Antibody 2F5 Research Articles

Antibody-based targeted delivery of interleukin-4 synergizes with dexamethasone for the reduction of inflammation in arthritis.

We have previously reported that F8-IL4, a fusion protein consisting of the F8 antibody specific to the alternatively-spliced extra domain A of fibronectin and of murine IL-4, cures mice with established arthritis, when used in combination with dexamethasone (DXM). The goal of this study was to assess whether other therapeutic agents, besides DXM, could induce cures in combination with F8-IL4 and to elucidate which leucocytes are most affected by the pharmacological treatment. We performed therapy experiments in mice with CIA, using intravenous administrations of F8-IL4 in combination with DXM, MTX, murine cytotoxic T-lymphocyte-associated protein 4 fused to the fragment crystallizable portion of murine IgG2a, as well as mAbs to murine IL17A or the p40 subunit of murine IL12/IL23. Histology and immunohistochemistry for the identification of the various leucocytes were performed on the paws of mice euthanized at different therapy time points. Only the use of F8-IL4 in combination with DXM induced complete remissions, while all other combinations did not lead to cures. The light microscopical evaluation of paws with arthritis revealed a predominant infiltration of neutrophils, which substantially decreased 24 h after treatment with F8-IL4 and DXM. The combination of F8-IL4 with DXM promotes a rapid anti-arthritic action by potently inhibiting neutrophil activity. A fully human analogue of F8-IL4 may find clinical utility for the treatment of neutrophil-driven chronic inflammatory conditions.

Mechanistic and Fc requirements for inhibition of Sudan virus entry and in vivo protection by a synthetic antibody

The Sudan virus (SUDV), an ebolavirus, causes severe hemorrhagic fever with human case fatality rates of ∼50%. Previous work from our lab demonstrated the synthetic antibody F4 potently inhibits viral entry and protects against lethal virus challenge in mice [Chen et al., ACS Chem. Biol., 2014, 9, 2263–2273]. Here, we explore mechanistic requirements as well as contribution of the Fc region and function on neutralization and in vivo protection. Live cell imaging demonstrates that the antibody colocalizes with vesicular stomatitis virus particles containing the Sudan virus glycoprotein (VSV-GPSUDV) and that the antibody is rapidly degraded within cellular endosomes. A viral escape mutant contained substitutions on the N-heptad repeat (NHR) segment of GP2, the fusion subunit. Truncation studies indicated that the size of the Fc impacts virus neutralization potential. Finally, we examined the protective efficacy of Fc-null mutants in mice, and found that Fc function was not required for high levels of protection. Altogether, these results indicate that neutralization of SUDV GP-mediated cell entry likely involves blockade of viral membrane fusion within endosomes, and that inhibition of viral entry is the likely mechanism of in vivo protection.

MYCN-amplified stage 2/3 neuroblastoma: excellent survival in the era of anti-GD2 immunotherapy.

High-risk neuroblastoma (HR-NB) includes MYCN-amplified stage 2/3, but reports covering anti-GD2 immunotherapy, which recently became standard for HR-NB, do not provide details on this subset. We now report on all 20 MYCN-amplified stage 2/3 patients who received induction chemotherapy at our center during the era of consolidation with anti-GD2 antibody 3F8/ granulocyte-macrophage colony-stimulating factor (GM-CSF) (2000-2015). Early in this period, consolidation included autologous stem-cell transplantation (ASCT). Event-free survival (EFS) and overall survival (OS) were estimated using Kaplan-Meier analyses.With induction, 19/20 (95%) patients achieved complete/very good partial remission (CR/VGPR) but one had progressive disease with early death. One responder did not receive consolidation and died of relapse. Five-year post-diagnosis EFS/OS rates for all 20 patients were 72%/84%. The 18 CR/VGPR patients who received consolidation had EFS/OS 81%/94% at five years from starting 3F8/GM-CSF: 4/4 ASCT patients remained relapse-free, while 11/14 non-ASCT patients remained relapse-free and two of the three relapsed patients achieved 2nd CR (consolidated by retreatment with 3F8/GM-CSF) and remained in 2nd CR at 36+ and 95+ months post-relapse. The 14 non-ASCT patients had EFS/OS 73.5%/93% at five years from starting 3F8/GM-CSF.This subset appears to have a good prognosis with contemporary multi-modality therapy, possibly even without ASCT.

Abstract 3984: Neuropilin-1 mediates neutrophil elastase uptake and antigen cross-presentation in breast cancer cells

Abstract We previously showed that soluble neutrophil elastase (NE) is rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer (BrCa). NE uptake led to cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapeutic target in hematologic malignancies. The mechanism of NE uptake, however, remains unknown. Here, we demonstrated that NE internalization was temperature- and time-sensitive, dose-saturation was observed, and uptake was partially blocked by chlorpromazine (CPZ) and wortmannin, supporting a receptor-mediated process and suggesting a role for clathrin- and PI3K-dependent mechanisms. To identify a candidate NE receptor, we performed mass spectrometry (MS) of proteins after anti-NE co-immunoprecipitation of NE-pulsed MDA-MB-231 BrCa cells and we identified neuropilin-1 (NRP1) as an NE co-associated protein. Binding of NE to NRP1 was confirmed by ELISA, and peptide epitope mapping studies indicated NE bound via it consensus sequence RRXR, which is also known to bind the b1b2 domain of NRP1. NE bound to NRP1 with high affinity (Kd=38.7 nM) as measured with biolayer interferometry. To confirm the role of NRP1 in cellular uptake of NE, we transfected MDA-MB-231 BrCa cells with siRNA or shRNA against NRP1 and showed that NRP1 knockdown resulted in a 2-fold decrease in NE uptake as determined with flow cytometry. Similarly, blocking with an NRP1-neutralizing antibody decreased NE uptake by 60% vs. isotype control antibody, an effect that was also observed in other breast cancer cell lines that expressed NRP1. Conversely, transient expression of NRP1 in the NRP1-deficient T47D BrCa cell line was sufficient to induce uptake of NE. Importantly, knockdown of NRP1 expression in MDA-231 cells also prevented PR1 cross-presentation as determined with the anti-PR1/HLA-A2 monoclonal antibody 8F4. This was confirmed by the loss of susceptibility of NE-pulsed MDA-MB-231 cells to lysis by PR1-specific cytotoxic T cells following knockdown of NRP1 expression. Our data support a novel function of NRP1 in the uptake and cross-presentation of neutrophil-derived proteins by non-hematopoietic cancer cells. Because 8F4 mediates killing of PR1/HLA-A2+ leukemia and NRP1 is broadly expressed on many tumors, our results suggest a role for immunotherapy strategies that target NE-derived peptides on NRP1+ tumors. Citation Format: Celine Kerros, Satyendra C. Tripathi, Dongxing Zha, Sergeeva Anna, Haley L. Peters, Hiroyuki Katayama, Pariya Sukhumalchandra, Kathryn R. Cox, Alexander A. Perakis, Lisa S. St John, Gheath Alatrash, Elizabeth A. Mittendorf, Karen C. Dwyer, Samir M. Hanash, Jeffrey J. Molldrem. Neuropilin-1 mediates neutrophil elastase uptake and antigen cross-presentation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3984. doi:10.1158/1538-7445.AM2017-3984

Exploiting Natural Cross-reactivity between Human Immunodeficiency Virus (HIV)-1 p17 Protein and Anti-gp41 2F5 Antibody to Induce HIV-1 Neutralizing Responses In Vivo.

Anti-p17 antibodies are able to neutralize human immunodeficiency virus (HIV) entry in a mouse model. In this study, we identified a region of sequence similarity between the epitopes of anti-p17 neutralizing antibodies and anti-gp41 neutralizing 2F5 antibody and verified cross-reactivity between p17 and 2F5 in vitro. The p17 sequence was modified to increase sequence identity between the p17 and 2F5 epitopes, which resulted in enhanced cross-reactivity in vitro. Immunogenicity of wild-type and modified p17 was characterized in a rabbit model. Both wild-type and mutated p17 induced anti-gp41 responses in rabbits; sera from these animals reacted with gp41 from different HIV clades. Moreover, introduction of the 2F5 sequence in p17 resulted in induction of antibodies with partially neutralizing activity. Based upon these data, we suggest that the natural cross-reactivity between HIV-1 p17 protein and 2F5 antibody can be exploited to induce antibodies with neutralizing activity in an animal model.

Anti-GD2 immunotherapy in adults with high-risk neuroblastoma (HR-NB): The Memorial Sloan Kettering Cancer Center (MSKCC) experience.

10550 Background: The diagnosis of NB in adulthood is rare and little is known about its biology and clinical course. There is no established therapy for adult NB. Anti-GD2 immunotherapy is now standard in children with HR-NB but its use has not been reported in adults. Methods: After obtaining IRB waiver, records of all patients with adult-onset (≥18 years) NB seen at MSKCC between 1983 and 2015 were reviewed. Overall survival (OS) was tested by log-rank test. Cox-regression was used for multivariate analysis. Results: The subjects were 42 adults (median: 25; range18-71 years); 23 male and 19 female. Five, 1, 1 and 35 patients had INSS stage 1, 2, 3 and 4 disease, respectively. Genetic abnormalities included somatic ATRX (59%) and ALK mutations (43%) but not MYCN-amplification. 16 patients remain alive at a median follow-up of 5.3 years. OS for non-stage 4 patients was superior to stage 4 (median survival 14.6 vs 5.3 years; p < 0.05). However 5/7 patients with < stage 4 NB progressed to stage 4. Among 35 stage 4 patients, 4 achieved complete remission (CR) after induction chemotherapy and surgery, 11 underwent autologous stem cell transplant (ASCT) and 15 received multiple cycles of anti-GD2 antibodies 3F8 or hu3F8 without complications. In univariate analysis, patients ≤ 29 years old (n = 24) at diagnosis, those achieving CR, and those receiving anti-GD2 antibodies had superior OS (p < 0.05 for each). ASCT was not beneficial (p = 0.3 for ASCT vs no ASCT). For stage 4 patients, anti-GD2 immunotherapy was associated with favorable OS in multivariate analysis (95% CI of anti-GD2 antibody: 1.270 to 7.990). Conclusions: Adult-onset stage 4 NB demonstrates a high incidence of somatic mutations and is only partially chemosensitive. However, 3F8/hu3F8-based anti-GD2 immunotherapy appears to improve long-term survival and is well tolerated.

Differential effects of immunotherapy with antibodies targeting α-synuclein oligomers and fibrils in a transgenic model of synucleinopathy

Disorders with progressive accumulation of α-synuclein (α-syn) are a common cause of dementia and parkinsonism in the aging population. Accumulation and propagation of α-syn play a role in the pathogenesis of these disorders. Previous studies have shown that immunization with antibodies that recognize C-terminus of α-syn reduces the intra-neuronal accumulation of α-syn and related deficits in transgenic models of synucleinopathy. These studies employed antibodies that recognize epitopes within monomeric and aggregated α-syn that were generated through active immunization or administered via passive immunization. However, it is possible that more specific effects might be achieved with antibodies recognizing selective species of the α-syn aggregates. In this respect we recently developed antibodies that differentially recognized various oligomers (Syn-O1, -O2, and -O4) and fibrilar (Syn-F1 and -F2) forms of α-syn. For this purpose wild-type α-syn transgenic (line 61) mice were immunized with these 5 different antibodies and neuropathologically and biochemically analyzed to determine which was most effective at reducing α-syn accumulation and related deficits. We found that Syn-O1, -O4 and -F1 antibodies were most effective at reducing accumulation of α-syn oligomers in multiple brain regions and at preventing neurodegeneration. Together this study supports the notion that selective antibodies against α-syn might be suitable for development new treatments for synucleinopathies such as PD and DLB.

Targeted drug distribution in tumor extracellular fluid of GD2-expressing neuroblastoma patient-derived xenografts using SN-38-loaded nanoparticles conjugated to the monoclonal antibody 3F8

Neuroblastoma is a pediatric solid tumor with high expression of the tumor associated antigen disialoganglioside GD2. Despite initial response to induction therapy, nearly 50% of high-risk neuroblastomas recur because of chemoresistance. Here we encapsulated the topoisomerase-I inhibitor SN-38 in polymeric nanoparticles (NPs) surface-decorated with the anti-GD2 mouse mAb 3F8 at a mean density of seven antibody molecules per NP. The accumulation of drug-loaded NPs targeted with 3F8 versus with control antibody was monitored by microdialysis in patient-derived GD2-expressing neuroblastoma xenografts. We showed that the extent of tumor penetration by SN-38 was significantly higher in mice receiving the targeted nano-drug delivery system when compared to non-targeted system or free drug. This selective penetration of the tumor extracellular fluid translated into a strong anti-tumor effect prolonging survival of mice bearing GD2-high neuroblastomas in vivo.

Dynamics of F1 antibody responses to Yersinia pestis infection in Rhombomys opimus

Objective: To observe the dynamics of antibody response in great gerbils infected with Yersinia pestis in experiment. Method: A total of 211 great gerbils were captured in the southern margin of plague natural focus of Junggar Basin of the Xinjiang Uygur Autonomous Region in 2011. Among them, there were 167 great gerbils without infection of Y. pestis and 44 great gerbils infected by Y.pestis. Y.pestis No. 2504 was employed for this experimental strain, which was strong toxic strain with negativity in the reduction experiment of nitrate. 35 great gerbils without the infection of Y. pestis were divided randomly and averagely into 7 groups including 6 experimental groups and 1 control group. Great gerbils in the 1st to 6th experimental groups were exposed first with 1 × 10(6)-1 × 10(11) CFU/ml of bacterial fluid with 10 times of gradient dilution; groin areas of great gerbils in the control group were injected subcutaneously with physiological saline; and the amount of infection was all 1 ml. 17 great gerbils infected with Y. pestis and the first detection of F1-antibody titer in 1∶256-1∶4 096 were grouped according to F1-antibody titer: group 1∶4 096 (n=4), group 1∶2 048 (n=4), group 1∶1 024 (n=3), group 1∶512 (n=3) and group 1∶256 (n=3); and blood in caudal regions was collected in asepsis for the detection of F1-antibody, with a total of 5 times. 9 great gerbils which were selected from the remaining great gerbils infected with Y. pestis and detected F1-antibody negative 2 times were exposed 1×10(6) CFU/ml of bacterial fluid for the second infection, with the amount of infection being 1 ml. Blood in caudal regions of great gerbils after the first and second infection were collected for the detection of plague F1-antibody on the 3rd, 5th, 7th, 15th, 30th, 60th, 90th and 120th day after infection. Declined regression models for great gerbils' antibodies were established with unary linear regression equation; declined change diagrams for the antibodies were drawn to observe the declined F1-antibody after great gerbils were exposed to Y. pestis. Results: In great gerbils with the first infection of Y. pestis, antibodies were detected in the 1 × 10(6)-1 × 10(8) CFU/ml of group on the 30th, 15th and 15th day, respectively; the positive rates of antibody were 1/4, 3/4 and 4/5, respectively; the group 1×10(7) and 1× 10(8) CFU/ml reached to the highest antibody titer with 1∶256 on the 120th day; antibodies were revealed in the group 1×10(9), 1×10(10) and 1×10(11) CFU/ml from the 5th to 7th day when the seroconversion of all antibodies was observed; group 1×10(11) CFU/ml reached to the highest antibody titer on the 120th day with 1∶4 096. In the great gerbils with the second exposure to Y.pestis, positive antibodies were detected on the 3rd day with the positive rate being 2/9; and the highest antibody titer with 1∶2 048 was noted on the 90th day. Unary linear regression equation of declined F1 antibody of great gerbils was y=0.045x- 0.321 (F=115.40, P< 0.001), and the shortest duration for F1-antibody titer declining from 1∶4 096 to 0 was 140 d and the longest duration 200 d. Conclusion: Great gerbils infected with the high concentration of Y. pestis fluid show shorter duration in producing F1-antibody, the antibody positive rate is also higher, and the highest antibody titer can reach 1∶4 096. The great gerbils could hold the plague F1 antibodies for a long time which was about 140 to 200 days from the highest titer.

Lipophilicity is a key factor to increase the antiviral activity of HIV neutralizing antibodies

The HIV broadly neutralizing antibody 2F5 targets the transiently exposed epitope in the membrane proximal external region (MPER) of HIV-1 gp41, by a two-step mechanism involving the viral membrane and this viral glycoprotein. It was recently shown that 2F5 conjugation with a cholesterol moiety outside of the antibody paratope substantially increases its antiviral activity. Additionally, the antiviral activity of D5, a human antibody that binds to the N-terminal heptad repeat (NHR) of gp41 and lacks membrane binding, was boosted by the same cholesterol conjugation. In this work, we evaluated the membrane affinity of both antibodies towards membranes of different compositions, using surface plasmon resonance. A correlation was found between membrane affinity and antiviral activity against HIV-1. We propose that the conjugation of cholesterol to 2F5 or D5 allows a higher degree of antibody pre-concentration at the viral membrane. This way, the antibodies become more available to bind efficiently to the gp41 epitope, blocking viral fusion faster than the unconjugated antibody. These results set up a relevant strategy to improve the rational design of therapeutic antibodies against HIV.

Analysis of the Phase 1a/b Study of Chimeric Fibril-Reactive Monoclonal Antibody 11-1F4 in Patients with AL Amyloidosis

Background: Mortality in patients with AL Amyloidosis remains high due to progressive organ damage from amyloid deposition. Current therapies eliminate the plasma cell clone that produces amyloidogenic light chains. However, there are no approved therapies that directly target amyloid deposits, a major culprit of progressive multi-organ dysfunction. To address this, a murine (Mu) amyloid fibril-reactive monoclonal antibody (mAb) 11-1F4 was developed that binds to a conformational epitope present on human light-chain amyloid fibrils and initiates cell-mediated phagocytosis. In vivo testing of the Mu mAb and later its chimeric (Ch) form in mice with induced human AL amyloidomas resulted in rapid amyloidolysis without any evidence of toxicity [Hrncic 2000; Solomon 2003]. Subsequent evaluation of an I-124 labeled Mu mAb confirmed that it specifically bound to amyloid-laden organs as evidenced by PET/CT imaging [Wall 2010]. Because of these favorable results, GMP-grade amyloid fibril-reactive Ch IgG1 mAb 11-1F4 was produced by NCI's Biological Resource Branch for a phase 1a/b trial. An analysis of Phase 1a was presented at the American Society of Hematology's 2015 annual meeting. Here we report data from the phase 1a/b trial.Methods: Patients with relapsed or refractory AL Amyloidosis were enrolled in this open-label, dose-escalation phase 1a/b study of Ch IgG1 mAb 11-1F4 (NCT02245867). The primary objective was to determine safety and tolerability of the antibody when given as a single intravenous infusion (phase 1a) or as a series of weekly infusions for 4 weeks (phase 1b). Secondary objectives included pharmacokinetics and efficacy as evidenced by organ response. For both phase 1a and 1b, a dose-escalation "up and down" design was used where sequential doses of 0.5, 5, 10, 50, 100, 250 and 500 mg/m2 were administered to successive patients. Assessment of organ response was based on the International Society of Amyloidosis' revised consensus criteria [Pallidini 2012] and the clinically validated renal staging and response criteria [Pallidini 2014].Results: As of July 15th, 2016, 8 (2 κ and 6 λ) patients completed phase 1a and 11 (4 κ and 7 λ) patients commenced treatment in phase 1b. Median age was 67 years (range: 34 - 77) and median number of organs involved was 2 (range: 1 - 4) with heart and kidney being the most common. All patients received prior anti-plasma cell systemic treatment and achieved at least partial hematologic response. All patients tolerated the given dose of mAb 11-1F4. The maximum tolerated dose (MTD) was 500mg/m2 for phase 1a and 1b. There were no grade 4 or 5 adverse events (AEs) related to the drug. In phase 1a, one patient at dose level 4 developed a grade 2 rash 4 days after infusion. Skin biopsy revealed a so far undiagnosed cutaneous amyloidosis and immunohistochemical staining showed the mAb surrounding amyloid fibrils with an accompanying neutrophilic infiltrate. The same patient and another patient developed a similar rash during treatment in phase 1b suggesting mAb 11-1F4 binding. Although the primary objective of the trial was to evaluate safety, 63% of patients (5 of 8) with measurable disease burden demonstrated organ response after one infusion of mAb 11-1F4 in phase 1a. In phase 1b, 83% of patients (5 of 6 who completed follow up) showed organ response. At the time of presentation, we will report a complete analysis of the phase 1a and 1b clinical trial.Conclusions: Treatment with mAb 11-1F4 is well tolerated and safe without grade 4 or 5 AEs or dose limiting toxicity up to an MTD of 500mg/m2. Clinical efficacy data shows early and sustained organ response when the mAb is administered as a single infusion or as a weekly infusion for 4 weeks. Based on these very encouraging results, a phase 2 SWOG trial for patients with newly diagnosed AL Amyloidosis will be launched. Overall, we posit that amyloid fibril-specific 11-1F4 mAb represents a novel and promising adjunct to the treatment of AL Amyloidosis by safely promoting amyloid resolution and subsequent improvement in organ function. This may result in improved outcomes for patients with this devastating disease. DisclosuresWall:Prothena Inc: Patents & Royalties. Lentzsch:Celgene: Consultancy, Honoraria; BMS: Consultancy.

CD20 Antibodies of Human IgA Isotype Mediate CDC, and ADCC By Myeloid Effector Cells

Introduction: Three unconjugated CD20 antibodies have been approved for the treatment of lymphoma patients. All three are of human IgG1 isotype, but nevertheless they differ in their modes of action: type I antibodies (e.g. rituximab and ofatumumab) trigger effective complement-dependent cytotoxicity (CDC), whereas type II antibodies (e.g. obinutuzumab) are potent inducers of direct cell death, while both type I and II antibodies can induce antibody-dependent cellular cytotoxicity (ADCC). Studies in syngeneic mouse models suggest that myeloid cells are the predominant effector cell type for CD20 antibodies (Uchida et al. J Exp Med 199:1659, 2004). However, human myeloid cells, particularly PMN, are activated more effectively by human IgA than by IgG1 antibodies (Dechant et al. Blood 100:4574, 2002) - especially when the latter are engineered for enhanced FcγRIII affinity (Peipp et al. Blood 112:2390, 2008). Antibodies of IgA isotype constitute an integral part of the mucosal immune system, and differ from IgG antibodies in their pharmacokinetic properties and immune effector mechanisms (Boross et al. EMBO Mol Med 5:1213, 2013, Lohse et al. Cancer Res 76:403, 2016). Here, we compared the efficiency of IgG1 and IgA2 isotype variants of the type I CD20 antibody 1F5 in killing lymphoma cells in vitro and in vivo.Methods: Recombinant antibodies against human CD20 were produced by co-transfecting BHK cells with vectors encoding the 1F5 variable, Igα2 or Igγ1 heavy, and κ light chain constant regions, respectively. The resulting isotype variants were compared for their biochemical characteristics as well as Fab- and Fc- mediated effector functions using human lymphoma cell lines as targets. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice xenotransplanted with human RAJI lymphoma cells were employed to investigate the therapeutic efficacy of human CD20 antibodies. Additionally, in vivo depletion of human CD20 transgenic B cells was evaluated in a syngeneic B cell depletion model using wildtype, human FcαRI transgenic and C3 knock-out mice.Results and Discussion:In vivo studies with xenotransplanted RAJI cells in NSG mice, which lack functional T and NK cells, demonstrated significantly prolonged survival of treated as compared to non-treated mice, indicating that myeloid effector cells may contribute to the therapeutic efficacy of CD20 antibodies against human lymphoma cells. In vitro, neither IgG1 nor IgA2 variants of 1F5 showed efficient Fab-mediated effects such as direct cell death induction and homotypic aggregation compared to type II antibodies. However, human IgA2 but not IgG1 antibody variants against CD20 effectively triggered ADCC by human PMN, the most numerous myeloid effector cell population. Although IgA does not bind C1q, CD20 IgA antibodies also triggered CDC against several lymphoma cell lines. CDC was predominantly mediated by the alternative pathway, as evidenced by the kinetics of lysis, the requirement for higher serum concentrations and inhibition by the C3 inhibitor compstatin. Further in vivo experiments demonstrated that 1F5-IgA2 effectively depleted B cells in a syngeneic human CD20 transgenic B cell depletion model. However, studies in human FcαRI transgenic or C3 knock-out mice indicated that B cell depletion was not mediated by FcαRI or complement - suggesting that other currently undefined mechanisms contribute to the in vivo efficacy of IgA antibodies against CD20.Conclusions: Together, the presented results suggest that CD20 antibodies of human IgA isotype constitute promising immunotherapeutic reagents with unique effector functions. Additional studies are required to further elucidate their effector mechanisms in vitro and in vivo. DisclosuresCragg:Roche: Consultancy, Research Funding; Gilead Sciences: Research Funding; Baxalta: Consultancy; Bioinvent International: Consultancy, Research Funding; GSK: Research Funding.

Antibody-Based Targeted Delivery of Interleukin-22 Promotes Rapid Clinical Recovery in Mice With DSS-Induced Colitis.

We have recently described the potential of the alternatively spliced extradomain A of fibronectin as a target for antibody-based pharmacodelivery applications in ulcerative colitis. Here, we report on the cloning and therapeutic properties of novel antibody-based fusion proteins, comprising the F8 antibody specific to extradomain A and murine interleukin (IL)-22, a globular cytokine belonging to the IL10 family. A protective function for IL22 in colitis has previously been described, as this cytokine induces antimicrobial, proliferative, and antiapoptotic pathways, preventing tissue damage and promoting epithelial repair. Two fusion proteins comprising IL22, fused at the N- or at the C-terminus of the F8 antibody in diabody format, were expressed in mammalian cells. The ability of radiolabeled preparations of the 2 fusion proteins to localize at sites of disease was assessed by autoradiography in a murine model of dextran sodium sulfate-induced colitis and by quantitative biodistribution analysis in a syngeneic mouse teratocarcinoma model. Therapeutic activity was assessed in mice with dextran sodium sulfate-induced colitis, which received intravenous injections of antibody-cytokine fusion proteins. Both fusion proteins were able to selectively accumulate at the site of disease. The fusion protein with the cytokine moiety at the N-terminal extremity (IL22-F8) exhibited better results than the C-terminal fusion, both in terms of targeting selectivity and therapeutic efficacy. Mice treated with IL22-F8 showed a more rapid recovery from clinical symptoms compared with controls and improved macroscopic and microscopic morphology of the colon. IL22-F8 is a promising biopharmaceutical drug candidate for the treatment of ulcerative colitis.

Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles.

The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17–172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17–172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675–683) in the 17–172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17–172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17–172 with a Kd of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17–172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane.

Different tissue distribution properties for glycosylation variants of fusion proteins containing the p40 subunit of murine interleukin-12.

Antibody-based fusion proteins are gaining increasing importance for therapeutic applications, but the impact of glycosylation on in vivo biopharmaceutical performance is not always completely understood. In this article, we have analyzed biochemical and pharmaceutical properties of fusion proteins, consisting of the F8 antibody (specific to the EDA domain of fibronectin, a marker of tissue remodeling and of angiogenesis) and of the p40 subunit of interleukin-12, an inhibitor of inflammation. The corresponding fusion protein (F8-IL12p40), which inhibits colitis development in mice, is a glycosylated protein with suboptimal disease targeting properties in vivo Since the protein was extensively glycosylated, as evidenced by PNGase F treatment and mass spectrometric analysis, we mutated four asparagine residues in various combinations. The corresponding proteins exhibited similar biochemical and antigen-binding properties, but differences in thermal stability and bioactivity. Asparagine mutations did not lead to recovery of disease targeting performance in vivo, as evidenced by quantitative biodistribution studies with radioiodinated protein preparations in tumor-bearing mice. By contrast, an almost complete recovery of targeting was achieved with an enzymatically deglycosylated protein preparation. These findings reinforce the concept that different glycostructures can have an impact on tissue distribution properties.

Abstract IA17: T cell receptor-like antibody 8F4 targets leukemia and non-hematopoietic cancer

Abstract Adaptive anti-tumor immune surveillance of leukemia, called the graft-versus-leukemia (GVL) effect in the setting of allogeneic stem cell transplantation (alloSCT), is responsible for inducing and maintaining long term remission. However, although donor T cells mediate GVL, they also mediate graft-versus-host disease (GVHD), which results in significant morbidity and can be fatal, and is thus a substantial barrier to the more widespread application of alloSCT for many patients with hematological malignancies. CD8 T cells are activated by cognate peptide/MHC-I target cell antigens through binding of clonally unique αβ-heterodimer T cell receptors (TCRs) on the cell surface. Thus, because GVL and GVHD target antigens can be unique, immunotherapy strategies that target GVL antigens such as adoptively transferred T cells could be developed to mediate GVL without GVHD. Likewise, TCR-like monoclonal antibodies (mAbs) that similarly bind to peptide/MHC-I also could be useful as novel immunotherapeutic agents to mediate GVL in the absence of cellular therapies. Moreover, if potent anti-tumor activity was mediated by such TCR-like mAbs, they could have advantages over adoptively transferred donor T cells, including easier standardized manufacturing, ease of dose and scheduled administration, and they could be given to patients in the absence of an alloSCT. We identified PR1, an HLA-A2-restricted 9-mer peptide a peptide derived from aberrantly expressed proteinase 3 (P3) and neutrophil elastase (NE) in myeloid leukemia, as a leukemia-associated target antigen for CD8 T cells that mediate GVL. PR1-specific CD8 T cells target HLA-A2+ AML, CML and MDS but not normal hematopoietic cells and they contribute to cytogenetic and molecular remission of myeloid leukemia. We developed a murine TCR-like mAb that binds to PR1/HLA-A2 (8F4) and showed that 8F4 mediated complement-dependent cytotoxicity (CDC) of AML, CML and MDS progenitor cells and blasts, but not normal bone marrow cells. Because 8F4 did not eliminate normal human hematopoietic cells in a xenograft model, we humanized 8F4 to an IgG1 isotype (h8F4). In AML PDX models, 8F4 and h8F4 eliminated HLA-A2+ AML and leukemia stem cells (LSCs). In preclinical safety models, only mild reversible blood cytopenia was observed after multiple-dose IV administration of high-dose 8F4. Based on these findings, h8F4 is being developed for a first-in-human phase I safety study in HLA-A2+ patients with high-risk AML. In addition, 8F4 also mediated lysis of many non-hematopoietic cancers due to PR1 cross-presentation on surface HLA-A2, which increased cancer cells susceptibility to 8F4-mediated lysis. Therefore, 8F4 also could be tested for the treatment of non-hematopoietic HLA-A2+ cancers such as breast cancer, non-small cell lung cancer, and colon cancer. Citation Format: Jeffrey J. Molldrem, Anna Sergeeva, Hong He, Amanda C. Herrmann, Tian-Hui Yang, Celine Kerros, Haley Peters, Jin Seon Im, Sapna Parshottam, Sijie Lu, Qing Ma, Karen C. Dwyer, Elizabeth Mittendorf, Gheath Alatrash. T cell receptor-like antibody 8F4 targets leukemia and non-hematopoietic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA17.

Abstract 2303: The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients

Abstract Background: Chimeric antigen receptor (CAR)-engineered T cells have demonstrated potent clinical efficacy in patients with B cell lymphoma. However, the application of CAR-T cell therapy targeting other solid tumors has been limited. The GD2 gangliosides are sialic acid-containing glycosphingolipids that play a role in signal transduction and cell-cell recognition. 1st generation gangliosides GD2 chimeric antigen receptor T cells in some patients with refractory neuroblastoma already studied in a phase I clinical trials, indicate that T cells expressing 1st generation anti-GD2 chimeric antigen receptors were safe and mediated modest antitumor activity. In a phase I study, the murine IgG3 monoclonal antibody (MoAb) 3F8, specific for the ganglioside GD2, was administered intravenously to 17 patients with metastatic GD2 positive neuroblastoma or malignant melanoma. Antitumor responses occurred in 7 of 17 patients. However, the expression of the gangliosides GD2 in Asian melanoma populations has not been reported. Methods: This study involved tumor samples from 228 melanoma patients (129 acral melanomas, 65 mucosal melanomas, 13 melanomas on skin with chronic sun-induced damage, 21 melanomas on skin without chronic sun-induced damage) in Peking University Cancer Hospital &amp; Institute. Clinical data were collected. Immunohistochemistry assays using antibodies against gangliosides GD2 were performed on formalin-fixed, paraffin-embedded specimens. The ability of gangliosides GD2 chimeric antigen receptor T cells to kill gangliosides GD2+ melanoma cell was evaluated by MTT in vitro. Results: Among the 228 samples,40.3% of the cases(92/228) demonstrated positive staining with gangliosides GD2. Expression of gangliosides GD2 were relatively more frequent in acral (48.1%) and mucosal (36.9%) melanomas than in CSD (15.4%) and Non-CSD (19.0%) melanomas. The median survival time for patients with expression of gangliosides GD2 was significantly shorter than patients with absence of gangliosides GD2 expression (21.4 vs 57.6 months, P&amp;lt;0.05). Statistical differences were found between primary melanomas with metastatic melanomas (34.9% vs 53.0%, P&amp;lt;0.05). In addition, the gangliosides GD2 chimeric antigen receptor T cells exhibited specific lysis of melanoma cell with gangliosides GD2 expression in vitro experiment. Conclusion: These data demonstrate the expression of ganglioside GD2 are more frequent in acral and mucosal melanomas than in cutaneous melanomas in Chinese patients. The higher expression of gangliosides GD2 on metastatic melanomas compared to primary melanomas was in conformity with the viewpoint that GD2 expression was related to increased metastatic potential. Gangliosides GD2 chimeric antigen receptor T cells represents a clinically appealing treatment strategy for melanoma patients with ganglioside GD2 expression. Citation Format: Yan Kong, Jiayi Yu, Lu Si, Zhihong Chi, Chuanliang Cui, Xinan Sheng, Jun Guo. The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2303.

Human mannose-binding lectin inhibitor prevents Shiga toxin–induced renal injury

Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in STEC HUS. Using novel human MBL2-expressing mice (MBL2 KI) that lack murine Mbls (MBL2(+/+)Mbl1(-/-)Mbl2(-/-)), anovel STEC HUS model consisted of an intraperitoneal injection with Shiga toxin-2 (Stx-2) with or without anti-MBL2 antibody (3F8, intraperitoneal). Stx-2 induced weight loss, anemia, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, butasignificantly decreased glomerular filtration rate compared with control/sham mice. Immunohistochemical staining revealed renal C3d deposition and fibrin deposition inglomeruli in Stx-2-injected mice. Treatment with 3F8 completely inhibited serum MBL2 levels and significantly attenuated Stx-2 induced-renal injury, free serum hemoglobin levels, renal C3d, and fibrin deposition and preserved the glomerular filtration rate. Thus, MBL2 inhibition significantly protected against complement activation and renal injury induced by Stx-2. This novel mouse model can beused to study the role of complement, particularly lectin pathway-mediated complement activation, in Stx-2-induced renal injury.

Oncotargets GD2 and GD3 are highly expressed in sarcomas of children, adolescents, and young adults.

GD2 and GD3 are the tumor-associated glycolipid antigens found in a broad spectrum of human cancers. GD2-specific antibody is currently a standard of care for high-risk neuroblastoma therapy. In this study, the pattern of GD2 and GD3 expression among pediatric/adolescent or young adult tumors was determined, providing companion diagnostics for targeted therapy. Ninety-two specimens of human osteosarcoma (OS), rhabdomyosarcoma (RMS), Ewing family of tumors, desmoplastic small round cell tumor (DSRCT), and melanoma were analyzed for GD2/GD3 expression by immunohistochemistry. Murine monoclonal antibody 3F8 was used for GD2 staining, and R24 for GD3. Staining was scored according to both intensity and percentage of positive tumor cells from 0 to 4. Both gangliosides were highly prevalent in OS and melanoma. Among other tumors, GD3 expression was higher than GD2 expression. Most OS samples demonstrated strong staining for GD2 and GD3, whereas expression for other tumors was highly variable. Mean intensity of GD2 expression was significantly more heterogeneous (P < 0.001) when compared to GD3 across tumor types. When assessing the difference between GD2 and GD3 expression in all tumor types combined, GD3 expression had a significantly higher score (P = 0.049). When analyzed within each cancer, GD3 expression was significantly higher only in DSRCT (P = 0.002). There was no statistical difference in either GD2 or GD3 expression between primary and recurrent sarcomas. GD2/GD3 expression among pediatric solid tumors is common, albeit with variable level of expression. Especially for patients with sarcoma, these gangliosides can be potential targets for antibody-based therapies.

How Tolerance Shapes the Development of the HIV-1 Broadly Neutralizing Antibody 2F5

Abstract An effective HIV-1 vaccine is likely to require induction of broadly neutralizing antibodies (bnAbs), which neutralize multiple HIV-1 strains. Generating HIV-1 bnAbs has been challenging due partly to a number of viral immune evasion mechanisms, including viral molecular mimicry of host in order to exploit immunological tolerance. Indeed, generation of neutralizing antibodies to the 2F5 epitope of HIV-1 gp41 is proscribed by immune tolerance. However, the specific mechanisms by which tolerance relieves the autoreactivity of B cells expressing the VH and VL regions (dKI) or the VH region (sKI) of the 2F5 bnAb are not well understood. To compare the reactivity and repertoire of 2F5 dKI and sKI B cells before and after immune tolerance checkpoints, we used a newly developed in vitro single B cell culture system that induces B cell expansion and differentiation into Ab-secreting plasmacytes. The reactivities of expanded B cell clones were determined by ELISA, while repertoire was determined by recovering the V(D)J rearrangements using RT-PCR and Sanger sequencing. In 2F5 dKI mice, the great majority of cells in the small-pre-B compartment express the knock in heavy- and light-chains, and maintain HIV-1 gp41-reactivity. However, 2F5 dKI B cells that survive tolerance checkpoints to become mature B cells have undergone extensive light-chain editing and are purged of gp41- and KYNU-reactivity. Importantly, the peripheral 2F5 dKI IgM−IgD+ (anergic) B cell subset retains a substantial fraction of HIV-1- and KYNU-reactive cells. In conclusion, our study provides mechanistic insights into how immunological tolerance impairs humoral responses to HIV-1, and suggests activation of anergic B cells as a potential target for HIV-1 vaccination.