Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection

Abstract

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Keywords: animal models: nonhuman primate; basic (laboratory) research/science; costimulation; fusion proteins and monoclonal antibodies: costimulation molecule specific; immunosuppressant; immunosuppression/immune modulation; rejection: T cell mediated (TCMR); translational research/science.

Figure 1. Anti-CD154 dAb does not activate human platelets in vitro

Platelets from human volunteers were incubated with hu5c8, hu5c8-mod with a mutated IgG construct, the F(ab)2 fragment of hu5c8, and the novel anti-CD154 dAb agent with the mutated IgG construct. Only hu5c8 resulted in platelet activation by PAC-1 ( ) or CD62P ( ) expression.

Figure 2. Treatment schedules for anti-CD154 dAb and conventional immunosuppression

(A) To determine the safety and efficacy of optimal dosing of anti-CD154 dAb, animals were treated with either 2, 10, or 20 mg/kg and underwent protocol sacrifice on day 77 after surgery while still on therapy. (B) To determine the potential survival benefit of anti- CD154 dAb, animals were treated with 30 mg/kg regimen of anti-CD154 dAb in combination with a conventional immunosuppression regimen of basiliximab, mycophenolate mofetil, and steroids.

Figure 3. 20mg/kg anti-CD154 dAb is a safe and effective dose

(A) Animals treated with 20mg/kg IV anti-CD154 dAb (n=5) (—) had significantly prolonged graft survival than those treated at lower doses (10mg/kg (n=1) ( ), 2mg/kg (n=2) ( )). (B) Animals treated with 20mg/kg maintained good renal function as measured by serum creatinine. Animals treated with lower doses had accelerated rises in their laboratory values consistent with their shortened rejection-free survival times. (C) After the first infusion on the date of transplant, the level of anti- CD154 dAb peaked on post-operative day 1 and subsequently decreased until day 7 when they received their next infusion. The 20mg/kg dose maintained trough levels >20ug/ml.

Figure 4. Coagulation factors and platelet counts were maintained in the normal range in animals treated with the optimal dosing of anti-CD154 dAb

(A–D) Serum levels of coagulation factors were measured from animals on the optimal dosing regimen of anti-CD154 dAb (n=5) for the first 30 days after transplant. Coagulation factors (PTT ,PT/Fibrinogen/D-Dimer/Antithrombin III ) were maintained in the normal range. Animals who did not receive anti-CD154 dAb as part of their treatment regimen were also tested as controls ( ). (E) Platelet counts were maintained above 100 (103/μL) with the 20mg/kg dosing regimen (20mg/kg —, 10mg/kg , 2mg/kg ) and comparable to control animals who did not receive anti-CD154 dAb ( ).

Figure 5. The addition of conventional immunosuppression to anti-CD154 dAb improves graft survival versus anti-CD154 dAb alone

(A) The combination of anti-CD154 dAb with basiliximab, mycophenolate mofetil, and steroids (n=5) (—)significantly improved rejection-free allograft survival than anti-CD154 dAb monotherapy (n=5) ( ) (MST 397 vs MST 103). (B) Animals treated with anti-CD154 and conventional immunosuppression had excellent renal function as assessed by serum creatinine (C) CMV viral load was used as a surrogate for overall level of immunosuppression Only one animal in the monotherapy group developed detectable CMV viremia which was quickly controlled with anti-viral therapy. No animal in the combined group developed detectable levels of virus.

Figure 6. Protocol biopsies revealed less infiltrate in the anti-CD154 dAb + conventional therapy group at both earlier and later time points

(A) Early protocol H&E stain biopsy of allograft at day 35 receiving only anti-CD154 dAb. (B) Late protocol H&E biopsy of allograft at day 70 receiving only anti-CD154 dAb. (C) Early protocol H&E biopsy of allograft at day 70 receiving anti-CD154 dAb + conventional therapy. (D) Late protocol H&E biopsy of allograft at day 140 receiving anti-CD154 dAb + conventional therapy. Monotherapy animals demonstrated greater severity in Banff scoring manifested by increased t and i scores compared to anti- CD154 dAb + conventional therapy animals. Monotherapy animals also had evidence of acute cellular rejection on earlier biopsies while those that received combination therapy did not.

Figure 7. A lower frequency of terminally differentiated CCR7-CD45RA+ CD4+ T cells was observed when conventional immunosuppression was added to anti-CD154 dAb

(A) No difference in the percentage of phenotypically naïve CD4+ or CD8+ T cells was observed between animals receiving anti-CD154 dAb alone (n=5) ( ) or anti-CD154 dAb + conventional therapy (n=5) ( ). (B) Animals receiving conventional immunosuppression in addition to anti-CD154 dAb demonstrated a significantly lower frequency of terminally differentiated CCR7-CD45RA+ CD4+ T cells during their time on therapy. Graphs depict mean ± standard error of the mean (SEM).

Figure 8. Treatment with anti-CD154 dAb increases the frequency of CD25+Foxp3+ regulatory T cells

For the duration of therapy with anti-CD154 dAb (n=5) ( ), the frequency of CD25+Foxp3+ phenotypically regulatory T cells was increased compared to standard costimulation blockade therapy (n=9) ( ). Differences between treatment groups were significant for time points over the entire duration of therapy. Graphs depict mean ± SEM.