William Kelton

High-resolution antibody dynamics of vaccine-induced immune responses

Significance The immune system must constantly adapt to combat infections and other challenges. This is accomplished by continuously evolving the antibody repertoire, and by maintaining memory of prior challenges. By using next-generation DNA sequencing technology, we have examined the shear amount of antibody made by individuals during a flu vaccination trial. We demonstrate one of the first characterizations of the fast antibody dynamics through time in multiple individuals responding to an immune challenge. The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge.

Bypassing glycosylation: engineering aglycosylated full-length IgG antibodies for human therapy

In recent years a number of aglycosylated therapeutic antibodies have entered the clinic. The clinical evaluation of these antibodies has served to dispel concerns that the absence of the ubiquitous N297 glycan in the Fc of IgG might result in immunogenicity, poor in vivo stability or unfavorable pharmacokinetics. Importantly, recent studies have now demonstrated that aglycosylated antibodies can be engineered to display novel effector functions and mechanisms of action that do not appear to be possible with their glycosylated counterparts. Moreover, the ability to manufacture aglycosylated antibodies in lower eukaryotes or in bacteria provides significant bioprocessing advantages in terms of shorter bioprocess development and running times and by completely bypassing the problems associated with the glycan heterogeneity of conventional antibodies. These advantages are poised to catapult aglycosylated antibodies to the forefront of protein therapeutics.

Effective Phagocytosis of Low Her2 Tumor Cell Lines with Engineered, Aglycosylated IgG Displaying High FcγRIIa Affinity and Selectivity

Glycans anchored to residue N297 of the antibody IgG Fc domain are critical in mediating binding toward FcγRs to direct both adaptive and innate immune responses. However, using a full length bacterial IgG display system, we have isolated aglycosylated Fc domains with mutations that confer up to a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous human inhibitory receptor, FcγRIIb. The mutant Fc domain (AglycoT-Fc1004) contained a total of 5 amino acid substitutions that conferred an activating to inhibitory ratio of 25 (A/I ratio; FcyRIIa-R131:FcγRIIb). Incorporation of this engineered Fc into trastuzumab, an anti-Her2 antibody, resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with both medium and low Her2-expressing cancer cells. A mathematical model has been developed to help explain how receptor affinity and the A/I ratio relate to improved antibody dependent cell-mediated phagocytosis. Our model provides guidelines for the future engineering of Fc domains with enhanced effector function.

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia.

Single-cell quantification of functional outcomes of CD16 ligation in NK cells by Fc-engineered antibodies

Humanized monoclonal antibodies (mAb) targeting tumor antigens have paved the way to complementary strategies in the treatment of cancer by harnessing the immune system towards a more specific response against tumors. Such mAb can mobilize natural killer (NK) cells function by mediating antibody-dependent cell cytotoxicity (ADCC). ADCC can be enhanced through Fc-region modification, but high affinity-Fc mAb have been associated with NK cell depletion. Here, we have quantified in vitro the improvement of NK cell-mediated ADCC brought by a humanized mAb targeting CD33 (HuM195) engineered in its Fc domain (DLE mutation). We used human NK cells in cytotoxicity assays against murine EL4 tumor cells transfected with human CD33 and pre-coated with the DLE HuM195 anti-CD33 antibody. In parallel, human K562 cells were used as targets in comparative assays to quantify kinetics of direct tumor recognition. A single-cell assay combining high throughput microscopy and immuno-printing of thousands of NK - target cell interactions allowed us to observe effector-mediated apoptosis and to measure cytokine secretion. We demonstrated that the DLE HuM195 anti-CD33 antibody increased NK cell-mediated ADCC up to 3 times compared with the non-mutated HuM195 anti-CD33 in a bulk cytotoxicity assay. In the single-cell assay, we confirmed that the DLE-engineered HuM195 potentiated the cytotoxicity of NK cells against CD33-expressing target cells by endowing more NK cells with killing function as compared to wild type HuM195 (19% with DLE HuM195 vs 1% with HuM195), by increasing their serial killing ability (5-19% involved in serial killing with DLE HuM195 vs 0-1% without antibody) and by stimulating their IFN-γ secretion. Dynamic timelapse monitoring showed that ADCC speeded up the kinetics of target conjugation, which was further increased by DLE HuM195. Secondly, DLE HuM195 necessitated fewer contacts to induce target apoptosis but also increased activation-induced cell death of NK cells. However, this increased AICD was independent of serial killing. Antibody-based therapies targeting tumor antigens will benefit from better understanding of cell-mediated tumor elimination. Single-cell analysis shows that Fc-modified mAb can significantly enhance NK cell killing of targets through improved recruitment of NK cells, conjugation kinetics, and serial killing. Enhanced target killing is also associated with increased AICD of NK cells, but the balance of effect favors the Fc modification for enhanced efficacy.