Periodic Reporting for period 1 - GlycoCAR (Targeting of glycosylation pathways to empower CAR-T therapy of solid tumors.)
Reporting period: 2023-07-01 to 2025-12-31
In a Chimeric Antigen Receptor (CAR), the tumor antigen-specificity of an antibody fragment is directly coupled with T cell (co)receptor intracellular domains, which trigger the cytotoxic effector functions of T cells (CD8 T-lymphocytes). T cells can be isolated from human blood buffy coats with ease and genome-engineered ex vivo, upon which they can be adoptively transferred back to patients (and modelled in the lab by mice) that carry a tumor that expresses the antigen for the CAR. This approach of antibody-mediated redirection of T cells to tumor-specific antigens expressed on the surface of tumor cells, is independent from T cell specificity being restricted to MHC-peptide recognition, which is patient-specific and very often selected to be lost in the course of tumor evolution in the face of a patient’s immune system.
However, enormous challenges remain in extending the therapeutic success of CAR-T therapy to solid tumors. The most frequent tumor types are carcinomas, and the heavily immunosuppressive environment that is selected for during cancer evolution up to the point of clinical manifestation, is fundamentally hampering success with adoptive cell therapy, incl. with CAR-T cells. Also, and possibly in part because of this, long-term persistence of CAR-T cells upon primary tumor load clearance has been very difficult to achieve, despite recent progress in mouse models. Most of the CAR-T engineering interventions to overcome this intratumoral immunosuppression that have been studied so far target only one of the multitude of immunosuppressive pathways. However, it is clear from early-stage clinical trials with such engineered CAR-T cells that multiple pathways will need to be tackled at the same time.
Manipulations of CAR-T cells that can overcome multiple key mechanisms of tumor immunosuppression with as few genetic engineering steps as possible are amongst the most sought-after medical biotechnological advances in oncology at this moment.
Glycan synthesis pathways almost always modify a large variety of cell surface proteins rather than just a single one, and the assembled properties of this ‘glycocalyx’ are often what determines its function11. Therefore, manipulation of these pathways most often has very pleiotropic effects on cell surface receptor biology. In the context situated above, we hypothesize that the pleiotropism of glycocalyx manipulation can actually be a strong advantage: one glyco-gene manipulation affects multiple receptor-triggered pathways at the same time. Glycans are known to regulate multiple key physiological aspects of T cell biology, such as T cell development and thymocyte selection, T cell receptor signaling sensitivity, sensitivity to cytokine signals, T cell differentiation, tissue homing and proliferation. We have recently reviewed the current state of knowledge in this field. The challenge of this ERC project is to investigate which CAR-T glycocalyx components can be engineered to enhance therapeutic efficacy.
We have found that MGAT5 knockout in CAR-T cells enhances their functional persistence upon tumor clearance in the highly immunosuppressive SKOV-3 carcinoma model and hence enhances their capability of clearing subsequent tumor rechallenges, with a majority of mice remaining tumor-free. (De Bousser et al., patent application ‘Glyco-engineered CAR-T cells’, WO/2023/111322). This is a very difficult to achieve result with any other known CAR-T manipulation and it provides strong proof of concept that glycocalyx engineering of CAR-T cells deserves in-depth study. Hence, we have defined a programme to build on this finding and to explore a candidate set of further glycosylation engineering concepts in CAR-T cells, to further improve CAR-T therapy of solid tumors.
However, enormous challenges remain in extending the therapeutic success of CAR-T therapy to solid tumors. The most frequent tumor types are carcinomas, and the heavily immunosuppressive environment that is selected for during cancer evolution up to the point of clinical manifestation, is fundamentally hampering success with adoptive cell therapy, incl. with CAR-T cells. Also, and possibly in part because of this, long-term persistence of CAR-T cells upon primary tumor load clearance has been very difficult to achieve, despite recent progress in mouse models. Most of the CAR-T engineering interventions to overcome this intratumoral immunosuppression that have been studied so far target only one of the multitude of immunosuppressive pathways. However, it is clear from early-stage clinical trials with such engineered CAR-T cells that multiple pathways will need to be tackled at the same time.
Manipulations of CAR-T cells that can overcome multiple key mechanisms of tumor immunosuppression with as few genetic engineering steps as possible are amongst the most sought-after medical biotechnological advances in oncology at this moment.
Glycan synthesis pathways almost always modify a large variety of cell surface proteins rather than just a single one, and the assembled properties of this ‘glycocalyx’ are often what determines its function11. Therefore, manipulation of these pathways most often has very pleiotropic effects on cell surface receptor biology. In the context situated above, we hypothesize that the pleiotropism of glycocalyx manipulation can actually be a strong advantage: one glyco-gene manipulation affects multiple receptor-triggered pathways at the same time. Glycans are known to regulate multiple key physiological aspects of T cell biology, such as T cell development and thymocyte selection, T cell receptor signaling sensitivity, sensitivity to cytokine signals, T cell differentiation, tissue homing and proliferation. We have recently reviewed the current state of knowledge in this field. The challenge of this ERC project is to investigate which CAR-T glycocalyx components can be engineered to enhance therapeutic efficacy.
We have found that MGAT5 knockout in CAR-T cells enhances their functional persistence upon tumor clearance in the highly immunosuppressive SKOV-3 carcinoma model and hence enhances their capability of clearing subsequent tumor rechallenges, with a majority of mice remaining tumor-free. (De Bousser et al., patent application ‘Glyco-engineered CAR-T cells’, WO/2023/111322). This is a very difficult to achieve result with any other known CAR-T manipulation and it provides strong proof of concept that glycocalyx engineering of CAR-T cells deserves in-depth study. Hence, we have defined a programme to build on this finding and to explore a candidate set of further glycosylation engineering concepts in CAR-T cells, to further improve CAR-T therapy of solid tumors.
There is currently an intensive search on how to further biotechnologically engineer the CAR T cells such that they keep up the fight vigorously and for sufficiently long to clear solid tumors, and with success for more patients. Ideally, after tumor clearance, such enhanced CAR T cells should also functionally persist in the systemic immune system to prevent relapses of the tumor. At the same time, the extra engineering should be easy to implement in already complex clinical CAR T manufacturing, and should not worsen the safety of CAR T treatment. It is a challenging biopharmaceutical design matrix indeed that requires new discovery and invention.
We may have found exactly such solution, and in a place that will likely come as a surprise to immunologists, i.e. in the CAR T layer of cell surface glycan structures, its glycocalyx, which so far has not at all been targeted for engineering in the CAR T field. Inactivation of a single glycosyltransferase (MGAT5) in human donor CAR T cells efficiently reduced the density of poly-LacNAc glycotopes and we find that this remodelling robustly enhanced CAR T tumor control. Excitingly, we observed this both in a highly immunosuppressive model of ovarium carcinoma and in the most aggressive model of metastatic B cell lymphoma.
For those human donors with weakly responding CAR T cells, it leads to stronger expansion of the CAR T cells throughout the fight with the tumor, leading to enhanced numbers of CAR T cells in the systemic circulation. These cells remained fully competent in killing multiple loads of tumor cells when we put them back in culture with those ex vivo.
We demonstrate that these cells still need the same tumor antigen density to trigger the degranulation that is key for tumor cell cytotoxicity, limiting the risk that they would get off-target activated more frequently. And we demonstrate that MGAT5 KO CAR T cells are still growth factor dependent for proliferation, another key safety requirement.
We have published these data on BioRxiv (https://www.biorxiv.org/content/10.1101/2023.01.23.525164v2(opens in new window)) and will submit the data for peer-review soon.
We may have found exactly such solution, and in a place that will likely come as a surprise to immunologists, i.e. in the CAR T layer of cell surface glycan structures, its glycocalyx, which so far has not at all been targeted for engineering in the CAR T field. Inactivation of a single glycosyltransferase (MGAT5) in human donor CAR T cells efficiently reduced the density of poly-LacNAc glycotopes and we find that this remodelling robustly enhanced CAR T tumor control. Excitingly, we observed this both in a highly immunosuppressive model of ovarium carcinoma and in the most aggressive model of metastatic B cell lymphoma.
For those human donors with weakly responding CAR T cells, it leads to stronger expansion of the CAR T cells throughout the fight with the tumor, leading to enhanced numbers of CAR T cells in the systemic circulation. These cells remained fully competent in killing multiple loads of tumor cells when we put them back in culture with those ex vivo.
We demonstrate that these cells still need the same tumor antigen density to trigger the degranulation that is key for tumor cell cytotoxicity, limiting the risk that they would get off-target activated more frequently. And we demonstrate that MGAT5 KO CAR T cells are still growth factor dependent for proliferation, another key safety requirement.
We have published these data on BioRxiv (https://www.biorxiv.org/content/10.1101/2023.01.23.525164v2(opens in new window)) and will submit the data for peer-review soon.
We have found that MGAT5 knockout (KO) in CAR-T cells enhances their functional persistence upon tumor clearance in the highly immunosuppressive SKOV-3 carcinoma model and hence enhances their capability of clearing subsequent tumor re-challenges, with a majority of mice remaining tumor-free. We were able to confirm the MGAT5 KO effects with CD70 nanoCAR-T cells in a highly aggressive Raji non-Hodgkin lymphoma (NHL) model. This is a very difficult to achieve result with any other known CAR-T manipulation, providing strong proof-of-concept that glycocalyx (i.e. the assembly of sugar-containing molecules that forms the layer outside the plasma membrane) engineering of CAR-T cells could be generally beneficial to combat cancer. We were the first to demonstrate the importance of this glycocalyx engineering to improve CAR-T efficacies.
For the industrial proof of concept and further valorisation path of our patented initial glyco-engineering invention deriving from our ERC Advanced Grant research program, we have applied for an ERC Proof of Concept grant (not granted) and an Industrial Research Fund (granted). The applications encompassed the preparation of our modular glycoengineering asset, which can in principle be incorporated in any CAR T product, to make it ready to enter clinical trials, as well as to further solidify the competitive IP position.
For the industrial proof of concept and further valorisation path of our patented initial glyco-engineering invention deriving from our ERC Advanced Grant research program, we have applied for an ERC Proof of Concept grant (not granted) and an Industrial Research Fund (granted). The applications encompassed the preparation of our modular glycoengineering asset, which can in principle be incorporated in any CAR T product, to make it ready to enter clinical trials, as well as to further solidify the competitive IP position.