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]

我们继续识别，工程和特征人类单克隆抗体（HMAB），包括针对HIV-1的纳米珠单抗，与HIV-1，新兴和生物化的相关病毒以及癌症以及癌症，以及其中一些与纳米颗粒共轭。我们已经广泛评估了我们最有前途的抗HIV抗体G41特异性M43，M44，M46，M46，M47和M48的中和活性和反应性。先前已经发现，这两个最佳特征的GP41特异性交叉反应中和人类单克隆抗体4E10和2F5靶向线性表位在膜近端外部区域（MPER）（MPER）（MPER），并对包括心脏抗原（包括心脏磷脂（CL）和磷酸磷脂的自我抗原（包括心脏抗原）表现出反应性。据推测，这种多反应性可能是未能基于MPER引起疫苗免疫剂的2F5和4E10样交叉反应中和HMAB的主要原因。 我们发现，在基于PBMC的测定中，我们的一种抗体M44中和来自不同进化枝的21个HIV-1主要分离株中的大多数，其平均效力明显高于4E10和Z13。与2F5，4E10和Z13相反，M44与源自GP41的变性GP140和线性肽之间没有任何显着结合，这表明表位的构象性质。与4E10和2F5不同，M44在ELISA和BIACORE分析中不与Cl和PS结合，也不与Luminex分析中的任何一组蛋白质和DNA自动抗体结合。这是第一个GP41特异性的交叉反应性HMAB，没有可检测到的多反应性。它的新型GP41上的保守构象位用于疫苗免疫原子的设计和治疗剂的靶标。我们还在GP120上开发针对CD4诱导的表位的纳米珠单抗。为了应对生物恐怖主义的威胁以及由SARS冠状病毒（SARS COV）造成的健康危机，我们继续识别和表征与生物浮雕相关的Hendra病毒（HEV）和NIPAH病毒（NIV）（NIV）和SARS COV的中和抗体。以前，我们报道了HENIPAVIRUS中和重组HMAB的分离。其中一种抗体之一M102表现出最高水平的交叉反应性并与NIV和HEV G蛋白结合，是通过轻链改组的成熟，并与HEV G蛋白的可溶性形式SGHEV相结合。最成熟的克隆（指定为M102.4）被转换为IgG1，并针对感染性NIV和HEV进行了测试。它表现出异常有效和交叉反应性抑制活性，分别低于0.04和0.6 g/ml，抑制浓度分别显示出50％。这是对HEV和NIV都非常有效的第一个也是唯一的HMAB。它的半衰期很长，在雪貂中没有毒性，并且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具有用于治疗HENIPAVIRAS引起的疾病的治疗性的潜力。它也可用于预防，诊断和研究试剂。 SARS COV在2002年底/2003年初引起了全球流行病，并在2003 - 2004年冬季进行了第二次爆发。据报道，GD03菌株是从第二次爆发的指数患者中分离出来的，据报道通过HMABS 80R和S3.1抵抗中和，可以从第一次爆发中有效中和分离株。我们最近报道说，从第一次SARS爆发（Urbani，Tor2）和Palm Civets（SZ3，SZ16）中，有效中和GD03的M396和S230.15和S230.15的两个HMAB。这些抗体还保护了对含有GD03和SZ16 S糖蛋白的重组病毒挑战的小鼠。 两种抗体都与SARS COV受体ACE2竞争，以与RBD结合，表明一种中和机制涉及干扰SARS COV-ACE2相互作用。 基于先前求解的RBD-M396结构，我们进行了广泛的位置，定向了RBD的诱变。在SARS COV Spike中发现了RBD（I489和Y491）中的两个假定的热点残留物，可能会导致大多数M396结合能。残基I489和Y491在SARS COV尖峰中高度保守，表明M396交叉反应性的可能机制。序列分析和诱变数据表明，M396可能用已知序列中和所有人畜共患病和流行病SARS COV分离株，除了源自蝙蝠的菌株。这些抗体是第一个鉴定出的HMAB，它们对两种SARS爆发和棕榈树枝的分离株表现出交叉反应性，并且可能在诊断，预防和对SARS COV感染的治疗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）系统的抗体，该系统在癌症中起着重要作用。目前，针对IGF受体I型（IGF-IR）的几种单克隆抗体正在实体瘤患者中进行测试。但是，全长抗体渗透到实体瘤中的效率缓慢且效率低下。我们一直在假设，靶向IGF-IR配体IGF-I和IGF-II，抗体可能不需要在肿瘤间质空间中存在抗体，但可以将复杂平衡和准稳态状态转移到导致ligand的血浆中的配体 - 抗体复合物中，以使血浆中的配体 - 抗体复合物在demoror in tumor in tumor in tumor in tumor中。为了检验该假设，我们使用了完全人类的单克隆抗体IgG1 M610，该抗体与IGF-II具有高（NM）亲和力结合，并抑制由IGF-II与IGF-IR相互作用介导的信号转导。我们选择了基于MCF-7细胞的小鼠异种移植模型，因为这些乳腺癌细胞会产生高水平的IGF-II，但不需要这种生长因子来增加其增殖。与没有抗体的对照相比，肿瘤中的IGF-II浓度从24 +/- 14 nm（范围11-37 nm）（范围11-37 nm）显着降低至8 +/- 3 nm（4-10 nm）（范围为4-10 nm）。下降取决于每只小鼠0.1 mg的抗体浓度，平均为1.4倍。间质性液中的IGF-II耗竭可能更高 - 基于30％间质流体分数的估计值和肿瘤中IGF-II均匀浓度的估计值表明至少降低了10倍。由于肿瘤中的IGF-I浓度不受影响，因此对IGF-II的耗竭是特异性的。这些结果支持这样的概念，即全长抗体可以降低肿瘤中可溶性配体的浓度。由于在实体瘤中获得间质液的获取是完整抗体功效的主要问题，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起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结合使用，以治疗IGF-II敏感的固体肿瘤。我们还针对IGF-IR开发了纳米珠单抗，并将我们的某些抗体与纳米型体结合。我们计划继续识别针对癌症，HIV-1和Biodef的新型NHMAB [汇总以7800个字符的截断]