Flow Cytometry-based Binding Assay Research Articles

RAGE Is a Receptor for SARS-CoV-2 N Protein and Mediates N Protein-induced Acute Lung Injury.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N-protein) increases early in body fluids during infection and has recently been identified as a direct inducer for lung injury. However, the signal mechanism of N-protein in the lung inflammatory response remains poorly understood. The goal of this study was to determine whether RAGE (receptor for advanced glycation endproducts) participated in N-protein-induced acute lung injury. The binding between N-protein and RAGE was examined via assays for protein-protein interaction. To determine the signaling mechanism invitro, cells were treated with recombinant N-protein and assayed for the activation of the RAGE/MAPK (mitogen-activated protein kinase)/NF-ĸB pathway. RAGE deficiency mice and antagonist were used to study N-protein-induced acute lung injury invivo. Binding between N-protein and RAGE was confirmed via flow cytometry-based binding assay, surface plasmon resonance, and ELISA. Pull-down and coimmunoprecipitation assays revealed that N-protein bound RAGE via both N-terminal and C-terminal domains. In vitro, N-protein activated the RAGE-ERK1/2-NF-ĸB signaling pathway and induced a proinflammatory response. RAGE deficiency subdued N-protein-induced proinflammatory signaling and response. In vivo, RAGE was upregulated in the BAL and lung tissue after recombinant N-protein insult. RAGE deficiency and small molecule antagonist partially protected mice from N-protein-induced acute lung injury. Our study demonstrated that RAGE is a receptor for N-protein. RAGE is partially responsible for N-protein-induced acute lung injury and has the potential to become a therapeutic target for treating coronavirus disease.

Inhibition of Respiratory Syncytial Virus Infection by Small Non-Coding RNA Fragments

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infection in infants, immunocompromised individuals and the elderly. As the only current specific treatment options for RSV are monoclonal antibodies, there is a need for efficacious antiviral treatments against RSV to be developed. We have previously shown that a group of synthetic non-coding single-stranded DNA oligonucleotides with lengths of 25–40 nucleotides can inhibit RSV infection in vitro and in vivo. Based on this, herein, we investigate whether naturally occurring single-stranded small non-coding RNA (sncRNA) fragments present in the airways have antiviral effects against RSV infection. From publicly available sequencing data, we selected sncRNA fragments such as YRNAs, tRNAs and rRNAs present in human bronchoalveolar lavage fluid (BALF) from healthy individuals. We utilized a GFP-expressing RSV to show that pre-treatment with the selected sncRNA fragments inhibited RSV infection in A549 cells in vitro. Furthermore, by using a flow cytometry-based binding assay, we demonstrate that these naturally occurring sncRNAs fragments inhibit viral infection most likely by binding to the RSV entry receptor nucleolin and thereby preventing the virus from binding to host cells, either directly or via steric hindrance. This finding highlights a new function of sncRNAs and displays the possibility of using naturally occurring sncRNAs as treatments against RSV.

326 The anti-TIGIT antibody M6223 induces significant anti-tumor efficacy and immune response via multiple mechanisms of action

BackgroundM6223 is a fully human antagonistic anti-T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) antibody in IgG1 format with Fc-mediated effector function.MethodsThe ability of M6223 to block the interaction of TIGIT with its ligands, CD155 and CD112, and the interaction of TIGIT with CD226 was determined by a flow cytometry-based binding assay. The anti-tumor efficacy, immune profile, and effector function of M6223 were investigated in syngeneic tumor models in huTIGIT knock-in mice. M6223 was either formatted with an effector competent mouse IgG2c constant region (M6223-muIgG2c) or formatted with effector null mouse IgG1-D256A constant region (M6223-muIgG1) as two versions of chimeric antibodies for the in vivo studies.ResultsM6223 dose-dependently blocked the binding of TIGIT to its ligands, including CD155 and CD112, thereby inhibiting a TIGIT-mediated immunosuppressive pathway. In addition, M6223 interrupted the interaction of TIGIT with the costimulatory receptor CD226. By blocking the interactions, the chimeric protein M6223-muIgG2c showed anti-tumor efficacy in multiple tumor models, including an MC38 tumor model (figure 1), and generated tumor antigen-specific long-term protective immunity in immunocompetent huTIGIT knock-in mice. M6223 monotherapy dose-dependently elevated the ratio of CD8+ cytotoxic T cells to regulatory T cells and the ratio of CD226 to TIGIT expression in immune cells in the tumor microenvironment. We also found that M6223 selectively depleted suppressive and exhausted TIGIT+ immune cell subsets and the anti-tumor activity of effector null M6223-muIgG1 was significantly lost (p<0.0001), suggesting that Fc-mediated effector function contributes to M6223 anti-tumor activity. Antibody depletion studies demonstrated that CD8+ T cells and natural killer cells contributed to the anti-tumor activity of M6223 in a complementary manner.Abstract 326 Figure 1M6223-muIgG2c displayed dose-dependent anti-tumor efficacy. M6223-muIgG2c displayed dose-dependent anti-tumor efficacy in an MC38 tumor model in hTIGIT knock-in mice.ConclusionsGiven that TIGIT blockade can inhibit an immunosuppressive pathway as well as remove the suppression on a costimulatory pathway, M6223 has the potential to induce an anti-tumor immune response by three complementary mechanisms: direct blockade of the TIGIT pathway, stimulation of CD226 dimerization/activation, and depletion of TIGIT+ immune subsets by Fc-mediated effector function. Our data demonstrate that these complementary mechanisms orchestrate the anti-tumor activity of M6223. A Phase I, first-in-human clinical trial (NCT04457778) is underway to determine the safety, tolerability, maximum tolerated dose and recommended dose for expansion of M6223 as a single agent (Part 1A) and in combination with bintrafusp alfa (Part 1B) in patients with metastatic or locally advanced solid unresectable tumors.Ethics ApprovalAll animal experiments were performed in accordance with EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA (protocol 17-008, 20-005) and Wuxi AppTec Animal Care and Use Committee (IACUC) guidelines.

Polymorphisms in LILRB-1 affects its ligand interactions

Abstract Leukocyte immunoglobulin-like receptor B-1 (LILRB-1) is an inhibitory receptor of the immunoglobulin super family expressed on a subset of natural killer (NK) cells. LILRB-1 binds a wide range of classical and non-classical MHC class I molecules, and a glycoprotein product of the human cytomegalovirus (HCMV), UL18. UL18 is a structural homolog of MHC class I expressed on the surface of infected cells. The two most distal domains of LILRB-1, denoted as D1 and D2, are the main contributors at the interaction interface of LILRB-1 with its ligands. Four non-synonymous polymorphisms within D1 and D2 domains produce LILRB-1’s natural variants. Interestingly, these variants have shown different expression and binding trends on NK cells and were suggested to affect susceptibility to HCMV infection in a cohort of kidney transplant recipients. Differences in the interaction of different LILRB-1 variants with their ligands such as, classical (HLA-Cw15) and non-classical MHC class I ligands (HLA-G) as well as UL18 have are assessed using a flowcytometry based binding assay and confirmed with functional assays. Our experiments show a trend of tight binding range of most LILRB-1 variants with HLA-G. However, the strength of binding is more variant with other classical MHC class I ligands, showing a constant trend based on the protein introduced by each of the four non-synonymous polymorphisms in different genotypes. Such findings may highlight an evolutionary pressure to maintain a relatively constant interaction of LILRB-1 with HLA-G, which is expressed on fetal trophoblasts to maintain maternal tolerance.

YB-1 Interferes with TNFα-TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling.

Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor α (TNFα) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNFα-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNFα to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNFα-mediated signaling, supporting the functionality with an NF-κB luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNFα to its receptors and influencing TNFα-mediated signaling via its interaction with progranulin.

Enhancing natural killer cell function with gp41-targeting bispecific antibodies to combat HIV infection.

The aim of this study was to develop and evaluate the activity of bispecific antibodies (bsAbs) to enhance natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC) against HIV-infected cells. These bsAbs are based on patient-derived antibodies targeting the conserved gp41 stump of HIV Env, and also incorporate a high-affinity single chain variable fragment (scFv) targeting the activating receptor CD16 on NK cells. Overall, we expect the bsAbs to provide increased affinity and avidity over their corresponding mAbs, allowing for improved ADCC activity against Env-expressing target cells. bsAbs and their corresponding mAbs were expressed in 293T cells and purified. The binding of bsAbs and mAbs to their intended targets was determined using Bio-Layer Interferometry, as well as flow cytometry based binding assays on in-vitro infected cells. The ability of these bsAbs to improve NK cell activity against HIV-infected cells was tested using in-vitro co-culture assays, using flow cytometry and calcein release to analyse NK cell degranulation and target cell killing, respectively. The bsAbs-bound gp41 with similar affinity to their corresponding mAbs had increased affinity for CD16. The bsAbs also bound to primary CD4 T cells infected in vitro with two different strains of HIV. In addition, the bsAbs induce increased NK cell degranulation and killing of autologous HIV-infected CD4 T cells. On the basis of their in-vitro killing efficacy, bsAbs may provide a promising strategy to improve NK-mediated immune targeting of infected cells during HIV infection.

Macrophage Class A Scavenger Receptors Bind Tumor‐Associated Carbohydrate Antigens and Promote Breast Cancer Growth and Metastases in Mice

Tumor‐associated macrophages (TAMs) play a key role in determining the immune response in cancer. Macrophages express pattern‐recognition receptors, such as Class A Scavenger Receptors (SR‐A), that bind a variety of different ligands and regulate macrophage immune phenotype. SR‐A expression by TAMs is thought to reflect a tumor‐promoting M2 macrophage phenotype, and the accumulation of SR‐A expressing macrophages is associated with increased tumor aggressiveness and poor patient prognosis in a variety of cancers, including breast cancer. Previous studies indicate that SR‐A ligands are present in the medium of cultured tumor cells. To test the possibility that SR‐A may directly bind to tumor cells, we developed a flow cytometry based binding assay using a soluble SR‐A (sSR‐A) protein and showed that SR‐A binds directly to breast cancer cells. This binding depended on glycosylation indicating SR‐A binds to cell‐surface glycans on tumor cells, which is important because tumor‐associated carbohydrate antigens (TACAs) are associated with immune suppression, tumor progression, and poor prognosis for breast cancer patients. The ability of SR‐A to bind TACAs was tested using carbohydrate mimetic peptides (CMPs) that are structurally similar to TACAs. sSR‐A bound to CMPs, and a CMP designed to react with mannose‐rich structures blocked sSR‐A binding to tumor cells. Because SR‐A induces an immune‐suppressive macrophage phenotype, we hypothesized that SR‐A would enhance the progression of breast cancer. Using mouse models of spontaneous breast cancer, our results show that development of palpable tumors was significantly delayed and the number of lung metastases significantly decreased in mice that do not express SR‐A (SR‐A−/−). These findings demonstrate that SR‐A binds to TACAs on breast cancer cells, and that SR‐A enhances breast cancer growth and metastases. Together with previous findings, our results indicate a model in which SR‐A binds to TACAs to induce an immune‐suppressive macrophage phenotype, which contributes to immune suppression, tumor growth, and metastases.Support or Funding InformationThis research was supported by grants from the NIH (R21CA185691), the Arkansas Breast Cancer Research Program, and a DEAP award from UAMS.

Digital Receptor Occupancy Assay in Quantifying On- and Off-Target Binding Affinities of Therapeutic Antibodies.

While monoclonal antibodies are the fastest-growing class of therapeutic agents, we lack a method that can directly quantify the on- and off-target binding affinities of newly developed therapeutic antibodies in crude cell lysates. As a result, some therapeutic antibody candidates could have a moderate on-target binding affinity but a high off-target binding affinity, which not only gives a reduced efficacy but triggers unwanted side effects. Here, we report a single-molecule counting method that precisely quantifies antibody-bound receptors, free receptors, and unbound antibodies in crude cell lysates, termed digital receptor occupancy assay (DRO). Compared to the traditional flow cytometry-based binding assay, DRO assay enables direct and digital quantification of the three molecular species in solution without the additional antibodies for competitive binding. When characterizing the therapeutic antibody, cetuximab, using DRO assay, we found the on-target binding ratio to be 65% and the binding constant (Kd) to be 2.4 nM, while the off-target binding causes the binding constant to decrease by 0.3 nM. Other than cultured cells, the DRO assay can be performed on tumor mouse xenograft models. Thus, DRO is a simple and highly quantitative method for cell-based antibody binding analysis which can be broadly applied to screen and validate new therapeutic antibodies.

TFPR1 acts as an immune regulator and an efficient adjuvant for proteins and peptides by activating immune cells, primarily through TLR2

Triflin, a non-toxic protein found in the venom of the Habu snake, belongs to the CRISP (cysteine-rich secretory protein) family, which comprises two domains: a C-terminal cysteine-rich domain (CRD) and an N-terminal pathogenesis-related-1 (PR-1) domain. The function of the highly structurally conserved PR-1 domain is unknown. Here, we successfully expressed the PR-1 domain of triflin (hereafter called TFPR1) in E. coli. Animal experiments showed that TFPR1 augmented Th1-biased antibody- and cell-mediated immune responses in mice immunized with two protein antigens (OVA and HBsAg) or a peptide antigen (HIV-1 pep). A flow cytometry-based binding assay and in vitro stimulation with TFPR1 showed that it triggered Th1-biased proinflammatory and immunoregulatory cytokine secretion primarily by binding to B cells and macrophages within the mouse splenocyte population. Quantitative RT-PCR, antibody blocking assays using a specific anti-mTLR2 antibody, and stimulatory experiments in vitro using splenocytes from TLR2-KO mice demonstrated that TFPR1 activated murine immune cells, primarily by stimulating toll-like receptor 2 (TLR2). These results suggest that TFPR1 acts as a novel immune modulator and potent adjuvant primarily by activating TLR2. Thus, the PR-1-based core domain might play a role in immune regulation.

Evaluation of the Physicochemical and Biological Stability of Cetuximab under Various Stress Condition.

Cetuximab is a chimeric monoclonal antibody against epidermal growth factor receptor (EGFR) and it is approved for treatment of human colorectal cancer and squamous cell carcinoma of head and neck. The aim of this research was to study the stability of cetuximab finish product (5 mg/mL) under various stress conditions including mechanical, thermal, light stress, and various freeze-thaw cycles. To determine the effects of environmental stresses on the physicochemical properties and bioactivity of cetuximab, a combination of physicochemical and cell-based biological methods including size exclusion chromatography (SEC), cation exchange chromatography (CEX), flow cytometry-based binding assay, and MTS cell viability/proliferation assay was used. The results obtained by the SEC and CEX methods revealed that incubation of cetuximab at 25 and 30 °C, shaking, and various freeze-thaw cycles caused no physicochemical instability. However, functional analysis of the samples exposed to the above-mentioned conditions revealed a significant decrease in the bioactivity of cetuximab indicated by a significant reduction in the cell binding and growth inhibitory effects of cetuximab in EGFR overexpressing cancer cell line (A431). Incubation of cetuximab at 40 and 50 °C led to polymerization and fragmentation of the mAb and resulted in a significant decrease in the bioactivity of the mAb. Our findings show that the light exposure had the most destructive effects on physicochemical and biological characteristics of cetuximab. In conclusion, we found that all mentioned stress conditions significantly affect the bioactivity of cetuximab. Our finding highlights the importance of bioactivity evaluation of biopharmaceuticals in their quality control assessment.

Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro

The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions.

Isoagglutinin reduction by a dedicated immunoaffinity chromatography step in the manufacturing process of human immunoglobulin products.

The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen-a 10% l-proline-stabilized IVIG product-that allows extensive reduction of isoagglutinin concentrations in the final product. An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry-based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures. The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process. Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.

Outer membrane protein U (OmpU) mediates adhesion of Vibrio mimicus to host cells via two novel N-terminal motifs.

Vibrio mimicus (V.mimicus) is a causative agent of ascites disease in aquatic animals. Our previous studies have demonstrated that the outer membrane protein U (OmpU) from V.mimicus is an immunoprotective antigen with six immunodominant linear B-cell epitopes. Although the N-terminus of OmpU contains potential binding motifs, it remained unclear whether OmpU possesses adhesion function. Here, the adhesive capacity of recombinant OmpU and V.mimicus to epithelioma papulosum cyprinid (EPC) cells was determined by immunofluorescence and adherence assay. The results showed that after co-incubated with rOmpU, an obvious visible green fluorescence could be observed on the EPC cell surface and the nuclei exhibited blue fluorescence; while the control cell surface did not show any signal, only nuclei exhibited blue fluorescence. The average number of wild-type strain adhered to each cell was 32.3 ± 4.5. The average adhesion number of OmpU gene deletion mutant was significantly reduced to 10.8 ± 0.5 (P < 0.01) and restored to 31.3 ± 2.8 by complement strain (P >0.05). Pretreatment of cells with rOmpU reduced the average adhesion number of wild-type strain to 9.7 ± 2.9 (P < 0.01). Likewise, binding was significantly decreased to 8.8 ± 3.2 (P < 0.01) due to blocking role of OmpU antibodies. To determine binding motifs of OmpU, six immunodominant B-cell epitope peptides labeled with FITC were employed in flow cytometry-based binding assay. Two FITC-labeled epitope peptides (aa90-101 and aa173-192) showed strong binding to EPC cells (the fluorescence positive cell rate was 99 ± 0.6% and 98 ± 0.3%, respectively), which could be specifically competed by excess corresponding unlabeled peptides, whereas the remaining four showed a low level of background binding. This is the first demonstration that OmpU possesses adhesion function and its N terminal 90–101 and 173–192 amino acid regions are critical sites for cell surface binding.

Novel TCR-like Bi-Specific T Cell Engaging Antibody Targeting the PR1/HLA-A2 Myeloid Leukemia Antigen

PR1 is a HLA-A2 restricted immune-dominant peptide derived from proteinase 3 and neutrophil elastase - leukemia associated self-antigens aberrantly over-expressed in myeloid leukemia. The persistent PR1/HLA-A2 specific cytotoxic T cell responses are associated with prolonged remission from myeloid leukemia, suggesting that PR1/HLA-A2 would be a potent target for T cell based immunotherapy. Previously, we have successfully developed a high affinity T cell receptor (TCR)-like antibody specific for the PR1/HLA-A2 complex (h8F4), and demonstrated that h8F4 mediated specific lyses of myeloid leukemia cells invitro via antibody dependent cell mediated cytotoxicity (ADCC), and significantly reduced leukemic blasts invivo in murine xenograft model of treatment refractory acute myeloid leukemia. We hypothesize that the therapeutic efficacy of h8F4 would be enhanced by combining T cell mediated cytotoxicity through a novel antibody engineering technology, called, Bi-Specific T cell Engager (BiTE) specific for T cells and tumor associated antigens. The BiTE molecules redirect effector function of polyclonal T cells against the tumor leading to lyses of tumor cells, and early clinical data show promising therapeutic efficacy of this approach. In the current study, we constructed Bi-Specific T cell Engaging antibody: h8F4-BiTE, a single chain Fragment of variable domain (scFv) of h8F4 conjugated with scFv of anti-CD3 antibody – OKT3, and produced h8F4-BiTE proteins using eukaryotic expression system. The h8F4-BiTE proteins retained the similar binding specificity to PR1/HLA-A2 as compared to the parent antibody (h8F4), demonstrated by flowcytometry based binding assays with a series of alanine substituted PR1 peptide/HLA-A2 complexes on T2 cells. The 8F4-BiTE bound to native and endogenous PR1/HLA-A2 complexes presented by HLA-A2 positive or transduced myeloid cell lines such as K562, THP-1, and HL-60. In addition, h8F4-BiTE showed comparable binding affinity to CD3 moiety of T cells. Lastly, h8F4-BiTE proteins activated T cells in a PR1/HLA-A2 specific and dose dependent manner, shown by increased expression of activation markers such as CD69 when co-cultured with PR1-pulsed T2 target cells in various concentrations of h8F4-BiTE proteins. Importantly, the PR1/HLA-A2 specific activation of T cells by h8F4-BiTE proteins was equally efficient among all subsets of T cells regardless of their phenotypes, e.g., CD4 vs CD8, and naïve vs memory, suggesting that h8F4-BiTE would be a highly potent inducer of cytotoxic T cells against PR1/HLA-A2 positive myeloid leukemia. Moreover, we generated multiple h8F4-BiTE variants with specific mutations in frameworks and/or complement determining regions (CDRs), and compared binding affinity and function to wildtype h8F4-BiTE. The h8F4-BiTE variants indeed improved specificity to PR1/HLA-A2 complex by complete elimination of residual non-specific binding to HLA-A2, and increased the binding affinity to CD3 by 10-folds resulting in dramatic 50 fold increase in potency to activate T cells. In conclusion, we have developed a novel immunetherapeutics: h8F4-BiTE targeting PR1/HLA-A2 myeloid leukemia antigen. We demonstrated high binding affinity and specificity of h8F4-BiTE to both PR1/HLA-A2 on myeloid leukemia cells and CD3 on T cells and in vitro activation of T cells in the presence of PR1/HLA-A2 expressing target cells in a dose dependent manner. Finally, we improved antigen specificity and functional activity of h8F4-BiTE via selective site-directed mutagenesis. Further preclinical evaluation of therapeutic efficacy is thus warranted using animal in vivo animal treatment model with NOD/SCID/IL2Rγ −/− mice xenografted with human myeloid leukemia cells. This unique TCR-like therapeutic agent adds an additional mechanism of action to PR1/HLA-A2 specific antibody (h8F4) and promises to enhance T cell function against myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Naturally mutated envelope protein domain I of Chinese B dengue virus attenuated human dendritic cell maturation

Dengue virus (DENV) can infect human dendritic cells (DCs), and cause a spectrum of clinical symptoms. Envelope protein of DENV contains three distinct domains, including domain I (DI), domain II (DII) and domain III (DIII), and plays important roles in receptor binding and induction of protective antibodies. Previously, a new DENV-2 type virus (named B strain) with eight gene mutations in DI of the envelope protein was isolated from a dengue hemorrhagic fever patient. BALB/c mice infected with DENV B strain showed more prolonged viremia than mice infected with the New Guinea C (NGC) strain. However, the mechanism of prolonged viremia was not determined. In this study, DI proteins derived from B and NGC strains of DENV were expressed in Rosetta (DE3) host bacteria and purified by affinity chromatography after refolding. A flow cytometry-based binding assay and confocal microscopy indicated that both proteins could bind to human DCs induced from peripheral blood mononuclear cells (PBMCs), but DI of the B strain had a lower affinity than DI of the NGC strain, and viable B virus also show less binding efficiency with DCs. In addition, DI of the NGC strain, but not the B strain, induced IL-12 secretion and phenotypic maturation of DCs, such as up-regulated expression of CD80, CD83, CD86 and HLA-DR. NGC strain could induce more virus specific IgM/IgG. These results suggest that the naturally mutated envelope protein DI of the Chinese B strain of DENV cannot induce DC maturation as high efficiency as that of NGC strain, which may be the partial reason that DENV B strain escapes immune recognition and induce prolonged viremia. The mutated B strain envelope protein is not a good candidate for subunit vaccine target.

Comparative structural analysis of the glycosylation of salivary and buccal cell proteins: innate protection against infection by Candida albicans

Mucosal epithelial surfaces, such as line the oral cavity, are common sites of microbial colonization by bacteria, yeast and fungi. The microbial interactions involve adherence between the glycans on the host cells and the carbohydrate-binding proteins of the pathogen. Saliva constantly bathes the buccal cells of the epithelial surface of the mouth and we postulate that the sugars on the salivary glycoproteins provide an innate host immune mechanism against infection by competitively inhibiting pathogen binding to the cell membranes. The structures of the N- and O-linked oligosaccharides on the glycoproteins of saliva and buccal cell membranes were analyzed using capillary carbon liquid chromatography-electrospray ionization MS/MS. The 190 glycan structures that were characterized were qualitatively similar, but differed quantitatively, between saliva and epithelial buccal cell membrane proteins. The similar relative abundance of the terminal glycan epitope structures (e.g. ABO(H) blood group, sialylation and Lewis-type antigens) on saliva and buccal cell membrane glycoproteins indicated that the terminal N- and O-linked glycan substructures in saliva could be acting as decoy-binding receptors to competitively inhibit the attachment of pathogens to the surface of the oral mucosa. A flow cytometry-based binding assay quantified the interaction between buccal cells and the commensal oral pathogen Candida albicans. Whole saliva and released glycans from salivary proteins inhibited the interaction of C. albicans with buccal epithelial cells, confirming the protective role of the glycans on salivary glycoproteins against pathogen infection.

A Novel HLA-A2 Restricted Peptide Derived From Cathepsin G Is An Effective Immunotherapeutic Target for Myeloid Leukemia

Abstract 2986 Background:Immunotherapy targeting aberrantly expressed leukemia associated antigens (LAA) has shown promising results in the management of myeloid leukemia. However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. PR1 is a well-characterized LAA, is derived from the azurophil granule proteases neutrophil elastase (NE) and proteinase-3 (P3), and was effectively targeted in myeloid leukemia. We have previously shown that NE and P3 are aberrantly localized outside azurophil granules in myeloid leukemia and that their aberrant expression facilitates PR1 antigen presentation. Similar to NE and P3, cathepsin G (CG) is a serine protease located within azurophil granules in neutrophils. Because azurophil granules are disrupted in myeloid leukemia and since CG was shown to be immunogenic through its association with autoimmune disease, we hypothesized that CG may be aberrantly expressed in myeloid leukemia, thereby facilitating its presentation on HLA class I molecules and rendering it a novel target for myeloid leukemia immunotherapy. Methods:SYFPEITHI and IEDB binding algorithms were used to identify CG peptides with highest binding affinities. Flow cytometry based binding assays were performed using the T2 cell line followed by HLA-A0201 staining to confirm peptide binding and determine affinities of CG-derived peptides for HLA-A0201. Peptide/HLA-A0201 complexes were released from HLA-A0201 transfected U937 cells and purified using immunoaffinity chromatography with the anti-HLA-A0201 antibody BB7.2. The peptides were dissociated from HLA-A0201 by weak acid elution and analyzed using reverse-phase HPLC-tandem mass spectrometry. Tandem mass spectra were analyzed using the Mascot sequencing algorithm. Western blots (WB) and immunoflouresence confocal microscopy were used to determine CG expression by primary leukemia. Peptide-pulsed T2 cells were used to expand CG1- and PR1-cytotoxic T lymphocytes (CTLs) for use in calcein AM cytotoxicity assays. Peptide-pulsed T2 cells and primary leukemia from patients were used as target cells. Tetramer staining and cytokine flow cytometry (CFC) assays were used to show frequency and function of CG1-CTLs, respectively. CG1- and PP65- pulsed T2 cells were used as stimulators in CFC assays. Patient samples were collected after informed consent. Results:Five CG derived nonameric peptides were identified using SYFPEITHI and IEDB binding algorithms. CG1 peptide (FLLPTGAEA) was identified as having the highest binding affinity to HLA-A0201 in comparison with other CG-derived peptides and with PR1 peptide (CG1 IC50=1 uM vs. PR1=9 uM). The dissociation t1/2 for CG1 and PR1 peptides was > 8 hours. CG1 peptide was eluted from the surface of U937-A2 cell line, and using reverse-phase HPLC-tandem mass spectrometry, we confirmed CG1 antigen presentation by leukemia. WB analysis for CG showed high CG expression in 9 of 11 AML patient samples and along with immunofluorescent cell imaging, showed localization of CG outside granules in compartments that could render CG susceptible to proteasomal degradation and antigen presentation. Killing of primary AML blasts by CG1-CTL was shown in 6 of 8 AML patient samples and directly correlated with CG expression and HLA-A0201 status. Blocking HLA-A0201 with BB7.2 antibody abrogated CG1-CTL mediated cytotoxicity, indicating HLA-A0201 dependent killing by CG1-CTLs. CG1-specific CTLs were detected in AML patient samples at frequencies ranging between 0.6% to 1.4% of total CD8+ cells. Furthermore, following stem cell transplant (SCT), 4–10 fold higher frequencies of functional CG1-CTLs were detected in patient samples when compared with pre-SCT samples from the same patient, as measured by IFN-gamma and/or TNF-alpha CFC. Conclusion:We demonstrate that CG is a novel immunotherapeutic target in myeloid leukemia. We show that CG is a LAA that is aberrantly expressed in leukemia, is presented on HLA-A0201 molecule and can be effectively targeted by CG1-CTLs. Since immunity to CG has been previously reported in autoimmune diseases, our findings show that the immunogenic potential of CG can be redirected therapeutically to target leukemia. Cumulatively, our data suggest that further development of CG-targeting therapies in myeloid leukemia is warranted. Disclosures:No relevant conflicts of interest to declare.

Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use.

Flow Cytometry-Based Binding Assay for GPR40 (FFAR1; Free Fatty Acid Receptor 1)

GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of approximately 3 microM. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium- to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca(2+)](i) responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca(2+)](i) response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.

Interaction of antiflammin-1 with uteroglobin-binding protein induces phosphorylation of ERK1/2 in NIH 3T3 cells

Previously, it has been suggested that uteroglobin (UG)-binding protein functions as a putative receptor of UG; however, the specific epitope of UG that interacts with this receptor has not yet been identified. The downstream events of UG-binding protein signaling remain unclear. Here we report that antiflammin-1 (AF-1, a bioactive C-terminal peptide of UG) specifically binds to UG-binding protein and has a cellular signaling consequence. We reduced the level of endogenous UG-binding protein expression in murine fibroblast cell line NIH 3T3 by RNA interference and found that knockdown of UG-binding protein inhibited AF-1-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Meanwhile, the interaction between AF-1 and UG-binding protein was confirmed by flow cytometry-based binding assays and co-localization of AF-1 and enhanced green fluorescent protein (EGFP)-tagged UG-binding protein. The present study provides evidence for the first time for AF-1 binding with UG-binding protein, and preliminarily characterized UG-binding protein as a point downstream of AF-1 in mediating ERK phosphorylation.