Furthermore, since the main focus of this study is the generation of mouse strains for the study of Fc effector function and subsequently direct Fc engineering of the antibody. human clinical activity. Rituximab contains a AZD2014 (Vistusertib) wild type hIgG1 heavy chain constant region, while obinutuzumab contains AZD2014 (Vistusertib) a hIgG1 heavy chain constant region with decreased levels of fucosylated oligosaccharides resulting in enhanced binding to hFcRIIIA and thus enhanced ADCC activity. A recent Phase III study in patients with CLL exhibited that this Fc enhanced obinutuzumab confers a significantly higher response rate and survival advantage over rituximab therapy9. We first investigated antibody pharmacokinetics. For both antibodies, Rag2?/? mouse FcR+ mice experienced prolonged antibody blood half-lives, while the RHuFR mice experienced extremely short antibody kinetics (Physique S5). Human FcRI is the only high affinity human ING4 antibody FcR that binds soluble, monomeric IgG. The high degree of homology between human and mouse FcRI, as well as their IgG binding affinity suggest that human FcRI has likely limited contribution to the cytotoxic activity of anti-tumor mAbs activity of anti-CD20 antibodies revealed that cytotoxicity against tumor targets is usually mediated exclusively through FcRIIIa-mechanisms. Therefore, we employed the RHuFR derivative strain that lacks human FcRI (RHuFR1?) to remedy this kinetic discrepancy (Physique S5), thereby allowing for an antibody-dosing regimen to compare therapeutic effects between the RHuFR1? and the Rag2?/? AZD2014 (Vistusertib) mouse FcR+ mice. Importantly, the RHuFR1? mouse expresses all other human FcRs in the same manner as in the RHuFR mice (Physique S6) and is functionally intact, showing identical platelet clearance to the RHuFR mouse in the ITP model (Physique S4B). Furthermore, since the main focus of this study is the AZD2014 (Vistusertib) generation of mouse strains for the study of Fc effector function and subsequently direct Fc engineering of the antibody. Furthermore, crossing these RHuFR knockout strains can quickly create mice made up of combinations of human FcR knockouts for probing receptor interactions and redundancy in function. Overall, these mice are tools to expand our understanding of the complexity of the human FcR network and how individual antibody targets (e.g. epitope, target cell type, microenvironment, etc.) can greatly shape the observed therapeutic effect for a given antibody. Supplementary Material 1Click here to view.(6.0M, docx) Acknowledgments Dr. Michael Kharas provided the Hemavet machine and Dr. Joseph Sun supplied the anti-NK1.1 (PK136) antibody. Dr. Dmitry Pankov, Dr. Katja Behling, and Aaron Chang assisted with mouse work. Funding: The work was supported by: a Ruth L. Kirschstein National Research Service Award (F31 CA189305 to E.C.), NIH R01 CA55349, P01 CA33049, CCSG P30-008749 to D.A.S., The Tudor Fund, Lymphoma Foundation, and The Center for Experimental Therapeutics to D.A.S.; American Foundation for AIDS Research Mathilde Krim Fellowship in Basic Biomedical Research (109519-60-RKVA) to S.B., and the NIH P01 CA190174 and R35 CA196620 to J.V.R. The content is usually solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Footnotes Author contributions: E.C. conceived and performed mouse breeding and colony maintenance, circulation cytometry characterization of new mice strains, ITP model experiments, lymphoma antibody therapy experiments, and authored the manuscript. S.B. contributed to the conception of breeding plan and experimental plans, contributed mice and antibody resources, performed the antibody-FcR SPR anaylsis, and contributed methods and edited the manuscript. G.M. and P.M contributed to mouse characterization experiments and edited the manuscript. K.S.T. performed statistical analysis, contributed to the methods, and edited the manuscript. J.V.R. contributed mice and other resources and edited the manuscript. D.A.S. contributed resources and helped guideline the planning and execution of the experiments and edited the manuscript. Competing interests: A patent has been filed by AZD2014 (Vistusertib) MSKCC encompassing these novel mouse strains..
Furthermore, since the main focus of this study is the generation of mouse strains for the study of Fc effector function and subsequently direct Fc engineering of the antibody