Abstract

Background

Although monoclonal antibodies binding the EGF-receptor (cetuximab, panitumumab) or CD38 (daratumumab) show therapeutic efficacy, many cancer patients still develop resistance. Therefore, new therapies are necessary. Nanobodies are highly soluble immunoglobulin domains derived from heavy chain antibodies (hcAbs) that naturally occur in camelids. Replication defective AAV (adeno associated virus) vectors, when equipped with a tumor targeting ligand, can be used for the delivery of (suicide) genes to tumor cells.

Methods

We generated recombinant chimeric nanobody-based human IgG1 hcAbs, by fusing EGFR- or CD38-specific nanobodies derived from immunized llamas to the hinge, CH2 and CH3 domains of human IgG1 (1, 2). To promote cytotoxic effector functions we introduced CDC (complement dependent cytotoxicity)-enhancing and ADCC (antibody-dependent cell-mediated cytotoxicity)-enhancing mutations into the CH2 and CH3 domains of human IgG1. As a second strategy to target CD38-overexpressing cells, we inserted CD38-specific nanobodies into the VP1 capsid protein of AAV2 (3). We analyzed the capacity of these constructs to target EGFR and CD38 in tumor cell lines and multiple myeloma (MM) patient bone marrow samples using proliferation, CDC, ADCC and AAV-transduction assays.

Results

EGFR-specific hcAbs effectively blocked the proliferation of tumor cell lines (HNSCC, head and neck squamous cell carcinoma and mCRC, metastatic colorectal cancer) expressing WT EGFR and cetuximab/panitumumab-escape variants of EGFR. EGFR- and CD38-specific hcAbs mediated effective ADCC towards tumor cell lines (HNSCC, mCRC and MM respectively). HcAbs carrying the hexabody mutation were more potent at inducing CDC than parental hcAbs. Display of CD38-specific nanobodies on the AAV capsid resulted in a 20- to 100-fold enhanced transduction of myeloma cells in patient bone marrow samples. Some of these results have recently been published (1, 2) or accepted for publication (3). We will present updated data on tumor cell lines and patient samples.

Conclusion

Nanobody-based hcAbs and nanobody-displaying AAV hold promise as novel tools to target solid and hematological tumors.

Legal entity responsible for the study

Friedrich Koch-Nolte.

Funding

DFG (German Research Foundation) and SFB (Collaborative Research Centres).

Disclosure

The author has declared no conflicts of interest.

中文翻译：

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114P用基于纳米抗体的重链抗体和AAV载体靶向固体和血液恶性肿瘤中的EGF受体和CD38

抽象的

背景

尽管结合EGF受体（西妥昔单抗，帕尼单抗）或CD38（达拉妥单抗）的单克隆抗体显示出治疗效果，但许多癌症患者仍产生耐药性。因此，新的疗法是必要的。纳米抗体是高度可溶性的免疫球蛋白结构域，其衍生自骆驼科动物中自然存在的重链抗体（hcAb）。当配备有肿瘤靶向配体时，复制缺陷型AAV（腺相关病毒）载体可用于将（自杀）基因传递给肿瘤细胞。

方法

通过将衍生自免疫的美洲驼的EGFR或CD38特异性纳米抗体融合到人IgG1的铰链，CH2和CH3域，我们生成了基于重组嵌合纳米抗体的人IgG1 hcAb（1、2）。为了促进细胞毒性效应子功能，我们将CDC（补体依赖性细胞毒性）增强和ADCC（抗体依赖性细胞介导的细胞毒性）增强突变引入人IgG1的CH2和CH3结构域。作为靶向CD38过表达细胞的第二种策略，我们将CD38特异性纳米抗体插入AAV2的VP1衣壳蛋白中（3）。我们使用增殖，CDC，ADCC和AAV转导分析法分析了这些构建体针对肿瘤细胞系和多发性骨髓瘤（MM）患者骨髓样品中的EGFR和CD38的能力。

结果

EGFR特异性hcAb可有效阻断表达WT EGFR和EGFR西妥昔单抗/帕尼单抗逃逸变体的肿瘤细胞系（HNSCC，头颈部鳞状细胞癌和mCRC，转移性结直肠癌）的增殖。EGFR和CD38特异性hcAb介导针对肿瘤细胞系（分别为HNSCC，mCRC和MM）的有效ADCC。携带六抗体突变的HcAb在诱导CDC方面比亲本hcAb更有效。在AAV衣壳上展示CD38特异性纳米抗体可导致患者骨髓样本中的骨髓瘤细胞转导提高20到100倍。其中一些结果最近已发布（1、2）或被接受发表（3）。我们将提供有关肿瘤细胞系和患者样品的最新数据。

结论

基于纳米抗体的hcAb和展示纳米抗体的AAV有望成为靶向实体瘤和血液肿瘤的新型工具。

负责研究的法人实体

弗里德里希·科赫·诺尔特（Friedrich Koch-Nolte）。

资金

DFG（德国研究基金会）和SFB（协作研究中心）。

揭露

作者宣称没有利益冲突。