Cell-based anti-cancer compositions with reduced toxicity and methods of making and using the same
Isolated pluralities of T cells which recognize at least one epitope of an intestinal cancer antigen or CNS cancer antigen and pharmaceutical compositions comprising the same are disclosed. Methods of making a plurality of T cells that recognize at least one epitope of an intestinal cancer antigen or CNS cancer antigen are also disclosed. Methods of treating an individual who has been diagnosed with cancer of a mucosal tissue or preventing such cancer in an individual at elevated risk are disclosed as are nucleic acid molecules that comprise a nucleotide sequence that encode proteins that recognize at least one epitope of an intestinal cancer antigen or CNS cancer antigen and T cells comprising such nucleic acid molecules.
1. A method of making a plurality of T cells that recognize at least one epitope of guanylyl cyclase C, and are modified to inhibit expression or activity of α 4 β 7 integrin and chemokine receptor 9 (CCR9), the method comprising the steps of:
a) isolating a sample from a cell donor that comprises at least one T cell;
b) transforming the T cell with
i) an expressible form of nucleic acid sequence that encodes a guanylyl cyclase C-binding membrane-bound fusion protein that comprises at least a functional fragment of an antibody that binds to at least one epitope of guanylyl cyclase C, wherein the guanylyl cyclase C-binding membrane-bound fusion protein is a membrane bound protein when expressed in the T cell; and
ii) an expressible form of nucleic acid sequence that inhibits expression or activity of α 4 β 7 integrin and chemokine receptor 9 (CCR9),
and
c) culturing said transformed T cell under conditions to promote its replication for a period sufficient to produce a plurality of T cells that recognize guanylyl cyclase C and are modified to inhibit expression or activity of α 4 β 7 integrin and chemokine receptor 9 (CCR9), wherein said i) the nucleic acid sequences that encode the guanylyl cyclase C-binding membrane-bound fusion protein that comprises at least a functional fragment of an antibody that binds to at least one epitope of guanylyl cyclase C, and ii) the nucleic acid sequence that inhibits expression or activity of α 4 β 7 integrin and chemokine receptor 9 (CCR9), are expressed in said cells to produce a plurality of T cells that recognize guanylyl cyclase C and are modified to inhibit expression or activity of α 4 β 7 integrin and chemokine receptor 9 (CCR9).
2. The method of claim 1 wherein the plurality of T cells are modified to inhibit expression of α 4 β 7 integrin and chemokine receptor 9 (CCR9) by delivering to the plurality of T cells, expressible forms of nucleic acid sequences selected from the group consisting of: nucleic acid sequences that express siRNA which inhibits expression of α 4 β 7 integrin, nucleic acid sequences that express microRNA which inhibits expression of α 4 β 7 integrin, nucleic acid sequences that express antisense sequences which inhibit expression of α 4 β 7 integrin, nucleic acid sequences that express siRNA which inhibits expression of CCR9, nucleic acid sequences that express microRNA which inhibits expression of CCR9, and nucleic acid sequences that express antisense sequences which inhibit expression of CCR9.
3. The method of claim 1 wherein said T cells are derived from clonal expansion of T cells that are isolated from an individual and transformed with a nucleic acid molecule which encodes a guanylyl cyclase C-binding membrane-bound fusion protein which comprises at least a functional antigen binding fragment of an antibody that recognizes at least one epitope of guanylyl cyclase C, wherein the fusion protein is expressed as a membrane bound protein such that the T cells recognize at least one epitope of guanylyl cyclase C.
4. The method of claim 1 , wherein the plurality of T cells are modified to inhibit expression of α 4 β 7 integrin and CCR9 by providing the plurality of T cells with nucleic acid sequences that express siRNA which inhibits expression of α 4 β 7 integrin and nucleic acid sequences that express siRNA which inhibits expression of CCR9.
5. The method of claim 1 wherein the guanylyl cyclase C-binding membrane-bound fusion protein comprises: a GCC specific scFv linked to a truncated CD28 extracellular domain to the intracellular part of the FcRIγ chain; a GCC specific scFv linked to a truncated CD28 extracellular domain to the intracellular part of the CD3zeta signaling chain; a GCC specific scFv linked to the intracellular part of the FcRIγ chain; or a GCC specific scFv linked to the intracellular part of the CD3zeta signaling chain.
6. The method of claim 1 wherein the plurality of T cells are modified to inhibit expression of α 4 β 7 integrin and chemokine receptor 9 (CCR9) by delivering to the plurality of T cells, expressible forms of nucleic acid sequences selected from the group consisting of: nucleic acid sequences that express siRNA which inhibits expression of α 4 β 7 integrin, nucleic acid sequences that express microRNA which inhibits expression of α 4 β 7 integrin, nucleic acid sequences that express antisense sequences which inhibit expression of α 4 β 7 integrin, nucleic acid sequences that express siRNA which inhibits expression of CCR9, nucleic acid sequences that express microRNA which inhibits expression of CCR9, and nucleic acid sequences that express antisense sequences which inhibit expression of CCR9.
7. The method of claim 5 , wherein the plurality of T cells are modified to inhibit expression of α 4 β 7 integrin and CCR9 by providing the plurality of T cells with nucleic acid sequences that express siRNA which inhibits expression of α 4 β 7 integrin and nucleic acid sequences that express siRNA which inhibits expression of CCR9.
8. The method of claim 1 wherein the guanylyl cyclase C-binding membrane-bound fusion protein comprises a GCC specific scFv linked to a truncated CD28 extracellular domain to the intracellular part of the CD3zeta signaling chain.
9. The method of claim 8 wherein the plurality of T cells are modified to inhibit expression of α 4 β 7 integrin and chemokine receptor 9 (CCR9) by delivering to the plurality of T cells, expressible forms of nucleic acid sequences selected from the group consisting of: nucleic acid sequences that express siRNA which inhibits expression of α 4 β 7 integrin, nucleic acid sequences that express microRNA which inhibits expression of α 4 β 7 integrin, nucleic acid sequences that express antisense sequences which inhibit expression of α 4 β 7 integrin, nucleic acid sequences that express siRNA which inhibits expression of CCR9, nucleic acid sequences that express microRNA which inhibits expression of CCR9, and nucleic acid sequences that express antisense sequences which inhibit expression of CCR9.
10. The method of claim 8 , wherein the plurality of T cells are modified to inhibit expression of α 4 β 7 integrin and CCR9 by providing the plurality of T cells with nucleic acid sequences that express siRNA which inhibits expression of α 4 β 7 integrin and nucleic acid sequences that express siRNA which inhibits expression of CCR9.