Panel Of mAbs Research Articles

Structural and antigenic characterization of a computationally-optimized H5 hemagglutinin influenza vaccine

Influenza A virus is a leading cause of death worldwide. Viruses of the H5 subtype have the potential to induce high mortality, and no vaccines are currently available to protect against H5 influenza viruses in the event of an outbreak. Experimental vaccination with one clade 2 virus does not protect against other subclades. The computationally optimized broadly reactive (COBRA) methodology was previously used to generate a H5 hemagglutinin (HA) antigen (COBRA2) that elicited increased serological breadth against multiple clade 2 H5N1 influenza viruses. In this report, we structurally and antigenically characterized the COBRA2 HA antigen. We examined the biochemical characteristics of the COBRA2 protein and determined the protein is correctly cleaved, properly folded into a trimeric structure, and antigenically correct by probing with HA head- and stem-specific monoclonal antibodies (mAbs). We further probed the antigenicity by examining binding of a panel of H5 mouse mAbs to the COBRA2 antigen, as well as several other HA antigens. We determined the X-ray crystal structure of the COBRA2 HA antigen to 2.8 Å and the protein was observed to be in the expected trimeric form. The COBRA2 HA was structurally similar to the naturally occurring H5 HA antigens and suggests the protein folds similar to known HA structures. Overall, our data allow us to formulate a hypothesis on the mechanism of increased breadth due to vaccination with the COBRA2 HA antigen, which is that the protein incorporates antigenic sites from numerous HA antigens, and elicits mAbs with limited breadth, but with diversity in targeted antigenic sites.

Functional selection of protease inhibitory antibodies

Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli-an antibody clone, a protease of interest, and a β-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified β-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), β-secretase 1 (BACE-1, an aspartic protease in Alzheimer's disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aβ40) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice.

Abstract 1555: Monoclonal antibodies targeting the TAM family of receptor tyrosine kinases

Abstract The TAM receptors (Tyro3/Axl/MerTK) family of receptor tyrosine kinases (RTKs) are important negative regulators of innate immunity. TAM receptor activation in myeloid cells by its ligands Gas6 or Protein S (PROS) promotes phosphatidylserine-dependent efferocytosis of apoptotic cells, inducing a tolerogenic state and mediating resolution of inflammation. TAM-deficient mice exhibit phenotypes consistent with systemic inflammation and autoimmunity. Importantly, individual ablation of TAM receptors can confer tumor immunity, increased pro-inflammatory cytokines and tumor lymphocyte infiltration, leading to the proposal that TAM receptors act as checkpoints of innate immunity. We hypothesize that pharmacological targeting of this family of receptors with monoclonal antibodies (mAbs) may lead to a similar pro-inflammatory response and recapitulate the antitumor effects observed in TAM-deficient mice. From a panel of human anti-MerTK, Axl or Tyro3 mAbs derived from phage-display libraries or human IgG expressing mice we identified unique mAbs that markedly enhanced cytokine and chemokine release from primary human immune cells, alone or in the presence of inflammatory stimuli. Interestingly, the qualitative and quantitative pattern of cytokine response from dendritic cells was very similar using antibodies targeting the individual TAM receptors, and also similar to activation of dendritic cells using a CD40 agonist mAb, suggesting that TAM-targeting mAbs can promote immune activation. We identified surrogate mAbs targeting mouse TAM receptors that elicited similar responses in vivo, and demonstrated antitumor activity when dosed alone, or in combination with PD-1/L1 blockade in syngeneic tumor models. In addition, human MerTK transgenic, and TAM knockout mice have been generated and characterized in order to establish in vivo proof-of-concept with human TAM mAbs. Overall, pharmacological modulation of TAM receptors with mAbs enhances cytokine production in human and murine model systems consistent with the published role of TAMs as negative regulators of innate immunity. Mechanistic and proof-of-concept studies support further development of these mAbs as novel approaches to overcome these checkpoints of the innate immune response. Citation Format: Diego Alvarado, Laura Vitale, Mike Murphy, Thomas O'Neill, Andrew Proffitt, Jay Lillquist, Gwenda Ligon, Komal Patel, Anna Wasiuk, Jeff Weidlick, Jenifer Widger, Laura Mills-Chen, Andrea Crocker, Colleen Patterson, Russell A. Hammond, Li-Zhen He, Joel Goldstein, Lawrence J. Thomas, Henry C. Marsh, Tibor Keler, Richard Gedrich. Monoclonal antibodies targeting the TAM family of receptor tyrosine kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1555.

Abstract 4121: Preclinical characterization of CS1003, a novel clinical-stage PD-1 monoclonal antibody

Abstract Background: The programmed cell death protein 1 receptor (PD-1) is an immune-checkpoint protein that negatively regulates the immune system to avoid collateral damage to self-tissues. Tumors hijack this mechanism as a way to avoid immune detection and destruction. Presently, the FDA has approved three anti-PD-1 antibodies for the treatment in more than ten cancer types. CS1003 is a novel anti-PD-1 mAb recognizing a unique epitope developed to disrupt the PD-1 interaction with PD-L1/PD-L2 to restore or improve T-cell function as stand-alone therapy or in combination with other approaches. Methods: A panel of rat PD-1 mAbs were generated through conventional hybridoma technology. Several mAbs with favorable characteristics were further humanized. CS1003, a humanized, hinge-stabilized IgG4 mAb, was selected as our lead candidate for further characterization including antigen binding, biophysical properties as well as functional characterization. Results: CS1003 binds mouse, cynomolgus monkey and human PD-1 with similar affinity. CS1003 bound PD-1-expressing cell lines and chronically-activated T cells, blocked PD-1 interactions with PD-L1/PD-L2, resulting in inhibition of PD-1 signaling, and enhanced T cell cytokine secretion and proliferation to levels comparable to those observed with nivolumab or pembrolizumab reference molecules. CS1003 showed significantly anti-tumor activity in conventional MC38 as well as in hPD-1 knock-in mice. CS1003 showed no unexpected cross-reactivity in human tissues, with specific staining observed primarily in lymphocytes of lymphoid organs. In a pharmacokinetic (PK) study in cynomolgus monkeys following single intravenous administration at multiple dose levels, PK properties were linear with the proportionally increasing exposures from 2-18 mg/kg. In a repeated-dose toxicity study, CS1003 was well tolerated in cynomolgus monkeys and the major finding was mononuclear infiltration in multiple organs, which was consistent with its pharmacologic activity. CS1003 demonstrated a favorable safety profile with the highest non-severely toxic dose (HNSTD) of 100 mg/kg. Conclusion: CS1003 is a novel anti-PD-1 mAb with favorable preclinical characteristics and safety profile in cynomolgus monkeys. The unique feature of CS1003 is its cross-reactivity with both human and murine PD-1. This should greatly facilitate further evaluation its potential for combination therapy in various syngeneic mouse tumor models. A phase I study for CS1003 is being conducted to evaluate the safety, tolerability, PK and anti-tumor activity in patients with advanced tumors [NCT03475251]. Citation Format: Fu Li, Jingrong Li, Ke Yin, Juan Zhang, Zhenhu Li, Liang Lu, Yuanwu Bao, Dan Pu, Zhen Qin, Yong Zheng, Baotian Yang, Jing Li, Xinzhong Jon Wang. Preclinical characterization of CS1003, a novel clinical-stage PD-1 monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4121.

Single-Cell Analysis Suggests that Ongoing Affinity Maturation Drives the Emergence of Pemphigus Vulgaris Autoimmune Disease

SUMMARYPemphigus vulgaris (PV) is an autoimmune disease characterized by blistering sores on skin and mucosal membranes, caused by autoantibodies primarily targeting the cellular adhesion protein, desmoglein-3 (Dsg3). To better understand how Dsg3-specific autoantibodies develop and cause disease in humans, we performed a cross-sectional study of PV patients before and after treatment to track relevant cellular responses underlying disease pathogenesis, and we provide an in-depth analysis of two patients by generating a panel of mAbs from single Dsg3-specific memory B cells (MBCs). Additionally, we analyzed a paired sample from one patient collected 15-months prior to disease diagnosis. We find that Dsg3-specific MBCs have an activated phenotype and show signs of ongoing affinity maturation and clonal selection. Monoclonal antibodies (mAbs) with pathogenic activity primarily target epitopes in the extracellular domains EC1 and EC2 of Dsg3, though they can also bind to the EC4 domain. Combining antibodies targeting different epitopes synergistically enhances in vitro pathogenicity.

Abstract 3001: Isolation of large and diverse monoclonal antibody panels against difficult targets in oncology

Abstract Although monoclonal antibodies (MAbs) are a well-established treatment approach in oncology, multipass membrane proteins are largely inaccessible as targets for MAb discovery due to their poor expression, membrane-dependent structure, small extracellular regions, and high sequence conservation between humans and rodents. We have developed a platform, MPS Antibody Discovery, that specifically addresses these challenges. We will present case studies of our successful isolation of large and diverse panels of MAbs, including functional (agonist/antagonist) MAbs, against oncology targets, including claudins, GLUT4, CXCR5, CCR7, and other chemokine receptors. Several key features of our MPS platform enabled our ability to isolate MAb panels against these targets. Antigen engineering allowed us to increase the expression levels of the target protein. Immunizing divergent animal species with a combination of DNA and Lipoparticles (high-concentration membrane proteins) allowed us to obtain high-titer immune responses against the native form of the membrane protein. Performing phage display with Lipoparticles allowed us to isolate diverse panels of MAbs with a success rate >95%. Our technology offers the potential for discovering MAbs against difficult targets in cancer, including neoantigens. Citation Format: Joseph Rucker. Isolation of large and diverse monoclonal antibody panels against difficult targets in oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3001.

Development and characterization of novel anti-C5 monoclonal antibodies capable of inhibiting complement in multiple species.

SummaryOver the last decade there has been an explosion in complement therapies; one‐third of the drugs in the clinic or in development target C5 protein. Eculizumab, a monoclonal antibody (mAb) that binds C5 and blocks its cleavage by the convertase, is the current reference standard treatment for atypical haemolytic uraemic syndrome (aHUS) and paroxysmal nocturnal haemoglobinuria (PNH) and in clinical trials for many other diseases. Here we describe a panel of novel anti‐C5 mAb, including mAb that, like Eculizumab, are efficient inhibitors of complement but, unlike Eculizumab, inhibit across species, including human, rat, rabbit and guinea pig. Several inhibitory anti‐C5 mAb were identified and characterized for C5 binding and lytic inhibitory capacity in comparison to current therapeutic anti‐C5 mAb; three clones, 4G2, 7D4 and 10B6, were selected and further characterized for ligand specificity and affinity and cross‐species inhibitory activity. The mAb 10B6 was human‐specific whereas mAb 4G2 and 7D4 efficiently inhibited lysis by human, rabbit and rat serum, and weakly inhibited guinea pig complement; 7D4 also weakly inhibited mouse complement in vitro The rat C5‐cross‐reactive mAb 4G2, when administered intraperitoneally in a rat model of myasthenia gravis, effectively blocked the disease and protected muscle endplates from destruction. To our knowledge this is the first report of an anti‐C5 function blocking mAb that permits preclinical studies in rats.

An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic mice.

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.

1177-P: Discovery of New Therapeutic Antibodies to Two Metabolically Relevant Targets—GPCR CB1 and Transporter GLUT4—Using Virus-Like Particles

Integral membrane proteins are important drug targets, and monoclonal antibodies (mAbs) against them are highly sought for therapeutic and diagnostic purposes. However, the complex structure of multipass membrane proteins makes discovery of these mAbs particularly challenging. By employing strategies such as sequential immunization with DNA (prime) and virus-like particles (boost), and using divergent species (chickens) as immunization hosts, we routinely isolate diverse panels of mAbs against structurally complex and highly conserved membrane protein targets. Here, we report the isolation and characterization of mAbs recognizing two distinct metabolically relevant targets: the 12-transmembrane insulin-responsive glucose transporter GLUT4 and the 7-transmembrane GPCR cannabinoid receptor-1 (CB1). In both cases, we isolated diverse panels of mAbs against native conformational epitopes. Affinities ranged from 2.6 nM (anti-CB1 IM-102) to 1 pM (anti-GLUT4 LM048). Two anti-GLUT4 mAbs discriminated between inward-open and outward-open conformational states, including one mAb (LM048) that inhibited transporter function. Similarly, we isolated several anti-CB1 mAbs that antagonized CB1-mediated calcium flux. Lead mAbs were confirmed to be specific for their target proteins, based on reactivity profiling in the Membrane Proteome Array of >5,600 human membrane proteins. Identified mAbs represent novel and valuable molecular tools for understanding and potentially treating common metabolic diseases, such as type 2 diabetes mellitus and nonalcoholic steatohepatitis/fatty liver disease (NASH/NAFLD). Furthermore, the technologies underpinning their discovery can be rapidly and routinely employed to facilitate discovery of mAbs against other therapeutically relevant membrane protein targets. Disclosure D. Tucker: None. T. Charpentier: None. N. Molino: None. E. Rosenberg: None. B. Screnci: None. L.J. Stafford: None. C. Sulli: None. R. Chambers: None. B.J. Doranz: None. J.B. Rucker: None. Funding National Institutes of Health

Development and characterization of monoclonal antibodies against p30 protein of African swine fever virus

Among the structural proteins that compose the virion of African swine fever virus (ASFV), p30 is one of the most immunogenic proteins and is produced during early stage of ASFV infection. These two characteristics make p30 a good target for diagnostic assays to detect ASFV infection. In this study, we describe a panel of newly generated p30-specific monoclonal antibodies (mAbs). The reactivity of these mAbs was confirmed by immunoprecipitation and Western blot analysis in Vero cells infected with alphavirus replicon particles that express p30 (RP-p30). Furthermore, this panel of mAbs recognized ASFV strains BA71 V (Genotype I) and Georgia/2007 (Genotype II) in immunofluorescence assays on virus-infected Vero cells and swine macrophages, respectively. These mAbs also detected p30 expression by immunohistochemistry in tissue samples from ASFV-infected pigs. Epitope mapping revealed that a selected mAb from the panel recognized a linear epitope within the 32-amino acid region, 61–93. In contrast, two of the mAbs recognize the C-terminal region of the protein, which is highly hydrophilic, enriched in glutamic acid residues, and predicted to contain an intrinsically disordered protein region (IDPR). This panel of mAbs and mAb-based diagnostic assays potentially represent valuable tools for ASFV detection, surveillance and disease control.

Change of phenotypic properties of escape mutants and readaptants of influenza virus A (H1N1)pdm09 under the influence of selected mutations in the molecule of hemagglutinin.

After the emergence and spread of pandemic H1N1 viruses in 2009, antigenic epitopes recognized by neutralizing antibodies against the hemagglutinin of influenza A/Moscow/01/09(H1N1)pdm09 viruses were studied. The purpose of the study was to obtain readapted variants of the virus from a low-virulent escapemutant that has an increased affinity of the avian and the human types cellular receptors compared to the wild type and the comparative study of their antigenic and receptor specificity. Viruses were accumulated in 10-day-old chicken embryos. The MAB panel against HA of influenza virus strain A/IIV-Moscow/01/09(H1N1)sw1 was used in the form of ascites fluids from mice. Immunization of mice, HI testing, elution of viruses from chicken erythrocytes, PCR and sequencing of readapted variants were performed by standard methods. The amino acid substitution A198E acquired in the process of readaptation leads to changes in the antigenic specificity. A correlation was found between a decrease in virulence of a low-virulent escape mutant associated with the substitution D190N in the hemagglutinin molecule and an increase in the hemagglutinating titer to inhibitors in normal mouse serum. Viruses with low affinity of cellular receptor analogs and carrying amino acid substitutions have an increased ability to elute from chicken erythrocytes. The results discuss the effect of mutations in the HA molecule of the influenza A(H1N1) pdm09 virus to the change in antigen specificity; virulence for mice, adsorption-elution at cellular receptors. A comparative study of the antigenic specificity and receptor-binding activity of the escape mutants was conducted for the hemagglutinin of the influenza virus A/Moscow/01/2009 (H1N1)swl, and the readapted variants obtained for one of the escape mutants with reduced virulence for mouse. Monitoring the pleiotropic effect of mutations in the hemagglutinin H1 molecule is necessary to predict variants of the virus with pandemic potential.

Establishment of Systems to Enable Isolation of Porcine Monoclonal Antibodies Broadly Neutralizing the Porcine Reproductive and Respiratory Syndrome Virus.

The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity.

Generation and Characterization of Anti-Filovirus Nucleoprotein Monoclonal Antibodies.

Filoviruses cause lethal hemorrhagic fever in humans. The filovirus nucleoprotein (NP) is expressed in high abundance in infected cells and is essential for virus replication. To generate anti-filovirus monoclonal antibodies (mAbs) against the NP, mice were immunized with peptides known as B-cell epitopes corresponding to different filovirus NPs, and hybridomas were screened using FLAG-tagged filovirus NP constructs. Numerous mAbs were identified, isotyped, and characterized. The anti-NP mAbs demonstrated different ranges of binding affinities to various filovirus NPs. Most of the clones specifically detected both recombinant and wild-type NPs from different filoviruses, including Ebola (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Marburg (MARV), Tai Forest (TAFV), and Reston (RESTV) viruses in western blot analysis. The mAbs were also able to detect native NPs within the cytoplasm of infected cells by immunofluorescence confocal microscopy. Thus, this panel of mAbs represents an important set of tools that may be potentially useful for diagnosing filovirus infection, characterizing virus replication, and detecting NP–host protein interactions.

Abstract A161: Targeting gastrointestinal tumors with constant region engineered anti-glycan antibodies

Abstract Post-translational modifications, for instance protein and lipid glycosylation, are attractive targets for therapeutic antibody (mAb) development. The altered tumor glyco-code drives oncogenic features such as the ability to proliferate, metastasize as well evade immune detection. Through immunizations with colorectal cancer cell membrane extracts we have generated a panel of anti-glycan mAbs with potential for application as cancer therapeutics. These mAbs selectively target Lewis or sialylated Lewis glycans on glycoproteins and/or glycolipids, binding to a large percentage of colorectal, pancreatic and gastric tumor tissues on tumor microarrays and induce a significant tumor volume reduction combined with a survival benefit in metastatic colorectal cancer xenograft models. Underlying these potent antitumor responses is the mAbs’ ability to induce direct tumor cell killing in the absence of complement or immune effector cells. In vitro, this direct cytotoxicity is characterized by mAb-induced cellular aggregation, pore formation, release of alarmins (ATP and high mobility group box 1 protein (HMGB1)), and maturation of immature dendritic cells, thereby constituting a form of inflammatory cell death (ICD). This mode of cell killing is linked to mAb cooperative binding on repeating antigen, a characteristic mostly associated with the murine mIgG3 isotype, thus human hIgG1 formats do not exhibit this form of direcT-cell killing. We have identified the residues required for the cooperative binding behaviour, through mAb constant region (CH2/CH3) screening, and have engineered improved (‘i’) hIgG1 variants, containing these selected residues. In silico immunogenicity prediction (IEDB) suggests limited immunogenicity, which requires further validation. Functional characterization of three improved anti-glycan hIgG1 mAbs in in vitro cell-based and Biacore assays demonstrates significant direct cancer cell killing, pore formation as well as increased functional glycan affinity. Classical immune effector functions (ADCC and CDC) were maintained or improved. Importantly, these improved hIgG1 variants now show significant tumor volume reduction in vivo in a mouse colorectal xenograft model.The multifaceted killing activity of our hIgG1 anti-glycan mAbs, has the potential to synergize with checkpoint blockade, thus holding tremendous therapeutic promise for the treatment of gastrointestinal tumors. Additionally, the activity of other therapeutic mAbs could be further enhanced with our Fc-engineering strategy for introducing cooperative binding. Citation Format: Mireille Vankemmelbeke, Thomas Kirk, Jia X. Chua, Richard McIntosh, Lindy G. Durrant. Targeting gastrointestinal tumors with constant region engineered anti-glycan antibodies [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A161.

Monoclonal antibodies to hemagglutinin of influenza b viruses victoria evolutionary lineage.

Co-circulation of two evolutionary distinct lineages of influenza virus in one epidemic season has led to development specifc reagents for rapid identifcation and typing of new isolates. Panel of MAbs to hemagglutinin of influenza virus B/Brisbane/46/15 belonging to Victoria evolutionary lineage was developed. All MAbs reacted in ELISA with B/Victoria-like strains only. There were no interactions with heterologous influenza viruses of B/Yamagata lineage, seasonal and potentially pandemic influenza A viruses. All MAbs reacted in hemagglutination inhibition and virus neutralization. MAbs interacted in hemagglutination inhibition only with B/Victoria-like viruses, but did not interacted B/Yamagata-like strains. Neutralization and hemagglutination inhibition studies of viruses isolated before 1983 with MAbs revealed that MAbs 6E11, 9G5, 9B5 and 6A4 had the ability to interact with the virus B/ Russia/69 which may evidence that B strains of early isolation period (before lineage separation) have common epitope with recent Victoria lineage viruses. MAbs 7C8, 7G9, 7H8 and 8D11 were directed to a conserved epitope (or epitopes) specifc for influenza hemagglutinin viruses of B/Victoria group. The presence of differences in the effectiveness of the interaction of MAbs 6A9, 7G9 and 8A8 in hemagglutination inhibition test allows the identifcation and differentiation of strains isolated in chicken embryos and MDCK cell culture. Thus, the developed MAbs can be successfully used for identifcation and antigenic analysis of B/Victoria-like strains.

Broad-spectrum monoclonal antibodies against chikungunya virus structural proteins: Promising candidates for antibody-based rapid diagnostic test development.

In response to the aggressive global spread of the mosquito-borne chikungunya virus (CHIKV), an accurate and accessible diagnostic tool is of high importance. CHIKV, an arthritogenic alphavirus, comprises three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. A previous rapid immunochromatographic (IC) test detecting CHIKV E1 protein showed promising performance for detection of the ECSA genotype. Unfortunately, this kit exhibited lower capacity for detection of the Asian genotype, currently in circulation in the Americas, reflecting the low avidity of one of the monoclonal antibodies (mAbs) in this IC kit for the E1 protein of the Asian-genotype because of a variant amino acid sequence. To address this shortcoming, we set out to generate a new panel of broad-spectrum mouse anti-CHIKV mAbs using hybridoma technology. We report here the successful generation of mouse anti-CHIKV mAbs targeting CHIKV E1 and capsid proteins. These mAbs possessed broad reactivity to all three CHIKV genotypes, while most of the mAbs lacked cross-reactivity towards Sindbis, dengue, and Zika viruses. Two of the mAbs also lacked cross-reactivity towards other alphaviruses, including O'nyong-nyong, Ross River, Mayaro, Western Equine Encephalitis, Eastern Equine Encephalitis, and Venezuelan Equine Encephalitis viruses. In addition, another two mAbs cross-reacted weakly only with most closely related O'nyong-nyong virus. Effective diagnosis is one of the keys to disease control but to date, no antibody-based rapid IC platform for CHIKV is commercially available. Thus, the application of the mAbs characterized here in the rapid diagnostic IC kit for CHIKV detection is expected to be of great value for clinical diagnosis and surveillance purposes.

Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.

Cross-reactive dengue virus-derived monoclonal antibodies to Zika virus envelope protein: Panacea or Pandora\u2019s box?

BackgroundDengue Virus (DENV) and Zika Virus (ZIKV) are closely related flaviviruses, circulating in overlapping geographical regions. The recent ZIKV epidemic has been linked to an explosion in reports of microcephaly and neurological defects. It is conceivable that our knowledge of DENV might potentiate the development of a ZIKV vaccine due to the close phylogenetic relationship between these flaviviruses and cross-reactive antibodies, principally to the envelope protein (E protein). Alternatively, cross-reactive antibodies that are generated following vaccination or infection, might become damaging during subsequent infections.Main bodyThe aims of this review are to collate and analyse data from a recent series of DENV-derived monoclonal antibody (mAb) panels from different research groups. These panels measured DENV-mAb activity against ZIKV in terms of antibody-dependent enhancement (ADE) and neutralisation. Methodology used across groups was compared and critiqued. Furthermore, the specific antibody targets on E protein were considered and their therapeutic potential evaluated. Shortcomings of hmAb panels suggest ADE may be over-estimated and neutralisation underestimated, as compared to clinical situations. It remains unknown whether preference of enhancement or neutralisation by antibodies to ZIKV E protein is dictated by quantitative aspects of antibody titre or epitope specific variation. Additionally, little is known about how duration between flavivirus reinfections affect secondary antibody response.ConclusionThis review concludes that our current knowledge of cross-reactive antibodies to E protein is inadequate to anticipate the outcome of deploying an E protein based vaccine to regions co-infected by DENV and ZIKV.

Multifaceted characterization of recombinant protein-based vaccines: An immunochemical toolbox for epitope-specific analyses of the hepatitis E vaccine

The integrity of functional epitopes is a critical quality attribute for recombinant protein based vaccines since the presence of these native-like epitopes is the structural basis for vaccines to elicit functional antibodies. To demonstrate the quality and quantity of functional epitopes on vaccine antigens, a toolbox of assessing antigen characteristics is essential. Among the physicochemical, biophysical, immunochemical and in vivo potency analyses, the epitope-specific assays are most critical assessment of the antigen functionality. In this study, we used hepatitis E virus vaccine as an example to illustrated how the monoclonal antibody (mAb) based immunochemical assays were established for in-depth and multifaceted antigen characterization. A large panel of mAbs were developed and characterized using epitope clustering analysis. A subset of these mAbs recognizing non-overlapping epitopes were chosen to be used for assay development. Orthogonal methods, including surface plasma resonance-based BIAcore, solution competitive ELISA and sandwich ELISA, were developed for the antigenicity assessment. The sandwich ELISA with a pair of mAbs, recognizing two different epitopes, was used to assess the accelerated antigen stability, showing enhanced stability with adjuvant adsorption. Such a sandwich ELISA with robust performance has the potentials to be used for in vitro potency analysis to replace animal-based potency assay as product release test. In summary, using hepatitis E vaccine as an example, we demonstrated the importance and establishment of a mAb-based immunochemical toolbox for multifaceted antigen characterization. This is particularly important to demonstrate the successful reconstruction of the native-like and functional epitopes on a recombinant antigen post expression and purification. These epitope-specific and multifaceted assays serve as critical tools for process monitoring or lot consistency tests in support of vaccine development and manufacturing.

Epitope clustering analysis for vaccine-induced human antibodies in relationship to a panel of murine monoclonal antibodies against HPV16 viral capsid

Human papillomavirus (HPV) type 16 is the most common type implicated as the etiological agent that causes cervical cancer. The marketed prophylactic vaccines against HPV infection are composed of virus-like particles (VLPs) assembled from the recombinant major capsid protein L1. Elicitation of functional and neutralizing antibodies by vaccination is the mode of action by which the vaccines prevent the viral infection. In this study, a panel of murine mAbs against HPV16 L1 were generated and comprehensively characterized with respect to their mapping to the epitope spectrum on the viral capsid. These mAbs were categorized into five epitope bins by two different methods based on the pairwise cross-inhibition and competition with human polyclonal antibodies. In addition, a preliminary demonstration of the spatial relationship of the epitopes recognized by these mAbs was performed using a cross-blocking assay with a well-characterized human mAb, 26D1. Interestingly, two mAbs recognizing different epitopes were found to act synergistically in the pseudovirion-based neutralization assay (PBNA). To facilitate cross-lab and cross-study comparison, the international standard (IS) serum 05/134 was used to calibrate the mAbs as well as the human serum samples from the HPV16/18 vaccine recipients. The neutralizing mAbs, particularly those that recognizing immunodominant epitopes, would be useful in developing epitope-specific assays for monitoring the vaccine production process and for serological assessment.