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Expansion of KRAS hotspot mutations reactive T cells from human pancreatic tumors using autologous T cells as the antigen-presenting cells

Cancer Immunology, Immunotherapy (2022)Cite this article

Abstract

Adoptive cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) or TCR gene-modified T cells (TCR-T) that recognize mutant KRAS neo-antigens can mediate tumor regression in patients with advanced pancreatic ductal adenocarcinoma (PDAC) (Tran et al in N Engl J Med, 375:2255–2262, 2016; Leidner et al in N Engl J Med, 386:2112–2119, 2022). The mutant KRAS-targeted ACT holds great potential to achieve durable clinical responses for PDAC, which has had no meaningful improvement over 40 years. However, the wide application of mutant KRAS-centric ACT is currently limited by the rarity of TIL that recognize the mutant KRAS. In addition, PDAC is generally recognized as a poorly immunogenic tumor, and TILs in PDAC are less abundant than in immunogenic tumors such as melanoma. To increase the success rate of TIL production, we adopted a well-utilized K562-based artificial APC (aAPC) that expresses 4-1BBL as the costimulatory molecules to enhance the TIL production from PDCA. However, stimulation with K562-based aAPC led to a rapid loss of specificity to mutant KRAS. To selectively expand neo-antigen-specific T cells, particularly mKRAS, from the TILs, we used tandem mini gene-modified autologous T cells (TMG-T) as the novel aAPC. Using this modified IVS protocol, we successfully generated TIL cultures specifically reactive to mKRAS (G12V). We believe that autologous TMG-T cells provide a reliable source of autologous APC to expand a rare population of neoantigen-specific T cells in TILs.

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Data availability

The data supporting this study's findings are available from the corresponding author, GY, upon reasonable request.

References

  1. Tran E, Robbins PF, Lu YC et al (2016) T-cell transfer therapy targeting mutant KRAS in cancer. N Engl J Med 375:2255–2262. https://doi.org/10.1056/NEJMoa1609279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Leidner R, Sanjuan Silva N, Huang H et al (2022) Neoantigen T-cell receptor gene therapy in pancreatic cancer. N Engl J Med 386:2112–2119. https://doi.org/10.1056/nejmoa2119662

    Article  CAS  PubMed  Google Scholar 

  3. McGuigan A, Kelly P, Turkington RC, Jones C, Coleman HG, McCain RS (2018) Pancreatic cancer: a review of clinical diagnosis, epidemiology, treatment and outcomes. World J Gastroenterol 24:4846–4861. https://doi.org/10.3748/wjg.v24.i43.4846

    Article  PubMed  PubMed Central  Google Scholar 

  4. Conroy T, Hammel P, Hebbar M et al (2018) FOLFIRINOX or gemcitabine as adjuvant therapy for pancreatic cancer. N Engl J Med 379:2395–2406. https://doi.org/10.1056/NEJMoa1809775

    Article  CAS  PubMed  Google Scholar 

  5. Morrison AH, Byrne KT, Vonderheide RH (2018) Immunotherapy and prevention of pancreatic cancer. Trends Cancer 4:418–428. https://doi.org/10.1016/j.trecan.2018.04.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Balachandran VP, Beatty GL, Dougan SK (2019) Broadening the impact of immunotherapy to pancreatic cancer: challenges and opportunities. Gastroenterology 156:2056–2072. https://doi.org/10.1053/j.gastro.2018.12.038

    Article  CAS  PubMed  Google Scholar 

  7. Dudley ME, Wunderlich JR, Shelton TE, Even J, Rosenberg SA (2003) Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J Immunother 26:332–342. https://doi.org/10.1097/00002371-200307000-00005

    Article  PubMed  PubMed Central  Google Scholar 

  8. Goff SL, Smith FO, Klapper JA et al (2010) Tumor infiltrating lymphocyte therapy for metastatic melanoma: analysis of tumors resected for TIL. J Immunother 33:840–847. https://doi.org/10.1097/CJI.0b013e3181f05b91

    Article  PubMed  PubMed Central  Google Scholar 

  9. Sakellariou-Thompson D, Forget MA, Creasy C et al (2017) 4–1BB agonist focuses CD8+ tumor-infiltrating T-cell growth into a distinct repertoire capable of tumor recognition in pancreatic cancer. Clin Cancer Res 23:7263–7275. https://doi.org/10.1158/1078-0432.CCR-17-0831

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Friedman KM, Devillier LE, Feldman SA, Rosenberg SA, Dudley ME (2011) Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement. J Immunother 34:651–661. https://doi.org/10.1097/CJI.0b013e31823284c3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ye Q, Loisiou M, Levine BL et al (2011) Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes. J Transl Med 9:131. https://doi.org/10.1186/1479-5876-9-131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Li Y, Bleakley M, Yee C (2005) IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8 T cell response. J Immunol 175:2261–2269. https://doi.org/10.4049/jimmunol.175.4.2261

    Article  CAS  PubMed  Google Scholar 

  13. Meng Q, Liu Z, Rangelova E et al (2016) Expansion of tumor-reactive T Cells from patients with pancreatic cancer. J Immunother 39:81–89. https://doi.org/10.1097/CJI.0000000000000111

    Article  CAS  PubMed  Google Scholar 

  14. Liu Z, Meng Q, Bartek J et al (2017) Tumor-infiltrating lymphocytes (TILs) from patients with glioma. Oncoimmunology 6:e1252894. https://doi.org/10.1080/2162402X.2016.1252894

    Article  CAS  PubMed  Google Scholar 

  15. Santegoets SJ, Turksma AW, Suhoski MM et al (2013) IL-21 promotes the expansion of CD27+ CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells. J Transl Med 11:37. https://doi.org/10.1186/1479-5876-11-37

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Denman CJ, Senyukov VV, Somanchi SS et al (2012) Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells. PLoS ONE 7:e30264. https://doi.org/10.1371/journal.pone.0030264

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Liu E, Tong Y, Dotti G et al (2018) Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity. Leukemia 32:520–531. https://doi.org/10.1038/leu.2017.226

    Article  CAS  PubMed  Google Scholar 

  18. Tran E, Ahmadzadeh M, Lu YC et al (2015) Immunogenicity of somatic mutations in human gastrointestinal cancers. Science 350:1387–1390. https://doi.org/10.1126/science.aad1253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jin J, Gkitsas N, Fellowes VS et al (2018) Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules. J Transl Med 16:13. https://doi.org/10.1186/s12967-018-1384-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cafri G, Yossef R, Pasetto A et al (2019) Memory T cells targeting oncogenic mutations detected in peripheral blood of epithelial cancer patients. Nat Commun 10:449. https://doi.org/10.1038/s41467-019-08304-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Altman JD, Moss PA, Goulder PJ et al (1996) Phenotypic analysis of antigen-specific T lymphocytes. Science 274:94–96. https://doi.org/10.1126/science.274.5284.94

    Article  CAS  PubMed  Google Scholar 

  22. Reithofer M, Rosskopf S, Leitner J et al (2021) 4–1BB costimulation promotes bystander activation of human CD8 T cells. Eur J Immunol 51:721–733. https://doi.org/10.1002/eji.202048762

    Article  CAS  PubMed  Google Scholar 

  23. Strizova Z, Snajdauf M, Stakheev D et al (2020) The paratumoral immune cell signature reveals the potential for the implementation of immunotherapy in esophageal carcinoma patients. J Cancer Res Clin Oncol 146:1979–1992. https://doi.org/10.1007/s00432-020-03258-y

    Article  CAS  PubMed  Google Scholar 

  24. June CH (2016) Drugging the undruggable ras — immunotherapy to the rescue. N Engl J Med 375:2286–2289. https://doi.org/10.1056/nejme1612215

    Article  CAS  PubMed  Google Scholar 

  25. Levin N, Paria BC, Vale NR et al (2021) Identification and validation of T-cell receptors targeting RAS hotspot mutations in human cancers for use in cell-based immunotherapy. Clin Cancer Res 27:5084–5095. https://doi.org/10.1158/1078-0432.CCR-21-0849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Parkhurst MR, Robbins PF, Tran E et al (2019) Unique neoantigens arise from somatic mutations in patients with gastrointestinal cancers. Cancer Discov 9:1022–1035. https://doi.org/10.1158/2159-8290.CD-18-1494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yossef R, Tran E, Deniger DC et al (2018) Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy. JCI Insight 3:122467. https://doi.org/10.1172/jci.insight.122467

    Article  PubMed  Google Scholar 

  28. Rius C, Attaf M, Tungatt K et al (2018) Peptide-MHC class I tetramers can fail to detect relevant functional T cell clonotypes and underestimate antigen-reactive T cell populations. J Immunol 200:2263–2279. https://doi.org/10.4049/jimmunol.1700242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Birrer DL, Golcher H, Casadei R et al (2021) Neoadjuvant therapy for resectable pancreatic cancer: a new standard of care. Pooled data from 3 randomized controlled trials. Ann Surg. 274:713–720. https://doi.org/10.1097/SLA.0000000000005126

    Article  PubMed  Google Scholar 

  30. Duhen T, Duhen R, Montler R et al (2018) Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nature Commun. https://doi.org/10.1038/s41467-018-05072-0

    Article  Google Scholar 

  31. Scheper W, Kelderman S, Fanchi LF et al (2019) Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. Nat Med 25:89–94. https://doi.org/10.1038/s41591-018-0266-5

    Article  CAS  PubMed  Google Scholar 

  32. Caushi JX, Zhang J, Ji Z et al (2021) Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature 596:126–132. https://doi.org/10.1038/s41586-021-03752-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang X, Lee DA, Wang Y et al (2013) Membrane-bound interleukin-21 and CD137 ligand induce functional human natural killer cells from peripheral blood mononuclear cells through STAT-3 activation. Clin Exp Immunol 172:104–112. https://doi.org/10.1111/cei.12034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Vidard L, Dureuil C, Baudhuin J et al (2019) CD137 (4–1BB) engagement fine-tunes synergistic IL-15- and IL-21-driven NK cell proliferation. J Immunol 203:676–685. https://doi.org/10.4049/jimmunol.1801137

    Article  CAS  PubMed  Google Scholar 

  35. Shah N, Martin-Antonio B, Yang H et al (2013) Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity. PLoS ONE 8:e76781. https://doi.org/10.1371/journal.pone.0076781

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Denman CJ, Senyukov VV, Somanchi SS et al (2012) Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells. PLoS ONE 7:e30264. https://doi.org/10.1371/journal.pone.0030264

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Liu E, Ang SOT, Kerbauy L et al (2021) GMP-compliant universal antigen presenting cells (uAPC) promote the metabolic fitness and antitumor activity of armored cord blood CAR-NK cells. Front Immunol 12:626098. https://doi.org/10.3389/fimmu.2021.626098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ojo EO, Sharma AA, Liu R et al (2019) Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells. Sci Rep 9:14916. https://doi.org/10.1038/s41598-019-51287-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yang Y, Badeti S, Tseng HC et al (2020) Superior expansion and cytotoxicity of human primary NK and CAR-NK cells from various sources via enriched metabolic pathways. Mol Ther Methods Clin Dev 18:428–445. https://doi.org/10.1016/j.omtm.2020.06.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Maus MV, Thomas AK, Leonard DG et al (2002) Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4–1BB. Nat Biotechnol 20:143–148. https://doi.org/10.1038/nbt0202-143

    Article  CAS  PubMed  Google Scholar 

  41. Forget MA, Malu S, Liu H et al (2014) Activation and propagation of tumor-infiltrating lymphocytes on clinical-grade designer artificial antigen-presenting cells for adoptive immunotherapy of melanoma. J Immunother 37:448–460. https://doi.org/10.1097/CJI.0000000000000056

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Chacon JA, Wu RC, Sukhumalchandra P et al (2013) Co-stimulation through 4–1BB/CD137 improves the expansion and function of CD8(+) melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy. PLoS ONE 8:e60031. https://doi.org/10.1371/journal.pone.0060031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Shah P, Forget MA, Frank ML et al (2022) Combined IL-2, agonistic CD3 and 4–1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes. J Immunother Cancer 10:e003082. https://doi.org/10.1136/jitc-2021-003082

    Article  PubMed  PubMed Central  Google Scholar 

  44. Reithofer M, Rosskopf S, Leitner J et al (2021) 4–1BB costimulation promotes bystander activation of human CD8 T cells. Eur J Immunol 51:721–733. https://doi.org/10.1002/eji.202048762

    Article  CAS  PubMed  Google Scholar 

  45. Poschke IC, Hassel JC, Rodriguez-Ehrenfried A et al (2020) The outcome of ex vivo TIL expansion is highly influenced by spatial heterogeneity of the tumor T-cell repertoire and differences in intrinsic in vitro growth capacity between T-cell clones. Clin Cancer Res 26:4289–4301. https://doi.org/10.1158/1078-0432.CCR-19-3845

    Article  CAS  PubMed  Google Scholar 

  46. Bear AS, Blanchard T, Cesare J et al (2021) Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting. Nat Commun 12:4365. https://doi.org/10.1038/s41467-021-24562-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Choi J, Goulding SP, Conn BP et al (2021) Systematic discovery and validation of T cell targets directed against oncogenic KRAS mutations. Cell Reports Methods 1:100084. https://doi.org/10.1016/j.crmeth.2021.100084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Levin N, Lowery FJ, et al (2022) Identification of T-cell receptors targeting RAS hotspot mutations using TIL IVS in human cancer in cell-based immunotherapy. Resistance mechanisms and new advances in immunotherapeutics. AACR 2022. #3576

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Acknowledgements

The authors would like to thank Drs. Eric Tran and Rom Leidner of Earle A. Chiles Research Institute for helpful discussions on topics related to this work. This work is supported by the Jiangsu Clinical Medical Center (innovation platform) construction project (No. YXZXA2016006, to XW) and Shanghai Pujiang Program (No.20PJ1417400, to GY).

Funding

It is sponsored by Jiangsu Clinical Medical Center (innovation platform) construction project YXZXA2016006 and Shanghai Pujiang Program 20PJ1417400.

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Author notes

  1. Rongxi Shen

    Present address: Research Institute of General Surgery, Jinling Hospital, Nanjing University Medical School, 305 Zhong Shan East Road, Nanjing, 210002, China

  2. Sizhen Wang, Xiaohui Zhang, Xuemei Zou, Maorong Wen have contributed equally to this work.

Authors and Affiliations

  1. Research Institute of General Surgery, Jinling Hospital, Nanjing University Medical School, 305 Zhong Shan East Road, Nanjing, 210002, China

    Sizhen Wang, Min Li, Daojun Zhu, Anlong Yao, Yu Fang & Xinbo Wang

  2. ImmuXell Biotech Ltd, 299 Kangwei Road, Shanghai, 201315, China

    Xiaohui Zhang, Xuemei Zou, Maorong Wen, Chi Gan, Xiaochun Jiang, Hong-Ming Hu & Guangjie Yu

  3. Ubivac Inc, 18183 SW Boones Ferry Rd, Portland, OR, 97224-7672, USA

    Bernard A. Fox & Hong-Ming Hu

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  1. Sizhen Wang
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Contributions

SW, XZ, XZ, and MW are joint first authors. XW and GY are PIs of research projects and study guarantors. XW, HH, and GY contributed to the study's concept and design. SW, XZ, XZ, CG, XJ, ML, RS, DZ, AY, and YF collected samples, performed the experiments, and analyzed the data. GY and HH drafted the manuscript. XW and BAF contributed to interpreting the results and critical revision of the manuscript and approved the final version. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Hong-Ming Hu, Guangjie Yu or Xinbo Wang.

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Competing interests

Xiaohui Zhang, Xuemei Zou, Maorong Wen, Chi Gan, Xiaochun Jiang, and Guangjie Yu are full-time employees of ImmuXell ltd. They have filed patent applications related to the TCR technology and could potentially receive licensing royalties. All other authors have no conflict of interest to declare. Hong-Ming Hu and Bernard A. Fox is the founder of UbiVAC, and UbiVAC is a potential licensee of technology developed in this study.

Conflict of interest

Xiaohui Zhang, Xuemei Zou, Maorong Wen, Chi Gan, Xiaochun Jiang, and Guangjie Yu are full-time employees of ImmuXell ltd. They have filed patent applications related to the TCR technology and could potentially receive licensing royalties. All other authors have no conflict of interest to declare. Hong-Ming Hu and Bernard A. Fox is the founder of UbiVAC, and UbiVAC is a potential licensee of technology developed in this study.

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This study was approved by the Ethics Committee of Jinling Hospital (Nanjing, China), reference number 2020NJKY-020–01.

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Wang, S., Zhang, X., Zou, X. et al. Expansion of KRAS hotspot mutations reactive T cells from human pancreatic tumors using autologous T cells as the antigen-presenting cells. Cancer Immunol Immunother (2022). https://doi.org/10.1007/s00262-022-03335-w

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