Role of anti‐CD40 antibody‐mediated costimulation blockade on non‐Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig‐to‐baboon model

Abstract

Background Recently, we have shown that an immunosuppression regimen including costimulation blockade via anti-CD154 antibody significantly prolongs the cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon heterotopic xenotransplantation model. Unfortunately, many coagulation disorders were observed with the use of anti-CD154 antibody, and recipient survival was markedly reduced by these complications. Material and Methods In this experiment, we replaced anti-CD154 antibody with a more clinically acceptable anti-CD40 antibody while keeping the rest of the immunosuppressive regimen and the donor pig genetics the same. This was carried out to evaluate the antibody's role in xenograft survival and prevention of coagulopathies. Two available clones of anti-CD40 antibody were tested. One mouse anti-human CD40 antibody, (clone 3A8), activated B lymphocytes in vitro and only modestly suppressed antibody production in vivo. Whereas a recombinant mouse non-human primate chimeric raised against macaque CD40, (clone 2C10R4), blocked B-cell activation in vitro and completely blocked antibody production in vivo. Results The thrombotic complications seen with anti-CD154 antibody were effectively avoided but the graft survival, although extended, was not as prolonged as observed with anti-CD154 antibody treatment. The longest survival for the 3A8 antibody group was 27 days, and the longest graft survival in the 2C10R4 antibody group was 146 days. All of the grafts except two rejected and were explanted. Only two recipient baboons had to be euthanized due to unrelated complications, and the rest of the baboons remained healthy throughout the graft survival period or after graft explantation. In contrast to our anti-CD 154 antibody-treated baboons, the non-Gal antibody levels started to rise after B cells made their appearance around 8 weeks post-transplantation. Conclusions Anti-CD40 antibody at the current dose does not induce any coagulopathies but while effective, had reduced efficacy to induce similar long-term graft survival as with anti-CD154 antibody perhaps due to ineffective control of B-cell function and antibody production at the present dose. More experiments are required to determine antibody affinity and effective dose for inducing long-term cardiac xenograft survival.

Genetically engineered pigs and target-specific immunomodulation provide significant graft survival and hope for clinical cardiac xenotransplantation

“…However, treatment with 4 weekly doses of anti CD20 effectively depleted all the circulating B cells and they stayed depleted for at least 60 days after which their number was slowly restored to pretreatment levels (6, 9). Although anti CD20 has no direct affect on plasma cells, the non Gal antibody production was maintained at a very low level by this treatment.…”

“…Neither clones had the thrombogenic characteristics seen with the anti CD154 (5C8) clone. The antibody from the 3A8 clone was not able to extend graft survival beyond 28 days (9). However, the graft survival was significantly prolonged with the 2C10R4 clone, irrespective of the pig genetics used.…”

In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs

“…Since neither 5C3 nor 3A8 can be used for future in vivo studies because 5C3 is limited to in vitro use and 3A8 has the capacity to be a B cell agonist [19, 23], only 2C10R4 was studied in subsequent in vitro functional assays.…”

“…Several anti-CD40mAbs, e.g., Chi220, 3A8, 5D12, 4D12, and 2C10, have shown promise in various transplant models without thromboembolic complication [4-6, 9-19, 21-24]. However, some of these anti-CD40 mAbs (i.e., Chi220, 3A8) have been associated with potentially adverse effects, e.g., as a B cell agonist or associated with substantial peripheral B cell depletion [9, 16, 19, 23]. Therefore, these specific anti-CD40mAbs may not be candidates for clinical translation.…”

“…Mohiuddin et al demonstrated prolonged xenograft survival of pig hearts in baboons receiving high doses of 2C10R4 (50mg/kg) in the absence of belatacept, but in combination with B cell depletion [23, 24], suggesting CD40/CD154 blockade alone at high dosage is sufficient to prevent rejection. Our own group has recently confirmed the good effect of this regimen after pig kidney transplantation in a baboon [47].…”