Compositions and methods for non-immunogenicity

Abstract

The present disclosure provides a polypeptide comprising a human signal-regulatory protein alpha (SIRPα) binding sequence, wherein the polypeptide elicits a substantially similar or reduced immune response when expressed on the surface of a cell compared to a polypeptide comprising a wild-type (WT) CD47 sequence. The polypeptide of the present disclosure may exhibit a regulated conformational ensemble.

Description

[Technical Field]

[0001] (cross reference) This application claims the benefit of UK Patent Application No. 2211117.3, filed July 29, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in SML format and is incorporated herein by reference in its entirety. The XML copy, created on July 27, 2023, is named 62600-735_601_SL.xml and is 765,489 bytes in size. [Background technology]

[0003] Cell therapy holds great promise for combating previously intractable diseases. The use of autologous cells can be very costly and burdensome. Allogeneic pluripotent stem cells (PSCs) offer greater scalability and cost savings, but their utility is limited by the need to evade the innate immune system (e.g., white blood cells such as natural killer cells and macrophages). Summary of the Invention

[0004] Aspects of the present disclosure provide engineered polypeptides comprising a signal regulatory protein alpha (SIRPα) binding sequence, wherein the engineered polypeptide is configured to elicit a reduced immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of residues 19-290 of any one of SEQ ID NOS: 1-4, wherein the reduced immune response is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduced relative to the reference polypeptide.

[0005] In some embodiments, the immune response comprises natural killer (NK) cell cytotoxicity. In some embodiments, the immune response comprises macrophage cytotoxicity.

[0006] An additional aspect of the present disclosure provides an engineered polypeptide comprising a signal-regulating protein alpha (SIRPα) binding sequence, wherein the polypeptide comprises a conformational ensemble comprising a first metastable state, the first metastable state configured to bind to SIRPα, the conformational ensemble comprising a greater proportion of the first metastable state than a conformational ensemble of a reference polypeptide, the reference polypeptide comprising the sequence of residues 19-290 of any one of SEQ ID NOs: 1-4. In some embodiments, the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by hydrogen-deuterium exchange (HDX), small-angle x-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD). In some embodiments, the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by MD. In some embodiments, the first metastable state is characterized by a bend angle between the transmembrane domain (TMD) and the extracellular domain (ECD) of the polypeptide. In some embodiments, the TMD comprises five alpha helices. In some embodiments, the ECD is configured to bind to SIRPα. In some embodiments, the bend angle is about 130 to about 180 degrees. In some embodiments, the first metastable state is characterized by a distance between the TMD and the ECD of the polypeptide. In some embodiments, the distance is about 10 to about 25 angstroms (Å).In some embodiments, the engineered polypeptide is configured to elicit a reduced immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of residue 19 through the last residue of any one of SEQ ID NOS: 1-4, wherein the reduced immune response is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduced relative to the reference polypeptide. In some embodiments, the immune response comprises natural killer (NK) cell cytotoxicity. In some embodiments, the immune response comprises macrophage cytotoxicity.

[0007] An additional aspect of the present disclosure provides an engineered polypeptide comprising a signal-regulatory protein alpha (SIRPα) binding sequence, wherein the engineered polypeptide is configured to elicit a decreased immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of residues 19-290 of any one of SEQ ID NOs: 1-4, wherein the SIRPα binding sequence comprises at least one mutation relative to any one of SEQ ID NOs: 1-4, and wherein the at least one mutation is associated with cancer.

[0008] In some embodiments, the at least one mutation is included in a database. In some embodiments, the database includes the Catalogue of Somatic Mutations in Cancer (COSMIC), the Genome Aggregation Database (gnomAD), or both. In some embodiments, the at least one mutation includes a mutation selected from the group consisting of the mutations listed in Table 3, and any combination thereof. In some embodiments, the at least one mutation comprises a mutation selected from the group consisting of M31, L40, C42, D47, D64, C75, E80, F97, G105, K106, F106, K111, S123, S127, K128, F131, C132, C136, K140, T142, G146, M153, L157, L160, E166, C170, D178, A203, S207, V210, D211, L214, S215, V262, L264, Y267, and any combination thereof. In some embodiments, the polypeptide comprises an additional mutation, wherein the additional mutation is selected from the group consisting of Y31, A32, R35, K35, P71, A77, A79, N80, L100, K138, L164, M185, A211, S259, E262, and any combination thereof.

[0009] An additional aspect of the present disclosure provides an engineered signal-regulatory protein alpha (SIRPα) binding polypeptide comprising an extracellular domain (ECD), a transmembrane domain (TMD), and an extracellular loop region (ECLR), wherein the extracellular loop region comprises a heterologous sequence compared to residue 19 through the last residue of any one of SEQ ID NOs: 1-4.

[0010] In some embodiments, the ECD, TMD, and ECLR are derived from at least two different organisms. In some embodiments, the ECLR comprises at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the ECLR of any one of SEQ ID NOs: 639 and 640. In some embodiments, the heterologous sequence comprises a pair of cysteine residues configured to form a disulfide pair. In some embodiments, the heterologous sequence comprises at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 641-647. In some embodiments, the engineered polypeptide is configured to elicit a reduced immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of any one of SEQ ID NOS: 1-4. In some embodiments, the polypeptide and the reference polypeptide are configured to adopt a first metastable state, and the first metastable state is configured to bind to SIRPα. In some embodiments, the conformational ensemble of the engineered polypeptide comprises a greater proportion of the first metastable state than the conformational ensemble of the reference polypeptide. In some embodiments, the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by hydrogen-deuterium exchange (HDX), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD). In some embodiments, the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by MD. In some embodiments, the first metastable state is characterized by a bend angle between the transmembrane domain (TMD) and the extracellular domain (ECD) of the polypeptide. In some embodiments, the TMD comprises five alpha helices. In some embodiments, the ECD is configured to bind to SIRPα. In some embodiments, the bend angle is about 130 to about 180 degrees.In some embodiments, the first metastable state is characterized by the distance between the TMD and ECD of the polypeptide. In some embodiments, the distance is about 10 to about 25 angstroms (Å). In some embodiments, the SIRPα binding sequence comprises at least about 70%, 80%, 90%, 95%, or 99%, or 100% identity to any one of SEQ ID NOs: 10-640 or 648-653. In some embodiments, the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to the extracellular domain (ECD) of any one of SEQ ID NOs: 1-640 and 648-653. In some embodiments, the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to the transmembrane domain (TMD) of any one of SEQ ID NOs: 1-640 and 648-653. In some embodiments, the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to the extracellular loop region (ECLR) of any one of SEQ ID NOs: 1-640 and 648-653. In some embodiments, the engineered polypeptide comprises at least one amino acid substitution as specified in Table 3. In some embodiments, the engineered polypeptide comprises one or more amino acid substitutions selected from the group consisting of M31, L40, C42, D47, D64, C75, E80, F97, G105, K106, F106, K111, S123, S127, K128, F131, C132, C136, K140, T142, G146, M153, L157, L160, E166, C170, D178, A203, S207, V210, D211, L214, S215, V262, L264, Y267, and any combination thereof. In some embodiments, the engineered polypeptide comprises at least one amino acid substitution specified in Table 4.In some embodiments, the engineered polypeptide comprises at least one amino acid substitution selected from the group consisting of Y31, A32, R35, K35, P71, A77, A79, N80, L100, K138, L164, M185, A211, S259, E262, and any combination thereof. In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the polypeptide comprises an N-terminal addition. In some embodiments, the TMD, ECD, and linker are from more than one organism. In some embodiments, the engineered polypeptide is configured to elicit a reduced integrin response compared to a reference polypeptide. In some embodiments, the polypeptide is configured to elicit a reduced thrombospondin 1 (TSP-1) response compared to a reference polypeptide.

[0011] Additional aspects of the present disclosure provide engineered polypeptides comprising a sequence having at least about 70%, 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, or 100% identity to any one of SEQ ID NOs: 1-640 and 648-653.

[0012] In some embodiments, the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to the extracellular domain (ECD) of any one of SEQ ID NOs: 1-640 and 648-653. In some embodiments, the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to the transmembrane domain (TMD) of any one of SEQ ID NOs: 1-640 and 648-653. In some embodiments, the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to the extracellular loop region (ECLR) of any one of SEQ ID NOs: 1-640 and 648-653. In some embodiments, the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to any one of SEQ ID NOs:641-647.

[0013] An additional aspect of the present disclosure provides an engineered polypeptide comprising a signal-regulatory protein alpha (SIRPα) binding sequence, wherein the engineered polypeptide comprises a conformational ensemble comprising a first metastable state, the first metastable state being configured to bind to SIRPα, the conformational ensemble comprising a greater proportion of the first metastable state than a conformational ensemble of a reference polypeptide, and the reference polypeptide comprises the sequence of residues 19-290 of any one of SEQ ID NOs: 1-4.

[0014] In some embodiments, the conformational ensemble proportion of the engineered polypeptide or the conformational ensemble proportion of the reference polypeptide is determined, at least in part, by hydrogen-deuterium exchange (HDX), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD). In some embodiments, the conformational ensemble proportion of the engineered polypeptide or the conformational ensemble proportion of the reference polypeptide is determined, at least in part, by MD. In some embodiments, the first metastable state is characterized by a bend angle between the transmembrane domain (TMD) and the extracellular domain (ECD) of the polypeptide. In some embodiments, the TMD comprises five alpha helices. In some embodiments, the ECD is configured to bind to SIRPα. In some embodiments, the bend angle is about 130 to about 180 degrees. In some embodiments, the first metastable state is characterized by a distance between the TMD and the ECD of the polypeptide. In some embodiments, the distance is about 10 to about 25 angstroms (Å). In some embodiments, the engineered polypeptide is configured to elicit a reduced immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of residues 19-290 of any one of SEQ ID NOS: 1-4, wherein the reduced immune response is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduced relative to the reference polypeptide. In some embodiments, the immune response comprises natural killer (NK) cell cytotoxicity. In some embodiments, the immune response comprises macrophage cytotoxicity. In some embodiments, the SIRPα binding sequence comprises at least about 70%, 80%, 90%, 95%, or 99%, or 100% identity to any one of SEQ ID NOS: 10-640 or 648-653. In some embodiments, the engineered polypeptide comprises at least one amino acid substitution specified in Table 3.In some embodiments, the engineered polypeptide comprises one or more amino acid substitutions selected from the group consisting of M31, L40, C42, D47, D64, C75, E80, F97, G105, K106, F106, K111, S123, S127, K128, F131, C132, C136, K140, T142, G146, M153, L157, L160, E166, C170, D178, A203, S207, V210, D211, L214, S215, V262, L264, Y267, and any combination thereof. In some embodiments, the engineered polypeptide comprises at least one amino acid substitution specified in Table 4. In some embodiments, the engineered polypeptide comprises at least one amino acid substitution selected from the group consisting of Y31, A32, R35, K35, P71, A77, A79, N80, L100, K138, L164, M185, A211, S259, E262, and any combination thereof. In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the polypeptide comprises an N-terminal addition. In some embodiments, the TMD, ECD, and linker are from more than one organism. In some embodiments, the engineered polypeptide is configured to elicit a reduced integrin response compared to a reference polypeptide. In some embodiments, the polypeptide is configured to elicit a reduced thrombospondin 1 (TSP-1) response compared to a reference polypeptide.

[0015] An additional aspect of the present disclosure provides an engineered cell comprising any of the engineered peptides described herein.

[0016] An additional aspect of the present disclosure provides an engineered cell comprising a plurality of SIRPα binding polypeptides, wherein the plurality of SIRPα binding polypeptides comprises any of the engineered peptides described herein.

[0017] In some embodiments, the plurality of SIRPα-binding polypeptides further comprises wild-type CD47. In some embodiments, the plurality of SIRPα-binding polypeptides comprises a plurality of any of the engineered peptides described herein. In some embodiments, the engineered cells are stem cells. In some embodiments, the stem cells are embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0018] An additional aspect of the present disclosure provides a nucleic acid molecule encoding any of the engineered polypeptides disclosed herein.

[0019] An additional aspect of the present disclosure provides a nucleic acid molecule encoding a plurality of SIRPα-binding polypeptides, wherein the plurality of SIRPα-binding polypeptides comprises wild-type CD47 and any of the engineered polypeptides disclosed herein.

[0020] An additional aspect of the present disclosure provides a nucleic acid molecule encoding a plurality of SIRPα-binding polypeptides, wherein the plurality of SIRPα-binding polypeptides comprises a plurality of any of the engineered polypeptides disclosed herein.

[0021] An additional aspect of the present disclosure provides a vector comprising any of the nucleic acids disclosed herein.

[0022] In some embodiments, the vector is a plasmid, a minicircle, a CELiD, an adeno-associated virus (AAV)-derived virion, a lentivirus, an adenovirus, or a herpes simplex virus (HSV).

[0023] An additional aspect of the present disclosure provides a method of generating hypoimmunogenic cells, the method comprising administering to the cells any of the vectors disclosed herein.

[0024] An additional aspect of the present disclosure provides an engineered polypeptide comprising a signal-regulatory protein alpha (SIRPα) binding sequence, wherein the polypeptide is configured to elicit a decreased macrophage response when expressed on the surface of a cell compared to a polypeptide comprising the sequence of any one of SEQ ID NOs: 1-4.

[0025] In some embodiments, the SIRPα binding sequence comprises at least about 70%, 80%, 90%, 95%, or 99% identity to any one of the sequences described herein. In some embodiments, the SIRPα binding sequence comprises at least one amino acid substitution specified in Table 3. In some embodiments, the SIRPα binding sequence, when optimally aligned to any one of SEQ ID NOS: 1-4, comprises at least one amino acid substitution specified in Table 3. In some embodiments, the SIRPα binding sequence, when optimally aligned to any one of SEQ ID NOS: 1-4, comprises at least one amino acid substitution specified in Table 4. In some embodiments, the SIRPα binding sequence, when optimally aligned to any one of SEQ ID NOS: 1-4, comprises at least one amino acid substitution specified in Table 4. In some embodiments, the cell comprises a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the polypeptide comprises an N-terminal addition. In some embodiments, the N-terminal addition comprises at least 1, 2, or 3 amino acids added to the N-terminus of the polypeptide. In some embodiments, the three amino acids comprise the formula X-3X-2X-1, where X-3 is W, X-2 is selected from Q, A, and G, and X-1 is selected from R, P, L, T, F, I, and M. In some embodiments, the three amino acids are selected from WQR, WAP, WQL, WQP, WQT, WQF, WQI, WGP, and WQM. In some embodiments, the polypeptide and the reference polypeptide are configured to adopt a first metastable state, wherein the first metastable state is configured to bind to SIRPα. In some embodiments, the reference polypeptide and optionally the polypeptide are configured to adopt a second metastable state, wherein the second metastable state does not substantially bind to SIRPα. In some embodiments, the conformational ensemble of the polypeptide comprises a greater proportion of the first metastable state than the conformational ensemble of the reference polypeptide. In some embodiments, the proportion of a conformational ensemble of a polypeptide is determined, at least in part, by hydrogen-deuterium exchange (HDX), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD).In some embodiments, the first metastable state is characterized by a first bend angle between the transmembrane domain (TMD) and the extracellular domain (ECD) of the polypeptide. In some embodiments, the TMD comprises five alpha helices. In some embodiments, the ECD is configured to bind to SIRPα. In some embodiments, the first bend angle is between about 130 and about 180 degrees. In some embodiments, the first bend angle is between about 150 and about 170 degrees. In some embodiments, the second metastable state is characterized by a second bend angle between the TMD and the ECD. In some embodiments, the second bend angle is between about 100 and about 120 degrees. In some embodiments, the first metastable state is characterized by a first distance between the TMD and the ECD of the polypeptide. In some embodiments, the first distance is between about 10 and about 25 angstroms (Å). In some embodiments, the second metastable state is characterized by a second distance between the TMD and the ECD. In some embodiments, the second distance is about 4 to about 9 angstroms (Å). In some embodiments, the TMD, ECD, and linker are all from one organism. In some embodiments, the TMD, ECD, and linker are from more than one organism. In some embodiments, at least one of the TMD, ECD, and linker is engineered. In some embodiments, the polypeptide is configured to elicit a reduced integrin response compared to a reference polypeptide. In some embodiments, the polypeptide is configured to elicit a reduced TSP-1 response compared to a reference polypeptide.

[0026] Another aspect of the present disclosure provides a non-transitory computer-readable medium containing machine-executable code that, when executed by one or more computer processors, performs any of the methods described above or elsewhere herein.

[0027] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto, the computer memory including machine-executable code that, when executed by the one or more computer processors, performs any of the methods described above or elsewhere herein.

[0028] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0029] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or supersede such conflicting material. [Brief explanation of the drawings]

[0030] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG."). [Figure 1] 1 shows several representative metastates of SIRPα binding peptides according to some embodiments of the present disclosure. [Figure 2A]1 shows exemplary free energy surfaces representing conformational ensembles of SIRPα-binding peptides, according to some embodiments of the present disclosure. [Figure 2B] 1 shows exemplary free energy surfaces representing conformational ensembles of SIRPα-binding peptides, according to some embodiments of the present disclosure. [Figure 3A] 1 shows an exemplary plot of a reaction coordinate monitoring transitions between metastates in a molecular dynamics simulation of a SIRPα binding polypeptide, according to some embodiments of the present disclosure. [Figure 3B] 1 shows an exemplary plot of a reaction coordinate monitoring transitions between metastates in a molecular dynamics simulation of a SIRPα binding polypeptide, according to some embodiments of the present disclosure. [Figure 4] 1 shows a molecular model of a SIRPα binding polypeptide described herein. [Figure 5A] 1 shows an exemplary plot of a reaction coordinate monitoring transitions between metastates in a molecular dynamics simulation of a SIRPα binding polypeptide, according to some embodiments of the present disclosure. [Figure 5B] 1 shows an exemplary plot of a reaction coordinate monitoring transitions between metastates in a molecular dynamics simulation of a SIRPα binding polypeptide, according to some embodiments of the present disclosure. [Figure 6] 1 shows vectors constructed for expression of the polypeptides disclosed herein. [Figure 7A] 1 shows a bar plot depicting the relative proportion of the meta state in simulated conformational ensembles of SIRPα binding polypeptides disclosed herein. [Figure 7B] 1 shows a bar plot depicting the relative proportion of the meta state in simulated conformational ensembles of SIRPα binding polypeptides disclosed herein. [Figure 7C] 1 shows exemplary free energy surfaces representing conformational ensembles of SIRPα-binding peptides, according to some embodiments of the present disclosure. [Figure 8]1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 9] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 10] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 11] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 12] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 13] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 14] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 15] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 16] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 17] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 18] 1 shows a graph quantifying the protection from natural killer cell cytotoxicity provided by SIRPα binding polypeptides of the disclosure. [Figure 19] 1 shows a bar graph depicting measurements of surface expression of SIRPα binding polypeptides of the present disclosure. [Figure 20]1 shows a bar graph depicting measurements of surface expression of SIRPα binding polypeptides of the present disclosure. [Figure 21] 1 shows a bar graph depicting measurements of surface expression of SIRPα binding polypeptides of the present disclosure. [Figure 22] 1 shows a graph quantifying the protection from macrophage phagocytosis afforded by SIRPα-binding polypeptides of the disclosure. [Figure 23] 1 shows a molecular model of a SIRPα binding polypeptide described herein. [Figure 24] 1 depicts a computer system that is programmed or otherwise configured to perform the methods provided herein.

[0031] Brief Description of Sequence Listing The Sequence Listing submitted herewith provides exemplary polynucleotide and polypeptide sequences for use in the methods, compositions, and systems according to the present disclosure. Below are representative descriptions of those sequences.

[0032] SEQ ID NOs: 1 to 4 show representative amino acid structures of wild-type human CD47 sequences.

[0033] SEQ ID NOs: 5-9 show representative cysteine mutations to SEQ ID NO: 1 that can be made to induce oligomerization.

[0034] SEQ ID NOs: 10-384 show representative engineered signal regulatory protein alpha (SIRPα) binding sequences according to some embodiments of the present disclosure.

[0035] SEQ ID NOs: 385-475 show representative SIRPα binding sequences that are based at least in part on mutations associated with cancer.

[0036] SEQ ID NOs: 476-633 and 648 show representative SIRPα binding sequences based at least in part on polymorphisms identified in one or more individuals.

[0037] SEQ ID NOs: 634-638 and 648-653 show representative SIRPα binding sequences based at least in part on one or more rational mutations.

[0038] SEQ ID NOs: 639-640 show representative SIRPα binding sequences that are chimeric and / or contain loop insertions as described herein.

[0039] SEQ ID NOs: 641-647 show the sequences of representative insertions in engineered SIRPα binding sequences of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0040] Cluster of differentiation 47 (CD47), also known as integrin-associated protein (IAP), is a transmembrane protein encoded by the cd47 gene in humans. CD47 belongs to the immunoglobulin superfamily, partners with membrane integrins, and also binds to the ligands thrombospondin 1 (TSP-1) and signal-regulatory protein alpha (SIRPα). It is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration. CD47 is a highly glycosylated, ubiquitously expressed membrane protein of approximately 50 kDa from the immunoglobulin superfamily. It has a single IgV-like domain at its N-terminus (its extracellular domain or ECD), a highly hydrophobic stretch with five membrane-spanning segments (also called transmembrane or TM domains), and an alternatively spliced cytoplasmic C-terminus (C-terminal domain or CTD). Each of the four alternatively spliced cytoplasmic tails (eg, the C-terminal portions of SEQ ID NOs: 1-4) is present at different frequencies in vivo, but all lack substantial signaling domains.

[0041] CD47 is an essential component of the innate immune system, and binding of the ECD to signal regulatory protein alpha (SIRPα) on immune cells inhibits phagocytosis and NK cell killing. CD47 is ubiquitously expressed on human cells and has been found to be overexpressed on many different tumor cells.

[0042] One particular attempt to engineer hypoimmunogenic cells has focused on avoiding recognition by the adaptive immune response through the abrogation or modification of major histocompatibility complex (MHC) proteins expressed by the cells. Nevertheless, such cells may remain susceptible to killing by agents of the innate immune system, including natural killer (NK) cells and macrophages, which target the cells through other cognate binding partners and signaling pathways (e.g., CD47).

[0043] Accordingly, disclosed herein are various engineered signal-regulatory protein alpha (SIRPα)-binding polypeptides that can overcome immune rejection in cell-based transplantation therapies using universal donor stem cell lines. Some aspects of the present disclosure provide engineered SIRPα-binding polypeptides and variants that retain or enhance native CD47 "don't eat me" signaling (e.g., relative to WT CD47). In some embodiments, the engineered SIRPα agonist constructs described herein exhibit enhanced CD47 / SIRPα pathway signaling relative to wild-type (WT) CD47, thus protecting cells expressing such molecules from killing by macrophages and / or NK cells. In some embodiments, the SIRPα-binding polypeptides and variants disclosed herein elicit a reduced immune response when expressed on the surface of cells compared to WT CD47. In some embodiments, the reduced immune response includes NK cell cytotoxicity. In some embodiments, the reduced immune response includes macrophage cytotoxicity. In some embodiments, engineered SIRPα agonist constructs exhibit "detuning" or reduced signaling through pathways other than SIRPα. In some embodiments, the engineered SIRPα agonist sequences disclosed herein comprise low sequence homology to human CD47 polypeptides and variants. In some embodiments, the engineered SIRPα agonist sequences disclosed herein comprise less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or less sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, engineered molecules can exhibit enhanced CD47 / SIRPα signaling function while retaining essential functions required for cellular homeostasis, and these SIRPα agonist constructs can be expressed as a single entity on the surface of cells (e.g., where the human CD47 gene has been knocked out or combined with endogenously expressed wild-type CD47).

[0044] Another aspect of the present disclosure includes rationally designed SIRPα binding sequences that retain binding to human SIRPα, exhibit sensitivity to macrophage phagocytosis and / or NK cell killing comparable to or less than WT CD47 splice variants 1-4 (corresponding to SEQ ID NOS: 1-4), and have the advantage of having "skewed" signaling function toward reduced macrophage phagocytosis and / or NK cell killing signaling, and optionally detuning the binding of one or more other endogenous binding partners. In some embodiments, the detuned endogenous binding partner comprises an integrin. In some embodiments, the detuned endogenous binding partner comprises thrombospondin-1 (TSP-1).

[0045] Another aspect of the present disclosure includes the co-expression of different engineered SIRPα binding polypeptides (eg, two or more), including the co-expression of any of the SIRPα binding polypeptides described herein.

[0046] Another aspect of the present disclosure includes rationally designed SIRPα-binding polypeptide sequences that retain binding to human SIRPα but exhibit reduced or abolished signaling function via other (e.g., non-SIRPα) binding partners, such as reduced or abolished binding and signaling via TSP-1, integrins, and other human CD47 endogenous binding partners. Engineered SIRPα-binding sequences or constructs that exhibit detuning of CD47 signaling to one or more endogenous binding partners other than CD47 may be referred to as engineered "selective" SIRPα-binding sequences or constructs. Another aspect of the present disclosure includes SIRPα-binding mutations based at least in part on any one of WT CD47 splice variants 1-4 that enhance "don't eat me" signaling, thereby preventing or reducing phagocytosis (e.g., by macrophages) and / or NK cell killing of cells (e.g., induced pluripotent stem cells (iPSCs)) expressing the mutant SIRPα-binding polypeptide. In some embodiments, the mutations are based at least in part on mutations such as those listed in Table 3 or Table 4, or are obtained from in silico computations (e.g., all-atom molecular dynamics, normal mode analysis, coarse-grained simulations, frustration analysis, or protein stability).

[0047] Another aspect of the present disclosure includes engineered SIRPα-binding sequences based at least in part on any one of SEQ ID NOs: 1-4 that retain binding of human SIRPα but contain a peptide insertion within the wild-type human CD47 RVVSWF peptide linker (e.g., residues 132-137 of any one of SEQ ID NOs: 1-4) connecting the extracellular domain (ECD) and transmembrane domain (TMD) of any one of SEQ ID NOs: 1-4, and may exhibit altered / detuned signaling function (e.g., no binding or reduced binding to endogenous protein partners other than SIRPα, or no signaling through protein partners other than SIRPα, or reduced transmembrane signaling through protein partners other than SIRPα). In some embodiments, the peptide insertion is based at least in part on one or more orthologs or homologs of SEQ ID NOs: 1-4. In some embodiments, the peptide insertion comprises at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity in sequence to any one of SEQ ID NOs: 641-647. In some embodiments, the peptide insertions described herein modulate the conformational ensemble of the SIRPα binding polypeptide relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence such as that contained in any one of SEQ ID NOs: 1-4).

[0048] Another aspect of the present disclosure includes engineered SIRPα-binding sequences based at least in part on any one of SEQ ID NOS: 1-4 that retain human SIRPα binding but contain a peptide insertion containing two cysteines (e.g., constrained by a disulfide bond) within a region adjacent to or located within the RVVSWF peptide linker (e.g., residues 132-137 of any one of SEQ ID NOS: 1-4) connecting the ECD and TMD of WT CD47, and that exhibit altered or detuned function (e.g., no binding to or reduced binding to endogenous protein partners other than SIRPα and / or no signaling through or reduced signaling function with protein partners other than SIRPα). In some embodiments, the cysteine residues configured to form disulfide bonds described herein modulate the conformational ensemble of the SIRPα-binding polypeptide relative to a reference polypeptide (e.g., including a wild-type human CD47 sequence such as that contained in any one of SEQ ID NOS: 1-4).

[0049] Another aspect of the present disclosure includes engineered SIRPα-binding sequences based at least in part on any one of SEQ ID NOS: 1-4 that retain human SIRPα binding but contain peptide insertions and additional mutations (single or in combination) within the RVVSWF peptide linker connecting the ECD and TMD of WT CD47 (e.g., residues 132-137 of any one of SEQ ID NOS: 1-4) and exhibit altered / detuned function (e.g., no binding or reduced binding to endogenous protein partners other than SIRPα and / or no signaling through or reduced signaling function with these other protein partners). In some embodiments, the peptide insertions combined with the additional mutations modulate the conformational ensemble of the SIRPα-binding polypeptide relative to a reference polypeptide (e.g., a wild-type CD47 sequence such as that contained in any one of SEQ ID NOS: 1-4). In some embodiments, the mutations modulate the conformational ensemble of the SIRPα-binding polypeptide relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence such as that contained in any one of SEQ ID NOs: 1-4).

[0050] Another aspect of the present disclosure includes mutations on SIRPα binding polypeptides that enhance dimer or other oligomer (e.g., trimer, tetramer, etc.) formation based at least in part on any one of SEQ ID NOs: 1-4 (e.g., one or more of the mutations detailed in Table 2 below). In some embodiments, SIRPα binding polypeptides with enhanced oligomerization exhibit altered or detuned function relative to the reference polypeptide (e.g., do not bind or have reduced binding to endogenous protein partners other than SIRPα, and / or do not signal through or have reduced signaling function with protein partners other than SIRPα).

[0051] Another aspect of the present disclosure includes SIRPα-binding sequences that have been computationally engineered based on in silico evolutionary strategies and / or trained algorithms (such as machine learning algorithms) to enhance the stability of SIRPα-binding polypeptides and retain binding and signaling via SIRPα, while reducing binding to other endogenous protein binding partners and associated signaling. In some embodiments, these constructs comprise less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or less sequence identity to any one of SEQ ID NOS: 1-4. In some embodiments, these constructs have a tailored conformational ensemble relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence, such as that contained in any one of SEQ ID NOS: 1-4).

[0052] Another aspect of the present disclosure includes compositions and methods for dimerization or oligomerization achieved by single or multiple mutations of SIRPα-binding sequences designed computationally based on in silico evolutionary strategies and / or trained algorithms (e.g., machine learning algorithms) to enhance stability relative to any one of SEQ ID NOS: 1-4, retain SIRPα binding and signaling, but reduce binding to other endogenous protein binding partners and associated signaling. In some embodiments, these constructs comprise less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or less sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, these constructs have a regulated conformational ensemble relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence, such as that contained in any one of SEQ ID NOS: 1-4).

[0053] Another aspect of the present disclosure includes computationally engineered SIRPα-binding polypeptides comprising a peptide insertion within a wild-type CD47 RVVSWF peptide linker (e.g., residues 132-137 of any one of SEQ ID NOS: 1-4) grafted onto a computationally designed SIRPα agonist sequence based on an in silico evolution strategy algorithm. Such insertions may enhance SIRPα agonist stability and preserve SIRPα binding and signaling while reducing binding to other endogenous protein binding partners and associated signaling. In some embodiments, these constructs comprise less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or less sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, these constructs have a tailored conformational ensemble relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence such as that contained in any one of SEQ ID NOS: 1-4).

[0054] Another aspect of the present disclosure includes computationally engineered SIRPα-binding sequences that include mutations (single point mutations or combinations thereof) relative to any one of SEQ ID NOs: 1-4 grafted onto computationally designed SIRPα-binding sequences based on in silico evolutionary strategies and / or trained algorithms (e.g., machine learning algorithms) to enhance the stability of the SIRPα-binding polypeptide, retain binding and signaling via SIRPα, but reduce binding to other endogenous protein binding partners and associated signaling. In some embodiments, these constructs comprise less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or less sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, these constructs have a regulated conformational ensemble relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence, such as that contained in any one of SEQ ID NOs: 1-4).

[0055] Another aspect of the present disclosure includes engineered SIRPα-binding polypeptides comprising one or more mutations in CD47 (e.g., one or more of those listed in Tables 3 and 4) grafted onto computationally designed SIRPα-binding sequences based on in silico evolutionary strategies and / or trained algorithms (such as machine learning algorithms) to enhance the stability of the SIRPα-binding polypeptide, retain binding and signaling via SIRPα, but reduce binding to other endogenous protein binding partners and associated signaling. In some embodiments, these constructs comprise less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or less sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, the computationally designed SIRPα-binding sequences comprising additional mutations are characterized by a modulated conformational ensemble relative to that of a reference polypeptide (e.g., a wild-type CD47 sequence such as that included in any one of SEQ ID NOS: 1-4).

[0056] Another aspect of the present disclosure includes a computationally engineered SIRPα binding polypeptide comprising a chimera comprising an ECD domain of residues 19-131 (1-113 after signal peptide cleavage) of any one of SEQ ID NOS: 1-4 and a TMD from a SIRPα agonist sequence that has been computationally designed based on an in silico evolution strategy algorithm and / or a trained algorithm (e.g., a machine learning algorithm) to enhance SIRPα agonist TMD stability. In some embodiments, the engineered SIRPα agonist comprises a chimera comprising residues 19-131 (1-113 after signal peptide cleavage) of any one of SEQ ID NOS: 1-4 and a TMD from another organism. In some embodiments, the engineered SIRPα agonist comprises a chimera comprising an engineered (e.g., rationally or computationally) ECD of residues 19-131 (1-113 after signal peptide cleavage) of any one of SEQ ID NOS: 1-4 and a TMD from another organism. In some embodiments, the SIRPα-binding polypeptide chimera comprising the engineered domain is characterized by a modulated conformational ensemble relative to that of the reference polypeptide (e.g., a wild-type CD47 sequence such as that contained in any one of SEQ ID NOs: 1-4).

[0057] Another aspect of the present disclosure includes computationally engineered SIRPα binding polypeptides, including chimeras encompassing residues 19-131 (1-113 after signal peptide cleavage) of any one of SEQ ID NOS: 1-4 bearing single or multiple mutations (such as those listed in Table 3, Table 4, and / or rationally / computationally engineered mutations) and a TMD from a SIRPα agonist sequence that has been computationally designed based on an in silico evolution strategy algorithm and / or a trained algorithm (such as a machine learning algorithm) to enhance SIRPα agonist TMD stability. In some embodiments, the computationally modified chimeras are combined with mutations to modulate the conformational ensemble of the SIRPα binding polypeptide relative to a reference polypeptide (e.g., a wild-type CD47 sequence such as that included in any one of SEQ ID NOS: 1-4).

[0058] definition The practice of some methods disclosed herein employs, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds.) series, Methods in Enzymology (Academic Press, Inc.) series, PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)), which are incorporated herein by reference in their entireties.

[0059] Whenever the terms "at least," "greater," or "greater than or equal to" precede the first number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to each of the numbers in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0060] Whenever the terms "no more than," "less than," or "less than or equal to" precede the first number in a series of two or more numbers, the terms "no more than," "less than," or "less than or equal to" apply to each and every number in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0061] As used herein, "cell" generally refers to a biological cell. A cell can be the basic structural, functional, and / or biological unit of a living organism. A cell can be from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, liverworts, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, etc.). C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes the cells are not derived from a natural organism (e.g., cells can be synthetically produced, sometimes referred to as artificial cells).

[0062] The term "nucleotide," as used herein, generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term "nucleotide," as used herein, may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled, such as with a moiety containing an optically detectable moiety (e.g., a fluorophore). Labeling may also be performed using quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.Fluorescent labels for nucleotides may include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif.; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; and Boehringer Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP available from Mannheim, Indianapolis, Ind., and chromosome-labeled nucleotides BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7- ... Examples of suitable nucleotides include Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides can also be labeled or marked by chemical modification.The chemically modified single nucleotide may be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs may include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP). The nucleotide may include a nucleotide analog. In some embodiments, the nucleotide analog may include a natural nucleotide structure modified at any position to alter certain chemical properties of the nucleotide but still retain the ability of the nucleotide analog to perform its intended function (e.g., hybridization to other nucleotides in RNA or DNA). Examples of positions of nucleotides that can be derivatized include the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, such as 6-(2-amino)propyluridine; the 8-position of adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, such as 7-deaza-adenosine:O and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or otherwise known in the art) nucleotides, as well as other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310. Nucleotide analogs can also include modifications to the sugar moiety of the nucleotide. For example, the 2'OH- group can be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH, NHR, NR, COOR, or OR, where R is a substituted or unsubstituted C-C alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.Examples of positions of nucleotides that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc., the 6-position, e.g., 6-(2-amino)propyluridine, and the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine:O and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or otherwise known in the art) nucleotides, and other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0063] Nucleotide analogs can also include modifications to the sugar portion of the nucleotide. For example, the 2'OH group can be replaced with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH, NHR, NR, COOR, or OR, where R is a substituted or unsubstituted C-C alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

[0064] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in either single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can be present in a cell-free environment. A polynucleotide can be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function. A polynucleotide can contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, locus(s) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0065] The term "transfection" or "transfected" generally refers to the introduction of nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecule may be a genetic sequence encoding an entire protein or a functional portion thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88, incorporated herein by reference in its entirety.

[0066] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and generally refer to a polymer of at least two amino acid residues joined by a peptide bond. The term does not denote a specific length of the polymer, nor is it intended to denote or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. The term applies to naturally occurring amino acid polymers and amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structure (e.g., domains). The term also encompasses amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. The terms "amino acid" and "amino acids," as used herein, generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids that have been chemically modified to include non-naturally occurring groups or chemical moieties on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0067] As used herein, the term "non-native" may generally refer to a nucleic acid or polypeptide sequence not found in a native nucleic acid or protein. Non-native may refer to an affinity tag. Non-native may refer to a fusion. Non-native may refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, and / or deletions. A non-native sequence may exhibit and / or encode an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that may also be exhibited by the nucleic acid and / or polypeptide sequence to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence may be linked by genetic engineering to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) to generate a chimeric nucleic acid and / or polypeptide sequence encoding the chimeric nucleic acid and / or polypeptide.

[0068] The term "promoter," as used herein, generally refers to a regulatory DNA region that controls the transcription or expression of a gene and may be located adjacent to or overlapping the nucleotide or region of nucleotide at which RNA transcription is initiated. A promoter may contain specific DNA sequences that bind protein factors, often referred to as transcription factors, which promote the binding of RNA polymerase to DNA, resulting in gene transcription. A "basal promoter," also referred to as a "core promoter," generally refers to a promoter that contains all of the elements basically required to promote the transcriptional expression of an operably linked polynucleotide. A eukaryotic basal promoter may contain a TATA box and / or a CAAT box.

[0069] The term "expression," as used herein, generally refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0070] As used herein, "operably linked," "operable linkage," "operatively linked," or their grammatical equivalents generally refer to the juxtaposition of genetic elements, e.g., promoters, enhancers, polyadenylation sequences, and the like, wherein these elements are in a relationship permitting them to operate in the expected manner. For example, a regulatory element, which may include a promoter and / or enhancer sequence, is operably linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There can be intervening residues between the regulatory element and the coding region so long as this functional relationship is maintained.

[0071] "Vector," as used herein, generally refers to a polymer or assembly of polymers that contains or associates with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. A vector generally contains genetic elements, such as regulatory elements, operably linked to a gene to promote expression of the gene in a target.

[0072] As used herein, "expression cassette" and "nucleic acid cassette" are used interchangeably and generally refer to a combination of nucleic acid sequences or elements that are expressed together or operably linked for expression. In some cases, an expression cassette refers to a combination of regulatory elements and one or more genes to which they are operably linked for expression.

[0073] A "functional fragment" of a DNA or protein sequence generally refers to a fragment that retains a biological activity (either functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence may be its ability to affect expression in a manner known to be attributed to the full-length sequence.

[0074] As used herein, "engineered" or an object generally refers to an object that has been modified by human intervention. By way of non-limiting example, a nucleic acid can be modified by changing its sequence to a non-naturally occurring sequence, a nucleic acid can be modified by ligating it to a nucleic acid with which it is not naturally associated such that the ligated product has a function not present in the original nucleic acid, an engineered nucleic acid can be synthesized in vitro using a non-naturally occurring sequence, a protein can be modified by changing its amino acid sequence to a non-naturally occurring sequence, and an engineered protein can acquire a new function or property. An "engineered" system includes at least one engineered component.

[0075] As used herein, "optimally aligned" generally refers to the alignment of two amino acid sequences that gives the highest percent identity score or maximizes the number of matching residues.

[0076] As used herein, "synthetic" and "artificial" are used interchangeably and generally refer to proteins or domains thereof that have low sequence identity to naturally occurring human proteins (e.g., less than 80% sequence identity, less than 70% sequence identity, less than 60% sequence identity, less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity). For example, VPR and VP64 domains are synthetic transactivation domains.

[0077] The term "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example, BLASTP, using the parameters of the BLOSUM62 scoring matrix, which sets a word length (W) of 3, an expectation (E) of 10, and presence of 11, a gap cost at an extension of 1, and using a conditional composition score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP, using the parameters of the PAM30 scoring matrix, which sets a word length (W) of 2, an expectation (E) of 1,000,000, and a gap cost at 9 for opening a gap and 1 for extending a gap for sequences of less than 30 residues (default parameters for BLASTP in the BLAST suite available at https: / / blast.ncbi.nlm.nih.gov); or CLUSTALW, with parameters for the Smith-Waterman homology search algorithm, with parameters of 2 matches, -1 mismatches, and -1 gaps; MUSCLE, with default parameters; MAFFT, with parameters retree of 2, a maximum iteration of 1,000; Novafold, with default parameters; and HMMER hmmalign, with default parameters.

[0078] As defined herein, the terms "inhibition," "inhibit," "inhibiting," and the like, with respect to protein-inhibitor interactions, generally refer to negatively affecting (e.g., decreasing) the activity or function of a protein relative to the activity or function of the protein in the absence of the inhibitor. Inhibition can refer to the reduction of a disease or disease symptoms. Inhibition can refer to the reduction of activity of a specific protein or nucleic acid target. The protein can be deoxycytidine kinase. Thus, inhibition includes at least partially, partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signal transduction or the amount of an enzymatic activity or protein.

[0079] The term "modulator" refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule or the physical state of a target of the molecule.

[0080] The term "modulate" generally refers to the act of changing or varying one or more properties. Generally, "modulation" refers to the process of changing or varying one or more properties. For example, a modulator of a target protein may alter the property or function of the target molecule by increasing or decreasing the amount of the target molecule. A modulator of a disease may reduce the symptoms, cause, or characteristics of the target disease.

[0081] As used herein, "immune checkpoint modulator" generally refers to an agent that results in the activation or inhibition of one or more immune checkpoint proteins. For example, immune checkpoint modulators may include, but are not limited to, CD47, PD-L1, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, TIM-3, VISTA, and SIGLEC7.

[0082] The term "pluripotent cells" generally refers to cells that are capable of self-renewal and proliferation while maintaining an undifferentiated state and that, under appropriate conditions, can be induced to differentiate into specialized cell types. As used herein, the term "pluripotent cells" encompasses embryonic stem cells and other types of stem cells, including fetal, amniotic, or somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Additional exemplary stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (described in Cowan, CA et. al, New England J. Med. 350:13. (2004), which is incorporated herein by reference in its entirety).

[0083] The term "pluripotent stem cells," as used herein, generally refers to cells that have the ability to differentiate into any of the three germ layers: endoderm (e.g., the gastric junction, gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term "pluripotent stem cells," as used herein, may also encompass "induced pluripotent stem cells" or "iPSCs," which are a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be generated by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the derivation of iPS cells are known in the art and are described further below. (See, e.g., Zhou et al., Stem Cells 27(11):2667-74(2009); Huangfu et al., Nature Biotechnol. 26(7):795(2008); Woltjen et al., Nature 458(7239):766-770(2009); and Zhou et al., Cell Stem Cell 8:381-384(2009), each of which is incorporated herein by reference in its entirety.)

[0084] The term "pluripotent stem cell characteristics" generally refers to cellular features that distinguish pluripotent stem cells from other cells. The ability to give rise to progeny that, under appropriate conditions, can undergo differentiation into cell types that collectively exhibit properties associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. The expression or non-expression of certain combinations of molecular markers is also a pluripotent stem cell characteristic. For example, human pluripotent stem cells express at least some, and in some embodiments, all, of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cell morphology associated with pluripotent stem cells is also a pluripotent stem cell characteristic. As described herein, cells do not need to go through pluripotency to be reprogrammed into endodermal progenitor cells and / or hepatocytes.

[0085] As used herein, the term "multipotent" or "multipotent cell" generally refers to a cell type that can give rise to a limited number of other specific cell types. For example, induced multipotent cells can form endoderm cells. Additionally, multipotent blood stem cells can differentiate themselves into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc.

[0086] As used herein, the term "oligopotent" generally refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells can form cells of either the lymphoid or myeloid lineage, respectively.

[0087] As used herein, the term "unipotent" generally refers to the ability of a cell to form a single cell type.

[0088] As used herein, the term "totipotent" generally refers to the ability of a cell to form an entire organism.

[0089] As used herein, the term "non-pluripotent cells" generally refers to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, cells from tissues selected from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells. Starting cells used to generate induced pluripotent cells, endodermal progenitor cells, and hepatocytes may be non-pluripotent cells.

[0090] Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In certain embodiments, a less potent cell is considered "differentiated" with respect to a more potent cell.

[0091] The term "somatic cells," as used herein, generally refers to cells that form the body of an organism. Somatic cells include cells that make up the organs, skin, blood, bone, and connective tissue in an organism, but do not include germ cells.

[0092] As used herein, the term "subject" or "patient" generally refers to any animal, such as a domestic animal, a zoo animal, or a human. A "subject" or "patient" can be a mammal, such as a dog, cat, bird, livestock, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) who have a disease or disorder associated with the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bone, bone marrow, and the like.

[0093] The term "low immunogenic pluripotent cells" or "HIP cells," as used herein, generally refers to pluripotent cells that retain their pluripotent characteristics but generate a reduced immunological rejection response when transferred into an allogeneic host. In some cases, HIP cells do not generate an immune response. Thus, "low immunogenic," as used herein, generally refers to a significantly reduced or eliminated immune response when compared to the immune response of the parent (e.g., wild-type) cells outlined herein. In some cases, the immune response includes natural killer (NK) cell cytotoxicity. In some cases, the immune response includes macrophage cytotoxicity. In some cases, the HIP cells described herein are immunologically silenced and still retain pluripotent capabilities.

[0094] The term "allogeneic," as used herein, generally refers to the genetic dissimilarity between the host organism and the cell graft.

[0095] "Inhibitors," "activators," and "modulators" generally refer to substances or conditions that affect the function or expression of biologically relevant molecules. The term "modulator" generally includes both inhibitors and activators, which may be identified using in vitro and in vivo assays for the expression or activity of the target molecule.

[0096] The term "inhibitor" generally refers to an agent that, for example, inhibits expression or binds to a target molecule or protein. They may partially or completely block stimulation or may have protease inhibitor activity. They may reduce, decrease, prevent, or delay activation, including inactivating, desensitizing, or downregulating the activity of the described target protein. A modulator may be an antagonist of the target molecule or protein.

[0097] "Activator" generally refers to an agent that induces or activates, for example, the function or expression of a target molecule or protein. They may bind to, stimulate, increase, open, activate, or promote target molecule activity. An activator may be an agonist of the target molecule or protein.

[0098] As used herein, the term "modification" generally refers to a change that physically distinguishes a modified molecule from a parent molecule. In one embodiment, the amino acid changes in a CD47, HSVtk, EC-CD, or iCasp9 variant polypeptide prepared according to the methods described herein distinguish it from a corresponding parent that has not been modified according to the methods described herein, such as a wild-type protein, a naturally occurring mutant protein, or another engineered protein that does not contain the modification of such a variant polypeptide. In another embodiment, the variant polypeptide contains one or more modifications that distinguish the function of the variant polypeptide from the unmodified polypeptide. For example, the amino acid changes in the variant polypeptide affect its receptor binding profile. In other embodiments, the variant polypeptide contains substitution, deletion, or insertion modifications, or a combination thereof. In another embodiment, the variant polypeptide contains one or more modifications that increase its affinity for a receptor compared to the affinity of the unmodified polypeptide.

[0099] In some cases, a variant polypeptide comprises one or more substitutions, insertions, or deletions relative to the corresponding native or parent sequence. In certain embodiments, a variant polypeptide comprises one or more substitutions, insertions, or deletions of 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more modifications.

[0100] 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.

[0101] It is understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0102] Whenever the terms "at least," "greater," or "greater than or equal to" precede the first number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to each of the numbers in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0103] SIPRα-binding polypeptide In one aspect, the present disclosure provides a signal-regulatory protein alpha (SIRPα)-binding polypeptide. In some cases, the SIRPα-binding peptide comprises the sequence of a wild-type (WT) CD47 variant. Exemplary WT CD47 splice variants include Q08722-1 (SEQ ID NO: 1), Q08722-2 (SEQ ID NO: 2), Q08722-3 (SEQ ID NO: 3), and Q08722-4 (SEQ ID NO: 4), which are shown in Table 1. In some cases, the SIRPα-binding peptide comprises one or more modifications (e.g., mutations) of a WT CD47 variant. In some cases, the SIRPα-binding peptide is an engineered SIRPα-binding peptide. The engineered SIRPα-binding peptide can be generated by one or more of the strategies disclosed herein. Relative to a reference polypeptide (e.g., a WT CD47 variant such as any one of SEQ ID NOs: 1-4), the engineered SIRPα-binding peptide can comprise different structural and / or functional characteristics described herein. In some embodiments, the reference polypeptide comprises residues 19 through the last residue of any one of SEQ ID NOs: 1-4. In some embodiments, the reference polypeptide comprises residues 19-290 of any one of SEQ ID NOs: 1-4.

[0104] The engineered SIRPα-binding peptide may comprise an extracellular domain (ECD). The ECD may comprise a sequence having at least about 50%, at least about 55%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the ECD of any one of SEQ ID NOs: 1-4, 10-640, or 648-653. The domain boundaries of the ECD can be identified, for example, by optimal alignment to any one of SEQ ID NOs: 1 to 4. In one example, the ECD of SEQ ID NO: 1 includes residues 19 to 131 of SEQ ID NO: 1 (residues 1 to 113 after cleavage of the leader peptide).

[0105] The engineered SIRPα-binding polypeptide may comprise an extracellular loop region (ECLR). The ECLR may comprise portions of the ECD and TMD that are on the extracellular side of the cell membrane when expressed by a cell. The ECLR may comprise, at least in part, the extracellular loop 1 (ECL1) region. The ECL1 region may comprise any of the extracellular residues of the loop region connecting helices II and III of the TMD described herein (e.g., as contained in wild-type CD47 or an engineered SIRPα-binding polypeptide of the disclosure). In one example, ECL1 comprises residues 198-207 of SEQ ID NO: 1. The ECLR may comprise, at least in part, the extracellular loop 2 (ECL2) region. The ECL2 region may comprise any of the extracellular residues of the loop region connecting helices VI and V of the TMD described herein (e.g., as contained in wild-type CD47 or an engineered SIRPα-binding polypeptide of the disclosure). In one example, ECL2 comprises residues 258-267 of SEQ ID NO: 1. The ECLR may comprise, at least in part, a peptide linker connecting the C-terminus of the ECD and the N-terminal extracellular tip of the TMD. In one example, the peptide linker comprises residues 132-137 of SEQ ID NO:1. The ECLR may comprise a sequence having at least about 50%, at least about 55%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the ECLR of any one of SEQ ID NOs: 1 to 4, 10 to 640, or 648 to 653. The domain boundaries of the ECLR may be identified, for example, by optimal alignment to any one of SEQ ID NOs: 1 to 4.In one example, the ECLR of SEQ ID NO:1 comprises residues 132-137, 198-207, and 258-267 of SEQ ID NO:1 (residues 114-119, 180-189, and 240-249 after cleavage of the leader peptide).

[0106] The engineered SIRPα-binding peptide may comprise a transmembrane domain (TMD). The TMD may comprise five transmembrane helices configured to span the lipid membrane of a cell expressing the SIRPα polypeptide. The TMD may comprise a first transmembrane helix (helix I). In one example, helix I comprises residues 142-162 of SEQ ID NO:1. The TMD may comprise a second transmembrane helix (helix II). In one example, helix II comprises residues 177-197 of SEQ ID NO:1. The TMD may comprise a third transmembrane helix (helix III). In one example, helix III comprises residues 208-228 of SEQ ID NO:1. The TMD may comprise a fourth transmembrane helix (helix IV). In one example, helix IV comprises residues 236-256 of SEQ ID NO:1. The TMD may comprise a fifth transmembrane helix (helix V). In one example, helix V comprises residues 269-289 of SEQ ID NO:1. The TMD can comprise a sequence having at least about 50%, at least about 55%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the TMD of any one of SEQ ID NOs: 1-4, 10-640, or 648-653. Domain boundaries of the TMD can be identified, for example, by optimal alignment to any one of SEQ ID NOs: 1-4. In one example, the TMD of SEQ ID NO: 1 comprises residues 142-162, 177-197, 208-228, 236-256, and 269-289 of SEQ ID NO: 1 (residues 124-144, 159-179, 190-210, 218-238, and 251-271 after cleavage of the leader peptide).

[0107] The SIRPα binding polypeptide may include a signal peptide. The signal peptide may include the first 18 residues of any one of SEQ ID NOs: 1-4. The signal peptide may target the protein to the endoplasmic reticulum for further processing and ultimate cell surface expression. In some embodiments, the signal peptide is cleaved to yield the mature polypeptide. In some embodiments, the engineered SIRPα binding polypeptide does not include a signal peptide.

[0108] The αSIRPα-binding polypeptide may comprise a C-terminal domain (CTD). The CTD may be present on the cytoplasmic side of the membrane of a cell expressing the SIRPα-binding polypeptide. The CTD may comprise residue 290 through the last residue of any one of SEQ ID NOs: 1-4. In some embodiments, the engineered SIRPα-binding peptide does not comprise a CTD.

[0109] [Table 1]

[0110] In some embodiments, a polypeptide described herein (e.g., an engineered SIRPα-binding polypeptide) comprises one or more cysteine mutations (e.g., relative to a reference polypeptide comprising the sequence of any one of SEQ ID NOs: 1-4). Examples of cysteine mutations to induce oligomerization (e.g., relative to a reference sequence such as SEQ ID NO: 1) are shown in Table 2 below.

[0111] [Table 2]

[0112] CD47 cancer variants In some embodiments, a polypeptide described herein (e.g., an engineered SIRPα binding polypeptide) comprises one or more mutations identified in or associated with cancer (e.g., relative to a reference polypeptide comprising the sequence of any one of SEQ ID NOs: 1-4).

[0113] In some aspects, the mutation may be a single CD47 point mutation. In some embodiments, the mutation may include multiple CD47 mutations. In some embodiments, the mutation has been identified in a cancer sample. In some embodiments, the mutation may be available in a public database (e.g., COSMIC, TCGA, cBioPortal, OncoDB). In some embodiments, the mutation may be present in a very low percentage of cancer samples or may be overexpressed in some cancer samples / types. Some of these mutations have been mapped on the crystal structure of CD47 and found to cluster in important functional regions of the receptor. For example, the cancer-associated CD47 mutations disclosed herein have been mapped to the extracellular loop region (ECLR) region, transmembrane helix III, and extracellular domain (ECD).

[0114] In some embodiments, the mutations map to different regions of the molecule. In some embodiments, SIRPα-binding polypeptides containing the cancer mutations described herein may have functional effects that depend on their location on the crystal structure of the polypeptide. Accordingly, these mutations can be subdivided based on where they occur in the molecule, such as in the extracellular domain (ECD), the transmembrane domain (TMD), the extracellular loop region (ECLR), the C-terminal domain (CTD), or between any two of these domains. Based on their relative location within the molecule, mutations can affect secondary, tertiary, or quaternary structure by creating opportunities for new non-covalent and / or covalent interactions or abolishing others. These structural changes can, in turn, affect functional aspects of SIRPα-binding polypeptide transmembrane signaling. For example, the cancer mutation W136C can be configured to form a cysteine residue with C259, resulting in an additional disulfide bond between the ECD and TMD interdomain linker and the extracellular portion of helix V. As discussed elsewhere herein, such changes in interdomain interactions can alter the conformational ensemble of the polypeptide, leading to various functional consequences, such as enhanced binding to SIRPα. In another example, mutations can affect or alter the quaternary assembly of SIRPα-binding polypeptides by promoting or preventing dimer or oligomer formation. In some embodiments, mutations (alone or in combination) can increase the stability of the polypeptide, and mutations can disrupt binding of endogenous protein partners (other than SIRPα). Thus, engineered polypeptides of the present disclosure can include one or more of the cancer mutations disclosed herein to confer a particular functional outcome (e.g., enhanced SIPRα binding) relative to a reference polypeptide (e.g., a wild-type human CD47 molecule). In another example, the cancer-associated mutations discussed herein were found to map to the ECD. In some embodiments, mutations in the ECD can alter / disrupt binding to TSP-1, an integrin, or increase affinity for SIRPα.In some embodiments, the CD47 ECD cancer mutation site represents a region of instability on CD47, and the mutation may increase receptor stability, which may enhance function.

[0115] Non-limiting examples of cancer-associated CD47 mutations that may appear in polypeptides of the present disclosure are listed in Table 3.

[0116] [Table 3-1]

[0117] [Table 3-2]

[0118] In some cases, the SIRPα binding polypeptide comprises at least one variant listed in Table 3. In some cases, the SIRPα binding polypeptide comprises any 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 mutations listed in Table 3. In some cases, the SIRPα binding polypeptide comprises at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 mutations listed in Table 3. In some cases, the SIRPα binding polypeptide includes at most 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation listed in Table 3.

[0119] In some embodiments, the SIRPα binding polypeptide comprises at least one mutation at M31, L40, C42, D47, D64, C75, E80, F97, G105, K106, F106, K111, S123, S127, K128, F131, C132, C136, K140, T142, G146, M153, L157, L160, E166, C170, D178, A203, S207, V210, D211, L214, S215, V262, L264, Y267, or any combination thereof, wherein the position of the mutation is relative to any one of SEQ ID NOs: 1-4. In some embodiments, the SIRPα binding polypeptide comprises the M31 residue. In some embodiments, the SIRPα binding polypeptide comprises the L40 residue. In some embodiments, a SIRPα binding polypeptide comprises a C42 residue. In some embodiments, a SIRPα binding polypeptide comprises a D47 residue. In some embodiments, a SIRPα binding polypeptide comprises a D64 residue. In some embodiments, a SIRPα binding polypeptide comprises a C75 residue. In some embodiments, a SIRPα binding polypeptide comprises an E80 residue. In some embodiments, a SIRPα binding polypeptide comprises an F97 residue. In some embodiments, a SIRPα binding polypeptide comprises a G105 residue. In some embodiments, a SIRPα binding polypeptide comprises a K106 residue. In some embodiments, a SIRPα binding polypeptide comprises an F106 residue. In some embodiments, a SIRPα binding polypeptide comprises a K111 residue. In some embodiments, a SIRPα binding polypeptide comprises an S123 residue. In some embodiments, a SIRPα binding polypeptide comprises an S127 residue. In some embodiments, a SIRPα binding polypeptide comprises a K128 residue. In some embodiments, a SIRPα binding polypeptide comprises an F131 residue. In some embodiments, the SIRPα binding polypeptide comprises a C132 residue. In some embodiments, the SIRPα binding polypeptide comprises a C136 residue. In some embodiments, the SIRPα binding polypeptide comprises a K140 residue. In some embodiments, the SIRPα binding polypeptide comprises a T142 residue.In some embodiments, a SIRPα binding polypeptide comprises a G146 residue. In some embodiments, a SIRPα binding polypeptide comprises a M153 residue. In some embodiments, a SIRPα binding polypeptide comprises a L157 residue. In some embodiments, a SIRPα binding polypeptide comprises a L160 residue. In some embodiments, a SIRPα binding polypeptide comprises an E166 residue. In some embodiments, a SIRPα binding polypeptide comprises a C170 residue. In some embodiments, a SIRPα binding polypeptide comprises a D178 residue. In some embodiments, a SIRPα binding polypeptide comprises an A203 residue. In some embodiments, a SIRPα binding polypeptide comprises a S207 residue. In some embodiments, a SIRPα binding polypeptide comprises a V210 residue. In some embodiments, a SIRPα binding polypeptide comprises a D211 residue. In some embodiments, a SIRPα binding polypeptide comprises a L214 residue. In some embodiments, a SIRPα binding polypeptide comprises a S215 residue. In some embodiments, a SIRPα binding polypeptide comprises a V262 residue. In some embodiments, the SIRPα binding polypeptide comprises the L264 residue.In some embodiments, the SIRPα binding polypeptide comprises the Y267 residue.

[0120] In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to any one of SEQ ID NOs: 385-475. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 389. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 391. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 419. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 393. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 395.In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 397. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 406. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 411. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 413. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 467. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 468.In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 469. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 469. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 470. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO:471. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 473. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO:474.In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 475. In some cases, the SIRPα binding polypeptide comprises a sequence at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 472.

[0121] CD47 polymorphism In some embodiments, a polypeptide (e.g., an engineered SIRPα-binding polypeptide) described herein contains one or more mutations identified as naturally occurring polymorphisms (e.g., relative to a reference polypeptide comprising any one of SEQ ID NOs: 1-4). Some of these mutations have been mapped to the crystal structure of human CD47 and found to cluster in important functional regions of the receptor. For example, the CD47 polymorphisms disclosed herein have been mapped to the extracellular loop region (ECLR) region, the transmembrane domain (TMD), and the extracellular domain (ECD).

[0122] In some embodiments, polymorphisms map to different regions of the molecule. In some embodiments, the polymorphisms described herein may have functional effects that depend on their location on the crystal structure of a molecule (e.g., a SIRPα-binding polypeptide). Thus, these polymorphisms can be subdivided based on where they occur in the molecule, such as in the extracellular domain (ECD), the transmembrane domain (TMD), the extracellular loop region (ECLR), or between any two of these molecules. Based on their relative location within the molecule, polymorphisms can affect secondary, tertiary, or quaternary structure by creating opportunities for new non-covalent and / or covalent interactions or nullifying others. These structural changes can then affect functional aspects of SIRPα-binding polypeptide transmembrane signaling. For example, the polymorphism C33Y can disrupt an interdomain disulfide bond, thus altering the conformational ensemble of the molecule. In another example, polymorphisms may affect or alter the quaternary assembly of SIRPα-binding polypeptides by promoting or preventing the formation of dimers or oligomers. In some embodiments, polymorphisms (alone or in combination) may increase receptor stability, and polymorphisms may disrupt binding of endogenous protein partners (other than SIRPα). Thus, engineered polypeptides of the present disclosure may contain one or more of the polymorphisms disclosed herein to confer a particular functional result (e.g., enhanced SIPRα binding) relative to wild-type CD47 molecules.

[0123] Non-limiting examples of CD47 polymorphisms that may appear in polypeptides of the present disclosure are listed in Table 4.

[0124] [Table 4-1]

[0125] [Table 4-2]

[0126]

Table 4-3

[0127]

Table 4-4

[0128] In some cases, the SIRPα binding polypeptide comprises at least one polymorphism listed in Table 4. In some cases, the SIRPα binding polypeptide comprises any 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 1 9, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 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, or 132 polymorphisms. In some cases, the SIRPα binding polypeptide is selected from the group consisting of at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 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, or 132 polymorphisms.In some cases, the SIRPα binding polypeptide is at most 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 8, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 polymorphism.

[0129] In some cases, the SIRPα binding polypeptide comprises a sequence that is at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to any one of SEQ ID NOs: 476-633 and 648. In some cases, the SIRPα binding polypeptide comprises a sequence that is at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO:648.

[0130] Rational mutation In some embodiments, a polypeptide (e.g., an engineered SIRPα-binding polypeptide) described herein comprises one or more mutations developed or determined based on knowledge of peptide functionality (e.g., relative to a reference polypeptide comprising the sequence of any one of SEQ ID NOS: 1-4). In some embodiments, the determination of potentially useful mutations may use tools and data, which may include bioinformatics models, protein structure data, crystallographic information, protein function data, and protein conformation models. The analysis may include algorithmic calculations, expert determination, or both.

[0131] In some embodiments, the polypeptide contains one or more mutations configured to generate new covalent or non-covalent interactions or to abolish existing covalent or non-covalent interactions. In one example, the SIRPα-binding sequence contains a D35R mutation (e.g., relative to any one of SEQ ID NOS: 1-4) and an A262E mutation. The arginine residue can form an interdomain ionic interaction with glutamate, thus altering the conformational ensemble and function of the polypeptide relative to wild-type CD47. In another example, the polypeptide contains a S138F mutation configured to interact with the T31M cancer mutation. The bulky aromatic side chain of the phenylalanine residue was selected to compensate for the loss of steric bulk near the methionine and further restrict movement between the TMD and ECD. As a result, the mutant polypeptide can exhibit an altered conformational ensemble relative to the wild-type sequence.

[0132] In some embodiments, the SIRPα binding polypeptide comprises at least one residue of Y31, A32, R35, K35, P71, A77, A79, N80, L100, K138, L164, M185, A211, S259, E262, and any combination thereof, wherein the mutation is relative to any one of SEQ ID NOs: 1-4. In some embodiments, the SIRPα binding polypeptide comprises the Y31 residue. In some embodiments, the SIRPα binding polypeptide comprises the A32 residue. In some embodiments, the SIRPα binding polypeptide comprises the R35 residue. In some embodiments, the SIRPα binding polypeptide comprises the K35 residue. In some embodiments, the SIRPα binding polypeptide comprises the P71 residue. In some embodiments, the SIRPα binding polypeptide comprises the A77 residue. In some embodiments, the SIRPα binding polypeptide comprises the A79 residue. In some embodiments, the SIRPα binding polypeptide comprises the N80 residue. In some embodiments, a SIRPα binding polypeptide comprises an L100 residue. In some embodiments, a SIRPα binding polypeptide comprises a K138 residue. In some embodiments, a SIRPα binding polypeptide comprises an L164 residue. In some embodiments, a SIRPα binding polypeptide comprises an M185 residue. In some embodiments, a SIRPα binding polypeptide comprises an A211 residue. In some embodiments, a SIRPα binding polypeptide comprises an S259 residue. In some embodiments, a SIRPα binding polypeptide comprises an E262 residue.

[0133] In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to any one of SEQ ID NOs: 634-638 and 648-653. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 634. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 635. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 636. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 637. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 638.In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 648. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 649. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 650. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 651. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 652. In some embodiments, the SIRPα binding polypeptide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical to SEQ ID NO: 653.

[0134] Chimera In some embodiments, a polypeptide described herein (e.g., an engineered SIRPα-binding polypeptide) comprises one or more regions or portions thereof from a non-human protein (e.g., relative to a reference polypeptide comprising the sequence of any one of SEQ ID NOs: 1-4). In some embodiments, the region is an extracellular domain (ECD). In some embodiments, the region is a transmembrane domain (TMD) or portion thereof. In some embodiments, the region is an extracellular loop region (ECLD). In some embodiments, the region is a C-terminal intracellular domain. In some embodiments, the region comprises any two of an ECD, a TMD, an ECLR, or a C-terminal intracellular domain, or portions thereof. In some embodiments, the region comprises any three of an ECD, a TMD, an ECLR, or a C-terminal intracellular domain, or portions thereof. In some embodiments, the region comprises all of an ECD, a TMD, an ECLR, or a C-terminal domain, or portions thereof.

[0135] In some embodiments, to avoid signal transduction of certain (e.g., non-SIRPα) binding partners upon binding, engineered SIRPα-binding polypeptides may comprise hybrid molecules with detuned human functions. In some embodiments, the TMD and C-terminal domains may be based on domains from non-human species. In some embodiments, the ECD domain may contain specific residues that can be replaced with residues from different species. In some embodiments, the TMD and C-terminal domains may contain specific residues that can be replaced with residues from different species. Polypeptides of such compositions may be prevented from binding to human TSP-1 and integrins and may abolish transmembrane signaling. In some embodiments, the chimeras may also contain modified TMD domains.

[0136] In some embodiments, the non-human sequence may be derived from any other non-human that expresses CD47. Representative, non-limiting species from which the sequence may be derived include Gallus gallus, Alligator mississippiensis, Accipiter gentilis, Anas platyrhynchos, Ficedula albicollis, Phasianus colchicus, and Chloebia gouldiae (Erythrura gouldiae). In some embodiments, the non-human sequence may comprise a sequence having at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOs: 641-647. In some embodiments, a polypeptide described herein (e.g., a hybrid SIRPα binding polypeptide) comprises a sequence having at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:648.

[0137] Loop Insertion In some embodiments, a polypeptide described herein (e.g., an engineered SIRPα binding polypeptide) comprises a modified peptide sequence such that a peptide linker is inserted into a loop or linker of the polypeptide structure (e.g., an RVVSWF linker comprised in residues 132-137 of any one of SEQ ID NOs: 1-4) (e.g., relative to a reference polypeptide comprising the sequence of any one of SEQ ID NOs: 1-4). In some embodiments, the loop is a polypeptide sequence between the ECD domain and the TMD domain of the polypeptide. In some embodiments, the loop comprises residues 132-137 of any one of SEQ ID NOs: 1-4. The loop insertion can be inserted between any two residues in the range 132-137, before residue 132, or after residue 137 of the reference polypeptide sequence of any one of SEQ ID NOs: 1-4. In some embodiments, a linker is added between the ECD domain of a reference peptide having an IgV-like protein fold and the first helix of a TMD comprised in a protein fold containing five transmembrane helices. In some embodiments, the linker sequence can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more residues long. The linker can include a pair of cysteine residues configured to form a disulfide bond. The pair of cysteine residues can be separated by any suitable number of residues. In some embodiments, the pair of cysteine residues is separated by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more residues. In some embodiments, the pair of cysteine residues is separated by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more residues. In some embodiments, pairs of cysteine residues are separated by no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or fewer residues.

[0138] In some embodiments, the polypeptides described herein comprise a sequence having at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:640. In some embodiments, a polypeptide described herein comprises a sequence having at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:639.

[0139] In some embodiments, a polypeptide described herein (e.g., a synthetic SIRPα binding polypeptide) comprises a sequence having at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOs: 10-384. In some embodiments, the percent identity is between any two of these values. In some embodiments, the polypeptide has about 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less identity to any one of the sequences of SEQ ID NOs: 10-384. In some embodiments, the percent identity is between any two of these values.

[0140] In some embodiments, a polypeptide described herein (e.g., a synthetic SIRPα binding polypeptide) comprises a sequence having at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOs: 47, 50, 81, 108, 141, 215, 286, 377, 381, or 638. In some embodiments, the percent identity is between any two of these values. In some embodiments, the polypeptide has about 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less identity to any one of the sequences in SEQ ID NOs: 47, 50, 81, 108, 141, 215, 286, 377, 381, or 638. In some embodiments, the percent identity is between any two of these values.

[0141] Conformational ensembles of SIRP.ALPHA.-binding polypeptide sequences In some embodiments, a polypeptide described herein (e.g., a synthetic SIRPα-binding polypeptide) comprises a conformational ensemble that is adjusted relative to the conformational ensemble of a reference polypeptide. In some cases, the reference polypeptide is a WT CD47 sequence (e.g., any one of SEQ ID NOs: 1-4).

[0142] The conformational ensemble of a polypeptide described herein (e.g., an engineered SIRPα-binding polypeptide, a reference polypeptide) can be determined by any suitable procedure. In some cases, the conformational ensemble can be observed or predicted by biophysical and molecular simulation techniques, such as x-ray crystallography (XRC), nuclear magnetic resonance (NMR), hydrogen-deuterium exchange (HDX), small-angle X-ray scattering (SAXS), neutron diffraction, electron paramagnetic resonance (EPR), cryogenic electron microscopy (cyroEM), molecular dynamics (MD), Monte Carlo (MC) methods, artificial intelligence and machine learning algorithms, and any combination thereof.

[0143] As described herein below, computational analysis of molecular dynamics (MD) simulations of WT CD47 led to the identification of four major metastable states ("metastates") that characterize most of the conformational ensemble of CD47. A superposition of these metastates is shown in Figure 1. Of these, the S1 and S2 metastates were found to comprise the majority of the conformational ensemble, and the primary motion of the molecule at equilibrium was the transition between these two metastates.

[0144] The S1 and S2 metastates (as well as other metastates of the polypeptides disclosed herein) may be characterized by points on a reaction coordinate. In some cases, the reaction coordinate includes an atomic coordinate, a set of atomic coordinates, an order parameter, or a combination or transformation thereof. Generally, any atomic coordinate, a set of atomic coordinates, an order parameter, or a transformation thereof may be used as a reaction coordinate if the coordinate, set of coordinates, order parameter, or transformation thereof adopts different values that readily distinguish metastable states from one another. In one example, the interatomic distance between two atoms, one on the ECD and one on the TMD, of a SIRPα binding protein described herein may be used as a reaction coordinate to identify a metastate (or a potential set of metastates) to which a particular conformation of the SIRPα binding protein may be assigned. In one example, the C of residues R132 and E151 α The interatomic distances between atoms are used as the reaction coordinate. In another example, the bending angle between the ECD and TMD can be used as the reaction coordinate to identify a metastate (or a potential set of metastates) to which a particular conformation of the binding protein can be assigned. The angle between domains for a given configuration can be determined as the angle of a vector between an xy plane set at the origin (e.g., the center of mass of one domain) and a vector between the origin and the center of mass of a subset of residues in the other domain. In a specific example, the first domain was the TMD, and the second vector was the vector between the origin and the center of mass of residues N50-V54 in the ECD.

[0145] Without wishing to be bound by any particular theory, certain SIRPα agonists described herein may elicit a reduced immune response when expressed on the surface of a cell by comprising conformational ensembles that contain different proportions of these metastable states compared to a reference polypeptide (e.g., comprising the sequence of any one of SEQ ID NOS: 1-4). In some embodiments, a SIRPα agonist of the present disclosure comprises a conformational ensemble with a greater proportion of the "S1" meta-state, as discussed above. In some embodiments, a cellular product contains one or more SIRPα agonists of the present disclosure that contain different distributions of conformational ensembles (e.g., some exhibiting a greater S1 proportion and some exhibiting a greater S2 proportion). The various strategies discussed herein (cancer mutations, polymorphisms, oligomerization, chimeras, loop insertions, rational mutations) may be used alone or in combination to engineer SIRPα agonist polypeptide sequences with conformational ensembles that contain a greater proportion of the S1 meta-state, thus enhancing SIRPα signaling by making a greater proportion of molecules available to interact with SIRPα. Alternatively, the (e.g., engineered) SIRPα-binding polypeptides of the present disclosure may enhance SIRPα-mediated signaling by mechanisms other than increasing the proportion of the S1 meta state in their conformational ensemble (e.g., relative to WT CD47), such as by reducing affinity for other endogenous binding partners and thus enhancing the amount of available "free" SIRPα-binding molecules or sites.

[0146] N-terminal modifications of engineered selective SIRPα binding peptides In some embodiments, the engineered SIRPα-binding peptides described herein further comprise N-terminal modifications that further enhance binding to SIRPα, hi some embodiments, these N-terminal modifications are made in addition to any one of the modifications (e.g., sequence modifications such as mutations or computational design strategies) described herein.

[0147] In some embodiments, the engineered SIRPα-binding peptide comprises at least one amino acid added to the N-terminus of the mature protein. In some embodiments, the engineered SIRPα-binding peptide comprises at least two amino acids added to the N-terminus of the mature protein. In some embodiments, the engineered SIRPα-binding peptide comprises at least three amino acids added to the N-terminus of the mature protein. In some embodiments, the three added amino acids have the formula X-3X-2X-1, where X-3 is W, X-2 is selected from Q, A, and G, and X-1 is selected from R, P, L, T, F, I, and M. In some embodiments, the three added amino acids are selected from WQR, WAP, WQL, WQP, WQT, WQF, WQI, WGP, and WQM.

[0148] nucleic acid molecule Also provided herein are nucleic acids encoding any of the peptides, polypeptides, fusion proteins, and compositions described herein.

[0149] In some embodiments, nucleic acid molecules can contain any length of nucleotides, either ribonucleotides or deoxyribonucleotides, in polymeric form. In some embodiments, nucleic acid molecules can refer only to the primary structure of the molecule. In some embodiments, nucleic acid molecules can be triple-stranded DNA, double-stranded DNA, and single-stranded DNA, as well as triple-stranded RNA, double-stranded RNA, and single-stranded RNA. In some embodiments, nucleic acid molecules can be modified by methylation and / or capping. In some embodiments, nucleic acid molecules can be natural, synthetic, or a combination of both.

[0150] Methods for generating hypoimmunogenic cells Also provided herein are methods of making any of the peptides, polypeptides, fusion proteins, and compositions of the present disclosure. Such methods may include introducing a nucleic acid sequence encoding the peptide into a cell to produce a recombinant cell, and culturing the recombinant cell under conditions sufficient for expression of the peptide. In some embodiments, the introducing step includes introducing into the cell an expression vector comprising the nucleic acid sequence encoding the peptide. In some embodiments, Also provided herein are vectors comprising any of the nucleic acids provided herein. A vector can refer to a polynucleotide capable of directing expression of a recombinant peptide in a host cell. In some embodiments, the vector further comprises a promoter and / or enhancer operably linked to any of the nucleic acids described herein. In some embodiments, In certain embodiments, vectors can be constructed to contain exogenous nucleic acid sequences for genetic modification of any cell used herein, particularly starting cells such as stromal cells or stem or progenitor cells in the culture methods or compositions. Those skilled in the art will be well equipped to construct vectors via standard recombinant techniques.

[0151] Vectors may also contain other components or functionalities that further modulate gene delivery and / or gene expression or that otherwise provide beneficial properties to the target cells, including, for example, components that affect cell binding or targeting (including components that mediate cell-type or tissue-specific binding), components that affect uptake of vector nucleic acid by cells, components that affect localization of the polynucleotide within the cell after uptake (such as agents that mediate nuclear localization), and components that affect expression of the polynucleotide.

[0152] Such components may also include markers, such as detectable and / or selectable markers, that can be used to detect or select cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (e.g., the use of certain viral vectors that have components or functionality that mediate binding and uptake), or the vector may be modified to provide such functionality. A wide variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, it can either be stably replicated by the cell during mitosis as an autonomous structure, integrated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0153] Genetic modification or introduction of exogenous nucleic acids into the starting cells of the culture composition or method can be performed using any suitable method for nucleic acid delivery for cell transformation, as described herein or known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA, such as ex vivo transfection, injection, including microinjection, calcium phosphate precipitation, DEAE-dextran followed by polyethylene glycol, direct sonication, liposome-mediated transfection and receptor-mediated transfection, particle bombardment, agitation with silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation of protoplasts, desiccation / inhibition-mediated DNA uptake, and any combination of such methods. Through the application of such techniques, organelles, cells, tissues, or organisms can be stably or transiently transformed.

[0154] In certain embodiments, cells can be engineered to contain one or more genetic modifications by genetic manipulation of the cells either before or after differentiation. A cell is said to be "genetically engineered," "genetically modified," or "transgenic" when an exogenous nucleic acid or polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or when the cell is the progeny of an originally engineered cell that inherits the polynucleotide. For example, a cell can be engineered to increase its replicative potential by genetically modifying the cell to express telomerase reverse transcriptase either before or after progression to a restricted developmental lineage or terminally differentiated cell.

[0155] In certain embodiments, cells containing an exogenous nucleic acid construct can be identified in vitro or in vivo by including a marker, such as a selectable or screenable marker, in the expression vector. Such markers confer an identifiable change to the cells, allowing for easy identification of cells containing the expression vector, or aid in enriching or identifying differentiated cells by using tissue-specific promoters. Generally, a selectable marker confers a property that allows for selection. A positive selectable marker is one whose presence allows for its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0156] Cells of the present disclosure may comprise one or more SIRPα binding polypeptides described herein. In some embodiments, the plurality of SIRPα binding polypeptides comprises wild-type CD47 (e.g., comprising any one of the sequences of SEQ ID NOs: 1-4). In some embodiments, the plurality of SIRPα binding polypeptides does not comprise a wild-type CD47 sequence. In some embodiments, the plurality of SIRPα binding polypeptides comprises multiple engineered SIRPα binding polypeptides. The multiple engineered SIRPα binding polypeptides may comprise any engineered SIRPα binding polypeptide disclosed herein.

[0157] Computer Systems The present disclosure provides a computer system programmed to carry out the methods of the present disclosure. Figure 24 shows a computer system 2401 programmed or otherwise configured to design a SIRPα agonist described herein (e.g., any one of the methods described herein). The computer system 2401 can regulate various aspects of the sequence design of the SIRPα agonist, such as stability and an enhanced "don't eat me" signal. The computer system 2401 can be a user's electronic device or a computer system located remotely with respect to the electronic device. The electronic device can be a mobile electronic device.

[0158] Computer system 2401 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 2405, which may be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 2401 also includes memory or memory locations 2410 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 2415 (e.g., a hard disk), a communication interface 2420 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 2425, such as cache, other memory, data storage devices, and / or electronic display adapters. Memory 2410, storage unit 2415, interface 2420, and peripheral devices 2425 communicate with CPU 2405 via a communication bus (solid lines), such as a motherboard. Storage unit 2415 may be a data storage unit (or data repository) for storing data. Computer system 2401 may be operably coupled to a computer network ("network") 2430 with the aid of communication interface 2420. Network 2430 may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. Network 2430, in some cases, is a telecommunications and / or data network. Network 2430 may include one or more computer servers, which may enable distributed computing such as cloud computing. Network 2430, in some cases with the help of computer system 2401, may implement a peer-to-peer network, which may enable devices coupled to computer system 2401 to act as clients or servers.

[0159] The CPU 2405 can execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 2410. The instructions may be directed to the CPU 2405, which may then program or otherwise configure the CPU 2405 to implement the methods of the present disclosure. Examples of operations performed by the CPU 2405 may include fetch, decode, execute, and write-back.

[0160] The CPU 2405 may be part of a circuit, such as an integrated circuit. One or more other components of the system 2401 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0161] The storage unit 2415 may store files such as drivers, libraries, and saved programs. The storage unit 2415 may store user data, such as user preferences and user programs. The computer system 2401 may, in some cases, include one or more additional data storage units external to the computer system 2401, such as located on a remote server that communicates with the computer system 2401 via an intranet or the Internet.

[0162] Computer system 2401 may communicate with one or more remote computer systems via network 2430. For example, computer system 2401 may communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a telephone, a smartphone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access computer system 2401 via network 2430.

[0163] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of computer system 2401, such as memory 2410 or electronic storage unit 2415. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by processor 2405. In some cases, the code may be retrieved from storage unit 2415 and stored on memory 2410 for easy access by processor 2405. In some situations, electronic storage unit 2415 can be eliminated, and machine-executable instructions are stored in memory 2410.

[0164] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled during run-time. The code may be supplied in a programming language that may be selected to allow the code to execute in a pre-compiled or compile-time manner.

[0165] Aspects of the systems and methods provided herein, such as computer system 2401, may be embodied in programming. Various aspects of the present technology can be considered a "product" or "article of manufacture," typically in the form of machine (or processor) executable code and / or associated data carried on or embodied in some type of machine-readable medium. The machine-executable code may be stored in an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media may include any or all of various semiconductor memories, tape drives, disk drives, etc., tangible memory of a computer, processor, etc., or their associated modules, which may provide non-transitory storage for software programming at any time. All or portions of the software may, from time to time, be communicated via the Internet or various other telecommunications networks. Such communication may, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, another type of medium that may carry software elements includes optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and over various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered software-bearing media. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0166] Thus, a machine-readable medium such as a computer-executable code may take many forms, including, but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices in any computer, such as may be used to implement the databases, etc., shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire, and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs, or DVD-ROMs, any other optical media, punched cards, paper tape, any other physical storage media with patterns of holes, RAM, ROM, PROMs, and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves that transmit data or instructions, cables or links that transmit such carrier waves, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0167] The computer system 2401 may include or be in communication with an electronic display 2435 that includes a user interface (UI) 2440 for providing, for example, the SIRPα agonist sequences described herein. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0168] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit 2405. The algorithms may, for example, design the sequences of the SIRPα agonists described herein. [Example]

[0169] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure, and by their illustrative nature it will be understood that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0170] Example 1. Molecular dynamics simulation of SIRPα-binding polypeptide To investigate the thermodynamics and kinetics of signal-regulatory protein alpha (SIRPα) binding to cluster of differentiation 47 (CD47), molecular dynamics (MD) simulations of wild-type (WT) CD47 alone and in complex with SIRPα were performed and analyzed. The MD simulations were generally performed according to the protocol of Fenalti, G; et al. Nat. Commun. 2021, 12, 5218. (doi.org / 10.1038 / s41467-021-25475-w, also referred to herein as "Fenalti"), which is incorporated herein by reference in its entirety. Three independent simulations were performed and concatenated. The concatenated trajectories were analyzed using principal component analysis (PCA) to identify the predominant metastable states present at equilibrium.

[0171] The equilibrium conformational ensemble of WT CD47 without SIRPα was found to contain several predominant metastable states, designated herein as S1, S2, S3, and S3'. Representative microstates (individual conformations) for each metastate are shown in Figure 1. Figure 2A shows a PCA plot illustrating the relative distribution of conformations along the first two principal components for WT CD47 without SIRPα. Figure 2B shows a PCA plot illustrating the relative distribution of conformations along the first two principal components for WT CD47 in complex with SIRPα. As indicated by the relative density (saturation) in each plot, SIRPα binding shifts the conformational ensemble from a predominantly S2 metastate to a predominantly S1 metastate. Based on this observation, we hypothesized that SIRPα preferentially binds to the S1 metastate of CD47.

[0172] To further explain the S1 and S2 meta states and the interconversion between them, several reaction coordinates were selected and investigated. Qualitative examination of representative conformations from the S1 and S2 meta states suggested that the states could be distinguished by the relative distance and angle between the extracellular domain (ECD) and transmembrane domain (TMD) of the molecule. Each of these was therefore selected as a potential reaction coordinate. The distance between the two domains was determined by the C of residues R132 and E141. α The interatomic distances were calculated by taking the distances between the domains. These distances were found to range from approximately 4-9 Å in the S2 state to approximately 10-25 Å in the S1 state (Figure 3A). The angles between the domains were also calculated and were found to range from approximately 100-120 degrees in the S2 state to approximately 130-180 degrees in the S1 state (Figure 3B). The angles between the domains were calculated by determining the angle of the vector between the xy plane set at the origin (the center of mass of the TMD bundle) and the vector between the origin and the center of mass of residues N50-V54 in the ECD.

[0173] Example 2: Investigation of mutations in CD47 Documented mutations (e.g., included in Table 3) and other polymorphisms (e.g., included in Table 4) found in human CD47 and associated with cancer samples were mapped onto different regions of the crystal structure of the molecule complexed with an antibody (PDB ID: 7MYZ). Certain mutations were found to cluster on important functional regions of the molecule, including the extracellular domain (ECD), transmembrane domain (TMD), and extracellular loop region (ECLR).

[0174] Cancer mutations located in the transmembrane domain (TMD) of CD47 Several cancer mutation sites (e.g., included in Table 3) were mapped to the TMD of CD47 (e.g., corresponding to any of positions 142-162, 177-197, 208-228, 236-256, or 269-289 of any of SEQ ID NOs: 1-4). These mutations were hypothesized to affect protein packing in the TMD and therefore transmembrane signaling through modulation of hydrophobic interactions (e.g., hydrophobic interactions between transmembrane helices in the core of the TMD, or hydrophobic interactions referring to the lipid membrane environment), non-covalent interactions including hydrogen bonding interactions, and covalent bonds (e.g., disulfide bonds such as between cancer mutants W136C and C259).

[0175] Cancer mutations located in the extracellular domain (ECD) of WT CD47 Several cancer mutation sites (e.g., those included in Table 3) were mapped to the ECD of CD47 (e.g., corresponding to any of positions 19-141 of any of SEQ ID NOS: 1-4). Computational analysis revealed that the ECD of CD47 contains regions predicted to be instability regions on CD47. It was therefore hypothesized that cancer mutations in the ECD of CD47 may act to increase receptor stability and thus enhance signaling through the SIRPα pathway, affect the distribution of metastable states, or affect interactions with the lipid membrane environment.

[0176] Other CD47 polymorphisms Analysis of CD47 polymorphisms (e.g., those included in Table 4) showed that these mutations, when mapped onto the CD47 crystal structure, are spread across different domains of CD47, which were hypothesized to map to the dimer surface and to regions that bind other endogenous binders of CD47, such as thrombospondin 1 (TSP-1), integrins, or other endogenous protein partners.

[0177] Example 3. Chimeric Polypeptides and Loop Insertions SIRPα agonist peptide insertions identified from analysis of receptor evolution across multiple species. SIRPα agonist peptide insertions were identified in the RVVSWF peptide linker (positions 132-137 of any of SEQ ID NOS: 1-4) connecting the ECD and TMD of human CD47. Several species were identified with peptides of varying amino acid length and constrained by disulfide bonds (e.g., SEQ ID NOS: 641-647). These peptides are predicted to affect ECD domain dynamics and can be tested for function (e.g., protection from NK cell cytotoxicity, protection from macrophage cytotoxicity) according to the procedures disclosed herein. They may also affect binding kinetics to other endogenous protein partners or affect oligomerization of SIRPα polypeptides.

[0178] Example 4. Conformational ensembles of SIRPα-binding polypeptides A synthetic SIRPα-binding polypeptide is generated according to the procedures described herein. The conformational ensemble of the polypeptide is characterized (e.g., at least in part, by one or more of hydrogen-deuterium exchange (HDX), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD)). The conformational ensemble of a reference polypeptide (e.g., comprising any one of SEQ ID NOS: 1-4) is similarly examined.

[0179] In each case, the conformational ensemble is found to include a first metastable state that is configured to bind to SIRPα. The first metastable state includes a conformation characterized by one or more of the following features: a bending angle between the transmembrane domain (TMD) and the extracellular domain (TMD) of about 130 to about 180 degrees, such as about 150 to about 170 degrees, and A distance between the TMD and the ECD of about 10 angstroms (Å) to about 25 Å, or greater than about 25 Å.

[0180] Metastable states characterized by these features may be referred to herein as "S1" or "S1-like" metastates.

[0181] The conformational ensemble of the synthetic SIRPα binding polypeptide is found to contain a higher proportion (e.g., as determined, estimated, or predicted by one or more of HDX, SAXS, NMR, or MD) of the first metastable state than the conformational ensemble of the reference polypeptide.

[0182] The conformational ensemble of the reference polypeptide is also found to include a second metastable state that does not substantially bind to SIRPα. The second metastable state is characterized by one or more of the following features: a bending angle between the transmembrane domain (TMD) and the extracellular domain (TMD) of about 100 degrees to about 120 degrees, or less than 100 degrees; and A distance between the TMD and the ECD of about 4 angstroms (Å) to about 9 Å, or less than about 4 Å.

[0183] The conformational ensemble of the polypeptide may also be found to include the second metastable state, but to a lesser extent than the conformational ensemble of the reference polypeptide.

[0184] Metastable states characterized by these features may be referred to herein as "S2" or "S2-like" metastates.

[0185] Example 5: Enhancement of S1 sampling via cancer mutations and polymorphisms To test the hypothesis that cancer-associated CD47 mutations (e.g., those included in Table 3) and certain CD47 polymorphisms (e.g., those included in Table 4) enhance the SIRPα-binding activity of CD47 by shifting the conformational ensemble of the molecule toward a higher proportion of the S1 meta state, constructs containing some of these mutations were simulated using MD according to the protocol outlined in Fenalti, and the resulting conformational ensembles were sampled and compared to wild-type CD47 (e.g., SEQ ID NO: 1).

[0186] A sequence containing the R132C mutation associated with cancer samples (SEQ ID NO: 419) was simulated using MD. Residue 132 is present in the ECLR of CD47, and therefore, it was hypothesized that the R132C mutant reduces the relative motion between the ECD and TMD, thereby increasing the relative proportion of the S1 meta state in its conformational ensemble. Another sequence containing T31M and S138F (SEQ ID NO: 648) was also simulated using MD. Residues 31 and 138 are also present in the ECLR. Therefore, it was hypothesized that these two hydrophobic substitutions fill the ECLR, restricting hinge motion between the ECD and TMD, and thus increasing the relative proportion of the S1 meta state. A representative model of the T31M / S138F mutant showing the mutated residues in ECLR is shown in Figure 4.

[0187] Two separate MD results for each construct are shown in Figure 5A (R132C) and Figure 5B (T31M, S138F). These figures show the distance between the ECD and TMD domains (top panel, calculated as described in Example 2) and the domain angles (bottom panel, calculated as described in Example 2). As shown in Figure 5A and Figure 5B, these mutations in each case increase the percentage of time the simulated molecule (conformational ensemble) spends in the S1 meta state compared to WT CD47 (Figures 3A and 3B).

[0188] Example 6. Macrophage assay This example outlines a macrophage killing assay to measure the protection from macrophage cytotoxicity conferred by engineered SIRPα binding polypeptides of the disclosure.

[0189] construct generation DNA encoding the engineered SIRPα-binding peptides described herein was synthesized by VectorBuilder Inc. and cloned into a mammalian gene expression lentiviral vector (pLV) under the EF1α promoter and blasticidin antibiotic selection. A representative diagram of the polypeptides disclosed herein is shown in Figure 6. The construct contains a T2A ribosomal skipping sequence, so the engineered polypeptide to GFP expression ratio is 1:1, and the expression product contains two unlinked proteins: the engineered SIRPα-binding polypeptide that is transported to the membrane, and cytoplasmic GFP.

[0190] Target cell line development K562 CD47 - / - Cell lines are thawed and allowed to recover for 1 week before transduction. Cell lines are maintained in RPMI + 10% FBS + 1% Pen-Strep at 0.2 x 10 in a working volume of 20 mL. 6 Seed cells into T75 flasks at 1 x 10 VC / mL. Cells are typically cultured every 2-3 days or until the cells reach 1 x 10 6 Passage when a concentration of VC / mL is reached. Record cell number and viability using a cell counter (e.g., NucloeCounter® NC-202™).

[0191] For lentiviral transduction, 0.25 x 10 cells in a 2 mL cell suspension of CD47 KO K562 per well of a 6-well plate 6 VC / mL (0.5 × 10 6 This is performed using an MOI of 1, such as by adding 10 μL of LV (total cells) followed by 5 μL and 10 μL of LV and polybrene for an engineered SIRPα binding polypeptide construct described herein.

[0192] The cells were continued to be cultured in 6-well plates for 3 days and then cultured in T25 (0.2 × 10 in 5 mL). 6 After scaling to 1000 VC / mL, apply antibiotic selection pressure (e.g., approximately 6 µg blasticidin). After adding the antibiotic, maintain the cells in the selection medium for 3-5 passages (generally 3 passages) before cell sorting.

[0193] Cell sorting and analysis for cell line characterization Cell sorting is performed using a Sony MA900 cell sorter. Double-positive GFP-CD47 cells are selected. After sorting, cells are grown in RPMI + 10% FBS + 1% PS + 6 μg BLAST medium. For FACS analysis, lentivirally transduced cells at early passages (2-4) are assessed for GFP and CD47 or engineered SIRPα binding protein expression.

[0194] Cell line characterization: SIRPα binding protein expression For each cell line generated, the amount of engineered SIRPα-binding polypeptide expressed on the cell surface is determined using monoclonal antibodies B6H12 and CC2C6 stained with Brilliant Violet™ 650 dye (BV605). Isotype controls stained with PerCP-Cyanine 5.5 are used for background subtraction, and surface expression levels are generally normalized using human WT CD47-transduced cells for comparison and analysis.

[0195] Functional assessment: macrophage cytotoxicity To assess protection, THP-1-derived macrophages were prepared according to documented procedures and cocultured with the K562-CD47 KO BFluc cell line (target cells) generated as described above, displaying various engineered SIRPα-binding polypeptides. Macrophages (effector cells) were titrated, and when the macrophage cell ratio was high, target cells were 100% susceptible to cytotoxic killing. Negative control CD47 knockout (K562-CD47-KO BFluc) target cells were virtually completely killed, and no luminescence signal was observed from luciferase-containing target cells. Titration of effector cells against target cells (E:T) resulted in fewer target cell killings and thus luciferase signals. Data can be expressed as a percentage of cell killing for each construct to allow comparison between different constructs.

[0196] Example 7. Natural Killer (NK) Cell Assay This example outlines an NK cell killing assay to measure the protection from macrophage cytotoxicity conferred by engineered SIRPα binding polypeptides of the present disclosure.

[0197] construct generation DNA encoding the engineered SIRPα-binding peptides described herein was synthesized by VectorBuilder Inc. and cloned into a mammalian gene expression lentiviral vector (pLV) under the EF1α promoter and blasticidin antibiotic selection. A representative diagram of the polypeptides disclosed herein is shown in Figure 6. The construct contains a T2A ribosomal skipping sequence, so the engineered polypeptide to GFP expression ratio is 1:1, and the expression product contains two unlinked proteins: the engineered SIRPα-binding polypeptide that is transported to the membrane, and cytoplasmic GFP.

[0198] Target cell line development The K562 CD47 KO cell line is thawed and allowed to recover for one week before transduction. The cell line is maintained in RPMI + 10% FBS + 1% Pen-Strep at 0.2 x 10 cells / ml in a working volume of 20 mL. 6 Seed cells into T75 flasks at 1 x 10 VC / mL. Cells are typically cultured every 2-3 days or until the cells reach 1 x 10 6 Passage when a concentration of VC / mL is reached. Record cell number and viability using a cell counter (e.g., NucloeCounter® NC-202™).

[0199] A first round of transduction is performed using a commercially available vector to incorporate the FLuc transgene, followed by a second round of transduction to incorporate the polypeptide of the present disclosure. KO and K562 cell lines containing the engineered polypeptides are typically maintained in RPMI + 10% FBS + 1% Pen-Strep at 0.2 x 10 in a 20 mL working volume. 6 Seed cells into a T75 flask at 10 VC / mL. Cells are typically cultured every 2-3 days or until the cells reach 10 6 Subculture is performed when the concentration reaches VC / mL.

[0200] Cell sorting and analysis for cell line characterization Cell sorting is performed using a Sony MA900 cell sorter. Double-positive GFP-CD47 cells are sorted and expanded in RPMI + 10% FBS + 1% PS + 6 μg BLAST medium after sorting. Before each assay, cell number and viability are recorded using an automated cell counter (e.g., NucloeCounter® NC-202™).

[0201] Cell line characterization: SIRPα binding protein expression For each cell line generated, the amount of engineered SIRPα-binding polypeptide expressed on the cell surface is determined using monoclonal antibodies B6H12 and CC2C6 stained with Brilliant Violet™ 650 dye (BV605). Mean fluorescence intensity (MFI) is quantified for cells expressing each construct. Isotype controls stained with PercCP-Cyanine 5.5 are used for background subtraction, and surface expression levels are generally normalized using human WT CD47-transduced cells for comparison and analysis.

[0202] Functional assessment: NK cell cytotoxicity To assess protection, IL-2-activated NK cells (approximately 20 nM) from healthy donors were cocultured with the K562-CD47 KO BFluc cell line (target cells) generated as described above and displaying various engineered SIRPα-binding polypeptides. The parental K562 cell line is used as a control because it represents the relative endogenous level of CD47. Activated NK cells (effector cells) were titrated, and at a high ratio of NK cells, target cells were 100% susceptible to cytotoxic killing. Negative control cells (K562-CD47-KO BFluc target cells) with wild-type CD47 knockout were virtually completely killed, and no luminescence signal was observed from luciferase-containing target cells. Effector-target titration (E:T) resulted in fewer target cell killings and thus a luciferase signal. Data can be expressed as the percentage of cell killing for each construct to allow comparison between various K562-CD47-KO BFluc constructs. Additional positive controls can include K562 cells engineered to overexpress WT CD47 (K562 CD47 T2A GFP) and a human leukocyte antigen E (HLA-E) construct (K562 HLA-E).

[0203] Example 8. Conformational ensembles of engineered SIRPα binding proteins Following the procedures described herein, several engineered SIRPα-binding polypeptide sequences containing cancer-associated mutations (e.g., one or more of the mutations listed in Table 3) were generated. A brief summary of the constructs is listed in Table 5. Conformational ensembles of the sequences were simulated by molecular dynamics (MD) according to the molecular dynamics procedures outlined in Fenalti and Examples 1 and 4 herein above. Control constructs used as a basis for comparison were wild-type human CD47 (WT CD47), WT CD47 complexed with signal regulatory peptide alpha (SIRPα), and WT CD47 complexed with the fragment antigen binding (Fab) region of a CD47-specific antibody (WT+Fab).

[0204] [Table 5]

[0205] The results of each simulation were sampled and analyzed as discussed in Examples 1 and 4. Figures 7A and 7B summarize the conformational ensembles for each construct and control tested. As shown in Figures 7A-7B, with the exception of construct 32 (SEQ ID NO: 472), nearly all constructs shifted the conformational ensemble toward a greater proportion of the S1 meta state relative to the wild type.

[0206] For construct 22 (SEQ ID NO: 419), which contains the R132C mutation relative to SEQ ID NO: 1, the free energy landscape was investigated by performing principal component analysis (PCA) on the downsampled trajectories and compared to that of the wild type. As shown in Figure 7C, the introduction of the cancer-associated R132C mutation shifts the conformational ensemble from primarily "S2-like" to primarily "S1-like."

[0207] Example 9. Reduced immunogenicity of engineered SIRPα binding proteins (cancer mutants) Following the procedures of Example 7, engineered SIRPα binding sequences (e.g., those included in Table 3) containing one or more mutations associated with cancer were generated and tested for NK cell killing and surface expression. The constructs tested are summarized below. Control constructs, including endogenous expression of wild-type CD47 (WT), CD47 knockout (CD47 KO), overexpression of wild-type CD47 (CD47 T2A GFP), and expression of HLA-E (HLA-E), were also tested.

[0208] [Table 6]

[0209] Figures 8-14 and 16 show the protection from NK cell cytotoxicity conferred by the constructs tested. As shown in the figures, each of the constructs provided protection from NK cell killing, demonstrating reduced NK cell cytotoxicity relative to endogenous expression of WT CD47. Additionally, construct 32 (SEQ ID NO: 472) showed enhanced protection from NK cell killing even beyond that of overexpression of WT CD47 (Figure 13).

[0210] Figures 19-21 show the measured surface expression of the constructs. Surface expression was measured according to the procedure in Example 7 using monoclonal antibodies CD47: B6H12 (Figures 19 and 20) and CC2C6 (Figure 21). Control constructs included endogenous expression of wild-type CD47 (WT), CD47 knockout (KO), and overexpression of wild-type CD47 (hCD47 T2A GFP). Figure 19 shows the measured mean fluorescence intensity (MFI) of the B6H12 monoclonal antibody specific for human CD47. Figure 20 shows the MFI of B6H12, except that each value is normalized to the value of endogenously expressed wild-type. Figure 21 shows the MFI of another CD47-specific monoclonal antibody, CC2C6, again normalized to endogenous expression. In general, constructs for which surface expression could be measured showed greater surface expression than those expressed endogenously. For some constructs (eg, construct 8 and construct 28), expression could not be measured, presumably because mutations had altered the epitope such that monoclonal antibody binding could no longer be observed.

[0211] Example 10. Reduced immunogenicity of engineered SIRPα binding proteins Sequences predicted to bind to SIRPα and, optionally, exhibit one or more of the following: (i) detuning binding of other CD47 cognate binding molecules (e.g., integrins, TSP-1), (ii) enhanced SIRPα signaling, (iii) enhanced binding to SIRPα, and (iv) increased stability were generated by a rational mutation approach. These sequences were then tested for NK cell killing and cell surface expression according to the procedures in Example 7. Control constructs including endogenous expression of wild-type CD47 (WT), CD47 knockout (CD47 KO), overexpression of wild-type CD47 (CD47 T2A GFP), and expression of HLA-E (HLA-E) were also tested.

[0212] [Table 7]

[0213] Figures 8, 10, and 13-15 show the protection from NK cell cytotoxicity conferred by the constructs tested. As shown in the figures, the constructs generally conferred protection from NK cell cytotoxicity and demonstrated reduced NK cell cytotoxicity relative to endogenous expression of wild-type CD47.

[0214] Figures 19-21 show the measured surface expression of the constructs. Surface expression was measured according to the procedure in Example 7 using monoclonal antibodies CD47: B6H12 (Figures 19 and 20) and CC2C6 (Figure 21). Control constructs included endogenous expression of wild-type CD47 (WT), CD47 knockout (KO), and overexpression of wild-type CD47 (hCD47 T2A GFP). Figure 19 shows the measured mean fluorescence intensity (MFI) of the B6H12 monoclonal antibody specific for human CD47. Figure 20 shows the MFI of B6H12, except that each value is normalized to the value of endogenously expressed wild-type. Figure 21 shows the MFI of another CD47-specific monoclonal antibody, CC2C6, again normalized to endogenous expression. In general, constructs for which surface expression could be measured showed greater surface expression than those expressed endogenously.

[0215] Construct 43 (SEQ ID NO: 286) was further investigated for protection from macrophage cytotoxicity according to the procedures of Example 6. Figure 22 shows the protection from macrophage phagocytosis (macrophage cytotoxicity) conferred by CD47 and construct 43. Relative to CD47 knockout cells (CD47), cells expressing construct 43 or cells overexpressing WT CD47 (CD47 T2A GFP) showed reduced macrophage susceptibility, suggesting that protection from macrophage cytotoxicity was conferred by expression of construct 43.

[0216] Example 11. Reduced immunogenicity of engineered SIRPα binding proteins (chimeras) Chimeric SIRPα binding sequences containing peptide loop inserts, and in some cases domains from non-human species, were generated according to the procedures in Example 7, and the constructs were tested for cell killing and surface expression. A model of Construct 3 is shown in Figure 23. The constructs tested are summarized below. Control constructs, including endogenous expression of wild-type CD47 (WT), CD47 knockout (CD47 KO), overexpression of wild-type CD47 (CD47 T2A GFP), and expression of HLA-E (HLA-E), were also tested.

[0217] [Table 8] Figures 8 and 16-18 show the protection from NK cell cytotoxicity conferred by the constructs tested. As shown in the figures, each of the constructs provided protection from NK cell killing, demonstrating reduced NK cell cytotoxicity relative to endogenous expression of WT CD47. Additionally, in one particular donor, construct 3 (SEQ ID NO: 640) demonstrated enhanced protection from NK cell killing even beyond that of overexpression of WT CD47 (Figure 8).

[0218] Figures 19-21 show the measured surface expression of the constructs. Surface expression was measured according to the procedure in Example 7 using monoclonal antibodies CD47: B6H12 (Figures 19 and 20) and CC2C6 (Figure 21). Control constructs included endogenous expression of wild-type CD47 (WT), CD47 knockout (KO), and overexpression of wild-type CD47 (hCD47 T2A GFP). Figure 19 shows the measured mean fluorescence intensity (MFI) of the B6H12 monoclonal antibody specific for human CD47. Figure 20 shows the MFI of B6H12, except that each value is normalized to the value of endogenously expressed wild-type. Figure 21 shows the MFI of another CD47-specific monoclonal antibody, CC2C6, again normalized to endogenous expression. In general, constructs for which surface expression could be measured showed greater surface expression than those expressed endogenously. Of note, construct 3, which showed better protection from NK cell killing than CD47 overexpression in some donors, also showed lower surface expression than overexpressed CD47.

[0219] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Those skilled in the art will recognize numerous variations, changes, and substitutions without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention. It is therefore contemplated that the present invention shall encompass any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. An engineered polypeptide comprising a signal-regulatory protein alpha (SIRPα) binding sequence, wherein the engineered polypeptide is configured to elicit a reduced immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of residues 19-290 of any one of SEQ ID NOs: 1-4, wherein the reduced immune response is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduced relative to the reference polypeptide.

2. 10. The engineered polypeptide of claim 1, wherein the immune response comprises natural killer (NK) cell cytotoxicity.

3. 3. The engineered polypeptide of claim 1 or 2, wherein the immune response comprises macrophage cytotoxicity.

4. 1. An engineered polypeptide comprising a signal-regulatory protein alpha (SIRPα) binding sequence, wherein the polypeptide comprises a conformational ensemble comprising a first metastable state, the first metastable state being configured to bind to SIRPα, the conformational ensemble comprising a greater proportion of the first metastable state than a conformational ensemble of a reference polypeptide, wherein the reference polypeptide comprises the sequence of residues 19-290 of any one of SEQ ID NOs: 1-4.

5. 5. The engineered polypeptide of claim 4, wherein the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by hydrogen-deuterium exchange (HDX), small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD).

6. 6. The engineered polypeptide of claim 5, wherein the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by MD.

7. 7. The engineered polypeptide of any one of claims 4 to 6, wherein the first metastable state is characterized by a bend angle between the transmembrane domain (TMD) and the extracellular domain (ECD) of the polypeptide.

8. The engineered polypeptide of claim 7 , wherein the TMD comprises five alpha helices.

9. 9. The engineered polypeptide of claim 7 or 8, wherein the ECD is configured to bind to SIRPα.

10. 10. The engineered polypeptide of any one of claims 7 to 9, wherein the bend angle is from about 130 to about 180 degrees.

11. 11. The engineered polypeptide of any one of claims 4 to 10, wherein the first metastable state is characterized by a distance between the TMD and the ECD of the polypeptide.

12. 12. The engineered polypeptide of claim 11, wherein the distance is from about 10 to about 25 angstroms (Å).

13. 13. The engineered polypeptide of any one of claims 4-12, wherein the engineered polypeptide is configured to elicit a reduced immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of residue 19 through the last residue of any one of SEQ ID NOS: 1-4, wherein the reduced immune response is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduced relative to the reference polypeptide.

14. 14. The engineered polypeptide of claim 13, wherein the immune response comprises natural killer (NK) cell cytotoxicity.

15. 15. The engineered polypeptide of claim 13 or 14, wherein the immune response comprises macrophage cytotoxicity.

16. 1. An engineered polypeptide comprising a signal regulatory protein alpha (SIRPα) binding sequence, wherein the engineered polypeptide is configured to elicit a decreased immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of residues 19-290 of any one of SEQ ID NOs: 1-4, wherein the SIRPα binding sequence comprises at least one mutation relative to any one of SEQ ID NOs: 1-4, and wherein the at least one mutation is associated with cancer.

17. 17. The engineered polypeptide of claim 16, wherein the at least one mutation is included in a database.

18. 18. The engineered polypeptide of claim 17, wherein the database comprises the Catalogue of Somatic Mutations in Cancer (COSMIC), the Genome Aggregation Database (gnomaD), or both.

19. 19. The engineered polypeptide of any one of claims 16-18, wherein the at least one mutation comprises a mutation selected from the group consisting of the mutations listed in Table 3, and any combination thereof.

20. 19. The engineered polypeptide of any one of claims 16-18, wherein the at least one mutation comprises a mutation selected from the group consisting of M31, L40, C42, D47, D64, C75, E80, F97, G105, K106, F106, K111, S123, S127, K128, F131, C132, C136, K140, T142, G146, M153, L157, L160, E166, C170, D178, A203, S207, V210, D211, L214, S215, V262, L264, Y267, and any combination thereof.

21. 21. The engineered polypeptide of any one of claims 16-20, wherein the polypeptide comprises an additional mutation selected from the group consisting of Y31, A32, R35, K35, P71, A77, A79, N80, L100, K138, L164, M185, A211, S259, E262, and any combination thereof.

22. 1. An engineered signal-regulatory protein alpha (SIRPα) binding polypeptide comprising an extracellular domain (ECD), a transmembrane domain (TMD), and an extracellular loop region (ECLR), wherein the extracellular loop region comprises a heterologous sequence compared to residue 19 through the last residue of any one of SEQ ID NOs: 1-4.

23. 23. The engineered polypeptide of claim 22, wherein the ECD, the TMD, and the ECLR are derived from at least two different organisms.

24. 24. The engineered polypeptide of claim 22 or 23, wherein the ECLR comprises at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the ECLR of any one of SEQ ID NOs: 639 and 640.

25. 25. The engineered polypeptide of any one of claims 22-24, wherein the heterologous sequence comprises a pair of cysteine residues that are configured to form a disulfide pair.

26. 26. The engineered polypeptide of claim 25, wherein the heterologous sequence comprises at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any one of SEQ ID NOs:641-647.

27. 27. The engineered polypeptide of any one of claims 22 to 26, wherein the engineered polypeptide is configured to elicit a decreased immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of any one of SEQ ID NOs: 1 to 4.

28. 28. The engineered polypeptide of any one of claims 1-3 or 27, wherein the polypeptide and the reference polypeptide are configured to adopt a first metastable state, and the first metastable state is configured to bind to SIRPα.

29. 29. The engineered polypeptide of claim 28, wherein the conformational ensemble of the engineered polypeptide comprises a greater proportion of the first metastable state than the conformational ensemble of the reference polypeptide.

30. 30. The engineered polypeptide of claim 29, wherein the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by hydrogen-deuterium exchange (HDX), small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD).

31. 31. The engineered polypeptide of claim 30, wherein the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by MD.

32. 31. The engineered polypeptide of any one of claims 28-30, wherein the first metastable state is characterized by a bend angle between the transmembrane domain (TMD) and the extracellular domain (ECD) of the polypeptide.

33. 33. The engineered polypeptide of claim 32, wherein the TMD comprises five alpha helices.

34. 34. The engineered polypeptide of Claim 32 or 33, wherein the ECD is configured to bind to SIRPα.

35. 35. The engineered polypeptide of any one of claims 32-34, wherein the bend angle is from about 130 to about 180 degrees.

36. 36. The engineered polypeptide of any one of claims 28-35, wherein the first metastable state is characterized by a distance between the TMD and the ECD of the polypeptide.

37. 37. The engineered polypeptide of claim 36, wherein the distance is from about 10 to about 25 angstroms (Å).

38. 38. The engineered polypeptide of any one of claims 1-37, wherein the SIRPα binding sequence comprises at least about 70%, 80%, 90%, 95%, or 99%, or 100% identity to any one of SEQ ID NOs: 10-640 or 648-653.

39. 39. The engineered polypeptide of any one of claims 1-38, wherein the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to the extracellular domain (ECD) of any one of SEQ ID NOs: 1-640 and 648-653.

40. 40. The engineered polypeptide of any one of claims 1-39, wherein the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to a transmembrane domain (TMD) of any one of SEQ ID NOs: 1-640 and 648-653.

41. 41. The engineered polypeptide of any one of claims 1-40, wherein the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to an extracellular loop region (ECLR) of any one of SEQ ID NOs: 1-640 and 648-653.

42. 42. The engineered polypeptide of any one of claims 1 to 41, wherein the engineered polypeptide comprises at least one amino acid substitution specified in Table 3.

43. 43. The engineered polypeptide of claim 42, wherein the engineered polypeptide comprises one or more amino acid substitutions selected from the group consisting of M31, L40, C42, D47, D64, C75, E80, F97, G105, K106, F106, K111, S123, S127, K128, F131, C132, C136, K140, T142, G146, M153, L157, L160, E166, C170, D178, A203, S207, V210, D211, L214, S215, V262, L264, Y267, and any combination thereof.

44. 44. The engineered polypeptide of any one of claims 1 to 43, wherein said engineered polypeptide comprises at least one amino acid substitution specified in Table 4.

45. 45. The engineered polypeptide of any one of claims 1-44, wherein the engineered polypeptide comprises at least one amino acid substitution selected from the group consisting of Y31, A32, R35, K35, P71, A77, A79, N80, L100, K138, L164, M185, A211, S259, E262, and any combination thereof.

46. 46. The engineered polypeptide of any one of claims 1 to 45, wherein the cell is a stem cell.

47. 47. The engineered polypeptide of claim 46, wherein the stem cell is an induced pluripotent stem cell (iPSC).

48. 48. The engineered polypeptide of any one of claims 1 to 47, wherein the polypeptide comprises an N-terminal addition.

49. 49. The engineered polypeptide of any one of claims 1-48, wherein the TMD, the ECD, and the linker are from more than one organism.

50. 50. The engineered polypeptide of any one of claims 1 to 49, wherein the engineered polypeptide is configured to elicit a reduced integrin response compared to the reference polypeptide.

51. 51. The engineered polypeptide of any one of claims 1 to 50, wherein the polypeptide is configured to elicit a reduced thrombospondin 1 (TSP-1) response compared to the reference polypeptide.

52. An engineered polypeptide comprising a sequence having at least about 70%, 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, or 100% identity to any one of SEQ ID NOs: 1-640 and 648-653.

53. 53. The engineered polypeptide of claim 52, wherein the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to the extracellular domain (ECD) of any one of SEQ ID NOs: 1-640 and 648-653.

54. 54. The engineered polypeptide of claim 52 or 53, wherein the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to a transmembrane domain (TMD) of any one of SEQ ID NOs: 1-640 and 648-653.

55. 55. The engineered polypeptide of any one of claims 52-54, wherein the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to an extracellular loop region (ECLR) of any one of SEQ ID NOs: 1-640 and 648-653.

56. 56. The engineered polypeptide of any one of claims 52-55, wherein the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to any one of SEQ ID NOs:641-647.

57. 1. An engineered polypeptide comprising a signal-regulatory protein alpha (SIRPα) binding sequence, wherein the engineered polypeptide comprises a conformational ensemble comprising a first metastable state, the first metastable state being configured to bind to SIRPα, the conformational ensemble comprising a greater proportion of the first metastable state than a conformational ensemble of a reference polypeptide, and the reference polypeptide comprising the sequence of residues 19-290 of any one of SEQ ID NOs: 1-4.

58. 58. The engineered polypeptide of claim 57, wherein said proportion of said conformational ensemble of said engineered polypeptide or said proportion of said conformational ensemble of said reference polypeptide is determined, at least in part, by hydrogen-deuterium exchange (HDX), small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD).

59. 59. The engineered polypeptide of claim 58, wherein the proportion of the conformational ensemble of the engineered polypeptide or the proportion of the conformational ensemble of the reference polypeptide is determined, at least in part, by MD.

60. 60. The engineered polypeptide of any one of Claims 57-59, wherein the first metastable state is characterized by a bend angle between the transmembrane domain (TMD) and the extracellular domain (ECD) of the polypeptide.

61. 61. The engineered polypeptide of claim 60, wherein the TMD comprises five alpha helices.

62. 62. The engineered polypeptide of claim 60 or 61, wherein the ECD is configured to bind to SIRPα.

63. 63. The engineered polypeptide of any one of claims 60-62, wherein the bend angle is from about 130 to about 180 degrees.

64. 64. The engineered polypeptide of any one of claims 57-63, wherein the first metastable state is characterized by a distance between the TMD and the ECD of the polypeptide.

65. 65. The engineered polypeptide of claim 64, wherein the distance is from about 10 to about 25 angstroms (Å).

66. 66. The engineered polypeptide of any one of claims 57-65, wherein the engineered polypeptide is configured to elicit a reduced immune response when expressed on the surface of a cell compared to a reference polypeptide comprising the sequence of residues 19-290 of any one of SEQ ID NOS: 1-4, wherein the reduced immune response is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduced relative to the reference polypeptide.

67. 67. The engineered polypeptide of claim 66, wherein the immune response comprises natural killer (NK) cell cytotoxicity.

68. 68. The engineered polypeptide of claim 66 or 67, wherein the immune response comprises macrophage cytotoxicity.

69. 69. The engineered polypeptide of any one of claims 57-68, wherein the SIRPα binding sequence comprises at least about 70%, 80%, 90%, 95%, or 99%, or 100% identity to any one of SEQ ID NOs: 10-640 or 648-653.

70. 70. The engineered polypeptide of any one of claims 57 to 69, wherein said engineered polypeptide comprises at least one amino acid substitution specified in Table 3.

71. 71. The engineered polypeptide of claim 70, wherein the engineered polypeptide comprises one or more amino acid substitutions selected from the group consisting of M31, L40, C42, D47, D64, C75, E80, F97, G105, K106, F106, K111, S123, S127, K128, F131, C132, C136, K140, T142, G146, M153, L157, L160, E166, C170, D178, A203, S207, V210, D211, L214, S215, V262, L264, Y267, and any combination thereof.

72. 72. The engineered polypeptide of any one of claims 57-71, wherein said engineered polypeptide comprises at least one amino acid substitution specified in Table 4.

73. 73. The engineered polypeptide of any one of claims 57-72, wherein the engineered polypeptide comprises at least one amino acid substitution selected from the group consisting of Y31, A32, R35, K35, P71, A77, A79, N80, L100, K138, L164, M185, A211, S259, E262, and any combination thereof.

74. 74. The engineered polypeptide of any one of claims 57 to 73, wherein the cell is a stem cell.

75. 75. The engineered polypeptide of Claim 74, wherein the stem cell is an induced pluripotent stem cell (iPSC).

76. 76. The engineered polypeptide of any one of claims 57 to 75, wherein the polypeptide comprises an N-terminal addition.

77. 77. The engineered polypeptide of any one of claims 57-76, wherein the TMD, the ECD, and the linker are from more than one organism.

78. 78. The engineered polypeptide of any one of claims 57 to 77, wherein the engineered polypeptide is configured to elicit a reduced integrin response compared to the reference polypeptide.

79. 79. The engineered polypeptide of any one of claims 57 to 78, wherein the polypeptide is configured to elicit a reduced thrombospondin 1 (TSP-1) response compared to the reference polypeptide.

80. 80. An engineered cell comprising an engineered polypeptide according to any one of claims 1 to 79.

81. 80. An engineered cell comprising a plurality of SIRPα binding polypeptides, wherein the plurality of SIRPα binding polypeptides comprises the engineered polypeptide of any one of claims 1 to 79.

82. 82. The engineered cell of claim 81 , wherein said plurality of SIRPα binding polypeptides further comprises wild-type CD47.

83. 83. The engineered cell of claim 81 or 82, wherein the plurality of SIRPα binding polypeptides comprises a plurality of the engineered polypeptides of any one of claims 1-79.

84. 84. The engineered cell of any one of claims 81 to 83, wherein the engineered cell is a stem cell.

85. 85. The engineered cell of claim 84, wherein the stem cell is an embryonic stem cell, a mesenchymal stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

86. A nucleic acid molecule encoding the engineered polypeptide of any one of claims 1 to 79.

87. 80. A nucleic acid molecule encoding a plurality of SIRPα binding polypeptides, said plurality of SIRPα binding polypeptides comprising wild-type CD47 and an engineered polypeptide of any one of claims 1 to 79.

88. 80. A nucleic acid molecule encoding a plurality of SIRPα binding polypeptides, said plurality of SIRPα binding polypeptides comprising a plurality of the engineered polypeptides of any one of claims 1-79.

89. A vector comprising the nucleic acid of any one of claims 86 to 88.

90. 90. The vector of claim 89, wherein the vector is a plasmid, a minicircle, a CELiD, an adeno-associated virus (AAV)-derived virion, a lentivirus, an adenovirus, or a herpes simplex virus (HSV).

91. 91. A method for producing a hypoimmunogenic cell, the method comprising administering to a cell the vector of claim 89 or 90.

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