Antibody Molecules Research Articles

Abstract 2125: Genetic analysis for classification and treatment of poorly differentiated sinonasal cancer

Abstract Introduction: The sinonasal cavities harbour a wide variety of rare tumour types, including squamous-cell carcinoma (SNSCC), intestinal-type adenocarcinoma (ITAC), olfactory neuroblastoma (ONB), neuroendocrine carcinoma (SNEC), undifferentiated carcinoma (SNUC) and malignant mucosal melanoma (MMM). Histopathological classification is related to clinical outcome, but correct diagnosis is difficult in cases with poor differentiation. Five-year overall survival ranges from 20% to 60% while therapeutic options are limited. This study aims to identify genetic alterations that may aid diagnosis and indicate targets for modern specific therapies. Experimental procedures: DNA was extracted from 80 frozen or paraffin-embedded tumor tissues collected from three different hospital centers. Mutations were screened by next generation sequencing of a panel of 120 cancer-related genes using the SureSelect QXT Target Enrichment Kit for Ilumina Multiplexed Sequencing, and recurrent hits were confirmed by Sanger sequencing and by analyzing their effect on protein expression. Results: Frequently mutated genes were TP53, KRAS, PI3K, NTRK1, ATM and BRCA1 and BRCA2. Several mutations were found exclusive to only one sinonasal tumor-type: APC and B-catenin in ITAC, EGFR exon 20 in SNSCC, IDH2 in SNUC and NF1 in MMM. With exception of ONB, a majority of cases showed a high mutational burden. Conclusions: A number of tumor-specific mutations were identified that may be used for differential diagnosis. In addition, many gene mutations may serve as targets for specific antibody or small molecule inhibitors. Finally, the finding that sinonasal tumors carry a high mutational burden suggests that therapy with immune checkpoint inhibitors may be effective in clinical management. Citation Format: Mario Hermsen, Cristina Riobello, Virginia N. Cabal, Rocío García-Marín, Sira Potes-Ares, Laura Suárez-Fernández, Blanca Vivanco, Alessandro Franchi, Reinhard Büttner, Fernando López, José Luis Llorente. Genetic analysis for classification and treatment of poorly differentiated sinonasal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2125.

Abstract 2132: Next generation sequencing for screening of clinically actionable mutations in intestinal-type sinonasal adenocarcinoma

Abstract Introduction: Sinonasal ITAC is a rare tumor commonly related to occupational wood or leather dust exposure. Clinical management has improved,however, recurrences are frequent, and the overall 5-year survival rateremains poor (60%). Therefore, there is a clear need for new therapeutic options. The aim of this study was to identify genetic alterations as specific targets for personalized treatment. Experimental procedures: Next Generation Sequencing using SureSelect QXT (Ilumina) was performed on a panel of 120 genes against which drugs have been approved by the FDA. Tumor and germline DNA of 25 patients was sequenced, for one case also the recurrence and metastasis. Pools were sequenced in a MiSeq system with a minumum coverage of 500X. To select somatic and pathogenic variants, the raw sequencing data were manually curated as follows: all silent mutations and variants with allele frequency >5% were discarded, only variants with tumor frequency >10 % of readings were considered; by comparing with the germline sample, only variants that appeared exclusively in the tumor sample were selected, as well as homozygous changes in the tumor that were heterozygous in the germline. Results: Five tumors (20%) did not show any somatic alteration in the 120 genes tested.In the remaining 20 tumors, mutations were found in 33 genes. The most relevant were involved in the EGFR-PI3K-AKT-MTOR, the MAPK-ERK and the Wnt signaling pathways, indicating an important role in ITAC tumorigenesis. The most frequently affected genes were tyrosin-kinase receptors (44%), especially the ERBB family (20% of cases with mutations) and FGFR1 (20% of tumors with gene copy number amplifications). Thirty-six percent and 28% of tumors showed alterations in the MAPK-ERK and PI3K-AKT-MTOR cascades respectively. PI3K mutations were particulary frequent (7 different variants in 5 tumors.) With regard to the Wnt pathway, both APC and CTNBB1 genes appeared mutated in 3 cases. In one patient, the primary tumor, recurrence and lymph node metastasis were investigated. All somatic alterations observed in the primary tumor (mutations in ATM, BRC2, KMT2B and amplifications of MET, ATM and FGF1R) remained present in both recurrence and metastasis, excluding the possibility of a second primary tumor. Additional mutations in the recurrence (NTRK1, TSC2 and PIK3R2) were also seen in the metastasis, while no alterations occurred in the metastasis exclusively. Confirmatory studies are underway correlating the most relevant sequencing results with gene and protein expression data of the affected pathways. Conclusions: By sequencing 120 clinically actionable genes in ITAC, several alterations were found that could be used as targets for new, personalized therapies. Examples include Dovitinib against FGFR1 amplification, antibody and small molecule inhibitors of ERBB, BRAF and MTOR, and tankyrase inhibitors targeting the Wnt pathway. Citation Format: Cristina Riobello, Virginia N. Cabal, Paula Sánchez Fernández, Rocío García-Marín, Blanca Vivanco, Fernando López, José L Llorente, Mario Hermsen. Next generation sequencing for screening of clinically actionable mutations in intestinal-type sinonasal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2132.

Abstract 1953: HER2 functionalized nanoparticles are safe and specific for in vivo HER2+ breast tumor cell detection

Abstract Superparamagnetic Relaxometry (SPMR) is a highly sensitive in vivo detection technology that can localize and quantify superparamagnetic iron oxide (Fe3O4) nanoparticles when bound to tumor cells. Nanoparticles that reach and bind to the target cells are measureable by superconducting quantum interference device (SQUID) magnetometers (MRX instrument developed in house), while unbound nanoparticles such as those freely circulating in the bloodstream are not detected and bone and normal tissue do not produce any magnetic signal. In this study we demonstrate that anti-HER2 antibody conjugated nanoparticles exhibit great specificity and selectivity towards HER2+ tumor cells, and are safe (non-toxic) in preclinical models. Anti-HER2 antibody conjugated nanoparticles consist of a 25nm Fe3O4 core covered by a polymer shell with PEG and antibody conjugated on the surface. The overall size of particles is 70-80nm with PDI <0.1 as measured by DLS. Each particle has between 3-5 antibody molecules covalently attached to the surface with PEGylation to reduce opsonization. Our results have shown that our antibody conjugated nanoparticles can distinguish high, medium and low HER2+ expression cell lines, such as BT474 (3+), ZR75 (2+), MCF7(0/1+) in an in vitro cell based assay. Furthermore, positive signal can be competed out by pre-incubation with free HER2 antibody. In additional, negative cell lines, such as MCF10 produce undetectable SPMR signal, demonstrating good sensitivity, specificity and selectivity. Incubation with PBMC indicates that the nanoparticles do not produce an SPMR signal, suggesting minimum non-specific interaction with lymphocytes. Extensive in vivo studies using xenograph tumor mouse models have shown the nanoparticles can selectively distinguish between MCF7 vs BT474 dual implanted tumors. In addition, as a control, we used PEG only nanoparticles and showed they do not generate measurable signal in tumor. Results are corroborated using other orthogonal detection methods, such as IHC and ICP-MS. The safety profile of the nanoparticles has been demonstrated by analyzing nanoparticle accumulation in organs when injected at 20mg/kg dose via intravenous, intraperitoneal and subcutaneous delivery routes. All major organs except liver do not generate measurable MRX signal at multiple time points post injection, indicating no significant accumulation of nanoparticles in major organs. A full GLP toxicity study using rats is being conducted to demonstrate the safety of the anti-HER2 antibody conjugated nanoparticle prior to first-in-human use. Together, these results suggest that our HER2 nanoparticles are safe; can provide targeted and specific delivery to cancerous tissue in vivo and generate measurable signal on our MRX detection instrument. These studies lay out ground work for our future human clinical study for breast cancer detection. Citation Format: Marie Zhang, Jose Vargas, Antimone Dewing, Farideh Bischoff, Kelsey Mathieu, John Hazle. HER2 functionalized nanoparticles are safe and specific for in vivo HER2+ breast tumor cell detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1953.

Mucosal delivery of Lactococcus lactis carrying an anti-TNF scFv expression vector ameliorates experimental colitis in mice

BackgroundAnti-Tumor Necrosis Factor-alpha therapy has become clinically important for treating inflammatory bowel disease. However, the use of conventional immunotherapy requires a systemic exposure of patients and collateral side effects. Lactic acid bacteria have been shown to be effective as mucosal delivering system for cytokine and single domain antibodies, and it is amenable to clinical purposes. Therefore, lactic acid bacteria may function as vehicles for delivery of therapeutic antibodies molecules to the gastrointestinal tract restricting the pharmacological effect towards the gut. Here, we use the mucosal delivery of Lactococcus lactis carrying an anti-TNFα scFv expression plasmid on a DSS-induced colitis model in mice.ResultsExperimental colitis was induced with DSS administered in drinking water. L. lactis carrying the scFv expression vector was introduced by gavage. After four days of treatment, animals showed a significant improvement in histological score and disease activity index compared to those of untreated animals. Moreover, treated mice display IL-6, IL17A, IL1β, IL10 and FOXP3 mRNA levels similar to health control mice. Therefore, morphological and molecular markers suggest amelioration of the experimentally induced colitis.ConclusionThese results provide evidence for the use of this alternative system for delivering therapeutic biopharmaceuticals in loco for treating inflammatory bowel disease, paving the way for a novel low-cost and site-specific biotechnological route for the treatment of inflammatory disorders.

Topoisomerase Inhibitors and Targeted Delivery in Cancer Therapy.

DNA topoisomerases are enzymes that catalyze the alteration of DNA topology with transiently induced DNA strand breakage, essential for DNA replication. Topoisomerases are validated cancer chemotherapy targets. Anticancer agents targeting Topoisomerase I and II have been in clinical use and proven to be highly effective, though with significant side effects. There are tremendous efforts to develop new generation of topoisomerase inhibitors. Targeted delivery of topoisomerase inhibitors is another way to reduce the side effects. Conjugates of topoisomerases inhibitors with antibody, polymer, or small molecule are developed to target these inhibitors to tumor sites.

Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3.

The variable domain of New Antigen Receptors (vNAR) from sharks, present special characteristics in comparison to the conventional antibody molecules such as: small size (12–15 kDa), thermal and chemical stability and great tissue penetration, that makes them a good alternative source as therapeutic or diagnostic agents. Therefore, it is essential to improve techniques used for the development and selection of vNAR antibodies that recognize distinct antigens. The development of synthetic antibody libraries offers a fast option for the generation of antibodies with the desired characteristics. In this work three synthetic antibody libraries were constructed; without cysteines (Cys), with one Cys and with two Cys residues within its CDR3, with the objective of determining whether the presence or absence of Cys in the CDR3 favors the isolation of vNAR clones from a synthetic library. The libraries were validated selecting against six mammalian proteins. At least one vNAR was found for each of the antigens, and a clone coming from the library without Cys in the CDR3 was selected with all the antigens. In vitro angiogenesis assay with the isolated anti-VEGF antibodies, suggest that these vNARs are capable of inhibiting in vitro angiogenesis. In silico analysis of anti-VEGF antibodies showed that vNARs from synthetic libraries could rival antibodies with affinity maturation by in silico modeling.

Controlled immobilization of IgG1 on carboxymethyl and amino-dextran SPR chips under external vertical electric fields

Aiming at producing surface plasmon resonance (SPR) sensor chips of improved performance, a novel approach for controlled immobilization of antibody (Ab) molecules has been developed through the implementation of surface functionalization and external vertical electric field (EVEF) assisted immobilization techniques. Carboxymethyl dextran (CMD) chips were reacted with ethylenediamine to create surface amino groups and subsequently reacted with the activated carboxyl group of the fragment crystallisable (Fc) region of Ab. An external vertical electric field was applied to control the movement of antibody molecules toward the chip and their orientation on the surface. The EVEF imparted a current density of 4.17 μA cm−2 through the solution. Immobilization was achieved via a carbodiimide reaction as the antibody molecules reached the functionalized surface. Physical characterization was performed through ATR-FTIR, AFM and surface plasmon resonance (SPR) analyses. Results show that the orientation of antibody molecule on the amino dextran (AmDex) surface is better than on the CMD surface in terms of antibody accessibility for interaction with toxin molecule. Moreover, applying EVEF during the antibody immobilization stage leads to an increased density of the antibody layer and enhanced accessibility of antibody binding domain due to improved orientation of antibody in the direction of the electric field. Discrete-Time-Fourier-Transformation analysis of the chip surface line profiles obtained by AFM analysis clearly show an increased order of Ab molecules after implementation of EVEF. The new findings obtained in this study are expected to be useful for controlling Ab molecule orientation on the immobilizing surface.

Suitability of a generic virus safety evaluation for monoclonal antibody investigational new drug applications.

Biologics produced from CHO cell lines with endogenous virus DNA can produce retrovirus-like particles in cell culture at high titers, and other adventitious viruses can find their way through raw materials into the process to make a product. Therefore, it is the industry standard to have controls to avoid introduction of viruses into the production process, to test for the presence of viral particles in unclarified cell culture, and to develop purification procedures to ensure that manufacturing processes are robust for viral clearance. Data have been accumulated over the past four decades on unit operations that can inactivate and clear adventitious virus and provide a high degree of assurance for patient safety. During clinical development, biological products are traditionally tested at process set points for viral clearance. However, the widespread implementation of platform production processes to produce highly similar IgG antibodies for many indications makes it possible to leverage historical data and knowledge from representative molecules to allow for better understanding and control of virus safety. More recently, individualized viral clearance studies are becoming the rate-limiting step in getting new antibody molecules to clinic, particularly in Phase 0 and eIND situations. Here, we explore considerations for application of a generic platform virus clearance strategy that can be applied for relevant investigational antibodies within defined operational parameters in order to increase speed to the clinic and reduce validation costs while providing a better understanding and assurance of process virus safety.

Hierarchically structured emulsions for brain therapy

In this paper, we developed a doxorubicin-loaded double emulsion, with a specific pH-triggered drug release mechanism, and with functionalized drops’ surfaces as an effective drug (doxorubicin-DOX) delivery implant for brain tumor therapy. The functionalization of double emulsions with antibodies at oil-water interfaces was performed for targeting brain tumor cells - glioblastoma multiforme (GBM). The functionalization involved physical adsorption of antibodies: anti-CD15 (GBM-targeting surface proteins) on oil drops of double water-in-oil-in-water emulsions. The interactions between molecules of antibodies and compounds of the oil drops were measured using isothermal titration calorimetry (ITC). Double emulsions with different structures and pH-sensitive polymer (sodium carboxymethylcellulose) in the external phase were prepared by a one-step method, in a Couette-Taylor flow contactor. The release rates of DOX from emulsions were analysed in phosphate buffers with a pH of 6.3 (acidic microenvironment of solid tumors) and a pH of 7.4 (normal cells). The release study demonstrated an expected morphology/structure, composition and pH-responsive controlled release behaviour. The ITC analysis showed that antibodies interacted with emulsion compounds, which was vital for functionalization of drops of emulsion for targeting cancer cells. This was demonstrated by changes in the heat rates reflecting molecular interactions and proved that proteins adhered to oil-drop interfaces.

Developing a flow cytometry-based quantitative ido assay to measure immune potency of mesenchymal stromal cells product for phase i clinical trial

Background & Aim Establishment of a potency assay in the manufacturing of clinical grade mesenchymal stromal cells (MSCs) has been a challenge due to the issues of variability in starting cellular material, complex mechanisms of action, and lack of reference cells or materials. Indoleamine 2, 3-dioxygenase (IDO) is one of the most important factors for human MSC-mediated immunosuppression. IDO is expressed in IFN-γ-licensed MSC, but not in resting MSC in vitro. This intracellular enzyme catalyzes tryptophan degradation toward kynurenine and depletion of tryptophan suppresses T cells proliferation. Methods, Results & Conclusion In this study, we have developed a flow cytometry-based IDO assay for quantitatively measuring the IDO expression level per cell, the IPBV (Intracellular Protein Binding Value) of the cells represents the relative quantity of IDO expressed in the MSC. IPBV is defined as the number of anti-IDO fluorescent antibody molecules bound to intracellular IDO and standardized by fluorescence intensity of beads. Two kinds of in-house quality control reference cells were running in parallel with product to ensure that the assay is performing as expected and the level of IDO expression. Validation of the quantitative IDO assay, including accuracy, precision, specificity, linearity, range and robustness, were presented in this study. Moreover, we presented the application of the quantitative IDO assay for analysis of MSC product stability, comparability, and compatibility in the manufacturing of clinical grade MSC-based product- RegStem® ASC. In this study, we demonstrated the flow cytometry-based quantitative MSC IDO assay correlates with MSC's inhibition of autologous T cells proliferation, as well as the correlation with preliminary clinical effects in the phase I autologous MSC-based clinical trial for the treatment of Osteoarthritis. As the results, we believe that our novel flow cytometry-based quantitative IDO assay can be a potential potency assay candidate for advanced phase clinical trial of MSCs products.

Fabrication of a polypropylene immunoassay platform by photografting reaction

The technology of an immunoassay detection platform is critical to clinical disease diagnoses, especially for developing a medical diagnostic system. A polymer-based immunoassay platform was fabricated on nonwoven fabric polypropylene (PP) using a photografting reaction to graft 2-hydroxyethyl methacrylate (HEMA) and sulfobetaine (SBMA). The antifouling properties of PP-g-P(HEMA-co-SBMA) were investigated by fibrinogen adsorption and platelet adhesion. Carbonyldiimidazole was employed to activate the pendant hydroxyl groups in HEMA moieties and covalently coupled antibody molecules. The detection of the limit of the immunoassay platform was as low as 10 pg/mL. Antibody amount and bioactivity affected the availability of antibody and the sensitivity of immunoassay. The immune efficiency was dependent on the strategies of antibody immobilization. The immune efficiency of Au-g-P(SBMA-co-HEMA) and Au-SH surfaces measured by QCM-D was 165% and 35.7%, respectively. The covalently binding antibody via hydrophilic polymer chains as spacers could retain fragment antigen-binding up orientation, maintain the bioactivity of antibody, and mainly improve the accessibility of antibody molecules via adjusting the conformations of polymer chains when the antibodies recognized the antigens. Therefore, grafting hydrophilic polymers, such as zwitterionic PSBMA and reactive PHEMA onto nonwoven fabric PP, and binding antibody by covalent strategy had the potential to be developed as a commercial immunoassay platform.

The Ligands for Human IgG and Their Effector Functions.

Activation of the humoral immune system is initiated when antibodies recognize an antigen and trigger effector functions through the interaction with Fc engaging molecules. The most abundant immunoglobulin isotype in serum is Immunoglobulin G (IgG), which is involved in many humoral immune responses, strongly interacting with effector molecules. The IgG subclass, allotype, and glycosylation pattern, among other factors, determine the interaction strength of the IgG-Fc domain with these Fc engaging molecules, and thereby the potential strength of their effector potential. The molecules responsible for the effector phase include the classical IgG-Fc receptors (FcγR), the neonatal Fc-receptor (FcRn), the Tripartite motif-containing protein 21 (TRIM21), the first component of the classical complement cascade (C1), and possibly, the Fc-receptor-like receptors (FcRL4/5). Here we provide an overview of the interactions of IgG with effector molecules and discuss how natural variation on the antibody and effector molecule side shapes the biological activities of antibodies. The increasing knowledge on the Fc-mediated effector functions of antibodies drives the development of better therapeutic antibodies for cancer immunotherapy or treatment of autoimmune diseases.

Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones

PfEMP1 is a family of adhesive proteins expressed on the surface of Plasmodium falciparum-infected erythrocytes (IEs), where they mediate adhesion of IEs to a range of host receptors. Efficient PfEMP1-dependent IE sequestration often depends on soluble serum proteins, including IgM. Here, we report a comprehensive investigation of which of the about 60 var gene-encoded PfEMP1 variants per parasite genome can bind IgM via the Fc part of the antibody molecule, and which of the constituent domains of those PfEMP1 are involved. We erased the epigenetic memory of var gene expression in three distinct P. falciparum clones, 3D7, HB3, and IT4/FCR3 by promoter titration, and then isolated individual IEs binding IgM from malaria-unexposed individuals by fluorescence-activated single-cell sorting. The var gene transcription profiles of sub-clones measured by real-time qPCR were used to identify potential IgM-binding PfEMP1 variants. Recombinant DBL and CIDR domains corresponding to those variants were tested by ELISA and protein arrays to confirm their IgM-binding capacity. Selected DBL domains were used to raise specific rat anti-sera to select IEs with uniform expression of candidate PfEMP1 proteins. Our data document that IgM-binding PfEMP1 proteins are common in each of the three clones studied, and that the binding epitopes are mainly found in DBLε and DBLζ domains near the C-terminus.

Lanthanide-Loaded Nanoparticles as Potential Fluorescent and Mass Probes for High-Content Protein Analysis.

Multiparametric and high-content protein analysis of single cells or tissues cannot be accomplished with the currently available flow cytometry or imaging techniques utilizing fluorophore-labelled antibodies, because the number of spectrally resolvable fluorochromes is limited. In contrast, mass cytometry can resolve more signals by exploiting lanthanide-tagged antibodies; however, only about 100 metal reporters can be attached to an antibody molecule. This makes the sensitivity of lanthanide-tagged antibodies substantially lower than fluorescent reporters. A new probe that can carry more lanthanide molecules per antibody is a desirable way to enhance the sensitivity needed for the detection of protein with low cellular abundance. Herein, we report on the development of new probes utilizing mesoporous silica nanoparticles (MSNPs) with hydroxyl, amine, or phosphonate functional groups. The phosphonated MSNPs proved to be best at loading lanthanides for up to 1.4 × 106 molecules per particle, and could be loaded with various lanthanide elements (Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, and Lu) at relatively similar molar extents. The modified MSNPs can also load a fluorescent dye, allowing bimodal mass and fluorescence-based detection. We achieved specificity of antibody-conjugated nanoparticles (at 1.4 × 103 antibodies per nanoparticle) for targeting proteins on the cell surface. The new materials can potentially be used as mass cytometry probes and provide a method for simultaneous monitoring of a large host of factors comprising the tumor microenvironment (e.g., extracellular matrix, cancer cells, and immune cells). These novel probes may also benefit personalized medicine by allowing for high-throughput analysis of multiple proteins in the same specimen.

Detection of the Causative Agent of Tularemia by Surface Plasmon Resonance

A method for detection of causative agent of tularemia in aqueous solutions by surface plasmon resonance spectrometry was proposed. The kinetics of immobilization of antibodies to Francisella tularensis on the surface of gold-plated glass chips was studied. Based on a quantitative analysis of the results, it was concluded that the antibody molecules were predominantly laterally oriented on the surface. This conclusion was confirmed by analysis of sensorgrams of the subsequent antigen–antibody interaction. It is shown that the proposed method can reliably determine the presence of the causative agent of tularemia in aqueous solutions with a sensitivity of 1.0 × 102 cell/mL for several minutes. Various options for improving this method for detection of the tularemia causative agent to increase its sensitivity have been proposed.

Development of Chemically Signal Amplified Nano-Biosensor Mediated by Poly-L-Lysine.

Amyloid β (Aβ) is considered to be one of a potential biomarker to monitor Alzheimer's Disease (AD) not only for diagnostic purposes but for early detection. Here we describe a novel nano-biosensor for Aβ mediated by poly-L-lysine (PLL) which was used for the amplification of detection signal for Aβ. The indirect enzyme-linked immunosorbent assay (ELISA) method was modified using PLL for the amplification of the Aβ detection signal. A commercially available ELISA plate was modified by PLL using chemical agent and the amplified amino groups were activated by a chemical agent for the detection of Aβ. The detection was carried out by the traditional immunochemistry using primary antibody and fluorescence molecules conjugated secondary antibody. In the result, the fluorescence intensity was increased by the increasing treated Aβ amount, and the sensitivity was approximately 2 times higher in the concentration of 2 ng/mL Aβ treatment, and approximately 4 times higher in the concentration of 200 ng/mL Aβ treatment compare with that of indirect ELISA detection method. We suggest our novel signal amplification method for the Aβ early detection.

Paired-agent imaging for detection of head and neck cancers.

Head and neck cancers overwhelmingly overexpress epidermal growth factor receptor (EGFR). This overexpression has been utilized for head and neck cancers using molecular targeted agents for therapy and cancer cell detection. Significant progress has been made in using EGFR-targeted fluorescent antibody and Affibody molecule agents for fluorescent guided surgery in head and neck cancers. Although success in achieving tumor-to-background ratio of 3-5 have been achieved, the field is limited by the non-specific fluorescence in normal tissues as well as EGFR specific fluorescence in the oral cavity. We propose that paired-agent imaging (PAI) could improve the contrast between tumor and normal tissue by removing the fluorescent signal arising from non-specific binding. Here, ABY-029 - an anti-EGFR Affibody molecule labeled with IRDye 800CW - and IRDye 680RD conjugated to Affibody Control Imaging Agent molecule (IR680-Affctrl) are used as targeted and untargeted control agents, respectively, in a panel of head and neck squamous cell carcinomas (HNSCC) to test the ability of PAI to increase tumor detection. Initial results demonstrate that binding potential, a value proportional to receptor concentration, correlates well to EGFR expression but experimental limitations prevented pixel-by-pixel analysis that was desired. Although promising, a more rigorous and well-defined experimental protocol is required to align ex vivo EGFR immunohistochemistry with in vivo binding potential and fluorescence intensity. Additionally, a new set of paired-agents, ABY-029 and IRDye 700DX, are successfully tested in naïve mice and will be carried forward for clinical translation.

Multimerization Increases Tumor Enrichment of Peptide⁻Photosensitizer Conjugates.

Photodynamic therapy (PDT) is an established therapeutic modality for the management of cancers. Conjugation with tumor-specific small molecule ligands (e.g., short peptides or peptidomimetics) could increase the tumor targeting of PDT agents, which is very important for improving the outcome of PDT. However, compared with antibody molecules, small molecule ligands have a much weaker affinity to their receptors, which means that their tumor enrichment is not always ideal. In this work, we synthesized multimeric RGD ligand-coupled conjugates of pyropheophorbide-a (Pyro) to increase the affinity through multivalent and cluster effects to improve the tumor enrichment of the conjugates. Thus, the dimeric and trimeric RGD peptide-coupled Pyro conjugates and the monomeric one for comparison were efficiently synthesized via a convergent strategy. A short polyethylene glycol spacer was introduced between two RGD motifs to increase the distance required for multivalence. A subsequent binding affinity assay verified the improvement of the binding towards integrin αvβ3 receptors after the increase in the valence, with an approximately 20-fold improvement in the binding affinity of the trimeric conjugate compared with that of the monomeric conjugate. In vivo experiments performed in tumor-bearing mice also confirmed a significant increase in the distribution of the conjugates in the tumor site via multimerization, in which the trimeric conjugate had the best tumor enrichment compared with the other two conjugates. These results indicated that the multivalence interaction can obviously increase the tumor enrichment of RGD peptide-conjugated Pyro photosensitizers, and the prepared trimeric conjugate can be used as a novel antitumor photodynamic agent with high tumor enrichment.

An origami paper-based electrochemical immunoassay for the C-reactive protein using a screen-printed carbon electrode modified with graphene and gold nanoparticles

An origami paper-based electrochemical immunoassay for C-reactive protein (CRP) detection is described. The assay integrates multiple steps of electrode modification into a single device. A graphene-modified screen-printed carbon electrode (G/SPCE) was employed to enhance sensitivity. Gold nanoparticles were first electrodeposited onto the G/SPCE, followed by a self-assembled monolayer of L-cysteine. The capture anti-CRP was then covalently immobilized on the modified electrode. CRP was quantified by measuring the changes in the charge-transfer resistance of the electrode by using hexacyanoferrate as the redox probe. Cyclic voltammetry and scanning electron microscopy were also applied to verify the successful modification of the electrode. Under optimal conditions, impedance increase in the 0.05-100μgmL-1 CRP concentration range, and the limit of detection is 15ngmL-1 (at S/N = 3). The immunoassay was successfully applied to the determination of CRP in a certified human serum sample. This method is simple, low-cost, portable and disposable. Graphical abstract An origami paper-based analytical device (oPAD) is described that integrates the multistep of electrode modification, immobilization and detection into a single device. The direct conjugation between the capture antibody and target molecule was allowed to use in this system. The C-reactive protein (CRP) concentration in serum samples was determined using electrochemical impedance spectroscopy.

Abstract B026: Development of tumor-targeted agonistic Immunomodulators using the DARPin® technology platform

Abstract Background: Antibodies against several agonistic immunomodulatory receptors proved to be highly efficacious in preclinical tumor models especially in combination with other immunomodulatory compounds and treatment modalities. However, their clinical application met with limited success and was associated with systemic toxicities which are believed to be driven by Fc-receptor-mediated cross-linking of antibody molecules. We developed a new class of biologics based on the DARPin® technology platform, which allow tumor-localized engagement of immunomodulatory agonists in the absence of Fc-receptors. Preclinical in vitro and in vivo results indicate that DARPin®-based compounds are at least comparable to conventional antibodies in agonistic potency. The localization of their activity to the tumor site relative to the systemic activity of agonistic MAbs should improve the safety and therefore the therapeutic index for drug candidates which exploit this mode of action. Methods: We generated multi-specific DARPin® therapeutics, which bind to human fibroblast activation protein (FAP) and either to 4-1BB, OX40 or CD40 receptors. Each of these receptors can effectively enhance immune responses through a set of distinct and non-redundant mechanisms. Functional properties of these molecules were characterized using appropriate sets of biochemical methods and cell-based assays utilizing reporter cells lines or various subsets of primary immune cells. The FAP-targeted 4-1BB agonist referred to as MP0310, which represents our most advanced drug candidate, was also characterized in vivo in terms of its pharmacokinetics and pharmacodynamics in mice as well as cynomolgus monkeys. The in vivo efficacy of MP0310 was analyzed both in monotherapy and in combination with tumor-specific T-cell engagers using a xenotransplantation human tumor model of immunodeficient mice engrafted with human PBMCs. Results and Conclusions: All analyzed FAP-targeted DARPin®-based agonistic immunomodulators efficiently induced activation of the NFkB pathway in reporter cell lines in a dose dependent manner but only upon crosslinking through binding to FAP. The same pattern of functional activity was exhibited by the molecules in assays with appropriately selected human primary immune cell subsets. We show that MP0310 promotes tumor regression in a dose dependent manner when co-administered with a tumor-specific T-cell engaging bi-specific antibody into tumor-bearing immunodeficient NGS mice engrafted with human PBMCs. The observed effect correlated well with 4-1BB receptor occupancy in blood and an increase of CD8 T lymphocytes in the tumors. Urelumab, a clinically tested 4-1BB agonistic antibody associated with liver toxicity, led to signs of severe graft-versus-host disease (GVHD) and weight loss in the humanized NOG mice. In contrast, mice treated with several doses of MP0310 showed only minor signs of GVHD and marginal weight loss. Thus, tumor targeted DARPin® agonists of co-stimulatory molecules can enhance T-cell activity in a manner independent of Fc-crosslinking and localized primarily to the tumor site, thereby avoiding the systemic toxicity seen with antibodies against the same immunologic targets. This approach with DARPin® therapeutics can be broadly applied to a range of tumor targets and immune receptors. Citation Format: Victor Levitsky, Alexand Link, Michael T. Stump, Elmar vom Baur. Development of tumor-targeted agonistic Immunomodulators using the DARPin® technology platform [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B026.