Tumor infiltrating lymphocytes and methods of therapy

Abstract

Genetically modified compositions, such as non-viral vectors and tumor infiltrating lymphocytes, for the treatment of gastrointestinal cancer are disclosed. Disclosed are methods of utilizing a CRISPR system to generate genetically modified compositions. Also disclosed are the methods of making and using the genetically modified compositions for the treatment of gastrointestinal cancer.

1 .- 193 . (canceled) 
     
     
         194 . A method of treating a cancer in a subject in need thereof, the method comprising:
 obtaining a population of human immune cells;   obtaining from said subject a population of cancer cells from said cancer;   exomic sequencing said cancer cells to identify mutations uniquely present in said cancer cells as compared to normal cells of said subject;   screening said human immune cells in vitro for reactivity to said mutations, thereby identifying cancer reactive immune cells;   genetically modifying said cancer reactive immune cells by disrupting a target sequence of an endogenous cytokine inducible SH2-containing protein gene that suppresses expression of a protein encoded by said cytokine inducible SH2-containing protein gene, thereby obtaining a population of genetically modified cancer reactive immune cells;   administering to said subject
 a preparative regime that comprises administration of at least one immunosuppressant to said subject in an amount sufficient to reduce an immune response in said subject; and 
 a pharmaceutical composition that comprises said population of genetically modified cancer reactive immune cells. 
   
     
     
         195 . The method of  claim 194 , further comprising administering an antibiotic to the subject. 
     
     
         196 . The method of  claim 194 , further comprising administering an antifungal agent to the subject. 
     
     
         197 . The method of  claim 194 , wherein said preparative regime comprises administration of said immunosuppressant from about 14 days to about 24 hours before administering said genetically modified cancer reactive immune cells. 
     
     
         198 . The method of  claim 194 , wherein said disrupting is by a system selected from the group consisting of CRISPR, Zinc Finger, TALEN, and any combination thereof. 
     
     
         199 . The method of  claim 198 , wherein said system is a CRISPR system. 
     
     
         200 . The method of  claim 199 , wherein said CRISPR system comprises an endonuclease selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, and Cas9HiFi. 
     
     
         201 . The method of  claim 200 , wherein said endonuclease is Cas9. 
     
     
         202 . The method of  claim 194 , wherein said disruption is within about 5 base pairs of a protospacer adjacent motif (PAM). 
     
     
         203 . The method of  claim 194 , wherein said pharmaceutical composition comprises genetically modified cancer reactive immune cells that are cryopreserved at a freeze density from about 7.0×10 7  cells/mL to about 2.0×10 8  cells/mL. 
     
     
         204 . The method of  claim 194 , wherein said genetically modified cancer reactive immune cells undergo pre-infusion testing prior to said administering, and wherein said pre-infusion testing comprises phenotypic testing, potency testing, endotoxin testing, viability testing, and tumor cell testing. 
     
     
         205 . The method of  claim 204 , wherein said phenotypic testing comprises detecting a presence of CD3 on said genetically modified cancer reactive immune cells. 
     
     
         206 . The method of  claim 204 , wherein said potency testing comprises detecting a level of IFNγ upon anti-CD3 stimulation of said genetically modified cancer reactive immune cells. 
     
     
         207 . The method of  claim 204 , wherein said endotoxin testing comprises performing a limulus assay. 
     
     
         208 . The method of  claim 204 , wherein said tumor cell testing comprises a cytopathology assay. 
     
     
         209 . The method of  claim 204 , wherein said genetically modified cancer reactive immune cells are administered to said subject when at least 70% of the cell population is determined to be viable cells in said viability testing. 
     
     
         210 . The method of  claim 204 , wherein said genetically modified cancer reactive immune cells are administered when said pre-infusion testing is at least about 80% CD3 positive for said phenotypic testing. 
     
     
         211 . The method of  claim 204 , wherein said genetically modified cancer reactive immune cells are administered when said pre-infusion testing is at least about 200 pg/mL per 10 5  cells of IFNγ upon anti-CD3 stimulation of said genetically modified cancer reactive immune cells in said potency testing. 
     
     
         212 . The method of  claim 204 , wherein said genetically modified cancer reactive immune cells are administered when said pre-infusion testing is negative for tumor cells per at least about 200 genetically modified cancer reactive immune cells examined in said cytopathology testing.