Modified natural killer cells and natural killer cell lines having increased cytotoxicity

Abstract

NK cells and NK cell lines are modified to increase cytotoxicity, wherein the cells and compositions thereof have a use in the treatment of cancer. Production of modified NK cells and NK cell lines is via genetic modification to add mutant (variant) TRAIL ligand expression.

1 - 20 . (canceled) 
     
     
         21 . A human natural killer (NK) cell or NK cell line modified to express a TRAIL mutant, wherein the TRAIL mutant has an increased affinity for a TRAIL receptor and/or a decreased affinity for a decoy TRAIL receptor, relative to wildtype TRAIL. 
     
     
         22 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL receptor is selected from DR4 and DR5 and/or the decoy TRAIL receptor is selected from DcR1 and DcR2. 
     
     
         23 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL mutant comprises at least one amino acid substitution at a position selected from the group consisting of 131, 149, 159, 160, 189, 191, 193, 195, 199, 200, 201, 203, 204, 212, 213, 214, 215, 218, 240, 251, 261, 264, 266, 267, 269, and 270. 
     
     
         24 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL mutant comprises at least one amino acid substitution selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251D, K251E, K251Q, T261L, H264R, I266L, D267Q, D269A, D269H, and H270D. 
     
     
         25 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL mutant comprises at least two amino acid substitutions selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251D, K251E, K251Q, T261L, H264R, I266L, D267Q, D269A, D269H, and H270D. 
     
     
         26 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL mutant comprises at least three amino acid substitutions selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251D, K251E, K251Q, T261L, H264R, I266L, D267Q, D269A, D269H, and H270D. 
     
     
         27 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL mutant comprises an amino acid substitution selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251D, K251E, K251Q, T261L, H264R, I266L, D267Q, D269A, D269H, H270D, T214R/E195R, T214R/D269H, Y189A/Q193S/N199V/K201R/Y213W/S215D, Y213W/S215D, N199R/K201H, N199H/K201R, G131R/N199R/K201H, G131R/N199R/K201H/R149I/S159R/S215D, G131R/R149I/S159R/S215D, G131R/N199R/K201H/R149I/S159R/S215D, G131R/D218H, Y189Q/R191K/Q193R/H264R/I266L/D267Q, T261L/G160E, T261L/H270D, T261L/G160E/H270D, and T261L/G160E/H270D/T200H. 
     
     
         28 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL mutant comprises an amino acid substitution selected from the group consisting of D269H, E195R, T214R, D269H/E195R, T214R/E195R, T214R/D269H, N199V, Y189A/Q193S/N199V/K201R/Y213W/S215D, Y213W/S215D, D269A, and Y240A. 
     
     
         29 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL mutant comprises an amino acid substitution selected from the group consisting of G131R, G131K, R149I, R149M, R149N, R149K, S159R, Q193H, W193K, N199R, N199R/K201H, N199H/K201R, G131R/N199R/K201H, G131R/N199R/K201H, G131R/N199R/K201H/R149I/S159R/S215D, G131R/R149I/S159R/S215D, G131R/D218H, K201R, K201H, K204E, K204D, K204L, K204Y, K212R, S215E, S215H, S215K, S215D, D218H, K251D, K251E, K251Q, and Y189Q/R191K/Q193R/H264R/I266L/D267Q. 
     
     
         30 . The NK cell or NK cell line of  claim 21 , wherein the TRAIL mutant comprises an amino acid substitution selected from the group consisting of T261L, H270D, T200H, T261L/G160E, T261L/H270D, T261L/G160E/H270D, T261L/G160E/H270D/T200H, D203A, and D218A. 
     
     
         31 . The NK cell or NK cell line of  claim 21  further modified to reduce or abolish expression of a checkpoint inhibitory receptor. 
     
     
         32 . The NK cell or NK cell line of  claim 31 , wherein the checkpoint inhibitory receptor is selected from the group consisting of CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT, and TIM-3. 
     
     
         33 . The NK cell or NK cell line of  claim 21 , wherein affinity of the TRAIL mutant for the TRAIL receptor is increased by at least 10%, relative to the affinity of wildtype TRAIL for the TRAIL receptor. 
     
     
         34 . The NK cell or NK cell line of  claim 21  in combination with a proteasome inhibitor. 
     
     
         35 . The NK cell or NK cell line of  claim 34 , wherein the proteasome inhibitor is bortezomib. 
     
     
         36 . The NK cell or NK cell line of  claim 21  further modified to express a chimeric antigen receptor (CAR). 
     
     
         37 . A natural killer (NK) cell or NK cell line that is genetically modified to express a TRAIL mutant, wherein the TRAIL mutant has an increased affinity for the DR5 receptor, relative to wildtype TRAIL. 
     
     
         38 . The NK cell or NK cell line of  claim 37 , wherein affinity of the TRAIL mutant for DR5 is increased by at least 10%, relative to wildtype TRAIL. 
     
     
         39 . A natural killer (NK) cell or NK cell line that is genetically modified to express a TRAIL mutant, wherein the TRAIL mutant has an increased affinity for the DR4 receptor, relative to wildtype TRAIL. 
     
     
         40 . The NK cell or NK cell line of  claim 39 , wherein affinity of the TRAIL mutant for DR4 is increased by at least 10%, relative to wildtype TRAIL.