Regulatory T Cell Depletion Using a CRISPR Fc-Optimized CD25 Antibody

Abstract

Regulatory T cells (T_regs) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. T_regs are characterized by a high expression of CD25, which is a potentially valuable target for T_reg depletion to alleviate immune suppression. The preclinical anti-CD25 (αCD25) antibody, clone PC-61, has met with modest anti-tumor activity due to its capacity to clear T_regs from the circulation and lymph nodes, but not those that reside in the tumor. The optimization of the Fc domain of this antibody clone has been shown to enhance the intratumoral T_reg depletion capacity. Here, we generated a stable cell line that produced optimized recombinant T_reg-depleting antibodies. A genome engineering strategy in which CRISPR-Cas9 was combined with homology-directed repair (CRISPR-HDR) was utilized to optimize the Fc domain of the hybridoma PC-61 for effector functions by switching it from its original rat IgG1 to a mouse IgG2a isotype. In a syngeneic tumor mouse model, the resulting αCD25-m2a (mouse IgG2a isotype) antibody mediated the effective depletion of tumor-resident T_regs, leading to a high effector T cell (T_eff) to T_reg ratio. Moreover, a combination of αCD25-m2a and an αPD-L1 treatment augmented tumor eradication in mice, demonstrating the potential for αCD25 as a cancer immunotherapy.

Keywords: CRISPR-HDR; Fc optimization; antibody; hybridoma; immunotherapy.

Figure 1

CRISPR-HDR-mediated Fc optimization of PC-61 hybridoma. (A) Schematic of CRISPR-HDR engineering to tune the expression of PC-61 hybridoma from rat IgG1 to mouse IgG2a. (B) The IgH locus of PC-61 was targeted with gRNA-R1 to create a double-stranded break that could be repaired through homology with the donor construct that inserted the murine IgG2a Fc domain with c-terminal sortag and his-tag (srt-his) and gene elements for blasticidin resistance. (C) Supernatant HDR and gRNA-R1-transfected cells that were resistant to blasticidin were taken and screened for secretion of his-tag-positive antibodies via dot-blot. (D) His-tag-positive supernatants were used to stain activated splenocytes in combination with secondary antibodies against mouse IgG2a (yellow) and rat IgG1 (violet) to select the engineered hybridoma that exclusively secreted m2a antibodies targeting CD25. The selected PC-61-m2a clone for antibody production is indicated (B2). (E) His-tag removal of PC-61-m2a via sortase-mediated hydrolysis with non-reducing SDS-PAGE and immunoblot. (F) αCD25-m2a efficiently depleted T_regs, resulting in an increased T_eff/T_reg ratio.

Figure 2

Effective depletion of T_regs in blood, lymph nodes, and tumors via Fc-optimized αCD25-m2a. (A) Schematic of tumor challenge in C57/Bl6 mice. On day 0, mice were subcutaneously inoculated with MC38 and on days 5 and 7 were treated with either αCD25-m2a or an isotype control. Blood, lymph nodes, and tumors were harvested on day 9. (B) Representative plots showing expression of CD25 and FoxP3 in CD3⁺CD4⁺ T cells isolated from tumors. Numbers show percentage of cells in each quadrant. (C) MFI of CD25 in T_regs, percentage of T_reg cells in CD3⁺CD4⁺ T cells, and T_eff/T_reg cell ratios in blood, lymph nodes, and tumors (n = 5, statistical difference determined via independent Student’s t-test: ** p ≤ 0.01; *** p ≤ 0.001; **** p < 0.0001).

Figure 3

CD25-m2a treatment synergizes with αPD-L1 treatment. (A) Schematic of treatment schedule of C57/BL6 mice inoculated with MC38. On day 0, mice were subcutaneously inoculated with MC38 and subsequently treated on days 5 and 7 with αCD25-m2a or an isotype control, or treated on days 5, 8, and 11 with αPD-L1, or received both αCD25-m2a and the αPD-L1 treatment. Mice were euthanized when tumors reached a volume of 1500 mm³. (B) Growth curves of MC38 tumors. Number of tumor-free survivors is shown in each graph. (C) Survival curves and (D) response rates of mice shown in (B) (n = 8–10, statistical difference determined via log-rank test). Significance is shown as * = p < 0.05, ** = p < 0.01.

Figure 4

Continued T_reg depletion does not affect therapeutic efficacy. (A) Schematic of treatment schedule of C57/BL6 mice inoculated with MC38. On day 0, mice were subcutaneously inoculated with MC38 and subsequently treated on days 5 and 7 with αCD25-m2a (short-term) or days 5, 7, 11, 16, 20, and 25 (continued) or an isotype control, or treated on days 5, 8, and 11 with αPD-L1, or received both αCD25-m2a and the αPD-L1 treatment. Mice were euthanized when tumors reached a volume of 1500 mm³. (B) Growth curves of MC38 tumors. Number of tumor-free survivors is shown in each graph. (C) Survival curves including significance table and (D) response rates of mice shown in (B) (n = 8–10, statistical difference determined via log-rank test). Significance is shown as * = p < 0.05, and *** = p < 0.001.