Modified stem cell memory t cells, methods of making and methods of using same

Abstract

The disclosure provides a method of producing modified stem memory T cells (e.g. CAR-T cells) for administration to a subject as, for example an adoptive cell therapy.

What is claimed is: 
     
         1 - 39 . (canceled) 
     
     
         40 . A method of producing a modified stem memory T cell (T SCM ) or a modified T SCM -like cell, comprising:
 (a) introducing into a primary human T cell a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same to produce a modified T-cell, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and   (b) contacting the modified T-cell and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce an activated modified T-cell,   wherein the activated modified T-cell expresses one or more cell-surface marker(s) of a stem memory T cell (T SCM ) or a T SCM -like cell,   thereby producing a modified stem memory T cell (T SCM ) or a modified T SCM -like cell.   
     
     
         41 . A method of producing a plurality of modified stem memory T cells (T SCM ) or modified T SCM -like cells, comprising:
 (a) introducing into a plurality of primary human T cells a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same to produce a plurality of modified T-cells, wherein the antigen receptor or the therapeutic protein is not contained in a transposon, and   (b) contacting the plurality of modified T-cells and a T-cell activator composition comprising one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex and an activation supplement to produce a plurality of activated modified T-cells,   wherein at least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the plurality of activated modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (T SCM ) or a T SCM -like cell,   thereby producing a plurality of modified stem memory T cells (T SCM ) or modified T SCM -like cells.   
     
     
         42 - 50 . (canceled) 
     
     
         51 . The method of  claim 41 , wherein a nucleic acid vector comprises the antigen receptor or the therapeutic protein. 
     
     
         52 - 53 . (canceled) 
     
     
         54 . The method of  claim 41 , wherein the introducing step comprises a homologous recombination. 
     
     
         55 . The method of  claim 54 , wherein the homologous recombination comprises contacting the composition comprising the antigen receptor or the therapeutic protein, a genomic editing construct, and a genomic sequence of at least one primary human T cell of the plurality of primary human T cells. 
     
     
         56 . The method of  claim 55 , wherein a vector comprises the antigen receptor or the therapeutic protein. 
     
     
         57 . The method of  claim 56 , wherein the vector is an adeno-associated vector (AAV). 
     
     
         58 . The method of  claim 55 , wherein the genomic editing construct comprises a guide RNA and a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) DNA endonuclease. 
     
     
         59 . The method of  claim 58 , wherein the genomic editing construct comprises a DNA binding domain and a type IIS endonuclease. 
     
     
         60 - 61 . (canceled) 
     
     
         62 . The method of  claim 55 , wherein the genomic editing construct comprises a sequence derived from a Cas9 endonuclease. 
     
     
         63 . The method of  claim 62 , wherein the sequence derived from a Cas9 endonuclease is the DNA binding domain. 
     
     
         64 . The method of  claim 62 , wherein the sequence derived from a Cas9 endonuclease encodes an inactive Cas9. 
     
     
         65 - 68 . (canceled) 
     
     
         69 . The method of  claim 55 , wherein the genomic editing construct comprises a sequence derived from a transcription activator-like effector nuclease (TALEN). 
     
     
         70 - 71 . (canceled) 
     
     
         72 . The method of  claim 55 , wherein the genomic editing construct comprises a sequence derived from a zinc-finger nuclease (ZFN). 
     
     
         73 - 75 . (canceled) 
     
     
         76 . The method of  claim 55 , wherein genomic editing construct targets a safe harbor site on a human chromosome. 
     
     
         77 . The method of  claim 76 , wherein the chromosome is in vivo, in situ, ex vivo or in vitro. 
     
     
         78 . (canceled) 
     
     
         79 . The method of  claim 55 , wherein genomic editing construct targets a sequence encoding a component of an endogenous T-cell receptor or a sequence encoding a component of an endogenous major histocompatibility complex (MHC) on a human chromosome. 
     
     
         80 . The method of  claim 41 , wherein the antigen receptor is a T-cell receptor. 
     
     
         81 - 84 . (canceled) 
     
     
         85 . The method of  claim 41 , wherein the antigen receptor is a Chimeric Antigen Receptor (CAR). 
     
     
         86 - 90 . (canceled) 
     
     
         91 . The method of  claim 41 , wherein the therapeutic protein is a secreted or secretable protein. 
     
     
         92 - 94 . (canceled) 
     
     
         95 . The method of  claim 41 , wherein the T-cell activator composition of (b) further comprises an anti-human CD2 monospecific tetrameric antibody complex. 
     
     
         96 . The method of  claim 41 , further comprising the step of:
 (c) contacting the activated modified T-cell and a T-cell expansion composition comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to produce a plurality of expanded modified T-cells,   wherein at least 2% of the plurality of expanded modified T-cells expresses one or more cell-surface marker(s) of a stem memory T cell (T SCM ) or a T SCM -like cell.   
     
     
         97 . The method of  claim 96 , wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of expanded modified T-cells expresses cell-surface marker(s) of a stem memory T cell (T SCM ) or a T SCM -like cell. 
     
     
         98 - 110 . (canceled) 
     
     
         111 . The method of  claim 41 , wherein the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. 
     
     
         112 . The method of  claim 111 , wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints. 
     
     
         113 . The method of  claim 111 , wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg/kg, palmitic acid at a concentration of about 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and a sterol at a concentration of about 1 mg/kg. 
     
     
         114 . The method of  claim 111 , wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of the endpoints. 
     
     
         115 . The method of  claim 111 , wherein the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at a concentration of about 7.5 μmol/kg and a sterol at a concentration of about 2.5 μmol/kg. 
     
     
         116 - 270 . (canceled) 
     
     
         271 . A composition comprising a modified-T SCM  or a modified T SCM -like cell produced by the method of  claim 41 . 
     
     
         272 - 304 . (canceled)