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

项目摘要/摘要 在拟议的研究中，GlycoT将使用其专有的趋化酶糖链重塑平台来 建立一种简便、健壮、可扩展的部位特异性抗体偶联技术。不同种类的共轭 抗体的功能分子在生命科学中经常被用于各种各样的应用 用于检测和成像的荧光标记抗体、抗体-药物结合物(ADC)等部门 对于癌症治疗，抗体-抗生素结合物用于传染病治疗，LYTAC用于靶向 退化。抗体偶联最典型的应用是ADC疗法的发展。 在过去的10年里，adc已经发展成为一种强大而成功的治疗途径之一。 癌症的威胁。对于FDA批准的11个ADC和其他临床试验中的ADC，有效载荷主要是共轭的 通过与半胱氨酸或精氨酸的非特异性随机连接而产生抗体，导致异源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。结果, 具有改进的药代动力学和增强的治疗指数的部位特异性ADC已经被开发出来。 在不同的产生位点特异性ADC的方法中，Fc-多糖在保守的ASN-297上的重塑 产生Fc-葡聚糖特异性ADC的位置特别吸引人。半乳糖基转移酶(GalT)的应用 能够将修饰的UDP-Gal衍生物作为供体底物的突变体使 在Fc糖链上掺入选定的标签，用于随后与修饰的细胞毒性的位点特异性结合 探员们。这一技术路线已被几家临床分期公司采用。然而，作为高尔特 突变体只能转移叠氮或酮基的小Gal-GDP衍生物，每个抗体的药物由这个提供 方法只有2。相比之下，另一种基于内切糖苷酶的转糖基化方法已经克服了这种 限制。这种收敛的方法由两个关键的酶步骤组成：抗体的脱糖基化 内切糖苷酶，以及随后标记的N-聚糖通过一种 内切糖苷酶突变体，作为功能分子的加载点。其核心酶 这个平台，内切糖苷酶突变体，可以转移二糖或大分子糖链底物 链接器。这种承印物尺寸可梳合性的灵活性是GALT或其他基于酶的平台无法比拟的。在这 随着该项目的实施，GlycoT将进一步优化和简化这一部位特异性抗体结合技术。我们会 扩展能够提供明确的药物/配体与抗体比率的葡聚糖功能化路线。我们会 此外，还建立了一种与不同功能分子相容的优化的可切割多肽。至 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。目标 1是多糖的官能化，以获得广泛和明确的结合载荷点。目标2是 用于exatecan有效载荷的新型可切割多肽连接物的开发。目标3是ADC的可伸缩合成 使用MMAE或Exatecan有效载荷。目的4进行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.