Human Monoclonal Antibodies and Antibody-Guided Nanoparticles

人单克隆抗体和抗体引导的纳米颗粒

• 批准号: 7592864
• 负责人: Dimiter S Dimitrov
• 金额: $ 135.06万
• 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/7592864
• 关键词: Affect; Affinity; Animal Model; Animals; Antibodies; Antigens; Antineoplastic Agents; Autoantigens; Binding; Biological Assay; Bioterrorism; Breast Cancer Cell; Cardiolipins; Cell Proliferation; Chiroptera; Communicable Diseases; Complex; Coronavirus Infections; DNA; Data; Development; Diagnosis; Diagnostic; Disease; Disease Outbreaks; Engineering; Enzyme-Linked Immunosorbent Assay; Epidemic; Epitopes; Equilibrium; Exhibits; Failure; Felis catus; Ferrets; GP 140; GTP-Binding Proteins; Glycoproteins; Goals; Growth Factor; HIV Antibodies; HIV Envelope Protein gp120; HIV-1; Half-Life; Health; Hendra Virus; Henipavirus; Hot Spot; Human; IgG1; Inhibitory Concentration 50; Insulin Receptor; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Insulin-Like Growth Factor Receptor; Intercellular Fluid; Knowledge; Length; Ligands; Light; MCF7 cell; Malignant Neoplasms; Mediating; Membrane; Monoclonal Antibodies; Mus; Mutagenesis; Nature; Nipah Virus; Pan Genus; Patients; Penetration; Peptides; Play; Prevention; Prophylactic treatment; Proteins; RNA Recognition Motif; Range; Reagent; Recombinants; Reporting; Research; Role; SARS coronavirus; Sequence Analysis; Severe Acute Respiratory Syndrome; Signal Transduction; Site-Directed Mutagenesis; Solid Neoplasm; Somatomedins; Structure; System; Testing; Therapeutic; Toxic effect; Vaccine Design; Vaccines; Viverridae; Week; Xenograft Model; antibody conjugate; base; biodefense; concept; coronavirus receptor; cross reactivity; human monoclonal antibodies; indexing; interstitial; nano; nanoparticle; neutralizing antibody; novel; plasma lead; prophylactic; receptor; recombinant virus; therapeutic target; transmission process; tumor; virus related cancer

We have continued to identify, engineer and characterize human monoclonal antibodies (hmAbs) including nanohumAbs against HIV-1, emerging and biodefense-related viruses, and cancer, and conjugate some of them with nanoparticles. We have extensively evaluated the neutralizing activity and reactivity to self antigens of our most promising anti-HIV antibodies g41-specific m43, m44, m46, m47 and m48. It has been previously found that the two best characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies 4E10 and 2F5 target linear epitopes in the membrane proximal external region (MPER) and exhibit reactivity to self antigens including cardiolipin (CL) and phosphatidyliserine (PS). It has been hypothesized that such polyreactivity could be a major reason for the failure to elicit 2F5 and 4E10-like cross-reactive neutralizing hmAbs by vaccine immunogens based on the MPER. We found that in PBMCs based assays one of our antibodies, m44, neutralized most of the 21 HIV-1 primary isolates from different clades with a significantly higher average potency than that of 4E10 and Z13. In contrast to 2F5, 4E10 and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. Unlike 4E10 and 2F5, m44 did not bind to CL and PS in ELISA and Biacore assays, nor bound to any of a panel of protein and DNA autoantigens in luminex assays. This is the first gp41-specific cross-reactive hmAb that does not have detectable polyreactivity. Its novel conserved conformational epitope on gp41 could be used in the design of vaccine immunogens and as a target for therapeutics. We are also developing nanohumAbs against CD4-induced epitopes on gp120. Responding to the threat of bioterrorism and to the health crisis caused by the SARS coronavirus (SARS CoV), we have continued to identify and characterize neutralizing antibodies to the biodefense-related Hendra virus (HeV) and Nipah virus (NiV), and to the SARS CoV. Previously, we reported on the isolation of henipavirus-neutralizing recombinant hmAbs. One of these antibodies, m102, which exhibited the highest level of cross-reactivity and binding to both NiV and HeV G protein, was affinity maturated by light chain shuffling, and further panning against a soluble form of the HeV G protein, sGHeV. The most matured clone, designated m102.4, was converted to IgG1 and tested against infectious NiV and HeV; it exhibited exceptionally potent and cross-reactive inhibitory activity with 50% inhibitory concentrations below 0.04 and 0.6 g/ml, respectively. This is the first and only hmAb that is exceptionally potent against both HeV and NiV. It has long (weeks) half-life and no toxicities in ferrets, and is being tested in animal models based on ferrets and cats. These results suggest that m102.4 has potential as a therapeutic for treatment of diseases caused by henipaviruses. It could be also used for prophylaxis, diagnosis and as a research reagent. The SARS CoV caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003-2004 by an independent animal to human transmission. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the hmAbs 80R and S3.1 which can potently neutralize isolates from the first outbreak. We have recently reported that two hmAbs, m396 and S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani, or recombinant viruses bearing the GD03 and SZ16 S glycoproteins. Both antibodies competed with the SARS CoV receptor, ACE2, for binding to the RBD suggesting a mechanism of neutralization that involves interference with the SARS CoV-ACE2 interaction. Based on the previously solved RBD-m396 structure we performed extensive site directed mutagenesis of the RBD. Two putative hot spot residues in the RBD (I489 and Y491) were identified within the SARS CoV spike that likely contribute to most of the m396 binding energy. Residues I489 and Y491 are highly conserved within the SARS CoV spike indicating a possible mechanism of the m396 cross-reactivity. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS CoV isolates with known sequences, except strains derived from bats. These antibodies are the first identified hmAbs, which exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets, and could have potential applications for diagnosis, prophylaxis and treatment of SARS CoV infections. We have also continued to characterize our antibodies against components of the insulin-like growth factor (IGF) system which plays an important role in cancer. Currently several monoclonal antibodies targeting the IGF receptor type I (IGF-IR) are being tested in patients with solid tumors. However, penetration of full-length antibodies into solid tumors is slow and inefficient. We have been hypothesizing that targeting the IGF-IR ligands, IGF-I and IGF-II, by antibodies may not require antibody presence in the tumor interstitial space but could shift the complex equilibrium and quasi-steady states to ligand-antibody complexes in the plasma leading to depletion of the ligand in the tumor. To test this hypothesis we used a fully human monoclonal antibody, IgG1 m610, which binds with high (nM) affinity to IGF-II and inhibits signal transduction mediated by the IGF-II interaction with the IGF-IR. We chose a mouse xenograft model based on MCF-7 cells because these breast cancer cells produce high levels of IGF-II but do not require this growth factor for their proliferation. The IGF-II concentration in tumors was significantly decreased from 24 +/-14 nM (range 11 - 37 nM) to 8 +/-3 nM (range 4-10 nM) after the fourth administration of the antibody at the highest concentration of 1 mg per mouse compared to the control without antibody. The decrease was dependent on the antibody concentration at 0.1 mg per mouse it was on average 1.4-fold. The IGF-II depletion in the interstitial fluid is likely to be much higher - an estimate based on 30% interstitial fluid fraction and uniform IGF-II concentration in the tumor indicates at least 10-fold decrease. The depletion was specific for IGF-II since the IGF-I concentration in the tumor was not affected. These results support the concept that full-length antibodies can decrease the concentration of soluble ligands in tumors. Because limited access to interstitial fluid in solid tumors is a major problem for the efficacy of intact antibodies, targeting ligands of growth factors could be an useful strategy for treatment of solid tumors. Targeting of IGF-II could inhibit not only its binding to the IGF-IR but also its binding to the insulin receptor and decrease cell proliferation caused by signals initiated by the IGF-II interaction with both receptors. Thus m610 could be used in combination with anti-IGF-IR antibodies and other anti-cancer drugs for treatment of IGF-II-sensitive solid tumors. We are also developing nanohumAbs against the IGF-IR and conjugating some of our antibodies with nanoliposomes. We plan to continue to identify novel potent nhmAbs against cancer, HIV-1, and biodef [summary truncated at 7800 characters]

我们继续鉴定、设计和表征人类单克隆抗体 (hmAb)，包括针对 HIV-1、新兴病毒和生物防御相关病毒以及癌症的 nanohumAb，并将其中一些与纳米颗粒结合。我们广泛评估了我们最有前途的抗 HIV 抗体 g41 特异性 m43、m44、m46、m47 和 m48 的中和活性和对自身抗原的反应性。此前已发现，两种最佳表征的 gp41 特异性交叉反应中和人单克隆抗体 4E10 和 2F5 靶向近膜外部区域 (MPER) 中的线性表位，并对包括心磷脂 (CL) 和磷脂酰丝氨酸 (PS) 在内的自身抗原表现出反应性）。据推测，这种多反应性可能是基于 MPER 的疫苗免疫原未能引发 2F5 和 4E10 样交叉反应中和 hmAb 的主要原因。 我们发现，在基于 PBMC 的检测中，我们的一种抗体 m44 中和了来自不同进化枝的 21 种 HIV-1 主要分离株中的大部分，其平均效力显着高于 4E10 和 Z13。与2F5、4E10和Z13相比，m44没有以任何显着程度结合变性的gp140和衍生自gp41的线性肽，表明表位的构象性质。与 4E10 和 2F5 不同，m44 在 ELISA 和 Biacore 测定中不与 CL 和 PS 结合，也不在 luminex 测定中与任何一组蛋白质和 DNA 自身抗原结合。这是第一个不具有可检测到的多反应性的 gp41 特异性交叉反应 hmAb。其 gp41 上的新型保守构象表位可用于疫苗免疫原的设计并作为治疗的靶点。我们还在开发针对 gp120 上 CD4 诱导表位的 nanohumAb。为了应对生物恐怖主义的威胁和 SARS 冠状病毒 (SARS CoV) 引起的健康危机，我们继续识别和表征生物防御相关的亨德拉病毒 (HeV) 和尼帕病毒 (NiV) 的中和抗体，以及针对SARS 冠状病毒。此前，我们报道了亨尼帕病毒中和重组 hmAb 的分离。其中一种抗体 m102 表现出最高水平的交叉反应性并与 NiV 和 HeVG 蛋白结合，通过轻链改组和进一步针对可溶形式的 HeVG 蛋白 sGHeV 进行淘选，使亲和力成熟。最成熟的克隆被命名为 m102.4，被转化为 IgG1，并针对传染性 NiV 和 HeV 进行测试；它表现出异常有效的交叉反应抑制活性，50% 抑制浓度分别低于 0.04 和 0.6 g/ml。这是第一个也是唯一一个对 HeV 和 NiV 都非常有效的 hmAb。它的半衰期长（数周），对雪貂没有毒性，目前正在基于雪貂和猫的动物模型中进行测试。这些结果表明m102.4具有作为治疗由亨尼帕病毒引起的疾病的治疗剂的潜力。它还可用于预防、诊断和作为研究试剂。 SARS 冠状病毒在 2002 年底/2003 年初引起了世界范围内的流行，并在 2003-2004 年冬季通过独立的动物向人类传播引起了第二次爆发。据报道，从第二次爆发的指示患者中分离出的 GD03 菌株能够抵抗 hmAb 80R 和 S3.1 的中和作用，而 hmAb 80R 和 S3.1 可以有效中和第一次爆发的分离株。我们最近报道了两种 hmAb，m396 和 S230.15，有效中和了 GD03 以及来自第一次 SARS 爆发（Urbani、Tor2）和果子狸（SZ3、SZ16）的代表性分离株。这些抗体还可以保护受到 Urbani 或带有 GD03 和 SZ16 S 糖蛋白的重组病毒攻击的小鼠。 这两种抗体都与 SARS 冠状病毒受体 ACE2 竞争与 RBD 的结合，这表明存在一种涉及干扰 SARS 冠状病毒-ACE2 相互作用的中和机制。 基于先前解决的 RBD-m396 结构，我们对 RBD 进行了广泛的定点诱变。在 SARS CoV 刺突中发现了 RBD 中两个假定的热点残基（I489 和 Y491），它们可能贡献了大部分 m396 结合能。残基 I489 和 Y491 在 SARS CoV 刺突内高度保守，表明 m396 交叉反应的可能机制。序列分析和诱变数据表明，m396 可能会中和所有具有已知序列的人畜共患和流行性 SARS 冠状病毒分离株，但源自蝙蝠的菌株除外。这些抗体是第一个被鉴定的 hmAb，对两次 SARS 爆发的分离株和果子狸表现出交叉反应，可能在诊断、预防和治疗 SARS 冠状病毒感染方面具有潜在的应用。我们还继续表征针对胰岛素样生长因子（IGF）系统成分的抗体，该系统在癌症中发挥着重要作用。目前，几种针对 I 型 IGF 受体 (IGF-IR) 的单克隆抗体正在实体瘤患者中进行测试。然而，全长抗体渗透到实体瘤中的速度缓慢且效率低下。我们一直假设，通过抗体靶向IGF-IR配体IGF-I和IGF-II，可能不需要肿瘤间隙中存在抗体，但可以将复合物平衡和准稳态转变为配体-抗体复合物。血浆导致肿瘤中的配体耗尽。为了检验这一假设，我们使用了全人单克隆抗体 IgG1 m610，它以高 (nM) 亲和力与 IGF-II 结合，并抑制 IGF-II 与 IGF-IR 相互作用介导的信号转导。我们选择基于 MCF-7 细胞的小鼠异种移植模型，因为这些乳腺癌细胞产生高水平的 IGF-II，但不需要这种生长因子来进行增殖。第四次施用最高浓度的抗体后，肿瘤中的 IGF-II 浓度从 24 +/-14 nM（范围 11 - 37 nM）显着降低至 8 +/-3 nM（范围 4-10 nM）。与没有抗体的对照相比，每只小鼠 1 mg。下降幅度取决于每只小鼠 0.1 mg 的抗体浓度，平均下降 1.4 倍。间质液中 IGF-II 的消耗可能要高得多 - 基于 30% 间质液分数和肿瘤中均匀 IGF-II 浓度的估计表明至少减少了 10 倍。由于肿瘤中的 IGF-I 浓度不受影响，因此 IGF-II 的消耗是特异性的。这些结果支持全长抗体可以降低肿瘤中可溶性配体浓度的概念。由于实体瘤中间质液的获取受限是完整抗体功效的一个主要问题，因此靶向生长因子的配体可能是治疗实体瘤的有用策略。 IGF-II 的靶向不仅可以抑制其与 IGF-IR 的结合，还可以抑制其与胰岛素受体的结合，并减少由 IGF-II 与两种受体相互作用引发的信号引起的细胞增殖。因此，m610可以与抗IGF-IR抗体和其他抗癌药物联合用于治疗IGF-II敏感的实体瘤。我们还在开发针对 IGF-IR 的 nanohumAb，并将我们的一些抗体与纳米脂质体结合。我们计划继续识别针对癌症、HIV-1 和 biodef 的新型有效 nhmAb [摘要被截断为 7800 个字符]