Chimeric antigen receptor T cell (CAR-T) therapy harnesses the body’s immune system to fight cancer.

CAR-T cells are made from a patient’s own T cells, key cells of the immune system. The T cells are isolated from a sample of the patient’s blood and then genetically modified to express a chimeric antigen receptor (CAR) on their surface. The CAR is targeted to recognise a specific marker on the surface of cancer cells.

CAR-T cell therapy has generated some outstanding results (read more) in clinical trials against blood cancers. However, similar results have not yet been seen in trials of CAR-T cells directed at solid cancers, which constitute the majority of cancers diagnosed.

At Carina, we are developing broad-spectrum CAR-T cells targeted at molecular markers that have been found on a number of solid cancer cells and with little to no expression on healthy cells.

We hope to produce a broad-spectrum CAR-T therapy that can be used to treat multiple solid cancers yet is patient-specific and has little, if any, off-cancer damage.

‘A new and unique way to treat cancer, CAR-T therapy is poised to transform the outlook for children and adults with certain otherwise incurable cancers.’

Chimeric Antigen Receptor T cell (CAR-T) therapy was named by the American Society of Clinical Oncology (ASCO) as its Advance of the Year for 2018.

CAR-T cell technology is an immunotherapy that harnesses potentially the most powerful weapon there is against cancer – the human immune system. CAR-T cells are made from a patient’s own T cells, and so are often referred to as a ‘personalised’ cancer treatment.

Named for the chimera, a fire-breathing monster in Greek mythology comprising the body parts of different animals, CAR-Ts have extra parts – genetically engineered receptors designed to recognise and bind to receptors on cancer cells.

So far CAR-T therapy has shown impressive and, in some cases, stunning success against blood cancers (read more).

However, solid tumours make up the majority of cancers diagnosed and, so far, clinical trials of CAR-T therapies targeted against solid cancers have not yielded the same results. Solid tumours present a set of new difficulties in designing effective CAR-T cells.

Most researchers agree there are two major challenges in designing effective CAR-T cell therapies for solid cancers.

1 Lack of ideal solid cancer antigen targets

None of the antigens being targeted in solid cancer clinical trials have been specific for solid cancer cells, and there has been little positive data for most of the antigens studied.

Significant on-target off-tumour effects have been recorded where the CAR-Ts have attacked both diseased and healthy cells.

2 Limited access of CAR-T cells to tumour tissues

So far it has proven a challenge to deliver clinically effective doses of CAR-T cells to solid tumours or resection (after surgery) sites. One of the issues to overcome is that CAR-T cells often move away from the tumour/resection site before they have had a chance to work.

The structure of a CAR construct

Chimeric Antigen Receptors (CARs) are human-made molecules expressed on the surface of T cells, key cells of the immune system.

These CARs are targeted at markers on cancer cells. T cells with CARs on their surface are then directed straight towards cancer cells, enabling for targeted, specific killing.

CARs are genetically engineered fusion proteins, proteins consisting of at least two domains encoded by separate genes that have been joined so they are transcribed and translated as a single unit, producing a single polypeptide.

It is useful to think of the CAR molecule as trying to get our T cells to react and activate in ways that mimic what would happen under natural conditions during an immune response, but armed with ‘unnatural’ targeting capacities that allow them to ‘home in’ directly on cancer cells.

T cells are distinguished as such due to the presence of T cell receptors (TCRs) on their surface, which bind to fragments of antigens on foreign cells and initiate their destruction.

CARs can be viewed as manufactured and altered T cell receptors.

They consist of an ectodomaintransmembrane domain and an endodomain.

The ectodomain sits outside of the T cell in the extracellular space and consists of a targeting element (signal peptide) of a single chain variable region domain (scFv) formed by variable regions of immunogloblin (antibody). The signal peptide is targeted to the cancer-specific marker on tumour cells (CARs are targeted to different markers).

The transmembrane domain is attached to the ectodomain by the spacer domain. The transmembrane spans the T cell membrane and connects to the endodomain, the functional end of the CAR molecule embedded inside the T cell.

CAR constructs are classified as being in one of 4 GENERATIONS based on the structure of their endodomain.

The first-generation CAR molecule, generated in 1993 by Dr Eshhar and colleagues, had an endodomain consisting of two CD3 zeta-chain molecules (or CD3 ζ), which is one of the four peptides that forms the T cell receptor CD3 complex initiating intracellular signaling that leads to T cell activation. It is still the most common component of CAR endodomains today.

After limited success with the first-generation CARs, scientists realised that the CAR constructs worked better when they were designed with a co-stimulatory molecule (as well as the CD3 zeta molecules on the first gen CARs) to help with intracellular signaling and encourages the CAR-T cells to remain active and proliferate once in the body.

Dr Michel Sadelain and colleagues went on to develop second-generation CAR-T cells with a co-stimulatory signaling domain CD28, one of the proteins expressed on T cells that provides co-stimulatory signals required for T cell activation and survival.

Third-generation CARs were produced by incorporating two or more co-stimulatory domains (usually CD28 and 41BB or OX40) into the CAR construct.

Fourth-generation CARs, known as ‘T cell redirected for universal cytokine-mediated killing’ (TRUCKs) were generated by adding IL-2 (Interleukin 2, a type of cytokine signaling molecule in the immune system) to the base of the second-generation constructs. TRUCKs have shown improvements in anti-tumour efficacy.

Now, researchers have many options to choose from when building their CAR constructs – adding some elements in, taking some out, modifying others.