Background
B-cells are abundant tumor-infiltrating lymphocytes (TILs) in various solid tumors and exhibit both pro- and anti-tumor roles. Regulatory B-cells secrete immunosuppressive cytokines in the tumor microenvironment (TME), counteracting T-cell responses. Conversely, B-cells can produce anti-tumor antibodies, present tumor-specific antigens, secrete inflammatory molecules, and form tertiary lymphoid structures (TLSs), Germinal Centre (GC)-like structures crucial for anti-tumor responses. Antigen-specific B-cells significantly influence cancer prognosis; for instance, in HER2+ breast cancer (BC) patients, HER2-specific B-cells correlate with reduced recurrence and improved chemotherapy response. Preclinical studies suggested that transferring tumor-specific B-cells inhibit tumor progression and boost T-cell immunity, though isolating/expanding these rare B-cells remains challenging.
Methods
By optimizing time of treatment, AAV serotype, and cytokine stimulation, we refined a CRISPR-based B-Cell Receptor (BCR)-editing strategy to insert with high efficiency (~60%) an antigen-binding fragment from a clinical anti-HER2 antibody into the endogenous heavy chain locus of mouse and human B-cells, mimicking natural VDJ recombination (figure 1A). To maximize B-cell editing/expansion and instruct an activation phenotype, we tailored culture conditions using a CD40L-based stimulation. The resulting B-cells upregulate MHC and costimulatory molecules (CD80, CD86, and ICOSL) and supported T-cell stimulation on an in-vitro antigen presentation assay. Adoptive transplants in both syngeneic C57BL/6 and PBMC-humanized NSG mice, orthotopically implanted with HER2+ BCs, were used to study the impact of edited B-cells on tumor progression (figure 1B).
Results
Edited B-cells expressed the HER2-BCR, responded to BCR-mediated signaling, performed class switching, and secreted the engineered antibody (figure 1C,D). Biodistribution and bioluminescent analyses showed that edited B-cells migrate to hematopoietic tissues and infiltrate the tumor in both the syngeneic and xenografted mice. In the murine BC model, a pre-transplant chemotherapy regimen increased homing to the tumor, boosted GC reactions of host B-cells in the tumor-draining lymph-node, inguinal lymph-node, and spleen, and slowed-down tumor growth (figure 2A,B). Similarly, the adoptive transplant of edited human B-cells into PBMC-humanized NSG mice resulted in significant tumor reduction (figure 2C), increased tumor infiltration by transplanted B-cells (figure 2D) and by CD4 T-cells, and production of anti-HER2 antibodies. Notably, tumor reduction directly correlates with T-cell expansion but not with antibody levels; we observed a marked increase in IFN-producing T-cells after in-vitro re-challenge with tumor cells, suggesting a boost in tumor-reactive T-cells.
Conclusions
This study provides the first proof-of-concept in mouse models that ex-vivo-engineered B-cells specific for a tumor-associated antigen can increase the pool of anticancer B-cells, opening new avenues for combined active and passive cancer immunotherapy strategies.
Abstract 1140 Figure 1
In vitro generation of engineered primary B-cells. (A) Editing strategy. (B) Transplantation regimen of mouse (top) and human (bottom) B-cells. (C) FACS plot of mouse (left) and human (right) edited B-cells (HER2 +Tag+). (D) Anti-HER2 IgM/IgG antibodies; ELISA on culture supernatant
Abstract 1140 Figure 2
In vivo anti-tumor efficacy of engineered B-cells. (A) Tumor growth overtime in C57BL/6. (B) Frequency of host Germinal-Center+ B-cells within the Tumor-draining lymph nodes (TDLNs). (C) Tumor growth overtime in NSG mice. (D) Bioluminescence scanning of Luciferase+ human B-cells (Tumor highlighted in yellow