Human Monoclonal Antibodies Against Cancer

抗癌人类单克隆抗体

• 批准号: 8763179
• 负责人: Dimiter S Dimitrov
• 金额: $ 75.83万
• 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/8763179
• 关键词: Adverse effects; Affect; Affinity; Animals; Antibodies; Antigen Receptors; Antigens; Avidity; B lymphoid malignancy; B-Cell Acute Lymphoblastic Leukemia; B-Lymphocytes; Binding; Binding Sites; Biological Assay; C-terminal; CD22 gene; CD28 gene; CD3 Antigens; Cancer Cell Growth; Cell Cycle Arrest; Cell surface; Cell-Mediated Cytolysis; Cells; Chimeric Proteins; Clinical; Complement-Dependent Cytotoxicity; Computer Simulation; Data; Development; Diagnosis; Diagnostic; Dimerization; Disease; Engineering; Epitopes; Escherichia coli; European Union; Excision; Exhibits; Exotoxins; Fc domain; Flow Cytometry; Fluorescence; Free Energy; G1 Phase; Generations; Goals; HIV-1; Half-Life; Human; Human Engineering; IgG1; Immune System Diseases; Immune Targeting; Immune system; Immunoglobulin G; Immunotoxins; In Vitro; Knowledge; Libraries; Magic; Malignant Neoplasms; Measures; Membrane; Mesothelioma; Molecular Conformation; Monoclonal Antibodies; N-terminal; Nude Mice; Oncogenic; Patients; Penetration; Phage Display; Pharmaceutical Preparations; Play; Primary carcinoma of the liver cells; Process; Property; Proteins; Pseudomonas; Recombinants; Resistance; Role; Series; Signal Transduction; Site; Solid; Solid Neoplasm; Structure; System; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic Uses; Thioflavin T; Time; Tissues; Treatment Efficacy; Variant; Xenograft procedure; anti-cancer therapeutic; antibody engineering; antibody-dependent cell cytotoxicity; antigen binding; base; cancer cell; cancer diagnosis; cancer therapy; cell killing; disulfide bond; folate-binding protein; glypican 3; human monoclonal antibodies; immunogenic; improved; killings; light scattering; lymphoblast; mesothelin; monolayer; neonatal Fc receptor; neoplastic cell; neutralizing monoclonal antibodies; novel; novel strategies; novel therapeutics; scaffold; screening; small molecule; synthetic peptide; therapeutic development; therapeutic target; tool; tumor; tumor growth; tumor xenograft; vector

This year we have continued to identify novel mAbs in several formats as Fabs, scFvs and eAds against cancer-related proteins. These mAbs were tested for their activity against cancer cells in vitro and used for development of novel approaches for multispecific targeting. We have also characterized drugability of some mAbs including their propensity for aggregation. The major accomplishments are summarized below. 1) Most of the therapeutic antibodies approved for clinical use are full-size IgG1 molecules. The interaction of the IgG1 Fc with the neonatal Fc receptor (FcRn) plays a critical role in maintaining their long half-life. We have hypothesized that isolated Fc domains could be engineered to functionally mimic full-size IgG1 (nanoantibodies) but with decreased (10-fold) size. We demonstrated for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). In contrast to the wild-type dimeric CH3, the mCH3 exhibited pH-dependent binding to FcRn similar to that of Fc. The binding free energy of mCH3 to FcRn was higher than that of isolated CH2 but lower than that of Fc. Therefore, CH3 may contribute a larger portion of the free energy of binding to FcRn than CH2. A fusion protein of mCH3 with an engineered antibody domain (m36.4) also bound to FcRn in a pH-dependent fashion, and exhibited significantly higher neutralizing activity against HIV-1 than m36.4-Fc fusion proteins. The m36.4-mCH3 fusion protein was monomeric, stable, soluble and expressed at a high level in E. coli. We also found that engineering an additional disulfide bond in mCH3 remarkably increased its thermal stability while the FcRn binding was not affected. These data suggest that mCH3 could not only help in the exploration of the dual mechanisms of the CH3 contribution to Fc functions (dimerization and FcRn interactions) but could also be used for the development of candidate therapeutics with optimized half-life, enhanced tissue penetration, access to sterically restricted binding sites and increased therapeutic efficacy. 2) Isolated human immunoglobulin G (IgG) G CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that removal of the CH2 N-terminal residues may not only increase itsthe stability but also itsthe aggregation resistance of the CH2. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak shFcRn binding to CH2 s as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins to increase their stability and aggregation resistance. 3) Glypican-3 (GPC3) has emerged as a candidate therapeutic target in hepatocellular carcinoma (HCC). However, the oncogenic role of GPC3 in HCC is poorly understood. A novel human engineered antibody domain (HN3) with high avidity (KD = 0.6 nM) for cell surface-associated GPC3 molecules was identified from one of our libraries. HN3 recognized a conformational epitope that requires both the amino and carboxy terminal domains of GPC3. It inhibited proliferation of GPC3-positive cells and exhibited significant HCC xenograft tumor growth inhibition in nude mice. The underlying mechanism of HN3 action may involve cell cycle arrest at G1 phase through yap signaling. This study suggests a novel mechanism for GPC3-targeted cancer therapy. 4) Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma and multiple forms of cancers, and show great promise for clinical development for solid cancers. Antibodies against mesothelin have been shown to act via immunotoxin-based inhibition of tumor growth and induction of antibody-dependent cellular cytotoxicity (ADCC). However, complement-dependent cytotoxicity (CDC), which is considered one of the most important cell killing mechanisms of therapeutic antibodies against tumors, is inactive for such antibodies. A phage display antibody engineering technology and synthetic peptide screening was used to identify SD1, an engineered human antibody domain to mesothelin. The SD1 human antibody domain recognizes a novel conformation epitope at the C terminal end (residues 539-588) of mesothelin close to the cell surface. To investigate SD1 as a potential therapeutic agent a recombinant human Fc (SD1-hFc) fusion protein with an immunotoxin (SD1-PE38) was generated by fusing SD1 to a truncated form of Pseudomonas exotoxin (PE38). Interestingly, the SD1-hFc protein exhibited strong CDC activity, in addition to ADCC, against mesothelin-expressing tumor cells. The SD1-PE38 immunotoxin inhibited both 2D monolayer and 3D spheroid growth of cancer cells in vitro. Furthermore, the SD1-hFc protein caused significant tumor growth inhibition of tumor xenografts in nude mice. These results demonstrate that the SD1 human antibody has potential as a novel cancer therapeutic candidate, and may significantly improve current antibody therapy targeting mesothelin-expressing tumors. 5) Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains plus/minus an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

今年，我们继续以几种格式鉴定出新型的mAB，因为它针对与癌症相关的蛋白质的Fab，SCFV和EADS。这些mAb在体外对癌细胞的活性进行了测试，并用于开发新的多特异性靶向方法。我们还表征了某些mAb的可药用，包括它们的聚集倾向。主要成就将在下面总结。 1）批准用于临床使用的大多数治疗抗体是全尺寸IgG1分子。 IgG1 FC与新生儿FC受体（FCRN）的相互作用在维持长期寿命中起着至关重要的作用。我们假设孤立的FC结构域可以设计成功能上模拟全尺寸IgG1（纳米抗体），但尺寸降低（10倍）。我们首次证明了成功产生可溶的单体CH3域（MCH3）。与野生型二聚体CH3相反，MCH3表现出与FCR相似的FCR的pH依赖性结合。 MCH3与FCRN的结合自由能高于孤立的CH2，但低于FC。因此，CH3可能比CH2贡献与FCRN结合的自由能的更大部分。具有工程抗体结构域（M36.4）的MCH3的融合蛋白也以pH依赖性的方式与FCRN结合，并且对HIV-1的中和活性明显高于M36.4-FC融合蛋白。 M36.4-MCH3融合蛋白是单体，稳定，可溶性的，并且在大肠杆菌中高水平表达。我们还发现，MCH3中的工程额外的二硫键显着提高了其热稳定性，而FCRN结合不受影响。这些数据表明，MCH3不仅可以帮助探索CH3对FC功能的双重机制（二聚化和FCRN相互作用），而且还可以用于开发具有优化的半衰期，增强组织渗透的候选疗法，访问处于空间限制的结合位点并提高治疗功效。 2）分离的人类免疫球蛋白G（IgG）G CH2结构域是新型候选疗法的有前途的脚手架。与其他人IgG结构域不同，CH2不参与强链相互作用，而孤立的CH2相对稳定。然而，孤立的单个CH2容易容易聚集。在天然IgG和FC分子中，来自两个重链的CH2的N末端残基相互相互作用并形成铰链区域。相比之下，N末端残基在孤立的CH2中高度无序。我们假设去除CH2 N末端残基不仅可以提高其稳定性，还可以提高其CH2的聚集抗性。为了检验该假设，我们构建了缩短IgG1 CH2（CH2）的变体，其中删除了N末端的前七个残基。我们发现，与CH2相比，CH2S的热稳定性增加了5C。重要的是，我们证明了CH2S比通过硫非硫醇T（THT）荧光，浊度和光散射测量的CH2明显易于聚集。我们还发现，CH2s表现出与可溶性的单链人类新生儿FC受体（SHFCRN）的结合，该结合比流式细胞仪测量的非常弱的SHFCRN结合与CH2 S的非常弱的SHFCRN结合。计算机建模提出了涉及其N末端残基的CH2聚集模式。因此，删除N末端残基可以增加基于CH2的治疗候选物的可药用性。这种提高稳定性和聚集抗性的策略也可以适用于其他与Ig相关的蛋白质，以提高其稳定性和抗聚合抗性。 3）Glypican-3（GPC3）已成为肝细胞癌（HCC）中的候选治疗靶标。但是，GPC3在HCC中的致癌作用知之甚少。从我们的一个库中鉴定出一种针对细胞表面相关的GPC3分子的新型人类工程抗体结构域（HN3），用于细胞表面相关的GPC3分子。 HN3识别出一个需要GPC3的氨基和羧基终端域的构象表位。它抑制了GPC3阳性细胞的增殖，并在裸鼠中表现出明显的HCC异种移植肿瘤生长抑制。 HN3作用的基本机制可能涉及通过YAP信号在G1期间的细胞周期停滞。这项研究提出了一种针对GPC3靶向癌症治疗的新型机制。 4）正在评估针对间皮素的单克隆抗体治疗间皮瘤和多种形式的癌症，并对固体癌症的临床发育显示出巨大的希望。对间皮素的抗体已显示通过基于免疫毒素的肿瘤生长抑制和抗体依赖性细胞毒性（ADCC）的诱导作用。然而，对于这种抗体而言，对肿瘤治疗性抗体的最重要细胞杀死机制之一被认为是补体依赖性细胞毒性（CDC）。噬菌体显示抗体工程技术和合成肽筛选用于识别SD1，这是一种对间皮素的工程人类抗体结构域。 SD1人类抗体结构域在靠近细胞表面的间皮素的C末端（残基539-588）处识别出一种新型的构象表位。为了研究SD1作为潜在的治疗剂，通过将SD1融合到截断形式的假单胞菌Exotoxin（PE38）的截断形式（PE38）中，生成了具有免疫毒素（SD1-PE38）的重组人FC（SD1-HFC）融合蛋白。有趣的是，除ADCC外，SD1-HFC蛋白还表现出强烈的CDC活性，对表达间皮素表达的肿瘤细胞。 SD1-PE38免疫毒素在体外抑制了癌细胞的2D单层和3D球体生长。此外，SD1-HFC蛋白在裸鼠中引起肿瘤异种移植物的显着抑制。这些结果表明，SD1人类抗体具有潜力作为新型癌症治疗候选者，并且可能显着改善靶向表达间皮素表达肿瘤的当前抗体疗法。 5）使用嵌合抗原受体（CAR）对B细胞恶性肿瘤的免疫靶向是一种有希望的新方法，但是影响汽车功效的关键因素尚不清楚。为了测试将CD22靶向前体B细胞急性淋巴细胞白血病（BCP-ALL）的适用性，对111例BCP all的患者的淋巴细胞进行了CD22表达，发现所有淋巴母细胞均为CD22阳性，并具有中位数CD22表达水平。 3500个站点/单元。靶向CD22的三个不同的结合域被融合到各种TCR信号域Plus/Minus a igG重链常数域（CH2CH3），以创建一系列适合于描述最佳汽车配置的向量构造。源自M971抗CD22 MAB的汽车，其靶向近端CD22表位与与其他结合结构域相比表现出优异的抗血肿活性，并且添加了4-1BB信号传导域中的CD28.CD3。抗CD22结合基序，并通过CH2CH3从膜上扩展CD22结合结构域没有影响。我们得出的结论是，第二代M971 mab衍生的抗CD22汽车是有希望的新型疗法，应在BCP-ALL中进行测试。