Genetic Modification of T Cells for the Immunotherapy of Cancer

Abstract

Immunotherapy is a beneficial treatment approach for multiple cancers, however, current therapies are effective only in a small subset of patients. Adoptive cell transfer (ACT) is a facet of immunotherapy where T cells targeting the tumor cells are transferred to the patient with several primary forms, utilizing unmodified or modified T cells: tumor-infiltrating lymphocytes (TIL), genetically modified T cell receptor transduced T cells, and chimeric antigen receptor (CAR) transduced T cells. Many clinical trials are underway investigating the efficacy and safety of these different subsets of ACT, as well as trials that combine one of these subsets with another type of immunotherapy. The main challenges existing with ACT are improving clinical responses and decreasing adverse events. Current research focuses on identifying novel tumor targeting T cell receptors, improving safety and efficacy, and investigating ACT in combination with other immunotherapies.

Keywords: adoptive cell transfer; cancer immunotherapy; chimeric antigen receptors; gene-modified TCR transduced T cells; tumor-infiltrating lymphocytes.

Figure 1

Adoptive T cell therapy strategies. Adoptive T cell therapy for treating cancer patients requires ex vivo expansion of autologous T cells for infusion back into the patient. Adoptive T cell transfer of TIL (left side of the Figure) occurs by first resecting tumor lesions from a patient and then isolating tumor-reactive T cells from that sample. The tumor-reactive T cells are then expanded ex-vivo and infused back into the patient. Adoptive T cell transfer of genetically engineered T cells (right side of the Figure) occurs by first isolating PBL-derived T cells from patient blood then genetically modifying them to express a specific TCR or CAR. The TCR or CAR engineered T cells are then expanded ex-vivo and infused back into the patient.

Figure 2

TCR structure compared to 1st, 2nd, and 3rd generation CARs. T cell receptors and chimeric antigen receptors differ significantly in their structure and how they recognize antigen. (A) T cell receptors are an αβ heterodimer that associates with the CD3 complex. CD3 consists of 6 chains, an ε-δ heterodimer, an ε-γ heterodimer, and a ζ-ζ homodimer. Chimeric antigen receptors consist of a scFv fused to a hinge (usually CD8), transmembrane region, and (B) CD3ζ (first generation CAR) or (C) CD28 or 4-1BB and CD3ζ (second generation CAR) or (D) CD28 and 4-1BB and CD3ζ (third generation CAR).

Figure 3

T cell subtypes. T cells are generally classified based on their cytokine production profiles and effector function. They are activated or respond to APCs or targets differently based on how antigen is presented and the other signals (cytokines, chemokines, and cell surface molecules) they receive. (A) CD8⁺ effector and CD4⁺ helper T cells each possess unique TCRs that interact with MHC class I or MHC class II molecules respectively on APC or target cells. In this panel, CD4⁺ T cells are providing help to CD8⁺ T cells in the form of IL-2 and other signals not shown. (B) CD4⁺ regulatory T cells suppress immune responses by inhibiting T activation and function. (C) CD8⁺ T cells are mainly effector T cells capable of inducing target cell destruction.

Figure 4

Transduction, expression, and function of TIL 1383I TCR transduced human T cells. We use retroviral and lentiviral vectors to engineer normal and cancer patient PBL-derived T cells to express TCR. (A) The general structure of our TIL 1383I TCR retroviral vector is shown as follows: 5′ LTR, the Ψ⁺ packaging signal, the TCR α chain fused to a P2A self-cleavage peptide fused to the TCR β chain fused to a T2A self-cleavage peptide fused to the CD34t marker gene and 3′ LTR. (B) Expression of the TIL 1383I TCR in PBL-derived T cells from 3 normal donors. The TIL 1383I TCR expression is based on Vβ12 expression (Y axis) and the CD34 marker gene expression (X axis). Transduction efficiency before CD34 purification (left panels) and after CD34 purification is shown (right panels). (C) The amount of IFN-γ released by the TIL 1383I TCR transduced T cells is shown. HLA-A2⁺ tyrosinase_(368–376)⁺ stimulator cells include T2 loaded with 10 µg/mL tyrosinase_(368–376) peptide, 624 MEL, 1300 MEL, and 1383 MEL. HLA-A2⁺ tyrosinase_(368–376)⁻ stimulator cells include T2 alone or loaded with 10 µg/mL HCV_{(1406–1415)} peptide. HLA-A2⁻ tyrosinase_(368–376)⁺ stimulator cells were 624-28 MEL. The amount of IFN-γ released was measured in triplicate wells via ELISA. (D) HLA-A2 restricted, tyrosinase reactive antigen recognition by TIL 1383I TCR transduced CD8⁺ and CD4⁺ T cells was measured using intracellular IFN-γ assays. As before, HLA-A2⁺ tyrosinase_(368–376)⁺ stimulator cells include T2 loaded with 10 µg/mL tyrosinase_(368–376) peptide, 624 MEL, and 1300 MEL. HLA-A2⁺ tyrosinase_(368–376)⁻ stimulator cells include T2 cells loaded with 10 µg/mL HCV_{(1406–1415)} peptide. HLA-A2⁻ tyrosinase_(368–376)⁺ stimulator cells were 624-28 MEL. Cells were also stained with anti-CD4, andti-CD8, and anti-CD34 (not shown) mAb. The histograms shown were gated on CD34⁺ T cells (transduced). CD4 vs. IFN-γ (top panels) and CD8 vs. IFN-γ (bottom panels) staining is shown.

Figure 5

Transduction, expression, and function of CD19 transduced human T cells. We use retroviral and lentiviral vectors to engineer normal and cancer patient PBL-derived T cells to express TCR. (A) The general structure of our CD19 CAR retroviral vector is shown as follows: 5′ LTR, the Ψ⁺ packaging signal, the CD19 CAR which consists of CD19 V_L fused to CD19 V_h by a flexible linker followed by CD8 hinge then a CD28 cassette followed by CD3ζ. The CAR is fused to a T2A self-cleavage peptide fused to the CD34t marker gene followed by the 3′ LTR. (B) Expression of the CD19 CAR in PBL-derived T cells from 3 normal donors. CD19 CAR expression is based on the CD34 marker gene expression. Transduction efficiency of untransduced (Negative Gate), pre-CD34 selection, and post-CD34 selection is shown. Histograms represent CD3 expression (Y axis) and CD34 expression (X axis). (C) The amount of IFN-γ released by the CD19 CAR transduced T cells is shown. CD19⁺ stimulators include the line EBV, 836 EBV, and SEM and CD19⁻ stimulators include 624 MEL and 624-28 MEL. The amount of IFN-γ released was measured in triplicate wells via ELISA.