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TMEFF2, a transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is primarily restricted in expression to healthy brain and prostate and is enriched in prostate cancer. JNJ-70218902 (JNJ-902) is a bispecific antibody that engages TMEFF2-expressing tumor cells and CD3 on T cells causing exposure dependent pro-inflammatory responses and target tumor cell lysis. Here we report the preclinical characterization of JNJ-902 using in vitro and in vivo prostate cancer models. Expression of TMEFF2 transcript and protein in brain and prostate was confirmed in healthy human tissue, as was its elevated expression in advanced prostate cancer tumors, including in 41/45 (91%) metastatic castration-resistant prostate cancer (mCRPC) samples. JNJ-902 bound to TMEFF2-positive LNCaP-AR cells (androgen-sensitive human prostate cancer cell line) in a concentration-dependent manner (EC50 = 9.6 nM); no binding to TMEFF2-negative DU145 prostate cancer cells was observed. The binding affinity (KD) of JNJ-902 for CD3 on primary human T cells was determined to be ~151 nM. T cell-mediated killing, as measured by caspase-3 activity, was induced upon incubation of JNJ-902 with healthy human donor T cells (n = 5) and LNCaP-AR cells at a 3:1 effector to target ratio (EC50 = 1.4 nM). No cytotoxicity was detected using DU145 cells or with control antibodies. Corresponding T cell activation was confirmed by concentration-dependent increases in cell surface CD8+CD25+ expression and proinflammatory cytokine production (eg, GM-CSF, IFN-γ, IL-10, TNF-α). Antitumor activity of JNJ-902 at increasing concentrations up to 5 mg/kg was observed in T cell humanized NSG mice bearing LNCaP xenografts (tumor growth inhibition [∆%TGI] of 75-122%) and in LuCaP 86.2 patient-derived xenografts (∆%TGI of 72-88%) as compared with control-treated mice. JNJ-902-dependent intratumoral infiltration of CD8+ T cells was detected in both models with increases in frequency of CD8+granzymeB+ effector cells. The data summarized here are the first report on the pharmacological activity of a novel TMEFF2 × CD3 bispecific antibody, supporting further investigation of TMEFF2 in mCRPC given its expression in advanced prostate cancer. Targeting TMEFF2 with the TMEFF2 × CD3 bispecific antibody JNJ-902 elicited robust T cell-mediated responses in both prostate cancer cells and xenograft models. Based on these data, a phase 1 dose escalation trial of JNJ-902 in patients with mCRPC is currently ongoing (NCT04397276). Citation Format: Subhasree Basu, Mike Russell, Katrin Sproesser, Bethany Mattson, Cassandra L. Lowenstein, Sathya Venkataramani, Maura Diamond, Robin E. Ernst, Stephen Jarantown, Jackson Wong, Gerald Chu, Kathryn Packman, Shaozhou Ken Tian, Scott R. Brodeur, Margaret Yu, Brent A. Rupnow. Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB087.

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.

Immunoglobulin E (IgE) isotype antibodies are associated with atopic disease, namely allergic rhinitis, asthma and atopic dermatitis, but are also involved in host immune defence mechanisms against parasitic infection. The commitment of a B cell to isotype class switch to an IgE-producing cell is a tightly regulated process, and our understanding of the regulation of IgE-antibody production is central to the prevention and treatment of atopic disease. Both those that are presently in use and potential future therapies to prevent IgE-mediated disease take advantage of our existing knowledge of the specific mechanisms that are required for IgE class switching.

We demonstrate that the alpha chain of human C4b binding protein (C4BP) binds directly to CD40 on human B cells at a site that differs from that used by CD40 ligand. C4BP induces proliferation, upregulation of CD54 and CD86 expression, and IL4-dependent IgE isotype switching in normal B cells but not in B cells from patients with CD40 or IKKgamma/NEMO deficiencies. Furthermore, C4BP colocalized with B cells in the germinal centers of human tonsils. These observations suggest that C4BP is an activating ligand for CD40 and establish a novel interface between complement and B cell activation.

To define the role of TRAF proteins in CD40-dependent isotype switching in B cells, we introduced wild-type (WT) and mutant CD40 transgenes that lacked the binding motifs for TRAF6 (CD40deltaTRAF6), TRAF2 and TRAF3 (CD40deltaTRAF2/3), or both (CD40deltaTRAFs) into B cells of CD40(-/-) mice. The in vivo isotype switch defect in CD40(-/-) mice was fully corrected by WT and CD40deltaTRAF6, partially by CD40deltaTRAF2/3, and not at all by CD40deltaTRAFs transgenes. CD40-mediated isotype switching, proliferation, and activation of p38, JNK, and NFkappaB in B cells were normal in WT and CD40deltaTRAF6 mice, severely impaired in CD40deltaTRAF2/3, and absent in CD40deltaTRAFs mice. These results suggest that binding to TRAF2 and/or TRAF3 but not TRAF6 is essential for CD40 isotype switching and activation in B cells.

Background: We recently described a murine model of atopic dermatitis (AD) elicited by epicutaneous sensitization with ovalbumin (OVA). The skin lesions in these mice were characterized by a dermal infiltrate consisting of eosinophils and T cells and by increased expression of the T H 2 cytokines IL-4 and IL-5. Epicutaneous sensitization induces a rise in the levels of serum total IgE and OVA-specific antibodies, further indicating that it elicits a predominantly T H 2 response. Objective: This study was undertaken to assess the roles of T cells, B cells, and CD40L-CD40 interactions in AD. Methods: Mice with targeted gene deletions were sensitized with OVA. Histologic and immunohistochemical examinations, as well as measurements of IL-4 mRNA, were performed on OVA-sensitized skin. Total and antigen-specific serum IgE levels were determined. Results: RAG2 –/– mice, which lack both T and B cells, did not exhibit cellular infiltration, induction of dermal IL-4 mRNA, or elevation of serum IgE after OVA sensitization; all of these features were present in B-cell–deficient IgH –/– mice. T-cell receptor α –/– mice did not display cellular infiltration, IL-4 mRNA expression, or increased IgE levels after OVA sensitization, but these responses were elicited in T-cell receptor δ –/– mice after sensitization. Absence of CD40 had no effect on these responses. Conclusion: These results suggest that αβ T cells, but not γδ T cells, B cells, or CD40L-CD40 interactions, are critical for skin inflammation and the T H 2 response in AD. (J Allergy Clin Immunol 2001;107:359-66.)

CD40 induces B cells to switch to IgE in the presence of IL-4 and up-regulates their expression of the low-affinity receptor for IgE, CD23, which promotes the immune response to allergen complexed with IgE antibody. CD40 binds to CD40L and to the C4b-binding protein (C4BP) using distinct sites. CD46 is a receptor for the product of activated complement C4b. Some microbial antigens bind both C4BP and CD46, potentially bridging CD40 to CD46. In addition, immune complexes containing both C4b and C4BP may cross-link CD40 to CD46. We demonstrate that cross-linking CD46 to CD40 on B cells inhibits CD40-mediated up-regulation of surface CD23 expression and induction of IL-4-dependent IgE isotype switching. This was associated with inhibition of induction of Ce germ line transcripts and of activation-induced cytidine deaminase mRNA expression. Furthermore, co-ligation of CD46 to CD40 blocked CD40-mediated NF-kB activation. These observations suggest that complement components may play an important role in regulating CD40 activation of B cells and the allergic response.

Background Many receptors rely on the appropriate activation of nuclear factor (NF) κB to induce cellular function. This process depends critically on the phosphorylation of the inhibitor of NF-κB (IκB) by the IκB kinase. This targets IκB for ubiquitination and degradation, allowing NF-κB to translocate to the nucleus, where it can direct transcription. Hypomorphic human mutations affecting one IκB kinase component, the NF-κB essential modulator (NEMO), result in impaired signaling from receptors required for ectodermal development and immune function. Male subjects with these mutant NEMO molecules have an X-linked syndrome known as ectodermal dysplasia with immunodeficiency, which is characterized by severe infections, with herpesviruses, bacteria, and mycobacterial susceptibility. Objective We sought to genetically and biochemically characterize a patient with a mutant NEMO molecule without ectodermal abnormalities. Methods We evaluated NEMO in a patient who had immunodeficiency and atypical mycobacterial infection but normal ectoderm. Results We identified a novel NEMO mutant causing immunodeficiency without ectodermal dysplasia. The mutation, which altered the exon 9 splice site, was present in cells of ectodermal and hematopoetic origin and resulted in a heterogeneous mixture of mutant and wild-type cDNA species. Immunologic function was variably impaired, with reduced CD40-induced B-cell proliferation, partially reduced NF-κB p65 nuclear translocation, and variable Toll-like receptor–induced TNF production. This variability might be explained by an inconsistent ratio of mutant to wild-type NEMO. The lack of any ectodermal phenotype, however, suggested a separation in the hematopoetic and ectodermal function of NEMO that leads to NF-κB activation. Conclusion Mutation of the gene encoding NEMO can result in immunodeficiency without ectodermal dysplasia.

Background: A substantial body of evidence exists to support the pivotal role of IL-13 in the pathogenesis of bronchial asthma. We recently found that a variant of the IL13 gene (Arg110Gln) is genetically associated with bronchial asthma, which is concordant with animal experiments using IL-13 in the development of asthma. Objective: To address whether the Gln110 variant of IL13 influences IL-13 function, contributing to the pathogenesis of bronchial asthma, we studied the functional properties of the variant. Methods: We generated 2 types of recombinant IL-13 proteins, the amino acids of which at 110 were arginine or glutamine, and analyzed the binding affinities with the IL-13 receptors, as well as the stability of the proteins. We further compared the relationship between the genotype and serum levels of IL-13. Results: The variant showed a lower affinity with the IL-13 receptor α2 chain, a decoy receptor, causing less clearance. The variant also demonstrated an enhanced stability in both human and mouse plasma. We further identified that asthmatic patients homozygous for the Gln110 variant have higher serum levels of IL-13 than those without the variant. Conclusion: These results suggested that the variant might act as a functional genetic factor of bronchial asthma with a unique mechanism to upregulate local and systemic IL-13 concentration in vivo. (J Allergy Clin Immunol 2002;109:980-7.)

IL-4 and CD40 ligation are essential for IgE synthesis by B cells. We have shown previously that hydrocortisone (HC) induces IgE synthesis in IL-4-stimulated human B cells. In this study we demonstrate that HC fails to induce IgE synthesis in B cells from CD40 ligand-deficient (CD40L-deficient) patients. Disruption of CD40L-CD40 interactions by soluble CD40-Ig fusion protein or anti-CD40L mAb blocked the capacity of HC to induce IgE synthesis in normal B cells. HC upregulated CD40L mRNA expression in PBMCs and surface expression of CD40L in PBMCs as well as in purified populations of T and B cells. Upregulation of CD40L mRNA in PBMCs occurred 3 hours after stimulation with HC and was inhibited by actinomycin D. Upregulation of CD40L mRNA and induction of IgE synthesis by HC were inhibited by the steroid hormone receptor antagonist RU-486. These results indicate that ligand-mediated activation of the glucocorticoid receptor upregulates CD40L expression in human lymphocytes.

The TRAF3 molecule interacts with the cytoplasmic carboxyl terminus (COOH terminus) of the Epstein-Barr virus-encoded oncogene LMP-1. NF-kappaB activation is a downstream signaling event of tumor necrosis factor receptor-associated factor (TRAF) molecules in other signaling systems (CD40 for example) and is an event caused by LMP-1 expression. One region capable of TRAF3 interaction in LMP-1 is the membrane-proximal 45 amino acids (188-242) of the COOH terminus. We show that this region contains the only site for binding of TRAF3 in the 200-amino acid COOH terminus of LMP-1. The site also binds TRAF2 and TRAF5, but not TRAF6. TRAF3 binds to critical residues localized between amino acids 196 and 212 (HHDDSLPHPQQATDDSG), including the PXQX(T/S) motif, that share limited identity to the CD40 receptor TRAF binding site (TAAPVQETL). Mutation of critical residues in the TRAF3 binding site of LMP-1 that prevents binding of TRAF2, TRAF3, and TRAF5 does not affect NF-kappaB-activating potential. Deletion mapping localized the major NF-kappaB activating region of LMP-1 to critical residues in the distal 4 amino acids of the COOH terminus (383-386). Therefore, TRAF3 binding and NF-kappaB activation occur through two separate motifs at opposite ends of the LMP-1 COOH-terminal sequence.

Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.