By convention, CD4 + T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with MHC class II molecules. Alternative pathways of epitope production have been identified but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4 + T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome, and gamma-interferon inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4 + T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses.The classical MHCII processing pathway, developed chiefly through work with stable, globular proteins, entails: 1) engulfment of extracellular material, 2) delivery of nascent MHC class II (MHCII) molecules to a late endosomal compartment via its transient partner invariant chain (Ii), 3) catabolism of both Ii and internalized material in the endocytic compartment. 4) exchange of the remaining class II-associated invariant chain peptide (CLIP) portion of Ii for high affinity peptides and 5) trafficking of peptide/MHCII Reprints and permissions information is available online at

B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiological conditions using healthy human whole blood, teclistamab mediated dose-dependent lysis of H929 cells and activation of T cells. Antitumor activity of teclistamab was also observed in 2 BCMA+ MM murine xenograft models inoculated with human T cells (tumor inhibition with H929 model and tumor regression with the RPMI 8226 model) compared with vehicle and antibody controls. The specific and potent activity of teclistamab against BCMA-expressing cells from MM cell lines, patient samples, and MM xenograft models warrant further evaluation of this bispecific antibody for the treatment of MM. Phase 1 clinical trials (monotherapy, #NCT03145181; combination therapy, #NCT04108195) are ongoing for patients with relapsed/refractory MM.

T-cell–mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein–coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells. Here, we demonstrate that GPRC5D protein is present on the surface of MM cells and describe JNJ-64407564, a GPRC5DxCD3 bispecific antibody that recruits CD3+ T cells to GPRC5D+ MM cells and induces killing of GPRC5D+ cells. In vitro, JNJ-64407564 induced specific cytotoxicity of GPRC5D+ cells with concomitant T-cell activation and also killed plasma cells in MM patient samples ex vivo. JNJ-64407564 can recruit T cells and induce tumor regression in GPRC5D+ MM murine models, which coincide with T-cell infiltration at the tumor site. This antibody is also able to induce cytotoxicity of patient primary MM cells from bone marrow, which is the natural site of this disease. GPRC5D is a promising surface antigen for MM immunotherapy, and JNJ-64407564 is currently being evaluated in a phase 1 clinical trial in patients with relapsed or refractory MM (NCT03399799).

CD33 is expressed in 90% of patients with acute myeloid leukemia (AML), and its extracellular portion consists of a V domain and a C2 domain. A recent study showed that a single nucleotide polymorphism (SNP), rs12459419 (C > T), results in the reduced expression of V domain–containing CD33 and limited efficacy of V domain–binding anti-CD33 antibodies. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33 and to CD3, to induce T-cell recruitment and CD33+ tumor cell cytotoxicity independently of their SNP genotype status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro along with T-cell activation and cytokine release. JNJ-67571244 also exhibited statistically significant antitumor activity in vivo in established disseminated and subcutaneous mouse models of human AML. Furthermore, this antibody depletes CD33+ blasts in AML patient blood samples with concurrent T-cell activation. JNJ-67571244 also cross-reacts with cynomolgus monkey CD33 and CD3, and dosing of JNJ-67571244 in cynomolgus monkeys resulted in T-cell activation, transient cytokine release, and sustained reduction in CD33+ leukocyte populations. JNJ-67571244 was well tolerated in cynomolgus monkeys up to 30 mg/kg. Lastly, JNJ-67571244 mediated efficient cytotoxicity of cell lines and primary samples regardless of their SNP genotype status, suggesting a potential therapeutic benefit over other V-binding antibodies. JNJ-67571244 is currently in phase 1 clinical trials in patients with relapsed/refractory AML and high-risk myelodysplastic syndrome.

Background: Blockade of the programmed death-1 (PD-1) pathway has shown efficacy across a range of tumor types. However, some tumors do not respond and a combination strategy with other treatments may be required. CD38, a leukocyte receptor and ectoenzyme, was recently shown to be upregulated following PD-1 pathway blockade in mouse tumor models (Chen et al. ASCO-SITC 2017). Daratumumab is a fully human monoclonal antibody (mAb) that binds to CD38. It has been shown to exhibit immunomodulatory properties in patients with multiple myeloma (Krejcik et al. Blood. 2016). Studies in syngeneic mouse models and in vitro assays were conducted to investigate the potential of targeting CD38 in combination with anti-PD-1 to increase antitumor response. Methods: mAbs targeting mouse CD38 (mCD38-mg2a; mCD38) and mouse PD-1 (mPD1-mIgG1-D265A; mPD-1) were tested alone or in combination in several syngeneic models: colon adenocarcinoma (MC38), plasmacytoma (J558), and lung carcinoma (M109). The effect of these mAbs on the phenotype and functionality of tumor-infiltrating and circulating lymphocytes was evaluated using flow cytometry and immunohistochemistry. In addition, the effect of daratumumab on human regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) was assessed. Results: Combined anti-mCD38/anti-mPD-1 mAb treatment enhanced tumor regression and extended survival compared with single-mAb treatment in mouse tumor models. In the MC38 model (n=10/group), 55% of mice were tumor free (TF) following combined treatment, whereas 0% and 25% were TF following individual administration of anti-mCD38 or anti-mPD-1 mAbs, respectively. Similar effects were observed in J558- and M109-bearing mice (n=10/group for both models): with combined treatment, 85% of J558 and 20% of M109 mice were TF, compared with 50% (J558) and 0% (M109) for anti-mCD38 and 25% (J558) and 0% (M109) for anti-mPD-1 mAbs, respectively. Enhanced antitumor activity with combination treatment was associated with increased infiltration and activation of intratumoral CD4+ and CD8+ T cells, as well as increased levels of Ki67 on circulating T cells in MC38 and J558 models. The antitumor activity of anti-mCD38 mAb was dependent on active Fc function, suggesting that CD38+ cells may limit antitumor response. Consistent with this hypothesis, anti-mCD38 mAb led to a reduction in the frequency of CD38+ immunosuppressive Tregs and MDSC populations in mice. In vitro, daratumumab mediated antibody-dependent cellular cytotoxicity of human Tregs and led to the depletion of human monocytic MDSCs in blood of patients with lung cancer (n=17). Conclusion: These results demonstrate the combined effect of targeting both CD38 and PD-1 pathways in regulating antitumor immunity, and suggest that dual targeting of CD38 with daratumumab and PD-1 with nivolumab may represent a promising combination strategy for treating cancer. Citation Format: Natalie A. Bezman, Michelle Kinder, Amy D. Jhatakia, Bethany K. Mattson, Darlene Pizutti, Edward W. Thompson, Dorie A. Capaldi, Mark W. Mendonca, Aravind Anandam, Gopal Dhar, Lavanya Kovvuri, Archana Devi, Swagatam Ray, Vivek Surse, Elizabeth Saravia, Robert F. Graziano, Natalie A. Hutnick, Alan Korman. Antitumor activity associated with dual targeting of CD38 and programmed death-1 (PD-1) pathways in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1727.

Background: The outcome of multiple myeloma (MM) patients has been dramatically improved by the monoclonal antibody (mAb) daratumumab (DARA). DARA is a human IgG1κ mAb specific for CD38 and approved for the treatment of newly diagnosed and relapsed/refractory MM as a monotherapy and in combination with standard of care. Isatuximab and TAK-079 are additional CD38 targeting mAbs in clinical development by Sanofi and Takeda, respectively. These mAbs are reported to bind different epitopes and induce lysis of MM tumor cells via multiple mechanisms. It is unclear how the pleiotropic mechanisms collectively impact tumor cytolysis and exhibit anti-tumor effects in a comprehensive ex vivo immune milieu. Methods: For research purposes, DARA was compared with representative mAbs based on the published patent sequences for isatuximab and TAK-079, these surrogate analogs will hereafter be referred to as Sanofi anti-CD38 and Takeda anti-CD38. Mechanism of action (MOA) studies were done to compare antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell phagocytosis (ADCP), complement mediated cytotoxicity (CDC), and early and late detection of apoptosis. In addition, fresh whole blood from healthy donors was used to assess the cumulative effect of the MOAs on MM cell lines. Results: We tested the CDC activity of the anti-CD38s on multiple MM cell lines with a range of CD38 surface expression and CDC sensitivity levels. In LP-1 and MOLP-8 MM cell lines, DARA resulted in higher levels CDC activity as compared to the other anti-CD38 mAbs. In ADCC and ADCP assays, all 3 anti-CD38 mAbs induced similar levels of MM cell death and phagocytosis. Apoptosis was also assessed in the presence and absence of FcR crosslinking. The Annexin V assay replicated published results that only the Sanofi anti-CD38 was able to induce phosphatidylserine translocation to the cell surface without FcR crosslinking. However, in a robust 5-day cytotoxicity assay detecting metabolically active cells, all 3 antibodies elicited comparable high levels of cell death in the presence of the FcR crosslinker and low levels in its absence. We also performed assays with fresh whole blood from healthy volunteers (n=6) and determined the cumulative effect of the anti-CD38 mAbs on LP-1 and MOLP-8 MM cell lines. DARA demonstrated a higher maximal cytotoxicity than the other mAbs. Moreover, DARA had a significantly lower EC50 than Takeda anti-CD38 in both cell lines and lower than Sanofi-anti-CD38 in MOLP-8. Conclusion: DARA and surrogate analogs of Sanofi anti-CD38 and Takeda anti-CD38 have similar MOAs with the exception of higher CDC demonstrated by DARA which may contribute to the observed improved efficacy of DARA in a comprehensive immune milieu in vitro. As DARA is the only approved CD38 mAb, it remains to be determined in clinical trials if these in vitro differences lead to differences in clinical benefit. Citation Format: Michelle Kinder, Mi Ta, Amy Axel, Amy Wong, Stephen Rudnick, Bart de Goeij, Jeroen Lammerts van Bueren, Alex Babich, Mark Mendonca, Jocelyn Sendecki, Christopher Chiu, Kevin Bellew, Colleen Kane. Comparison of anti-CD38 antibodies in vitro mechanisms of action in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2970.

CD4+ T cells play critical roles in defending against poxviruses, both by potentiating cellular and humoral responses and by directly killing infected cells. Despite this central role, the basis for pox-specific CD4+ T cell activation, specifically the origin of the poxvirus-derived peptides (epitopes) that activate CD4+ T cells, remains poorly understood. In addition, because the current licensed poxvirus vaccines can cause serious adverse events and even death, elucidating the requirements for MHC class II (MHC-II) processing and presentation of poxviral Ags could be of great use. To address these questions, we explored the CD4+ T cell immunogenicity of ectromelia, the causative agent of mousepox. Having identified a large panel of novel epitopes via a screen of algorithm-selected synthetic peptides, we observed that immunization of mice with inactivated poxvirus primes a virtually undetectable CD4+ T cell response, even when adjuvanted, and is unable to provide protection against disease after a secondary challenge. We postulated that an important contributor to this outcome is the poor processability of whole virions for MHC-II–restricted presentation. In line with this hypothesis, we observed that whole poxvirions are very inefficiently converted into MHC-II–binding peptides by the APC as compared with subviral material. Thus, stability of the virion structure is a critical consideration in the rational design of a safe alternative to the existing live smallpox vaccine.

B-cell maturation antigen (BCMA) is a tumor necrosis factor (TNF) family surface protein predominantly expressed on terminally differentiated B-cells. BCMA signals through P38/NF-κB pathway upon binding to its ligands; a proliferation inducing ligand (APRIL) and B-cell activator of the TNF family (BAFF) and promote anti-apoptotic gene expression. BCMA expression is elevated in plasma blasts, plasma cells from spleen and bone marrow and correlates with disease progression in multiple myeloma (MM). BCMA expression in premalignant MM settings such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) also gives an opportunity for early cancer interception. To target cancer cells expressing BCMA, we developed a BCMAxCD3 bispecific antibody using the Genmab DuoBody® technology (Ab-957) to recruit T cells to BCMA-expressing MM cells so that T cells could be activated and induced to kill BCMA+ cancer cells. This antibody can induce cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells: EC50=0.15nM, MM1.R cells: EC50=0.06nM, RPMI8226 cells: EC50=0.45nM) with a concomitant T cell activation (H929 cells: EC50=0.21nM, MM1.R cells: EC50=0.1nM, RPMI8226 cells: EC50=0.28nM). In contrast, this antibody was unable to kill BCMA- cancer cell line (MV4-11), demonstrating the specificity of the cytotoxicity. Ab-957 also inhibited tumor development or growth in two BCMA+ MM murine xenograft models inoculated with human T cells. Furthermore, this antibody could deplete BCMA+ cells in bone marrow samples from MM patient's in an ex-vivo assay with an average EC50 value of 2.5 nM. Lastly, Ab-957 is well-tolerated in cynomolgus monkey and is being developed for Phase I clinical trial in patients with multiple myeloma. Disclosures Pillarisetti: Janssen: Employment. Baldwin:Janssen: Employment. Babich:Janssen: Employment. Majewski:Janssen: Employment. Barone:Janssen: Employment. Li:Janssen: Employment. Zhang:Janssen: Employment. Chin:Janssen: Employment. Luistro:Janssen: Employment. Mendonça:Janssen: Employment. Nanjunda:Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Bellew:Janssen: Employment. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.