For hematologic malignancies treated with CAR T-cells for which efficacy is quite impressive, a current major limitation is patient access to the product, and the financial burden placed on the healthcare system due to the high cost of manufacturing a single product from a single patient using their own autologous T-cells. The current list price for the approved CAR T-cell therapies is $373,000, and the process takes roughly 3 weeks to manufacture. These 3 weeks are a vulnerable time for patients; they have already progressed on the most effective known chemotherapy regimens so keeping their disease controlled can be a challenge, and progression of disease in this time sometimes renders them ineligible to receive the manufactured CAR T-cells. Even if they remain eligible, progression in this time may make the disease less controllable by the CAR T-cells. Allogeneic, or “off-the-shelf” CAR T-cells would provide a solution to these problems. Of course, the challenging question remains, can allogeneic CAR T-cells address these limitations while still providing the same level of efficacy as autologous CAR T-cells?
Theoretically, cancer patients who have anergic, exhausted, or senescent T-cells as a result of their cancer, their age, or their multiple prior lines of cytotoxic therapy, may enjoy improved disease response if given a CAR product from a healthy donor with optimally-functioning T-cells. However the inclination of the host immune system to reject the foreign T-cells tends to limit their efficacy, and the inclination of the foreign T-cells to reject the host’s tissues or organs (i.e., graft- versus- host disease or GVHD) introduces the potential for additional toxicities with this strategy.
Overcoming these obstacles requires first understanding the mechanisms by which cells are rejected by either the host or the donor. Since GVHD is a major cause of death in allogeneic stem cell transplant, much research has been devoted to the subject, and αβ T cells have been found to be central to its pathogenesis. The αβ TCR recognizes peptides presented by MHC molecules, which in humans are comprised of HLA proteins. HLA genes have more polymorphisms than any other human genes, leading to thousands of slightly different HLA protein variants. Of these many different variants, up to six MHC-I and six MHC-II variants are present in each individual. Through a process of negative selection in the thymus, T-cells are educated to not react against any self-MHC molecule, or even any self-MHC molecule loaded with a peptide derived from any of the thousands of proteins encoded in that individual’s genome. However, a new peptide presented by a self-MHC, or a different MHC variant, may still be recognized and lead to alloreactivity. Further, amino acid differences in the peptide-binding region of a given MHC molecule determine the specific sequence of peptides that are capable of being presented, and thus simply changing the MHC results in a wide variety of different peptides presented that were not present in the thymus’s negative selection process, even if the full proteins themselves are identical.
While it is nearly impossible to match all HLA variants among donors and recipients, matching HLA-A, HLA-B and HLA-DR is sufficient to reduce the incidence of allogeneic graft rejection. However, establishing a matched HLA CAR T-cell program would require a large bank from a wide range of donors, and would inevitably exclude a number of patients, especially ethnic minorities or those with less common HLA variants. Further, even though partially-matched CAR T-cells may not react strongly against the host, it is likely that these infused allogeneic CAR T-cells will be more rapidly eliminated by either host T-cells or antibodies recognizing remaining discordant peptide-MHC complexes, or other surface proteins.
Thus, achieving allogeneic CAR T-cell persistence is a major challenge. In addition to HLA matching, more intense lymphodepletion therapy may be required to further immunosuppress the host response against the infused CAR T-cells; however, this approach also compromises other aspects of host immunity, such as effective responses to infectious agents. Repeating intensive lymphodepletion for subsequent CAR T-cell administration carries additional risk and morbidity. A more specific, genetic approach to improving CAR T-cell persistence is to knock out (genetically delete) MHC-I, which is a potential major target of rejection of CAR T-cells by the host immune system. The most efficient way to do this is to delete β2-microglobulin, which is essential for forming functional MHC-I molecules on the cell surface. MHC-II is also expressed on activated T-cells and may also likely be a target of host immune mediated rejection of allogeneic CAR T-cells. Therefore, knocking out or blocking MHC-II on CAR T-cells would also be necessary to maximize their persistence. A relatively efficient way of doing this is to knock out the master regulator of all MHC-II molecule expression, CIITA. Knocking both MHC-I and MHC-II molecules in CAR T-cells has been achieved in mouse studies.
Since MHC molecules are major inhibitors of NK cell cytotoxicity as they signify self, depleting MHC-I and MHC-II makes the CAR T-cells more susceptible to NK-mediated elimination. To circumvent this problem, the additional expression of non-classical HLA molecules (HLA-E or HLA-G), which can inhibit NK cells but are less common targets for T-cell rejection, can be added to the CAR vector.
An option available to the relatively small number of patients who have previously had an allogeneic stem cell transplant and have subsequently relapsed is to generate CAR T-cells from the original stem cell donor. In this way, the host immune system, which has already been reconstituted with a different donor, will be genetically identical to the infused CAR-modified cells. In a series of 20 patients who underwent this strategy, eight had a response to CAR T-cells (six complete responses [CRs] and two partial responses [PRs]), and none developed new-onset GVHD after CAR T-cell infusion.
In addition to solving the problem of allogeneic CAR T-cell persistence, the problem of GVHD needs to be addressed. Beyond HLA matching, preventing GVHD can be successfully achieved through a variety of other means, such as knocking out the TCR from the infused cells, using a cell product with a restricted TCR profile (such as only those that recognize a viral antigen), or using cell types that naturally lack an αβ TCR. The TCR is a heterogeneous group of proteins consisting of either an α-chain and a β-chain (in αβ Τ cells) or a γ-chain and a δ-chain (in γδ T cells), as well as four separate CD3 transmembrane proteins (CD3δ, CD3γ, CD3ε and CD3ζ). The β-chain contains two possible constant regions, while the α-chain has one, making it logistically easier to abolish the αβ TCR by targeting the single α-chain (TRAC). Methods have progressed from successfully knocking out the TRAC in CAR T-cells, to knocking the CAR gene into the TRAC locus, put ting the CAR under the natural transcriptional regulation of the TCRα and ameliorating some of the exhaustive effects of tonic signaling from constitutive high CAR expression. TCR knockout CAR T-cells (UCART19) have shown feasibility in two clinical trials with relapsed B-cell leukemia, demonstrating a 67% CR, and a 6-month PFS of 27% [9]. In these studies, the UCART19 product has both the TCR and the mature lymphocyte marker CD52 knocked out, which allows for additional lymphodepletion but not CAR T-cell depletion using the monoclonal anti-CD52 antibody alemtuzumab. This strategy thus both prevents the CAR T-cells from recognizing the host through its TCR (deleted by CRISPR/Cas9), and reduces the host’s ability to eliminate the CAR T-cells by depleting host lymphocytes (via infusion of alemtuzumab). How this approach will compare to autologous CAR T-cell efficacy remains to be seen in further studies.
While knocking out the TCR is an eloquent approach, it requires sophisticated and relatively expensive techniques. Another potential strategy, which still utilizes intact αβ T-cells, is to selectively employ memory T-cells that recognize a viral antigen for CAR transduction. Since the risk of GVHD is proportional to the diversity of the TCRs present, selecting for a smaller number of TCR clones against a known, non-human target should greatly reduce the risk. However, since TCRs are degenerate, the possibility remains that a particular antiviral TCR may still cross- react with a host tissue antigen. Additionally, since these are all T-cells that have previously encountered the viral antigen at least once, the baseline phenotype will be different; the effect of using this population of prior antigen-exposed cells is unknown. This approach has been demonstrated in glioblastoma patients targeting HER2 in a clinical trial, which showed feasibility and one partial response among 17 patients. Whether this approach is will be effective against a more responsive tumor like ALL remains to be reported in clinical trials.
