Re-adapting T cells for cancer therapy: from mouse models to clinical trials

Abstract

Adoptive T-cell therapy involves the isolation, expansion, and reinfusion of T lymphocytes with a defined specificity and function as a means to eradicate cancer. Our research has focused on specifying the requirements for tumor eradication with antigen-specific T cells and T cells transduced to express a defined T-cell receptor (TCR) in mouse models and then translating these strategies to clinical trials. Our design of T-cell-based therapy for cancer has reflected efforts to identify the obstacles that limit sustained effector T-cell activity in mice and humans, design approaches to enhance T-cell persistence, develop methods to increase TCR affinity/T-cell functional avidity, and pursue strategies to overcome tolerance and immunosuppression. With the advent of genetic engineering, a highly functional population of T cells can now be rapidly generated and tailored for the targeted malignancy. Preclinical studies in faithful and informative mouse models, in concert with knowledge gained from analyses of successes and limitations in clinical trials, are shaping how we continue to develop, refine, and broaden the applicability of this approach for cancer therapy.

Keywords: TCR gene therapy; adoptive T-cell therapy; clinical trials; high affinity; preclinical models.

Fig. 1. Lessons learned from the clinic

(A) Mean effective concentrations of peptide required to achieve 50% lysis of TAP-deficient HLA A*0201⁺ B-lymphoblastoid cell lines (T2 B-LCL) pulsed with titrating doses of peptide at an effector to target ratio (E:T) of 10:1 by the CD8⁺ T-cell clones infused into each patient. Arrows indicate the avidity of the clones infused into patients described in Fig. 1B (1) and Fig. 1C (2), respectively. (B) Percent of leukemic blasts in the blood (grey bars) measured at indicated timepoints before and after the infusion of 3.3 × 10⁹ cells/m² WT1-specific CD8⁺ T cells (vertical arrow). (C) Percent multimer⁺CD8⁺ T cells (y-axis) among total CD8⁺ T cells (solid circles) and in BM (open circles) collected before and after infusions. Vertical arrows indicate time of infusions.

Fig. 2. Peripheral T cells transduced with enhanced-affinity TCRs surpass the threshold of thymic selection

(A) Splenocytes from P14 mice were transduced to express the wildtype-affinity WT1-specific TCR from clone 3D (3D-wt), which was the highest affinity clone that could be isolated from the normal peripheral T cell pool, or one of two CD8-independent enhanced affinity variants of 3D-wt: 3D-NYH (intermediate affinity), and 3D-PYY (highest affinity). (B) Splenocytes were isolated from retrogenic mice generated by transducing RAG2−/− bone marrow HSC with the 3D-wt or enhanced-affinity TCR constructs, followed by transfer into lethally irradiated B6 recipients. TCR-transduced P14 T cells (A) or retrogenic T cells (B) were analyzed for relative TCR transgene expression as measured by Vβ10 staining, and for staining with WT1 tetramer. Transduced P14 T cells or retrogenic T cells were also stimulated with WT1 peptide, expanded for 1 week in vitro, and then assayed for functional avidity by measuring interferon-γ production in response to antigen-presenting cells pulsed with decreasing concentrations of WT1 peptide.

Fig. 3. Preclinical modeling to maximize therapeutic efficacy of T cells during adoptive immunotherapy of PDA

(A) Histopathology of PDA in representative sections from KPC mice demonstrates extensive desmoplasia as indicated by Masson’s trichrome staining of collagen in the ECM. (B) Influx of distinct subsets of CD11b⁺ myeloid cells to the spleen and tumors of KPC mice compared to non-tumor bearing mice. Plot is gated on CD45⁺ cells. (C) CD4⁺FoxP3⁺ Tregs accumulate in PDA in KPC mice. CD45⁺CD4⁺ cells isolated from collagenase-digested KPC tumors were analyzed for expression of CD25 and FoxP3. (D) Frequency of tetramer-binding MSLN-specific CD8⁺ T cells in wildtype mice before and after immunization with Ad-MSLN, and following two cycles of in vitro expansion of MSLN-reactive splenocytes from immunized mice stimulated with MSLN and IL-2. (E) A comparison of the mean fluorescence intensity (MFI) of TCR chain (Vβ) staining and tetramer staining of MSLN-specific T-cell clones isolated from T cell lines derived from immunized wild type (black bars) and MSLN−/− (red bars) mice. (F) Representative high-resolution ultrasound (Vevo 2100) of a pancreatic mass in the body of the pancreas of a 4 month old KPC mouse that met the enrollment criteria for T-cell therapy. PV, portal vein; T, tumor.