Human Monoclonal Antibodies Against Cancer

抗癌人类单克隆抗体

• 批准号: 7965549
• 负责人: Dimiter S Dimitrov
• 金额: $ 64.91万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965549
• 关键词: Affect; Affinity; Animals; Antibodies; Antibody Affinity; Antibody Repertoire; Antigens; Antineoplastic Agents; B-Cell Lymphomas; B-Lymphocytes; Binding; Breast Cancer Cell; C-terminal; CD22 gene; CDR1 gene; Cancer cell line; Cells; Chemicals; Chimeric Proteins; Circular Dichroism; Clinical; Complement; Complementarity Determining Region II; Complementarity Determining Regions; Confocal Microscopy; Coupling; Cysteine; Data; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Differential Scanning Calorimetry; Disease; Diversity Library; Drug Delivery Systems; ERBB2 gene; Engineering; Exhibits; Fluorescence; Galactose; Generations; Goals; High temperature of physical object; Human; Immune response; Immune system; Immunization; Immunoglobulin Fragments; Immunoliposome; In Vitro; Individual; Investigation; Knowledge; Label; Large-Scale Sequencing; Length; Libraries; Ligands; Light; Liposomes; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Methods; Modality; Monoclonal Antibodies; Mus; NMR Spectroscopy; Nature; Pan Genus; Patients; Penetration; Phage Display; Physiological; Positioning Attribute; Prevention; Process; Property; Protein Binding; Proteins; Recombinants; Reporting; Research; Science; Sequence Analysis; Side; Site; Solid Neoplasm; Somatomedins; Source; Structure; System; Systems Biology; TNFRSF10A gene; TNFRSF10B gene; Temperature; Testing; Therapeutic; Therapeutic antibodies; Threonine; Tissues; Treatment Efficacy; Urea; Uridine Diphosphate Sugars; Work; anti-cancer therapeutic; antibody conjugate; antibody engineering; antigen binding; base; cancer cell; cancer diagnosis; cancer therapy; design; disulfide bond; flexibility; human disease; human monoclonal antibodies; hydroxyl group; immunogenic; improved; in vitro activity; killings; mesothelin; mutant; nanodrug; nanoparticle; neonatal Fc receptor; neutralizing monoclonal antibodies; novel; novel diagnostics; novel strategies; novel therapeutics; overexpression; polypeptide; receptor; receptor mediated endocytosis; scaffold; targeted delivery; therapeutic development; tool

This year we have continued to identify novel antibodies and engineered antibody domains against cancer-related proteins. These antibodies are tested for their activity against cancer cells in vitro and some of them are also used for conjugation with nanoliposomes and for development of novel approaches for multiple site-specific conjugations of bioactive molecules to antibody fragments. We have also developed several large libraries containing billions of dAbs and nAbs which can be used as a source of binders to various targets. The major accomplishments are summarized below. 1) We have proposed to use isolated CH2 domains as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions (nAbs). However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (Tm = 54.1C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41C). One mutant (m01) was remarkably stable (Tm = 73.8C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 M vs 4.2 M). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases. 2) Several large (size 1010) phage-displayed libraries of engineered CH2 domains were constructed and are being tested for selection of binders. We are also expressing human neonatal Fc receptor (FcRn) to test binding of some of the engineered CH2 domains with the specific aim to generate nAbs that can bind specifically to a cancer-related protein and simultaneously to the FcRn. 3) We have previously constructed a large (size 2.5 1010) dAb library by grafting human antibody heavy chain complementarity determining regions (CDRs) 2 and 3 (H2s, H3s) into their cognate positions in a human heavy chain variable domain (VH) scaffold and mutagenizing the CDR1 (H1). High-affinity binders against some antigens were selected from this library but panning against others was not very successful likely due to limited diversity. We have hypothesized that by grafting highly variable, both in length and composition, human CDRs into non-cognate positions, the dAb library diversity could be significantly increased and the library would allow for more efficient selection of high-affinity antibodies against some targets. To test this hypothesis we designed a novel type of dAb library containing CDRs in non-cognate positions. It is based on our previous library where H1 was replaced by a library of human light chain CDR3s (L3s) thus combining three most diversified fragments (L3, H3 and H2) in one VH scaffold. This large (size 1010) phage-displayed library was highly diversified as determined by analyzing the sequences of randomly selected clones. The new library could be used not only for selection of such dAbs thus complementing existing libraries but also as a research tool for exploration of the mechanisms determining folding and stability of human antibody domains. 4) Novel high-affinity dAbs against the components (receptor and ligands) of the human insulin-like growth factor (IGF) system were selected from the new library that could not be selected from the previously constructed library. The newly identified dAbs were highly soluble, expressible, monomeric and may have potential as candidate cancer therapeutics. 5) We have continued to characterize our previously identified human mAbs against mesothelin and CD22. We showed their high activity in vitro. Further studies are needed to find whether they could be used as candidate therapeutics. 6) We constructed human anti HER2 single chain variable fragments (scFvs) with a C-terminal fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. The C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins were used as acceptor substrates for human polypeptide-alpha-Nu-acetylgalactosaminyltransferase II (h-ppGalNAc-T2) that transfers either GalNAc or 2-keto-Gal, a modified galactose with a chemical handle, from their respective UDP-sugars to the side-chain hydroxyl group of the Thr residue(s). These fusion scFv proteins with the modified galactose were then conjugated with a fluorescence probe, Alexa488, that carries an orthogonal reactive group. The fluorescence labeled scFv proteins bound specifically to a human breast cancer cell line (SK-BR-3) that overexpresses HER2, indicating that the in vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen. This novel method for conjugation to specific sites could be used for construction of targeted nanoparticles guided by antibodies. 7) We have hypothesized that coupling of the current treatment modalities with a nano-drug delivery vehicle (immunoliposomes) could significantly improve the drug delivery efficiency thereby achieving better treatment efficacies. To test this hypothesis we generated a CD22 specific scFv which has an engineered cysteine at the C-terminus, conjugated it to liposomes and targeted these liposomes to CD22-expressing cells. We found that the targeted liposomes recognize specifically CD22 on BJAB cells but does not bind to cells that do not express CD22. Intracellular localization of the targeted liposomes at 370C but not at 40C (observed by confocal microscopy) indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis. Therefore, these 2nd-generation liposomes may serve as promising carriers for targeted delivery of anticancer agents to treat patients suffering from B-cell lymphoma. 8) We initiated a high-throughput sequencing of large portions of the antibody repertoires of humans (the human antibodyome). We believe that knowledge of the complete antibodyome will have implications for research, diagnosis, prevention and treatment of cancer. It can help for deeper understanding of the B cell (system) biology and diseases, help develop new diagnostic methods based on individual antibodyomes, help predict individual immune responses to immunization and therapeutics as well as provide information for the design of novel therapeutics. Most implications can not be predicted due to the very nature of an omic science.

今年，我们继续确定针对癌症相关蛋白的新型抗体和工程抗体结构域。这些抗体在体外对癌细胞的活性进行了测试，其中一些抗体也用于与纳米脂质体结合，并开发出新的方法，用于多种位点特异性的生物活性分子与抗体片段的结合。我们还开发了几个大型文库，其中包含数十亿个DAB和NAB，这些库可用作各种目标的粘合剂来源。主要成就将在下面总结。 1）我们建议将孤立的CH2域用作构造包含各种粘合剂的库的支架，这些库也可以赋予某些效应子功能（NABS）。但是，以前的工作表明，一个孤立的鼠CH2结构域对于热诱导的展开相对不稳定。为了探索分离的人类CH2的展开机制，并提高其稳定性＆＃947; 1 CH2被克隆，并构建了一系列半胱氨酸突变体。人＆＃947; 1 CH2在更高的温度（TM = 54.1C，通过圆形二色性测量）下展开，比以前报道的小鼠CH2（41c）的温度下展出。一个突变体（M01）非常稳定（TM = 73.8C）。通过差异扫描量热法获得了相似的结果。对于尿素引起的展开，该突变体也比野生型CH2明显更稳定（尿素浓度为6.8 m vs 4.2 m）。 M01高度溶于单体。分别通过质谱和核磁共振光谱证明了M01中第二二硫键及其正确位置的存在。在CH2和M01中，循环平均比框架更灵活，并且整个二级结构不受额外的二硫键影响。这些数据表明，人类CH2结构域在生理温度下相对稳定，而CH2和高度稳定的突变体M01都有希望为针对人类疾病开发治疗疗法提供新的支架。 2）构建了几个大型（尺寸1010）噬菌体斑点的CH2域的库，并正在测试以选择粘合剂。我们还表达人类新生儿FC受体（FCRN），以测试某些工程CH2结构域的结合，其特定目的是生成可以专门与癌症相关蛋白并同时与FCRN结合的NAB。 3）以前，我们以前已经通过接纳人类抗体重链互补性确定区域（CDRS）2和3（H2S，H3S）来构建了一个大的（2.5 1010）DAB库，以在人类重型链可变域（VH）支架中的同源位置并诱变CDR1（H1）。从该图书馆中选择了针对某些抗原的高亲和力粘合剂，但由于多样性有限，针对其他抗原的固定器可能并不是很成功。我们假设，通过将高度可变的长度和组成可变，人类的CDR置于非认知位置，可以显着提高DAB文库多样性，并且该库将允许更有效地选择针对某些目标的高亲和力抗体。为了检验这一假设，我们设计了一种新型的DAB库，其中包含非认知位置的CDR。它基于我们以前的库，其中H1被人体轻链CDR3S（L3S）的库取代，因此将三个最多样化的片段（L3，H3和H2）组合在一个VH脚手架中。通过分析随机选择的克隆的序列确定，这个大型（尺寸1010）噬菌体播放的文库被高度多样化。新库不仅可以用于选择此类DAB，从而补充现有库，还可以用作探索确定人类抗体域折叠和稳定性的机制的研究工具。 4）对人类胰岛素样生长因子（IGF）系统的成分（受体和配体）系统的新型高亲和力DAB被从新文库中选择，这些系统无法从先前构建的文库中选择。新近鉴定的DABS高度可溶，表达，单体，并且可能具有候选癌症治疗剂的潜力。 5）我们继续表征我们先前鉴定出的针对间皮素和CD22的人物mab。我们在体外表现出了很高的活性。需要进一步的研究来发现它们是否可以用作候选治疗剂。 6）我们用含有1、3或17苏氨酸（THR）残基的C末端融合多肽构建了人类抗HER2单链可变片段（SCFV）。这些重组SCFV融合蛋白的C末端扩展融合多肽用作人多肽 - α-NU-乙酰乳乙酰乳糖苷转移酶II（H-PPGALNAC-T2）的受体底物（H-PPGALNAC-T2），可转移与GalNAC或2-Keto-Gal一起，或2-Keto-gal，从各自的UDP-糖到THR残基的侧链羟基的化学手柄。然后将这些融合的SCFV蛋白与修饰的半乳糖与荧光探针Alexa488结合，该荧光探针带有正交反应性基团。荧光标记为SCFV蛋白过度表达HER2的人类乳腺癌细胞系（SK-BR-3），表明其体外折叠折叠的SCFV融合蛋白具有生物活性，并且在其C-端子中存在共轭多个ALEXA488探针。末端不会干扰它们与抗原的结合。这种与特定位点结合的新方法可用于构建受抗体引导的靶向纳米颗粒。 7）我们假设，当前治疗方式与纳米药物输送车（免疫脂质体）的耦合可以显着提高药物递送效率，从而实现更好的治疗效率。为了检验该假设，我们生成了CD22特异性SCFV，该SCFV在C端具有工程性半胱氨酸，将其偶联为脂质体，并将这些脂质体靶向CD22表达细胞。我们发现，靶向脂质体在Bjab细胞上特别识别CD22，但与不表达CD22的细胞没有结合。 靶向脂质体的细胞内定位在370C而不是在40C处（通过共聚焦显微镜观察）表明，通过受体介导的内吞作用，通过依赖能量的过程将我们的靶向脂质体通过能量依赖于。因此，这些第二代脂质体可能是针对抗癌药的靶向递送来治疗患有B细胞淋巴瘤患者的有前途的载体。 8）我们启动了大部分人类抗体库（人类抗体组）的高通量测序。我们认为，对完整抗体组的了解将对癌症的研究，诊断，预防和治疗有影响。它可以帮助您深入了解B细胞（系统）生物学和疾病，有助于基于单个抗体组的新诊断方法，有助于预测个人免疫反应对免疫和治疗剂的免疫反应，并为新型治疗剂的设计提供信息。由于OMIC科学的本质，无法预测大多数含义。