A library of cancer testis specific T cell receptors for T cell receptor gene therapy

Abstract

To increase the number of cancer patients that can be treated with T cell receptor (TCR) gene therapy, we aimed to identify a set of high-affinity cancer-specific TCRs targeting different melanoma-associated antigens (MAGEs). In this study, peptides derived from MAGE genes with tumor-specific expression pattern were identified by human leukocyte antigen (HLA) peptidomics. Next, peptide-HLA tetramers were generated, and used to sort MAGE-specific CD8⁺ T cell clones from the allogeneic (allo) HLA repertoire of healthy donors. To evaluate the clinical potential, most potent TCRs were sequenced, transferred into peripheral blood-derived CD8⁺ T cells, and tested for antitumor efficacy. In total we identified, seven MAGE-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6, and MAGE-A9 in the context of HLA-A∗01:01, -A∗02:01, -A∗03:01, -B∗07:02, -B∗35:01, or -C∗07:02. TCR gene transfer into CD8⁺ T cells resulted in efficient reactivity against a variety of different tumor types, while no cross-reactivity was detected. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8⁺ T cells were observed in the orthotopic xenograft model for established multiple myeloma. The identification of seven MAGE-specific TCRs expands the pool of cancer patients eligible for TCR gene therapy and increases possibilities for personalized TCR gene therapy.

Keywords: CD8+ T cells; Cancer Testis genes; MAGE; T cell receptor; TCR gene therapy.

Graphical abstract

Figure 1

Selection of MAGE-reactive T cells (A) Flowchart describing the four steps for isolation of MAGE-reactive T cell clones. T cell clones were selected based on peptide titration experiment and specific tumor cell line recognition. (B) Six of 18 T cell clones recognizing MAGE-A1 KVLEYVIKV in HLA-A∗02:01 (MAGE-A1 KVL/A2) are depicted in this graph that were overnight co-cultured with KVL peptide loaded Raji cells. These six T cell clones are a representative for T cell clone selection process of all 187 MAGE-specific T cell clones. (C and D) T cell clones were stimulated with tumor cell lines of different origin, including multiple myeloma (RPMI8226, U266, L363, OPM-2, UM9), osteosarcoma (U2-OS, ZK-58, Saos-2), melanoma (88.23, MEL01.14, SK2.3, 518A2, OPM-2), chronic myeloid leukemia k562, cervix carcinoma Caski, and prostate carcinoma cell line PC-3M-PRO4. T cell reactivity was demonstrated by IFN-γ production, as measured by ELISA, after an overnight co-culture. MAGE-gene expression levels, measured by qPCR, are depicted between brackets as percentage relative to HKGs. Targeted HLA was naturally expressed or transduced (+) into the target cell. (C) Four of 18 MAGE-A1 KVL/A2 recognizing T cell clones are depicted as representatives. T cell clones 4A2 and 4F7 strictly recognize the MAGE-expressing tumor cell lines efficiently and therefore selected for further analysis. (D) Results of all selected T cell clones reactive against MAGE-A1 LTQDLVQEKYLEY in HLA-A∗01:01 (MAGE-A1 LTQ/A1), MAGE-A1 KVL/A2, MAGE-A1 SLFRAVITK in HLA-A∗03:01 (MAGE-A1 SLF/A3), MAGE-A1 RVRFFFPSL in HLA-B∗07:02 (MAGE-A1 RVR/B7), MAGE-A1 VRFFFPSL in HLA-C∗07:02 (MAGE-A1 VRF/C7), MAGE-A3/A6 EVDPIGHLY/EVDPIGHVY in HLA-B∗35:01 (MAGE-A3/A6 EVD/B35), and MAGE-A1 YVGKEHMFY in HLA-A∗01:01 (MAGE-A9 YVG/A1). The Burkitt lymphoma Raji, negative for all MAGE antigens, was included as negative control. To confirm proper HLA expression and recognition capacity of the targets allo-HLA reactive T cell clones (white bars) were included for each HLA specificity. Values and error bars represent mean and standard deviations of technical duplicates. Experiments are representative of at least two independent experiments.

Figure 2

Recognition of specific MAGE-A family members by our selected T cell clones (A) T cell clones were stimulated with Raji transduced with the different MAGE-A family members, including MAGE-A1 (A1), MAGE-A2 (A2), MAGE-A3 (A3), MAGE-A4 (A4), MAGE-A6 (A6), MAGE-A8 (A8), MAGE-A9 (A9), MAGE-A10 (A10), MAGE-A11 (A11), and MAGE-A12 (A12). Cytokine production was measured by IFN-γ ELISA after an overnight co-culture. In all experiments, an allo-HLA reactive T-cell clone (white bars) was included to confirm proper HLA expression and stimulatory capacity of the targets. Values and error bars represent means and standard deviations of technical duplicates. (B) In peptide titration experiments the three selected MAGE-A3/A6 EVD/B35 T cell clones were separately stimulated with MAGE-A3^EVD or MAGE-A6^EVD peptide loaded on Raji cells. T cell reactivity was determined by IFN-γ production, as measured by ELISA.

Figure 3

Cross-reactivity against a peptide in the context of the targeted HLA Cross-reactivity was determined by an overnight co-culture of T cells with MAGE-gene negative tumor cell lines (left side) and healthy subsets (right side) from different tissue origin. The cell lines included originated from acute myeloid leukemia (AML), bile duct carcinoma (BILE), colon carcinoma (CC), cervical carcinoma (CER), T cell leukemia (LT), melanoma (MEL), multiple myeloma (MM), ovarian carcinoma (OC), prostate carcinoma (PC), and pulmonary carcinoma (PUL) and fibroblast cell lines (FC). The healthy subsets included were CD19-positive cells (CD19⁺), immature dendritic cells (imDC), mature dendritic cells (mDC), activated T cells (CD4/CD8), fibroblasts (Fibro), and keratinocytes (Kera). Each dot in the healthy subset panel represents the average of at least two of these cell subsets derived from different healthy donors. All included targets expressed the MAGE-A genes of interest <1% relative to HKGs and naturally expressed the HLA of interest or were HLA-Td (+HLA). IFN-γ production was measured by ELISA after an overnight co-culture assay. Proper HLA presentation and recognition capacity of the target cells was confirmed by allo-HLA reactive T cell clones (data not shown). Raji transduced or naturally expressing the HLA and MAGE gene of interest was included as a positive control. Error bars depict standard deviations.

Figure 4

TCR expression and cytotoxicity of transduced primary CD8⁺ T cells against malignant cell lines (A) The TCR expression of selected TCRs (6G4 (MAGE-A1 LTQ/A1), 4F7 (MAGE-A1 KVL/A2), 3H4 (MAGE-A1 SLF/A3), 3G2 (MAGE-A1 RVR/B7), 10C1 (MAGE-A1 VRF/C7), 2H9 (MAGE-A3/A6 EVD/B35), and 2D8 (MAGE-A9 YVG/A1) was determined after transduction in primary CD8⁺ T cells. TCR-T cells were stained with mTCRβ APC (left) and pHLA tetramer PE (right). In the graph, the delta mean fluorescent intensity (MFI) (sample MFI – control MFI) of the tetramer and mTCRβ stain are depicted. Untransduced or CMV TCR-T cells were included as negative control (depicted in gray). (B) Cytotoxicity of the TCR-T cells against multiple tumor cell lines was determined by 6-h ⁵¹Cr-release assay with E:T ratio of 9:1 or 1:1. T cell clones were stimulated with tumor cell lines of different origin, including multiple myeloma (L363, U266, RPMI8226, UM9), melanoma (518A2, SK2.3), osteosarcoma cell line Saos-2, mammary carcinoma cell line CAMA-1, and prostate carcinoma cell line PC-3M-PRO4. Target cells naturally express target HLA or were transduced with target HLA alleles (+HLA). The MAGE-gene expression levels, measured by qPCR, are depicted between brackets as percentage relative to HKGs. CMV TCR-T cells were included as negative control and allo-HLA reactive T cell clones as positive control for HLA expression and killing capacity. Values and error bars represent means and standard deviations of technical triplicates, and experiments are representative of at least two independent experiments.

Figure 5

Reactivity of the MAGE-specific TCRs against early-passage melanoma cell lines (A) To determine recognition of early-passage melanoma cell lines (passage ≤10), TCR-T cells were stimulated. All melanomas (MEL) naturally expressed the HLA of interest except for MEL18.07 that was transduced with HLA-A∗01:01. IFN-γ production, as measured by ELISA, was determined after an overnight stimulation in at least two independent experiments. An allo-HLA reactive T cell clone was included for each target HLA as a positive control for HLA expression and stimulatory capacity of target cells. The MAGE-gene expression levels, measured by qPCR, are depicted between brackets as percentage relative to HKGs. Values and error bars represent means and standard deviations of technical duplicates. (B) Cytotoxicity of the TCR-T cells was analyzed by a 6-h ⁵¹Cr-release assay in at least two independent experiments. In Figure S5, the positive (allo-HLA reactive clones) and negative (CMV TCR) are shown. Values and error bars represent means and standard deviations of technical triplicates.

Figure 6

In vivo antitumor efficacy of MAGE-A1 specific HLA-A2, -A3, and -C7 restricted TCRs NSG mice engrafted with 2 × 10⁶ U266 cells were i.v. injected with 5 × 10⁶ TCR-T cells 14 days after tumor injection. T cells were transduced with the 4F7 (MAGE-A1 KVL/A2) TCR, 3H4 (MAGE-A1 SLF/A3) TCR, 10C1 (MAGE-A1 VRF/C7) TCR, or CMV (pp65 NLV/A2) TCR and enriched for mTCRβ expression by MACS. Tumor growth was visualized by bioluminescence imaging 1–2 times per week. (A) Mean of average tumor outgrowth of the dorsal and ventral side of 6 × 4F7 TCR-T cell (black), 6 × 10C1 TCR-T cell (red), and 4 × CMV TCR-T cell (white) treated mice. (B) Tumor outgrowth of representative individual 4F7 TCR-T cell, 10C1 TCR-T cell, and CMV TCR-T cell treated mice measured on the ventral side. (C) Mean of average tumor outgrowth of the dorsal and ventral side of 5 × 3H4 TCR-T cell (red) and 4 × CMV TCR-T cell (white). (D) Kaplan-Meier plot of 3H4 TCR-T cell and CMV TCR-T cell treated mice.