Devices and methods for isolating tumor infiltrating lymphocytes and uses thereof

Abstract

The present invention provides methods for isolating and cryopreserving tumor infiltrating lymphocytes (TILs) and producing therapeutic populations of TILs, including methods via use of a kit and a semi-automatic device for aseptic disaggregation, enrichment, and cryopreservation of a resected tumor prior to expansion of the TIL population. The present invention also provides methods for expansion, and/or stabilization of TILs, for instance UTILs, compositions involving the same and methods of treatment involving the same.

1 - 30 . (canceled) 
     
     
         31 . A method for preparing a therapeutic population of tumor infiltrating lymphocytes (TILs) comprising:
 (a) aseptically disaggregating a tumor resected from a subject thereby preparing a disaggregated tumor product, wherein the disaggregation comprises repeated physical pressure applied 120 to 360 times per minute at up to 6 N/cm 2  in the presence of a media enzyme solution, wherein the tumor is sufficiently disaggregated into a cell suspension so that the disaggregated tumor product can be cryopreserved;   (b) within 24 hours of preparing the disaggregated tumor product, cooling the disaggregated tumor product to a suitable cryopreservation temperature to prepare a cryopreserved disaggregated tumor product;   (c) storing the cryopreserved disaggregated tumor product in a frozen state;   (d) thawing the cryopreserved disaggregated tumor product;   (e) performing a first expansion by culturing the cryopreserved disaggregated tumor product in a cell culture medium comprising IL-2 to produce a first population of TILs;   (f) performing a second expansion by culturing the first population of TILs in a cell culture medium with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a second population of TILs; and   (g) cryopreserving the second population of TILs to prepare a cryopreserved therapeutic population of TILs;   wherein steps (a), (b), (c), (d), (e), (f) and (g) are performed in a closed system.   
     
     
         32 . The method of  claim 31 , wherein the resected tumor is not fragmented prior to disaggregation. 
     
     
         33 . The method of  claim 31 , wherein the media enzyme solution comprises DNase and Collagenase. 
     
     
         34 . The method of  claim 31 , wherein the disaggregated tumor product is filtered by an in-line filter prior to the cooling the disaggregated tumor product. 
     
     
         35 . The method of  claim 34 , wherein the filtered disaggregated tumor product constituents have an average size of less than 200 μm. 
     
     
         36 . The method of  claim 34 , wherein the filtered disaggregated tumor product constituents have an average size of less than 170 μm. 
     
     
         37 . The method of  claim 31 , wherein the performing a second expansion by culturing the first population of TILs is performed on about 1 to about 20 million TILs from the first population. 
     
     
         38 . The method of  claim 37 , wherein the about 1 to about 20 million TILs from the first population comprises a mixture of resident and emergent T cells. 
     
     
         39 . The method of  claim 31 , wherein step (b) comprises cooling the disaggregated tumor product directly to the cryopreservation temperature. 
     
     
         40 . The method of  claim 31 , wherein the disaggregation period is 90 min or less. 
     
     
         41 . The method of  claim 31 , which comprises cooling the disaggregated tumor product in a controlled temperature device programmed to reduce temperature at a controlled rate. 
     
     
         42 . The method of  claim 41 , wherein the cryopreservation temperature is −80° C.±10° C. and the device is programmed to reduce temperature by about 1° C./min or about 1.5° C./min or about 2° C./min. 
     
     
         43 . The method of  claim 31 , wherein the TILs comprise UTILs or wherein the TILs comprise MTILs. 
     
     
         44 . The method of  claim 31 , wherein the second expansion comprises a rapid expansion. 
     
     
         45 . The method of  claim 31 , wherein the first expansion is performed over about two weeks; and the second expansion is performed over about two weeks. 
     
     
         46 . The method of  claim 31 , wherein the first expansion is performed over about two weeks; and the second expansion is performed over about 7 days. 
     
     
         47 . The method of  claim 31 , wherein the media in the culturing in the first expansion and/or in the second expansion further comprises IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof. 
     
     
         48 . The method of  claim 31 , wherein the APCs are artificial APCs. 
     
     
         49 . The method of  claim 31 , further comprising:
 after the second expansion and before the cryopreserving the second population of TILs, identifying in the second population of TILs, the presence of T-cells expressing a combination of markers, the combination comprising two or more of:   CD107a;   CD137;   TNF-α; or,   IFN-γ.   
     
     
         50 . The method of  claim 49 , wherein the combination comprises:
 CD107a and CD137, or   CD107a and TNF-α, or   CD107a and IFN-γ, or   CD137 and TNF-α, or   CD137 and IFN-γ, or   TNF-α and IFN-γ.   
     
     
         51 . The method of  claim 49 , wherein the combination comprises three or more of:
 CD107a;   CD137;   TNF-α; and,   IFN-γ.   
     
     
         52 . The method of  claim 51 , wherein the combination comprises:
 CD107a, CD137 and TNF-α, or   CD107a, CD137 and IFN-γ, or   CD107a, TNF-α and IFN-γ, or   CD137, TNF-α and IFN-γ.   
     
     
         53 . The method of  claim 49 , wherein the combination comprises each of CD107a, CD137, TNF-α and IFN-γ. 
     
     
         54 . The method of  claim 31 , further comprising:
 after the second expansion and before the cryopreserving the second population of TILs, identifying in the second population of TILs, the presence of CD2+ T-cells expressing a combination of markers, the combination comprising two or more of:   a T-cell expressing CD107a;   a T-cell expressing CD137;   a T-cell expressing TNF-α; or,   a T-cell expressing IFN-γ.   
     
     
         55 . The method of  claim 54 , wherein the combination comprises:
 CD107a and CD137, or   CD107a and TNF-α, or   CD107a and IFN-γ, or   CD137 and TNF-α, or   CD137 and IFN-γ, or   TNF-α and IFN-γ.   
     
     
         56 . The method of  claim 54 , wherein the combination comprises three or more of:
 CD107a;   CD137;   TNF-α; and,   IFN-γ.   
     
     
         57 . The method of  claim 56 , wherein the combination comprises:
 CD107a, CD137 and TNF-α, or   CD107a, CD137 and IFN-γ, or   CD107a, TNF-α and IFN-γ, or   CD137, TNF-α and IFN-γ.   
     
     
         58 . The method of  claim 54 , wherein the combination comprises each of CD107a, CD137, TNF-α and IFN-γ. 
     
     
         59 . A method for preparing a therapeutic population of tumor infiltrating lymphocytes (TIL) comprising:
 (a) aseptically disaggregating a tumor resected from a subject thereby preparing a disaggregated tumor product, wherein the disaggregation comprises repeated physical pressure applied 120 to 360 times per minute at up to 6 N/cm 2  in the presence of a media enzyme solution at a temperature up to 35° C., wherein the tumor is sufficiently disaggregated into a cell suspension so that the disaggregated tumor product can be cryopreserved;   (b) within 24 hours of preparing the disaggregated tumor product, cooling the disaggregated tumor product to a suitable cryopreservation temperature to prepare a cryopreserved disaggregated tumor product;   (c) storing the cryopreserved disaggregated tumor product in a frozen state;   (d) thawing the cryopreserved disaggregated tumor product;   (e) performing a first expansion by culturing the cryopreserved disaggregated tumor product in a cell culture medium comprising IL-2 to produce a first population of TILs, wherein the first expansion is performed over about two weeks;   (f) performing a second expansion by culturing the first population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), wherein the second expansion is performed over about two weeks, to produce a second population of TILs;   (g) removing a sample from the second population of TILs;   (h) identifying in the sample from the second population of TILs, the presence of CD2+ T-cells expressing a combination of markers, the combination comprising two or more of:   a T-cell expressing CD107a;   a T-cell expressing CD137;   a T-cell expressing TNF-α; or,   a T-cell expressing IFN-γ.   (i) cryopreserving the second population of TILs to prepare a cryopreserved therapeutic population of TILs;   wherein the steps (a), (b), (c), (d), (e), (f), (g) and (i) are performed in a closed system.   
     
     
         60 . A therapeutic population of cryopreserved tumor infiltrating lymphocytes (TILs) obtained by the method of  claim 31 .