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

项目摘要/概要 在拟议的研究中，GlycoT将采用其专有的化学酶聚糖重塑平台， 建立一种简便、稳健和可扩展的位点特异性抗体偶联技术。各种共轭 抗体的功能分子经常用于生命科学中的各种应用 部门，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，用于感染性疾病治疗的抗体-抗生素缀合物，用于靶向治疗的LYTAC 降解抗体偶联的最典型应用是ADC治疗剂的开发。 在过去的10年里，ADC已经发展成为治疗该疾病的有效和成功的途径之一 癌症。对于FDA批准的11种ADC和临床试验中的其他ADC，有效载荷主要是缀合的， 通过与半胱氨酸或精氨酸的非特异性随机连接与抗体结合，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，在本发明中， 已经开发了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。 在产生位点特异性ADC的不同方法中，保守的Asn-297上的Fc-聚糖的重构是一种新的途径。 用于产生Fc-聚糖特异性ADC的位置是特别有吸引力的。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已经使得 在Fc聚糖处掺入选定的标签，用于随后与修饰的细胞毒性缀合 剂.该技术路线已被多家临床阶段公司采用。然而，作为Galt 突变体只能转移叠氮基或酮基的小Gal-GDP衍生物，这种抗体提供的药物 方法只有2。相比之下，另一种基于内切糖基化酶的转糖基化方法已经克服了这种缺陷。 限制.这种聚合方法由两个关键的酶促步骤组成： 将标记的N-聚糖与去糖基化的抗体连接，随后通过内切糖苷酶将标记的N-聚糖与去糖基化的抗体连接。 内切糖苷酶突变体，其充当功能分子的加载点。核心酶 这个平台，内切糖苷酶突变体，可以转移二糖或大聚糖底物， 连接器。底物大小可梳理性的这种灵活性是GalT或其他基于酶的平台所无法比拟的。在这 GlycoT将进一步优化和简化这种位点特异性抗体偶联技术。我们将 扩展聚糖功能化途径，可提供明确的药物/配体与抗体比率。我们将 还建立了与不同功能分子相容的优化的可切割肽。到 为达致研究目的，我们建议在第I期研究中达致以下四个具体目的。目的 1是聚糖的官能化，以获得广泛和确定的缀合加载点。目标2是 开发用于依沙替康有效载荷的新型可裂解肽接头。目标3是ADC的可扩展合成 携带MMAE或艾西特扫描器目的4是体内毒性和抗肿瘤疗效试验。

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.