ASH 2024 | Development and evaluation of a first-in-class CAR-T therapy against calreticulin-mutant neoplasms

Alex Rampotas:

So in myelofibrosis we had a few treatments that became available very recently, but none of these treatments have had a curative effect, none of these treatments have been disease-modifying, and the only treatment option available for patients is an allogeneic stem cell transplant. However, this treatment is only available for a small number of patients and even in those cases up to 25% of patients they will have treatment-related mortality. This is a very toxic treatment. So we do need better treatments and targeted therapies. 

And mutated calreticulin is a fascinating molecule because the mutation enables the mutated protein to escape the endoplasmic reticulum, to bind to the thrombopoietin receptor and to be expressed on the surface. And this leads to abberant activation of megakaryocytes and stem cells and drives the disease. The other important thing is that the mutated protein is very unique because the C-Terminus is novel and that means that we can develop specific immunotherapies against it. 

And there have been a few immunotherapies recently. There is a bi-specific T-cell engager and a blocking antibody against it. But during our project we developed a novel second-generation CAR T-cell therapy because CAR immunotherapies is the strongest immunotherapy that we can develop. And CAR T-cells are able to have direct cytotoxicity. They can bypass the need for MHC antigen-dependent presentation. So potentially, they are able to overcome some of the immune suppression of myelofibrosis and directly eliminate the malignant stem cells. 

So during the first phase of the project we developed this CAR T-cell and we tested it against cell lines that they were carrying the mutated calreticulin target and we have identified excellent results that CAR-T was able to eliminate those cell lines specifically and it was able to do that only in vitro and in vivo in a NSG urine xenograft model and also we have identified excellent proliferation of the CAR T-cells. They maintain a quite naive phenotype after multiple rounds of stimulation and overall we’re very quite happy. But on the next stage of the project we want to test this CAR T-cell therapy against primary cells, CD34 positive cells isolated, from actual patients that they have myelofibrosis and this is when we formed collaboration with the SiLA lab in Oxford and Zoe Wong performed some of this work. 

Zoe Wong:

Yeah, so as Alex said, we have taken this first-in-class novel CAR-T and tested it against primary patient cells. And we’ve tested this CAR-T against cells from patients with type 1, type 2, and type 1 and type 2-like mutations and seen great results. We’ve seen highly specific cytotoxicity with the CAR-T with very limited off-target effects. The other exciting thing that we’ve been able to do in this project is look at the efficacy of the CAR-T in a relevant human tissue microenvironment. A major problem for preclinical validation of immunotherapies is the fact that a lot of our preclinical testing is done in a murine model, which is nonspecific and also done in a dish. So, with our human bone marrow organoid model, what we’ve been able to do is test the efficacy of the CAR-Ts against primary patient cells in a 3D microenvironment or a 3D rendering or model of what the actual patient’s myelofibrotic niche would be like. 

And we’ve been really excited to dive into how CAR-Ts and hopefully other immunotherapies like bispecific antibodies or inhibitory antibodies as well perform in a niche or a microenvironment that is fibrotic or immunoregulatory which is a major problem for patients as well. 

Alex Rampotas:

So what do you think are the advantages of this organoid model in comparison to using mice? 

Zoe Wong:

The organoid model is scalable, so we can make many organoids and test different doses of CAR-T’s, different timing, synergy with other existing therapies, and that would be very limited in a mouse model. And also before moving on to patient or clinical studies, we can kind of refine what types of dosing and timing we want to move forward. So that’s a huge benefit. 

Another benefit in comparison to mice is that in a lot of the mouse models we actually have difficulty setting up models that faithfully represent the human microenvironment and models that are immunocompetent as well. So this model allows us to really carefully think about what elements of patient microenvironment we want to recapitulate and we can test efficacy of immunotherapies with and without certain immunoregulatory factors as well. 

Alex Rampotas:

I think this is making a very good point because if we were to test, for example, our CAR T-cell in a murine immunocompetent model, we would have to use murine CAR T-cells. And although it would have been another complex model, actually the CAR T-cells would have been different from the final product that might go into humans. So I definitely agree that organoids may be the future of drug development because of all this versatile ability to test all of these different things and to recapitulate the actual human bone marrow rather than testing drugs in a murine environment. 

Zoe Wong:

Yeah, we can also look at off-target effects and see how the different therapies that we’re testing are having cytotoxic effects or not on non-specific cells. And we can directly compare immunotherapies like the bispecific antibodies and inhibitory antibodies and the CAR-Ts against each other and see how they’re affecting stromal cells or other cells that are around the malignant cells. 

Alex Rampotas:

And I would like to take that point to highlight maybe the advantage of CAR T-cell therapy over other immunotherapies because a CAR T-cell is not a fixed immunotherapy. It can be engineered to enhance its efficacy. We can potentially armor it against some of the inhibitory effects of this inflammatory microenvironment or make it secrete therapeutic payload or a cytokine to further enhance the potency and the immunotherapeutic effect. 

So we’re very excited because I think, calreticulin, as we discussed, is a fascinating target and it is possible that some of the other immunotherapeutic approaches would work, but we think that our CAR-T would be more suitable and able to overcome even the worst cases of myelofibrosis, where there is a lot of inflammation and fibrosis and more simple approaches may fail to tackle this challenging environment.

Zoe Wong:

CAR-Ts are a very promising field and we’ve been really excited by this collaboration between University of Oxford and UCL, combining the expertise of both of our laboratories, so it’s been a really exciting project.

Alex Rampotas:

It has been a great collaboration, again Martin Pule was my host lab and Claire Roddie’s lab provide the CAR T-cell expertise, and the SiLA Lab has huge experience in working with MPN and developing the organoid model and hopefully we will be able to move into a Phase I trial where all the questions about safety and efficacy can be answered and hopefully provide another treatment option for patients.

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