CD80 variant immunomodulatory proteins and uses thereof

Abstract

To provide immunomodulatory proteins, including soluble proteins or transmembrane immunomodulatory proteins capable of being expressed on cells.SOLUTION: A variant CD80 polypeptide comprises an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant CD80 polypeptide comprises one or more amino acid modifications in an unmodified CD80 or specific binding fragment thereof, corresponding to specific positions, with reference to numbering of specific amino acid sequences. The present invention also provides immunomodulatory proteins comprising variant CD80 polypeptides, and nucleic acids encoding such proteins. Compositions and methods for making and using such proteins are also provided.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 353,595, filed April 15, 2016, entitled "CD80 Ligand Variant Immunomodulatory Proteins and Uses Thereof," U.S. Provisional Patent Application No. 62 / 394,743, filed September 14, 2016, entitled "CD80 Ligand Variant Immunomodulatory Proteins and Uses Thereof," U.S. Provisional Patent Application No. 62 / 410,844, filed October 20, 2016, entitled "CD80 Ligand Variant Immunomodulatory Proteins and Uses Thereof," U.S. Provisional Patent Application No. 62 / 410,844, filed March 16, 2017, entitled "CD80 Ligand Variant Immunomodulatory Proteins and Uses Thereof," U.S. Provisional Patent Application No. This application claims priority to U.S. Provisional Patent Application No. 62 / 472,570, entitled "CD80 Ligand Variant Immunomodulatory Proteins and Uses Thereof," filed March 22, 2017, and U.S. Provisional Patent Application No. 62 / 475,201, entitled "CD80 Ligand Variant Immunomodulatory Proteins and Uses Thereof," filed March 22, 2017, the contents of each of which are incorporated by reference in their entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format, which is provided as a file entitled 761612000440SeqList.txt, created on April 13, 2017, and is 815,991 bytes in size. The information in the electronic format of the Sequence Listing is 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 immunological diseases. In some aspects, the present disclosure relates to particular variants of CD80 that exhibit altered binding affinity for their cognate binding partners. [Background technology]

[0004] background There is growing medical interest in modulating immune responses by intervening in processes occurring at the immune synapse (IS), the region formed between antigen-presenting cells (APCs) or target cells and lymphocytes. Mechanistically, cell surface proteins within the IS can engage in coordinated, often simultaneous, interactions between multiple protein targets and a single protein to which they bind. Interactions at the IS occur in close association with the junction of two cells, and a single protein within this structure can interact (possibly simultaneously) with proteins on both the same cell (cis) and interacting cells (trans). While therapeutic agents capable of modulating the IS are known, improved therapeutic agents are needed. We provide immunomodulatory proteins, including soluble or transmembrane immunomodulatory proteins that can be expressed on cells, that meet this need. Summary of the Invention

[0005] overview In some embodiments, a variant CD80 polypeptide comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, is provided, comprising SEQ ID Positions based on the numbering of NO:28: 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 27, 28, 29, 30, 31, 33, 36, 37, 38, 40, 41, 42, 43, 44, 47, 48, 50, 52, 53, 54, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 72, 74, 76, 77, 80, 81, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 102, 103, 104, 108, 107, 1 Provided herein are variant CD80 polypeptides containing one or more amino acid modifications in unmodified CD80 or a specific-binding fragment thereof corresponding to 09, 110, 114, 115, 116, 117, 118, 120, 121, 122, 126, 127, 128, 129, 130, 133, 137, 140, 142, 143, 144, 148, 149, 152, 154, 160, 162, 164, 168, 169, 174, 175, 177, 178, 183, 178, 185, 188, 190, 192, 193, or 199. In some embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions. In some embodiments, the unmodified CD80 is a mammalian CD80 or a specific-binding fragment thereof. In some embodiments, the unmodified CD80 is human CD80 or a specific-binding fragment thereof. In some embodiments, the variant CD80 polypeptide comprises an IgV domain, or a specific-binding fragment thereof; and an IgC domain, or a specific-binding fragment thereof. In some embodiments, the unmodified CD80 comprises (i) the amino acid sequence set forth in SEQ ID NO:28, (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:28; or (iii) a portion thereof, wherein the portion comprises the IgV domain or the IgC domain or a specific-binding fragment thereof.

[0006] In some embodiments of any one of the variant CD80 polypeptides, the specific binding fragment of the IgV domain or IgC domain has a length of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; the specific binding fragment of the IgV domain comprises at least 80% of the length of the IgV domain set forth as amino acids 35-135 of SEQ ID NO: 1; or the specific binding fragment of the IgC domain comprises at least 80% of the length of the IgC domain set forth as amino acids 145-230 of SEQ ID NO: 1. In some embodiments, the variant CD80 polypeptide contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations (optionally amino acid substitutions). In some embodiments, the variant CD80 polypeptide comprises an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:28 or a specific-binding fragment thereof. In some embodiments, the variant CD80 has altered binding specificity for the ectodomain of CD28, PD-L1, or CTLA-4 compared to unmodified CD80. In some cases, the altered binding is altered binding affinity and / or altered binding selectivity.

[0007] In some embodiments of any one of the variant CD80 polypeptides, the one or more amino acid substitutions are TIFF2024099752000002.tif106165 or a conservative amino acid substitution thereof.

[0008] In some embodiments, the variant CD80 polypeptide contains an IgV or IgC domain or a specific-binding fragment thereof, which comprises one or more amino acid deletions in unmodified CD80 or a specific-binding fragment thereof. In some embodiments, the deletion corresponds to position 43 of SEQ ID NO:28.

[0009] In some embodiments, the one or more amino acid substitutions are TIFF2024099752000003.tif19151TIFF2024099752000004.tif231166TIFF2024099752000005.tif231166TIFF2024099752000006.tif230166TIFF2024099752000007.tif231166TIFF2024099752000008.tif231164TIFF2024099752000009.tif231166TIFF2024099752000010.tif172166

[0010] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide contains an IgV domain or a specific fragment thereof and / or an IgC domain or a specific fragment thereof. In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide contains an IgV domain or a specific binding fragment thereof. In some embodiments, the IgV domain or a specific binding fragment thereof is the only CD80 portion of the variant CD80 polypeptide. In some embodiments, the IgC domain or a specific binding fragment thereof is the only CD80 portion of the variant CD80 polypeptide.

[0011] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide comprises an amino acid sequence set forth in any of SEQ ID NOs:55-108, 280-346, 414-475, or a specific-binding fragment thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs:55-108, 280-346, 414-475, or a specific-binding fragment thereof, and containing one or more amino acid substitutions. In some embodiments, the variant CD80 polypeptide comprises an IgV domain or a specific-binding fragment thereof. In some embodiments, the IgV domain or a specific fragment thereof is the only CD80 portion of the variant CD80 polypeptide. In some embodiments, the IgC domain or a specific fragment thereof is the only CD80 portion of the variant CD80 polypeptide. In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 153-195, 347, 373-386, 476-477, or a specific binding fragment thereof, an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 153-195, 347, 373-386, 476-477, or a specific binding fragment thereof, and contains one or more amino acid substitutions.

[0012] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of CD28, PD-L1, or CTLA-4 with improved affinity compared to the unmodified CD80 polypeptide. In some of any such embodiments, the variant polypeptide specifically binds to the ectodomain of CD28, PD-L1, or CTLA-4 with improved selectivity compared to the binding of unmodified CD80 to the ectodomain. In some aspects, the improved selectivity comprises a greater ratio of binding to one cognate binding partner selected from among CD28, PD-L1, and CTLA-4 to another of the cognate binding partners compared to the ratio of binding of the unmodified CD80 polypeptide to the one cognate binding partner to the other of the cognate binding partners.

[0013] In some embodiments, the variant polypeptide specifically binds to the CD28 ectodomain with improved selectivity compared to the binding of unmodified CD80 to the CD28 ectodomain. In some aspects, the improved selectivity comprises a greater ratio of binding to CD28 to binding to PD-L1 or CTLA-4 compared to the ratio of binding of the unmodified CD80 polypeptide to CD28 to binding to PD-L1 or CTLA-4. In some cases, the ratio is at least or at least about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more.

[0014] In some embodiments, the variant CD80 polypeptide specifically binds to the ectodomain of CD28 with improved affinity compared to the unmodified CD80 polypeptide, hi some embodiments, the improved affinity for the ectodomain 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, 50-fold, or 60-fold improved compared to the binding affinity of unmodified CD80 for the ectodomain.

[0015] In some embodiments of any one of the variant CD80 polypeptides, the one or more amino acid substitutions correspond to positions 12, 18, 20, 29, 31, 36, 40, 41, 43, 52, 59, 60, 63, 67, 70, 77, 81, 87, 88, 89, 90, 91, 92, 93, 107, 109, 114, 117, 118, 120, 122, 127, 130, 144, 169, 177, or 199 relative to the numbering of SEQ ID NO:28. TIFF2024099752000011.tif34160 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: The file is TIFF2024099752000012.tif106161.

[0016] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of PD-L1 with improved affinity compared to the unmodified CD80 polypeptide. In some cases, the improved affinity for the ectodomain 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, 50-fold, or 60-fold improved compared to the binding affinity of unmodified CD80 for the ectodomain. In some of any such embodiments, the variant polypeptide specifically binds to the ectodomain of PD-L1 with improved selectivity compared to the binding of unmodified CD80 for the ectodomain. In some cases, the improved selectivity comprises a ratio of binding to PD-L1 to binding to CD28 or CTLA-4 that is greater than the ratio of binding of the unmodified CD80 polypeptide to PD-L1 to binding of the unmodified CD80 polypeptide to CD28 or CTLA-4, which in some cases is at least or at least about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more greater.

[0017] In some embodiments, the one or more amino acid substitutions correspond to positions 12, 18, 29, 31, 33, 36, 38, 40, 41, 42, 43, 47, 59, 67, 70, 77, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 109, 118, 120, 122, 144, 148, 149, or 177 relative to the numbering of SEQ ID NO:28. TIFF2024099752000013.tif26157 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: The file is TIFF2024099752000014.tif77164.

[0018] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the CD28 ectodomain and the PD-L1 ectodomain with improved affinity compared to an unmodified CD80 polypeptide. In some embodiments, the one or more amino acid substitutions correspond to positions 12, 18, 36, 40, 43, 59, 77, 88, 89, 90, 91, 92, 93, 109, 118, 122, 177, based on the numbering of SEQ ID NO:28 ... TIFF2024099752000015.tif11158 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: The file is TIFF2024099752000016.tif18137.

[0019] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of CTLA-4 with improved affinity compared to the unmodified CD80 polypeptide. In some embodiments, the improved affinity for the ectodomain 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 or more improved compared to the binding affinity of unmodified CD80 for the ectodomain. In some of any such embodiments, the variant polypeptide specifically binds to the ectodomain of CTLA-4 with improved selectivity compared to the binding of unmodified CD80 to the ectodomain. In some cases, the improved selectivity comprises a greater ratio of binding to CTLA-4 to binding to CD28 or PD-L1 compared to the ratio of binding of the unmodified CD80 polypeptide to CTLA-4 to binding to CD28 or PD-L1, in some embodiments, the ratio is at least or at least about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more greater.

[0020] In some embodiments, the one or more amino acid substitutions correspond to positions 4, 29, 31, 36, 40, 41, 52, 67, 68, 70, 87, 88, 89, 90, 91, 92, 93, 107, 109, 110, 118, 120, 130, 144, or 169 based on the numbering of SEQ ID NO:28. TIFF2024099752000017.tif18170 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: The file is TIFF2024099752000018.tif55162.

[0021] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of CD28 and the ectodomain of CTLA-4 with improved affinity compared to an unmodified CD80 polypeptide. In some embodiments, the one or more amino acid substitutions correspond to positions 36, 40, 52, 70, 88, 89, 90, 91, 92, 93, 107, 118, 120, 130, 144, or 169 of SEQ ID NO:28 ... TIFF2024099752000019.tif11167 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: The file is TIFF2024099752000020.tif26159.

[0022] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of PD-L1 and the ectodomain of CTLA-4 with improved affinity compared to an unmodified CD80 polypeptide. In some embodiments, the one or more amino acid substitutions correspond to positions 29, 31, 36, 40, 41, 67, 70, 87, 88, 89, 90, 91, 92, 93, 109, 118, 120, 122, or 178 of SEQ ID NO:28 ... TIFF2024099752000021.tif19156 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: The file is TIFF2024099752000022.tif19140.

[0023] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the CD28 ectodomain, the PD-L1 ectodomain, and the CTLA-4 ectodomain with improved affinity compared to an unmodified CD80 polypeptide. In some embodiments, the one or more amino acid substitutions correspond to positions 36, 40, 88, 89, 90, 91, 92, or 93 of SEQ ID NO:28. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of K36G, L40M, E88D, K89R, D90K, A91G, F92Y, K93R, and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are The file is TIFF2024099752000023.tif4170.

[0024] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of CD28 or the ectodomain of PD-L1 with improved affinity compared to the unmodified CD80 polypeptide, and the variant CD80 polypeptide specifically binds to the ectodomain of CTLA-4 with decreased affinity compared to the unmodified CD80 polypeptide. In some embodiments, the one or more amino acid substitutions correspond to positions 29, 31, 33, 36, 38, 41, 42, 43, 47, 63, 67, 70, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 109, 114, 118, 120, 127, 130, 144, 148, or 149 of SEQ ID NO:28 ... TIFF2024099752000024.tif19167 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: The file is TIFF2024099752000025.tif11167.

[0025] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of CD28 with improved affinity compared to the unmodified CD80 polypeptide, and the variant CD80 polypeptide specifically binds to the ectodomain of CTLA-4 with reduced affinity compared to the unmodified CD80 polypeptide. In some embodiments, the one or more amino acid substitutions correspond to positions 63, 70, 81, 87, 91, 114, 118, 120, 127, or 130 of SEQ ID NO:28. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of N63S, L70Q, E81A, Y87N, A91G, S114T, I118V, T120S, I127T, and T130A, and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are The file is TIFF2024099752000026.tif77164.

[0026] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of PD-L1 with improved affinity compared to the unmodified CD80 polypeptide, and the variant CD80 polypeptide specifically binds to the ectodomain of CTLA-4 with decreased affinity compared to the unmodified CD80 polypeptide. In some embodiments, the one or more amino acid substitutions correspond to positions 29, 31, 33, 36, 38, 41, 42, 43, 47, 67, 70, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 109, 118, 120, 144, 148, 149 of SEQ ID NO:28 ... TIFF2024099752000027.tif18163 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: The file is TIFF2024099752000028.tif63164.

[0027] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide specifically binds to the ectodomain of CD28 and the ectodomain of PD-L1 with improved affinity compared to the unmodified CD80 polypeptide, and the variant CD80 polypeptide specifically binds to the ectodomain of CTLA-4 with reduced affinity compared to the unmodified CD80 polypeptide. In some embodiments, the one or more amino acid substitutions correspond to positions 70, 81, 87, 91, or 120 of SEQ ID NO:28. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of L70Q, Y87N, A91G, and T120S, and conservative amino acid substitutions thereof.

[0028] In some embodiments of any one of the variant CD80 polypeptides, the CD28 is human CD28. In some embodiments, the PD-L1 is human PD-L1. In some embodiments, the CTLA-4 is human CTLA-4.

[0029] In some embodiments of any one of the variant CD80 polypeptides, the binding activity is altered (improved or decreased) by more 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 relative to the unmodified CD80 polypeptide.

[0030] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide is a soluble protein.

[0031] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide is linked to a multimerization domain. In some embodiments, the variant CD80 polypeptide is a multimeric polypeptide, optionally a dimeric polypeptide, comprising a first variant CD80 polypeptide linked to a multimerization domain and a second variant CD80 polypeptide linked to a multimerization domain. In some embodiments, the first variant CD80 polypeptide and the second variant CD80 polypeptide are the same or different. In some embodiments, the multimerization domain is an Fc domain or a variant thereof having reduced effector function.

[0032] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide is linked to a moiety that increases the biological half-life of the polypeptide. In some embodiments, the variant CD80 polypeptide is linked to an Fc domain or variant thereof that has reduced effector function. In some embodiments, the Fc domain is mammalian (optionally human); or the variant Fc domain contains one or more amino acid modifications compared to an unmodified Fc domain that is mammalian (optionally human). In some embodiments, the Fc domain or variant thereof contains the amino acid sequence set forth in SEQ ID NO:226 or SEQ ID NO:227, or contains an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:226 or SEQ ID NO:227. In some embodiments, the variant CD80 polypeptides are indirectly linked via a linker.

[0033] In some embodiments of any one of the variant CD80 polypeptides that are transmembrane immunomodulatory proteins, the variant CD80 polypeptide further contains a transmembrane domain linked to the extracellular domain (ECD) or a specific-binding fragment thereof of the variant CD80 polypeptide. In some embodiments, the transmembrane domain contains the amino acid sequence set forth as residues 243-263 of SEQ ID NO:1, or a functional variant thereof that exhibits at least 85% sequence identity to residues 243-263 of SEQ ID NO:1. In some embodiments, the variant CD80 polypeptide further contains a cytoplasmic signaling domain linked to the transmembrane domain. In some embodiments, the cytoplasmic signaling domain contains the amino acid sequence set forth as residues 264-288 of SEQ ID NO:1, or a functional variant thereof that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to residues 264-288 of SEQ ID NO:1.

[0034] In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 increases IFN-γ (interferon-γ) expression relative to unmodified CD80 in an in vitro primary T cell assay. In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 decreases IFN-γ (interferon-γ) expression relative to unmodified CD80 in an in vitro primary T cell assay. In some embodiments of any one of the variant CD80 polypeptides, the variant CD80 polypeptide is deglycosylated.

[0035] In some embodiments, provided herein is an immunomodulatory polypeptide comprising a variant CD80 according to any one of the embodiments described herein linked to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain. In some embodiments, the IgSF domain has modified affinity and altered binding to one or more of its cognate binding partners compared to the unmodified or wild-type IgSF domain. In some embodiments, the IgSF domain has improved binding to one or more of its cognate binding partners compared to the unmodified or wild-type IgSF domain. In some embodiments, the variant CD80 is a first variant CD80, and the IgSF domain of the second polypeptide is an IgSF domain derived from a second variant CD80 according to any one of the embodiments described herein, wherein the first and second variant CD80s are the same or different. In some embodiments, the variant CD80 polypeptide is capable of specifically binding to CD28, PD-L1, or CTLA-4, and the IgSF domain of the second polypeptide is capable of binding to a cognate binding partner other than that to which the variant CD80 polypeptide specifically binds. In some embodiments, the variant CD80 polypeptide is capable of specifically binding to CD28 or PD-L1, and the IgSF domain is capable of binding to a cognate binding partner other than that to which the variant CD80 polypeptide specifically binds. In some embodiments, the variant CD80 polypeptide is capable of specifically binding to CD28 or CTLA-4, and the IgSF domain is capable of binding to a cognate binding partner other than that to which the variant CD80 polypeptide specifically binds. In some embodiments, the variant CD80 polypeptide is capable of specifically binding to CTLA-4 or PD-L1, and the IgSF domain is capable of binding to a cognate binding partner other than that to which the variant CD80 polypeptide specifically binds. In some embodiments, the IgSF domain is derived from a member of the B7 family.In some embodiments, the IgSF domain is a tumor-localizing moiety that binds to a ligand expressed on a tumor. In some embodiments, the ligand is B7H6. In some embodiments, the IgSF domain is derived from NKp30. In some embodiments, the IgSF domain has modified affinity and improved binding to one or more of its cognate binding partners compared to an unmodified or wild-type IgSF domain. In some embodiments, the IgSF domain is or contains an IgV domain. In some embodiments, the variant CD80 polypeptide is or contains an IgV domain.

[0036] In some embodiments of any one of the immunomodulatory proteins, the immunomodulatory protein contains a multimerization domain linked to one or both of the variant CD80 polypeptide and the second polypeptide (comprising an IgSF domain). In some embodiments, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the immunomodulatory protein is a dimer. In some embodiments, the immunomodulatory protein is a homodimer. In some cases, the immunomodulatory protein is a heterodimer.

[0037] In some embodiments, provided herein is a conjugate comprising a variant CD80 according to any one of the embodiments described herein or an immunomodulatory polypeptide according to any one of the embodiments described herein linked to a moiety. In some embodiments, the moiety is a targeting moiety that specifically binds to a molecule on the surface of a cell. In some embodiments, the targeting moiety specifically binds to a molecule on the surface of an immune cell. In some embodiments, the immune cell is an antigen-presenting cell or a lymphocyte. In some embodiments, the targeting moiety is a tumor-localizing moiety that binds to a molecule on the surface of a tumor. In some embodiments, the moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle. In some embodiments, the moiety is an antibody or antigen-binding fragment. In some of any such embodiments, the conjugate is bivalent, tetravalent, hexavalent, or octavalent.

[0038] In some embodiments, provided herein is a nucleic acid molecule encoding a variant CD80 according to any one of the embodiments described herein or an immunomodulatory polypeptide according to any one of the embodiments described herein. In some embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some embodiments, the nucleic acid is a cDNA.

[0039] In some embodiments, provided herein is a vector comprising the nucleic acid of any one of the embodiments described herein. In some embodiments, the vector is an expression vector.

[0040] In some embodiments, the vector is a mammalian vector or a viral vector.

[0041] In some embodiments, provided herein is a cell comprising a vector according to any one of the embodiments described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0042] In some embodiments, provided herein is a method of producing a variant CD80 polypeptide or immunomodulatory protein, the method comprising introducing a nucleic acid molecule according to any one of the embodiments described herein or a vector according to any one of the embodiments described herein into a host cell under conditions allowing expression of the protein in the cell. In some embodiments, the method further comprises isolating or purifying the variant CD80 polypeptide or immunomodulatory protein from the cell.

[0043] In some embodiments, provided herein is a method of modifying a cell to express a variant CD80 polypeptide, the method comprising introducing into a host cell a nucleic acid molecule encoding a variant CD80 polypeptide according to any one of the embodiments described herein under conditions such that the polypeptide is expressed in the cell.

[0044] In some embodiments, provided herein is a modified cell expressing a variant CD80 polypeptide according to any one of the embodiments described herein, an immunomodulatory protein according to any one of the embodiments described herein, a nucleic acid molecule according to any one of the embodiments described herein, or a vector according to any one of the embodiments described herein. In some embodiments, the variant CD80 polypeptide or immunomodulatory polypeptide contains a signal peptide. In some aspects, the variant CD80 polypeptide or immunomodulatory polypeptide does not contain a transmembrane domain and / or is not expressed on the surface of the cell. In some embodiments, the variant CD80 polypeptide or immunomodulatory polypeptide is secreted from the modified cell.

[0045] In some embodiments, the engineered cell contains a variant CD80 polypeptide that contains a transmembrane domain and / or is a transmembrane immunomodulatory protein according to any one of the embodiments described herein. In some embodiments, the variant CD80 polypeptide is expressed on the surface of the cell.

[0046] In some embodiments, the modified cells are immune cells. In some embodiments, the immune cells are antigen-presenting cells (APCs) or lymphocytes. In some embodiments, the modified cells are primary cells. In some embodiments, the modified cells are mammalian cells. In some embodiments, the modified cells are human cells. In some embodiments, the lymphocytes are T cells. In some embodiments, the APCs are artificial APCs.

[0047] In some of any such embodiments, the modified cells further contain a chimeric antigen receptor (CAR) or a modified T cell receptor.

[0048] Also provided is an infectious agent comprising a nucleic acid molecule encoding a variant CD80 polypeptide according to any one of the embodiments described herein or an immunomodulatory polypeptide according to any one of the embodiments described herein. In some cases, the encoded variant CD80 polypeptide or immunomodulatory polypeptide does not contain a transmembrane domain and / or is not expressed on the surface of the cell in which it is expressed. In some embodiments, the encoded variant CD80 polypeptide or immunomodulatory polypeptide is secreted from the cell in which it is expressed. In some aspects, the encoded variant CD80 polypeptide contains a transmembrane domain. In some aspects, the encoded variant CD80 polypeptide is expressed on the surface of the cell in which it is expressed.

[0049] In some of any such embodiments, the infectious agent is a bacterium or a virus. In some cases, the virus is an oncolytic virus. In some embodiments, the oncolytic virus is an adenovirus, an adeno-associated virus, a herpes virus, a herpes simplex virus, a vesicular stomatic virus, a reovirus, a Newcastle disease virus, a parvovirus, a measles virus, a vesicular stomatitis virus (VSV), a coxsackievirus, or a vaccinia virus. In some embodiments, the virus specifically targets dendritic cells (DCs) and / or is dendritic cell-tropic. In some cases, the virus is a lentiviral vector pseudotyped with a modified Sindbis virus envelope product.

[0050] In some of any such embodiments, the infectious agent further contains a nucleic acid molecule encoding an additional gene product that can cause death of a target cell or enhance or potentiate an immune response. In some embodiments, the additional gene product is selected from an anti-cancer agent, an anti-metastatic agent, an anti-angiogenic agent, an immunomodulatory molecule, an immune checkpoint inhibitor, an antibody, a cytokine, a growth factor, an antigen, a cytotoxic gene product, a pro-apoptotic gene product, an anti-apoptotic gene product, a cell matrix degrading gene, a gene for tissue regeneration, or a gene for reprogramming human somatic cells to pluripotency.

[0051] In some embodiments, provided herein is a pharmaceutical composition comprising a variant CD80 polypeptide according to any one of the aspects described herein, an immunomodulatory protein according to any one of the aspects described herein, a conjugate according to any one of the aspects described herein, or a modified cell according to any one of the aspects described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterilized.

[0052] In some embodiments, provided herein is an article of manufacture comprising a pharmaceutical composition according to any one of the embodiments described herein in a vial. In some embodiments, the vial is sealed.

[0053] In some embodiments, provided herein is a kit comprising a pharmaceutical composition according to any one of the embodiments described herein and instructions for use. In some embodiments, provided herein is a kit comprising an article of manufacture according to any one of the embodiments described herein and instructions for use.

[0054] In some embodiments, provided herein is a method for modulating an immune response in a subject, the method comprising administering to the subject a pharmaceutical composition according to any one of the embodiments described herein. In some embodiments, the method comprises administering to the subject a modified cell according to any one of the embodiments described herein. In some embodiments, the modified cell is autologous to the subject. In some embodiments, the modified cell is allogeneic to the subject.

[0055] In some embodiments, modulating the immune response treats a disease or condition in a subject. In some embodiments, the immune response is enhanced. In some embodiments, an immunomodulatory protein or conjugate comprising a variant CD80 polypeptide linked to a tumor-localizing moiety is administered to a subject. In some cases, the tumor-localizing moiety is or contains a binding molecule that recognizes a tumor antigen. In some cases, the binding molecule contains an antibody or an antigen-binding fragment thereof, or contains a wild-type IgSF domain or a variant thereof.

[0056] In some embodiments, a pharmaceutical composition comprising an immunomodulatory protein according to any one of the embodiments described herein or a conjugate according to any one of the embodiments described herein is administered to the subject. In some embodiments, modified cells comprising a variant CD80 polypeptide that is a transmembrane immunomodulatory protein are administered to the subject, and / or modified cells according to any one of the embodiments described herein are administered to the subject.

[0057] In some embodiments, an infectious agent encoding a variant CD80 polypeptide that is a transmembrane immunomodulatory protein is administered to a subject, optionally under conditions where the infectious agent infects tumor cells or immune cells and the transmembrane immunomodulatory protein is expressed on the surface of the infected cells. In some aspects, the transmembrane immunomodulatory protein is a transmembrane immunomodulatory protein according to any one of the embodiments described herein.

[0058] In some embodiments, the disease or condition is a tumor or cancer, hi some embodiments, the disease or condition is selected from melanoma, lung cancer, bladder cancer, hematological malignancies, liver cancer, brain cancer, kidney cancer, breast cancer, pancreatic cancer, colon cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric cancer, musculoskeletal cancer, head and neck cancer, gastrointestinal cancer, germ cell cancer, or endocrine and neuroendocrine cancer.

[0059] In some embodiments, the immune response is reduced by the provided methods of modulating an immune response.

[0060] In some embodiments, a soluble variant CD80 polypeptide or immunomodulatory protein is administered to the subject. In some cases, the soluble polypeptide or immunomodulatory protein is an Fc-fusion protein. In some of any such embodiments, a pharmaceutical composition comprising a variant CD80 polypeptide according to any one of the embodiments described herein or an immunomodulatory protein according to any one of the embodiments described herein is administered to the subject. In some embodiments, modified cells comprising a secretable variant CD80 polypeptide are administered to the subject. In some embodiments, modified cells according to any one of the embodiments described herein are administered to the subject.

[0061] In some embodiments, an infectious agent encoding a variant CD80 polypeptide that is a secreted immunomodulatory protein is administered to a subject, optionally under conditions where the infectious agent infects tumor cells or immune cells and the secreted immunomodulatory protein is secreted from the infected cells.

[0062] In some of any such embodiments, the disease or condition is an inflammatory or autoimmune disease or condition. In some embodiments, the disease or condition is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatic disease, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory nervous system disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune hematological disease. In some embodiments, the disease or condition is selected from inflammatory bowel disease, transplantation, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis. [Brief explanation of the drawings]

[0063] [Figure 1A]1A-1C show various formats of the provided variant IgSF domain molecules. Figure 1A shows a soluble molecule comprising: (1) a variant IgSF domain (vIgD), e.g., variant CD80, fused to an Fc chain; (2) a stacked molecule containing a first variant IgSF domain (first vIgD), e.g., variant CD80, and a second IgSF domain, e.g., second variant IgSF domain (second vIgD); (3) a tumor-targeted IgSF molecule containing a first variant IgSF domain (vIgD), e.g., variant CD80, and an IgSF domain that targets a tumor antigen, e.g., NkP30 IgSF domain; and (4) a variant IgSF domain (vIgD), e.g., variant CD80, linked to an antibody (V-Mab). [Figure 1B] 1B shows a transmembrane immunomodulatory protein (TIP) containing a variant IgSF domain (vIgD) expressed on the surface of a cell. In an exemplary embodiment, the cognate binding partner of the transmembrane-bound vIgD is a costimulatory receptor, and the TIP containing vIgD (e.g., CD80 vIgD) engages the costimulatory receptor such that the TIP induces a positive signal in cells expressing the costimulatory receptor. [Figure 1C] 1C shows a secreted immunomodulatory protein (SIP) in which a variant IgSF domain (vIgD) is secreted from a cell, e.g., a first T cell (e.g., a CAR T cell). In an exemplary embodiment, the cognate binding partner of the secreted vIgD is an activating receptor, e.g., CD28, which can be expressed on the first cell (e.g., a T cell) and / or a second cell (e.g., a T cell; either endogenous or modified, e.g., a CAR T cell). Binding of the SIP to its cognate binding partner blocks signaling through the activating receptor. In all cases, the vIgD can be simply the V-domain (IgV), a combination of the V-domain (IgV) and the C-domain (IgC) including the entire extracellular domain (ECD), or any combination of Ig domains of an IgSF superfamily member. [Figure 2]

[0023] Figure 1 shows an exemplary schematic of the activity of a variant IgSF domain (vIgD) fused to Fc (vIgD-Fc), where vIgD is a variant of the IgSF domain of CD80. As shown, soluble vIgD of CD80 interacts with its cognate binding partner and blocks the interaction of CD28, thereby blocking costimulation by the CD28 costimulatory receptor. [Figure 3] 1 shows an exemplary schematic diagram of a stack molecule for localizing a variant IgSF domain (vIgD) to tumor cells. In this format, the stack molecule contains a first variant IgSF domain (first vIgD) and a second IgSF domain (e.g., second vIgD), where the second IgSF domain (e.g., second vIgD) is a tumor-targeting IgSF domain that binds to a tumor antigen. An exemplary tumor-targeting IgSF domain is the IgSF domain of NkP30, which binds to the tumor antigen B7-H6. In this depiction, vIgD is a variant of the IgSF domain of CD80. As shown, binding of the tumor-targeting IgSF domain to the surface of tumor cells localizes the first vIgD on the tumor cell surface, where it can interact with one or more of its cognate binding partners (e.g., CD28) expressed on the surface of adjacent immune cells (e.g., T cells) to stimulate costimulatory receptors. [Figure 4]

[0023] Figures 1A-1C show various exemplary configurations of stacked molecules containing a first variant IgSF domain (first vIgD) and a second IgSF domain, e.g., a second variant IgSF domain (second vIgD). As shown, the first vIgD and second IgSF domain are independently linked, directly or indirectly, to the N- or C-terminus of an Fc subunit. To generate homodimeric Fc molecules, the Fc subunits are matched and capable of forming homodimers upon coexpression of the individual Fc subunits in a cell. To generate heterodimeric Fc molecules, the individual Fc subunits contain mutations (e.g., "knob-into-hole" mutations in the CH3 domain) that favor heterodimer formation over homodimer formation when the individual Fc subunits are coexpressed in a cell. [Figure 5] 1 shows an exemplary schematic diagram of the activity of a variant IgSF domain (vIgD) conjugated to an antibody (V-Mab), where the antibody (e.g., an anti-HER2 antibody) binds to an antigen on the surface of a tumor cell. In this depiction, vIgD is a variant of the IgSF domain of CD80. As shown, binding of the antibody to the surface of the tumor cell localizes vIgD on the tumor cell surface, where it can interact with one or more of its cognate binding partners expressed on the surface of adjacent immune cells (e.g., T cells) to activate receptor signaling. In the exemplary embodiment shown, the variant IgSF domain (vIgD) is a variant of the IgSF domain of CD80. Binding of CD80 vIgD to the CD28 costimulatory receptor provides an agonistic or costimulatory signal. [Figure 6A] Figures 6A-6C show various exemplary configurations of variant IgSF domains (vIgD) conjugated to antibodies (V-Mabs). Figure 6A shows various configurations in which vIgD is linked directly or indirectly to the N-terminus and / or C-terminus of the antibody light chain. [Figure 6B]FIG. 6B shows various configurations in which vIgD is linked directly or indirectly to the N-terminus and / or C-terminus of the antibody heavy chain. [Figure 6C] FIG. 6C shows the resulting V-Mab configuration when the light chain of FIG. 6A and the heavy chain of FIG. 6B are co-expressed in cells. [Figure 7A] Figure 1 shows the results of a competitive binding assay for the binding of biotinylated recombinant CD28 Fc fusion protein (rCD28.Fc) to immobilized variant CD80 A91G ECD-Fc fusion molecules in the presence of unlabeled recombinant human PD-L1-his, human CTLA-4-his, or human-PD-L2-Fc fusion proteins. [Figure 7B] Figure 1 shows the results of a competitive binding assay for the binding of biotinylated recombinant human PD-L1-his monomer protein to immobilized variant CD80 A91G ECD-Fc fusion molecules in the presence of unlabeled recombinant human rCD28.Fc, human CTLA-4.Fc, or human PD-L2.Fc. [Figure 8] 1 shows impedance results reflecting the cytotoxic killing activity of cells engineered with anti-CD19 chimeric antigen receptor (CAR) alone or with exemplary transmembrane immunomodulatory TIP (CD80-TIP or ICOSL-TIP) or the corresponding CD80 or ICOSL wild-type transmembrane proteins, followed by co-culture with target antigen-expressing cells. Impedance was assessed using an Acea Real-Time Cell Analyzer (RTCA), which measures impedance fluctuations in the culture medium of a 96-well microelectronic plate (E-plate). DETAILED DESCRIPTION OF THE INVENTION

[0064] Detailed Description Provided herein are immunomodulatory proteins that are or comprise CD80 variants or mutants of CD80 and specific-binding fragments thereof, which have altered binding activity or affinity for at least one target ligand cognate binding partner (also referred to as a counter-structure ligand protein). In some embodiments, the variant CD80 polypeptide contains one or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions) compared to the unmodified or wild-type CD80 polypeptide. In some embodiments, the variant CD80 polypeptide contains one or more amino acid modifications (e.g., substitutions) compared to the unmodified or wild-type CD80 polypeptide. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgSF domain (e.g., IgV) of the unmodified or wild-type CD80 polypeptide. In some embodiments, the altered binding activity or affinity (e.g., improved or decreased binding activity or affinity) is for at least one of the cognate binding partner proteins CD28, PD-L1, or CTLA-4. In some embodiments, the variant CD80 polypeptide has altered (e.g., improved or decreased) binding activity or affinity for one or more of CD28, PD-L1, or CTLA-4 compared to unmodified or wild-type CD80 that does not contain the one or more modifications. In some embodiments, the immunomodulatory protein is soluble. In some embodiments, the immunomodulatory protein is a transmembrane immunomodulatory protein that can be expressed on the surface of a cell. In some embodiments, also provided herein are one or more other immunomodulatory proteins that are conjugates or fusions containing a variant CD80 polypeptide provided herein and one or more other moieties or polypeptides.

[0065] In some embodiments, the variant CD80 polypeptide has improved binding affinity to one or more of CD28, PD-L1, or CTLA-4 compared to unmodified or wild-type CD80 that does not contain one or more modifications. In some embodiments, the variant CD80 polypeptide has decreased binding affinity to one or more of CD28, PD-L1, or CTLA-4 compared to unmodified or wild-type CD80 that does not contain one or more modifications. In some embodiments, the variant CD80 polypeptide has improved binding affinity to one or more of CD28, PD-L1, or CTLA-4 and decreased binding affinity to another one or more of CD28, PD-L1, or CTLA-4 compared to unmodified or wild-type CD80 that does not contain one or more modifications.

[0066] In some embodiments, also provided herein is one or more other immunomodulatory proteins that are conjugates or fusions containing a variant CD80 polypeptide provided herein and one or more other moieties or polypeptides. In some embodiments, the variant CD80 polypeptides and immunomodulatory proteins modulate the immunological immune response, e.g., enhance or reduce the immune response. In some embodiments, the variant CD80 polypeptides and immunomodulatory proteins provided herein can be used to treat diseases or conditions associated with a dysregulated immune response.

[0067] In some embodiments, the provided variant CD80 polypeptides regulate T cell activation through interaction with costimulatory signaling molecules. In general, antigen-specific T cell activation generally requires two distinct signals. The first signal is provided by the interaction of the T cell receptor (TCR) with major histocompatibility complex (MHC)-associated antigens present on antigen-presenting cells (APCs). The second signal is costimulatory for TCR engagement and is required to avoid T cell apoptosis or anergy.

[0068] In some embodiments, under normal physiological conditions, T cell-mediated immune response is initiated by antigen recognition by T cell receptor (TCR), and is regulated by the balance of costimulatory signal and inhibitory signal (e.g., immune checkpoint protein).The immune system relies on immune checkpoint to prevent autoimmunity (i.e., self-tolerance) and protect tissue from excessive damage during immune response (e.g., during attack against pathogenic infection).However, in some cases, these immune-regulating proteins may be dysregulated in diseases and pathologies, including tumors, as a mechanism to evade the immune system.

[0069] In some embodiments, CD28 is a known T cell costimulatory receptor for the ligands B7-1 (CD80) and B7-2 (CD86), both 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 CD28; binding to CTLA-4 acts to downregulate immune responses. Similarly, CD80 can bind to programmed cell death ligand 1 (PD-L1). PD-L1 is also a negative regulator of immune activation and can downregulate immune responses through interaction with the programmed cell death 1 (PD-1) receptor. Binding of CD80 to PD-L1 can block the interaction between PD-L1 and PD-1, thereby strengthening or enhancing the immune response. Thus, in some cases, the interaction of CD80 with CD28 and PD-L1 results in overlapping and complementary effects.

[0070] In some embodiments, CD28 and PD-L1 may play complementary roles in regulating immune responses. Enhancing or inhibiting the activity of these receptors has clinical significance for the treatment of inflammatory and autoimmune disorders, cancer, and viral infections. However, in some cases, therapies that intervene in and alter the costimulatory effects of both receptors are constrained by the spatial location requirements and size limitations imposed by the localization of the immune synapse. In some aspects, existing therapeutic agents, including antibody drugs, cannot simultaneously interact with multiple target proteins involved in regulating these interactions. In addition, in some cases, existing therapeutic agents only have the ability to antagonize immune responses, but not to stimulate them. Additionally, the pharmacokinetic differences between drugs that independently target one or the other of these two receptors can make it difficult to adequately maintain the desired blood concentration of such drug combinations throughout the treatment process.

[0071] In some embodiments, the provided variant CD28 polypeptides or immunomodulatory proteins modulate (e.g., enhance or reduce) immune activity induced by or associated with the costimulatory receptors CD28 or PD-L1, and in some cases, CTLA-4. Thus, in some embodiments, the provided polypeptides overcome these limitations by providing variant CD80s that have independent binding affinities for both CD28 and PD-L1, and in some cases, CTLA-4, thereby stimulating or antagonizing the complementary effects of costimulation by the receptors. Also provided are methods of making and using these CD80 variants.

[0072] All publications (including patents, patent applications, scientific articles, and databases) mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication (including patents, patent applications, scientific articles, and databases) was specifically and individually indicated to be incorporated by reference. To the extent that a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0073] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0074] I. definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or related terms used herein 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 having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as making a significant difference from what is commonly understood in the art.

[0075] Terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. As 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 herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, abbreviations and symbols for chemical and biochemical names are in accordance with IUPAC-IUB nomenclature. Unless otherwise specified, all numerical ranges include not only the values ​​defining the range but also all integer values ​​therebetween.

[0076] The term "affinity-modified (affinity-engineered)," when used in the context of immunoglobulin superfamily domains, refers to a mammalian immunoglobulin superfamily (IgSF) domain having an amino acid sequence that has been altered to improve or decrease (relative to a corresponding wild-type parent or unmodified IgSF domain) the binding affinity or avidity for at least one of its cognate binding partners (or "counterstructures") relative to a parent wild-type or unmodified (i.e., non-affinity-modified) IgSF control domain. Included in this context are affinity-modified CD80 IgSF domains. In some embodiments, an 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) in a wild-type or unmodified IgSF domain. The improvement or decrease in binding affinity or avidity can be determined using well-known binding assays, such as flow cytometry. Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005). See also Linsley et al., Immunity, 1: 7930801 (1994). An improvement in the binding affinity or avidity of a protein for its cognate binding partner is an improvement that is at least 10% greater than the wild-type IgSF domain control, and in some embodiments, is at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type IgSF domain control value. The reduction in binding affinity or avidity of the protein for at least one of its cognate binding partners is a reduction of 90% or less of the control but 10% or more of the wild-type IgSF domain control value, and in some embodiments, a reduction of 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the wild-type IgSF domain control value but 10% or more.Affinity-modified proteins have altered primary amino acid sequences due to substitution, addition, or deletion of amino acid residues. The term "affinity-modified IgSF domain" should not be construed as imposing any requirement on any particular starting composition or method by which the affinity-modified IgSF domain is generated. Thus, the affinity-modified IgSF domains of the present invention are not limited to converting a wild-type IgSF domain into an affinity-modified IgSF domain by any particular affinity modification 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 binding to their cognate binding partners, and finally recombinantly or chemically synthesized to generate the subject affinity-modified IgSF domain compositions. As another example only, affinity-modified IgSF domains can be generated by site-directed mutagenesis of wild-type IgSF domains. Thus, affinity-modified IgSF domains represent products, but not necessarily, produced by any given process. A variety of techniques, including recombinant methods, chemical synthesis, or a combination thereof, may be used.

[0077] The term "allogeneic," as used herein, refers to cells or tissues that are removed from one organism and then injected or adoptively transferred into a genetically different organism of the same species. In some embodiments of the invention, the species is murine or human.

[0078] The term "autologous" as used herein refers to cells or tissues that are extracted from the same organism and then injected or adoptively transferred into the organism. Autologous cells or tissues can be modified, for example, by recombinant DNA techniques, so that they are no longer genetically identical to the natural cells or natural tissues extracted 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 involves modifying T cells or TILs (tumor-infiltrating lymphocytes). The modified cells are then infused into the patient from which the natural T cells were isolated. In some embodiments, the organism is a human or a mouse.

[0079] The terms "binding affinity" and "binding avidity," as used herein, refer to the specific binding affinity and specific binding avidity of a protein to its counterstructure under specific binding conditions, respectively. In biochemical kinetics, avidity refers to the cumulative strength of the affinities of multiple individual noncovalent interactions, such as between CD80 and its counterstructures PD-L1, CD28, and / or CTLA-4. Thus, avidity differs from affinity, which represents the strength of a single interaction. The improved or decreased binding affinity of an affinity-modified CD80 variant containing an IgSF domain to its counterstructure is determined relative to the binding affinity of unmodified CD80 (e.g., unmodified CD80 containing a native or wild-type IgSF domain (e.g., an 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 CD80 (e.g., a CD80 containing an affinity-modified IgSF domain) specifically binds to CD28, PD-L1 and / or CTLA-4 with a binding affinity that results in a mean fluorescence intensity (MFI) value that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than an unmodified CD80 control, as measured by flow cytometry, e.g., in a binding assay described in Example 6.

[0080] The term "biological half-life" refers to the time required for a substance (e.g., an immunomodulatory polypeptide, including a variant CD80 polypeptide of the invention) to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by the substance's elimination, excretion, degradation (e.g., enzymatic degradation), or absorption and concentration in specific organs or tissues in the body. In some embodiments, biological half-life can be assessed by determining the time required for the substance's plasma concentration to reach half of its steady-state level ("plasma half-life"). Conjugates that can be used to derivatize the polypeptides of the 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 peptides; see WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylated).

[0081] The term "chimeric antigen receptor" or "CAR," as used herein, refers to an artificial (i.e., man-made) transmembrane protein expressed on mammalian cells, comprising at least an ectodomain, a transmembrane domain, and an endodomain. Optionally, the CAR protein includes a "spacer" covalently linking the ectodomain to the transmembrane domain. The spacer is often a polypeptide that links the ectodomain to the transmembrane domain via a peptide bond. CARs are typically expressed on mammalian lymphocytes. In some embodiments, CARs are expressed on mammalian cells such as T cells or tumor-infiltrating lymphocytes (TILs). CARs expressed on T cells are referred to herein as "CAR T cells" or "CAR-T." In some embodiments, CAR-Ts are helper T cells, cytotoxic T cells, natural killer T cells, memory T cells, regulatory T cells, or γδ T cells. When used clinically, for example, in adoptive cell transfer, CAR-Ts with antigen-binding specificity for a patient's tumor are typically engineered to be expressed on natural T cells obtained from the patient. The engineered T cells expressing the CAR are then infused back into the patient. Thus, while CAR-Ts are often autologous, allogeneic CAR-Ts are also within the scope of the present invention. The ectodomain of the CAR comprises an antigen-binding region (e.g., an antibody or its antigen-binding fragment (e.g., scFv)) that specifically binds to a target antigen (e.g., a tumor-specific antigen) under physiological conditions. Upon specific binding, a series of biochemical events (i.e., signal transduction) results in the modulation of the immune activity of the CAR-T. Thus, for example, specific binding of the antigen-binding region of the CAR-T to its target antigen can lead to changes in the immune activity of T cell activity, as reflected by changes in cytotoxicity, proliferation, or cytokine production. In some embodiments, signal transduction upon CAR-T activation is achieved by the CD3 zeta chain ("CD3-z"), which is involved in signal transduction in natural mammalian T cells. CAR-Ts can further comprise multiple signaling domains (e.g., CD28, 41BB, or OX40) that further regulate the immune regulatory response of T cells.CD3-z contains a conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM) that is involved in T cell receptor signaling.

[0082] The terms "collectively" or "total," when used in reference to cytokine production induced by the presence of two or more variant CD80 polypeptides in an in vitro assay, refer to the overall cytokine expression level regardless of cytokine production induced by individual variant CD80 polypeptides. In some embodiments, the cytokine assayed is IFN-γ, e.g., in the in vitro primary T cell assay described in Example 7.

[0083] The term "cognate binding partner" (used interchangeably with "counterstructure"), with respect to a polypeptide (e.g., an IgSF domain of a variant CD80), refers to at least one molecule (typically a native mammalian protein) to which the referenced polypeptide specifically binds under specific binding conditions. In some aspects, a variant CD80 containing an affinity-modified IgSF domain specifically binds to the counterstructure of the corresponding native or wild-type CD80 with improved or reduced affinity. A species of ligand that is recognized and specifically binds to its cognate receptor under specific binding conditions is an example of a counterstructure or cognate binding partner for that receptor. A "cell surface cognate binding partner" is a cognate binding partner expressed on the surface of a mammalian cell. A "cell surface molecular species" is a cognate binding partner of a ligand of the immune synapse (IS) that is expressed on or by cells (e.g., mammalian cells) that form the IS.

[0084] As used herein, "conjugate," "conjugation," or grammatical variations thereof, refers to the joining or linking of two or more compounds together by any connection or linking method known in the art, resulting in the formation of another compound. It can also refer to a compound produced by joining or linking two or more compounds together. For example, a variant CD80 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.

[0085] 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 simultaneously bind to two cognate binding partners. Generally, competitive binders contain the same or overlapping binding sites for specific binding, although this is not a requirement. In some embodiments, competitive binding causes 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 are known, such as ELISA (enzyme-linked immunosorbent assay).

[0086] The term "conservative amino acid substitution," as used herein, refers to an amino acid substitution in which one amino acid residue is replaced with 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 with 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. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0087] The term "corresponding" with respect to a protein position, e.g., a statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence (e.g., as shown in the sequence listing), refers to a nucleotide or amino acid position identified by alignment with the disclosed sequence based on 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 of wild-type CD80 shown in SEQ ID NO:28 (ECD domain) or the sequence shown in SEQ ID NO:152 or 372 (IgV domain) according to the structural alignment methods described herein. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as criteria.

[0088] The terms "reduce" or "attenuate" or "inhibit," as used herein, refer to a statistically significant decrease. The decrease can be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease.

[0089] The term "derivative" or "derivatized" refers to the modification of a protein by directly or indirectly covalently attaching the protein to a composition to alter properties such as biological half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy, while retaining or enhancing its therapeutic benefit. Derivatives of the immunomodulatory polypeptides of the invention are within the scope of the invention and can be produced, for example, by glycosylation, pegylation, lipidation, or Fc fusion.

[0090] As used herein, a domain (typically a sequence of three or more, generally five or seven or more amino acids, e.g., 10-200 amino acid residues) refers to a portion of a molecule (e.g., a protein or encoding nucleic acid) that is structurally and / or functionally distinct from and identifiable with the rest of the molecule. For example, a domain includes a portion of a polypeptide chain that can form an independently folded structure within a protein composed of one or more structural motifs and / or is recognized by a functional activity, such as binding activity. A protein can have one or more distinct domains. For example, a domain can be identified, defined, or distinguished by primary sequence or structural homology to related family members, e.g., homology to a motif. In another example, a domain can be distinguished by its function (e.g., ability to interact with a biomolecule, such as a cognate binding partner). A domain can exhibit an independent biological function or activity, such that it can perform an activity (e.g., binding) independently or fused to another molecule. A domain can be a linear or nonlinear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. For purposes of illustration herein, definitions are provided, but it is understood that recognizing a particular domain by name is well within the skill of the art. If necessary, appropriate software can be employed to identify the domains.

[0091] The term "ectodomain," as used herein, refers to the region of a membrane protein (e.g., a transmembrane protein) that is outside the vesicle membrane. Ectodomains often include a binding domain that specifically binds to a ligand or cell surface receptor, e.g., via a binding domain that specifically binds to the ligand or cell surface receptor. The ectodomain of a cellular transmembrane protein is alternatively referred to as the extracellular domain.

[0092] The term "effective amount" or "therapeutically effective amount" refers to an amount and / or concentration of a therapeutic composition (including a protein or cell composition) of the present invention that, when administered ex vivo (by contact with cells from a patient) or in vivo (by administration to a patient), either alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, results in a statistically significant reduction in disease progression, e.g., by ameliorating or eliminating the symptoms and / or pathogenesis of the disease. An effective amount can be an amount that alleviates, reduces, or relieves at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of the disease or disorder, or improves the patient's physical function. In the case of cell therapy, an effective amount is an effective dose or number of cells administered to a patient via adoptive cell therapy. In some embodiments, the patient is a mammalian patient, e.g., a non-human primate or a human patient.

[0093] The term "endodomain," as used herein, refers to a region found in some membrane proteins (e.g., 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. Within the cell, the endodomain can interact with intracellular components and play a role in signal transduction, and thus, in some cases, can be an intracellular signaling domain. The endodomain of a cellular transmembrane protein is alternatively referred to as a cytoplasmic domain, which, in some cases, can be a cytoplasmic signaling domain.

[0094] The terms "enhanced" or "increased (improved)," as used herein in the context of enhancing the immune activity of mammalian lymphocytes, refer to enhancing one or more activities of the lymphocytes. Enhanced activity can be an enhancement (e.g., a statistically significant amount) of one or more of cell survival, cell proliferation, cytokine production, or T-cell cytotoxicity. In some embodiments, reference to enhanced immune activity refers to increasing interferon-γ (IFNγ) production (e.g., by a statistically significant amount). In some embodiments, immune activity can be assessed in a mixed lymphocyte reaction (MLR) assay. Methods for performing MLR assays are known in the art. Wang et al., Cancer Immunol Res. 2014 Sep: 2(9):846-56. Other methods for assessing lymphocyte activity are known in the art, including any of the assays described herein. In some embodiments, an enhancement can be an increase (improvement) of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than a non-zero control value.

[0095] The term "modified cell" as used herein refers to a mammalian cell that has been genetically modified (altered) by human intervention (e.g., recombinant DNA or viral transduction). In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g., T cell, B cell, NK cell) or an antigen-presenting cell (e.g., dendritic cell). The cell may be a primary cell derived from a patient or a cell line. In some embodiments, the modified cell of the invention comprises a variant CD80 polypeptide of the invention that has been modified to modulate the immune activity of T cells expressing CD28, PD-L1, and / or CTLA-4, to which the variant CD80 specifically binds, or APCs expressing PD-L1. In some embodiments, the variant CD80 is a transmembrane immunomodulatory protein (hereinafter referred to as "TIP") that contains an extracellular domain or portion thereof that contains an IgV domain linked to a transmembrane domain (e.g., a CD80 transmembrane domain), and optionally contains an intracellular signaling domain. In some cases, TIP is formed as a chimeric receptor that contains heterologous cytoplasmic signaling domain or endodomain. In some embodiments, the modified cell can express and secrete the immunomodulatory protein as described herein. Some of the modified cells provided further contain modified T cell receptor (TCR) or chimeric antigen receptor (CAR).

[0096] The term "engineered T cells," as used herein, refers to T cells (e.g., helper T cells, cytotoxic T cells (or cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or γδ T cells) that have been genetically modified (engineered) by human intervention (e.g., by recombinant DNA or viral transduction). The engineered T cells comprise a variant CD80 transmembrane immunomodulatory protein (TIP) of the invention expressed on the T cell, which has been engineered to modulate the immune activity of the engineered T cell itself or of a mammalian cell to which the variant CD80 expressed on the T cell specifically binds.

[0097] The term "modified T cell receptor" or "modified TCR" refers to a T cell receptor (TCR) that has been modified to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen that is selected, cloned, and / or subsequently introduced into a population of T cells, which are often used in adoptive immunotherapy. In contrast to modified TCRs, CARs are modified to bind target antigens in an MHC-dependent manner.

[0098] The term "expressed on," as used herein, is used in reference 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 expressed protein is a transmembrane protein. In some embodiments, the protein is conjugated to a small molecule moiety (e.g., a drug or a detectable label). A protein expressed on the surface of a cell can include a cell surface protein (e.g., a cell surface receptor) expressed on a mammalian cell.

[0099] The term "half-life extending moiety" refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum compared to the half-life of the protein not so conjugated to the moiety. In some embodiments, the half-life is extended by more than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold, or by more than 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. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety. Half-life refers to the time required for a protein to lose half of its concentration, amount, or activity. Half-life can be determined, for example, by using ELISA assay or activity assay. Exemplary half-life extending moieties include Fc domain, multimerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see WO 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipid (acylation), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylation).

[0100] The term "immune synapse," as used herein, refers to the interface between a mammalian cell (e.g., an antigen-presenting cell or tumor cell) that expresses MHC I (major histocompatibility complex) or MHC II, and a mammalian lymphocyte (e.g., an effector T cell or natural killer (NK) cell).

[0101] The Fc (fragment crystallizable) region or domain of an immunoglobulin molecule (also referred to as an Fc polypeptide) primarily corresponds to the constant region of the immunoglobulin heavy chain and is involved in various functions, including antibody effector functions. The Fc domain contains part or all of the hinge domain and the CH2 and CH3 domains of an immunoglobulin molecule. 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 effector function-promoting activity (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater reduction). In some embodiments, references to amino acid substitutions in the Fc region are in accordance with the EU numbering system, unless otherwise specified with reference to a specific SEQ ID NO. 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 (created May 17, 2001, last updated January 10, 2013) and Kabat, EA et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0102] Immunoglobulin Fc fusions ("Fc fusions"), e.g., immunomodulatory Fc fusion proteins, are molecules comprising one or more polypeptides (or one or more small molecules) operably linked to the Fc region of an immunoglobulin. An Fc fusion can comprise, for example, the Fc region of an antibody (to enhance pharmacokinetics) and a variant CD80 polypeptide. The immunoglobulin Fc region can be indirectly or directly linked to one or more variant CD80 polypeptides 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 the Fc fusion. Fc fusions of the same species can be dimerized to form Fc fusion homodimers, or non-identical species can be used to form Fc fusion heterodimers. In some embodiments, the Fc is a mammalian Fc, e.g., a murine or human Fc.

[0103] The term "host cell" refers to a cell that can be used to express a protein encoded by a recombinant expression vector. Host cells can be prokaryotic, e.g., Escherichia coli (E. coli), or they can be eukaryotic, e.g., unicellular eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or hybridomas. Examples of host cells include Chinese hamster ovary (CHO) cells or their derivatives, e.g., Veggie CHO and related cell lines grown in serum-free medium, or the CHO line DX-B11, which is DHFR-deficient. In some embodiments, host cells can be mammalian cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells).

[0104] The term "immunoglobulin" (abbreviated "Ig"), as used herein, refers to mammalian immunoglobulin proteins, including any of the 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 refers to immunoglobulins that are less than full-length, whether wholly or partially synthetic (e.g., recombinantly or chemically synthesized) or naturally produced, such as antigen-binding fragments (Fab), V, and VI. H and V L variable fragments (Fv) containing V linked together in one chain; H and V L Bispecific antibodies include single-chain variable fragments (scFv) containing the V region fragments (Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc, and Fd polypeptide fragments). Homo- and hetero-bispecific bispecific antibodies are included within the scope of the term.

[0105] The term "immunoglobulin superfamily" or "IgSF" as used herein refers to a group of cell surface and soluble proteins involved in cellular recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on structural features shared with immunoglobulins (i.e., antibodies); they all possess domains known as immunoglobulin domains or folds. IgSF members include cell surface antigen receptors, coreceptors, and costimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors, and intracellular muscle proteins. These are typically associated with roles in the immune system. Proteins in the immune synapse are often members of IgSF. IgSF can also be divided into "subfamilies" based on shared characteristics, such as function. Such subfamilies typically consist of 4 to 30 IgSF members.

[0106] The terms "IgSF domain" or "immunoglobulin domain" or "Ig domain" as used herein refer to the structural domain of an IgSF protein. Ig domains are named after immunoglobulin molecules. They contain approximately 70 to 110 amino acids and are classified according to their size and function. Ig domains possess a characteristic Ig fold, a sandwich-like structure formed by two sheets of antiparallel beta strands. Interactions between hydrophobic amino acids within the sandwich and highly conserved disulfide bonds formed between cysteine ​​residues in the B and F strands stabilize the Ig fold. One end of an Ig domain contains a region called the complementarity-determining region, which is important for the specificity of antibodies for their ligands. Ig-like domains can be classified as IgV, IgC1, IgC2, or IgI. Most Ig domains are either variable (IgV) 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 amino acid sequence, yet are similar in size to IgC domains. These are called IgC2 domains, while standard IgC domains are called IgC1 domains. T cell receptor (TCR) chains contain two Ig domains in their extracellular portions: one IgV domain at the N-terminus and one IgC1 domain adjacent to the cell membrane. CD80 contains two Ig domains: IgV and IgC.

[0107] The term "IgSF species" as used herein refers to a group of IgSF member proteins that have identical or substantially identical primary amino acid sequences. Each mammalian immunoglobulin superfamily (IgSF) member defines a unique identity for all IgSF species belonging to that IgSF member. 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 other IgSF family member species. 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, minor sequence differences within a single IgSF species due to genetic polymorphism, as well as wild-type truncated forms of an IgSF species due to, for example, proteolytic cleavage, also constitute differences between other forms within a single IgSF species. A "cell surface IgSF species" is an IgSF species expressed on the surface of a cell (generally a mammalian cell).

[0108] The term "immune activity," as used herein in the context of mammalian lymphocytes such as T cells, refers to one or more of cell survival, cell proliferation, cytokine production (e.g., interferon-γ), or T-cytotoxic activity. In some cases, immune activity can refer to the expression of cytokines such as chemokines or interleukins. Assays for determining enhanced or suppressed immune activity include the MLR (mixed lymphocyte reaction) assay, which measures interferon-γ cytokine levels in culture supernatants (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). Because T cell activation is associated with the secretion of IFN-γ cytokines, detection of IFN-γ levels in culture supernatants 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 immune activity compared to resting lymphocytes. In some embodiments, immunomodulatory proteins (e.g., variant CD80 polypeptides containing affinity-engineered IgSF domains) as provided herein can increase, or alternatively decrease, IFN-γ (interferon-γ) expression in primary T cell assays compared to wild-type IgSF members or IgSF domain controls. Those skilled in the art will recognize that the format of the primary T cell assay used to determine increased IFN-γ expression differs from the format employed to assay for decreased IFN-γ expression.When assaying for the ability of an immunomodulating protein or affinity-modified IgSF domain of the 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. Conveniently, a soluble form of an affinity-modified IgSF domain of the invention can be employed to determine its ability to reduce IFN-γ expression by antagonizing it in an MLR, also as described in Example 6. Alternatively, when assaying for the ability of an immunomodulating protein or affinity-modified IgSF domain of the invention to increase IFN-γ expression in a primary T cell assay, a co-immobilization assay can be used, in which a T cell receptor signal (in some embodiments, provided by an anti-CD3 antibody) is used in combination with a co-immobilized affinity-modified IgSF domain, such as a variant CD80, to determine the ability to increase IFN-γ expression relative to a wild-type IgSF domain control. Methods for assaying the immune activity of modified cells, including assessing the activity of variant CD80 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 appropriate animal models. Assays are also standard. 51 Assays to assess cytotoxicity include Cr release assays (see, e.g., Milone et al., (2009) Molecular Therapy 17: 1453-1464) or flow-based cytotoxicity assays, or impedance-based cytotoxicity assays (Peper et al. (2014) Journal of Immunological Methods, 405:192-198).

[0109] An "immunomodulating polypeptide" is a polypeptide that modulates immune activity. "Modulating" an immune response means either enhancing or suppressing immune activity. An immunomodulating polypeptide 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 an interchain disulfide bond). Thus, monomeric, dimeric, and higher order multimeric polypeptides are within the scope of the defined term. Multimeric polypeptides can be homomultimers (of the same polypeptide chain) or heteromultimers (of different polypeptide chains). An immunomodulating polypeptide can include a variant CD80.

[0110] The term "increase" as used herein means to increase by a statistically significant amount. The increase can be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, or greater than a non-zero control value.

[0111] An "isoform" of CD80 is one of multiple naturally occurring CD80 polypeptides that differ in amino acid sequence. Isoforms can be the product of splice variants of an RNA transcript expressed by a single gene, or the expression product of highly similar but different genes that produce functionally similar proteins, such as can result from gene duplication. As used herein, the term "isoform" of CD80 also refers to the products of different alleles of the CD80 gene.

[0112] The term "lymphocyte," as used herein, refers to 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 (which are involved in cell-mediated cytotoxic adaptive immunity), and B cells (which are involved in humoral antibody-mediated adaptive 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 lymphocyte are innate lymphocytes (ILCs).

[0113] The term "mammal" or "patient" specifically includes reference to at least one of a human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.

[0114] The term "membrane protein," as used herein, refers to a protein that directly or indirectly attaches (binds) to a lipid bilayer under physiological conditions. The lipid bilayer that forms the membrane can be a biological membrane, such as a cell membrane of a eukaryote (e.g., a mammal), or an artificial (i.e., man-made) membrane, such as a membrane found on a liposome. The membrane protein can be bound to the lipid bilayer by a covalent bond or by a non-covalent interaction (e.g., a hydrophobic interaction or an electrostatic interaction). The 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 a non-covalent interaction or to an integral membrane protein by a non-covalent interaction. The peripheral membrane protein forms a temporary bond to the lipid bilayer so that the peripheral membrane protein can interact with and / or dissociate from the lipid bilayer under physiological conditions in mammals. In contrast to peripheral membrane proteins, integral membrane proteins form a substantially permanent bond to the lipid bilayer of the membrane, so that they do not dissociate from the lipid bilayer under physiological conditions in mammals.Membrane proteins can form a bond to the membrane through one layer of the lipid bilayer (monotopic) or through both layers of the membrane (polytopic).Integral membrane proteins that interact with only one lipid bilayer are "integral monotopic proteins."Integral membrane proteins that interact with both lipid bilayers are "integral polytopic proteins."Alternatively, they are referred to herein as "transmembrane proteins."

[0115] The terms "modulation" or "modulating," as used herein in the context of an immune response (e.g., a mammalian immune response), refer to any change (e.g., enhancement or reduction) in an existing or potential immune response that occurs as a result of administration of an immunomodulatory polypeptide comprising a variant CD80 of the invention or as a result of administration of modified cells expressing an immunomodulatory protein of the invention (e.g., a variant CD80 transmembrane immunomodulatory protein). Thus, modulation refers to a change (e.g., enhancement or reduction) in an immune response compared to an immune response that occurs or exists in the absence of administration of an immunomodulatory protein comprising a variant CD80. 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 made to an existing, developing, or potential immune response, or to the ability to induce, regulate, influence, 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 elimination, deletion, or sequestration of immune cells; the 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; the induction of an unresponsive state in immune cells (i.e., anergy); and the enhancement or suppression of immune cell activity or function (including, but not limited to, altering the pattern of proteins expressed by these cells). Examples include alterations in the production and / or secretion of specific classes of molecules, such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors, or any combination of these regulatory events.Modulation can be assessed, for example, by changes in IFN-γ (interferon gamma) expression compared to wild-type or unmodified CD80 controls in primary T cell assays (see Zhao and Ji, Exp Cell Res. 2016 Jan1;340(1) 132-138). Modulation can be assessed, for example, by changes in the immune activity of the modified cells compared to cells modified with wild-type CD80 transmembrane protein, such as changes in the cytotoxic activity of the modified cells or changes in cytokine secretion of the modified cells.

[0116] The term "multimerization domain" refers to an amino acid sequence that promotes stable interaction between a polypeptide molecule and one or more additional polypeptide molecules, each containing complementary multimerization domains, which can be the same or different multimerization domains that form stable multimers with the first domain. Generally, the polypeptide is directly or indirectly connected to the multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain can be, for example, an immunoglobulin constant region or domain, such as an Fc domain or portion thereof, derived from IgG (including IgG1, IgG2, IgG3, or IgG4 subtypes), IgA, IgE, IgD, and IgM, and modified forms thereof.

[0117] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form. Unless otherwise specified, the terms encompass nucleic acids containing known natural nucleotide analogs, nucleic acids with similar binding properties, 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 sequence explicitly set forth ("reference sequence"), but also conservatively modified variants thereof (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 mixed-base and / or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

[0118] The term "molecular species" as used herein refers to a population of proteins with identical or substantially identical primary amino acid sequences. Each mammalian immunoglobulin superfamily (IgSF) member defines a collection of identical or substantially identical molecular species. Thus, for example, human CD80 is an IgSF member, and each human CD80 molecule is a molecular species of CD80. 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, minor sequence differences within a single molecular species due to genetic polymorphism, as well as wild-type truncated forms of a single molecular species due to, for example, proteolytic cleavage, also constitute differences between other forms within a single molecular species. A "cell surface molecular species" is a molecular species expressed on the surface of a mammalian cell. Two or more different protein species, each present in only one or the other (but not both) of two mammalian cells forming an IS, are said to be in "cis" or "cis configuration" with respect to each other. Two different protein species, the first of which is present only in the first of two mammalian cells that form an IS and the second of which is present only in the second of two mammalian cells that form an IS, are said to be in "trans" or in a "trans configuration." Two different protein species, each of which is present in both two mammalian cells that form an IS, are in both cis and trans configurations on those cells.

[0119] The term "non-competitive binding," as used herein, refers to the ability of a protein to simultaneously specifically bind to at least two cognate binding partners. Thus, a protein can simultaneously bind to at least two different cognate binding partners, but the binding interactions do not necessarily have to be of the same duration; in some cases, the protein specifically binds to only one of the cognate binding partners. In some embodiments, binding occurs under specific binding conditions. In some embodiments, simultaneous binding is such that binding of one cognate binding partner does not substantially inhibit simultaneous binding to a second cognate binding partner. In some embodiments, non-competitive binding means that binding of a second cognate binding partner to its binding site on the protein does not displace 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 those described in Perez de La Lastra et al., Immunology, 1999 Apr: 96(4): 663-670. In some cases, in a non-competitive interaction, a first cognate binding partner specifically binds at an interaction site that does not overlap with the interaction site of a 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 via a mechanism other than direct interference with the binding of the first cognate binding partner. For example, in the context of enzyme-substrate interactions, 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 only binds to the second interaction site when the first interaction site is occupied by the first cognate binding partner.

[0120] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject, often a human. Pharmaceutical compositions typically comprise an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising a variant CD80 or engineered cells expressing a variant CD80 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0121] 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 term does not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. The term includes post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. The term also includes molecules that contain one or more amino acid analogs or non-standard or unnatural amino acids, which can be synthesized or recombinantly expressed using known protein modification techniques. In addition, proteins may be derivatized.

[0122] The term "primary T cell assay," as used herein, refers to an in vitro assay for measuring interferon-γ ("IFN-γ") expression. Various such primary T cell assays are known in the art, such as the assay described in Example 6. In a preferred embodiment, the assay used is an anti-CD3 co-fixation assay. In this assay, primary T cells are stimulated with immobilized anti-CD3, with or without additional recombinant proteins. Culture supernatants are collected at a certain time point (usually 24-72 hours). In another embodiment, the assay used is a mixed lymphocyte reaction (MLR). In this assay, primary T cells are stimulated with allogeneic APCs. Culture supernatants are collected at a certain time point (usually 24-72 hours). Human IFN-γ levels in the culture supernatants are measured by standard ELISA techniques. Commercial kits are available from suppliers, and the assay is performed according to the manufacturer's recommendations.

[0123] The term "purified," as applied to nucleic acids (e.g., nucleic acids encoding immunomodulatory proteins of the invention), generally refers to nucleic acids or polypeptides that are 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 gives rise to essentially one band in an electrophoretic gel is "purified." Purified nucleic acids or proteins of the invention are at least about 50% pure, typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., on a weight percent or molar basis).

[0124] 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. The alteration can be performed on a substance in or removed from its natural environment or state. For example, a "recombinant nucleic acid" is one produced by recombining nucleic acids, e.g., during cloning, affinity engineering, DNA shuffling, or other well-known molecular biological procedures. A "recombinant DNA molecule" is composed of segments of DNA joined together by such molecular biological techniques. The term "recombinant protein" or "recombinant polypeptide," as used herein, refers to a protein molecule expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid, or a cell that has otherwise 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, and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. The terms "in functional combination," "in functional order," and "operably linked," as used herein, refer to the linking of nucleic acid sequences in a manner or orientation that produces a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule.

[0125] The term "recombinant expression vector," as used herein, refers to a DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host cell. 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, and termination and polyadenylation signals. If desired, a secretory signal peptide sequence may also be encoded by the recombinant expression vector operably linked to the coding sequence of the 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 autonomously replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Such vectors include viral vectors, such as lentiviral vectors.

[0126] The term "selectivity" refers to the preference for specific binding of a protein or polypeptide of interest to one substrate (e.g., one cognate binding partner) compared to the specific binding of the protein of interest to another substrate (e.g., a different cognate binding partner). Selectivity refers to the binding activity (e.g., binding affinity) (e.g., K) of a protein of interest to a first substrate (e.g., a first cognate binding partner). d1 ) and the binding activity (e.g., binding affinity) (e.g., K d2 ) can be reflected as a ratio.

[0127] The term "sequence identity" as used herein refers to the 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 between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequence at a given position in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned. Methods for aligning 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).

[0128] The term "soluble" when used herein in reference to a protein 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 containing an IgSF domain or its specific binding fragment, but does not contain a transmembrane domain. In some cases, the solubility of a protein can be improved by linking or binding to an Fc domain directly or indirectly via a linker, which in some cases can also improve the stability and / or half-life of the protein. In some aspects, the soluble protein is an Fc fusion protein.

[0129] The term "species," as used herein with respect to a polypeptide or nucleic acid, refers to a group of molecules having identical or substantially identical sequences. 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. Slightly truncated polypeptide sequences that differ from the full-length species by (or encode) only one, two, or three amino acid residues at the amino or carboxy terminus are considered to be sequences of a single species. Such microheterogeneity is a common feature of manufactured proteins.

[0130] The term "specific binding fragment," as used herein with reference to a full-length wild-type mammalian CD80 polypeptide or its IgV or IgC domain, refers to a polypeptide that has a subsequence of the IgV and / or IgC domain and specifically binds to mammalian CD28, mammalian PD-L1, and / or mammalian CTLA-4 (e.g., human or murine CD28, PD-L1, and / or CTLA-4) in vitro and / or in vivo. In some embodiments, a specific binding fragment of CD80 IgV or CD80 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 altered to form a variant CD80.

[0131] The term "specifically binds" as used herein refers to 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 times greater than the average affinity or avidity of the same protein for a population of random peptides or polypeptides of sufficient statistical size, but optionally at least 10, 20, 30, 40, 50, 100, 250, or 500 times greater, or even at least 1000 times greater. A specifically binding protein need not bind only to a single target molecule, but may specifically bind to a non-target molecule (e.g., a paralog or ortholog) due to the similarity in configuration between the target and the non-target. Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs) or to non-target molecules (e.g., paralogs) with substantially similar epitopes to the target molecule is possible and does not impair the specificity of binding determined for a statistically valid population of unique non-targets (e.g., random polypeptides). Therefore, due to cross-reactivity, the polypeptides of the present invention can specifically bind to multiple distinct target molecular species. Solid-phase ELISA immunoassays or Biacore measurements can be used to determine the specific binding between two proteins. Generally, the interaction between two binding proteins is greater than 1×10 -5 Less than M, often 1 × 10 -12 In certain embodiments of the present disclosure, the interaction between two binding proteins has a dissociation constant (Kd) as low as 1×10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M or 1×10 -11 It has a dissociation constant of M.

[0132] With respect to mammalian cells expressing a polypeptide, the term "surface-expressed" or "surface expression" means that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.

[0133] As used herein, "synthetic" refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant and / or chemical synthesis methods, e.g., with respect to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide.

[0134] The term "targeting moiety," as used herein, refers to a composition that is covalently or non-covalently attached to or physically encapsulates a polypeptide comprising a variant CD80. A targeting moiety has specific binding affinity for a desired counterstructure, such as a cell surface receptor (e.g., the B7 family member PD-L1) or a tumor antigen (e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA), such as B7-H6). Typically, the desired counterstructure is localized on a specific tissue or cell type. A targeting moiety can be an antibody, an antigen-binding fragment (Fab), a V-cell, or a mAb. H and V L variable fragments (Fv) containing V linked together in one chain; H and V L and other antibody V region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, nanobodies, soluble receptors, receptor ligands, affinity matured receptors or ligands, and small molecule (<500 dalton) compositions (e.g., specific binding receptor compositions). Targeting moieties can also be covalently or non-covalently bound (attached) to the lipid membrane of liposomes that encapsulate the polypeptides of the invention.

[0135] As used herein, the term "transmembrane protein" refers to a membrane protein that substantially or completely spans a lipid bilayer, which is found in biological membranes, such as mammalian cells, or in artificial constructs, such as liposomes. A transmembrane protein comprises a transmembrane domain ("transmembrane domain") that is integrated into the lipid bilayer and whose integration is thermodynamically stable under physiological conditions. The transmembrane domain can generally be predicted from the amino acid sequence of the transmembrane domain through several commercially available bioinformatics software applications, based on the fact that the hydrophobicity of the transmembrane domain is higher than that of the region of the protein that interacts with the aqueous environment (e.g., cytosol, extracellular fluid). The transmembrane domain is often a hydrophobic α-helix that spans the membrane. A transmembrane protein may span both layers of the lipid bilayer once or multiple times. The transmembrane immunomodulatory proteins provided herein are included in transmembrane proteins. In addition to the transmembrane domain, the transmembrane immunomodulatory proteins of the present invention further comprise an ectodomain, and in some embodiments, an endodomain.

[0136] The term "treatment" or "therapy" of a disease or disorder, as used herein, means slowing, halting, or reversing the progression of the disease or disorder, as evidenced by the reduction, arrest, or elimination of any clinical or diagnostic symptoms by administering a therapeutic composition of the invention (e.g., containing an immunomodulatory protein or modified cells), either alone or in combination with another compound as described herein. "Treating" or "treatment" also means reducing the severity of symptoms in acute or chronic diseases or disorders, or reducing the rate of relapse (e.g., as in relapsing or remitting autoimmune disease processes), or reducing inflammation in the case of inflammatory aspects of autoimmune diseases. As used herein in the context of cancer, the terms "treating" cancer or "inhibiting" cancer or "inhibition" of cancer refer to at least one of a statistically significant reduction in tumor growth rate, halting tumor growth, or a reduction 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, but not limited to, Response Evaluation Criteria for Solid Tumors (RECIST). "Preventing" a disease or disorder or "prevention" of a disease or disorder, as used in the context of the present invention, refers to the administration of an immunomodulatory polypeptide or modified cell of the present invention, either alone or in combination with another compound, to prevent the appearance or development of a disease or disorder or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of developing a disease or disorder.

[0137] The term "tumor-specific antigen" or "TSA," as used herein, refers to a counter structure that is primarily present on tumor cells of a mammalian subject but is not generally found on normal cells of the mammalian subject. A tumor-specific antigen need not be present exclusively on tumor cells, but the proportion of cells in a particular mammal that have the tumor-specific antigen or the level of the tumor-specific antigen on the surface of the tumor must be sufficiently high so that it can be targeted with an anti-tumor therapeutic agent (e.g., an immunomodulatory polypeptide of the present invention) and provide protection or treatment for the mammal from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammal with a tumor, at least 50% of the cells that display the TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells that display the TSA are cancerous.

[0138] The term "variant" (also "modified" or "mutant"), when used with reference to variant CD80, refers to a CD80 created by human intervention, e.g., mammalian (e.g., human or murine) CD80. A variant CD80 is a polypeptide having an altered amino acid sequence compared to unmodified or wild-type CD80. A variant CD80 is a polypeptide that differs from the wild-type CD80 isoform sequence by one or more amino acid substitutions, deletions, additions, or a combination thereof. For purposes herein, a variant CD80 contains at least one affinity-altered domain, whereby one or more of the amino acid differences occur in the IgSF domain (e.g., the IgV domain). A variant CD80 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. Variant CD80 polypeptides generally exhibit at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified CD80 (e.g., the sequence of SEQ ID NO:1), its mature sequence, or a portion thereof containing the extracellular domain or IgSF domain. In some embodiments, the variant CD80 polypeptide exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the corresponding wild-type or unmodified CD80, including the sequence set forth in SEQ ID NO:28, SEQ ID NO:152, or SEQ ID NO:372. Both non-naturally occurring and naturally occurring amino acids are included within the scope of permissible substitutions or additions. The variant CD80 is not limited to any particular method of production, including, for example, de novo chemical synthesis, de novo recombinant DNA techniques, or a combination thereof.The variant CD80 of the present invention specifically binds to at least one or more of CD28, PD-L1, and / or CTLA-4 of a mammalian species. In some embodiments, the change in amino acid sequence results in a change (i.e., an improvement or decrease) in binding affinity or avidity to CD28, PD-L1, and / or CTLA-4 compared to the unmodified or wild-type CD80 protein. The improvement or decrease in binding affinity or avidity can be determined using well-known binding assays, such as flow cytometry. Larsen et al., American Journal of Transplantation, Vol. 5: 443-453 (2005). See also Linsley et al., Immunity, 1: 7930801 (1994). The improved binding affinity or avidity of the variant CD80 for CD28, PD-L1, and / or CTLA-4 can be at least 5% greater than that of the unmodified or wild-type CD80, and in some embodiments can be at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than that of the unmodified or wild-type CD80 control value. The decreased binding affinity or avidity of CD80 for CD28, PD-L1, and / or CTLA-4 can be at most 95% of the unmodified or wild-type CD80 control value, and in some embodiments at most 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the binding affinity or avidity of the unmodified or wild-type CD80 control value, or to an undetectable value. Variant CD80 polypeptides have altered primary amino acid sequence due to substitution, addition, or deletion of amino acid residues. The term "variant" in the context of variant CD80 polypeptides should not be construed as imposing any requirement on any particular starting composition or method by which the variant CD80 is made.A variant CD80 can be generated, for example, starting from wild-type mammalian CD80 sequence information, then modeled in silico for binding to CD28, PD-L1, and / or CTLA-4, and finally recombinantly or chemically synthesized to generate the variant CD80. As just another example, a variant CD80 can be made by site-directed mutagenesis of unmodified or wild-type CD80. Thus, a variant CD80 represents a composition, but not necessarily a product, produced by any given process. A wide variety of techniques may be employed, including recombinant methods, chemical synthesis, or a combination thereof.

[0139] The terms "wild-type" or "natural" or "native" as used herein in connection with biological materials such as nucleic acid molecules, proteins (e.g., CD80), IgSF members, host cells, etc., refer to those found in nature and unmodified by human intervention.

[0140] II. Variant CD80 Polypeptides Provided herein are variant CD80 polypeptides that have altered (enhanced or decreased) binding activity or affinity for one or more CD80 cognate binding partners. In some embodiments, the CD80 cognate binding partner is CD28, PD-L1, or CTLA-4. In some embodiments, the variant CD80 polypeptide contains one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "exchanges"), deletions, or additions, in the immunoglobulin superfamily (IgSF) domain (IgD) compared to a wild-type or unmodified CD80 polypeptide, or an IgD-containing portion of a wild-type or unmodified CD80, or a specific-binding fragment thereof.

[0141] Thus, the provided variant CD80 polypeptides are or include variant IgDs (hereinafter referred to as "vIgD") in which one or more amino acid modifications (eg, substitutions) are in the IgD.

[0142] 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 an IgC (e.g., IgC2) domain, or a combination thereof. In some embodiments, the IgD can be simply IgV, a combination of IgV and IgC including the entire extracellular domain (ECD), or any combination of the Ig domains of CD80. Table 2 provides exemplary residues corresponding to the IgV or IgC regions of CD80. In some embodiments, the variant CD80 polypeptide contains an IgV domain or an IgC domain, or a specific-binding fragment thereof, with at least one amino acid modification (e.g., substitution) in the IgV domain or IgC domain or a specific-binding fragment thereof. In some embodiments, the variant CD80 polypeptide contains an IgV domain or a specific-binding fragment thereof, with at least one amino acid modification (e.g., substitution) in the IgV domain or IgC domain or a specific-binding fragment thereof. In some embodiments, the altered IgV or IgC domain is an affinity-modified IgSF domain with altered avidity or affinity.

[0143] In some embodiments, the variant has one more IgSF domain modified compared to the sequence of the unmodified CD80 sequence. In some embodiments, the unmodified CD80 sequence is wild-type CD80. In some embodiments, the unmodified or wild-type CD80 has the sequence of a native CD80 or its orthologue. In some embodiments, the unmodified CD80 is or comprises the extracellular domain (ECD) of CD80, or a portion thereof, containing one or more IgSF domains (see Table 2). In some embodiments, the extracellular domain of an unmodified or wild-type CD80 polypeptide comprises an IgV domain and an IgC domain. However, a variant CD80 polypeptide need not comprise both an IgV domain and an IgC domain. In some embodiments, a variant CD80 polypeptide comprises or consists essentially of an IgV domain or a specific-binding fragment thereof. In some embodiments, a variant CD80 polypeptide comprises or consists essentially of an IgC domain or a specific-binding fragment thereof. In some embodiments, a variant CD80 is soluble and lacks a transmembrane domain. In some embodiments, the variant CD80 further comprises a transmembrane domain, and in some cases, a cytoplasmic domain.

[0144] In some embodiments, the wild-type or unmodified CD80 polypeptide is a mammalian CD80 polypeptide, such as, but not limited to, a human, mouse, cynomolgus monkey, or rat CD80 polypeptide. In some embodiments, the wild-type or unmodified CD80 sequence is human.

[0145] In some embodiments, the wild-type or unmodified CD80 polypeptide (i) has the amino acid sequence set forth in SEQ ID NO:1 or a mature form thereof lacking the signal sequence, (ii) has an amino acid sequence that exhibits 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 a mature form thereof, or (iii) is a portion of (i) or (ii) that contains an IgV domain or an IgC domain or a specific-binding fragment thereof.

[0146] In some embodiments, the wild-type or unmodified CD80 polypeptide is or comprises the extracellular domain of CD80 or a portion thereof. For example, in some embodiments, the unmodified or wild-type CD80 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:28, or an ortholog thereof. For example, the unmodified or wild-type CD80 polypeptide can (i) comprise the amino acid sequence set forth in SEQ ID NO:28, (ii) comprise 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:28, or (iii) is a specific-binding fragment of (i) or (ii) comprising the IgV domain or the IgC domain. In some embodiments, the wild-type or unmodified extracellular domain of CD80 is capable of binding to one or more CD80 cognate binding proteins, e.g., one or more of CD28, PD-L1, or CTLA-4.

[0147] In some embodiments, a wild-type or unmodified CD80 polypeptide contains an IgV domain or an IgC domain, or a specific-binding fragment thereof. In some embodiments, the IgV domain of a wild-type or unmodified CD80 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 152 or 372, or an ortholog thereof. For example, the IgV domain of an unmodified or wild-type CD80 polypeptide can (i) contain the amino acid sequence set forth in SEQ ID NO: 152 or 372, or (ii) contain 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: 152 or 372, or (iii) is a specific-binding fragment of (i) or (ii). In some embodiments, the wild-type or unmodified IgV domain is capable of binding to one or more CD80 cognate binding proteins, such as one or more of CD28, PD-L1, or CTLA-4.

[0148] In some embodiments, the IgC domain of a wild-type or unmodified CD80 polypeptide comprises the amino acid sequence set forth as residues 145-230 or 154-232 of SEQ ID NO: 1, or an ortholog thereof. For example, the IgC domain of an unmodified or wild-type CD80 polypeptide can (i) contain the amino acid sequence set forth as residues 145-230 or 154-232 of SEQ ID NO: 1, (ii) contain 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 residues 145-230 or 154-232 of SEQ ID NO: 1, or (iii) is a specific-binding fragment of (i) or (ii). In some embodiments, the wild-type or unmodified IgC domain is capable of binding to one or more CD80 cognate binding proteins.

[0149] In some embodiments, the wild-type or unmodified CD80 polypeptide contains a specific-binding fragment of CD80, e.g., a specific-binding fragment of the IgV domain or the IgC domain. In some embodiments, the specific-binding fragment can bind to CD28, PD-L1, and / or CTLA4. The specific-binding fragment can have a length of at least 50 amino acids, e.g., 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%, or 99% of the length of the IgV domain set forth as amino acids 35-135 or 37-138 of SEQ ID NO:1. In some embodiments, a specific binding fragment of an IgC domain comprises 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 IgC domain set forth as amino acids 145-230 or 154-232 of SEQ ID NO:1.

[0150] In some embodiments, the variant CD80 polypeptide comprises an ECD domain or a portion thereof comprising one or more affinity-altered IgSF domains. In some embodiments, the variant CD80 polypeptide can comprise an IgV domain or an IgC domain, or a specific-binding fragment of an IgV domain or a specific-binding fragment of an IgC domain, wherein at least one of the IgV or IgC domains contains one or more amino acid modifications (e.g., substitutions). In some embodiments, the variant CD80 polypeptide can comprise an IgV domain and an IgC domain, or a specific-binding fragment of an IgV domain and a specific-binding fragment of an IgC domain. In some embodiments, the variant CD80 polypeptide comprises a full-length IgV domain. In some embodiments, the variant CD80 polypeptide comprises a full-length IgC domain. In some embodiments, the variant CD80 polypeptide comprises a specific-binding fragment of an IgV domain. In some embodiments, the variant CD80 polypeptide comprises a specific-binding fragment of an IgC domain. In some embodiments, the variant CD80 polypeptide comprises a full-length IgV domain and a full-length IgC domain. In some embodiments, the variant CD80 polypeptide comprises a full-length IgV domain and a specific-binding fragment of the IgC domain. In some embodiments, the variant CD80 polypeptide comprises a specific-binding fragment of the IgV domain and a full-length IgC domain. In some embodiments, the variant CD80 polypeptide comprises a specific-binding fragment of the IgV domain and a specific-binding fragment of the IgC domain.

[0151] In any of such embodiments, the one or more amino acid modifications (e.g., substitutions) of the variant CD80 polypeptide can be located in any one or more of the CD80 polypeptide domains. For example, in some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the extracellular domain of the variant CD80 polypeptide. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the IgV domain or a specific-binding fragment of the IgV domain. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are located in the IgC domain or a specific-binding fragment of the IgC domain.

[0152] Generally, each of the various attributes of the polypeptides is disclosed separately below (e.g., soluble and membrane-bound polypeptides, affinity of CD80 for CD28, PD-L1, and CTLA-4, number of differences per polypeptide chain, number of linked polypeptide chains, number and nature of amino acid alterations per variant CD80, etc.). However, as will be apparent to one of skill in the art, any particular polypeptide can include a combination of these independent attributes. It will be understood that references to amino acids, including references to specific sequences set forth as SEQ ID NOs, used to describe the domain organization of IgSF domains are for illustrative purposes and are not meant to limit the scope of the provided embodiments. It will be understood that descriptions of polypeptides and their domains are theoretically derived based on homology analysis and alignment with similar molecules. Thus, the exact locus may vary and will not necessarily be the same from protein to protein. Thus, a particular IgSF domain, e.g., a particular IgV or IgC domain, may be several amino acids longer or shorter (e.g., 1, 2, 3, or 4).

[0153] Furthermore, various aspects of the present invention as discussed below are often provided within the meaning of defined terms as disclosed above. Therefore, aspects described in a particular definition should be construed as being incorporated by reference when the defined terms are utilized in the discussion of various aspects and attributes described herein. Thus, the headings, the order of presentation of various aspects and embodiments, and the separate disclosure of each independent attribute are not meant to limit the scope of the present disclosure.

[0154] Exemplary Modifications Provided herein are variant CD80 polypeptides containing at least one affinity-altered IgSF domain (e.g., IgV or IgC) or a specific-binding fragment thereof, relative to the IgSF domain contained in a wild-type or unmodified CD80 polypeptide, wherein the variant CD80 polypeptide has altered (enhanced or decreased) binding activity or affinity for one or more ligands, CD28, PD-L1, or CTLA-4, relative to the wild-type or unmodified CD80 polypeptide. In some embodiments, the variant CD80 polypeptide has a binding affinity for CD28, PD-L1, and / or CTLA-4 that is different from that of a wild-type or unmodified CD80 polypeptide control sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, or Biacore assay. In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28, PD-L1, and / or CTLA-4. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28, PD-L1, and / or CTLA-4 compared to a wild-type or unmodified CD80 polypeptide. CD28, PD-L1, and / or CTLA-4 can be mammalian proteins, e.g., human or murine proteins.

[0155] The binding affinity for each of the cognate binding partners is independent; i.e., in some embodiments, the variant CD80 polypeptide has improved binding affinity for one, two, or three of CD28, PD-L1, and CTLA-4, and / or decreased binding affinity for one, two, or three of CD28, PD-L1, and CTLA-4, compared to a wild-type or unmodified CD80 polypeptide.

[0156] In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has improved binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has improved binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides.

[0157] In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28 and PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28 and reduced binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28 and PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28 and improved binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides.

[0158] In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28 and CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28 and reduced binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28 and CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28 and improved binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides.

[0159] In some embodiments, the variant CD80 polypeptides have improved binding affinity for PD-L1 and CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have improved binding affinity for PD-L1 and reduced binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have reduced binding affinity for PD-L1 and CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptides have reduced binding affinity for PD-L1 and improved binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides.

[0160] In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28, PD-L1, and CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28 and PD-L1 and reduced binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28 and CTLA-4 and reduced binding affinity for PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28 and PD-L1 and improved binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28 and PD-L1 and improved binding affinity for CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28 and reduced binding affinity for PD-L1 and CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28, CTLA-4, and PD-L1 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, the variant CD80 polypeptide has reduced binding affinity for CD28 and improved binding affinity for PD-L1 and CTLA-4 compared to wild-type or unmodified CD80 polypeptides.

[0161] In some embodiments, a variant CD80 polypeptide with improved or higher binding affinity for CD28, PD-L1, and / or CTLA-4 will have at least about a 5%, e.g., at least about a 10%, 15%, 20%, 25%, 35%, or 50% improvement in binding affinity for CD28, PD-L1, and / or CTLA-4 compared to a wild-type or unmodified CD80 polypeptide control. In some embodiments, the improved binding affinity 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, or 50-fold greater than the wild-type or unmodified CD80 polypeptide. In such examples, the wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0162] In some embodiments, a variant CD80 polypeptide with reduced or decreased binding affinity for CD28, PD-L1, and / or CTLA-4 will have at least a 5%, e.g., at least about a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more decrease in binding affinity for CD28, PD-L1, and / or CTLA-4 compared to a wild-type or unmodified CD80 polypeptide control. In some embodiments, the decrease in binding affinity is greater than a 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 decrease compared to a wild-type or unmodified CD80 polypeptide. In such examples, the wild-type or unmodified CD80 polypeptide has the same sequence as the variant CD80 polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0163] In some embodiments, the equilibrium dissociation constant (K d ) is 1×10 -5 M, 1 x 10 -6M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M or 1×10 -11 M, or 1 x 10 -12 It can be less than M.

[0164] A wild-type or unmodified CD80 sequence need not necessarily be used as the starting composition to generate the variant CD80 polypeptides described herein. Thus, the use of the term "substitution" does not imply that the provided embodiments are limited to a particular method of producing the variant CD80 polypeptide. Variant CD80 polypeptides can be produced, for example, by de novo peptide synthesis and thus do not necessarily require a "substitution" in the sense of altering a codon to encode the substitution. This principle also extends to the terms "addition" and "deletion" of amino acid residues, which also do not imply a particular method of production. The means by which variant CD80 polypeptides are designed or generated are not limited to any particular method. However, in some embodiments, nucleic acids encoding wild-type or unmodified CD80 are mutagenized from wild-type or unmodified CD80 genetic material and screened for the desired specific binding affinity and / or induction of IFN-γ expression or other functional activity according to the methods disclosed in the Examples or other methods known to those of skill in the art. In some embodiments, variant CD80 polypeptides are synthesized de novo using protein or nucleic acid sequences available in a number of publicly available databases, which are then subsequently screened. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, as is the UniProtKB database, as discussed above.

[0165] Unless otherwise specified, as indicated throughout this disclosure, amino acid substitutions are designated by amino acid position numbers that correspond to the numbering of positions in the unmodified ECD sequence shown in SEQ ID NO:28, or, where applicable, the unmodified IgV sequence shown in SEQ ID NO:152 or 372 (containing residues 1-101 or 1-107, respectively, of SEQ ID NO:28, depending on annotation and convention), as follows: TIFF2024099752000029.tif76161

[0166] It is within the level of ordinary skill in the art to identify the corresponding position of a modification (e.g., amino acid substitution) in a CD80 polypeptide (including a portion thereof containing its IgSF domain (e.g., IgV)) by alignment with a reference sequence having, for example, SEQ ID NO:28 or SEQ ID NO:152 or SEQ ID NO:372. In descriptions of modifications throughout this disclosure, the amino acid position is centered, the corresponding unmodified (e.g., wild-type) amino acid is listed before the number, and the amino acid substitution of the identified variant is listed after the number. If the modification is a deletion at that position, it is designated "del," and if the modification is an insertion at that position, it is designated "ins."

[0167] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) in a wild-type or unmodified CD80 sequence. The one or more amino acid modifications (e.g., substitutions) can be in the ectodomain (e.g., extracellular domain) of the wild-type or unmodified CD80 sequence. In some embodiments, the one or more amino acid modifications (e.g., substitutions) are in the IgV domain or a specific-binding fragment thereof. In some embodiments of the variant CD80 polypeptide, some of the one or more amino acid modifications (e.g., substitutions) are in the IgV domain or a specific-binding fragment thereof, and some of the one or more amino acid modifications (e.g., substitutions) are in the IgC domain or a specific-binding fragment thereof.

[0168] In some embodiments, the variant CD80 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions). The modifications (e.g., substitutions) can be in the IgV domain or the IgC domain. In some embodiments, the variant CD80 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the IgV domain or a specific-binding fragment thereof. In some embodiments, the variant CD80 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the IgC domain or specific-binding fragment thereof. In some embodiments, the variant CD80 polypeptide has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type or unmodified CD80 polypeptide or specific-binding fragment thereof, e.g., to the amino acid sequence of SEQ ID NO:28, 152, or 372.

[0169] In some embodiments, the variant CD80 polypeptide is located at positions 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 27, 28, 29, 30, 31, 33, 36, 37, 38, 40, 41, 42, 43, 44, 47, 48, 50, 52, 53, 54, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 72, 74, 76, 77, 80, 81, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 14 160, 162, 164, 168, 169, 174, 175, 177, 178, 183, 178, 185, 188, 190, 192, 193, or 199. In some embodiments, such variant CD80 polypeptides have altered binding affinity for one or more of CD28, PD-L1, or CTLA-4 compared to wild-type or unmodified CD80 polypeptides. For example, in some embodiments, variant CD80 polypeptides have improved binding affinity for CD28, PD-L1, and / or CTLA-4 compared to wild-type or unmodified CD80 polypeptides. In some embodiments, variant CD80 polypeptides have decreased binding affinity for CD28, PD-L1, and / or CTLA-4 compared to wild-type or unmodified CD80 polypeptides.

[0170] In some embodiments, the variant CD80 polypeptide is In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from: TIFF2024099752000031.tif107165 or conservative amino acid substitutions thereof.

[0171] In some embodiments, the one or more amino acid modifications, e.g., substitutions, are TIFF2024099752000032.tif77154TIFF2024099752000033.tif230166TIFF2024099752000034.tif230166TIFF2024099752000035.tif230166TIFF2024099752000036.tif231166TIFF2024099752000037.tif231164TIFF2024099752000038.tif231166TIFF2024099752000039.tif142166

[0172] In some embodiments, the variant CD80 polypeptide is located at position 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 27, 28, 29, 31, 33, 36, 37, 38, 40, 41, 42, 43, 44, 47, 48, 50, 52, 53, 54, 59, 60, 61, 62, 63, 64, 66, 67, 68, 69, 70, 72, 74, 76, 77, 80, 81, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 147, 148, 149, , 103, 104, 108, 109, 110, 115, 116, 117, 118, 120, 121, 122, 126, 127, 128, 129, 130, 133, 137, 140, 142, 143, 144, 148, 149, 152, 154, 160, 162, 168, 169, 174, 175, 177, 178, 183, 178, 188, 190, 192, or 193. In some embodiments, such variant CD80 polypeptides have altered binding affinity for one or more of CD28, PD-L1, or CTLA-4 compared to a wild-type or unmodified CD80 polypeptide. For example, in some embodiments, the variant CD80 polypeptide has improved binding affinity for CD28, PD-L1, and / or CTLA-4 compared to a wild-type or unmodified CD80 polypeptide, hi some embodiments, the variant CD80 polypeptide has decreased binding affinity for CD28, PD-L1, and / or CTLA-4 compared to a wild-type or unmodified CD80 polypeptide.

[0173] In some embodiments, the variant CD80 polypeptide is In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from: TIFF2024099752000041.tif99165 or conservative amino acid substitutions thereof.

[0174] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) in unmodified CD80 or a specific-binding fragment thereof corresponding to positions 30, 65, 107, 114, 164, 185, or 199 based on the numbering of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from I30V, N48D, L65H, E99D, D107N, S114T, I126V, N144D, Y164H, and T199S. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of N48D, L65H, E99D, D107N, S114T, I126V, N144D, Y164H, K185E, T199S, or conservative amino acid substitutions thereof.

[0175] Conservative amino acid substitutions are any amino acids that belong to the same class of amino acids as the substituted amino acid, except for wild-type or unmodified amino acids. The 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). Thus, for example, conservative amino acid substitutions for V4M substitution include V4S, V4C, and V4T amino acid substitutions.

[0176] In some embodiments, the variant CD80 polypeptide comprises any of the substitutions (mutations) listed in Table 1. Table 1 also provides exemplary sequences by reference to SEQ ID NOs for the extracellular domain (ECD) or IgV domain of wild-type CD80 or exemplary variant CD80 polypeptides. As indicated, the exact locus or residues corresponding to a given domain may vary depending, for example, on the method used to identify or classify the domain. In addition, in some cases, flanking amino acids N- and / or C-terminal to a given domain (e.g., IgV) may also be included in the sequence of the variant IgSF polypeptide, for example, to ensure proper folding of the domain when expressed. Thus, it is understood that the exemplary SEQ ID NOs in Table 1 should not be construed as limiting. For example, a particular domain (e.g., IgV domain) of a variant CD80 polypeptide may be several amino acids longer or shorter than the amino acid sequence set forth in the respective SEQ ID NO, e.g., 1 to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0177] In some embodiments, the variant CD80 polypeptide comprises any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs:55-108, 280-346, 414-475). In some embodiments, the variant CD80 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, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs:55-108, 280-346, 414-475), and contains an amino acid modification (e.g., a substitution) that is not present in wild-type or unmodified CD80. In some embodiments, the variant CD80 polypeptide comprises a specific-binding fragment of any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOs:55-108, 280-346, 414-475) and contains an amino acid modification (e.g., a substitution) that is not present in wild-type or unmodified CD80. In some embodiments, the variant CD80 polypeptide comprises any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOs:153-195, 347, 373-386, 476-477). In some embodiments, the variant CD80 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, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOs:153-195, 347, 373-386, 476-477), and contains an amino acid modification (e.g., a substitution) that is not present in wild-type or unmodified CD80.In some embodiments, the variant CD80 polypeptide comprises a specific-binding fragment of any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOs:153-195, 347, 373-386, 476-477) and contains an amino acid substitution (e.g., a substitution) that is not present in wild-type or unmodified CD80.

[0178] Table 1 also provides exemplary sequences by reference to SEQ ID NOs for the extracellular domain (ECD) or IgV domain of wild-type CD80 or exemplary variant CD80 polypeptides. As indicated, the exact locus or residues corresponding to a given domain may vary depending, for example, on the method used to identify or classify the domain. In addition, in some cases, adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., ECD) may also be included in the sequence of the variant IgSF polypeptide, for example, to ensure proper folding of the domain when expressed. Thus, it is understood that the exemplary SEQ ID NOs in Table 1 should not be construed as limiting. For example, a particular domain (e.g., IgV domain) of a variant CD80 polypeptide may be several amino acids longer or shorter, e.g., 1 to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter, than the amino acid sequence set forth in the respective SEQ ID NO.

[0179] Table 1. Exemplary variant CD80 polypeptides TIFF2024099752000042.tif210168TIFF2024099752000043.tif230168TIFF2024099752000044.tif229168 TIFF2024099752000045.tif229168TIFF2024099752000046.tif230168TIFF2024099752000047.tif212168

[0180] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 12, 18, 20, 29, 31, 36, 40, 41, 43, 52, 59, 60, 63, 67, 70, 77, 81, 87, 88, 89, 90, 91, 92, 93, 107, 109, 114, 117, 118, 120, 122, 127, 130, 144, 169, 178, or 199 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from: TIFF2024099752000049.tif26165 or conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are: TIFF2024099752000050.tif113161. In some embodiments, the variant CD80 polypeptide has improved affinity for and / or selectivity for the CD28 ectodomain, compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0181] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 12, 18, 21, 22, 28, 29, 31, 33, 36, 38, 40, 41, 42, 43, 47, 48, 59, 64, 67, 68, 70, 77, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 97, 104, 109, 115, 117, 118, 120, 122, 126, 130, 133, 140, 144, 148, 149, 168, 169, 177, 183, 188, or 193 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2024099752000052.tif33163 and one or more conservative amino acid substitutions thereof.

[0182] In some embodiments, the one or more amino acid substitutions are TIFF2024099752000053.tif99165TIFF2024099752000054.tif237166. In some embodiments, the variant CD80 polypeptide has improved affinity for and / or selectivity for PD-L1 compared to a wild-type or unmodified CD80 polypeptide comprising, for example, a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0183] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 12, 18, 36, 40, 43, 59, 77, 88, 89, 90, 91, 92, 93, 109, 118, 122, or 177 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2024099752000056.tif18161 and conservative variations thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: TIFF2024099752000057.tif26128. In some embodiments, the variant CD80 polypeptide has improved affinity for the CD28 ectodomain and the PD-L1 ectodomain compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0184] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 4, 29, 31, 36, 40, 41, 52, 67, 68, 70, 87, 88, 89, 90, 91, 92, 93, 107, 109, 110, 118, 120, 130, 144, or 169 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2024099752000059.tif19165 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are: TIFF2024099752000060.tif47162. In some embodiments, the variant CD80 polypeptide has improved affinity and / or selectivity for the ectodomain of CTLA-4 compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372. In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 36, 40, 52, 70, 88, 89, 90, 91, 92, 93, 107, 118, 120, 130, 144, or 169 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2024099752000062.tif11167 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are: TIFF2024099752000063.tif26159. In some embodiments, the variant CD80 polypeptide has improved affinity for the ectodomain of CD28 and the ectodomain of CTLA-4 compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0185] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 29, 31, 36, 40, 41, 67, 70, 87, 88, 89, 90, 91, 92, 93, 109, 118, 120, 122, 130, or 178 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2024099752000065.tif11132 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are: TIFF2024099752000066.tif19140. In some embodiments, the variant CD80 polypeptide has improved affinity for the ectodomain of PD-L1 and the ectodomain of CTLA-4 compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0186] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 36, 40, 88, 89, 90, 91, 92, or 93 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of K36G, L40M, E88D, K89R, D90K, A91G, F92Y, and K93R. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of K36G, L40M, E88D, K89R, D90K, A91G, F92Y, K93R, and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are TIFF2024099752000067.tif4170. In some embodiments, the variant CD80 polypeptide has improved affinity for the CD28 ectodomain, the PD-L1 ectodomain, and the CTLA-4 ectodomain compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0187] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 12, 18, 29, 31, 33, 36, 38, 40, 41, 42, 43, 47, 48, 63, 59, 64, 67, 68, 70, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 97, 104, 109, 114, 115, 117, 118, 120, 122, 126, 127, 130, 133, 140, 144, 148, 149, 168, 169, 177, 183, 188, or 193 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2024099752000069.tif18169 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are: TIFF2024099752000070.tif11155. In some embodiments, the one or more amino acid substitutions are TIFF2024099752000071.tif158166TIFF2024099752000072.tif179166. In some embodiments, the variant CD80 polypeptide has improved affinity for the CD28 ectodomain or the PD-L1 ectodomain and decreased affinity for the CTLA-4 ectodomain, compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0188] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 63, 70, 81, 87, 91, 114, 118, 120, 127, or 130 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2024099752000074.tif4138 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are: TIFF2024099752000075.tif77164. In some embodiments, the variant CD80 polypeptide has improved affinity for the CD28 ectodomain and reduced affinity for the CTLA-4 ectodomain, as compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0189] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 12, 18, 29, 31, 33, 36, 38, 40, 41, 42, 43, 47, 48, 59, 64, 67, 68, 70, 77, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 97, 104, 109, 115, 117, 118, 120, 122, 126, 130, 133, 140, 144, 148, 149, 168, 169, 177, 183, 188, or 193 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of: TIFF2024099752000077.tif25161 and conservative amino acid substitutions thereof. In some embodiments, the one or more amino acid substitutions are: TIFF2024099752000078.tif63164. In some embodiments, the one or more amino acid substitutions are TIFF2024099752000079.tif136166TIFF2024099752000080.tif201166. In some embodiments, the variant CD80 polypeptide has improved affinity for the ectodomain of PD-L1 and decreased affinity for the ectodomain of CTLA-4 compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence as set forth in SEQ ID NO:28, 152, or 372.

[0190] In some embodiments, the variant CD80 polypeptide has one or more amino acid modifications (e.g., substitutions) corresponding to positions 70, 81, 87, 91, or 120 of SEQ ID NO:28. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of L70Q, Y87N, A91G, and T120S. In some embodiments, the variant CD80 polypeptide has one or more amino acid substitutions selected from the group consisting of L70Q, Y87N, A91G, and T120S, and conservative amino acid substitutions thereof. In some embodiments, the variant CD80 polypeptide has improved affinity for the CD28 ectodomain and the PD-L1 ectodomain, and decreased affinity for the CTLA-4 ectodomain, compared to a wild-type or unmodified CD80 polypeptide comprising, e.g., a sequence set forth in SEQ ID NO:28, 152, or 372.

[0191] III. Variant Polypeptide Formats Immunomodulating polypeptides, including variant CD80s provided herein containing vIgD, can be formatted in a variety of ways, for example, for delivery as soluble proteins, fusions or conjugates, membrane-bound proteins, secreted proteins, or in infectious agents. In some embodiments, a particular format can be selected for a desired therapeutic application. In some cases, immunomodulating polypeptides, including variant CD80 polypeptides, are provided in a format that antagonizes or blocks the activity of their cognate binding partners (e.g., CD28, PD-L1, and / or CTLA-4). In some embodiments, antagonism against CTLA-4 can be useful for promoting immunity in tumors. In some embodiments, antagonism against CD28 can be useful for treating inflammation or autoimmunity. In some cases, immunomodulating polypeptides, including variant CD80 polypeptides, are provided in a format that agonizes or stimulates the activity of their cognate binding partners (e.g., CD28, PD-L1, and / or CTLA-4). In some embodiments, agonism against CTLA-4 can be useful for treating inflammation or autoimmunity. In some embodiments, agonism against CD28 can be useful for treating tumors.Those skilled in the art can easily determine the activity of a particular format, for example, to antagonize or agonize one or more specific cognate binding partners.Exemplary methods for assessing such activity are provided herein, including in the Examples.

[0192] In some aspects, immunomodulatory proteins comprising the vIgD of CD80 are provided, which are soluble, e.g., fused to an Fc chain. In some aspects, one or more additional IgSF domains (e.g., one or more additional vIgDs) can be linked to the vIgD of CD80 as provided herein (hereinafter referred to as "stacked" or "stacked" immunomodulatory proteins). In some embodiments, the modular format of the provided immunomodulatory proteins provides flexibility for modifying or generating immunomodulatory proteins to modulate the activity of multiple counter structures (multiple cognate binding partners). In some embodiments, such "stacked" molecules can be provided in a soluble format, and in some cases may be provided as membrane-bound or secreted proteins. In some embodiments, the variant CD80 immunomodulatory protein is provided as a conjugate containing vIgD of CD80 linked, directly or indirectly, to a targeting agent or moiety (e.g., an antibody or other binding molecule) that specifically binds to a ligand (e.g., an antigen), e.g., to target or localize vIgD to a particular environment or cell when administered to a subject. In some embodiments, the targeting agent, e.g., an antibody or other binding molecule, binds to a tumor antigen, thereby localizing the vIgD-containing variant CD80 to the tumor microenvironment, e.g., modulating the activity of tumor-infiltrating lymphocytes (TILs) specific to the tumor microenvironment.

[0193] In some embodiments, the provided immunomodulatory proteins are expressed in cells and provided as part of engineered cell therapy (ECT). In some embodiments, the variant CD80 polypeptide is expressed in a membrane-bound form in a cell, e.g., an immune cell (e.g., a T cell or an antigen-presenting cell), thereby providing a transmembrane immunomodulatory protein (also referred to hereinafter as "TIP"). In some embodiments, depending on the cognate binding partner recognized by TIP, the engineered cell expressing TIP can engage the cognate binding partner by providing either a positive or negative costimulatory signal to other engineered cells and / or endogenous T cells. In some aspects, the variant CD80 polypeptide is expressed in a secreted form in a cell, e.g., an immune cell (e.g., a T cell or an antigen-presenting cell), thereby producing a secreted or soluble form of the variant CD80 polypeptide (also referred to hereinafter as "SIP"), e.g., when the cell is administered to a subject. In some aspects, the SIP can antagonize its cognate binding partner in the environment into which it is secreted (e.g., the tumor microenvironment). In some embodiments, the variant CD80 polypeptide is expressed in an infectious agent (e.g., a viral or bacterial agent) that, upon administration to a subject, is capable of infecting cells, e.g., immune cells (e.g., T cells or antigen-presenting cells), in vivo for delivery or expression of the variant polypeptide into cells as a TIP or SIP.

[0194] In some embodiments, a soluble immunomodulatory polypeptide, such as a variant CD80 containing vIgD, can be encapsulated in a liposome, which can itself be conjugated to any one or any combination of the provided conjugates (e.g., targeting moieties). In some embodiments, the soluble or membrane-bound immunomodulatory polypeptides of the present invention are deglycosylated. In more specific embodiments, the variant CD80 sequence is deglycosylated. In even more specific embodiments, the IgV and / or IgC (e.g., IgC2) domains of the variant CD80 are deglycosylated.

[0195] Non-limiting examples of the formats provided are set forth in Figures 1A-1C and further described below.

[0196] A. Soluble Proteins In some embodiments, the immunomodulatory protein containing a variant CD80 polypeptide is a soluble protein. Those skilled in the art will recognize that cell surface proteins typically have an intracellular domain, a transmembrane domain, and an extracellular domain (ECD), and that the extracellular domain or an immunologically active sequence thereof can be used to produce a soluble form of such a protein. Thus, in some embodiments, the immunomodulatory protein containing a variant CD80 polypeptide lacks the transmembrane domain or a portion of the transmembrane domain. In some embodiments, the immunomodulatory protein containing a variant CD80 lacks the intracellular (cytoplasmic) domain or a portion of the intracellular domain. In some embodiments, the immunomodulatory protein containing a variant CD80 polypeptide contains only a vIgD portion containing an ECD domain or a portion thereof containing an IgV domain and / or an IgC (e.g., IgC2) domain or a specific-binding fragment thereof containing an amino acid modification.

[0197] In some embodiments, an immunomodulatory polypeptide comprising a variant CD80 can comprise one or more variant CD80 polypeptides of the invention. In some embodiments, a polypeptide of the invention comprises exactly one, two, three, four, or five variant CD80 sequences. In some embodiments, at least two of the variant CD80 sequences are the same variant CD80 sequence.

[0198] In some embodiments, the immunomodulatory polypeptides provided comprise two or more vIgD sequences of CD80. Multiple variant CD80 polypeptides within a polypeptide chain can be identical (i.e., homologous) or non-identical (i.e., heterologous) variant CD80 sequences. In addition to single polypeptide chain embodiments, in some embodiments, two, three, four, or more polypeptides of the invention may be covalently or non-covalently linked to one another. Thus, monomeric, dimeric, and higher (e.g., 3-, 4-, 5-, or higher) multimeric proteins are provided herein. In some embodiments, for example, exactly two polypeptides of the invention can be covalently or non-covalently linked to one another to form a dimer. In some embodiments, the linkage is via an interchain cysteine ​​disulfide bond. Compositions comprising two or more polypeptides of the invention can be compositions of polypeptides of the same or substantially the same species (e.g., homodimers) or polypeptides of different species (e.g., heterodimers). Compositions having multiple linked polypeptides of the invention can have one or more identical or non-identical variant CD80 polypeptides of the invention in each polypeptide chain, as described above.

[0199] In some embodiments, the immunomodulatory protein comprises a variant CD80 polypeptide linked to an immunoglobulin Fc (resulting in an "immunomodulatory Fc fusion," e.g., a "variant CD80-Fc fusion," also referred to as a CD80 vIgD-Fc fusion). In some embodiments, the variant CD80 polypeptide is linked at the N-terminus of the Fc. In some embodiments, the variant CD80 polypeptide is linked at the C-terminus of the Fc. In some embodiments, two or more (the same or different) CD80 variant polypeptides are independently linked at the N-terminus and C-terminus.

[0200] In some embodiments, the Fc is a murine Fc or a 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 an IgG1, e.g., a human IgG1. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:226, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:226.

[0201] In some embodiments, the Fc region contains additional modifications that alter (e.g., reduce) one or more of its normal functions. Generally, the Fc region is involved in effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), in addition to antigen binding, which is the primary function of immunoglobulins. Additionally, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by extending in vivo half-life through binding to the in vivo FcRn receptor. In some embodiments, such functions can be reduced or modified in the Fc for use with the provided Fc fusion proteins.

[0202] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the CD80-Fc variant fusion provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has reduced effector function. There are many examples of changes or mutations to the Fc sequence that can alter effector function. For example, WO 00 / 42072, WO 2006019447, WO 2012125850, WO 2015 / 107026, US 2016 / 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 contents of these publications are specifically incorporated herein by reference.

[0203] In some embodiments, the provided variant CD80-Fc fusions comprise an Fc region that exhibits reduced effector function, making them desirable candidates for applications where the in vivo half-life of the CD80-Fc variant fusion is important but certain effector functions (e.g., CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the CD80-Fc variant fusion lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. 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. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., ACTI™ Non-Radiotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96™ Non-Radiotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for 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 assessed in vivo, for example, in an animal model such as that 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 CD80-Fc variant fusion is unable to bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0204] CD80-Fc variant fusions with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region according to EU numbering (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0205] In some embodiments, the Fc region of the CD80-Fc variant fusion comprises an Fc region in which any 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) have been substituted with a different amino acid compared to the native Fc region. Such Fc region modifications are not limited to the above modifications and may include, for example, deglycosylated chains as described in Current Opinion in Biotechnology (2009) 20 (6), 685-691. TIFF2024099752000081.tif41165; modifications such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R described in WO 2008 / 092117; amino acid insertions at positions 233, 234, 235, and 237 (denoted by EU numbering); and modifications at sites described in WO 2000 / 042072.

[0206] Certain Fc variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, WO 2006019447, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0207] In some embodiments, CD80-Fc variant fusions are provided that comprise a variant Fc region comprising one or more amino acid substitutions that extend half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.) or WO 2015107026. These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues according to EU numbering: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at residue 434 of the Fc region (U.S. Patent No. 7,371,826).

[0208] In some embodiments, the Fc region of the CD80-Fc variant fusion comprises one or more amino acid substitutions E356D and M358L. In some embodiments, the Fc region of the CD80-Fc variant fusion comprises one or more amino acid substitutions C220S, C226S, C229S. In some embodiments, the Fc region of the CD80 variant fusion comprises one or more amino acid substitutions R292C and V302C. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0209] In some embodiments, modifications are made in the Fc region that result in reduced C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0210] In some embodiments, a CD80-Fc variant fusion is provided comprising a variant Fc region comprising one or more amino acid modifications, wherein the variant Fc region is derived from an IgG1, e.g., human IgG1. In some embodiments, the variant Fc region is derived from the amino acid sequence set forth in SEQ ID NO:226. In some embodiments, the Fc contains at least one amino acid substitution that is N82G according to the numbering of SEQ ID NO:226 (corresponding to N297G according to EU numbering). In some embodiments, the Fc further contains at least one amino acid substitution that is R77C or V87C according to the numbering of SEQ ID NO:226 (corresponding to R292C or V302C according to EU numbering). In some embodiments, the variant Fc region further comprises a C5S (corresponding to C220S according to EU numbering) amino acid modification according to the numbering of SEQ ID NO:226. For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: N82G and one or more of the amino acid modifications C5S, R77C, or V87C relative to SEQ ID NO:226.

[0211] In some embodiments, a CD80-Fc variant fusion is provided comprising a variant Fc region, wherein the variant Fc comprises an amino acid sequence set forth in any of SEQ ID NOs: 389, 392-394, and 413, or an amino acid sequence that exhibits 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 NOs: 389, 392-394, and 413.

[0212] In some embodiments, the Fc is derived from IgG2, e.g., human IgG2. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:227, or an amino acid sequence that exhibits 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:227.

[0213] In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 411, or an amino acid sequence that exhibits 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: 411. In some embodiments, the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is replaced with the CH3 domain of human IgG1 and exhibits inhibited aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are replaced with the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which arginine at position 409 of human IgG4 as defined by the EU index proposed by Kabat et al. is replaced with lysine and 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, which has been shown to prevent recombination between therapeutic antibodies 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:412, or an amino acid sequence exhibiting 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:412.

[0214] In some embodiments, the variant CD80 polypeptide is directly linked to the Fc sequence. In some embodiments, the variant CD80 polypeptide is indirectly linked to the Fc sequence, e.g., via a linker. In some embodiments, one or more "peptide linkers" link the variant CD80 polypeptide and the Fc domain. In some embodiments, the peptide linker can be a single amino acid residue or longer in length. In some embodiments, the peptide linker has at least one amino acid residue but is no longer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length. In some embodiments, the linker is (in the single-letter amino acid code) GGGGS ("4GS") or a multimer of a 4GS linker, e.g., two, three, four, or five repeats of the 4GS linker.

[0215] In some embodiments, the variant CD80-Fc fusion protein is a dimer formed by two variant CD80 Fc polypeptides linked to Fc domains. In some specific embodiments, CD80-Fc variant fusion polypeptides of the same or substantially the same species (allowing for three or fewer N-terminal or C-terminal amino acid sequence differences) are dimerized to produce a homodimer. In some embodiments, the dimer is a homodimer in which the two variant CD80 Fc polypeptides are the same. Alternatively, CD80-Fc variant fusion polypeptides of different species can be dimerized to produce a heterodimer. Thus, in some embodiments, the dimer is a heterodimer in which the two variant CD80 Fc polypeptides are different.

[0216] Also provided are nucleic acid molecules encoding variant CD80-Fc fusion proteins. In some embodiments, the nucleic acid molecules encoding the variant CD80-Fc fusion proteins are inserted into an appropriate expression vector for production of the Fc fusion protein. The resulting variant CD80-Fc fusion proteins can be expressed in host cells transformed with the vector, where the interchain disulfide bonds formed between the Fc moieties result in assembly between the Fc domains to produce dimers, such as bivalent variant CD80-Fc fusion proteins.

[0217] The resulting Fc fusion protein can be easily purified by affinity chromatography on a Protein A or Protein G column. For heterodimer formation, additional purification steps may be required. For example, when cells are transformed with two nucleic acids encoding different variant CD80 polypeptides, the variant CD80 molecules bearing the Fc domain are also expressed as disulfide-linked homodimers, so heterodimer formation must be achieved biochemically. Thus, homodimer formation can be reduced under conditions that favor the disruption of interchain disulfides but do not affect the interchain disulfides. In some cases, different variant CD80 Fc monomers are mixed in equimolar amounts and oxidized to form a mixture of homodimers and heterodimers. The components of this mixture are separated by chromatographic techniques. Alternatively, the formation of this type of heterodimer can be biased by genetically modifying and expressing Fc fusion molecules containing variant CD80 polypeptides using the knob-into-hole method described below.

[0218] B. Stacked molecules with additional IgSF domains In some embodiments, the immunomodulatory protein can contain any of the variant CD80 polypeptides provided herein linked, directly or indirectly, to one or more other immunoglobulin superfamily (IgSF) domains ("stacked" immunomodulatory protein constructs, also referred to as "type II" immunomodulatory proteins). In some aspects, this can create unique multidomain immunomodulatory proteins that provide multitargeted modulation of the immune synapse by binding to two or more, e.g., three or more, cognate binding partners.

[0219] In some embodiments, the immunomodulatory protein comprises a combination ("non-wild-type combination") and / or arrangement ("non-wild-type arrangement" or "non-wild-type permutation") of a variant CD80 domain with one or more other affinity-modified and / or affinity-unmodified IgSF domain sequences of another IgSF family member (e.g., a mammalian IgSF family member) that is not found in a wild-type IgSF family member. In some embodiments, the immunomodulatory protein contains two, three, four, five, or six immunoglobulin superfamily (IgSF) domains, wherein at least one of the IgSF domains is a variant CD80 IgSF domain (vIgD of CD80) according to the description provided.

[0220] In some embodiments, the sequence of the additional IgSF domain can be a modified IgSF domain containing one or more amino acid modifications (e.g., substitutions) compared to a wild-type or unmodified IgSF domain. In some embodiments, the IgSF domain can be affinity-unmodified (e.g., wild-type) or affinity-modified. In some embodiments, the unmodified or wild-type IgSF domain can be from mouse, rat, cynomolgus monkey, or human origin, or a combination thereof. In some embodiments, the additional IgSF domain can be an IgSF domain of an IgSF family member shown in Table 2. In some embodiments, the additional IgSF domain can be an affinity-modified IgSF domain containing one or more amino acid modifications (e.g., substitutions) compared to an IgSF domain contained in an IgSF family member shown in Table 2.

[0221] In some embodiments, the additional IgSF domain is an affinity-modified or unmodified IgSF domain contained in an IgSF family member of a family selected from the following: the signal-regulatory receptor (SIRP) family, the triggering receptor-like expressed on myeloid cells (TREML) family, the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, the sialic acid-binding Ig-like lectin (SIGLEC) family, the butyrophilin family, the B7 family, the CD28 family, the V-set and immunoglobulin domain-containing (VSIG) family, the V-set transmembrane domain (VSTM) family, the major histocompatibility complex (MHC) family, the signaling lymphocyte activation molecule (SLAM) family, the leukocyte immunoglobulin-like receptor (LIR), the nectin (Nec) family, the nectin-like (NECL) family, the poliovirus receptor-related (PVR) family, the natural cytotoxicity-inducing receptor (NCR) family, the T-cell immunoglobulin and mucin (TIM) family, or the killer cell immunoglobulin-like receptor (KIR) family. In some embodiments, the additional IgSF domains are independently selected from the group consisting of CD80 (B7-1), CD86 (B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2 (PD-L2, CD273), ICOSLG (B7RP1, CD275, ICOSL, B7-H2), CD276 (B7-H3), VTCN1 (B7-H4), CD28, CTLA4, PDCD1 (PD-1), ICOS, BTLA (CD272), CD4, CD8A (CD8α), CD8B (CD8β), LAG3, HAVCR2 (TIM-3), CEACAM1, TIGIT, PVR (CD155), PVRL2 (CD112), CD226, CD2, CD160, CD200, CD200R1 (CD200R), and NC R3 (NKp30).

[0222] The first column of Table 2 provides the name and, in some cases, several possible aliases for that particular IgSF member. The second column provides the protein identifier from the UniProtKB database, a publicly accessible database via the Internet at uniprot.org, and in some cases, provides 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 primarily supported by grants from the US National Institutes of Health (NIH). GenBank is the NIH's gene sequence database, a collection of all publicly available, annotated DNA sequences (Nucleic Acids Research, 2013 Jan;41(D1):D36-42). Column 3 provides the region in which the indicated IgSF domain is located. The region is identified as the range encompassing the residues that the domain defines. Column 3 also indicates the IgSF domain class of the identified IgSF region. Column 4 provides the region in which the indicated additional domains are located (signal peptide, S; extracellular domain, E; transmembrane domain, T; cytoplasmic domain, C). It should be understood that the domain descriptions may vary depending on the method used to identify or classify the domains and may be independently identified from different sources. The descriptions of residues corresponding to domains in Table 2 are exemplary only and may be several amino acids longer or shorter (e.g., 1, 2, 3, or 4).Column 5 indicates some of the IgSF members listed (ie, some of their cell surface cognate binding partners).

[0223] Table 2. IgSF members of the present disclosure TIFF2024099752000082.tif104170TIFF2024099752000083.tif230167TIFF2024099752000084.tif222167TIFF2024099752000085.tif62167

[0224] The number of such affinity-unmodified or affinity-modified IgSF domains (whether in non-wild-type combinations or non-wild-type arrangements) present in a "stacked" immunomodulatory protein construct is at least two, three, four, or five, and in some embodiments exactly two, three, four, or five IgSF domains (whereby the determination of the number of affinity-modified IgSF domains ignores any non-specific binding split sequences thereof and / or split sequences thereof that are substantially immunologically inactive).

[0225] In some embodiments of the stacked immunomodulatory proteins provided herein, the number of IgSF domains is at least two, and wherein the number of affinity-modified IgSF domains and the number of affinity-unmodified IgSF domains are each independently at least 0, 1, 2, 3, 4, 5, or 6. Thus, the number of affinity-modified IgSF domains and the number of affinity-unmodified IgSF domains, respectively (affinity-modified IgSF domains:affinity-unmodified IgSF domains), can be exactly or at least 2:0 (affinity-modified:wild-type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2, 2:4, 4:2, 1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1.

[0226] In some embodiments of the stacked immunomodulatory proteins, at least two of the non-affinity-modified and / or affinity-modified IgSF domains are identical IgSF domains.

[0227] In some embodiments, the stacked immunomodulatory proteins provided herein comprise at least two affinity-modified and / or affinity-unmodified IgSF domains derived from a single IgSF member but in a non-wild-type arrangement (or "permutation"). One example of a non-wild-type arrangement or permutation is an immunomodulatory protein of the invention comprising affinity-modified and / or affinity-unmodified IgSF domain sequences of a non-wild-type series compared to those found in wild-type CD80, which IgSF domain sequences serve as the source of the variant IgSF domains as provided herein. Thus, in one example, the immunomodulatory protein can comprise an IgV proximal to the transmembrane domain and an IgC distal to the transmembrane domain, regardless of whether the affinity-unmodified and / or affinity-modified form is present. It is also within the scope of the provided subject matter that both non-wild-type combinations and non-wild-type arrangements of affinity-unmodified and / or affinity-modified IgSF domains are present in the immunomodulatory proteins provided herein.

[0228] In some embodiments of stacked immunomodulatory proteins, the affinity-unmodified and / or affinity-modified IgSF domains are non-identical (i.e., different) IgSF domains. Non-identical affinity-modified IgSF domains specifically bind to different cognate binding partners under specific binding conditions and are "non-identical" regardless of whether the wild-type or unmodified IgSF domains to which they are modified are the same. Thus, for example, a non-wild-type combination of at least two non-identical IgSF domains in an immunomodulatory protein can include at least one IgSF domain sequence that originates from one CD80 and is unique, and at least one second IgSF domain sequence that originates from another IgSF family member other than CD80 and is unique, where the IgSF domains of the immunomodulatory protein are in their affinity-unmodified and / or affinity-modified forms. However, in alternative embodiments, the two non-identical IgSF domains originate from the same IgSF domain sequence, but at least one has been affinity-modified so that it specifically binds to a different cognate binding partner.

[0229] In some embodiments, the provided immunomodulatory proteins, in addition to containing a variant CD80 polypeptide, also contain at least two, three, four, five, or six additional immunoglobulin superfamily (IgSF) domains (e.g., the IgD domain of an IgSF family member set forth in Table 2). In some embodiments, the provided immunomodulatory proteins contain at least one additional IgSF domain (e.g., a second IgSF domain), wherein the at least one additional or second IgSF domain is a wild-type or unmodified IgSF domain contained in the amino acid sequence set forth in any of SEQ ID NOs:1-27 and 348, or a specific-binding fragment thereof. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, e.g., an IgC1 or IgC2 domain.

[0230] In some embodiments, the provided immunomodulatory proteins, in addition to containing a variant CD80 polypeptide, also contain at least one additional IgSF domain (e.g., a second IgSF domain) that is vIgD that contains one or more amino acid modifications (e.g., substitutions, deletions, or mutations) compared to an IgSF domain within a wild-type or unmodified IgSF domain, e.g., an IgSF domain within an IgSF family member set forth in Table 2. In some embodiments, the additional or second affinity-modified IgSF domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a wild-type or unmodified IgSF domain, or a specific-binding fragment thereof, contained in the amino acid sequence set forth in any of SEQ ID NOs: 1-27 and 348. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, e.g., an IgC1 or IgC2 domain. In some embodiments, the additional or second IgSF domain is an affinity-modified IgV domain or an IgC domain.

[0231] In some embodiments, one or more additional IgSF domains (e.g., a second IgSF domain) are IgSF domains of another IgSF family member (e.g., IgV) that bind to or recognize a tumor antigen. In such embodiments, the IgSF family member serves as a tumor-localizing moiety, thereby bringing the vIgD of CD80 into close proximity with immune cells within the tumor microenvironment. In some embodiments, the additional IgSF domain (e.g., a second IgSF domain) is an IgSF domain of NkP30 that binds to or recognizes B7-H6 expressed on tumor cells. In some embodiments, at least one additional (e.g., a second) IgSF domain, e.g., NkP30, is a vIgD containing one or more amino acid modifications (e.g., substitutions, deletions, or additions). In some embodiments, the one or more amino acid modifications improve binding affinity and / or selectivity for B7-H6, e.g., by at least or at least 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, or 50-fold, compared to an unmodified IgSF domain (e.g., NkP30). Among exemplary polypeptides is an NKp30 variant containing the mutations L30V / A60V / S64P / S86G relative to positions in the NKp30 extracellular domain corresponding to the positions set forth in SEQ ID NO:54.

[0232] Tables 3-5 provide exemplary polypeptides containing one or more affinity-engineered IgSF domains that can be used in the stack constructs provided herein.

[0233] Table 3: Exemplary variant ICOSL polypeptides TIFF2024099752000086.tif166170

[0234] Table 4: Exemplary variant NKp30 polypeptides TIFF2024099752000087.tif43170

[0235] Table 5. Exemplary variant CD86 polypeptides TIFF2024099752000088.tif35170

[0236] In some embodiments, two or more IgSF domains, including the vIgD of CD80 and one or more additional IgSF domains (e.g., a second variant IgSF domain) from another IgSF family member, are covalently or non-covalently linked. Multiple affinity-unmodified and / or affinity-modified IgSF domains within a stacked immunomodulatory protein polypeptide chain need not be directly covalently linked to each other. In some embodiments, two or more IgSF domains are directly or indirectly linked, for example, via a linker. In some embodiments, an intervening span of one or more amino acid residues indirectly covalently links the IgSF domains to each other. Linkage can be via residues from the N-terminus to the C-terminus. In some embodiments, linkage can be via the side chain of an amino acid residue not located at the N-terminus or C-terminus of the IgSF domain. Thus, linkage can be via terminal or internal amino acid residues, or a combination thereof.

[0237] In some embodiments, one or more "peptide linkers" connect the vIgD of CD80 to an additional IgSF domain (e.g., a second variant IgSF domain). In some embodiments, the peptide linker can be a single amino acid residue or longer in length. In some embodiments, the peptide linker has at least one amino acid residue but is no longer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in length. In some embodiments, the linker is (in the single-letter amino acid code): GGGGS ("4GS") or a multimer of 4GS linkers, e.g., 2, 3, 4, or 5 repeats of the 4GS linker. In some embodiments, the peptide linker is (GGGGS)2 or (GGGGS)3. In some embodiments, the linker can also include a series of alanine residues, alone or in addition to another peptide linker (e.g., a 4GS linker or multimer thereof). In some embodiments, the number of alanine residues in each stretch is 2, 3, 4, 5, or 6 alanines.

[0238] In some embodiments, the affinity-unmodified and / or affinity-modified IgSF domains are linked by a "wild-type peptide linker" inserted at the N-terminus and / or C-terminus of the first and / or second affinity-unmodified and / or affinity-modified IgSF domains. These linkers are also referred to as leading sequences (N-terminus of the affinity-unmodified or affinity-modified IgSF domain) or trailing sequences (C-terminus of the affinity-unmodified or affinity-modified IgSF domain), and sequences present in the wild-type that extend just outside the predicted structure of the IgSF Ig fold. In some embodiments, a "wild-type linker" is an amino acid sequence that occurs after the signal sequence but before the IgSF domain (e.g., a defined IgV domain) in the amino acid sequence of the wild-type protein. In some embodiments, a "wild-type" linker is an amino acid sequence that occurs immediately after the IgSF domain (e.g., immediately after the defined IgV domain) but before the IgC domain in the amino acid sequence of the wild-type protein. These linker sequences can contribute to the proper folding and function of adjacent IgSF domains. In some embodiments, there is a leading peptide linker inserted at the N-terminus of a first IgSF domain and / or a trailing sequence inserted at the C-terminus of a first affinity-unmodified and / or affinity-modified IgSF domain. In some embodiments, there is a second leading peptide linker inserted at the N-terminus of a second IgSF domain and / or a second trailing sequence inserted at the C-terminus of a second affinity-unmodified and / or affinity-modified IgSF domain. When the first and second affinity-unmodified and / or affinity-modified IgSF domains are derived from the same parent protein and are connected in the same orientation, the wild-type peptide linkers between the first and second affinity-unmodified and / or affinity-modified IgSF domains do not overlap.For example, when the first trailing wild-type peptide linker and the second leading wild-type peptide linker are the same, the type II immunomodulatory protein does not include either the first trailing wild-type peptide linker or the second leading wild-type peptide linker.

[0239] In some embodiments, the type II immunomodulatory protein comprises a first leading wild-type peptide linker inserted at the N-terminus of a first affinity-unmodified and / or affinity-modified IgSF domain, wherein the first leading wild-type peptide linker comprises at least five (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) contiguous amino acids from an intervening sequence in the wild-type protein from which the first affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (e.g., signal peptide or IgSF domain). In some embodiments, the first leading wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the first affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (e.g., signal peptide or IgSF domain).

[0240] In some embodiments, the type II immunomodulatory protein further comprises a first trailing wild-type peptide linker inserted at the C-terminus of the first affinity-unmodified and / or affinity-modified IgSF domain, wherein the first trailing wild-type peptide linker comprises at least five (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) contiguous amino acids from an intervening sequence in the wild-type protein from which the first affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately following domain (e.g., the IgSF domain or the transmembrane domain). In some embodiments, the first trailing wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the first affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately following domain (e.g., the IgSF domain or the transmembrane domain).

[0241] In some embodiments, the type II immunomodulatory protein further comprises a second leading wild-type peptide linker inserted at the N-terminus of the second affinity-unmodified and / or affinity-modified IgSF domain, wherein the second leading wild-type peptide linker comprises at least five (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) contiguous amino acids from an intervening sequence in the wild-type protein from which the second affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (e.g., signal peptide or IgSF domain). In some embodiments, the second leading wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the second affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately preceding domain (e.g., signal peptide or IgSF domain).

[0242] In some embodiments, the type II immunomodulatory protein further comprises a second trailing wild-type peptide linker inserted at the C-terminus of the second affinity-unmodified and / or affinity-modified IgSF domain, wherein the second trailing wild-type peptide linker comprises at least five (e.g., at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) contiguous amino acids from an intervening sequence in the wild-type protein from which the second affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately following domain (e.g., IgSF domain or transmembrane domain). In some embodiments, the second trailing wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the second affinity-unmodified and / or affinity-modified IgSF domain is derived, between the parent IgSF domain and the immediately following domain (e.g., IgSF domain or transmembrane domain).

[0243] An exemplary trailing sequence for a type II protein containing a CD80 IgSF domain may contain the amino acid sequence set forth in SEQ ID NO:232. Exemplary trailing sequences for a type II protein containing a CD80 IgSF domain are set forth in SEQ ID NOs:231, 232, and 371. Exemplary trailing sequences for a type II protein containing an ICOSL IgSF domain are set forth in SEQ ID NOs:233 and 234. Exemplary leading and trailing sequences for a type II protein containing a CD86 IgSF domain are set forth in SEQ ID NOs:236-238. An exemplary trailing sequence for a type II protein containing an NKp30 IgSF domain is set forth in SEQ ID NO:235.

[0244] In some embodiments, two or more IgSF domains, including the vIgD of CD80 and one or more additional IgSF domains (e.g., a second variant IgSF domain) from another IgSF family member, are linked or attached to Fc to form a dimeric multidomain stacked immunomodulatory protein. In some embodiments, the variant CD80 polypeptide and the second IgSF domain are independently linked, directly or indirectly, to the N- or C-terminus of the Fc subunit. In some embodiments, the variant CD80 polypeptide and the second IgSF domain are linked, directly or indirectly, and one of the variant CD80 or the second IgSF domain is also linked, directly or indirectly, to the N- or C-terminus of the Fc subunit. In some embodiments, the link to the Fc is via a peptide linker (e.g., a peptide linker as described above). In some embodiments, the link between the variant CD80 and the second IgSF domain is via a peptide linker (e.g., a peptide linker as described above). In some embodiments, the vIgD, one or more additional IgSF domains, and the Fc domain of CD80 can be linked together in any of a number of configurations as shown in Figure 4. Exemplary configurations are described in the Examples.

[0245] In some embodiments, the stacked immunomodulatory protein is a dimer formed by two stacked immunomodulatory Fc fusion polypeptides. Also provided are nucleic acid molecules encoding any of the stacked immunomodulatory proteins. In some embodiments, the dimeric multidomain stacked immunomodulatory protein can be produced in cells by expression, or in some cases, co-expression, of the stacked immunomodulatory Fc fusion polypeptides described above, following the production of the dimeric Fc fusion protein.

[0246] In some embodiments, the dimeric multidomain stack immunomodulatory protein is bivalent for each Fc subunit, monovalent for each subunit, or bivalent for one subunit and tetravalent for the other.

[0247] In some embodiments, the dimeric multidomain stacked immunomodulatory protein is a homodimeric multidomain stacked Fc protein. In some embodiments, the dimeric multidomain stacked immunomodulatory protein comprises a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first and second polypeptides are the same. In some embodiments, the Fc portion of the polypeptide can be any Fc, as described above.

[0248] In some embodiments, the multidomain stack molecule is a heterodimer comprising two different Fc polypeptides, at least one of which is an Fc polypeptide containing at least one variant CD80 polypeptide and / or at least one second IgSF domain (e.g., a second variant IgSF domain). In some embodiments, the multidomain stack molecule contains a first Fc polypeptide containing a variant CD80 and a second IgSF domain and a second Fc polypeptide containing a variant CD80 and a second IgSF domain. In some embodiments, the multidomain stack molecule contains a first Fc polypeptide containing a variant CD80 polypeptide and a second IgSF domain and a second Fc polypeptide that is not linked to either the variant CD80 polypeptide or the second IgSF domain.

[0249] In some embodiments, the multidomain stack molecule contains a first Fc polypeptide containing one, two, three, four, or more variant CD80 polypeptides and one, two, three, four, or more second IgSF domains, such that the total number of IgSF domains in the first stacked Fc polypeptide is two, three, four, five, six, or more. In one example of such an embodiment, the second stacked Fc polypeptide contains one, two, three, four, or more variant CD80 polypeptides and one, two, three, four, or more second IgSF domains, such that the total number of IgSF domains in the first stacked Fc polypeptide is two, three, four, five, six, or more. In another example of such an embodiment, the second Fc polypeptide is not linked to either the variant CD80 polypeptide or the second IgSF domains.

[0250] In some embodiments, the heterodimeric stack molecule contains a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first and second polypeptides are different. In some embodiments, the heterodimeric stack molecule contains a first Fc subunit containing a first variant CD80 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain), and a second Fc subunit containing the other of the first variant CD80 polypeptide or the second IgSF domain. In some embodiments, the heterodimeric stack molecule contains a first stacked immunomodulatory Fc fusion polypeptide and a second stacked immunomodulatory Fc fusion polypeptide, wherein the first and second polypeptides are different. In some embodiments, the heterodimeric stack molecule contains a first Fc subunit containing a first variant CD80 polypeptide and / or a second IgSF domain (e.g., a second variant IgSF domain), and a second Fc subunit containing both the first variant CD80 polypeptide and a second IgSF domain (e.g., a second variant IgSF domain), but in a different orientation or configuration than the first Fc subunit.

[0251] In some embodiments, the Fc domain of one or both of the first and second stacked immunomodulatory Fc fusion polypeptides comprises a modification (e.g., a substitution) such that the interface of the Fc molecules is altered to facilitate and / or promote heterodimerization. In some embodiments, the modification comprises the introduction of a protrusion (knob) into the first Fc polypeptide and a cavity (hole) into the second Fc polypeptide, thereby facilitating complexation of the first and second Fc-containing polypeptides by allowing the protrusion to be positioned within the cavity. The amino acids targeted for replacement and / or modification to create the protrusion or cavity within the polypeptide are typically interface amino acids that interact or contact one or more amino acids at the interface of the second polypeptide.

[0252] In some embodiments, the first polypeptide modified to contain a protrusion (hole) amino acid comprises a replacement of a natural or original amino acid with an amino acid having at least one side chain that protrudes from the interface of the first polypeptide and can therefore be positioned in a compensatory cavity (hole) in the adjacent interface of the second polypeptide. In most cases, the replacement amino acid has a larger side chain volume than the original amino acid residue. Those skilled in the art will know how to determine and / or evaluate the properties of an amino acid residue to identify an ideal replacement amino acid to create the protrusion. In some embodiments, the replacement residue for forming the protrusion is a naturally occurring amino acid residue, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the original residue identified for replacement is an amino acid residue with a small side chain, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0253] In some embodiments, the second polypeptide to be modified to contain a cavity (hole) comprises a natural or original amino acid replaced with an amino acid having at least one side chain that is recessed from the interface of the second polypeptide and can accommodate a corresponding protrusion from the interface of the first polypeptide. In most cases, the replacement amino acid has a smaller side chain volume than the original amino acid residue. Those skilled in the art know how to determine and / or evaluate the characteristics of amino acid residues to identify ideal replacement residues for cavity formation. Generally, replacement residues for cavity formation are naturally occurring amino acids, including, for example, alanine (A), serine (S), threonine (T) and valine (V). In some examples, the original amino acid identified for replacement is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0254] The CH3 interface of human IgG1, for example, involves 16 residues on each domain located on four antiparallel β-strands, buried 1090 Å from each surface (see, e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168). The modification of CH3 domain to create protrusion or cavity is described in, for example, U.S. Patent No. 5,731,168; International Patent Application WO 98 / 50431 and WO 2005 / 063816; and Ridgway et al., (1996) Prot. Engin., 9: 617-621.In some examples, the modification of CH3 domain to create protrusion or cavity is typically targeted to the residue located on the two central antiparallel β-strands.The purpose is to minimize the risk that the protrusion created may be accommodated by protruding into the surrounding solvent, rather than being accommodated by the compensatory cavity in the partner CH3 domain.

[0255] In some embodiments, the heterodimeric molecule contains a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A, or Y407V mutation in the CH3 domain of the "hole chain." In some cases, an additional interchain disulfide bridge between the CH3 domains (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681) can also be used, for example, by introducing a Y349C mutation in the CH3 domain of the "knob" or "hole" chain and an E356C or S354C mutation in the CH3 domain of the other chain. In some embodiments, the heterodimeric molecule contains an S354C or T366W mutation in one of the two CH3 domains and a Y349C, T366S, L368A, or Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises an E356C, T366W mutation in one of the two CH3 domains and an Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises a Y349C, T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises a Y349C, T366W mutation in one of the two CH3 domains and an S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. Other examples of knob-in-hole technology are known in the art, for example, as described in EP 1 870 459 A1.

[0256] In some embodiments, the Fc subunit of the heterodimeric molecule can additionally contain one or more other Fc mutations, such as any of those described above, hi some embodiments, the heterodimeric molecule contains an Fc subunit with a mutation that reduces effector function.

[0257] In some embodiments, an Fc variant containing a CH3 protrusion / cavity modification can be connected anywhere in the stacked immunomodulatory polypeptide, but typically via its N- or C-terminus, to the N- or C-terminus of a first and / or second stacked immunomodulatory polypeptide, for example, to form a fusion polypeptide. The linkage can be direct or indirect via a linker. Typically, the knob and hole molecules are produced by coexpression of a first stacked immunomodulatory polypeptide linked to an Fc variant containing a CH3 protrusion modification and a second stacked immunomodulatory polypeptide linked to an Fc variant containing a CH3 cavity modification.

[0258] C. Conjugates and Fusions of Variant Polypeptides and Immunomodulatory Proteins In some embodiments, the variant polypeptides provided herein, which are immunomodulatory proteins comprising a variant of the IgSF family Ig domain (vIgD), can be conjugated or fused, directly or indirectly, to a moiety, e.g., an effector moiety, e.g., another protein, to form a conjugate ("IgSF conjugate"). In some embodiments, the attachment can be covalent or non-covalent (e.g., via a biotin-streptavidin non-covalent interaction). In some embodiments of the CD80-Fc variant fusion, any one or combination of any two or more of the aforementioned conjugates can be attached to the Fc or variant CD80 polypeptide, or both.

[0259] In some embodiments, the moiety can be a targeting moiety, a small molecule drug (a non-polypeptide drug of less than 500 Dalton molar mass), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressant, a radioactive substance suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug-activating enzyme, an agent that extends biological half-life, or a diagnostic or detectable agent.

[0260] In some embodiments, the effector moiety is a therapeutic agent that provides cytotoxicity, cytostaticity, or otherwise some therapeutic benefit, e.g., a cancer therapeutic agent. In some embodiments, the effector moiety is a targeting moiety or agent, e.g., an agent that targets a cell surface antigen, e.g., an antigen on the surface of a tumor cell. In some embodiments, the effector moiety is a label that can generate a detectable signal either directly or indirectly. In some embodiments, the effector moiety is a toxin. In some embodiments, the effector moiety is a protein, peptide, nucleic acid, small molecule, or nanoparticle.

[0261] In some embodiments, one, two, three, four, five, or more effector moieties, which may be the same or different, are conjugated, linked, or fused to a variant polypeptide or protein to form an IgSF conjugate. In some embodiments, such effector moieties can be attached to the variant polypeptide or immunomodulatory protein using various molecular biological or chemical conjugation and linking methods known in the art and described below. In some embodiments, the effector moiety can be linked or conjugated to the variant polypeptide or immunomodulatory protein using a linker, such as a peptide linker, a cleavable linker, a non-cleavable linker, or a linker that facilitates the conjugation reaction.

[0262] In some embodiments, the IgSF conjugate comprises the following components: (protein or polypeptide), (L) q and (effector part) mwherein the protein or polypeptide is any of the described variant polypeptides or immunomodulatory proteins capable of binding to one or more cognate counterstructure ligands as described; L is a linker for joining the protein or polypeptide to the moiety; m is at least 1; q is 0 or greater; and the resulting IgSF conjugate binds to one or more counterstructure ligands. In certain embodiments, m is 1 to 4 and q is 0 to 8.

[0263] In some embodiments, an IgSF conjugate is provided that includes a variant polypeptide or immunomodulatory protein provided herein conjugated with a targeting agent that binds to a cell surface molecule, e.g., for targeted delivery of the variant polypeptide or immunomodulatory protein to a specific cell. In some embodiments, the targeting agent is a molecule that has the ability to localize and bind to a molecule present on normal cells / tissues and / or tumor cells / tumors in a subject. In other words, the IgSF conjugate including the targeting agent can bind (directly or indirectly) to a ligand present on cells, e.g., tumor cells. Targeting agents of the present invention that are contemplated for use include antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof, that can bind to a component of a target cell or molecule.

[0264] In some embodiments, the targeting agent can bind to tumor cells or to the vicinity of tumor cells (e.g., tumor vasculature or tumor microenvironment) after administration to a subject. The targeting agent can bind to a receptor or ligand on the surface of cancer cells. In another aspect of the present invention, a targeting agent is selected that is specific to non-cancerous cells or tissues. For example, the targeting agent can be specific to a molecule normally present on a particular cell or tissue. Furthermore, in some embodiments, the same molecule can be present on normal cells and cancer cells. Various cellular components and molecules are known. For example, if the targeting agent is specific to EGFR, the resulting IgSF conjugate can target both EGFR-expressing cancer cells and EGFR-expressing normal skin epithelial cells. Therefore, in some embodiments, the IgSF conjugates of the present invention can act by two separate mechanisms (targeting cancer cells and non-cancerous cells).

[0265] In various aspects of the invention disclosed herein, the IgSF conjugates of the invention comprise a targeting agent capable of binding to / targeting a cellular component, such as a tumor antigen, a bacterial antigen, a viral antigen, a mycoplasmal antigen, a fungal antigen, a prion antigen, or an antigen from a parasite. In some aspects, the cellular component, antigen, or molecule can each be used to refer to the desired target of the targeting agent. For example, in various embodiments, the targeting agent is selected from the group consisting of epidermal growth factor receptor (EGFR, ErbB-1, HER1), ErbB-2 (HER2 / neu), ErbB-3 / HER3, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF-binding proteins (IGFBPs), IGFR ligand family; platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGF) family, and VEGF-binding proteins. long-acting factor receptor (VEGFR) family, VEGF family; HGF receptor family; TRK receptor family; ephrin (EPH) receptor family; AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family, e.g., ROR1; CD171 (L1CAM); B7-H6 (NCR3LG1); PD-L1, tumor glycosylated antigens, e.g., sTn or Tn, e.g., sTn of MUC1Ag; LHR (LHCGR); phosphatidylserine, discoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; transforming growth factor-α (TGF-α) receptor, TGF-β; cytokine receptors, class I (hematopoietin family) and class II (interferon / IL-10 family) receptors, tumor necrosis factor (TNF) receptor superfamily (TNFRSF), death receptor family; cancer-testis (CT) antigen, lineage-specific antigen, differentiation antigen, alpha-actinin-4, ARTCl, breakpoint cluster region-Abelson (Bcr-abl) fusion product, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNBl), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-I, dek-can fusion protein, EFTUD-2, elongation factor 2 (ELF2), Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), e.g., fibronectin ectodomain A (EDA), GPNMB, low-density lipid receptor / GDP-L-fucose:β-D-galactose 2-α-L-fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2 arginine-isoleucine exchange at residue 170 of the α-helix of the α2 domain in the HLA-A2 gene (HLA-A*201-R170I), HLA-A*201-R170I, ...1, heat shock protein 70-2 mutant (HSP70-2M), K1AA0205, MART2, melanoma ubiquitous mutant 1, 2, 3 (MUM-I, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, myosin class I, NFYC, OGT, OS-9, pml-RAR alpha fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPDl, SYT-SSXl or -SSX2 fusion protein, triosephosphate isoform Melase, BAGE, BAGK-1, BAGE-2,3,4,5, GAGE-1,2,3,4,5,6,7,8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGAT5), HERV-K-MEL, KK-LC, KM-HN-I, LAGE, LAGE-I, CTL-recognized antigen on melanoma (CAMEL), MAGE-Al (MAGE-I), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-AlO, MAGE-AIl, MAGE-A12, MAGE-3, MAGE-Bl, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-Cl, MAGE-C2, Mucin 1 (MUCl), MART-1 / Melan-A (M LANA), gplOO, gplOO / Pmell7(SILV), tyrosinase (TYR), TRP-I, HAGE, NA-88, NY-ESO-I, NY-ESO-l / LAGE-2, SAGE, Spl 7, SSX-1, 2, 3, 4, TRP2-INT2, carcinoembryonic antigen (CEA), kallikrein 4, mammaglobin-A, OAl, prostate-specific antigen (PSA), TRP-1 / gp75, TRP-2, adipophilin, interferon-inducible protein 2 (AIM-2) absent in melanoma, BING-4, CPSF, cyclin Dl, epithelial cell adhesion molecule (Ep-CAM), EphA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250), EGFR (ERBB1), HER-2 / neu (ERBB2), interleukin-13 receptor α2 chain (IL13Rα2), IL-6 receptor, intestinal carboxylesterase (iCE), alpha-fetoprotein (AFP), M-CSF, mdm-2, MUCl, p53 (TP53), PBF, PRAME, PSMA, RAGE-I, RNF43, RU2AS , SOXlO, STEAPl, survivin (BIRC5), human telomerase reverse transcriptase (hTERT), telomerase, Wilms tumor gene (WTl), SYCPl, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIPl, CTAGE-I, CSAGE, MMAl, CAGE, BORIS, HOM-TES-85, AF15ql4, HCA661, LDHC, MORC, SGY-I, SPOl 1, TPXl, NY-SAR-35, FTHL17, NXF2, TDRDl, TEX15, FATE, TPTE, immunoglobulin idiotype, Bence-Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD30, CD20, CD19, CD33, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA19-9), beta-human chorionic gonadotropin, beta-2 microglobulin, squamous cell carcinoma antigen, neurospecific enolase, heat shock protein gp96, GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinoembryonic antigen peptide-1 (CAP-I), calcium activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), human papillomavirus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsid antigen, etc.), Epstein-Barr virus (EBV) proteins (EBV latent membrane protein-LMP1, LMP2; etc.), hepatitis B or C virus proteins, and HIV proteins.

[0266] In some embodiments, the IgSF conjugate, through its targeting agent, binds to tumor cells, tumor vasculature, or cellular components of the tumor microenvironment, thereby promoting killing of the targeted cells through modulation of the immune response (e.g., by activating costimulatory molecules or inhibiting negative regulatory molecules of immune cell activation), inhibition of survival signals (e.g., growth factor or cytokine or hormone receptor antagonists), activation of death signals, and / or immune-mediated cytotoxicity (e.g., by antibody-dependent cellular cytotoxicity). Such IgSF conjugates can function by several mechanisms to suppress, reduce, or eliminate tumor cells, e.g., to facilitate delivery of a conjugated effector moiety to the tumor target, e.g., by receptor-mediated endocytosis of the IgSF conjugate; or such conjugates can recruit, bind, and / or activate immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells). Moreover, in some cases, one or more of the aforementioned pathways may be affected by administration of one or more IgSF conjugates of the invention.

[0267] In some embodiments, the targeting agent allows the IgSF conjugate to localize to, e.g., bind to, tumor cells, tumor vasculature, or cellular components of the tumor microenvironment, thereby modulating cells of the immune response near the tumor. In some embodiments, the targeting agent facilitates delivery of the conjugated IgSF (e.g., vIgD) to tumor targets, e.g., to interact with its cognate binding partner and alter signaling of immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells) bearing the cognate binding partner. In some embodiments, localized delivery mediates costimulatory signaling via CD28 or ICOS.

[0268] In some embodiments, the targeting agent is an immunoglobulin. As used herein, the term "immunoglobulin" includes natural or artificial monovalent or polyvalent antibodies, including, but not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by an Fab expression library, single-chain Fv (scFv); anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the present invention), and epitope-binding fragments of any of the above. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules containing an antigen-binding site that immunospecifically binds to an antigen. The immunoglobulin molecules of the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0269] In some embodiments, the IgSF conjugate, through its antibody targeting moiety, binds to tumor cells, tumor vasculature, or cellular components of the tumor microenvironment, thereby promoting apoptosis of the targeted cells through modulation of the immune response (e.g., by activating costimulatory molecules or inhibiting negative regulatory molecules of immune cell activation), inhibition of survival signals (e.g., growth factor or cytokine or hormone receptor antagonists), activation of death signals, and / or immune-mediated cytotoxicity (e.g., by antibody-dependent cellular cytotoxicity). Such IgSF conjugates can function through several mechanisms to suppress, reduce, or eliminate tumor cells, e.g., to facilitate delivery of a conjugated effector moiety to the tumor target, e.g., through receptor-mediated endocytosis of the IgSF conjugate; or such conjugates can recruit, bind, and / or activate immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells).

[0270] In some embodiments, the IgSF conjugate, through its antibody targeting moiety, binds to tumor cells, tumor vasculature, or cellular components of the tumor microenvironment, thereby modulating the immune response (e.g., by activating costimulatory molecules or inhibiting negative regulatory molecules of immune cell activation). In some embodiments, such conjugates are capable of recognizing, binding to, and / or modulating (e.g., inhibiting or activating) immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells, T cells, B cells).

[0271] Antibody targeting moieties of the present invention include antibody fragments, including, but not limited to, Fab, Fab', and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and fragments comprising either the VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, can comprise the variable region alone or in combination with all or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the present invention are antigen-binding fragments that also comprise any combination of the variable region and hinge region, CH1, CH2, and CH3 domains. Also included in the present invention are Fc fragments, antigen-Fc fusion proteins, and Fc-targeting moiety conjugates or fusion products (Fc-peptides, Fc-aptamers). The antibody targeting moieties of the present invention can be derived from any animal origin, including birds and mammals. In one aspect, the antibody targeting moiety is human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Furthermore, such antibodies may be humanized versions of animal antibodies. The antibody targeting moieties of the present invention may be monospecific, bispecific, trispecific, or of higher order multispecificity.

[0272] In various embodiments, the antibody / targeting moiety recruits, binds, and / or activates immune cells (e.g., NK cells, monocytes / macrophages, dendritic cells) via interactions between Fc (in the antibody) and Fc receptors (on the immune cells) and via a conjugated variant polypeptide or immunomodulatory protein provided herein. In some embodiments, the antibody / targeting moiety recognizes or binds to tumor material via a conjugated variant polypeptide or immunomodulatory protein provided herein, localizing to tumor cells and facilitating modulation of immune cells in the vicinity of the tumor.

[0273] Examples of antibodies that can be incorporated into the IgSF conjugate include, but are not limited to, antibodies such as cetuximab (IMC-C225; Erbitux®), trastuzumab (Herceptin®), rituximab (Rituxan®; MabThera®), bevacizumab (Avastin®), alemtuzumab (Campath®; Campath-1H®; Mabcampath®), panitumumab (ABX-EGF; Vectibix®), ranibizumab (Lucentis®), ibritumomab, ibritumomab tiuxetan (Zevalin®), tositumomab, iodine I 131 Tositumomab (BEXXAR®), catumaxomab (Removab®), gemtuzumab, gemtuzumab ozogamicin (Mylotarg®), abatacept (CTLA4-Ig; Orencia®), belatacept (L104EA29YIg; LEA29Y; LEA), ipilimumab (MDX-010; MDX-101), tremelimumab (ticilimumab; CP-675,206), PRS-010, PRS-050, aflibercept (VEGF Trap, AVE005), volociximab (M200), F200, MORAb-009, SS1P (CAT-5001), cixutumumab (IMC-A12), matuzumab (EMD72000), nimotuzumab (h-R3), zalutumumab (HuMax-EGFR), necitumumab IMC-11F8, mAb806 / ch806, Sym004, mAb-425, Panorex@(17-1A) (murine monoclonal antibody); Panorex@(17-1A) (chimeric murine monoclonal antibody); IDEC-Y2B8 (murine, anti-CD2O MAb); BEC2 (anti-idiotypic MAb, mimicking the GD epitope) (containing BCG); Oncolym (Lym-1 monoclonal antibody); SMART MI95 Ab, humanized 13'I LYM-I (Oncolym), Ovarex (B43.13, anti-idiotypic murine MAb; MDX-210 (humanized anti-HER-2 bispecific antibody); 3622W94 MAb binding to EGP40 (17-1A) pancarcinoma antigen on adenocarcinoma; anti-VEGF, Zenapax (SMART anti-Tac (IL-2 receptor); SMART MI95 Ab, humanized Ab, humanized); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptor bispecific antibody); NovoMAb-G2 (pancarcinoma-specific Ab); TNT (chimeric MAb against histone antigen); TNT (chimeric MAb against histone antigen); Gliomab-H (Monoclon s-humanized Ab); GNI-250 These include Mab; EMD-72000 (chimeric EGF antagonist); Lymphoside (humanized LL2 antibody); and MDX-260 bispecific, targeting GD-2, ANA Ab, SMART ID10 Ab, SMART ABL 364 Ab, or ImmuRAIT-CEA. As exemplified in the foregoing list, it is routine to produce antibodies against specific target epitopes.

[0274] In some embodiments, the antibody targeting moiety is a full-length antibody or antigen-binding fragment thereof containing an Fc domain. In some embodiments, the variant polypeptide or immunomodulatory protein is conjugated to the Fc portion of the antibody targeting moiety, for example, by conjugation to the N-terminus of the Fc portion of the antibody.

[0275] In some embodiments, vIgD is linked, directly or indirectly, to the N- or C-terminus of the antibody light and / or heavy chain. In some embodiments, the linkage can be via a peptide linker, such as any of those described above. Various configurations can be constructed. Figures 6A-6C show exemplary configurations. In some embodiments, the antibody conjugate can be produced by coexpression of the antibody heavy and light chains in a cell.

[0276] In one aspect of the present invention, the targeting agent is an aptamer molecule. For example, in some embodiments, the aptamer is composed of a nucleic acid that functions as a targeting agent. In various embodiments, the IgSF conjugates of the present invention comprise an aptamer specific to a molecule on tumor cells, tumor vasculature, and / or tumor microenvironment. In some embodiments, the aptamer itself can comprise a biologically active sequence in addition to a targeting module (sequence), where the biologically active sequence can induce an immune response against the target cell. In other words, such an aptamer molecule is a dual-use agent. In some embodiments, the IgSF conjugates of the present invention comprise an aptamer conjugated to an antibody, where the aptamer and the antibody are specific for binding to different molecules on tumor cells, tumor vasculature, tumor microenvironment, and / or immune cells.

[0277] The term "aptamer" includes DNA, RNA, or peptides selected based on their specific binding properties to a particular molecule. For example, aptamers can be selected to bind to specific genes or gene products in tumor cells, tumor vasculature, tumor microenvironment, and / or immune cells as disclosed herein, where selection is performed by methods known in the art and familiar to those skilled in the art.

[0278] In some aspects of the present invention, the targeting agent is a peptide.For example, the variant polypeptide or immunomodulatory protein provided herein can be conjugated to a peptide that can bind to components of cancer or tumor cells.Therefore, such IgSF conjugates of the present invention include peptide targeting agents that bind to cellular components of tumor cells, tumor vasculature, and / or components of the tumor microenvironment.In some embodiments, the targeting agent peptide can be an integrin antagonist or agonist.Integrins containing alpha and beta subunits include many types well known to those skilled in the art.

[0279] In one embodiment, the targeting substance is Vvβ3. Integrin Vvβ3 is expressed on a wide variety of cells and has been shown to mediate several biologically relevant processes, including osteoclast adhesion to bone matrix, vascular smooth muscle cell migration, and angiogenesis. Suitable targeting molecules for integrins include RGD peptides or peptidomimetics and non-RGD peptides or peptidomimetics (see, for example, U.S. Patent Nos. 5,767,071 and 5,780,426) for other integrins, such as V4.βi (VLA-4), V4-P7 (see, for example, U.S. Patent No. 6,365,619; Chang et al., Bioorganic & Medicinal Chem Lett, 12:159-163 (2002); Lin et al., Bioorganic & Medicinal Chem Lett, 12:133-136 (2002)).

[0280] In some embodiments, an IgSF conjugate is provided, comprising a variant polypeptide or immunomodulatory protein provided herein conjugated to a therapeutic agent. In some embodiments, the therapeutic agent includes, for example, daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother. 21:183-187, 1986). In some embodiments, the therapeutic agent has intracellular activity. In some embodiments, the IgSF conjugate is internalized, and the therapeutic agent is a cytotoxin that blocks cellular protein synthesis, leading to cell death therein. In some embodiments, the therapeutic agent is a cytotoxin, including a polypeptide with ribosome-inactivating activity, including, for example, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin, Pseudomonas exotoxin A, and variants thereof. In some embodiments, when the therapeutic agent is a cytotoxin comprising a polypeptide with ribosome-inactivating activity, the IgSF conjugate must be internalized upon binding to the target cell in order for the protein to be cytotoxic to the cell.

[0281] In some embodiments, an IgSF conjugate is provided that comprises a variant polypeptide or an immunomodulatory protein provided herein conjugated to a toxin. In some embodiments, the toxin is, for example, a bacterial toxin, e.g., diphtheria toxin, a plant toxin, e.g., ricin, a small molecule toxin, e.g., geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92 (19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)). Toxins can exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0282] In some embodiments, IgSF conjugates are provided, which comprise the variant polypeptide or immunomodulatory protein provided herein conjugated with a label that can indirectly or directly generate detectable signals.These IgSF conjugates can be used for research or diagnostic purposes, for example, for in vivo cancer detection.Preferably, the label can generate detectable signals either directly or indirectly.For example, the label can be a radiopaque or radioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase; an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom, such as 99Tc or 123I, for scintigraphy studies, or a spin label, such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI). Zirconium-89 may be complexed to various metal chelators and conjugated to antibodies, for example, for PET imaging (WO 2011 / 056983). In some embodiments, the IgSF conjugate is indirectly detectable. For example, the IgSF conjugate can be detected using a secondary antibody specific for the IgSF conjugate and containing a detectable label.

[0283] IgSF conjugates may be prepared using any method known in the art, see, for example, WO 2009 / 067800, WO 2011 / 133886, and U.S. Patent Application Publication No. 2014322129, which are incorporated by reference in their entireties.

[0284] The variant polypeptide or immunomodulatory protein of an IgSF conjugate can be "conjugated" to the effector moiety by any means that allows the variant polypeptide or immunomodulatory protein to associate with or link to the effector moiety. For example, the variant polypeptide or immunomodulatory protein of an IgSF conjugate can be conjugated to the effector moiety by chemical or recombinant means. Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare IgSF conjugates. The method used to conjugate the variant polypeptide or immunomodulatory protein to the effector moiety must be capable of connecting the variant polypeptide or immunomodulatory protein to the effector moiety without interfering with the ability of the variant polypeptide or immunomodulatory protein to bind to its one or more counterstructure ligands.

[0285] The IgSF-conjugated variant polypeptide or immunomodulatory protein may be indirectly linked to an effector moiety. For example, the IgSF-conjugated variant polypeptide or immunomodulatory protein may be directly linked to a liposome containing one of several effector moieties. Alternatively, the effector moiety and / or the variant polypeptide or immunomodulatory protein may be bound to a solid surface.

[0286] In some embodiments, the variant polypeptide or immunomodulatory protein and the effector moiety of the IgSF conjugate are both proteins, and they can be conjugated using techniques well known in the art. There are hundreds of crosslinkers available that can conjugate two proteins (see, for example, "Chemistry of Protein Conjugation and Crosslinking," 1991, Shans Wong, CRC Press, Ann Arbor). Crosslinkers are generally selected based on the reactive functional groups available on or inserted into the variant polypeptide or immunomodulatory protein and / or effector moiety. In addition, if there are no reactive groups, photoactivatable crosslinkers can be used. In certain cases, it may be desirable to include a spacer between the variant polypeptide or immunomodulatory protein and the effector moiety. Cross-linking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyladipimidate, and bis(diazobenzidine), and heterobifunctional agents: m-maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.

[0287] In some embodiments, the variant polypeptide or immunomodulatory protein of the IgSF conjugate may be modified at specific residues for chemical conjugation of the effector moiety. Specific residues known in the art for use in chemical conjugation of molecules include lysine and cysteine. The crosslinker is selected based on the reactive functional groups inserted on the variant polypeptide or immunomodulatory protein and the reactive functional groups available on the effector moiety.

[0288] IgSF conjugates can also be prepared using recombinant DNA technology. In such cases, the DNA sequence encoding the variant polypeptide or immunomodulatory protein is fused to the DNA sequence encoding the effector portion to obtain a chimeric DNA molecule. The chimeric DNA sequence is transfected into host cells that express the fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.

[0289] Examples of conjugating a labeling effector moiety to a variant polypeptide or immunomodulatory protein include those described in Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Radioimmunoimaging And Radioimmunotherapy, Elsevier, NY (1983); and Colcher et al., "Use Of Monoclonal Antibodies As Radiopharmaceuticals For The Localization Of Human Carcinoma Xenografts In Athymic Mice", Meth. Enzymol., 121:802-16 (1986).

[0290] Radiolabels or other labels may be incorporated into the conjugates by known methods. For example, peptides may be biosynthesized or synthesized by chemical amino acid synthesis using suitable amino acid precursors, e.g., containing fluorine-19 instead of hydrogen. Labels such as 99Tc or 123I, 186Re, 188Re, and 111In can be attached via cysteine ​​residues in the peptide. Yttrium-90 can be attached via lysine residues. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun. 80:49-57 (1978)). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0291] Conjugates of variant polypeptides or immunomodulatory proteins and cytotoxic agents can be prepared using a wide variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), activated esters ( For example, radioactive nucleotides may be prepared using disuccinimidyl suberate, aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See, e.g., WO 94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0292] The IgSF conjugates of the present invention expressly contemplate, but are not limited to, drug conjugates prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), available commercially (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA). See pages 467-498 of the Applications Handbook and Catalog 2003-2004.

[0293] D. Transmembrane and Secreted Immunomodulatory Proteins and Modified Cells Provided herein are engineered cells (alternatively, "engineered cells") that express an immunomodulatory variant CD80 polypeptide. In some embodiments, the expressed immunomodulatory variant CD80 polypeptide is a transmembrane protein and is expressed on the surface. In some embodiments, the expressed immunomodulatory variant CD80 polypeptide is expressed in and secreted from the cell.

[0294] 1. Transmembrane immunomodulatory proteins In some embodiments, the immunomodulatory polypeptide comprising a variant CD80 can be a membrane-bound protein. As described in more detail below, the immunomodulatory polypeptide can be a transmembrane immunomodulatory polypeptide comprising a variant CD80 containing an ectodomain containing at least one affinity-engineered IgSF domain (IgV or IgC), a transmembrane domain, and optionally, a cytoplasmic domain. In some embodiments, the transmembrane immunomodulatory protein can be expressed on the surface of an immune cell, e.g., a mammalian cell, including the surface of a lymphocyte (e.g., a T cell or NK cell) or an antigen-presenting cell. In some embodiments, the transmembrane immunomodulatory protein is expressed on the surface of a mammalian T cell, including a T cell such as a helper T cell, a cytotoxic T cell (alternatively, a cytotoxic T lymphocyte or CTL), a natural killer T cell, a regulatory T cell, a memory T cell, or a γδ T cell. In some embodiments, the mammalian cell is an antigen-presenting cell (APC). Typically, but not exclusively, the ectodomain (or alternatively, the "extracellular domain") comprises one or more amino acid modifications (e.g., amino acid substitutions) of a variant CD80 of the invention. Thus, for example, in some embodiments, a transmembrane protein comprises an ectodomain that comprises one or more amino acid substitutions of a variant CD80 of the invention.

[0295] In some embodiments, the engineered cells express a variant CD80 polypeptide that is a transmembrane immunomodulatory polypeptide (TIP), which can be a membrane protein such as a transmembrane protein. In typical embodiments, the ectodomain of the membrane protein comprises the extracellular domain of a variant CD80 provided herein, or an IgSF domain thereof, containing one or more amino acid substitutions in at least one IgSF domain as described. The transmembrane immunomodulatory proteins provided herein further contain a transmembrane domain linked to the ectodomain. In some embodiments, the transmembrane domain provides the encoded protein for cell surface expression on the cell. In some embodiments, the transmembrane domain is directly linked to the ectodomain. In some embodiments, the transmembrane domain is indirectly linked to the ectodomain via one or more linkers or spacers. In some embodiments, the transmembrane domain contains predominantly hydrophobic amino acid residues, such as leucine and valine.

[0296] In some embodiments, a full-length transmembrane anchor domain can be used to ensure that TIP is expressed on the surface of engineered cells, e.g., engineered T cells. Conveniently, this can be derived from the particular native protein (e.g., CD80 or other native IgSF protein) for which affinity is being engineered, and can be simply fused to the sequence of the first membrane-proximal domain in the same manner as a native IgSF protein (e.g., CD80). In some embodiments, the transmembrane immunomodulatory protein comprises the transmembrane domain of a corresponding wild-type or unmodified IgSF member (e.g., the transmembrane domain contained in the amino acid sequence shown in SEQ ID NO:1) (Table 2). In some embodiments, the membrane-bound form comprises the transmembrane domain of a corresponding wild-type or unmodified polypeptide, e.g., corresponding to residues 243-263 of SEQ ID NO:1.

[0297] In some embodiments, the transmembrane domain is a non-native transmembrane domain that is not the transmembrane domain of native CD80. In some embodiments, the transmembrane domain is derived from a transmembrane domain from another non-CD80 family member polypeptide that is a membrane-bound or transmembrane protein. In some embodiments, a transmembrane anchor domain from another protein on T cells can be used. In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, the transmembrane domain can further contain the extracellular portion of CD8 that serves as a spacer domain. Exemplary CD8-derived transmembrane domains are set forth in SEQ ID NO:246 or 399 or portions thereof that contain the CD8 transmembrane domain. In some embodiments, the transmembrane domain is a synthetic transmembrane domain.

[0298] In some embodiments, the transmembrane immunomodulatory protein further comprises an endodomain, such as a cytoplasmic signaling domain, linked to the transmembrane domain. In some embodiments, the cytoplasmic signaling domain induces cell signaling. In some embodiments, the endodomain of the transmembrane immunomodulatory protein comprises the cytoplasmic domain of the corresponding wild-type or unmodified polypeptide, such as the cytoplasmic domain contained in the amino acid sequence set forth in SEQ ID NO:5 (see Table 2).

[0299] In some embodiments, the provided transmembrane immunomodulatory protein is or comprises a variant CD80 and comprises an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:253, and contains an ectodomain and a transmembrane domain that includes at least one affinity-modified CD80 IgSF domain as described. In some embodiments, the transmembrane immunomodulatory protein contains any one or more amino acid substitutions in the IgSF domain (e.g., IgV domain) as described, including any shown in Table 1. In some embodiments, the transmembrane immunomodulatory protein can further comprise a cytoplasmic domain as described. In some embodiments, the transmembrane immunomodulatory protein can further contain a signal peptide. In some embodiments, the signal peptide is the native signal peptide of a wild-type IgSF member, for example, as contained in the amino acid sequence set forth in SEQ ID NO:1 (see, eg, Table 2).

[0300] Also provided are nucleic acid molecules encoding such transmembrane immunomodulatory proteins. In some embodiments, the nucleic acid molecule encoding the transmembrane immunomodulatory protein comprises a nucleotide sequence encoding an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 253, and contains an ectodomain comprising at least one affinity-modified IgSF domain as described, a transmembrane domain, and optionally, a cytoplasmic domain. In some embodiments, the nucleic acid molecule can further comprise a sequence of nucleotides encoding a signal peptide. In some embodiments, the signal peptide is the native signal peptide of the corresponding wild-type IgSF member (see, for example, Table 2).

[0301] An exemplary transmembrane immunomodulatory protein is CD80 TIP, comprising i) the amino acid sequence set forth in SEQ ID NO:241, or ii) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:241 and includes the affinity-modified domain of SEQ ID NO:241 or the amino acid substitutions contained therein. Also provided are i) the nucleotide sequence set forth in SEQ ID NO:242, ii) a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:242 and encodes a TIP comprising the affinity-modified domain of SEQ ID NO:241 or a polypeptide containing amino acid substitutions of SEQ ID NO:241, or iii) a sequence of i) or ii) with degenerate codons.

[0302] In some embodiments, a CAR-associated transmembrane immunomodulatory protein is provided, wherein the endodomain of the transmembrane immunomodulatory protein comprises a cytoplasmic signaling domain that includes at least one ITAM (immunoreceptor tyrosine-based activation motif)-containing signaling domain. ITAM is a conserved motif found in many protein signaling domains involved in immune cell signaling, including the CD3-zeta chain ("CD3-z"), which is involved in T cell receptor signaling. In some embodiments, the endodomain comprises a CD3-zeta signaling domain. In some embodiments, the CD3-zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 333, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 247 and retains T cell signaling activity. In some embodiments, the endodomain of the CAR-associated transmembrane immunomodulatory protein can further comprise a costimulatory signaling domain that further modulates the immunomodulatory response of T cells. In some embodiments, the costimulatory signaling domain is CD28, ICOS, 41BB, or OX40. In some embodiments, the costimulatory signaling domain is derived from CD28 or 4-1BB and comprises an amino acid sequence set forth in any of SEQ ID NOs:400-403, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NOs:400-403 and retains T cell costimulatory signaling activity. In some embodiments, the provided CAR-associated transmembrane immunomodulatory proteins have the characteristic of CARs that stimulate T cell signaling through binding of the affinity-engineered IgSF domain to a cognate binding partner or counter-structure. In some embodiments, specific binding of the affinity-engineered IgSF domain to its counter-structure can lead to altered immune activity of T cell activity, as reflected by changes in cytotoxicity, proliferation, or cytokine production.

[0303] In some embodiments, the transmembrane immunomodulatory protein does not contain an endodomain capable of mediating cytoplasmic signal transduction. In some embodiments, the transmembrane immunomodulatory protein lacks the signal transduction mechanism of the wild-type or unmodified polypeptide and therefore does not induce cell signal transduction by itself. In some embodiments, the transmembrane immunomodulatory protein lacks the intracellular (cytoplasmic) domain or a portion of the intracellular domain of the corresponding wild-type or unmodified polypeptide, for example, the cytoplasmic signal transduction domain contained in the amino acid sequence shown in SEQ ID NO:5 (see Table 2). In some embodiments, the transmembrane immunomodulatory protein does not contain an ITIM (immunoreceptor tyrosine-based inhibitory motif), which is contained in certain inhibitory receptors, including, for example, inhibitory receptors of the IgSF family (e.g., PD-1 or TIGIT). Thus, in some embodiments, the transmembrane immunomodulatory protein contains only an ectodomain and a transmembrane domain (e.g., any of those described).

[0304] 2. Secreted immunomodulatory proteins and modified cells In some embodiments, a CD80 variant immunomodulatory polypeptide containing any one or more of the amino acid mutations described herein is secretable, for example, when expressed in a cell. Such a variant CD80 immunomodulatory protein does not contain a transmembrane domain. In some embodiments, the variant CD80 immunomodulatory protein is not conjugated to a half-life extending moiety (e.g., an Fc domain or a multimerization domain). In some embodiments, the variant CD80 immunomodulatory protein contains a signal peptide, such as an antibody signal peptide or other efficient signal sequence, that directs the domain outside the cell. When the immunomodulatory protein contains a signal peptide and is expressed by a modified cell, the signal peptide causes the immunomodulatory protein to be secreted by the modified cell. Generally, the signal peptide, or a portion of the signal peptide, is cleaved from the immunomodulatory protein upon secretion. The immunomodulatory protein can be encoded by a nucleic acid (which can be part of an expression vector). In some embodiments, the immunomodulatory protein is expressed and secreted by a cell (e.g., an immune cell, e.g., a primary immune cell).

[0305] Thus, in some embodiments, a variant CD80 immunomodulatory protein is provided that further comprises a signal peptide. In some embodiments, provided herein is a nucleic acid molecule encoding a variant CD80 immunomodulatory protein operably linked to a secretory sequence encoding a signal peptide.

[0306] A signal peptide is a sequence at the N-terminus of an immunomodulatory protein that signals secretion of the immunomodulatory protein from a cell. In some embodiments, the signal peptide is about 5 to about 40 amino acids in length (e.g., about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids).

[0307] In some embodiments, the signal peptide is a native signal peptide from the corresponding wild-type CD80 (see Table 2). In some embodiments, the signal peptide is a non-native signal peptide. For example, in some embodiments, the non-native signal peptide is a variant native signal peptide from the corresponding wild-type CD80 and can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutions, insertions, or deletions. In some embodiments, the non-native signal peptide is a signal peptide of a family member from the same IgSF family as the wild-type IgSF family member, or a variant thereof. In some embodiments, the non-native signal peptide is a signal peptide of an IgSF family member from a different IgSF family than the wild-type IgSF family member, or a variant thereof. In some embodiments, the signal peptide is a signal peptide or variant thereof from a non-IgSF protein family, such as a signal peptide from an immunoglobulin (e.g., IgG heavy chain or IgGκ light chain), a cytokine (e.g., interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, luciferase, a trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide capable of efficiently secreting proteins from cells. Exemplary signal peptides include any listed in Table 6.

[0308] Table 6. Exemplary signal peptides TIFF2024099752000089.tif137170

[0309] In some embodiments of the secreted variant CD80 immunomodulatory protein, the immunomodulatory protein includes a signal peptide when expressed, and the signal peptide (or a portion thereof) is cleaved from the immunomodulatory protein upon secretion.

[0310] In some embodiments, the engineered cells express a variant CD80 polypeptide that is secreted from the cells. In some embodiments, such a variant CD80 polypeptide is encoded by a nucleic acid molecule encoding an immunomodulatory protein under the operable control of a signal sequence for secretion. In some embodiments, the encoded immunomodulatory protein is secreted when expressed from the cells. In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule does not include a transmembrane domain. In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule does not include a half-life extending moiety (e.g., an Fc domain or a multimerization domain). In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule includes a signal peptide. In some embodiments, the nucleic acids of the invention further include a nucleotide sequence encoding a secretory or signal peptide operably linked to the nucleic acid encoding the immunomodulatory protein, thereby allowing for secretion of the immunomodulatory protein.

[0311] 3. Cells and Cell Modifications Provided herein are modified cells that express any of the provided immunomodulatory polypeptides. In some embodiments, the modified cells express any of the provided transmembrane immunomodulatory polypeptides on their surface. In some embodiments, the modified cells express the immunomodulatory protein and are capable of secreting it from the cell under conditions suitable for protein secretion, or are capable of secreting it. In some embodiments, the immunomodulatory protein is expressed on lymphocytes, such as tumor-infiltrating lymphocytes (TILs), T cells, or NK cells, or on myeloid cells. In some embodiments, the modified cells are antigen-presenting cells (APCs). In some embodiments, the modified cells are modified mammalian T cells or modified mammalian antigen-presenting cells (APCs). In some embodiments, the modified T cells or APCs are human or murine cells.

[0312] In some embodiments, the modified T cells include, but are not limited to, helper T cells, cytotoxic T cells (alternatively, cytotoxic T lymphocytes or CTLs), natural killer T cells, regulatory T cells, memory T cells, or γδ T cells. + or CD8 + In addition to MHC signals, modified T cells also require costimulatory signals, which in some embodiments are provided by the variant CD80 transmembrane immunomodulatory polypeptide, which is expressed in a membrane-bound form as discussed above.

[0313] In some embodiments, modified APCs include, for example, APCs that express MHC II, such as macrophages, B cells, and dendritic cells, as well as aAPCs, including both cellular and acellular (e.g., biodegradable polymeric microparticles) artificial APCs (aAPCs). Artificial APCs (aAPCs) are synthetic versions of APCs that can act in a similar manner to APCs in that they present antigens to T cells and activate them. Antigen presentation is carried out by MHC (class I or class II). In some embodiments, in modified APCs such as aAPCs, the antigen loaded on the MHC is, in some embodiments, a tumor-specific antigen or tumor-associated antigen. The antigen loaded on the MHC is recognized by the T cell receptor (TCR) of T cells, which in some cases can express CTLA-4 or CD80 or other molecules recognized by the variant CD80 polypeptides provided herein. Materials that can be used to modify aAPCs include poly(glycolic acid), poly(lactic-co-glycolic acid), iron oxide, liposomes, lipid bilayers, sepharose, and polystyrene.

[0314] In some embodiments, the immunomodulatory proteins provided herein, e.g., transmembrane immunomodulatory proteins or secreted immunomodulatory proteins, are co-expressed or engineered in cells expressing an antigen-binding receptor, e.g., a recombinant receptor, e.g., a chimeric antigen receptor (CAR) or T cell receptor (TCR). In some embodiments, the engineered cells, e.g., engineered T cells, recognize a desired antigen associated with cancer, inflammatory and autoimmune disorders, or viral infection. In specific embodiments, the antigen-binding receptor contains an antigen-binding moiety that specifically binds to a tumor-specific antigen or a tumor-associated antigen. In some embodiments, the engineered T cells are CAR (chimeric antigen receptor) T cells that contain an antigen-binding domain (e.g., scFv) that specifically binds to an antigen, e.g., a tumor-specific antigen or a tumor-associated antigen. In some embodiments, the antigen-binding domain (e.g., scFv) is specific for CD19. An exemplary CAR is an anti-CD19 CAR, e.g., a CAR containing the anti-CD19 scFv set forth in SEQ ID NO: 245. In some embodiments, a TIP protein is expressed in engineered T cell receptor cells or engineered chimeric antigen receptor cells. In such embodiments, the modified cells co-express TIP and a CAR or TCR.

[0315] In some embodiments, the CAR further contains an intracellular signaling domain (endodomain) comprising a spacer or hinge, a transmembrane domain, and an ITAM signaling domain (e.g., a CD3ζ signaling domain). In some embodiments, the CAR further comprises a costimulatory signaling domain. In some embodiments, the spacer or hinge is located between the antigen-binding domain and the transmembrane domain, e.g., between the antigen-binding domain and the plasma membrane when expressed on a cell. In some embodiments, the spacer or hinge is derived from an IgG subclass (e.g., IgG1 and IgG4), IgD, or CD8 (see, e.g., Qin et al. (2017) J. Hematol. Oncol., 10:68). In some embodiments, the spacer or hinge is derived from IgG1.

[0316] In some embodiments, the spacer and transmembrane domains are hinge and transmembrane domains derived from CD8, e.g., as set forth in SEQ ID NO:246 or 399, or in an amino acid sequence exhibiting 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:332 or 364. In some embodiments, the endodomain comprises a CD3-zeta signaling domain. In some embodiments, the CD3-zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO:247, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:247 and retains T cell signaling activity. In some embodiments, the endodomain of the CAR-associated transmembrane immunomodulatory protein can further comprise a costimulatory signaling domain that further modulates the immunomodulatory response of T cells. In some embodiments, the costimulatory signaling domain is CD28, ICOS, 41BB, or OX40. In some embodiments, the costimulatory signaling domain is derived from CD28 or 4-1BB and comprises an amino acid sequence set forth in any of SEQ ID NOs:400-403, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs:400-403 and retains T cell costimulatory signaling activity.

[0317] In another embodiment, the modified T cell possesses a TCR comprising a recombinant or modified TCR. In some embodiments, the TCR can be a natural TCR. Those skilled in the art will recognize that natural mammalian T cell receptors generally comprise alpha and beta chains (or gamma and delta chains) involved in antigen-specific recognition and binding. In some embodiments, the TCR is a modified TCR that has been modified. In some embodiments, the TCR of the modified T cell specifically binds to a tumor-associated or tumor-specific antigen presented by an APC.

[0318] In some embodiments, immunomodulatory polypeptides, such as transmembrane immunomodulatory polypeptides or secreted immunomodulatory polypeptides, can be incorporated into engineered cells, such as engineered T cells or engineered APCs, by a variety of strategies, such as those used in recombinant host cells. A variety of methods for introducing DNA constructs into primary T cells are known in the art. In some embodiments, viral transduction or plasmid electroporation is used. In typical embodiments, the nucleic acid molecule encoding the immunomodulatory protein or the expression vector contains a signal peptide that localizes the expressed transmembrane immunomodulatory protein to the cell membrane or for secretion. In some embodiments, the nucleic acid encoding the transmembrane immunomodulatory protein of the present invention is subcloned into a viral vector, such as a retroviral vector, that allows expression in host mammalian cells. The expression vector can be introduced into mammalian host cells, and under host cell culture conditions, the immunomodulatory protein is expressed on the surface or secreted.

[0319] In an illustrative example, primary T cells can be purified ex vivo (CD4 cells or CD8 cells, or both) and stimulated with an activation protocol consisting of beads coated with various TCR / CD28 agonists, such as anti-CD3 / anti-CD28. After a two- or three-day activation process, recombinant expression vectors containing immunomodulatory polypeptides can be stably introduced into primary T cells using standard lentiviral or retroviral transduction protocols or plasmid electroporation strategies. Cells can be monitored for immunomodulatory polypeptide expression, for example, by flow cytometry using anti-epitope tags or antibodies that cross-react with the native parent molecule and polypeptide (including variant CD80). T cells expressing immunomodulatory polypeptides can be enriched by sorting with anti-epitope tag antibodies or enriched for high or low expression, depending on the application.

[0320] Expression of immunomodulatory polypeptides allows modified T cells to be assayed for appropriate function by a variety of means. Coexpression of the modified CAR or TCR can be verified to show that this portion of the modified T cells was largely unaffected by the expression of the immunomodulatory protein. Once verified, standard in vitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN-γ expression) can be used to evaluate the functionality of the modified T cells. Exemplary standard endpoints are the rate of lysis of a tumor line, the proliferation of modified T cells, or IFN-γ protein expression in the culture supernatant. Modified constructs that result in a statistically significant increase in tumor line lysis, increased proliferation of modified T cells, or increased IFN-γ expression relative to a control construct can be selected. Additionally, unmodified cells, such as natural primary or endogenous T cells, can also be incorporated into the same in vitro assay to measure the ability of the immunomodulatory polypeptide constructs expressed on modified cells, such as modified T cells, to modulate the activity of bystander natural T cells (including, in some cases, activating and generating effector functions). Increased expression of activation markers, such as CD69, CD44, or CD62L, can also be monitored on endogenous T cells, and increased proliferation and / or cytokine production can indicate the desired activity of the immunomodulatory protein expressed on the modified T cells.

[0321] In some embodiments, similar assays can be used to compare the function of engineered T cells containing CAR or TCR alone with those containing CAR or TCR and TIP constructs. Typically, these in vitro assays are performed by plating various ratios of engineered T cells and "tumor" cell lines containing cognate CAR or TCR antigens in culture. Standard endpoints are tumor line lysis rate, engineered T cell proliferation, or IFN-γ production in the culture supernatant. Engineered immunomodulatory proteins that resulted in statistically significant increases in tumor line lysis, engineered T cell proliferation, or IFN-γ production relative to the same TCR or CAR construct alone can be selected. Engineered human T cells can be analyzed in immunodeficient mice, such as the NSG strain, which lacks T, NK, and B cells. Engineered human T cells in which CAR or TCR bind to a target counter structure on the xenograft and coexpress the affinity-engineered IgSF domain of TIP can be adoptively transferred in vivo at different cell numbers and ratios compared to the xenograft. For example, CD19 containing a luciferase / GFP vector can be used. + The engraftment of leukemia tumor lines can be monitored by bioluminescence or ex vivo by flow cytometry. In one common embodiment, xenografts are introduced into murine models, followed by the introduction of modified T cells several days later. Modified T cells containing immunomodulatory proteins can be assayed for increased survival, tumor clearance, or the proliferation number of modified T cells compared to modified T cells containing CAR or TCR alone. Similar to in vitro assays, endogenous natural (i.e., unmodified) human T cells can also be co-adoptively transferred to predict the success of epitope spreading in the population, which leads to better survival or tumor clearance.

[0322] E. Infectious Agents Expressing Variant Polypeptides and Immunomodulatory Proteins Also provided are infectious agents containing nucleic acids encoding any of the variant polypeptides described herein, including secreted or transmembrane immunomodulatory proteins, e.g., CD80 vIgD polypeptides. In some embodiments, such infectious agents are capable of delivering nucleic acids encoding variant immunomodulatory polypeptides described herein, e.g., CD80 vIgD polypeptides, to target cells of a subject, e.g., immune cells and / or antigen-presenting cells (APCs) or tumor cells of the subject. Also provided are nucleic acids contained in such infectious agents and / or nucleic acids, e.g., vectors and / or plasmids, for generating or modifying such infectious agents, as well as compositions containing such infectious agents.

[0323] In some embodiments, the infectious agent is a microorganism or pathogen. In some embodiments, the infectious agent is a virus or bacteria. In some embodiments, the infectious agent is a virus. In some embodiments, the infectious agent is a bacteria. In some embodiments, such an infectious agent can deliver a nucleic acid sequence encoding any of the variant polypeptides, including secreted or transmembrane immunomodulatory proteins described herein, such as a CD80 vIgD polypeptide. Thus, in some embodiments, cells of a subject infected with or contacted by an infectious agent can express or secrete the variant immunomodulatory polypeptide on the cell surface. In some embodiments, the infectious agent can also deliver nucleic acids encoding one or more other therapeutic agents or other therapeutic agents to cells and / or the environment within the subject. In some embodiments, other therapeutic agents that can be delivered by the infectious agent include cytokines or other immunomodulatory molecules.

[0324] In some embodiments, the infectious agent, e.g., a virus or bacterium, contains a nucleic acid sequence encoding any of the variant polypeptides comprising a secreted or transmembrane immunomodulatory protein described herein, e.g., a CD80 vIgD polypeptide, and upon contact and / or infection of cells of a subject, the cells express the variant polypeptide comprising a secreted or transmembrane immunomodulatory protein, e.g., a CD80 vIgD polypeptide, encoded by the nucleic acid sequence contained in the infectious agent. In some embodiments, the infectious agent can be administered to a subject. In some embodiments, the infectious agent can be transduced into cells from a subject ex vivo.

[0325] In some embodiments, variant polypeptides comprising transmembrane immunomodulatory proteins, e.g., CD80 vIgD polypeptides, expressed by cells infected with an infectious agent are transmembrane proteins and expressed on the surface. In some embodiments, variant polypeptides comprising secreted immunomodulatory proteins, e.g., CD80 vIgD polypeptides, expressed by cells infected with an infectious agent are expressed and secreted from the cells. The transmembrane immunomodulatory proteins or secreted immunomodulatory proteins can be any of those described herein.

[0326] In some embodiments, the cells of interest targeted by the infectious agent include tumor cells, immune cells, and / or antigen-presenting cells (APCs). In some embodiments, the infectious agent targets cells in the tumor microenvironment (TME). In some embodiments, the infectious agent delivers a nucleic acid encoding a variant polypeptide comprising a secreted or transmembrane immunomodulatory protein, e.g., a CD80 vIgD polypeptide, to an appropriate cell (e.g., an APC, e.g., a cell that presents a peptide / MHC complex on its cell surface, e.g., a dendritic cell) or tissue (e.g., lymphoid tissue), thereby achieving a desired effect, e.g., inducing and / or enhancing an immunomodulatory and / or specific cell-mediated immune response, e.g., a CD4 and / or CD8 T cell response, which may include a cytotoxic T cell (CTL) response. In some embodiments, the infectious agent targets an APC, e.g., a dendritic cell (DC). In some embodiments, the nucleic acid molecule delivered by the infectious agent described herein comprises appropriate nucleic acid sequences, e.g., regulatory elements, e.g., a promoter, necessary for expression in a particular target cell of an operably linked coding sequence encoding a variant immunomodulatory polypeptide.

[0327] In some embodiments, an infectious agent containing a nucleic acid sequence encoding an immunomodulatory polypeptide can also contain nucleic acid sequences encoding one or more additional gene products, e.g., cytokines, prodrug-converting enzymes, cytotoxins, and / or detectable gene products. For example, in some embodiments, the infectious agent is an oncolytic virus, and the virus can include nucleic acid sequences encoding additional therapeutic gene products (e.g., Kirn et al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al., (2010) Curr Opin Mol Ther 12:403-411; U.S. Patent Nos. 7,588,767, 7,588,771, 7,662,398, and 7,754,221, and U.S. Patent Application Publication Nos. 2007 / 0202572, 2007 / 0212727, 2010 / 0062016, 2009 / 0098529, 2009 / 0053244, See Nos. 2009 / 0155287, 2009 / 0117034, 2010 / 0233078, 2009 / 0162288, 2010 / 0196325, 2009 / 0136917, and 2011 / 0064650. In some embodiments, the additional gene product is capable of causing death of a target cell (e.g., a tumor cell). The gene product may be a therapeutic gene product capable of enhancing, strengthening, or modulating an immune response (e.g., a cytokine). Exemplary gene products also include anti-cancer agents, anti-metastatic agents, anti-angiogenic agents, immunomodulatory molecules, immune checkpoint inhibitors, antibodies, cytokines, growth factors, antigens, cytotoxic gene products, pro-apoptotic gene products, anti-apoptotic gene products, cell matrix degrading genes, genes for tissue regeneration, and genes for reprogramming human somatic cells to pluripotency, as well as other genes described herein or known to those skilled in the art. In some embodiments, the additional gene product is granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0328] 1. Viruses In some embodiments, the infectious agent is a virus. In some embodiments, the infectious agent is an oncolytic virus or a virus that targets specific cells, such as immune cells. In some embodiments, the infectious agent targets tumor cells and / or cancer cells of the subject. In some embodiments, the infectious agent targets immune cells or antigen-presenting cells (APCs).

[0329] In some embodiments, the infectious agent is an oncolytic virus. An oncolytic virus is a virus that accumulates in tumor cells and replicates in tumor cells. Replication in cells and optional delivery of nucleic acids encoding the variant immunomodulatory variant CD80 polypeptides or immunomodulatory proteins described herein can lyse tumor cells and shrink and eliminate tumors. Oncolytic viruses can have a wide range of host and cell types. For example, oncolytic viruses accumulate in immune-privileged cells or immune-privileged tissues, including tumors and / or metastases, and also wound tissues and cells, thus allowing delivery and expression of nucleic acids encoding the variant immunomodulatory polypeptides described herein in a wide range of cell types. Oncolytic viruses can also replicate specifically in tumor cells, resulting in tumor cell lysis and efficient tumor regression.

[0330] Exemplary oncolytic viruses include adenovirus, adeno-associated virus, herpes virus, herpes simplex virus, vesicular oral virus, reovirus, Newcastle disease virus, parvovirus, measles virus, vesicular stomatitis virus (VSV), coxsackievirus, and vaccinia virus. In some embodiments, oncolytic viruses can specifically colonize solid tumors without infecting other organs and can be used as infectious agents to deliver nucleic acids encoding the variant immunomodulatory polypeptides described herein to such solid tumors.

[0331] Oncolytic viruses for use in delivering nucleic acids encoding variant CD80 polypeptides or immunomodulatory proteins described herein can be any known to those of skill in the art, for example, vesicular stomatitis virus (VSV). virus) (see, e.g., U.S. Pat. Nos. 7,731,974, 7,153,510, 6,653,103, and U.S. Patent Application Publication Nos. 2010 / 0178684, 2010 / 0172877, 2010 / 0113567, 2007 / 0098743, 20050260601, 20050220818, and European Patent Nos. 1385466, 1606411, and 1520175); herpes simplex virus (see, e.g., U.S. Pat. Nos. 7,897,146, 7,731,952, 7,5 Nos. 50,296, 7,537,924, 6,723,316, and 6,428,968, and U.S. Patent Application Publication Nos. 2014 / 0154216, 2011 / 0177032, 2011 / 0158948, 2010 / 0092515, 2009 / 0274728, 2009 / 0285860, 2009 / 0215147, 2009 / 0010889, 2007 / 0110720, 2006 / 0039894, 2004 / 0009604, and 2004 / 0063094, and International Patent Publication WO 2007 / 052029, WO 1999 / 038955); retroviruses (see, e.g., U.S. Patent Nos. 6,689,871, 6,635,472, 5,851,529, 5,716,826, 5,716,613 and U.S. Patent Application Publication No. 20110212530); vaccinia viruses (see, e.g., U.S. Patent No. 2016 / 0339066), and adeno-associated viruses (see, e.g., U.S. Patent Nos. (See Patent Nos. 8,007,780, 7,968,340, 7,943,374, 7,906,111, 7,927,585, 7,811,814, 7,662,627, 7,241,447, 7,238,526, 7,172,893, 7,033,826, 7,001,765, 6,897,045, and 6,632,670).

[0332] Oncolytic viruses also include viruses that have been genetically modified to attenuate their pathogenicity, improve their safety profile, enhance their tumor specificity, and are equipped with additional genes, such as cytotoxins, cytokines, prodrug-converting enzymes, that also improve the overall efficacy of the virus (see, e.g., Kirn et al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al., (2010) Curr Opin Mol Ther 12:403-411; U.S. Patent Nos. 7,588,767, 7,588,771, 7,662,398, and 7,754,221, and U.S. Patent Application Publication Nos. 2007 / 0202572, 2007 / 0212727, 2010 / 0062016, 2009 / 00 (See, e.g., Nos. 98529, 2009 / 0053244, 2009 / 0155287, 2009 / 0117034, 2010 / 0233078, 2009 / 0162288, 2010 / 0196325, 2009 / 0136917, and 2011 / 0064650). In some embodiments, the oncolytic virus can be a virus that has been modified to selectively replicate in cancerous cells and is therefore oncolytic. For example, the oncolytic virus is an adenovirus that has been modified to have an altered tropism for tumor therapy and as a gene therapy vector. Examples are ONYX-015, H101 and Ad5ΔCR (Hallden and Portella (2012) Expert Opin Ther Targets, 16:945-58) and TNFerade (McLoughlin et al. (2005) Ann. Surg. Oncol., 12:825-30), or the conditionally replicating adenovirus Oncorine®.

[0333] In some embodiments, the infectious agent is a modified herpes simplex virus. In some embodiments, the infectious agent is a modified version of Talimogene laherparepvec (also known as T-Vec, Imlygic, or OncoVex GM-CSF) that has been modified to contain a nucleic acid encoding any of the variant immunomodulatory polypeptides described herein (e.g., any of the variant CD80 polypeptides or immunomodulatory proteins described herein). In some embodiments, the infectious agent is a modified herpes simplex virus, or a variant thereof, as described, for example, in WO 2007 / 052029, WO 1999 / 038955, US 2004 / 0063094, or US 2014 / 0154216.

[0334] In some embodiments, the infectious agent is a virus that targets a specific type of cell in the subject to which the virus is administered, for example, a virus that targets immune cells or antigen-presenting cells (APCs). Dendritic cells (DCs) are APCs essential for initiating and controlling immune responses. DCs can capture and process antigens, migrate from the periphery to lymphoid organs, and present antigens to resting T cells in a major histocompatibility complex (MHC)-restricted manner. In some embodiments, the infectious agent is a virus that can specifically target DCs and deliver nucleic acids encoding variant CD80 polypeptides or immunomodulatory proteins for expression in DCs. In some embodiments, the virus is a lentivirus or a variant thereof. In some embodiments, the virus is a lentivirus pseudotyped to efficiently bind to and productively infect cells, such as DCs, that express the cell surface marker dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin (DC-SIGN). In some embodiments, the virus is a lentivirus pseudotyped with Sindbis virus E2 glycoprotein, such as that described in WO 2013 / 149167.In some embodiments, the virus allows the delivery and expression of the sequence of interest (for example, the nucleic acid encoding any of the variant CD80 polypeptides or immunomodulatory proteins described herein) to DC.In some embodiments, the virus comprises those described in WO 2008 / 011636 or US 2011 / 0064763, or variants thereof.

[0335] 2. Bacteria In some embodiments, the infectious agent is a bacterium. For example, in some embodiments, the bacterium can deliver any of the variant immunomodulatory polypeptides described herein, e.g., a variant CD80 polypeptide or a nucleic acid encoding an immunomodulatory protein, to target cells of a subject, such as tumor cells, immune cells, antigen-presenting cells, and / or phagocytes. In some embodiments, the bacterium can be preferentially targeted to a particular environment within a subject, such as the tumor microenvironment (TME), for expression and / or secretion of the variant immunomodulatory polypeptide and / or to target specific cells of the environment for expression of the variant immunomodulatory polypeptide.

[0336] In some embodiments, bacteria deliver nucleic acids to cells via bacterially mediated transfer of plasmid DNA into mammalian cells (also referred to as "bactofection"). For example, in some embodiments, delivery of genetic material is achieved by invasion of whole bacteria into target cells. In some embodiments, spontaneous or induced bacterial lysis can lead to the release of the plasmid for subsequent eukaryotic expression. In some embodiments, bacteria can deliver nucleic acids to non-phagocytic mammalian cells (e.g., tumor cells) and / or phagocytic cells, such as certain immune cells and / or APCs. In some embodiments, the nucleic acid delivered by the bacteria can be transferred to the nucleus of the target cell for expression. In some embodiments, the nucleic acid also contains appropriate nucleic acid sequences, e.g., regulatory elements, such as promoters or enhancers, required for expression in a specific host cell of the operably linked sequence encoding the variant immunomodulatory polypeptide. In some embodiments, a bacterial infectious agent can deliver nucleic acids encoding immunomodulatory proteins in the form of RNA, e.g., pre-manufactured translation-competent RNA, which is delivered to the cytoplasm of the target cell for translation by the target cell's machinery.

[0337] In some embodiments, the bacteria can replicate in target cells, such as tumor cells, and lyse the cells. In some embodiments, the bacteria can contain and / or release nucleic acid sequences and / or gene products in the cytoplasm of the target cells, thereby killing the target cells, such as tumor cells. In some embodiments, the infectious agent is a bacterium that can specifically replicate in a particular environment of a subject, such as the tumor microenvironment (TME). For example, in some embodiments, the bacteria can specifically replicate in anaerobic or hypoxic microenvironments. In some embodiments, conditions or factors present in a particular environment, such as aspartate, serine, citrate, ribose, or galactose produced by cells in the TME, can act as chemoattractants to attract the bacteria to the environment. In some embodiments, the bacteria can express and / or secrete the immunomodulatory proteins described herein in the environment, such as the TME.

[0338] In some embodiments, the infectious agent is a bacterium that is a Listeria sp., Bifidobacterium sp., Escherichia sp., Closteridium sp., Salmonella sp., Shigella sp., Vibrio sp., or Yersinia sp. In some embodiments, the bacteria is selected from the group consisting of Listeria monocytogenes, Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli, Vibrio cholera, Clostridium perfringens, Clostridium butyricum, Clostridium novyi, Clostridium acetobutylicum, Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium adolescentis. In some embodiments, the bacterium is selected from one or more of:In some embodiments, the bacterium is a modified bacterium, e.g., Seow and Wood (2009) Molecular Therapy 17(5):767-777; Baban et al. (2010) Bioengineered Bugs 1:6, 385-394; Patyar et al. (2010) J Biomed Sci 17:21; Tangney et al. (2010) Bioengineered Bugs 1:4, 284-287; van Pijkeren et al. (2010) Hum Gene Ther. 21(4):405-416; WO 2012 / 149364; WO 2014 / 198002; US 9103831; US ​​9453227; US 2014 / 0186401; US ​​2004 / 0146488; 2011 / 0293705; US 2015 / 0359909 and EP 3020816. The bacteria can be modified to deliver nucleic acid sequences encoding any of the variant...

Claims

1. A purified homodimer of CD80-Fc fusion, the CD80-Fc fusion comprises one polypeptide operably linked to the Fc region of an immunoglobulin, the one polypeptide is a variant CD80 polypeptide that contains an IgV domain; the variant CD80 polypeptide comprises the amino acid substitution H18Y, based on the amino acid numbering of SEQ ID NO:28; the variant CD80 polypeptide is a polypeptide having an amino acid sequence that exhibits at least 93% sequence identity to (i) the amino acid sequence set forth in SEQ ID NO:28, or (ii) the amino acid sequence of positions 35 to 141 of SEQ ID NO:1; and the variant CD80 polypeptide specifically binds to the ectodomain of PD-L1 with improved affinity compared to an unmodified CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

28. Purified homodimer of CD80-Fc fusion.

2. 2. The purified homodimer of CD80-Fc fusion of claim 1, wherein said variant CD80 polypeptide binds non-competitively to the ectodomains of CD28 and PD-L1.

3. the variant CD80 polypeptide comprising: Amino acid substitutions K9E / E10R / V11S / A12G / T13N / K14A / S15V / C16L / G17W / H18Y / Y53C / L70Q / D90G / T130A / N149D / N152T / H188D Including, A purified homodimer of the CD80-Fc fusion of claim 1 or 2.

4. 4. The purified homodimer of CD80-Fc fusion of any one of claims 1 to 3, wherein said variant CD80 polypeptide comprises said IgV domain and an IgC domain.

5. 4. The purified homodimer of CD80-Fc fusion of any one of claims 1 to 3, wherein said variant CD80 polypeptide comprises only said IgV domain.

6. the variant CD80 polypeptide comprising: The amino acid sequence set forth in SEQ ID NO: 314, or An amino acid sequence that exhibits at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 314 and contains the amino acid substitution.

5. The purified homodimer of CD80-Fc fusion of any one of claims 1 to 4, comprising:

7. 7. The purified homodimer of CD80-Fc fusion of any one of claims 1-6, wherein the improved affinity for the ectodomain of PD-L1 is more than two-fold improved compared to the binding affinity of the unmodified CD80 for the ectodomain.

8. 8. The purified homodimer of CD80-Fc fusion of any one of claims 1 to 7, wherein the Fc region is a variant Fc region having reduced effector function.

9. The Fc region is selected from the group consisting of amino acid substitutions N297A, N297Q, 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 / 9. The purified homodimer of CD80-Fc fusion of any one of claims 1 to 8, comprising G236del / S267K, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, or IgG4-L236E.

10. 8. The purified homodimer of CD80-Fc fusion of any one of claims 1 to 7, wherein the Fc region is an Fc region derived from IgG1 or a variant IgG1 Fc region having reduced effector function.

11. 11. The purified homodimer of CD80-Fc fusion of claim 10, wherein the Fc region comprises an amino acid sequence as set forth in SEQ ID NO:226, or an amino acid sequence that exhibits at least 85% sequence identity to SEQ ID NO:

226.

12. 12. The purified homodimer of CD80-Fc fusion of claim 10 or 11, wherein said variant IgG1 Fc region comprises the amino acid substitutions R292C / N297G / V302C.

13. 12. The purified homodimer of CD80-Fc fusion of claim 10 or 11, wherein said variant IgG1 Fc region comprises the amino acid substitutions L234A / L235E / G237A.

14. The purified homodimer of CD80-Fc fusion according to any one of claims 1 to 7, wherein the Fc region is an IgG2 Fc region.

15. 15. The purified homodimer of CD80-Fc fusion of any one of claims 1 to 7 and 14, wherein said Fc region comprises an amino acid sequence as set forth in SEQ ID NO:227, or an amino acid sequence that exhibits at least 85% sequence identity to SEQ ID NO:

227.

16. 9. The purified homodimer of CD80-Fc fusion according to any one of claims 1 to 8, wherein said Fc region is an IgG4 Fc region or a variant IgG4 Fc region containing the mutation S228P.

17. 17. The purified homodimer of CD80-Fc fusion of any one of claims 1 to 16, wherein said one polypeptide is indirectly linked to the Fc region via a linker.

18. 20. An in vitro method for producing a purified homodimer of a CD80-Fc fusion of any one of claims 1 to 17, comprising the step of introducing a nucleic acid or a nucleic acid molecule encoding said Fc fusion polypeptide into a host cell under conditions whereby said Fc fusion polypeptide is expressed in the host cell.

19. The method of claim 18, further comprising isolating or purifying said CD80-Fc fusion from said cells to produce a purified homodimer of said CD80-Fc fusion.

20. 18. A pharmaceutical composition comprising a purified homodimer of CD80-Fc fusion according to any one of claims 1 to 17, and a pharma- ceutically acceptable excipient.

21. 21. An article of manufacture or kit comprising the pharmaceutical composition of claim 20.

22. 21. Use of the pharmaceutical composition of claim 20 in the manufacture of a medicament for modulating an immune response in a subject.

23. 21. The pharmaceutical composition of claim 20 for use in modulating an immune response in a subject.

24. 24. The use of claim 22 or the pharmaceutical composition of claim 23, wherein the modulation of the immune response treats a disease or condition in the subject.

25. 25. The use or pharmaceutical composition of claim 24, wherein the disease or condition is cancer.

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