Applications of molecular engineering in T-cell-based immunotherapies

Abstract

Harnessing an individual's immune cells to mediate antitumor and antiviral responses is a life-saving option for some patients with otherwise intractable forms of cancer and infectious disease. In particular, T-cell-based engineered immune cells are a powerful new class of therapeutics with remarkable efficacy. Clinical experience has helped to define some of the major challenges for reliable, safe, and effective deployment of T-cells against a broad range of diseases. While poised to revolutionize immunotherapy, scalable manufacturing, safety, specificity, and the development of resistance are potential roadblocks in their widespread usage. The development of molecular engineering tools to allow for the direct or indirect engineering of T-cells to enable one to troubleshoot delivery issues, amplify immunomodulatory effects, integrate the synergistic effects of different molecules, and home to the target cells in vivo. In this review, we will analyze thus-far developed cell- and material-based tools for enhancing T-cell therapies, including methods to improve safety and specificity, enhancing efficacy, and overcoming limitations in scalable manufacturing. We summarize the potential of T-cells as immune modulating therapies and the potential future directions for enabling their adoption for a broad range of diseases. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Nanotechnology Approaches to Biology > Cells at the Nanoscale.

Keywords: T-cells; adoptive cell transfer; cell engineering; immunotherapy.

Fig. 1. Methodologies for T-cell Engineering.

Examples of T-cell manufacturing techniques for generating immunotherapies derived from hematopoietic stem and progenitor cells (HSPCs) or mature T-cells. (A) HSPCs are transduced with viral vectors coding for transgenic T-cell receptor (TCR) or chimeric antigen receptor (CAR). Transduced HSPCs are expanded ex vivo and intravenously administered to a conditioned patent resulting in sustained in vivo differentiation into TCR-transduced T-cells or CAR T-cells. (B) Isolated HSPCs are transduced with viral vectors coding for transgenic TCR or CAR-T and differentiated on feeder cell layer allowing for ex vivo differentiation of TCR-transduced T-cells or CAR-T cells for adoptive immune transfer. (C) Isolated mature T-cells are transduced with viral vectors coding for transgenic TCR or CAR. TCR transduced T-cells or CAR-T cells are subsequently expanded for adoptive immune transfer. (D) Isolated mature T-cells are transduced with viral vectors coding for pluripotent stem cell reprogramming factors. Induced pluripotent stem cells (ISPCs) are expanded and seeded onto feeder cell layer to induce T-cell differentiation for adoptive immune transfer.

Fig. 2. Design of CAR T cells.

T cells can be redirected to have specificity for tumors by the introduction of chimeric antigen receptors (CAR) proteins. CARs targeting is controlled via an extracellular ectodomain that is comprised of light and heavy variable regions (VL and VH) from an antibody bound together with a peptide linker and attached to the transmembrane region with a hinge peptide. The endodomain controls intracellular signaling and activation and is comprised of conserved modules. First-generation CAR endodomains use the CD3-ζ ITAM, whereas second generation CARs include one costimulatory domain and third generation CARs contain multiple costimulatory domains. Fourth generation CARs termed “TRUCKs” that include an inducible pathway for the expression of a transgenic product.

Fig. 3. Schematic illustration of novel high-throughput approach for enrichment, culturing, and screening strategy of TILs.

(A) Tumor cell digests were thawed and rested overnight in complete media in the absence of exogenous cytokines. (B) A piece of the tumor underwent whole-exome sequencing (WES) and RNA sequencing to identify nonsynonymous mutations. Based on mutation calls, 25mer peptides encompassing the mutations at position 13 were synthesized. (C) Cells were washed, labeled, and sorted based on PD-1 and/or activation markers (CD134 or CD137) expression. Sorted cells were cultured in 96-well plates at 3 cells/well in the presence of irradiated allogeneic feeder cells, 3,000 IU/ml IL-2, and anti-CD3ε (OKT3) for expansion. (D) Peptide pools were pulsed on autologous APCs that served as a target in a coculture with sorted cells that grow in the microwell cultures. To minimize the assays, cells from 2 or 3 cultures were combined in the assay wells. (E) Cells from coculture assay were labeled and reactive T cells were single-cell sorted into 96-well plates containing lysis buffer and PCR primers for TCR sequencing. Adapted from Lu, Y.-C. et al. (2018). Molecular Therapy, 26(2), 379–389.

Fig. 4. Data showing function of ON-switch CAR demonstrating antigen-specific and titratable killing of target cell population by engineered primary cytotoxic (CD8⁺) T cells.

Schematic illustrating the variety of control mechanisms for CAR T-cells as well as their respective mechanisms for activation or deactivation. (A) ON CARs and AND CARs replicate AND gated Boolean logic, requiring multiple signals to initiate T-cell function, thereby improving specificity. (B) Dual CARs and TanCARs replicate OR gated Boolean logic, requiring one or both signals for T-cell activation to prevent tumor escape. (C) Suicide CARs and iCARs replicate Boolean logic in which a there is NOT gate on the inhibitory signal followed by an AND gate. This means that the T-cells will only function in the condition where there is stimulatory signal with no inhibitory signal. iCARs may be used to prevent activation when encountering epitopes found on healthy tissue, while suicide CARs represent the possibility of eliminating transferred T-cells after therapy via the administration of a small molecule.

Fig. 5. Generation of Higher Affinity TCRs.

(A) Schematic depicting transduction of antigen specific TCRα chain into T-cell progenitor cells and differentiation into SP T-cells after interaction with peptide loaded APC. Only TCRα chain coding region is transduced into progenitor cells allowing for normal rearrangement of TCRβ coding region. (B) The newly generated TCRs with endogenous TCRβ and transduced TCRα are subsequently screened for relative affinity for the cognate peptide by titrating the amounts of peptide/MHC tetramer and analyzing by flow cytometry. The relative change in affinity compared to the parental TCR is listed in parenthesis. (C) To measure off-target reactivity to a subset of antigens, Clone#1 from (B) is stained with WT1 specific tetramer, as well as several non-specific H-2Db tetramers.

Fig. 6. Reprogramming of peripheral blood T-cells into CAR-T-iPSC-T Cells.

(A)Schematic of study. Peripheral blood lymphocytes are reprogrammed to pluripotency by transduction with retroviruses encoding reprogramming factors c-MYC, SOX2, KLF4 and OCT-4. The resulting T-iPSCs are genetically engineered to express a CAR and are then differentiated into T cells that express both the CAR and an endogenous TCR. (B) In vitro lymphoid differentiation protocol. T-iPSCs were stably transduced with a lentiviral vector encoding the 19–28z CAR and the fluorescent marker mCherry. Differentiation in three steps: (i) mesoderm formation (days 1–4), (ii) hematopoietic specification and expansion (days 5–10) and (iii) T-lymphoid commitment (days 10–30). Fluorescence microscopy images (below) show mCherry expression was maintained throughout the differentiation process. Scale bars=100 μM.

Fig. 7. Data and methodology for optimization of electroporation based incorporation of CRISPR/Cas9 and DNA for gene editing.

(A) Schematic illustrating CRISPR/Cas9 integration of a GFP fusion tag to the housekeeping gene RAB11A. (B) Conditions considered and strategy used for development and optimization of non-viral genome targeting for both cell viability and HDR efficiency. (C) The ability to target multiple sites was confirmed by inserting a GFP fusion tag into various endogenous genes using non-viral targeting in primary human gated CD4⁺ and CD8⁺ T cells using HDRT, HDR template. (D) Average efficiency with the RAB11A–GFP HDR template was 33.7% and 40.3% in CD4⁺ and CD8⁺ cells, respectively. (E) Viability (number of live cells relative to non-electroporated control) after non-viral genome targeting averaged 68.6%. Efficiency and viability were measured 4 days after electroporation. Mean values of n = 12 independent healthy donors are shown (horizontal bars, d, e). Adapted from Roth TL et al. (2018) Nature 2018, 559:405 with permission from Springer Nature.

Fig. 8. Expansion of T-cells using antigen presenting cell mimic scaffolds.

(A) Schematic showing a cross-sectional view of the biomimetic scaffold components. Notably, a liposomal coating allows the APC-ms to better mimic natural antigen presentation. (B) T-cell activation cues used to prepare APC-ms were either anti-CD3 and anti-CD28 or pMHC and CD28 allowing for both polyclonal and antigen specific expansion. (C) Illustration of cell based antigen presentation for T-cell expansion. K562 may be engineered to express a variety of additional surface ligands to enhance expansion. (D) Illustration of a Dynabead aAPC. Beads are generally coated with anti-CD3 and anti-CD28 to promote T-cell expansion and require administration exogenous IL-2 to achieve optimal expansion. Adapted from Cheung et al. (2018) Nature biotechnology 36:160 with permission from Springer Nature