The survival of transitional and mature B cells requires both the B cell antigen receptor (BCR) and BLyS receptor 3 (BR3), which suggests that these receptors send signals that are nonredundant or that engage in crosstalk with each other. Here we show that BCR signaling induced production of the nonclassical transcription factor NF-κB pathway substrate p100, which is required for transmission of BR3 signals and thus B cell survival. The capacity for sustained p100 production emerged during transitional B cell differentiation, the stage at which BCR signals begin to mediate survival rather than negative selection. Our findings identify a molecular mechanism for the reliance of primary B cells on continuous BR3 and BCR signaling, as well as for the gradual resistance to negative selection that is acquired during B cell maturation.Primary B cells rely on signals from both the B cell antigen receptor (BCR) and B lymphocyte stimulator (BLyS1; also called BAFF2; A000383) receptor 3 (BR3; also called BAFFr; A000374) for survival. Most peripheral B cells die after BCR ablation regardless of BR3 sufficiency, which indicates a need for continuous 'tonic' signals through the BCR3. Conversely, the lack of either BLyS or BR3, both of which are members of the tumor necrosis factor (TNF) family, results in B cell deficiency despite normal BCR function4-6. The requirement for both BCR and BR3 becomes apparent during transitional B cell differentiation and affects survival at the transitional 2 (T2) and T3 differentiation stages, such that the BCR signaling thresholds for negative and positive selection are modulated by BLyS availability7, 8. The molecular mechanism that underlies this codependence on BCR and BR3 is poorly understood.Correspondence should be addressed to M.P.C. (cancro@mail.med.upenn.edu). Accession codes. UCSD-Nature Signaling Gateway (http://www.signaling-gateway.org): A000383, A000374, A002936, A002248, A000374 and A000305.Note: Supplementary information is available on the Nature Immunology website. AUTHOR CONTRIBUTIONS J.E.S., M.K., F.G.K., J.L.S., J.P.M., W.J.Q., R.J.B., L.S.T. and K.A.J. did research, analyzed data and generated key reagents; J.E.S., M.K., J.G.M., R.S. and M.P.C. designed research and analyzed data; and J.E.S., R.S., M.K. and M.P.C. wrote the paper.Published online at http://www.nature.com/natureimmunology/ Reprints and permissions information is available online at

Edited by Peter CresswellThe antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-␥ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-␥-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.

Therapeutic monoclonal antibodies are among the most effective biotherapeutics to date. An important aspect of antibodies is their ability to bind antigen while at the same time recruit immune effector functions. The majority of approved recombinant monoclonal antibody therapies are of the human IgG1 subclass, which can engage both humoral and cellular components of the immune system. The wealth of information generated about antibodies has afforded investigators the ability to molecularly engineer antibodies to modulate effector functions. Here, we review various antibody engineering efforts intended to improve efficacy and safety relative to the human IgG isotype. Further, we will discuss proposed mechanisms by which engineering approaches led to modified interactions with immune components and provide examples of clinical studies using next generation antibodies.

Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/ receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC.Cancer Res; 76(13); 3942-53. Ó2016 AACR.

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