A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.

Background:The protein AID generates nucleotide insertions and deletions (indels) critical for antibody affinity maturation. Results: The location, diversity, and evolution of indels were examined. Conclusion: AID generates diverse sequence-related indels that localize to antigen binding regions in vitro and in vivo. Significance: AID is sufficient to form indels that combine with point mutations to form a robust system for antibody evolution.

Among the most promising approaches in the treatment of cancer is the activation of antitumor immunity by blockade of immune checkpoints. These inhibitory immune checkpoints are crucial for maintaining self-tolerance in the normal immune system but can be co-opted in cancer to allow tumor escape from immune surveillance. Therapeutic validation has been provided using antibodies that inhibit the CTLA-4 and PD-1 signaling pathways, which have shown significant clinical activity. Interestingly, blockade of other T-cell inhibitory signaling molecules TIM-3 and LAG-3 has also been shown to be effective in mouse models of cancer. Potential therapeutic molecules that inhibit the negative signaling of TIM-3 or LAG-3 were identified by screening the AnaptysBio Evolvable Library (ABEL) of fully human germline antibodies. The initial ABEL screens used the soluble extra cellular domains (ECD) of either TIM-3 or LAG-3, followed by screening on cell-surface expressed antigens. The resulting panels of human antibodies were matured to high affinity and potency using SHM-XEL™ which uses mammalian cell display of human IgG followed by in vitro somatic hypermutation (SHM). We have explored the activity of these antibodies both as single agents as well as simultaneous blockade of multiple pathways in in vitro cell-based assays. Inhibition of each pathway in isolation demonstrated immune stimulatory activity as evidenced by increased secretion of IL-2 in a mixed lymphocyte reaction or an activated T-cell assay. Combination of an anti LAG-3 or an anti TIM-3 antibody with an anti-PD-1 inhibitory antibody could increase IL-2 secretion over that seen with blockade of a single checkpoint alone. The magnitude of this effect was donor cell dependent. These data are suggestive that combination immunotherapy towards these targets is worthy of clinical evaluation and may lead to increased efficacy. Citation Format: H. Toni Jun, Patricia A. McNeeley, Andrew Lassen, Brandon Hynes, Larry Altobell, Mark Chhoa, Jesus Olvera, Josh MacLaren, Minjee Do, Michael Brown, Jean da Silva Correia, David King. Generation of antagonistic anti-TIM-3 and anti-LAG-3 monoclonal antibodies for potential novel immunotherapy combinations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-266. doi:10.1158/1538-7445.AM2014-LB-266