Chimeric engulfment receptor molecules

Abstract

The present disclosure relates to chimeric engulfment receptor molecules, host cells modified to include the phagocytic engulfment molecules, and methods of making and using such receptor molecules and modified cells.

1 . A chimeric engulfment receptor (CER) comprising a single chain chimeric protein, the single chain chimeric protein comprising:
 an extracellular domain comprising a binding domain that binds to phosphatidylserine (PtdSer);   an engulfment signaling domain; and   a transmembrane domain positioned between and connecting the extracellular domain and the engulfment signaling domain.   
     
     
         2 . The CER of  claim 1 , wherein the binding domain comprises an scFv specific to PtdSer, or a PtdSer binding domain from Tim1, Tim4, Tim3, stabilin-2, receptor for advanced glycation endproducts (RAGE), brain-specific angiogenesis inhibitor 1 (BAI1), Milk Fat Globule-EGF Factor 8 Protein (MFG-E8), Growth Arrest Specific 6 (GAS6), protein S, protein C, Factor II, Factor VII, Factor IX, Factor X, Beta 2-glycoprotein I, α5β3 integrin and other integrins, CR3 complement receptor, CR4 complement receptor, CD14, CD93, annexin V, phosphatidylserine receptor (PSr), prothrombin, or a scavenger receptor. 
     
     
         3 . (canceled) 
     
     
         4 . The CER of  claim 1 , wherein the extracellular domain further comprises an extracellular spacer domain positioned between the binding domain and transmembrane domain. 
     
     
         5 . The CER of  claim 4 , wherein the extracellular spacer domain comprises an immunoglobulin hinge region, a hinge region of a type 1 membrane proteins, a stalk region of a type II C-lectin, an immunoglobulin constant domain, or a fragment thereof. 
     
     
         6 .- 7 . (canceled) 
     
     
         8 . The CER of  claim 1 , wherein the transmembrane domain comprises a Tim1, Tim4, Tim3, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, CD8a, CD28, MERTK, Axl, Tyro3, BAI1, CD4, DAP12, or MRC1 transmembrane domain. 
     
     
         9 .- 11 . (canceled) 
     
     
         12 . The CER of claim  11 , wherein the engulfment signaling domain is-comprises an ItgB5, MERTK, Tyro3, Axl, BAI1, ELMO, MRC1, PI3K Traf6, Syk, MyD88, Zap70 FcγRI, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, or CD79b signaling domain. 
     
     
         13 .- 19 . (canceled) 
     
     
         20 . The CER of  claim 1 , wherein the engulfment signaling domain comprises a primary engulfment signaling domain and a secondary engulfment signaling domain. 
     
     
         21 .- 24 . (canceled) 
     
     
         25 . The CER of  claim 20 , wherein the primary engulfment signaling domain and secondary engulfment signaling domain are the same or different. 
     
     
         26 . The CER of  claim 20 , wherein the primary engulfment signaling domain comprises an ItgB5, MERTK, Tyro3, Axl, BAI1, ELMO, MRC1, PI3K, Traf6, Syk, MyD88, Zap70, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, or CD79b signaling domain. 
     
     
         27 . (canceled) 
     
     
         28 . The CER of  claim 20 , wherein the secondary engulfment signaling domain comprises an ItgB5, MERTK, Tyro3, Axl, BAI1, ELMO, MRC1, PI3K, Traf6, Syk, MyD88, Zap70, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, or CD79b signaling domain. 
     
     
         29 .- 43 . (canceled) 
     
     
         44 . A chimeric engulfment receptor (CER) comprising a single chain chimeric protein, the single chain chimeric protein comprising:
 an extracellular domain comprising a binding domain that binds to a pro-engulfment marker or target antigen;   an engulfment signaling domain comprising a PI3K, Traf6, Syk, MyD88, Zap70, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R DAP12, NFAM1, or CD79b signaling domain; and   a transmembrane domain positioned between and connecting the extracellular domain and the Engulfment signaling domain.   
     
     
         45 . The CER of  claim 44 , wherein:
 (i) the binding domain binds to a pro-engulfment marker selected from phosphatidylserine (PtdSer), ICAM-3, oxidized low density lipoprotein, calreticulin, annexin I, complement C1q, and thrombospondin;   (ii) the binding domain comprises a scFv specific for a tumor antigen; or   (iii) the binding domain comprises a scFv specific for an antigen of a microbe that is capable of establishing a persistent infection in a host.   
     
     
         46 . The CER of  claim 45 , wherein:
 (i) the binding domain binds to PtdSer and comprises an scFv specific to PtdSer, or comprises a PtdSer binding domain from Tim1, Tim4, Tim3, stabilin-2, receptor for advanced glycation endproducts (RAGE), brain-specific angiogenesis inhibitor 1 (BAI1), Milk Fat Globule-EGF Factor 8 Protein (MFG-E8), Growth Arrest Specific 6 (GAS6), protein S, protein C, Factor II, Factor VII, Factor IX, Factor X, Beta 2-glycoprotein I, α5β3 integrin and other integrins, CR3 complement receptor, CR4 complement receptor, CD14, CD93, annexin V, phosphatidylserine receptor (PSr), prothrombin, or a scavenger receptor; or   (ii) the binding domain comprises a scFv specific for a tumor antigen selected from CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor α CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13R1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor B, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD2, and GD2.   
     
     
         47 .- 49 . (canceled) 
     
     
         50 . The CER of  claim 44 , wherein the extracellular domain further comprises an extracellular spacer domain positioned between the binding domain and transmembrane domain. 
     
     
         51 . The CER of  claim 50 , wherein the extracellular spacer domain comprises an immunoglobulin hinge region, an extracellular region of a type 1 membrane proteins, a stalk region of a type II C-lectin, an immunoglobulin constant domain, or a fragment thereof. 
     
     
         52 .- 53 . (canceled) 
     
     
         54 . The CER of  claim 44 , wherein the transmembrane domain comprises a Tim1, Tim4, Tim3, FcR, CD8a, CD28, MERTK, Axl, Tyro3, BAI1, CD4, MRC1, or DAP12 transmembrane domain. 
     
     
         55 .- 60 . (canceled) 
     
     
         61 . A chimeric engulfment receptor (CER) comprising a single chain chimeric protein, the single chain chimeric protein comprising:
 an extracellular domain comprising a binding domain that binds to a pro-engulfment marker or target antigen;   an engulfment signaling domain comprising a primary engulfment signaling domain and a secondary engulfment signaling domain; and   a transmembrane domain positioned between and connecting the extracellular domain and the primary engulfment signaling domain.   
     
     
         62 . The CER of  claim 61 , wherein:
 (i) the binding domain binds to a pro-engulfment marker selected from phosphatidylserine (PtdSer), ICAM-3, oxidized low density lipoprotein, calreticulin, annexin I, complement C1q, and thrombospondin;   (ii) the binding domain comprises a scFv specific for a tumor antigen; or   (iii) the binding domain comprises a scFv specific for a microbial antigen.   
     
     
         63 . The CER of  claim 62 , wherein:
 (i) the binding domain that binds to PtdSer and comprises a scFv specific to PtdSer, or comprises a PtdSer binding domain from Tim1, Tim4, Tim3, stabilin-2, receptor for advanced glycation endproducts (RAGE), brain-specific angiogenesis inhibitor 1 (BAI1), Milk Fat Globule-EGF Factor 8 Protein (MFG-E8), Growth Arrest Specific 6 (GAS6), protein S, protein C, Factor II, Factor VII, Factor IX, Factor X, Beta 2-glycoprotein I, α5β3 integrin and other integrins, CR3 complement receptor, CR4 complement receptor, CD14, CD93, annexin V, phosphatidylserine receptor (PSr), prothrombin, or a scavenger receptor; or   (ii) the binding domain comprises a scFv specific for a tumor antigen selected from CD138, CD38, CD33, CD123, CD72, Cd79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor α, CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13Rα2, IL-13Rα1, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor β, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD2, and GD2.   
     
     
         64 .- 66 . (canceled) 
     
     
         67 . The CER of  claim 61 , wherein the extracellular domain further comprises an extracellular spacer domain positioned between the binding domain and transmembrane domain. 
     
     
         68 . The CER of  claim 67 , wherein the extracellular spacer domain comprises an immunoglobulin hinge region, an extracellular region of a type 1 membrane proteins, a stalk region of a type II C-lectin, an immunoglobulin constant domain, or a fragment thereof. 
     
     
         69 .- 70 . (canceled) 
     
     
         71 . The CER of  claim 61 , wherein the transmembrane domain comprises a Tim1, Tim4, Tim3, FcR, CD8a, CD28, MERTK, Axl, Tyro3, BAI1, CD4, MRC1, or DAP12 transmembrane domain. 
     
     
         72 .- 77 . (canceled) 
     
     
         78 . The CER of  claim 61 , wherein the primary engulfment signaling domain and secondary engulfment signaling domain are the same or different. 
     
     
         79 . The CER of  claim 61 , wherein the primary engulfment signaling domain comprises an ItgB5, MERTK, Tyro3, Axl, BAI1, ELMO, MRC1, PI3K, Traf6, Syk, MyD88, Zap70, FcαR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R DAP12, NFAM1, or CD79b signaling domain. 
     
     
         80 . (canceled) 
     
     
         81 . The CER of any one of  claim 61 , wherein the secondary engulfment signaling domain comprises an ItgB5, MERTK, Tyro3, Axl, BAI1, ELMO, MRC1, PI3K, Traf6, Syk, MyD88, Zap70, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, or CD79b signaling domain. 
     
     
         82 . (canceled) 
     
     
         83 . A chimeric engulfment receptor (CER) comprising a single chain chimeric protein, the single chain chimeric protein comprising:
 an extracellular domain comprising a scFv that binds to a pro-engulfment marker or target antigen;   an engulfment signaling domain; and   a transmembrane domain positioned between and connecting the extracellular domain and the engulfment signaling domain,   wherein the transmembrane domain and engulfment signaling domain are each derived from a different molecule.   
     
     
         84 . The CER of  claim 83 , wherein:
 (i) the scFv binds to a pro-engulfment marker selected from phosphatidylserine (PtdSer), ICAM-3, oxidized low density lipoprotein, calreticulin, annexin I, complement C1q, and thrombospondin,   (ii) the scFv binds to a tumor antigen selected from CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor α, CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor β, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, o-acetyl GD2, and GD2; or   (iii) the scFv binds to a microbial antigen.   
     
     
         85 .- 87 . (canceled) 
     
     
         88 . The CER of  claim 83 , wherein
 the extracellular domain further comprises an extracellular spacer domain positioned between the binding domain and transmembrane domain.   
     
     
         89 . The CER of  claim 88 , wherein the extracellular spacer domain comprises an immunoglobulin hinge region, an extracellular region of a type 1 membrane protein, a stalk region of a type II C-lectin, an immunoglobulin constant domain, or a fragment thereof. 
     
     
         90 .- 91 . (canceled) 
     
     
         92 . The CER of  claim 83 , wherein the transmembrane domain comprises a Tim1, Tim4, Tim3, FcR, CD8a, CD28, MERTK, Axl, Tyro3, BAI1, CD4, MRC1, or DAP12 transmembrane domain. 
     
     
         93 .- 95 . (canceled) 
     
     
         96 . The CER of  claim 83 , wherein the engulfment signaling domain is-comprises an ItgB5, MERTK, Tyro3, Axl, BAI1, ELMO, MRC1, PI3K, Traf6, Syk, MyD88, Zap70 FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, or CD79b signaling domain. 
     
     
         97 .- 105 . (canceled) 
     
     
         106 . A nucleic acid molecule encoding a CER according to  claim 1 . 
     
     
         107 .- 112 . (canceled) 
     
     
         113 . A vector comprising a nucleic acid molecule according to  claim 106 . 
     
     
         114 .- 116 . (canceled) 
     
     
         117 . A genetically modified cell comprising a CER according to  claim 1 . 
     
     
         118 .- 123 . (canceled) 
     
     
         124 . The genetically modified cell according to  claim 117 , wherein the genetically modified cell is a T cell, including CD4 + , CD8 + , naïve (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO−), central memory (CD45RO + , CD62L + , CD8 + ), effector memory (CD45RA+, CD45RO−, CCR7−, CD62L−, CD27−), virus-specific, mucosal-associated invariant, γδ (gd), natural killer, and tissue resident T cells, a natural killer cell, a B cell, or a lymphoid precursor cell, including common lymphocyte precursor cells, an antigen presenting cell, including dendritic cells, a Langerhans cell, a myeloid precursor cell, or a mature myeloid cell. 
     
     
         125 .- 126 . (canceled) 
     
     
         127 . The modified host cell of  claim 117 , wherein the modified host cell exhibits:
 (i) engulfment activity when the extracellular domain of the CER binds to PtdSer;   (ii) phagocytic activity when the extracellular domain of the CER binds to PtdSer; or   (iii) a phagocytic index of at least 20 towards a target cell.   
     
     
         128 .- 132 . (canceled) 
     
     
         133 . A composition comprising a plurality of genetically modified cells according to  claim 117 , and a pharmaceutically acceptable excipient. 
     
     
         134 . A method of treating a subject with cancer or treating a subject having a disease, disorder, or undesired condition associated with an overexpression of a tumor antigen, the method comprising administering to the subject an effective amount of composition of  claim 133 . 
     
     
         135 .- 151 . (canceled) 
     
     
         152 . A nucleic acid molecule encoding a CER according to  claim 44 . 
     
     
         153 . A vector comprising a nucleic acid molecule according to  claim 152 . 
     
     
         154 . A genetically modified cell comprising a CER according to  claim 44   
     
     
         155 . A composition comprising a plurality of genetically modified cells according to  claim 154  and a pharmaceutically acceptable excipient. 
     
     
         156 . A method of treating a subject with cancer or treating a subject having a disease, disorder, or undesired condition associated with an overexpression of a tumor antigen, the method comprising administering to the subject an effective amount of a composition of  claim 155 . 
     
     
         157 . A method of treating a subject having an autoimmune disease, disorder, or undesired condition, the method comprising administering to the subject an effective amount of a composition of  claim 155 . 
     
     
         158 . A method of treating or preventing an infectious disease in a subject, the method comprising administering to the subject an effective amount of a composition of  claim 155 . 
     
     
         159 . A nucleic acid molecule encoding a CER according to  claim 61 . 
     
     
         160 . A vector comprising a nucleic acid molecule according to  claim 159 . 
     
     
         161 . A genetically modified cell comprising a CER according to  claim 61 . 
     
     
         162 . A composition comprising a plurality of genetically modified cells according to  claim 161  and a pharmaceutically acceptable excipient. 
     
     
         163 . A method of treating a subject with cancer or treating a subject having a disease, disorder, or undesired condition associated with an overexpression of a tumor antigen, the method comprising administering to the subject an effective amount of a composition of  claim 162 . 
     
     
         164 . A method of treating a subject having an autoimmune disease, disorder, or undesired condition, the method comprising administering to the subject an effective amount of a composition of  claim 162 . 
     
     
         165 . A method of treating or preventing an infectious disease in a subject, the method comprising administering to the subject an effective amount of a composition of  claim 162 . 
     
     
         166 . A nucleic acid molecule encoding a CER according to  claim 83 . 
     
     
         167 . A vector comprising a nucleic acid molecule according to  claim 166 . 
     
     
         168 . A genetically modified cell comprising a CER according to  claim 83 . 
     
     
         169 . A composition comprising a plurality of genetically modified cells according to  claim 168  and a pharmaceutically acceptable excipient. 
     
     
         170 . A method of treating a subject with cancer or treating a subject having a disease, disorder, or undesired condition associated with an overexpression of a tumor antigen, the method comprising administering to the subject an effective amount of a composition of  claim 169 . 
     
     
         171 . A method of treating a subject having an autoimmune disease, disorder, or undesired condition, the method comprising administering to the subject an effective amount of a composition of  claim 169 . 
     
     
         172 . A method of treating or preventing an infectious disease in a subject, the method comprising administering to the subject an effective amount of a composition of  claim 169 .