:59 or SEQ ID NO:61. In an embodiment, 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 an embodiment, the chimeric protein comprises an amino acid sequence identical to SEQ ID NO:59 or SEQ ID NO:61.

The dosing frequency of the first phase and the dosing frequency of the second phase are the same or different. In an embodiment, the dosing frequency of the first phase and the dosing frequency of the second phase are each independently about every 3 days, about twice a week, about every week, about every 10 days, about twice every 3 weeks, or about 2 weeks. Every, about every 3 weeks, about every 4 weeks, about every month, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, and about every 2 months. In embodiments, the dosing frequency of the first phase is from about every 3 days to about every 10 days, from about every week to about every 2 weeks, from about every 10 days to about every 3 weeks, from about every 2 weeks to about every 4 weeks, From about every 3 weeks to about every 5 weeks, from about every 4 weeks to about every 6 weeks, from about every 5 weeks to about every 7 weeks, from about every 6 weeks to about every 8 weeks, and from about every 6 weeks to about every 2 months. is selected.

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

The effective amounts for the first phase, second phase and third phase are the same or different. In an embodiment, the effective amounts for the first phase, second phase and third phase each independently comprise from about 0.01 mg/kg to about 10 mg/ml. In embodiments, the effective amount for the first phase is about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, or about 1 mg/kg. mg/kg to about 10 mg/kg; The effective amount for the second phase is about 0.01 mg/kg to about 0.1 mg/kg, about 0.03 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 10 mg/kg. In an embodiment, the chimeric protein disclosed herein is a human CD172a(SIRPα)-Fc-CD40L chimeric protein.

In an embodiment, the human CD172a(SIRPα)-Fc-CD40L chimeric protein can provide sustained immunomodulatory effects.

In an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, 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 an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG. In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from an IgG selected from IgG1 and IgG4. In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from human IgG1 or human IgG4. In an embodiment, the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4. In an embodiment, the hinge-CH2-CH3 Fc domain is derived from human IgG4.

Additionally, or alternatively, in an embodiment, the extracellular domain of human signal regulatory protein α (CD172a(SIRPα)) is at least 90%, or 93%, or 95%, or 97% identical to the amino acid sequence of SEQ ID NO:57. , or 98%, or 99% identical amino acid sequences. In an embodiment, the extracellular domain of human signal regulatory protein α (CD172a(SIRPα)) 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 regulatory protein α (CD172a(SIRPα)) 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 regulatory protein α (CD172a(SIRPα)) 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 regulatory protein α (CD172a(SIRPα)) 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 regulatory protein α (CD172a(SIRPα)) comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO:57.

Additionally, or alternatively, in an embodiment, the extracellular domain of human CD40 ligand (CD40L) is at least 90%, or 93%, or 95%, or 97%, or 98%, the amino acid sequence of SEQ ID NO:58, or contain 99% identical amino acid sequences. In an embodiment, the extracellular domain of human CD40 ligand (CD40L) 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 (CD40L) 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 (CD40L) 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 (CD40L) 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 (CD40L) comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO:58.

Additionally, or alternatively, in an embodiment, the human CD172a(SIRPα)-Fc-CD40L chimeric protein is at least 90%, or 93%, or 95%, or 97%, or 98% identical to SEQ ID NO:59 or SEQ ID NO:61. %, or 99% identical amino acid sequences. In an embodiment, the human CD172a(SIRPα)-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 an embodiment, the human CD172a(SIRPα)-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 an embodiment, the human CD172a(SIRPα)-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 an embodiment, the human CD172a(SIRPα)-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 an embodiment, the human CD172a(SIRPα)-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 an embodiment, the human CD172a(SIRPα)-Fc-CD40L chimeric protein comprises an amino acid sequence identical to SEQ ID NO:59 or SEQ ID NO:61.

In one aspect, the present disclosure provides: (i) the general structure of the N terminus - (a) - (b) - (c) - C terminus, where (a) is a cell of human signal regulatory protein α (CD172a (SIRPα)) (b) is a linker adjacent to the first domain and the second domain, wherein the linker includes at least one cysteine residue capable of forming a disulfide bond and/or a hinge-CH2-CH3 administering to a human subject a chimeric protein comprising an Fc domain, and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L); and (ii) administering a second therapeutic agent. In an embodiment, the chimeric protein is administered at a dose of about 0.0001 mg/kg to about 10 mg/kg. In an embodiment, the dose of chimeric protein administered is at least about 0.3 mg/kg, such as at least about 0.3 mg/kg, or about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4 mg. /kg, or about 6 mg/kg, or about 8 mg/kg, or about 10 mg/kg. In an embodiment, the dose of chimeric protein administered is at least about 1 mg/kg, such as at least about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4 mg/kg, or about 6 mg. /kg, or about 8 mg/kg, or about 10 mg/kg. In an embodiment, the chimeric protein is, for example, from about 0.3 mg/kg to about 3 mg/kg, or from about 0.5 mg/kg to about 3 mg/kg, or from about 0.7 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 3 mg/kg, or about 2.5 mg/kg to about 3 mg/kg. kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about 0.3 mg/kg to about 2 mg/kg, or about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 It exhibits a linear dose response in a dose range of mg/kg, or about 0.3 mg/kg to about 0.5 mg/kg. In an embodiment, the chimeric protein does not exhibit a longitudinal dose response.

In an embodiment, administration of the chimeric protein results in a receptor occupancy (RO) of at least about 30%, or at least about 40%, relative to the receptor occupancy (RO) of the second subject and/or external control to which the chimeric protein was not administered prior to administration of the chimeric protein. 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 CD47 receptor occupancy on leukocytes, which is 95%, also causes RO. In an embodiment, administration of the chimeric protein results in a receptor occupancy (RO) 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 CD47 receptor occupancy on B cells, which is 95%, also causes RO. In an embodiment, administration of the chimeric protein modifies IL-12, MCP-1, MIP-1β, MIP-1α, and MDC relative to the RO of a second subject and/or external control group not administered the chimeric protein prior to administration of the chimeric protein. Causes an increase in the amount or activity of one or more of the %.

In one aspect, the present disclosure provides a general structure of the N terminus - (a) - (b) - (c) - C terminus, where (a) comprises the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)). (b) is a linker adjacent to the first domain and the second domain, wherein the linker includes at least one cysteine residue capable of forming a disulfide bond and/or includes a hinge-CH2-CH3 Fc domain. and (c) is a second domain comprising the extracellular domain of human CD40 ligand (CD40L)) to a subject in need of cancer treatment. Regarding this, the subject is receiving or has received treatment with a second therapeutic agent. In an embodiment, the chimeric protein is administered at a dose of about 0.0001 mg/kg to about 10 mg/kg. In an embodiment, the dose of chimeric protein administered is at least about 0.3 mg/kg, such as at least about 0.3 mg/kg, or about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4 mg. /kg, or about 6 mg/kg, or about 8 mg/kg, or about 10 mg/kg. In an embodiment, the dose of chimeric protein administered is at least about 1 mg/kg, such as at least about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4 mg/kg, or about 6 mg. /kg, or about 8 mg/kg, or about 10 mg/kg. In an embodiment, the chimeric protein is, for example, from about 0.3 mg/kg to about 3 mg/kg, or from about 0.5 mg/kg to about 3 mg/kg, or from about 0.7 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 3 mg/kg, or about 2.5 mg/kg to about 3 mg/kg. kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about 0.3 mg/kg to about 2 mg/kg, or about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 It exhibits a linear dose response in a dose range of mg/kg, or about 0.3 mg/kg to about 0.5 mg/kg. In an embodiment, the chimeric protein does not exhibit a longitudinal dose response.

In one aspect, the disclosure relates to a method of treating cancer in a human subject comprising administering a second anti-cancer therapeutic agent to a subject in need of cancer treatment, wherein the subject has an N-terminal - (a) - (b) - (c) - is receiving or has received treatment with a chimeric protein of the general structure of the C terminus, where (a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)); , (b) is a linker adjacent to 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-CH2-CH3 Fc domain, and (c) ) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L). In an embodiment, the chimeric protein is administered at a dose of about 0.0001 mg/kg to about 10 mg/kg. In an embodiment, the chimeric protein is, for example, from about 0.3 mg/kg to about 3 mg/kg, or from about 0.5 mg/kg to about 3 mg/kg, or from about 0.7 mg/kg to about 3 mg/kg, or about 1 mg/kg to about 3 mg/kg, or about 1.5 mg/kg to about 3 mg/kg, or about 2 mg/kg to about 3 mg/kg, or about 2.5 mg/kg to about 3 mg/kg. kg, or about 0.3 mg/kg to about 2.5 mg/kg, or about 0.3 mg/kg to about 2 mg/kg, or about 0.3 mg/kg to about 1.5 mg/kg, or about 0.3 mg/kg to about 1.0 It exhibits a linear dose response in a dose range of mg/kg, or about 0.3 mg/kg to about 0.5 mg/kg. In an embodiment, the chimeric protein does not exhibit a longitudinal dose response.

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

In an embodiment, the second therapeutic agent is selected from antibodies and chemotherapeutic agents. In embodiments, the antibody may be antibody-dependent cellular cytotoxic (ADCC). In an embodiment, the antibody is selected from cetuximab, rituximab, obinutuzumab, Hul4.18K322A, Hu3F8, dinituximab, and trastuzumab. In an embodiment, the antibody may be antibody-dependent cellular phagocytosis (ADCP). In an embodiment, the antibody is selected from cetuximab, daratumumab, rituximab, and trastuzumab. In an embodiment, the antibody is capable of binding a molecule selected from carcinoembryonic antigen (CEA), EGFR, HER-2, epithelial cell adhesion molecule (EpCAM), and human epithelial mucin-1, CD20, CD30, CD38, CD40, and CD52. there is. In embodiments, the antibody is capable of binding EGFR. In an embodiment, the antibody is Mab A13, AMG595, Cetuximab (Erbitux, C225), Panitumumab (ABX-EGF, Vectibix), Depatuxizumab (ABT 806), Depatuxizumab Mab, mafodotin, duligotuzumab (MEHD7945A, RG7597), futuximab (Sym004), GC1118, imgatuzumab (GA201), matuzumab (EMD 72000), necitumumab (Portrazza), Nemo It is selected from tuzumab (h-R3), anitumumab (Vectibix, ABX-EGF), zalutumumab, humMR1 and tomuzotuximab. In an embodiment, the antibody is cetuximab.

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

In an embodiment, the dose of chimeric protein administered is at least about 0.0001 mg/kg, such as about 0.0001 mg/kg to about 10.0 mg/kg. The chimeric protein may be administered at an initial dose (e.g., 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 can be administered in one or more subsequent administrations (e.g., 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 an embodiment, the dose of chimeric protein administered is at least about 0.3 mg/kg, such as at least about 0.3 mg/kg, or about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4 mg. /kg, or about 6 mg/kg, or about 8 mg/kg, or about 10 mg/kg. In an embodiment, the dose of chimeric protein administered is at least about 1 mg/kg, such as at least about 1.0 mg/kg, or about 2 mg/kg, or about 3, about 4 mg/kg, or about 6 mg. /kg, or about 8 mg/kg, or about 10 mg/kg. In an embodiment, the initial dose is less than the dose for at least one of the subsequent administrations (e.g., each of the subsequent administrations), or the initial dose is the same as the dose for at least one of the subsequent administrations (e.g., each of the subsequent administrations). do. In embodiments, the starting dose and/or subsequent doses are at or below the maximum tolerated dose. In an embodiment, the chimeric protein is administered at least about 1 time per month, such as at least about 2 times per month, at least about 3 times per month, and at least about 4 times per month. In an embodiment, the chimeric protein is initially administered once weekly for three weeks, and then the chimeric protein is administered about once every three weeks or once every four weeks; Alternatively, the chimeric protein is initially administered once a week for 3 weeks, then the chimeric protein is administered about twice per month, e.g., once a week for 3 weeks, and then the chimeric protein is administered It is administered approximately once every two weeks.

In an embodiment, the cancer comprises an advanced solid tumor (localized and/or metastatic) or lymphoma. In an embodiment, the cancer is selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous squamous cell carcinoma (CSCC), and squamous cell carcinoma of the head and neck (SCCHN). In an embodiment, the cancer comprises an advanced solid tumor (local and/or metastatic) or an advanced lymphoma.

Example

The examples herein illustrate the advantages and benefits of the present technology and are provided to further assist those skilled in the art in making or using chimeric proteins of the present technology. The examples herein are presented to more fully illustrate preferred aspects of the present technology. The embodiments, if defined by the appended claims, should in no way be construed as limiting the scope of the present technology. The embodiments may include or utilize any variations, aspects or embodiments of the technology described above. The variations, aspects or embodiments described above may also each further include or invoke any or all other variations, aspects or embodiments of the subject technology.

Example 1: Phase 1 clinical trial of SIRPα-Fc-CD40L chimeric protein (SL-172154)

This first-in-human Phase 1 dose-escalation study is currently evaluating SL-172154 as monotherapy in subjects with platinum-resistant ovarian cancer. The primary objectives of the study were to evaluate safety; The purpose is to confirm the maximum tolerated dose or maximum administered dose of SL-172154. Secondary objectives of the study are to confirm the dose and schedule (i.e., recommended phase 2 dose [RP2D]), characterize PK and immunogenicity, and evaluate antitumor activity according to RECISTv1.1 in solid tumors. The exploratory objective is to evaluate receptor occupancy of SIRPα and CD40 on PBMCs and study pharmacodynamic (PD) effects in blood and tumors.

The planned dose escalation is in 1/2 log increments ( Figure 1 ). At least three subjects were enrolled at sequential dose levels (DL) and assessed for dose-limiting toxicities (DLTs) in the first cycle of treatment. Subjects will receive intravenous (IV) administration of SL-172154 on Schedule 1 or Schedule 2 until disease progression, unacceptable toxicity, or withdrawal of consent. Currently 10mg/kg is being registered.

Important inclusion criteria are as follows:

국소 진행 또는 전이성 난소암, 원발성 복막암 또는 나팔관암.

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

기존 요법(들)에 불응성이며 백금 요법에 부적합하다. 상동 재조합 결핍 양성 질환이 있는 대상체는 베바시주맙 유무와 상관없이 사전 PARPi를 받은 적이 있어야 한다.

Refractory to existing therapy(s) and unsuitable for platinum therapy. Subjects with homologous recombination deficiency benign disease must have received prior PARPi with or without bevacizumab.

18세 이상

18 years or older

0 또는 1의 ECOG 수행도

ECOG performance status of 0 or 1

RECIST v1.1당 측정 가능한 질환

Measurable disease per RECIST v1.1

Important exclusion criteria are as follows:

선행 백금 요법 중 또는 1개월 이내에 진행되는 것으로 정의되는 1차 백금 불응성

Primary platinum refractoriness, defined as progression during or within 1 month of neoadjuvant platinum therapy.

항-CD47 또는 항-SIRPα 표적화제 또는 CD40 작용제에 의한 사전 치료.

Prior treatment with anti-CD47 or anti-SIRPα targeting agents or CD40 agonists.

자가면역질환 또는 활성 폐렴의 기록된 병력

Documented history of autoimmune disease or active pneumonia

전신 코르티코스테로이드 또는 다른 면역억제 의약의 동시 사용

Concomitant use of systemic corticosteroids or other immunosuppressive medications

Subject characteristics are shown in Table 4 :

Fifteen subjects with a median age of 67 years (range 33 to 79) who had received a median of 5 prior lines of systemic therapy (range 2 to 7) were treated with IV SL-172154 over 4 dose levels for 2 schedules. did. Thirteen of 15 subjects had an ECOG PS of 1 at baseline. Six subjects were treated with 0.1 mg/kg (n=3) and 0.3 mg/kg (n=3) on Schedule 1 (days 1, 8, 15, and 29, q2wks). Nine subjects were treated with 0.3 mg/kg (n=3), 1.0 mg/kg (n=3), and 3.0 mg/kg (n=3) on Schedule 2 (once a week). Most subjects had primary ovarian cancer (n=9; 60%), FIGO stage IV (n=9; 60%), high-grade disease (n=11; 73%), and severe cancerous tissue (n=11; 73%), as shown in Table 1 n=11; 73%). Adverse events are listed in Table 5 .

No DLTs were observed with SL-172154 for any of the schedules at doses ranging from 0.1 mg/kg to 3.0 mg/kg. Fifteen subjects (100%) experienced adverse events (AEs) during treatment. The most common AEs occurring in 3 or more subjects (all causalities) were fatigue, infusion-related reactions (IRR), nausea, diarrhea, decreased appetite, dehydration, and pruritus ( Table 2 ). Fourteen subjects (93%) had treatment-related AEs (TRAEs). The most common TRAEs occurring in 2 or more subjects were IRR (n=8; 53%) fatigue (n=7; 47%), nausea (n=4; 27%), and decreased appetite (n=3; 20%). , chills (n=2; 13%), diarrhea (n=2; 13%), and dyspnea (n=2; 13%). G3 TRAE abnormalities have never been reported. Eight subjects (53%) had an infusion-related reaction (IRR) dosed at 0.3 mg/kg (n=3), 1.0 mg/kg (n=2), and 3.0 mg/kg (n=3); One IRR event was G3 and believed to be secondary to the iron infusion, 14 IRR events were G2 in severity, and 2 IRRs were G1 in severity. IRR was manageable with premedication and did not prevent completion of IV medication or lead to discontinuation of SL-172154. Thirteen subjects (87%) discontinued SL-172154; Twelve discontinued due to radiological or clinical progression, and one subject elected to discontinue treatment.

Figure 3 shows tumor response and duration of treatment. Among the 14 subjects evaluable for efficacy via post-baseline scan, the best responses were:

- SD, n=4

-PD, n=9

- NE, n=1 (subject had SD, but did not meet protocol-specified minimum interval for SD)

- 1 subject (3 mg/kg) did not reach initial on-treatment disease assessment at week 8

Pharmacokinetics:

최대 농도(C_max) 및 곡선하면적, 0시간에서 무한대로 외삽(AUC_inf)은 용량이 증가함에 따라 불균형적으로 증가되었다.

Maximal concentration (C _max ) and area under the curve, extrapolated from 0 hours to infinity (AUC _inf ), increased disproportionately with increasing dose.

As the dose increased, the clearance rate decreased.

PK was found to be biphasic at 1.0 and 3.0 mg/kg.

The elimination phase of the curve was not fully characterized at the study dose.

Figures 4A - 4D show reproducible increases in serum cytokines following repeated administration of SL-172154. The subject's blood was collected at the designated time and plasma was prepared. Cytokine levels were analyzed using a multiplex ECL ELISA method, and selected dose levels of cytokines are indicated. Pro-inflammatory chemokines, CCL2(MCP-1) ( Figure 4A ), CCL4(MIP-1β) ( Figure 4B ), CCL3(MIP-1α) ( Figure 4C ), and CCL22(MDC) ( Figure 4D ) were measured after all injections. showed a dose-dependent increase in plasma levels. This increase was of equal duration and magnitude across all injection intervals.

Figures 5A and 5B show dose-dependent and reproducible increases in serum IL-12. Figure 5A shows that subject levels interleukin 12 (IL-12), a mediator of the TH1 pro-inflammatory response over time, is a hallmark of the cyclic effector cytokine response observed in study subjects. Figure 5B shows the median response (horizontal bars) at the first injection and appears preliminary to be dose dependent.

Figures 6A and 6B demonstrate that SL-172154 preferentially binds to CD47 on leukocytes but not RBCs. Figure 6A shows CD47 receptor occupancy (RO) assessed by fluorescence activated cell sorting (FACS) analysis using whole blood for both red blood cells (RBCs) and white blood cells (WBCs). One hour after infusion on Cycle 1 Day 1 (C1D1), the median CD47 RO (horizontal bar) for leukocytes is approximately 80%. Figure 6B shows that CD47 RO on RBC is less than 5% for all dose levels.

Figures 7A - 7C demonstrate that SL-172154 stimulates dose-dependent B cell marginalization and activation. B cells represent a large pool of circulating immune cells that express high levels of CD40. A fluorescence activated cell sorting (FACS) panel was designed to obtain information on CD40 receptor occupancy and activation status and maturation. In C1D1, almost all (approximately 80%) CD40+ B cells are demarcated, or exit the circulation, within 1 hour after injection. Figure 7A shows that the median frequency of marginal cells increases in a dose-dependent manner (horizontal bars). Receptor engagement is approximately 100% at all dose levels (data not shown). Figure 7B shows that the median B cell frequency returns to pre-infusion levels by the next infusion and returns to each infusion cycle, maintaining the breakaway cycle pattern. Figure 7C shows that reverting B cells show an increase in the costimulatory marker CD86 as well as the maturation marker CD95, suggesting that SL-172154 can induce phenotypic changes. Similarly, CD14+ monocytes demarcate, or exit the circulation, within 1 hour of injection; It returns to pre-infusion levels by the next infusion, maintaining a cyclical pattern of shedding and returning during each infusion cycle. The observed pattern of demargination is largely driven by CD86+ classical and nonclassical monocytes (data not shown).

Figures 8A and 8B show distinct profiles of TNFα and interleukin-6 (IL-6) for CD40 mAb. Figure 8A shows the induction of TNFα at various doses of CP-870,893 (left panel) or SL-172154 (right panel). Figure 8B shows the induction of IL-6 at various doses of CP-870,893 (left panel) or SL-172154 (right panel). CP-870,893 data is derived from Vonderheide et al ., J Clin Oncol 25:876-883 (2007). These results specifically demonstrate that SL-172154 does not induce TNFα and interleukin-6 (IL-6) compared to CD40 mAb. Dose limiting toxicity (DLT) was due to cytokine release syndrome (CRS) limiting dose escalation of the CD40 agonist mAb. Interestingly, no notable increase in TNFα and IL-6 was observed with SL-172154. As a result, SL-172154 is currently being administered at a dose of 10×CP-870,893.

Figures 9A and 9B demonstrate that SL-172154 induces an innate immune response in the tumor microenvironment (TME). Figure 9A shows immunohistochemical analysis of a biopsy sample from Patient A before and after administration of SL-172154. Monocytes were detected by staining for CD68 (a protein highly expressed by cells of the monocyte lineage). Figure 9B demonstrates upregulation of CD40 and MHC class II, activation markers of the TME, in tumor biopsy samples following treatment with SL-172154 compared to pre-treatment biopsy samples.

Figures 10A and 10B demonstrate that SL-172154 induces an adaptive immune response in the tumor microenvironment (TME). Figure 9A shows CD8+ cells, granzyme B+ cells, CD68+ cells, and Ki67+ cells in a biopsy sample from Patient A before and after administration of SL-172154. CD8+ cells, granzyme B+ cells, CD68+ cells and Ki67+ cells are increased in post-treatment biopsy samples compared to pre-treatment biopsy samples. Figure 9B is a plot comparing tumor proportion score (TPS) and combined positive score (CPS). Induction of PD-L1 on immune cells is a result of CD8+ T cell activation.

These results suggest that SL-172154 was well tolerated with no evidence of DLTs or anemia, thrombocytopenia, liver dysfunction, cytokine release syndrome, or pneumonitis. Dose escalation continues at 10 mg/kg. Preliminary PK parameters for SL-172154 suggest target-mediated drug disposition through receptor binding. High receptor occupancy was observed for SL-172154 on CD47+ leukocytes at the doses studied with minimal binding to RBCs. Binding of SL-172154 to CD40+ B cells and monocytes resulted in rapid activation and demargination after injection. Cyclic increases in innate and adaptive serum cytokines coincided with CD40 receptor engagement and activation. There was no increase in IL-6 or TNFα and no evidence of a longitudinal dose response. SL-172154 was well tolerated at doses that saturated both CD40 and CD47, and there was evidence of on-target PD activity that had not yet plateaued, requiring further dose escalation. These results, without being bound by theory, further demonstrate that SL-172154 induces innate as well as adaptive immune responses in the tumor microenvironment (TME).

Figure 11 depicts the planned clinical development strategy for SL-172154. The strategies are SL-172154 monotherapy in ovarian cancer, SL-172154 + liposomal doxorubicin combination in ovarian cancer, SL-172154 + azacitidine + venetoclax in AML, and SL-172154 + aza in HR-MDS. Includes trials of combination therapy with cytidine and SL-172154 plus azacytidine in TP53 mutant AML.

Example 2: Increased expression of CD80 in tumors following administration of SIRPα-Fc-CD40L chimeric protein (SL-172154)

SL-172154 was administered intratumorally to ovarian cancer patients. Tumor biopsy samples were obtained from patients before and after administration of SL-172154. Biopsy samples were analyzed for expression of CD80 in the tumor. As shown in Figure 12 , when compared to biopsy samples obtained before administration of SL-172154, samples obtained after administration of SL-172154 showed increased abundance of CD80+ cells and/or CD80 expression in the tumor after administration of SL-172154.

USE BY REFERENCE

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

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

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

equality theory

Although the invention has been disclosed with respect to specific embodiments, further modifications are possible and the application generally follows the principles of the invention, is within known or customary practice in the art to which the invention pertains, and is described herein as hereinbefore described. It will be understood that it is intended to cover any changes, uses or adaptations of the invention, including such departures from the disclosure as may apply to the essential features and as come within the scope of the appended claims.

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

Sequence list electronic file attached

Claims (111)

1. A method of treating cancer in a human subject, comprising:
(i) administering to said human subject a first dose of a chimeric protein having the general structure:
N terminus - (a) - (b) - (c) - C terminus
(here,
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent to the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L),
Background levels of cells were measured in a pre-dose biological sample obtained from the subject prior to administration of the first dose, and the cells were among CD80+ cells, CD8+ cells, granzyme B+ cells, CD68+ cells, Ki67+ cells and PD-L1+ immune cells. administering the first dose, selected from one or more, to the human subject;
(ii) administering a second dose of the chimeric protein to the human subject when the post-administration level of the cells is greater than the background level of the cells,
administering the second dose to the human subject, wherein the second dose is administered at least about 48 hours after administration of the first dose.
Method, including. The method of claim 1, wherein the biological sample is blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing nucleic acids, sputum, saliva, urine, cerebrospinal fluid. , a method selected from peritoneal fluid, pleural fluid, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, irrigation or irrigation, aspirates, scrapings, 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. The method of claim 3, wherein the tumor biopsy sample or tumor surgical specimen derived from a tumor is selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous squamous cell carcinoma (CSCC), and squamous cell carcinoma of the head and neck (SCCHN). . The method of any one of claims 1 to 4, wherein the cellular level is determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or Method, measured by a combination of these. 6. The method of claim 5, wherein the level of cells is measured by contacting the sample with an agent that specifically binds to one or more of CD80, CD8, granzyme B, CD68, Ki67, and PD-L1. The method of claim 6, wherein the agent that specifically binds to the one or more molecules is an antibody or fragment thereof. The method of claim 7, wherein the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. 6. The method of claim 5, wherein the level of 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, CD68, Ki67, and PD-L1. , method. 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. 1. A method of treating cancer in a human subject, comprising:
(i) administering to said human subject a first dose of a chimeric protein having the general structure:
N terminus - (a) - (b) - (c) - C terminus
(here:
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L),
Background levels and/or activity of B cells and/or CD40+ cells were measured in a first biological sample obtained from the subject prior to administration of the first dose;
The level and/or activity of B cells and/or CD40+ cells at N hours after administration was measured in a second biological sample obtained from the subject after administration of the first dose, where N is a number from 1 to 24, and after administration of the first dose. The level and/or activity of B cells and/or CD40+ cells at hour N is less than the background level and/or activity of said B cells and/or CD40+ cells;
The level and/or activity of B cells and/or CD40+ cells at M days post-dose were measured in a third biological sample obtained from the subject after administration of the first dose, wherein M is a number from 1 to 28. administering to the human subject; and
(ii) a chimera if the level and/or activity of B cells and/or CD40+ cells at M days post-administration is at least about 50% higher than the level and/or activity of B cells and/or CD40+ cells at N hours post-administration. administering a second dose of protein to the human subject, comprising:
administering the second dose to the human subject, wherein the second dose is administered at least about 48 hours after administration of the first dose.
Method, including. According to clause 11,
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
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. According to clause 11,
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
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. 14. The method of any one of claims 11 to 13, wherein the level and/or activity of B cells and/or CD40+ cells at N hours after administration is greater than the background level and/or activity of said B cells and/or CD40+ cells. Big, way. 14. The method of any one of claims 11 to 13, wherein the level and/or activity of B cells and/or CD40+ cells at N hours after administration is greater than the background level and/or activity of said B cells and/or CD40+ cells. Less, way. 14. The method of any one of claims 11 to 13, wherein the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than or equal to the background level and/or activity of said B cells and/or CD40+ cells. A method that is within about 10%, or about 20%, or about 30%, or about 40%. 12. The method of claim 11, wherein the first biological sample, the second biological sample and the third biological sample are blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy sample, cells- Contains body fluids, free-flowing nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, excretions, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, scrapings, A method independently selected from a bone marrow specimen, a biopsy specimen, and a surgical specimen. 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 determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence 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 are measured by contacting the sample with an agent that specifically binds to CD40 and/or B-cell markers. 21. The method of claim 20, wherein the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. 22. The method of claim 20 or 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, monoclonal antibody, polyclonal antibody, or fragment thereof. 20. The method of claim 19, wherein the level and/or activity of B cells and/or CD40+ cells are measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding CD40 and/or B-cell markers. , method. 25. The method of claim 24, wherein the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD and CD40. 26. The method of claim 24 or 25, wherein the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe. 1. A method of treating cancer in a human subject, comprising:
(i) administering to said human subject a first dose of a chimeric protein having the general structure:
N terminus - (a) - (b) - (c) - C terminus
(here:
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L),
Background amounts and/or activities of cytokines selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α and MDC are determined in the subject prior to administration of the first dose. It was measured in a first biological sample obtained from,
The amount and/or activity of the cytokine at N hours after administration was measured in a second biological sample obtained from the subject after administration of the first dose, N being a number from 1 to 24, and The amount and/or activity of the cytokine is greater than the background amount and/or activity of the cytokine;
The amount and/or activity of the cytokine at M days after administration was measured in a third biological sample obtained from the subject after administration of the first dose, where M is a number from 1 to 28. administering to; and
(ii) a second dose of the chimeric protein when the amount and/or activity of the cytokine at M days after the administration is at least about 30% lower than the amount and/or activity of the cytokine at N hours after the administration. administering to the human subject,
administering the second dose to the human subject, wherein the second dose is administered at least about 48 hours after administration of the first dose.
Method, including. According to clause 27,
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
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. According to clause 27,
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
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. 30. The method of any one of claims 27-29, wherein the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine. 30. The method of any one of claims 27-29, wherein the amount and/or activity of the cytokine at N hours after administration is less than the background amount and/or activity of the cytokine. 30. The method of any one of claims 27-29, wherein the amount and/or activity of said cytokine at N hours after said administration is about 10%, or about 20% of the background amount and/or activity of said cytokine; or within about 30%, or within about 40%. 28. The method of claim 27, wherein the first biological sample, the second biological sample and the third biological sample are blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy sample, cells- Contains body fluids, free-flowing nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, excretions, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, scrapings, A method independently selected from a bone marrow specimen, a biopsy specimen, and a surgical specimen. 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 determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activation. Measured by 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 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, monoclonal antibody, polyclonal antibody or fragment 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 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 The method is administered over 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. A method of evaluating the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject has cancer,
(i) obtaining a biological sample from said subject who has received a first dose of the chimeric protein,
The chimeric protein has the following general structure;
N terminus - (a) - (b) - (c) - C terminus
(here
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent to the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L);
The background level and/or activity of the cells was measured in a biological sample obtained from the subject prior to administration of the first dose, and the cells were CD80+ cells, CD8+ cells, granzyme B+ cells, CD68+ cells, Ki67+ cells, and PD-L1+ cells. Obtaining said biological sample selected from one or more immune cells;
(ii) determining the post-administration level and/or activity of said cells in said biological sample; and
(iii) determining that the chimeric protein is efficacious if the level and/or activity of the cell after administration is greater than the background level and/or activity of the cell.
Method, including. 1. A method of selecting a subject for treatment with cancer therapy, comprising:
(i) obtaining a biological sample from said subject who has received a first dose of the chimeric protein,
The chimeric protein has the following general structure:
N terminus - (a) - (b) - (c) - C terminus
(here
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L);
Background levels and/or activity of cells were measured in biological samples obtained from the subject prior to administration of the first dose, wherein the cells included CD80+ cells, CD8+ cells, granzyme B+ cells, CD68+ cells, Ki67+ cells, and PD-L1+ immune cells. Obtaining the biological sample selected from one or more 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 cells is greater than the background level and/or activity of the cells, selecting the subject for treatment with the chimeric protein.
Method, including. 43. The method of claim 41 or 42, wherein the biological sample is blood, plasma, serum, blood cells, tear fluid, tears, bone marrow, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free flowing nucleic acids, sputum, saliva. , selected from urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, washes or irrigations, aspirates, scrapings, bone marrow specimens, biopsy specimens and surgical specimens. How to become. 44. The method of claim 43, wherein the biological sample is a biopsy sample or surgical specimen. 45. The method of claim 44, wherein the biological sample is a tumor biopsy sample or a tumor surgical specimen. 46. The method of claim 45, wherein said tumor biopsy sample or said tumor surgical specimen derived from said tumor is selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, cutaneous squamous cell carcinoma (CSCC), and squamous cell carcinoma of the head and neck (SCCHN). method. 47. The method of any one of claims 41 to 46, wherein the cellular level is determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS) or Method, measured by a combination of these. 48. The method of claim 47, wherein the level and/or activity of the cells is measured by contacting the sample with an agent that specifically binds to one or more molecules selected from CD80, CD8, granzyme B, CD68, Ki67, and PD-L1. , method. 49. The method of claim 48, wherein the agent that specifically binds to the one or more molecules is an antibody or fragment thereof. The method of claim 49, wherein the antibody is a recombinant antibody, monoclonal antibody, polyclonal antibody, or fragment thereof. 48. The method of claim 47, wherein the level and/or activity of said cells is measured by comparing said sample with an agent that specifically binds to one or more nucleic acids encoding one or more of CD80, CD8, granzyme B, CD68, Ki67 and PD-L1. Method, measured by contact. 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. A method of evaluating the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject has cancer,
(i) obtaining a first biological sample obtained from a subject receiving a first dose of the chimeric protein,
Obtaining the first biological sample, wherein the chimeric protein has the following general structure:
N terminus - (a) - (b) - (c) - C terminus,
(here:
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent to the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L);
(ii) determining the background level and/or activity of B cells and/or CD40+ cells in the first biological sample;
(iii) obtain a second biological sample from the subject at N hours after administration, where N is 1 to 24, and the level and/or activity of B cells and/or CD40+ cells in the second biological sample at N hours after administration. determining;
(iv) Obtain a third biological sample obtained from the subject on M days after administration, where M is 1 to 28, and determine the level and/or activity of B cells and/or CD40+ cells in the third biological sample on M days after administration. deciding step; and
(v) if the level and/or activity of B cells and/or CD40+ cells at N hours after said administration is less than the background level and/or activity of B cells and/or CD40+ cells, and/or at M days after said administration. Determining that the chimeric protein is efficacious if the level and/or activity of B cells and/or CD40+ cells is at least about 50% higher than the level and/or activity of B cells and/or CD40+ cells at N hours after administration. step
Method, including. 1. A method of selecting a subject for treatment with cancer therapy, comprising:
(i) obtaining a biological sample obtained from a subject receiving a first dose of the chimeric protein, comprising:
Obtaining the biological sample, wherein the chimeric protein has the following general structure:
N terminus - (a) - (b) - (c) - C terminus,
(here,
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L);
(ii) determining the background level and/or activity of B cells and/or CD40+ cells in the first biological sample;
(iii) obtain a second biological sample from the subject at N hours after administration, where N is 1 to 24, and determine the level and/or activity of B cells and/or CD40+ cells in the second biological sample at N hours after administration. deciding step;
(iv) Obtain a third biological sample obtained from the subject on M days after administration, where M is 1 to 28, and determine the level and/or activity of B cells and/or CD40+ cells in the third biological sample on M days after administration. deciding step; and
(v) the level and/or activity of B cells and/or CD40+ cells at N hours after said administration is less than the background level and/or activity of said B cells and/or CD40+ cells, and/or at M days after said administration. for treatment with the chimeric protein, if the level and/or activity of B cells and/or CD40+ cells is at least about 50% higher than the level and/or activity of B cells and/or CD40+ cells at N hours after administration. Steps to select an object
Method, including. The method of claim 53 or 54,
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
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. The method of claim 53 or 54,
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
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. 57. The method of any one of claims 53 to 56, wherein the level and/or activity of B cells and/or CD40+ cells at N hours after administration is greater than the background level and/or activity of said B cells and/or CD40+ cells. Big, way. 57. The method of any one of claims 53 to 56, wherein the level and/or activity of B cells and/or CD40+ cells at N hours after administration is greater than the background level and/or activity of said B cells and/or CD40+ cells. Less, way. 57. The method of any one of claims 53 to 56, wherein the level and/or activity of B cells and/or CD40+ cells at N hours after administration is less than or equal to the background level and/or activity of said B cells and/or CD40+ cells. A method that is within about 10%, or about 20%, or about 30%, or about 40%. 55. The method of claim 53 or 54, wherein the first biological sample, the second biological sample and the third biological sample are blood, plasma, serum, blood cells, lacrimal fluid, tears, bone marrow, ascites, tissue or fine needle biopsy. Samples, cell-containing body fluids, free-flowing nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, A method independently selected from scrapings, bone marrow specimens, biopsy specimens and surgical specimens. 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 determined by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence 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 are measured by contacting the sample with an agent that specifically binds to CD40 and/or B-cell markers. 64. The method of claim 63, wherein the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. 65. The method of claim 63 or 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, monoclonal antibody, polyclonal antibody, or fragment thereof. 63. The method of claim 62, wherein the level and/or activity of B cells and/or CD40+ cells are measured by contacting the sample with an agent that specifically binds to one or more nucleic acids encoding CD40 and/or B-cell markers. , method. 68. The method of claim 67, wherein the B-cell marker is selected from CD19, CD20, CD24, CD27, CD34, CD38, CD45R, CD86, CD95, IgM, IgD, and CD40. 69. The method of claim 67 or 68, wherein the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe. A method of evaluating the efficacy of cancer treatment in a subject in need of cancer treatment, wherein the subject has cancer,
(i) obtaining a first biological sample from the subject who has received the first dose of the chimeric protein,
Obtaining the first biological sample, wherein the chimeric protein has the following general structure:
N terminus - (a) - (b) - (c) - C terminus
(here:
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent to the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L);
(ii) background amount and/or activity of cytokines selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α and MDC in said first biological sample. deciding step;
(iii) obtaining a second biological sample from the subject at N hours after administration, where N is 1 to 24, and determining the amount and/or activity of the cytokine in the second biological sample at N hours after administration;
(iv) obtaining a third biological sample from the subject on M days after administration, where M is 1 to 28, and determining the amount and/or activity of the cytokine in the third biological sample on M days after administration; and
(v) if the amount and/or activity of the cytokine at N hours after the administration is greater than the background amount and/or activity of the cytokine, and/or the amount and/or the amount and/or activity of the cytokine at M days after the administration determining that the chimeric protein is efficacious if the activity is at least about 50% less than the amount and/or activity of the cytokine at N hours after administration.
Method, including. 1. A method of selecting a subject for treatment with cancer therapy, comprising:
(i) obtaining a biological sample obtained from a subject receiving a first dose of the chimeric protein, comprising:
The chimeric protein has the following general structure:
N terminus - (a) - (b) - (c) - C terminus,
(here,
(a) is the first domain comprising the extracellular domain of human signal regulatory protein α (CD172a (SIRPα)),
(b) is a linker adjacent the first and second domains, wherein the linker comprises a hinge-CH2-CH3 Fc domain, and
(c) is the second domain comprising the extracellular domain of human CD40 ligand (CD40L);
(ii) background amount and/or activity of cytokines selected from CCL2, CXCL9, CXCL10, IFNα, IL15, IL23, IL-12, MCP-1, MIP-1β, MIP-1α and MDC in said first biological sample. deciding step;
(iii) obtaining a second biological sample from the subject at N hours after administration, wherein N is 1 to 24, and determining the amount and/or activity of the cytokine in the second biological sample at N hours after administration. ;
(iv) obtaining a third biological sample from the subject on M days after administration, wherein M is 1 to 28, and determining the amount and/or activity of the cytokine in the third biological sample on M days after administration. ; and
(v) if the amount and/or activity of said cytokine at N hours after administration is greater than the background amount and/or activity of said cytokine, and/or the amount and/or activity of said cytokine at M days after administration Selecting the subject for treatment with the chimeric protein if the activity is at least about 50% less than the amount and/or activity of the cytokine at N hours after the administration.
Method, including. According to claim 70 or 71,
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
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. According to claim 70 or 71,
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
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. 74. The method of any one of claims 70-73, wherein the amount and/or activity of the cytokine at N hours after administration is greater than the background amount and/or activity of the cytokine. 74. The method of any one of claims 70-73, wherein the amount and/or activity of the cytokine at N hours after administration is less than the background amount and/or activity of the cytokine. 74. The method of any one of claims 70-73, wherein the amount and/or activity of said cytokine at N hours after said administration is about 10%, or about 20% of the background amount and/or activity of said cytokine; or within about 30%, or within about 40%. 72. The method of claim 70 or 71, wherein the first biological sample, the second biological sample and the third biological sample are blood, plasma, serum, blood cells, lacrimal fluid, tears, bone marrow, ascites, tissue or fine needle biopsy. Samples, cell-containing body fluids, free-flowing nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, cleansing or irrigation, aspirates, A method independently selected from scrapings, bone marrow specimens, biopsy specimens and surgical specimens. 78. The method of claim 77, wherein the first biological sample, the second biological sample and the third biological sample are blood. 79. The method of any one of claims 70 to 78, wherein the amount and/or activity of the cytokine is measured by RNA sequencing, immunohistochemical staining, Western blotting, intracellular Western, immunofluorescence staining, ELISA, and fluorescence activation. Measured by 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 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, monoclonal antibody, polyclonal antibody, or fragment 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 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 ranges from about 0.03 mg/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 10 mg/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 administered on day 8, 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. 89. The method of claim 87 or 88, wherein the second dose of the chimeric protein ranges from about 0.03 mg/kg to 10 mg/kg. 89. The method of any one of claims 87-89, 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 10 mg/kg. 91. The method of any one of claims 1-90, wherein the first domain is capable of binding CD172a (SIRPα) ligand. 92. The method of any one of claims 1-91, wherein the first domain comprises substantially all of the extracellular domain of CD172a(SIRPα). 93. The method of any one of claims 1-92, wherein the second domain is capable of binding 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 CD40L. 95. The method of any one of claims 1-94, wherein the linker comprises a hinge-CH2-CH3 Fc domain derived from IgG4. 96. The method of claim 95, wherein the linker comprises a hinge-CH2-CH3 Fc domain derived from human IgG4. 97. The method of any one of claims 1 to 96, wherein the linker is at least about 90%, or at least about 95%, or at least about 96%, or A method comprising amino acid sequences that are at least about 97% identical, or at least about 98% identical, or at least about 99% identical. 98. The method of any one of claims 1 to 97, wherein the first domain is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or A method comprising amino acid sequences that are at least about 98% identical, or at least about 99% identical. 99. The method of any one of claims 1 to 98, wherein the second domain is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or A method comprising amino acid sequences that are at least about 98% identical, or at least about 99% identical. According to any one of claims 1 to 99,
(a) the first domain includes 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 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%, with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or comprising amino acid sequences that are at least about 99% identical. 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 to 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 is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97% identical to the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61. %, or at least about 98%, or at least about 99% identical amino acid sequences. 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. The method of claim 103 or 104, wherein the chimeric protein has 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8 amino acids relative to the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 61. A method comprising a mutation. 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 to 106, wherein the human subject is suffering from a cancer selected from ovarian cancer, fallopian tube cancer, peritoneal cancer, squamous cell carcinoma of the skin (CSCC), and squamous cell carcinoma of the head and neck (SCCHN), or Suspected of having these cancers, how. 108. The method of any one of claims 1-107, wherein the human subject has failed at least one platinum-based therapy. 109. The method of claim 108, wherein the human subject is ineligible for additional platinum therapy. 109. The method of any one of claims 1-108, wherein the human subject is ineligible for platinum therapy. 101. The method of any one of claims 1 to 100, wherein the human subject is not receiving concurrent chemotherapy, immunotherapy, biological or hormonal therapy, and/or the human subject has received standard therapy or has received standard therapy. and/or the cancer is intolerant to or ineligible for and/or the cancer has no approved therapy considered standard of care.