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US-20260126440-A1 - METHODS FOR MEASURING LYMPHOCYTE ACTIVITY

US20260126440A1US 20260126440 A1US20260126440 A1US 20260126440A1US-20260126440-A1

Abstract

Provided herein are methods for measuring antigen recognition response by lymphocytes upon exposure to a target antigen. Methods are disclosed for functionalizing red blood cells with antigens recognized by lymphocytes. By contacting antigen functionalized red blood cells with lymphocytes having specificity for the antigen, lymphocyte recognition response can be measured. The recognition response can be in the form of cytokine release by the lymphocytes and/or the expression of activation and/or differentiation markers on the surface of the lymphocytes.

Inventors

  • Chad M. Williams

Assignees

  • KITE PHARMA, INC.

Dates

Publication Date
20260507
Application Date
20251031

Claims (20)

  1. 1 . A method for measuring the antigen recognition response of a population of lymphocytes wherein at least one lymphocyte of the population of lymphocytes expresses a molecule having binding specificity for an antigen comprising: (a) affixing the antigen on a red blood cell (RBC) to create a functionalized RBC; (b) contacting the functionalized RBC with the population of lymphocytes; and (c) measuring for a response associated with antigen recognition exhibited by the population of lymphocytes, wherein the response is selected from the group consisting of (1) an increased cytokine release in comparison to a control population of the lymphocytes not contacted with the functionalized RBC; (2) the response is a greater proliferation of the population of lymphocytes in comparison to a control population of the lymphocytes not contacted with the functionalized RBC; (3) the response is an increased expression of cell surface activation markers of the population of lymphocytes in comparison to a control population of the lymphocytes not contacted with the functionalized RBC; (4) the response is an increased expression of cell surface differentiation markers of the population of lymphocytes in comparison to a control population of the lymphocytes not contacted with the functionalized RBC and any combination thereof.
  2. 2 . The method of claim 1 , wherein the population of lymphocytes comprises a cell type selected from the group consisting of a T cell, a B cell, a NK cell, a monocyte, a macrophage and any combination thereof.
  3. 3 . The method of claim 2 wherein the population of lymphocytes comprises a T cell.
  4. 4 . The method of claim 3 , wherein the population of lymphocytes comprises a cell expressing a chimeric antigen receptor (CAR).
  5. 5 . (canceled)
  6. 6 . The method of claim 4 , wherein the antigen is affixed to the RBC by: 1) providing a RBC, 2) biotinylating the RBC, 3) coating the biotinylated RBC with streptavidin, and 4 ) contacting the streptavidin-coated RBC of step 3 with a biotinylated, avi-tagged antigen.
  7. 7 . The method of claim 6 , wherein the RBC is contacted with a biotin solution having a biotin concentration of 0.1 μM, 0.2 M, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 AM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, 200 μM, 220 μM, 240 μM, 260 μM, 280 HM, 300 μM, 320 μM, 340 μM, 360 μM, 380 μM, 400 μM, 420 μM, 440 μM, 460 μM, 480 μM, 500 μM, 520 μM, 540 μM, 560 μM, 580 μM, 600 μM, 620 μM, 640 μM, 660 μM, 680 μM, 700 μM, 720 μM, 740 μM, 760 μM, 780 μM, 800 μM, 820 μM, 840 μM, 860 μM, 880 μM, 900 μM, 920 μM, 940 μM, 960 μM, 980 μM, or 1000 μM to provide for a biotinylated RBC.
  8. 8 . The method of claim 7 , wherein the biotinylated RBC is further contacted with a streptavidin solution having a streptavidin concentration of 0.5-2 mg/mL to provide for a biotin and streptavidin coated RBC.
  9. 9 . The method of claim 8 , wherein the biotin and streptavidin coated RBC is contacted with a biotinylated, avi-tagged antigen.
  10. 10 . The method of claim 9 , wherein the antigen is selected from the group consisting of DAP-10, CD 28, OX- 40, 4-1BB (CD 137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ICAM1, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), ITGAX (CD11c), ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG (Cbp), CD19a, a ligand that specifically binds with CD83, 5T4, alphafetoprotein (AFP), B-human chorionic gonadotropin, B7-1 (CD80), B7-2 (CD86), BCMA, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD20, CD22, CD23, CD24, CD25, CD33, CD34, CD44, CD56, CD79b, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal-epithelial mucine, EB V-specific antigen, EGFR variant III (EGFR vIII), ELF2M, endoglin, ephrin B2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2/neu), fibroblast associated protein (fap), FLT3, folate binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipids, gp36, GPC3, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 in combination, HERV-K, high molecular weight-melanoma associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11Ralpha, IL-13R-a2, Influenza Virus-specific antigen; CD38, insulin growth factor (IGF1)-1, intestinal carboxyl esterase, kappa chain, LAGA-la, lambda chain, Lassa Virus-specific antigen, lectin-reactive AFP, lineage-specific or tissue specific antigen such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecule, major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mut hsp70-2, mutated p53, mutated ras, neutrophil elastase, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate specific antigen (PSA), prostate-carcinoma tumor antigen-1 (PCTA-1), prostate-specific antigen protein, PSMA, RAGE-1, ROR1, RUI, RU2 (AS), STEAP1, STEAP2, surface adhesion molecule, surviving and telomerase, TAG-72, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stromal antigens, vascular endothelial growth factor receptor-2 (VEGFR2), virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20) and any combinations thereof.
  11. 11 . The method of claim 10 , wherein the antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CLL1, GPC3, and BCMA.
  12. 12 - 15 . (canceled)
  13. 16 . The method of claim 11 wherein antigen comprises a combination of two or more antigens.
  14. 17 . The method of claim 9 , wherein the concentration of the antigen is selected to provide an average number of antigens affixed to an RBC between 100 antigen molecules per RBC to 500,000 antigen molecules per RBC.
  15. 18 - 23 . (canceled)
  16. 24 . The method of claim 17 , wherein the functionalized RBC and the population of lymphocytes are contacted for up to 12 hours.
  17. 25 - 32 . (canceled)
  18. 33 . The method of claim wherein the cytokine is selected from the group consisting of IL-2, IFNγ, Granzyme B, GM-CSF, TNFα, Perforin, IL-4, IL-5, IL-13, IL-17, IL-10, MIP-1a, MIP-1b, IL-6, sFasL, s4-1BB, Granzyme K and any combination thereof.
  19. 34 . The method of claim 1 , wherein the response is an increased cytokine release in comparison to a control population of the lymphocytes not contacted with the functionalized RBC.
  20. 35 . The method of claim 1 , wherein the response is a greater proliferation of the population of lymphocytes in comparison to a control population of the lymphocytes not contacted with the functionalized RBC.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Ser. No. 63/716,067 , filed on Nov. 4, 2024, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD The present disclosure relates to the field of cell therapy, and more specifically, compositions and methods for measuring antigen recognition response in lymphocytes engineered to express a T-cell receptor and/or a chimeric antigen receptor. BACKGROUND Cell therapy products prepared from lymphocytes have varying capacity for response to antigens, including but not limited to cytotoxic activity. One indicator of cytotoxic capacity of cell therapy products is cytokine release upon exposure to a target antigen. There is a need in the art for methods to characterize and quantify antigen recognition response by cell therapy products, e.g. cytokine release activity. SUMMARY Described herein are methods and compositions for characterization and quantification of antigen recognition response by cell therapy products upon exposure to a target antigen, e.g. cytokine release. In certain aspects, cell therapy products are contacted with red blood cells which are functionalized with one or more target antigens specifically recognized by a T-cell receptor or chimeric antigen receptor that the lymphocytes of a cell therapy product are genetically engineered to express. In accordance with a further embodiment of the present disclosure, therefore, provided is a method for measuring the antigen recognition response of a population of lymphocytes wherein at least one lymphocyte of the population of lymphocytes expresses a molecule having binding specificity for an antigen comprising: (a) affixing the antigen on a red blood cell (RBC) to create a functionalized RBC; (b) contacting the functionalized RBC with the population of lymphocytes; and (c) measuring for a response associated with antigen recognition exhibited by the population of lymphocytes, wherein the response is selected from the group consisting of (1) an increased cytokine release in comparison to a control population of the lymphocytes not contacted with the functionalized RBC; (2) the response is a greater proliferation of the population of lymphocytes in comparison to a control population of the lymphocytes not contacted with the functionalized RBC; (3) the response is an increased expression of cell surface activation markers of the population of lymphocytes in comparison to a control population of the lymphocytes not contacted with the functionalized RBC; (4) the response is an increased expression of cell surface differentiation markers of the population of lymphocytes in comparison to a control population of the lymphocytes not contacted with the functionalized RBC and any combination thereof. In some embodiments, the population of lymphocytes comprises a cell type selected from the group consisting of a T cell, a B cell, a NK cell, a monocyte, a macrophage and any combination thereof. In some embodiments, the population of lymphocytes comprises a T cell. In some embodiments, the population of lymphocytes comprises a cell expressing a chimeric antigen receptor (CAR). In some embodiments, the population of lymphocytes comprises a cell expressing a T cell receptor. In some embodiments, the antigen is affixed to the RBC by: 1) providing a RBC, 2) biotinylating the RBC, 3) coating the biotinylated RBC with streptavidin, and 4) contacting the streptavidin-coated RBC of step 3 with a biotinylated, avi-tagged antigen. In some embodiments, the RBC is contacted with a biotin solution having a biotin concentration of 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, 200 μM, 220 μM, 240 μM, 260 μM, 280 μM, 300 μM, 320 μM, 340 μM, 360 μM, 380 μM, 400 μM, 420 μM, 440μM, 460 μM, 480 μM, 500 μM, 520 μM, 540 μM, 560 μM, 580 μM, 600 μM, 620 μM, 640 μM, 660 μM, 680 μM, 700 μM, 720 μM, 740 μM, 760 μM, 780 μM, 800 μM, 820 μM, 840 μM, 860 M, 880 μM, 900 μM, 920 μM, 940 μM, 960 μM, 980 μM, or 1000 μM to provide for a biotinylated RBC. In some embodiments, the biotinylated RBC is further contacted with a streptavidin solution having a streptavidin concentration of 1 mg/mL to provide for a biotin and streptavidin coated RBC. In some embodiments, the biotin and streptavidin coated RBC is contacted with a biotinylated, avi-tagged antigen. In some embodiments, the antigen is selected from the group consisting of DAP-10, CD 28, OX-40, 4-1BB (CD 137), CD 2,CD7,CD27,CD30,CD40, CD 70, OX40L, PD-L 1, CD 155 (PVR), 4-1BB ligand (4-1BBL), CD58, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis