Nondepleting anti-CD40-based therapy prolongs allograft survival in nonhuman primates

Abstract

Costimulation blockade of the CD40/CD154 pathway has been effective at preventing allograft rejection in numerous transplantation models. This strategy has largely depended on mAbs directed against CD154, limiting the potential for translation due to its association with thromboembolic events. Though targeting CD40 as an alternative to CD154 has been successful at preventing allograft rejection in preclinical models, there have been no reports on the effects of CD40-specific agents in human transplant recipients. This delay in clinical translation may in part be explained by the presence of cellular depletion with many CD40-specific mAbs. As such, the optimal biologic properties of CD40-directed immunotherapy remain to be determined. In this report, we have characterized 3A8, a human CD40-specific mAb and evaluated its efficacy in a rhesus macaque model of islet cell transplantation. Despite partially agonistic properties and the inability to block CD40 binding of soluble CD154 (sCD154) in vitro, 3A8-based therapy markedly prolonged islet allograft survival without depleting B cells. Our results indicate that the allograft-protective effects of CD40-directed costimulation blockade do not require sCD154 blockade, complete antagonism or cellular depletion, and serve to support and guide the continued development of CD40-specific agents for clinical translation.

Figure 1. 3A8 binds to rhesus CD40, but does not block sCD154

PFC was used to determine the specificity of 3A8 for rhesus CD40. (A) A competitive CD40-specific antibody was used to detect saturation of CD40 by 3A8 on CD20⁺ lymphocytes incubated with 3A8 (10 μg/ml). (B) CD40 saturation was measured using a competitive CD40-specific antibody as above at the indicated doses of 3A8 and IgG2b isotype control. (C) To test the ability of 3A8 to block sCD154 binding to CD40, lymphocyteswere incubated with 3A8, IgG2b isotype control or Chi220 (chimeric anti-CD40 mAb) at 10 μg/ml and then recombinant sCD154 at 1 μg/ml. Cells were then secondarily stained for sCD154. (D) Ability of various concentrations of 3A8, IgG2b and Chi220 to interfere with sCD154 binding. Data is representative of 3 independent experiments.

Figure 2. 3A8 inhibits alloreactive T cell proliferation

CFSE-labeled responder T cells were allo-stimulated in MLCs for 5 days in the presence of 3A8 or IgG2b isotype control and analyzed by PFC. (A) The percentage of proliferating cells (top left PFC plot corner) is depicted in the presence of 3A8 at the indicated doses. CD3⁺CD8⁺ PFC plots shown are representative. Similar findings were observed with CD4⁺ T cells. % Maximum (B)CD4⁺ and (C) CD8⁺ T cell proliferation (n = 5) at increasing doses of 3A8 and IgG2b isotype control. % Maximum is the percentage of proliferating cells at the indicated dose of 3A8 or IgG2b divided by the percentage of cells proliferating in the absence of antibody. Data represent mean ± SEM from 4 independent experiments.

Figure 3. 3A8 induces upregulation of CD80 and CD86on B cells in vitro

PFC was used to analyze the surface expression of CD80 and CD86 on CD20⁺ lymphocytes incubated for 24 hourswith3A8 or IgG2b isotype control at 10 μg/ml. (A) Percentage of CD80^hi and CD86^hi CD20⁺ cells in untreated, IgG2b-and 3A8-treated lymphocytes. (B) Fold change in the % of CD80^hi and CD86^hi B cells (n = 3). (C) Fold change in MFI of CD80 and CD86 on untreatedand treated B cells (n = 3). Data represent mean ± SEM from 3 independent experiments.

Figure 4. 3A8 prolongs islet allograft survival

STZ-induced diabetic rhesus monkeyswere transplanted allogeneic islets under cover of 3A8 (5–20 mg/kg) plus basiliximab (0.3 mg/kg) and sirolimus (target trough levels 5–15 ng/ml). Rejectionwas defined as FBG > 130 mg/dL on two consecutive daysafter initial islet engraftment. (A) Kaplan-Meier survival curves of 3A8 plus basiliximab/sirolimus-, 3A8-, and basiliximab/sirolimus-treated groups (n = 5, 2 and 3, respectively). 3A8 was discontinued on POD 35 (white arrow) and sirolimus on POD 134 (black arrow). Statistical analysis using the logrank test for graft survival between groups showed superiority of 3A8 plus basiliximab/sirolimus as compared to 3A8-and basiliximab/sirolimus-treated controls (P= 0.0082 and 0.0046, respectively). (B–D) Representative FBG graphs of islet recipients. Survival times (days) are listed for each corresponding graph.

Figure 5. 3A8 is non-depleting in vivo

Peripheral leukocyte counts were serially monitored using PFC in 3A8-treated monkeys. (A) Absolute numbers of CD3⁺ and CD20⁺ cells during and after 3A8 administration (n = 5). (B) Depletion of CD20⁺ lymphocytes observed in a previous studywith the depleting CD40-specific mAb Chi220 (n = 5) (18). The black bars depict 3A8 and Chi220 dosing. Data represent mean ± SEM.

Figure 6. 3A8 achieves CD40 saturation in vivo

CD40 saturation was assessed in 3A8-treated animals by serial pharmacodynamic monitoring of peripheral blood using PFC. (A) Representative PFC plots of unbound CD40 on recipient CD20⁺ lymphocytes pre-transplant (PreTx, red) and while on 3A8 (blue) on POD 7 are shown, as determined by staining cells with a competitive anti-CD40 mAb (as described in Figure 1). (B) CD20⁺ lymphocytes were stained with anti-mouse IgG2b to demonstrate the presence of 3A8 on targeted cells after treatment. Data shown was obtained on POD 7 (4 days after last 3A8 dose). (C) Representative longitudinal graph of CD40 saturation by 3A8 on CD20⁺ lymphocytes in a 3A8-treated recipient, as determined by the competitive binding assay described above (3A8 dosing depicted by the black bars). The possibility that less unbound CD40 represents a combination of 3A8 saturation and lower CD40 expression levels cannot be excluded.

Figure 7. 3A8-based therapy attenuates DSA formation

Donor lymphocytes were incubated with corresponding islet recipient seraat the indicated time pointsand then analyzed by PFC to test for donor-specific IgG antibody formation. Each line represents one individual recipient (n = 5) and the order is maintained amongst the designated time points. Detectable antibody levels are shown pre-transplant (yellow),during therapy (green), and at immunosuppression withdrawal (blue) and rejection (red).