James Rush

Deficiency of MALT1 Paracaspase Activity Results in Unbalanced Regulatory and Effector T and B Cell Responses Leading to Multiorgan Inflammation

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1PD/PD) and compared their phenotype with that of MALT1 knockout animals (Malt1−/−). Malt1PD/PD mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10–producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1−/− animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1PD/PD mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1PD/PD animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1PD/PD animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

A Novel, Blocking, Fc-Silent Anti-CD40 Monoclonal Antibody Prolongs Nonhuman Primate Renal Allograft Survival in the Absence of B Cell Depletion

CD40–CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti‐CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti‐CD40 antibody can still promote graft survival. The parent anti‐CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC‐mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc‐silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98–100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well‐tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc‐silent anti‐CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.

Nonclinical Safety Assessment of CFZ533, a Fc-Silent Anti-CD40 Antibody, in Cynomolgus Monkeys

CFZ533 is a pathway blocking, nondepleting anti-CD40 antibody that is in clinical development for inhibition of transplant organ rejection and therapy for autoimmune diseases. A 26-week GLP toxicity study in sexually mature Cynomolgus monkeys was conducted in order to support chronic application of CFZ533. CFZ533 was subcutaneously administered at doses up to 150 mg/kg/week and was safe and generally well tolerated. CFZ533 showed no adverse effects for cardiovascular, respiratory, and neurobehavioral endpoints, and no changes were observed for blood lymphocyte and platelet counts or blood coagulation markers. In line with the nondepleting nature of CFZ533, CD20+ B cells in the blood were only marginally reduced. A complete suppression of germinal center (GC) development in lymph nodes and spleen was the most prominent result of post-mortem histological investigations. This was corroborated by an abrogated T-dependent antibody response (TDAR) to the antigen Keyhole Limpet Hemocyanin (KLH) as well as an absence of anti-drug antibodies (ADAs) in the absence of B cell depletion as seen with immunophenotyping and histology. When serum levels of CFZ533 in recovery animals dropped levels necessary for full CD40 occupancy on B cells, all animals were able to mount a TDAR to KLH. All histological changes also reverted to normal appearance after recovery. In summary, CFZ533 was shown to be well tolerated and safe in the 26-week toxicity study with a distinct pharmacodynamic profile in histology and immune function.

Characterization of the in vitro and in vivo properties of CFZ533, a blocking and non‐depleting anti‐CD40 monoclonal antibody

The CD40‐CD154 costimulatory pathway is essential for T cell–dependent immune responses, development of humoral memory, and antigen presenting cell function. These immune functions have been implicated in the pathology of multiple autoimmune diseases as well as allograft rejection. We have generated CFZ533, a fully human, pathway blocking anti‐CD40 monoclonal antibody that has been modified with a N297A mutation to render it unable to mediate Fcγ‐dependent effector functions. CFZ533 inhibited CD154‐induced activation of human leukocytes in vitro, but failed to induce human leukocyte activation. Additionally, CFZ533 was unable to mediate depletion of human CD40 expressing B cells. In vivo, CFZ533 blocked primary and recall T cell‐dependent antibody responses in nonhuman primates and abrogated germinal formation without depleting peripheral blood B cells. We also established a relationship between plasma concentrations of CFZ533 and CD40 pathway‐relevant pharmacodynamic effects in tissue. Collectively these data support the scientific rationale and posology for clinical utility of this antibody in select autoimmune diseases and solid organ transplantation.