Antibody Candidates Research Articles

Abstract 1826: Comprehensive preclinical study on GI-101, a novel CD80-IgG4-IL2 variant protein, as a therapeutic antibody candidate with bispecific immuno-oncology target

Abstract Introduction: GI-101 was designed to address the significant unmet needs in immunotherapy for noninflamed tumor. The harmonized mechanisms of action consist of the extracellular domain of CD80 acting as a CTLA-4 inhibitor, together with a long-acting IL-2 variant that preferentially binds to the IL2Rβ. Therefore, GI-101 can play a role in the activation of cytotoxic immune cells and inhibition of CTLA-4-B7.1 axis-based immune suppression. Methods: The binding affinity of GI-101 to IL-2Rs, CTLA-4, and CD28 was performed by SPR and immune cell proliferation was analyzed by CFSE assay in vitro. GI-101 induced dose-dependent pharmacodynamics effects with consistent magnitude following repeat administration in monkeys. Direct anti-tumor effect of GI-101 was tested by single or combination treatment manner in multiple syngeneic and humanized models. Immune profiling in TME was analyzed by flow cytometry, IHC and IFN-γ ELISPOT assay. To mimic the standard care (SOC) in clinic, TC1 lung cancer model was involved in evaluating the efficacy of both 1st line and maintenance therapy of GI-101 with or without immuno-chemotherapy (Cisplatin, Pemetrexed, and anti-PD-1). Results: CD80 of GI-101 highly binds to CTLA-4 (Kd, 2.9nM), acting as a decoy ligand. IL-2 variant induces CD8+ T and NK cell proliferation. However, GI-101 had no evidence of toxicity related to IL-2 activity in the non-GLP monkey study, including vascular leakage syndrome and cytokine storm. GI-101 elicits improved restoration of immune functions in human PBMCs co-cultured setting with PD-L1/CTLA-4 co-expressed tumor cells. A dose-dependent (3 to 12 mg/kg) single inhibition of tumor growth was observed in CT26 syngeneic model. Immune profiling revealed a robust increase of M1 macrophages, CD8+ central memory T (Tcm) and NK cells but not Tregs in TME. Splenocyte tumor-specific immune cells were strongly proliferated when stimulated with CT26 neoantigens (gp70). IFN-γ+ T cells were significantly increased in draining lymph nodes from GI-101 treated mice. Furthermore, GI-101 was superior at inhibiting tumor growth when co-treated with anti-PD-1 in syngeneic (MC38, TC1, and B16F10) and MDA-MB-231 humanized mice models. Finally, the combination of GI-101 and immuno-chemotherapy showed not only suppressed tumor growth but also improved survival compared to immuno-chemotherapy alone. Conclusion: The complementary modes of action of GI-101 via checkpoint blockade and IL-2 activity to enhance the proliferation and activation of Tcm and NK cells are projected to translate into superior clinical efficacy and safety as indicated even in ‘cold tumor' models. GI-101 has promising potential to replace the first-generation ICBs as a monotherapy or in combination with other immunotherapies. Our findings provide a rationale for further clinical investigations. Citation Format: Kyoung-Ho Pyo, Young Jun Koh, Chun-Bong Synn, Jae Hwan Kim, Youngseon Byeon, Ha Ni Jo, Young Seob Kim, Wongeun Lee, Do Hee Kim, Seul Lee, Dong Kwon Kim, Eun ji Lee, Beung-Chul Ahn, Min Hee Hong, Myoung Ho Jang, Sun Min Lim, Hye Ryun Kim, Su Youn Nam, Byoung Chul Cho. Comprehensive preclinical study on GI-101, a novel CD80-IgG4-IL2 variant protein, as a therapeutic antibody candidate with bispecific immuno-oncology target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1826.

Developing a multivariate prediction model of antibody features associated with protection of malaria-infected pregnant women from placental malaria.

Plasmodium falciparum causes placental malaria, which results in adverse outcomes for mother and child. P. falciparum-infected erythrocytes that express the parasite protein VAR2CSA on their surface can bind to placental chondroitin sulfate A. It has been hypothesized that naturally acquired antibodies towards VAR2CSA protect against placental infection, but it has proven difficult to identify robust antibody correlates of protection from disease. The objective of this study was to develop a prediction model using antibody features that could identify women protected from placental malaria. We used a systems serology approach with elastic net-regularized logistic regression, partial least squares discriminant analysis, and a case-control study design to identify naturally acquired antibody features mid-pregnancy that were associated with protection from placental malaria at delivery in a cohort of 77 pregnant women from Madang, Papua New Guinea. The machine learning techniques selected 6 out of 169 measured antibody features towards VAR2CSA that could predict (with 86% accuracy) whether a woman would subsequently have active placental malaria infection at delivery. Selected features included previously described associations with inhibition of placental binding and/or opsonic phagocytosis of infected erythrocytes, and network analysis indicated that there are not one but multiple pathways to protection from placental malaria. We have identified candidate antibody features that could accurately identify malaria-infected women as protected from placental infection. It is likely that there are multiple pathways to protection against placental malaria. This study was supported by the National Health and Medical Research Council (Nos. APP1143946, GNT1145303, APP1092789, APP1140509, and APP1104975).

Ultradilute Measurements of Self-Association for the Identification of Antibodies with Favorable High-Concentration Solution Properties.

There is significant interest in formulating antibody therapeutics as concentrated liquid solutions, but early identification of developable antibodies with optimal manufacturability, stability, and delivery attributes remains challenging. Traditional methods of identifying developable mAbs with low self-association in common antibody formulations require relatively concentrated protein solutions (>1 mg/mL), and this single challenge has frustrated early-stage and large-scale identification of antibody candidates with drug-like colloidal properties. Here, we describe charge-stabilized self-interaction nanoparticle spectroscopy (CS-SINS), an affinity-capture nanoparticle assay that measures colloidal self-interactions at ultradilute antibody concentrations (0.01 mg/mL), and is predictive of antibody developability issues of high viscosity and opalescence that manifest at four orders of magnitude higher concentrations (>100 mg/mL). CS-SINS enables large-scale, high-throughput selection of developable antibodies during early discovery.

A Bioluminescent Biosensor for Quantifying the Interaction of SARS-CoV-2 and Its Receptor ACE2 in Cells and In Vitro.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus–host interactions in order to quickly evaluate the infectious ability of mutant viruses and to develop or validate virus-inhibiting drugs. Here, we developed an ultrasensitive bioluminescent biosensor to evaluate virus–cell interactions by quantifying the interaction between the SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid, and HTP biosensor tool will significantly expedite the detection of viral mutants and the anti-COVID-19 drug discovery process.

Modeling the impact of amino acid substitution in a monoclonal antibody on cation exchange chromatography.

A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early-phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate. While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time-to-market pressure and an abundance of monoclonal antibodies (mAbs) in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform. This study investigates the influence of amino acid substitutions in the complementarity-determining region (CDR) of a full-length IgG1 mAb on the elution behavior in preparative cation exchange chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared with the wild-type antibody. Furthermore, the substitution of tryptophan with lysine in the H-CDR3 increased charge heterogeneity of the product. A multiscale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing.

Exploring antibody repurposing for COVID-19: beyond presumed roles of therapeutic antibodies

The urgent need for a treatment of COVID-19 has left researchers with limited choice of either developing an effective vaccine or identifying approved/investigational drugs developed for other medical conditions for potential repurposing, thus bypassing long clinical trials. In this work, we compared the sequences of experimentally verified SARS-CoV-2 neutralizing antibodies and sequentially/structurally similar commercialized therapeutic monoclonal antibodies. We have identified three therapeutic antibodies, Tremelimumab, Ipilimumab and Afasevikumab. Interestingly, these antibodies target CTLA4 and IL17A, levels of which have been shown to be elevated during severe SARS-CoV-2 infection. The candidate antibodies were evaluated further for epitope restriction, interaction energy and interaction surface to gauge their repurposability to tackle SARS-CoV-2 infection. Our work provides candidate antibody scaffolds with dual activities of plausible viral neutralization and immunosuppression. Further, these candidate antibodies can also be explored in diagnostic test kits for SARS-CoV-2 infection. We opine that this in silico workflow to screen and analyze antibodies for repurposing would have widespread applications.

Development of an Antigen-Antibody Co-Display System for Detecting Interaction of G-Protein-Coupled Receptors and Single-Chain Variable Fragments.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.

A method for systematically ranking therapeutic drug candidates using multiple uncertain screening criteria.

Multiple different screening tests for candidate leads in drug development may often yield conflicting or ambiguous results, sometimes making the selection of leads a nontrivial maximum-likelihood ranking problem. Here, we employ methods from the field of multiple criteria decision making (MCDM) to the problem of screening candidate antibody therapeutics. We employ the SMAA-TOPSIS method to rank a large cohort of antibodies using up to eight weighted screening criteria, in order to find lead candidate therapeutics for Alzheimer’s disease, and determine their robustness to both uncertainty in screening measurements, as well as uncertainty in the user-defined weights of importance attributed to each screening criterion. To choose lead candidates and measure the confidence in their ranking, we propose two new quantities, the Retention Probability and the Topness, as robust measures for ranking. This method may enable more systematic screening of candidate therapeutics when it becomes difficult intuitively to process multi-variate screening data that distinguishes candidates, so that additional candidates may be exposed as potential leads, increasing the likelihood of success in downstream clinical trials. The method properly identifies true positives and true negatives from synthetic data, its predictions correlate well with known clinically approved antibodies vs. those still in trials, and it allows for ranking analyses using antibody developability profiles in the literature. We provide a webserver where users can apply the method to their own data: http://bjork.phas.ubc.ca.

Similarity assessment by multivariate statistics method based on the distance between biosimilar and originator

The development of biosimilar products or follow-on biologics has been flourishing in recent years because of their lower price than the originators. In this study, a multivariate data analysis method based on JMP software was proposed to assess the glycosylation pattern similarity of antibody candidates from different conditions in optimization experiments with a reference. A specific distance was generated by this method and indicated the glycoform similarity between the biosimilar and the reference. This method can be applied to analyze the similarity of other physicochemical and functional characteristics between follow-on biologics and originators. Then, the design of experimental methods can be realized to optimize the conditions of cell culture to attain similar antibody candidates. A higher concentration of GlcNAc added to the basal media made the glycan of the antibody more similar to the glycan of the reference in this study.

CHOK1SV GS-KO SSI expression system: A combination of the Fer1L4 locus and glutamine synthetase selection.

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates. Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene. The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1.

Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-dependent phospholipid-binding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.

Potential neutralizing antibodies discovered for novel corona virus using machine learning

The fast and untraceable virus mutations take lives of thousands of people before the immune system can produce the inhibitory antibody. The recent outbreak of COVID-19 infected and killed thousands of people in the world. Rapid methods in finding peptides or antibody sequences that can inhibit the viral epitopes of SARS-CoV-2 will save the life of thousands. To predict neutralizing antibodies for SARS-CoV-2 in a high-throughput manner, in this paper, we use different machine learning (ML) model to predict the possible inhibitory synthetic antibodies for SARS-CoV-2. We collected 1933 virus-antibody sequences and their clinical patient neutralization response and trained an ML model to predict the antibody response. Using graph featurization with variety of ML methods, like XGBoost, Random Forest, Multilayered Perceptron, Support Vector Machine and Logistic Regression, we screened thousands of hypothetical antibody sequences and found nine stable antibodies that potentially inhibit SARS-CoV-2. We combined bioinformatics, structural biology, and Molecular Dynamics (MD) simulations to verify the stability of the candidate antibodies that can inhibit SARS-CoV-2.

Accelerating target deconvolution for therapeutic antibody candidates using highly parallelized genome editing

Therapeutic antibodies are transforming the treatment of cancer and autoimmune diseases. Today, a key challenge is finding antibodies against new targets. Phenotypic discovery promises to achieve this by enabling discovery of antibodies with therapeutic potential without specifying the molecular target a priori. Yet, deconvoluting the targets of phenotypically discovered antibodies remains a bottleneck; efficient deconvolution methods are needed for phenotypic discovery to reach its full potential. Here, we report a comprehensive investigation of a target deconvolution approach based on pooled CRISPR/Cas9. Applying this approach within three real-world phenotypic discovery programs, we rapidly deconvolute the targets of 38 of 39 test antibodies (97%), a success rate far higher than with existing approaches. Moreover, the approach scales well, requires much less work, and robustly identifies antibodies against the major histocompatibility complex. Our data establish CRISPR/Cas9 as a highly efficient target deconvolution approach, with immediate implications for the development of antibody-based drugs.

Plasmid DNA encoding neutralizing human monoclonal antibody without enhancing activity protects against dengue virus infection in mice

Objective: To evaluate the expression of DNA plasmid-harboring modified antibody gene that produces neutralizing human monoclonal antibodies against four serotypes of dengue virus (DENV) without enhancing activity in BALB/c mice.Methods: We constructed pFUSE-based vectors (pFUSE_1G7C2_ hVH and pFUSE_1G7C2_hVL) containing genes encoding the variable domains of the heavy or light chain of the anti-dengue virus antibody 1G7C2, a human IgG1 that has been characterized for its neutralizing activity to DENV-1-4. Leucine (L) at positions 234 and 235 on the Fc CH2 domain in pFUSE_1G7C2_hVH was mutated to alanine (A) (LALA mutation) by site direct mutagenesis, and the new plasmid was termed pFUSE_1G7C2_hVH_LALA. An equal amount of pFUSE_1G7C2_hVL and 1G7C2_hG1-LALA plasmids were co-transfected into Chinese hamster ovary cells (CHO-K1) and a single dose of 100 μg 1G7C2_hG1-LALA plasmid was intramuscularly injected, followed by electroporation in BALB/c mice. The secreted 1G7C2_hG1-LALA antibodies in cell culture supernatant and mouse serum were examined for their biological functions, neutralization and enhancing activity.Results: The co-transfection of heavy- and light-chain 1G7C2_ hG1-LALA plasmids in CHO-K1 cells produced approximately 3 900 ng/mL human IgG and neutralized 90%-100% all four DENV, with no enhancing activity. Furthermore, the modified human IgG was produced more than 1 000 ng/mL in mouse serum on day 7 post plasmid injection and showed cross-neutralization to four DENV serotypes. Subsequently, antibody production and neutralization decreased rapidly. Nevertheless, the secreted neutralizing 1G7C2_ hG1-LALA in mouse serum demonstrated complete absence of enhancing activities to all DENV serotypes.Conclusions: These findings reveal that a new modified 1G7C2_ hG1-LALA expressing plasmid based on gene transfer is a possible therapeutic antibody candidate against DENV infection.

Optimization of therapeutic antibodies.

In this review, we have summarized the current landscape of therapeutic antibody optimization for successful development. By engineering antibodies with display technology, computer-aided design and site mutagenesis, various properties of the therapeutic antibody candidates can be improved with the purpose of enhancing their safety, efficacy and developability. These properties include antigen binding affinity and specificity, biological efficacy, pharmacokinetics and pharmacodynamics, immunogenicity and physicochemical developability features. A best-in-class strategy may require the optimization of all these properties to generate a good therapeutic antibody.

Therapeutic Antibodies Targeting Potassium Ion Channels.

Monoclonal antibodies combine specificity and high affinity binding with excellent pharmacokinetic properties and are rapidly being developed for a wide range of drug targets including clinically important potassium ion channels. Nonetheless, while therapeutic antibodies come with great promise, K+ channels represent particularly difficult targets for biologics development for a variety of reasons that include their dynamic structures and relatively small extracellular loops, their high degree of sequence conservation (leading to immune tolerance), and their generally low-level expression in vivo. The process is made all the more difficult when large numbers of antibody candidates must be screened for a given target, or when lead candidates fail to cross-react with orthologous channels in animal disease models due to their highly selective binding properties. While the number of antibodies targeting potassium channels in preclinical or clinical development is still modest, significant advances in the areas of protein expression and antibody screening are converging to open the field to an avalanche of new drugs. Here, the opportunities and constraints associated with the discovery of antibodies against K+ channels are discussed, with an emphasis on novel technologies that are opening the field to exciting new possibilities for biologics development.

Comparative analysis of candidate vaccines to prevent covid 19 pandemic

Covid-19 which is a SARS-CoV-2 (severe acute respiratory syndrome) has caused the new overall pandemic moreover is an arising virus profoundly contagious namely coronavirus. The unavailability of a particular antiviral treatment had led to the utmost destruction of life because of this virus. During this time, impressive efforts were placed into creating safe medications and immunizations against SARS-CoV-2. Just 56 vaccines made it at the different clinical stages from more than 80 clinical trials which had started including 23 antibody candidates got looked at moreover had affirmed for the use. Various types of variables are utilized for the production of such vaccines to start the immune reaction to produce antibodies in a person for killing the virus. The countries which are top of the race for producing vaccines are Russia, India, U.S.A., China as well U.K. Among the vaccines produced by these countries are Covaxin (India), Sputnik V (Russia), CoronaVac (China), AZD1222 (United Kingdom), BNT162b2 (Germany), and mRNA-1273(United States of America). We bring forth the certain potential factors that are required when creating vaccines as well as a comparative analysis of data obtained from SARS-CoV-2 vaccine trials for different vaccines as well as environmental impacts of its preparation.

Screening and Identification of a Novel Anti-siglec-15 Human Antibody 3F1 and the Research on Its Antitumor Activity

Siglec-15, as an important immunosuppressive molecule, is considered as an important target in the next generation of tumor immunotherapy. In this study, we screened multiple high affinity antibodies that specifically recognized human siglec-15 by using human phage antibody library with large capacity, and verified their binding, blocking and biological activities in vitro and in vivo. The results showed that the selected antibodies could effectively bind to siglec-15 overexpressed cell lines. As the reported ligands of siglec-15, the binding activity of siglec-15 and sialyl-Tn, CD44, MAG and LRRC4C can be blocked by 3 of the candidate antibodies, among them, 3F1 has a competitive advantage, which indicate 3F1 can effectively breaking down the tumor microenvironment by siglec-15 immunosuppressive signals. In addition, 3F1 antibody could reverse the inhibitory effect of siglec-15 on lymphocyte proliferation (especially CD4+T, CD8+T) and cytokine release (IFN-γ). In the tumor model of Balb/c Nude mice, mice were subcutaneously inoculated with genetically modified human non-small-cell lung carcinoma cell line H157 (overexpressed with siglec-15), compared with PBS group, 3F1 showed certain anti-tumor effect, moreover, the combination of 3F1 and ERBITUX showed significant antitumor effect (p<0.05). In conclusion, a novel human antibody 3F1 targeting human siglec-15 was obtained from phage display. The antibody has certain anti-tumor activity and is expected to be an innovative candidate drug targeting siglec-15 for tumor immunotherapy. Funding: This research was supported by the National Natural Sciences Foundation of China grant (No. 31771010, No. 81700122). Declaration of Interests: The authors declare no conflict of interest Animal Care Committee Approval Statement: The Experimental Animal Management and use committee of my institute has reviewed the Animal Experiment Program and approved it.

Western Blot as a Support Technique for Immunohistochemistry to Detect Programmed Cell Death Ligand 1 Expression.

Antibody selection and optimization are crucial to guarantee accurate and reproducible results when using such antibodies for applications such as western blot analysis and immunohistochemistry (IHC). This is especially important when selecting good candidate antibodies that will be used for cancer immunotherapy diagnostics and research. In this chapter, we describe a Western Blot technique as support methodology for the selection and validation of Programmed Cell Death Ligand 1 (PD-L1) antibodies that can be subsequently used in immunohistochemistry applications. Western Blot is a sensitive, specific, and widely available protein characterization technique, used for the detection of specific antigens. PD-L1 is a major immune checkpoint protein that mediates antitumor immune suppression and response, which is routinely detected using IHC in formalin-fixed and paraffin-embedded tissues as part of cancer clinical diagnostic workflows. For this reason, it is critical to define and select the best antibody clones and validate them using different techniques in order to have a reliable detection of positive staining when these antibodies are used in IHC.

EPEN-14. GENERATION OF A C11orf95-RELA FUSION TARGETING ANTIBODY AS A DIAGNOSTIC TOOL FOR SUPRATENTORIAL EPENDYMOMA

Ependymomas account for 10% of paediatric brain tumours and arise in the ventricular walls of the central nervous system. Ependymomas were previously classified as one tumour type and all patients received similar treatment. However, recent genomic studies have identified nine different molecular subgroups of the disease, including one supratentorial subtype characterized by a novel fusion gene C11ORF95-RELA. When introduced into neural stem cells, this fusion is a potent driver of tumorigenesis and its presence in patient samples has previously also been shown to negatively correlate with overall survival. Accurate diagnosis of this subgroup is currently limited to sophisticated approaches such as break-apart FISH or RNA sequencing. Here, we report the generation of a C11ORF95-RELA Fusion-specific antibody that can be used for routine immunohistochemistry (IHC). Candidate antibodies were first selected using phage display and favourable leads were subjected to affinity maturation using ribosome display after a screening process involving immunoblotting and IHC. Further IHC-based screening of affinity-matured candidates using fusion-positive and -negative mouse tissue as well as human fusion-negative ependymoma tumour tissue produced one lead antibody. The antibody detects fusion-specific nuclear staining pattern on fusion-positive tissue and does not react with fusion-negative tissues. This candidate antibody is currently being tested on human fusion-positive ependymoma tissue. This accurate diagnostic tool holds great promise to transform the management of patients with supratentorial ependymoma.