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Introduction: Response to trastuzumab in metastatic breast cancer correlates with expression of the high binding variant (158V) of the activating Fcγ receptor IIIA (CD16A). We engineered MGAH22, a chimeric anti-HER2 monoclonal antibody with specificity and affinity similar to trastuzumab, with an Fc domain engineered for increased binding to both alleles of human CD16A.Methods: MGAH22 was compared to an identical anti-HER2 mAb except for a wild type Fc domain. Antibody-dependent cell cytotoxicity (ADCC) assays were performed with HER2-expressing cancer cells as targets and human PBMC or purified NK cells as effectors. Xenograft studies were conducted in mice with wild type murine FcγRs; in mice lacking murine CD16; or in mice lacking murine CD16 but transgenic for human CD16A-158F, the low-binding variant. The latter model reproduces the differential binding between wild type and the Fc-optimized mAb for human CD16A. The JIMT-1 human breast tumor line, derived from a patient that progressed on trastuzumab therapy, was used in these studies. Single and repeat dose toxicology studies with MGAH22 administered intravenously at high dose were conducted in cynomolgus monkeys.Results: The optimized Fc domain confers enhanced ADCC against all HER2-positive tumor cells tested, including cells resistant to trastuzumab's anti-proliferative activity or expressing low HER2 levels. The greatest improvement occurs with effector cells isolated from donors homozygous or heterozygous for CD16A-158F, the low-binding allele. MGAH22 demonstrates increased activity against HER2-expressing tumors in mice transgenic for human CD16A-158F. In single and repeat-dose toxicology studies in cynomolgus monkeys, a species with a HER2 expression pattern comparable to that in humans and Fcγ receptors that exhibit enhanced binding to the optimized Fc domain, MGAH22 was well tolerated at all doses tested (15-150 mg/kg) and exhibited pharmacokinetic parameters similar to that of other anti-HER2 antibodies. Induction of cytokine release by MGAH22 in vivo or in vitro was similar to that induced by the corresponding wild type mAb or trastuzumab.Conclusions: The data support the clinical development of MGAH22, which may have utility in patients with low HER2 expressing tumors or carrying the CD16A low-binding allele. [ABSTRACT FROM AUTHOR]

Background: Bispecific antibodies represent a promising class of biologics for cancer treatment. However, their dual specificity and complex structure pose challenges in the engineering process, often resulting in molecules with good functional but poor physicochemical properties. Method: To overcome limitations in the properties of an anti-5T4 x anti-CD3 (α5T4 x αCD3) DART molecule, a phage-display method was developed, which succeeded in simultaneously engineering cross-reactivity to the cynomolgus 5T4 ortholog, improving thermostability and the elevating expression level. Results: This approach generated multiple DART molecules that exhibited significant improvements in all three properties. The lead DART molecule demonstrated potent in vitro and in vivo anti-tumor activity. Although its clearance in human FcRn-transgenic mice was comparable to that of the parental molecule, faster clearance was observed in cynomolgus monkeys. The lead α5T4 x αCD3 DART molecule displayed no evidence of off-target binding or polyspecificity, suggesting that the increased affinity for the target may account for its accelerated clearance in cynomolgus monkeys. Conclusions: This may reflect target-mediated drug disposition (TMDD), a potential limitation of targeting 5T4, despite its limited expression in healthy tissues.

Introduction: B7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Vobramitamab duocarmazine (also referred to as MGC018 and herein referred to as vobra duo) is an investigational duocarmycin-based antibody-drug conjugate (ADC) directed against the B7-H3 antigen. It is composed of an anti-B7-H3 humanized IgG1/kappa monoclonal antibody chemically conjugated through a cleavable valine-citrulline linker to a du ocarmycin-hydroxy b enzamide a zaindole (vc-seco-DUBA). Vobra duo has shown preliminary clinical activity in B7-H3-expressing tumors., Methods: B7-H3 expression was evaluated by flow-cytometry in a panel of human NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid (MCTS) models by the water-soluble tetrazolium salt,MTS, proliferation assay and Cell Titer Glo 3D cell viability assay, respectively. Apoptotic cell death was investigated by annexin V staining. Orthotopic, pseudometastatic, and resected mouse NB models were developed to mimic disease conditions related to primary tumor growth, metastases, and circulating tumor cells with minimal residual disease, respectively., Results: All human NB cell lines expressed cell surface B7-H3 in a unimodal fashion. Vobra duo was cytotoxic in a dose-dependent and time-dependent manner against all cell lines (IC50 range 5.1-53.9 ng/mL) and NB MCTS (IC50 range 17.8-364 ng/mL). Vobra duo was inactive against a murine NB cell line (NX-S2) that did not express human B7-H3; however, NX-S2 cells were killed in the presence of vobra duo when co-cultured with human B7-H3-expressing cells, demonstrating bystander activity. In orthotopic and pseudometastatic mouse models, weekly intravenous treatments with 1 mg/kg vobra duo for 3 weeks delayed tumor growth compared with animals treated with an irrelevant (anti-CD20) duocarmycin-ADC. Vobra duo treatment for 4 weeks further increased survival in both orthotopic and resected NB models. Vobra duo compared favorably to TOpotecan-TEMozolomide (TOTEM), the standard-of-care therapy for NB relapsed disease, with tumor relapse delayed or arrested by two or three repeated 4-week vobra duo treatments, respectively. Further increased survival was observed in mice treated with vobra duo in combination with TOTEM. Vobra duo treatment was not associated with body weight loss, hematological toxicity, or clinical chemistry abnormalities., Conclusion: Vobra duo exerts relevant antitumor activity in preclinical B7-H3-expressing NB models and represents a potential candidate for clinical translation., Competing Interests: Competing interests: EB and DL are employees of MacroGenics and receive salary and stocks as part of their compensations. The other authors do not have competing interests to declare., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Acute myeloid leukemia (AML) relapse is one of the most common and significant adverse events following allogeneic hematopoietic cell transplantation (HCT). Downregulation of major histocompatibility class II (MHC-II) surface expression on AML blasts may represent a mechanism of escape from the graft-versus-malignancy effect and facilitate relapse. We hypothesized that T-cell immunotherapies targeting AML antigens would upregulate MHC-II surface expression via localized release of interferon gamma (IFN-γ), a protein known to upregulate MHC-II expression via JAK-STAT signaling. We demonstrate that flotetuzumab (FLZ), a CD123 × CD3 bispecific DART molecule, and chimeric antigen receptor expressing T cells targeting CD123, CD33, or CD371 upregulate MHC-II surface expression in vitro on a THP-1 AML cell line with intermediate MHC-II expression and 4 primary AML samples from patients relapsing after HCT with low MHC-II expression. We additionally show that FLZ upregulates MHC-II expression in a patient-derived xenograft model and in patients with relapsed or refractory AML who were treated with FLZ in a clinical trial. Finally, we report that FLZ-induced MHC-II upregulation is mediated by IFN-γ. In conclusion, we provide evidence that T-cell immunotherapies targeting relapsed AML can kill AML via both MHC-independent mechanisms and by an MHC-dependent mechanism through local release of IFN-γ and subsequent upregulation of MHC-II expression., (© 2023 by The American Society of Hematology.)

ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non-small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding. Subsequent analysis revealed anti-ADAM9 antibodies were efficiently internalized and processed by tumor cells making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe the preclinical evaluation of IMGC936, a novel ADC targeted against ADAM9. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21-C, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker, at a drug antibody ratio of approximately 2.0. In addition, the YTE mutation (M252Y/S254T/T256E) was introduced into the CH2 domain of the antibody Fc to maximize in vivo plasma half-life and exposure. IMGC936 exhibited cytotoxicity toward ADAM9-positive human tumor cell lines, as well as bystander killing, potent antitumor activity in human cell line-derived xenograft and patient-derived xenograft tumor models, and an acceptable safety profile in cynomolgus monkeys with favorable pharmacokinetic properties. Our preclinical data provide a strong scientific rationale for the further development of IMGC936 as a therapeutic candidate for the treatment of ADAM9-positive cancers. A first-in-human study of IMGC936 in patients with advanced solid tumors has been initiated (NCT04622774)., (©2022 American Association for Cancer Research.)

Background: Availability of checkpoint inhibitors has created a paradigm shift in the management of patients with solid tumors. Despite this, most patients do not respond to immunotherapy, and there is considerable interest in developing combination therapies to improve response rates and outcomes. B7-H3 (CD276) is a member of the B7 family of cell surface molecules and provides an alternative immune checkpoint molecule to therapeutically target alone or in combination with programmed cell death-1 (PD-1)-targeted therapies. Enoblituzumab, an investigational anti-B7-H3 humanized monoclonal antibody, incorporates an immunoglobulin G1 fragment crystallizable (Fc) domain that enhances Fcγ receptor-mediated antibody-dependent cellular cytotoxicity. Coordinated engagement of innate and adaptive immunity by targeting distinct members of the B7 family (B7-H3 and PD-1) is hypothesized to provide greater antitumor activity than either agent alone., Methods: In this phase I/II study, patients received intravenous enoblituzumab (3-15 mg/kg) weekly plus intravenous pembrolizumab (2 mg/kg) every 3 weeks during dose-escalation and cohort expansion. Expansion cohorts included non-small cell lung cancer (NSCLC; checkpoint inhibitor [CPI]-naïve and post-CPI, programmed death-ligand 1 [PD-L1] <1%), head and neck squamous cell carcinoma (HNSCC; CPI-naïve), urothelial cancer (post-CPI), and melanoma (post-CPI). Disease was assessed using Response Evaluation Criteria in Solid Tumors version 1.1 after 6 weeks and every 9 weeks thereafter. Safety and pharmacokinetic data were provided for all enrolled patients; efficacy data focused on HNSCC and NSCLC cohorts., Results: Overall, 133 patients were enrolled and received ≥1 dose of study treatment. The maximum tolerated dose of enoblituzumab with pembrolizumab at 2 mg/kg was not reached. Intravenous enoblituzumab (15 mg/kg) every 3 weeks plus pembrolizumab (2 mg/kg) every 3 weeks was recommended for phase II evaluation. Treatment-related adverse events occurred in 116 patients (87.2%) and were grade ≥3 in 28.6%. One treatment-related death occurred (pneumonitis). Objective responses occurred in 6 of 18 (33.3% [95% CI 13.3 to 59.0]) patients with CPI-naïve HNSCC and in 5 of 14 (35.7% [95% CI 12.8 to 64.9]) patients with CPI-naïve NSCLC., Conclusions: Checkpoint targeting with enoblituzumab and pembrolizumab demonstrated acceptable safety and antitumor activity in patients with CPI-naïve HNSCC and NSCLC., Trial Registration Number: NCT02475213., Competing Interests: Competing interests: CA reports personal fees from AstraZeneca, BluPrint Oncology, Celgene, Eli Lilly, Merck, Daiichi Sankyo for advisory boards, and research funding to the institution from Merck, MacroGenics, Novartis, and AstraZeneca; SA reports consulting fees from Bristol-Myers Squibb, Merck, CBMG, AstraZeneca, Memgen, RAPT, Venn, Achilles Therapeutics, Celsius, Samyang Biopharma, GlaxoSmithKline, Amgen, travel expenses from Bristol-Myers Squibb, Merck, Rapt, Achilles Therapeutics, Celsius, GlaxoSmithKline, Amgen, and grants from Novartis; VA reports personal fees and non-financial support from Bristol-Myers Squibb, and personal fees from Merck, MSD, Novartis, QBiotics, Nektar, Roche, Pierre Fabre; RC reports grants paid to the institution from MacroGenics, Merck, personal fees from AstraZeneca, grants to the institution and personal fees from Innate Pharma, Genocea Biosciences, Heat Biologics, advisory boards for Innate Pharma, Genocea Biosciences, AstraZeneca, Heat Biologics, Cantargia, research funding from Celldex, Kyn Therapeutics, Merck, Genocea Biosciences, Xencor, Innate Pharma, Heat Biologics, and travel expenses from Heat Biologics, Innate Pharma, Genocea Biosciences; RLF reports consulting fees from Aduro Biotech, Novasenta, Sanofi, Zymeworks, honoraria from Bristol-Myers Squibb, EMD Serono, MacroGenics, Merck, Numab Therapeutics, Pfizer for advisory boards, research funds to institution from AstraZeneca/MedImmune, Bristol-Myers Squibb, Novasenta, Tesaro, clinical trial support from AstraZeneca/MedImmune, Bristol-Myers Squibb, Merck, and stocks in Novasenta; VG reports honoraria from AstraZeneca, Roche/Genentech; ARK reports holding stocks in ECOM Medical, receiving consulting fees from Exelixis, AstraZeneca, Bayer, Pfizer, Novartis, Genentech, Bristol-Myers Squibb, EMD Serono, serving on Speakers’ Bureau at Janssen, Astellas Medivation, Pfizer, Novartis, Sanofi, Genentech/Roche, Eisai, AstraZeneca, Bristol-Myers Squibb, Amgen, Exelixis, EMD Serono, Merck, Seattle Genetics/Astellas, receiving research funding to the institution from Genentech, Exelixis, Janssen, AstraZeneca, Bayer, Bristol-Myers Squibb, Eisai, MacroGenics, Astellas Pharma, beyond spring, BioClin Therapeutics, Clovis Oncology, Bavarian Nordic, Seattle Genetics, Immunomedics, Epizyme, and receiving travel expenses from Genentech, Prometheus Laboratories, Astellas Medivation, Janssen, Eisai, Bayer, Pfizer, Novartis, Exelixis, AstraZeneca; SK reports consulting fees from MacroGenics and is a stock holder of Abbott, Aqua, Bank of America, Baxter, Betterment, Bristol-Myers Squibb, Etrade, ExxonMobil, Invesco, Johnson & Johnson, Lockheed Martin Corp, Marathon Oil, Marathon Petroleum, Pfizer, Takeda Pharm Co, Verizon, Welltower, Vanguard; NL reports research funding from ALX Therapeutics, Ascentage, Asana, BeiGene, Constellation Pharma, Alexion, Cerulean, Forty Seven, Alpine, Merck, Pfizer, Regeneron, TaiRx, Apexian, Formation Biologics (Forbius), Symphogen, CytomX, InhibRx, Incyte, Jounce, Livzon, Northern Biologics, Innovent Biologics, Ikena, Odonate, Loxo, Alpine Biosciences, Ikena, Mersana, and serves on advisory boards for Innovent Biologics; YL reports research funding from Merck, MacroGenics, Tolero Pharmaceuticals, AstraZeneca, Vaccinex, Blueprint Medicines, Harpoon Therapeutics, Sun Pharma Advanced Research, Kyowa Pharmaceuticals, Tesaro, Bayer HealthCare, honorarium from Clarion Healthcare, served on the advisory board for AstraZeneca, Novocure, and consultant for AstraZeneca; OR reports research support from Merck, Speakers’ Bureau activities supported by educational grants from Bristol-Myers Squibb and Merck, and honoraria for consulting activities for Merck, Celgene, Five Prime, GlaxoSmithKline, Bayer, Roche/Genentech, Puretech, Imvax, Sobi. In addition, OR has a patent pending for 'Methods of using pembrolizumab and trebananib'; MS received honoraria from Deciphera Pharmaceuticals and Daiichi Sankyo for serving on advisory boards; AT reports consulting fees from AbbVie, Adagene, Agenus, Aro Biotherapeutics, Ascentage, Aximmune, Bayer, BioInvent, Birdie, Cello Health, Ellipses, EMD Serono, Five Prime, Forbius, Gilde Healthcare Partners, HBM Partners, Immunomet, Karma Oncology, Mekanistic, Menarini, Mersana, Nanobiotix, Nuvalent, OSI, Partner Therapeutics, Pfizer, Pieris, Pierre Fabre, Ridgeway, RYVU Therapeutics, Scitemex, Seattle Genetics, Symphogen, Syneos, TFS Trial Form Support, and Trillium Therapeutics, and honoraria from Abgenomics, Adagene, ADC Therapeutics, Aro Biotherapeutics, BioInvent, Elucida, Immunome, Jazz, NBE Therapeutics, Pelican, Innate Pharma, TFS Trial Form Support International, and Zymeworks for serving on advisory boards. In addition, AT serves on the Independent Data Monitoring Committees for Mirati and Genentech; NV reports non-financial support for conducting trials with Amgen, Aravive, Arrowhead, Arvinas, AstraZeneca, Bayer, Bristol-Myers Squibb, Calithera, Clovis, Corvus, Dendreon, eFFECTOR, Eisai, Endocyte, Epizyme, ESSA, Exelixis, Genentech, Immunomedics, Kangpu, Kinter Suzhou, MacroGenics, Merck, Mirati, Nektar, Novartis, Seattle Genetics, Tolero, advisory boards for Amgen, Astellas, AstraZeneca, Aveo, Caris, Clovis, Corvus, Eisai, Exelixis, GeneCentric, Genentech, Janssen, Merck, Myovant, OnQuality Pharma, Pfizer, Sanofi-Genzyme, Tolero, serves on the Speakers’ Bureau at Bayer, Caris, Clovis, Sanofi-Genzyme, Seattle Genetics, consulting services to Cancer Expert Now, vice-chair responsibilities at SWOG, section editor at Up-To-Date, and legal defense advisor to Novartis, Sanofi-Genzyme; AW reports grants from Merck, grants and personal fees from Bristol-Myers Squibb, personal fees from Ipsen, Pfizer; DPZ received institutional research support from Merck, Bristol-Myers Squibb, GlaxoSmithKline, AstraZeneca, Aduro Biotech, Lilly, Astellas, MacroGenics for his role as a PI of clinical trials sponsored by these companies and received honoraria from Blueprint Medicines for serving on an advisory board; SMG is a former employee of MacroGenics serving as medical monitor; XZ, RC, and LP are employees of MacroGenics; JB was an employee of MacroGenics; PAM is an employee of MacroGenics, holds stock in MacroGenics, and holds a patent with all rights assigned to MacroGenics for 'Antibodies reactive with B7-H3, immunologically active fragments thereof' US Patent 9,150,656; EB is an employee of MacroGenics, holds stock in MacroGenics, and has a patent US20190389952 pending to MacroGenics; FA was an employee of MacroGenics and is now an employee of AstraZeneca; AP, AAA, AB, RH, and AMJ have nothing to disclose., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Children with acute myeloid leukemia (AML) have a poor prognosis despite the intensification of chemotherapy. Future efforts to improve outcomes should focus on more precise targeting of leukemia cells. CD123, or IL3RA, is expressed on the surface of nearly all pediatric AML samples and is a high-priority target for immunotherapy. The efficacy of an investigational dual-affinity retargeting antibody (DART) molecule (CD123 × CD3; MGD006 or flotetuzumab) was assessed in two distinct patient-derived xenograft (PDX) models of pediatric AML. MGD006 simultaneously binds to CD123 on target cells and CD3 on effector T cells, thereby activating T cells and redirecting them to induce cytotoxicity in target cells. The concurrent treatment of cytarabine and MGD006 was performed to determine the effect of cytarabine on T-cell counts and MGD006 activity. Treatment with MGD006 along with an allogeneic human T-cell infusion to act as effector cells induced durable responses in both PDX models, with CD123 positivity. This effect was sustained in mice treated with a combination of MGD006 and cytarabine in the presence of T cells. MGD006 enhanced T-cell proliferation and decreased the burden of AML blasts in the peripheral blood with or without cytarabine treatment. These data demonstrate the efficacy of MGD006 in prolonging survival in pediatric AML PDX models in the presence of effector T cells and show that the inclusion of cytarabine in the treatment regimen does not interfere with MGD006 activity.

Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: the following authors were employed by Eli Lilly and Company at the time of conducting the research and may own stock: JC, YTK, NH, ALG, APM, and RJB. The following authors were employed by Macrogenics at the time of conducting the research and may own stock: PAM and EB. APM is an employee of Boehringer Ingelheim Pharmaceuticals Inc. However, this affiliation was not held at the time the study was conducted. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Approximately 50% of acute myeloid leukemia (AML) patients do not respond to induction therapy (primary induction failure [PIF]) or relapse after <6 months (early relapse [ER]). We have recently shown an association between an immune-infiltrated tumor microenvironment (TME) and resistance to cytarabine-based chemotherapy but responsiveness to flotetuzumab, a bispecific DART antibody-based molecule to CD3ε and CD123. This paper reports the results of a multicenter, open-label, phase 1/2 study of flotetuzumab in 88 adults with relapsed/refractory AML: 42 in a dose-finding segment and 46 at the recommended phase 2 dose (RP2D) of 500 ng/kg per day. The most frequent adverse events were infusion-related reactions (IRRs)/cytokine release syndrome (CRS), largely grade 1-2. Stepwise dosing during week 1, pretreatment dexamethasone, prompt use of tocilizumab, and temporary dose reductions/interruptions successfully prevented severe IRR/CRS. Clinical benefit accrued to PIF/ER patients showing an immune-infiltrated TME. Among 30 PIF/ER patients treated at the RP2D, the complete remission (CR)/CR with partial hematological recovery (CRh) rate was 26.7%, with an overall response rate (CR/CRh/CR with incomplete hematological recovery) of 30.0%. In PIF/ER patients who achieved CR/CRh, median overall survival was 10.2 months (range, 1.87-27.27), with 6- and 12-month survival rates of 75% (95% confidence interval [CI], 0.450-1.05) and 50% (95% CI, 0.154-0.846). Bone marrow transcriptomic analysis showed that a parsimonious 10-gene signature predicted CRs to flotetuzumab (area under the receiver operating characteristic curve = 0.904 vs 0.672 for the European LeukemiaNet classifier). Flotetuzumab represents an innovative experimental approach associated with acceptable safety and encouraging evidence of activity in PIF/ER patients. This trial was registered at www.clinicaltrials.gov as #NCT02152956., (© 2021 by The American Society of Hematology.)

Combination immunotherapy with antibodies directed against PD-1 and CTLA-4 shows improved clinical benefit across cancer indications compared to single agents, albeit with increased toxicity. Leveraging the observation that PD-1 and CTLA-4 are co-expressed by tumor-infiltrating lymphocytes, an investigational PD-1 x CTLA-4 bispecific DART molecule, MGD019, is engineered to maximize checkpoint blockade in the tumor microenvironment via enhanced CTLA-4 blockade in a PD-1-binding-dependent manner. In vitro , MGD019 mediates the combinatorial blockade of PD-1 and CTLA-4, confirming dual inhibition via a single molecule. MGD019 is well tolerated in non-human primates, with evidence of both PD-1 and CTLA-4 blockade, including increases in Ki67 + CD8 and ICOS + CD4 T cells, respectively. In the ongoing MGD019 first-in-human study enrolling patients with advanced solid tumors (NCT03761017), an analysis undertaken following the dose escalation phase revealed acceptable safety, pharmacodynamic evidence of combinatorial blockade, and objective responses in multiple tumor types typically unresponsive to checkpoint inhibitor therapy., Competing Interests: A.B., B.J.S., K.S., D.L., J.L., S.-S.H., A.D., K.S., F.C., H.L., E.B., G.D., and P.A.M. are contracted or employed by MacroGenics, and received stock options as a condition of employment. A.B., B.J.S., K.S., E.B., G.D., and P.A.M. are inventors on MacroGenics patent applications based on the work described herein., (© 2020.)

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline- seco duocarmycin hydroxybenzamide azaindole (vc- seco -DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg., (©2020 American Association for Cancer Research.)

Purpose: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system., Experimental Design: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models., Results: B7-H3 CAR T cells mediate significant antitumor activity in vivo , causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3., Conclusions: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials., (©2019 American Association for Cancer Research.)

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 ., (©2018 American Association for Cancer Research.)

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.

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 ., (©2016 American Association for Cancer Research.)

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART ® ) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (T m 1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML., (© 2016 by The American Society of Hematology.)

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.

Protégé 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 >0.2 nmol/L, those randomized ≤6 weeks after diagnosis, HbA1c <7.5% (58 mmol/mol), insulin use <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.

Elevations in C-reactive protein (CRP) are associated with an increased risk of insulin resistance. Whether CRP plays a causal role is unknown. Here we show that CRP transgenic mice and wild-type mice administered recombinant CRP are insulin resistant. Mice lacking the inhibitory Fcγ receptor IIB (FcγRIIB) are protected from CRP-induced insulin resistance, and immunohistochemistry reveals that FcγRIIB is expressed in skeletal muscle microvascular endothelium and is absent in skeletal muscle myocytes, adipocytes, and hepatocytes. The primary mechanism in glucose homeostasis disrupted by CRP is skeletal muscle glucose delivery, and CRP attenuates insulin-induced skeletal muscle blood flow. CRP does not impair skeletal muscle glucose delivery in FcγRIIB(-/-) mice or in endothelial nitric oxide synthase knock-in mice with phosphomimetic modification of Ser1176, which is normally phosphorylated by insulin signaling to stimulate nitric oxide-mediated skeletal muscle blood flow and glucose delivery and is dephosphorylated by CRP/FcγRIIB. Thus, CRP causes insulin resistance in mice through FcγRIIB-mediated inhibition of skeletal muscle glucose delivery.

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.

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.

Objective: To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications., Methods: DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model., Results: DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice., Conclusion: This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.

Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor FcgammaRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcgammaRIIB in NIH3T3IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domain-containing inositol 5'-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via FcgammaRIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes.

Despite advances in therapy, many patients with systemic light-chain amyloidosis (AL) die within 3 years from diagnosis. The humanized 2B6 monoclonal antibody (MoAb) is specific for the low-affinity IgG Fc receptor CD32B and effective in a human CD32B+ B-cell lymphoma murine xenograft model. Because MoAb therapy could improve outcomes in AL, we studied CD32B expression by clonal plasma cells obtained from 48 patients with AL. Transcript profiling showed that expression of CD32B was significantly higher than expression of all other Fc receptor family members. Reverse-transcriptase polymerase chain reaction (RT-PCR) using double-enriched CD138+ plasma cells showed uniform expression of the stable cell surface CD32B1 isoform at diagnosis and relapse, and flow cytometry showed intense CD32B cell surface staining on 99% of CD138+ plasma cells at diagnosis and relapse. These data provide a rationale for the novel therapeutic targeting of CD32B using the humanized 2B6 MoAb in patients with systemic AL-amyloidosis.

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 Fcgamma regions to low-affinity Fcgamma receptors (FcgammaR) 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 (FcgammaRIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (FcgammaRIIB). Identification of new amino acid residues important for FcgammaR 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 FcgammaRIV, a unique mouse activating FcgammaR 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 FcgammaR, 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-158(phe) 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 FcgammaR present on the surface of tumor-infiltrating effector cell populations.

Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) is a key enzyme that governs cellular functions such as gene transcription, secretion, proliferation, motility, and development. Here, we show that PLC-gamma1 is regulated via a novel autoinhibitory mechanism involving its carboxy-terminal Src homology (SH2C) domain. Mutation of the SH2C domain tyrosine binding site led to constitutive PLC-gamma1 activation. The amino-terminal split pleckstrin homology (sPHN) domain was found to regulate the accessibility of the SH2C domain. PLC-gamma1 constructs with mutations in tyrosine 509 and phenylalanine 510 in the sPHN domain no longer required an intact amino-terminal Src homology (SH2N) domain or phosphorylation of tyrosine 775 or 783 for activation. These data are consistent with a model in which the SH2C domain is blocked by an intramolecular interaction(s) that is released upon cellular activation by occupancy of the SH2N domain.

Objective: Fcgamma receptors (FcgammaRs) recognize immune complexes (ICs) and coordinate the immune response by modulating the functions of dendritic cells (DCs). The purpose of this study was to unravel the role of the inhibitory FcgammaRIIb in rheumatoid arthritis (RA) by studying the effect of the FCGR2B 695T>C polymorphism on susceptibility to RA, severity of the disease, and DC function., Methods: Genotyping was performed in RA patients (n = 246) and healthy blood donors (n = 269). The patients' demographic data, disease severity, and disease progression were assessed over a followup of 6 years. DCs were cultured for flow cytometry to determine the expression of FcgammaRs. For detection of FcgammaRIIb (CD32B), a unique anti-FcgammaRIIb antibody (2B6-fluorescein isothiocyanate [FITC]) was used. The capacity for antigen uptake by DCs was studied by assessing the uptake of FITC-labeled ICs. Levels of cytokine production by DCs were measured during lipopolysaccharide-mediated cell activation in the presence and absence of ICs., Results: Although no role of the FCGR2B variant in RA susceptibility was demonstrated, this variant was associated with a nearly doubled rate of radiologic joint damage during the first 6 years of RA. Multiple regression analysis showed that FCGR2B was by far the strongest predictor of joint damage identified to date. DCs from patients carrying this variant failed to display the inhibitory phenotype normally observed upon IC-mediated triggering of inflammation and displayed diminished FcgammaRII-mediated antigen uptake compared with wild-type DCs. However, the levels of FcgammaRs were not affected, suggesting that the FCGR2B variant alters the function rather than regulation of proteins., Conclusion: This study is the first to show that a single genetic variant, the FCGR2B 695T>C polymorphism, is a critical determinant of disease severity in RA and radically changes DC behavior. Our results underscore the key role of DCs in the progression of RA and reveal FcgammaRIIb as an important potential therapeutic target in RA and other autoimmune conditions.