shp-2 gene knockout upregulates CAR-driven cytotoxicity of YT NK cells

In Russia, cancer is the second leading cause of death following cardiovascular diseases. Adoptive transfer of NK cells is a promising approach to fight cancer; however, for their successful use in cancer treatment, it is necessary to ensure their robust accumulation at tumor foci, provide resistance to the immunosuppressive tumor microenvironment, and to engineer them with higher cytotoxic activity. NK lymphocytes are known to kill cancer cells expressing a number of stress ligands; and the balance of signals from inhibitory and activating receptors on the surface of the NK cell determines whether a cytotoxic reaction is triggered. We hypothesized that stronger cytotoxicity of NK cells could be achieved via gene editing aimed at enhancing the activating signaling cascades and/or weakening the inhibitory ones, thereby shifting the balance of signals towards NK cell activation and target cell lysis. Here, we took advantage of the CRISPR/Cas9 system to introduce mutations in the coding sequence of the shp-2 (PTPN11) gene encoding the signaling molecule of inhibitory pathways in NK cells. These shp-2 knock-out NK cells were additionally transduced to express a chimeric antigen receptor (CAR) that selectively recognized the antigen of interest on the target cell surface and generated an activating signal. We demonstrate that the combination of shp-2 gene knockout and CAR expression increases the cytotoxicity of effector NK-like YT cells against human prostate cancer cell line Du-145 with ectopic expression of PSMA protein, which is specifically targeted by the CAR.

1. Becker P.S.A., Suck G., Nowakowska P., Ullrich E., Seifried E., Bader P., Tonn T., Seidl C. Selection and expansion of natural killer cells for NK cell-based immunotherapy. Cancer Immunol. Immunother. 2016;65(4):477-484. DOI 10.1007/s00262-016-1792-y.

2. Cerwenka A., Baron J.L., Lanier L.L. Ectopic expression of retinoic acid early inducible-1 gene (RAE-1) permits natural killer cell-mediated rejection of a MHC class I-bearing tumor in vivo. Proc. Natl. Acad. Sci. USA. 2001;98(20):11521-11526. DOI 10.1073/pnas.201238598.

3. Chang S.S. Overview of prostate-specific membrane antigen. Rev. Urol. 2004;6(Suppl.10):S13-S18.

4. Chester C., Fritsch K., Kohrt H.E. Natural killer cell immuno-modulation: targeting activating, inhibitory, and co-stimulatory receptor signaling for cancer immunotherapy. Front. Immunol. 2015;6:601. DOI 10.3389/fimmu.2015.00601.

5. Childs R.W., Carlsten M. Therapeutic approaches to enhance natural killer cell cytotoxicity against cancer: the force awakens. Nat. Rev. Drug Discov. 2015;14:487. DOI 10.1038/nrd4506.

6. De Charette M., Marabelle A., Houot R. Turning tumour cells into antigen presenting cells: the next step to improve cancer immunotherapy? Eur. J. Cancer. 2016;68:134-147. DOI 10.1016/j.ejca.2016.09.010.

7. Deaglio S., Zubiaur M., Gregorini A., Bottarel F., Ausiello C.M., Dianzani U., Sancho J., Malavasi F. Human CD38 and CD16 are functionally dependent and physically associated in natural killer cells. Blood. 2002;99(7):2490-2498.

8. Del Zotto G., Marcenaro E., Vacca P., Sivori S., Pende D., Della Chiesa M., Moretta F., Ingegnere T., Mingari M.C., Moretta A., Moretta L. Markers and function of human NK cells in normal and pathological conditions. Cytometry B. Clin. Cytom. 2017; 92(2):100-114. DOI 10.1002/cyto.b.21508.

9. Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., Virgin H.W., Listgarten J., Root D.E. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016;34(2):184-191. DOI 10.1038/nbt.3437.

10. Edsparr K., Speetjens F.M., Mulder-Stapel A., Goldfarb R.H., Basse P.H., Lennernas B., Kuppen P. J.K., Albertsson P. Effects of IL-2 on MMP expression in freshly isolated human NK cells and the IL-2-independent NK cell line YT. J. Immunother. 2010;33(5): 475-481. DOI 10.1097/CJI.0b013e3181d372a0.

11. Freund-Brown J., Chirino L., Kambayashi T. Strategies to enhance NK cell function for the treatment of tumors and infections. Crit. Rev. Immunol. 2018;38(2):105-130. DOI 10.1615/CritRevImmunol.2018025248.

12. Golubovskaya V, Berahovich R., Zhou H., Xu S., Harto H., Li L., Chao C.C., Mao M.M., Wu L. CD47-CAR-T cells effectively kill target cancer cells and block pancreatic tumor growth. Cancers (Basel). 2017;9(10):139. DOI 10.3390/cancers9100139. Gorchakov A.A., Kulemzin S.V., Kochneva G.V., Taranin A.V. Challenges and prospects of chimeric antigen receptor T-cell therapy for metastatic prostate cancer. Eur. Urol. 2019. DOI 10.1016/j.eururo.2019.08.014.

13. Hanke T., Takizawa H., Mcmahon C.W., Busch D.H., Pamer E.G., Miller J.D., Altman J.D., Liu Y, Cado D., Lemonnier F.A., Bjorkman PJ., Raulet D.H. Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity. 1999;11(1):67-77. DOI 10.1016/S1074-7613(00)80082-5.

14. Hewitt E.W. The MHC class I antigen presentation pathway: strategies for viral immune evasion. Immunology. 2003;110(2):163-169. DOI 10.1046/j.1365-2567.2003.01738.x.

15. Hood S.P., Foulds G.A., Imrie H., Reeder S., Mcardle S.E.B., Khan M., Pockley A.G. Phenotype and function of activated natural killer cells from patients with prostate cancer: patient-dependent responses to priming and IL-2 activation. Front. Immunol. 2019;9:3169. DOI 10.3389/fimmu.2018.03169.

16. Igarashi T., Wynberg J., Srinivasan R., Becknell B., McCoy J.P., Takahashi Y, Suffredini D.A., Linehan W.M., Caligiuri M.A., Childs R.W. Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells. Blood. 2004; 104(1):170. DOI 10.1182/blood-2003-12-4438.

17. Imai C., Iwamoto S., Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood. 2005;106(1):376-383. DOI 10.1182/blood-2004-12-4797.

18. Ingegnere T., Mariotti F.R., Pelosi A., Quintarelli C., De Angelis B., Tumino N., Besi F., Cantoni C., Locatelli F., Vacca P., Moretta L. Human CAR NK cells: a new non-viral method allowing high efficient transfection and strong tumor cell killing. Front. Immunol. 2019;10:957. DOI 10.3389/fimmu.2019.00957.

19. Karre K., Ljunggren H.G., Piontek G., Kiessling R. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature. 1986;319(6055):675-678. DOI 10.1038/319675a0.

20. Kulemzin S.V, Matvienko D.A., Sabirov A.H., Sokratyan A.M., Chernikova D.S., Belovezhets T.N., Chikaev A.N., Taranin A.V, Gorchakov A.A. Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and CAR NK-cell lines. BMC Med. Genomics. 2019;12(Suppl.2):44. DOI 10.1186/s12920-019-0489-4.

21. Kutner R.H., Zhang X.-Y., Reiser J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 2009;4(4):495-505. DOI 10.1038/nprot.2009.22.

22. Lee S.K., Gasser S. The role of natural killer cells in cancer therapy. Front. Biosci. (Elite Ed). 2010;2:380-391.

23. Malarkannan S. The balancing act: inhibitory Ly49 regulate NKG2D-mediated NK cell functions. Semin. Immunol. 2006; 18(3):186-192. DOI 10.1016/j.smim.2006.04.002.

24. Mamessier E., Sylvain A., Thibult M.-L., Houvenaeghel G., Jac-quemier J., Castellano R., Gonęalves A., Andre P, Romagne F., Thibault G., Viens P, Birnbaum D., Bertucci F., Moretta A., Olive D. Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity. J. Clin. Invest. 2011;121(9):3609-3622. DOI 10.1172/JCI45816.

25. Moreno-Mateos M.A., Vejnar C.E., Beaudoin J.D., Fernandez J.P., Mis E.K., Khokha M.K., Giraldez A.J. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat. Methods. 2015;12(10):982-988. DOI 10.1038/nmeth.3543.

26. Nayyar G., Chu Y, Cairo M.S. Overcoming resistance to natural killer cell based immunotherapies for solid tumors. Front. Oncol. 2019;9:51. DOI 10.3389/fonc.2019.00051.

27. O’Doherty U., Swiggard W.J., Malim M.H. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J. Virol. 2000;74(21):10074-10080. DOI 10.1128/jvi.74.21.10074-10080.2000.

28. Pasero C., Gravis G., Granjeaud S., Guerin M., Thomassin-Pi-ana J., Rocchi P, Salem N., Walz J., Moretta A., Olive D. Highly effective NK cells are associated with good prognosis in patients with metastatic prostate cancer. Oncotarget. 2015;6(16):14360-14373. DOI 10.18632/oncotarget.3965.

29. Pasero C., Gravis G., Guerin M., Granjeaud S., Thomassin-Pi-ana J., Rocchi P, Paciencia-Gros M., Poizat F., Bentobji M., Azario-Cheillan F., Walz J., Salem N., Brunelle S., Moretta A., Olive D. Inherent and tumor-driven immune tolerance in the prostate microenvironment impairs natural killer cell antitumor activity. Cancer Res. 2016;76(8):2153. DOI 10.1158/0008-5472.CAN-15-1965.

30. Paul S., Lal G. The molecular mechanism of natural killer cells function and its importance in cancer immunotherapy. Front. Immunol. 2017;8:1124. DOI 10.3389/fimmu.2017.01124.

31. Purdy A.K., Campbell K.S. SHP-2 expression negatively regulates NK cell function. J. Immunol. 2009;183(11):7234-7243. DOI 10.4049/jimmunol.0900088.

32. Quintarelli C., Sivori S., Caruso S., Carlomagno S., Boffa I., Orlando D., Guercio M., Cembrola B., Pitisci A., Di Cecca S., Li Pira G., Vinti L., De Angelis B., Moretta L., Locatelli F. CD19 redirected CAR NK cells are equally effective but less toxic than CAR T cells. Blood. 2018;132(Suppl.1):3491. DOI 10.1182/blood-2018-99-118005.

33. Rehman A.U., Rahman M.U., Khan M.T., Saud S., Liu H., Song D., Sultana P., Wadood A., Chen H.F. The landscape of protein tyrosine phosphatase (Shp2) and cancer. Curr. Pharm. Des. 2018;24(32):3767-3777. DOI 10.2174/1381612824666181106100837.

34. Rezvani K., Rouce R., Liu E., Shpall E. Engineering natural killer cells for cancer immunotherapy. Mol. Ther. 2017;25(8):1769-1781. DOI 10.1016/j.ymthe.2017.06.012.

35. Rusakiewicz S., Semeraro M., Sarabi M., Desbois M., Locher C., Mendez R., Vimond N., Concha A., Garrido F., Isambert N., Chaigneau L., Le Brun-Ly V, Dubreuil P, Cremer I., Caig-nard A., Poirier-Colame V, Chaba K., Flament C., Halama N., Jager D., Eggermont A., Bonvalot S., Commo F., Terrier P, Opolon P, Emile J.-F., Coindre J.- M., Kroemer G., Chaput N., Le Cesne A., Blay J.-Y, Zitvogel L. Immune infiltrates are prognostic factors in localized gastrointestinal stromal tumors. Cancer Res. 2013;73(12):3499. DOI 10.1158/0008-5472.CAN-13-0371.

36. Sanjana N.E., Shalem O., Zhang F. Improved vectors and genomewide libraries for CRISPR screening. Nat. Methods. 2014;11(8): 783-784. DOI 10.1038/nmeth.3047.

37. Sivori S., Vacca P, Del Zotto G., Munari E., Mingari M.C., Moret-ta L. Human NK cells: surface receptors, inhibitory checkpoints, and translational applications. Cell. Mol. Immunol. 2019;16(5): 430-441. DOI 10.1038/s41423-019-0206-4.

38. Suen W.C.W., Lee W.Y.W., Leung K.T., Pan X.H., Li G. Natural killer cell-based cancer immunotherapy: a review on 10 years completed clinical trials. Cancer Invest. 2018;36(8):431-457. DOI 10.1080/07357907.2018.1515315.

39. Yang L., Shen M., Xu L.J., Yang X., Tsai Y., Keng PC., Chen Y, Lee S.O. Enhancing NK cell-mediated cytotoxicity to cispla-tin-resistant lung cancer cells via MEK/Erk signaling inhibition. Sci. Rep. 2017;7(1):7958. DOI 10.1038/s41598-017-08483-z.

40. Yodoi J., Teshigawara K., Nikaido T., Fukui K., Noma T., Honjo T., Takigawa M., Sasaki M., Minato N., Tsudo M., Uchiyama T., Maeda M. TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL-2 receptor on a natural-killer-like cell-line (YT-cells). J. Immunol. 1985;134(3):1623-1630.

41. Yusa S.-I., Campbell K.S. Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function of killer cell Ig-like receptors in human NK cells. J. Immunol. 2003;170(9):4539. DOI 10.4049/jimmunol.170.9.4539.