Tolerance induction remains the “Holy Grail” in transplant medicine given the disillusioning long-term graft survival rates. The establishment of hematopoietic chimerism is a promising tolerance strategy, with encouraging results having been reported from clinical proof-of-concept trials in kidney transplant recipients.1 However, despite these results, full translational success is hampered by various factors, including the remaining toxicity of the recipient conditioning and the restricted applicability to the living donor setting. Thus, novel regimens are sought to overcome these issues. The use of costimulation blockers as part of chimerism regimens has long appeared to be a step toward this goal. For 2 decades, inhibition of the CD40/CD40L (CD154) pathway has yielded promising results in murine studies. Anti-CD40L treatment—with or without additional CTLA4Ig—allowed conditioning requirements to be substantially reduced in mouse models of chimerism-based tolerance.2,3 Tolerance was achieved through a unique interplay of deletional and regulatory mechanisms, and anti-CD40L became a mainstay of many experimental chimerism models. In parallel, anti-CD40L monoclonal antibodies (mAbs) were developed as immunosuppressive drugs due to the attractive immunomodulatory properties observed in preclinical studies.4 However, because conventional anti-CD40L mAbs cause thromboembolic adverse effects, development of this class of antibodies was halted.5 Only recently, monovalent anti-CD40L antibodies (domain antibodies or Fab fragments) have entered development. As an alternative approach, mAbs specific for CD40—the receptor of CD40L—have emerged that are free of thrombotic complications and several clones of anti-CD40 mAbs have been tested in preclinical and clinical studies.
In this issue of Transplantation Oura and colleagues present their experience with an anti-CD40 mAb in a nonhuman primate (NHP) model of chimerism-based tolerance.6 They added anti-CD40 (clone 2C10R4) to a delayed bone marrow transplantation (BMT) regimen. Kidney or kidney/islet recipients received immunosuppression (including anti-CD40) for 4 months, when they were conditioned with nonmyeloablative irradiation and received donor bone marrow. At the time of BMT hATG, belatacept (a CTLA4Ig derivative) and cyclosporine were started, and anti-CD40 therapy was continued. All immunosuppressions were stopped 1 month after BMT. Chimerism levels were observed that were superior to previous regimens. However, unexpectedly, all islet and kidney grafts were rejected soon after the withdrawal of immunosuppression. This is in stark contrast to a delayed BMT protocol based on belatacept and rATG without anti-CD40 that had previously been reported by the same group in which long-term immunosuppression-free graft acceptance was achieved. Collectively, the data presented suggest that anti-CD40 mAb impeded rather than promoted tolerance induction (Figure 1).
Several factors could account for the unexpected detrimental effect of anti-CD40. Anti-CD40 was associated with higher frequencies of memory B cells and the early appearance of donor-specific antibodies. Notably, donor-specific regulation—an important feature of chimerism-based tolerance in previous NHP protocols—was impaired and anti-donor CD8 reactivity failed to diminish. Thus, anti-CD40 therapy seemed to inhibit regulation and the tolerization of CD8 T cells. This finding is in contrast to the evidence that anti-CD40L potently promotes regulation.7,8 A potential explanation offered by the authors might be that APC maturation, which is necessary for regulatory T (Treg) cell generation, is hampered by anti-CD40 therapy. Because of the sample size limitations inherent in NHP studies, historical groups served as controls. As acknowledged by the authors, these differed compared with the current study group also with regard to the potency of the ATG preparation used (rat vs horse ATG), and thus, the degree of lymphocyte depletion. A direct control group differing solely with respect to the addition of anti-CD40 is not available. This is a caveat when interpreting the observed higher frequencies of memory B cells and donor-specific antibodies, which theoretically might also be influenced by the less potent ATG preparation used in the anti-CD40 group of the present study.
Anti-CD40 abrogated tolerance induction even though it led to higher and more extended levels of chimerism. The relationship between chimerism and tolerance remains incompletely understood. Although (at least transient) chimerism is a prerequisite for tolerance in BMT-based transplant models (in NHP and mice), chimerism development does not guarantee organ graft acceptance. Chimerism without organ-specific tolerance has been observed in a variety of protocols, including an NHP study relying on CD40 blockade and belatacept.9 Chimerism quantity (ie, levels) is generally a poor predictor of tolerance. Lymphocyte, or more specifically, T-cell chimerism, and thus the quality of chimerism, is usually a better predictor for tolerance. However, in the present study, lymphocyte chimerism was also superior to previous protocols, nevertheless, the kidney grafts were rejected (T-cell chimerism was not reported). From rodent studies, it has become increasingly clear that the tolerance mechanisms established through the specific conditioning cocktail in connection with the donor BMT are the decisive factor whether graft tolerance ensues or not. In particular, there is a strong association between regulatory mechanisms which are capable of tolerizing tissue-specific antigens and durable tolerance.10 Regulation might be perturbed by anti-CD40, as CD40 is involved in Treg cell generation in several ways,11,12 which might be the critical factor in tolerance failure. In this respect, it is important to note that kidney recipients in the present NHP study had already received 4 months of anti-CD40 before they received the BMT. If anti-CD40 indeed negatively interferes with Treg cell generation, prolonged anti-CD40 therapy might lead to untoward changes in the recipient T-cell repertoire negatively affecting the balance of regulatory and effector T cells and potentially making tolerance induction more difficult. It would thus be of interest to test if anti-CD40 had a similarly negative effect when started at the time of BMT.
In the murine setting of mixed chimerism, chronic absence of CD40 (using CD40 knock-out donor and recipient mice) led to results comparable to the treatment with anti-CD40L.13 It remains puzzling why findings are so strikingly different in NHP models using anti-CD40 (or anti-CD40L, for that matter14). The physiological differences in the immune systems of young protected laboratory animals versus adult NHP, with respect to memory T-cell frequencies and many other factors, might explain most of the discrepancies. Whether pharmacokinetic properties of anti-CD40L and anti-CD40 mAbs (ie, ligand/receptor saturation) used in mouse versus NHP might also play a role or whether various anti-CD40 clones differ in their ability to block CD40 without any partial agonistic activity15 remains unclear. In this respect, there remains the concern that binding affinities of anti-CD40 mAbs might differ between humans and nonhuman primates.
Currently, the clinical experience with CD40 blockade remains limited and studies in transplant patients are scarce. In a phase II trial of kidney transplant recipients, ASKP1240 (bleselumab) was associated with a very high rate of biopsy-proven acute rejections when given as part of a CNI-free regimen.16 Besides, ASKP1240 has been tested in patients with psoriasis,17 where it proved to be safe, but the study was not powered to detect a difference in disease activity. A recent study compared another (Fc-silenced) anti-CD40 clone (CFZ533) with tacrolimus in de novo renal transplant recipients (in combination with basiliximab, MMF, and corticosteroids). Preliminary results reported so far suggest that CFZ533 had comparable efficacy with regard to the composite endpoint of treated biopsy-proven acute rejection, graft loss, or death and was associated with superior graft function.18 Future and more definitive trial results are awaited to allow a full assessment of the potential of anti-CD40 therapy as primary immunosuppression in organ transplantation.
The group of Tatsuo Kawai and colleagues are to be commended for their persistent and methodical efforts to develop improved chimerism-based tolerance protocols. Although mouse models are important for developing proof-of-concept, results from NHP studies such as the one discussed herein, are critical for guiding clinical translation. The present study cautions against the use of anti-CD40 in chimerism protocols relying on Treg-dependent tolerance mechanisms. The search for widely applicable chimerism protocols continues.
1. Sasaki H, Oura T, Spitzer TR, et al. Preclinical and clinical studies for transplant tolerance via the mixed chimerism approach. Hum Immunol
2. Wekerle T, Sayegh MH, Hill J, et al. Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance. J Exp Med
3. Durham MM, Bingaman AW, Adams AB, et al. Cutting edge: administration of anti-CD40 ligand and donor bone marrow leads to hemopoietic chimerism and donor-specific tolerance without cytoreductive conditioning. J Immunol
4. Kirk AD, Harlan DM, Armstrong NN, et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci U S A
5. Kawai T, Andrews D, Colvin RB, et al. Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand. Nat Med
6. Oura T, Hotta K, Rosales I, et al. Addition of anti-CD40 monoclonal antibody to nonmyeloablative conditioning with belatecept abrogated allograft tolerance despite induction of mixed chimerism. Transplantation
7. Kingsley CI, Karim M, Bushell AR, et al. CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. J Immunol
8. Ferrer IR, Wagener ME, Song M, et al. Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade. Proc Natl Acad Sci U S A
9. Ramakrishnan SK, Page A, Farris AB 3rd, et al. Evidence for kidney rejection after combined bone marrow and renal transplantation despite ongoing whole-blood chimerism in rhesus macaques. Am J Transplant
10. Pilat N, Mahr B, Unger L, et al. Incomplete clonal deletion as prerequisite for tissue-specific minor antigen tolerization. JCI Insight
11. Oh J, Wu N, Barczak AJ, et al. CD40 mediates maturation of thymic dendritic cells driven by self-reactive CD4(+) thymocytes and supports development of natural regulatory T cells. J Immunol
12. Akiyama T, Shimo Y, Yanai H, et al. The tumor necrosis factor family receptors RANK and CD40 cooperatively establish the thymic medullary microenvironment and self-tolerance. Immunity
13. Pilat N, Klaus C, Schwarz C, et al. Rapamycin and CTLA4Ig synergize to induce stable mixed chimerism without the need for CD40 blockade. Am J Transplant
14. Larsen CP, Page A, Linzie KH, et al. An MHC-defined primate model reveals significant rejection of bone marrow after mixed chimerism induction despite full MHC matching. Am J Transplant
15. Badell IR, Thompson PW, Turner AP, et al. Nondepleting anti-CD40-based therapy prolongs allograft survival in nonhuman primates. Am J Transplant
16. Harland R, Klintmalm G, Yang H. ASKP1240 in de novo kidney transplant recipients. American Transplant Congress
17. Anil Kumar MS, Papp K, Tainaka R, et al. Randomized, controlled study of bleselumab (ASKP1240) pharmacokinetics and safety in patients with moderate-to-severe plaque psoriasis. Biopharm Drug Dispos
18. Nashan B, Tedesco H, van den Hoogen M, et al. CFZ533, a new anti-CD40 mAB demonstrates comparable efficacy and better renal function versus tacrolimus in de-novo CNI-free kidney transplantation. Am J Transplant
. 2018;18(suppl 4).