Human Monoclonal Antibodies Against Cancer

抗癌人类单克隆抗体

• 批准号: 9343676
• 负责人: Dimiter S Dimitrov
• 金额: $ 90.97万
• 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/9343676
• 关键词: Acute Myelocytic Leukemia; Adverse effects; Affect; Affinity; Animal Model; Animals; Antibodies; Antibody Affinity; Antibody-drug conjugates; Antigens; B-Lymphocytes; Binding; Bispecific Antibodies; CD14 gene; CD22 gene; CD28 gene; CD34 gene; Cell Line; Cell physiology; Cells; Cessation of life; Childhood Solid Neoplasm; Chimeric Proteins; Clinical; Clinical Trials; Computer Simulation; Cytolysis; Dependency; Detection; Development; Diagnosis; Diagnostic; Disease remission; Dissociation; Dominant Negative Receptor; Dose; Down-Regulation; Dreams; Electroporation; Engineering; Epithelial; Epitopes; Equilibrium; European Union; Ewings sarcoma; Exhibits; Fibronectins; Generations; Goals; Growth; Half-Life; Hematopoietic; Hematopoietic stem cells; Hour; Human; IgG1; Immune; Immune System Diseases; Immune system; Immunoglobulin Fragments; Immunoglobulin G; In Vitro; Insulin Receptor; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Insulin-Like-Growth Factor I Receptor; Knowledge; Libraries; Ligand Binding; Ligands; Lymphoma; Macaca; Magic; Malignant Childhood Neoplasm; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Measures; Mediating; Membrane; Messenger RNA; Methodology; Monoclonal Antibodies; Multiple Myeloma; Mus; Myelogenous; N-terminal; NCAM1 gene; Neoplasm Metastasis; Neural Cell Adhesion Molecules; Neuroblastoma; Neurosecretory Systems; PDCD1LG1 gene; Pathway interactions; Pediatric Neoplasm; Pediatric Oncology; Penetration; Peptide/MHC Complex; Phage Display; Pharmaceutical Preparations; Phosphorylation; Pleural Mesothelioma; Process; Property; Proteins; Reporting; Resistance; Rhabdomyosarcoma; Risk; Solid; Solid Neoplasm; Somatomedins; Structure; System; T cell therapy; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; Therapeutic Uses; Time; Tissues; Toxic effect; Up-Regulation; Work; Xenograft Model; anti-cancer therapeutic; antibody engineering; antigen binding; base; cancer cell; chemotherapeutic agent; chemotherapy; chimeric antigen receptor; cytokine; cytotoxic; cytotoxicity; design; dimer; exhaustion; folate-binding protein; human monoclonal antibodies; immunogenic; improved; in vitro activity; in vivo; insight; killings; lung small cell carcinoma; mTOR Inhibitor; mesothelin; monocyte; mouse model; neonatal Fc receptor; neoplastic cell; neuroblastoma cell; novel; novel strategies; osteosarcoma; overexpression; pediatric patients; programs; pyrrolobenzodiazepine; receptor; small hairpin RNA; small molecule; therapeutic development; tool; transcytosis; tumor; tumor eradication; tumor microenvironment

This year we have continued to identify novel mAbs in several formats as Fabs, scFvs and eAds against cancer-related proteins mostly for pediatric patients. These mAbs were tested for their activity against cancer cells in vitro and in vivo and used for development of novel approaches for multispecific targeting. Two of the mAbs in CAR format, against CD22 and mesothelin, continued to be tested in clinical trials. We have also characterized drugability of some mAbs including their propensity for aggregation. The major accomplishments are summarized below. 1) Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Our collaborators and we investigated whether programmed death-1-mediated (PD-1-mediated) T cell exhaustion affects mesothelin-targeted CAR T cells using our antibody m912, and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, it was determined that relatively high doses of both CD28- and 4-1BB-based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. 2) CD56 (NCAM, neural cell adhesion molecule) is over-expressed in many tumor types, including neuroblastoma, multiple myeloma, small cell lung cancer, ovarian cancer, acute myeloid leukemia, NK-T lymphoma, neuroendocrine cancer and pancreatic cancer. Using phage display, we identified 2 high-affinity anti-CD56 human monoclonal antibodies (mAbs), m900 and m906, which bound to spatially separated non-overlapping epitopes with similar affinity (equilibrium dissociation constant 2.9 and 4.5 nM, respectively). m900 bound to the membrane proximal fibronectin type III-like domains, whereas m906 bound to the N-terminal IgG-like domains. m906 induced significant down-regulation of CD56 in 4 neuroblastoma cell lines tested, while m900-induced downregulation of CD56 was much lower. Antibody-drug conjugates (ADCs) made by conjugation with a highly potent pyrrolobenzodiazepine dimer (PBD) exhibited killing activity that correlated with CD56 down-regulation, and to some extent with in vivo binding ability of the antibodies. The m906PBD ADC was much more potent than m900PBD, likely due to higher CD56-mediated downregulation and stronger binding to cells. Treatment with m906PBD ADC resulted in very potent cytotoxicity (IC50: 0.05-1.7 pM). These results suggest a novel approach for targeting CD56-expressing neuroblastoma cells. Further studies in animal models and in humans are needed to find whether these antibodies and their drug conjugates are promising candidate therapeutics. 3) Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. We isolated a high-affinity (HA) folate receptor beta (FRbeta)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRbeta-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRbeta CAR exhibited greatly enhanced antitumor activity against FRbeta(+) AML in vitro and in vivo compared with a low-affinity FRbeta CAR (54.3 nm KD). Using the HA-FRbeta immunoglobulin G, FRbeta expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRbeta CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRbeta CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRbeta CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. 4) Engineered antibody domains (eAds) are promising candidate therapeutics but their half-life is relatively short partly due to weak or absent binding to the neonatal Fc receptor (FcRn). We developed a novel approach to increase the eAd binding to FcRn based on a combination of structure-based design, computational modeling and phage display methodologies. By using this approach, we identified 2 IgG1 CH2-derived eAds fused to a short FcRn-binding motif derived from IgG1 CH3 that exhibited greatly enhanced FcRn binding with strict pH dependency. Importantly, the increased affinity resulted in significantly enhanced FcRn-mediated epithelial transcytosis and prolonged elimination half-life (mean 44.1 hours) in cynomolgus macaques. These results demonstrate for the first time that the half-life of isolated eAds can be prolonged (optimized) by increasing their binding to FcRn while maintaining their small size, which has important implications for development of therapeutics, including eAd-drug conjugates with enhanced penetration in solid tissues. 5) The insulin-like growth factors (IGFs), IGF-1 and IGF-2, have been implicated in the growth, survival and metastasis of a broad range of malignancies including pediatric tumors. They bind to the IGF receptor type 1 (IGF-1R) and the insulin receptor (IR) which are overexpressed in many types of solid malignancies. Activation of the IR by IGF-2 results in increased survival of tumor cells. We have previously identified a novel human monoclonal antibody, m708.5, which binds with high (pM) affinity to both human IGF-1 and IGF-2, and potently inhibits phosphorylation of the IGF-1R and the IR in tumor cells. m708.5 exhibited strong antitumor activity as a single agent against most cell lines derived from neuroblastoma, Ewing family of tumor, rhabdomyosarcoma and osteosarcoma. When tested in neuroblastoma cell lines, it showed strong synergy with temsirolimus and synergy with chemotherapeutic agents in vitro. In xenograft models, the combination of m708.5 and temsirolimus significantly inhibited neuroblastoma growth and prolonged mouse survival. Taken together, these results support the clinical development of m708.5 for pediatric solid tumors with potential for synergy with chemotherapy and mTOR inhibitors. 6) Currently, we participate in the work of the Pediatric Oncology Dream team where we identified and performed initial characterization of several mAbs against targets of importance for childhood cancers. Some of the results were described above and others will be reported in the next year report after additional characterization of those antibodies including in animal models.

今年，我们继续以几种格式鉴定出新型的mAB，因为针对癌症相关蛋白的Fab，SCFV和EADS主要用于小儿患者。这些mAb在体外和体内测试了它们针对癌细胞的活性，并用于开发新的多特异性靶向方法。在临床试验中，对CD22和间皮素的CAR格式的两个mAB进行了测试。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）免疫攻击后，实体瘤上调与T细胞上抑制受体结合的共抑制性配体。这种自适应耐药性损害了嵌合抗原受体（CAR）T细胞疗法的功效，后者将T细胞重定向到实体瘤。我们的合作者，我们研究了使用我们的抗体M912的编程死亡-1介导的T细胞耗尽（PD-1介导的T细胞耗尽）会影响间皮蛋白靶向的CAR T细胞，并探索了克服抑制CAR T细胞的细胞内部策略。使用胸膜间皮瘤的原位小鼠模型，确定基于CD28-和4-1BB的第二代汽车T细胞相对较高剂量可以消除肿瘤。 CAR介导的CD28和4-1BB共刺激导致低T细胞剂量治疗的动物的T细胞持久水平相似。但是，肿瘤微环境中的PD-1上调抑制了T细胞功能。在较低剂量下，4-1BB CAR T细胞保留其细胞毒性和细胞因子分泌的功能比CD28 CAR T细胞更长。 4-1BB CAR T细胞的延长功能与改善的存活率相关。 PD-1/PD-1配体[PD-L1]途径干扰，通过PD-1抗体检查点阻断，细胞中性PD-1 SHRNA阻断或PD-1显性负受体，恢复了CD28 CAR T细胞的效应函数。这些发现为实体瘤中的人类汽车T细胞耗尽提供了机械见解，并表明PD-1/PD-L1封锁可能是提高CAR T细胞疗法效力的有效策略。 2）在许多肿瘤类型中，CD56（NCAM，神经细胞粘附分子）过表达，包括神经母细胞瘤，多发性骨髓瘤，小细胞肺癌，卵巢癌，急性髓性白血病，NK-T淋巴瘤，神经内泌酸酯癌和panccreatic癌。使用噬菌体显示，我们确定了2个高亲和力抗CD56人单克隆抗体（MABS），M900和M906，它们注定会在空间分离具有相似亲和力的空间分离的非重叠表位（平衡分离常数2.9和4.5 nm）。 M900与膜近端纤连蛋白型III样结构域结合，而M906与N末端IgG样结构域结合。 M906在4个神经母细胞瘤细胞系中诱导了CD56的显着下调，而M900诱导的CD56下调的下调要低得多。抗体 - 药物结合物（ADC）与高度有效的吡咯苯二氮卓二聚二聚体（PBD）产生的抗体表现出与CD56下调相关的杀伤活性，并且在某种程度上与抗体的体内结合能力相关。 M906PBD ADC比M900pBD更有效，这可能是由于CD56介导的下调较高，并且与细胞的结合更强。用M906PBD ADC治疗导致非常有效的细胞毒性（IC50：0.05-1.7 pm）。这些结果表明了一种用于靶向表达CD56神经母细胞瘤细胞的新方法。需要在动物模型和人类中进行进一步的研究，以查找这些抗体及其药物缀合物是否有希望的候选治疗疗法。 3）急性髓样白血病（AML）是一种侵略性恶性肿瘤，有必要开发新的治疗方法。嵌合抗原受体（CAR）T细胞疗法似乎很有希望，但在健康组织中对抗原的靶向，肿瘤识别仍然是一个令人关注的问题。我们分离了一个高亲和力（HA）叶酸受体β（FRBETA）特异性单链可变片段（2.48 nm kD），以优化FRBETA重新定向的AML CAR T细胞治疗。与低亲和力的FRBETA汽车（54.3 nm kD）相比，稳定表达Ha-frbeta汽车的T细胞在体外和体内表现出大大增强的抗肿瘤活性。使用HA-FRBETA免疫球蛋白G，可以在髓样细胞造血细胞中检测到FRBETA表达。但是，几乎无法检测到CD34（+）造血干细胞（HSC）中的表达。因此，Ha-frbeta Car T细胞裂解成熟的CD14（+）单核细胞，而HSC菌落形成不受影响。由于消除成熟的髓样谱系的潜力，评估了瞬时CAR表达的mRNA CAR电穿孔。 mRNA - 电饰HA-FRBETA CAR T细胞在体外和体内保留有效的抗肿瘤活性。总之，我们的结果强调了抗体亲和力在靶蛋白检测和CAR开发中的重要性，并表明有效的HA-FRBETA CAR T细胞的瞬时输送对AML非常有效，并降低了长期髓样毒性的风险。 4）工程抗体结构域（EADS）是有希望的候选治疗剂，但其半衰期相对较短，部分原因是与新生儿FC受体（FCRN）较弱或不存在结合。我们开发了一种新型方法，以基于基于结构设计，计算建模和噬菌体显示方法的组合来增加与FCRN的结合。通过使用这种方法，我们确定了2个IgG1 CH2衍生的EADS融合到源自IgG1 CH3的短FCRN结合基序中，这些基序具有严格的pH依赖性，其表现出极大的增强FCRN结合。重要的是，增加的亲和力导致FCRN介导的上皮转胞膜增长显着增强，并在cynomolgus猕猴中长期消除半衰期（平均44.1小时）。这些结果首次表明，可以通过增加其与FCRN的结合，同时保持其小尺寸，从而延长（优化）孤立的EAD的半衰期，这对疗法的发展具有重要意义，包括EAD-Drug conjugates具有增强固体组织中的渗透率。 5）胰岛素样生长因子（IGF），IGF-1和IGF-2已与包括小儿肿瘤在内的广泛恶性肿瘤的生长，生存和转移有关。它们与IGF受体1型（IGF-1R）和胰岛素受体（IR）结合，在许多类型的固体恶​​性肿瘤中都过表达。 IGF-2激活IR会导致肿瘤细胞的存活率增加。我们先前已经鉴定出一种新型的人类单克隆抗体M708.5，该抗体与人IGF-1和IGF-2都具有高（PM）亲和力，并在肿瘤细胞中有效抑制IGF-1R和IR的磷酸化。 M708.5作为对大多数源自神经母细胞瘤，肿瘤尤林家族，横纹肌肉瘤和骨肉瘤的单一剂表现出强大的抗肿瘤活性。当在神经母细胞瘤细胞系中进行测试时，它在体外与Temsirolimus和与化学治疗剂的协同作用显示出很强的协同作用。在异种移植模型中，M708.5和Temsirolimus的组合显着抑制神经母细胞瘤的生长和延长的小鼠生存率。综上所述，这些结果支持M708.5的临床发展，用于儿童实体瘤，具有与化学疗法和MTOR抑制剂协同作用的潜力。 6）目前，我们参加了儿科肿瘤学团队的工作，在那里我们确定并对几个mAB进行了对儿童癌症重要性的初步表征。上面描述了一些结果，而另一些结果将在明年的报告中进行报告，此这些抗体包括动物模型中的这些抗体。