WO2024026495A1 - Compositions et procédés de non-immunogénicité - Google Patents

Compositions et procédés de non-immunogénicité Download PDF

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WO2024026495A1
WO2024026495A1 PCT/US2023/071272 US2023071272W WO2024026495A1 WO 2024026495 A1 WO2024026495 A1 WO 2024026495A1 US 2023071272 W US2023071272 W US 2023071272W WO 2024026495 A1 WO2024026495 A1 WO 2024026495A1
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polypeptide
engineered polypeptide
engineered
sirpa
sequence
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PCT/US2023/071272
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English (en)
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Adrian Woolfson
Herman Waldmann
Ashley BUCKLE
Gustavo Fenalti
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Replay Holdings, Inc.
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Publication of WO2024026495A1 publication Critical patent/WO2024026495A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • PSCs pluripotent stem cells
  • An aspect of the present disclosure provides an engineered polypeptide comprising a signal regulatory protein alpha (SIRPa) binding sequence, wherein the engineered polypeptide is configured to elicit a decreased immune response when expressed on a surface of a cell as compared to a reference polypeptide comprising a sequence of residues 19-290 of any one of SEQ ID NOs: 1-4, wherein the decreased 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% decreased relative to the reference polypeptide.
  • SIRPa signal regulatory protein alpha
  • the immune response comprises natural killer (NK) cell cytotoxicity.
  • the immune response comprises macrophage cytotoxicity.
  • An additional aspect of the present disclosure provides for an engineered polypeptide comprising a signal regulatory protein alpha (SIRPa) binding sequence, wherein the polypeptide comprises a conformational ensemble comprising a first metastable state, which first metastable state is configured to bind to SIRPa, and wherein the conformational ensemble comprises a greater proportion of the first metastable state than a conformational ensemble of a reference polypeptide, wherein the reference polypeptide comprises a sequence of residues 19-290 of any one of SEQ ID NOs: 1-4.
  • SIRPa signal regulatory protein alpha
  • 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).
  • 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.
  • the first metastable state is characterized by a bending angle between a transmembrane domain (TMD) and an extracellular domain (ECD) of the polypeptide.
  • the TMD comprises five alpha helices.
  • the ECD is configured to bind SIRPa. In some embodiments, the bending angle is from 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 from about 10 to about 25 angstroms (A).
  • the engineered polypeptide is configured to elicit a decreased immune response when expressed on a surface of a cell as compared to a reference polypeptide comprising a sequence of residue 19 through the final residue of any one of SEQ ID NOs: 1 -4, wherein the decreased 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% decreased relative to the reference polypeptide.
  • the immune response comprises natural killer (NK) cell cytotoxicity.
  • the immune response comprises macrophage cytotoxicity.
  • An additional aspect of the present disclosure provides an engineered polypeptide comprising a signal regulatory protein alpha (SIRPa) binding sequence, wherein the engineered polypeptide is configured to elicit a decreased immune response when expressed on a surface of a cell as compared to a reference polypeptide comprising a sequence of residues 19-290 of any one of SEQ ID NOs: 1 -4, wherein the SIRPa binding sequence comprises at least one mutation relative to any one of SEQ ID NOs: 1 -4, wherein the at least one mutation is associated with a cancer.
  • SIRPa signal regulatory protein alpha
  • the at least one mutation is comprised in a database.
  • the database comprises the Catalogue of Somatic Mutations in Cancer (COSMIC), the Genome Aggregation Database (gnomAD), or both.
  • the at least one mutation comprises a mutation selected from the group consisting of : the mutations listed in Table 3, and any combination thereof.
  • 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, Kl ll, S123, S127, K128, F131, C132, C136, K140, T142, G146, Ml 53, LI 57, LI 60, El 66, Cl 70, DI 78, A203, S207, V210, D211, L214, S215, V262, L264, Y267, and any combination thereof.
  • 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.
  • An additional aspect of the present disclosure provides an engineered signal regulatory protein alpha (SIRPa) 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 residues 19 through the final residue of any one of SEQ ID NOs: 1-4.
  • SIRPa engineered signal regulatory protein alpha
  • the ECD, the TMD, and the ECLR are derived from at least two different organisms.
  • 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 an ECLR of any one of SEQ ID NOs: 639 and 640.
  • the heterologous sequence comprises a pair of cysteine residues configured to form a disulfide pair.
  • 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.
  • the engineered polypeptide is configured to elicit a decreased immune response when expressed on a surface of a cell as compared to a reference polypeptide comprising a sequence of any one of SEQ ID NO: 1 -4.
  • the polypeptide and the reference polypeptide are configured to adopt a first metastable state, wherein the first metastable state is configured to bind to SIRPa.
  • a conformational ensemble of the engineered polypeptide comprises a larger proportion of the first metastable state than a conformational ensemble of the reference polypeptide.
  • 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).
  • 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.
  • the first metastable state is characterized by a bending angle between a transmembrane domain (TMD) and an extracellular domain (ECD) of the polypeptide.
  • TMD transmembrane domain
  • ECD extracellular domain
  • the TMD comprises five alpha helices.
  • the ECD is configured to bind SIRPa.
  • the bending angle is from about 130 to about 180 degrees.
  • the first metastable state is characterized by a distance between the TMD and the ECD of the polypeptide. In some embodiments, the distance is from about 10 to about 25 angstroms (A).
  • the SIRPa binding sequence comprises at least about 70%, 80%, 90%.
  • the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to an 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 a transmembrane domain (TMD) of any one of SEQ ID NOs: 1 -640 and 648- 653.
  • ECD extracellular domain
  • TMD transmembrane domain
  • 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. In some embodiments, the engineered polypeptide comprises at least one amino acid substitution specified in Table 3.
  • 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, KI 11, 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.
  • the engineered polypeptide comprises at least one amnio acid substitution specified in Table 4.
  • the engineered polypeptide comprises at least one amino acid substitution selected from the group consisting of: Y31, A32, R35, K35, P71, A77, A79, N80, LI 00, KI 38, LI 64, M185, A211, S259, E262, and any combination thereof.
  • the cell is a stem cell.
  • the stem cell is an induced pluripotent stem cell (iPSC).
  • the polypeptide comprises an N-terminal addition.
  • the TMD, the ECD, and the linker are from more than one organism.
  • the engineered polypeptide is configured to elicit a reduced integrin response as compared to the reference polypeptide.
  • the polypeptide is configured to elicit a reduced thrombospondin 1 (TSP-1) response as compared to the reference polypeptide.
  • TSP-1 reduced thrombospondin 1
  • An additional aspect of the present disclosure provides an engineered polypeptide comprising a sequence with 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.
  • the engineered polypeptide comprises a sequence at least about 70%, 80%, 80%, 95%, or 99%, or 100% identical to an 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 a transmembrane domain (TMD) of any one of SEQ ID NOs: 1 -640 and 648-653.
  • ECD extracellular domain
  • TMD transmembrane domain
  • 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. 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.
  • ECLR extracellular loop region
  • An additional aspect of the present disclosure provides an engineered polypeptide comprising a signal regulatory protein alpha (SIRPa) binding sequence, wherein the engineered polypeptide comprises a conformational ensemble comprising a first metastable state, wherein the first metastable state is configured to bind SIRPa, and wherein the conformational ensemble comprises a greater proportion of the first metastable state than a conformational ensemble of a reference polypeptide, wherein the reference polypeptide comprises a sequence of residues 19- 290 of any one of SEQ ID NOs: 1 -4.
  • SIRPa signal regulatory protein alpha
  • 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).
  • 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.
  • the first metastable state is characterized by a bending angle between a transmembrane domain (TMD) and an extracellular domain (ECD) of the polypeptide.
  • the TMD comprises five alpha helices.
  • the ECD is configured to bind SIRPa.
  • the bending angle is from about 130 to about 180 degrees.
  • the first metastable state is characterized by a distance between the TMD and the ECD of the polypeptide. In some embodiments, the distance is from about 10 to about 25 angstroms (A).
  • the engineered polypeptide is configured to elicit a decreased immune response when expressed on a surface of a cell as compared to a reference polypeptide comprising a sequence of residues 19-290 of any one of SEQ ID NOs: 1 -4, wherein the decreased 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% decreased relative to the reference polypeptide.
  • the immune response comprises natural killer (NK) cell cytotoxicity.
  • the immune response comprises macrophage cytotoxicity.
  • the SIRPa binding sequence comprises at least about 70%, 80%, 90%.
  • 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, KI 06, F106, KI 11, 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.
  • the engineered polypeptide comprises at least one amnio 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, LI 00, KI 38, LI 64, M185, A211, S259, E262, and any combination thereof.
  • 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, the ECD, and the linker are from more than one organism.
  • the engineered polypeptide is configured to elicit a reduced integrin response as compared to the reference polypeptide. In some embodiments, the polypeptide is configured to elicit a reduced thrombospondin 1 (TSP-1) response as compared to the reference polypeptide.
  • TSP-1 reduced thrombospondin 1
  • An additional aspect of the present disclosure provides an engineered cell comprising any of the engineered peptides described herein.
  • An additional aspect of the present disclosure provides an engineered cell comprising a plurality of SIRPa binding polypeptides, wherein the plurality of SIRPa binding polypeptide comprises any of the engineered peptides described herein.
  • the plurality of SIRPa binding polypeptide further comprises a wild type CD47. In some embodiments, the plurality of SIRPa binding polypeptide comprises a plurality of any of the engineered peptides described herein.
  • the engineered cell is a stem cell. In some embodiments, the stem cell is an embryonic stem cell, a mesenchymal stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.
  • An additional aspect of the present disclosure provides a nucleic acid molecule encoding the any of the engineered polypeptides disclosed herein.
  • An additional aspect of the present disclosure provides a nucleic acid molecule encoding a plurality of SIRPa binding polypeptides, wherein the plurality of SIRPa binding polypeptides comprises a wild type CD47 and any of the engineered polypeptides disclosed herein.
  • An additional aspect of the present disclosure provides a nucleic acid molecule encoding a plurality of SIRPa binding polypeptides, wherein the plurality of SIRPa binding polypeptides comprises a plurality of any of the engineered polypeptide disclosed herein.
  • an additional aspect of the present disclosure provides a vector comprising any of the nucleic acids disclosed herein.
  • 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).
  • AAV adeno-associated virus
  • An additional aspect of the present disclosure provides a method of generating a hypoimmunogenic cell comprising administering to a cell any of the vectors disclosed herein.
  • An additional aspect of the present disclosure provides an engineered polypeptide comprising a signal regulatory protein alpha (SIRPa) binding sequence, wherein the polypeptide is configured to elicit a decreased macrophage response when expressed on a surface of a cell as compared to a polypeptide comprising a sequence of any one of SEQ ID NOs: 1-4.
  • SIRPa signal regulatory protein alpha
  • the SIRPa 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 SIRPa binding sequence comprises at least one amino acid substitution specified in Table 3. In some embodiments, the SIRPa binding sequence comprises at least one amino acid substitution specified in Table 3 when optimally aligned to any one of SEQ ID NOs: 1 -4. In some embodiments, the SIRPa binding sequence comprises at least one amnio acid substitution specified in Table 4. In some embodiments, the SIRPa binding sequence comprises at least one amino acid substitution specified Table 4 when optimally aligned to any one of SEQ ID NOs: 1 - 4. In some embodiments, the cell comprises a stem cell.
  • the stem cell comprises an induced pluripotent stem cell (iPSC).
  • the polypeptide comprises an N-terminal addition.
  • the N-terminal addition comprises at least one, two, or three amino acids added to an N-terminus of the polypeptide.
  • the three amino acids comprise a formula X-3X-2X-1 , where X-3 is W; X-2 is selected from Q, A and G; and X-l is selected from R, P, L, T, F, I, and M.
  • the three amino are selected from: WQR, WAP, WQL, WQP, WQT, WQF, WQI, WGP, and WQM.
  • the polypeptide and the reference polypeptide are configured to adopt a first metastable state, wherein the first metastable state is configured to bind to SIRPa.
  • the reference polypeptide, and optionally the polypeptide is configured to adopt a second metastable state, wherein the second metastable state does not substantially bind to SIRPa.
  • a conformational ensemble of the polypeptide comprises a larger proportion of the first metastable state than a conformational ensemble of the reference polypeptide.
  • the proportion of the conformational ensemble of the polypeptide is determined at least in part by hydrogendeuterium exchange (HDX), small-angle x-ray scattering (SAXS), nuclear magnetic resonance (NMR), or molecular dynamics (MD).
  • the first metastable state is characterized by a first bending angle between a transmembrane domain (TMD) and an extracellular domain (ECD) of the polypeptide.
  • TMD transmembrane domain
  • ECD extracellular domain
  • the ECD is configured to bind SIRPa.
  • the first bending angle is from about 130 to about 180 degrees. In some embodiments, the first bending angle is from about 150 to about 170 degrees.
  • the second metastable state is characterized by a second bending angle between the TMD and the ECD. In some embodiments, the second bending angle is from about 100 degrees to about 120 degrees.
  • 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 from about 10 to about 25 angstroms (A).
  • the second metastable state is characterized by a second distance between the TMD and the ECD. In some embodiments, the second distance is from about 4 to about 9 angstroms (A).
  • the TMD, the ECD, and the linker are all from one organism.
  • the TMD, the ECD, and the linker are from more than one organism. In some embodiments, at least one of the TMD, the ECD, and the linker is engineered. In some embodiments, the polypeptide is configured to elicit a reduced integrin response as compared to the reference polypeptide. In some embodiments the polypeptide is configured to elicit a reduced TSP-1 response as compared to the reference polypeptide.
  • Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.
  • Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto.
  • the computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.
  • FIG. 1 shows several representative metastates of a SIRPa binding peptide in accordance with some embodiments of the present disclosure.
  • FIGs. 2A-2B show example free energy surfaces representative of conformational ensembles of SIRPa binding peptides in accordance with some embodiments of the present disclosure.
  • FIGs. 3A-3B show example plots of reaction coordinates monitoring a transition between metastates in a molecular dynamics simulation of a SIRPa binding polypeptide in accordance with some embodiments of the present disclosure.
  • FIG. 4 shows a molecular model of a SIRPa binding polypeptide as described herein.
  • FIGs. 5A-5B show example plots of reaction coordinates monitoring a transition between metastates in a molecular dynamics simulation of a SIRPa binding polypeptide in accordance with some embodiments of the present disclosure.
  • FIG. 6 illustrates a vector configured for expression of polypeptides as disclosed herein.
  • FIGs. 7A-7B depict bar plots showing relative proportions of metastates in simulated conformational ensembles of SIRPa binding polypeptides as disclosed herein.
  • FIG. 7C shows example free energy surfaces representative of conformational ensembles of SIRPa binding peptides in accordance with some embodiments of the present disclosure.
  • FIGs. 8-18 show graphs quantifying protection from natural killer cell cytotoxicity afforded by SIRPa binding polypeptides of the disclosure.
  • FIGs. 19-21 depict bar graphs showing measure surface expression of SIRPa binding polypeptides of the present disclosure.
  • FIG. 22 show a graph quantifying protection from macrophage phagocytosis afforded by SIRPa binding polypeptides of the disclosure.
  • FIG. 23 depicts a molecular model of a SIRPa binding polypeptide as described herein.
  • FIG. 24 shows a computer system that is programmed or otherwise configured to implement methods provided herein.
  • SEQ ID NOs: 1 -4 show representative amino acid constructs of wildtype human CD47 sequences.
  • SEQ ID NOs: 5-9 show representative cysteine mutations to SEQ ID NO: 1 that may be made to induce oligomerization.
  • SEQ ID Nos: 10-384 show representative engineered signal regulatory protein alpha (SIRPa) binding sequences in accordance with some embodiments of the disclosure.
  • SEQ ID NOs: 385-475 show representative SIRPa binding sequences based at least in part on mutations associated with cancer.
  • SEQ ID NOs: 476-633 and 648 show representative SIRPa binding sequences based at least in part on polymorphisms identified in one or more individuals.
  • SEQ ID NOs: 634-638 and 648-653 show representative SIRPa binding sequences based at least in part on one or more rational mutations.
  • SEQ ID NOs: 639-640 show representative SIRPa binding sequences which are chimeric and/or comprise loop insertions as described herein.
  • SEQ ID NOs: 641-647 show sequences of representative insertions in engineered SIRPa binding sequences of the disclosure.
  • CD47 Cluster of Differentiation 47
  • IAP integrin associated protein
  • CD47 belongs to the immunoglobulin superfamily and partners with membrane integrins and also binds the ligands thrombospondin 1 (TSP- 1) and signal-regulatory protein alpha (SIRPa). It is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration .
  • CD47 is a ⁇ 50 kDa heavily glycosylated, ubiquitously expressed membrane protein of the immunoglobulin superfamily with a single IgV-like domain at its N-terminus (its extracellular domain or ECD), a highly hydrophobic stretch with five membrane-spanning segments (also referred to as the transmembrane or TM domain), and an alternatively spliced cytoplasmic C-terminus (C-terminal domain or CTD).
  • ECD extracellular domain
  • TM domain membrane-spanning segments
  • C-terminal domain or CTD alternatively spliced cytoplasmic C-terminus
  • Each of the four alternatively spliced cytoplasmic tails e.g., the C-terminal portions of SEQ ID NOs: 1 -4) exists in vivo at different frequencies, but all lack a substantial signaling domain.
  • CD47 is an integral component of the innate immune system, and binding of the ECD to signal regulatory protein alpha (SIRPa) on immune cells suppresses phagocytosis and NK- cell killing. CD47 is ubiquitously expressed in human cells and has been found to be overexpressed in many different tumor cells.
  • SIRPa signal regulatory protein alpha
  • MHC major histocompatibility complex
  • SIRPa signal-regulatory protein alpha
  • Some aspects of the present disclosure provide engineered SIRPa binding polypeptides and variants that retain or enhance the native CD47 “don’t eat me” signaling (e.g., relative to WT CD47).
  • the engineered SIRPa agonist constructs described herein display enhanced signaling for the CD47/SIRPa pathway over wild-type (WT) CD47, thus protecting cells expressing such molecules from killing by macrophages and/or NK cells.
  • the SIRPa binding polypeptides and variants disclosed herein elicit a reduced immune response when expressed on a surface of a cell as compared to WT CD47.
  • the reduced immune response comprises NK cell cytotoxicity.
  • the reduced immune response comprises macrophage cytotoxicity.
  • the engineered SIRPa agonist constructs display “detuning” or decreased signaling through pathways other than SIRPa.
  • the engineered SIRPa agonist sequences disclosed herein comprise low sequency homology with human CD47 polypeptides and variants.
  • the engineered SIRPa agonist sequences disclosed herein comprise less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about20%, or less sequence identity with any one of SEQ ID NOs: 1 -4.
  • the engineered molecules may retain basal functions required for cellular homeostasis while displaying enhanced CD47/SIRPa signaling functions; these SIRPa agonist constructs can be expressed as a single entity on the surface of cells (e.g., wherein a human cd47 gene is knocked out or in combination with endogenously expressed wild-type CD47).
  • Another aspect of the present disclosure comprises rationally designed SIRPa binding sequences that retain binding to human SIRPa and display susceptibility to macrophage phagocytosis and/or NK cell killing comparable to or lower than WT CD47 splice variants 1 -4 (corresponding to SEQ ID NOs: 1 -4), with the advantage of having ’’skewed” signaling function towards reduced macrophage phagocytosis and/or NK cell killing signaling and optionally detuned binding of one or more other endogenous binding partners.
  • the detuned endogenous binding partners comprises integrins.
  • the detuned endogenous binding partners comprise thrombospondin- 1 (TSP-1).
  • Another aspect of the present disclosure comprises co -expression of different engineered SIRPa binding polypeptides (e.g., 2 or more) comprising co-expression of any of the SIRPa binding polypeptides described herein.
  • Another aspect of the present disclosure comprises rationally designed SIRPa binding polypeptide sequences that retain binding to human SIRPa but display reduced or abrogated signaling function though other (e.g., non-SIRPa) binding partners, such as reduced or abrogated binding and signaling through TSP-1, integrins and other human CD47 endogenous binding partners.
  • Engineered SIRPa binding sequences or constructs which exhibit detuned CD47 signaling for one or more endogenous binding partners other than CD47 may be referred to as engineered “selective” SIRPa binding sequences or constructs.
  • Another aspect of the present disclosure comprises SIRPa binding mutations based at least in part on any one of WT CD47 splice variants 1 -4 that enhance “don’t eat me” signaling, thus preventing or reducing phagocytosis (e.g., by macrophages) and/or NK cell killing of cells which express the mutated SIRPa binding polypeptides (e.g., induced pluripotent stem cells (iPSCs)).
  • the mutations are based at least in part on mutations such as those listed in Table 3 or Table 4, or result from computational in silico calculations (e.g., all-atom molecular dynamics, normal mode analysis, coarse grain simulations, frustration analysis, or protein stability).
  • Another aspect of the present disclosure comprises engineered SIRPa binding sequences based at least in part on any one of SEQ ID NOs: 1-4 that retain binding of human SIRPa but contain peptide insertions within the wild-type human CD47 RVVSWF peptide linker (e.g., residues 132-137 of any one of SEQ ID NOs: 1 -4) connectingthe extracellular domain (ECD) and transmembrane domain (TMD) of any one of SEQ ID NOs: 1 -4 and can display altered/detuned signaling function (e.g., do not bind or have reduced binding of endogenous protein partners other than SIRPa or do not signal through or have reduced transmembrane signaling through protein partners other than SIRPa).
  • altered/detuned signaling function e.g., do not bind or have reduced binding of endogenous protein partners other than SIRPa or do not signal through or have reduced transmembrane signaling through protein partners other than SIRPa.
  • the peptide insertions are based at least in part on one or more orthologs or homologs of SEQ ID NOs: 1-4.
  • the peptide insertion comprises at sequence at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ IDNOs: 641 - 647.
  • a peptide insertion as described herein modulates the conformational ensemble of a SIRPa binding polypeptide relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence such as comprised in any one of SEQ ID NOs: 1-4).
  • Another aspect of the present disclosure comprises engineered SIRPa binding sequences based at least in parton any one of SEQ IDNOs: 1-4 that retain binding of human SIRPa but contain peptide insertions that contain two cysteines (e.g., constrained by a disulfide bond) within a region adjacentto or disposed within the RVVSWF peptide linker connectingthe ECD and TMD ofWT CD47 (e.g., residues 132-137 of any one of SEQ ID NOs: 1 -4) and display altered or detuned function (e.g., do not bind or have reduced binding of endogenous protein partners other than SIRPa and/or do not signal through or have decreased signaling functions with protein partners other than SIRPa).
  • cysteines e.g., constrained by a disulfide bond
  • cysteine residues configured to form a disulfide bond as described herein modulate the conformational ensemble of a SIRPa binding polypeptide relative to a reference polypeptide (e.g., comprising a wild -type human CD47 sequence such as comprised in any one of SEQ ID NOs: 1 -4).
  • a reference polypeptide e.g., comprising a wild -type human CD47 sequence such as comprised in any one of SEQ ID NOs: 1 -4.
  • Another aspect of the present disclosure comprises engineered SIRPa binding sequences based at least in parton any one of SEQ IDNOs: 1-4 that retain binding of human SIRPa but contain peptide insertions within the RVVSWF peptide linker connectingthe ECD and TMD of WT CD47 (e.g., residues 132-137 of any one of SEQ ID NOs: 1 -4) and additional mutations (single point or combinations thereof) and display altered/detuned function (e.g., do not bind or have reduced binding of endogenous protein partners other than SIRPa and/or do not signal through or have decreased signaling functions with these other protein partners).
  • the peptide insertions combined with additional mutations(s) modulates the conformational ensemble of a SIRPa binding polypeptide relative to a reference polypeptide (e.g., a wild-type CD47 sequence, such as comprised in any one of SEQ ID NOs: 1 -4).
  • the mutations(s) modulate the conformational ensemble of a SIRPa binding polypeptide relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence such as comprised in any one of SEQ ID NOs: 1 -4).
  • SIRPa binding polypeptides that enhance dimer or other oligomer (e.g., trimer, tetramer, etc.) formation based at least in parton any one of SEQ ID NOs: 1-4 (such as one or more mutations detailed in Table 2 hereinbelow).
  • SIRPa binding polypeptides with enhanced oligomer formation display altered or detuned function relative to a reference polypeptide (e.g., do not bind or have reduced binding of endogenous protein partners other than SIRPa and/or do not signal through or have decreased signaling functions with protein partners other than SIRPa).
  • Another aspect of the present disclosure comprises computationally engineered SIRPa binding sequences based on in silico evolutionary strategy algorithms and/or trained algorithms (such as machine learning algorithms) to enhance SIRPa binding polypeptide stability and retain binding and signaling through SIRPa, while reducing binding to other endogenous protein binding partners and associated signaling.
  • these constructs have less than about 90%, about 80%, about 70%, about 60%, about 50%, about40%, about 30%, about20%, or less sequence identity with any one of SEQ ID NOs: 1-4.
  • these constructs have a modulated conformational ensemble relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence such as comprised in any one of SEQ ID NOs: 1 - 4).
  • these constructs have 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.
  • these constructs have a modulated conformational ensemble relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence such as comprised in any one of SEQ ID NOs: 1 -4).
  • Another aspect of the present disclosure comprises computationally engineered SIRPa binding polypeptides comprising peptide insertions within the wild-type human CD47 RVVSWF peptide linker (e.g., residues 132-137 of any one of SEQ ID NOs: 1 -4) grafted on computationally designed SIRPa agonist sequencesbased on in silico evolutionary strategy algorithms.
  • Such insertions may enhance SIRPa agonist stability and retain binding and signaling of SIRPa, while reducing binding to other endogenous protein binding partners and associated signaling.
  • these constructs have 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.
  • these constructs have a modulated conformational ensemble relative to a reference polypeptide (e.g., comprising a wild - type human CD47 sequence such as comprised in any one of SEQ ID NOs: 1 -4).
  • Another aspect of the present disclosure comprises computationally engineered SIRPa binding sequences comprising mutations (single point mutations or combinations thereof) relative to any one of SEQ ID NOs: 1 -4 grafted on computationally designed SIRPa binding sequences based on in silico evolutionary strategy algorithms and/or trained algorithms (such as machine learning algorithms) to enhance SIRPa binding polypeptide stability and retain binding and signaling of SIRPa, while reducing binding to other endogenous protein binding partners and associated signaling.
  • these constructs have 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.
  • these constructs have a modulated conformational ensemble relative to a reference polypeptide (e.g., comprising a wild-type human CD47 sequence such as comprised in any one of SEQ ID NOs: 1 -4).
  • Another aspect of the present disclosure comprises engineered SIRPa binding polypeptides comprising one or more mutations in CD47 (e.g., one or more of those listed in Table 3 and Table 4) grafted on computationally designed SIRPa binding sequences based on in silico evolutionary strategy algorithms and/or trained algorithms (such as machine learning algorithms) to enhance SIRPa binding polypeptide stability and retain binding and signaling of SIRPa, while reducing binding to other endogenous protein binding partners and associated signaling.
  • these constructs have 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.
  • a computationally designed SIRPa binding sequence comprising additional mutation(s) is characterized by a conformational ensemble modulated relative to that of a reference polypeptide (e.g., a wild -type CD47 sequence, such as comprised in any one of SEQ ID NOs: 1 -4).
  • a reference polypeptide e.g., a wild -type CD47 sequence, such as comprised in any one of SEQ ID NOs: 1 -4.
  • Another aspect of the present disclosure comprises computationally engineered SIRPa binding polypeptides comprising chimeras encompassing the ECD domain of residues 19-131 (1-113 following cleavage of the signal peptide) of any one of SEQ ID NOs: 1 -4 and the TMD from computationally designed SIRPa agonist sequencesbased on in silico evolutionary strategy algorithms and/or trained algorithms (such as machine learning algorithms) to enhance SIRPa agonist TMD stability.
  • the engineered SIRPa agonists comprising chimeras encompassing residues 19-131 (1-113 following cleavage of the signal peptide) of any one of SEQ ID NOs: 1-4 and the TMD from another organism.
  • the engineered SIRPa agonists comprising chimeras encompassing an engineered (e.g., rationally or computationally) ECD of residues 19-131 (1-113 following cleavage of the signal peptide) of any one of SEQ ID NOs: 1 -4 and the TMD from another organism.
  • a SIRPa binding polypeptide chimera comprising an engineered domain is characterized by a conformational ensemble modulated relative to that of a reference polypeptide (e.g., a wild-type CD47 sequence, such as comprised in any one of SEQ ID NOs: 1 -4).
  • Another aspect of the present disclosure comprises computationally engineered SIRPa binding polypeptides comprising chimeras encompassing residues 19-131 (1-113 following cleavage of the signal peptide) of any one of SEQ ID NOs: 1 -4 carrying single or multiple mutations (such as those listed in Table 3, Table 4, and/or rationally/computationally engineered mutations) and the TMD from computationally designed SIRPa agonist sequences based on in silico evolutionary strategy algorithms and/or trained algorithms (such as machine learning algorithms) to enhance SIRPa agonist TMD stability.
  • the computationally modified chimeras combines with mutation(s) modulates the conformational ensemble of a SIRPa binding polypeptide relative to a reference polypeptide (e.g., a wild -type CD47 sequence, such as comprised in any one of SEQ ID NOs: 1 -4).
  • a reference polypeptide e.g., a wild -type CD47 sequence, such as comprised in any one of SEQ ID NOs: 1 -4.
  • a “cell” generally refers to a biological cell.
  • a cell may be the basic structural, functional and/or biological unit of a living organism.
  • a cell may originate from any organism having one or more cells.
  • Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosse s, homworts, liverworts, mosses), an algal cell, (e.g. context Botryococcusbraunii, Chlamydomonas reinhardtii, Nannochloropsis ga
  • seaweeds e.g., kelp
  • a fungal cell e.g. commonly a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruitfly, cnidarian, echinoderm, nematode, etc.)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a cell is not originating from a natural organism (e.g., a cell can be synthetically made, sometimes termed an artificial cell).
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • a nucleotide may comprise a synthetic nucleotide.
  • a nucleotide may comprise a synthetic nucleotide analog.
  • Nucleotides may be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • nucleotide may 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.
  • ATP ribonucleoside triphosphates adenosine triphosphate
  • UDP uridine triphosphate
  • CTP cytosine triphosphate
  • GTP guanosine triphosphate
  • deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof.
  • derivatives may include, for example, [aS]dATP, 7-deaza-dGTP and 7 -deaza-d ATP, and nucle
  • nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • Illustrative examples of dideoxy ribonucleoside triphosphates may include, but are not limited to, dd ATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fluorophores). Labeling may also be carried out with quantum dots.
  • Detectable labels may include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Fluorescent labels of nucleotides may include but are not limited fluorescein, 5 -carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6- carboxyfluorescein (JOE), rhodamine, 6 -carb oxyrhodamine (R6G),N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4 -(4 'dimethylaminophenylazo) benzoic acid (DABCYL), CascadeBlue, Oregon Green, Texas Red, Cyanine and 5 -(2 - aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS).
  • FAM fluor
  • fluorescently labeled nucleotides can 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, [dRl 10]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.; Fluorescein-15-
  • Nucleotides can also be labeled or marked by chemical modification.
  • a chemically -modified single nucleotide can be biotin-dNTP.
  • biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin- 14-d ATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin- 14-dCTP), and biotin-dUTP (e.g., biotin- 11-dUTP, biotin- 16 -dUTP, biotin-20-dUTP).
  • biotin-dATP e.g., bio-N6-ddATP, biotin- 14-d ATP
  • biotin-dCTP e.g., biotin-11-dCTP, biotin- 14-dCTP
  • biotin-dUTP e.g., biotin- 11-dUTP
  • nucleotide analogs may comprise structures of natural nucleotides that are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function (e.g. hybridization to other nucleotides in RNA orDNA).
  • positions of the nucleotide which may be derivatized include the 5 position, e.g., 5-(2- amino)propyl uridine, 5 -bromo uridine, 5 -propyne uridine, 5 -propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8- bromo guanosine, 8 -chloro guanosine, 8 -fluoroguanosine, etc.
  • 5 position e.g., 5-(2- amino)propyl uridine, 5 -bromo uridine, 5 -propyne uridine, 5 -propenyl uridine, etc.
  • the 6 position e.g., 6-(2-amino)propyl uridine
  • Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine: O- and N-modified(e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310. Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides.
  • the 2' OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, wherein R is substituted or un substituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc.
  • R is substituted or un substituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc.
  • Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438.
  • positions of the nucleotide which maybe derivatized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5- bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2- amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8 -bromo guanosine, 8-chloro guanosine, 8 -fluoroguanosine, etc.
  • 5 position e.g., 5-(2-amino)propyl uridine, 5- bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.
  • the 6 position e.g., 6-(2- amino)propyl uridine
  • the 8-position for adenosine and/or guanosines e.g., 8
  • Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine: O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocy disruptally modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297 - 310.
  • Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides.
  • the 2' OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, wherein R is substituted or un substituted Cl -C6 alkyl, alkenyl, alkynyl, aryl, etc.
  • R is substituted or un substituted Cl -C6 alkyl, alkenyl, alkynyl, aryl, etc.
  • Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438.
  • polynucleotide oligonucleotide
  • nucleic acid a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form.
  • a polynucleotide may be exogenous or endogenous to a cell.
  • a polynucleotide may exist in a cell-free environment.
  • a polynucleotide may be a gene or fragment thereof.
  • a polynucleotide may be DNA.
  • a polynucleotide may be RNA.
  • a polynucleotide may have any three-dimensional structure and may perform any function.
  • a polynucleotide may comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • analogs include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores(e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine.
  • Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro- RNA (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.
  • loci locus
  • locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short inter
  • the sequence of nucleotides maybe interrupted by non -nucleotide components.
  • transfection or “transfected” generally refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods.
  • the nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18. 1-18.88 (which is entirely incorporated by reference herein).
  • peptide “polypeptide,” and “protein” are used interchangeably herein to generally refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains).
  • amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component.
  • amino acid and amino acids generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues.
  • Modified amino acids may include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid.
  • Amino acid analogues may refer to amino acid derivatives.
  • amino acid includes both D-amino acids andL-amino acids.
  • non-native can generally refer to a nucleic acid or polypeptide sequence that is not found in a native nucleic acid or protein.
  • Non-native may refer to affinity tags.
  • Non-native may refer to fusions.
  • Non-native may refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions.
  • a non-native sequence may exhibit and/or encode for an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating 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 to a naturally -occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and/or polypeptide sequence encoding a chimeric nucleic acid and/or polypeptide.
  • promoter generally refers to the regulatory DNA region which controls transcription or expression of a gene and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated.
  • a promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription.
  • a “basal promoter,” also referred to as a “core promoter,” may generally refer to a promoter that contains all the basic necessary elements to promote transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters can contain a TATA-box and/or a CAAT box.
  • expression generally refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides maybe collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • operably linked As used herein, “operably linked,” “operable linkage,” “operatively linked,” or grammatical equivalents thereof generally refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc. , wherein the elements are in a relationship permitting them to operate in the expected manner.
  • a regulatory element which may comprise promoter and/or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.
  • a “vector” as used herein generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which maybe used to mediate delivery of the polynucleotide to a cell.
  • vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles.
  • the vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.
  • an expression cassette and “a nucleic acid cassette” are used interchangeably generally to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression.
  • an expression cassette refers to the combination of regulatory elements and a gene or genes to which they are operably linked for expression.
  • a “functional fragment” of a DNA or protein sequence generally refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence.
  • a biological activity of a DNA sequence may be its ability to influence expression in a manner known to be attributed to the full-length sequence.
  • an “engineered” or object gen erally indicates that the object has been modified by human intervention.
  • a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid may be modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid may synthesized in vitro with a sequence that does not exist in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein may acquire a new function or property.
  • An “engineered” system comprises at least one engineered component.
  • optically aligned generally refers to an alignment of two amino acid sequences that give the highest percent identity score or maximizes the number of matched residues.
  • synthetic and “artificial” are used interchangeably to generally refer to a protein or a domain thereof that has low sequence identity (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) to a naturally occurring human protein.
  • sequence identity 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
  • VPR and VP64 domains are synthetic transactivation domains.
  • sequence identity in the context of two or more nucleic acids 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, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11 , extension of 1 , and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https://blast.ncbi.nlm.nih.gov); or CLUSTALW with parameters of the Smith -Waterman homology search algorithm with parameters of a match of 2, a mismatch of -1, and a gap of -1 ; MUSCLE with default parameters; MAFFT with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters; HMMER
  • the term “inhibition,” “inhibit,” “inhibiting” and the like in reference to a protein-inhibitor interaction generally means negatively affecting (e.g. decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor.
  • Inhibition may refer to reduction of a disease or symptoms of disease.
  • Inhibition may refer to a reduction in the activity of a particular protein or nucleic acid target.
  • the protein may be deoxy cytidine kinase.
  • inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down -regulating signal transduction or enzymatic activity or the amount of a protein.
  • modulator generally 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 the target of the molecule.
  • modulate generally refers to the act of changing or varying one or more properties. “Modulation” generally to the process of changing or varying one or more properties. For example, a modulator of a target protein can change by increasing or decreasing a property or function of the target molecule or the amount of the target molecule. A modulator of a disease can decrease a symptom, cause, or characteristic of the targeted disease.
  • immune checkpoint modulator generally refers to an agent which results in the activation or inhibition of one or more immune checkpoint proteins.
  • immune checkpoint modulators may include, but are not limited to, CD47, PD-L1, A2AR, B7- H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, N0X2, PD-1, TIM-3, VISTA, and SIGLEC7.
  • pluralitripotent cells generally refers to cells that can self -renew and proliferate while remainingin an undifferentiated state and that can, under the proper conditions, be induced to differentiate into specialized cell types.
  • pluripotent cells encompass 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 made available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (as describedin Cowan, C. A. et. al, New England J. Med. 350:13. (2004), incorporated by reference herein in its entirety.)
  • pluripotent stem cells generally refers to cell which have the potential to differentiate into any of the three germ layers: endoderm (e.g. the stomach linking, gastrointestinal tract, lungs, etc), mesoderm (e.g. muscle, bone, blood, urogenital tissue, etc) or ectoderm (e.g. epidermal tissues and nervous system tissues).
  • endoderm e.g. the stomach linking, gastrointestinal tract, lungs, etc
  • mesoderm e.g. muscle, bone, blood, urogenital tissue, etc
  • ectoderm e.g. epidermal tissues and nervous system tissues.
  • the term “pluripotent stem cells,” as used herein, can also encompass “induced pluripotent stem cells,” or “iPSCs,” a type of pluripotent stem cell derived from a non-pluripotent cell.
  • 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 created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the induction of iPS cells are known in the art and are further described below. (See, e.g., Zhou et al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al., Nature Biotechnol.
  • pluripotent stem cell characteristics generally refers to characteristics of a cell that distinguish pluripotent stem cells from other cells.
  • the ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic.
  • Expression or non-expression of certain combinations of molecular markers are also pluripotent stem cell characteristics.
  • human pluripotent stem cells express at least several, and in some embodiments, all of the markers from the following non -limiting list: SSEA-3, S SEA- 4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rexl, and Nanog.
  • Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics. As described herein, cells do not need to pass through pluripotency to be reprogrammed into endodermal progenitor cells and/or hepatocytes.
  • multipotent or “multipotent cell” generally refer to a cell type that can give rise to a limited number of other particular cell types.
  • induced multipotent cells are capable of forming endodermal cells.
  • multipotent blood stem cells can differentiate itself into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc.
  • oligopotent generally refers to the ability of an adult stem cell to differentiate into only a few different cell types.
  • lymphoid or myeloid stem cells are capable of forming cells of either the lymphoid or myeloid lineages, respectively.
  • the term “unipotent” generally refers to the ability of a cell to form a single cell type.
  • the term “totipotent” generally refers to the ability of a cell to form an entire organism.
  • the term “non-pluripotent cells” generally refers to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, but are not limited to, cells from a tissue selected from bone marrow, skin, skeletal muscle, fat tissue and peripheral blood. Example cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells. The starting cells employed for generating the induced multipotent cells, the endodermal progenitor cells, and the hepatocytes can be non -pluripotent cells.
  • Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells.
  • a less potent cell is considered “differentiated” in reference to a more potent cell.
  • Somatic cell generally refers to a cell forming the body of an organism. Somatic cells include cells making up organs, skin, blood, bones and connective tissue in an organism, but not germ cells.
  • the terms “subject” or “patient” generally refer to any animal, such as a domesticated animal, a zoo animal, or a human.
  • the “subject” or “patient” can be a mammal like a dog, cat, bird, livestock, or a human.
  • Specific examples of “subjects” and “patients” include, but are not limited to, individuals (particularly human) with a disease or disorder related to the liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bone, bone marrow, and the like.
  • hypo-immunogenic pluripotent cell or “HIP cell” as used herein generally refer to a pluripotent cell that retains its pluripotent characteristics and yet gives rise to a reduced immunological rejection response when transferred into an allogeneic host. In some cases, HIP cells do not give rise to an immune response. Thus, “hypo-immunogenic” as used herein generally refers to a significantly reduced or eliminated immune response when compared to the immune response of a parental (e.g., wild-type) cell as outlined herein. In some cases, the immune response comprises natural killer (NK) cell cytotoxicity. In some cases, the immune response comprises macrophage cytotoxicity. In some cases, HIP cells as described herein are immunologically silent and yet retain pluripotent capabilities.
  • NK natural killer
  • HIP cells as described herein are immunologically silent and yet retain pluripotent capabilities.
  • allogeneic as used herein generally refers to the genetic dissimilarity of a host organism and a cellular transplant.
  • Inhibitors generally refer to substances or conditions which impact a function or expression of a biologically -relevant molecule.
  • the term “modulator” generally includes both inhibitors and activators. They maybe identified using in vitro and in vivo assays for expression or activity of a target molecule.
  • the term “Inhibitors” generally refers to agents that, e.g., inhibit expression or bind to target molecules or proteins. They may partially or totally block stimulation or have protease inhibitor activity. They may reduce, decrease, prevent, or delay activation, including inactivation, desensitization, or down regulation of the activity of the described target protein. Modulators may be antagonists of the target molecule or protein.
  • Activators generally refers to agents that, e.g., induce or activate the function or expression of a target molecule or protein. They may bind to, stimulate, increase, open, activate, or facilitate the target molecule activity. Activators may be agonists of the target molecule or protein.
  • a variant polypeptide includes one or more modifications that differentiates the function of the variant polypeptide from the unmodified polypeptide.
  • an amino acid change in a variant polypeptide affects its receptor binding profile.
  • a variant polypeptide comprises substitution, deletion, or insertion modifications, or combinations thereof.
  • a variant polypeptide includes one or more modifications that increasesits affinity for a receptor compared to the affinity of the unmodified polypeptide.
  • a variant polypeptide includes one or more substitutions, insertions, or deletions relative to a corresponding native or parent sequence.
  • a variant polypeptide comprises 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, or more modifications
  • a SIRPa binding polypeptide comprises a sequence of a wild-type (WT) CD47 variant.
  • 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.
  • a SIRPa binding peptide comprises one or more modifications (e.g., mutations) of a WT CD47 variant.
  • a SIRPa binding peptide is an engineered SIRPa binding peptide.
  • Engineered SIRPa binding peptides can be generated by one or more of the strategies disclosed herein.
  • a reference polypeptide e.g., a WT CD47 variant, such as any one of SEQ ID NOs: 1-4
  • an engineered SIRPa binding peptide may comprise different structural and/or functional properties as described herein.
  • the reference polypeptide comprises from residue 19 to the final residue of any one of SEQ ID NOs: 1 -4.
  • the reference polypeptide comprises residues 19-290 of any one of SEQ ID Nos: 1 -4.
  • the engineered SIRPa binding peptide may comprise an extracellular domain (ECD).
  • ECD can comprise a sequence with 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%, atleast 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 an ECD of any one of SEQ ID NOs: 1-4, 10-640, or 648-653.
  • an ECD of SEQ ID NO: 1 comprises residues 19-131 of SEQ ID NO: 1 (residues 1 -113 following cleavage of the leader peptide).
  • the engineered SIRPa binding polypeptide may comprise an extracellular loop region (ECLR).
  • the ECLR may comprise portions of the ECD and TMD which are on the extracellular side of a cell membrane when expressed by a cell.
  • the ECLR can comprise, at least in part, an extracellular loop 1 (ECL1) region.
  • the ECL1 region can comprise any of the extracellular residues of the loop region connecting helices II and III of a TMD as described herein (e.g., as comprised in wild type CD47 or an engineered SIRPa binding polypeptide of the disclosure).
  • the ECL1 comprises residues 198-207 of SEQ ID NO: 1 .
  • the ECLR can comprise, at least in part, an extracellular loop 2 (ECL2) region.
  • the ECL2 region can comprise any of the extracellular residues of the loop region connecting helices VI and V of a TMD as described herein (e.g., as comprised in wild type CD47 or an engineered SIRPa binding polypeptide of the disclosure).
  • the ECL2 comprises residues 258-267 of SEQ ID NO: 1 .
  • the ECLR can comprise, at least in part, a peptide linker connecting the C-terminal end of the ECD and the N-terminal extracellular tip of the TMD.
  • the peptide linker comprises residues 132-137 of SEQ ID NO: 1.
  • the ECLR can comprise a sequence with 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%, atleast 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%, atleast about 98%, atleast about 99%, or 100% sequence identity to an ECLR of any one of SEQ ID NOs: 1 -4, 10-640, or 648-653.
  • an 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 following cleavage of the leader peptide).
  • the engineered SIRPa binding peptide may comprise a transmembrane domain (TMD).
  • TMD can comprise five transmembrane helices which are configured to span the lipid membrane of a cell expressing the SIRPa polypeptide.
  • the TMD can comprise a first transmembrane helix (helix I).
  • the helix I comprises residues 142-162 of SEQ ID NO: 1 .
  • the TMD can comprise a second transmembrane helix (helix II).
  • the helix II comprises residues 177-197 of SEQ ID NO: 1.
  • the TMD can comprise a third transmembrane helix (helix III).
  • the helix III comprises residues 208-228 of SEQ ID NO: 1.
  • the TMD can comprise a fourth transmembrane helix (helix IV).
  • the helix IV comprises residues 236-256 of SEQ ID NO: 1.
  • the TMD can comprise a fifth transmembrane helix (helix V).
  • the helix V comprises residues 269-289 of SEQ ID NO: L
  • the TMD can comprise a sequence with atleast 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%, atleast 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 an TMD of any one of SEQ ID NOs: 1 -4, 10-640, or 648-653.
  • an TMD of SEQ ID NO: 1 comprises residues 142-162, 177-197, 208-228, 236-256, and 269-289 SEQ ID NO: 1 (residues 124-144, 159-179, 190-210, 218-238, and 251-271 following cleavage of the leader peptide).
  • the SIRPa binding polypeptide may comprise a signal peptide.
  • the signal peptide may comprise 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.
  • the signal peptide is cleaved to give a mature polypeptide.
  • the engineered SIRPa binding polypeptide does not comprise a signal peptide.
  • the a SIRPa binding polypeptide may comprise a C-terminal domain (CTD).
  • CTD may be present on the cytoplasmic side of a membrane of a cell expressing the SIRPa binding polypeptide.
  • the CTD may comprise residue 290 through the final residue of any one of SEQ ID NOs: 1 -4.
  • the engineered SIRPa binding peptide doesnot comprise a CTD.
  • a polypeptide as described herein comprises one or more cysteine mutations (e.g., relative to a reference polypeptide comprising a sequence of any one of SEQ ID NOs: 1 -4). Cysteine residues may be introduced into Examples of cysteine mutations to induce oligomerization (e.g., relative to a reference sequence, such as SEQ ID NO: 1) are shown below in Table 2.
  • a polypeptide e.g., engineered SIRPa binding polypeptide as described herein comprises one or more mutations that have been identified in a cancer or associated with a cancer, (e.g., relative to a reference polypeptide comprising a sequence of any one of SEQ ID NOs: 1 -4).
  • the mutation may be a single CD47 point mutations. In some embodiments, the mutation may comprise a plurality of 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 onto the crystal structure of CD47 and found to cluster on critical functional regions of the receptor. For example, CD47 mutations associated with cancer as disclosed herein have been mapped to the extra cellular loop region (ECLR) region, transmembrane helix III, and the extracellular domain (ECD).
  • ECLR extra cellular loop region
  • ECD extracellular domain
  • the mutation is mapped on a distinct region of the molecule.
  • a SIRPa binding polypeptide comprising a cancer mutation as described herein may have functional effects that depend on its location on the crystal structure of the polypeptide. Accordingly, these mutations may be subdivided based on where they occur in the molecule, such as in the extracellular domain (ECD), in the transmembrane domain (TMD), in the extracellular loop region (ECLR), in the C-terminal domain (CTD) or between any two of these domains.
  • ECD extracellular domain
  • TMD transmembrane domain
  • ECLR extracellular loop region
  • CTD C-terminal domain
  • cancer mutant W136C can be configured to form a cysteine residue with C259, resultingin an additional disulfide bond between the ECD and TMD interdomain linker and the extracellular portion of helix V.
  • changes in the interactions between domains can alter the conformational ensembles of polypeptides, leading to various functional consequences, such as enhanced binding to SIRPa.
  • mutations may influence or alter the quaternary assembly of SIRPa binding polypeptides by encouraging or disrupting formation of dimers or oligomers.
  • mutations may increase stability of the polypeptide (alone or in combination), and mutations that may disrupt binding of endogenous protein partners (other than SIRPa).
  • engineered polypeptides of the disclosure may comprise one or more of the cancer mutations disclosed herein to impart a certain functional consequence (e.g., enhanced SIPRa binding) relative to a reference polypeptide (e.g., wild-type human CD47 molecule).
  • mutations associated with cancer as discussed herein were found to map to the ECD.
  • a mutation in the ECD may alter/disrupt binding to TSP-1, integrins or increase affinity for SIRPa.
  • CD47 ECD cancer mutation sites represent regions of instability on CD47, and a mutation may increase receptor stability which may enhance function.
  • CD47 mutations associated with cancer which may appear in polypeptides of the disclosure are listed in Table 3.
  • a SIRPa binding polypeptide comprises at least one mutant listed in Table 3.
  • the SIRPa 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.
  • the SIRPa 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.
  • the SIRPa binding polypeptide comprises atmost 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.
  • a SIRPa binding polypeptide comprises at least one mutation of: M31, L40, C42, D47, D64, C75, E80, F97, G105, K106, F1O6,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, where the mutation position is relative to any one of SEQ ID NOs: 1 -4.
  • a SIRPa binding polypeptide comprises an M31 residue.
  • a SIRPa binding polypeptide comprises an L40 residue. In some embodiments, a SIRPa binding polypeptide comprises a C42 residue. In some embodiments, a SIRPa binding polypeptide comprises a D47 residue. In some embodiments, a SIRPa binding polypeptide comprises a D64 residue. In some embodiments, a SIRPa binding polypeptide comprises a C75 residue. In some embodiments, a SIRPa binding polypeptide comprises an E80 residue. In some embodiments, a SIRPa binding polypeptide comprises an F97 residue. In some embodiments, a SIRPa binding polypeptide comprises a G105 residue. In some embodiments, a SIRPa binding polypeptide comprises a KI 06 residue.
  • a SIRPa binding polypeptide comprises an Fl 06 residue. In some embodiments, a SIRPa binding polypeptide comprises a KI 11 residue. In some embodiments, a SIRPa binding polypeptide comprises an SI 23 residue. In some embodiments, a SIRPa binding polypeptide comprises an S127 residue. In some embodiments, a SIRPa binding polypeptide comprises a K128 residue. In some embodiments, a SIRPa binding polypeptide comprises an F131 residue. In some embodiments, a SIRPa binding polypeptide comprises a C132 residue. In some embodiments, a SIRPa binding polypeptide comprises a Cl 36 residue. In some embodiments, a SIRPa binding polypeptide comprises a KI 40 residue.
  • a SIRPa binding polypeptide comprises a T142 residue. In some embodiments, a SIRPa binding polypeptide comprises a G146 residue. In some embodiments, a SIRPa binding polypeptide comprises an Ml 53 residue. In some embodiments, a SIRPa binding polypeptide comprises an L157 residue. In some embodiments, a SIRPa binding polypeptide comprises an LI 60 residue. In some embodiments, a SIRPa binding polypeptide comprises an El 66 residue. In some embodiments, a SIRPa binding polypeptide comprises a C170 residue. In some embodiments, a SIRPa binding polypeptide comprises a DI 78 residue. In some embodiments, a SIRPa binding polypeptide comprises a A203 residue.
  • a SIRPa binding polypeptide comprises an S207 residue. In some embodiments, a SIRPa binding polypeptide comprises a V210 residue. In some embodiments, a SIRPa binding polypeptide comprises a D211 residue. In some embodiments, a SIRPa binding polypeptide comprises an L214 residue. In some embodiments, a SIRPa binding polypeptide comprises an S215 residue. In some embodiments, a SIRPa binding polypeptide comprises a V262 residue. In some embodiments, a SIRPa binding polypeptide comprises an L264 residue. In some embodiments, a SIRPa binding polypeptide comprises a Y267 residue.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPabinding 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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 .
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a polypeptide e.g., engineered SIRPa binding polypeptide as described herein comprises one or more mutations that have been identified as a naturally occurring polymorphism (e.g., relative to a reference polypeptide comprising a sequence of any one of SEQ ID NOs: 1 -4). Some of these mutations have been mapped onto the crystal structure of human CD47 and found to cluster on critical functional regions of the receptor. For example, CD47 polymorphisms as disclosed herein have been mapped to the extra cellular loop region (ECLR) region, transmembrane domain (TMD), and the extracellular domain (ECD).
  • ECLR extra cellular loop region
  • TMD transmembrane domain
  • ECD extracellular domain
  • the polymorphism is mapped on a distinct region of the molecule.
  • a polymorphism described herein may have functional effects that depend on its location on the crystal structure of the molecule (e.g., SIRPa binding polypeptide). Accordingly, these polymorphisms may be subdivided based on where they occur in the molecule, such as in the extracellular domain (ECD), in the transmembrane domain (TMD), in the extracellular loop region (ECLR), or between any two of these molecules.
  • ECD extracellular domain
  • TMD transmembrane domain
  • ECLR extracellular loop region
  • polymorphisms may impact secondary, tertiary, or quaternary structure by creating opportunities for new non -covalent and/or covalent interactions or abrogating others.
  • These structural changes may in turn affect functional aspects of the SIRPa binding polypeptide transmembrane signaling.
  • polymorphism C33Y can disrupt an interdomain disulfide bond, thus alteringthe conformational ensemble of the molecule.
  • polymorphisms may influence or alter the quaternary assembly of SIRPa binding polypeptides by encouraging or disrupting formation of dimers or oligomers.
  • polymorphisms may increase stability of the receptor (alone or in combination), and polymorphisms that may disrupt binding of endogenous protein partners (other than SIRPa). Accordingly, engineered polypeptides of the disclosure may comprise one or more of the polymorphisms disclosed herein to impart a certain functional consequence (e.g., enhanced SIPRa binding) relative to a wild type CD47 molecule.
  • CD47 polymorphisms which may appear in polypeptides of the disclosure are listed in Table 4.
  • a SIRPa binding polypeptide comprises at least one polymorphism listed in Table 4.
  • the SIRPa 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,
  • the SIRPa binding polypeptide comprises atmost 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, 78, 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,
  • a SIRPa 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: 476-633 and 648.
  • a SIRPa 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: 648.
  • a polypeptide e.g., engineered SIRPa binding polypeptide as described herein comprises one or more mutations that have been developed or determined based on knowledge of the peptide functionality (e.g., relative to a reference polypeptide comprising a sequence of any one of SEQ ID NOs: 1 -4).
  • determination of the potentially useful mutations may use tools and data that may include bioinformatic models, protein structure data, crystallographic information, protein functional data, and protein conformation models. Analysis may include algorithmic calculations, expert determinations, or both.
  • a polypeptide comprises one or more mutations configured to generate a new covalent or non -covalent interaction or to abrogate an existing covalent or non- covalent interaction.
  • a SIRPa binding sequence comprises a D35R mutation (e.g., relative to any one of SEQ ID NOs: 1 -4) and an A262E mutation.
  • the arginine residue may form an inter-domain ionic interaction with the glutamate and thus alter the conformational ensemble and function of the polypeptide relative to a wild-type CD47.
  • a polypeptide comprises a S138F mutation configured to interact with a T3 IM cancer mutation.
  • the bulky aromatic sidechain 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.
  • the mutated polypeptide can display an altered conformational ensemble relative to the wild type sequence.
  • the SIRPa binding polypeptide comprises at least one residue of: Y31 , A32, R35, K35, P71 , A77, A79, N80, LI 00, KI 38, LI 64, Ml 85, A211 , S259, E262, and any combination thereof, wherein the mutation is relative to any one of SEQ ID NOs: 1 -4.
  • the SIRPa binding polypeptide comprises a Y31 residue.
  • the SIRPa binding polypeptide comprises a A32 residue.
  • the SIRPa binding polypeptide comprises a R35 residue.
  • the SIRPa binding polypeptide comprises a K35 residue.
  • the SIRPa binding polypeptide comprises a P71 residue. In some embodiments, the SIRPa binding polypeptide comprises a A77 residue. In some embodiments, the SIRPa binding polypeptide comprises a A79 residue. In some embodiments, the SIRPa binding polypeptide comprises a N80 residue. In some embodiments, the SIRPa binding polypeptide comprises a LI 00 residue. In some embodiments, the SIRPa binding polypeptide comprises a KI 38 residue. In some embodiments, the SIRPa binding polypeptide comprises a LI 64 residue. In some embodiments, the SIRPa binding polypeptide comprises a Ml 85 residue. In some embodiments, the SIRPa binding polypeptide comprises a A211 residue. In some embodiments, the SIRPa binding polypeptide comprises a S259 residue. In some embodiments, the SIRPa binding polypeptide comprises a E262 residue.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a SIRPa 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.
  • a polypeptide as described herein comprises one or more regions, or portions thereof, derived from non-human proteins (e.g., relative to a reference polypeptide comprising a sequence of any one of SEQ ID NOs: 1-4).
  • the region is an extracellular domain (ECD).
  • the region is a transmembrane domain (TMD), or portion thereof.
  • the region is an extracellular loop region (ECLD).
  • the region is a C-terminal intracellular domain.
  • the region comprises any two of the ECD, TMD, ECLR, or C-terminal intracellular domain, or potion thereof. .In some embodiments, the region comprises any three of the ECD, TMD, ECLR, or C-terminal intracellular domain, or portion thereof. In some embodiments, the region comprises all of the ECD, TMD, ECLR, or C-terminal domain, or portion thereof.
  • engineered SIRPa binding polypeptides may comprise hybrid molecules with detuned human function.
  • the TMD and C-terminal domains may be based on a domain from a non-human species.
  • the ECD domains may contain specific residues that may have been replaced with a residue from a different species.
  • the TMD and C-terminal domains may contain specific residues that may have been replaced with a residue from a different species.
  • a polypeptide of such a composition maybe prevented from binding to human TSP-1 and integrins and abrogate transmembrane signaling.
  • chimeras may also contain a modified TMD domain.
  • the non-human sequence may be derived from any other non- human that expresses CD47.
  • Representative, non -limiting species from which sequences may be derived include Gallus gallus, Alligator mississippiensis, Accipiter genii Us., Anas platyrhynchos, Ficedula albicollis. Phasianus colchicus. and Chloebia gouldiae (Erythruragouldiae).
  • the non-human sequence may comprise a sequence that has 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.
  • a polypeptide as described herein comprises a sequence that has 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.
  • a polypeptide e.g., engineered SIRPa binding polypeptide as described herein comprises a modified peptide sequence such that a peptide linker is inserted into a loop or linker (e.g. the RVVSWF linker comprised in residues 132-137 of any one of SEQ ID NOs: 1-4) of the polypeptide structure (e.g., relative to a reference polypeptide comprising a sequence of any one of SEQ ID NOs: 1 -4).
  • the loop is a polypeptide sequence between the ECD domain and the TMD domain of the polypeptide.
  • the loop comprises residues 132-137 of any one of SEQ ID NOs: 1 -4.
  • the loop insertion may be inserted between any two residues in the range of 132-137, prior to residue 132, or subsequentto residue 137, relative to a reference polypeptide sequence of any one of SEQ ID NOs: 1 -4.
  • the linker is added between the ECD domain of the reference peptide with a IgV-like protein fold and the first helix of the TMD comprised by a protein fold containing 5 transmembrane helices.
  • the linker sequence may be between 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more residues in length.
  • the linker may comprise a pair of cysteine residues configured to form a disulfide bond.
  • the pair of cysteine residues may be separated by any suitable number of residues. In some embodiments, the pair of cysteine residues are 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 are 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, the pair 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.
  • a polypeptide as described herein comprises a sequence that has 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%, ormore identity to SEQ ID NO: 640.
  • a polypeptide as described herein comprises a sequence that has atleast about20%, 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.
  • a polypeptide as described herein comprises a sequence that has 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.
  • the polypeptide has no more than 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 IS NOs: 10-384. In some embodiments, the percent identity is between any two of these values.
  • a polypeptide as described herein comprises a sequence that has 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.
  • the polypeptide has no more than 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 sequencesin 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.
  • a polypeptide as described herein comprises a conformational ensemble which has been modulated relative to a conformational ensemble of a reference polypeptide.
  • the reference polypeptide is a WT CD47 sequence (e.g., any one of SEQ ID NOs: 1 -4).
  • Conformational ensembles of polypeptides as described herein can be determined by any suitable operation.
  • a conformational ensemble may be observed or predicted by techniques of biophysics and molecular simulation such as x-ray crystallography (XRC), nuclear magnetic resonance (NMR), hydrogen-deuterium exchange (HDX), small-angle e-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.
  • XRC x-ray crystallography
  • NMR nuclear magnetic resonance
  • HDX hydrogen-deuterium exchange
  • SAXS small-angle e-ray scattering
  • EPR electron paramagnetic resonance
  • CMR cryogenic electron microscopy
  • MD molecular dynamics
  • Monte Carlo Monte Carlo
  • metastates primary metastable states
  • FIG. 1 A superposition of these metastates is illustrated in FIG. 1.
  • SI and S2 metastates were found to comprise a majority of the conformational ensemble and the primary motion of the molecule at equilibrium was a transition between these two metastates.
  • the SI and S2 metastates may be characterized in terms of a reaction coordinate.
  • the reaction coordinate comprises an atomic coordinate, set of atomic coordinates, order parameter, or a combination or transformation thereof.
  • any atomic coordinate, set of atomic coordinates, order parameters, or transformations thereof may be used as a reaction coordinate if the coordinate, set of coordinates, order parameter, or transformation thereof adopts differing values that readily distinguish the metastable states from one another.
  • an interatomic distance between two atoms, one on the ECD and one on the TMD, of a SIRPa binding protein as described herein can be used as a reaction coordinate to identify the metastate (or potential set of metastates) to which a particular conformation of the SIRPa binding protein may be assigned.
  • interatomic distance between the C a atoms of residues R132 and El 51 is used as a reaction coordinate.
  • a bending angle between an ECD and TMD can be used as a reaction coordinate to identify the metastate (or potential set of metastates) to which a particular conformation of the SIRPa binding protein may be assigned.
  • the angle between the domains for a given configuration can be determined as the angle of the vector between an x-y plane set at an origin (e.g., center of mass of one domain) and a vector between the origin and the center of mass of a subset of residues of the other domain.
  • the first domain was the TMD and the second vector was the vector between the origin and the center of mass of residues N50 through V54 of an ECD.
  • SIRPa agonists as described herein may elicit a reduced immune response when expressed on the surface of a cell due to comprising a conformational ensemble which comprises a different proportion of these metastable states as compared to a reference polypeptide (e.g., comprising a sequence of any one of SEQ ID NOs; 1 -4).
  • a SIRPa agonist of the present disclosure comprises a conformational ensemble with a greater proportion of the “SI” metastate as discussed above.
  • a cell product would contain one or more SIRPa agonist of the present disclosure containing different distributions of conformational ensembles (e.g.
  • SI proportion some may display greater SI proportion, some may display greater S2 proportion.
  • Various strategies as discussed herein cancer mutations, polymorphisms, oligomerization, chimera, loop insertions, rational mutations) maybe employed singly or in combination to engineer SIRPa agonist polypeptide sequences with a conformational ensembles that comprise a larger proportion of the SI metastate and thus enhance SIRPa signaling by having a larger proportion of the molecule(s) available to interact with SIRPa.
  • SIRPa binding polypeptides of the disclosure may enhance signaling through SIRPa by a mechanism other than increasing the proportion of the SI metatstate 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 “free” SIRPa binding molecules or sites available.
  • the engineered SIRPa binding peptides described herein further comprise N-terminal modifications that further enhance binding to SIRPa.
  • 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.
  • the engineered SIRPa binding peptide comprises at least 1 amino acid added to the N-terminus of the mature protein. In some embodiments, the engineered SIRPa binding peptide comprises at least 2 amino acids added to the N-terminus of the mature protein. In some embodiments the engineered SIRPa binding peptide comprises at least 3 amino acids added to the N-terminus of the mature protein. In some embodiments, the 3 amino acids added have the formula X-3X-2X-1 , where X-3 is W; X-2 is selected from Q, A and G; and X-l is selected from R, P, L, T, F, I, and M. In some embodiments, the three amino acids added are selected from: WQR, WAP, WQL, WQP, WQT, WQF, WQI, WGP, and WQM.
  • nucleic acids that encode any of the peptides, polypeptides, fusion proteins, and compositions described herein.
  • nucleic acid molecules may include polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. In some embodiments, nucleic acid molecules may only to the primary structure of the molecule. In some embodiments, nucleic acid molecules may be triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. In some embodiments, nucleic acid molecules may be modified by methylation and/or by capping. In some embodiments, nucleic acid molecules may be natural, synthetic, or a combination of both.
  • the introducing step includes introducing into a cell an expression vector including a nucleic acid sequence encoding the peptide.
  • vectors that include any of the nucleic acids provided herein.
  • the vector may refer to a polynucleotide capable of inducing the expression of a recombinant peptide in a host cell.
  • the vector further comprises a promoter and/or enhancer operably linked to any of the nucleic acids described herein.
  • vectors could be constructed to comprise exogenous nucleic acid sequences for genetic modification of any cells used herein, particularly the starting cells, such as stroma cells or stem or progenitor cells in the culturing methods or compositions.
  • One of skill in the art would be well equipped to construct a vector through standard recombinant techniques.
  • Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell -type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
  • Such components also might include markers, such as detectable and/or selection markers that can be used to detector select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • vectors can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • a large variety of such vectors are known in the art and are generally available.
  • the vector When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell's nucleus or cytoplasm.
  • Genetic modification or introduction of exogenous nucleic acids into starting cells of the culturing composition or methods may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art.
  • Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection, including microinjection; by calcium phosphate precipitation; by using DEAE-dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection and receptor-mediated transfection; by microprojectile bombardment; by agitation with silicon carbide fibers; by Agrobacterium -mediated transformation; by PEG- mediated transformation of protoplasts; by desiccati on/inhibition -mediated DNA uptake, and any combination of such methods.
  • organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.
  • the cells in certain embodiments can be made to contain one or more genetic alterations by genetic engineering of the cells either before or after differentiation.
  • a cell is said to be “genetically altered”, “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 where the cell is a progeny of the originally altered cell that has inherited the polynucleotide.
  • the cells can be processed to increase their replication potential by genetically altering the cells to express telomerase reverse transcriptase, either before or after they progress to restricted developmental lineage cells or terminally differentiated cells.
  • cells containing an exogenous nucleic acid construct may be identified in vitro or in vivo by including a marker in the expression vector, such as a selectable or screenable marker.
  • a marker in the expression vector such as a selectable or screenable marker.
  • Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector, or help enrich or identify differentiated cells by using a tissue-specific promoter.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • Cells of the present disclosure may comprise one or a plurality of SIRPa binding polypeptide as described herein.
  • the plurality of SIRPa binding polypeptides comprises a wild type CD47 (e.g., comprising any one of the sequences of SEQ ID NOs: 1-4).
  • the plurality of SIRPa binding polypeptides does not comprise a wildtype CD47 sequence.
  • the plurality of SIRPa binding polypeptides comprises a plurality of engineered SIRPa binding polypeptides.
  • the plurality of engineered SIRPa binding polypeptides may comprise any engineered SIRPa binding polypeptides disclosed herein.
  • FIG. 24 shows a computer system 2401 that is programmed or otherwise configured to design the SIRPa agonists described herein (e.g. any one of the methods described herein).
  • the computer system 2401 can regulate various aspects of sequence design of the present SIRPa agonists, such as, for example, stability and an enhanced “don’t eat me” signal.
  • the computer system 2401 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system 2401 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 2405, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 2401 also includes memory or memory location 2410 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 2415 (e.g., hard disk), communication interface 2420 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 2425, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory 2410, storage unit 2415, interface 2420 and peripheral devices 2425 are in communication with the CPU 2405 through a communication bus (solid lines), such as a motherboard.
  • the storage unit 2415 can be a data storage unit (or data repository) for storing data.
  • the computer system 2401 can be operatively coupled to a computer network (“network”) 2430 with the aid of the communication interface 2420.
  • the network 2430 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network 2430 in some cases is a telecommunication and/or data network.
  • the network 2430 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network 2430, in some cases with the aid of the computer system 2401, can implement a peer-to-peer network, which may enable devices coupled to the computer system 2401 to behave as a client or a server.
  • the CPU 2405 can execute a sequence of machine-readable instructions, which canbe embodied in a program or software.
  • the instructions maybe stored in a memory location, such as the memory 2410.
  • the instructions canbe directed to the CPU 2405, which can subsequently program or otherwise configure the CPU 2405 to implement methods of the present disclosure. Examples of operations performed by the CPU 2405 can include fetch, decode, execute, and writeback.
  • the CPU 2405 can be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the system 2401 can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • ASIC application specific integrated circuit
  • the storage unit 2415 can store files, such as drivers, libraries and saved programs.
  • the storage unit 2415 can store user data, e.g., user preferences and user programs.
  • the computer system 2401 in some cases can include one or more additional data storage units that are external to the computer system 2401, such as located on a remote server that is in communication with the computer system 2401 through an intranet or the Internet.
  • the computer system 2401 can communicate with one or more remote computer systems through the network 2430. For instance, the computer system 2401 can communicate with a remote computer system of a user.
  • remote computer systems examples include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smartphones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants.
  • the user can access the computer system 2401 via the network 2430.
  • Methods as described herein can be implemented byway of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 2401, such as, for example, on the memory 2410 or electronic storage unit 2415.
  • the machine executable or machine readable code can be provided in the form of software.
  • the code can be executed by the processor 2405.
  • the code can be retrieved from the storage unit 2415 and stored on the memory 2410 for ready access by the processor 2405.
  • the electronic storage unit 2415 can be precluded, and machine-executable instructions are stored on memory 2410.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.
  • “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tap e drives, disk drives and the like, which may provide non -transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • a machine readable medium such as computer-executable code
  • a tangible storage medium such as computer-executable code
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory, such as 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.
  • RF radio frequency
  • IR infrared
  • Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may 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.
  • the computer system 2401 can include or be in communication with an electronic display 2435 that comprises a user interface (UI) 2440 for providing, for example, SIRPa agonist sequences described herein.
  • UI user interface
  • Examples of UI’ s include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • Methods and systems of the present disclosure can be implemented by way of one or more algorithms.
  • An algorithm can be implemented by way of software upon execution by the central processingunit 2405.
  • the algorithm can, for example, design the sequences for the SIRPa agonists described herein.
  • FIG. 2 A depicts a PCA plot showing the relative distribution of conformations along the first two principal components of WT CD47 without SIRPa.
  • FIG. 2B depicts a PCA plot showing the relative distribution of conformations along the first two principal components for WT CD47 in complex with SIRPa.
  • binding of SIRPa shifts the conformational ensemble from predominantly the S2 metastate to predominantly the SI metastate. Based on this observation, it was hypothesized that SIRPa preferentially binds the SI metastate of CD47.
  • the angle between the domains was also calculated and found to range from about 100-120 degrees in the S2 state to about 130-180 degrees in the SI state (FIG. 3B).
  • the angle between the domains was calculated by determining the angle of the vector between an x-y plane set at the origin (center of mass of the TMD bundle) and a vector between the origin and the center of mass of residues N50 through V54 in the ECD.
  • Example 2 Investigation of mutations in CD47
  • TMD transmembrane domain
  • Some cancer mutations sites (e.g., as comprisedin 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).
  • Some cancer mutation sites (e.g., as comprisedin 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).
  • ECD of CD47 comprises regions predicted to be regions of instability on CD47. Accordingly, it was hypothesized that cancer mutations in the ECD of CD47 may act to increase stability of the receptor and thus enhance signaling through the SIRPa pathway, affect distribution of metastable states, or affect interactions with the lipidic membrane environment.
  • CD47 polymorphisms e.g., as comprisedin Table 4
  • TSP-1 thrombospondin 1
  • integrins or other endogenous protein partners.
  • SIRPa agonist peptide insertions identified from analysis of the receptor evolution across multiple species.
  • SIRPa agonist peptide insertions were identified in the RVVSWF peptide linker (positions 132-137 of any of SEQ ID NOs; 1-4) linking the ECD and the TMD of human CD47.
  • RVVSWF peptide linker positions 132-137 of any of SEQ ID NOs; 1-4
  • SEQ ID NOs: 641-647 Several species were identified to have peptides that vary in amino acid length and are constrained by a disulfide bond (e.g., SEQ ID NOs: 641-647). These peptides are predicted to influence 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 SIRPa polypeptides.
  • a synthetic SIRPa binding polypeptide is generated in accordance with the operations 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., comprisingthe sequence of any one of SEQ ID NOs: 1-4 is examined similarly.
  • the conformational ensemble is found to comprise a first metastable state which is configured to bind to SIRPa.
  • the first metastable state comprises conformations characterized by one or more of the following features:
  • TMD transmembrane domain
  • TMD extracellular domain
  • a distance between the TMD and the ECD of from about 10 angstroms (A) to about 25 A, or more than about 25 A.
  • a metastable state characterized by these features may be referred to as an “SI” or “SI -like” metastate herein.
  • the conformational ensemble of the synthetic SIRPa binding polypeptide is found to comprise a higher proportion (e.g., as determined, estimated, or predicted by one of more of HDX, SAXS, NMR, or MD) of the first metastable state than the conformational ensemble of the reference polypeptide.
  • the conformational ensemble of the reference polypeptide is also found to comprise a second metastable state which doesnot substantially bind SIRPa.
  • the second metastable state is characterized by one or more of the following features:
  • TMD transmembrane domain
  • TMD extracellular domain
  • a distance between the TMD and the ECD of from about 4 angstroms (A) to about 9 A, or less than about 4 A.
  • the conformational ensemble of the polypeptide may also be found to comprise the second metastable state but at a smaller proportion than the conformational ensemble of the reference polypeptide.
  • a metastable state characterized by these features may be referred to as an “S2” or “S2-like” metastate herein.
  • CD47 mutations associated with cancer e.g., as comprised in Table 3
  • certain CD47 polymorphisms e.g., as comprised in Table 4
  • constructs comprising some ofthese mutations were simulated using MD in accordance with the protocols outlinedin Fenalti and the resulting conformational ensembles sampled and compared to wild type CD47 (e.g., SEQ ID NO: 1).
  • SEQ ID NO: 419 A sequence comprising an R132C mutation (SEQ ID NO: 419), which is associated with cancer samples, was simulated using MD. Residue 132 occurs in the ECLR of CD47, and thus the R132C mutant was hypothesized to reduce relative motion between the ECD and TMD, thus increasing the relative proportion of the SI metastate in its conformational ensemble.
  • T3 IM and S138F (SEQ ID NO: 648) was also simulated using MD. Residues 31 and 138 also occur in the ECLR. Accordingly, it was hypothesized that these two hydrophobic substitutions would fill the ECLR and restrict hinge motion between the ECD and TMD, thus increasing the relative proportion of the SI metastate.
  • a representative model of the T31M/S138F mutant showingthe mutated residues in the ECLR is depicted in FIG. 4. [00192] Results of two separate MD for each construct are shown in FIG. 5 A (R132C) and FIG. 5B (T3 IM, S138F).
  • FIGs. 5A and 5B depict the distance between the ECD and TMD domains (top panels; calculated as described in Example 2) and the angle of the domains (bottom panels; calculated as described in Example 2). As shown in FIGs. 5A and 5B, these mutation in each case increase the proportion of time the simulated molecule (the conformational ensemble) spends in the SI metastate as compared to WT CD47 (FIGs. 3A and 3B)
  • This example outlines a macrophage killing assay to measure protection from macrophage cytotoxicity conferred by the engineered SIRPa binding polypeptides of the present disclosure.
  • DNA encoding engineered SIRPa binding peptides as described herein are synthesized by VectorBuilder Inc. and cloned into a mammalian gene expression lentiviral vector (pLV) under the EFl a promotor and blasticidin antibiotic selection.
  • pLV mammalian gene expression lentiviral vector
  • FIG. 6 A representative diagram for a polypeptide as disclosed herein is illustrated in FIG. 6.
  • the constructs comprise a T2A ribosomal skipping sequence so the engineered polypeptide to GFP expression ratio is 1 :1 and the expression product comprises two unlinked proteins, engineered SIRPa binding polypeptides that traffic to the membrane and cytosolic GFP.
  • a K562 CD47 _/ - cell line is thawed and allowed to recover for 1 week before transductions.
  • the cell line is seeded into a T75 flask at 0.2 * 10 6 VC/mL in 20 mL working volume for maintenance in RPMI + 10% FBS + 1% Pen-Strep.
  • Cells are typically passaged every 2-3 days or when they reach a concentration of 1 x 10 6 VC/mL.
  • Cell counts and viability are recorded with a cell counter (e.g., NucloeCounter®NC-202TM).
  • Cell sorting is performed in a Sony MA900 cell sorter. Double positive GFP -CD47 cells are sorted. Cells are expanded post-sorting in RPMI + 10% FBS +1 % PS + 6 pg BLAST media. For FACS analysis, cells transduced with Lentivirus on early passages (2-4) are assessed for GFP and CD47 or engineered SIRPa binding protein expression .
  • the amount of engineered SIRPa binding polypeptide expressed on the cell surface is determined using the monoclonal antibodies B6H12 and CC2C6 dyed with Brilliant VioletTM 650 Dye (BV605).
  • An isotype control dyed with PerCP-Cyanine5.5 is used forbackground subtraction, and surface expression levels are generally normalized using human WT CD47 transduced cells for comparison and analysis.
  • THP-1 derived macrophages are prepared according to documented procedures and are co -cultured with K562-CD47 KO BFluc cell lines generated as described above displaying different engineered SIRPa binding polypeptides (target cells).
  • Macrophages effector cells
  • target cells are 100% susceptibleto cytotoxic killing.
  • Negative control CD47 knockout K562-CD47-KO BFluc target cells will be substantially all killed, and no luminescence signal will be observed from Luciferase containing target cells.
  • Titration of effector to target cells E:T results in less killing of target cells and thus a luciferase signal.
  • the data can be expressed as a percentage of cell killing for each construct to enable comparison between different constructs.
  • This example outlines an NK cell killing assay to measure protection from macrophage cytotoxicity conferred by the engineered SIRPa binding polypeptides of the present disclosure.
  • DNA encoding engineered SIRPa binding peptides as described herein are synthesized by VectorBuilder Inc. and cloned into a mammalian gene expression lentiviral vector (pLV) under the EFl a promotor and blasticidin antibiotic selection.
  • pLV mammalian gene expression lentiviral vector
  • FIG. 6 A representative diagram for a polypeptide as disclosed herein is illustrated in FIG. 6.
  • the constructs comprise a T2A ribosomal skipping sequence so the engineered polypeptide to GFP expression ratio is 1 :1 and the expression product comprises two unlinked proteins, engineered SIRPa binding polypeptides that traffic to the membrane and cytosolic GFP.
  • K562 CD47 KO cell lines are thawed and allowed to recover for 1 week before transductions.
  • the cell line is seeded into a T75 flask at 0.2 x 10 6 VC/mL in 20 mL working volume for maintenance in RPMI + 10% FBS + 1% Pen -Strep.
  • Cells are typically passaged every 2-3 days or when they reach a concentration of 1 x 10 6 VC/mL.
  • Cell counts and viability are recorded with a cell counter (e.g., NucloeCounter®NC-202TM).
  • the first round of transductions are performed with a commercial vector to integrate a FLuc transgene, followed by a second round of transductions to integrate polypeptides of the disclosure.
  • KO and K562 cell lines containing engineered polypeptides are typically seeded into a T75 flask at 0.2 x 10 6 VC/mL in 20 mL working volume for maintenance in RPMI + 10% FBS + 1% Pen-Strep. Cells are typically passaged every 2-3 days or when they reach a concentration of 10 6 VC/mL.
  • Cell sorting is performed in a Sony MA900 cell sorter. Double positive GFP-CD47 cells are sorted and expanded post-sort in RPMI + 10% FBS + 1% PS + 6 pg BLAST media. Cell counts and viability are recorded with an automated cell counter (e.g., NucloeCounter® NC-202TM) prior to each assay. Characterization of cell lines: SIRPa binding protein expression
  • the amount of engineered SIRPa binding polypeptide expressed on the cell surface is determined using the monoclonal antibodies B6H12 and CC2C6 dyed with Brilliant VioletTM 650 Dye (BV605). Mean fluorescence intensity (MFI) is quantified for cells expressing each construct. An isotype control dyed with PercCP-Cyanine5.5 is used for background subtraction and surface expression levels are generally normalized using human WT CD47 transduced cells for comparison and analysis.
  • IL-2 activated NK cells (about 20 nM) from healthy donors are co-cultured with K562-CD47 KO BFluc cell lines generated as described above displaying different engineered SIRPa binding polypeptides (target cells).
  • a parental K562 cell line is used as control as it is representative of the relative endogenous levels of CD47.
  • Activated NK cells effector cells
  • target cells are 100% susceptible to cytotoxic killing.
  • Negative control cells with wild type CD47 knocked out (K562-CD47-KO BFluc target cells) will be substantially all killed, and no luminescence signal will be observed from Luciferase containing target cells.
  • E:T Titration of effector to target cells results in less killing of target cells and thus a luciferase signal.
  • the data can be expressed as a percentage of cell killing for each construct to enable comparison between different K562 -CD47 -KO BFluc constructs.
  • Additional positive controls can include K562 cells engineered to overexpress WT CD47 (K562 CD47 T2A GFP) and human leukocyte antigen E (HLA-E) constructs (K562 HLA-E).
  • SIRPa binding polypeptide sequences comprising mutations associated with cancer (e.g., one or more mutations listed in Table 3) were generated.
  • a brief summary of the constructs are listed in Table 5.
  • the conformational ensembles of the sequences were simulated by molecular dynamics (MD) in accordance with the molecular dynamics procedures outlined in Fenalti and Examples 1 and 4 herein above.
  • Control constructs used as a basis of comparison were wild type human CD47 (WT CD47), WT CD47 in complex with signal regulatory peptide alpha (SIRPa), and WT CD47 in complex with the fragment antigen binding (Fab) region of a CD47 -specific antibody (WT + Fab).
  • FIG. 7 A and FIG. 7B summarize the conformational ensembles of each construct tested as well as the controls. As illustrated in FIGs. 7A-7B, almost every construct shifted the conformational ensemble toward a greater proportion of the SI metastate relative to wild type, with the exception of Construct 32 (SEQ ID NO: 472).
  • Construct22 (SEQ ID NO: 419), which comprises an R132C mutation relative to SEQ ID NO: 1, the free energy landscape was investigated by performing principal component analysis (PC A) on the downsampled trajectory and compared to that of the wild type. As illustrated in FIG. 7C, the introduction of the R132C mutation associated with cancer shifts the conformational ensemble from predominantly “S2-like” to predominantly “SI -like.”
  • Engineered SIRPa binding sequences comprising one or more mutations associated with cancer (e.g., comprised in Table 3) were generated and tested forNK-cell killing and surface expression in accordance with the procedures of Example 7.
  • the constructs tested are summarized below.
  • Control constructs included endogenous expression of wild type CD47 (WT), CD47 knockout (CD47 KO), overexpression of wildtype CD47 (CD47 T2A GFP), and expression of HLA-E (HLA-E), which were similarly tested.
  • FIGs. 8-14, and 16 illustrate the protection fromNK-cell cytotoxicity conferred by the tested constructs. As illustrated in the Figures, each of the constructs provided protection from NK-cell killing, showing reduced NK-cell cytotoxicity relative to endogenous expression ofWT CD47. Additionally, Construct 32 (SEQ ID NO: 472) showed protection fromNK-cell killing enhanced beyond even that of overexpression of WT CD47 (FIG. 13).
  • FIGs. 19-21 illustrate the measured surface expression for the constructs. Surface expression was measured in accordance with the procedures of Example 7 using monoclonal antibodies for CD47: B6H12 (FIGs. 19 and 20) and CC2C6 (FIG. 21). Control constructs included endogenous expression of wild type CD47 (WT), CD47 knockout (KO), and overexpression of wild type CD47 (hCD47 T2A GFP).
  • FIG. 19 illustrates measured mean fluorescence intensity (MFI) of a B6H12 monoclonal antibody specific for human CD47.
  • FIG. 20 illustrates MFI of B6H12 except that each value has been normalized relative to the endogenously expressed wild type value.
  • FIG. 1 mean fluorescence intensity
  • 21 illustrates the MFI of another CD47-specific monoclonal antibody, CC2C6, again with each value normalized relative to endogenous expression.
  • constructs for which surface expression could be measured showed surface expression greater than that of endogenously expressed.
  • expression could not be measured, presumably because the mutations altered the epitope such that monoclonal antibody binding could no longer be observed.
  • WT wild type CD47
  • CD47 knockout CD47 knockout
  • HLA-E HLA-E
  • FIGs. 8, 10, and 13-15 illustrate the protection from NK-cell cytotoxicity conferred by the tested constructs. As illustrated in the Figures, constructs generally conferred protection from NK cell cytotoxicity, showing reduced NK-cell cytotoxicity relative to endogenous expression of wild type CD47.
  • FIGs. 19-21 illustrate the measured surface expression for the constructs. Surface expression was measured in accordance with the procedures of Example 7 using monoclonal antibodies for CD47: B6H12 (FIGs. 19 and 20) and CC2C6 (FIG. 21). Control constructs included endogenous expression of wild type CD47 (WT), CD47 knockout (KO), and overexpression of wild type CD47 (hCD47 T2A GFP).
  • FIG. 19 illustrates measured mean fluorescence intensity (MFI) of a B6H12 monoclonal antibody specific for human CD47.
  • FIG. 20 illustrates MFI of B6H12 except that each value has been normalized relative to the endogenously expressed wild type value.
  • FIG. 21 illustrates the MFI of another CD47-specific monoclonal antibody, CC2C6, again with each value normalized relative to endogenous expression.
  • constructs for which surface expression could be measured showed surface expression greater than that of endogenously expressed.
  • FIG. 22 illustrates protection from macrophage phagocytosis (macrophage cytotoxicity) conferred by CD47 and Construct 43.
  • CD47 knockout cells CD47
  • cells expressing Construct 43 or overexpressing WT CD47 CD47 T2A GFP
  • WT CD47 CD47 T2A GFP
  • FIGs. 8 and 16-18 illustrate the protection fromNK-cell cytotoxicity conferred by the tested constructs. As illustrated in the Figures, each of the constructs provided protection from NK-cell killing, showing reduced NK-cell cytotoxicity relative to endogenous expression of WT CD47. Additionally, in certain donors, Construct 3 (SEQ ID NO: 640) showed protection from NK-cell killing enhanced beyond even that of overexpression of WT CD47 (FIG. 8).
  • FIGs. 19-21 illustrate the measured surface expression for the constructs. Surface expression was measured in accordance with the procedures of Example 7 using monoclonal antibodies for CD47: B6H12 (FIGs. 19 and 20) and CC2C6 (FIG. 21). Control constructs included endogenous expression of wild type CD47 (WT), CD47 knockout (KO), and overexpression of wild type CD47 (hCD47 T2A GFP).
  • FIG. 19 illustrates measured mean fluorescence intensity (MFI) of aB6H12 monoclonal antibody specific for human CD47.
  • FIG. 20 illustrates MFI of B6H12 except that each value has been normalized relative to the endogenously expressed wild type value.
  • FIG. 21 illustrates the MFI of another CD47-specific monoclonal antibody, CC2C6, again with each value normalized relative to endogenous expression.
  • constructs for which surface expression could be measured showed surface expression greater than that of endogenously expressed.

Abstract

La présente divulgation concerne des polypeptides comprenant une séquence de liaison de protéine alpha régulatrice de signal humaine (SIRPα), le polypeptide déclenchant une réponse immunitaire sensiblement similaire ou réduite lorsqu'il est exprimé sur une surface d'une cellule par comparaison avec un polypeptide comprenant une séquence CD47 de type sauvage (WT). Les polypeptides selon la divulgation peuvent afficher des ensembles conformationnels modulés par rapport à.
PCT/US2023/071272 2022-07-29 2023-07-28 Compositions et procédés de non-immunogénicité WO2024026495A1 (fr)

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Citations (5)

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WO2002000677A1 (fr) * 2000-06-07 2002-01-03 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps
WO2003054152A2 (fr) * 2001-12-10 2003-07-03 Nuvelo, Inc. Nouveaux acides nucleiques et polypeptides
WO2018132783A1 (fr) * 2017-01-13 2018-07-19 The Regents Of The University Of California Cellules pluripotentes immunologiquement modifiées
US10968426B2 (en) * 2015-05-08 2021-04-06 President And Fellows Of Harvard College Universal donor stem cells and related methods
WO2022036150A1 (fr) * 2020-08-13 2022-02-17 Sana Biotechnology, Inc. Méthodes de traitement de patients sensibilisés avec des cellules hypo-immunogènes, ainsi que méthodes et compositions associés

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000677A1 (fr) * 2000-06-07 2002-01-03 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps
WO2003054152A2 (fr) * 2001-12-10 2003-07-03 Nuvelo, Inc. Nouveaux acides nucleiques et polypeptides
US10968426B2 (en) * 2015-05-08 2021-04-06 President And Fellows Of Harvard College Universal donor stem cells and related methods
WO2018132783A1 (fr) * 2017-01-13 2018-07-19 The Regents Of The University Of California Cellules pluripotentes immunologiquement modifiées
WO2022036150A1 (fr) * 2020-08-13 2022-02-17 Sana Biotechnology, Inc. Méthodes de traitement de patients sensibilisés avec des cellules hypo-immunogènes, ainsi que méthodes et compositions associés

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Title
BARCLAY, A. N. ET AL.: "The Interaction Between Signal Regulatory Protein Alpha (SIRPalpha) and CD 47: Structure, Function, and Therapeutic Target", ANNUAL REVIEW OF IMMUNOLOGY, vol. 32, no. 1, 2014, pages 25 - 50, XP055166307, DOI: 10.1146/annurev-immunol-032713-120142 *
DATABASE PROTEIN ANONYMOUS : "leukocyte surface antigen CD47 [Carlito syrichta]", XP093141848, retrieved from NCBI *
DATABASE PROTEIN ANONYMOUS : "leukocyte surface antigen CD47 isoform X7 [Aotus nancymaae]", XP093141847, retrieved from NCBI *
DATABASE UNIPROTKB ANONYMOUS : "A0A2I2Z240 · A0A2I2Z240_GORGO", XP093136195, retrieved from UNIPROT *

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