The Establishment Of PSMA-Targeted Chimeric Antigen Receptors Engineered Natural Killer Cells For Castration-Resistant Prostate Cancer

BackgroundAndrogen deprivation therapy is commonly used for advanced PCa.However,almost all patients become less sensitive to the treatment and evolve into castrationresistant prostate cancer(CRPC)after hormone deprivation treatment.Without effective treatment options,the mortality of CRPC is high.Chimeric antigen receptor(CAR)based therapy is a promising immunotherapeutic strategy,and further research on its application in CRPC needs to be conducted.Prostate-specific membrane antigen(PSMA)is significantly overexpressed in prostate cancer.We aimed to design a novel PSMAtargeted CAR NK cell with clinically significant tumoricidal effect on CRPC.MethodsWe constructed novel CAR NK92MI cells with a CD244-based recombinant lentiviral vector.This study was divided into two parts.In the first part,PSMA-targeted CAR NK92MI cells were constructed.The intracellular signaling region and extracellular antigen-binding segment of CAR were screened.Different intracellular segments(CD244,NKG2D or CD3ζ)were screened to find the best candidate according to cell lysis assay and CD 107a expression levels.Then,to enhance the affinity of CAR for the tumor antigen,we compared the antibody specific for prostate-specific membrane antigen(anti-PSMA)to the PSMA-targeting polypeptide(p-PSMA),which was identified via phage display combinatorial library screening.A CAR NK92MI cell with both a high affinity for PSMA and a strong tumoricidal ability was thus generated.In the second part,we verified the tumor-killing effect in vitro and in vivo.The release of cytokines by NK92MI was compared to that from CAR NK92MI by flow cytometry and ELISA assay.Ferroptosis-related cell death was identified as a possible underlying mechanism.ResultsIn the first part,three different CAR intracellular regions CAR1(CD244),CAR2(CD244,NKG2D)and CAR3(CD244,NKG2D and CD3ζ)were constructed.The results showed CAR2 and CAR3mediated strong antigen-specific NK92MI cells signaling,when compared to CAR1(CD244).CAR2 had a shorter costimulatory domain than CAR3,so CAR2 was chosen to confer CAR-NK92MI cells with an enhanced tumoricidal ability.Compared to anti-PSMA,p-PSMA exhibited stronger affinity for the tumor antigen.Thus,peptides targeting PSMA were selected to construct CAR’s extracellular segments.In the second part,it was demonstrated that p-PSMA-CAR NK92MI cells were lethal to CRPC cells in vitro and in vivo and that p-PSMA CAR NK92MI cells could selectively kill PSMA-positive tumor cells.p-PSMA CAR NK92MI cells showed time-and dose-dependent lethality,with an efficacy-target ratio of 15:1 at which CRPC cell lysis rate reached 77.87%.Additionally,granzyme B was the major tumoricidal mediator,degranulating upon contact with CRPC cells.Finally,ferroptosis was a potential mechanism for CAR NK92MI cells to attack cancer cells,triggered by IFN-γ.ConclusionsWe developed a novel PSMA-targeted CAR NK92MI cell with a strong antitumor effect against CRPC.p-PSMA-CAR-NK92MI cells can effectively kill CRPCPSMA+cells in vitro and in vivo.We revealed that it affects tumor cells primarily by releasing granzyme B and explored into the possibilities of it inducing ferroptosis in tumor cells.