By Sarah Stubington
Introduction
Cancer is responsible for nearly 1 in 6 deaths every year. This figure is evidence that the antitumour immune response is far from perfect: in the biological game of hide and seek, it appears that cancer is winning. Despite T cells constantly monitoring the body for cells presenting danger-associated antigens, cancer cells find many ways of evading detection. These include downregulating the MHC molecules that present tumour-associated antigens or expressing immune checkpoint molecules (e.g. PD-L1) that can suppress T cell activity. The tumour microenvironment also limits the immune response to solid tumours: a fibrous matrix limits immune cell infiltration, and the cells that manage to penetrate it are often suppressed by tumour-resident immunosuppressive cells such as Tregs and Myeloid-Derived Suppressor Cells (MDSCs). When cancerous cells evade the immune system, they are able to develop into a tumour. Thus, one way to solve this problem is to artificially design better T cells. Great progress has been made in the last 40 years with manipulating the antitumour immune response by transfusing carefully selected or designed tumour-responsive T cells into the patient. This is known as Adoptive Cell Transfer Therapy and it provides a highly personalised approach by using a patient’s own cells and editing them to selectively target the patient’s unique tumour.
Adoptive Cell Transfer Therapy
The earliest forms of adoptive cell transfer (ACT) therapy used naturally occurring, tumour-infiltrating lymphocytes (TILs) extracted by tumour resection. Tumour-responsive T cells were then selected for before in-vitro activation and culturing. Performing this in vitro removed the cells from the immunosuppressive tumour microenvironment and allowed for the addition of various combinations of cytokines to favour survival, rapid proliferation and differentiation. The expanded activated TIL population could then be infused back into the patient to target the tumour. However, traditional ACT was limited to the T cells already present in the patient. These T cells had a propensity to express TCRs with a very low affinity for tumour antigens, and, as antigen recognition is MHC-dependent, downregulation of MHC expression would prevent an effective antitumour response. These two limitations (affinity and MHC-dependence) were overcome by gene engineering approaches to ACT.
Engineered T Cell Therapy
Gene-engineered T cells involve introducing receptors that target tumour-associated antigens which have been artificially designed to optimise the antitumour response. Engineered T Cell therapy can be divided into two types: TCR-T and CAR-T Cell therapy.
A key issue with the immune response to cancer is that cancerous cells are very similar to healthy cells. The majority of the antigens being presented by MHC complexes are also expressed by healthy tissues, thus making it difficult for T cells to distinguish between the two. Any T cells that do recognise the tumour-associated antigens will likely have TCRs with a very low affinity. This is because negative selection during T cell development ensures that T cells with a high affinity for self-proteins get destroyed; the subsequent low affinity prevents an effective anti-tumour response. TCR-T Cell Therapy uses a conventional T cell receptor which has been genetically edited to increase specificity and affinity for the tumour antigen, thus allowing for improved recognition and destruction of the tumour. TCRs provide MHC-restricted recognition of both intracellular and extracellular antigens.
TCRs and antibodies are both well-adapted for antigen recognition, but they each have limitations. TCRs effectively activate T cells through a CD3 ζ coreceptor but the binding of a CD4 coreceptor to MHC restricts their antigen recognition to MHC-presented epitopes. This poses a problem as cancer cells often downregulate their MHC expression to hide from immune detection. Conversely, the variable region of an antibody can bind to a specific, complementary antigen independent of MHC complexes, but they cannot activate T cells.
CAR-T Cell therapy overcomes these limitations by using Chimeric Antigen Receptors (CAR) which are designed from the variable region of an antibody (for antigen recognition) fused to the CD3 ζ coreceptor of the TCR (to facilitate T cell activation). This provides antigen-dependent but MHC-independent T Cell activation which bypasses the issue of MHC downregulation but restricts the antigen repertoire to surface molecules, a small fraction of the antigens normally accessible to T cells.
There have been four generations of CAR T cell development so far: the first generation contained only a CD3ζ signalling domain, while the second and third generations contained one or more costimulatory domains in addition. The fourth generation then included inducible cytokine genes allowing for the localised secretion of pro-inflammatory cytokines once the T cell has been activated by a CAR-antigen interaction. The cytokines can then recruit innate immune cells to target cancer cells that would otherwise be invisible to the CAR-T cells.
CAR-T cell therapy can be autologous using T cells extracted from the patient, or it can be allogeneic using T cells from a healthy donor. Allogeneic CAR-T cell therapy has the benefit of treating patients with defective T cells (e.g. due to chemotherapy) which is a major cause of tumour recurrence after ACT, and it has the potential of providing off-the-shelf CAR-T Cells which would allow for faster and potentially cheaper administration. However, allogeneic CAR-T Cell therapy comes with a risk of graft-versus-host disease (GVHD) if the T-cells recognise the patient as foreign and a risk of rejection if the patient's immune system recognises the allogeneic T cells as foreign. Both of these issues can be managed with careful HLA haplotype matching or genetic engineering of the T cells to remove receptors that could lead to an unwanted immune response.
Neoantigens and Personalisation
The potential of ACT has been limited by severe toxicity associated with on-target, off-tumour toxicity. This occurs because many tumour-associated antigens are proteins that are overexpressed by tumours but still expressed at a low level by normal tissues. Subsequently, the transplanted T cells can target healthy cells as well. This was seen in a severe reaction trialling CAR-T cells against HER2 which led to fatal lung T cell infiltration due to the expression of HER2 in pulmonary epithelial cells. Unexpected toxicity can also come from cross-reactivity when a TCR/CAR recognises a different but very similar epitope. For example, TCRs designed to recognise MAGE-A3 (a cancer-testes antigen not thought to be expressed by normal tissue) can show cross-reactivity with the related epitope MAGE-A12 expressed in the brain, resulting in damage to healthy grey matter. Furthermore, there is a much higher risk of toxicity with TCR-T Cell therapy as the short, MHC-presented epitopes are more likely to resemble other epitopes than a folded surface antigen recognised by a CAR-T Cell.
One way around these side effects has been to include “safety switches” in the engineered T cells. TCR-T cell therapy can use an inducible caspase 9 safety switch, making the T cells lethally sensitive to an exogenous ligand such that the T cell reaction can be terminated should side effects occur. Switchable CARs have also been designed whereby the antigen-binding domain is separated from the signal transduction domain - subsequently, they can only contact each other in the presence of an antibody. This process allows for better control of T cell activity – and, thus, the risk of toxicity - by controlling the dose of antibody. Mass-spectrometry HLA peptidomics studies could also help to identify the epitopes responsible for cross-reactivity and on-target/off-tumour toxicity, allowing patients at high risk of severe side effects to be identified through HLA-haplotyping. For example, patients with a HLA-A*0201 haplotype express MHC molecules that would present the cross-reactive MAGE-A12 epitope. These patients would therefore not be offered MAGE-A3 targeted treatments. The risk of toxicity can also be minimised for allogeneic CAR-T cell therapy by using TCRs with a CD4 coreceptor that cannot interact with the MHC molecule capable of presenting a cross-reactive epitope.
An alternative approach would be to use natural killer (NK) cells instead of T cells. NK cells lack TCRs and thus cannot cause GVHD. While NK cells would naturally have a shorter lifespan than T cells, they can be artificially encouraged to proliferate and persist in the body using IL-15 treatment or the deletion of TGFβ receptors, thus helping them to provide a longer-lasting immune response.
The main focus of ACT development is currently identifying high-specificity neoantigens. The mutations that drive tumour development produce proteins with an altered amino acid sequence and, thus, unique peptide epitopes that are specific to the tumour and not expressed by any healthy cells. Developing CARs/TCRs which target these neoantigens will minimise on-target/off-tumour toxicity. Neoantigens can be identified by whole-exome sequencing of a tumour sample to identify mutated proteins before screening the candidate epitopes to determine which are presented on the surface of the tumour cell (and thus suitable for CAR targeting) and which are presented by MHC molecules (necessary for TCR-T Cell therapy). As part of this screening process, neoantigens are presented by antigen-presenting cells and T cells expressing activation markers are selected through flow cytometry. The neoantigens expressed by a tumour will be highly patient-specific, and the T cells engineered will also need to be patient-specific to ensure antigen recognition and prevent rejection. The most effective neoantigens are often those derived from the driving mutation in cancer development (often mutations to p53, KRAS, MYD88, etc) meaning that all cells descended from this initial mutated cell will express the neoantigen. This is especially important when targeting solid tumours which show considerable heterogeneity in antigen expression.
The future of Adoptive Cell Transfer Therapy
Adoptive cell transfer therapy is one of the most personalised forms of cancer treatment available, but for ACT to reach its full potential, it is important to balance maximising specificity with increasing accessibility and minimising toxicity. At the most personal level, T cells are extracted from the patient and CARs are designed to target the specific neoantigens presented by the patient's tumour. However, this approach is currently too time-consuming and expensive for wide application. Finding neoantigens shared by multiple cancer types could help to provide rational therapy to groups of patients identified by tumour genome sequencing and HLA haplotyping. Combining this approach with multi-gene edited allogeneic CAR-T Cells (“universal CAR-T Cells”) could streamline the process to make it quick and cheap. For TCR T-cell therapy to play a role in this future, it is important to find ways of minimising the toxicity risk with careful HLA haplotyping and restriction. Efficacy can then be maximised with combination treatments that deliver ACT alongside immune checkpoint inhibitors to ensure the infused cells do not get suppressed by the tumour microenvironment.
Further reading
1. Rosenberg, S. A. & Restifo, N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62-68 (2015).
2. Zhao, L. & Cao, Y. J. Engineered T Cell Therapy for Cancer in the Clinic. Frontiers in Immunology 10, 2250 (2019).
3. King, A. The Less Personal Touch. Precision Oncology 585, 4-6 (2020).