Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy

Abstract

Nine cancer patients were treated with adoptive cell therapy using autologous anti-MAGE-A3 T-cell receptors (TCR)-engineered T cells. Five patients experienced clinical regression of their cancers including 2 on-going responders. Beginning 1-2 days postinfusion, 3 patients (#'s 5, 7, and 8) experienced mental status changes, and 2 patients (5 and 8) lapsed into comas and subsequently died. Magnetic resonance imagining analysis of patients 5 and 8 demonstrated periventricular leukomalacia, and examination of their brains at autopsy revealed necrotizing leukoencephalopathy with extensive white matter defects associated with infiltration of CD3(+)/CD8(+) T cells. Patient 7, developed Parkinson-like symptoms, which resolved over 4 weeks and fully recovered. Immunohistochemical staining of patient and normal brain samples demonstrated rare positively staining neurons with an antibody that recognizes multiple MAGE-A family members. The TCR used in this study recognized epitopes in MAGE-A3/A9/A12. Molecular assays of human brain samples using real-time quantitative-polymerase chain reaction, Nanostring quantitation, and deep-sequencing indicated that MAGE-A12 was expressed in human brain (and possibly MAGE-A1, MAGE-A8, and MAGE-A9). This previously unrecognized expression of MAGE-A12 in human brain was possibly the initiating event of a TCR-mediated inflammatory response that resulted in neuronal cell destruction and raises caution for clinical applications targeting MAGE-A family members with highly active immunotherapies.

Trial registration: ClinicalTrials.gov NCT01273181.

Figure 1

Phenotype of infused cell products. Shown are FACS dot plots for patients 1–8 for cells surface marker proteins CD45RO and CD62L with the percentages of positive cells indicated in each quadrant. T cells subsets are defined as follows, T effector cells (Teff, CD45RO⁺/CD62L⁻), T central memory cells (Tcm, CD45RO⁺/CD62L⁺), and T naïve cells (Tn, CD45RO⁻/CD62L⁺).

Figure 2

Persistence of MAGE-A3 TCR post-infusion. Shown are the FACS density dot plots from patients PBMC isolated approximately one-month post-infusion. Scatter plots depict CD3⁺ and murine MAGE -A3 TCR beta chain expression on lymphoid cells, with the percentages of positive cells indicated in each quadrant. muTCR, murine beta chain TCR.

Figure 3

Biological activity of MAGE-A3 TCR engineered T cells in patient PBMC. A. PBMC samples from patients 1–8 were obtained approximately one month post-infusion and co-cultured with control tumor line H1299 (MAGE-A3⁺/HLA-A*0201⁻) or reactive tumor line H1299-A2 (MAGE-A3⁺/HLA-A*0201⁺). Shown are the resultant FACS density plots gated on CD3⁺/mouse-TCR beta⁺ for effector cytokines IFN-γ and TNF. The percentages of positive cells were as indicated in each quadrant. B. PBMC samples from patients 1–9 were obtained approximately one month post-infusion and subject to Elispot analysis. Number of Elispot reactive cells are plotted following PBMC co-culture with MAGE-A3-peptide pulsed cells (left side) or MAGE-A3⁺/HLA-A*0201⁺ tumor cell line (right side). Results from control peptide pulsed cells and MAGE-A3⁺/HLA-A*0201⁻ tumor cell lines were <10 spots/1 × 10⁵ PBMC (not shown).

Figure 4

Clinical anti-tumor response. Shown are CT scans for patients 1 and 5 and CT/PET scans for patient 7. The timing of the images were as listed.

Figure 5

Neurological imaging studies. Shown are MRI scans for patients 5, 7 and 8, with the timing of the images as listed.

Figure 6

Neurologic toxicity. Autopsy samples from patients 5 and 8 had microscopic sections that displayed similar changes consistent with necrotizing leukoencephalopathy, multifocal. There was diffuse white matter damage with sparing of gray matter (panel A, hematoxylin and eosin (HE) stain, patient 8, 10X). Marked white matter vaccuolation (patient 8, panel B, HE-20X) to frank areas of infarct/necrosis with mineralization (patient 5, panel C, HE -20X) were identified. Inflammatory infiltrates were present around small vessels and parenchyma and consisted of CD3⁺/CD8⁺ lymphocytes and KP1⁺ histiocytes (patient 5, panel D: CD3 immunostain at 2x, panel E: CD8 immunostain at 2x, panel F : KP-1 immunostain at 2× and patient 8; panel G: CD3 immunostain at 2x, panel H: CD8 immunostain at 2x)

Figure 7

MAGE-A3 TCR engineered T cells in CSF. A. Shown are FACS dot plots for patients 5, 7, and 8 for T cells obtained from CSF and expanded ex vivo (and PBL for patient 7). Samples were stained and plotted for murine TCR beta (muTCR) and CD8. B. Ex vivo expanded cells from patient 5’s CSF sample were co-cultured with control tumor lines 888 mel and 938 mel (MAGE-A3⁺/HLA-A*0201⁻) or reactive tumor cell lines 526 mel, 624 mel, and H1299-A2 (MAGE-A3⁺/HLA-A*0201⁺). Shown is the resultant IFN-γ production following overnight culture. C. Samples taken directly from the CSF of patient 8 were subject to rapid amplification of cDNA ends (5′-RACE) PCR to amplify the variable region of human TCR beta chain genes. Shown are the results of DNA sequence analysis as a pie chart showing the specific TCR vbeta gene (color) as well as its frequency (size of pie slice).

Figure 8

Serum cytokine production. Shown are the gamma-interferon (IFN-γ) levels (pg/ml) found in the serum of patients 1–8 during the times indicated (time = 0 is the day of infusion).

Figure 9

Recognition of MAGE-A3-like peptides. The human genome was searched for peptide similar to MAGE-A3 that were predicted to have physiologically relevant HLA-A*0201 binding affinities and were expressed in human brain. These eight peptides were synthesized and pulsed onto T2 cells along with MAGE-A3 and MAGE-A12 peptides at the indicated concentrations. Shown is the resultant gamma-interferon (IFN-γ) production (pg/ml) following overnight co-culture.

Figure 10

Analysis of human peptidome. The 114 most highly prevalent peptides found on the surface of HLA-A*0201+ normal tissues including brain were used to test for reactivity of MAGE-A3 TCR transduced T cells. Shown are the resultant IFN-γ levels produced in overnight co-cultures with peptide pulsed cells, open bars-untransduced T cells, solid bars-MAGE-A3 TCR transduced T cells. Shown is data for patient 5, similar results were obtained with patient 8 and a patient treated with NY-ESO-1 TCR transduced T cells. Co-cultures with melanoma cells mel 624.38 (+) and stimulation with PMA/ionomycin (P) served as positive controls per 96 -well plate to exclude differences between plates. Medium alone, PBL alone, T2 cells alone and PBL co-cultured with unpulsed T2 cells (−) were used as negative controls.

Figure 11

Test for recognition of neuronal cells. A. Fetal-derived neural progenitor cells were transduced with a lentiviral vector expressing GFP or HLA-A*0201 and induced to differentiate into neural cells. Image of resultant GFP-transduced neural cells was as shown on the left. These cells were co-cultured with MAGE-A3 TCR transduced or untransduced (UnTd) T cells. As control for recognition, neural cell cultures were pulsed with MAGE-A3 peptide or as a negative control, SSX-2 peptide. B and C. Autologous dendritic cells were prepared from patient 5 and loaded with cell lysates from neuroblastoma cell line SK-N-SH, whole brain homogenate (BH), or pulsed with MAGE-A3 peptide (MAGE-A3). These were then co-cultured with T cells derived from patient 5; pre-treatment PBMC (periph), T cells derived from the ex vivo expanded CSF (CSF), the MAGE-A3 TCR transduced infusion cell product (TCR), or ex vivo cultured, untransduced T cells (UnTd). Reactivity was measured by CD107 amobilization (B) or 8-day cell proliferation measured by CFSE dilution (C). Reactivity in both CD8+ (left) and CD4+ (right) T cells was as shown.

Figure 12

MAGE-A expression in human brain. MAGE-A family reactive antibody 6C1 was used for immunohistochemical staining of brain sections. Positive staining was observed in neuronal cell bodies and processes in three control patients (A at 10x, B at 20x, and C at 10x), in patient 5 (D at 20x, E at 40x, and F at 40x), and patient 8 (G at 10x, H at 20x, I at 20x).