Panel Of mAbs Research Articles

Development of new immune reagents for swine health, vaccine and disease studies

Abstract Current research efforts require a broad range of immune reagents; those available for pigs are limited. The goal of the US-UK Collaborative Swine Immune Toolkit Initiative is to generate priority immune reagents, based on international input, and pipeline them for marketing. UK researchers focus on mucosal targets, including production of monoclonal antibodies (mAbs) to chemokine receptors and IgE. Plans for mAbs to each 7-transmembrane G-protein coupled receptor (CCR3, CCR9, CCR10, CXCR1, CXCR6, F4/80) are to target peptides from extracellular loop 2 (ECL-2) based on threaded protein structures. These peptides have been designed for CCR3 and CCR9 and mAb plans are underway. US efforts are aimed at expression of soluble proteins and swine CD molecules, and production of panels of mAbs. The team has set up collaborations with commercial partners for protein expression and mAb production, and updated protocols to evaluate reagent specificity. New panels of mAbs reactive with IL-6, IFNβ and IFNγ are currently being evaluated. Immunizations are underway to produce panels of mAbs for CXCL10, CX3CL1 (fractalkine), IL-13, IL-17A, IL-17F, IFNω1 and IFNω5. Plans for mAbs to porcine CD1d using 4 synthetic peptides are in progress using CD1d knockout (KO) mice and screening on CD1d KO pig cells. Plans continue to use reagents produced to develop new assays for swine immune markers, e.g., multiplex assays and intracellular staining to provide the veterinary community with new commercial reagents and techniques for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine immune, disease and biomedical research efforts. Supported by USDA ARS, NIFA grant #2015-67015-23216 and BBSRC grant BB/M028232/1.

Antigenic diversity of H5 highly pathogenic avian influenza viruses of clade 2.3.4.4 isolated in Asia.

H5 highly pathogenic avian influenza viruses (HPAIV) have spread in both poultry and wild birds since late 2003. Continued circulation of HPAIV in poultry in several regions of the world has led to antigenic drift. In the present study, we analyzed the antigenic properties of H5 HPAIV isolated in Asia using four neutralizing mAbs recognizing hemagglutinin, which were established using A/chicken/Kumamoto/1-7/2014 (H5N8), belonging to clade 2.3.4.4 and also using polyclonal antibodies. Viruses of clades 1.1, 2.3.2.1, 2.3.4, and 2.3.4.4 had different reactivity patterns to the panel of mAbs, thereby indicating that the antigenicity of the viruses of clade 2.3.4.4 were similar but differed from the other clades. In particular, the antigenicity of the viruses of clade 2.3.4.4 differed from those of the viruses of clades 2.3.4 and 2.3.2.1, which suggests that the recent H5 HPAIV have further evolved antigenically divergent. In addition, reactivity of antiserum suggests that the antigenicity of viruses of clade 2.3.4.4 differed slightly among groups A, B, and C. Vaccines are still used in poultry in endemic countries, so the antigenicity of H5 HPAIV should be monitored continually to facilitate control of avian influenza. The panel of mAbs established in the present study will be useful for detecting antigenic drift in the H5 viruses that emerge from the current strains.

Cross-reactive dengue human monoclonal antibody prevents severe pathologies and death from Zika virus infections.

Zika virus (ZIKV) infections have been linked with neurological complications and congenital Zika syndrome. Given the high level of homology between ZIKV and the related flavivirus dengue virus (DENV), we investigated the level of cross-reactivity with ZIKV using a panel of DENV human mAbs. A majority of the mAbs showed binding to ZIKV virions, with several exhibiting neutralizing capacities against ZIKV in vitro. Three of the best ZIKV-neutralizing mAbs were found to recognize diverse epitopes on the envelope (E) glycoprotein: the highly conserved fusion-loop peptide, a conformation-specific epitope on the E monomer, and a quaternary epitope on the virion surface. The most potent ZIKV-neutralizing mAb (SIgN-3C) was assessed in 2 type I interferon receptor–deficient (IFNAR–/–) mouse models of ZIKV infection. Treatment of adult nonpregnant mice with SIgN-3C rescued mice from virus-induced weight loss and mortality. The SIgN-3C variant with Leu-to-Ala mutations in the Fc region (SIgN-3C-LALA) did not induce antibody-dependent enhancement (ADE) in vitro but provided similar levels of protection in vivo. In pregnant ZIKV-infected IFNAR–/– mice, treatment with SIgN-3C or SIgN-3C-LALA significantly reduced viral load in the fetal organs and placenta and abrogated virus-induced fetal growth retardation. Therefore, SIgN-3C-LALA holds promise as a ZIKV prophylactic and therapeutic agent.

Monoclonal Antibodies Specific for the V2, V3, CD4-Binding Site, and gp41 of HIV-1 Mediate Phagocytosis in a Dose-Dependent Manner.

In light of the weak or absent neutralizing activity mediated by anti-V2 monoclonal antibodies (MAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP), which is an important element of anti-HIV-1 immunity. We tested six anti-V2 MAbs and compared them with 21 MAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from chronically HIV-1-infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664, or BG505 DS-SOSIP.664 complexed with MAbs. The measurement of ADCP activity by the area under the curve showed significantly higher activity of anti-gp41 MAbs than of the members of the three other groups of MAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 MAbs were dominant compared to anti-V3 and anti-CD4bs MAbs against clade C gp120ZM109 ADCP activity mediated by V2 and V3 MAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting that a closed envelope conformation better exposes the variable loops. Two IgG3 MAbs against the V2 and V3 regions displayed dominant ADCP activity compared to a panel of IgG1 MAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 MAbs were compared to their IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers, with some higher activity of anti-V2 MAbs than of anti-V3 and anti-CD4bs MAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy.IMPORTANCE Anti-V2 antibodies (Abs) correlated with reduced risk of HIV-1 infection in recipients of the RV144 vaccine, suggesting that they play a protective role, but a mechanism providing such protection remains to be determined. The rare and weak neutralizing activities of anti-V2 MAbs prompted us to study Fc-mediated activities. We compared anti-V2 MAbs with other MAbs specific for V3, CD4bs, and gp41 for Ab-dependent cellular phagocytosis (ADCP) activity, implicated in protective immunity. The anti-V2 MAbs displayed stronger activity than other anti-gp120 MAbs in screening against one of two gp120s and against DS-SOSIP, which mimics the native trimer. The activity of anti-gp41 MAbs was superior in targeting monomeric gp41 but was comparable to that seen against trimers, which may not adequately expose gp41 epitopes. While anti-envelope MAbs in general mediated ADCP activity, anti-V2 MAbs displayed some dominance compared to other MAbs. Our demonstration that anti-V2 MAbs mediate ADCP activity suggests a functional mechanism for their contribution to protective efficacy.

Monoclonal antibodies that bind to the Ly6 domain of GPIHBP1 abolish the binding of LPL

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1's Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1's Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease.

The Analysis of CD83 Expression on Human Immune Cells Identifies a Unique CD83+-Activated T Cell Population.

CD83 is a member of the Ig gene superfamily, first identified in activated lymphocytes. Since then, CD83 has become an important marker for defining activated human dendritic cells (DC). Several potential CD83 mRNA isoforms have been described, including a soluble form detected in human serum, which may have an immunosuppressive function. To further understand the biology of CD83, we examined its expression in different human immune cell types before and after activation using a panel of mouse and human anti-human CD83 mAb. The mouse anti-human CD83 mAbs, HB15a and HB15e, and the human anti-human CD83 mAb, 3C12C, were selected to examine cytoplasmic and surface CD83 expression, based on their different binding characteristics. Glycosylation of CD83, the CD83 mRNA isoforms, and soluble CD83 released differed among blood DC, monocytes, and monocyte-derived DC, and other immune cell types. A small T cell population expressing surface CD83 was identified upon T cell stimulation and during allogeneic MLR. This subpopulation appeared specifically during viral Ag challenge. We did not observe human CD83 on unstimulated human natural regulatory T cells (Treg), in contrast to reports describing expression of CD83 on mouse Treg. CD83 expression was increased on CD4+, CD8+ T, and Treg cells in association with clinical acute graft-versus-host disease in allogeneic hematopoietic cell transplant recipients. The differential expression and function of CD83 on human immune cells reveal potential new roles for this molecule as a target of therapeutic manipulation in transplantation, inflammation, and autoimmune diseases.

Anti-CD27 Enhances Lymphoma Immunotherapy through Profound Myeloid Cell Recruitment

Direct-targeting monoclonal antibodies (mAb) such as anti-CD20 mAb are thought to elicit their anti-tumor function through antibody-dependent cellular phagocytosis (ADCP) mediated by myeloid cells (monocytes and macrophages), with little involvement of T cells. In contrast, immunomodulatory mAbs to TNFR superfamily members, CD27, OX40 and CD137, function by augmenting T-cell responses.We examined the therapeutic potential of combining anti-CD20 mAb with a panel of immunomodulatory mAbs (OX40, CD137, CD27, TIGIT, GITR, CTLA4, PD-1). In the syngeneic BCL1 B-cell lymphoma mouse model only an agonistic mAb to CD27, provided a synergistic effect when combined with anti-CD20. Anti-CD20 and anti-CD27 mAb individually provided modest therapeutic benefit (median survival 33 days and 62 days, respectively), but mice treated with the combination survived beyond 100 days. Similar synergistic survival benefit was observed in another B-cell lymphoma model, A31, and in BCL1-bearing human CD27 transgenic mice, when anti-CD20 was combined with varlilumab, an anti-human CD27 mAb currently under clinical investigation.We observed that in mice treated with anti-CD27, there was an early and substantial increase in intra-tumoral monocyte, neutrophil and macrophage infiltration. CD27 is expressed constitutively on T and NK cells but not myeloid cells or the tumor itself. To investigate whether CD27 agonism promotes intra-tumoral myeloid cell infiltration through T cells, we depleted T cells in the BCL1model. Surprisingly, CD4 or CD8 T-cell depletion had no effect on the survival of anti-CD20 and anti-CD27-treated mice, suggesting that the remaining CD27+ immune effector cells, NK cells, are required. To further probe the relative importance of these two sub-sets, we performed experiments in γ chain knockout mice, where activatory FcγR are not expressed. Here, anti-CD27 mediated T-cell activation can still occur via crosslinking from the inhibitory FcγRII, but effector function through NK cells, mediated through activatory FcγR, is abrogated. In this model, the therapeutic benefit of anti-CD27 was completely abolished, thereby supporting the requirement for NK cells.We hypothesize that anti-CD27 stimulates CD27+ NK cells to release chemokines that draw myeloid cells into the tumor, where they subsequently perform augmented anti-CD20 mediated ADCP.These data demonstrate the clear therapeutic potential of combining direct targeting and immunomodulatory mAb but that the therapeutic mechanism of action may differ to that expected; here involving a previously unheralded effect of anti-CD27 on myeloid infiltration. Based upon these data, we have implemented a phase II clinical trial examining rituximab and varlilumab in B-cell lymphoma, which will commence recruitment shortly. DisclosuresKeler:Celldex Therapeutics: Employment, Equity Ownership. Johnson:Celldex Therapeutics: Research Funding. Al-Shamkhani:Celldex Therapeutics: Patents & Royalties: On therapeutic use of antibodies targeting anti-CD27 and another applied for anti-CD20/anti-CD27 use, Research Funding. Glennie:Celldex Therapeutics: Patents & Royalties: Patent on therapeutics use of antibodies targeting human CD27 and patent for anti-CD20+anti-CD27 applied. Cragg:Baxalta: Consultancy; Gilead Sciences: Research Funding; GSK: Research Funding; Roche: Consultancy, Research Funding; Bioinvent International: Consultancy, Research Funding. Lim:Celldex Therapeutics: Patents & Royalties: Patent for anti-CD20+anti-CD27 applied, Research Funding.

Non-Invasive Genetic Profiling Is Highly Applicable in Multiple Myeloma (MM) through Characterization of Circulating Tumor Cells (CTCs)

Chromosomal abnormalities (CA) play a pivotal role in predicting MM patients risk. Accordingly, well-defined CA have been associated with higher risk of transformation in smoldering MM (SMM), have been recently incorporated to the revised ISS, and may even guide treatment decisions; furthermore, because high-risk CA can be present in minor subclones at diagnosis it is recommended to reassess them at the time of relapse. Altogether, this implies that throughout their clinical course MM patients go through several bone marrow (BM) aspirates for cytogenetic risk assessment, and less invasive methods to screen for CA [ie. peripheral blood (PB) liquid biopsies] would be mostly welcomed providing they are fully comparable to its assessment in BM clonal plasma cells (PCs).Here, using the recently developed and sensitive next generation flow (NGF) method to monitor MRD in MM (median limit of detection, 3x10-6), we assessed the applicability of screening for CA in the PB, by determining the frequency of MGUS and MM patients with detectable CTCs and their respective absolute numbers. Using NGF, we detected CTCs in 22/37 (60%) MGUS cases, 9/12 (75%) SMM, 24/25 (96%) newly diagnosed MM, and 22/26 (85%) relapsed MM. Median numbers of CTCs/µL in the PB of MGUS, SMM, newly diagnosed and relapsed MM were 0.04, 0.1, 0.5 and 0.6, respectively. Thus, we noted highly significant differences (P<.001) between benign and malignant disease stages, with a 12.5-fold increment in CTCs/µL between MGUS and MM, and 5-fold between SMM and MM.By using whole exome sequencing, we reported recently that the concordance in CA between CTCs and BM clonal PCs was typically <50% due to the limited amount of DNA extracted from low numbers of CTCs, which required whole genome amplification. Accordingly, we investigated in 18 patient paired BM and PB (~5mL) samples (2 MGUS, 2 SMM and 14 MM) whether after NGF sorting and using the Affymetrix CytoScan HD (which requires significantly less DNA to provide molecular karyotypes), we could determine the pattern of CA in BM clonal PCs by screening that of CTCs. In 10 patients in which CTCs were successfully characterized, we obtained 100% concordance between BM clonal PCs and CTCs at the chromosome level (including trisomies of chromosomes 3, 5 and 21 which have been described to modulate the prognosis of patients with high-risk MM), for chromosomal arms, and for interstitial gains or losses (Panel A). Importantly, there was also 100% concordance with FISH performed on CD138+ BM clonal PCs for del(1p), +1q, del(13q) and del(17p) CA. In the remaining cases, ultra-low numbers of sorted CTCs (≤5.000) precluded their successful characterization, which could be readily overcome by using larger volumes of PB (ie. >5mL). Because the CytoScan HD does not assess the t(4;14), we included FGFR3 into the NGF panel of mAb in order to detect its surface expression in patients harboring the t(4;14). Similarly to that described above, we observed 100% concordance between FGFR3 expression in CTCs and the presence of t(4;14) in CD138+ BM clonal PCs, as confirmed by FISH (Panel B).Since gene expression profiling (GEP) has also been used to predict risk in MM, we investigated in 12 patient paired BM and PB samples whether after NGF sorting and using the Affymetrix Human Gene 2.0 ST Array, we could determine patients risk by screening the GEP of CTCs. Unsupervised analysis showed correct clustering between GEP of BM clonal PCs and CTCs in 9/12 cases, demonstrating that individual patient signatures typically outweighed GEP of MM clones located in different tissues (Panel C). Thus, CTCs could be used to predict risk according to well-established GEP signatures.In conclusion, by using sensitive NGF we showed that CTCs are present in 60% of MGUS and 87% of MM, which unravels high feasibility for liquid biopsying these patients. By also using NGF to sort CTCs, as well as commercially available, cost-effective and standardized arrays to determine CA and gene expression profiles, we demonstrated for the first time that the genetic features of BM clonal PCs can be determined with great accuracy by studying CTCs. Altogether, this combined approach could offer to many MM patients a less invasive cytogenetic characterization at certain stages of their clinical course. [Display omitted] DisclosuresPaiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Epitope-dependent Functional Effects of Celiac Disease Autoantibodies on Transglutaminase 2

Transglutaminase 2 (TG2) is a Ca2+-dependent cross-linking enzyme involved in the pathogenesis of CD. We have previously characterized a panel of anti-TG2 mAbs generated from gut plasma cells of celiac patients and identified four epitopes (epitopes 1–4) located in the N-terminal part of TG2. Binding of the mAbs induced allosteric changes in TG2. Thus, we aimed to determine whether these mAbs could influence enzymatic activity through modulation of TG2 susceptibility to oxidative inactivation and Ca2+ affinity. All tested epitope 1 mAbs, as well as 679-14-D04, which recognizes a previously uncharacterized epitope, prevented oxidative inactivation and increased Ca2+ sensitivity of TG2. We have identified crucial residues for binding of 679-14-D04 located within a Ca2+ binding site. Epitope 1 mAbs and 679-14-D04, although recognizing separate epitopes, behaved similarly when assessing their effect on TG2 conformation, suggesting that the shared effects on TG2 function can be explained by induction of the same conformational changes. None of the mAbs targeting other epitopes showed these effects, but epitope 2 mAbs reduced the rate of TG2-catalyzed reactions. Collectively, these effects could be relevant to the pathogenesis of CD. In A20 B cells transduced with TG2-specific B-cell receptor, epitope 2-expressing cells had poorer uptake of TG2-gluten complexes and were less efficient in gluten epitope presentation to T cells than cells expressing an epitope 1 receptor. Thus, the ability of epitope 1-targeting B cells to keep TG2 active and protected from oxidation might explain why generation of epitope 1-targeting plasma cells seems to be favored in celiac patients.

Systematic evaluation of monoclonal antibodies and immunoassays for the detection of Interferon-γ and Interleukin-2 in old and new world non-human primates

Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species.A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-γ and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-γ and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset). Pan-reactive mAbs, recognizing cytokines from all NHP species, were further analyzed in capture assays and flow cytometry with NHP peripheral blood mononuclear cells (PBMC).Pan-reactive mAb pairs for IFN-γ well as IL-2 were identified and used in ELISA to measure IFN-γ and IL-2, respectively, in Old and New World NHP PBMC supernatants. The same mAb pairs displayed high functionality in ELISpot and FluoroSpot for the measurement of antigen-specific IFN-γ and IL-2 responses using cynomolgus PBMC. Functionality of pan-reactive mAbs in flow cytometry was also verified with cynomolgus PBMC.The development of well-defined immunoassays functional with a panel of NHP species facilitates improved analyses of cellular immunity and enables inclusion in multiplex cytokine assays intended for a variety of NHP.

Identification of an IgG CDR sequence contributing to co-purification of the host cell protease cathepsin D.

Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb-1. The current work was focused on identification of a primary sequence in mAb-1 responsible for the binding and consequent co-purification of trace levels of CHO cathepsin D. Surface plasmon resonance (SPR) was used to detect binding between immobilized CHO cathepsin D and a panel of mAbs. Out of 13 mAbs tested, only mAb-1 and mAb-6 bound to cathepsin D. An LYY motif in the HC CDR2 was common, yet unique, to only these two mAbs. Mutation of LYY to AAA eliminated binding of mAb-1 to cathepsin D providing confirmation that this sequence motif was involved in the binding to CHO cathepsin D. Interestingly, the binding between mAb-1 and cathepsin D was weaker than that of mAb-6, which may be related to the fact that two aspartic acid residues near the LYY motif in mAb-1 are replaced with neutral serine residues in mAb-6. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:140-145, 2017.

Autoantibodies to protein S in patients with recurrent pregnancy loss recognize EGF-like domains

Pseudomonas aeruginosa is a serious human pathogen in a variety of patient groups including those with burns, hospitalized in intensive care, cystic fibrosis and neutropenia. Since there is no vaccine available, passive antibody prophylaxis against protective epitopes is an alternative strategy to prevent P. aeruginosa infection. However, immunoglobulin derived from multiple donors has variable anti-pseudomonas antibody titers, and human Mab are difficult to make from patient samples. We previously reported the use of XenoMouse® mice, Ig-inactivated transgenic mice reconstituted with human immunoglobulin loci, to generate human Mab against a single serotype of P. aeruginosa lipopolysaccharide O-specific side chain (PS). We now report the creation of a panel of anti-PS human IgG2 Mab against nine additional O-specific side chain P. aeruginosa serotypes. The majority of the Mab were highly opsonic for uptake and killing of homologous P. aeruginosa by human PMN in the presence of human complement, and all the Mab protected cyclophosphamide-induced neutropenic mice from fatal P. aeruginosa sepsis with homologous serotypes. DNA sequence analysis showed that the Mab used VH3, VH4, VH5 and VH6 and Vκ2, 3 and 4 variable region genes consistent with the heterogeneity of P. aeruginosa LPS O-side chain structure. We conclude that human Mab made in these transgenic mice against common pathogenic serotypes of P. aeruginosa are opsonic and highly protective, and that a high titer, multi-valent human Mab preparation against the majority of circulating O-side chain serotypes of P. aeruginosa could be used as prophylaxis against invasive infections in selected patient groups.

Abstract B092: Therapeutic targeting of tumorigenic EphA2+/EphA3+ brain tumor initiating cells with bi-specific antibody in glioblastoma

Abstract Glioblastoma (GBM), the most aggressive primary human brain tumor, carrier a dismal prognosis and is increasingly characterized by cellular and genetic intra-tumoral heterogeneity (ITH). Many of the 14 members of the erythropoietin-producing hepatocellular carcinoma receptor (EphR) family and their ephrin ligands are expressed in GBM cells and constitute potential molecular targets for novel therapeutic agents. We hypothesize that multiple members of the EphR family play a critical role in orchestrating the clonal evolution of GBM progression. Individual Eph receptor targeting strategies have shown only modest pre-clinical success, likely because single agent therapy cannot target the degree of ITH in GBM. Using a highly specific human Eph receptor monoclonal antibody (mAb) panel (EphR profiler), we identified five Eph receptors with dysregulated expression in recurrent GBM as compared to primary GBM. With our unique chemoradiotherapy-adapted, patient-derived xenograft model of GBM, we identified EphA2 and EphA3 expression to be upregulated after therapy. Here we show that EphA2 and EphA3 co-expression marks a highly tumorigenic cell population in recurrent GBM with higher in vitro and in vivo self-renewal and proliferation capacity as compared to EphA2+/EphA3-, EphA2-/EphA3+ or EphA2-/EphA3- cells. Lentiviral mediated knockdown of EphA2 and EphA3 blocks this self-renewal and proliferation capacity in recurrent GBM. Through further characterization using mass cytometry (CyTOF) assay, we find that EphA2 and EphA3 is co-expressed with multiple brain tumor initiating cell (BTIC) markers (CD133, CD15, Bmi1, Sox2, Integrin α6 and FoxG1). Considering the important role of EphA2+/EphA3+ cells in GBM tumorigenesis and recurrence, we generated a bi-specific antibody (bsIgG) that co-targets EphA2 and EphA3. In vitro treatment of GBM with bsEphA2/A3 IgG led to pharmacological blockade of phosphorylated EphA2. We then assessed the in vivo efficacy of the bsEphA2/A3 IgG to block GBM tumor growth in our PDX model, and found that treatment with intracranial bsIgG resulted in non-invasive and significantly smaller lesions. The striking reduction in tumor burden in recurrent GBM after co-targeting of EphA2 and EphA3 validates the premise of our therapeutic strategy of targeting multiple EphRs. Discovering the clonal composition of recurrent GBM will enable us to target cellular subpopulations, and this ITH, with selective compounds that inhibit BTIC and Eph receptor activity with minimal off-target effects. Comprehensive Eph receptor profiling of individual patient-derived GBM will allow us to develop a therapeutic strategy for each patient's tumor, employing polytherapy with mAbs against Eph receptors expressed at recurrence. Citation Format: Parvez Vora, Maleeha Qazi, Chirayu Chokshi, Chitra Venugopal, Max London, Amy Hu, Nicole McFarlane, Minomi Subapanditha, Mohini Singh, Sujeivan Mahendram, Jarrett Adams, Jason Moffat, Sachdev Sidhu, Sheila Singh. Therapeutic targeting of tumorigenic EphA2+/EphA3+ brain tumor initiating cells with bi-specific antibody in glioblastoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B092.

Biological function analysis of monoclonal antibodies against human granulins in vitro using U251 cells as a model

Progranulin (PGRN), a highly glycosylated, secreted 593 amino acid precursor protein, is a multifunctional molecule that is critical for early embryogenesis, wound repair, inflammatory and tumorigenesis. PGRN can be proteolytically cleaved into seven cysteine-rich granulin (Grn) peptides: G, F, B, A, C, D and E. Both PGRN and its constituent Grn peptides have been implicated in a wide variety of biological activities. However, their functions are far from clear, and the lack of granulin domain-specific antibodies has hindered the progress of the functional study of PGRN and Grns. Monoclonal antibodies against GrnB, GrnA, GrnC and GrnF have been previously developed by our laboratory. In this study, we generated monoclonal antibodies (MAbs) against GrnD, GrnG and GrnE by using recombinant proteins HSA-GrnG, HSA-GrnD and HSA-GrnE as immunogens, and characterized them by indirect ELISA, Western blot and immunocytochemistry. Furthermore, the neutralizing activities of the MAbs against seven Grns were tested in vitro using the U251 cell line. This full antibody panel of MAbs against seven Grns will be a valuable tool for elucidating the biological roles of PGRN and Grns in different physiopathological processes, which will further promote the development of PGRN-based clinical diagnosis and therapy.

Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy linked to a deficiency in the metalloprotease ADAMTS13. In the current study, a novel mouse model for acquired TTP was generated to facilitate development and validation of new therapies for this disease. Therefore, a large panel (n = 19) of novel anti-mouse ADAMTS13 (mADAMTS13) monoclonal antibodies (mAbs) of mouse origin was generated. Inhibitory anti-mADAMTS13 mAbs were identified using the FRETS-VWF73 assay. Four mAbs strongly inhibited mADAMTS13 activity in vitro (∼68–90% inhibition). Injecting a combination of 2 inhibitory mAbs (13B4 and 14H7, 1.25 mg/kg each) in Adamts13+/+ mice resulted in full inhibition of plasma ADAMTS13 activity (96 ± 4% inhibition, day 1 post injection), leading to the appearance of ultra-large von Willebrand factor (UL-VWF) multimers. Interestingly, the inhibitory anti-mADAMTS13 mAbs 13B4 and 14H7 were ideally suited to induce long-term ADAMTS13 deficiency in Adamts13+/+ mice. A single bolus injection resulted in full ex vivo inhibition for more than 7 days. As expected, the mice with the acquired ADAMTS13 deficiency did not spontaneously develop TTP, despite the accumulation of UL-VWF multimers. In line with the Adamts13-/- mice, TTP-like symptoms could only be induced when an additional trigger (rVWF) was administered. On the other hand, the availability of our panel of anti-mADAMTS13 mAbs allowed us to further develop a sensitive ELISA to detect ADAMTS13 in mouse plasma. In conclusion, a novel acquired TTP mouse model was generated through the development of inhibitory anti-mADAMTS13 mAbs. Consequently, this model provides new opportunities for the development and validation of novel treatments for patients with TTP. In addition, these newly developed inhibitory anti-mADAMTS13 mAbs are of great value to specifically study the role of ADAMTS13 in mouse models of thrombo-inflammatory disease.

Neutralization and Binding Profile of Monoclonal Antibodies Generated Against Influenza A H1N1 Viruses.

Monoclonal antibodies (MAbs) provide scope for the development of better therapeutics and diagnostic tools. Herein, we describe the binding and neutralization profile(s) for a panel of murine MAbs generated against influenza A H1N1 viruses elicited by immunization with pandemic H1 recombinant hemagglutinin (rHA)/whole virus or seasonal H1 rHA. Neutralizing MAbs, MA-2070 and MA-M, were obtained after pandemic A/California/07/2009 (H1N1) virus/rHA immunization(s). Both MAbs reacted specifically with rHA from A/California/07/2009 and A/England/195/2009 in ELISA. MA-2070 bound rHA of A/California/07/2009 with high affinity (KD = 51.36 ± 9.20 nM) and exhibited potent in vitro neutralization (IC50 = 2.50 μg/mL). MA-2070 bound within the stem domain of HA. MA-M exhibited both hemagglutination inhibition (HI, 1.50 μg/mL) and in vitro neutralization (IC50 = 0.66 μg/mL) activity against the pandemic A/California/07/2009 virus and showed higher binding affinity (KD = 9.80 ± 0.67 nM) than MA-2070. MAb, MA-H generated against the seasonal A/Solomon Islands/03/2006 (H1N1) rHA binds within the head domain and bound the seasonal H1N1 (A/Solomon Islands/03/2006 and A/New Caledonia/20/1990) rHAs with high affinity (KD; 0.72-8.23 nM). MA-H showed high HI (2.50 μg/mL) and in vitro neutralization (IC50 = 2.61 μg/mL) activity against the A/Solomon Islands/03/2006 virus. All 3 MAbs failed to react in ELISA with rHA from various strains of H2N2, H3N2, H5N1, H7N9, and influenza virus B, suggesting their specificity for either pandemic or seasonal H1N1 influenza virus. The MAbs reported here may be useful in developing diagnostic assays.

Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There isan increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG).

Selection of a monoclonal antibody by ELISA, immunoblotting and Quartz Crystal Microbalance technology for immunohistochemical detection of Mycoplasma mycoides subsp. mycoides.

An immunohistochemical (IHC) technique was optimised using a monoclonal antibody (MAb) to detect Mycoplasma mycoides subsp. mycoides (Mmm), the agent of Contagious Bovine Pleuropneumonia (CBPP), in sections of lung tissue. A panel of MAbs was produced and screened for Mmm speci city and for cross-reactivity against other mycoplasmas belonging and not belonging to the Mycoplasma mycoides cluster, using in parallel indirect ELISA (i-ELISA) and Immunoblotting (IB). Based on i-ELISA and IB characterization data, 1 MAb (clone 3G10E7) was selected and its highest a nity vs Mmm was con rmed by the Quartz Crystal Microbalance (QCM) technology. Afterwards, IHC analyses were conducted to compare MAb 3G10E7 vs rabbit Mmm speci c hyperimmune serum using lung tissue sections of CBPP infected and CBPP negative animals. Results suggest that screening of MAbs using in parallel ELISA, IB, and QCM technology enables to select high a nity target speci c MAbs. Immunohistochemical results demonstrated that MAb 3G10E7 improved IHC performances, showing reduced background staining and no cross-reactivity against Mycoplasma bovis, which is responsible of pneumonia in cattle.

Abstract 1498: Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors

Abstract Introduction. IL13Rα2 is a membrane-bound protein expressed on a number of malignant tumors, but not at significant levels in most normal tissues. Clinically tested therapies to treat cancer by targeting IL13Rα2 to date have included a ligand toxin conjugate (IL13-PE) and IL13Rα2 directed CAR-T cells. Both strategies appear to be well tolerated, although with mixed results in tumor eradication. An alternative strategy for targeting IL13Rα2 is an IL13Rα2 x CD3 Dual-Affinity Re-Targeting (DART®) bispecific molecule designed to co-engage IL13Rα2 on tumor cells and cytotoxic T cells through CD3, resulting in killing of tumor cells. Methods. Mouse monoclonal antibodies (mAbs) were generated using standard immunization protocols followed by binding and epitope binning analyses. Cell binding was performed by flow cytometry and frozen normal and tumor tissues analyses by immunohistochemistry (IHC). In vitro studies were performed with cancer cell lines and primary human T cells or peripheral blood mononuclear cells (PBMCs). In vivo studies were performed in immune-deficient tumor-bearing mice co-implanted with activated human T cells or reconstituted with human PBMCs. Results. A panel of mAbs that bind human and cynomolgus monkey IL13Rα2 proteins were selected, representing a range of binding characteristics and epitope diversity. IHC showed favorable normal vs tumor tissue reactivity, with high levels of IL13Rα2 expression in glioblastoma and melanoma. The mAb panel was converted to DART molecules with an anti-CD3 arm, assessed for the ability to mediate cytotoxic T lymphocyte (CTL) activity and cytokine release and a lead selected for humanization and engineering into an IL13Rα2 x CD3 DART molecule incorporating an Fc domain to prolong circulating half-life. The IL13Rα2 x CD3 DART protein bound human and cynomolgus IL13Rα2 (KD = 0.64 nM and 0.5 nM, respectively) and mediated CTL activity against LOX-IMVI (melanoma) and SK-MES-1 (lung adenocarcinoma) target cells. Cytokines (IFN-γ, IL-10, and TNF-α) were released in the presence of IL13Rα2-expressing target cells and human PBMC, but not with PBMCs alone. Administration of the IL13Rα2 x CD3 DART molecule (≤50 μg/kg IV for 4 consecutive days) prevented tumor growth when tumor cells (A375 and LOX-IMVI melanoma, U87 glioblastoma, or DAOY medulloblastoma cell lines) were co-mixed with activated human T cells and implanted subcutaneously in NOD/SCID/IL2gamma-chain null mice. Tumor eradication was also observed in NOD/SCID/IL2gamma-chain/MHCI KO mice reconstituted with human PBMCs and bearing established LOX-IMVI melanoma tumors following 2 weekly IV doses of 50 μg/kg IL13Rα2 x CD3 DART molecule. Conclusions: Robust in vitro and in vivo antitumor activity for an IL13Rα2 x CD3 DART protein was demonstrated. Additional studies are underway to further characterize the molecule as a development candidate for treatment of IL13Rα2-positive cancers. Citation Format: Jill Rillema, Monica Licea, Shereen Saini-Lal, Doug Smith, Francine Chen, Annie Lam, Yinhua Yang, Liqin Liu, James Tamura, Ralph Alderson, Ezio Bonvini, Syd Johnson, Paul Moore. Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors. [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 1498.

Abstract 586: Discovery of a novel TIM3 binding partner and a key role for TIM3 on macrophages: Identification of specific antibodies capable of converting immune-suppressive macrophages to immune-enhancing

Abstract Our Translational Science Platform uses an unbiased bioinformatics-based approach to interrogate particular cell types within the tumor microenvironment (TME). Given the correlation between high levels of immune-suppressive macrophages within the tumor TME and poor patient prognosis across a number of solid tumor types we focused initially on developing novel immunotherapies to modify this cell type. We identified 10 targets as candidates for converting tumor-associated macrophages from immune-suppressing to immune-enhancing. One of these targets was TIM3. To date, TIM3 has been pursued mainly as a checkpoint target for T cell-directed immunotherapies based on its expression on exhausted T cells. Anti-TIM3 mAbs, generated by multiple groups, induce responsiveness in T cells and demonstrate anti-tumor benefit in certain mouse models. However, our macrophage-centric approach has identified a previously unrecognized protein-protein interaction between TIM3 and one of our additional macrophage targets. Based on knowledge of this interaction, we were able to generate and select for panels of mAbs to TIM3 and to its binding partner capable of converting macrophages from an “M2” to an “M1” pro-inflammatory phenotype. In contrast to published anti-TIM3 mAbs, our particular anti-TIM3 mAbs lacked activity in T cell-based assays, but promoted an increase in pro-inflammatory cytokines with a reduction or no effect in anti-inflammatory cytokines in a macrophage activity assay. In this assay, monocytes were prepared from human peripheral blood and cultured in the presence of M-CSF to bias toward an M2 phenotype. Under sub-optimal stimulation with LPS or CD40L or HMGB1, treatment of these cells with the anti-TIM3 mAbs led to increases in pro-inflammatory cytokines including IL-1β and TNFα. The conversion to an “M1” macrophage by anti-TIM3 mAbs had downstream consequences on T cells as demonstrated by mixed lymphocyte reaction experiments. In these studies, the addition of anti-TIM3 led to a macrophage-dependent increase in IFNγ from the T cells. To assess the impact of our anti-TIM3 mAbs in the tumor setting, tumor histoculture experiments were performed. Tumor tissue slices from ovarian cancer patients treated with anti-TIM3 showed an increase in a range of cytokines and in this tumor setting the initial sub-optimal stimulus was not required. Specific antibodies to TIM3 and its binding partner that are able to promote a pro-inflammatory macrophage phenotype have been generated. We are developing these as modulators of the TME, to be assessed either as single agents or in combination with other therapies such as checkpoint inhibitors. Citation Format: Jamie Wong, Ryan Phennicie, Igor Feldman, Sriram Sathyanarayanan, Don Shaffer, Mohammad Zafari, Steve Sazinsky, Kenneth Crook, Debbie Law. Discovery of a novel TIM3 binding partner and a key role for TIM3 on macrophages: Identification of specific antibodies capable of converting immune-suppressive macrophages to immune-enhancing. [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 586.