*Purpose: Humoral Alloimmune memory is a main barrier for successful kidney transplantation and is orchestrated by a compartmentalized cellular immune response driven by bone-marrow LLPCs and Germinal-center memory B (mBc) and T follicular helper (TFH) cells through key co-stimulatory signals such as CD28-CD80/86 and PD1-PDL2. Therefore, we hypothesized that by using a newly developed recombinant bi-specific fusion protein (Hybri2) that concomitantly blocks the CD80/86 pathway with a human CTLA4Ig extracellular domain and stimulates the PD1 with a human PD-L2 extracellular domain, it would confer a robust and sustained inhibition of the GC immune compartment thus, effectively inhibiting a donor-specific humoral memory immune response.

*Methods: To investigate the efficacy of this novel dual targeting as desensitization therapy, we used a stringent sensitization experimental MHC-mismatch rat model with two sequential skin transplants. Sensitized animals received Belatacept (10mg/kg, n=4), Hybri2 (20mg/kg, n=4) or bortezomib (1.33 mg/m2, n=4) IV, twice weekly for two weeks after sensitization. Desensitization efficacy was monitored with numbers and functionality of donor-specific mBc, short-live plasmablast, and TFH from the spleen and LLPC in the BM using both flow-cytometry and B-cell ELISPOT assays.

*Results: Dual Hybri2 desensitization therapy treated group showed significantly reduced frequencies of IgG-secreting mBC (1,5±2) than belatacept (15,5±6, p=0.054), the control group (22,5±6, p=0.04 vs Hibry2 and p=0.07 vs bela) and bortezomib (20,5±5, p=0.045 vs Hybri and p=0.08 vs bela). Significantly reduced frequencies on IgG-secreting plasmablasts were similarly observed between the Hybri2 (7,25±4) and belatacept (9.5±4; p=0.09), than the control group (51±16, 0.034 vs Hybri2 and p=0.045 vs bela). A significant reduction in the proportion of CXCR5+PD1+ICOS+ TFH cells among CD4+ T cells was detected in Hybri2 (8.01±2) and belatacept (8.55±3) as compared to the control (13,1±3, p=0.053) and bortezomib (14,3±2, p=0.048 vs Hybri2 and p=0.05 vs bela). A numerically lower but not statistically different numbers and frequencies of IgG-secreting LLPC in the BM were observed in the Hybri2 and belatacept groups than the control group, whereas bortezomib showed a significantly less LLPC than the other groups.

*Conclusions: Our data strongly suggest that dual targeting at the CD28-CD80/86 and PD1-PDL2 costimulatory level effectively abrogates GC humoral memory immune responses. This approach combined with central LLPC inhibition may be a new effective therapy strategy for successful desensitization.

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