Phase 1 evaluation of CRISPR-CAR gene edited T cells in relapsed refractory B cell acute lymphoblastic leukaemia

New treatments for leukaemia using gene therapy to re-program white bloods cells against cancer cells are showing a great deal of promise. In some trials, over 8 out of every 10 patient treated had a good response. Most of the trials so far have relied in collecting a patients own white cells and then using a disabled virus to fit new genes into a group of cells called T cells. This process then arms the cells against leukaemia, so when they are given back to the patients they can attack the disease. We recently showed that its possible not only to arm cells against leukaemia but also disarm them to stop side effects if they are taken from a healthy donor and transplanted to a patient without any matching. A technique called gene editing was used to to achieve this, and we are currently running a clinical trial to test the approach in more patients. In the meantime, improvements in the way we modify cells have led to the development of new versions of the treatment that we think are going to be more widely used. This project will test the new versions, made using CRISPR gene editing, to see if we can treat leukaemia in children where all other treatments have failed. We aim to treat 10 children over two years, and if they can be cleared of leukaemia, they will receive a vote marrow transplant to improve their chances of cure.

Universal T cells aim to circumvent HLA barriers and provide an 'off-the-shelf' alternative to autologous cell therapies. We undertook the first application of genome modified T cells expressing a chimeric antigen receptor (CAR) against CD19 to successfully treat infant B cell acute lymphoblastic leukaemia (B-ALL) in 2015. Cells were transduced with a lentiviral vector to express CAR19 and then further modified using TALENS to disrupt T cell receptor (TCR) and CD52. Genome editing technology is evolving rapidly and we have developed a self-inactivating lentiviral vector that tightly couples CAR expression with CRISPR guides through a hybrid 3' long terminal repeat region (LTR). Subsequent electroporation of Cas9 mRNA results in transient CRISPR mediated DNA cleavage and highly efficient disruption of target loci. Further downstream processing enables depletion of residual TCR+ cells and yields highly homogenous (>99% CAR+TCR-) populations. Furthermore, we have developed an automated process with selection techniques to generate CAR-T cells from umbilical cord blood T cells as well as conventional peripheral blood lymphocyte harvests from healthy donors. In vivo, human-murine anti-leukaemia modelling has confirmed functional integrity of CAR+TCR- cells, and found these were less prone to exhaustion compared to TCR+ cells. Extensive molecular screening for off-target events using Digenome analysis, targeted next-generation and whole-genome sequencing was undertaken. We now propose a Phase 1 study to treat children with relapsed refractory CD19+ leukaemia as a proof of concept application of CRISPR genome modified T cells. The therapy will aim to induce molecular remission & thereby secure eligibility for allogeneic stem cell transplantation. We will assess disease responses, relapse, survival as well as complications including cytokine release syndrome and graft versus host disease. Multiple additional therapeutic applications are envisaged upon completion.

Patient Benefit
The first beneficiaries of this project will be children with relapsed B-ALL who have treatment-refractory disease and have exhausted conventional approaches.
There are over 350 new cases of B-ALL in children the UK annually. Around 10-15% go on to develop relapsed refractory cases and may fail to respond to aggressive treatment, including allogeneic stem cell transplantation. A number of these children may be suitable for autologous CAR therapy, but manufacturing is often problematic, especially in small infants. The TTCAR19 trial will provide access to treatment for 10-13 children over 24 months, with dosing intervals to allow safety profiling in each subject. We anticipate that this should meet the immediate paediatric need in the UK.

Older patients (>16yrs) are expected to be benefit in subsequent applications, and we are liaising with the relevant adult care clinicians. Furthermore, alternative target antigens are being included for patients with B-ALL but exhibiting leukaemic escape through loss of CD19 expression. Other leukaemia including T cell malignancies and acute myeloid leukaemia should be amenable to similar approaches. Specialised patient groups/leukaemia charities such as Bloodwise and Children with cancer will be kept informed of progress with the study.

Beyond T cell/CAR therapies, the terminal-CRISPR vector configuration is being applied to a correction of single gene disorders of the haematopoietic system and skin disorders, and will be of broad interest to the field. Alternative CRISPR/Cas platforms, including those exploiting deactivated Cas or cytidine deaminase base editing configurations are also being adapted into the same vector delivery system.

Industry benefit
Industry interest form gene editing companies is expected. The experience will be of interest to CAR-T cell companies.

Funder and sponsor benefit
Experience in issues relating to clinical gene editing and transfer of emerging technology to clinic. The institutions (GOSH/UCL) and trials sponsors will also generate expertise in hosting an innovative application of CRISPR.