Methods and uses of variant ICOS ligand (ICOSL) fusion proteins

Abstract

To provide methods and therapeutic compositions for modulating immune response in the treatment of cancer and immunological diseases.SOLUTION: Immunomodulatory proteins comprising ICOS ligand (ICOSL) variants and nucleic acids encoding the proteins are provided herein. The immunomodulatory proteins provide therapeutic usefulness for various immunological and oncological conditions. Compositions and methods for producing and using the proteins are provided.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 835,488, filed Apr. 17, 2019; U.S. Provisional Patent Application No. 62 / 855,830, filed May 31, 2019; U.S. Provisional Patent Application No. 62 / 931,212, filed Nov. 5, 2019; U.S. Provisional Patent Application No. 62 / 945,071, filed Dec. 6, 2019; U.S. Provisional Patent Application No. 62 / 962,832, filed Jan. 17, 2020; and U.S. Provisional Patent Application No. 62 / 987,854, filed Mar. 10, 2020, the entire contents of each of which are hereby incorporated by reference.

[0002] Incorporation by reference of the Sequence Listing This application is filed together with a Sequence Listing in electronic format. The Sequence Listing is provided as a 116,543-byte file entitled 761612003240SeqList.txt created on Apr. 16, 2020. The information on the electronic format of the Sequence Listing is hereby incorporated by reference in its entirety.

[0003] Field The present disclosure relates to therapeutic compositions for modulating immune responses in the treatment of cancer and immune diseases. In some aspects, the present disclosure relates to certain ICOS ligand (ICOSL) variants that exhibit improved binding (e.g., improved affinity or selectivity) to one or both of the cognate binding partner proteins ICOS or CD28.

Background Art

[0004] Background There is growing medical interest in regulating the immune response by intervening in the processes that occur in the immune synapse (IS) formed between antigen-presenting cells (APCs) or target cells and lymphocytes and between these cells. Mechanistically, cell surface proteins in the IS can be involved in coordinated, often simultaneous interactions of multiple protein targets with one type of protein to which they bind. The interactions in the IS occur in close association with the junction of the two cells, and one type of protein in this structure can interact with both a protein on the same cell (cis) and a protein on the binding cell (trans), presumably simultaneously. Therapeutic agents capable of modulating the IS are known, but improved therapeutic agents are needed. Provided are immunomodulatory proteins, including soluble proteins or transmembrane immunomodulatory proteins that can be expressed on cells, that meet such needs.

Summary of the Invention

[0005] Summary In some aspects, provided herein is a method of treating an autoimmune or inflammatory disease in a subject, the method comprising administering to a subject having an autoimmune or inflammatory disease a variant ICOS-L fusion protein, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain. In some aspects, provided herein is a method of treating an autoimmune or inflammatory disease in a subject, the method comprising administering to a subject having an autoimmune or inflammatory disease a variant ICOS-L fusion protein during a treatment period, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain. In some aspects, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a portion thereof that comprises an IgV domain or a specific binding fragment thereof, and comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R relative to the sequence shown in SEQ ID NO:1 in a reference ICOS-L polypeptide.

[0006] In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 20 mg / kg or about 20 mg / kg. In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 10 mg / kg or about 10 mg / kg. In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 6 mg / kg or about 6 mg / kg. In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 3 mg / kg or about 3 mg / kg. In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 1 mg / kg or about 1 mg / kg. In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 0.3 mg / kg or about 0.3 mg / kg. In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 0.1 mg / kg or about 0.1 mg / kg. In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.1 mg / kg or about 0.1 mg / kg to 20 mg / kg or about 20 mg / kg. In some aspects, each dose of the variant ICOS-L fusion protein is administered in an amount from 0.1 mg / kg or about 0.1 mg / kg to 15 mg / kg or about 15 mg / kg. In some aspects, each dose is administered in an amount from 0.1 mg / kg or about 0.1 mg / kg to 10 mg / kg or about 10 mg / kg. In some aspects, each dose is administered in an amount from 0.1 mg / kg or about 0.1 mg / kg to 6 mg / kg or about 6 mg / kg. In some aspects, each dose is administered in an amount from 0.1 mg / kg or about 0.1 mg / kg to 3 mg / kg or about 3 mg / kg.In some embodiments, each dose is administered in an amount from 0.1 mg / kg or about 0.1 mg / kg to 1 mg / kg or about 1 mg / kg. In some embodiments, each dose is administered in an amount from 0.1 mg / kg or about 0.1 mg / kg to 0.3 mg / kg or about 0.3 mg / kg. In some embodiments, each dose is administered in an amount from 0.3 mg / kg or about 0.3 mg / kg to 20 mg / kg or about 20 mg / kg. In some embodiments, each dose is administered in an amount from 0.3 mg / kg or about 0.3 mg / kg to 15 mg / kg or about 15 mg / kg. In some embodiments, each dose is administered in an amount from 0.3 mg / kg or about 0.3 mg / kg to 10 mg / kg or about 10 mg / kg. In some embodiments, each dose is administered in an amount from 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg. In some embodiments, each dose is administered in an amount from 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg. In some embodiments, each dose is administered in an amount from 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg. In some embodiments, each dose is administered in an amount from 1 mg / kg or about 1 mg / kg to 20 mg / kg or about 20 mg / kg. In some embodiments, each dose is administered in an amount from 1 mg / kg or about 1 mg / kg to 15 mg / kg or about 15 mg / kg. In some embodiments, each dose is administered in an amount from 1 mg / kg or about 1 mg / kg to 10 mg / kg or about 10 mg / kg. In some embodiments, each dose is administered in an amount from 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg. In some embodiments, each dose is administered in an amount from 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg. In some embodiments, each dose is administered in an amount from 3 mg / kg or about 3 mg / kg to 20 mg / kg or about 20 mg / kg. In some embodiments, each dose is administered in an amount from 3 mg / kg or about 3 mg / kg to 15 mg / kg or about 15 mg / kg.In some embodiments, each dose is administered in an amount from 3 mg / kg or about 3 mg / kg to 10 mg / kg or about 10 mg / kg. In some embodiments, each dose is administered in an amount from 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg. In some embodiments, each dose is administered in an amount from 6 mg / kg or about 6 mg / kg to 20 mg / kg or about 20 mg / kg. In some embodiments, each dose is administered in an amount from 6 mg / kg or about 6 mg / kg to 15 mg / kg or about 15 mg / kg. In some embodiments, each dose is administered in an amount from 6 mg / kg or about 6 mg / kg to 10 mg / kg or about 10 mg / kg. In some embodiments, each dose is administered in an amount from 10 mg / kg or about 10 mg / kg to 20 mg / kg or about 20 mg / kg. In some embodiments, each dose is administered in an amount from 10 mg / kg or about 10 mg / kg to 15 mg / kg or about 15 mg / kg.

[0007] In some embodiments, only a single dose of the variant ICOS-L fusion protein is administered during the treatment period. In some embodiments, multiple doses of the variant ICOS-L fusion protein are administered during the treatment period. In some embodiments, the multiple doses are two, three, four, or five doses. In some embodiments, the treatment period is at least 20 days. In some embodiments, the treatment period is 20 to 40 days.

[0008] In some aspects, provided herein is a method of treating an autoimmune or inflammatory disease in a subject, the method comprising administering a variant ICOS-L fusion protein to a subject having an autoimmune or inflammatory disease, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain. In some aspects, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a portion thereof that comprises an IgV domain or a specific binding fragment thereof, and comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R relative to the sequence set forth in SEQ ID NO:1 in a reference ICOS-L polypeptide. In some aspects, the variant ICOS-L fusion protein is administered in multiple doses selected from three, four, and five over a treatment period of 20 to 40 days. In some aspects, each dose is in an amount from 0.1 mg / kg or about 0.1 mg / kg to 10 mg / kg or about 10 mg / kg.

[0009] In some aspects, provided herein is a method of treating an autoimmune or inflammatory disease or condition in a subject, the method comprising administering to a subject having an autoimmune or inflammatory disease a variant ICOS-L fusion protein, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain. In some aspects, the variant ICOS-L polypeptide is the ICOS-L extracellular domain or a portion thereof that comprises an IgV domain or a specific binding fragment thereof, and in a reference polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R relative to the sequence set forth in SEQ ID NO:1. In some aspects, the variant ICOS-L fusion protein is administered in multiple doses over a treatment period of at least 20 days. In some aspects, each dose is in an amount from 0.1 mg / kg or about 0.1 mg / kg to 10 mg / kg or about 10 mg / kg. In some aspects, each dose is over a maximum treatment period of 30 days. In some aspects, each dose is such that each of the multiple doses is spaced apart from each other by at least 5 days. In some aspects, the treatment period is 21 days or about 21 days. In some aspects, the treatment period is 28 days or about 28 days. In some aspects, each of the multiple doses is administered no more than once per week.

[0010] In some aspects, provided herein is a method of treating an autoimmune or inflammatory disease in a subject, the method comprising administering a variant ICOS-L fusion protein to a subject having an autoimmune or inflammatory disease, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain. In some aspects, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a portion thereof that comprises an IgV domain or a specific binding fragment thereof, and in the reference polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R relative to the sequence set forth in SEQ ID NO:1. In some aspects, the variant ICOS-L fusion protein is administered in multiple doses within a treatment period of at least 4 weeks. In some aspects, each dose is an amount from 0.1 mg / kg or about 0.1 mg / kg to 10 mg / kg or about 10 mg / kg and is administered less than once a week.

[0011] In some aspects, a method of preventing or reducing acute graft-versus-host disease (aGVHD) comprising administering to a subject one or more doses of a variant ICOS ligand fusion protein during a treatment period, said variant ICOS ligand fusion protein comprising a variant ICOS ligand polypeptide linked to a multimerization domain, said variant ICOS ligand polypeptide being an ICOS ligand extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and having in the reference ICOS polypeptide one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1, and wherein the dose of the variant ICOS ligand fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 20 mg / kg or about 20 mg / kg, is provided herein. In some aspects, the aGVHD is associated with a stem cell transplant and at least one dose of the ICOS ligand fusion protein is administered to the subject prior to the subject undergoing a stem cell transplant. In some aspects, the aGVHD is associated with a stem cell transplant and at least one dose of the ICOS ligand fusion protein is administered to the subject prior to the subject undergoing a stem cell transplant and at least one dose of the ICOS ligand fusion protein is administered to the subject simultaneously with or after the subject undergoes a stem cell transplant. In some aspects, the aGVHD is associated with a stem cell transplant and at least one dose of the ICOS ligand fusion protein is administered to the subject prior to the subject undergoing a stem cell transplant and at least one dose of the ICOS ligand fusion protein is administered to the subject simultaneously with the subject undergoing a stem cell transplant. In some aspects, the aGVHD is associated with a stem cell transplant and at least one dose of the ICOS ligand fusion protein is administered to the subject prior to the subject undergoing a stem cell transplant and at least one dose of the ICOS ligand fusion protein is administered to the subject after the subject undergoes a stem cell transplant.

[0012] In some of any provided aspects, a method of treating graft-versus-host disease (GVHD) in a subject, comprising administering to the subject a single dose of a variant ICOS-L fusion protein, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1, and the single dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 20 mg / kg or about 20 mg / kg, is provided herein. In some of any provided aspects, the GVHD is acute GVHD (aGVHD). In some of any provided aspects, the GVHD is chronic GVHD.

[0013] In some aspects of any provided aspect, a method of treating graft-versus-host disease (GVHD) in a subject, comprising administering to the subject a single dose of a variant ICOS-L fusion protein, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1, the single dose of the variant ICOS-L fusion protein is administered in an amount from 0.001 mg / kg or about 0.001 mg / kg to 20 mg / kg or about 20 mg / kg, and the subject is resistant or refractory to an immunosuppressive agent, is provided herein. In some aspects, the immunosuppressive agent is a corticosteroid, such as a glucocorticoid. In some aspects, the immunosuppressive agent is cyclosporine. In some aspects of any provided aspect, the GVHD is acute GVHD (aGVHD). In some aspects of any provided aspect, the GVHD is chronic GVHD.

[0014] In some aspects, the treatment period is 4 weeks or about 4 weeks. In some aspects, each of the multiple doses is administered once a week (Q1W). In some aspects, each dose is from 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, from 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, from 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, from 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, from 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, from 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg in amount. In some aspects, the dose is from 0.3 mg / kg or about 0.3 mg / kg to 10 mg / kg or about 10 mg / kg, from 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, from 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, from 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, from 1 mg / kg or about 1 mg / kg to 10 mg / kg or about 10 mg / kg, from 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, from 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, from 3 mg / kg or about 3 mg / kg to 10 mg / kg or about 10 mg / kg, from 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg, or from 6 mg / kg or about 6 mg / kg to 10 mg / kg or about 10 mg / kg in amount. In some aspects, each dose is an amount of 0.3 mg / kg or about 0.3 mg / kg. In some aspects, each dose is an amount of 1 mg / kg or about 1 mg / kg. In some aspects, each dose is an amount of 3 mg / kg or about 3 mg / kg. In some aspects, each dose is an amount of 6 mg / kg or about 6 mg / kg. In some aspects, each dose is an amount of 10 mg / kg or about 10 mg / kg. In some aspects, at least one dose is an amount of 0.3 mg / kg or about 0.3 mg / kg.In some embodiments, at least one dose is in an amount of 1 mg / kg or about 1 mg / kg. In some embodiments, at least one dose is in an amount of 3 mg / kg or about 3 mg / kg. In some embodiments, at least one dose is in an amount of 6 mg / kg or about 6 mg / kg. In some embodiments, at least one dose is in an amount of 10 mg / kg or about 10 mg / kg.

[0015] In some embodiments, administration is by subcutaneous administration. In some embodiments, administration is by intravenous administration. In some embodiments, at least one dose is administered via subcutaneous administration. In some embodiments, at least one dose is administered via intravenous administration. In some embodiments, the treatment period is repeated. In some embodiments, the treatment period is repeated until remission is achieved, until partial remission is achieved, or until the disease or condition no longer progresses in the subject.

[0016] A method of treating an autoimmune or inflammatory disease of the eye in a subject, comprising intravitreally administering a dose of a variant ICOS-L fusion protein comprising a variant ICOS-L polypeptide linked to a multimerization domain, wherein the variant ICOS-L polypeptide is an ICOS extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and wherein the reference ICOS-L polypeptide comprises one or more amino acid substitutions relative to the sequence set forth in SEQ ID NO:1. A method of reducing or preventing an autoimmune or inflammatory disease of the eye in a subject, comprising intravitreally administering a dose of a variant ICOS-L fusion protein comprising a variant ICOS-L polypeptide linked to a multimerization domain, wherein the variant ICOS-L polypeptide is an ICOS extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and wherein the reference ICOS-L polypeptide comprises one or more amino acid substitutions relative to the sequence set forth in SEQ ID NO:1. In some embodiments, the variant ICOS-L fusion protein has one or more amino acid modifications (e.g., one or more amino acid substitutions) that improve binding to CD28 and / or ICOS, and may comprise a variant ICOS-L polypeptide fused to a multimerization domain, e.g., an Fc domain. In some embodiments, the one or more amino acid modifications are the substitutions N52H / N57Y / Q100R.

[0017] In some aspects, the variant ICOS-L polypeptide comprises the amino acid substitutions N52H / N57Y / Q100R relative to the numbering of SEQ ID NO:1. In some aspects, the binding affinity of the variant ICOS-L polypeptide for the extracellular domain of CD28 is improved as compared to the binding of the ICOS-L reference polypeptide for the same extracellular domain. In some aspects, the binding affinity of the variant ICOS-L polypeptide for the extracellular domain of ICOS is substantially the same as or improved as compared to the binding of the ICOS-L reference polypeptide for the same extracellular domain. In some aspects, the binding affinity is 80% or about 80% or greater than the binding affinity of the ICOS-L reference polypeptide for the same extracellular domain. In some aspects, the binding of the variant ICOS-L polypeptide for the extracellular domains of ICOS and CD28 is improved as compared to the binding of the ICOS-L reference polypeptide for the same extracellular domains. In some aspects, the binding is 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold greater or about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 60-fold greater improved.

[0018] In some aspects, each dose of the variant ICOS-L fusion protein is C maxreaches or is predicted to reach target binding saturation of 25% or greater than about 25% to CD28; and / or each dose of the variant ICOS-L fusion protein reaches or is predicted to reach target binding saturation of 10% or greater than about 10% to CD28 within 24 hours of dosing. In some embodiments, each dose of the variant ICOS-L fusion protein reaches or is predicted to reach target binding saturation of 85% or greater than about 85% to CD28 within 24 hours, within 2 days, or within 7 days of dosing. In some embodiments, each dose of the variant ICOS-L fusion protein reaches or is predicted to reach target binding saturation of 90% or greater than about 90% to CD28 within 24 hours, within 2 days, or within 7 days of dosing. In some embodiments, each dose of the variant ICOS-L fusion protein reaches or is predicted to reach target binding saturation of 95% or greater than about 95% to CD28 within 24 hours, within 2 days, or within 7 days of dosing. In some embodiments, each dose of the variant ICOS-L fusion protein reaches or is predicted to reach target binding saturation of 10% or greater than about 10% to CD28 within 21 days or within 28 days of dosing. In some embodiments, each dose of the variant ICOS-L fusion protein reaches or is predicted to reach target binding saturation of 20% or greater than about 20% to CD28 within 21 days or within 28 days of dosing.

[0019] In some aspects, CD28 is human CD28. In some aspects, ICOS is human ICOS. In some aspects, the ICOS-L reference polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1, an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, or a portion of the amino acid sequence set forth in SEQ ID NO:1 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1 that includes an IgV domain or an IgC domain or a specific binding fragment thereof or both. In some aspects, the variant ICOS-L polypeptide comprises an IgV domain or a specific binding fragment thereof. In some aspects, the IgV domain or a specific binding fragment thereof is the only ICOS-L portion of the variant ICOS-L polypeptide or the variant ICOS-L fusion protein. In some aspects, the ICOS-L reference polypeptide is a continuous amino acid sequence comprising amino acids 1 to 112 based on the ICOS-L extracellular domain sequence set forth in SEQ ID NO:1 and a truncated ICOS-L extracellular domain comprising a C-terminal truncation of at least 25 amino acids. In some aspects, the C-terminal truncation is a C-terminal truncation of at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125 amino acid residues.

[0020] In some aspects, the ICOS-L reference polypeptide is altered at or lacks the protease cleavage site shown as amino acids 204 - 209 of SEQ ID NO:1. In some aspects, the ICOS-L reference polypeptide comprises the amino acid sequence shown in SEQ ID NO:3. In some aspects, the ICOS-L reference polypeptide consists of the amino acid sequence shown in SEQ ID NO:3. In some aspects, the variant ICOS-L polypeptide has the sequence shown in SEQ ID NO:36, or at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the sequence shown in SEQ ID NO:36, and comprises an amino acid substitution selected from N52H, N57Y, and Q100R.

[0021] In some aspects, the variant ICOS-L polypeptide has the sequence shown in SEQ ID NO:36. In some aspects, protein cleavage of the variant ICOS-L polypeptide when expressed from a cell is reduced compared to the full-length extracellular domain of the variant ICOS-L polypeptide when expressed from the same cell. In some aspects, the cell is a Chinese hamster ovary (CHO) cell.

[0022] In some aspects, the multimerization domain is or comprises the Fc region of an immunoglobulin. In some aspects, the variant ICOS-L polypeptide is linked to the multimerization domain via a linker. In some aspects, the linker is a peptide linker. In some aspects, the linker comprises from 1 to 10 amino acids. In some aspects, the linker is AAA. In some aspects, the linker is G4S (SEQ ID NO:52). In some aspects, the linker is (G4S)2 (SEQ ID NO:53). In some aspects, the linker is a GSGGGGS linker (SEQ ID NO:58).

[0023] In some aspects, the variant ICOS-L fusion protein is a multimer comprising a first variant ICOS-L polypeptide linked to a first multimerization domain and a second variant ICOS-L polypeptide linked to a second multimerization domain. In some aspects, the first multimerization domain and the second multimerization domain are the same. In some aspects, the first multimerization domain and the second multimerization domain are the Fc regions of an immunoglobulin. In some aspects, the multimer is a dimer. In some aspects, the dimer is a homodimer.

[0024] In some embodiments, the Fc region is a variant Fc region that exhibits reduced one or more effector functions compared to the Fc of wild-type human immunoglobulins. In some embodiments, the Fc region is a variant IgG1 Fc region that contains one or more amino acid substitutions compared to wild-type human IgG1. In some embodiments, the Fc region contains a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:37. In some embodiments, the variant Fc region contains one or more amino acid substitutions selected from N297G, E233P / L234V / L235A / G236del / S267K, or L234A / L235E / G237A, and the residues are numbered according to the Kabat EU index. In some embodiments, the variant Fc region further contains the amino acid substitution C220S, and the residues are numbered according to the Kabat EU index. In some embodiments, the Fc region contains K447del, and the residues are numbered according to the Kabat EU index. In some embodiments, the Fc region contains the amino acid sequence shown in SEQ ID NO:43 or SEQ ID NO:46. In some embodiments, the Fc region contains the amino acid sequence shown in SEQ ID NO:44 or SEQ ID NO:47. In some embodiments, the Fc region contains the amino acid sequence shown in SEQ ID NO:40. In some embodiments, the Fc region contains the amino acid sequence shown in SEQ ID NO:39. In some embodiments, the Fc region contains the amino acid sequence shown in SEQ ID NO:41. In some embodiments, the Fc region contains the amino acid sequence shown in SEQ ID NO:42.

[0025] In some aspects, the variant ICOS-L fusion protein reduces the immune response in a subject. In some aspects, the inflammatory or autoimmune disease or condition is anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatic disease, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory neurological disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune blood disease. In some aspects, the inflammatory or autoimmune disease or condition is inflammatory bowel disease, transplantation, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, psoriatic arthritis, or psoriasis. In some aspects, the inflammatory or autoimmune disease or condition is psoriatic arthritis. In some aspects, the inflammatory or autoimmune disease or condition is rheumatoid arthritis. In some aspects, the inflammatory or autoimmune disease or condition is Crohn's disease. In some aspects, the inflammatory disease or autoimmune disease is ulcerative colitis. In some aspects, the inflammatory disease or autoimmune disease is uveitis. In some aspects, the inflammatory or autoimmune disease or condition is systemic lupus erythematosus (SLE). In some aspects, the inflammatory or autoimmune disease or condition is Sjögren's syndrome. In some aspects, the inflammatory or autoimmune disease or condition is graft-versus-host disease (GVHD). In some aspects, the GVHD is acute GVHD. In some aspects, the GVHD is chronic GVHD. In some aspects, the GVHD is resistant or refractory. In some aspects, the GVHD is chronic GVHD. In some aspects, the GVHD is resistant or refractory to immunosuppressive agents. In some aspects, the immunosuppressive agent is a corticosteroid. In some aspects, the corticosteroid is a glucocorticoid. In some aspects, the immunosuppressive agent is cyclosporine.

[0026] In some aspects, the method of treatment includes prophylactic treatment. In some aspects, at least one dose is administered prior to the onset of an inflammatory or autoimmune disease or condition. In some aspects, at least one dose of the ICOS-L fusion protein is administered at the time of, or after, the onset of an inflammatory or autoimmune disease or condition. In some aspects, at least one dose of the ICOS-L fusion protein is administered prior to the onset of an inflammatory or autoimmune disease or condition, and at least one dose of the ICOS-L fusion protein is administered at the time of, or after, the onset of the inflammatory or autoimmune disease or condition.

[0027] In some aspects, the subject is human.

[0028] The following are exemplary numbered aspects of the disclosure: Aspect 1. A method of preventing or reducing acute graft-versus-host disease (aGVHD), comprising administering to a subject one or more doses of a variant ICOS-L fusion protein during a treatment period, the variant ICOS-L fusion protein comprising a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide being an ICOS-L extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and the variant ICOS-L polypeptide comprising one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R relative to the sequence shown in SEQ ID NO:1 in a reference ICOS-L polypeptide, wherein each of the one or more doses of the variant ICOS-L fusion protein is administered in an amount of 0.1 mg / kg or about 0.1 mg / kg to 20 mg / kg or about 20 mg / kg. Aspect 2. The method of Aspect 1, wherein the subject has previously received an allogeneic hematopoietic stem cell transplant (HSCT), and aGVHD occurs in the subject after receiving the allogeneic HSCT. Aspect 3. The method of Aspect 1 or Aspect 2, wherein the subject has grade II-IV aGVHD. Aspect 4. The method according to any one of Aspects 1 to 3, wherein the aGVHD of the subject is resistant or refractory to treatment with an immunosuppressive agent. Aspect 5. The method according to Aspect 4, wherein the immunosuppressive agent comprises a corticosteroid. Aspect 6. The method according to Aspect 4 or Aspect 5, wherein the immunosuppressive agent comprises cyclosporine. Aspect 7. A method for preventing or reducing inflammation secondary to a viral infection, comprising administering to a subject one or more doses of a variant ICOS-L fusion protein during a treatment period, the variant ICOS-L fusion protein comprising a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide being an ICOS-L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, comprising one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1, and each dose of the one or more doses of the variant ICOS-L fusion protein being administered in an amount of 0.1 mg / kg or about 0.1 mg / kg to 20 mg / kg or about 20 mg / kg. Aspect 8. The method according to Aspect 7, wherein the virus is a coronavirus. Aspect 9. The method according to Aspect 8, wherein the coronavirus is SARS-CoV-2 and the infection is COVID-19. Aspect 10. The method according to any one of Aspects 7 to 9, wherein the inflammation is related to cytokine release syndrome (CRS). Aspect 11. The method according to Aspect 10, wherein the CRS is severe CRS or CRS of grade 3 or higher. Aspect 12. The method according to any one of Aspects 7 to 11, wherein the subject has severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, or septic shock related to or caused by the viral infection at or immediately before the time of the administration. Aspect 13. A method for treating an autoimmune or inflammatory disease or condition in a subject, comprising administering, during a treatment period, one or more doses of a variant ICOS-L fusion protein to a subject having the autoimmune or inflammatory disease or condition, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1, and each dose of the one or more doses of the variant ICOS-L fusion protein is administered in an amount of 0.001 mg / kg or about 0.001 mg / kg to 20 mg / kg or about 20 mg / kg. Aspect 14. The method according to any one of Aspects 1 to 13, wherein each dose of the one or more doses is administered in an amount of 0.1 mg / kg or about 0.1 mg / kg to 10 mg / kg or about 10 mg / kg. Aspect 15. The method of Aspect 14, wherein the autoimmune or inflammatory disease or condition is an acute condition. Aspect 16. The method according to any one of Aspects 1 to 15, wherein only a single dose of the variant ICOS-L fusion protein is administered to the subject. Aspect 17. The method of Aspect 14, wherein the autoimmune or inflammatory disease or condition is a chronic condition. Aspect 18. The method according to Aspect 13, Aspect 14, or Aspect 17, wherein the inflammatory or autoimmune disease or condition is systemic lupus erythematosus (SLE). Aspect 19. The method according to Aspect 13, Aspect 14, or Aspect 17, wherein the inflammatory or autoimmune disease or condition is Sjögren's syndrome. Aspect 20. The method according to Aspect 13, Aspect 14, or Aspect 17, wherein the inflammatory or autoimmune disease or condition is psoriatic arthritis. Aspect 21. The method of any one of Aspects 13, 14, or 17, wherein the inflammatory or autoimmune disease or condition is rheumatoid arthritis. Aspect 22. The method of any one of Aspects 13, 14, or 17, wherein the inflammatory or autoimmune disease or condition is Crohn's disease. Aspect 23. The method of any one of Aspects 13, 14, or 17, wherein the inflammatory or autoimmune disease or condition is ulcerative colitis. Aspect 24. The method of any one of Aspects 1 to 15 and 17 to 23, wherein a plurality of doses of the variant ICOS-L fusion protein are administered to the subject. Aspect 25. The method of Aspect 24, wherein each of the plurality of doses is administered at a frequency of once per week or less. Aspect 26. The method of Aspect 24 or 25, wherein each of the plurality of doses is administered once a week (Q1W). Aspect 27. The method of Aspect 24 or 25, wherein each of the plurality of doses is administered once every two weeks (Q2W). Aspect 28. The method of Aspect 24 or 25, wherein each of the plurality of doses is administered once a month (Q4W). Aspect 29. Each dose of the one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg to 10 mg / kg or about 10 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, 1 mg / kg or about 1 mg / kg to 10 mg / kg or about 10 mg / kg, 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, 3 mg / kg or about 3 mg / kg to 10 mg / kg or about 10 mg / kg, 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg, or 6 mg / kg or about 6 mg / kg to 10 mg / kg or about 10 mg / kg, the method of any one of Aspects 1 to 28. Aspect 30. The method according to any one of Aspects 1 to 29, wherein each dose of the one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, or 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg. Aspect 31. The method according to any one of Aspects 1 to 30, wherein each dose of the one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg. Aspect 32. The method according to any one of Aspects 1 to 30, wherein each dose of the one or more doses is administered in an amount of 1 mg / kg or about 1 mg / kg. Aspect 33. The method according to any one of Aspects 1 to 30, wherein each dose of the one or more doses is administered in an amount of 3 mg / kg or about 3 mg / kg. Aspect 34. The method according to any one of Aspects 1 to 30, wherein each dose of the one or more doses is administered in an amount of 6 mg / kg or about 6 mg / kg. Aspect 35. The method according to any one of Aspects 1 to 30, wherein each dose of the one or more doses is administered in an amount of 10 mg / kg or about 10 mg / kg. Aspect 36. The method according to any one of Aspects 1 to 28, wherein each dose of the one or more doses is administered in an amount of 15 mg / kg or about 15 mg / kg. Aspect 37. The method according to any one of Aspects 1 to 28, wherein each dose of the one or more doses is administered in an amount of 20 mg / kg or about 20 mg / kg. Aspect 38. The method according to any one of Aspects 1 to 37, wherein the treatment period is repeated. Aspect 39. The method according to any one of Aspects 1 to 38, wherein the administration is subcutaneous administration. Aspect 40. The method according to any one of Aspects 1 to 38, wherein the administration is intravenous administration. Aspect 41. A method of treating an autoimmune or inflammatory disease of the eye in a subject, comprising the step of intravitreally administering a dose of a variant ICOS-L fusion protein, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. Aspect 42. The method of Aspect 41, wherein the autoimmune or inflammatory disease of the eye is uveitis. Aspect 43. The variant ICOS-L fusion protein is administered at a dose of about 0.01 mg to about 10 mg, 0.05 mg or about 0.05 mg to 10 mg or about 10 mg, 0.1 mg or about 0.1 mg to 10 mg or about 10 mg, 0.5 mg or about 0.5 mg to 10 mg or about 10 mg, 1 mg or about 1 mg to 10 mg or about 10 mg, 1.5 mg or about 1.5 mg to 10 mg or about 10 mg, 2 mg or about 2 mg to 10 mg or about 10 mg, 3 mg or about 3 mg to 10 mg or about 10 mg, 4 mg or about 4 mg to 10 mg or about 10 mg, 5 mg or about 5 mg to 10 mg or about 10 mg, 6 mg or about 6 mg to 10 mg or about 10 mg, 7 mg or about 7 mg to 10 mg or about 10 mg, 8 mg or about 8 mg to 10 mg or about 10 mg, 9 mg or about 9 mg to 10 mg or about 10 mg (including both end values), the method of Aspect 41 or Aspect 42. Aspect 44. The method according to any one of Aspects 41 to 43, wherein the variant ICOS-L fusion protein is administered in a volume of less than 0.2 mL, optionally less than 0.1 mL. Aspect 45. The method according to any one of Aspects 41 to 44, wherein the variant ICOS-L fusion protein is administered in a volume of 0.05 mL or about 0.05 mL. Aspect 46. The method according to any one of Aspects 1 to 45, wherein the ICOS-L reference polypeptide comprises (i) the amino acid sequence shown in SEQ ID NO: 32, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 32, or (iii) a part of (i) and / or (ii) that includes an IgV domain or an IgC domain or a specific binding fragment thereof. Aspect 47. The method according to any one of Aspects 1 to 46, wherein the variant ICOS-L polypeptide comprises the IgV domain or a specific binding fragment thereof. Aspect 48. The method according to any one of Aspects 1 to 47, wherein the IgV domain or a specific binding fragment thereof is the only ICOS-L part of the variant ICOS-L polypeptide or the variant ICOS-L fusion protein. Aspect 49. The method according to any one of Aspects 1 to 48, wherein the ICOS-L reference polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3. Aspect 50. The method according to any one of Aspects 1 to 48, wherein the ICOS-L reference polypeptide consists of the amino acid sequence shown in SEQ ID NO: 3. Aspect 51. The method according to any one of Aspects 1 to 50, wherein the variant ICOS-L polypeptide has a sequence shown in SEQ ID NO: 36, or a sequence showing at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the sequence shown in SEQ ID NO: 36, and includes one or more amino acid substitutions selected from N52H, N57Y, and Q100R. Aspect 52. The method according to any one of Aspects 1 to 51, wherein the variant ICOS-L polypeptide has the sequence shown in SEQ ID NO: 36. Aspect 53. A method according to any one of Aspects 1 to 52, wherein the multimerization domain is or comprises the Fc region of an immunoglobulin. Aspect 54. The method of Aspect 53, wherein the Fc region is a variant Fc region that exhibits reduced effector function compared to the Fc of wild-type human immunoglobulin. Aspect 55. The method of Aspect 53 or 54, wherein the Fc region is a variant IgG1 Fc region that contains one or more amino acid substitutions compared to wild-type human IgG1. Aspect 56. The method of Aspect 54 or 55, wherein the variant Fc region contains one or more amino acid substitutions selected from N297G, E233P / L234V / L235A / G236del / S267K, or L234A / L235E / G237A, and the residues are numbered according to the Kabat EU index. Aspect 57. The method according to any one of Aspects 54 to 56, wherein the variant Fc region further contains the amino acid substitution C220S, and the residue is numbered according to the Kabat EU index. Aspect 58. The method according to any one of Aspects 53 to 57, wherein the Fc region contains K447del, and the residue is numbered according to the Kabat EU index. Aspect 59. A pharmaceutical composition comprising a variant ICOS-L fusion protein for use in a method of preventing or reducing acute graft-versus-host disease (aGVHD) in a subject, wherein the variant ICOS-L fusion protein is or comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, contains one or more amino acid substitutions corresponding to the amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. Use of a variant ICOS-L fusion protein in the preparation of a medicament for use in a method of preventing or reducing acute graft-versus-host disease (aGVHD), wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. Aspect 61. The use according to aspect 59 or the use according to aspect 60, wherein the method comprises administering to the subject one or more doses of the variant ICOS-L fusion protein during a treatment period, and each of the one or more doses of the variant ICOS-L fusion protein is administered in an amount of 0.1 mg / kg or about 0.1 mg / kg to 20 mg / kg or about 20 mg / kg. Aspect 62. The pharmaceutical composition for use according to aspect 59 or aspect 61 or the use according to aspect 60 or aspect 61, wherein the aGvHD is grade II-IV aGVHD. Aspect 63. The pharmaceutical composition for use according to any one of aspects 59, 61, and 62 or the use according to any one of aspects 60, 61, and 62, wherein the aGVHD is resistant or refractory to treatment with an immunosuppressive agent. Aspect 64. The pharmaceutical composition for use according to any one of aspects 59 and 61-63 or the use according to any one of aspects 60 and 61-63, wherein the immunosuppressive agent comprises a corticosteroid. Aspect 65. The pharmaceutical composition for use according to aspect 63 or aspect 64 or the use according to aspect 63 or aspect 64, wherein the immunosuppressive agent comprises cyclosporine. Aspect 66. A pharmaceutical composition comprising a variant ICOS-L fusion protein for use in a method of preventing or reducing inflammation secondary to a viral infection in a subject, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. Aspect 67. Use of a variant ICOS-L fusion protein in the preparation of a medicament for use in a method of preventing or reducing inflammation secondary to a viral infection, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. Aspect 68. The method comprises administering to the subject one or more doses of the variant ICOS-L fusion protein during a treatment period, each of the one or more doses of the variant ICOS-L fusion protein being administered in an amount of 0.1 mg / kg or about 0.1 mg / kg to 20 mg / kg or about 20 mg / kg. A pharmaceutical composition for use according to aspect 66 or use according to aspect 67. Aspect 69. A pharmaceutical composition for use according to aspect 66 or aspect 68 or use according to aspect 67 or aspect 68, wherein the virus is a coronavirus. Aspect 70. A pharmaceutical composition for use according to aspect 69 or use according to aspect 69, wherein the coronavirus is SARS-CoV-2 and the infection is COVID-19. Aspect 71. A pharmaceutical composition for use according to any one of Aspects 66, 68, and 69 or use according to any one of Aspects 67 - 69, wherein the inflammation is related to cytokine release syndrome (CRS). Aspect 72. A pharmaceutical composition for use according to Aspect 71 or use according to Aspect 71, wherein the CRS is severe CRS or CRS of grade 3 or higher. Aspect 73. A pharmaceutical composition for use according to any one of Aspects 68, and 69 - 72 or use according to any one of Aspects 68, and 69 - 72, wherein the subject has severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, or septic shock related to or caused by the viral infection at the time of or immediately before the administration. Aspect 74. A pharmaceutical composition comprising a variant ICOS - L fusion protein for use in the treatment of an autoimmune or inflammatory disease or condition in a subject, wherein the variant ICOS - L fusion protein comprises a variant ICOS - L polypeptide linked to a multimerization domain, the variant ICOS - L polypeptide is an ICOS extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS - L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. Aspect 75. Use of a variant ICOS - L fusion protein in the preparation of a medicament for use in the treatment of an autoimmune or inflammatory disease or condition, wherein the variant ICOS - L fusion protein comprises a variant ICOS - L polypeptide linked to a multimerization domain, the variant ICOS - L polypeptide is an ICOS extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS - L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. Aspect 76. The method includes a step of administering to the subject one or more doses of the variant ICOS-L fusion protein during the treatment period, and each of the one or more doses of the variant ICOS-L fusion protein is administered in an amount of 0.001 mg / kg or about 0.001 mg / kg to 20 mg / kg or about 20 mg / kg. A pharmaceutical composition for the use of Aspect 74 or the use of Aspect 75. Aspect 77. Each dose of the one or more doses is administered in an amount of 0.1 mg / kg or about 0.1 mg / kg to 10 mg / kg or about 10 mg / kg. A pharmaceutical composition for the use of any one of Aspects 61-65, 68-73, and 76 or the use of any one of Aspects 61-65, 68-73, and 76. Aspect 78. The autoimmune or inflammatory disease or condition is in an acute state. A pharmaceutical composition for the use of Aspect 77 or the use of Aspect 77. Aspect 79. Only a single dose of the variant ICOS-L fusion protein is administered to the subject. A pharmaceutical composition for the use of any one of Aspects 61-65, 68-73, and 76-78 or the use of any one of Aspects 61-65, 68-73, and 76-78. Aspect 80. The autoimmune or inflammatory disease or condition is a chronic disease or chronic condition. A pharmaceutical composition for the use of Aspect 77 or the use of Aspect 77. Aspect 81. The inflammatory or autoimmune disease or condition is systemic lupus erythematosus (SLE). A pharmaceutical composition for the use of any one of Aspects 74, 77, and 80 or the use of any one of Aspects 75, 77, and 80. Aspect 82. The inflammatory or autoimmune disease or condition is Sjögren's syndrome. A pharmaceutical composition for the use of any one of Aspects 74, 77, and 80 or the use of any one of Aspects 75, 77, and 80. Aspect 83. The inflammatory or autoimmune disease or condition is psoriatic arthritis. A pharmaceutical composition for the use of any one of Aspects 74, 77, and 80 or the use of any one of Aspects 75, 77, and 80. Aspect 84. A pharmaceutical composition for use according to any one of aspects 74, 77, and 80, or use according to any one of aspects 75, 77, and 80, wherein the inflammatory or autoimmune disease or condition is rheumatoid arthritis. Aspect 85. A pharmaceutical composition for use according to any one of aspects 74, 77, and 80, or use according to any one of aspects 75, 77, and 80, wherein the inflammatory or autoimmune disease or condition is Crohn's disease. Aspect 86. A pharmaceutical composition for use according to any one of aspects 74, 77, and 80, or use according to any one of aspects 75, 77, and 80, wherein the inflammatory or autoimmune disease or condition is ulcerative colitis. Aspect 87. A pharmaceutical composition for use according to any one of aspects 61 - 65, 68 - 73, and 76 - 78, or use according to any one of aspects 61 - 65, 68 - 73, and 76 - 78, wherein a plurality of doses of the variant ICOS - L fusion protein are administered to the subject. Aspect 88. A pharmaceutical composition for use according to aspect 87, or use according to aspect 87, wherein each of the plurality of doses is administered at a frequency of once per week or less. Aspect 89. A pharmaceutical composition for use according to aspect 87 or 88, or use according to aspect 87 or 88, wherein each of the plurality of doses is administered once a week (Q1W). Aspect 90. A pharmaceutical composition for use according to aspect 87 or 88, or use according to aspect 87 or 88, wherein each of the plurality of doses is administered once every two weeks (Q2W). Aspect 91. A pharmaceutical composition for use according to aspect 87 or 88, or use according to aspect 87 or 88, wherein each of the plurality of doses is administered once a month (Q4W). Aspect 92. For use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 91, the pharmaceutical composition or use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 91, wherein each dose of the one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg to 10 mg / kg or about 10 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, 1 mg / kg or about 1 mg / kg to 10 mg / kg or about 10 mg / kg, 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, 3 mg / kg or about 3 mg / kg to 10 mg / kg or about 10 mg / kg, 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg, or 6 mg / kg or about 6 mg / kg to 10 mg / kg or about 10 mg / kg. Aspect 93. For use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 92, the pharmaceutical composition or use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 92, wherein each dose of the one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, or 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg. Aspect 94. A pharmaceutical composition for use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, or use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, wherein each dose of said one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg. Aspect 95. A pharmaceutical composition for use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, or use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, wherein each dose of said one or more doses is administered in an amount of 1 mg / kg or about 1 mg / kg. Aspect 96. A pharmaceutical composition for use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, or use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, wherein each dose of said one or more doses is administered in an amount of 3 mg / kg or about 3 mg / kg. Aspect 97. A pharmaceutical composition for use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, or use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, wherein each dose of said one or more doses is administered in an amount of 6 mg / kg or about 6 mg / kg. Aspect 98. A pharmaceutical composition for use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, or use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 93, wherein each dose of said one or more doses is administered in an amount of 10 mg / kg or about 10 mg / kg. Aspect 99. A pharmaceutical composition for use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 91, or use according to any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 91, wherein each dose of said one or more doses is administered in an amount of 15 mg / kg or about 15 mg / kg. Aspect 100. A pharmaceutical composition for use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 91, or use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 91, wherein each dose of the one or more doses is administered in an amount of 20 mg / kg or about 20 mg / kg. Aspect 101. A pharmaceutical composition for use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 100, or use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 100, wherein the treatment period is repeated. Aspect 102. A pharmaceutical composition for use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 101, or use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 101, wherein the variant ICOS - L fusion protein is administered subcutaneously. Aspect 103. A pharmaceutical composition for use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 101, or use in any one of Aspects 61 - 65, 68 - 73, 76 - 78, and 87 - 101, wherein the variant ICOS - L fusion protein is administered intravenously. Aspect 104. A pharmaceutical composition comprising a variant ICOS - L fusion protein for use in a method of treating an autoimmune or inflammatory disease of the eye in a subject, wherein the variant ICOS - L fusion protein comprises a variant ICOS - L polypeptide linked to a multimerization domain, the variant ICOS - L polypeptide is an ICOS - L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS - L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. Use of a variant ICOS-L fusion protein in the preparation of a medicament for use in a method of treating an autoimmune or inflammatory disease of the eye in a subject, wherein the variant ICOS-L fusion protein comprises a variant ICOS-L polypeptide linked to a multimerization domain, the variant ICOS-L polypeptide is an ICOS-L extracellular domain or a part thereof comprising an IgV domain or a specific binding fragment thereof, and in the reference ICOS-L polypeptide, comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:1. The pharmaceutical composition for use according to aspect 104 or the use according to aspect 105, wherein the autoimmune or inflammatory disease of the eye is uveitis. The method according to aspect 104 or aspect 106 or the use according to aspect 105 or aspect 106, wherein the method comprises administering to the subject one or more doses of the variant ICOS-L fusion protein, and each of the one or more doses of the variant ICOS-L fusion protein is administered at a dose of from about 0.01 mg to about 10 mg, 0.05 mg or from about 0.05 mg to 10 mg or about 10 mg, 0.1 mg or from about 0.1 mg to 10 mg or about 10 mg, 0.5 mg or from about 0.5 mg to 10 mg or about 10 mg, 1 mg or from about 1 mg to 10 mg or about 10 mg, 1.5 mg or from about 1.5 mg to 10 mg or about 10 mg, 2 mg or from about 2 mg to 10 mg or about 10 mg, 3 mg or from about 3 mg to 10 mg or about 10 mg, 4 mg or from about 4 mg to 10 mg or about 10 mg, 5 mg or from about 5 mg to 10 mg or about 10 mg, 6 mg or from about 6 mg to 10 mg or about 10 mg, 7 mg or from about 7 mg to 10 mg or about 10 mg, 8 mg or from about 8 mg to 10 mg or about 10 mg, 9 mg or from about 9 mg to 10 mg or about 10 mg (including the values at both ends). Aspect 108. A pharmaceutical composition for use according to aspect 107 or the use according to aspect 107, wherein the variant ICOS-L fusion protein is administered in a volume of less than 0.2 mL, optionally less than 0.1 mL. Aspect 109. A pharmaceutical composition for use according to aspect 107 or 108 or the use according to aspect 107 or 108, wherein the variant ICOS-L fusion protein is administered in a volume of 0.05 mL or about 0.05 mL. Aspect 110. The ICOS-L reference polypeptide is (i) the amino acid sequence shown in SEQ ID NO: 32, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 32; or (iii) a part of (i) and / or (ii) comprising an IgV domain or an IgC domain or a specific binding fragment thereof. A pharmaceutical composition for use according to any one of aspects 59, 61-65, 66, 68-73, 74, 76-103, 104, and 106-109 or the use according to any one of aspects 60, 61-65, 67, 68-73, 75, 76-103, 105, and 106, 107-109. Aspect 111. A pharmaceutical composition for use according to any one of aspects 59, 61-65, 66, 68-73, 74, 76-103, 104, and 106-110 or the use according to any one of aspects 60, 61-65, 67, 68-73, 75, 76-103, and 105-110, wherein the variant ICOS-L polypeptide comprises an IgV domain or a specific binding fragment thereof. Aspect 112. A pharmaceutical composition for use according to any one of aspects 59, 61-65, 66, 68-73, 74, 76-103, 104, and 106-111 or the use according to any one of aspects 60, 61-65, 67, 68-73, 75, 76-103, and 105-111, wherein the IgV domain or the specific binding fragment thereof is the only ICOS-L part of the variant ICOS-L polypeptide or the variant ICOS-L fusion protein. Aspect 113. A pharmaceutical composition for use in any one of Aspects 59, 61 - 65, 66, 68 - 73, 74, 76 - 103, 104, and 106 - 112, or use in any one of Aspects 60, 61 - 65, 67, 68 - 73, 75, 76 - 103, and 105 - 112, wherein the ICOS-L reference polypeptide comprises the amino acid sequence shown in SEQ ID NO:3. Aspect 114. A pharmaceutical composition for use in any one of Aspects 59, 61 - 65, 66, 68 - 73, 74, 76 - 103, 104, and 106 - 113, or use in any one of Aspects 60, 61 - 65, 67, 68 - 73, 75, 76 - 103, and 105 - 113, wherein the ICOS-L reference polypeptide consists of the amino acid sequence shown in SEQ ID NO:3. Aspect 115. A pharmaceutical composition for use in any one of Aspects 59, 61 - 65, 66, 68 - 73, 74, 76 - 103, 104, and 106 - 114, or use in any one of Aspects 60, 61 - 65, 67, 68 - 73, 75, 76 - 103, and 105 - 114, wherein the variant ICOS-L polypeptide has a sequence shown in SEQ ID NO:36, or a sequence having at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the sequence shown in SEQ ID NO:36, and comprises one or more amino acid substitutions selected from N52H, N57Y, and Q100R. Aspect 116. A pharmaceutical composition for use in any one of Aspects 59, 61 - 65, 66, 68 - 73, 74, 76 - 103, 104, and 106 - 115, or use in any one of Aspects 60, 61 - 65, 67, 68 - 73, 75, 76 - 103, and 105 - 115, wherein the variant ICOS-L polypeptide has the sequence shown in SEQ ID NO:36. Aspect 117. A pharmaceutical composition for use according to any one of Aspects 59, 61 - 65, 66, 68 - 73, 74, 76 - 103, 104, and 106 - 116, or use according to any one of Aspects 60, 61 - 65, 67, 68 - 73, 75, 76 - 103, and 105 - 116, wherein the multimerization domain is an Fc region of an immunoglobulin or comprises said region. Aspect 118. A pharmaceutical composition for use according to Aspect 117 or use according to Aspect 117, wherein the Fc region is a variant Fc region that exhibits reduced effector function as compared to the Fc of wild - type human immunoglobulin. Aspect 119. A pharmaceutical composition for use according to Aspect 117 or 118 or use according to Aspect 117 or 118, wherein the Fc region is a variant IgG1 Fc region that contains one or more amino acid substitutions as compared to wild - type human IgG1. Aspect 120. A pharmaceutical composition for use according to Aspect 118 or 119 or use according to Aspect 118 or 119, wherein the variant Fc region contains one or more amino acid substitutions selected from N297G, E233P / L234V / L235A / G236del / S267K, or L234A / L235E / G237A, and the residues are numbered according to the Kabat EU index. Aspect 121. A pharmaceutical composition for use according to any one of Aspects 118 - 120 or use according to any one of Aspects 118 - 120, wherein the variant Fc region further contains the amino acid substitution C220S, and the residue is numbered according to the Kabat EU index. Aspect 122. A pharmaceutical composition for use according to any one of Aspects 117 - 121 or use according to any one of Aspects 117 - 121, wherein the Fc region contains K447del, and the residue is numbered according to the Kabat EU index. BRIEF DESCRIPTION OF THE DRAWINGS

[0029]

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Mode for Carrying Out the Invention

[0030] Detailed description An immunomodulatory protein is provided herein that is a variant or mutant of ICOS ligand (ICOSL) or a specific binding fragment thereof, or comprises the same, and exhibits activity to bind to at least one target ligand homophilic binding partner (also referred to as a counter-structure protein). In some embodiments, the variant ICOSL polypeptide contains one or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions) compared to a reference (e.g., unmodified) ICOSL or wild-type ICOSL polypeptide. In some embodiments, one or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions) are within the immunoglobulin superfamily (IgSF) domain (e.g., IgV) of the reference (e.g., unmodified) ICOSL or wild-type ICOSL polypeptide. In some embodiments, the variant ICOSL polypeptide exhibits altered, e.g., enhanced or reduced, binding activity or affinity to at least one homophilic binding partner, e.g., at least one of ICOS, CD28, or CTLA-4. In some embodiments, the immunomodulatory protein is soluble. In some embodiments, one or more other immunomodulatory proteins are provided herein that are conjugates or fusions containing the variant ICOSL polypeptide provided herein and one or more other moieties or polypeptides.

[0031] In some embodiments, the variant ICOSL polypeptide and the immunomodulatory protein modulate an immunological immune response, e.g., enhance or reduce an immune response. In some embodiments, the variant ICOSL polypeptide and the immunomodulatory protein provided herein can be used to treat diseases or conditions associated with a dysregulated immune response.

[0032] In some embodiments, the provided variant ICOS-L polypeptides modulate T cell activation through interaction with co-stimulatory signaling molecules. Generally, antigen-specific T cell activation requires two distinct signals. The first signal is provided by the interaction of the T cell receptor (TCR) with major histocompatibility complex (MHC)-bound antigen present on antigen-presenting cells (APCs). The second signal is co-stimulation of TCR binding and is required to avoid T cell apoptosis or anergy.

[0033] In some embodiments, under normal physiological conditions, the T cell-mediated immune response is initiated by antigen recognition by the T cell receptor (TCR) and is regulated by the balance of co-stimulatory and inhibitory signals (e.g., immune checkpoint receptors). The immune system relies on immune checkpoint receptors to prevent autoimmunity (i.e., self-tolerance) and to protect tissues from excessive damage during an immune response, e.g., during an attack against a pathogenic infection. However, in some cases, in diseases and conditions including tumors, these immune regulatory proteins can become dysregulated as a mechanism to escape the immune system.

[0034] In some embodiments, among the known T cell co-stimulatory receptors is CD28, a T cell co-stimulatory receptor for both ligands B7-1 (CD80) and B7-2 (CD86) present on APCs. These same ligands can also bind to the inhibitory T cell receptor CTLA4 (cytotoxic T lymphocyte-associated protein 4) with higher affinity than to CD28. Binding to CTLA-4 serves to downregulate the immune response. ICOS (inducible co-stimulator) is another T cell co-stimulatory receptor that binds to the ICOS ligand (ICOS-L) on APCs. In some cases, CD28 and CTLA-4 are also known to interact with ICOS-L at binding sites that overlap with the binding of ICOS-L to the T cell co-stimulatory receptor ICOS (Yao et al. (2011) Immunity, 34:729-740).

[0035] CD28 and ICOS are closely related T cell co-stimulatory molecules that, when bound by their ligands CD80 and CD86, and ICOS ligand (ICOSL), respectively, induce cell signaling that results in partially overlapping roles in immunity. Thus, CD28 and ICOS are related CD28 family activating receptors that share some intracellular signaling motifs, although the co-stimulatory effects between CD28 and ICOS may differ in some respects. For example, CD28 is expressed on both naive and activated T cells, and its signaling is important for IL-2 production and subsequent T cell effector functions, and CD28 is involved in naive T cell activation. Therapeutic inhibitors of the CD28 pathway (e.g., abatacept, CTLA4-Ig; and belatacept, a second-generation CTLA4-Ig) have been found to be useful for the treatment of some inflammatory arthritis conditions (e.g., rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis) and the prevention of kidney allograft rejection (Wekerle et al. Transpl. Int., 2012, 25:139-50). However, the therapeutic blockade of the CD28 pathway, studied mainly using abatacept, has been found not to work well in several other inflammatory diseases (e.g., Crohn's disease, lupus nephritis, multiple sclerosis), even when T cells are involved in the pathogenesis of such disease indications. Even among these approved indications, treatment with CTLA4-Ig compounds does not lead to complete remission and / or suppression of inflammatory activity in the majority of patients (Masson, Cochrane Database Sys. Rev., 2014, CD010699; Singh, Cochrane Database Sys. Rev., 2017, CD012657). This is consistent with additional co-stimulatory pathways of pathogenesis that remain unaddressed by these treatments.

[0036] ICOS is generally not expressed on the T cell surface until after T cell activation, and signaling through ICOS on activated T cells supports the differentiation of specialized T cell subsets. Thus, in some cases, costimulation by CD28 and ICOS provides overlapping and complementary effects. Therefore, ICOS may represent a distinct pathogenic pathway not addressed by therapeutic agents targeting only CD28.

[0037] In some contexts, T cells express the costimulatory molecules CD28 and ICOS, and the costimulatory molecules CD28 and ICOS interact with CD80 / CD86 and ICOS ligand (ICOSL), respectively, on antigen-presenting cells (APCs). In lymphoid organs, professional APCs (i.e., dendritic cells, macrophages, and B cells) express CD80, CD86, and ICOS-L and bind to CD28+ / ICOS+ T cells. ICOS is not expressed on naive T cells but is rapidly upregulated upon activation. Then, in some embodiments, activated T cells can differentiate into effector cells such as CD8+ cytotoxic T lymphocytes (CTLs), IL-17A / F-secreting CD4+ Th17 cells, or CD4+ follicular helper (T FH ) cells. CD40L-expressing T FH cells bind to B cells within lymphoid follicles and secrete cytokines (e.g., IL-21) that induce the differentiation of B cells into antibody (Ab)-secreting plasma cells. Plasma cells may produce tissue-damaging antibodies such as rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA) in humans, and anti-collagen (CII) antibodies in mice, which can form immune complexes and deposit in joints and other tissues. ICOS-L is also expressed on non-professional APCs and can activate T cells in non-lymphoid tissues, further damaging tissues and joints.

[0038] ICOS appears to play a role in the function of several activated and / or effector T cell subsets, such as differentiated type 1, 2 and 17, and follicular helper (Wikenheiser, Front. Immunol., 2016 7:304). Indeed, activated T cells often downregulate CD28 and / or become less dependent on CD28 co-stimulation, and CD28-negative T cells accumulate in various inflammatory diseases, which correlates with disease activity and lack of responsiveness to abatacept (Garin et al., Eur J Haematol, 1996, 56:119-23; Schmidt et al., J. Clin. Invest., 1996, 97:2027-37; Yang et al., Chin J Microbiol Immunol, 2005, 25:248-251; Scarsi et al., J. Rheumatol., 2011, 38:2105-11; Mou et al., Am J Transplant, 2014, 14:2460-6; Zabinska et al., J Immunol Res., 2016, 2016:1058165; Piantoni et al., Lupus, 2018, 27:143-149). Inhibiting the combination of CD28 and ICOS, for example, via the molecules provided herein, may result in improved therapies for autoimmune and / or inflammatory diseases.

[0039] In some contexts, CD4+ Th1-, Th9-, and Th17-cells are implicated as major causes of multiple sclerosis (MS) by increasing inflammation within the CNS in both MS and experimental autoimmune encephalomyelitis, and CD4+ ICOS+ CXCR5+ T follicular helper cells are increased in PBMCs in relapsing-remitting MS and are correlated with disease progression in secondary progressive MS. In some embodiments, ICOS gene expression is significantly increased in cerebrospinal fluid cells in secondary progressive MS, and an increase in the percentage (%) of total monocytes and monocytes expressing ICOS ligand (ICOSL) is observed. ICOS ligand is also expressed on non-professional APCs and results in T cell activation and further tissue damage in non-lymphoid tissues.

[0040] Among the variant ICOS ligand polypeptides provided are polypeptides that exhibit improved binding affinity for CD28 and / or ICOS when modified by one or more amino acid modifications in the IgSF domain of the ICOS ligand reference polypeptide. In some cases, since it has already been demonstrated that the binding affinity of wild-type ICOS ligand for ICOS is significantly higher than that for CD28, the total increase in ICOS binding in the variants provided is less than the increase in CD28 binding. Various formats of the variant polypeptides provided are also provided. For example, it is shown herein that delivery of an enhanced ICOS ligand protein in a soluble format antagonizes T cell activation by inhibiting CD28 and / or ICOS signaling.

[0041] In particular, the variant ICOS ligand polypeptides are provided, for example, as Fc fusion proteins, in a format that antagonizes or blocks the activity of its cognate binding partners, such as ICOS and / or CD28. In some embodiments, blocking or inhibiting co-stimulatory signaling via CD28 or ICOS may be useful for suppressing the immune response, which may be useful in the treatment of inflammatory or autoimmune disorders (e.g., multiple sclerosis or encephalitis) or organ transplantation.

[0042] In some embodiments, the modulation of immune signaling achieved by the provided variant ICOS-L polypeptides and immunomodulatory polypeptides provides benefits in the treatment of inflammatory and autoimmune disorders and other diseases and conditions as compared to other treatments. In some cases, therapies that intervene in and alter the costimulation of both receptors are limited by the spatial orientation requirements and size constraints imposed by the extent of the immune synapse. In some aspects, existing therapeutic agents, including antibody drugs, may not be able to interact simultaneously with multiple target proteins involved in the regulation of these interactions. Furthermore, because of pharmacokinetic differences between drugs that target one or the other of these two receptors independently, it can be difficult to correctly maintain the desired blood concentrations of such drug combinations over the entire course of treatment.

[0043] In some embodiments, the provided variant ICOS-L polypeptide or immunomodulatory protein modulates (e.g., enhances) the immunological activity induced by the costimulatory receptors CD28 or ICOS. Thus, in some embodiments, the provided polypeptide provides a variant ICOS-L (inducible costimulatory molecule ligand) with altered (e.g., increased or decreased) binding affinity for both CD28 and ICOS and, in some cases, for CTLA-4, thereby overcoming these limitations by agonizing or antagonizing the complementary action of costimulation by the receptor. Methods of making and using these variant ICOS-Ls are also provided.

[0044] In some situations, the molecules provided may also be more effective than other soluble therapeutic protein agents. For example, abatacept (CTLA-4-Fc) has been shown to interfere with T cell co-stimulation to attenuate T cell responses when treating autoimmune disease situations such as rheumatoid arthritis, psoriatic arthritis, and juvenile idiopathic arthritis, while belatacept, a variant CTLA-4-Fc molecule, interferes with T cell co-stimulation to attenuate T cell responses when treating graft rejection. However, these CTLA-4-Fc proteins bind to CD80 and CD86 and prevent these co-stimulatory ligands from binding only to CD28 and inducing only CD28. The variant ICOS-L polypeptides provided herein exhibit improved binding affinity and activity for both CD28 and ICOS in some cases.

[0045] All publications (including patents, patent applications, scientific papers, and databases) referred to herein are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication (including patents, patent applications, scientific papers, and databases) were specifically and individually indicated to be incorporated by reference. If the definitions set forth herein are contrary to or otherwise inconsistent with the definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein shall control.

[0046] The headings of the items used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0047] I. Definitions Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or related terms used in this specification are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or for ready reference, and the inclusion of such definitions in this specification should not necessarily be construed as making a significant difference from what is generally understood in the art.

[0048] As used throughout this specification, terms are defined as follows unless otherwise limited in a particular instance. When used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms, acronyms, and abbreviations used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Unless otherwise indicated, chemical and biochemical abbreviations and symbols are in accordance with the IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include not only the values defining the range but also all integer values therebetween.

[0049] As used in the context of immunoglobulin superfamily domains, the term "affinity-modified" means a mammalian immunoglobulin superfamily (IgSF) domain having an amino acid sequence that has been altered such that the binding affinity or avidity for at least one of its cognate binding partners (or "counterstructures") is changed (compared to the parental wild-type or unmodified (i.e., unaltered in affinity) IgSF control domain) to be either increased or decreased (compared to the corresponding wild-type parental or unmodified IgSF domain). In this context, affinity-modified ICOS-L IgSF domains are included. In some embodiments, the affinity-modified IgSF domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid differences (e.g., amino acid substitutions) compared to a reference (e.g., unmodified) IgSF domain or wild-type IgSF domain. The increase or decrease in binding affinity or avidity can be determined using well-known binding assays such as flow cytometry. See Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005). See also Linsley et al., Immunity, Vol. 1: 793-801 (1994). An increase in the binding affinity or avidity of a protein for its cognate binding partner is a value that is at least 10% greater than the wild-type IgSF domain control value, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type IgSF domain control value. A decrease in the binding affinity or avidity of a protein for at least one of its cognate binding partners is a value that is 90% or less of the control but 10% or more of the wild-type IgSF domain control value, and in some embodiments, 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the wild-type IgSF domain control value but 10% or more of the value.The protein with modified affinity has an altered primary amino acid sequence due to substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain" should not be construed as imposing any conditions of any particular starting composition or method by which the affinity-modified IgSF domain was made. Thus, the affinity-modified IgSF domains of the present invention are not limited to being converted from wild-type IgSF domains by any particular affinity-modifying process. Affinity-modified IgSF domain polypeptides can be generated, for example, starting from wild-type mammalian IgSF domain sequence information, then modeled in silico for their binding to their cognate binding partner, and finally recombinantly or chemically synthesized to produce the subject affinity-modified IgSF domain composition. As another mere example, affinity-modified IgSF domains can be made by site-directed mutagenesis of wild-type IgSF domains. Thus, an affinity-modified IgSF domain represents a product that can be produced by any given process but is not necessarily so. Various techniques may be used, including recombinant methods, chemical synthesis, or combinations thereof.

[0050] As used herein, the term "allogeneic" means cells or tissues that are removed from one organism and then injected or transplanted into a genetically different organism of the same species. In some aspects of the present invention, the species is murine or human.

[0051] As used herein, the term "autologous" means cells or tissues that are removed from the same organism and later injected or transplanted into that organism. Autologous cells or tissues can be modified, for example, by recombinant DNA methods, such that they are no longer genetically identical to the natural cells or natural tissue removed from the organism. For example, natural autologous T cells can be genetically modified by recombinant DNA techniques to become autologous modified cells that express transmembrane immunomodulatory proteins and / or chimeric antigen receptors (CARs), which, in some cases, includes modifying T cells or tumor infiltrating lymphocytes (TILs). The modified cells are then injected into the patient from whom the natural T cells were isolated. In some embodiments, the organism is a human or a mouse.

[0052] As used herein, the terms "binding affinity" and "binding avidity" each refer to the specific binding affinity and specific binding avidity of a protein for its counterstructure under specific binding conditions. In biochemical kinetics, avidity refers to the cumulative strength of multiple affinities of individual non-covalent interactions, such as between ICOS-L and its counterstructures ICOS and / or CD28. Thus, avidity is different from the affinity that represents the strength of a single interaction. The improvement or decrease in the binding affinity of a variant ICOS-L containing an IgSF domain with modified affinity for its counterstructure is determined compared to the binding affinity of unmodified ICOS-L (e.g., unmodified ICOS-L containing a native or wild-type IgSF domain (e.g., IgV domain)). Methods for determining binding affinity or avidity are known in the art. See, for example, Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005). In some embodiments, the variant ICOS-L of the present invention (i.e., an ICOS-L protein containing an affinity-modified IgSF domain) specifically binds to CD28 and / or ICOS with a binding affinity that results in an average fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than that of the wild-type ICOS-L control when measured by flow cytometry, e.g., in the binding assay described in Example 6.

[0053] The term "biological half-life" refers to the time required for a substance (e.g., an immunomodulatory polypeptide including a variant ICOS-L of the present invention) to lose half of its pharmacological or physiological activity or concentration. The biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic degradation) of the substance, or absorption and concentration in specific organs or tissues in the body. In some embodiments, the biological half-life can be evaluated by determining the time required for the plasma concentration of the substance to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used to derivatize the polypeptides of the present invention to extend their biological half-life are known in the art and include, but are not limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

[0054] The term "chimeric antigen receptor" or "CAR", as used herein, refers to an artificial (i.e., man-made) transmembrane protein expressed on mammalian cells that includes at least an ectodomain, a transmembrane domain, and an endodomain. Optionally, the CAR protein includes a "spacer" that covalently links the ectodomain to the transmembrane domain. The spacer is often a polypeptide that links the ectodomain to the transmembrane domain via peptide bonds. CARs are typically expressed on mammalian lymphocytes. In some embodiments, the CAR is expressed on mammalian cells such as T cells or tumor-infiltrating lymphocytes (TILs). A CAR expressed on a T cell is referred to herein as a "CAR T cell" or "CAR-T". In some embodiments, the CAR-T is a helper T cell, a cytotoxic T cell, a natural killer T cell, a memory T cell, a regulatory T cell, or a γδ T cell. For example, when clinically used in adoptive cell transfer, the CAR-T having antigen-binding specificity for a patient's tumor is typically modified to be expressed on natural T cells obtained from the patient. The modified T cells expressing the CAR are then returned to the patient by infusion. Thus, CAR-Ts are often autologous CAR-Ts, but allogeneic CAR-Ts are also included within the scope of the invention. The ectodomain of the CAR includes an antigen-binding region (e.g., an antibody or an antigen-binding fragment thereof (e.g., scFv)) that specifically binds to a target antigen (e.g., a tumor-specific antigen) under physiological conditions. Specific binding results in a series of biochemical events (i.e., signal transduction) that lead to the modulation of the immune activity of the CAR-T. Thus, for example, specific binding of the CAR-T to its target antigen by the antigen-binding region of the CAR-T can lead to a change in the immune activity of the T cell, as reflected by a change in cytotoxicity, proliferation, or cytokine production. In some embodiments, signal transduction by CAR-T activation is achieved by the CD3ζ chain ("CD3-z"), which is involved in signal transduction in natural mammalian T cells. The CAR-T can further include multiple signal transduction domains (e.g., CD28, 4-1BB, or OX40) that further modulate the immune regulatory response of the T cell.CD3-z contains a conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM) that is involved in T cell receptor signaling.

[0055] The term "collectively" or "collective" when used with respect to cytokine production induced by the presence of two or more of the variant ICOS-Ls of the invention in an in vitro assay means the overall cytokine expression levels regardless of the cytokine production induced by the individual variant ICOS-Ls. In some embodiments, the cytokine being assayed is IFN-γ in an in vitro primary T cell assay.

[0056] The term "cognate binding partner" (used interchangeably with "counterstructure") with respect to a polypeptide (e.g., an IgSF domain of a variant ICOS-L) refers to at least one molecule (typically a native mammalian protein) to which the polypeptide being referred to specifically binds under specific binding conditions. In some aspects, a variant ICOS-L containing an affinity-modified IgSF domain specifically binds to the counterstructure of the corresponding native or wild-type ICOS-L with enhanced or reduced affinity. One kind of ligand that is recognized under specific binding conditions and specifically binds to its cognate receptor is an example of a counterstructure or cognate binding partner of that receptor. A "cell surface cognate binding partner" is a cognate binding partner that is expressed on the surface of a mammalian cell. A "cell surface molecular species" is a cognate binding partner of a ligand of an immunological synapse (IS) that is expressed on or by a cell (e.g., a mammalian cell) that forms the immunological synapse.

[0057] As used herein, "conjugate", "conjugation" or their grammatical variations refer to connecting or linking two or more compounds together by any method of connection or linkage known in the art to result in the formation of another compound. It can also refer to a compound produced by connecting or linking two or more compounds together. For example, a variant ICOS-L polypeptide directly or indirectly linked to one or more chemical moieties or polypeptides is an exemplary conjugate. Such conjugates include fusion proteins, those produced by chemical conjugation, and those produced by any other method.

[0058] The term "competitive binding", as used herein, means that a protein can specifically bind to at least two cognate binding partners, but the specific binding of one cognate binding partner inhibits (e.g., interferes with or prevents) the simultaneous binding of a second cognate binding partner. Thus, in some cases, a protein cannot bind to two cognate binding partners simultaneously. Generally, competitive binders contain the same or overlapping binding sites for specific binding, but this is not an essential requirement. In some embodiments, competitive binding causes a measurable (partial or complete) inhibition of the specific binding of a protein to one of its cognate binding partners due to the specific binding of a second cognate binding partner. Various methods for quantifying competitive binding, such as ELISA (enzyme-linked immunosorbent assay), are known.

[0059] The term "conservative amino acid substitution" as used herein means an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. The group of conservative amino acid substitutions is valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0060] The term "corresponding" with respect to the position of a protein, e.g., the description that a nucleotide or amino acid position "corresponds" to a nucleotide or amino acid position in a disclosed sequence (e.g., as shown in a Sequence Listing), refers to the position of a nucleotide or amino acid identified by alignment with the disclosed sequence based on a structural sequence alignment or using a standard alignment algorithm (e.g., the GAP algorithm). For example, corresponding residues can be identified by alignment with a reference sequence having the sequence shown in SEQ ID NO:1 (ECD domain) or the sequences shown in SEQ ID NO:2 or 3 (IgV domain) by the structural alignment method described herein. By aligning the sequences, one of ordinary skill in the art can identify corresponding residues, e.g., using conserved amino acid residues and identical amino acid residues as a criterion.

[0061] The terms "reduce" or "attenuate" or "inhibit", as used herein, mean a statistically significant amount of reduction. The reduction can be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction relative to a control value (e.g., a non-zero control value).

[0062] The terms "reduced" or "decreased", as used herein in the context of reducing the immunological activity of mammalian lymphocytes, mean that one or more activities of the lymphocytes are reduced as compared to a control, e.g., an untreated control, or a control used under the same conditions as a treatment using an unmodified control or non-variant control. The reduction in activity can refer to, for example, inhibiting the cell cycle, reducing cell survival, reducing cell proliferation, reducing cytokine production, or reducing the cytotoxicity of T cells, by a statistically significant amount. In some embodiments, reference to a reduction in immunological activity means reducing interferon gamma (IFN-γ) production, e.g., by a statistically significant amount, as compared to no treatment. In some embodiments, the immunological activity can be evaluated in a mixed lymphocyte reaction (MLR) assay. Methods of performing an MLR assay are known in the art. Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56. Other methods of evaluating lymphocyte activity are known in the art, including any assay described herein. In some embodiments, the reduction can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% reduction as compared to a control value, e.g., an untreated control value or a non-zero control value.

[0063] The term "derivative" or "derivatized" refers to modifying a protein by covalently attaching it directly or indirectly to a composition so as to change characteristics such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, efficacy, or potency while retaining or improving the therapeutic benefit of a protein. Derivatives of the immunomodulatory polypeptides of the present invention are within the scope of the present invention and can be made, for example, by glycosylation, pegylation, lipid addition, or Fc fusion.

[0064] When a domain (typically three or more, generally five or seven or more amino acids, e.g., a sequence of 10 to 200 amino acid residues) is used herein, it refers to a portion of a molecule, e.g., a protein or coding nucleic acid, that is structurally and / or functionally distinct from other parts of the molecule and can be identified. For example, a domain can include a polypeptide chain portion that forms a structure that folds independently within a protein and is composed of one or more structural motifs and / or is recognized by a functional activity such as a binding activity. A protein may have one or more distinct domains. For example, a domain can be identified, defined, or distinguished by its primary sequence or homology to related family members in the primary structure, e.g., homology to a motif. In another example, a domain can be distinguished by its function, e.g., its ability to interact with a biomolecule, e.g., a cognate binding partner. A domain can exhibit a biological function or activity independently so as to be able to perform an activity, e.g., binding, either independently or in a state fused to another molecule. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. Definitions are provided herein for purposes of illustration, but it is understood that recognizing a particular domain by name is well within the level of those skilled in the art. If necessary, domains can be identified using appropriate software.

[0065] As used herein, the term "ectodomain" refers to the region of a membrane protein that is outside the cell membrane, such as the region of a transmembrane protein. The ectodomain often contains a binding domain that specifically binds to a ligand or cell surface receptor, for example, via a binding domain that specifically binds to the ligand or cell surface receptor. The ectodomain of a transmembrane protein of a cell is also sometimes referred to as the extracellular domain.

[0066] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a therapeutic composition of the invention comprising a protein composition or cell composition that, when administered alone (i.e., as monotherapy), or in combination with additional therapeutic agents, ex vivo (by contacting cells derived from a patient), or in vivo (by administering to a patient), statistically significantly retards disease progression, for example, by alleviating or eliminating the symptoms and / or cause of the disease, or statistically significantly retards disease progression. An effective amount may also be an amount that alleviates, reduces, or mitigates at least one symptom or biological response or effect associated with a disease or disorder, prevents progression of the disease or disorder, or improves the physical function of a patient. In the case of cell therapy, the effective amount is the effective dose or number of cells administered to a patient by adoptive cell therapy. In some embodiments, the patient is a mammal such as a non-human primate or a human patient.

[0067] As used herein, the term "endodomain" refers to the region of some membrane proteins, such as transmembrane proteins, that extends into the internal space defined by the cell surface membrane. In mammalian cells, the endodomain is the cytoplasmic region of the membrane protein. Inside the cell, the endodomain can interact with intracellular components and play a role in signal transduction, and thus in some cases may be an intracellular signal transduction domain. The endodomain of a transmembrane protein of a cell is also sometimes referred to as the cytoplasmic domain and in some cases may also be a cytoplasmic signal transduction domain.

[0068] As used herein in the context of enhancing or improving the immunological activity of mammalian lymphocytes, the terms "enhanced" or "improved" mean that one or more activities of the lymphocytes are improved as compared to a control, e.g., an untreated control, or a control used under the same conditions as a treatment using an unmodified or non-variant control. The improvement in activity may be one or more of an improvement in cell survival, cell proliferation, cytokine production, or cytotoxicity of T cells, e.g., by a statistically significant amount. In some embodiments, reference to an improvement in immune activity means an improvement in interferon gamma (IFNγ) production, e.g., by a statistically significant amount. In some embodiments, the immunological activity can be evaluated in a mixed lymphocyte reaction (MLR) assay. Methods of performing the MLR assay are known in the art. Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56. Other methods of evaluating lymphocyte activity are known in the art, including any of the assays described herein. In some embodiments, the enhancement may be at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% improvement over a non-zero control value.

[0069] As used herein, the term "modified cell" refers to a mammalian cell that has been genetically engineered by human intervention such as recombinant DNA methods or viral transduction. In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., a T cell, B cell, NK cell) or an antigen-presenting cell (e.g., a dendritic cell). The cell may be a primary cell derived from a patient or a cell line. In some embodiments, the modified cells of the invention comprise a variant ICOS-L provided herein. In some embodiments, the variant ICOS-L is a transmembrane immunomodulatory protein (hereinafter referred to as "TIP") expressed on the modified cell. In some embodiments, the TIP contains an extracellular domain or a portion thereof that contains an IgV domain linked to a transmembrane domain (e.g., the ICOS-L transmembrane domain) and optionally an intracellular signaling domain. In some cases, the TIP is arrayed as a chimeric receptor that contains a heterologous cytoplasmic signaling domain or endodomain. In some embodiments, the modified cells can express and secrete an immunomodulatory protein as described herein. Some of the modified cells provided further contain a modified T cell receptor (TCR) or chimeric antigen receptor (CAR).

[0070] As used herein, the term "modified T cell" refers to a T cell (e.g., a helper T cell, a cytotoxic T cell (or cytotoxic T lymphocyte or CTL), a natural killer T cell, a regulatory T cell, a memory T cell, or a γδ T cell) that has been genetically modified by human intervention (e.g., by recombinant DNA methods or viral transduction methods). The modified T cell comprises a variant ICOS ligand transmembrane immunomodulatory protein (TIP) or a secreted immunomodulatory protein (SIP) of the invention that is expressed on the T cell, and the TIP or SIP is modified to regulate the immune activity of the modified T cell itself or to regulate the immune activity of mammalian cells to which the variant ICOS ligand specifically binds that is expressed on the T cell. The modified T cell may comprise a variant ICOS ligand secreted immunomodulatory protein (SIP) of the invention that is expressed and / or secreted by the T cell and modified to regulate the immune activity of the modified T cell itself or of mammalian cells to which the variant ICOS ligand specifically binds when secreted by the T cell.

[0071] The term "modified (T cell) receptor" or "modified TCR" refers to a T cell receptor (TCR) that has been selected, cloned, and / or subsequently introduced into a population of T cells (which population is often used in adoptive immunotherapy) and modified to specifically bind to a major histocompatibility complex (MHC) / peptide target antigen with a desired affinity. In contrast to a modified TCR, a CAR is modified to bind to a target antigen in an MHC-independent manner.

[0072] The term "expressed on" as used herein refers to a protein expressed on the surface of a cell (e.g., a mammalian cell). Thus, the protein is expressed as a membrane protein. In some embodiments, the protein being expressed is a transmembrane protein. In some embodiments, the protein is conjugated to a moiety (e.g., a drug or a detectable label). The protein expressed on the surface of the cell can include cell surface proteins (e.g., cell surface receptors) expressed on mammalian cells.

[0073] The term "half-life extension moiety" refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum as compared to the half-life of the protein not conjugated to such a portion. In some embodiments, the half-life is greater than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold, or about 1.2-fold, about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold, or about 6.0-fold extended. In some embodiments, the half-life is extended by more than 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, or 1 week after in vivo administration as compared to a protein having no half-life extension moiety. Half-life refers to the time it takes for a protein to lose half of its concentration, amount, or activity. The half-life can be determined, for example, by using an ELISA assay or an activity assay. Exemplary half-life extension moieties include the Fc domain, multimerization domains, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylation).

[0074] As used herein, the term "immune synapse" refers to the interface between a mammalian cell expressing MHC I (major histocompatibility complex) or MHC II (e.g., an antigen-presenting cell or a tumor cell) and a mammalian lymphocyte (e.g., an effector T cell or a natural killer (NK) cell).

[0075] The Fc (fragment crystallizable) region or domain of an immunoglobulin molecule (also referred to as the Fc polypeptide) corresponds primarily to the constant region of the immunoglobulin heavy chain and is involved in various functions including the effector functions of the antibody. The Fc domain contains a portion or all of the hinge domain of the immunoglobulin molecule, as well as the CH2 and CH3 domains. The Fc domain can form a dimer of two polypeptide chains connected by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc with reduced activity to promote effector functions (e.g., reduced by 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more). In some embodiments, references to amino acid substitutions in the Fc region are made according to the EU numbering system unless otherwise specified based on a particular SEQ ID NO. The EU numbering is known and follows the EU index as reported in the recently updated IMGT Scientific Chart (IMGT®, i.e., international ImMunoGeneTics information system® http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (creation date: May 17, 2001, last update date: January 10, 2013)) and Kabat, E.A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0076] An immunoglobulin Fc fusion (an "Fc fusion"), such as an immunomodulatory Fc fusion protein, is a molecule that contains one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. The Fc fusion can include, for example, the Fc region of an antibody (which promotes effector function and pharmacokinetics) and variant ICOS-L. The immunoglobulin Fc region can be linked indirectly or directly to one or more variant ICOS-Ls or small molecules (fusion partners). Various linkers are known in the art and can optionally be used to link the Fc to the fusion partner to generate an Fc fusion. The same type of Fc fusion can be dimerized to form an Fc fusion homodimer, or non-identical types can be used to form an Fc fusion heterodimer. In some embodiments, the Fc is a mammalian Fc, such as murine or human Fc. In some embodiments, the Fc is an Fc lacking effector function.

[0077] The term "host cell" refers to a cell that can be used to express a protein encoded by a recombinant expression vector. The host cell can be a prokaryote, such as Escherichia coli (E. coli), or the host cell can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Examples of host cells include Chinese hamster ovary (CHO) cells or derivatives thereof, such as Veggie CHO and related cell lines that grow in serum-free media or the CHO line DX-B11 that is DHFR-deficient. In some embodiments, the host cell is a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell).

[0078] The term "immunoglobulin" (abbreviated as "Ig") as used herein refers to mammalian immunoglobulin proteins including any of five human classes of antibodies: IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also includes immunoglobulins that are less than full length whether fully or partially synthetic (e.g., recombinant or chemically synthesized) or naturally produced, such as antigen-binding fragments (Fab), V H and V L containing variable fragments (Fv), V H and V L linked together in one chain containing single-chain variable fragments (scFv), as well as other antibody V-region fragments (Fab’, F(ab)2, F(ab’)2, dsFv diabodies, Fc, and Fd polypeptide fragments). Bispecific antibodies, both homodimeric and heterodimeric, are included within the scope of the term.

[0079] The term "immunoglobulin superfamily" or "IgSF" as used herein means a group of cell surface proteins and soluble proteins that are involved in cell recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on structural features common to immunoglobulins (i.e., antibodies); all of these possess domains known as immunoglobulin domains or folds. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors as well as intracellular muscle proteins. These are usually associated with roles in the immune system. Proteins in the immune synapse are often members of the IgSF. The IgSF can also be classified into "subfamilies" based on common properties such as function. Such subfamilies typically consist of 4 - 30 IgSF members.

[0080] The term "IgSF domain" or "immunoglobulin domain" or "Ig domain", as used herein, refers to the structural domain of an IgSF protein. Ig domains are named after the immunoglobulin molecule. They contain approximately 70 to 110 amino acids and are classified according to their size and function. Ig domains possess a characteristic Ig fold with a sandwich-like structure formed by two sheets of antiparallel β-strands. Interactions between the hydrophobic amino acids inside the sandwich and highly conserved disulfide bonds formed between cysteine residues in the B and F strands stabilize the Ig fold. One end of the Ig domain has a portion called the complementarity-determining region, which is important for the specificity of the antibody for its ligand. Ig-like domains can be classified (into classes) as IgV, IgC1, IgC2, or IgI. Most Ig domains are either variable (IgV) domains or constant (IgC) domains. IgV domains with nine β-strands are generally longer than IgC domains with seven β-strands. The Ig domains of some members of the IgSF resemble IgV domains in the amino acid sequence but still have a size similar to that of IgC domains. These are called IgC2 domains, while the standard IgC domain is called the IgC1 domain. The T cell receptor (TCR) chain contains two Ig domains in its extracellular portion (one IgV domain at the N-terminus and one IgC1 domain adjacent to the cell membrane). ICOS-L contains two Ig domains: IgV and IgC.

[0081] As used herein, the term "IgSF species" means a population of IgSF member proteins having the same or substantially the same primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines an identity unique to all IgSF species to which that IgSF member belongs. Thus, each IgSF family member is unique compared to other IgSF family members, and thus each species of a particular IgSF family member is unique compared to species of another IgSF family member. Nevertheless, differences between molecules of the same IgSF species can arise due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Furthermore, small sequence differences within a single IgSF species due to genetic polymorphism also constitute differences in other forms within a single IgSF species, similar to the wild-type truncated form of an IgSF species due to proteolytic cleavage, for example. A "cell surface IgSF species" is an IgSF species that is expressed on the surface of a cell (generally a mammalian cell).

[0082] As used herein in the context of mammalian lymphocytes such as T cells, the term "immunoreactivity" refers to one or more of cell survival, cell proliferation, cytokine production (e.g., interferon-γ), or T cell cytotoxic activity. In some cases, immunoreactivity can mean the expression of cytokines such as chemokines or interleukins in cells. Assays for determining enhancement or suppression of immunoreactivity include the MLR (mixed lymphocyte reaction) assay that measures interferon-γ cytokine levels in the culture supernatant (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), the SEB (staphylococcal enterotoxin B) T cell stimulation assay (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), and the anti-CD3 T cell stimulation assay (Li and Kurlander, J Transl Med. 2010: 8: 104). Since T cell activation is associated with the secretion of IFN-γ cytokine, detection of IFN-γ levels in the culture supernatant from these in vitro human T cell assays can be assayed using commercially available ELISA kits (Wu et al, Immunol Lett 2008 Apr 15; 117(1): 57-62). Induction of an immune response results in enhanced immunoreactivity compared to resting lymphocytes. Immunomodulatory proteins as provided herein (e.g., variant ICOS-L polypeptides containing affinity-modified IgSF domains) can, in some embodiments, increase IFN-γ (interferon-γ) expression in primary T cell assays compared to wild-type IgSF members or IgSF domain controls, or, in alternative embodiments, decrease it. One of ordinary skill in the art will recognize that the format of the primary T cell assay used to determine an increase in IFN-γ expression is different from the format employed to assay for a decrease in IFN-γ expression.When assaying the ability of the immunomodulatory protein or affinity-modified IgSF domain of the present invention to reduce IFN-γ expression in a primary T cell assay, a mixed lymphocyte reaction (MLR) assay can be used as described in Example 6. Advantageously, the soluble form of the affinity-modified IgSF domain of the present invention can be employed to determine its ability to antagonize IFN-γ expression and thereby reduce its expression in the MLR, as also described in Example 6. Alternatively, when assaying the ability of the immunomodulatory protein or affinity-modified IgSF domain of the present invention to increase IFN-γ expression in a primary T cell assay, a co-stimulation assay can be used. In the co-stimulation assay, T cell receptor signals (provided by anti-CD3 antibody in some embodiments) are combined with a co-stimulated affinity-modified IgSF domain, such as variant ICOS-L, to determine the ability to increase IFN-γ expression compared to a wild-type IgSF domain control. Methods for assaying the immunological activity of modified cells, including evaluating the activity of variant ICOS-L transmembrane immunomodulatory proteins, are known in the art and include, but are not limited to, the ability to proliferate T cells after antigen stimulation, the ability to sustain T cell proliferation in the absence of restimulation, and anti-cancer activity in a suitable animal model. Assays are also standard. 51 Assays for evaluating cytotoxicity include, but are not limited to, Cr release assays (see, for example, Milone et al., (2009) Molecular Therapy 17: 1453-1464), flow-based cytotoxicity assays, or impedance-based cytotoxicity assays (Peper et al. (2014) Journal of Immunological Methods, 405:192-198).

[0083] An "immunomodulatory polypeptide" or "immunomodulatory protein" is a polypeptide or protein molecule that modulates immune activity. "Modulation" of the immune response means either enhancement or suppression of immune activity. An immunomodulatory protein can be a single polypeptide chain or a multimer (dimer or higher-order multimer) of at least two polypeptide chains covalently linked to each other (e.g., by interchain disulfide bonds). Thus, monomers, dimers, and higher-order multimer polypeptides are within the scope of the defined term. A multimer polypeptide can be a homomultimer (of the same polypeptide chain) or a heteromultimer (of different polypeptide chains). The immunomodulatory proteins of the present invention include variant ICOS-L.

[0084] The term "improve" as used herein means to improve by a statistically significant amount. The improvement can be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, or greater improvement over a non-zero control value.

[0085] An "isoform" of ICOS-L (inducible costimulator ligand; CD275) is one of a plurality of naturally occurring ICOS-L polypeptides having different amino acid sequences. An isoform can be the product of a splice variant of an RNA transcript expressed by a single gene or the product of highly similar but different genes that give rise to functionally similar proteins such as can result from gene duplication. As used herein, the term "isoform" of ICOS-L also refers to the products of different alleles of the ICOS-L gene (e.g., ICOS-LG).

[0086] As used herein, the term "lymphocyte" means any of three subtypes of white blood cells of the mammalian immune system. These include natural killer cells (NK cells) (which function in cell-mediated cytotoxic innate immunity), T cells (related to cell-mediated cytotoxic acquired immunity), and B cells (related to humoral antibody-mediated acquired immunity). T cells include helper T cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. Also included within the definition of lymphocytes are innate lymphoid cells (ILCs).

[0087] The term "mammal" or "patient" specifically includes reference to at least one of human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.

[0088] As used herein, the term "membrane protein" means a protein that attaches (binds) directly or indirectly to a lipid bilayer under physiological conditions. The lipid bilayer forming the membrane can be a biological membrane, such as the cell membrane of a eukaryote (e.g., a mammal), or an artificial (i.e., man-made) membrane, such as the membrane found on a liposome. The binding of a membrane protein to the lipid bilayer can be by covalent bonding or by non-covalent interactions (e.g., hydrophobic or electrostatic interactions). A membrane protein can be an integral membrane protein or a peripheral membrane protein. A membrane protein that is a peripheral membrane protein is bound to the lipid bilayer by non-covalent interactions or is bound to an integral membrane protein by non-covalent interactions. A peripheral membrane protein forms a transient binding to the lipid bilayer such that under physiological range conditions in a mammal, the peripheral membrane protein can interact with and / or dissociate from the lipid bilayer. In contrast to a peripheral membrane protein, an integral membrane protein forms a substantially permanent binding to the lipid bilayer of the membrane such that under physiological range conditions in a mammal, the integral membrane protein does not dissociate from its binding to the lipid bilayer. A membrane protein can form a binding to the membrane by one layer of the lipid bilayer (monotopic type) or can be bound by both layers of the membrane (polytopic type). An integral membrane protein that interacts with only one lipid bilayer is an "integral monotopic protein". An integral membrane protein that interacts with both lipid bilayers is an "integral polytopic protein". Alternatively, it is referred to herein as a "transmembrane protein".

[0089] As used herein, the term "modulation" or "modulating", when used in the context of an immune response (e.g., a mammalian immune response), refers to any change (e.g., enhancement or reduction) of an existing or potential immune response that occurs as a result of administration of an immunomodulatory polypeptide comprising a variant ICOS-L of the invention or as a result of administration of a modified cell expressing an immunomodulatory protein of the invention (e.g., a variant ICOS-L transmembrane immunomodulatory protein). Thus, modulation refers to a change (e.g., enhancement or reduction) of an immune response as compared to an immune response that occurs or exists in the absence of administration of an immunomodulatory protein comprising a variant ICOS-L or a cell expressing such an immunomodulatory polypeptide. Such modulation includes any induction, activation, suppression, or change in the degree or extent of immune activity of immune cells. Immune cells include B cells, T cells, NK (natural killer) cells, NK T cells, professional antigen-presenting cells (APCs), and non-professional antigen-presenting cells, as well as inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change imparted to an existing immune response, an immune response in the developmental stage, a potential immune response, or the ability to induce, modulate, affect, or respond to an immune response. Modulation includes any change in the expression and / or function of genes, proteins and / or other molecules in immune cells as part of an immune response. Modulation of an immune response or modulation of immune activity includes, for example, the following: elimination, deletion, or sequestration of immune cells; induction or generation of immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen-presenting cells, or inflammatory cells; induction of anergy (i.e., unresponsiveness) in immune cells; enhancing or suppressing the activity or function of immune cells (including, without limitation, changing the pattern of proteins expressed by these cells). Examples include changes in the production and / or secretion of specific molecular classes such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors, or any combination of these regulatory events.The modulation can be evaluated by, for example, changes in IFN-γ (interferon γ) expression compared to wild-type ICOS-L controls in a primary T cell assay (see Zhao and Ji, Exp Cell Res. 2016 Jan 1; 340(1): 132-138). The modulation can be evaluated by, for example, changes in the immunological activity of the modified cells, such as changes in the cytotoxic activity of the modified cells or changes in the cytokine secretion of the modified cells, compared to cells modified with wild-type ICOS-L transmembrane protein.

[0090] As used herein, the term "molecular species" means a population of proteins having the same or substantially the same primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines a collection of the same or substantially the same molecular species. Thus, for example, human ICOS-L is an IgSF member, and each human ICOS-L molecule is a molecular species of ICOS. Differences between molecules of the same molecular species can occur due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Furthermore, small sequence differences within a single molecular species due to genetic polymorphism also constitute differences in another form within a single molecular species, similar to the wild-type truncated form of a single molecular species due to proteolytic cleavage. A "cell surface molecular species" is a molecular species expressed on the surface of mammalian cells. Two or more different protein species that are present in only one (but not both) of two mammalian cells that each form an IS are said to be in "cis" or "cis configuration" to each other. Two different protein species, where the first is present only in the first of two mammalian cells that form an IS and the second is present only in the second of two mammalian cells that form an IS, are said to be in "trans" or "trans configuration". Two different protein species that are present in both of two mammalian cells that each form an IS are in both cis-type and trans-type configurations on these cells.

[0091] The term "oligomerization domain" refers to an amino acid sequence that promotes stable interaction between a polypeptide molecule and one or more additional polypeptide molecules, wherein each polypeptide molecule contains complementary oligomerization domains (e.g., a first oligomerization domain and a second oligomerization domain) that may be the same or different oligomerization domains. Interaction between complementary oligomerization domains, e.g., interaction between a first oligomerization domain and a second oligomerization domain, forms a stable protein-protein interaction, resulting in an oligomer of the polypeptide molecule and the additional polypeptide molecule. In some cases, the oligomerization domains are the same and interact with themselves to form a stable protein-protein interaction between two polypeptide chains. Generally, the polypeptide is directly or indirectly linked to the oligomerization domain. Exemplary oligomerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The oligomerization domain may be, for example, an Fc domain or a portion thereof derived from an immunoglobulin constant region or domain, such as IgG (including IgG1, IgG2, IgG3, or IgG4 subtypes), IgA, IgE, IgD, and IgM, and modified forms thereof. Generally, the polypeptide is directly or indirectly connected to the oligomerization domain. Exemplary oligomerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The oligomerization domain can be, for example, an Fc domain or a portion thereof derived from an immunoglobulin constant region or domain, such as IgG (including IgG1, IgG2, IgG3, or IgG4 subtypes), IgA, IgE, IgD, and IgM, and modified forms thereof.

[0092] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to polymers of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing analogs of known natural nucleotides, nucleic acids having similar binding properties thereto, and nucleic acids that are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses not only the explicitly recited sequence ("reference sequence") but also its conservatively modified variants (e.g., degenerate codon substitutions) and complementary nucleotide sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0093] The term "non-competitive binding", as used herein, means the ability of a protein to specifically bind simultaneously to at least two cognate binding partners. Thus, a protein can bind simultaneously to at least two different cognate binding partners, although the binding interactions need not be during the same period, so that in some cases, the protein is specifically bound to only one of the cognate binding partners. In some embodiments, the binding occurs under specific binding conditions. In some embodiments, the co-binding is such that the binding of one cognate binding partner does not substantially inhibit the co-binding to a second cognate binding partner. In some embodiments, non-competitive binding means that the binding of a second cognate binding partner to its binding site on the protein does not replace the binding of a first cognate binding partner to its binding site on the protein. Methods for assessing non-competitive binding are well known in the art, such as the method described in Perez de La Lastra et al., Immunology, 1999 Apr: 96(4): 663-670. In some cases, in non-competitive interactions, the first cognate binding partner specifically binds at an interaction site that does not overlap with the interaction site of the second cognate binding partner, such that the binding of the second cognate binding partner does not directly interfere with the binding of the first cognate binding partner. Thus, any effect of the binding of the second cognate binding partner on the binding of the cognate binding partner is through a mechanism other than direct interference with the binding of the first cognate binding partner. For example, in the context of an enzyme-substrate interaction, a non-competitive inhibitor binds to a site other than the active site of the enzyme. Non-competitive binding encompasses non-competitive binding interactions in which a second cognate binding partner specifically binds at an interaction site that does not overlap with the binding of the first cognate binding partner, but binds to the second interaction site only when the first interaction site is occupied by the first cognate binding partner.

[0094] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammalian subjects, often humans. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising a variant ICOS-L or a modified cell expressing a variant ICOS-L transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is each typically a pharmaceutically acceptable carrier, excipient, or diluent.

[0095] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked via peptide bonds. The terms do not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. The terms include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. The terms also include molecules containing one or more amino acid analogs or non-standard or unnatural amino acids that can be synthesized or recombinantly expressed using known protein modification techniques. In addition, a protein may be derivatized.

[0096] The term "primary T cell assay" as used herein refers to an in vitro assay for measuring interferon-γ ("IFN-γ") expression. The assay used is an anti-CD3 co-stimulation assay. In this assay, primary T cells are stimulated by anti-CD3 immobilized with or without additional recombinant protein. Culture supernatant is collected at a certain time point (usually 24 - 72 hours). In another embodiment, the assay used is MLR. In this assay, primary T cells are stimulated with allogeneic APCs. Culture supernatant is collected at a certain time point (usually 24 - 72 hours). Human IFN-γ levels in the culture supernatant are measured by standard ELISA techniques. Commercially available kits are obtainable from suppliers and the assay is performed according to the manufacturer's recommendations.

[0097] When applied to a nucleic acid (e.g., a nucleic acid encoding an immunomodulatory protein of the present invention), the term "purified" generally refers to a nucleic acid or polypeptide that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that yields essentially one band in an electrophoretic gel is "purified". A purified nucleic acid or protein of the present invention is at least about 50% pure, usually at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., on a weight percent or molar basis).

[0098] The term "recombinant" indicates that a substance (e.g., a nucleic acid or polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. Such alteration can be carried out on a substance within or removed from its natural environment or state. For example, a "recombinant nucleic acid" is produced, for example, by recombining nucleic acids during cloning, affinity modification, DNA shuffling, or other well-known molecular biology procedures. A "recombinant DNA molecule" is composed of segments of DNA joined together by such molecular biology techniques. The terms "recombinant protein" or "recombinant polypeptide", as used herein, refer to protein molecules expressed using recombinant DNA molecules. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid or, alternatively, a cell that has been genetically engineered (e.g., by introducing into the cell a nucleic acid molecule encoding a recombinant protein (e.g., a transmembrane immunomodulatory protein provided herein)). Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, as well as viruses (similar control elements, i.e., promoters, are also found in prokaryotes). The choice of a particular promoter and enhancer depends on what cell type should be used to express the protein of interest. The terms "in functional combination", "in functional order", and "functionally linked", as used herein, refer to the linkage of nucleic acid sequences in such a manner or orientation that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced.

[0099] As used herein, the term "recombinant expression vector" refers to a DNA molecule containing a desired coding sequence and the appropriate nucleic acid sequences necessary for expression in a particular host cell of the coding sequence operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, as well as termination and polyadenylation signals. Optionally, a secretion signal peptide sequence may also be encoded by a recombinant expression vector that is operably linked to the coding sequence of a recombinant protein (e.g., a recombinant fusion protein) so that the expressed fusion protein can be secreted by the recombinant host cell for easier isolation of the fusion protein from the cell. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of the host cell into which it has been introduced. Such vectors include viral vectors, such as lentiviral vectors.

[0100] The term "selectivity" refers to the preference of specific binding of a target protein or polypeptide to one substrate (e.g., one cognate binding partner) as compared to the specific binding of the target protein to another substrate (e.g., a different cognate binding partner). Selectivity can be reflected as the ratio of the binding activity (e.g., binding affinity) (e.g., K d1 ) of the target protein to a first substrate (e.g., a first cognate binding partner) to the binding activity (e.g., binding affinity) (e.g., K d2 ) of the same target protein to a second cognate binding partner.

[0101] As used herein, the term "sequence identity" refers to sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. "Sequence identity" is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequences at a given position in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequences at a given position in each sequence when the sequences are aligned. Methods for alignment of sequences for comparison are well known in the art and include GAP, BESTFIT, BLAST, FASTA, and TFASTA. The BLAST algorithm calculates the percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).

[0102] As used herein with respect to a protein, the term "soluble" means that the protein is not a membrane protein. Generally, a soluble protein contains only the extracellular domain or a portion thereof of an IgSF family member receptor that contains an IgSF domain or a specific binding fragment thereof, does not contain a transmembrane domain, and / or cannot be expressed on the surface of a cell. In some cases, the solubility of a protein can be improved by directly or indirectly linking or conjugating it to an Fc domain, either directly or via a linker, which in some cases can also improve the stability and / or half-life of the protein. In some aspects, a soluble protein is an Fc fusion protein.

[0103] As used herein with respect to polypeptides or nucleic acids, the term "species" means a population of molecules having the same or substantially the same sequence. Differences between polypeptides of the same species can occur due to differences in post-translational modifications such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. A polypeptide sequence that is only slightly shorter (or encodes a difference) and that differs from the full-length species by only 1, 2, or 3 amino acid residues at the amino or carboxy terminus is considered to be a single species sequence. Such minor heterogeneity is a common feature of manufactured proteins.

[0104] As used herein with respect to a full-length wild-type mammalian ICOS-L polypeptide or its IgV or IgC domain, the term "specific binding fragment" means a polypeptide having a subsequence of the IgV and / or IgC domain and that specifically binds to mammalian ICOS and / or mammalian CD28 (e.g., human or murine ICOS or CD28) in vitro and / or in vivo. In some embodiments, the specific binding fragment of ICOS-L IgV or ICOS-L IgC is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence length of the full-length wild-type sequence. The sequence of the specific binding fragment can be varied to form variant ICOS-Ls of the invention.

[0105] As used herein, the term "specifically binds" means the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or avidity is at least 5-fold greater, and in some cases at least 10, 20, 30, 40, 50, 100, 250, or 500-fold greater, or even at least 1000-fold greater, than the average affinity or avidity of the same protein for a collection of random peptides or polypeptides of sufficient statistical size. A protein that specifically binds need not bind to only a single target molecule, but can specifically bind to a non-target molecule due to conformational similarity between the target and the non-target (e.g., paralog or ortholog). One of ordinary skill in the art will recognize that specific binding to a molecule having the same function in different animal species (i.e., ortholog) or to a non-target molecule having an epitope substantially similar to the target molecule (e.g., paralog) is possible and does not compromise the binding specificity determined against a statistically valid collection of native non-targets (e.g., random polypeptides). Thus, the polypeptides of the present invention can specifically bind to multiple distinct target molecule species due to cross-reactivity. Specific binding between two proteins can be determined using a solid-phase ELISA immunoassay or Biacore measurements. Generally, the interaction between two binding proteins has a dissociation constant (Kd) as low as less than 1×10 -5 M, often as low as 1×10 -12 M. In certain embodiments of the present disclosure, the interaction between two binding proteins has a dissociation constant of 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M.

[0106] With respect to mammalian cells expressing a polypeptide, the terms "surface-expresses", "surface expression" or "expresses on the surface" mean that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.

[0107] As used herein, "synthetic" refers to nucleic acid molecules or polypeptide molecules produced by recombinant and / or chemical synthesis methods, for example, with respect to synthetic nucleic acid molecules or synthetic genes or synthetic peptides.

[0108] The term "targeting moiety", as used herein, refers to a composition that is covalently or non-covalently bound to, or physically encapsulates, a polypeptide comprising a variant ICOS-L of the invention. In some embodiments, the targeting moiety has specific binding affinity for a target molecule (e.g., a target molecule expressed on the surface of a cell). Typically, the target molecule is localized on a particular tissue or cell type. Targeting moieties include antibodies, antigen-binding fragments (Fab), variable fragments (Fv) containing V H and V L and variable fragments (Fv) containing V H and V L linked together in one chain, single-chain variable fragments (scFv) containing V

[0109] As used herein, the term "transmembrane protein" means a membrane protein that substantially or completely traverses a lipid bilayer, which can be found, for example, in a biological membrane such as a mammalian cell or in an artificial construct such as a liposome. A transmembrane protein contains a transmembrane domain ("transmembrane domain") that is integrated into the lipid bilayer and whose integration is thermodynamically stable under physiological conditions. The transmembrane domain is generally predictable from the amino acid sequence of the transmembrane domain based on the fact that the hydrophobicity of the transmembrane domain is high compared to regions that interact with the aqueous environment (e.g., the cytosol, extracellular fluid) in the protein, through several commercially available bioinformatics software applications. The transmembrane domain is often a hydrophobic α-helix that traverses the membrane. A transmembrane protein may traverse both layers of the lipid bilayer one or more times. The provided transmembrane immunomodulatory protein described herein is included within the transmembrane proteins. The transmembrane immunomodulatory protein of the present invention further comprises an ectodomain in addition to the transmembrane domain and, in some embodiments, further comprises an endodomain.

[0110] As used herein, the terms "treatment" or "therapy" of a disease or disorder means delaying, interrupting, or reversing the progression of a disease or disorder, as demonstrated by reduction, cessation, or elimination of any clinical or diagnostic symptom, by administration of a therapeutic composition of the invention (e.g., one containing an immunomodulatory protein or modified cell), either alone or in combination with another compound described herein. "Treatment" or "therapy" also means reduction in the severity of symptoms in acute or chronic diseases or disorders, or reduction in the recurrence rate (e.g., as in the case of relapsing or remitting autoimmune disease courses), or reduction of inflammation in the case of the inflammatory aspects of autoimmune diseases. As used herein in the context of cancer, the terms "treatment" of cancer, "inhibiting" cancer, or "inhibition" of cancer mean, but are not limited to, a statistically significant decrease in tumor growth rate, cessation of tumor growth, or a decrease in tumor size, mass, metabolic activity or volume, or a statistically significant improvement in progression-free survival (PFS) or overall survival (OS), as measured by standard criteria such as Response Evaluation Criteria for Solid Tumors (RECIST). "Prevention" of a disease or disorder, as used in the context of the present invention, means administering an immunomodulatory polypeptide or modified cell of the invention, either alone or in combination with another compound, to prevent the appearance or onset of a disease or disorder, or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of onset of a disease or disorder.

[0111] The term "tumor specific antigen" or "TSA", as used herein, refers to a counter structure that is primarily present on the tumor cells of a mammalian subject but is generally not found on the normal cells of the mammalian subject. Tumor specific antigens need not be present only on tumor cells, but the proportion of cells of a particular mammalian subject having tumor specific antigens is high enough or the level of tumor specific antigens on the surface of the tumor is high enough such that they can be targeted with anti-tumor therapeutic agents (e.g., the immunomodulatory polypeptides of the present invention) and can provide prophylaxis or treatment of a mammal from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammal having a tumor, at least 50% of the cells presenting TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells presenting TSA are cancerous.

[0112] The term "variant" (also referred to as "modified" or "mutant") when used with respect to variant ICOS-L means an ICOS-L created by human intervention, e.g., a mammalian (e.g., human or mouse) ICOS-L. A variant ICOS-L is a polypeptide having an amino acid sequence that varies compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L. A variant ICOS-L is a polypeptide that differs from a reference ICOS-L, e.g., a wild-type ICOS-L isoform sequence, by one or more modifications, e.g., one or more amino acid substitutions, deletions, additions, or combinations thereof. For purposes herein, a variant ICOS-L contains at least one domain with a modified affinity, and one or more of the amino acid differences occur in an IgSF domain (e.g., an IgV domain). A variant ICOS-L may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid differences, e.g., amino acid substitutions. A variant ICOS-L polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to a corresponding reference (e.g., unmodified) or wild-type, e.g., the sequence of SEQ ID NO:4, its mature sequence, or a portion thereof containing its extracellular or IgSF domain. In some embodiments, a variant ICOS-L polypeptide exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to a corresponding reference (e.g., unmodified) ICOS-L or wild-type ICOS-L, e.g., the reference ICOS-L shown in SEQ ID NO:1 or SEQ ID NO:2 or 3.Non-natural amino acids as well as natural amino acids are included within the scope of acceptable substitutions or additions. Variant ICOS-L is not limited to any particular production method and includes, for example, novel chemical synthesis, novel recombinant DNA methods, or combinations thereof. The variant ICOS-L of the present invention specifically binds to CD28, ICOS, and / or CTLA-4 of mammalian species. In some embodiments, changing the amino acid sequence results in a change (i.e., an increase or decrease) in the binding affinity or avidity for ICOS and / or CD28 as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L protein. The increase or decrease in binding affinity or avidity can be confirmed using well-known binding assays such as flow cytometry. See also Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005); Linsley et al., Immunity, Vol. 1(9): 793-801(1994). An increase in the binding affinity or avidity of variant ICOS-L for ICOS and / or CD28 is an increase to a value at least 5% greater than the binding affinity or avidity of a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L, and in some embodiments, an increase to a value at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than the binding affinity or avidity of a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L control value. A decrease in the ICOS-L binding affinity or avidity for ICOS and / or CD28 is a decrease to a value of 95% or less of the wild-type control value, and in some embodiments, a decrease to a value of 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less of the wild-type ICOS and / or CD28 control value, or no detectable binding affinity or avidity. The variant ICOS-L has an altered primary amino acid sequence by substitution, addition, or deletion of amino acid residues. The term "variant" in the context of variant ICOS-L is not construed as imposing any conditions of any particular starting composition or method by which the variant ICOS-L is created.To obtain a variant ICOS-L of the present invention, the variant ICOS-L can be made, for example, starting from the sequence information of a reference ICOS-L or a wild-type mammalian ICOS-L, then modeled in silico to determine whether it binds to ICOS and / or CD28, and finally synthesized recombinantly or chemically synthesized. In just one alternative, the variant ICOS-L may be created by site-directed mutagenesis of a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L. Thus, a variant ICOS-L represents a composition and does not necessarily represent a product produced by a specific process. A variety of techniques can be used, including recombinant methods, chemical synthesis, or a combination thereof.

[0113] The terms "wild-type" or "natural" or "native", when used herein in connection with biological materials such as nucleic acid molecules, proteins (e.g., ICOS-L), IgSF members, host cells, etc., refer to those found in nature and not modified by human intervention.

[0114] Furthermore, the various aspects of the present invention discussed below are frequently provided within the scope of the meaning of the defined terms disclosed above. Accordingly, aspects described in a particular definition should be construed as being incorporated by reference when the defined terms are utilized in the discussion of the various aspects and attributes described herein. Thus, the headings, the order of presentation of the various aspects and embodiments, and the separate disclosure of each independent attribute do not mean a limitation on the scope of this disclosure.

[0115] II. Variant ICOS-L Polypeptide Fusion Proteins Provided herein are variant ICOS-L polypeptides having an altered (increased or decreased) binding activity or affinity for one or more of the ICOS-L cognate binding partners. In some embodiments, the ICOS-L cognate binding partner is one or more of CD28, ICOS, or CTLA-4. In some embodiments, the variant ICOS-L polypeptide contains one or more amino acid modifications, such as one or more substitutions (or "mutations" or "exchanges"), deletions, or additions, in the immunoglobulin superfamily (IgSF) domain compared to a wild-type or unmodified ICOS-L polypeptide, or a portion of a wild-type or unmodified ICOS-L containing an IgSF domain, or a specific binding fragment thereof. Accordingly, the provided variant ICOS-L polypeptide is or comprises a variant IgD (hereinafter referred to as "vIgD") in which one or more amino acid modifications (e.g., substitutions) are in the IgD.

[0116] A. Variant ICOS-L polypeptide In some embodiments, the IgD comprises an IgV domain, or an IgC (e.g., IgC2) domain, or a specific binding fragment of an IgV domain or IgC (e.g., IgC2) domain, or a combination thereof. In some embodiments, the IgD may be IgV only, a combination of IgV and IgC that includes the entire extracellular domain (ECD), or any combination of the Ig domains of ICOS-L. Table 2 shows exemplary residues corresponding to the IgV region or IgC region of ICOS-L. In some embodiments, the variant ICOS-L polypeptide contains an IgV domain or IgC domain or a specific binding fragment thereof, wherein at least one of the amino acid modifications (e.g., substitutions) is in the IgV domain or IgC domain or a specific binding fragment thereof. In some embodiments, due to the change in binding activity or affinity, the IgV domain or IgC domain is an IgSF domain with modified affinity.

[0117] In some embodiments, the variant is modified in one or more IgSF domains as compared to the sequence of a reference (e.g., unmodified) ICOS-L sequence. In some embodiments, the reference (e.g., unmodified) ICOS-L sequence is wild-type ICOS-L. In some embodiments, the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L has the sequence of a native ICOS-L or its ortholog. In some embodiments, the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L is or comprises the extracellular domain (ECD) of ICOS-L, or a portion thereof, that contains one or more IgSF domains (see Table 1). In some embodiments, the extracellular domain of the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide comprises an IgV domain and an IgC domain. However, the variant ICOS-L polypeptide need not contain both an IgV domain and an IgC domain. In some embodiments, the variant ICOS-L polypeptide comprises or consists essentially of an IgV domain or a specific binding fragment thereof. In some embodiments, the variant ICOS-L polypeptide comprises or consists essentially of an IgC domain or a specific binding fragment thereof. In some embodiments, the variant ICOS-L is soluble and lacks a transmembrane domain. In some embodiments, the variant ICOS-L further comprises a transmembrane domain and, in some cases, further comprises a cytoplasmic domain.

[0118] Variant ICOS-L polypeptides are provided herein that contain an IgSF domain (e.g., IgV or IgC) or a specific binding fragment thereof in which at least one affinity has been modified in an IgSF domain contained in a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide such that the variant ICOS-L polypeptide exhibits altered (e.g., improved) binding activity or affinity for one or more ligand ICOS and / or CD28 as compared to the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide. In some embodiments, the variant ICOS-L polypeptide has a binding affinity for CD28 and / or ICOS that is different from a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide control sequence as determined, for example, by a solid-phase ELISA immunoassay, flow cytometry, or Biacore assay. In some embodiments, the binding affinity of the variant ICOS-L polypeptide for CD28 and / or ICOS is improved as compared to a reference (unmodified) or wild-type ICOS-L polypeptide. CD28 and / or ICOS may be mammalian proteins, e.g., human or mouse proteins.

[0119] The binding affinity for each of the cognate binding partners is independent. That is, in some embodiments, the binding affinity of the variant ICOS-L polypeptide for one or both of CD28 and / or ICOS is improved as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide. In some embodiments, the binding affinity of the variant ICOS-L polypeptide for both CD28 and ICOS is improved as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide.

[0120] In some embodiments, the binding affinity of the variant ICOS-L polypeptide for CD28 is improved compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide. In some embodiments, the binding affinity of the variant ICOS-L polypeptide for ICOS is improved compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide. In some embodiments, the binding affinity of the variant ICOS-L polypeptide for CD28 and ICOS is improved compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide.

[0121] In some embodiments, a variant ICOS-L polypeptide with improved or greater binding affinity for CD28 and / or ICOS has a binding affinity for CD28 and / or ICOS that is at least about 5% improved compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide control, e.g., at least about 10%, 15%, 20%, 25%, 35%, or 50% improved. In some embodiments, the improvement in binding affinity compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide is greater than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In such examples, the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide has the same sequence as the variant ICOS-L polypeptide except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0122] In some embodiments, the equilibrium dissociation constant (K d ) for any of the foregoing embodiments for CD28 and / or ICOS is 1 × 10 -5 M, 1 × 10 -6 M, 1 × 10 -7 M, 1 × 10 -8 M, 1 × 10 -9 M, 1 × 10 -10 M, or 1 × 10 -11 M, or may be less than 1 × 10 -12 M.

[0123] In some embodiments, the variant ICOS-L polypeptide has an improved or increased binding affinity for CD28. In some embodiments, the variant ICOS-L polypeptide having an improved or increased binding affinity for CD28 has a binding affinity for CD28 that is at least about 25% improved, such as at least about 30%, 40%, 50%, or 60% improved compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide control. In some embodiments, the equilibrium dissociation constant (K d ) for the variant ICOS-L polypeptide having an improved or increased binding affinity for CD28 is less than 200 pM, 300 pM, 400 pM, 500 pM, or 600 pM for CD28.

[0124] In some embodiments, the variant ICOS-L polypeptide has an improved or increased binding affinity for ICOS. In some embodiments, the variant ICOS-L polypeptide having an improved or increased binding affinity for ICOS has a binding affinity for ICOS that is at least about 10% improved, such as at least about 15%, 20%, 25%, 30%, 40%, 50%, or 60% improved compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide control. In some embodiments, the equilibrium dissociation constant (K d ) for the variant ICOS-L polypeptide having an improved or increased binding affinity for ICOS is less than 200 pM, 300 pM, 400 pM, 500 pM, or 600 pM for ICOS.

[0125] In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications, such as substitutions, in a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L sequence. The one or more amino acid modifications, such as substitutions, may be in the extracellular domain of the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L sequence. In some embodiments, the one or more amino acid modifications, such as substitutions, are in the IgV domain or a specific binding fragment thereof.

[0126] In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications, e.g., substitutions, corresponding to positions 52, 57, and / or 100 based on the numbering of SEQ ID NO:1 in the reference ICOS-L or its specific binding fragment. In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications, e.g., substitutions selected from N52H, N57Y, and / or Q100R, or conservative amino acid modifications thereof, e.g., substitutions, based on the numbering of SEQ ID NO:1.

[0127] Conservative amino acid modifications, e.g., substitutions, are any amino acids classified in the same amino acid class as the substituted amino acid other than the reference (e.g., unmodified) or wild-type amino acid. Amino acid classes are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartic acid, glutamic acid, asparagine, and glutamine).

[0128] In some embodiments, the one or more amino acid modifications (e.g., substitutions) are N52H / N57Y / Q100R based on the numbering of SEQ ID NO:1.

[0129] Unless otherwise specified, as shown throughout this disclosure, amino acid modifications are specified by the amino acid position numbers corresponding to the numbering of the positions in the reference ECD sequence shown in SEQ ID NO:1. Identifying the corresponding positions of modifications, e.g., amino acid substitutions, in an ICOS-L polypeptide, including a portion of the ICOS-L polypeptide containing an IgSF domain (e.g., IgV) of the ICOS-L polypeptide, is within the level of skill in the art, e.g., by alignment of a reference sequence (e.g., SEQ ID NOs:2, 3, 5 - 33) with SEQ ID NO:1. In the list of modifications throughout this disclosure, the amino acid position is shown in the middle, the corresponding reference (e.g., unmodified or wild-type) amino acid is listed before the number, and the amino acid substitution of the specified variant is listed after the number. When the modification is a deletion at that position, it is designated "del", and when the modification is an insertion at that position, it is designated "ins". In some cases, an insertion is described, the amino acid position is shown in the middle, the corresponding reference amino acid is listed before and after the number, and the amino acid insertion of the specified variant is listed after the unmodified (e.g., wild-type) amino acid.

[0130] In some embodiments, the variant is modified in one or more IgSF domains as compared to the sequence of a reference (e.g., unmodified) ICOSL sequence. In some embodiments, the reference (e.g., unmodified) ICOSL sequence is wild-type ICOSL. In some embodiments, the reference (e.g., unmodified) ICOSL or wild-type ICOSL has the sequence of a native ICOSL or its ortholog. In some embodiments, the reference (e.g., unmodified) ICOSL or wild-type ICOSL is or comprises an ICOSL extracellular domain (ECD) containing one or more IgSF domains (see Table 1) or a portion thereof. In some embodiments, the extracellular domain of the reference (e.g., unmodified) ICOSL or wild-type ICOSL polypeptide comprises an IgV domain and an IgC domain. However, the variant ICOSL polypeptide need not contain both an IgV domain and an IgC domain. In some embodiments, the variant ICOSL polypeptide comprises or consists essentially of an IgV domain or a specific binding fragment thereof. In some embodiments, the variant ICOSL is soluble and lacks a transmembrane domain.

[0131] In some embodiments, the reference (e.g., unmodified) ICOSL or wild-type ICOSL sequence is a mammalian ICOSL sequence. In some embodiments, the reference (e.g., unmodified) ICOSL or wild-type ICOSL sequence may be a mammalian ICOSL including, but not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the reference (e.g., unmodified) ICOSL or wild-type ICOSL sequence is human.

[0132] Various features of a reference (e.g., unmodified) ICOS or wild-type ICOS sequence are shown in Table 1. The first column of Table 1 shows the name of this particular IgSF member and, optionally, the name of any possible alternative aliases. The second column shows the protein identifier of the UniProtKB database, a publicly available database accessible via the Internet at uniprot.org, and in some cases shows the GenBank number. The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data. The UniProt database includes the UniProt Knowledgebase (UniProtKB). UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Institute of Bioinformatics, and the Protein Information Resource (PIR), and is mainly supported by grants from the US National Institutes of Health (NIH). GenBank is the NIH's gene sequence database, in which all publicly available DNA sequences are collected with annotations (Nucleic Acids Research, 2013 Jan;41(D1):D36-42). The third column shows the region in which the indicated IgSF domain is located. This region is specified as a range that includes the residues defining the domain. The third column also shows the IgSF domain class of the specified IgSF region. The fourth column shows the region in which the indicated additional domain is located (signal peptide, S; extracellular domain, E; transmembrane domain, T; cytoplasmic domain, C). It should be understood that the description of the domain may vary depending on the method used for domain identification or classification and may be identified differently from different sources. The description of the residues corresponding to the domains in Table 1 is illustrative only and may be longer or shorter by several amino acids (e.g., 1, 2, 3, or 4). The fifth column shows some of its cell surface cognate binding partners.

[0133] (Table 1) IgSF members of the present disclosure TIFF2025090714000002.tif67166

[0134] In some embodiments, a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L sequence is (i) the amino acid sequence shown in SEQ ID NO: 4, or its mature form lacking the signal sequence, (ii) has an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 4 or its mature form, or (iii) is a part of (i) or (ii) containing an IgV domain or an IgC domain or a specific binding fragment thereof.

[0135] In some embodiments, the reference ICOS-L sequence is or includes the ICOS-L extracellular domain or a part thereof. In some embodiments, the reference or wild-type ICOS-L polypeptide includes the amino acid sequence shown in SEQ ID NO: 1 or its ortholog. TIFF2025090714000003.tif33161

[0136] In some cases, a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide may include (i) the amino acid sequence shown in SEQ ID NO: 1, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1, or (iii) a specific binding fragment of the sequence of (i) or (ii) containing an IgV domain or an IgC domain.

[0137] In some embodiments, the reference ICOS-L polypeptide comprises a truncated extracellular domain that includes a C-terminal truncation relative to the reference ICOS-L extracellular domain sequence set forth in SEQ ID NO:1. In some embodiments, the C-terminal truncation is a C-terminal truncation of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125 amino acid residues. In some embodiments, the C-terminal truncation is a C-terminal truncation of at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35 amino acid residues. In some embodiments, the ICOS-L polypeptide comprising the C-terminal truncation does not contain contiguous amino acid residues of wild-type ICOS-L beyond the C-terminus of the truncation point. Thus, among the provided ICOS-L reference sequences, there are some that are shorter than the full extracellular domain of wild-type ICOS-L (e.g., wild-type ICOS-L set forth in SEQ ID NO:1). In some embodiments, the ICOS-L polypeptide comprising the C-terminal truncation does not contain or is not fused to amino acid residues of the ICOS-L domain beyond the extracellular domain.

[0138] In some embodiments, the ICOS-L reference polypeptide is altered (e.g., mutated or deleted) at one or more protease cleavage sites. As found herein, the wild-type ICOS-L polypeptide contains, in some cases, a protease cleavage site that results in cleavage of the protein when expressed in cells (e.g., Chinese hamster ovary cells), thereby generating a heterogeneous product of multiple species, including species of different lengths or sizes. For example, when the ICOS-L polypeptide is cleaved, an LQQN / LT protease cleavage site may occur between residues 207 and 208 of SEQ ID NO:1 (" / " indicates a potential cleavage site). In some embodiments, the ICOS-L reference polypeptide is altered at or lacks the protease cleavage site represented as amino acids 204-209 of SEQ ID NO:1. In some embodiments, the truncated ICOS-L polypeptide has higher protease cleavage resistance compared to the wild-type or non-truncated ICOS-L polypeptide. Exemplary truncated ICOS-L polypeptide ECD truncations (named Truncation #2, #3, #4, #5, #6, #7, or #8) that lack all or part of the LQQN / LT protease cleavage site are shown in SEQ ID NOs:5-11. TIFF2025090714000004.tif54160TIFF2025090714000005.tif193160

[0139] In some embodiments, the ICOS-L reference polypeptide is altered at one or more amino acids corresponding to amino acids 204-209 relative to SEQ ID NO:1. In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications, e.g., substitutions, corresponding to position 207 and / or 208 relative to the numbering of SEQ ID NO:1 in the reference ICOS-L or its specific binding fragment. In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications, e.g., substitutions, selected from N207A, N207G, L208G, or a conservative amino acid modification thereof, e.g., a substitution. In some embodiments, the one or more amino acid modifications, e.g., substitutions are N207A / L208G or N207G / L208G. In some embodiments, the full-length reference ECD or truncated reference ECD of the variant ICOS-L polypeptide is modified to contain one or more amino acid modifications, e.g., substitutions, selected from N207A, N207G, L208G, or a conservative amino acid modification. Exemplary full-length or truncated reference ECDs having one or more modifications are shown in SEQ ID NOs:12-33. Exemplary reference sequences containing mutations at cleavage sites N207 and / or L208 relative to the positions shown in SEQ ID NO:1 are shown in SEQ ID NOs:29-33. In some cases, the modifications provided may reduce proteolytic cleavage of the ICOS-L polypeptide, e.g., cleavage that may occur at the LQQN / LT protease cleavage site.

[0140] In some embodiments, a combination of the shortening strategy and the modification strategy described above can be used in the reference ICOS-L ECD sequence. In some embodiments, modifications, such as substitutions, are added to the shortened reference ICOS-L polypeptide, for example, the exemplary reference ICOS-L sequences shown in SEQ ID NOs: 5-11. Exemplary variant ICOS-L polypeptide sequences with modifications at the potential protease cleavage sites N207 and / or L208 are shown in SEQ ID NOs: 12-33. In some embodiments, the variant ICOS-L polypeptide exhibits reduced protease cleavage compared to the wild-type ICOS-L polypeptide (e.g., the wild-type ICOS-L polypeptide containing the ECD sequence shown in SEQ ID NO: 1).

[0141] In some embodiments, a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide comprises an IgV domain or an IgC domain or a specific binding fragment thereof. In some embodiments, an ICOS-L reference polypeptide containing an IgV domain comprises the amino acid sequence shown in SEQ ID NO: 2 (corresponding to amino acid residues 19-129 of SEQ ID NO: 4), or an ortholog thereof. TIFF2025090714000006.tif11160

[0142] In some embodiments, an ICOS-L reference polypeptide containing an IgV domain contains at least amino acids 1-112, 1-113, 1-114, 1-115, 1-116, 1-117, 1-118, 1-119, 1-120, 1-121, 1-122 based on the numbering shown in SEQ ID NO: 1. In some embodiments, an ICOS-L reference polypeptide containing an IgV domain comprises the amino acid sequence shown in SEQ ID NO: 3 (corresponding to amino acid residues 19-140 of SEQ ID NO: 4), or an ortholog thereof. In some embodiments, the IgV domain is the only IgSF domain of the ICOS-L reference polypeptide. TIFF2025090714000007.tif17160

[0143] In some embodiments, the IgV domain of a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide may contain (i) the amino acid sequence shown in SEQ ID NO:2, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2, or (iii) a specific binding fragment of the amino acid sequence shown in SEQ ID NO:2, or a specific binding fragment of the sequence of (i) or (ii). In some embodiments, the reference (e.g., unmodified) IgV domain can bind to one or more ICOS-L cognate binding proteins, such as one or both of CD28 and ICOS.

[0144] In some embodiments, the IgV domain of a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide may contain (i) the amino acid sequence shown in SEQ ID NO:3, (ii) an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:3, or (iii) a specific binding fragment of the amino acid sequence shown in SEQ ID NO:3, or a specific binding fragment of the sequence of (i) or (ii). In some embodiments, the reference (e.g., unmodified) IgV domain can bind to one or more ICOS-L cognate binding proteins, such as one or both of CD28 and ICOS.

[0145] In some embodiments, a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide contains a specific binding fragment of ICOS-L, such as a specific binding fragment of the IgV domain. In some embodiments, the specific binding fragment is capable of binding to CD28 and / or ICOS. The specific binding fragment may have an amino acid length of at least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, the specific binding fragment of the IgV domain contains an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the length of the IgV domain shown as amino acids 19-129 of SEQ ID NO:4.

[0146] In some embodiments, the variant ICOS-L polypeptide comprises an ECD domain, a truncated ECD domain, or a portion thereof, comprising one or more IgSF domains with modified affinities. In some embodiments, the variant ICOS-L polypeptide may comprise an IgV domain or an IgV domain containing one or more amino acid modifications (e.g., substitutions) in a specific binding fragment of the IgV domain. In some embodiments, the variant ICOS-L polypeptide may comprise an IgV domain and an IgC domain, or a specific binding fragment of the IgV domain and a specific binding fragment of the IgC domain. In some embodiments, the variant ICOS-L polypeptide comprises a full-length IgV domain. In some embodiments, the variant ICOS-L polypeptide comprises a full-length IgC domain. In some embodiments, the variant ICOS-L polypeptide comprises a specific binding fragment of the IgV domain. In some embodiments, the variant ICOS-L polypeptide comprises a specific binding fragment of the IgC domain. In some embodiments, the variant ICOS-L polypeptide comprises a full-length IgV domain and a full-length IgC domain. In some embodiments, the variant ICOS-L polypeptide comprises a full-length IgV domain and a specific binding fragment of the IgC domain. In some embodiments, the variant ICOS-L polypeptide comprises a specific binding fragment of the IgV domain and a full-length IgC domain. In some embodiments, the variant ICOS-L polypeptide comprises a specific binding fragment of the IgV domain and a specific binding fragment of the IgC domain

[0147] In any such embodiment, one or more amino acid modifications (e.g., substitutions) of the variant ICOS-L polypeptide can be located in any one or more of the ICOS-L polypeptide domains. For example, in some embodiments, one or more amino acid substitutions are located in the extracellular domain (ECD) of the variant ICOS-L polypeptide, as shown in, for example, SEQ ID NO:1. In some embodiments, one or more amino acid substitutions are located in the IgV domain or a specific binding fragment of the IgV domain

[0148] In some embodiments, the variant ICOS-L polypeptide has from 1 to 20 amino acid modifications, such as substitutions. The modifications, such as substitutions, may be in the IgV domain or the IgC domain. In some embodiments, the variant ICOS-L polypeptide has from 1 to 20 amino acid substitutions in the IgV domain or a specific binding fragment thereof. In some embodiments, the variant ICOS-L polypeptide has from 1 to 20 amino acid substitutions in the IgC domain or a specific binding fragment thereof. In some embodiments, the variant ICOS-L polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide or a specific binding fragment thereof, such as SEQ ID NO: 1, 2, or 3.

[0149] In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications, e.g., substitutions, corresponding to position 10, 11, 13, 16, 18, 20, 25, 27, 30, 33, 37, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 77, 78, 75, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107, 109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126, 129, 130, 132, 133, 135, 138, 139, 140, 142, 143, 144, 146, 151, 152, 153, 154, 155, 156, 158, 161, 166, 168, 172, 173, 175, 190, 192, 193, 194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224, 225, or 227 in the reference ICOS-L or its specific binding fragment, based on the numbering of SEQ ID NO:1. In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications, e.g., substitutions, corresponding to position 10, 11, 13, 16, 18, 20, 25, 26, 27, 30, 33, 37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75, 77, 78, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107, 109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126, 129, 130, 132, 133, 135, 137, 138, 139, 140, 142, 143, 144, 146, 151, 152, 153, 154, 155, 156, 158, 161, 164, 166, 168, 172, 173, 175, 190, 192, 193, 194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224, 225, or 227 in the reference ICOS-L or its specific binding fragment, based on the numbering of SEQ ID NO:1.

[0150] In some embodiments, the variant ICOS-L polypeptide is has one or more amino acid modifications selected from TIFF2025090714000008.tif99164, for example, substitutions, or conservative amino acid modifications, such as those substitutions. In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications selected from TIFF2025090714000009.tif106164, for example, substitutions, or conservative amino acid substitutions thereof.

[0151] In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications selected from TIFF2025090714000010.tif99166, for example, substitutions. In some embodiments, the variant ICOS-L polypeptide has one or more amino acid modifications selected from TIFF2025090714000011.tif106165, for example, substitutions, or conservative amino acid substitutions thereof.

[0152] In some embodiments, one or more amino acid modifications, for example, substitutions are TIFF2025090714000012.tif201166TIFF2025090714000013.tif245166TIFF2025090714000014.tif106157.

[0153] In some embodiments, one or more amino acid modifications are selected from TIFF2025090714000015.tif128166TIFF2025090714000016.tif245166TIFF2025090714000017.tif171170.

[0154] In some embodiments, one or more amino acid modifications (e.g., substitutions) are N52H / N57Y / Q100R based on the numbering of SEQ ID NO:1. In some embodiments, one or more amino acid modifications, e.g., substitutions, are N52L / N57H / Q100R based on the numbering of SEQ ID NO:1. In some embodiments, one or more amino acid modifications, e.g., substitutions, are N52H / Q100R based on the numbering of SEQ ID NO:1. In some embodiments, one or more amino acid modifications, e.g., substitutions, are N52D based on the numbering of SEQ ID NO:1.

[0155] Generally, various properties of the polypeptide (e.g., the affinity of ICOS-L for CD28 and / or ICOS, the number of variations per polypeptide chain, the number of polypeptide chains linked, the number and nature of the amino acid changes per variant ICOS-L, etc.) are each disclosed separately. However, as will be apparent to those skilled in the art, any particular polypeptide may include a combination of these independent properties. It is understood that references to amino acids, including references to the specific sequences shown by SEQ ID NOs used to describe the domain composition of the IgSF domain, are for purposes of illustration and do not mean to limit the scope of the provided embodiments. It is understood that the description of the polypeptide and its domains is derived theoretically based on homology analysis and alignment with similar molecules. Accordingly, the exact locus may vary and is not necessarily identical for each protein. Thus, a particular IgSF domain, e.g., a particular IgV domain or IgC domain, may be longer or shorter by only a few (e.g., 1, 2, 3, or 4) amino acids.

[0156] A reference (e.g., unmodified) ICOS-L or wild-type ICOS-L sequence is not necessarily required as a starting composition to generate the variant ICOS-L polypeptides described herein. Thus, the use of "modifications" such as the term "substitution" does not mean that this aspect is limited to a particular method of generating variant ICOS-L polypeptides. Variant ICOS-L polypeptides may be generated, for example, by de novo peptide synthesis and thus do not necessarily require "modifications" such as "substitution" in the sense of changing codons to encode modifications, such as substitutions. This principle also extends to the terms "addition" and "deletion" of amino acid residues and similarly does not mean a particular method of generation. The means by which variant ICOS-L polypeptides are designed or created are not limited to any particular method. However, in some aspects, a nucleic acid encoding a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L is mutagenized from the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L genetic material and screened for the presence of a desired specific binding affinity and / or modulation of IFN-γ expression or other functional activity. In some aspects, variant ICOS-L polypeptides are newly synthesized using protein or nucleic acid sequences available in any number of publicly available databases and then screened subsequently. Such information is provided by the National Center for Biotechnology Information and its website can be accessed publicly via the Internet, such as the UniProtKB database already discussed.

[0157] In some embodiments, the variant ICOS-L polypeptide comprises the mutations listed in Table 2. Table 2 also shows exemplary sequences by reference to the SEQ ID NOs of the extracellular domain (ECD) or IgV domain of wild-type ICOS-L (i.e., reference (e.g., unmodified)) or exemplary variant ICOS-L polypeptides. As shown, the exact locus or residues corresponding to a particular domain may vary depending, for example, on the method used to identify or classify the domain. Also, in some cases, adjacent N-terminal and / or C-terminal amino acids of a particular domain (e.g., IgV) may also be included in the sequence of the variant IgSF polypeptide, for example, to ensure proper domain folding upon expression. Thus, it is understood that the exemplification of SEQ ID NOs in Table 2 should not be construed as limiting. For example, a particular domain of a variant ICOS-L polypeptide, such as the ECD domain, may be several amino acids longer or shorter than the amino acid sequence shown in each respective SEQ ID NO, for example, 1-10, for example, 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0158] (Table 2) Exemplary variant ICOS-L polypeptides TIFF2025090714000018.tif26166

[0159] The variant ICOS-L polypeptide may contain any amino acid substitution (mutation) described herein. In some embodiments, the variant ICOS-L polypeptide comprises any of the mutations listed in Table 2. In some examples, a mutation is added to a reference ICOS-L containing the amino acid sequence shown in SEQ ID NO:1, a reference ICOS-L containing the IgV domain of ICOS-L shown in SEQ ID NO:2 or 3, or a truncated and / or modified reference ICOS-L containing the amino acid sequence shown in any of SEQ ID NOs:5-33.

[0160] In some embodiments, the variant ICOS-L polypeptide comprises the extracellular domain (ECD) sequence shown in SEQ ID NO:34. In some embodiments, the variant ICOS-L polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, for example, at least 96% identity, 97% identity, 98% identity, or 99% identity to the extracellular domain (ECD) sequence shown in SEQ ID NO:34, and contains amino acid modifications, for example, substitutions, for example, N52H / N57Y / Q100R, that are not present in the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L. In some embodiments, the variant ICOS-L polypeptide comprises a specific binding fragment of the extracellular domain (ECD) sequence shown in SEQ ID NO:34, and contains amino acid modifications, for example, substitutions, for example, N52H / N57Y / Q100R, that are not present in the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L.

[0161] In some embodiments, the variant ICOS-L polypeptide comprises the IgV sequence shown in SEQ ID NO:35. In some embodiments, the variant ICOS-L polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, for example, at least 96% identity, 97% identity, 98% identity, or 99% identity to the IgV sequence shown in SEQ ID NO:35, and contains amino acid modifications, for example, substitutions, for example, N52H / N57Y / Q100R, that are not present in the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L. In some embodiments, the variant ICOS-L polypeptide comprises a specific binding fragment of the IgV sequence shown in SEQ ID NO:35, and contains amino acid modifications, for example, substitutions, for example, N52H / N57Y / Q100R, that are not present in the reference (e.g., unmodified) ICOS-L or wild-type ICOS-L.

[0162] In some embodiments, the variant ICOS-L polypeptide comprises the IgV sequence shown in SEQ ID NO:36. In some embodiments, the variant ICOS-L polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, for example, at least 96% identity, 97% identity, 98% identity, or 99% identity to the IgV sequence shown in SEQ ID NO:36, and contains amino acid modifications that are not present in a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L, for example, substitutions, for example, N52H / N57Y / Q100R. In some embodiments, the variant ICOS-L polypeptide comprises a specific binding fragment of the IgV sequence shown in SEQ ID NO:36, and contains amino acid substitutions that are not present in a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L, for example, N52H / N57Y / Q100R.

[0163] In some embodiments, the binding affinity of the variant ICOS-L polypeptide for the extracellular domain of CD28 is improved as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide, for example, as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide comprising the sequence shown in SEQ ID NO:1, 2, or 3. In some embodiments, the affinity of the ICOS-L polypeptide for the extracellular domain of ICOS is improved as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L, for example, as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide comprising the sequence shown in SEQ ID NO:1, 2, or 3. In some embodiments, the affinity of the ICOS-L polypeptide for the extracellular domains of CD28 and ICOS is improved as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L, for example, as compared to a reference (e.g., unmodified) ICOS-L or wild-type ICOS-L polypeptide comprising the sequence shown in SEQ ID NO:1, 2, or 3.

[0164] B. Multimerization Domain The variant ICOS-L polypeptide fusion proteins provided herein that contain vIgD can be arrayed in various ways as soluble proteins. In some embodiments, the variant ICOS-L fusion proteins contain a multimerization domain. In some cases, the variant ICOS-L fusion proteins can antagonize or block the activity of its cognate binding partners, e.g., CD28 and / or ICOS-L. In some embodiments, the antagonism of CD28 and / or ICOS-L may be useful for treating inflammation or autoimmunity. Those skilled in the art will understand that cell surface proteins typically have intracellular, transmembrane, and extracellular domain (ECD), and that soluble forms of such proteins can be made using the extracellular domain or an immunologically active subsequence thereof. Thus, in some embodiments, the immunomodulatory proteins containing the variant ICOS-L polypeptide lack a transmembrane domain or a portion of the transmembrane domain. In some embodiments, the immunomodulatory proteins containing the variant ICOS-L lack an intracellular (cytoplasmic) domain or a portion of the intracellular domain. In some embodiments, the immunomodulatory proteins containing the variant ICOS-L polypeptide contain only the ECD domain or a portion thereof containing the IgV domain and / or IgC (e.g., IgC2) domain containing amino acid modifications, the vIgD portion, or a specific binding fragment thereof.

[0165] In some embodiments, the conjugate is a fusion protein of a variant ICOS-L polypeptide directly or via a linker linked to another protein or polypeptide moiety. In some embodiments, the fusion protein is an ICOS-L-Fc variant fusion, in which case any two or more of the foregoing variant polypeptides can be attached to the Fc. In some embodiments, the variant ICOS-L polypeptide has the structure of the soluble protein shown in FIG. 1.

[0166] In some embodiments, the fusion protein comprising variant ICOS-L may comprise one or more variant ICOS-L polypeptides of the invention. In some embodiments, the polypeptide of the invention comprises exactly one, two, three, four, five variant ICOS-L sequences. In some embodiments, at least two of the variant ICOS-L sequences are the same variant ICOS-L sequence.

[0167] In some embodiments, the provided immunomodulatory polypeptide comprises two or more vIgD sequences of ICOS-L. The multiple variant ICOS-L polypeptides within this polypeptide chain may be identical to each other (i.e., the same species), or may be variant ICOS-L sequences that are not identical (i.e., different species). In addition to the embodiment of a single polypeptide chain, in some embodiments, two, three, four, or more polypeptides of the invention can be attached to each other by covalent or non-covalent bonds. Thus, monomeric, dimeric, and even higher order (e.g., three, four, five, or more) multimeric proteins are provided herein. For example, in some embodiments, exactly two polypeptides of the invention can be attached to each other by covalent or non-covalent bonds to form a dimer. In some embodiments, the attachment is effected by an interchain cysteine disulfide bond. A composition comprising two or more polypeptides of the invention may be a composition of the same polypeptide species or substantially the same polypeptide species (e.g., a homodimer), or a composition of non-identical polypeptide species (e.g., a heterodimer). A composition having multiple linked polypeptides of the invention may, as described above, have one or more identical or non-identical variant ICOS-L polypeptides of the invention in each polypeptide chain.

[0168] In some embodiments, the immunomodulatory protein containing variant ICOS-L is multivalent, e.g., bivalent. In aspects, the immunomodulatory protein is directly or indirectly linked to a multimerization domain via a linker. In some aspects, the multimerization domain extends the half-life of this molecule.

[0169] The interaction of two or more variant ICOS-L polypeptides can be facilitated by directly or indirectly linking them to any moiety, or to other polypeptides that can interact with each other to form a stable structure. For example, separately encoded variant ICOS-L polypeptide chains can be joined by multimerization, which is mediated by a multimerization domain. Typically, a stable protein-protein interaction is formed between a first variant ICOS-L polypeptide and a second variant ICOS-L polypeptide by the multimerization domain. Homo- or hetero-multimeric polypeptides can be made from co-expressing separate variant ICOS-L polypeptides. The first and second variant ICOS-L polypeptides may be the same or different.

[0170] In some embodiments, the multimerization domain includes anything that can form stable protein - protein interactions. The multimerization domain can include immunoglobulin sequences (e.g., Fc domain; see, e.g., International Patent Publication Nos. WO93 / 10151 and WO2005 / 063816 US; US Patent Application Publication No. 2006 / 0024298; US Patent No. 5,457,035); leucine zippers (e.g., derived from the nuclear transforming proteins fos and jun, or from the oncogene c - myc or General Control of Nitrogen (GCN4)) (see, e.g., Busch and Sassone - Corsi (1990) Trends Genetics, 6:36 - 40; Gentz et al., (1989) Science, 243:1695 - 1699); hydrophobic regions; hydrophilic regions; or can interact via free thiols that form intermolecular disulfide bonds between homomultimer or heteromultimer chimeric molecules. Further, the multimerization domain can include, for example, amino acid sequences containing protrusions complementary to amino acid sequences containing holes as described in US Patent No. 5,731,168; International Patent Publication Nos. WO98 / 50431 and WO2005 / 063816; Ridgway et al. (1996) Protein Engineering, 9:617 - 621. Such multimerization regions can be modified such that the steric interactions not only promote stable interactions, but also further promote the formation of more heterodimers than homodimers from a mixture of chimeric monomers. Generally, the protrusions are constructed by exchanging small amino acid side chains from the interface of the first polypeptide for larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protrusions are optionally created at the interface of the second polypeptide by exchanging large amino acid side chains for smaller side chains (e.g., alanine or threonine). Exemplary multimerization domains are described below.

[0171] The variant ICOS-L polypeptide can be linked anywhere, but typically, it can be linked to the N-terminus or C-terminus of the multimerization domain via its N-terminus or C-terminus to form a chimeric polypeptide. The linkage can be direct or indirect via a linker. Also, the chimeric polypeptide can be a fusion protein and can be formed by chemical linkage, for example, via covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide containing a multimerization domain, the nucleic acid encoding all or part of the variant ICOS-L polypeptide can be functionally linked to the nucleic acid encoding the multimerization domain sequence directly, indirectly, or optionally via a linker domain. In some cases, the construct encodes a chimeric protein in which the C-terminus of the variant ICOS-L polypeptide is linked to the N-terminus of the multimerization domain. In some cases, the construct may encode a chimeric protein in which the N-terminus of the variant ICOS-L polypeptide is linked to the N-terminus or C-terminus of the multimerization domain.

[0172] The polypeptide multimer contains two chimeric proteins created by directly or indirectly linking two of the same or different variant ICOS-L polypeptides directly or indirectly to the multimerization domain. In some examples where the multimerization domain is a polypeptide, a gene fusion of the variant ICOS-L polypeptide and the multimerization domain is inserted into a suitable expression vector. The resulting chimeric or fusion protein can be expressed in host cells transformed with the recombinant expression vector and assembled to construct a multimer. In this case, the multimerization domains interact to form a multivalent polypeptide. The multimerization domain and the variant ICOS-L polypeptide can be chemically linked using a heterobifunctional linker.

[0173] The resulting chimeric polypeptides, such as fusion proteins, and multimers formed therefrom can be purified by any suitable method, such as by affinity chromatography on a protein A column or a protein G column. When two nucleic acid molecules encoding different polypeptides are introduced into cells by transformation, homodimers and heterodimers form. The expression conditions can be adjusted to favor heterodimer formation over homodimer formation.

[0174] In some embodiments, the immunomodulatory protein comprises a variant ICOS ligand (ICOSL) polypeptide attached to the Fc region of an immunoglobulin (“immunomodulatory Fc fusion,” such as an “ICOSL-Fc variant fusion”; also referred to as an ICOS vIgD-Fc fusion). In some embodiments, the ICOSL-Fc variant fusion also comprises one or more additional IgSF domains, such as one or more additional vIgDs linked to the vIgD of ICOS. In some embodiments, the attachment of the variant ICOS polypeptide or additional IgSF domain is made at the N-terminus of the Fc. In some embodiments, the attachment of the variant ICOS or additional IgSF domain polypeptide is made at the C-terminus of the Fc. In some embodiments, two or more ICOS or additional IgSF domain variant polypeptides (same or different) are attached independently at the N-terminus and C-terminus.

[0175] In some embodiments, the Fc is a mouse or human Fc. In some embodiments, the Fc is a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc is derived from IgG1, such as human IgG1. In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO:37.

[0176] In some embodiments, the Fc region contains another modification such that one or more of its normal functions are altered (e.g., reduced). Generally, in addition to the antigen-binding ability which is the main function of immunoglobulins, the Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Further, the FcRn sequence present in the Fc region plays a role in regulating IgG levels in serum by extending the in vivo half-life through conjugation with the in vivo FcRn receptor. In some embodiments, such functions may be reduced or altered in the Fc for use with the provided Fc fusion proteins.

[0177] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the ICOS-LFc variant fusion provided herein, thereby creating an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are numerous examples of changes or mutations to Fc sequences that can alter effector function. For example, WO2000 / 42072, WO2006 / 019447, WO2012 / 125850, WO2015 / 107026, US2016 / 0017041, and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001) describe exemplary Fc variants with improved or reduced binding to FcR. The content of such publications is specifically incorporated herein by reference.

[0178] In some embodiments, the provided variant ICOS-L-Fc fusion exhibits reduced effector function, and for this reason the in vivo half-life of the ICOS-L-Fc variant fusion is important, but is a desirable candidate for applications where certain effector functions (e.g., CDC and ADCC) are unwanted or harmful, including an Fc region (also referred to as inert Fc or Fc without effector) that lacks the ability to engage Fc receptors. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / abrogation of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the ICOS-L-Fc variant fusion lacks FcγR binding (and thus is likely to lack ADCC activity), but retains FcRn binding ability. NK cells, which are primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)).Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest may be evaluated in vivo, for example, in an animal model, such as the animal models disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the ICOS-L-Fc variant fusion cannot bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. A CDC assay may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996); Cragg, M. S. et al, Blood 101: 1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance / half-life can also be determined using methods known in the art (see, for example, Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

[0179] ICOSL-Fc variant fusions with reduced effector function include those having one or more substitutions of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region according to EU numbering (U.S. Patent No. 6,737,056). Such Fc variants include so-called "DANA" Fc variants having substitutions of residues 265 and 297 with alanine (U.S. Patent No. 7,332,581), and Fc variants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering.

[0180] In some embodiments, the Fc region of the ICOS-L-Fc variant fusion has an Fc region in which one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (as indicated by EU numbering) are substituted with different amino acids compared to the native Fc region. Such changes in the Fc region are not limited to the above changes, for example, changes such as the deglycosylated chains (N297A and N297Q) described in Current Opinion in Biotechnology (2009) 20 (6), 685-691, IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-E233P / L234V / L235A / G236del / S267K, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E; changes described in WO2008 / 092117, for example, G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R; amino acid insertions at positions 233, 234, 235, and 237 (as indicated by EU numbering); and changes at the sites described in WO2000 / 042072.

[0181] Particular Fc variants with improved or reduced binding to FcR have been described (see, for example, U.S. Patent No. 6,737,056; WO2004 / 056312, WO2006 / 019447, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0182] In some embodiments, an ICOS-LFc variant fusion comprising a variant Fc region that includes one or more amino acid substitutions that extend the half-life and / or improve the binding to the neonatal Fc receptor (FcRn) is provided. Antibodies with an extended half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO2015107026. These antibodies include an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants having a substitution at one or more of residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 of the Fc region according to EU numbering, for example, a substitution at residue 434 of the Fc region (U.S. Patent No. 7,371,826).

[0183] In some embodiments, the Fc region of the ICOS-LFc variant fusion includes one or more amino acid substitutions E356D and M358L according to EU numbering. In some embodiments, the Fc region of the ICOS-LFc variant fusion includes one or more amino acid substitutions C220S, C226S, and / or C229S according to EU numbering. In some embodiments, the Fc region of the ICOS variant fusion includes one or more amino acid substitutions R292C and V302C. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0184] In some embodiments, the wild-type IgG1 Fc may be the Fc shown in SEQ ID NO:37, having an allotype (e.g., the f allotype) containing the residues Glu (E) and Met (M) at positions 356 and 358 according to the EU numbering. In other embodiments, the wild-type IgG1 Fc contains residues containing Asp (D) and Leu (L) at positions 356 and 358, for example, amino acids of the human G1m1 allotype, as shown in SEQ ID NO:38. Thus, in some cases, the Fc provided herein may contain the amino acid substitutions E356D and M358L to reconstruct the residues of allotype G1m1 (e.g., the α allotype). In some aspects, the wild-type Fc is modified by one or more amino acid substitutions to reduce effector activity or to render the Fc inactive with respect to Fc effector functions. Exemplary effectorless or inactive mutations include those described herein. Among the effectorless mutations that can be included in the Fc of the constructs provided herein are L234A, L235E, and G237A according to the EU numbering. In some embodiments, the wild-type Fc is further modified by removal of one or more cysteine residues, for example, by exchanging the cysteine residue at position 220 with a serine residue (C220S) according to the EU numbering. Exemplary inactive Fc regions with reduced effector function are shown in SEQ ID NO:39 or 40 and SEQ ID NO:41 or 42. These are based on the allotypes shown in SEQ ID NO:37 or SEQ ID NO:38, respectively. In some embodiments, the Fc region used in the constructs provided herein may further lack the C-terminal lysine residue.

[0185] In some embodiments, for example, as described in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000), changes are made to the Fc region to reduce C1q binding and / or complement-dependent cytotoxicity (CDC).

[0186] In some embodiments, there is provided an ICOS-L-Fc variant fusion comprising a variant Fc region comprising one or more amino acid modifications, wherein the variant Fc region is derived from IgG1 such as human IgG1. In some embodiments, the variant Fc region is derived from the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the Fc exhibits reduced effector function. In some embodiments, the Fc contains at least one amino acid substitution that is N82G (corresponding to N297G according to EU numbering) according to the numbering of SEQ ID NO:37. In some embodiments, the Fc further contains at least one amino acid substitution that is R77C or V87C (corresponding to R292C or V302C according to EU numbering) according to the numbering of SEQ ID NO:37. In some embodiments, the variant Fc region further comprises a C5S amino acid modification (corresponding to C220S according to EU numbering) according to the numbering of SEQ ID NO:37. For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: V297G according to EU numbering and one or more of the following amino acid modifications C220S, R292C, or V302C (corresponding to N82G based on SEQ ID NO:37 and one or more of the following amino acid modifications C5S, R77C, or V87C). For example, the Fc region comprises the sequence set forth in SEQ ID NO:43. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L234A, L235E, or G237A. For example, the Fc region comprises the sequence set forth in SEQ ID NO:40. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, E233P, L234V, L235A, G236del, or S267K. For example, the Fc region comprises the sequence set forth in SEQ ID NO:44. In some embodiments, the variant Fc comprises one or more of the amino acid modifications C220S, L234A, L235E, G237A, E356D, or M358L. For example, the Fc region comprises the sequence set forth in SEQ ID NO:41.

[0187] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the reference (e.g., unmodified) or wild-type Fc shown in SEQ ID NO: 45 (corresponding to K447del according to EU numbering). In some embodiments, since the C-terminal lysine may be removed differently during biosynthesis, a more homogeneous product is obtained when the C-terminal lysine residue is removed when the protein is expressed in cells. In some aspects, such Fc regions may further include one or more additional modifications, such as amino acid substitutions, such as any of the amino acid substitutions described. Examples of such Fc regions are shown in SEQ ID NO: 46, 39, 47, or 42.

[0188] In some embodiments, provided is an ICOS-L-Fc variant fusion comprising a variant Fc region, wherein the variant Fc comprises an amino acid sequence shown in any of SEQ ID NO: 41, 43, 40, 44, 48, 46, 39, 47, or 42, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to any of SEQ ID NO: 41, 43, 40, 44, 48, 46, 39, 47, or 42. In some embodiments, the Fc exhibits reduced effector function.

[0189] In some embodiments, the Fc is derived from IgG2, such as human IgG2. In some embodiments, the Fc comprises an amino acid sequence shown in SEQ ID NO: 49, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 49.

[0190] In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:50, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO:50. In some embodiments, the IgG4 Fc has the CH3 domain of human IgG4 replaced with the CH3 domain of human IgG1, a stabilized Fc that exhibits inhibited aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced with the CH3 domain and CH2 domain of human IgG1, respectively, or an antibody in which the arginine at position 409 of human IgG4 as indicated by the EU index proposed by Kabat et al. is replaced with lysine and that exhibits inhibited aggregate formation (see, e.g., U.S. Patent No. 8,911,726). In some embodiments, the Fc is an IgG4 containing the S228P mutation that has been shown to prevent recombination between a therapeutic antibody and endogenous IgG4 by Fab arm exchange (see, e.g., Labrijin et al. (2009) Nat. Biotechnol., 27(8)767-71). In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:51 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO:51.

[0191] In some embodiments, the variant ICOS-L polypeptide is directly linked to the Fc sequence. In some embodiments, the variant ICOS-L polypeptide is indirectly linked to the Fc sequence, for example, via a linker. In some embodiments, one or more "peptide linkers" link the variant ICOS-L polypeptide and the Fc domain. In some embodiments, the peptide linker may be 1 amino acid residue or longer in length. In some embodiments, the peptide linker has at least 1 amino acid residue, but is 20 amino acid residues or less, 19 amino acid residues or less, 18 amino acid residues or less, 17 amino acid residues or less, 16 amino acid residues or less, 15 amino acid residues or less, 14 amino acid residues or less, 13 amino acid residues or less, 12 amino acid residues or less, 11 amino acid residues or less, 10 amino acid residues or less, 9 amino acid residues or less, 8 amino acid residues or less, 7 amino acid residues or less, 6 amino acid residues or less, 5 amino acid residues or less, 4 amino acid residues or less, 3 amino acid residues or less, 2 amino acid residues or less, or 1 amino acid residue or less in length. In some embodiments, the linker is three alanines (AAA). In some embodiments, the linker is (single-letter amino acid code): GGGGS ("4GS"; SEQ ID NO:52) or a multimer of the 4GS linker, for example, a repeat of 2, 3, 4, 5, or 6 4GS linkers, such as the repeat shown in SEQ ID NO:53 (2xGGGGS) or the repeat shown in SEQ ID NO:54 (3xGGGGS). In some embodiments, the linker is a rigid linker. For example, the linker is an α-helix linker. In some embodiments, the linker is (single-letter amino acid code): EAAAK or a multimer of the EAAAK linker, for example, a repeat of 2, 3, 4, or 5 4GS linkers, such as the repeat shown in SEQ ID NO:55 (EAAAK) or the repeat shown in SEQ ID NO:56 (3xEAAAK) or the repeat shown in SEQ ID NO:57 (5xEAAAK). In some embodiments, the linker can start with one or more EAAAK units and be extended by the addition of A, AA, AAA, AAAA, EAAAA, and EAAAK sequences.In some embodiments, the linker may further comprise amino acids derived from amino acids introduced by cloning and / or restriction sites. For example, the linker may comprise the amino acids GS (in single-letter amino acid code) introduced by using the restriction site BAMHI. In some embodiments, the linker (in single-letter amino acid code) is GSGGGGS (SEQ ID NO:58). In some examples, the linker is a linker (GGGGSGGGGSAAA; SEQ ID NO:59) having three alanines after 2xGGGGS.

[0192] In some embodiments, the variant ICOS-L-Fc fusion protein is a dimer formed by two variant ICOS-L Fc polypeptides linked to the Fc domain. In some particular embodiments, the same or substantially the same species of the ICOS-L-Fc variant fusion polypeptide (tolerating differences in the N-terminal or C-terminal amino acid sequences of three or fewer) are dimerized to form a homodimer. In some embodiments, the dimer is a homodimer in which the two variant ICOS-L Fc polypeptides are identical. Alternatively, different species of the ICOS-L-Fc variant fusion polypeptide can be dimerized to form a heterodimer. Thus, in some embodiments, the dimer is a heterodimer in which the two variant ICOS-L Fc polypeptides are different.

[0193] In some embodiments, provided is a variant ICOS-L-Fc fusion protein containing a variant ICOS-L polypeptide that is directly or indirectly linked to an Fc region and contains one or more amino acid modifications in the reference ICOS-L as described herein. In some cases, the C-terminus of the variant ICOS-L polypeptide is linked to the N-terminus of the Fc region. In some embodiments, the variant ICOS-L of the ICOS-L-Fc fusion contains one or more amino acid modifications in the amino acid sequence of the reference IgV domain shown in SEQ ID NO:3. In certain cases, such an immunomodulatory protein contains an IgV domain, e.g., the IgV domain shown in SEQ ID NO:35, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:35 and contains a variant ICOS-L polypeptide containing one or more amino acid modifications of the respective SEQ ID NO, e.g., N52H / N57Y / Q100R. In certain cases, such an immunomodulatory protein contains an IgV domain, e.g., the IgV domain shown in SEQ ID NO:36, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:36 and contains a variant ICOS-L polypeptide containing one or more amino acid modifications of the respective SEQ ID NO, e.g., N52H / N57Y / Q100R. In some embodiments, the variant ICOS-L polypeptide contains the amino acid modifications N52H / N57Y / Q100R (e.g., is or includes the IgV domain shown in SEQ ID NO:36).

[0194] In certain embodiments of such variant ICOS-L-Fc fusion proteins, the Fc polypeptide is a variant of the human IgG1 Fc region that exhibits reduced effector function, e.g., any of those described. In some embodiments, the Fc region is a human IgG1 containing the amino acid modifications N297G, E233P / L234V / L235A / G236del / S267K, or L234A / L235E / G237A, with residues numbered according to the Kabat EU index. In some embodiments, the variant IgG1 Fc region further contains the amino acid substitution C220S, with residues numbered according to the Kabat EU index. In some embodiments, the Fc region contains K447del, with residues numbered according to the Kabat EU index. In some aspects, the Fc region has an amino acid sequence shown in any of SEQ ID NO:41, 43, 40, 44, 39, or 42, or exhibits at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to any of SEQ ID NO:41, 43, 40, 44, 39, or 42, and contains an amino acid sequence containing the amino acid substitutions of each SEQ ID NO. The linkage between the variant ICOS-L IgSF (e.g., IgV) polypeptide and the Fc may be through a peptide linker, e.g., any peptide linker as described. In some embodiments, the linker is GGGGS ("4GS"; SEQ ID NO:52), SEQ ID NO:53 (2xGGGGS), or SEQ ID NO:54 (3xGGGGS). In certain examples, the C-terminus of the variant ICOS-L polypeptide is linked to the N-terminus of the Fc region such that the order of the components is variant ICOS-L-linker-Fc.

[0195] In some embodiments, a variant ICOS-L-Fc fusion protein is provided that contains the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:52, and the Fc polypeptide shown in SEQ ID NO:42, e.g., variant ICOS-L-linker-Fc. In some embodiments, a variant ICOS-L-Fc fusion protein is provided that contains the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:52, and the Fc polypeptide shown in SEQ ID NO:41, e.g., variant ICOS-L-linker-Fc. In some embodiments, a variant ICOS-L-Fc fusion protein is provided that contains the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:52, and the Fc polypeptide shown in SEQ ID NO:40, e.g., variant ICOS-L-linker-Fc. In some embodiments, a variant ICOS-L-Fc fusion protein is provided that contains the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:52, and the Fc polypeptide shown in SEQ ID NO:39, e.g., variant ICOS-L-linker-Fc.

[0196] In some embodiments, a variant ICOS-L-Fc fusion protein is provided that contains the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:53, and the Fc polypeptide shown in SEQ ID NO:42, e.g., variant ICOS-L-linker-Fc. In some embodiments, a variant ICOS-L-Fc fusion protein is provided that contains the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:53, and the Fc polypeptide shown in SEQ ID NO:41, e.g., variant ICOS-L-linker-Fc. In some embodiments, a variant ICOS-L-Fc fusion protein is provided that contains the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:53, and the Fc polypeptide shown in SEQ ID NO:40, e.g., variant ICOS-L-linker-Fc. In some embodiments, a variant ICOS-L-Fc fusion protein is provided that contains the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:53, and the Fc polypeptide shown in SEQ ID NO:39, e.g., variant ICOS-L-linker-Fc.

[0197] In some embodiments, there is provided a variant ICOS-L-Fc fusion protein containing the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:54, and the Fc polypeptide shown in SEQ ID NO:42, e.g., variant ICOS-L-linker-Fc. In some embodiments, there is provided a variant ICOS-L-Fc fusion protein containing the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:54, and the Fc polypeptide shown in SEQ ID NO:41, e.g., variant ICOS-L-linker-Fc. In some embodiments, there is provided a variant ICOS-L-Fc fusion protein containing the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:54, and the Fc polypeptide shown in SEQ ID NO:40, e.g., variant ICOS-L-linker-Fc. In some embodiments, there is provided a variant ICOS-L-Fc fusion protein containing the variant ICOS-L IgV domain shown in SEQ ID NO:36, the linker shown in SEQ ID NO:54, and the Fc polypeptide shown in SEQ ID NO:39, e.g., variant ICOS-L-linker-Fc.

[0198] In some embodiments, there is provided a variant ICOS-L IgSF Fc fusion protein having the amino acid sequence shown in SEQ ID NO:60, or an amino acid sequence showing at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to SEQ ID NO:60. In some embodiments, the variant ICOS-L IgSF Fc fusion protein binds to CD28 and ICOS with improved binding affinity as compared to a reference (wild-type) ICOS-L-Fc fusion protein. In some embodiments, the variant ICOS-L IgSF Fc fusion shows reduced Fc effector function as compared to a fusion with the Fc of wild-type human IgG1.

[0199] III. Nucleic Acids, Vectors, and Methods for Producing Polypeptides or Cells Isolated or recombinant nucleic acids, collectively referred to herein as "nucleic acids", are provided herein that encode any of the various provided aspects of the variant ICOS-L polypeptides or immunomodulatory fusion polypeptides provided herein. In some aspects, the nucleic acids provided herein, including all that are described below, are useful in the recombinant production (e.g., expression) of the variant ICOS-L polypeptides or immunomodulatory fusion polypeptides provided herein. The nucleic acids provided herein may be in the form of RNA or DNA, including mRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleic acids provided herein are typically DNA molecules, usually double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA, and hybrid DNA / RNA nucleic acids, or combinations thereof, containing any nucleotide sequence of the invention are also provided.

[0200] Also provided herein are recombinant expression vectors and recombinant host cells useful in producing the variant ICOS-L polypeptides or immunomodulatory fusion polypeptides provided herein.

[0201] In any of the aspects provided above, the nucleic acid encoding the variant polypeptide or immunomodulatory fusion polypeptide provided herein can be introduced into cells using recombinant DNA and cloning techniques. To do so, recombinant DNA molecules encoding the immunomodulatory polypeptide are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the sequence encoding the peptide can also be excised from the DNA using suitable restriction enzymes. Alternatively, DNA molecules can be synthesized using chemical synthesis techniques such as the phosphoramidite method. Combinations of these techniques can also be used. In some cases, recombinant or synthetic nucleic acids may be generated by polymerase chain reaction (PCR). In some aspects, a DNA insert encoding one or more variant ICOS-L polypeptides containing at least one affinity-modified IgSF domain can be generated according to the provided description. This DNA insert can be cloned into a suitable transduction / transfection vector as known to those skilled in the art. Also provided is an expression vector containing the nucleic acid molecule.

[0202] In some aspects, the expression vector can express the immunomodulatory protein in a suitable cell under conditions suitable for protein expression. In some respects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding an immunomodulatory protein operably linked to a suitable expression control sequence. Methods for achieving this operable linkage, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, initiation signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation. In some aspects, the expression of the immunomodulatory protein is controlled by a promoter or enhancer to control or regulate the expression. The promoter is operably linked to a portion of the nucleic acid molecule encoding the variant polypeptide or immunomodulatory protein.

[0203] In some embodiments, the inducible promoter is operably linked to a nucleic acid molecule encoding a variant polypeptide or an immunomodulatory protein such that the expression of the nucleic acid can be controlled by controlling the presence or absence of an appropriate inducer of transcription. For example, the promoter can be a regulated promoter and transcription factor expression system that allows for regulated expression of the encoded polypeptide, such as the publicly available tetracycline-regulated system or other regulatable systems (see, e.g., the publicly available international PCT application WO 01 / 30843). An exemplary regulatable promoter system is the Tet-On (and Tet-Off) system available, for example, from Clontech (Palo Alto, CA). This promoter system allows for regulated expression of the transgene, which is controlled by tetracycline or a tetracycline derivative, such as doxycycline. Other regulatable promoter systems are known (see, e.g., the published U.S. patent application No. 2002-0168714, entitled "Regulation of Gene Expression Using Single-Chain, Monomeric, Ligand Dependent Polypeptide Switches," which describes a gene switch containing a ligand binding domain and a transcriptional regulatory domain, such as those derived from a hormone receptor).

[0204] An appropriate host is transformed using the resulting recombinant expression vector having the DNA molecule thereon. This transformation can be carried out using methods well known in the art. In some embodiments, the nucleic acids provided herein further comprise a nucleotide sequence encoding a secretory or signal peptide operably linked to the nucleic acid encoding the immunomodulatory polypeptide such that the resulting soluble immunomodulatory polypeptide is recovered from the culture medium, host cell, or host cell periplasm. Additionally, commercially available kits and contract manufacturing companies can be utilized to produce the modified or recombinant host cells provided herein.

[0205] In some embodiments, an appropriate cell is transformed, e.g., transduced, using an obtained expression vector having a DNA molecule thereon. The introduction can be carried out using methods well known in the art. Exemplary methods include those for the transfer of nucleic acids encoding receptors, including via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation. In some embodiments, the expression vector is a viral vector. In some embodiments, the nucleic acid is transferred into the cell by a lentiviral or retroviral transduction method.

[0206] Any of a number of publicly available and well-known mammalian host cells can be used in preparing the polypeptide. The choice of cell depends on a number of factors recognized by those skilled in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation efficiency, ease of peptide recovery, expression characteristics, biosafety, and cost. It must be understood that the balance of these factors does not mean that all cells are equally effective in expressing a particular DNA sequence.

[0207] In some embodiments, the host cell may be various eukaryotic cells, such as yeast cells, or mammalian cells, such as Chinese hamster ovary (CHO) or HEK293 cells. In some embodiments, the host cell is a suspension cell, and the polypeptide is modified or produced in a culture suspension, for example, in a culture suspension of CHO cells, such as CHO-S cells. In some examples, the cell line is a CHO cell line lacking dihydrofolate reductase (DHFR-), such as DG44 and DUXB11. In some embodiments, the cells are deficient in glutamine synthetase (GS), such as CHO-S cells, CHOK1 SV cells, and CHOZN((R))GS- / - cells. In some embodiments, the CHO cells, such as suspension CHO cells, may be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells.

[0208] In some embodiments, expressing the provided ICOS-L polypeptide from CHO cells results in a more homogeneous composition of the produced protein. In some embodiments, the provided ICOS-L polypeptide, when expressed from CHO cells, contains the full ECD reference sequence and / or results in a more homogeneous product compared to an ICOS-L polypeptide containing a protease cleavage site (e.g., LQQN / LT). In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the composition of the produced protein containing the ICOS-L variant polypeptide produced herein has the same amino acid length and is the same size. Techniques for assessing size homogeneity include high performance liquid chromatography (HPLC), size exclusion chromatography, SDS-PAGE, or sequencing.

[0209] In some embodiments, the host cell can be a variety of eukaryotic cells such as yeast cells, or mammalian cells such as Chinese hamster ovary (CHO) or HEK293 cells. In some embodiments, the host cell can also be a prokaryotic cell such as Escherichia coli (E. coli). The transformed recombinant host is cultured under conditions that express the polypeptide and then purified to obtain the soluble protein. The recombinant host cell can be cultured under conventional fermentation conditions such that the desired polypeptide is expressed. Such fermentation conditions are well known in the art. Finally, the polypeptides provided herein can be recovered and purified from the recombinant cell culture by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography. If desired, a protein refolding step can be used in the completion of the conformation of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification step.

[0210] In some embodiments, the recombinant vector is a viral vector. Exemplary recombinant viral vectors include lentiviral vector genomes, poxviral vector genomes, vaccinia viral vector genomes, adenoviral vector genomes, adeno-associated viral vector genomes, herpes viral vector genomes, and alphaviral vector genomes. The viral vector can be a live viral vector, an attenuated viral vector, a replication conditional or replication defective viral vector, a non-pathogenic (defective) viral vector, a replication competent viral vector, and / or modified to express a heterologous gene product, e.g., a variant immunomodulatory polypeptide provided herein. The vector for virus production can also be modified to alter the attenuation of the virus, including any method that increases or decreases the transcriptional or translational load.

[0211] Exemplary viral vectors that can be used include modified vaccinia virus vectors (e.g., Guerra et al., J. Virol. 80:985-98 (2006); Tartaglia et al., AIDS Research and Human Retroviruses 8: 1445-47 (1992); Gheradi et al., J. Gen. Virol. 86:2925-36 (2005); Mayr et al., Infection 3:6-14 (1975); Hu et al., J. Virol. 75: 10300-308 (2001); see U.S. Pat. Nos. 5,698,530, 6,998,252, 5,443,964, 7,247,615, and 7,368,116); adenovirus vectors or adeno-associated virus vectors (e.g., Molin et al., J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol. Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA 101:6188-93 (2004); see U.S. Pat. No. 6,143,290; 6,596,535; 6,855,317; 6,936,257; 7,125,717; 7,378,087; 7,550,296); murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), amphotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and retroviral vectors based on combinations thereof (e.g., Buchscher et al., J. Virol. 66:2731-39 (1992); Johann et al., J. Virol. 66: 1635-40 (1992); Sommerfelt et al., Virology 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-78 (1989); Miller et al., J. Virol.See 65:2220-24 (1991); Miller et al., Mol. Cell Biol. 10:4239 (1990); Kolberg, NIH Res. 4:43 1992; Cornetta et al., Hum. Gene Ther. 2:215 (1991); lentiviral vectors, including those based on human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, simian immunodeficiency virus (SIV), and maedi / visna virus (e.g., see Pfeifer et al., Annu. Rev. Genomics Hum. Genet. 2: 177-211 (2001); Zufferey et al., J. Virol. 72: 9873, 1998; Miyoshi et al., J. Virol. 72:8150, 1998; Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al., J. Virol. 69: 2729, 1995; Nightingale et al., Mol. Therapy, 13: 1121, 2006; Brown et al., J. Virol. 73:9011 (1999); WO 2009 / 076524; WO 2012 / 141984; WO 2016 / 011083; McWilliams et al., J. Virol. 77: 11150, 2003; Powell et al., J. Virol. 70:5288, 1996) or any variant thereof, and / or vectors that can be used to generate any of the above viruses. In some embodiments, the recombinant vector can include regulatory sequences, such as promoter or enhancer sequences, that can regulate the expression of the viral genome in a packaging cell line, for example, in the case of an RNA virus (e.g., see U.S. Pat. Nos. 5,385,839 and 5,168,062).

[0212] In some embodiments, the polypeptides provided herein can also be produced by synthetic methods. Solid-phase synthesis is a preferred technique for producing individual peptides as it is the most cost-effective way to produce small peptides. For example, well-known solid-phase synthesis techniques include the use of protecting groups, linkers, and solid-phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, the use of scavengers, and other aspects of solid-phase peptide synthesis. The peptides can then be assembled into the polypeptides as provided herein.

[0213] IV. Methods for Evaluating the Immunomodulatory Activity of Variant ICOSL Polypeptides and Immunomodulatory Proteins In some embodiments, the variant ICOSL polypeptides provided herein (e.g., full-length and / or specific binding fragments or immunomodulatory fusions thereof) exhibit immunomodulatory activity that regulates T cell activation. In some embodiments, the ICOSL polypeptide regulates IFN-γ expression in a primary T cell assay compared to a reference (e.g., unmodified) or wild-type ICOSL control. In some cases, the regulation of IFN-γ expression can increase or decrease IFN-γ expression compared to the control. Assays for determining specific binding and IFN-γ expression are well known in the art and include the MLR (mixed lymphocyte reaction) assay (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), which measures interferon-γ cytokine levels in the culture supernatant, the SEB (Staphylococcus enterotoxin B) T cell stimulation assay (Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56), and the anti-CD3 T cell stimulation assay (Li and Kurlander, J Transl Med. 2010: 8: 104).

[0214] In some embodiments, the variant ICOS-L polypeptide can increase IFN-γ (interferon-γ) expression in some embodiments, or decrease it in alternative embodiments, compared to the wild-type ICOS-L control in a primary T cell assay. In some embodiments of the provided polypeptides containing a soluble variant ICOS-L sequence, the polypeptide can increase IFN-γ expression in a primary T cell assay compared to the wild-type ICOS-L control, or decrease IFN-γ expression in alternative embodiments. In some embodiments of the provided polypeptides containing multiple variant ICOS-L sequences, the polypeptide can increase IFN-γ expression in a primary T cell assay compared to the wild-type ICOS-L control, or decrease IFN-γ expression in alternative embodiments.

[0215] One of ordinary skill in the art will recognize that the format of the primary T cell assay used to determine an increase in IFN-γ expression can be different from that employed to assay a decrease in IFN-γ expression. When assaying the ability of a variant ICOS-L to decrease IFN-γ expression in a primary T cell assay, a mixed lymphocyte reaction (MLR) assay can be used. In some cases, a soluble form of the variant ICOS-L can be employed to determine its ability to antagonize IFN-γ expression in the MLR and thereby decrease that expression. Alternatively, when assaying the ability of a variant ICOS-L to increase IFN-γ expression in a primary T cell assay, a co-stimulation assay can be used. In the co-stimulation assay, a TCR signal (provided by an anti-CD3 antibody in some embodiments) is used in combination with the co-stimulated variant ICOS-L to determine the ability to increase IFN-γ expression compared to an ICOS-L control.

[0216] In some embodiments, a T cell reporter assay can be used to assay the ability of variant ICOS-L to modulate increases or decreases in IFN-γ expression. In some embodiments, the T cells are a Jurkat T cell line or are derived from a Jurkat T cell line. In the reporter assay, a reporter cell line (e.g., a Jurkat reporter cell) is also generated to overexpress a cognate binding partner of the variant IgSF domain polypeptide. In some embodiments, the reporter T cells also contain a reporter construct containing an inducible promoter that responds to T cell activation operably linked to the reporter. In some embodiments, the reporter is a fluorescent or luminescence reporter. In some embodiments, the reporter is luciferase. In some embodiments, the promoter responds to CD3 signaling. In some embodiments, the promoter is an NFAT promoter. In some embodiments, the promoter responds to co-stimulatory signaling, e.g., CD28 co-stimulatory signaling. In some embodiments, the promoter is an IL-2 promoter.

[0217] In the context of the reporter assay, the reporter cell line is stimulated by co-incubating it, for example, with an antigen presenting cell (APC) that expresses a wild-type ligand of an inhibitory receptor, such as ICOS-L. In some embodiments, the APC is an artificial APC. Artificial APCs are well known to those of skill in the art. In some embodiments, the artificial APC is derived from one or more mammalian cell lines, such as K562, CHO, or 293 cells.

[0218] In some embodiments, the jackett reporter cells are co-incubated with an artificial APC that overexpresses a ligand in the presence of a variant IgSF domain molecule or an immunomodulatory protein, such as a variant ICOS-L polypeptide or an immunomodulatory protein. In some embodiments, reporter expression is monitored by, for example, ascertaining the luminescence or fluorescence of the cells. In some embodiments, when its receptor and ligand interact normally, for example, compared to reporter expression by co-incubation of control, for example, control T cells and APCs in which there is no interaction between the receptor and ligand, such as APCs that do not overexpress ICOS-L, the reporter signal is suppressed or reduced. In some embodiments, the variant ICOS-L polypeptide or immunomodulatory protein provided herein antagonizes the interaction, for example, when provided in soluble form as variant ICOS-L-Fc, and as a result, the reporter signal is enhanced compared to in the absence of the variant ICOS-L polypeptide or immunomodulatory protein.

[0219] The use of appropriate controls is known to those of skill in the art, but in the foregoing embodiments, the control typically involves the use of a reference ICOS-L, such as a wild-type native ICOS-L isoform from the same mammalian species from which the variant ICOS-L was derived or generated. Regardless of whether the binding affinity to one or both of ICOS and CD28 is improved or decreased, the variant ICOS-L increases IFN-γ expression compared to the wild-type ICOS-L control in some embodiments and decreases IFN-γ expression in alternative embodiments in a primary T cell assay.

[0220] In some embodiments, variant ICOS-L increases IFN-γ expression (i.e., protein expression) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a reference (e.g., unmodified) or wild-type ICOS-L control. In other embodiments, variant ICOS-L decreases IFN-γ expression (i.e., protein expression) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a wild-type or unmodified ICOS-L control. In some embodiments, the wild-type ICOS-L control is murine ICOS-L, such as is typically used for variant ICOS-L whose sequence varies from the wild-type murine ICOS-L sequence. In some embodiments, the wild-type ICOS-L control is human ICOS-L, such as is typically used for variant ICOS-L whose sequence varies from a wild-type human ICOS-L sequence, e.g., an ICOS-L sequence comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or 3.

[0221] V. Pharmaceutical Formulations Compositions containing any of the variant ICOS-L polypeptides (e.g., immunomodulatory fusion proteins) described herein are provided herein. The pharmaceutical compositions can further include pharmaceutically acceptable excipients. For example, the pharmaceutical compositions can contain one or more excipients for adjusting, maintaining, or preserving, e.g., the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. In some aspects, one of ordinary skill in the art will understand that a pharmaceutical composition containing cells can differ from a pharmaceutical composition containing a protein.

[0222] In some embodiments, the pharmaceutical composition is a solid, such as a powder, capsule, or tablet. For example, the components of the pharmaceutical composition can be lyophilized. In some embodiments, the solid pharmaceutical composition is reconstituted or dissolved in a liquid prior to administration.

[0223] In some embodiments, the pharmaceutical composition is a variant ICOS-L polypeptide dissolved in a liquid, such as an aqueous solution (e.g., saline or Ringer's solution). In some embodiments, the pH of the pharmaceutical composition is from about 4.0 to about 8.5 (e.g., about 4.0 to about 5.0, about 4.5 to about 5.5, about 5.0 to about 6.0, about 5.5 to about 6.5, about 6.0 to about 7.0, about 6.5 to about 7.5, about 7.0 to about 8.0, or about 7.5 to about 8.5).

[0224] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, such as a filler, binder, coating agent, preservative, lubricant, flavoring agent, sweetening agent, coloring agent, solvent, buffer, chelating agent, or stabilizer. Examples of pharmaceutically acceptable fillers include cellulose, dibasic calcium phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch, or potato starch. Examples of pharmaceutically acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, or cellulose. Examples of pharmaceutically acceptable coating agents include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, or sodium starch glycolate. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate, or stearic acid. Examples of pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, or sorbic acid. Examples of pharmaceutically acceptable sweetening agents include sucrose, saccharin, aspartame, or sorbitol. Examples of pharmaceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

[0225] In some embodiments, the pharmaceutical composition further comprises a substance for controlled or sustained release of the product, such as injectable microspheres, biodegradable particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes.

[0226] In some embodiments, the pharmaceutical composition is sterilized. Sterilization may be achieved by filtration through a sterilizing filter membrane or by irradiation. If the composition is lyophilized, sterilization using this method may be performed before or after lyophilization and reconstitution. The parenteral composition may be stored in lyophilized form or in solution. Additionally, the parenteral composition is generally placed in a container having a sterile access port, such as an intravenous fluid bag or vial having a stopper penetrable by a hypodermic needle.

[0227] Such a formulation may be, for example, in a form suitable for intravenous infusion. A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition, or vehicle involved in the conveyance or transport of the cells of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other active ingredients of the formulation. It must also be suitable for contact with any tissue, organ, or part of the body that it may encounter in the sense that it should not have a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that unduly outweighs its therapeutic benefit.

[0228] In some embodiments, the pharmaceutical composition is prepared to contain a variant ICOS ligand polypeptide, such as variant ICOS IgSF-Fc, in an amount from 1 mg or about 1 mg to 100 mg or about 100 mg, for example, from 1 mg or about 1 mg to 75 mg or about 75 mg, from 1 mg or about 1 mg to 50 mg or about 50 mg, from 1 mg or about 1 mg to 25 mg or about 25 mg, from 1 mg or about 1 mg to 10 mg or about 10 mg, from 1 mg or about 1 mg to 5 mg or about 5 mg, from 5 mg or about 5 mg to 100 mg or about 100 mg, from 5 mg or about 5 mg to 75 mg or about 75 mg, from 5 mg or about 5 mg to 50 mg or about 50 mg, from 5 mg or about 5 mg to 25 mg or about 25 mg, from 5 mg or about 5 mg to 10 mg or about 10 mg, from 10 mg or about 10 mg to 100 mg or about 100 mg, from 10 mg or about 10 mg to 75 mg or about 75 mg, from 10 mg or about 10 mg to 50 mg or about 50 mg, from 10 mg or about 10 mg to 25 mg or about 25 mg, from 25 mg or about 25 mg to 100 mg or about 100 mg, from 25 mg or about 25 mg to 75 mg or about 75 mg, from 25 mg or about 25 mg to 50 mg or about 50 mg, from 50 mg or about 50 mg to 100 mg or about 100 mg, from 50 mg or about 50 mg to 75 mg or about 75 mg, or from 75 mg or about 75 mg to 100 mg or about 100 mg. In some embodiments, the pharmaceutical composition is prepared to contain a variant ICOS ligand polypeptide, such as variant ICOS IgSF-Fc, in an amount of 10 mg or about 10 mg, 25 mg or about 25 mg, 50 mg or about 50 mg, 75 mg or about 75 mg, or 100 mg or about 100 mg.

[0229] In some embodiments, the pharmaceutical composition is prepared in a volume of from 0.5 mL or about 0.5 mL to 10 mL or about 10 mL, such as from 0.5 mL or about 0.5 mL to 5 mL or about 5 mL, from 0.5 mL or about 0.5 mL to 2 mL or about 2 mL, from 0.5 mL or about 0.5 mL to 1 mL or about 1 mL, from 1 mL or about 1 mL to 10 mL or about 10 mL, from 1 mL or about 1 mL to 5 mL or about 5 mL, or from 5 mL or about 5 mL to 10 mL or about 10 mL. In some embodiments, the pharmaceutical composition is prepared in a volume of 0.5 mL or about 0.5 mL, 1 mL or about 1 mL, 2 mL or about 2 mL, 2.5 mL or about 2.5 mL, 3 mL or about 3 mL, 4 mL or about 4 mL, 5 mL or about 5 mL, 6 mL or about 6 mL, 7 mL or about 7 mL, 8 mL or about 8 mL, 9 mL or about 9 mL, or 10 mL or about 10 mL. In some embodiments, the concentration of the composition is from 1 mg or about 1 mg / mL to 50 mg or about 50 mg / mL, such as from 1 mg or about 1 mg / mL to 25 mg / mL or about 25 mg / mL, from 1 mg or about 1 mg / mL to 15 mg or about 15 mg / mL, from 1 mg or about 1 mg / mL to 5 mg or about 5 mg / mL, from 5 mg or about 5 mg / mL to 50 mg or about 50 mg / mL, from 5 mg or about 5 mg / mL to 25 mg or about 25 mg / mL, from 5 mg or about 5 mg / mL to 15 mg or about 15 mg / mL, from 15 mg or about 15 mg / mL to 50 mg or about 50 mg / mL, from 15 mg or about 15 mg / mL to 25 mg or about 25 mg / mL, or from 25 mg or about 25 mg / mL to 50 mg or about 50 mg / mL. In some embodiments, the concentration of the composition is 1 mg or about 1 mg / mL, 5 mg or about 5 mg / mL, 10 mg or about 10 mg / mL, 15 mg or about 15 mg / mL, 20 mg or about 20 mg / mL, 25 mg or about 25 mg / mL, 30 mg or about 30 mg / mL, 40 mg or about 40 mg / mL, or 50 mg or about 50 mg / mL.Provided herein is such an arbitrary composition contained in a container such as a vial. In certain aspects, the container, e.g., the vial, is sterile.

[0230] In some embodiments, the pharmaceutical composition is administered to a subject. Generally, the dosage and route of administration of the pharmaceutical composition are determined according to standard medical practice, depending on the size and condition of the subject. For example, first, a therapeutically effective dose can be estimated in either a cell culture assay or an animal model such as a mouse, rat, rabbit, dog, pig, or monkey. Also, an animal model may be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful dosages and routes of administration in humans. The exact dosage will be determined in light of factors relevant to the treatment the subject requires. The dosage and administration are adjusted to provide a sufficient level of the active compound or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the overall health of the subject, the age, weight, and gender of the subject, the time and frequency of administration, drug combinations, sensitivity to response, and response to treatment.

[0231] The long-acting pharmaceutical composition may be administered every 3 to 4 days, weekly, or bi-weekly, depending on the half-life and clearance rate of the specific formulation. The dosing frequency will depend on the pharmacokinetic parameters of the molecule in the formulation used. Typically, the composition is administered until a dosage that achieves the desired effect is reached. Therefore, the composition may be administered as a single dose, administered over time as multiple doses (at the same or different concentrations / dosages), or administered as a continuous infusion. Further refinement of the appropriate dosage is routinely performed. The appropriate dosage may be determined through the use of appropriate dose-response data. A number of biomarkers or physiological markers for the therapeutic effect can be monitored, including T cell activation or proliferation, cytokine synthesis or production (e.g., production of TNF-α, IFN-γ, IL-2), induction of various activation markers (e.g., CD25, IL-2 receptor), inflammation, joint swelling or tenderness, serum levels of C-reactive protein, anti-collagen antibody production, and / or T cell-dependent antibody response.

[0232] In some embodiments, the pharmaceutical composition is administered to the subject via any route including oral, transdermal, inhalation, intravenous, intraarterial, intramuscular, direct application to a wound site, application to a surgical site, intraperitoneal, by suppository, subcutaneous, intradermal, transdermal, by spray, intrapleural, intracerebroventricular, intra-articular, intraocular, or intrathecal. In some embodiments, the pharmaceutical composition is administered intravitreally to the subject.

[0233] In some embodiments, administration of the pharmaceutical composition is by single or repeated dosing. In some embodiments, it is administered to the subject once a day, twice a day, three times a day, or four or more times a day. In some embodiments, a dose of about once or more per week (e.g., about twice or more per week, about three times or more per week, about four times or more per week, about five times or more per week, about six times or more per week, or about seven times or more per week) is given. In some embodiments, multiple doses are given over several days, weeks, months, or years. In some embodiments, a series of treatments is about one dose or more (e.g., about two doses or more, about three doses or more, about four doses or more, about five doses or more, about seven doses or more, about ten doses or more, about fifteen doses or more, about twenty-five doses or more, about forty doses or more, about fifty doses or more, or about one hundred doses or more).

[0234] In some embodiments, the dosage of the pharmaceutical composition administered is at least about 1 μg of protein per kg of the subject's body weight (e.g., at least about 2 μg of protein per kg of the subject's body weight, at least about 5 μg of protein per kg of the subject's body weight, at least about 10 μg of protein per kg of the subject's body weight, at least about 25 μg of protein per kg of the subject's body weight, at least about 50 μg of protein per kg of the subject's body weight, at least about 100 μg of protein per kg of the subject's body weight, at least about 250 μg of protein per kg of the subject's body weight, at least about 500 μg of protein per kg of the subject's body weight, at least about 1 mg of protein per kg of the subject's body weight, at least about 2 mg of protein per kg of the subject's body weight, or at least about 5 mg of protein per kg of the subject's body weight). In some embodiments, the dosage of the pharmaceutical composition administered is 0.3 mg of protein / kg of subject body weight or about 0.3 mg of protein / kg of subject body weight. In some embodiments, the dosage of the pharmaceutical composition administered is 1 mg of protein / kg of subject body weight or about 1 mg of protein / kg of subject body weight. In some embodiments, the dosage of the pharmaceutical composition administered is 3 mg of protein / kg of subject body weight or about 3 mg of protein / kg of subject body weight. In some embodiments, the dosage of the pharmaceutical composition administered is 6 mg of protein / kg of subject body weight or about 6 mg of protein / kg of subject body weight. In some embodiments, the dosage of the pharmaceutical composition administered is 10 mg of protein / kg of subject body weight or about 10 mg of protein / kg of subject body weight. In some embodiments, the dosage of the pharmaceutical composition administered is 15 mg of protein / kg of subject body weight or about 15 mg of protein / kg of subject body weight. In some embodiments, the dosage of the pharmaceutical composition administered is 20 mg of protein / kg of subject body weight or about 20 mg of protein / kg of subject body weight.

[0235] Eliminating, isolating, or inactivating immune cells that mediate or can mediate an unwanted immune response; inducing, generating, or stimulating immune cells that mediate or can mediate a defensive immune response; altering the physical or functional properties of immune cells; or determining whether administration of a therapeutic composition of the present invention sufficiently regulates immune activity by a combination of these effects are known by a variety of means. Examples of measuring immune activity regulation include, but are not limited to, examination of the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measurement of the functional capabilities of immune cells, including the ability to proliferate or differentiate in response to a signal or resistance to proliferation or differentiation (e.g., T cell proliferation assay, and peps scan analysis based on 3H-thymidine incorporation after stimulation with antigen-presenting cells loaded with anti-CD3 antibody, anti-T cell receptor antibody, anti-CD28 antibody, calcium ionophore, PMA (phorbol 12-myristate 13-acetate), peptide or protein antigen; using a B cell proliferation assay); measurement of the ability to kill or lyse other cells (e.g., cytotoxic T cell assay); measurement of cytokines, chemokines, cell surface molecules, antibodies, and other products of cells (e.g., by flow cytometry, enzyme-linked immunosorbent assay, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measurement of biochemical markers of activation of signal transduction pathways within immune cells or immune cells (e.g., Western blot and immunoprecipitation analysis of tyrosine, serine or threonine phosphorylation, polypeptide cleavage, and formation or dissociation of protein complexes; protein array analysis; DNA transcription profiling using DNA array or subtractive hybridization); measurement of cell death by apoptosis, necrosis, or other mechanisms (e.g., annexin V staining, TUNEL assay, gel electrophoresis measuring DNA laddering, histology; fluorescence-generating caspase assay, Western blot analysis of caspase substrates);Measurement of genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarray, protein microarray, two-dimensional gel electrophoresis, Western blot analysis, enzyme-linked immunosorbent assay, flow cytometry); and measurement (clinical score, requirements for using additional therapies, functional status, imaging studies) of clinical symptoms or clinical outcomes (e.g., improvement) of autoimmune diseases, neurodegenerative diseases, and other diseases involving self-proteins or self-polypeptides, e.g., by measuring the relapse rate or disease severity in the case of multiple sclerosis (using clinical scores known to those skilled in the art), blood glucose in the case of type I diabetes, or joint inflammation in the case of rheumatoid arthritis. ;

[0236] VI. Manufactured Articles and Kits Also provided herein are manufactured articles comprising a pharmaceutical composition described herein (including a pharmaceutical composition comprising a variant ICOS Ligand IgSF domain fusion protein) in a suitable package. Suitable packages for the manufactured articles include one or more containers, typically multiple containers, packaging materials, and labels or inserts on or attached to the surface of the containers and / or the packaging, and generally the label or insert includes instructions for administering the composition to a subject. Suitable containers for packaging the compositions described herein are known in the art and include, for example, vials (e.g., sealed vials), vessels, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These manufactured articles may further be sterilized and / or sealed.

[0237] Furthermore, the manufactured article may comprise an attached document or label with one or more pieces of identification information and / or instructions for use. In some embodiments, the information or instructions indicate that the content can or must be used to treat a particular condition or disease and / or provide instructions therefor. The label or attached document may indicate that the contents of the manufactured article are used to treat a disease or condition. In some embodiments, the label or attached document provides instructions for treating a subject, for example, according to any aspect of the provided method. In some embodiments, the instructions specify administering one or more unit doses to the subject.

[0238] There is further provided a kit comprising the pharmaceutical composition (or manufactured article) described herein, which kit may further comprise instructions for using the composition, for example, instructions regarding the use described herein. The kits described herein may also include other buffers, diluents, filters, needles, syringes, and other commercially available and user-desirable materials including attached documents with instructions for performing any of the methods described herein.

[0239] VII. Therapeutic Uses Provided herein are methods of using and uses of molecules provided that contain a variant ICOS-L IgSF domain fusion protein described herein and pharmaceutical compositions containing the same. Such methods and uses include methods for modulating an immune response, including methods for modulating an immune response in a subject, e.g., a human patient, in connection with the treatment of a disease or condition. Among such molecules in the methods of use and uses herein are immunomodulatory fusion protein formats in which the extracellular domain or a portion of an ICOS-L variant polypeptide containing an IgSF domain (e.g., IgV) with modified affinity is directly or indirectly linked to a multimerization domain, e.g., an Fc domain or region. In some embodiments, such therapeutic agents are variant ICOS-L-Fc fusion proteins, e.g., variant ICOS-L IgV-Fv fusion proteins. In some embodiments, the pharmaceutical composition is used to treat an inflammatory disorder or an autoimmune disorder or in connection with organ transplantation in a mammal.

[0240] In some situations, the pharmaceutical composition can modulate (e.g., reduce) the immune response to treat a disease. Various means are known to determine whether the administration of the therapeutic composition of the present invention sufficiently modulates the immunological activity by reducing or decreasing the activity of immune cells, by changing the physical or functional properties of immune cells, or by a combination of these effects.Examples of measuring the regulation of immunological activity include: examination of the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); T cell proliferation assays and peps scan analysis based on the uptake of 3H-thymidine after stimulation with antigen-presenting cells loaded with, for example, anti-CD3 antibody, anti-T cell receptor antibody, anti-CD28 antibody, calcium ionophore, PMA (phorbol 12-myristate 13-acetate), peptide or protein antigen; measurement of the functional ability of immune cells to be able to proliferate or divide in response to signals (using, for example, B cell proliferation assays), or to be less able to proliferate or divide; measurement of the ability to kill or lyse other cells (e.g., cytotoxic T cell assays); measurement of cytokines, chemokines, cell surface molecules, antibodies, and other cell products (by, for example, flow cytometry, enzyme-linked immunosorbent assay, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measurement of biochemical markers of activation of signal transduction pathways within immune cells or immune cells (e.g., Western blot and immunoprecipitation analysis of tyrosine, serine, or threonine phosphorylation, polypeptide cleavage, and formation or dissociation of protein complexes; protein array analysis; DNA transcription profiling using DNA arrays or subtractive hybridization); measurement of cell death by apoptosis, necrosis, or other mechanisms (e.g., annexin V staining, TUNEL assay, gel electrophoresis to measure DNA laddering, histology; fluorescent caspase assay, Western blot analysis of caspase substrates); measurement of genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarray, protein microarray, two-dimensional gel electrophoresis, Western blot analysis, enzyme-linked immunosorbent assay, flow cytometry); and measurement of clinical symptoms or outcomes, for example, measurement of clinical symptoms or outcomes by measurement of recurrence rate or disease severity (using clinical scores known to those of skill in the art), including, but not limited to, such measurements.

[0241] Among the methods and uses provided are therapeutic methods and uses, such as therapeutic methods and uses involving the administration of said molecule or a composition containing said molecule to a subject having a disease, condition, or disorder in need of treatment. In some embodiments, the methods provided also include prophylactic treatments involving the administration of said molecule or a composition containing said molecule to a subject prior to the onset of a disease, condition, or disorder, or to a subject at high risk of developing a disease, condition, or disorder. The pharmaceutical compositions described herein (including pharmaceutical compositions containing a variant ICOS L IgSF domain fusion protein) can be used, for example, in a variety of therapeutic or prophylactic treatment applications, including the treatment of autoimmune or inflammatory diseases or disorders, such as the therapeutic or prophylactic treatment applications described. In some embodiments, said molecule is administered in an amount effective to effect the treatment of a disease or disorder. Uses include the use of a variant ICOS L polypeptide, such as a molecule containing an immunomodulatory fusion protein, in such methods and treatments and in the preparation of a medicament for carrying out such therapeutic methods. In some embodiments, the method is carried out by administering to a subject having or suspected of having a disease or condition a variant ICOS L polypeptide, such as an immunomodulatory fusion protein, or a composition containing the same. In some embodiments, thereby, the method treats a disease or condition or disorder in the subject.

[0242] In some embodiments, the methods provided can be applied to the therapeutic administration of variant ICOS-L polypeptides, such as the immunomodulatory fusion proteins described herein. In some embodiments, the pharmaceutical composition suppresses an immune response, which may be useful in the treatment of inflammatory or autoimmune disorders or organ transplantation. In some embodiments, the pharmaceutical composition contains a variant ICOS-L polypeptide in a format that exhibits antagonist activity against its cognate binding partner CD28 or ICOS and / or blocks or inhibits costimulatory signaling through CD28 or ICOS. Exemplary formats of ICOS-L polypeptides for use related to such therapeutic applications include, for example, soluble variant ICOS-L polypeptides (such as variant ICOS-L-Fc fusion proteins).

[0243] The provided methods and uses include the treatment of diseases or conditions involving the CD28 and / or ICOS pathways. In some aspects, the provided methods and uses include the treatment of diseases or conditions involving the CD28 and / or ICOS pathways. CD28 and ICOS supply co-stimulatory signals necessary for optimal T cell activation when bound to their respective ligands CD80 (B7-1) and CD86 (B7-2) and ICOS ligand. CD28 is involved in the initiation of the pathogenesis process in certain inflammatory diseases, such as graft-versus-host disease (GVHD). In some aspects, targeting CD28 by use of a CD28 pathway inhibitor may provide therapeutic utility against such diseases and conditions, including prevention and / or treatment. However, in some cases, CD28 pathway inhibition alone may not be sufficient to manage established disease in many patients. In this regard, CD28 is often downregulated after initial activation, whereas ICOS, its most closely related family member, is upregulated and recognized to provide additional T cell co-stimulation that can sustain diseases, such as GVHD, including gastrointestinal symptoms (Adom et al. Blood 2018; 132-355). The provided aspects are based on the superior utility of variant ICOS ligand molecules, such as variant ICOS ligand vIgD-Fc fusion proteins, that exhibit combined blockade of CD28 and ICOS as compared to agents that exhibit separate blockade of the CD28 or ICOS pathways alone. As described herein, variant ICOS ligand molecules, such as variant ICOS ligand vIgD-Fc fusion proteins, are provided that exhibit high affinity binding to CD28 and ICOS, potent T cell activation, and suppression of disease activity in various inflammatory animal models, such as the human xenogeneic GVHD model in mice. In such examples, such effects are observed after just a single dose of the variant ICOS ligand molecule.

[0244] A. Disease or Condition In some embodiments, the provided method is a method for treating an inflammatory disorder or an autoimmune disorder, such as any of the inflammatory or autoimmune disorders described herein. In some embodiments, the treatment is administered at the onset of the symptoms of the disorder, or approximately at the onset of the symptoms of the disorder. In some embodiments, the treatment is administered at a time delayed relative to the onset of the disease and / or the onset of the symptoms. The delay in treatment administration may be understood to be relative to a transplanation, such as the transplanation described herein. In some embodiments, the amount of th...

Claims

1. 1. A method for preventing or reducing acute graft-versus-host disease (aGVHD), comprising administering to a subject one or more doses of a variant ICOSL fusion protein during a treatment period, the variant ICOSL fusion protein comprising a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R with respect to the sequence set forth in SEQ ID NO:1, wherein each of the one or more doses of the variant ICOSL fusion protein is administered in an amount of at or about 0.1 mg / kg to at or about 20 mg / kg.

2. The method of claim 1, wherein the subject has previously undergone an allogeneic hematopoietic stem cell transplant (HSCT) and aGVHD occurs in the subject after receiving the allogeneic HSCT.

3. The method of claim 1 or claim 2, wherein the subject has aGVHD of grade II to IV.

4. The method of any one of claims 1 to 3, wherein the subject's aGVHD is resistant or refractory to treatment with an immunosuppressant.

5. 5. The method of claim 4, wherein the immunosuppressant comprises a corticosteroid.

6. 6. The method of claim 4 or claim 5, wherein the immunosuppressant comprises cyclosporine.

7. 1. A method for preventing or reducing inflammation secondary to a viral infection, comprising administering to a subject one or more doses of a variant ICOSL fusion protein during a treatment period, the variant ICOSL fusion protein comprising a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R with respect to the sequence set forth in SEQ ID NO:1, wherein each dose of the one or more doses of the variant ICOSL fusion protein is administered in an amount of at or about 0.1 mg / kg to at or about 20 mg / kg.

8. The method of claim 7, wherein the virus is a coronavirus.

9. 9. The method of claim 8, wherein the coronavirus is SARS-CoV-2 and the infectious disease is COVID-19.

10. The method of any one of claims 7 to 9, wherein the inflammation is associated with cytokine release syndrome (CRS).

11. 11. The method of claim 10, wherein the CRS is severe CRS or grade 3 or higher CRS.

12. The method of any one of claims 7 to 11, wherein the subject has severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, or septic shock associated with or caused by the viral infection at or immediately prior to said administering.

13. 1. A method of treating an autoimmune or inflammatory disease or condition in a subject, comprising administering to a subject having an autoimmune or inflammatory disease or condition one or more doses of a variant ICOSL fusion protein during a treatment period, the variant ICOSL fusion protein comprising a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R with respect to the sequence set forth in SEQ ID NO:1, wherein each dose of the one or more doses of the variant ICOSL fusion protein is administered in an amount of at or about 0.001 mg / kg to at or about 20 mg / kg.

14. 14. The method of any one of claims 1-13, wherein each dose of the one or more doses is administered in an amount of at or about 0.1 mg / kg to at or about 10 mg / kg.

15. 15. The method of claim 14, wherein the autoimmune or inflammatory disease or condition is an acute condition.

16. The method of any one of claims 1 to 15, wherein only a single dose of the variant ICOSL fusion protein is administered to the subject.

17. 15. The method of claim 14, wherein the autoimmune or inflammatory disease or condition is a chronic condition.

18. 18. The method of claim 13, claim 14, or claim 17, wherein the inflammatory or autoimmune disease or condition is systemic lupus erythematosus (SLE).

19. 18. The method of claim 13, claim 14, or claim 17, wherein the inflammatory or autoimmune disease or condition is Sjogren's syndrome.

20. 18. The method of claim 13, claim 14, or claim 17, wherein the inflammatory or autoimmune disease or condition is psoriatic arthritis.

21. 18. The method of claim 13, claim 14, or claim 17, wherein the inflammatory or autoimmune disease or condition is rheumatoid arthritis.

22. 18. The method of claim 13, claim 14, or claim 17, wherein the inflammatory or autoimmune disease or condition is Crohn's disease.

23. 18. The method of claim 13, claim 14, or claim 17, wherein the inflammatory or autoimmune disease or condition is ulcerative colitis.

24. The method of any one of claims 1 to 15 and 17 to 23, wherein multiple doses of the variant ICOSL fusion protein are administered to the subject.

25. 25. The method of claim 24, wherein each of the multiple doses is administered no more than once a week.

26. 26. The method of claim 24 or claim 25, wherein each of said multiple doses is administered once a week (Q1W).

27. 26. The method of claim 24 or claim 25, wherein each of said multiple doses is administered once every two weeks (Q2W).

28. 26. The method of claim 24 or claim 25, wherein each of said multiple doses is administered once a month (Q4W).

29. Each dose of the one or more doses is from 0.3 mg / kg or about 0.3 mg / kg to 10 mg / kg or about 10 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, 1 mg / kg or about 1 mg / kg to 10 mg / kg or about 10 mg / kg 29. The method of any one of claims 1-28, wherein the patient is administered in an amount of 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, 3 mg / kg or about 3 mg / kg to 10 mg / kg or about 10 mg / kg, 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg, or 6 mg / kg or about 6 mg / kg to 10 mg / kg or about 10 mg / kg.

30. 30. The method of any one of claims 1-29, wherein each dose of the one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, or 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg.

31. 31. The method of any one of claims 1-30, wherein each dose of the one or more doses is administered in an amount of at or about 0.3 mg / kg.

32. 31. The method of any one of claims 1-30, wherein each dose of the one or more doses is administered in an amount of 1 mg / kg or about 1 mg / kg.

33. 31. The method of any one of claims 1-30, wherein each dose of the one or more doses is administered in an amount of 3 mg / kg or about 3 mg / kg.

34. 31. The method of any one of claims 1-30, wherein each dose of the one or more doses is administered in an amount of 6 mg / kg or about 6 mg / kg.

35. 31. The method of any one of claims 1-30, wherein each dose of the one or more doses is administered in an amount of at or about 10 mg / kg.

36. 29. The method of any one of claims 1-28, wherein each dose of the one or more doses is administered in an amount of at or about 15 mg / kg.

37. 29. The method of any one of claims 1-28, wherein each dose of the one or more doses is administered in an amount of at or about 20 mg / kg.

38. The method of any one of claims 1 to 37, wherein the treatment period is repeated.

39. The method of any one of claims 1 to 38, wherein said administering is by subcutaneous administration.

40. The method of any one of claims 1 to 38, wherein said administering is by intravenous administration.

41. A method for treating an ocular autoimmune or inflammatory disease in a subject, comprising the step of intravitreally administering a dose of a variant ICOSL fusion protein, the variant ICOSL fusion protein comprising a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R, based on the sequence set forth in SEQ ID NO:

1.

42. 42. The method of claim 41, wherein the ocular autoimmune or inflammatory disease is uveitis.

43. The variant ICOSL fusion protein is from about 0.01 mg to about 10 mg, 0.05 mg or about 0.05 mg to 10 mg or about 10 mg, 0.1 mg or about 0.1 mg to 10 mg or about 10 mg, 0.5 mg or about 0.5 mg to 10 mg or about 10 mg, 1 mg or about 1 mg to 10 mg or about 10 mg, 1.5 mg or about 1.5 mg to 10 mg or about 10 mg, 2 mg or about 2 mg to 10 mg or about 10 mg, 3 mg or 43. The method of claim 41 or claim 42, wherein the compound is administered in a dose of about 3 mg to 10 mg or about 10 mg, 4 mg or about 4 mg to 10 mg or about 10 mg, 5 mg or about 5 mg to 10 mg or about 10 mg, 6 mg or about 6 mg to 10 mg or about 10 mg, 7 mg or about 7 mg to 10 mg or about 10 mg, 8 mg or about 8 mg to 10 mg or about 10 mg, 9 mg or about 9 mg to 10 mg or about 10 mg, inclusive.

44. 44. The method of any one of claims 41 to 43, wherein the variant ICOSL fusion protein is administered in a volume of less than 0.2 mL, optionally less than 0.1 mL.

45. The method of any one of claims 41 to 44, wherein the variant ICOSL fusion protein is administered in a volume of 0.05 mL or about 0.05 mL.

46. 46. ​​The method of any one of claims 1 to 45, wherein the ICOSL reference polypeptide comprises: (i) the amino acid sequence shown in SEQ ID NO:32; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:32; or (iii) a portion of (i) and / or (ii) comprising an IgV domain or an IgC domain or a specific binding fragment thereof.

47. The method of any one of claims 1 to 46, wherein the variant ICOSL polypeptide comprises the IgV domain or a specific binding fragment thereof.

48. The method of any one of claims 1 to 47, wherein the IgV domain or a specific binding fragment thereof is the only ICOSL portion of the variant ICOSL polypeptide or variant ICOSL fusion protein.

49. The method of any one of claims 1 to 48, wherein the ICOSL reference polypeptide comprises the amino acid sequence shown in SEQ ID NO:

3.

50. The method of any one of claims 1 to 48, wherein said ICOSL reference polypeptide consists of the amino acid sequence shown in SEQ ID NO:

3.

51. 51. The method of any one of claims 1 to 50, wherein the variant ICOSL polypeptide has a sequence as set forth in SEQ ID NO:36, or a sequence exhibiting at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% sequence identity to the sequence as set forth in SEQ ID NO:36, and comprises one or more amino acid substitutions selected from N52H, N57Y, and Q100R.

52. The method of any one of claims 1 to 51, wherein the variant ICOSL polypeptide has the sequence shown in SEQ ID NO:

36.

53. The method of any one of claims 1 to 52, wherein the multimerization domain is or comprises an Fc region of an immunoglobulin.

54. The method of claim 53, wherein the Fc region is a variant Fc region that exhibits reduced effector function compared to the Fc of a wild-type human immunoglobulin.

55. 55. The method of claim 53 or claim 54, wherein the Fc region is a variant IgG1 Fc region comprising one or more amino acid substitutions compared to wild-type human IgG1.

56. 56. The method of claim 54 or claim 55, wherein the variant Fc region comprises one or more amino acid substitutions selected from N297G, E233P / L234V / L235A / G236del / S267K, or L234A / L235E / G237A, wherein the residues are numbered according to the EU index of Kabat.

57. 57. The method of any one of claims 54-56, wherein said variant Fc region further comprises the amino acid substitution C220S, said residues being numbered according to the EU index of Kabat.

58. The method of any one of claims 53 to 57, wherein the Fc region comprises K447del, said residues being numbered according to the EU index of Kabat.

59. A pharmaceutical composition comprising a variant ICOSL fusion protein for use in a method for preventing or reducing acute graft-versus-host disease (aGVHD) in a subject, wherein the variant ICOSL fusion protein is or comprises a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:

1.

60. Use of a variant ICOSL fusion protein in the preparation of a medicament for use in a method for preventing or reducing acute graft-versus-host disease (aGVHD), wherein the variant ICOSL fusion protein comprises a variant ICOSL polypeptide linked to a multimerization domain, wherein the variant ICOSL polypeptide is an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprises one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R, based on the sequence shown in SEQ ID NO:

1.

61. 61. A pharmaceutical composition for use as described in claim 59 or a use as described in claim 60, wherein the method comprises administering to a subject one or more doses of the variant ICOSL fusion protein during a treatment period, each of the one or more doses of the variant ICOSL fusion protein being administered in an amount of 0.1 mg / kg or about 0.1 mg / kg to 20 mg / kg or about 20 mg / kg.

62. The pharmaceutical composition for use according to claim 59 or claim 61 or the use according to claim 60 or claim 61, wherein the aGvHD is aGVHD of grades II to IV.

63. A pharmaceutical composition for use according to any one of claims 59, 61 and 62 or a use according to any one of claims 60, 61 and 62, wherein the aGVHD is resistant or refractory to treatment with immunosuppressants.

64. A pharmaceutical composition for use according to any one of claims 59 and 61 to 63 or a use according to any one of claims 60 and 61 to 63, wherein said immunosuppressant comprises a corticosteroid.

65. 65. A pharmaceutical composition for use according to claim 63 or claim 64 or a use according to claim 63 or claim 64, wherein the immunosuppressant comprises cyclosporine.

66. A pharmaceutical composition comprising a variant ICOSL fusion protein for use in a method for preventing or reducing inflammation secondary to a viral infection in a subject, the variant ICOSL fusion protein comprising a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:

1.

67. Use of a variant ICOSL fusion protein in the preparation of a medicament for use in a method for preventing or reducing inflammation secondary to a viral infection, wherein the variant ICOSL fusion protein comprises a variant ICOSL polypeptide linked to a multimerization domain, wherein the variant ICOSL polypeptide is an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprises one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R, based on the sequence shown in SEQ ID NO:

1.

68. A pharmaceutical composition for use as described in claim 66 or a use as described in claim 67, wherein the method comprises administering to a subject one or more doses of the variant ICOSL fusion protein during a treatment period, each of the one or more doses of the variant ICOSL fusion protein being administered in an amount of at or about 0.1 mg / kg to at or about 20 mg / kg.

69. 69. A pharmaceutical composition for use according to claim 66 or claim 68 or a use according to claim 67 or claim 68, wherein the virus is a coronavirus.

70. 70. The pharmaceutical composition for use according to claim 69 or the use according to claim 69, wherein the coronavirus is SARS-CoV-2 and the infectious disease is COVID-19.

71. A pharmaceutical composition for use according to any one of claims 66, 68 and 69 or a use according to any one of claims 67 to 69, wherein said inflammation is associated with cytokine release syndrome (CRS).

72. 72. The pharmaceutical composition for use according to claim 71 or the use according to claim 71, wherein the CRS is severe CRS or grade 3 or higher CRS.

73. A pharmaceutical composition for use according to any one of claims 68 and 69 to 72 or a use according to any one of claims 68 and 69 to 72, wherein the subject has severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, or septic shock associated with or caused by the viral infection at or shortly before the administration.

74. A pharmaceutical composition comprising a variant ICOSL fusion protein for use in treating an autoimmune or inflammatory disease or condition in a subject, the variant ICOSL fusion protein comprising a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:

1.

75. Use of a variant ICOSL fusion protein in the preparation of a medicament for use in the treatment of an autoimmune or inflammatory disease or condition, wherein the variant ICOSL fusion protein comprises a variant ICOSL polypeptide linked to a multimerization domain, wherein the variant ICOSL polypeptide is an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprises one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R, based on the sequence shown in SEQ ID NO:

1.

76. 76. A pharmaceutical composition for use as described in claim 74 or a use as described in claim 75, wherein the method comprises administering to a subject one or more doses of the variant ICOSL fusion protein during a treatment period, each of the one or more doses of the variant ICOSL fusion protein being administered in an amount of at or about 0.001 mg / kg to at or about 20 mg / kg.

77. 77. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, and 76, or the use according to any one of claims 61 to 65, 68 to 73, and 76, wherein each dose of said one or more doses is administered in an amount of at or about 0.1 mg / kg to at or about 10 mg / kg.

78. 78. The pharmaceutical composition for use according to claim 77 or the use according to claim 77, wherein the autoimmune or inflammatory disease or condition is an acute condition.

79. A pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73 and 76 to 78 or a use according to any one of claims 61 to 65, 68 to 73 and 76 to 78, wherein only a single dose of said variant ICOSL fusion protein is administered to said subject.

80. 78. The pharmaceutical composition for use according to claim 77 or the use according to claim 77, wherein the autoimmune or inflammatory disease or condition is a chronic disease or condition.

81. A pharmaceutical composition for use according to any one of claims 74, 77 and 80 or a use according to any one of claims 75, 77 and 80, wherein the inflammatory or autoimmune disease or condition is systemic lupus erythematosus (SLE).

82. A pharmaceutical composition for use according to any one of claims 74, 77 and 80 or a use according to any one of claims 75, 77 and 80, wherein the inflammatory or autoimmune disease or condition is Sjogren's syndrome.

83. A pharmaceutical composition for use according to any one of claims 74, 77 and 80 or a use according to any one of claims 75, 77 and 80, wherein the inflammatory or autoimmune disease or condition is psoriatic arthritis.

84. A pharmaceutical composition for use according to any one of claims 74, 77 and 80 or a use according to any one of claims 75, 77 and 80, wherein the inflammatory or autoimmune disease or condition is rheumatoid arthritis.

85. 81. A pharmaceutical composition for use according to any one of claims 74, 77 and 80 or a use according to any one of claims 75, 77 and 80, wherein said inflammatory or autoimmune disease or condition is Crohn's disease.

86. A pharmaceutical composition for use according to any one of claims 74, 77 and 80 or a use according to any one of claims 75, 77 and 80, wherein the inflammatory or autoimmune disease or condition is ulcerative colitis.

87. A pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73 and 76 to 78 or a use according to any one of claims 61 to 65, 68 to 73 and 76 to 78, wherein multiple doses of said variant ICOSL fusion protein are administered to said subject.

88. 88. The pharmaceutical composition for use of claim 87 or the use of claim 87, wherein each of said multiple doses is administered no more than once a week.

89. 89. The pharmaceutical composition for use of claim 87 or claim 88, or the use of claim 87 or claim 88, wherein each of said multiple doses is administered once a week (Q1W).

90. 89. The pharmaceutical composition for use of claim 87 or claim 88, or the use of claim 87 or claim 88, wherein each of said multiple doses is administered once every two weeks (Q2W).

91. 89. The pharmaceutical composition for use of claim 87 or claim 88, or the use of claim 87 or claim 88, wherein each of said multiple doses is administered once a month (Q4W).

92. Each dose of the one or more doses is from 0.3 mg / kg or about 0.3 mg / kg to 10 mg / kg or about 10 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, 1 mg / kg or about 1 mg / kg to 10 mg / kg or about 10 mg / kg, 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg 91. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 91 or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 91, wherein the pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 91 is administered in an amount of from 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, from 3 mg / kg or about 3 mg / kg to 10 mg / kg or about 10 mg / kg, from 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg, or from 6 mg / kg or about 6 mg / kg to 10 mg / kg or about 10 mg / kg.

93. 92. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 92, or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 92, wherein each dose of said one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg to 6 mg / kg or about 6 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 3 mg / kg or about 3 mg / kg, 0.3 mg / kg or about 0.3 mg / kg to 1 mg / kg or about 1 mg / kg, 1 mg / kg or about 1 mg / kg to 6 mg / kg or about 6 mg / kg, 1 mg / kg or about 1 mg / kg to 3 mg / kg or about 3 mg / kg, or 3 mg / kg or about 3 mg / kg to 6 mg / kg or about 6 mg / kg.

94. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93 or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93, wherein each dose of the one or more doses is administered in an amount of 0.3 mg / kg or about 0.3 mg / kg.

95. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93 or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93, wherein each dose of the one or more doses is administered in an amount of 1 mg / kg or about 1 mg / kg.

96. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93 or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93, wherein each dose of the one or more doses is administered in an amount of 3 mg / kg or about 3 mg / kg.

97. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93 or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93, wherein each dose of the one or more doses is administered in an amount of 6 mg / kg or about 6 mg / kg.

98. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93 or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 93, wherein each dose of the one or more doses is administered in an amount of 10 mg / kg or about 10 mg / kg.

99. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 91 or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 91, wherein each dose of the one or more doses is administered in an amount of 15 mg / kg or about 15 mg / kg.

100. The pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 91 or the use according to any one of claims 61 to 65, 68 to 73, 76 to 78, and 87 to 91, wherein each dose of the one or more doses is administered in an amount of 20 mg / kg or about 20 mg / kg.

101. A pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78 and 87 to 100 or a use according to any one of claims 61 to 65, 68 to 73, 76 to 78 and 87 to 100, wherein said treatment period is repeated.

102. A pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78 and 87 to 101 or a use according to any one of claims 61 to 65, 68 to 73, 76 to 78 and 87 to 101, wherein said variant ICOSL fusion protein is administered subcutaneously.

103. A pharmaceutical composition for use according to any one of claims 61 to 65, 68 to 73, 76 to 78 and 87 to 101 or a use according to any one of claims 61 to 65, 68 to 73, 76 to 78 and 87 to 101, wherein said variant ICOSL fusion protein is administered intravenously.

104. A pharmaceutical composition comprising a variant ICOSL fusion protein for use in a method for treating an ocular autoimmune or inflammatory disease in a subject, the variant ICOSL fusion protein comprising a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:

1.

105. Use of a variant ICOSL fusion protein in the preparation of a medicament for use in a method for treating an ocular autoimmune or inflammatory disease in a subject, wherein the variant ICOSL fusion protein comprises a variant ICOSL polypeptide linked to a multimerization domain, the variant ICOSL polypeptide being an ICOSL extracellular domain or a portion thereof comprising an IgV domain or a specific binding fragment thereof, and comprising one or more amino acid substitutions in a reference ICOSL polypeptide corresponding to amino acid substitutions selected from N52H, N57Y, and Q100R based on the sequence shown in SEQ ID NO:

1.

106. 106. The pharmaceutical composition for use according to claim 104 or the use according to claim 105, wherein the ocular autoimmune or inflammatory disease is uveitis.

107. The method comprises administering to a subject one or more doses of the variant ICOSL fusion protein, each of the one or more doses of the variant ICOSL fusion protein being from about 0.01 mg to about 10 mg, 0.05 mg or about 0.05 mg to 10 mg or about 10 mg, 0.1 mg or about 0.1 mg to 10 mg or about 10 mg, 0.5 mg or about 0.5 mg to 10 mg or about 10 mg, 1 mg or about 1 mg to 10 mg or about 10 mg, 1.5 mg or about 1.5 mg to 10 mg or about 10 mg, 2 mg or about 2 mg to 10 mg, 106. A pharmaceutical composition for use according to claim 104 or claim 106 or use according to claim 105 or claim 106 to be administered in a dose of 3 mg or about 3 mg to 10 mg or about 10 mg, 4 mg or about 4 mg to 10 mg or about 10 mg, 5 mg or about 5 mg to 10 mg or about 10 mg, 6 mg or about 6 mg to 10 mg or about 10 mg, 7 mg or about 7 mg to 10 mg or about 10 mg, 8 mg or about 8 mg to 10 mg or about 10 mg, 9 mg or about 9 mg to 10 mg or about 10 mg, inclusive.

108. 108. A pharmaceutical composition for use as described in claim 107 or a use as described in claim 107, wherein the variant ICOSL fusion protein is administered in a volume of less than 0.2 mL, optionally less than 0.1 mL.

109. A pharmaceutical composition for use as claimed in claim 107 or claim 108 or a use as claimed in claim 107 or claim 108, wherein the variant ICOSL fusion protein is administered in a volume of 0.05 mL or about 0.05 mL.

110. A pharmaceutical composition for use according to any one of claims 59, 61 to 65, 66, 68 to 73, 74, 76 to 103, 104 and 106 to 109 or a use according to any one of claims 60, 61 to 65, 67, 68 to 73, 75, 76 to 103, 105 and 106, 107 to 109, wherein said ICOSL reference polypeptide comprises: (i) the amino acid sequence as set forth in SEQ ID NO:32; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:32; or (iii) a portion of (i) and / or (ii) comprising an IgV domain or an IgC domain or a specific binding fragment thereof.

111. A pharmaceutical composition for use according to any one of claims 59, 61 to 65, 66, 68 to 73, 74, 76 to 103, 104 and 106 to 110 or a use according to any one of claims 60, 61 to 65, 67, 68 to 73, 75, 76 to 103 and 105 to 110, wherein said variant ICOSL polypeptide comprises an IgV domain or a specific binding fragment thereof.

112. A pharmaceutical composition for use according to any one of claims 59, 61 to 65, 66, 68 to 73, 74, 76 to 103, 104 and 106 to 111 or a use according to any one of claims 60, 61 to 65, 67, 68 to 73, 75, 76 to 103 and 105 to 111, wherein the IgV domain or a specific binding fragment thereof is the only ICOSL part of the variant ICOSL polypeptide or variant ICOSL fusion protein.

113. A pharmaceutical composition for use according to any one of claims 59, 61-65, 66, 68-73, 74, 76-103, 104 and 106-112 or a use according to any one of claims 60, 61-65, 67, 68-73, 75, 76-103 and 105-112, wherein said ICOSL reference polypeptide comprises the amino acid sequence shown in SEQ ID NO:

3.

114. A pharmaceutical composition for use according to any one of claims 59, 61 to 65, 66, 68 to 73, 74, 76 to 103, 104 and 106 to 113 or a use according to any one of claims 60, 61 to 65, 67, 68 to 73, 75, 76 to 103 and 105 to 113, wherein said ICOSL reference polypeptide consists of the amino acid sequence shown in SEQ ID NO:

3.

115. The variant ICOSL polypeptide has the sequence shown in SEQ ID NO:36, or A pharmaceutical composition for use according to any one of claims 59, 61-65, 66, 68-73, 74, 76-103, 104, and 106-114, or a use according to any one of claims 60, 61-65, 67, 68-73, 75, 76-103, and 105-114, having a sequence exhibiting at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the sequence shown in SEQ ID NO:36, and comprising one or more amino acid substitutions selected from N52H, N57Y, and Q100R.

116. A pharmaceutical composition for use according to any one of claims 59, 61 to 65, 66, 68 to 73, 74, 76 to 103, 104 and 106 to 115 or a use according to any one of claims 60, 61 to 65, 67, 68 to 73, 75, 76 to 103 and 105 to 115, wherein said variant ICOSL polypeptide has the sequence shown in SEQ ID NO:

36.

117. A pharmaceutical composition for use according to any one of claims 59, 61 to 65, 66, 68 to 73, 74, 76 to 103, 104 and 106 to 116 or a use according to any one of claims 60, 61 to 65, 67, 68 to 73, 75, 76 to 103 and 105 to 116, wherein said multimerization domain is or comprises an Fc region of an immunoglobulin.

118. 118. The pharmaceutical composition for use according to claim 117 or the use according to claim 117, wherein the Fc region is a variant Fc region that exhibits reduced effector function compared to the Fc of a wild-type human immunoglobulin.

119. 119. A pharmaceutical composition for use according to claim 117 or claim 118, or a use according to claim 117 or claim 118, wherein the Fc region is a variant IgG1 Fc region comprising one or more amino acid substitutions compared to wild-type human IgG1.

120. A pharmaceutical composition for use as described in claim 118 or claim 119 or a use as described in claim 118 or claim 119, wherein the variant Fc region comprises one or more amino acid substitutions selected from N297G, E233P / L234V / L235A / G236del / S267K, or L234A / L235E / G237A, said residues being numbered according to the EU index of Kabat.

121. 121. The pharmaceutical composition for use according to any one of claims 118 to 120 or the use according to any one of claims 118 to 120, wherein said variant Fc region further comprises the amino acid substitution C220S, said residues being numbered according to the EU index of Kabat.

122. 122. A pharmaceutical composition for use according to any one of claims 117 to 121 or a use according to any one of claims 117 to 121, wherein said Fc region comprises K447del, said residues being numbered according to the EU index of Kabat.

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