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. 2024 Sep;300(9):107651.
doi: 10.1016/j.jbc.2024.107651. Epub 2024 Aug 8.

Fast on-rates of chimeric antigen receptors enhance the sensitivity to peptide MHC via antigen rebinding

Hiroyuki Hiratsuka  1 , Yasushi Akahori  2 , Shingo Maeta  3 , Yuriko Egashira  3 , Hiroshi Shiku  4
Affiliations

Fast on-rates of chimeric antigen receptors enhance the sensitivity to peptide MHC via antigen rebinding

Hiroyuki Hiratsuka et al. J Biol Chem. 2024 Sep.

Erratum in

Abstract

Chimeric antigen receptor (CAR) is a synthetic receptor that induces T cell-mediated lysis of abnormal cells. As cancer driver proteins are present at low levels on the cell surface, they can cause weak CAR reactivity, resulting in antigen sensitivity defects and consequently limited therapeutic efficacy. Although affinity maturation enhances the efficacy of CAR-T cell therapy, it causes off-target cross-reactions resulting in adverse effects. Preferentially expressed antigen in melanoma (PRAME) is an intracellular oncoprotein that is overexpressed in various tumors and restricted in normal tissues, except the testis. Therefore, PRAME could be an ideal target for cancer immunotherapy. In this study, we developed an experimental CAR system comprising six single-chain variable fragments that specifically recognizes the PRAMEp301/HLA-A∗24:02 complex. Cell-mediated cytotoxicity was demonstrated using a panel of CARs with a wide range of affinities (KD = 10-10-10-7 M) and affinity modulation. CAR-T cells with fast on-rates enhance antigen sensitivity by accelerating the killing rates of these cells. Alanine scanning data demonstrated the potential of genetically engineered CARs to reduce the risk of cross-reactivity, even among CARs with high affinities. Given the correlation between on-rates and dwell time that occurs in rebinding and cell-mediated cytotoxicity, it is proposed that CAR-binding characteristics, including on-rate, play a pivotal role in the lytic capacity of peptide-major histocompatibility complex-targeting CAR-T cells, thus facilitating the development of strategies whereby genetically engineered CARs target intracellular antigens in cancer cells to lyse the cells.

Keywords: T cell; binding affinity; cancer; cell engineering; chimeric antigen receptor; immunotherapy; major histocompatibility complex (MHC); peptides; receptor.

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Conflict of interest statement

Conflicts of interest Hiroyuki Hiratsuka and Yasushi Akahori have filed provisional patent applications for the sequence data of WT98 (WO 2022124282-A/1–WO 2022124282-A/8) and WT163 (JP2024–115711). The late Hiroshi Shiku was involved in the patent application for WT98. Part of this research was funded by a cooperative research grant from Sysmex Corporation. Shingo Maeta and Yuriko Egashira are affiliated with Sysmex Corporation.

Figures

Figure 1
Figure 1
Characterization of PRAME-specific CARs.A, schematic representation of the PRAME CAR construct containing the germline signal sequence. B, dot plots showing anti-lambda light chain staining for CAR expression in untransduced cells as background staining and WT163, WT98, 98A, 98G, 98J, and 98B CARs by flow cytometry. The left column of the dot plots shows CD8+ CAR+, whereas the right column shows CD4+ CAR+ in the representative data. C, expansion of PBMCs, including T cells, transduced with the six CARs. Data are presented as mean ± SDs of three independent experiments. D, PRAMEp301/HLA-A∗24:02-dependent degranulation of CD8+ T cells. CAR-T cells were cocultured with T2A24 cells pulsed with 10 μM PRAMEp301 or CMVpp65 peptides for 5 h, and then degranulated CD107a was measured by flow cytometry. Data are representative of two independent experiments and are shown as means ± SDs of duplicates. C and D, ∗∗∗∗p < 0.0001; ns, no significance was observed using two-way ANOVA with Tukey’s test. CAR, chimeric antigen receptor; CMV, cytomegalovirus; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cell; PRAME, preferentially expressed antigen in melanoma.
Figure 2
Figure 2
CAR-T cells induce functional activity in endogenous PRAME-expressing tumor cells.A, PRAME mRNA expression was determined using qPCR. The COS-7 cell line was used as a negative control. B, HLA-A∗24:02 cell surface expression was detected using flow cytometry. Cells stained with Alexa488 alone indicate the background. C, the indicated cell lines were stained with or without soluble WT98 along with the secondary antibody, and then with the tertiary antibody conjugated with PE. D, schematic representation of in vitro real-time killing system. E, the tumor-killing capacity of CAR-T cells was measured by the continuous decrease in impedance of the xCELLigence system against SK-MEL-124 and SK-MEL-128 cell lines at an E:T ratio of 5:1. F, killing rates were calculated as rate constants using a one-phase exponential decay curve fit. Data are representative of the duplicates. G, killing rates of CAR-T cells against the SK-MEL-124 and SK-MEL-128 cell lines. A, E, and G, data represent the mean ± SDs of the duplicates. E and G, data are representative of two independent experiments. G, ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; ns, no significance observed using one-way ANOVA with Tukey’s test. CAR, chimeric antigen receptor; HLA, human leukocyte antigen; PRAME, preferentially expressed antigen in melanoma; qPCR, quantitative PCR.
Figure 3
Figure 3
Alternative specificity for CAR-T cells containing six unique scFvs.A, relative HLA-A∗24:02 binding stability of each peptide to determine the amino acid residues that bind HLA-A∗24:02. T2A24 cells were loaded with peptides (50 μM) overnight, and HLA-A∗24:02 expression on the cell surface was measured using flow cytometry and compared with that of unloaded T2A24. B, alanine scanning of LYVDSLFFL identified significant peptide residues for recognition by CAR-T cells that carry six unique scFvs. CAR-T cells were incubated for 24 h with T2A24 cells pulsed with each peptide (300 nM) and the ratio of IFN-γ to PRAMEp301 was measured in the culture supernatants. Based on the significance of the effector alone and the positive control PRAMEp301, amino acids with gray arrows were identified as those that abolished CAR binding. Data are shown as mean ± SDs of triplicate experiments. Data are representative of two independent experiments. B, ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; ns, no significance observed using one-way ANOVA with Tukey’s test. CAR, chimeric antigen receptor; HLA, human leukocyte antigen; IFN-γ, interferon gamma; PRAME, preferentially expressed antigen in melanoma; scFV, single-chain variable fragment.
Figure 4
Figure 4
Relationship between CAR-T cell ligand potency and antibody binding affinity.A, dose-response curve based on cytotoxicity induced by LDH release. Lysis% of CAR-T cells coincubated for 4 h with T2A24 cells pulsed with the indicated PRAMEp301 peptide. B, dose-response curves of granzyme B secretion by CAR-T cells compared with T2A24 cells pulsed with the indicated PRAMEp301 peptide. ELISA was used to measure the levels of secreted granzyme B. C, EC50 for lysis% from the LDH-based cytotoxicity assay plotted over the KD, on-rate, off-rate, and ta in log-log-transformed values. D, EC50 of secreted granzyme B of CAR-T cells plotted over the KD, on-rate, off-rate, and ta on log-log-transformed values. The solid lines represent the fit to the simple linear regression. Data represent two independent experiments and are presented as mean ± SDs of duplicates. CAR, chimeric antigen receptor; LDH, lactate dehydrogenase; PRAME, preferentially expressed antigen in melanoma.
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