Presence Of Monoclonal Antibody Research Articles

C1q modulation of antibody-dependent enhancement of dengue virus infection in human myeloid cell lines is dependent on cell type and antibody specificity

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is one of the mechanisms contributing to increased severity during heterotypic, secondary infection. The complement protein C1q has been shown to reduce the magnitude of ADE in vitro. Therefore, we investigated the mechanisms of C1q modulation of ADE, focusing on processes of viral entry.Using a model of ADE of DENV-1 infection in human myeloid cell lines in the presence of monoclonal antibodies, 4G2 and 2H2, we found that C1q produced nearly a 40-fold reduction of ADE of DENV-1 in K562 cells, but had no effect in U937 cells. In K562 cells, C1q reduced adsorption of DENV-1/4G2 and exerted a dual inhibitory effect on adsorption and internalization of DENV-1/2H2. Distinct endocytic pathways in the presence of antibody corresponded to conditions where C1q produced a differential action. Also, C1q did not affect the intrinsic cell response mediated by FcγR in human myeloid cells.The modulation of ADE of DENV-1 by C1q is dependent on the FcγR expressed on immune cells and the specificity of the antibody comprising the immune complex. Understanding protective and pathogenic mechanisms in the humoral response to DENV infections is crucial for the successful design of antivirals and vaccines.

Impact of mAb-induced A475V substitution on viral fitness and antibody neutralization of SARS-CoV-2 omicron variants in the presence of monoclonal antibodies and human convalescent sera.

The emergence and rapid evolution of SARS-CoV-2 variants have posed a major challenge to the global efforts to control the COVID -19 pandemic. In this study, we investigated the potential of two SARS-CoV-2 variants, BA.2 and BA.5, to evade neutralization by a human monoclonal antibody targeting the virus's spike RBD (mAb 1D1). By subjecting the viruses to serial propagation in the presence of the antibody, we found that BA.2 exhibited poor growth, whereas BA.5 regained robust growth with significantly higher kinetics than the parental virus. Genetic analysis identified a single mutation, A475V, in the spike protein of BA.5 that substantially reduced the neutralizing activities of monoclonal antibodies and convalescent sera. In addition, the A475V mutation alone in BA.2 moderately reduced the neutralizing activity but completely abolished the neutralizing effect of mAb 1D1 when F486V or L452R were also present. Our results shed light on the possible evolutionary development of SARS-CoV-2 variants under selection pressure by monoclonal antibodies and have implications for the development of effective antibody therapies and vaccines against the virus.

On the essentiality of the angiotensin converting enzyme 2 receptor for SARS-CoV-2 infection and the potential of soluble angiotensin converting enzyme 2 proteins as universal approach for variants causing COVID-19.

On the essentiality of the angiotensin converting enzyme 2 receptor for SARS-CoV-2 infection and the potential of soluble angiotensin converting enzyme 2 proteins as universal approach for variants causing COVID-19.

Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.1 In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and FcγR. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.

Metformin sensitizes leukemic cells to cytotoxic lymphocytes by increasing expression of intercellular adhesion molecule-1 (ICAM-1)

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The anti-diabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention, although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1), a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of anti-apoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels, which favors cytotoxic lymphocytes binding to tumor cells. Finally, metformin decreases the growth of human hematological tumor cells in xenograft models, mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy.

Preparation of high concentration protein powder suspensions by milling of lyophilizates

Pharmaceutical formulations utilizing protein drugs as powders can be used as drug delivery systems in various ways. Besides powders for inhalation, another promising approach is their use as suspensions in non-aqueous liquids for subcutaneous administration providing high protein stability and good injectability. In this study protein powder suspensions were prepared using a swing-mill. Milling of lyophilizates containing a model monoclonal antibody in presence of the suspension vehicle was compared to cryogenic dry milling. Wet media milling led to injectable suspensions, but resulted in monomer loss and increase in protein aggregation. When the lyophilizates were cryogenic dry ball milled less aggregation and monomer loss were detected. Differences related to protein integrity were found for different process parameters, which were successfully optimized. If not cooled with liquid nitrogen, dry milling resulted in increased damage to the mAb. The type of polyol stabilizer, as well as the protein to stabilizer ratio, did not affect the preservation of protein integrity.As finding the right milling duration is time and resource intensive, a correlation between lyophilizate cake hardness and milling duration was established. Based on this approach high concentration lyophilizates were successfully micronized. Suspensions of cryogenic milled powders lead to clogging of 25G needles, which could be prevented by an additional sieving step. Depending on the suspension vehicle, low viscosity formulations (<10 mPa·s) even at high concentrations (≥100 mg/ml protein concentration) were obtained featuring good injectability.

Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium subsp. paratuberculosis peptide vaccine

Studies in cattle show CD8 cytotoxic T cells (CTL), with the ability to kill intracellular bacteria, develop following stimulation of monocyte-depleted peripheral blood mononuclear cells (mdPBMC) with antigen presenting cells (APC, i.e. conventional dendritic cells [cDC] and monocyte-derived DC [MoDC]) pulsed with MMP, a membrane protein from Mycobacterium avium subsp. paratuberculosis (Map) encoded by MAP2121c. CTL activity was diminished if CD4 T cells were depleted from mdPBMC before antigen (Ag) presentation by APC, suggesting simultaneous cognate recognition of MMP epitopes presented by MHC I and MHC II molecules to CD4 and CD8 T cells is essential for development of CTL activity. To explore this possibility, studies were conducted with mdPBMC cultures in the presence of monoclonal antibodies (mAbs) specific for MHC class I and MHC class II molecules. The CTL response of mdPBMC to MMP-pulsed APC was completely blocked in the presence of mAbs to both MHC I and II molecules and also blocked in the presence of mAbs to either MHC I or MHC II alone. The results demonstrate simultaneous cognate recognition of Ag by CD4 and CD8 T cells is essential for delivery of CD4 T cell help to CD8 T cells to elicit development of CTL.

Abstract 1775: Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate cancer

Abstract Enzalutamide (ENZ) is an androgen receptor (AR) antagonist that targets various stages of the AR signaling pathway, and approved for the treatment of castration resistant prostate cancer (CRPC). Studies show increased survival in men with metastatic CRPC after chemotherapy when treated with ENZ. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance and resistance to AR inhibitors may lead to increase in N-cadherin. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR positive and negative prostate cancers. Co-targeting AR and N-cadherin with ENZ and mAb respectively show promise in addressing not only castration resistant tumor progression but also ENZ resistance. Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with ENZ. In vivo studies on promising candidates were performed using castration resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to ENZ, mAb alone, or in combination. At end-point, tumors were harvested and further analyzed. Final candidate mAbs were then selected and re-formatted into fully humanized N-cadherin monoclonal antibodies and further evaluated. A molecular model of the mouse chimeric antibodies were built based on structure of similar antibodies and the CDR loops were grafted to the human framework region and back-translated into nucleotide sequences. Codon-optimized DNA encoding the humanized variable domains connected by a 15 glycine-rich amino acid linker was synthesized by GeneArt and sub-cloned into expression vectors containing light and heavy chain constant regions. Clones were then screened and selected. Candidates were transiently expressed in 293 cells and supernatant were collected and purified for further analysis. These newly generated humanized mAbs engineered from the promising human anti-mouse N-cadherin antibody candidates exhibited similar outcomes to their chimeric counterparts, revealing potential in providing methods for diagnosis and treatment of advanced diseases. Citation Format: Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Keyu Li, Joyce Yamashiro, Chun Wang, Anna Wu, Robert E. Reiter. Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1775.

Abstract A52: Efficacy of new developed N-cadherin monoclonal antibodies in combination with enzalutamide against castration-resistant prostate cancer

Abstract Introduction: Recent advances improved survival in men with metastatic castration-resistant prostate cancer (CRPC) after chemotherapy when treated with androgen ablation therapies, including the well-known androgen receptor (AR) antagonist enzalutamide. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next-generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR-positive and -negative prostate cancers. Here we report the further development of effective N-cadherin specific monoclonal antibodies against CRPC and their value in combination therapies. Method: Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen-dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with enzalutamide. Further, enzalutamide-resistant cell lines were also generated and evaluated. In vivo studies were performed using castration-resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to enzalutamide or mAb alone or in combination. When the tumors reached 100-200 mm3 in volume the mice were castrated and treated with 5-10 mg/kg enzalutamide and/or 5-10 mg/kg mAb candidates. Tumors were monitored for growth, local invasion, and metastasis. At endpoint, tumors were harvested and further analyzed. Promising mAb candidates, as well as new epitope targets, were then selected and reformatted into full humanized N-cadherin monoclonal antibodies and further analyzed. Results: Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. In vitro studies showed that combination treatment of N-cadherin positive and AR-positive cells reduced invasion and cell proliferation than either treatment alone. Certain enzalutamide-resistant cell lines generated showed an increase in N-cadherin expression. Such cells responded to N-cadherin antibodies in androgen-deprived conditions, suggesting a role for N-cadherin in a subset of enzalutamide resistance. In vivo studies using CRPC xenograft models expressing N-cadherin and AR showed inhibition of tumor growth when the animals were castrated and treated with a combination of enzalutamide and N-cadherin mAb than either treatment alone. Androgen-dependent xenograft models in castrated mice also significantly delayed castration-resistant tumor growth when treated in combination than either treatment alone. The newly generated humanized mAbs showed similar outcomes. Conclusion: Previous studies suggested that therapeutic targeting of N-cadherin with monoclonal antibodies have significant effect in inhibiting tumor progression. We have verified that humanized N-cadherin mAb exhibit significant efficacy, fostering our search for increasingly effective immunotherapy in the clinic. Further, the antibodies showed synergy in combination with enzalutamide, suggesting promise in potential combination therapies for CRPC. Other androgen receptor antagonists and other drugs targeting those potentially involved in downstream pathways may prove to have additional clinical benefit. Citation Format: Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Joyce Yamashiro, Wang Chun, Della Z. Hu, Anna M. Wu, Robert E. Reiter. Efficacy of new developed N-cadherin monoclonal antibodies in combination with enzalutamide against castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A52.

BRAF, KRAS and PIK3CA Mutation and Sensitivity to Trastuzumab in Breast Cancer Cell Line Model

Studies show that approximately 20% of all breast cancer patients have a breast tumor that tests positive for Human Epidermal Growth Factor Receptor 2, otherwise known as the HER2 gene. As such, treatments for breast cancer usually include drugs that target HER2. The drug Trastuzumab is a recombinant antibody that has been approved by the FDA for the treatment of these HER2 positive breast cancers. However, researchers have found that mutations in associated genes, PIK3CA and KRAS, can cause the tumor to become resistant to Trastuzumab. The purpose of this article is to evaluate the sensitivity of the cancer cell lines to the drug Trastuzumab and investigate how this sensitivity is compromised by the PIK3CA, KRAS and BRAF gene mutations. Trastuzumab responsiveness was evaluated in breast cancer cell lines by treating the lines with an optimal concentration of the drug followed by a proliferation assay of the cell lines in the presence of monoclonal antibodies. We determined the optimum concentration of Trastuzumab to be 7 μg/well. The BRAF and KRAS mutated cell line, MDA-MB-231, showed the least sensitivity after being treated with trastuzumab when compared to the sensitivity of the PIK3CA mutated cell lines, MCF-7 and MDA-MB-361, and the KRAS/ BRAF/ PIK3CA cell line, MDA-MB-453. Clinical observations show that mutations in BRAF and KRAS genes in breast cancer cells do lower the responsiveness of Trastuzumab drug treatments.

Abstract 3287: Expression of CD14 and SIRP-α defines distinct populations of intratumoral monocytes/macrophages in B-cell non-Hodgkin lymphomas

Abstract BACKGROUND Monocytes and macrophages (mo/mΦ) are part of composition of tumor microenvironment in B-cell non-Hodgkin lymphoma (B-NHL). CD14+HLA-DRlow monocytes are immunosuppressive and this phenotype is promoted by increased expression of IL-10 in lymphoma. Despite their association with poor outcome, monocytes retain phagocytic function particularly in the presence of monoclonal antibodies and can be associated with an improved outcome in patients treated with rituximab. The aim of this study was to determine the prevalence of CD14+HLA-DRlow mo/mΦ in B-NHL tissue compared to normal tissue, and to determine whether their phenotype suggests that they are capable of phagocytic function. METHODS Tissue mo/mΦ were isolated by negative selection from 13 diagnostic B-NHL biopsy specimens and 6 normal tissues using a monocyte enrichment kit. Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by flow cytometry, immunocytochemistry and cytometry by time-of-flight (CyTOF). The purity of isolated tissue mo/mΦ was more than 95%, being confirmed by Giemsa stain and flow cytometry. RESULTS Increased numbers of CD68+ cells in initial B-NHL biopsies when compared to normal tissue was confirmed by immunohistochemistry and flow cytometry. However, CD14 expression was substantially lower than CD68 expression in the tissues suggesting that many CD68+ mo/mΦ are CD14 negative. Using a monocyte enrichment kit to isolate all mo/mΦ, we found that CD14 negative mo/mΦ constituted half the tissue mo/mΦ. Furthermore, we found by flow cytometry, CyTOF and Giemsa stain that CD14- mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. While all cells expressed CD68, the larger cells had increased expression of CD32, CD64 and HLA-DR. The CD14+ cells typically also expressed CD163 and CD33 and were a subset of the larger monocyte population, while the population of small cells was positive only for CD68 and CD45. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αlow and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αlow cells typically constitute the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. Interestingly, while the CD14-SIRP-α- cells lack the typical phenotypic markers of mo/mΦ, they morphologically have the appearance of monocytic cells and this population appears expanded in lymphoma tissue. CONCLUSIONS We have identified a unique population of small mo/mΦ that have an immature phenotype and lack expression of CD14, SIRP-α, CD163, and other FcγR markers. This subset of mo/mΦ is prevalent in lymphoma and may have limited phagocytic function. This CD68+CD14-SIRP-α- mo/mΦ subpopulation may account for prognostic differences in the outcome of lymphoma patients treated with or without monoclonal antibodies. Citation Format: Ya-Ping Chen, Zhi-Zhang Yang, Jose C. Villasboas, Tammy Price-Troska, Anne J. Novak, Stephen M. Ansell. Expression of CD14 and SIRP-α defines distinct populations of intratumoral monocytes/macrophages in B-cell non-Hodgkin lymphomas. [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 3287.

Generation and Characterisation of HCV Genotype 1 Viruses Most Recent Common Ancestor

Background: Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope glycoproteins, E1 and E2, interact with host cell receptors and provide a target for neutralising antibodies. Past vaccine studies using unmodified E2 proteins have failed to convincingly generate broadly neutralising antibody responses. Objectives: This study sought to generate and evaluate an immune-focused, vaccine candidate for HCV. Methodology: A synthetic construct based on most recent common ancestral sequence (MRCA) of HCV genotype 1 viruses was generated using sequences available from the Los Alamos HCV database [720 sequences (360 subtype 1a and 360 subtype 1b sequences)], after exclusion of epidemiologically-related sequences. Soluble E2 (sE2) proteins were generated by stably transfected S2 cells and purified using Strep- tag purification and size exclusion chromatography. The MRCA construct was subsequently interrogated using a linear (AP33) and conformational (1:7) monoclonal antibodies directed at E2. A full length E1E2 construct was used for production of HCV pseudoparticles (HCVpp). The infectivity of the HCVpp was measured in the presence of monoclonal antibodies; AP33 1:7 and AR3A. Results: Monomeric proteins of the MRCA generated using a Drosophila expression system were conformationally intact when examined by the monoclonal antibody 1:7 that targets the conformational epitope on E2 responsible for interaction with the CD81 receptors. The full length MRCA E1E2 construct showed functionality in the HCV pseudo-particle (HCVpp) system. The MRCA HCVpp construct was susceptible to neutralisation by AP33, 1:7 and AR3A, in dose- dependant manner. Conclusion: This study demonstrates the generation of a functional construct that could be used as a vaccine candidate in a potential vaccine approach to minimise the problem of genetic diversity between the vaccine construct and contemporary viruses.

Mapping the AAV Capsid Host Antibody Response toward the Development of Second Generation Gene Delivery Vectors.

The recombinant adeno-associated virus (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2). Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies (MAbs) or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion, or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from MAbs, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed.

Multiple mechanisms mediate enhanced immunity generated by mAb‐inactivated F. tularensis immunogen

We have previously demonstrated that immunization with inactivated Francisella tularensis, a Category A intracellular mucosal pathogen, combined with IgG2a anti-F. tularensis monoclonal antibody, enhances protection against subsequent F. tularensis challenge. To understand the mechanism(s) involved, we examined the binding, internalization, presentation, and in vivo trafficking of inactivated F. tularensis in the presence and absence of opsonizing monoclonal antibody. We found that when inactivated F. tularensis is combined with anti-F. tularensis monoclonal antibody, presentation to F. tularensis-specific T cells is enhanced, this enhancement is Fc receptor-dependent, and requires a physical linkage between the monoclonal antibody and the inactivated F. tularensis immunogen. This enhanced presentation is due, in part, to enhanced binding and internalization of inactivated F. tularensis by antigen presenting cells, and involves interactions with multiple Fc receptor types. Furthermore, targeting inactivated F. tularensis to Fc receptors enhances dendritic cell maturation and extends the time period over which antigen presenting cells stimulate T cells. In vivo trafficking studies reveal enhanced transport of inactivated F. tularensis immunogen to the Nasal Associated Lymphoid Tissue in the presence of monoclonal antibody, which is FcRn-dependent. In summary, these are the first comprehensive studies using a single vaccine protection model/immunogen to establish the array of mechanisms involved in enhanced immunity/protection mediated by an Fc receptor-targeted mucosal immunogen. These results demonstrate that multiple cellular/immune mechanisms contribute to Fc receptor-enhanced immunity.

In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

BackgroundIFNγ-producing CD4+CD25+Foxp3+ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins.MethodsPBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ+ iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry. Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4+CD25+CD127-IFNγ+ PBL.ResultsHigh monoclonal antibody concentrations inhibited the induction of CD4+CD25+Foxp3+IFNγ+ PBL (anti-CD152, anti-CD279, anti-CD95: p < 0.05) and CD4+CD25+CD127-IFNγ+ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p < 0.05). Effector cell proliferation increased with increasing antibody concentrations in culture medium (anti-CD178 and anti-CD279: p < 0.05). Conversely, high concentrations of recombinant proteins induced formation of CD4+CD25+Foxp3+IFNγ+ PBL (rCD152 and rCD95: p < 0.05) and decreased cell proliferation dose-dependently (rCD178 and rCD95: p < 0.05). Our data suggest an inverse association of iTreg induction with effector cell proliferation in cell culture which is dependent on the concentration of monoclonal antibodies against iTreg surface determinants. 3-day co-cultures of polyclonally stimulated PBL with separated CD4+CD25+CD127-IFNγ+ PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-IFNγ- PBL (p < 0.05). Cell proliferation increased strongly in CD4+CD25+CD127-IFNγ- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p < 0.05) but remained low in co-cultures with CD4+CD25+CD127-IFNγ+ PBL (with the exception anti-CD28 monoclonal antibody: p < 0.05). Monoclonal antibodies prevent iTreg induction in co-cultures with CD4+CD25+CD127-IFNγ- PBL but do not efficiently block suppressive iTreg function in co-cultures with CD4+CD25+CD127-IFNγ+ PBL.ConclusionsCD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ+ iTreg.

Effects of Hydrophobic Amino Acids and Antibodies to Nerve Growth Factor Receptors on the Development of Splenic Tissue Culture from Young and Old Rats

The effects of hydrophobic L-amino acids alone and in the presence of monoclonal antibodies to nerve growth factor receptors NGFRp75 (apoptosis inductors) were studied on organotypic culture of splenic lymphoid tissue from young (3 months) and old (24 months) rats. Nine amino acids inhibited cell proliferation in splenic explants from young rats. This was paralleled by hyperexpression of p53 proapoptotic protein. Only two amino acids stimulated apoptosis in explants from old rats. The inhibitory effects on the development of splenic explants from young and old rats were abolished in the presence of antibodies to NGFRp75. Hence, the group of hydrophobic amino acids mediates the proapoptotic effect in the lymphoid tissue of old and young rats through nerve growth factor low affinity receptors.

RAD001 Exerts Anti-Tumor Activity in Waldenstrom Macroglobulinemia.

Abstract 3732Poster Board III-668 BackgroundWaldenstrom Macroglobulinema (WM) is an incurable B-cell disorder characterized by the presence of IgM monoclonal gammopathy and bone marrow infiltration of lymphoplasmacytic cells. Apart from promising advances in therapeutic strategy, response rates and treatment-free survival remain inconsistent across prescribed regimens. As we have previously shown, the PI3K/Akt pathway actively mediates tumor growth, survival, migration, and cell cycle within primary WM cells. An active target of this pathway includes the downstream mammalian target of rapamycin (mTOR). By inhibiting the mTOR pathway with RAD001 (Afinitor™, Novartis Pharmaceuticals), we hoped to constructively regulate aforementioned cellular function, and also understand from a preclinical perspective its specificity of action in our microenvironmental models, both alone and the presence of monoclonal antibody against CD20 and proteasome inhibitor. MethodsWM cell lines (BCWM1) and IgM secreting cell lines MEC1 and RL were used. Bone marrow stromal cells (BMSC) were obtained from patients with WM. Peripheral blood mononuclear cells were harvested from healthy donors. Agents tested included RAD001, Rituximab and Bortezomib. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [3H]-thymidine uptake, and flow cytometry/PI staining, respectively. Antibody-dependent cellular cytoxicity (ADCC), transwell, and Matrigel assays were preformed to measure cell lysis, SDF-1 chemotaxis-induced migration, and angiogenesis, respectively. Adhesion to fibronectin has been evaluated with WM cells and BMSC in the presence of RAD001, with and without Bortezomib. ResultsRAD001 induced cytotoxicity and inhibition of DNA synthesis with an IC50 of 1-10 nM in BCWM1 cells at 48 hours. Similar effects were seen in IgM cell lines with an effective dose of 0.1-1 nM. In contrast, at IC50 treatment level donor cells displayed no significant cytotoxicity (>85% survival). Cell cycle analysis showed corresponding G1 arrest, and angiogenesis was also inhibited. RAD001, Bortezomib, and Rituxan combinations showed synergistic cytotoxicity, which was attenuated when WM cells were co-cultured with BMSC. Migration of BCWM1 and adhesion of BCWM1 to BMSC was reduced under presence of RAD001. ConclusionsThese functional assays therefore show that RAD001 has significant and synergistic antitumor activity in WM which will inform the design of future clinical trials. Disclosures:Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Antigenic structure of the hemagglutinin of H9N2 influenza viruses

The hemagglutinins (HAs) of H9 influenza viruses isolated from birds and mammals of different species were antigenically and genetically analyzed. Antigenic variants were selected from A/swine/Hong Kong/10/98 (H9N2) and A/duck/Hokkaido/13/00 (H9N2) in the presence of monoclonal antibodies (MAbs). Based on the reactivity patterns of these mutants with a panel of MAbs, at least five non-overlapping antigenic sites were defined using eight MAbs which recognized seven distinct epitopes on the H9 HA molecule. Based on the reactivity patterns with the panel of monoclonal antibodies, 21 H9N2 virus strains isolated from birds and mammals were divided into 7 antigenically distinct groups. The present findings indicate that it is important to monitor the antigenic variation in H9 influenza viruses. The panel of MAbs in the present study, thus, should be useful for detailed antigenic analysis of the H9 HAs for epidemiological studies, the selection of vaccine strains, and diagnosis.

Identification of a neutralization epitope in the VP1 capsid protein of SV40

Three SV40 escape mutants were identified by selection in the presence of monoclonal antibodies with neutralizing activity. The VP1 amino acid alterations in these mutants were: (1) K73 → E (in loop BC); (2) D77 → E (in loop BC); (3) K171 → R (in loop EF); and (4) Q175 → H (in loop EF). These residues are clustered in close proximity to each other on the surface of the native capsid protein, strongly suggesting that they form a conformational epitope directly recognized by the neutralizing antibody. To our knowledge, the present study represents the first experimental mapping of a neutralization epitope of a polyomavirus family member. Structural information regarding the neutralization epitope should be useful for clarifying the extent of cross-reactivity exhibited by the humoral immune response towards related primate polyomaviruses (e.g., SV40, BKV, and JCV).

Development of a label-free electrochemical immunosensor based on carbon nanotube for rapid determination of clenbuterol

We have fabricated a label-free electrochemical immunosensor for the detection of clenbuterol, a kind of β-agonist. Clenbuterol was covalently linked to multi-wall carbon nanotubes (MWCNTs) through a two-step process using 1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide and N-hydroxysulfo-succinimide as crosslinkers. The clenbuterol-MWCNT conjugates were cast on a glassy carbon electrode. Cyclic voltammetry and differential pulse voltammetry were employed to monitor the fabrication steps of immunoreaction system using the redox probe of K 3Fe(CN) 6. In the presence of monoclonal antibody against clenbuterol, the redox peak current of [Fe(CN) 6] 3−/4− was decreased, presumably due to that antibody in solution could adsorb on the electrode surface modified clenbuterol-MWCNT conjugates. The selected monoclonal antibody showed very high sensitivity and specificity for clenbuterol, and was used for the detection and quantitative determination of clenbuterol in solution with a competitive mechanism. This approach provided a detection limit of 0.32 ng mL −1. Accurate detection of clenbuterol in spiked animal feeds was demonstrated by comparison with conventional ELISA assays and LC–MS method.