Engineered t cells and uses therefor

Abstract

Lipocalin muteins specific to a predetermined antigen can be transduced into a T cell to bring therapeutic benefits to patients in need. In one example, a lipocalin mutein specific to a predetermined antigen (e.g., a target differentially expressed on the surface of a tumor cell) can be transduced into a T cell membrane to serve as an antigen receptor, offering benefits over conventionally deployed antibody-derived protein moieties such as a single chain variable fragment (scFv). Benefits include a more stable structure, leading to superior target engagement, for example. Further, lipocalin muteins specific to a predetermined antigen (e.g. an immunomodulatory target such as an immune checkpoint or costimulatory molecule) can be transduced into a T cell for secretion thereby, bringing an added therapeutic benefit. Specific examples of such modified T cells and methods of making and using the same are provided herein.

1 . A modified T cell expressing and secreting a specific binding polypeptide comprising a lipocalin mutein selected from the group consisting of a mutein of human tear lipocalin (hTlc) and a mutein of human neutrophil gelatinase-associated lipocalin (hNGAL), wherein the lipocalin mutein is modified such that it is specific for a target other than the natural target of hTlc and hNGAL, respectively. 
     
     
         2 . The modified T cell of  claim 1 , wherein the modified T cell secretes the hTlc or the hNGAL mutein upon activation. 
     
     
         3 . The modified T cell of  claim 1 , wherein the lipocalin mutein is an hNGAL mutein. 
     
     
         4 . The modified T cell of  claim 3 , wherein the lipocalin mutein is an hNGAL mutein having at least 80% sequence identity to mature, wild-type hNGAL (SEQ ID NO: 30). 
     
     
         5 . The modified T cell of  claim 4 , wherein the hNGAL mutein retains a supersecondary or tertiary structure of mature, wild-type hNGAL (SEQ ID NO: 30). 
     
     
         6 . The modified T cell of  claim 4 , wherein the hNGAL mutein comprises an amino acid substitution at each of A40, Q49, L70, R72, K73, D77, W79, R81, L103, K125, S127, and Y132 relative to mature, wild-type hNGAL (SEQ ID NO: 30). 
     
     
         7 . The modified T cell of  claim 4 , wherein the hNGAL mutein comprises the amino acid sequence of mature, wild-type hNGAL (SEQ ID NO: 30) but for from 13 to 26 amino acid substitutions relative to mature, wild-type hNGAL, wherein at least A40, Q49, L70, R72, K73, D77, W79, R81, C87, L103, K125, S127, and Y132 are replaced with a different amino acid residue. 
     
     
         8 . The modified T cell of  claim 7 , wherein the hNGAL mutein further comprises at least one additional amino acid replacement at a position selected from E44, K46, D47, K50, N65, F71, P101, G102, T104, V126, Q128, R130, and K134 relative to mature, wild-type hNGAL (SEQ ID NO: 30). 
     
     
         9 . The modified T cell of  claim 1 , wherein the lipocalin mutein is an hNGAL mutein having at least 80% sequence identity to SEQ ID NO: 3. 
     
     
         10 . The modified T cell of  claim 9 , wherein the hNGAL mutein comprises an amino acid substitution at each of A40, Q49, L70, R72, K73, D77, W79, R81, L103, K125, S127, and Y132 relative to mature, wild-type hNGAL (SEQ ID NO: 30). 
     
     
         11 . The modified T cell of  claim 9 , wherein the hNGAL mutein comprises the amino acid sequence of mature, wild-type hNGAL (SEQ ID NO: 30) but for from 13 to 26 amino acid substitutions relative to mature, wild-type hNGAL, wherein at least A40, Q49, L70, R72, K73, D77, W79, R81, C87, L103, K125, S127, and Y132 are replaced with a different amino acid residue. 
     
     
         12 . The modified T cell of  claim 1 , wherein the modified T cell further comprises a chimeric antigen receptor. 
     
     
         13 . The modified T cell of  claim 12 , wherein the chimeric antigen receptor comprises: i. an extracellular ligand binding domain; ii. at least one costimulatory domain selected from a CD28 domain, a CD137 domain, an ICOS domain, an OX40 domain, a CD27 domain, and a CD40 domain; and iii. a cytoplasmic domain comprising at least one intracellular signaling domain, wherein the at least one intracellular signaling domain comprises a CD3zeta signaling domain. 
     
     
         14 . A modified T cell comprising: (a) single-domain, monomeric polypeptide comprising a mutein of human neutrophil gelatinase-associated lipocalin (hNGAL), wherein the hNGAL mutein has at least 80% sequence identity to mature, wild-type hNGAL (SEQ ID NO: 30); and (b) a chimeric antigen receptor. 
     
     
         15 . The modified T cell of  claim 14 , wherein the chimeric antigen receptor comprises: i. an extracellular ligand binding domain; ii. at least one costimulatory domain selected from a CD28 domain, a CD137 domain, an ICOS domain, an OX40 domain, a CD27 domain, and a CD40 domain; and iii. a cytoplasmic domain comprising at least one intracellular signaling domain, wherein the at least one intracellular signaling domain comprises a CD3zeta signaling domain. 
     
     
         16 . A pharmaceutical composition comprising a modified T cell of  claim 1  and optionally a pharmaceutically acceptable carrier or excipient. 
     
     
         17 . A pharmaceutical composition comprising a modified T cell of  claim 14  and optionally a pharmaceutically acceptable carrier or excipient.