Antibody engineering to generate anti-tumor-associated glycoprotein 72 mouse recombinant CC49 IgG with improved solubility, purity, and thermal stability

Article

作者: Muerhoff, A Scott ; Tu, Bailin ; Lin, Zhihong ; Hemken, Philip M

Tumor-associated glycoprotein 72 (TAG-72) is a mucin that is overexpressed heterogeneously on the surface of cancer cells, and is a potential target for immunotherapies for various cancer types. As a tumor marker, TAG-72 is measured with the cancer antigen (CA) 72-4 immunoassay. The murine monoclonal antibody (mAb) CC49 is a second-generation IgG that targets an antigen on TAG-72; however, CC49 has an unfavorable propensity to aggregate, which results in antibody impurity, instability, and low solubility and thus low potency and efficacy for therapeutic and diagnostic applications. Sequence analysis of CC49 revealed aggregation-prone motifs in the variable domain of the light chain. Using antibody engineering approaches, we developed three aggregation-resistant CC49 mIgG2a mutants (CC49M1, CC49M2, and CC49M3). The engineered CC49 mIgG2a mutants retained compatible binding performance with a significantly higher thermal stability. The CC49 mIgG2a mutants also demonstrated an almost 15-fold improvement in solubility, with 97% purity vs 70% purity of the parent molecule at 0.3 mg/mL. The enhanced stability and improved solubility of engineered CC49 could have significant advantages for diagnostic and therapeutic applications.

2020-08-01·The Journal of surgical research4区 · 医学

Humanized Anti–Tumor-Associated Glycoprotein–72 for Submillimeter Near-Infrared Detection of Colon Cancer in Metastatic Mouse Models

4区 · 医学

Article

作者: Michael Bouvet ; Paul J. Yazaki ; Robert M. Hoffman ; Justin Molnar ; Siamak Amirfakhri ; Filemoni Filemoni ; Hannah M. Hollandsworth

BACKGROUND:

Tumor-associated glycoprotein (TAG)-72 is a pancarcinoma antigen that is overexpressed in greater than 80% of colorectal adenocarcinomas. CC49 is a TAG-72-specific antibody. The aim of the present study was to demonstrate selective imaging of colon tumors and metastases with the humanized TAG-72 antibody (anti-huCC49) conjugated to a near-infrared fluorophore in orthotopic mouse models.

METHODS:

Anti-huCC49 was conjugated to near-infrared dye IR800CW. Mouse imaging was performed with the Pearl Trilogy Small Animal and FLARE Imaging Systems. Subcutaneous mouse models of colon cancer cell line LS174T were used to determine the optimal dose of administration and timing of imaging. Orthotopic mouse models of LS174T were established by surgical orthotopic implantation of LS174T tumors onto the serosa of the cecum. Peritoneal carcinomatosis models were established by injection of LS174T cells into the peritoneum of nude mice. Mice were administered anti-huCC49-IR800 via tail vein injection. Mice were euthanized 72 h later and imaged after laparotomy.

RESULTS:

Subcutaneous LS174T xenografts demonstrated optimal tumor detection 72 h after administration with 50 μg anti-huCC49-IR800CW. Tumors were visualized with fluorescence imaging with a mean tumor-to-liver ratio of 7.39 (standard deviation: 2.76). In the orthotopic model, metastases smaller than 1 mm were fluorescently visualized that were invisible with bright light.

CONCLUSIONS:

Anti-huCC49-IR800CW provides sensitive and specific imaging of colon cancer and metastases at a submillimeter resolution in metastatic nude mice models. This provides a promising near-infrared probe for the imaging of colon cancer and metastases for preoperative diagnosis and fluorescence-guided surgery.

2020-07-01·Cancer medicine3区 · 医学

Improved targeting of an anti‐TAG‐72 antibody drug conjugate for the treatment of ovarian cancer

3区 · 医学

ArticleOA

作者: Li, Lin ; Poku, Erasmus ; Minnix, Megan ; Shively, John E. ; Colcher, David ; Yazaki, Paul ; Chea, Junie

Abstract:

Introduction:

Ovarian cancer has only a 17% 5‐year survival rate in patients diagnosed with late stage disease. Tumor‐associated glycoprotein‐72 (TAG72), expressed in 88% of all stages of ovarian cancer, is an excellent candidate for antibody‐targeted therapy, as it is not expressed in normal human adult tissues, except in the secretory endometrium.

Methods:

Using the clinically relevant anti‐TAG72 murine monoclonal antibody CC49, we evaluated antibody drug conjugates (ADCs) incorporating the highly potent, synthetic antimitotic agent monomethylauristatin E (MMAE). MMAE was conjugated to CC49 via reduced disulfides in the hinge region, using three different types of linker chemistry, vinylsulfone (VS‐MMAE), bromoacetamido (Br‐MMAE), and maleimido (mal‐MMAE).

Results:

The drug antibody ratios (DARs) of the three ADCs were 2.3 for VS‐MMAE, 10 for Br‐MMAE, and 9.5 for mal‐MMAE. All three ADCs exhibited excellent tumor to blood ratios on PET imaging, but the absolute uptake of CC49‐mal‐MMAE (3.3%ID/g) was low compared to CC49‐Br‐MMAE (6.43%ID/g), at 142 hours. Blood clearance at 43 hours was 38% for intact CC49, about 24% for both CC49‐VS‐MMAE and CC49‐Br‐MMAE, and 7% for CC49‐mal‐MMAE. CC49‐VS‐MMAE was not further studied due to its low DAR, while CC49‐mal‐MMAE was ineffective in the OVCAR3 xenograft likely due to its rapid blood clearance. In contrast, CC49‐Br‐MMAE treated mice exhibited an average of a 15.6 day tumor growth delay and a 40% increase in survival vs controls with four doses of 7.5 or 15 mg/kg of CC49‐Br‐MMAE.

Conclusion:

We conclude that CC49‐Br‐MMAE with a high DAR and stable linker performs well in a difficult to treat solid tumor model.

项与 131I-CC49-SCA 相关的新闻（医药）

2023-12-15

·智慧芽新药科讯

肿瘤领域早期靶点sTn，未来能否收获惊喜？

肿瘤领域潜力靶点的寻找，是新药研发者持续进行的狩猎行为。早期靶点虽然风险系数高，但往往更具研究价值和探索的乐趣。对此，sTn在一定程度上符合该特征，且仅有极少品种在研及早期技术布局，可关注跟进。01sTn-靶点清楚&通路清晰sTn抗原，即唾液酸Tn（Sialyl Tn）抗原，是Tn抗原（GalNAc-α-Ser/Thr）的唾液酸化形式。sTn抗原具有明确的生理机制并关联多种疾病，其中一个非常重要的概念是“O-糖基化”。O-糖基化，常见于肿瘤的发生与发展。正常细胞内成熟的O-聚糖，先是形成Tn抗原，而sTn抗原是由Tn抗原过早发生2,6-唾液酸化，是不完全糖基化过程的产物。该过程详细如下：1）N-乙酰基半乳糖转移酶将UDP-GalNAc上的N-乙酰半乳糖氨（GalNAc）以链接的方式链接到丝氨酸/苏氨酸（Ser/Thr）上，形成Tn抗原（Tn antigen），即GalNAc-α-Ser/Thr；2）Tn抗原在T抗原合成酶（T-synthase）的辅助下获得一个来自UDP-Gal的Gal并合成核心1型结构（T抗原），但是当体内缺乏T抗原合成酶或少数情况下，Tn抗原在ST6GalNAc-I的作用下获得一个来自CMP-Neu5Ac的Neu5Ac并形成唾液酸Tn抗原（sTn抗原），也就是被截断的O-聚糖。研究还证实，sTn抗原的生物合成还与唾液酸转移酶ST6GalNAc1的表达以及伴侣蛋白Cosmic基因的突变和杂合性（如甲基化）的缺失相关。图1-1O-糖基化及Tn-T-sTn-sT相关抗原合成途径（Biomolecules 2015, 5, 1783-1809. DOI:10.3390/biom5031783）02sTn-肿瘤&高表达现阶段，对于sTn的研究倾向于肿瘤适应症领域。有文献报道，超过80%的癌细胞中有sTn抗原的表达，并且sTn抗原与癌症患者的不良预后相关。相对而言，在许多上皮类型的肿瘤中有sTn抗原的过度表达，如胃癌、结肠癌、乳腺癌、肺癌、食管癌、胰腺癌、前列腺癌、膀胱癌和子宫内膜癌等。以膀胱癌为例，sTn抗原在膀胱癌中呈现阳性表达，且主要是在肿瘤侵袭区，也可见于临近侵袭区的癌旁组织中。与未发生转移的肿瘤比较，sTn抗原过表达在伴有淋巴结转移的原发肿瘤患者中更常见。另，sTn抗原还与膀胱癌的分期、分级相关，有望成为膀胱癌患者非侵入性检查的一种选择，在膀胱癌患者的诊疗及预后发挥重要作用。图2-1肿瘤细胞与免疫细胞的sTn特异性相互作用（Oncology. 2023. DOI 10.3389/fonc.2022.1093496）03sTn-相关药物开发进展以sTn作为药物靶标来进行新药开发，整体还处于早期阶段。有文献报道：1）1981年，第一个针对Tn抗原开发的单抗，源自小鼠模型，过程中相继发现一些抗体对Tn和sTn抗原均产生作用。如CC49单抗，该品种曾开发为CC49 I-131和90Y-CC49等，临床前/临床用于肿瘤（如卵巢癌）的诊断与治疗，但目前总体进展缓慢，部分研究已暂停。2）另，Theratope sTn-KLH为sTn方向疫苗，可产生sTn免疫应答，曾开发进入到临床，适应症为转移性乳腺癌，一定程度上提高了患者的生存率，早年曾吸引多家公司共同开发（如默克），但现今处于无进展。在上述研究历史背景基础上，结合智慧芽新药情报库（英文名：Synapse）sTn靶点下的研发机构热点分析，当前在研品种总体上受关注度较大的为Seagen公司开发的SGN-STNV，以及Siamab Therapeutics布局的技术及品种。➣ SGN-STNV（Seagen Inc，临床I期）SGN-STNV，是sTn领域值得关注的品种之一。开发公司为Seagen，通过使用vedotin(ADC)技术开发，用于晚期实体瘤的潜在静脉治疗，目前处于临床I期。2020年11月递交IND；2021年该品种的临床前数据于AACR年会公开，显示SGN-STNV在多个异种移植模型中（如胃癌、卵巢癌、食管癌、非小细胞肺癌）存在较好的活性。注：Seagen Inc是全球ADC药物开发的龙头企业，有多款ADC药物上市，且在研临床阶段ADC品种十余款。图3-1 SGN-STNV临床信息https://clinicaltrials.gov/study/NCT04665921?tab=table➣ Siamab Therapeutics（2款临床前品种）另，综合使用智慧芽新药情报库（英文名：Synapse）和智慧芽专利数据库查询发现，Siamab Therapeutics同样深耕sTn药物开发。进入Synapse，靶点选项输入“sTn”，共搜索出3个品种，除上述Seagen Inc的SGN-STNV，另两个品种均来自Siamab Therapeutics，为抗体类药物，在研适应症暂锚定卵巢癌等实体瘤，且当前均处于临床前阶段。其中，ST1为ADC药物，部分临床前试验数据已在AACR年会进行公开，初步证实了早期较好的安全性、耐受性、药代动力学特性；而药效方面，在卵巢癌异种瘤模型中显示了较好的肿瘤抑制作用。图3-2智慧芽-新药情报库（Synapse）-sTn搜索结果再，进入智慧芽-patsnap数据库，输入“Siamab”，共计发现118条专利，受理局排序依次为美国、欧洲、WIPO、日本、中国、澳大利亚等，进入中国的专利共计6件。有趣的是，授权专利CN 108463248 B原始申请（专利权）人为Siamab Therapeutics, Inc.，当前申请（专利权）人为Seagen Inc，产生了权利转移。专利CN 108463248 B，当前已于中国授权，申请日为2016.11.10，发明名称为“聚糖相互作用化合物及使用方法”。技术方面，权利要求共计60项，其中：权1“一种抗体，其结合唾液酸(a2,6)N-乙酰半乳糖胺(STn),其中所述抗体包含序列如选自SEQ ID NO:237-241的氨基酸序列所示的重链可变结构域(VH)和序列如SEQ ID NO:235或236的氨基酸序列所示的轻链可变结构域”；权8“一种抗体-药物缀合物，其包含缀合至治疗剂的权利要求1-7任一项所述的抗体”；权22“权利要求1-7任一项所述的抗体或权利要求8-13任一项所述的抗体-药物缀合物在制备用于治疗受试者中癌症的药物中的用途”。图3-3专利CN 108463248 B附图内容小结从肿瘤细胞及其微环境特点来看，sTn呈现出不同于正常细胞的特征，具备成药靶点的潜力。ADC龙头企业Seagen Inc对sTn领域品种的布局开发，进一步证实该靶点的成药价值。同时，Seagen Inc还对Siamab Therapeutics的专利进行介入，也足见sTn靶点的潜在价值以及Seagen Inc对该方向的早期垄断。而此时，国内药企尚未对sTn形成具竞争力的布局，赛道开发环境良好，值得尝试跟进。参考资料1.医学研究杂志2022年11月第51卷第11期. DOI10.11969/j.issn.1673-548X.2022.11.0052.Chinese Journal of Clinical Research，May 2017，Vol．30，No．5. DOI:10.13429/j.cnki.cjcr.2017.05.0383.生命的化学，2016，36（6）:945-949.doi:10.13488/j.smhx.201606314.Int. J. Mol. Sci. 2016, 17, 275; doi:10.3390/ijms170302755.Biomolecules 2015, 5, 1783-1809; doi:10.3390/biom50317836.DOI 10.3389/fonc.2022.10934967.https://synapse.zhihuiya.com/drug/9d2c4bfbda804092820c6f269848f0268.https://www.cn-healthcare.com/articlewm/20220529/content-1366145.html9.https://synapse.zhihuiya.com/patent-list?query_id=5e0c67e6-a2be-41e3-9d82-61758a14e714&tab=analysis10.https://synapse.zhihuiya.com/target/5bbe07bab825404dad84b924d806386c点击图片，免费GET海量新药信息 👇👇👇

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100 项与 131I-CC49-SCA 相关的药物交易

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KEGG	Wiki	ATC	Drug Bank
-	131I-CC49-SCA	-	DB15470

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适应症	最高研发状态	国家/地区	公司	日期
心血管疾病	临床1期	美国	SCA Ventures, Inc.	-
心血管疾病	临床1期	美国	National Cancer Institute	-
肿瘤	临床1期	美国	National Cancer Institute	-
肿瘤	临床1期	美国	SCA Ventures, Inc.	-
结肠癌	临床前	加拿大	Toronto General Hospital	2004-04-01
结肠癌	临床前	加拿大	Ontario Cancer Institute	2004-04-01