The development of chimeric antigen receptor T (CAR-T) cell therapy is a major breakthrough in cancer therapy due to the remarkable clinical responses observed in certain hematological cancer patients infused with cancer-targeting CAR-T cells. However, the widespread application of CAR-T cell therapy for the treatment of cancer has encountered several challenges, including (1) the potential for on-target off-tumor toxicity and/or cytokine release syndrome (CRS); (2) relapses due to: (i) antigen escape and/or antigen heterogeneity of solid tumor, resulting in outgrowth of non-targeted tumor cells and/or (ii) a lack of CAR-T cell persistence; (3) lack of potency for the treatment of solid tumors; and (4) the high costs associated with CAR-T cell manufacturing.

Therefore, the potency and persistence of CAR-T cells are key to successful CAR-T cell therapy. The TSCM cells are characterized by stem-like properties with high capacity for self-renewal, resulting in long-term persistence. Furthermore, TSCM cells have the highest therapeutic efficacy due to enhanced metabolic fitness as well as low level of senescence and exhaustion (Figure 1 ). Consistent with these desired therapeutic properties, it has been clinically-proven that high levels of CAR+ TSCM cells lead to better clinical outcomes.

CAR-T cell therapy faces several challenges in the context of solid tumor, including (1) lack of CAR-T cell trafficking to the tumor; (2) antigen heterogeneity of solid tumors; and (3) immunosuppressive tumor microenvironment (TME) which can adversely affect T cell fitness (e.g. differentiation, exhaustion, senescence and survival). A technological breakthrough needs to be developed to overcome these hurdles.

Viral vector has been widely used for the development of CAR-T cell therapy due to its high efficiency in gene delivery and integration, resulting in stable long-term gene expression. However, CAR-T cells generated virally have several limitations. These include (1) virus-associated safety concerns; (2) limited payload capacity of viral vectors; (3) low expansion capacity and low percentage of CAR+ T cells following transduction; (4) low CAR-T cell persistence; and (5) high costs of virally-produced CAR-T cells. 

A virus-free system can overcome most if not all of these hurdles, but electroporation-associated damages in virus-free systems present an important challenge. In order for a virus-free system to be effective, we need: (1) a robust gene delivery system; (2) an effective system for integrating multiple genes into CAR-T cells; and (3) a robust cell expansion system for producing clinical-scale CAR-T cells.

GenomeFrontier Therapeutics has successfully developed Quantum CART ( qCART™ ), which is a virus-free Quantum Engine™ for developing CAR-T cell therapy that synergistically integrates four major platforms:

(1) G-Tailor™ : a rapid multiplex gene design, construction, and screening system for designing CAR-T cells with (i) the ability to bind to multiple cancer targets; (ii) modulators for effective CAR-T cell trafficking and TME resistance; and (iii) a safety control to terminate treatment as needed.

(2) Quantum Nufect™ : a robust gene delivery system for introducing therapeutic genes into T cells, resulting in (i) reliable CAR-T cell production while (ii) preserving high cell viability.

(3) Quantum pBac : a virus-free vector system with (i) a large payload gene integration capacity and (ii) high preference for TSCM genome integration.

(4) iCellar™ : a robust cell expansion system for producing clinical-scale CAR-T cells with (i) high percentage of CAR+ TSCM cells and (ii) enhanced fitness.

Collectively, qCART™ enables timely (~10 days) and cost-effective manufacturing of clinical-scale CAR-T cells (1-3.5E9/L) (Table 1, Figure 2 and Figure 3 ).

As shown, virus-free qCART™ robustly produces clinical-scale CAR-T cells comprising a high percentage of CAR+ TSCM cells (Table 1 ) capable of mediating high tumor cytolytic activities (Figure 2 ).

As shown, virus-free qCART™ reliably produces CAR+ TSCM cells with high expansion capacity against different targets and with various transgene sizes (Table 2 ), thereby demonstrating the feasibility of the multiplex CAR-T construct design.