Discussion

For any CAR T cell therapy, generating a product with high safety and efficacy in terms of longevity, engraftment and antitumor-effector function is the ultimate goal. CAR T cell design and cellular composition of the CAR T cell product are essential parameters defining these key therapeutic features. Parameters affecting CAR T cell function are e.g. the choice of costimulation, ratio between CD4+ and CD8+ CAR T cells, CAR T cell differentiation status and amount of exhausted CAR T cells. This paper describes a novel gene transfer vector, termed 62L-LV, which specifically transduces CD62L-positive cells, thus offering the potential to preferentially generate CD62L+ CAR T cells without the need of preselection of defined T cell subsets. Importantly, the newly generated 62L-LV vector could be robustly produced with regard to particle size, concentration and functional titer. With an average size of 142 nm and 1011particles/mL, size and concentrations of 62L-LV stocks lay in the expected ranges of previously established RT-LVs. Functional titers of concentrated 62L-LV batches encoding the αCD19-CAR were on average above 1x106 t.u./mL on HT1080αHis cells and about one log higher on PBMC. This difference in gene transfer activity illustrates that titer determination depends on the particular experimental conditions including the cell type, used transgene and transduction condition. Functional titers can therefore not be compared to those of other vector types. Gene transfer into primary human PBMC with 62L-LV was as efficient as with VSV-LV using same amount of vector volume (Suppl. Fig. 3), yet still resulted in a significantly higher proportion of less differentiated CAR T cells upon long term cultivation.
As CD62L is a T cell differentiation marker, its expression changes throughout T cell life time and activation status. CD62L is regulated by transcriptional shutdown of the CD62L gene as well as shedding from the cell surface upon T cell activation. Therefore, direct proof for the selectivity of 62L-LV on primary cells is difficult, since transduced CD62L+ cells might have turned negative for CD62L when gene expression becomes detectable. Yet, T cells transduced with 62L-LV contained significantly higher proportions of CD62L+CAR T cells than those generated with VSV-LV. While this already indicated that CD62L was used as entry receptor, we provide further evidence for its selectivity from transduction of engineered CD62L expressing cells as well as vector particle binding assays to primary T cells. Binding of 62L-LV to primary T lymphocytes was specifically blocked by the parental CD62L-specific antibody from which the targeting domain of 62L-LV was derived.
An interesting finding of our study was that 62L-LV particles were not blocked by shed CD62L. This was not expected, as it is known that binding capacities of CD62L to target molecules are retained after cleavage. Various reasons might be causative for this finding. The concentration of sCD62L in cell culture supernatants were lower (50 ng/mL) than those in serum of healthy individuals (0.8 – 2.3 µg/mL). In addition, sCD62L is known to aggregate which further reduces the amounts of molecules available for binding of 62L-LV. Even more relevant, it has been suggested that sCD62L is conformationally different from the membrane-associated full-length CD62L as a monoclonal antibody directed against an epitope in the EGF-like domain of CD62L was able to bind to the cell-surface associated CD62L but not the soluble form. The same may hold true for the 145/15 antibody. As a consequence, 62L-LV would be specific for CD62L but not the conformational different sCD62L. Regardless of the exact mechanism, we have proven that 62L-LV transduces T lymphocytes also in presence of sCD62L.
Currently approved CAR T cell products available on the US and EU market include Yescarta, Kymriah, Abecma, Tecartus and Breyanzi. Genetic modification of those products is accomplished by transduction with VSV-LV or γ-retroviral vectors. According to information provided on the companies’ homepages, between 2-5 weeks are required for CAR T cell production and release. To reduce production times, shorter T cell cultivation and expansion could be beneficial. In this regard, we show here that CAR T cells generated within three days of ex vivohandling control the tumor burden in a mouse model. This result is well in line with the previous observation of Ghassemi and colleagues, who have shown that functional CAR T cells cannot be only generated within three days, but also outperform conventionally generated CAR T cells in xenogeneic mouse tumor models. In difference to the published results, we stimulated our cells with IL-7 and IL-15 instead of IL-2, activated the PBMC for only two days with αCD3 and αCD28 and administered the cells already 24 hours after vector incubation. Additionally, using 62L-LV less differentiated T cells were directly targeted. It remains to be analyzed in more sophisticated mouse models whether targeting these cells provides a survival-advantage over VSV-LV incubated CAR T cells.
While shortening the manufacturing time for CAR T cells appears feasible and desirable, certain safety concerns arise with this procedure. During conventional CAR T cell manufacturing, transduced cells undergo several washing and expansion steps reducing the amounts of residual vector particles to negligible concentrations. In contrast, it can be assumed that particle uptake and gene transfer are not completed for CAR T cell products injected as early as 24 or 48 hours after vector incubation. Vector particles still bound to the T cells may transduce other cells upon infusion. This risk is expected to be higher for VSV-G pseudotyped vectors that have a broad cell tropism. That such a scenario can be fatal was demonstrated 2018 in a clinical trial investigating the CAR T cell product Kymriah. In this trial, an accidental transfer of a CD19-CAR into a single leukemic cell during manufacturing led to relapse and death of a patient. Causative for this event was that a CAR construct present in tumor cells can bind in cis to the CAR specific epitope on the surface of the tumor cell, in this case CD19, mask the epitope from recognition by CAR T cells, conferring resistance to the CAR T cell product and enabling its proliferation. In contrast to VSV-LV, RT-LVs, like 62L-LV, have a more restricted tropism which is controlled by the specificity of the used targeting domain. Theoretically, 62L-LV might bind and transduce all kinds of CD62L+ cells, like B lymphocytes, neutrophils, monocytes, eosinophils, hematopoietic progenitor cells, immature thymocytes and a subset of NK cells. Yet, it has to be assessed whether those cells will be modified by 62L-LV in vivo and what consequence the potential CAR expression in these CD62L-positive cell types could have. Importantly, it has to be ensured that tumor cells do not express CD62L. In order to reduce this potential safety concern, the exact time-point of completed transduction after short-term incubation should be investigated and additional washing steps could be implemented to remove residual particles from the cells prior to adoptive transfer.
Taken together, the newly established 62L-LV has shown great potential for the generation of less differentiated CAR T cells without the need of prior or later T cell subtype selection. It is thus a suitable alternative to VSV-G pseudotyped LV vectors. One immediate application may be its use for short-term generated CAR T cells within few days, which may substantially simplify CAR T cell production. Although promising, this approach will need further investigation with regard to safety concerns and scalibility of receptor targeted lentiviral vector production before implemented into clinical studies.