Methods for selective expansion of gamma delta t-cell populations and compositions thereof
The present invention relates to methods for the selective expansion of γδ T-cell population(s), compositions and admixtures thereof and methods for using the same as a therapeutic. Non-engineered and engineered, enriched γδ T-cell populations of the disclosure are useful in the treatment of various cancers, infectious diseases, and immune disorders.
1. An ex vivo method for producing an enriched γδ T-cell population from an isolated mixed cell population, comprising directly contacting the mixed cell population with one or more agents which selectively expand δ2 γδ T-cells by binding to an activating epitope specific to a δ2 TCR to provide clinically-relevant levels of the enriched γδ T-cell population;
wherein the one or more agents which selectively expand δ2 γδ T-cells are selected from an antibody that specifically binds to an epitope comprising a δ2 variable region of a human δ2 TCR and comprising the six complementarity determining regions (CDRs) of δ2-14, δ2-17, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, or δ2-37.
2. The method of claim 1 , wherein the one or more agents which selectively expand δ2 T-cells are selected from an antibody comprising a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 207/216, 208/217, 209/219, 210/220, 211/221, 212/222, 213/223, 214/224, and 215/225.
3. The method of claim 1 , wherein the percentage of δ2 T-cells in the enriched γδ T-cell population is greater than 60% δ2 T-cells of total viable cells.
4. The method of claim 3 , wherein the percentage of δ2 T-cells in the enriched γδ T-cell population is greater than 80% δ2 T-cells of total viable cells.
5. The method of claim 1 , wherein:
(i) the enriched γδ T-cell population is not expanded by an antigen presenting cell or an aminophosphate;
(ii) the one or more agents which selectively expand δ2 T-cells are immobilized on a surface;
(iii) the isolated mixed cell population is selected from a peripheral blood sample, a cord blood sample or a tumor;
(iv) the enriched γδ T-cell population comprises polyclonal TCR diversity;
(v) the enriched γδ T-cell population is formulated for administration to a subject;
(vi) the enriched γδ T-cell population includes a therapeutically effective amount of γδ T-cells; and/or
(vii) the enriched γδ T-cell population is engineered to stably express one or more antigen recognition moieties.
6. The method of claim 5 , wherein the γδ T-cell population is engineered to express two or more antigen recognition moieties.
7. The method of claim 6 , wherein the two or more antigen recognition moieties are different, and wherein each different antigen recognition moiety is engineered to recognize different epitopes of the same antigen or to recognize different epitopes of different antigens.
8. The method of claim 5 , wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogenic antigen.
9. The method of claim 8 , wherein said antigen recognition moiety is selected from the group consisting of a TCR, αβ TCR, γδ TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab) 2 , or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide-MHC complex).
10. The method of claim 8 , wherein the pathogenic antigen is a bacterial antigen or a viral antigen.
11. The method of claim 5 , wherein the engineered γδ T-cells are further engineered to lack gene expression from at least one HLA loci.
12. The method of claim 5 , wherein the engineered γδ T-cells are universal donor cells.
13. The method of claim 1 , wherein the method comprises serum-free culture conditions and/or suspension cell culture conditions.
14. The method of claim 1 , wherein the method achieves at least 10 8 γδ T-cells expanded from the isolated mixed population of cells within less than 90 days, less than 60 days, less than 30 days, less than 21 days, or less than 19 days from:
initiation of a first expansion,
a step of providing a donor sample, or
a first step of directly contacting the mixed cell population with the one or more agents.
15. The method of claim 1 , wherein the isolated mixed cell population comprises a whole PBMC population without prior depletion of monocytes, αβ T-cells, B-cells, and NK cells.
16. The method of claim 1 , further comprising removing one or more of red blood cells, NK cells, αβ cells, B-cells, monocytes, and macrophages prior to selectively expanding the δ2 γδ T-cells.
17. The method of claim 1 , wherein contacting the mixed cell population with one or more agents which selectively expand δ2 T-cells further comprises culture conditions comprising a cytokine selected from IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, and IL-33.
18. The method of claim 17 , wherein the cytokine is selected from the group consisting of IL-2, IL-7, and IL-15.
19. The method of claim 1 , further comprising directly contacting at least a portion of the enriched γδ T-cell population with one or more agents that (a) expand γδ T-cells, and/or (b) deplete αβ T-cells.
20. The method of claim 19 , comprising:
(i) selectively expanding δ2 T-cells by binding to an activating epitope specific of a δ2 TCR in a first γδ T-cell expansion, thereby producing a first enriched γδ T-cell population; and then
(ii) directly contacting at least a portion of the first enriched γδ T-cell population with antigen presenting cells (APCs) in a second γδ T-cell expansion, thereby producing a second enriched γδ T-cell population,
wherein the second enriched γδ T-cell population comprises a clinically relevant number of γδ T-cells.
21. The method of claim 1 , wherein the activating epitope is selected from a δ2 TCR Bin 1 δ2 epitope, a Bin 3 δ2 epitope, and a Bin 4 δ2 epitope of a human δ2 TCR.
22. The method of claim 1 , comprising:
(i) directly contacting the mixed cell population with one or more first activating agents which selectively expand δ2 T-cells by binding to an activating epitope specific of a δ2 TCR in a first γδ T-cell expansion, thereby producing a first enriched γδ T-cell population; and then
(ii) directly contacting at least a portion of the first enriched γδ T-cell population with one or more second activating agents that (a) expand γδ T-cells, or (b) selectively expand
δ1 T-cells by binding to an activating epitope specific of a δ1 TCR;
δ2 T-cells by binding to an activating epitope specific of a δ2 TCR;
δ1 T-cells, δ3 T-cells, δ4 T-cells, and δ5 T-cells by binding to an activating epitope specific of a δ1 TCR, a δ3 TCR, a δ4 TCR, and a δ5 TCR; or
δ1 T-cells and δ4 T-cells by binding to an activating epitope specific of a δ1 TCR and a δ4 TCR
in a second γδ T-cell expansion, thereby producing a second enriched γδ T-cell population.
23. The method of claim 20 , wherein: (a) one or more of the second activating agents are structurally different from one or more of the activating agents of the first γδ T-cell expansion.
24. The method of claim 22 , wherein: (a) one or more of the second activating agents are structurally different from one or more of the activating agents of the first γδ T-cell expansion.
25. The method of claim 1 , comprising:
(i) directly contacting the mixed cell population with one or more activating agents which selectively expand δ2 T-cells by binding to an activating epitope specific of a δ2 TCR in a first γδ T-cell expansion, thereby producing a first enriched γδ T-cell population; and then
(ii) directly contacting at least a portion of the enriched γδ T-cell population with one or more agents that deplete αβ T-cells, thereby producing a second enriched γδ T-cell population.
26. The method of claim 17 , wherein the culture conditions do not comprise IL-4, and wherein the mixed cell population has not been exposed to exogenous IL-4 prior to expansion.
27. The method of claim 18 , wherein the culture conditions do not comprise IL-4, and wherein the mixed cell population has not been exposed to exogenous IL-4 prior to expansion.
28. The method of claim 8 , wherein the antigen recognition moiety recognizes an antigen selected from the group consisting of CD19, CD20, CD30, CD22, CD37, CD38, CD56, CD33, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-16, ST4, NaPi2b, ROR1, ROR2, PLIF, Her2/Neu, EGFRvIII, GPMNB, LIV-1, glycolipidF77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c-Met, CSPG4, PVRL-4, VEGFR2, PSCA, CLEC12a, LICAM, FPC2, GPC3, folate binding protein/receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, 13-3Rα2, SLTRK6, gp100, MARTI, Tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes, KKLC1, mutated ras, p53, MHC class I chain-related molecule A (MICA), or MHC class I chain-related molecule B (MICB), or one or more antigens of HPV, CMV, or EBV.
29. The method of claim 20 , wherein the second γδ T-cell expansion further comprises directly contacting at least a portion of the enriched γδ T-cell population with one or more second activating agents which:
selectively expand δ1 T-cells by binding to an activating epitope specific of a δ1 TCR;
selectively expand δ1 T-cells, δ3 T-cells, δ4 T-cells, and δ5 T-cells by binding to an activating epitope specific of a δ1 TCR, a δ3 TCR, a δ4 TCR, and a δ5 TCR;
selectively expand δ1 T-cells and δ4 T-cells by binding to an activating epitope specific of a δ1 TCR and a δ4 TCR; or
selectively expand δ2 T-cells by binding to an activating epitope specific of a δ2 TCR.
30. The method of claim 6 , wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogenic antigen.
31. The method of claim 30 , wherein the pathogenic antigen is a bacterial antigen or a viral antigen.