Measuring the Impact of Targeting FcRn-Mediated IgG Recycling on Donor-Specific Alloantibodies in a Sensitized NHP Model.

Background: In transplantation, plasmapheresis and IVIg provide the mainstay of treatment directed at reducing or removing circulating donor-specific antibody (DSA), yet both have limitations. We sought to test the efficacy of targeting the IgG recycling mechanism of the neonatal Fc receptor (FcRn) using anti-FcRn mAb therapy in a sensitized non-human primate (NHP) model, as a pharmacological means of lowering DSA. Methods: Six (6) rhesus macaque monkeys, previously sensitized by skin transplantation, received a single dose of 30mg/kg anti-RhFcRn IV, and effects on total IgG, as well as DSA IgG, were measured, in addition to IgM and protective immunity. Subsequently, 60mg/kg IV was given in the setting of kidney transplantation from skin graft donors. Kidney transplant recipients received RhATG, and tacrolimus, MMF, and steroid for maintenance immunosuppression. Results: Circulating total IgG was reduced from a baseline 100% on D0 to 32.0% (mean, SD ± 10.6) on d4 post infusion (p<0.05), while using a DSA assay. T-cell flow cross match (TFXM) was reduced to 40.6±12.5% of baseline, and B-cell FXCM to 52.2±19.3%. Circulating total IgM and DSA IgM were unaffected by treatment. Pathogen-specific antibodies (anti-gB and anti-tetanus toxin IgG) were significantly reduced for 14d post infusion. Post-transplant, circulating IgG responded to anti-FcRn mAb treatment, but DSA increased rapidly. Conclusion: Targeting the FcRn-mediated recycling of IgG is an effective means of lowering circulating donor-specific IgG in the sensitized recipient, although in the setting of organ transplantation mechanisms of rapid antibody rise post-transplant remains unaffected.

A comparative study of human-and rhesus-specific antithymocyte globulins in Rhesus macaques.

Rabbit antithymocyte globulin (RATG) preparations are widely used in transplantation. They are developed in vivo against thymocytes and contain polyclonal antibodies specific for myriad cellular targets. The rhesus monkey is commonly used as a preclinical transplant model, but the fidelity of commercially available human-specific RATGs to anticipate the effects of RATGs in rhesus has not been established. We therefore developed two rhesus-specific ATGs (rhATG) and compared them to human-specific RATG (huATG, Thymoglobulin® ) in rhesus monkeys, assessing the magnitude and phenotype of depletion peripherally and in lymph nodes. Four primates were assigned to each group and received 20 mg/kg of drug. Depletion, repopulation, and changes in lymphocyte subsets were evaluated in peripheral blood and lymph nodes by flow cytometry over four months. We observed similar qualitative changes in lymphocyte subsets, but a generally more profound depletion with huATG compared to either rhATG. Peripheral homeostatic proliferation rather than thymic output was the major mechanism for repopulation with all RATGs. Repopulation was slower but qualitatively similar when examining RATGs in additional animals receiving concomitant chronic immunosuppression. Depletional induction is similar to human- and rhesus-specific RATGs in rhesus macaques. Both rhesus- and human-specific agents appear appropriate for preclinical modeling of clinical RATG use.

Non-neutralizing Antibodies May Contribute to Suppression of SIVmac239 Viremia in Indian Rhesus Macaques.

The antiviral properties of broadly neutralizing antibodies against HIV are well-documented but no vaccine is currently able to elicit protective titers of these responses in primates. While current vaccine modalities can readily induce non-neutralizing antibodies against simian immunodeficiency virus (SIV) and HIV, the ability of these responses to restrict lentivirus transmission and replication remains controversial. Here, we investigated the antiviral properties of non-neutralizing antibodies in a group of Indian rhesus macaques (RMs) that were vaccinated with vif, rev, tat, nef, and env, as part of a previous study conducted by our group. These animals manifested rapid and durable control of viral replication to below detection limits shortly after SIVmac239 infection. Although these animals had no serological neutralizing activity against SIVmac239 prior to infection, their pre-challenge titers of Env-binding antibodies correlated with control of viral replication. To assess the contribution of anti-Env humoral immune responses to virologic control in two of these animals, we transiently depleted their circulating antibodies via extracorporeal plasma immunoadsorption and inhibition of IgG recycling through antibody-mediated blockade of the neonatal Fc receptor. These procedures reduced Ig serum concentrations by up to 80% and temporarily induced SIVmac239 replication in these animals. Next, we transferred purified total Ig from the rapid controllers into six vaccinated RMs one day before intrarectal challenge with SIVmac239. Although recipients of the hyperimmune anti-SIV Ig fraction were not protected from infection, their peak and chronic phase viral loads were significantly lower than those in concurrent unvaccinated control animals. Together, our results suggest that non-neutralizing Abs may play a role in the suppression of SIVmac239 viremia.

Anti-CD40 antibody 2C10 binds to a conformational epitope at the CD40-CD154 interface that is conserved among primate species.

The antagonistic anti-CD40 antibody, 2C10, and its recombinant primate derivative, 2C10R4, are potent immunosuppressive antibodies whose utility in allo- and xenotransplantation have been demonstrated in nonhuman primate studies. In this study, we defined the 2C10 binding epitope and found only slight differences in affinity of 2C10 for CD40 derived from four primate species. Staining of truncation mutants mapped the 2C10 binding epitope to the N-terminal portion of CD40. Alanine scanning mutagenesis of the first 60 residues in the CD40 ectodomain highlighted key amino acids important for binding of 2C10 and for binding of the noncross-blocking anti-CD40 antibodies 3A8 and 5D12. All four 2C10-binding residues defined by mutagenesis clustered near the membrane-distal tip of CD40 and partially overlap the CD154 binding surface. In contrast, the overlapping 3A8 and 5D12 epitopes map to an opposing surface away from the CD154 binding domain. This biochemical characterization of 2C10 confirms the validity of nonhuman primate studies in the translation of this therapeutic antibody and provides insight its mechanism of action.

IL-10 Impairs Local Immune Response in Lung Granulomas and Lymph Nodes during Early Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major global health problem. Lung granulomas are organized structures of host immune cells that function to contain the bacteria. Cytokine expression is a critical component of the protective immune response, but inappropriate cytokine expression can exacerbate TB. Although the importance of proinflammatory cytokines in controlling M. tuberculosis infection has been established, the effects of anti-inflammatory cytokines, such as IL-10, in TB are less well understood. To investigate the role of IL-10, we used an Ab to neutralize IL-10 in cynomolgus macaques during M. tuberculosis infection. Anti-IL-10-treated nonhuman primates had similar overall disease outcomes compared with untreated control nonhuman primates, but there were immunological changes in granulomas and lymph nodes from anti-IL-10-treated animals. There was less thoracic inflammation and increased cytokine production in lung granulomas and lymph nodes from IL-10-neutralized animals at 3-4 wk postinfection compared with control animals. At 8 wk postinfection, lung granulomas from IL-10-neutralized animals had reduced cytokine production but increased fibrosis relative to control animals. Although these immunological changes did not affect the overall disease burden during the first 8 wk of infection, we paired computational modeling to explore late infection dynamics. Our findings support that early changes occurring in the absence of IL-10 may lead to better bacterial control later during infection. These unique datasets provide insight into the contribution of IL-10 to the immunological balance necessary for granulomas to control bacterial burden and disease pathology in M. tuberculosis infection.