CD40-specific costimulation blockade enhances neonatal porcine islet survival in nonhuman primates

Abstract

The widespread clinical implementation of alloislet transplantation as therapy for type 1 diabetes has been hindered by the lack of suitable islet donors. Pig-to-human islet xenotransplantation is one strategy with potential to alleviate this shortage. Long-term survival of porcine islets has been achieved using CD154-specific antibodies to interrupt the CD40/CD154 costimulation pathway; however, CD154-specific antibodies seem unlikely candidates for clinical translation. An alternative strategy for CD40/CD154 pathway interruption is use of CD40-specific antibodies. Herein, we evaluate the ability of a chimeric CD40-specific monoclonal antibody (Chi220) to protect islet xenografts. Neonatal porcine islets (~50,000 IEQ/kg) were transplanted intraportally into pancreatectomized diabetic macaques. Immunosuppression consisted of induction therapy with Chi220 and the IL-2 receptor-specific antibody basiliximab, and maintenance therapy with sirolimus and the B7-specific fusion protein belatacept. Chi220 effectively promoted xenoislet engraftment and survival, with five of six treated recipients achieving insulin-independent normoglycemia (median rejection-free survival 59 days; mean 90.8 days, maximum 203 days). No thromboembolic phenomena were observed. CD40 represents a promising alternative to CD154 as a therapeutic target, and the efficacy of CD40-specific antibodies in islet xenotransplantation warrants further investigation.

Figure 1. Anti-CD40 therapy facilitates xenoislet engraftment and function

Induction therapy with Chi220 resulted in prolonged insulin-independent normoglycemia in xenoislet recipients. Reason for experimental termination is indicated by the superscript following each ID. (a) Cohort 1, animals that received immunosuppression starting on the day of transplant. (b) Cohort 2, animals that received immunosuppression starting 5 days prior to transplant. (c) Cohort 3, animals that received a Chi220-free immunosuppressive regimen starting on day of transplant. Daily total insulin requirement is shown in red; daily FBG is shown in blue. ^a failure of engraftment; ^b weight loss / failure to thrive; ^c rejection

Figure 2. Weight loss in islet recipients

Change in weight over time for individual recipients in cohort 1 (a) and cohort 2 (b) shown as percentage of weight on day of transplant. All recipients demonstrated significant rapid weight loss following pancreatectomy, with a decrease in the rate of weight loss following islet transplantation. Horizontal line represents weight at transplant. POD – Post-operative day.

Figure 3. Improved glucose tolerance following islet transplant

Representative graph. An intravenous glucose bolus was given immediately after time 0 and blood glucose levels measured at the intervals shown. Recipients had poor glycemic control following pancreatectomy that improved following xenoislet transplant. Pre-transplant IVGTT was performed one week after pancreatectomy and two weeks prior to transplant. Post-transplant IVGTT was performed approximately one month following islet transplantation.

Figure 4. Confirmation of xenograft function with measurement of porcine c-peptide

(a) Serial c-peptide measurement. Porcine c-peptide was measured in serum samples from fasting recipients taken at regular intervals. Values were pooled by cohort, averaged, and arranged by number of weeks following transplant. While all recipients had detectable c-peptide levels until experimental endpoint, measurements taken in animals following rejection approached the lower threshold of detection. (b) Stimulated c-peptide production, representative graph. Porcine c-peptide was measured at the time intervals shown following an intravenous glucose bolus. Each line represents a different post-transplant day (legend inset) for a single animal with long-term graft function, ROf9. POD – post-operative day. IVGTT – intravenous glucose tolerance test.

Figure 5. Cellular infiltration and insulin staining in rejected vs. functional islet

Staining of intrahepatic islets with anti-insulin antibody (brown), performed on liver sections obtained at recipient necropsy. (a) Representative section from recipient experiencing loss of graft function (RZy9), showing dense cellular infiltrate with minimal insulin positivity. (b) Representative section from recipient euthanized in the setting of persistent normoglycemia (RIu9), showing minimal cellular infiltrate with strong insulin positivity.

Figure 6. Cellular infiltrate surrounding rejected islets consists mainly of T cells

Immunohistochemical assessment of intrahepatic islets from recipient experiencing acute rejection (RZy9 - left), compared with recipient undergoing euthanasia prior to loss of graft function (RIu9 - right). Brown areas represent staining positivity. The rejected islet displays a significant cellular infiltrate staining strongly for CD3 (T cell marker) and moderately for CD68 (macrophage marker), with minimal CD20 and C4d staining (B cell and complement markers, respectively). The cellular infiltrate also stained strongly for both CD4 and CD8 T cell subsets, but did not stain for neutrophil elastase (data not shown).