"Ready-to-use" CAR-T therapy: an expedient or future direction?
Release date: 2020-01-14 Views: 0
Since the first approval of CAR-T therapy in 2017, the preclinical and clinical development of this breakthrough therapy has seen explosive growth. Last year, CAR-T therapies developed by Janssen / Nanjing Legends, Bristol-Myers Squibb (BMS), Gilead Sciences / Kite Pharma, and many Chinese biopharmaceutical companies showed stunning data. This year, it is more likely that more than two CAR-T therapies will be approved by the FDA, which will double the number of approved therapies.
However, although approved CAR-T therapy has shown excellent and long-lasting efficacy in the treatment of blood cancer, its promotion faces many challenges. The currently approved CAR-T therapy is autologous CAR-T therapy, which means that doctors need to obtain T cells from patients and express the chimeric antigen receptor against cancer antigens on the surface of T cells by genetic engineering in vitro. (CAR), and then the proliferated cells are returned to the patient. This tedious production process can take up to 3 weeks and is costly. Some patients are unable to provide enough T cells due to their health, pre-treatment and other reasons, which makes them unable to benefit from this breakthrough technology.
Obtaining cells from healthy donors to make allogeneic CAR-T cells has the potential to address these challenges for autologous CAR-T cells. At the beginning of the new year, a review published in Nature Reviews Drug Discovery provided an in-depth inventory of the development status of "ready-to-use" allogeneic CAR-T therapy.
Advantages and challenges of allogeneic CAR-T cells
The manufacturing process of allogeneic CAR-T cells begins with T cells or other cell types obtained from healthy donors. These cells are genetically engineered to express CAR that can target cancer cells on the cell surface, and then undergo further processing. Gene editing reduces the risk of allogeneic CAR-T cells attacking the host and the possibility of host cells rejecting allogeneic CAR-T cells. These cells are then cultured, propagated, purified, and aliquoted into products that can be used at any time and stored frozen.
▲ Manufacturing process of autologous CAR-T and allogeneic CAR-T therapy (Image source: Allogene's official website)
The advantages of this CAR-T therapy are:
A large number of CAR-T cells can be generated from cells provided from a single healthy donor, meeting the needs of a large number of patients. For example, Allogene, which is committed to developing allogeneic CAR-T cells, says that cells obtained from a healthy donor could be used as a CAR-T therapy for 100 cancer patients.
These cells have been generated and frozen in stock, and patients who need to be treated need only thaw the stock therapy to receive immediate treatment, eliminating the delays in the manufacturing process of autologous CAR-T therapy.
Moreover, cancer patients usually cannot receive multiple CAR-T therapies due to the limitation of the number of their own T cells. The allogeneic CAR-T therapy makes it easier to repeatedly treat patients, and changes the antigens targeted by the CAR-T therapy to overcome the resistance to the CAR-T therapy.
However, the development of allogeneic CAR-T therapy also needs to face two unique challenges: First, the allogeneic cells introduced into the patient may attack the host, leading to life-threatening graft-versus-host disease (GVHD); and Allogeneic cells may be quickly recognized and eliminated by the host's immune cells, limiting their antitumor activity.
Strategies to address graft versus host disease
The reason why allogeneic CAR-T cells attack host tissues is because the T cell receptors (TCRs) expressed on the surface of these T cells can recognize alloantigens on the host tissues, thus triggering T-cell attacks on the host tissues. At present, strategies for overcoming graft-versus-host disease are mainly divided into two categories: one uses gene editing methods to eliminate natural TCR expression on T cells, and the other uses other cell types that do not cause GVHD.
Gene editing method eliminates natural TCR expression in T cells
The TCR naturally expressed on the surface of commonly used αβ-type T cells is the key to mediate the attack of these T cells on the host. Researchers have developed multiple methods to prevent TCR expression on the surface of these cells. One of the fastest growing methods is the use of gene editing technology. The TCR protein complex on the surface of αβ-type T cells is composed of α and β chains, and only one gene encodes constant regions of the α chain. Therefore, disrupting genes encoding the T-cell receptor alpha chain constant region (TRAC) is a direct and effective way to prevent the expression of alpha β-type TCR. Allogeneic CAR-T therapies generated using this strategy have entered clinical trials.
The advantage of this strategy is that CAR-T cells can be made using a large number of αβ-type T cells in healthy donors as raw materials. There are a variety of gene editing technologies that can be used to specifically disrupt the genes encoding TRAC, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), MegaTAL, and the CRISPR gene editing system. In the latest study, the researchers used the cell's homologous recombination mechanism to direct the CAR-expressing transgene into the TRAC gene site. This strategy has the effect of "two birds with one stone". While destroying the natural TCR expression of T cells, the CAR transgene is expressed at the TRAC gene site and regulated by the natural TCR gene promoter. Animal experiments show that CAR-T therapy targeted to CD19 generated by this strategy has better anti-cancer activity than CAR-T therapy generated by random insertion of CAR transgenes.
One hidden danger of using gene editing strategies is the potential risks of off-target effects of gene editing technology. However, since allogeneic cells are eventually eliminated by the host, the impact of this potential risk may be relatively low.
Use of cell types that reduce GVHD risk
Another strategy to reduce the risk of allogeneic CAR-T cells attacking the host is to use cell types that do not produce GVHD or have a lower risk of producing GVHD. They include natural killer cells (NK cells), γδ type T cells, NK T cells (T cells expressing NK cell surface markers), and virus-specific memory T cells.
Strategies to improve allogeneic CAR-T cell persistence
Although the above-mentioned multiple methods can reduce the risk of allogeneic CAR-T being injected into the patient to attack the host, allogeneic CAR-T therapy needs to solve another major challenge, that is, the patient's immune system will recognize these cells as "Foreign" cells, which cause an immune rejection of them. This immune rejection will eventually destroy allogeneic CAR-T cells introduced into the patient. The immune response mediated by the patient's own T cells may begin to work as soon as the allogeneic CAR-T therapy is introduced, reducing the efficacy of the allogeneic CAR-T therapy. Therefore, how to improve the durability of allogeneic CAR-T therapy is an urgent problem in this field. The current research to solve this problem is in the following directions:
More effective lymphocyte clearance
Existing research has shown that in order to allow the imported CAR-T cells to proliferate in the patient, it is necessary to use chemotherapy and other means to clear the existing lymphocytes in the patient before importing the cells, so as to provide proliferative space. For allogeneic CAR-T cells, the step of removing lymphocytes may be more important, because the remaining host T cells will immediately attack the "foreign" CAR-T cells.
A current method under study is to make allogeneic CAR-T cells resistant to drugs that clear T cells through gene editing, and these cells will not be affected by drugs that clear T cells. For example, Allogene's UCART19 uses gene editing to knock out genes that express the CD52 protein in cells. This makes these cells resistant to alemtuzumab, a monoclonal antibody that kills T cells by binding to CD52. Combining alemtuzumab with chemotherapy can more effectively clear the host's mature T cells and maintain the host's mature T cells at low levels. At the same time, allogeneic CAR-T therapy can still work.
▲ Strategies to improve CAR-T persistence by inhibiting the expression of CD52 protein in CAR-T cells (Image source: Allogene official website)
Gene editing can also make allogeneic CAR-T cells resistant to chemotherapeutics that clear T-cells, allowing them to work properly in an environment that inhibits T-cell growth. However, the disadvantage of this strategy is that the endogenous T cell levels in patients will be suppressed to a very low level, resulting in a significantly increased risk of their infection.
Reduce the immunogenicity of allogeneic CAR T cells
Experience with organ transplantation and hematopoietic stem cell transplantation has shown that if the donor's main human leukocyte antigen (HLA) phenotype is similar to the host's HLA phenotype, it can significantly reduce immune rejection of the graft. Based on this experience, in theory, it is possible to construct a donor cell bank by discovering donors with a specific HLA phenotype, and select donor cells that match the patient to generate allogeneic CAR-T cells.
Because class 1 HLA protein is a key molecule that mediates immune rejection factors. Another strategy is to use genetic engineering to eliminate the type 1 HLA protein expressed on the surface of allogeneic CAR-T cells. The expression of HLA on the cell surface requires a subunit called β2-microglobulin. Knocking out the expression of β2-microglobulin by genetic engineering can prevent the expression of HLA protein on the cell surface, thereby reducing the immunogenicity of these cells.
▲ HLA structure (Image source: User atropos235 on en.wikipedia [CC BY-SA (http://creativecommons.org/licenses/by-sa/3.0/)])
Choose the best T cell subset
The T cell population contains multiple T cell subpopulations with different functions and phenotypes. Different T cell subpopulations are also very different in their ability to attack cancer cells and persistence in patients. For example, preclinical studies have shown that CAR-T cells produced by CD8-positive and CD4-positive naïve T cells (TN) and central memory T cells (TCM) are more capable of killing cancer cells.
Clinical studies using autologous CAR-T therapy to treat chronic lymphocytic leukemia have shown that the quality of TCM and stem-like memory T cells (TSCM) with self-replicating ability, and cell depletion biomarkers Expression levels (including PD-1, TIM3, LAG3, etc.) are important indicators of CAR-T therapy activity and persistence.
Therefore, by improving screening and cell culture techniques, specific T cell subpopulations can be enriched to improve the durability of the therapy. In this regard, T cells obtained from healthy donors may have potential advantages because they have not been treated with previous therapies that may affect the T cell population, and therefore can provide T cells that are rich in TN, TCM, and TSCM .
Clinical progress of allogeneic CAR-T therapy
Currently, a number of allogeneic CAR-T therapies under investigation have entered clinical trials to treat blood cancers and solid tumors such as patients with acute lymphoblastic leukemia and acute myeloid leukemia (ALL) (see the table below).
In 2018, the initial clinical results of UCART19 developed by Allogene showed that UCART19 achieved a complete response of 82% / complete response of incomplete hematological recovery (CR / CRi) when treating patients with relapsed refractory ALL. Due to the limited duration of UCART19 in patients, pediatric patients receiving UCART19 subsequently received hematopoietic stem cell transplantation.
The future of CAR-T and cell therapy
Regarding "ready-to-use" CAR-T therapy, the industry also has two different views. Many people believe that it can solve the pain points of difficult to prepare CAR-T therapy and long production cycle, which brings new hope for more patients around the world; but some experts believe that "ready-to-use" CAR-T therapy is only temporary "Expedience". In the final analysis, it is our insights on "autologous" CAR-T therapy.
Recently, WuXi AppTec's content team had a dialogue with several leading experts in the field of cell therapy. Among them, Dr. Rick Klausner, founder and CEO of Lyell Immunopharma, looks forward to the future of cell therapy. The former head of the National Cancer Center (NCI) pointed out that one of the current motivations for developing "ready-to-use" CAR-T therapies lies in the bottlenecks in creating "autologous" CAR-T therapy. However, we need to realize that the current technology for manufacturing CAR-T therapy is far from mature. 5-10 years later, the development and manufacturing of cell therapies is bound to be very different from today.
Dr. Klausner mentioned that the reason why current cell therapy production is time consuming and laborious is that we do not know which of these cells are truly therapeutic. Therefore, we can only continuously expand T cells, and hope that among the astronomical cells, the number of cells with curative effect can reach the standard. In the future, if we can isolate those subsets of cells that have curative effects, we can drastically reduce the number of cells needed to treat cancer. This can greatly reduce the number of cells we need to culture and shorten the production time of cell therapies from weeks to just days.
This sounds like heaven and earth, but it represents a way forward. You know, in an era when a calculator takes up an entire house, not many people can expect the advent of smart phones. There is only a few decades between the two.
MD Anderson Cancer Center's director of cancer medicine, Dr. Patrick Hwu, pointed out different application prospects. For some more sensitive cancers, "ready-to-use" CAR-T therapy can work well in the short term, so you don't have to worry too much about it being rejected by the immune system. For solid tumors, we hope that T cells stay in the vicinity of the tumor for as long as possible, and the "autologous" CAR-T therapy will have more room to play.
We need to know that the hot discussion about "ready-to-use" and "autologous" CAR-T therapy is just the tip of the iceberg of cell therapy. Yes, since its approval, the field of CAR-T therapy has attracted a lot of attention and attention. But two experts pointed out that cell therapy is not the only CAR-T therapy. Cell therapy other than T cell therapy is also the future development direction. Gene and cell therapy is expected to bring new breakthroughs to patients around the world under the tide of biotechnology.
 Depil et al., (2020). 'Off- the-shelf' allogeneic CAR T cells: development and challenges. Nature Reviews Drug Discovery, http://doi.org/10.1038/s41573-019-0051-2
 Allogen Corporate Presentation. Retrieved January 5, 2020, from http://ir.allogene.com/static-files/f44b1f83-83eb-4cd0-b0fa-4f327b17fefb
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