Genetic engineering of T cells for immunotherapy

Abstract

Genetically engineered T cell immunotherapies have provided remarkable clinical success to treat B cell acute lymphoblastic leukaemia by harnessing a patient's own T cells to kill cancer, and these approaches have the potential to provide therapeutic benefit for numerous other cancers, infectious diseases and autoimmunity. By introduction of either a transgenic T cell receptor or a chimeric antigen receptor, T cells can be programmed to target cancer cells. However, initial studies have made it clear that the field will need to implement more complex levels of genetic regulation of engineered T cells to ensure both safety and efficacy. Here, we review the principles by which our knowledge of genetics and genome engineering will drive the next generation of adoptive T cell therapies.

Fig. 1 |. Autologous and allogeneic T cell immunotherapy.

a | Manufacturing process. Blood is collected by venipuncture or apheresis, and T cells are isolated either from the patient (autologous donor) or from an allogeneic donor. Purified T cells undergo engineering to introduce a transgene using viral vectors or non-viral strategies and/or genome editing to eliminate protein expression. Following stimulation, engineered T cells are expanded to increase cellular dosage and infused into the patient. Site-specific transgene insertion may be preferred for allogeneic products to reduce batch-to-batch variation (not shown). T cells can undergo genetic engineering (middle right), for example, to endow antigen specificity, evade the host immune response or confer resistance to the tumour microenvironment. From an efficacy perspective, allogeneic T cell products are vulnerable to immune-mediated rejection. This can be ameliorated by genetic deletion of both HLA class I and HLA class II expression by targeting B2M and CIITA, respectively, which extends the survival of chimeric antigen receptor (CAR) T cells in preclinical models. However, allogeneic CAR T cells without HLA molecules can be killed by the recipient’s natural killer cells, which may necessitate the overexpression of HLA-E or other non-classical major histocompatibility complex (MHC) molecules as a remedy. Deleting multiple genes and adding HLA-E in addition to a CAR or T cell receptor (TCR) is fairly complex, and thus recently an alloimmune defence receptor (ADR) approach was described whereby a CAR is used to trigger cytotoxicity of alloreactive host T cells and natural killer cells. b | Composition of CARs, chimeric autoantibody receptors (CAARs) and transgenic TCRs. CARs and CAARs recognize antigen via a binder domain — typically a single-chain variable fragment (scFv) or a protein target of a B cell receptor (BCR), respectively. Each also has a flexible hinge domain, a transmembrane domain and a CD3ζ activation domain (signal 1), along with a choice of co-stimulatory domains (signal 2) tailored to fit the therapeutic task. Alternatively, a pair of TCRα and TCRβ chains dimerize to form a transgenic TCR, which recognizes MHC-presented antigens. Wild-type chains can be engineered to facilitate their dimerization preferentially over cross-pairing with the T cell’s endogenous chains.

Fig. 2 |. The genetic outcome of modifying the T cell genome.

A | Viral vectors (top) or co-delivery of transposase and transposon (bottom) can integrate transgenes into the genome in a non-targeted fashion. Ba | Nuclease-targeted DNA double-strand breaks in the presence of homology directed repair (HDR) template DNA facilitates integration of template DNA at a specific genomic locus. Depending on the design of the template and the location of the DNA double-strand breaks, the transgene can be placed under the control of an endogenous promoter and/or can knock out expression of a gene into which it integrates. For example, integrating a transgene expressing a chimeric antigen receptor (CAR) into the TRAC locus, which encodes the T cell receptor (TCR) α-chain constant region, eliminates endogenous TCR expression. Bb | Cytidine deaminase (CDA) or adenine deaminase tethered to catalytically inactive (‘dead’) Cas9 (dCas9) forms a cytosine base editor (CBE) or adenosine base editor, respectively. Base editing can repair damaged genes or generate functional knockouts through introduction of a novel stop codon (shown) or by disrupting RNA splice acceptor–donor pairs. 2A, ribosomal skip peptide; ITR, inverted terminal repeat; LHA, left homology arm; pA, poly(A) tail; RHA, right homology arm; TCR, T cell receptor.

Fig. 3 |. Gene-engineered T cell products to enhance efficacy.

A | Overcoming barriers to T cell trafficking to tumour. The addition of an appropriate chemokine receptor matched to sense chemokines released by the target tumour has increased T cell infiltration in animal models. Addition of a dominant negative (DN) FAS receptor has enabled engineered T cells to avoid FAS ligand (FASL)-mediated apoptosis induction. Equipping T cells to secrete heparanase has enabled them to counter the dense extracellular matrix (ECM) of tumours in preclinical models. B | Overcoming antigen loss or diversity can be achieved by transduction with two or more chimeric antigen receptors (CARs) (OR gate logic) or via tandem CARs (TanCARs) (panel Ba), using a universal CAR and separate tumour-targeting ligands (panel Bb) or equipping T cells to express both growth factors and chemokines to enhance infiltration of tumours by T cells and dendritic cells (DCs) (panel Bc). Co-stim., co-stimulatory domain; scFv, single-chain variable fragment; TIL, tumour-infiltrating lymphocyte; TM, transmembrane domain; WT, wild type.

Fig. 4 |. Addition of armour or subtraction of suppressive genes or their transcripts enables TME resistance.

a | Many potential design solutions are available, including gene deletion (for example, using CRISPR–Cas9 genome editing to knock out PDCD1), RNA interference (for example, knockdown of the adenosine A_2A receptor gene ADORA2A to confer adenosine resistance) and addition of genes to enable metabolic self-sufficiency (for example, addition of ASS and OTC, which encode the enzymes argininosuccinate synthetase and ornithine transcarbamylase, respectively) or that counter specific checkpoints and suppressive cytokines. b | Receptor-based approaches to resist tumour microenvironment (TME) suppression include expression of a dominant negative form of an inhibitory receptor to act as a sink. Alternatively, the receptor’s inhibitory domain can be switched to a co-stimulatory domain to change the inhibitory signal into an activating one. Chimeric antigen receptor (CAR) design considerations include the choice of co-stimulatory domains; both natural and mutant co-stimulatory domains may enhance the ability of the engineered T cell to survive in the suppressive TME. Multiple strategies may be used in parallel and should be customized to the specific TME roadblocks encountered. LTR, long terminal repeat; shRNA, short hairpin RNA; TM, transmembrane domain; T_reg cell, regulatory T cell.

Fig. 5 |. Toxicity risks associated with gene-engineered T cells.

a | Product risks are the inherent liabilities associated with the manufactured product such as unwanted contaminants or by-products of the process, including replication competent vectors, chimeric antigen receptor (CAR) transformed residual tumour cells, T cells with DNA damage that may become cancerous themselves and allogeneic T cells that have escaped T cell receptor (TCR) ablation and thus pose a graft-versus-host disease risk. These risks also include the potential immunogenicity of the transgenic construct, which may provoke infusion reactions. b | On-target toxicity derives from a surplus of target cell killing resulting in tumour lysis syndrome (TLS) or a surplus of cytokine and chemokine production by the therapeutic T cells alone or in concert with myeloid cells that results in cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), macrophage activation syndrome (MAS) or prolonged cytopenia. c | On-target off-tumour toxicity occurs if the target antigen is also found on healthy cells at levels sufficient to trigger the therapeutic T cells. d | Off-target toxicity occurs if the transgenic immune receptor cross-reacts with an antigen found on healthy tissues. IFNγ interferon-γ; TNF, tumour necrosis factor.

Fig. 6 |. Gene-engineered T cell products for enhanced safety.

a | Exogenous control of gene-engineered T cells can be achieved via transient delivery of the transgene using mRNA electroporation or via application of a variety of on, off or suicide switches. The transgene may be switched on or off at the level of transcription or via induced protein degradation, or the therapeutic T cell can be eliminated either by a caspase-activation system or via the inclusion of specific tags recognized by approved therapeutic monoclonal antibodies (mAbs). b | Exogenous control can also be achieved via a ‘universal’ chimeric antigen receptor (CAR) system in which infusion of separate antigen-specific targeting ligands redirects the CAR T cells to the target, while the dose and schedule of the antigen-specific targeting ligand can be modified as required. c | Endogenous control can be achieved via Boolean logic-type CAR designs that require CAR T cells to recognize two antigens for full activation (AND gate, via a split CAR) or inhibit CAR T cell activation via a second inhibitory CAR (NOT gate). The synthetic Notch (synNotch) and hypoxia-inducible systems provide conditional ‘IF/THEN’ logic for endogenous control of T cell activity. In the synNotch system, the first CAR is an artificial Notch-type receptor wherein antigen recognition stimulates proteolytic cleavage of a transcription factor (TF), which in turn triggers expression of a second ‘traditional’ CAR recognizing a separate antigen, making sensing of the second antigen dependent on first sensing the first antigen. In the hypoxia-inducible system, the CAR includes oxygen-sensing elements of hypoxia inducible factor 1α, which promote its degradation via the ubiquitin pathway under normoxia but allow full expression in hypoxic conditions such as those found in solid tumours. d | Affinity tuning of the immune receptor provides another form of endogenous control whereby the affinity is set to enable the therapeutic T cell to be activated by tumours expressing high levels of the antigen but not by healthy tissues that express low levels. e | Extending the hinge and transmembrane (TM) domain of a CAR enabled maintenance of cytotoxicity but a significant reduction in the production of cytokines and chemokines, and subsequent cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Similarly, engineering CARs to express the CD3ε domain recruited the negative regulator CSK and resulted in more transient signalling with maintained cytotoxicity but reduced cytokine and chemokine expression. Co-stim., co-stimulatory domain; Inh., inhibitory domain; ODD, oxygen-dependent degradation domain.