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We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 μg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 μg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761–72. ©2018 AACR.

Supplementary Table 1: Sixteen-locus STR analysis of tumor tissue and banked CSLC lines Supplementary Table 2: CSLC derivation and characteristics Supplementary Table 3: Immunohistochemical Analysis of Colorectal Cancer Tumor Specimens Supplementary Table 4: Equilibrium Dissociation Constants (KD) for Binding of MGD007 to Human CD3 or gpA33 Supplementary Table 5: MGD007 Pharmacokinetic Parameters Derived from Two-compartment Analysis in Cynomolgus Monkeys Supplementary Figure 1: In vitro CSLC differentiation in MatrigelTM 3D cultures Supplementary Figure 2: RECA47 mAb binding restricted to intestinal epithelium Supplementary Figure 3: MGD007 Flow Cytometry Analyses on Tumor Cell Lines Supplementary Figure 4: Expanded Treg population exhibits suppressive T-cell properties Supplementary Figure 5: Cytokine Levels in Monkeys Following Treatment with MGD007

PDF file, 81KB, Kinetic and Equilibrium binding constants, determined by surface plasmon resonance, for binding of murine BRCA84D, RES240 and MGA271 to human and cynomolgus monkey B7-H3.

MGD011 mediated concentration-dependent cytotoxicity against CD19+ HBL-2 and Jeko-1 lymphoma cell lines

Supplemental figure legends for supplemental figures S1, S2, S3, S4, S5, S6

PDF file, 75KB, Expression level of B7-H3 on ATCC cancer cell lines as determined by FLOW cytometry with anti-B7-H3 mAb BRCA84D. Solid curves represent isotype control staining; open curves represent staining with FI.

PDF file, 78KB, Measurement of the absolute level of circulating B cells (CD20+; Panel A), T cells (CD3+; Panel B) and NK cells (CD159a+)Panel C) in blood samples collected from cynomolgus monkeys at the indicated days prior to administration (Day -2), or following intravenous administration of PBS vehicle on Day 1 (all animals received PBS vehicle on Day 1) or MGA271 on Day 8 at the indicated dose. The panels on the left for each group represent all animals (N=6) in each dosing group, while the panels on the right represent only the recovery group animals (N=2) in each dosing group.

PDF file, 89KB, Measurement of IL-5 (Panel A), IL-6 (Panel B) and TNF-B (Panel C) cytokine levels in serum samples collected from cynomolgus monkeys prior to infusion (Pre) or at the indicated time points following intravenous administration of PBS vehicle (Day 1) or MGA271 at the indicated dose (Day 8). The top two panels of each cytokine data set represent all animals (N=6) in each dosing group, while the bottom three panels represent only the recovery group animals (N=2) in each dosing group. Sera from the samples were analyzed for cytokine levels using the Becton Dickinson Cytometric Bead Array Non-Human Primate Th1/Th2 Cytokine kit.

Purpose: CD19, a B-cell lineage-specific marker, is highly represented in B-cell malignancies and an attractive target for therapeutic interventions. MGD011 is a CD19 x CD3 DART bispecific protein designed to redirect T lymphocytes to eliminate CD19-expressing cells. MGD011 has been engineered with a modified human Fc domain for improved pharmacokinetic (PK) properties and designed to cross-react with the corresponding antigens in cynomolgus monkeys. Here, we report on the preclinical activity, safety and PK properties of MGD011.Experimental Design: The activity of MGD011 was evaluated in several in vitro and in vivo models. PK, safety and pharmacodynamic activity was also assessed in dose-escalation and repeat-dose studies of MGD011 administered once weekly in cynomolgus monkeys.Results: MGD011 mediated killing of human B-cell lymphoma lines by human or cynomolgus monkey PBMCs as well as autologous B-cell depletion in PBMCs from both species. MGD011-mediated killing was accompanied by target-dependent T-cell activation and expansion, cytokine release and upregulation of perforin and granzyme B. MGD011 demonstrated antitumor activity against localized and disseminated lymphoma xenografts reconstituted with human PBMCs. In cynomolgus monkeys, MGD011 displayed a terminal half-life of 6.7 days; once weekly intravenous infusion of MGD011 at doses up to 100 μg/kg, the highest dose tested, was well tolerated and resulted in dose-dependent, durable decreases in circulating B cells accompanied by profound reductions of B lymphocytes in lymphoid organs.Conclusions: The preclinical activity, safety and PK profile support clinical investigation of MGD011 as a therapeutic candidate for the treatment of B-cell malignancies. Clin Cancer Res; 23(6); 1506–18. ©2016 AACR.

Supplemental Table 1: MGD011 repeat-dose and dose-escalation study design in non-human primates; Supplemental Table 2: PK properties of MGD011 derived from non-human primate studies

PDF file, 204KB, Panel A: Representative reactivity of anti-B7-H3 mAb BRCA84D with frozen tissue sections of prostate, breast, colon, lung, gastric and renal cancer. Panel B: Reactivity of anti-B7-H3 mAb BRCA69D on formalin-fixed paraffin embedded normal human spleen and lymph node tissues.

PDF file, 78KB.

Purpose: The goal of this research was to harness a monoclonal antibody (mAb) discovery platform to identify cell-surface antigens highly expressed on cancer and develop, through Fc optimization, potent mAb therapies toward these tumor-specific antigens.Experimental Design: Fifty independent mAbs targeting the cell-surface immunoregulatory B7-H3 protein were obtained through independent intact cell-based immunizations using human tissue progenitor cells, cancer cell lines, or cell lines displaying cancer stem cell properties. Binding studies revealed this natively reactive B7-H3 mAb panel to bind a range of independent B7-H3 epitopes. Immunohistochemical analyses showed that a subset displayed strong reactivity to a broad range of human cancers while exhibiting limited binding to normal human tissues. A B7-H3 mAb displaying exquisite tumor/normal differential binding was selected for humanization and incorporation of an Fc domain modified to enhance effector-mediated antitumor function via increased affinity for the activating receptor CD16A and decreased binding to the inhibitory receptor CD32B.Results: MGA271, the resulting engineered anti–B7-H3 mAb, mediates potent antibody-dependent cellular cytotoxicity against a broad range of tumor cell types. Furthermore, in human CD16A-bearing transgenic mice, MGA271 exhibited potent antitumor activity in B7-H3–expressing xenograft models of renal cell and bladder carcinoma. Toxicology studies carried out in cynomolgus monkeys revealed no significant test article-related safety findings.Conclusions: This data supports evaluation of MGA271 clinical utility in B7-H3–expressing cancer, while validating a combination of a nontarget biased approach of intact cell immunizations and immunohistochemistry to identify novel cancer antigens with Fc-based mAb engineering to enable potent antitumor activity. Clin Cancer Res; 18(14); 3834–45. ©2012 AACR.

PDF file, 59KB, Expanded normal tissue IHC analysis of B7-H3 on frozen tissues with MGA271 at optimal staining concentration (4 ug/ml) and at 5x the optimal concentration (20 ug/ml). The optimal staining concentration was determined by titration on the CaKi2 and Hs700T positive control cell lines to determine the lowest MGA271 concentration that yielded the greatest specific staining. (c) = cytoplasmic; (m) = membrane; (c,m) = cytoplasmic plus membrane.

Monoclonal antibodies (mAb) are widely used in the treatment of non-Hodgkin's lymphoma and autoimmune diseases. Although the mechanism of action in vivo is not always known, the therapeutic activity of several approved mAbs depends on the binding of the Fcγ regions to low-affinity Fcγ receptors (FcγR) expressed on effector cells. We did functional genetic screens to identify IgG1 Fc domains with improved binding to the low-affinity activating Fc receptor CD16A (FcγRIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (FcγRIIB). Identification of new amino acid residues important for FcγR binding guided the construction of an Fc domain that showed a dramatically enhanced CD16A binding and greater than a 100-fold improvement in antibody-dependent cell-mediated cytotoxicity. In a xenograft murine model of B-cell malignancy, the greatest enhancement of an Fc-optimized anti-human B-cell mAb was accounted for by improved binding to FcγRIV, a unique mouse activating FcγR that is expressed by monocytes and macrophages but not natural killer (NK) cells, consistent with experimental and clinical data suggesting that mononuclear phagocytes, effector cells expressing both activating and inhibitory FcγR, are critical mediators of B-cell depletion in vivo. By using mice transgenic for human CD16A, enhanced survival was observed due to expression of CD16A-158phe on monocytes and macrophages as well as on NK cells in these mice. The design of new generations of improved antibodies for immunotherapy should aim at Fc optimization to increase the engagement of activating FcγR present on the surface of tumor-infiltrating effector cell populations. [Cancer Res 2007;67(18):8882–90]

Supplementary Methods from Fc Optimization of Therapeutic Antibodies Enhances Their Ability to Kill Tumor Cells In vitro and Controls Tumor Expansion In vivo via Low-Affinity Activating Fcγ Receptors

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 μg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 μg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761–72. ©2018 AACR.

Many patients with B-cell malignancies can be successfully treated, although tumor eradication is rarely achieved. T-cell-directed killing of tumor cells using engineered T-cells or bispecific antibodies is a promising approach for the treatment of hematologic malignancies. We investigated the efficacy of CD19xCD3 DART bispecific antibody in a broad panel of human primary B-cell malignancies. The CD19xCD3 DART identified 2 distinct subsets of patients, in which the neoplastic lymphocytes were eliminated with rapid or slow kinetics. Delayed responses were always overcome by a prolonged or repeated DART exposure. Both CD4 and CD8 effector cytotoxic cells were generated, and DART-mediated killing of CD4+ cells into cytotoxic effectors required the presence of CD8+ cells. Serial exposures to DART led to the exponential expansion of CD4 + and CD8 + cells and to the sequential ablation of neoplastic cells in absence of a PD-L1-mediated exhaustion. Lastly, patient-derived neoplastic B-cells (B-Acute Lymphoblast Leukemia and Diffuse Large B Cell Lymphoma) could be proficiently eradicated in a xenograft mouse model by DART-armed cytokine induced killer (CIK) cells. Collectively, patient tailored DART exposures can result in the effective elimination of CD19 positive leukemia and B-cell lymphoma and the association of bispecific antibodies with unmatched CIK cells represents an effective modality for the treatment of CD19 positive leukemia/lymphoma.

Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. While vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic agent that is highly efficacious at low doses and is broadly specific against antigenically drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific Fc fusion protein (the Fc dual-affinity retargeting [FcDART] molecule). In mice, a single therapeutic or prophylactic dose of either monoclonal antibody at 2.5 mg/kg of body weight provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1-mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5-mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, on the basis of analysis of mutants with antibody escape mutations. While it was possible to generate escape mutants in vitro , they were neutralized by the antibodies in vivo , as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutant in vitro . In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART molecule, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses. IMPORTANCE Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and are a pandemic threat. A vaccine is available, but other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. The variability of the virus means that such an approach must be broad spectrum. To achieve this, we developed two antibodies that neutralize H5N1 influenza viruses. In mice, these antibodies provided complete protection against a spectrum of H5N1 influenza viruses at a single low dose. We then combined the two antibodies into a single molecule, FcDART, which combined the broad-spectrum activity and protective efficacy of both antibodies. This treatment provides a novel and effective therapeutic agent or prophylactic with activity against highly pathogenic H5N1 avian influenza viruses.

Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1 −/− mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle. IMPORTANCE Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.

Dysregulation of ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression correlates with poor prognosis in multiple cancers. We have shown that ADAM9 is overexpressed in multiple solid tumor indications and that anti-ADAM9 antibodies are efficiently internalized and degraded by tumor cell lines making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe IMGC936, the first ADAM9-targeting ADC to enter preclinical development. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable peptide linker at a drug-antibody ratio of two. To maximize the potential for IMGC936 activity, the M252Y/S254T/T256E (YTE) mutation was introduced into the CH2 domain of the antibody to increase in vivo plasma half-life and exposure. In vitro studies demonstrated targeted cytotoxicity of IMGC936 across a panel of ADAM9-positve tumor cell lines with activity at least 2 logs greater than a non-targeting conjugate. Consistent with the in vitroactivity, an anti-ADAM9-DM21 conjugate displayed compelling anti-tumor activity in multiple xenograft models representing non-small cell lung, gastric and colorectal cancers. For example, in the EBC-1 non-small cell lung cancer subcutaneous xenograft model with only moderate ADAM9 expression (H-score of 130), anti-ADAM9-DM21 not only induced tumor growth delay but produced complete and durable remissions in 6/6 mice following a single intravenous dose of 8.6 mg Ab/kg (100 ug DM21/kg). IMGC936 demonstrated a favorable pharmacokinetic profile with good conjugate stability in non-human primates. Importantly, IMGC936 was well-tolerated following repeat dosing in cynomolgus monkeys with no ADAM9 target-related toxicities identified at doses exceeding the levels required for anti-tumor activity in murine xenograft models. Based on the totality of the preclinical data, IMGC936 represents a promising therapeutic candidate to target a wide range of ADAM9-expressing tumors. Citation Format: Stuart Hicks, Deryk Loo, Kerstin Sinkevicius, Juniper Scribner, Bhaswati Barat, Nicholas Yoder, Christopher Espelin, Marian Themeles, Francine Chen, Jacquelynn Lucas, Jennifer Brown, Bahar Matin, Megan Fuller, Jenny Lee, Paulin Salomon, Juliet Costoplus, Sadiqa Yancey, Gundo Diedrich, Sergey Gorlatov, Thomas Son, Michael Chiechi, Pam Li, Michael Spliedt, Valentina Ciccarone, Jeff Hooley, Nadia Gantt, James Tamura, Kerry Donahue, Paul Moore, Syd Johnson, Thomas Chittenden, Richard Gregory, Ezio Bonvini. IMGC936, a first-in-class ADAM9-targeting antibody-drug conjugate, demonstrates promising anti-tumor activity [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 1533.

Introduction: CD137 (4-1BB) signaling provides co-stimulation of CD8 or NK cells following antigen or FcγR engagement, respectively. Efforts to leverage CD137 co-stimulation via agonistic monoclonal antibodies (mAbs) have been thwarted by limited clinical efficacy or unacceptable toxicity. Bispecific targeting strategies linking CD137 activation to a tumor-targeting moiety provides an approach to localize CD137 activation to the tumor microenvironment. Here we evaluate a panel of Fc-bearing HER2 x CD137 bispecific molecules incorporating different valency and geometry to define the format providing optimal CD137 co-stimulation in a tumor-cell anchor-dependent manner. Methods: An anti-HER2 mAb specificity that does not cross compete with margetuximab, trastuzumab or pertuzumab and a proprietary anti-CD137 mAb were utilized to assemble a set of HER2 x CD137 bispecific molecules in bivalent and tetravalent DART® or trivalent TRIDENTTM configurations. The resulting molecules were compared in binding, signaling and co-stimulation assays in the presence or absence of tumor cells expressing HER2. Combination studies were performed in vitro and in immune deficient mice reconstituted with human PBMCs. Results: TRIDENT molecules bearing bivalent CD137 and monovalent HER2 binding achieve optimal HER2-dependent tumor-cell anchored CD137 immune cell co-stimulation. CD137 co-stimulation increases proportionally with the level of HER2 expression as observed with HER2 1+ (MCF7 breast), HER2 2+ (JIMT1 breast) and 3+ (N87 gastric) tumor cells and was paralleled with increased HER2+/CD137+ cell association. No CD137 activation is observed in the absence of HER2-expressing tumor cells. HER2 x CD137 bispecifics enhance NK-cell proliferation and IFN-γ release induced by margetuximab, an Fc-optimized anti-HER2 mAb that up-regulates CD137 expression on NK-cells concomitant with enhanced ADCC against HER2-positive cells. Similarly, HER2 x CD137 bispecific molecules enhanced the in vitro activity of orlotamab, a B7-H3 x CD3 bispecific DART molecule that up-regulates CD137 during T-cell redirected killing. Finally, in vivo mouse studies demonstrate the ability of HER2 x CD137 molecules to expand tumor-associated CD8 cells when co-administered with a tumor targeted CD3 bispecific molecule and support enhance anti-tumor activity. Conclusion: An optimal HER2 x CD137 bispecific format providing maximal CD137 activation in a HER2- dependent manner was identified as a trivalent TRIDENT molecule bearing bivalent CD137 and monovalent HER2 binding. Combinatorial activity with HER2 x CD137 bispecifics was observed with both a HER2-directed therapeutic mAb and a CD3-engaging tumor-targeted bispecific molecule. HER2 x CD137 TRIDENT molecules have therapeutic potential and provide a structural template for incorporating alternate tumor- and/or co-stimulatory-targeting arms. Citation Format: Liqin Liu, Chia-Ying K. Lam, Ralph Alderson, Vatana Long, Yinhua Yang, Robert Burns, Lusiana Widjaja, Jonathan Li, Christina Wolf, Valentina Ciccarone, James Tamura, Gundo Diedrich, Ezio Bonvini, Syd Johnson, Paul A. Moore. Selection of a bispecific trivalent HER2 x CD137 TRIDENT format providing optimal tumor-anchored immune co-stimulation [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 1560.

Introduction: Trophoblast glycoprotein 5T4 is expressed on the cell surface of multiple cancers yet sparingly on normal adult tissues. CD137 (4-1BB) is a co-stimulatory molecule expressed by activated T and NK cells that support cell activation, proliferation and survival. Previously we described bispecific tumor associated antigen x CD137 DART® molecules limiting CD137 mediated immunostimulation to the tumor microenvironment (AACR2017) as an approach to reduce the unwanted systemic CD137 effects associated with agonistic anti-CD137 mAbs. We subsequently identified a bispecific TRIDENT format bearing bivalent CD137 and monovalent tumor antigen engagement that provides maximal CD137 activation in a tumor antigen anchored manner. Here we apply that format to generate a 5T4 x CD137 x CD137 TRIDENT molecule that promotes CD137 activation in proximity to 5T4-expressing tumor cells leading to enhanced T-cell co-stimulation, proliferation and tumor cell killing activity when combined with CD3-engaging tumor-targeting bispecific DART molecules. Methods: TRIDENT molecules were constructed comprising bivalent CD137 binding and monovalent 5T4 binding. Binding properties were evaluated by surface plasmon resonance (SPR) and flow cytometry. Signaling was assessed using a NF-κB luciferase reporter cell line expressing CD137. Co-stimulatory activity was characterized with primary human or cynomolgus monkey T cells. T cells were incubated with or without antigen-expressing cells and submaximal concentrations of either anti-CD3 bead or CD3-engaging tumor-targeting bispecific DART molecules. Results: SPR and flow cytometry analyses demonstrate that 5T4 x CD137 x CD137 TRIDENT molecule binds human and monkey target antigens. The 5T4 x CD137 x CD137 TRIDENT molecule induces CD137 signaling in a reporter cell line and promotes cytokine release in primary human & cynomolgus monkey T-cell assays in the presence of 5T4+ tumor cells. In the absence of 5T4+ tumor cells, the TRIDENT molecule lacks agonistic activity. Furthermore, 5T4 x CD137 x CD137 TRIDENT molecule enhances T-cell proliferation and tumor cell cytolysis induced by CD3-targeted DART molecules. Consistent with preferential induction of CD137 by CD8 T-cells, 5T4 x CD137 x CD137 TRIDENT increases the fraction of CD8+ central memory and effector memory T cells in the presence 5T4+ tumor cells. Conclusions: 5T4 x CD137 x CD137 TRIDENT molecule promotes T-cell co-stimulation in a tumor antigen-dependent manner offering an opportunity to target CD137 immunostimulation, while potentially limiting non-specific systemic T-cell activation and related side effects. Citation Format: Liqin Liu, Ling Huang, Vatana Long, Yinhua Yang, Robert Burns, Jonathan Li, Jennifer DiChiara, Qihong Xu, Arin Whiddon, Chia-Ying K Lam, Jim Tamura, Valentina Ciccarone, Syd Johnson, Ezio Bonvini, Paul Moore. Tumor-antigen 5T4-dependent activation of the CD137 costimulatory pathway by bispecific 5T4 x CD137 x CD137 TRIDENT™ molecules [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 554.

Protege was a phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety of teplizumab immunotherapy at 2 years and to identify characteristics associated with therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years of follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean area under the curve (AUC), a prespecified secondary end point, at 2 years versus placebo. In analyses of prespecified and post hoc subsets at entry, U.S. residents, patients with C-peptide mean AUC andgt;0.2 nmol/L, those randomized 6 weeks after diagnosis, HbA(1c) andlt;7.5% (58 mmol/mol), insulin use andlt;0.4 units/kg/day, and 8-17 years of age each had greater teplizumab-associated C-peptide preservation than their counterparts. Exogenous insulin needs tended to be reduced versus placebo. Antidrug antibodies developed in some patients, without apparent change in drug efficacy. No new safety or tolerability issues were observed during year 2. In summary, anti-CD3 therapy reduced C-peptide loss 2 years after diagnosis using a tolerable dose. Funding Agencies|MacroGenics, Inc (Rockville, MD)||Johnson Johnson||Tolerx||MacroGenics||JDRF|2008-502|Le Bonheur Foundation (Memphis, TN)

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo . Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity.

Dengue viruses are the most common arthropod-transmitted viral infection, with an estimated 390 million human infections annually and ∼3.6 billion people at risk. Currently, there are no approved vaccines or therapeutics available to control the global dengue virus disease burden. In this study, we demonstrate the binding, neutralizing activity, and therapeutic capacity of a novel bispecific d ual- a ffinity r e t argeting molecule (DART) that limits infection of all four serotypes of dengue virus.

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

Fc receptors are a critical component of the innate immune system responsible for the recognition of cross-linked antibodies and the subsequent clearance of pathogens. However, in autoimmune diseases, these receptors play a role in the deleterious action of self-directed antibodies and as such are candidate targets for treatment. GMA161 is an aglycosyl, humanized version of the murine antibody 3G8 that targets the human low-affinity Fcγ receptor III (CD16). As CD16 expression and sequence have high species specificity, preclinical assessments were conducted in mice transgenic for both isoforms of human CD16, CD16A, and CD16B. This transgenic mouse model was useful in transitioning into phase I clinical trials, as it generated positive efficacy data in a relevant disease model and an acceptable single-dose safety profile. However, when GMA161 or its murine parent 3G8 were dosed repeatedly in transgenic mice having both human CD16 isoforms, severe reactions were observed that were not associated with significant cytokine release, nor were they alleviated by antihistamine administration. Prophylactic dosing with an inhibitor of platelet-activating factor (PAF), however, completely eliminated all signs of hypersensitivity. These findings suggest that (1) GMA161 elicits a reaction that is target dependent, (2) immunogenicity and similar adverse reactions were observed with a murine version of the antibody, and (3) the reaction is driven by the atypical hypersensitivity pathway mediated by PAF.

Summary Background Findings of small studies have suggested that short treatments with anti-CD3 monoclonal antibodies that are mutated to reduce Fc receptor binding preserve β-cell function and decrease insulin needs in patients with recent-onset type 1 diabetes. In this phase 3 trial, we assessed the safety and efficacy of one such antibody, teplizumab. Methods In this 2-year trial, patients aged 8–35 years who had been diagnosed with type 1 diabetes for 12 weeks or fewer were enrolled and treated at 83 clinical centres in North America, Europe, Israel, and India. Participants were allocated (2:1:1:1 ratio) by an interactive telephone system, according to computer-generated block randomisation, to receive one of three regimens of teplizumab infusions (14-day full dose, 14-day low dose, or 6-day full dose) or placebo at baseline and at 26 weeks. The Protege study is still underway, and patients and study staff remain masked through to study closure. The primary composite outcome was the percentage of patients with insulin use of less than 0·5 U/kg per day and glycated haemoglobin A 1c (HbA 1C ) of less than 6·5% at 1 year. Analyses included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00385697. Findings 763 patients were screened, of whom 516 were randomised to receive 14-day full-dose teplizumab (n=209), 14-day low-dose teplizumab (n=102), 6-day full-dose teplizumab (n=106), or placebo (n=99). Two patients in the 14-day full-dose group and one patient in the placebo group did not start treatment, so 513 patients were eligible for efficacy analyses. The primary outcome did not differ between groups at 1 year: 19·8% (41/207) in the 14-day full-dose group; 13·7% (14/102) in the 14-day low-dose group; 20·8% (22/106) in the 6-day full-dose group; and 20·4% (20/98) in the placebo group. 5% (19/415) of patients in the teplizumab groups were not taking insulin at 1 year, compared with no patients in the placebo group at 1 year (p=0·03). Across the four study groups, similar proportions of patients had adverse events (414/417 [99%] in the teplizumab groups vs 98/99 [99%] in the placebo group) and serious adverse events (42/417 [10%] vs 9/99 [9%]). The most common clinical adverse event in the teplizumab groups was rash (220/417 [53%] vs 20/99 [20%] in the placebo group). Interpretation Findings of exploratory analyses suggest that future studies of immunotherapeutic intervention with teplizumab might have increased success in prevention of a decline in β-cell function (measured by C-peptide) and provision of glycaemic control at reduced doses of insulin if they target patients early after diagnosis of diabetes and children. Funding MacroGenics, the Juvenile Diabetes Research Foundation, and Eli Lilly.

W1-mAb is a chimpanzee-derived monoclonal antibody to protective antigen that improved survival when administered before anthrax lethal toxin challenge in rats. To better define W1-mAb's efficacy for anthrax, we administered it after initiation of 24-hr infusions of edema toxin and lethal toxin either alone or together in rats or following anthrax spore challenge in mice.W1-mAb or placebo treatment.In toxin-challenged rats treated with placebo, survival rates were lower with edema toxin (500 μg/kg) compared to lethal toxin either alone (175 μg/kg) or with edema toxin (175 μg/kg each) (8%, 33%, and 32%, respectively), but the median time to death was longer (36, 11, and 9 hrs, respectively) (p ≤ .01 for all comparisons). W1-mAb administered up to 12 hrs after edema toxin and 6 hrs after lethal toxin increased survival and reduced hypotension (p ≤ .01). However, only administration of W1-mAb at 0 hrs improved these variables with lethal toxin and edema toxin together (p ≤ .0002). In C57BL/6J mice challenged with anthrax spores subcutaneously, compared to placebo treatment (0 of 15 animals survived), W1-mAb administered beginning 24 hrs after challenge increased survival (13 of 15 survived) (p ≤ .0001).While rapidity of lethality may influence the effectiveness of delayed W1-mAb treatment, these rat and mouse studies provide a basis for further exploring this agent's usefulness for anthrax.

We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell–specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19xTCR bispecific DART molecule revealed equivalent potency with the CD19xCD3 DART molecule, demonstrating flexibility of the DART structure to support T-cell/B-cell associations for redirected T cell–killing applications. The enhanced level of killing mediated by DART molecules was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19− cells. Cell-association studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19xTCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART molecules for the treatment of B-cell malignancies.

Background: CD3-engaging DART molecules direct T cells to tumor-expressed antigens. Flotetuzumab, a CD123 x CD3 bispecific DART molecule that targets the differential expression of the IL-3 receptor alpha chain (CD123) on acute myeloid leukemia (AML) blasts and leukemic stem cells, is currently being investigated in a phase 1 study in relapsed/refractory AML and myelodysplastic syndrome (MDS), with initial evidence of clinical activity1. A common adverse event in redirected T-cell killing interventions is cytokine release syndrome (CRS), a Cmax-associated event that may limit their therapeutic window. Owing to its short circulating half-life, flotetuzumab is administered as continuous infusion, which affords prolonged exposure with limited Cmax excursions. In contrast, long-acting Fc-bearing, CD3-engaging molecules would eliminate the need for continuous dosing, but would be especially impacted by CRS, given the high Cmax levels required to maintain adequate trough concentrations over several days or weeks. We previously reported the development of a CD3-engaging DART molecule with reduced affinity for CD3 that maintained maximal target cell killing and T-cell proliferation, albeit at higher concentrations than its wild-type (WT) counterpart, but with reduced cytokine release2. We report here further optimization and validation of the approach. Methods: Anti-CD3 variants spanning a range of affinities were formatted as Fc-bearing DART molecules with CD123- or CD19-targeting arms and evaluated for cell binding as well as for antigen-dependent induction of T-cell proliferation, cytolytic activity and cytokine release. A therapeutic index (TI) was determined from the ratio of in vitro cell killing activity to that of target cell-induced cytokine release. In vivo anti-tumor activity was assessed in human immune cell-reconstituted mouse tumor models or in mice expressing the epitope recognized by the CD3 arm of the DART proteins (human CD3 k/i mice). Safety, pharmacokinetic (PK) and biological activity of selected molecules was evaluated in cynomolgus monkeys. Results: Six Fc-bearing DART molecules (from a panel of 23 CD123 x CD3 DART variants) were selected for characterization. These molecules spanned a relatively small interval of low CD3 affinities, but varied greatly in activity, from inactive at all concentrations tested to being capable of lysing tumor cells, including primary leukemic and MDS blasts, to the same maximal level of killing observed with the parental WT DART molecule. Higher concentrations of the active variants were needed, owing to a deliberate design incorporating decreased CD3 binding. At levels capable of complete tumor cell lysis, active DART variants showed greatly reduced pro-inflammatory cytokine release (e.g., IL-2, IFN-γ and TNF-α) in vitro and in human PBMC-reconstituted mice compared to the WT version. Molecules were ranked based on their TI and a version with a highly favorable index was engineered as a B-cell targeting CD19 x CD3 DART molecule to conveniently ascertain its biological activity in cynomolgus monkeys. The CD19 x CD3 DART variant demonstrated good tolerability at single doses up to 30 mg/kg. B-cell depletion in the circulation and lymphoid tissues occurred at 1 mg/kg and achieved maximal depletion at ≥10 mg/kg, equivalent to the degree of reduction observed with 0.1 mg/kg of the WT molecule. However, only minimal circulating pro-inflammatory cytokines (including IL-6) were observed at all doses of the DART variant, while a substantial increase in IL-2, IL-6 and IFN-γ (among others) was observed with 0.1 mg/kg of the WT counterpart. The PK profile of the DART variant in cynomolgus monkeys appeared linear in the 1-30 mg/kg interval, with a half-life of 5 to 10 days, consistent with possible clinical dosing at weekly or longer intervals. Conclusions: A next-generation CD3-engaging Fc-bearing DART platform was generated through CD3 arm affinity modulation. These DART variants feature extended PK and an expanded TI suitable for potential clinical development in hematological malignancies. 1. Uy et al, Blood 2017, vol. 130, Suppl 1 637 2. Huang et al, Keystone Symposia: Antibodies as Drugs, 2/25-3/1/2018, Whistler, BC, Canada Disclosures Bonvini: MacroGenics: Employment, Equity Ownership. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Huang:MacroGenics, Inc.: Employment, Equity Ownership. Lam:MacroGenics, Inc.: Employment, Equity Ownership. Kaufman:MacroGenics, Inc.: Employment, Equity Ownership. Liu:MacroGenics, Inc.: Employment, Equity Ownership. Alderson:MacroGenics, Inc.: Employment, Equity Ownership. Stahl:MacroGenics, Inc.: Employment, Equity Ownership. Brown:MacroGenics, Inc.: Employment, Equity Ownership. Li:MacroGenics, Inc.: Employment, Equity Ownership. Sharma:MacroGenics, Inc.: Employment, Equity Ownership. Tamura:MacroGenics, Inc.: Employment, Equity Ownership. Johnson:MacroGenics, Inc.: Consultancy, Equity Ownership. Moore:MacroGenics, Inc.: Employment, Equity Ownership.

Introduction MGD007 (glycoprotein A33 x CD3), a DART protein designed to redirect T cells to target gpA33-expressing colon cancer, is presently undergoing clinical evaluation (NCT02238805). The gpA33 target was selected based on its universal expression profile across primary and metastatic CRC, including expression on putative cancer stem cell (CSC) populations. MGD007 activity in CRC cell cytolysis and its prolonged PK in non-human primates have previously been reported (Cancer Res 2014;74(19 Suppl): Abstract nr 669.1). Here we further characterize MGD007 cellular mechanisms associated with redirected T-cell killing, cytokine responses and modulation with steroids. Method Redirected killing assays were performed using luciferase labelled gpA33+ve Colo205 or RECA0201-GF colorectal cancer stem-like cells (CSLC) with freshly isolated PBMC or fractionated T-cell populations; Treg cells (CD4+, CD127lo, CD25+) were expanded for 14 days in presence of IL-2 and rapamycin and confirmed to be suppressive; steroids (budesonide and dexamethasone) were evaluated at pharmacologically relevant concentrations; multi-parameter FACS and ELISA were performed to determine cell surface marker expression and cytokine levels respectively. Results MGD007 displays potent redirected T-cell killing of gpA33+ve CRC cells, including complete lysis of CRC stem cell-like models. Importantly, MGD007 mediated cytolysis can be supported by various T-cell populations, including Treg cells. Following prolonged in vitro exposure to MGD007 and gpA33+ve tumor cells, expanded T cells acquire a memory phenotype and retain potent CTL activity when challenged with fresh gpA33+ve target cells; however, much decreased cytokine release was observed compared to that observed following initial T-cell exposure. The addition of dexamethasone or budesonide to freshly isolated effector cells and gpA33+ve target cells also reduces cytokine release levels to baseline in the presence of MGD007, with minimal impact observed on MGD007-mediated killing. Discussion MGD007 supports targeted lysis of CRC, including CSC subpopulations, and can leverage suppressive Tregs in addition to conventional T cells for cytolytic activity. Biological activity modulation is also feasible through induction of cytolytic Tmem cells with diminished cytokine release potential via sequential exposure to MGD007 or the simultaneous exposure to low-dose steroids. These data support further clinical development of MGD007 for the treatment of CRC patients. Citation Format: Paul A. Moore, Yinhua Yang, Kalpana Shah, Daorong Liu, Vatana Long, Douglas H. Smith, Zhuangyu Pan, Ralph Alderson, Liqin Liu, Syd Johnson, Ezio Bonvini. Immunotherapy of colorectal cancer by the T-cell targeted DART® protein MGD007: Cellular mechanisms of action [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 1772.

TPS2601Background: T cells naturally undergo activation-induced upregulation of co-inhibitory pathways, which may limit the antitumor immune response. Blocking these inhibitory pathways may enhance...

The majority of human subjects who receive subcutaneous allergen immunotherapy (IT) develop decreased sensitivity to their allergens. Multiple factors may explain the efficacy of IT, some evidence support a role for allergen specific IgG antibodies. There is controversy whether such antibodies act by blocking allergen binding to IgE or initiation of active inhibitory signaling through low affinity IgG receptors (FcgammaRIIB) on mast cells and basophils. In this study, we addressed this question using peripheral blood from cat non-allergic, cat allergic, and immunotherapy-treated cat allergic subjects. Blood from subjects who received IT contain IgG antibodies that mediate inhibition of basophil activation by a mechanism that is blocked by antibodies specific for the inhibitory IgG receptor FcgammaRIIB. Surprisingly, inhibition was also blocked by aglycosylated, putatively non-FcR binding, antibodies that are specific for the FcgammaRIIA, suggesting a contribution of this receptor to the observed effect. Consistent with a cooperative effect, ex vivo basophils were found to express both IgG receptors. In other studies we found that basophils from subjects who were both chronically exposed to allergen and were producing both cat allergen specific IgE and IgG, are hyporesponsive to allergen. These studies confirm that IgG antibodies produced during IT act primarily by stimulation of inhibitory signaling, and suggest that FcgammaRIIA and FcgammaRIIB function cooperatively in activation of inhibitory signaling circuit. We suggest that under normal physiologic conditions in which only a small proportion of FcepsilonRI are occupied by IgE of a single allergen specificity, FcgammaRIIA co-aggregation may, by providing activated Lyn, be required to fuel activation of inhibitory FcgammaRIIB function.

Over the past decade, West Nile virus (WNV) has spread to all 48 of the lower United States as well as to parts of Canada, Mexico, the Caribbean, and South America, with outbreaks of neuroinvasive disease occurring annually. At present, no therapeutic or vaccine is available for human use. Epidemics of WNV and other emerging infectious disease threats demand cost-efficient and scalable production technologies that can rapidly transfer effective therapeutics into the clinical setting. We have previously reported that Hu-E16, a humanized anti-WNV mAb, binds to a highly conserved epitope on the envelope protein, blocks viral fusion, and shows promising postexposure therapeutic activity. Herein, we generated a plant-derived Hu-E16 mAb that can be rapidly scaled up for commercial production. Plant Hu-E16 was expressed at high levels within 8 days of infiltration in Nicotiana benthamiana plants and retained high-affinity binding and potent neutralizing activity in vitro against WNV. A single dose of plant Hu-E16 protected mice against WNV-induced mortality even 4 days after infection at rates that were indistinguishable from mammalian-cell-produced Hu-E16. This study demonstrates the efficacy of a plant-produced mAb against a potentially lethal infection several days after exposure in an animal challenge model and provides a proof of principle for the development of plant-derived mAbs as therapy against emerging infectious diseases.

Severe dengue virus (DENV) infection is epidemiologically linked to pre-existing anti-DENV antibodies acquired by maternal transfer or primary infection. A possible explanation is that DENV immune complexes evade neutralization by engaging Fcgamma receptors (FcgammaR) on monocytes, natural targets for DENV in humans. Using epitope-matched humanized monoclonal antibodies (mAbs) and stable FcgammaR-transfected CV-1 cells, we found that DENV neutralization by IgG1, IgG3, and IgG4 mAbs was enhanced in high-affinity FcgammaRIA transfectants and diminished in low-affinity FcgammaRIIA transfectants, whereas neutralization by IgG2 mAbs (low-affinity ligands for both FcgammaRs) was diminished equally. In FcgammaR-negative Vero cells, IgG3 mAbs exhibited the strongest neutralizing activity and IgG2, the weakest. Our results demonstrate that DENV neutralization is modulated by the Fc region in an IgG subclass manner, likely through effects on virion and FcgammaR binding. Thus, the IgG antibody subclass profile generated by DENV infection or vaccination may independently influence the magnitude of the neutralizing response.

Summary Virus neutralization is governed by the number of antibodies that bind a virion during the cellular entry process. Cellular and serum factors that interact with antibodies have the potential to modulate neutralization potency. Although the addition of serum complement can increase the neutralizing activity of antiviral antibodies in vitro, the mechanism and significance of this augmented potency in vivo remain uncertain. Herein, we show that the complement component C1q increases the potency of antibodies against West Nile virus by modulating the stoichiometric requirements for neutralization. The addition of C1q does not result in virolysis but instead reduces the number of antibodies that must bind the virion to neutralize infectivity. For IgG subclasses that bind C1q avidly, this reduced stoichiometric threshold falls below the minimal number of antibodies required for antibody-dependent enhancement (ADE) of infection of cells expressing Fc-γ receptors (CD32) and explains how C1q restricts the ADE of flavivirus infection.