Our lead product candidate, DSG3-CAART, is being evaluated for the treatment of mPV, a subtype of PV that affects mucosal surfaces. mPV is caused by autoantibodies against the cell adhesion protein desmoglein 3, or DSG3. DSG3-CAART is designed to selectively target B cells expressing autoantibodies specific for DSG3, which may prevent these B cells from producing DSG3 antibodies that are the cause of mPV while preserving general B cell immune function.
We submitted an IND for DSG3-CAART to the FDA in August 2019, and our IND was accepted by the FDA in September 2019.
In Vitro Studies
A variety of in vitro studies were conducted to evaluate DSG3-CAART from a preclinical activity and toxicity perspective. These studies included an evaluation of DSG3-CAART against proteins that are known to bind the DSG3 antigen, a screen of DSG3-CAART against an array of other membrane proteins and a set of studies designed to evaluate the potential effects of soluble DSG3 antibodies against DSG3-CAART. The results of these studies are summarized below.
Evaluation of DSG3-CAART reactivity against primary human cells expressing known or putative DSG3 binding proteins. The data demonstrated an absence of inflammatory T cell cytokines after being exposed to these cells, indicating an absence of T cell activation. No cytotoxicity was detected except at very high, non-pharmacologically feasible doses. Collectively, we believe there is sufficient evidence to suggest that the DSG3 protein in the context of a CAAR does not interact with other desmosomal proteins.
Evaluation of DSG3-CAART off-target binding against membrane proteins. The broad screen against over 5,300 proteins yielded no off-target signals, except for one weak signal against a protein that is designed to bind to glycoproteins, and which was detected in both the test and control conditions. Further evaluation of this protein in cell-based assays indicated that DSG3-CAART does not recognize and activate against this protein.
Evaluation of the effect of soluble antibodies on DSG3-CAART function. While DSG3 antibodies may have a variable effect on CAAR function, there was no systematic effect to enhance or reduce CAAR function. These dynamics were evaluated in a series of in vitro studies as follows:
- Soluble monoclonal and polyclonal DSG3 antibodies were added to CAAR T cell cytotoxicity assays, at a range of concentrations likely to be encountered in patients, to assess the impact on CAAR function. In all cases when the CAAR T cells were tested in the presence of soluble DSG3 antibodies, they retained their killing function. In addition, the presence of antibodies did not demonstrate a systematic effect to enhance or reduce the CAAR T cells’ cytotoxic ability. Therefore, we believe that removal of circulating soluble DSG3 antibodies from patients prior to infusions may not be necessary to enable potential benefit.
- Antibodies purified from PV patients were added to DSG3 CAAR T cells at a range of concentrations known to commonly occur in patients in order to evaluate the extent to which patient serum may activate CAAR T cells. These antibodies induced a dose dependent increase in interferon gamma.
- Potential for off-target toxicity due to antibody-mediated bridging of DSG3 CAAR T cell cytotoxicity against hematopoietic cells that express receptors designed to bind to antibodies, known as Fc receptors. To evaluate this, we loaded DSG3 antibodies onto cells expressing antibody-binding receptors, and evaluated the ability of DSG3 CAAR T cells to bind and activate against these targets in vitro. No evidence of cytotoxicity was observed, suggesting potentially low risk of off-target killing mediated by this mechanism.
In Vivo Studies
Three murine models were used to evaluate DSG3-CAART in vivo. These models were designed to directly compare the potency of DSG3-CAART in comparison with CART19 cells; evaluate the potential for on-target skin toxicity; and measure the activity of DSG3-CAART against a polyclonal mixture of anti-DSG3 B cells in the presence of soluble anti-DSG3 antibodies.
- Evaluation of potency of DSG3-CAART in a human B cell tumor line compared to CART19 cells. In this model, DSG3-CAART was found to result in a similar reduction of B cells compared to the CART19 cells.
- Evaluation of on-target skin toxicity mediated by DSG3-CAART. In this model, DSG3-CAART cells showed an absence of skin toxicity compared to the positive control, which did demonstrate skin toxicity.
- Evaluation of DSG3-CAART in the presence of soluble antibodies. In this model, we observed amelioration of disease, reduction of DSG3 antibody titers, as well as control of the pathogenic B cells. These results suggest the functional activity of DSG3-CAART in the presence of soluble antibody.