Preclinical evaluation of a novel CAR-T therapy utilizing a scFv antibody highly specific to MAGE-A4p230-239/HLA-A∗02:01 complex

Abstract

Despite the revolutionary success of chimeric antigen receptor (CAR)-T therapy for hematological malignancies, successful CAR-T therapies for solid tumors remain limited. One major obstacle is the scarcity of tumor-specific cell-surface molecules. One potential solution to overcome this barrier is to utilize antibodies that recognize peptide/major histocompatibility complex (MHCs) in a T cell receptor (TCR)-like fashion, allowing CAR-T cells to recognize intracellular tumor antigens. This study reports a highly specific single-chain variable fragment (scFv) antibody against the MAGE-A4_p230-239/human leukocyte antigen (HLA)-A∗02:01 complex (MAGE-A4 pMHC), screened from a human scFv phage display library. Indeed, retroviral vectors encoding CAR, utilizing this scFv antibody as a recognition component, efficiently recognized and lysed MAGA-A4⁺ tumor cells in an HLA-A∗02:01-restricted manner. Additionally, the adoptive transfer of T cells modified by the CAR-containing glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related receptor (GITR) intracellular domain (ICD), but not CD28 or 4-1BB ICD, significantly suppressed the growth of MAGE-A4⁺ HLA-A∗02:01⁺ tumors in an immunocompromised mouse model. Of note, a comprehensive analysis revealed that a broad range of amino acid sequences of the MAGE-A4p_230-239 peptide were critical for the recognition of MAGE-A4 pMHC by these CAR-T cells, and no cross-reactivity to analogous peptides was observed. Thus, MAGE-A4-targeted CAR-T therapy using this scFv antibody may be a promising and safe treatment for solid tumors.

Keywords: CAR; GITR; ICD; MAGE-A4 peptide; MHC complex; pMHC.

Graphical abstract

Figure 1

scFv antibody #17 recognizes MAGE-A4_p230-239/HLA-A∗02:01 with high specificity (A) The culture supernatant of scFv #17 specifically reacted with MAGE-A4 pMHC as detected by anti-cp3 (capsid protein 3) antibody followed by HRP-conjugated anti-mouse IgG. (B) MAGE-A4_p230-239/HLA-A∗02:01 was immobilized on a CAP sensor chip as the ligand, and 100, 200, 400, and 600 nM scFv #17-conjugated with His-FLAG for MAGE-A4_p230-239/HLA-A∗02:01 was applied as the analyte. After 120 s of association reaction, the dissociation reaction was measured, and the K_D value was calculated by global fitting. (C) Alanine substitution analysis identified MAGE-A4_p230-239 peptide residues essential for recognition by scFv #17. The MAGE-A4_p230-239 peptide sequence was substituted with alanine from residues 1 through 10. T2 was pulsed with the indicated peptides (see Table S1 for sequences) at 10 μM for 1 h. Subsequently, the cells were stained with an anti-DDDDK mouse monoclonal antibody for 1 h at R.T. and then with an Alexa Fluor 488-conjugated goat anti-mouse polyclonal antibody for 1 h at room temperature (RT) in the dark. Finally, the cells were analyzed by flow cytometry. (D) The peptides in (C) were subjected to an HLA-stabilizing assay to determine the critical amino acids required for HLA-A∗02:01 binding. T2 cells were used as peptide-loading cells and their HLA-A∗02:01 expression was determined by flow cytometry. (E) Validation of potential risk peptides derived from the human proteome database. T2 cells were pulsed with the indicated peptides (see Table S2 for sequences) at 10 μM for 1 h and analyzed by flow cytometry as described in (C). (F) The peptides in (E) were subjected to an HLA-stabilizing assay. T2 cells were used as peptide-loading cells and their HLA-A∗02:01 expression was determined by FACS.

Figure 2

MAGE CAR-T cells show functional capacities in vitro (A) Schematic representation of retroviral vectors encoding MAGE-A4_p230-239/HLA-A∗02:01-specific CAR. The transgene contains the 5′ long terminal repeat (LTR) region, a packaging signal (Ψ), the IgG leader sequence region, the scFv (VH-VL sequence) derived from the MAGE A4-pMHC complex-specific monoclonal antibody #17, the CL region, the human CD28 transmembrane region (CD28 TM), human CD3ζ, the intracellular domain of human GITR (GITR ICD), and the 3′-LTR region. (B) IFN-γ production of MAGE CAR-T in response to the indicated tumor cell lines. Culture supernatants were harvested at 24 h and subjected to an IFN-γ ELISA in triplicate. Error bars represent the standard deviation (SD) of the mean. NGMC, mock-transduced T cells; MAGE-T2, T2 cells pulsed with MAGE-A4_p230-239; ESO1-T2, T2 cells pulsed with NY-ESO-1_p157-165. A representative result of three independent experiments is shown. (C) Cytotoxic activity of CAR⁺ T cells against the indicated tumor cell lines assessed by 2-h non-radioactive system in triplicate as described in section “materials and methods.” Error bars represent the SD of the mean. A representative result of three independent experiments is shown. (D) HLA-A2 expression on the indicated tumor cell lines. Values of net mean fluorescence intensity (MFI) were calculated by subtracting the MFI of cells stained by isotype control from that stained by anti-HLA-A2 monoclonal antibody (mAb). (E) Production of IFN-γ by zG CAR-T cells or NGMCs stimulated with 397mel or HLA-A∗02:01 transduced-397mel. Culture supernatants were harvested at 24 h and subjected to IFN-γ ELISA in triplicate. Error bars represent the SD of the mean. A representative result of three independent experiments is shown. (F) Production of IFN-γ by zG CAR-T cells or NGMCs stimulated with HCT116 or MAGE-A4 transduced-397mel. Error bars represent the SD of the mean. A representative result of three independent experiments is shown.

Figure 3

zG.CAR-T cells show superior in vivo function in suppressing tumor growth in a NOG mouse model (A) Schematic representation of retroviral vectors encoding each type of MAGE-A4_p230-239/HLA-A∗02:01-specific CAR. (B) Expression of CARs on human T cells. Cells were stained with phycoerythrin-labeled HLA-A∗02:01 tetramers presenting MAGE-A4_p230-239 along with PE-cy7 anti-CD8 and APC anti-CD4. NGMCs served as background staining. (C) Schematic representation of the adoptive transfer experiment using NOG mice (upper). Growth curves of HCT116 and NW-MEL-38 tumors, and body weight of NOG mice (n = 3) transferred with CAR-T cells or NGMCs. Error bars represent the SD of the mean. ∗∗p < 0.01. Representative results from three independent experiments are shown. (D) Six days after intravenous injection of mock-transduced T cells (left) or zG.CD8⁺CAR⁺ T cells (right), NW-MEL-38 tumor tissues were harvested and subjected to immunohistochemical analysis. A representative result from two independent experiments is shown. (E) Production of IFN-γ by 28z, 4-1BBz, zG CAR-T cells, or NGMCs stimulated with T2 cells loaded with serially diluted MAGE peptide. Culture supernatants were harvested at 24 h and subjected to IFN-γ ELISA in triplicate. A representative result from two independent experiments is shown.

Figure 4

CD4⁺ zG.CAR⁺ T cells attenuate the antitumor efficacy of CD8⁺ zG.CAR⁺ T cells in a NOG mice model (A) Schematic representation of the adoptive transfer experiment using NOG mice. (B) Tumor growth curves of NW-MEL-38 tumors of NOG mice (n = 4) treated with PBS and CARs. Error bars represent the SD of the mean. ∗p < 0.05, ∗∗p < 0.01. Representative results from three independent experiments are shown. (C) Immunofluorescence analysis of NW-MEL38 tumor tissues at day 5 and day 10 after tumor inoculation. (D) Immunofluorescence analysis using HALO (Indica Labs) at day10. Error bars represent the SD of the mean. ∗p < 0.05.

Figure 5

Lack of cross-reactivity of MAGE CAR-T cells to analogous peptides on HLA-A∗0201 (A) Comprehensive amino acid substitution analysis identified amino acid residues that could be substituted at each position of the MAGE-A4_p230-239 peptide sequence for recognition by CD8⁺ MAGE CAR-T cells. An IFN-γ ELISPOT assay was conducted to examine the reactivity of #17zG.CD8⁺CAR⁺ T cells to T2 cells pulsed sequentially with 200 peptides, comprising MAGE-A4_p230-239 peptide substituted with all 20 amino acids. The percentage data shown in the heatmap in the upper column were calculated as follows: (experimental spot counts)/(spot counts of parental MAGE-A4_p230-239). Permissible amino acid residues were defined (lower column) based on a 0.1 (10%) cutoff value. (B) The indicated peptides (see Table 1 for sequences) were subjected to an HLA-stabilizing assay to determine the capacity binding to HLA-A∗02:01. T2 cells were used as peptide-loading cells and their HLA-A∗02:01 expression was determined by FACS. (C) Validation of potential risk peptides derived from the human proteome by BLAST search. An IFN-γ ELISPOT assay was conducted to examine the reactivity of #17zG.CD8⁺CAR⁺ T cells to T2 cells pulsed with the indicated peptides in triplicate.