CAR T cells are T cells genetically modified to combat cancer cells, which are leveraged in treatment modalities commonly referred to as immunotherapy. CAR T cells may be developed by genetically modifying T cells originally obtained from cancer patients to express recombinant chimeric antigen receptors or CARs. Expression of CARs allows these modified T cells to interact with tumor antigens independently of MHC presentation, resulting in their activation and cancer cell killing.
The first generation of CARs was introduced in 1991, with the recombinant receptor consisting of three structural domains: an antibody like antigen-binding region, a transmembrane domain, and an intracellular signaling domain, CD3 zeta chain. Successive generations of CARs were develop to incorporate co-stimulatory domains, one (CD28 or 4-1BB) or two (CD28 and 4-1BB) co-stimulatory domains in the second and third generation, respectively (Zhao et al. 2018). Therefore, the second and third generation CAR T cells show enhanced activation and expansion (Subklewe et al. 2019).
CAR T cell based therapies represent a breakthrough in the treatment of hematological malignancies including aggressive B cell lymphoma and B cell precursor acute lymphoblastic leukemia. CAR T cells developed against CD19, first approved by the FDA in 2017, have shown significant activity in the treatment of aggressive acute lymphoblastic leukemia, leading to complete remission in ~76% of patients (Liu et al. 2019, Nie et al. 2020).
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Traditionally, CAR T cells have been generated via the use of viral vectors, where CAR sequences are delivered by lentivirus (LV) for genome integration. This approach ensures efficient integration and stable expression of CAR constructs. However, use of viral vectors in CAR T cell generation carries disadvantages related to the random genome integration of CAR sequences, such as the potential for insertional mutagenesis, heterogeneous CAR expression and silencing of CAR expression (Chicaybam et al. 2020, Zhang et al. 2020).
The CRISPR/Cas9 system has made possible the targeting of CARs to specific genes of interest. Briefly, guide RNAs recognize specific DNA sequences through their crRNA component, a ~20 nucleotide sequence which anneals with a tracrRNA forming a ribonucleoprotein complex with Cas9. Targeting of genome loci by the CRISPR complex leads to double strand breaks, catalyzed by the associated Cas9 endonuclease. Lastly, cell repair mechanisms, such as non-homologous end joining (NHEJ) and homology-directed recombination (HDR), are subsequently activated to mend the sequences.
For CAR T cell generation, HDR may be exploited to achieve targeted gene insertions of large DNA sequences. For example, Eyquem et al. leveraged CRISPR/Cas9 tools, Cas9 mRNA and gRNA, to target a CD19-specific CAR for insertion in the TRAC (encoding the TCR alpha chain) locus (Eyquem et al. 2017). For this application, the CD19-CAR DNA donor template was delivered as part of an adeno associated virus (AAV6) vector.
Recently Zhang et al., in a new medRxiv preprint, targeted two genome loci, the AAVS1 safe harbor and PD1,to developed CAR T cells through the use of CRISPR/Cas9 tools. Targeting of a non-viral CAR DNA template, consisting of a CD19 antigen recognition domain and 4-1BB-CD3ζ signaling domain (19bbz), to the AAV1 locus via CRISPR/Cas9, resulted in the development of CAR T cells with similar cell expansion and tumor killing properties as those developed with a lentivirus. Similarly, targeting the same CAR cassette, 19bbz, to the PD1 locus resulted in CAR T cells with improved expansion properties and greater efficacy in eliminating PDL1 expressing tumor cells, when compared to those developed with lentivirus.
Next, a clinical trial (NCT04213469) was designed to evaluate the safety and efficacy of CRISPR/Cas9 PD1 targeted 19bbz for the treatment of relapsed/refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL). To this end, Zhang et al. electroporated patient derived T cells with the ribonucleoprotein complex (spCas9/ PD1-crRNA:tracRNA) and a non-viral CAR DNA template. Treatment with PD1-19bbz CAR T cells was not associated with severe adverse events and resulted in complete remission in two representative patients.
Overall, the specificity of the CRISPR/Cas gene editing system is rapidly being leveraged in the development of both autologous and allogenic CAR T cells. Relying on a non-viral knockin strategy for CAR T cell engineering is ensuring the generation of a therapeutic product that is both efficacious and safe.
