Glycotope (Germany)

Abstract Introduction: Recent advances in antibody engineering have created a portfolio of highly potent therapeutic approaches like antibody-drug conjugates (ADCs), radioimmunoconjugates (RITs), chimeric antigen receptors (CARs) or bi-specifics. Due to the increased potency, these drugs are more than ever dependent on very clean targets to prevent side effects. We aim to address this major problem through our approach to develop antibodies with improved tumor specificity and reduced on-target/off-tumor binding utilizing the aberrant O-glycosylation on tumor cells. Altered glycosylation of proteins and lipids is one of the most drastic changes in cancer, giving rise to truncated or highly fucosylated and highly sialylated glycans which are almost absent on normal cells. Thus, developing antibodies against protein/carbohydrate combined epitopes (GlycoTargets) comprising these tumor-specific glycans enables highly potent therapies with reduced off-tumor toxicity. Experimental procedures: A workflow and corresponding database was established to evaluate the O-glycosylation of proteins. The first and most basic criterion is the presence of suitable O-glycosylated peptide stretches in the extracellular domain of a protein based on predictions. For proteins fulfilling this criterion cancer-relevant expression is analyzed in a second step. This is done either using publicly available data or experimentally (immunohistochemistry or protein expression data). During the final major step, the theoretical existence of suitable O-glycosylation is confirmed by our own experimental data. This includes analysis of cellularly expressed proteins by anti-glycan antibodies and mass spectrometric studies. Results summary: Case studies will be presented that highlight our workflow to identify suitable GlycoTargets based on publicly available data analysis and experimental confirmation. Additionally, the subsequent development of antibodies against the identified GlycoTargets will be shown. Conclusion: A standardized process was developed for the data collection and prioritization of potential GlycoTargets. The obtained information is subsequently used for targeted discovery of antibodies that bind to protein/carbohydrate combined glycoepitopes (GlycoTargets) offering increased tumor-specificity compared to simple protein targets. Citation Format: Patrik Kehler, Johanna Gellert, Lisa Kalfhues, Sophie Marinoff, Manon Weis, Andreas Franz, Naomi Kast, Stephanie Gurka, Marianne Morche, Evelyn Hartung, Timo Lischke, Sarah Mayer-Hain, Antje Danielczyk. Platform approach to develop antibodies specifically recognizing cancer-associated glycoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5896.

Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody-toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.

Abstract Background: Highly potent therapeutic approaches require very clean targets. However, the majority of antibodies in clinical development or approved for cancer therapy address protein targets that are only overexpressed on cancer cells and yet often show significant expression in healthy organs. Glycans tend to elicit superior tumor specificity as compared to proteins, since glycosylation is strongly altered in cancer, reflecting the drastic changes in tumor metabolism. The changes in glycosylation are mostly due to mutated or mislocated glycosyltransferases and glycosidases, giving rise to highly fucosylated, highly sialylated and truncated glycans. Therefore, proteins expressed in cancer cells can carry tumor-associated carbohydrates like the Thomsen-Friedenreich (TF) and the Thomsen novelle (Tn) antigen as well as their respective sialylated forms sTF and sTn. To increase the tumor-specificity of protein-targeting antibodies, we have developed antibodies against several different protein/carbohydrate combined epitopes (GlycoTargets). Targeting these specific antigens offers reduced on-target/off tumor toxicity, which is key for highly potent therapies. Methods: The specificity and glycosylation dependency of our antibodies were shown using differentially glycosylated proteins or carbohydrate PAA-conjugates in an ELISA format. Binding to several tumor cell lines and healthy human leukocytes was analyzed via flow cytometry. Binding to different cancer indications as well as related healthy tissues was analyzed by immunohistochemistry. Results: We have generated antibodies that bind to their target protein only if a specific tumor-associated carbohydrate is present and do not cross-react with the non-glycosylated protein itself, or the carbohydrate structure on different carrier-proteins. Due to the carbohydrate-dependent binding, our antibodies show markedly decreased on-target/off-tumor binding. We will show that our antibodies mediate no binding of different healthy immune cell subsets in comparison to simple protein binding antibodies in flow cytometric analyses. Further, IHC studies reveal that our antibodies stain tumor tissue of different cancer indications but do not stain related normal tissues. Conclusions: We successfully used the fact that glycosylation is strongly altered in cancer for the generation of highly tumor-specific antibodies that target neither glycan nor protein alone, but the potent combination thereof - a tumor-specific protein/carbohydrate epitope. We could show that by using this approach we were able to reduce on-target/off-tumor binding towards different healthy tissues and healthy immune cell subsets making these antibodies ideal candidates for a therapeutic development. Citation Format: Patrik Kehler, Anika Jaekel, Antje Danielczyk, Christoph Goletz, Timo Lischke, Evelyn Hartung, Lisa Weiß, Manon Weis, Stephanie Gurka, Sophie Marinoff, Naomi Kast, Manon Weiske, Marianne Morche, Johanna Gellert, Theresa Neumann. Carbohydrate-dependent protein binding antibodies with superior tumor-specificity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 316.

BackgroundIL-15 is a potent pro-inflammatory cytokine that enhances the differentiation, proliferation and cytolytic activity of NK cells and T cells. Due to the huge potential of IL-15 to activate both innate and adaptive anti-tumor immunity, several IL-15-based immunocytokines are currently in clinical development. However, all of them preferentially act in the periphery and not locally within the tumor. To further increase the efficacy and safety of IL-15-based immunocytokines, we developed GT-00A x IL15, an immunocytokine targeting a tumor-associated, glycosylated epitope of MUC-1 (TA-MUC1). GT-00A x IL15 was designed to induce anti-tumor responses directly within the tumor microenvironment for the treatment of solid tumors.MethodsGT-00A x IL15 was extensively studied in vitro and in vivo to adequately characterize its complex mechanisms of action and to analyze its anti-tumor efficacy. The relevance of TA-MUC1 binding as differentiation criteria against untargeted IL-15 (super)agonists was investigated in detail. In vitro cytotoxicity and 3D tumor spheroid immune cell infiltration mediated by GT-00A x IL15 was compared to the parental antibody GT-00A and an untargeted IL-15 control. In vivo, several pharmacokinetic, pharmacodynamic, biodistribution and efficacy studies were performed in tumor-free or tumor-bearing mice.ResultsWe could show in vitro that GT-00A x IL15 increased the cytotoxic activity of PBMC against TA-MUC1-positive tumor cell lines compared to parental GT-00A and an untargeted IL-15 control construct. Additionally, dose-dependent infiltration of NK and T cells into MCF-7 tumor spheroids is mediated by GT-00A x IL15 but not the untargeted IL15 control construct or parental GT-00A. In vivo single agent efficacy of GT-00A x IL15 was shown in different tumor models by means of tumor growth inhibition and increased survival. Subsequent flow cytometric analysis of tumor samples confirmed activation and expansion of tumor-infiltrating NK and CD8+ T cells. Furthermore, in a biodistribution study using radioactively labelled GT-00A x IL15 we observed significantly increased enrichment in the tumor compared to the untargeted IL-15 control construct.ConclusionsOur results confirm the relevance of TA-MUC1-mediated tumor cell binding for the mechanisms of action of our immunocytokine. GT-00A x IL15 shows increased accumulation in the tumor and mediates enhanced cytotoxicity and immune cell infiltration compared to an untargeted IL-15 control construct highlighting the potential to increase the efficacy and safety of IL-15-based immunocytokines by tumor targeting. GT-00A x IL15 shows great promise for the treatment of TA-MUC1-positive solid tumors either as monotherapeutic agent or as valuable combination partner.

Surfactant protein D (SP-D) is a C-type lectin that participates in the innate immune defense of lungs. It binds pathogens through its carbohydrate recognition domain in a calcium-dependent manner. Human surfactant protein D (hSP-D) has been routinely obtained from bronchoalveolar lavage of patients suffering from pulmonary alveolar proteinosis (PAP) and from amniotic fluid (AF). As a consequence of the disease, hSP-D obtained from PAP is found in higher amounts and is mainly composed of higher order oligomeric forms. However, PAP-hSP-D has never been directly compared with nonpathological human protein in terms of structure and biological activity. Moreover, the quantitative distribution of the different hSP-D oligomeric forms in human protein obtained from a natural source has never been evaluated. In this work, we have determined the quantitative distribution of AF-hSP-D oligomers, characterized the sugars attached through the N-glycosylation site of the protein, and compared the activity of hSP-D from AF and PAP with respect to their ability to bind and agglutinate bacteria. We have found that fuzzy balls (40%) are the most abundant oligomeric form in AF-hSP-D, very closely followed by dodecamers (33%), with both together constituting 73% of the protein mass. The glycan attached to the N-glycosylation site was found to be composed of fucose, galactose, sialic acid, and N-acetylglucosamine. Finally, in the functional assays performed, hSP-D obtained from PAP showed higher potency, probably as a consequence of its higher proportion of large oligomers compared with hSP-D from AF.

3008 Background: Epidermal growth factor receptor (EGFR) is a validated target in cancer. EGFR antagonists in clinical use do not exploit the full potential of this target. CetuGEX is an IgG1 mAb against EGFR. Fully human and optimized glycosylation lead to a 10- to 250-fold improvement of ADCC-mediated tumor cell killing in all FcγRIIIa allotypes and lack of immunogenic carbohydrate-chains, compared to cetuximab. Methods: Eligible patients with advanced solid tumors, progressing after standard treatment, were enrolled into this phase I, first-in-human, multicenter, single agent dose escalation trial. PK, PD and immunological parameters were assessed. Endpoints were safety and tolerability and secondarily pharmacokinetics, immunogenicity and anti-tumor activity. Results: 41 patients were treated on a q1w (8 dose levels from 12 to 1,370 mg flat dose), or q2w (990 mg flat) schedule. 25 pts had received at least 8 weekly doses (per protocol population [PP]).The most frequently observed drug-related AE were nausea (20%), vomiting (20%), hypertension (20%), almost all low grade and acneiform dermatitis (25%), only grade 1 or 2. Infusion-related reactions (IRR), virtually restricted to the first infusion, were associated with cytokine secretion: IL-6, IL-8, TNFα, IFNγ and IP-10 as marker of macrophage activation. Optimization of infusion scheme and premedication reduced IRRs in severity and frequency from 76% to 57% mainly of low grade. Blood NK cells were reduced as sign of redistribution. Activity was seen over all dose levels. One patient with NSCLC achieved a complete response. One patient with metastatic colorectal cancer had a partial response, another 2 patients with esophageal and gastric cancer without measurable disease at study entry had marked improvement of symptoms and normalization of tumor markers. Additional 15 pts had stable disease lasting from 8 weeks to over a year, including several minor responses, leading to a clinical benefit rate of 46% (19/41) in the overall and 76% (19/25) in the PP population. PK supports q1w and q2w dosing. Conclusions: CetuGEX shows clear signs of activity and acceptable toxicity. Phase II will soon be initiated. Clinical trial information: NCT01222637.

The Thomsen-Friedenreich disaccharide (TF(alpha)) is a promising antigen for tumor immunotargeting, since it is almost exclusively expressed on carcinoma tissues. So far, an obstacle preventing the exploitation of TF for immunotargeting has been the lack of suitable (non-IgM) antibodies with high affinity and specificity. Recently we reported on a novel strategy for generating antibodies toward small uncharged carbohydrates and the generation of recombinant antibodies toward TF. Among them, two multivalent scFv antibodies showed sub-micromolar functional affinities and appeared well suited for immunotargeting. In the present study, the trimeric scFv(1aa) and the tetrameric scFv(0aa) have been further developed for radioimmunotargeting. The scFvs were radiolabeled with (111)In using DTPA as chelator without losing binding activity or molecular stoichiometry. Binding affinities as high as 1 x 10(-7) M toward TF displayed on living cells were determined. Antibody biodistribution and tumor targeting efficacy were studied in TF-positive human breast cancer (ZR-75-1) bearing mice. TF was successfully targeted in vivo with tumor uptakes of approximately 11 and 8% ID/g after 24 h for the trimeric and tetrameric scFv, respectively. These results validate TF as a potent antigen for tumor targeting. The biodistribution of the scFvs was comparable to that reported for IgGs. In contrast to the IgGs, the serum clearance of the scFvs was very fast, which could be an advantage in a therapeutic setting. Furthermore, kidney uptake, which is often critical for small recombinant antibodies labeled with radio-metals, was low with the tetramer (11% ID/g). We conclude that the multimeric anti-TF scFvs are promising candidates to be further developed toward therapeutic application.

Einleitung: Galectin-1 (Gal-1), ist ein Vertreter der humanen β-Galaktosid-bindenden Proteine, welches bevorzugt Galβ1–4GlcNAc Sequenzen in Oligosaccharidstrukturen erkennt, die mit einer Reihe von sezernierten und Zellmembran-Glykoproteinen assoziiert sind.