(174z) Optimizing Site-Specific Protein Conjugation through Free Cysteine Engineering to Improve Artificial Antigen Presenting Cell Technology

Traditional cancer chemotherapy, although sometimes effective, is aggressive and leads to death of healthy cells in addition to cancer cells. Immunotherapies such as adoptive T cell therapy, wherein a patient's own T cells are expanded ex vivo and subsequently reinfused into the patient, present promising approaches in cancer therapy. Through antigen-specific stimulation of T cells, adoptive cell therapy allows for targeted and individualized treatment specific to an individual's patients cancer antigen. However, more effective technologies for ex vivo expansion via antigen-specific stimulation are still needed to support adoptive T cell therapy’s widespread implementation. Moreover, the ability to directly stimulate a patient’s own T cells in vivo using an immunostimulatory platform would represent a revolutionary advance for cancer treatment.

Artificial antigen presenting cell (aAPC) technology represents a platform that consist of T-cell activating antibodies and a peptide-loaded MHC molecules loaded on a polymeric or metallic nanoparticle. This technology induces robust T cell activation and antitumor activity in cancer models, but there is significant room for optimization. In particular, traditional antibody-particle conjugation strategies are nonspecific and inefficient, as they result in antibody orientations with respect to the particle that are incompatible with target interactions.

Conjugation strategies which allow for only correctly oriented antibodies would significantly enhance particle loading and improve the potency of aAPC technologies. We have developed site specific mutations in the heavy chain constant region of T cell-activating antibodies that encode for a cysteine with high steric availability for selective reduction and subsequent conjugation. We designed and expressed anti-CD3, anti-CD28, and peptide-loaded human major histocompatibility complex (MHC) immunoglobulin G Fc fusion proteins with engineered free cysteines. We showed that these engineered molecules can be reduced using low molar equivalents of tris(2-carboxyethyl) phosphine (TCEP), without affecting antibody integrity. This reduction yields “uncapped” sulfur residues which react with maleimide on particles via sulfo- succinimidyl 4-[N-maleimidomethyl]cyclohexane-1- carboxylate (Sulfo-SMCC) chemistry, resulting in site-specific conjugation that enforces availability of the antibody binding interface. Notably a variety of human MHC-IgG Fc fusion proteins loaded with a variety of peptides could be integrated into this technology, and the modular format allows for versatile reformulation in response to newly identified antigens. Overall, this conjugation strategy enables more efficient conjugation and improved particle function, representing a promising advance in aAPC technology.