Manufacture of an Allogeneic CAR-T Stem Cell Memory Product Candidate for Multiple Myeloma, P-Bcma-ALLO1, Is Robust, Reproducible and Highly Scalable

Chimeric Antigen Receptor (CAR) T cell therapy has generated unprecedented efficacy in the treatment of multiple hematologic malignancies. For relapsed/refractory Multiple Myeloma (MM), autologous CAR-T products directed against the B cell maturation antigen (BCMA), such as Poseida's P-BCMA-101, have demonstrated significant efficacy. P-BCMA-101 is comprised of a high-percentage of stem cell memory T cells (T_SCM), resulting in a product that is much safer and potentially more durable than other anti-BCMA autologous product candidates. However, as individualized products, all autologous CAR-T products are expensive to manufacture and dependent upon patient T-cells of variable quality. We are developing P-BCMA-ALLO1, an off-the-shelf allogeneic (allo) BCMA-specific CAR-T product candidate derived from healthy donor material, which provides numerous advantages over autologous products, increasing patient access by being immediately available and greatly reducing manufacturing cost and variability.

P-BCMA-ALLO1 is produced using two key platform technologies: the nonviral piggyBac® (PB) DNA Modification System and the high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System. The mRNA coding for hyperactive, or "Super PB" transposase (SPB), and CC enzymes are codelivered with the P-BCMA-ALLO1 PB-based DNA transgene via electroporation to healthy donor T cells to stably integrate the transgene, as well as to knockout (KO) several mediators of allo graft-versus-host and host-versus-graft responses to maximize patient safety and durability of response. The P-BCMA-ALLO1 transgene encodes three genes, a BCMA-specific single-domain variable heavy chain (VH)-CAR (VCAR) gene, a drug selection gene to generate a ~100% CAR+ product, as well as a caspase-based safety switch gene to reduce or eliminate the product in vivo, if desired. The CC System is used to KO the endogenous T Cell Receptor (TCR) and beta-2 microglobulin, thereby decreasing Major Histocompatibility Complex (MHC) class I expression. KO of these key targets is aimed to prevent graft-versus-host disease, as well as reduce host-versus-graft rejection of the product. The CC System can efficiently edit resting T cells, thereby maintaining a high-percentage of T_SCM cells, and does not create unwanted off-target mutations, another important consideration when creating an allo product candidate. To maximize the number of doses produced from a single manufacturing run, we have developed a proprietary "booster molecule" that allows for significant expansion of TCR-KO CAR-T_SCM cells to potentially produce hundreds of doses.

To date, large-scale manufacturing of significant doses of potent allo CAR-T products has been challenging for the field. P-BCMA-ALLO1 manufacturing uses a potentially unlimited number of individual serial donors. We have currently produced P-BCMA-ALLO1 at both research and near-commercial scale from >35 donors with >97% manufacturing success. While a range of TCR-KO efficiencies was observed (~50-90%), the final product was always >99% homozygous TCR-KO after a purification step. Overall expansion of TCR-KO cells ranged from ~2-20 fold, and after removal of unedited TCR+ cells ~0.42-7.04x10e9 TCR-KO cells were recovered from 0.75x10e9 starting cells. However, working at clinical production scale (starting with ~3x10e9 cells), up to 250 doses of P-BCMA-ALLO1 could be manufactured per run, at a dose of 150x10e6 cells/patient. Importantly, with this level of donor and manufacturing robustness, no significant prior screening of donor material, other than to meet standard FDA requirements, would be needed.

P-BCMA-ALLO1 made from multiple donors were comprised of an exceptionally high-percentage of the desirable T_SCM cells (CD45RA+CD62L+CD45RO-) and had minimal to no expression of exhaustion markers, such as PD-1 or Lag3. Furthermore, P-BCMA-ALLO1 demonstrated potent efficacy in the RPMI-8226 xenograft model in NSG mice across multiple products generated from separate individual healthy donors. Altogether, these data demonstrate a robust, reproducible and highly scalable manufacturing process. Moreover, this manufacturing process can easily be expanded for use with additional CAR targets for treatment of other hematologic or solid tumor malignancies.