Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2

Abstract

In an attempt to treat cancer patients with ERBB2 overexpressing tumors, we developed a chimeric antigen receptor (CAR) based on the widely used humanized monoclonal antibody (mAb) Trastuzumab (Herceptin). An optimized CAR vector containing CD28, 4-1BB, and CD3zeta signaling moieties was assembled in a gamma-retroviral vector and used to transduce autologous peripheral blood lymphocytes (PBLs) from a patient with colon cancer metastatic to the lungs and liver, refractory to multiple standard treatments. The gene transfer efficiency into autologous T cells was 79% CAR(+) in CD3(+) cells and these cells demonstrated high-specific reactivity in in vitro coculture assays. Following completion of nonmyeloablative conditioning, the patient received 10(10) cells intravenously. Within 15 minutes after cell infusion the patient experienced respiratory distress, and displayed a dramatic pulmonary infiltrate on chest X-ray. She was intubated and despite intensive medical intervention the patient died 5 days after treatment. Serum samples after cell infusion showed marked increases in interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10, consistent with a cytokine storm. We speculate that the large number of administered cells localized to the lung immediately following infusion and were triggered to release cytokine by the recognition of low levels of ERBB2 on lung epithelial cells.

Figure 1

Expression of the ERBB2 CAR. Diagram of the ERBB2-CAR vector (MSGV1-4D5-CD8-28BBZ) used in this trial is as shown on the top of the figure. As described in Materials and Methods section, patient PBMC were stimulated to induced T-cell division and then transduced with the CAR vector. Four days before cell infusion samples were removed for analysis of ERBB2-CAR gene expression by FACS (along with the CD3, CD4, or CD8 T-cell markers). CAR, chimeric antigen receptor; LTR, long terminal repeats; PBMC, peripheral blood mononuclear cell; scFv, single-chain Fv fragment.

Figure 2

Serum cytokine levels. Serum samples obtained at the approximate times indicated after cell infusion were assayed for cytokine expression using commercial ELISA kits for cytokines IL-6, TNF-α, GM-CSF, and IFN-γ. The levels of cytokine IL-10 were independently determined by cytokine array (SearchLight) assay. All samples were diluted as necessary as to be in the linear range of the assay. ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte macrophage-colony stimulating factor; IFN-γ, interferon-γ IL, interleukin; TNF-α, tumor necrosis factor-α.

Figure 3

Cytokine genotype. DNA extracted for patient PBMC was subject to PCR with sequence-specific primers (PCR-SSP) as described in Materials and Methods section. Primer pairs are designed to have perfect matches only with a single allele or group of alleles. Matched primer pairs result in the amplification of target sequences (i.e., a positive amplification band), whereas mismatched primer pairs do not result in amplification (i.e., a negative result). The specific genotypes detected by the SSP are shown above each lane (Neg cntl, reaction without cytokine primers). Shown is one of duplicate determinations. IFN-γ, interferon-γ IL, interleukin; SNP, single-nucleotide polymorphism; TNF-α, tumor necrosis factor-α.

Figure 4

In vitro cytokine production. An aliquot of the ErbB2-CAR transduced (CAR) T lymphocytes infused into the patient or untransduced control T cells (UnTd) where assayed for IFN-γ cytokine production following overnight coculture with the indicated cell lines. ErbB2⁺ target cells were SK-OV3, SK-BR3, and MDA361. ErbB2⁻ tumor lines were MDA468 and CCRF-CEM (CEM). Primary cells adapted for growth in culture were HDF, human diploid fibroblast, HUVEC, human umbilical vein endothelial cells; EBV-B-EBV transformed B cell line, NHBE, normal human bronchial/tracheal epithelial cells; PrEC, human prostate epithelial cells, HSMM, human skeletal muscle myoblasts, keratinocytes-human keratinocytes, HMEC, human mammary epithelial cells, and HRE, human renal epithelial cells. Autologous patient cells were auto-DC-patient 6-day dendritic cell culture, and auto-mac-patient 6-day macrophage culture. All samples were diluted as necessary as to be in the linear range of the assay (sample SK-OV3 was off-scale in this assay and the value from a repeat determination was as indicated). CAR, chimeric antigen receptor; IFN-γ, interferon-γ.