CRG-023 Is a Novel Tri-Specific CAR T Product Candidate Engineered to Prevent Antigen Escape and Sustain Durable Anti-Tumor Functionality Against B-Cell Malignancies
Authors: Sagiv Barfi Idit , Liao Eric , Mannon Michael S , Quach Boi , Wang Lingyan , Koo-McCoy Samantha , Yi Michael C , Byers James S , Nguyen Nhung , Bentley Trevor M , Yung Savannah , Huang Yu , Bethune Michael T , Siegel Matthew , Ports Michael O 
Published: December 2024

Introduction:
Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment landscape for B-cell lymphomas and leukemias. However, most patients do not achieve durable response. Mechanisms that facilitate resistance and impact survival outcomes include antigen escape, loss of CD58 co-stimulation, and CAR T exhaustion. Novel CAR T-cell designs are critical to address these challenges.
Construct design and product characterizations:
CRG-023 is a tri-specific CAR T that targets the B-cell lineage antigens CD19, CD20, and CD22 via tri-cistronic expression of 3 distinct second-generation CARs from a single lentiviral vector. The CD19- and CD20-targeting CARs employ novel, human single-chain variable fragment (scFv) binders selected using methods to assess CAR-mediated T-cell functionality. The CD22-targeting CAR employs the human scFv m971 (Frank M et al. The Lancet 2024).
Each CAR incorporates a CD3ζ signaling domain and a distinct costimulatory domain derived from 4-1BB (CD22-targeting CAR), CD28 (CD19-targeting CAR), or CD2 (CD20-targeting CAR). CD2 is the costimulatory receptor required for CD58 engagement. Each CAR sequence and arrangement within the tri-cistronic vector were engineered to achieve optimal CAR T-cell activity. Further codon optimization and removal of splice sites were performed to limit potential recombination and to ensure stable CAR expression. For the tri-specific construct, flow cytometry analysis demonstrated that each CAR expressed well and the aggregate surface expression of all 3 receptors was comparable to levels measured on monospecific CAR T cells. Importantly, the tri-cistronic lentiviral vector was evaluated for manufacturability to ensure that sufficient titers of transducing virus were generated.
In vitro results:
The tri-specific CAR T cells sustained durable anti-tumor control when repeatedly challenged with new tumor cells expressing all three antigens. Moreover, durable functionality was demonstrated with repeat challenges by tumor cells expressing a single cognate antigen, indicating that the activity of CRG-023 is not dependent on any single CAR specificity. Superior tumor control was also observed against target cells expressing low CD19 antigen levels (~1000-6000 molecules per cell) as compared to FMC63 scFv CD19-targeting CAR T-cell benchmarks. In these assays, the tri-specific CAR T cells were less differentiated and sustained proliferation of both CD4 and CD8 T-cell subsets. Importantly, the expression of three distinct CARs did not result in excessive cytokine secretion or activation marker expression relative to the monospecific CAR T-cell benchmark controls.
In vivo results:
CRG-023 CAR T cells generated from healthy donor material in a full-scale manufacturing process demonstrated superior anti-tumor activity in mouse models of disseminated lymphoma compared to monospecific CAR T cells. Additional dose-response studies showed complete tumor clearance was achieved in Raji lymphoma models with the lowest dose tested (105 CRG-023 CAR T cells per mouse). We also modeled in vivo CD19 antigen escape using a mixture of wild-type Raji cells and CD19 knockout Raji (CD19ko) cells to address the potential for CD19 antigen loss to limit durable response as seen in CD19-targeting CAR T cells (Zurko J el al. Blood Advances 2023). Using this model, the tri-specific CAR T cells effectively eradicated all tumors, in contrast to FMC63-containing CD19 CAR T cells, for which tumor escape was observed. Collectively the data show that CRG-023 exhibits enhanced, durable clearance of lymphoma tumors in vivo when using low doses and in antigen loss models relative to benchmark controls.
Conclusions:
Significant engineering and screening were undertaken to develop CRG-023, a potent tri-specific CAR T-cell product candidate with differentiated pre-clinical activity. CRG-023 demonstrated durable potency across a range of antigen levels in stringent in vitro and in vivo models, with each of the three CARs contributing to its functionality. By addressing factors known to be associated with inferior CAR T-cell response, CRG-023 may improve outcomes for patients afflicted with B-cell malignancies, paving the way for further clinical development and translation into the clinic.