Human B-cell Ontogeny in Humanized NOD/SCID γcnull^{null} Mice Generates a Diverse Yet Auto/Poly- and HIV-1 Reactive Antibody Repertoire

Characterization of the human antibody (Ab) repertoire in mouse models of the human immune system is essential to establish their relevance in translational studies. Single human B-cells were sorted from bone marrow and periphery of humanized NOD/SCID γc$^{null}$ mice at 8–10 months post-engraftment with human cord blood-derived CD34$^+$ stem cells. Human immunoglobulin variable heavy (V$_H$) and kappa (V$_κ$) genes were amplified, cognate V$_H$-V$_κ$ gene-pairs assembled as single-chain variable fragment-Fc antibodies (scFvFcs) and functional studies performed. Although overall distribution of V$_H$ genes approximated the normal human Ab repertoire, analysis of the V$_H$-third complementarity determining regions (H-CDR3) in the mature B-cell subset demonstrated an increase in length and positive charges suggesting autoimmune characteristics. Additionally, >70% of Vκ sequences utilized V$_κ$4-1, a germline gene associated with autoimmunity. The mature B-cell subset-derived scFvFcs displayed the highest frequency of autoreactivity and polyspecificity, suggesting defects in checkpoint control mechanisms. Furthermore, these scFvFcs demonstrated binding to recombinant HIV envelope corroborating previous observations of poly/autoreactivity in anti-HIVgp140 antibodies. These data lend support to the hypothesis that anti-HIV BnAbs may be derived from auto/polyspecific Abs that escaped immune elimination and that the hNSG mouse could provide a new experimental platform for studying the origin of anti-HIV neutralizing Ab responses

Humanized mouse G6 anti-idiotypic monoclonal antibody has therapeutic potential against IGHV1-69

In 10–20% of the cases of chronic lymphocytic leukemia of B-cell phenotype (B-CLL), the IGHV1-69 germline is utilized as VH gene of the B cell receptor (BCR). Mouse G6 (MuG6) is an anti-idiotypic monoclonal antibody discovered in a screen against rheumatoid factors (RFs) that binds with high affinity to an idiotope expressed on the 51p1 alleles of IGHV1-69 germline gene encoded antibodies (G6-id(+)). The finding that unmutated IGHV1-69 encoded BCRs are frequently expressed on B-CLL cells provides an opportunity for anti-idiotype monoclonal antibody immunotherapy. In this study, we first showed that MuG6 can deplete B cells encoding IGHV1-69 BCRs using a novel humanized GTL mouse model. Next, we humanized MuG6 and demonstrated that the humanized antibodies (HuG6s), especially HuG6.3, displayed ∼2-fold higher binding affinity for G6-id(+) antibody compared to the parental MuG6. Additional studies showed that HuG6.3 was able to kill G6-id(+) BCR expressing cells and patient B-CLL cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Finally, both MuG6 and HuG6.3 mediate in vivo depletion of B-CLL cells in NSG mice. These data suggest that HuG6.3 may provide a new precision medicine to selectively kill IGHV1-69-encoding G6-id(+) B-CLL cells