Development of a facile, robust, scalable, and versatile chemoenzymatic glycan-remodeling approach for site-specific antibody conjugation

开发一种简便、稳健、可扩展且多功能的化学酶聚糖重塑方法，用于位点特异性抗体缀合

• 批准号: 10615237
• 负责人: Qiang Yang
• 金额: $ 8.97万
• 依托单位: GLYCOT THERAPEUTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10615237
• 关键词: Adopted; Affinity; Antibiotics; Antibodies; Antibody-drug conjugates; Antigens; Arginine; Azides; Basic Science; Biological Sciences; Biotechnology; Breast Cancer Model; Cells; Circulation; Clinical; Clinical Trials; Custom; Cysteine; Cytotoxic agent; Development; Diagnostic; Disaccharides; Drug Kinetics; Elements; Endoglycosidases; Enzymes; Evaluation; Exatecan; FDA approved; Freedom; Galactosyltransferases; Glycine; Goals; Half-Life; Image; Industrialization; Label; Length; Licensing; Ligands; Methods; Modeling; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Polysaccharides; Positioning Attribute; Research; Research Personnel; Route; Serum; Services; Sialic Acids; Site; System; Technology; Therapeutic; Therapeutic Index; Toxic effect; Toxicity Tests; Uridine Diphosphate Galactose; Washington; Xenograft procedure; antibody conjugate; antibody detection; cancer therapy; dosage; efficacy study; efficacy testing; flexibility; glycosylation; improved; in vitro testing; in vivo; infectious disease treatment; mouse model; mutant; novel; operation; phase 1 study; screening; sialylation; sound; therapeutic development; tumor

Project abstract/summary In the proposed research, GlycoT will employ its proprietary chemoenzymatic glycan-remodeling platform to establish a facile, robust, and scalable site-specific antibody conjugation technology. Conjugation of various functional molecules to antibodies are frequently used for a wide variety of applications within the life science sectors, such as fluorescent labeled antibodies for the detection and imaging, antibody-drug conjugate (ADC) for cancer therapy, antibody-antibiotic conjugate for infectious disease treatment, LYTAC for targeted degradation. The most exemplified application of antibody conjugation is the development of ADC therapeutics. Over the past 10 years, ADC has been developed as one of powerful and successful avenues for the treatment of cancer. For FDA-approved 11 ADCs and others in clinical trials, the payloads have been mainly conjugated to antibody by non-specific random linkage to either cysteine or arginine, resulting in heterogenous ADC regioisomers, with varied antigen affinity, aggregation potential, serum half-life, and other limitation. As a result, site-specific ADCs with improved pharmacokinetics, and enhanced therapeutic index have been developed. Among different approached to generate site-specific ADCs, remodeling of Fc-glycan on the conserved Asn-297 position to generate Fc-glycan specific ADC is particularly attractive. The use of the galactosyltransferase (GalT) mutants capable of accommodating modified UDP-Gal derivatives as the donor substrates has enabled the incorporation of a selected tag at the Fc glycans for subsequent site-specific conjugation with modified cytotoxic agents. This technology route has been adopted by several clinical stage companies. However, as the GalT mutant can only transfer azide or keto based small Gal-GDP derivative, the drug per antibody provided by this method is just 2. In contrast, another endoglycosidase-based transglycosylation method has overcome such limitation. This convergent approach consists of two key enzymatic steps: deglycosylation of the antibody by an endoglycosidase, and subsequent attachment of a tagged N-glycan to the deglycosylated antibody by an endoglycosidase mutant, which serves as the loading points for the functional molecules. The core enzyme of this platform, endoglycosidase mutant, can transfer either disaccharide or large glycan substrate with extended linker. Such flexibility of substrate size combability is unparallel by GalT, or other enzyme-based platform. In this project, GlycoT will further optimize and streamline this site-specific antibody conjugation technology. We will expand the glycan functionalization routes that can provide well-defined drug/ligand to antibody ratios. We will also establish an optimized cleavable peptide that is compatible with different functional molecules. To accomplish the research goals, we propose to pursue the following four specific aims in the Phase I study. Aim 1 is the functionalization of glycan to obtain broad and defined conjugation loading points. Aim 2 is the development of novel cleavable peptide linkers for the exatecan payload. Aim 3 is scalable synthesis of ADC with MMAE or exatecan payload. Aim 4 is the in vivo toxicity and anti-tumor efficacy tests.

项目摘要/总结 在拟议的研究中，GlycoT 将利用其专有的化学酶聚糖重塑平台来 建立一种简便、稳健且可扩展的位点特异性抗体缀合技术。各种共轭 功能分子到抗体经常用于生命科学领域的各种应用 领域，例如用于检测和成像的荧光标记抗体、抗体药物偶联物 (ADC) 用于癌症治疗、用于传染病治疗的抗体-抗生素偶联物、用于靶向治疗的 LYTAC 降解。抗体偶联最典型的应用是 ADC 疗法的开发。 在过去的 10 年里，ADC 已发展成为一种强大且成功的治疗途径 癌症。对于 FDA 批准的 11 种 ADC 和其他处于临床试验中的 ADC，有效负载主要是缀合的 通过与半胱氨酸或精氨酸非特异性随机连接来连接抗​​体，从而产生异源 ADC 区域异构体，具有不同的抗原亲和力、聚集潜力、血清半衰期和其他限制。因此， 已经开发出具有改善的药代动力学和增强的治疗指数的位点特异性 ADC。 在生成位点特异性 ADC 的不同方法中，在保守的 Asn-297 上重构 Fc-聚糖 生成 Fc-聚糖特异性 ADC 的地位特别有吸引力。半乳糖基转移酶 (GalT) 的用途 能够容纳修饰的 UDP-Gal 衍生物作为供体底物的突变体使得 在 Fc 聚糖上掺入选定的标签，以便随后与修饰的细胞毒性进行位点特异性缀合 代理。该技术路线已被多家临床阶段公司采用。然而，正如 GalT 突变体只能转移基于叠氮化物或酮基的小 Gal-GDP 衍生物，该抗体提供的药物 方法就是2。相比之下，另一种基于内切糖苷酶的转糖基方法克服了这种问题 局限性。这种聚合方法由两个关键的酶促步骤组成： 糖苷内切酶，然后通过 糖苷内切酶突变体，作为功能分子的装载点。核心酶为 该平台，糖苷内切酶突变体，可以转移二糖或大聚糖底物，延长 链接器。底物尺寸可组合性的这种灵活性是 GalT 或其他基于酶的平台无法比拟的。在这个 项目中，GlycoT 将进一步优化和简化这种位点特异性抗体缀合技术。我们将 扩展聚糖功能化途径，可以提供明确的药物/配体与抗体的比例。我们将 还建立了与不同功能分子兼容的优化可裂解肽。到 为了实现研究目标，我们建议在第一阶段研究中追求以下四个具体目标。目的 图 1 是聚糖的官能化以获得广泛且明确的缀合负载点。目标 2 是 开发用于 exatecan 有效负载的新型可裂解肽接头。目标 3 是 ADC 的可扩展综合 带有 MMAE 或 exatecan 有效负载。目的4是体内毒性和抗肿瘤功效试验。