A protein-based method for the generation of allogeneic chimeric antigen receptor T-cells
Lead Research Organisation:
University College London
Department Name: Haematology
Abstract
Chimeric Antigen Receptor (CAR) T-cell therapy has recently been approved in the USA and Europe for the treatment of relapsed B-cell malignancies, with potential further application in a broad range of tumour types. Currently, most CAR T-cell products are generated as autologous bespoke products for each patient. This has several disadvantages: each patient must undergo leukapheresis; production failure rate is ~10%; product quality is not guaranteed; there is a 3-4 week lag time to treatment; and economies of manufacturing scale cannot be applied.
A potential solution is allogeneic CAR T-cells from healthy donors, which could be manufactured in advance, quality-checked and cryopreserved for timely administration to any potential patient. However, unrelated donor CAR T-cells would cause severe or fatal graft-versus-host-disease (taGvHD) mediated by the T-cell receptor (TCR) of donor T-cells. To deliver allogeneic CAR T-cells, TCR signalling must be prevented.
To date, allogeneic CAR T-cell strategies have used genome-editing to delete the TCR. Indeed, we were the first to describe clinical use of anti-CD19 CAR T-cells generated from allogeneic TALEN-edited cells. While some clinical efficacy has been seen, a complex multi-stage manufacturing process is required, with highly stringent depletion of TCR positive T-cells. This difficult manufacture obviates much of the advantage in terms of practicality / cost of goods for allogeneic cells.
We have since developed an alternative platform for allogeneic CAR T-cell manufacturing without genome-editing. By co-expressing a TCR-specific single-chain variable fragment with a Golgi retention signal (TCR-KDEL) along with the CAR, TCR-negative CAR T-cells can be generated with a single viral transduction. Further, additional co-expression of a GMP compliant marker gene facilitates facile highly stringent selection of TCR-negative, CAR-positive cells in a single sorting step.
With this grant, we propose to test the TCR-KDEL allogeneic CAR T platform in patients with advanced B-cell malignancies, using a next-generation CD19 CAR (CAT19) currently in several studies at our centre. While we do not expect TCR-KDEL usage to be limited to CD19 CAR, testing with CAT19 allows direct comparison with ongoing autologous CAR studies, and acts as a 'proof of concept' for potential future studies. We anticipate our approach could reduce cost and broaden access to CAR T-cell therapy.
A potential solution is allogeneic CAR T-cells from healthy donors, which could be manufactured in advance, quality-checked and cryopreserved for timely administration to any potential patient. However, unrelated donor CAR T-cells would cause severe or fatal graft-versus-host-disease (taGvHD) mediated by the T-cell receptor (TCR) of donor T-cells. To deliver allogeneic CAR T-cells, TCR signalling must be prevented.
To date, allogeneic CAR T-cell strategies have used genome-editing to delete the TCR. Indeed, we were the first to describe clinical use of anti-CD19 CAR T-cells generated from allogeneic TALEN-edited cells. While some clinical efficacy has been seen, a complex multi-stage manufacturing process is required, with highly stringent depletion of TCR positive T-cells. This difficult manufacture obviates much of the advantage in terms of practicality / cost of goods for allogeneic cells.
We have since developed an alternative platform for allogeneic CAR T-cell manufacturing without genome-editing. By co-expressing a TCR-specific single-chain variable fragment with a Golgi retention signal (TCR-KDEL) along with the CAR, TCR-negative CAR T-cells can be generated with a single viral transduction. Further, additional co-expression of a GMP compliant marker gene facilitates facile highly stringent selection of TCR-negative, CAR-positive cells in a single sorting step.
With this grant, we propose to test the TCR-KDEL allogeneic CAR T platform in patients with advanced B-cell malignancies, using a next-generation CD19 CAR (CAT19) currently in several studies at our centre. While we do not expect TCR-KDEL usage to be limited to CD19 CAR, testing with CAT19 allows direct comparison with ongoing autologous CAR studies, and acts as a 'proof of concept' for potential future studies. We anticipate our approach could reduce cost and broaden access to CAR T-cell therapy.
Technical Summary
Chimeric Antigen Receptor (CAR) T-cell therapy is growing in application. Currently, CAR T-cell products are generated as individual autologous products for each patient. This has several disadvantages: each patient must undergo leukapheresis; production failure rate is ~10%; product quality is not guaranteed; there is a 3-4 week lag time to treatment; and economies of manufacturing scale cannot be applied.
A potential solution is allogeneic CAR T-cells from healthy donors. However, unrelated donor CAR T-cells would cause severe/ fatal graft-versus-host-disease (taGvHD) mediated by the T-cell receptor (TCR) of donor T-cells. To deliver allogeneic CAR T-cells, TCR signalling must be prevented.
To date, allogeneic CAR T-cell strategies have used genome-editing to delete the TCR. Indeed, we were the first to describe clinical use of anti-CD19 CAR T-cells generated from allogeneic TALEN-edited cells. While some clinical efficacy has been seen, a complex multi-stage manufacturing process is required with highly stringent depletion of TCR positive T-cells. This difficult manufacture negates much of the advantage in terms of practicality/ cost of goods for allogenic cells.
We have since developed an alternative platform for allogeneic CAR T-cell manufacturing without genome-editing. By co-expressing a TCR-specific single-chain variable fragment with a Golgi retention signal along with the CAR (TCR-KDEL), TCR-negative CAR T-cells can be generated with a single retroviral transduction. Further, additional co-expression of a GMP-usable marker gene facilitates facile highly stringent selection of TCR-negative, CAR-positive cells in a single sorting step.
With this grant, we propose to test the TCR-KDEL allogeneic CAR-T platform using a next-generation CD19 CAR (CAT19) currently in several studies at UCL. While we do not expect TCR-KDEL to be limited to CD19 CAR, testing with CAT19, testing with CAT19 allows direct comparison with ongoing autologous CAR studies.
A potential solution is allogeneic CAR T-cells from healthy donors. However, unrelated donor CAR T-cells would cause severe/ fatal graft-versus-host-disease (taGvHD) mediated by the T-cell receptor (TCR) of donor T-cells. To deliver allogeneic CAR T-cells, TCR signalling must be prevented.
To date, allogeneic CAR T-cell strategies have used genome-editing to delete the TCR. Indeed, we were the first to describe clinical use of anti-CD19 CAR T-cells generated from allogeneic TALEN-edited cells. While some clinical efficacy has been seen, a complex multi-stage manufacturing process is required with highly stringent depletion of TCR positive T-cells. This difficult manufacture negates much of the advantage in terms of practicality/ cost of goods for allogenic cells.
We have since developed an alternative platform for allogeneic CAR T-cell manufacturing without genome-editing. By co-expressing a TCR-specific single-chain variable fragment with a Golgi retention signal along with the CAR (TCR-KDEL), TCR-negative CAR T-cells can be generated with a single retroviral transduction. Further, additional co-expression of a GMP-usable marker gene facilitates facile highly stringent selection of TCR-negative, CAR-positive cells in a single sorting step.
With this grant, we propose to test the TCR-KDEL allogeneic CAR-T platform using a next-generation CD19 CAR (CAT19) currently in several studies at UCL. While we do not expect TCR-KDEL to be limited to CD19 CAR, testing with CAT19, testing with CAT19 allows direct comparison with ongoing autologous CAR studies.
Planned Impact
* Goals of this proposal:
The goals of this proposal are to move part of the way towards a simple allogeneic CAR T-cell platform. Such a platform would allow cheaper and easier CAR T-cell manufacture.
* Who might benefit from this research?
The potential beneficiaries of this work are: (1) Patients with relapsed / refractory cancers; (2) Clinicians and medical institutions who treat such patients; (3) The UK biotechnology industry.
* How might they benefit from this research?
(1) Patients: currently, access to CAR T-cell therapy is restricted to a small number of patients. CAR T-cell therapy is expensive and treatment is cumbersome. Part of the reasons for this is that currently, most CAR T-cell products are autologous and are generated on a bespoke basis. The proposed work should help to make CAR T-cells manufacture more "off-the-shelf" hopefully making CAR T-cell therapy more accessible. As well as this cost / convenience aspect, many patients with cancer, particularly those who have received chemotherapy have T-cells which do not function optimally. It may be difficult to manufacture CAR T-cell products for such patients, and even if manufactured, CAR T-cells from such patients may not work. An allogeneic approach where the CAR T-cells are generated from cord blood will allow these patients to be more effectively treated with CAR T-cell therapy.
(2) Clinicians / medical institutions continue to look for new treatments for patients with relapsed / refractory cancers. Reducing costs and increasing accessibility of CAR T-cell therapies will increase the numbers of patients who can be treated. The possibility of "off-the-shelf" CAR T-cells mean that patients can be treated quickly and without the need for a leukapheresis.
(3) Cellular therapy is a burgeoning new therapeutic modality which is expected to become a multi-billion dollar industry. It is also widely expected that CAR T-cell technology will start moving towards allogeneic approaches in the next few years. The UK biotech sector would benefit from having the opportunity to develop an allogeneic platform as described herein.
The goals of this proposal are to move part of the way towards a simple allogeneic CAR T-cell platform. Such a platform would allow cheaper and easier CAR T-cell manufacture.
* Who might benefit from this research?
The potential beneficiaries of this work are: (1) Patients with relapsed / refractory cancers; (2) Clinicians and medical institutions who treat such patients; (3) The UK biotechnology industry.
* How might they benefit from this research?
(1) Patients: currently, access to CAR T-cell therapy is restricted to a small number of patients. CAR T-cell therapy is expensive and treatment is cumbersome. Part of the reasons for this is that currently, most CAR T-cell products are autologous and are generated on a bespoke basis. The proposed work should help to make CAR T-cells manufacture more "off-the-shelf" hopefully making CAR T-cell therapy more accessible. As well as this cost / convenience aspect, many patients with cancer, particularly those who have received chemotherapy have T-cells which do not function optimally. It may be difficult to manufacture CAR T-cell products for such patients, and even if manufactured, CAR T-cells from such patients may not work. An allogeneic approach where the CAR T-cells are generated from cord blood will allow these patients to be more effectively treated with CAR T-cell therapy.
(2) Clinicians / medical institutions continue to look for new treatments for patients with relapsed / refractory cancers. Reducing costs and increasing accessibility of CAR T-cell therapies will increase the numbers of patients who can be treated. The possibility of "off-the-shelf" CAR T-cells mean that patients can be treated quickly and without the need for a leukapheresis.
(3) Cellular therapy is a burgeoning new therapeutic modality which is expected to become a multi-billion dollar industry. It is also widely expected that CAR T-cell technology will start moving towards allogeneic approaches in the next few years. The UK biotech sector would benefit from having the opportunity to develop an allogeneic platform as described herein.
