Establishment Of A CAR-T Immunogenicity Detection Method And Its Clinical Application

Objective:Chimeric antigen receptor T(CAR-T)cell therapy has shown remarkable therapeutic efficacy in various hematological malignancies.However,as a type of genetically engineered cell,CAR-T cells possess a certain degree of immunogenicity.This immunogenicity leads to the development of specific immune reactions against CAR-T cells in some patients undergoing CAR-T therapy,including both cellular and humoral immune responses.There is a correlation between this immune response and poor CAR-T efficacy in some patients.However,the current methods used to detect this immune response have certain limitations.By analyzing the correlation between CAR-T immunogenicity and therapeutic efficacy using CAR copy number variation and efficacy data from patients,we aim to investigate the impact of CAR-T immunogenicity on CAR-T therapy.Our findings may shed light on the development of more effective CAR-T treatment strategies.Methods:1)Peptide sequences of CAR were synthesized using solid-phase peptide synthesis technique.The synthesized peptides were used as stimuli to establish a cellular immunoassay using enzyme-linked immunospot assay(ELISPOT assay),and tested in a healthy population to verify the stability of the method.2)The NetMHCpan model was integrated to predict the stronger immunogenicity in CAR peptides.3)A rabbit monoclonal antibody targeting single-chain variable fragment(scFv)was synthesized as a standard for CAR-specific anti-drug antibody(ADA)and used in the bridging method with electrochemiluminescence(ECL).4)The above-established assay was applied to patients with hematological tumors treated with CD 19/22 CAR-T and multiple myeloma treated with BCMA CAR-T.The method was validated in healthy human serum.We collected CAR copy numbers from the patients and followed up their treatment efficacy to investigate the correlation between CAR-T immunogenicity and CAR-T expansion and efficacy.Results:1)CAR peptides were successfully synthesized,with an amino acid coverage of 98.699%.The stability and reliability of the method were validated in healthy volunteers using ELISPOT to establish a specific cellular immunoassay.2)ADA standards were obtained through four rounds of screening and purification.The assay included three levels of screening,confirmatory and titration,and was performed in normal human serum to calculate parameters such as screening cut-point,confirmatory cut-point,and titration cutpoint.The methodological validation of the bridging ECL assay was demonstrated to be qualified according to FDA standards.3)Sixteen patients treated with murine-derived CD 19/22 CAR-T and 25 patients treated with human-derived BCMA CAR-T in our center were included and tested with the two established assays.Antigenic peptides were individually predicted using models such as NetMHCpan.Combined with efficacy analysis,the results revealed no significant effect of humoral immunity(i.e.,ADA)on efficacy.Conclusions:The two CAR-T-specific immunoreactivity assays established in this study have undergone rigorous methodological validation and offer notable advantages over previous commonly used assays,including ease of localizing antigenic peptides and reduced susceptibility to matrix influence.CAR-T immunogenicity potentially impacts CAR-T efficacy.The assay established in this study can be used for individualized prediction of the effect of CAR immunogenicity on patient efficacy before CAR-T treatment.Moreover,it can be employed to monitor immune responses in patients after CAR-T infusion to predict efficacy and adjust subsequent treatment regimens accordingly.Part Ⅰ:Detection of CAR-T-Specific Cellular ImmunityObjective:Currently,the main method for detecting CAR-T cell immune specificity is the chromium release assay.However,this method poses a risk to the health of researchers and it is difficult to localize the immunogenicity to specific peptides.Therefore,it is necessary to establish safer and more accurate detection methods.Methods:We extracted the peptide sequence of CAR from the plasmid file using Snapgene software and synthesized multiple CAR peptides using solid-phase peptide synthesis technology.We established a cellular immune detection method using ELISPOT technology,using the extracted CAR peptide as a stimulus to detect specific T lymphocytes in the sample being tested.We performed ELISPOT detection in peripheral blood samples of healthy volunteers to verify the method’s effectiveness,reliability,and stability.We also integrated NetMHCpan and other models to predict peptides with strong immunogenicity in CAR peptides.Results:We successfully synthesized CAR peptides,with an amino acid coverage of 98.699%.ELISPOT was performed on 16 healthy volunteers,and we found that CAR peptides induced only low-level activation of T cells in healthy people.The activation levels of each peptide were similar in different people,and the standard deviation of each peptide test result was below 1.985.Conclusions:In summary,this part established a CAR-T-specific cellular immune detection method based on ELISPOT technology and carried out methodological validation in healthy individuals.The method demonstrated stability and repeatability,indicating its potential use for detecting CAR-T-specific cellular immunity in patients.Part Ⅱ:Detection of CAR-T-Specific Humoral ImmunityObjective:The detection of CAR-T-specific anti-drug antibodies(ADAs)is important for assessing the immunogenicity of CAR-T therapy.Flow cytometry is commonly used for this purpose,but it has limitations due to interference from other antibodies in serum.Therefore,we aimed to develop a bridging method based on electrochemiluminescence(ECL)technology to detect CAR-T-specific humoral immunity.Methods:To generate recombinant plasmids expressing scFv antibodies,B cells were isolated from the spleen of rabbits that had been immunized with scFv.Positive clones with high affinity for scFv were identified by specific screening,and ADA standard products were obtained after purification.A three-level screening,confirmation,and titration process was established to detect CAR-T-specific ADAs,which was validated by healthy human serum and ADA standard products.The verification included evaluating method sensitivity,drug tolerance,hook effect,matrix effect,precision,and sample stability.Results:After multiple rounds of ELISA positive and negative screening,a clone was finally selected as the ADA standard.Various parameters in the detection method were calculated,including screening cut-point(1.27),confirmatory cut-point(39.1%),and titration cut-point(1.52).The established detection method met the FDA standard and passed methodological verification.Conclusions:The CAR-T-specific ADA detection method based on bridging ECL established in this study has good reliability and stability.Part Ⅲ:Clinical Application of CAR-T Specific Immune Response DetectionObjective:This part of the study aimed to explore the relationship between CAR-T immunogenicity and efficacy by applying the detection methods established in the first two parts to clinical samples of patients with hematological malignancies who received CAR-T therapy.Methods:The study included patients with hematological malignancies who received CD 19/22 CAR-T cell therapy or human-derived BCMA CAR-T cell therapy.The established detection methods were used to detect specific immunity in these patients,and the results were analyzed in relation to copy number changes after CAR-T infusion.Results:Sixteen patients treated with murine CD 19/22 CAR-T and 25 patients treated with human BCMA CAR-T were included in the study.In the first group,2 out of 16 patients(12.5%)tested positive for specific cellular immunity.The peptides that elicited the immune response of the first positive patient were located in the hinge region of CD8α,CD28,and CD3ζ.The second positive patient had peptides localized to CD 19 scFv and CD22 scFv,and experienced relapse due to poor CAR-T cell performance and decreased copy numbers of the two CARs,which is consistent with the generation of scFv against both CARs.In the second group,only 1 out of 25 patients(4%)was confirmed positive for specific humoral immunity.The decline of CAR copy number in this patient was synchronous with the increase of ADA,which negatively impacted the survival of CAR-T cells.Conclusions:Cellular immunity against CAR-T cells may impact the efficacy of CAR-T therapy,while humoral immunity targeting CAR-T cells in vivo may affect the persistence of the cells.However,the effect of humoral immunity on efficacy was not significant.