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，我们继续鉴定多种形式的新型单克隆抗体，例如针对癌症相关蛋白的 Fab、scFv 和 eAd，主要用于儿科患者。这些单克隆抗体在体外和体内测试了其对抗癌细胞的活性，并用于开发多特异性靶向的新方法。其中两种 CAR 形式的单克隆抗体（针对 CD22 和间皮素）继续在临床试验中进行测试。我们还表征了一些单克隆抗体的成药性，包括它们的聚集倾向。主要成就总结如下。 1) 免疫攻击后，实体瘤上调与 T 细胞上的抑制性受体结合的共抑制配体。这种适应性耐药性损害了嵌合抗原受体 (CAR) T 细胞疗法的功效，该疗法将 T 细胞重定向至实体瘤。我们的合作者和我们使用我们的抗体 m912 研究了程序性死亡 1 介导的（PD-1 介导的）T 细胞耗竭是否影响间皮素靶向的 CAR T 细胞，并探索了克服 CAR T 细胞抑制的细胞内在策​​略。使用胸膜间皮瘤原位小鼠模型，确定相对高剂量的基于 CD28 和 4-1BB 的第二代 CAR T 细胞实现了肿瘤根除。 CAR 介导的 CD28 和 4-1BB 共刺激导致低 T 细胞剂量治疗的动物中 T 细胞持久性水平相似；然而，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 细胞比 CD28 CAR T 细胞保留细胞毒性和细胞因子分泌功能的时间更长。 4-1BB CAR T 细胞功能的延长与生存率的提高相关。 PD-1/PD-1配体[PD-L1]通路干扰，通过PD-1抗体检查点阻断、细胞固有的PD-1 shRNA阻断或PD-1显性失活受体，恢复CD28 CAR T的效应器功能细胞。这些发现为实体瘤中人类 CAR T 细胞耗竭的机制提供了见解，并表明 PD-1/PD-L1 阻断可能是提高 CAR T 细胞疗法效力的有效策略。 2）CD56（NCAM，神经细胞粘附分子）在许多肿瘤类型中过度表达，包括神经母细胞瘤、多发性骨髓瘤、小细胞肺癌、卵巢癌、急性髓系白血病、NK-T淋巴瘤、神经内分泌癌和胰腺癌。利用噬菌体展示，我们鉴定了 2 种高亲和力抗 CD56 人单克隆抗体 (mAb) m900 和 m906，它们以相似的亲和力（平衡解离常数分别为 2.9 和 4.5 nM）与空间分离的非重叠表位结合。 m900 与膜近端纤连蛋白 III 型样结构域结合，而 m906 与 N 端 IgG 样结构域结合。 m906 在测试的 4 个神经母细胞瘤细胞系中诱导 CD56 显着下调，而 m900 诱导的 CD56 下调要低得多。通过与高效吡咯并苯二氮卓二聚体 (PBD) 缀合制成的抗体-药物缀合物 (ADC) 表现出与 CD56 下调相关的杀伤活性，并在一定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 介导的下调程度更高以及与细胞的结合更强。使用 m906PBD ADC 处理会产生非常有效的细胞毒性 (IC50: 0.05-1.7 pM)。这些结果提出了一种靶向表达 CD56 的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，以确定这些抗体及其药物缀合物是否是有前途的候选疗法。 3）急性髓系白血病（AML）是一种侵袭性恶性肿瘤，有必要开发新的治疗方法来延长缓解期。嵌合抗原受体 (CAR) T 细胞疗法似乎很有前景，但健康组织中抗原的靶向、肿瘤外识别仍然令人担忧。我们分离了高亲和力 (HA) 叶酸受体 β (FRbeta) 特异性单链可变片段 (2.48 nm KD)，用于优化针对 AML 的 FRbeta 重定向 CAR T 细胞疗法。与低亲和力 FRbeta CAR (54.3 nm KD) 相比，稳定表达 HA-FRbeta CAR 的 T 细胞在体外和体内表现出大大增强的针对 FRbeta(+) AML 的抗肿瘤活性。使用 HA-FRbeta 免疫球蛋白 G，可在骨髓系造血细胞中检测到 FRbeta 表达；然而，CD34(+)造血干细胞(HSC)中的表达几乎检测不到。因此，HA-FRbeta CAR T细胞裂解成熟的CD14(+)单核细胞，而HSC集落形成不受影响。由于具有消除成熟骨髓谱系的潜力，因此评估了用于瞬时 CAR 表达的 mRNA CAR 电穿孔。 mRNA 电穿孔的 HA-FRbeta CAR T 细胞在体外和体内保留了有效的抗肿瘤活性。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，并表明强效 HA-FRbeta CAR T 细胞的瞬时递送对于 AML 非常有效，并降低了长期骨髓毒性的风险。 4) 工程化抗体结构域 (eAd) 是有前途的候选疗法，但其半衰期相对较短，部分原因是与新生儿 Fc 受体 (FcRn) 的结合较弱或不存在。我们开发了一种基于结构的设计、计算建模和噬菌体展示方法相结合的新方法来增加 eAd 与 FcRn 的结合。通过使用这种方法，我们鉴定了 2 个 IgG1 CH2 衍生的 eAd，与衍生自 IgG1 CH3 的短 FcRn 结合基序融合，该基序表现出极大增强的 FcRn 结合并具有严格的 pH 依赖性。重要的是，亲和力的增加导致食蟹猴中 FcRn 介导的上皮转胞吞作用显着增强并延长了消除半衰期（平均 44.1 小时）。这些结果首次证明，通过增加与 FcRn 的结合同时保持其小尺寸，可以延长（优化）分离的 eAd 的半衰期，这对治疗药物的开发具有重要意义，包括具有增强渗透性的 eAd-药物缀合物在固体组织中。 5) 胰岛素样生长因子 (IGF)、IGF-1 和 IGF-2 与包括儿童肿瘤在内的多种恶性肿瘤的生长、存活和转移有关。它们与 1 型 IGF 受体 (IGF-1R) 和胰岛素受体 (IR) 结合，这些受体在许多类型的实体恶性肿瘤中过度表达。 IGF-2 激活 IR 会增加肿瘤细胞的存活率。我们之前发现了一种新型人单克隆抗体 m708.5，它以高 (pM) 亲和力与人 IGF-1 和 IGF-2 结合，并有效抑制肿瘤细胞中 IGF-1R 和 IR 的磷酸化。 m708.5 作为单一药物对大多数源自神经母细胞瘤、尤文家族肿瘤、横纹肌肉瘤和骨肉瘤的细胞系表现出强大的抗肿瘤活性。在神经母细胞瘤细胞系中进行测试时，它显示出与替西罗莫司的强烈协同作用以及与体外化疗药物的协同作用。在异种移植模型中，m708.5 和替西罗莫司的组合显着抑制神经母细胞瘤生长并延长小鼠存活期。总而言之，这些结果支持 m708.5 用于儿童实体瘤的临床开发，并具有与化疗和 mTOR 抑制剂协同作用的潜力。 6) 目前，我们参与了儿科肿瘤梦想团队的工作，在该团队中，我们针对儿童癌症的重要靶标确定了几种单克隆抗体并进行了初步表征。其中一些结果如上所述，其他结果将在对这些抗体（包括在动物模型中）进行进一步表征后在明年的报告中报告。