Anti-CD71 immunotherapy to restore T cell homeostasis in autoimmunity

The transferrin receptor, CD71, is a receptor that brings iron into cells. The immune cells known as T lymphocytes (T cells) also rely on CD71 for iron, which they use for different metabolic processes. My previous work has shown that different types of T cells rely on this receptor for different reasons. By targeting or “blocking” the receptor with an antibody, we can limit the pro-inflammatory types of T cells and give a boost to the anti-inflammatory T cells. This method of immunotherapy was successful in mouse models of system lupus erythematosus (SLE), and may have similar benefits in other autoimmune diseases including type 1 diabetes (T1D) and multiple sclerosis (MS). Although there is evidence that CD71 and iron metabolism is dysregulated in T cells in patients with SLE, validation of patient samples with these other diseases needs to be done. Thus, this pathway could be a common target to multiple diseases that would benefit from anti-CD71 immunotherapy. We vaccinated an alpaca with human CD71 receptors to discover human-specific versions of antibodies that could accomplish this in people. Alpacas also make a smaller type of antibody known as nanobodies that are highly stable and can cross the blood-brain barrier. In this proposal, we will test the anti-CD71 nanobodies we discovered from this alpaca in mouse models of autoimmune disease.

We used two approaches to discover Nanobody (Nb) sequences in the alpaca we vaccinated with human CD71 receptors: phage display and LIBRA-seq. For LIBRA-Seq, we optimized flow cytometry conditions to sort alpaca B cells that were producing Nbs specific to CD71. This was accomplished by incubating the the PBMC sample with fluorescently labeled CD71 receptors as “bait” for the B cells to bind. Single cell sequencing yielded ~350 B cell receptor sequences; the four best candidates were then selected. We have confirmed two Nbs demonstrated binding to human CD71 by ELISA and will proceed to disease model testing. In total, we have five candidates for further testing including both Nbs from LIBRA-Seq and phage display. Experiments 1 and 2 focus on analysis of specimens from patients with T1D and MS. We will look at T cell iron levels and CD71 expression by flow cytometry. We also plan to measure a protein in patient serum which was previously found elevated in SLE samples, called hemochromatosis protein (HFE). HFE regulates CD71 endocytosis and may be a common mechanism to dysregulated iron metabolism in autoimmune diseases. Next, we will activate patient T cells in culture with or without our Nbs and test whether they alter the T cell activation or cytokine production. Experiments 3 and 4 will test these Nbs in humanized mouse models of autoimmunity. First, we will use a model of NSG mice where healthy control or patient PBMCs are injected into mice to induce autoimmunity similar to the patient phenotype(s). Next, we will test the Nbs in a new experimental autoimmune encephalomyelitis (EAE) model of MS, which also uses patient PBMCs to induce disease. Using a secondary antibody specific for alpaca Nbs, brains will be analyzed to determine whether our anti-CD71 Nbs crossed the blood-brain barrier.