GPR160 antibody development for cancer treatment
GPR160 antibody development for cancer treatment
批准号:
10711206
负责人:
James D Brien
金额:
$21.25万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2023
资助国家:
美国
项目状态:
未结题
起止时间:
2023-06-16 至 2025-05-31
关键词:
AffinityAffinity ChromatographyAlloysAmino Acid SequenceAntibodiesAntibody TherapyAvidityBindingBiological AssayBiological ProductsBreastBreast Cancer CellBreast Cancer cell lineCancer cell lineCause of DeathCell Culture TechniquesCell LineCell SurvivalCellsChemosensitizationCisplatinClinicalColonColon CarcinomaCombined Modality TherapyCytotoxinDataDevelopmentDoseDose LimitingElementsEsophagusFlow CytometryG-Protein-Coupled ReceptorsGenesGoalsHumanHuman Cell LineHybridomasImmunizeImmunodeficient MouseIn VitroKeyhole Limpet HemocyaninLigandsLightLungMDA MB 231Malignant NeoplasmsMalignant neoplasm of lungMalignant neoplasm of pancreasMalignant neoplasm of prostateMeasuresMediatingModelingMonoclonal AntibodiesMonoclonal Antibody TherapyMusOryctolagus cuniculusPaclitaxelPatientsProstatePublicationsRoleSW480StomachTestingTherapeuticTherapeutic Monoclonal AntibodiesToxic effectTransgenic MiceTranslatingTrastuzumabTreatment-Related CancerTreatment-related toxicityUncertaintyWorkXenograft ModelXenograft procedureanti-canceranticancer treatmentcancer cellcancer therapyclinical effectclinical efficacyclinical predictorscolon cancer cell linecytotoxicexperimental studyextracellularhuman monoclonal antibodiesimprovedin vitro Modelin vivomalignant breast neoplasmmouse modelneoplastic celloverexpressionoxaliplatinpharmacologicpolyclonal antibodyprogramsreceptorresponsescale upside effectsmall moleculesuccesstherapy developmenttriple-negative invasive breast carcinomatumor
中文摘要
我们发现,一种兔多克隆抗体针对人G蛋白的第二个细胞外环,
偶联受体160(hGPR 160/ECL 2)具有两个显著的作用:(1)直接抗肿瘤作用
结肠癌和三阴性乳腺癌(TNBC)细胞系,其表达受体而不改变细胞的免疫原性。
正常结肠癌细胞系的存活力,和(2)小分子的细胞毒性作用的极大增强
化疗剂奥沙利铂,其剂量本身没有显著活性。这种增强表明
与hGPR 160/ECL 2抗体的组合可能在临床效果上产生相当大的改善
细胞毒素的剂量比目前使用的剂量更低,产生的副作用更少。翻译这些
为了将观察转化为实际治疗,我们提出开发人单克隆hGPR 160/ECL 2抗体,用于
进一步表征其抗癌作用,并作为开发治疗上有用的人
单克隆hGPR 160/ECL 2抗体。目前的提案是对PAR-22-216的回应,旨在制定
并测试一种既能治疗癌症又能减轻癌症治疗相关毒性的新生物制剂。我们
不知道任何市售的GPR 160的小分子配体。我们最近的出版物1和
本文提供的初步数据表明,hGPR 160/ECL 2单克隆抗体是一种可行的
化疗。然而,我们的初步研究使用了兔多克隆抗体,其具有不可还原的
关于结合位点的不确定性因素。因此,人单克隆hGPR 160/ECL 2
抗体将解决这个问题,并允许明确的体外实验抗癌作用在人类
癌细胞系和免疫缺陷小鼠中人细胞系来源的异种移植物(CDX)的体内实验。
在三个目标中提出的工作的成功将导致进一步的工作,以开发一种治疗药物。
单克隆抗体目的一:通过免疫人源性hGPR 160/ECL 2,制备人源性hGPR 160/ECL 2单克隆抗体,
表达具有hGPR 160的ECL 2氨基酸序列的人重链和轻链基因的转基因小鼠(或
其亚序列)缀合至KLH并通过PEG融合形成杂交瘤。我们将筛选克隆
通过流式细胞术检测抗体,并使用结合阻断试验向下选择约10个抗体
用于进一步体外和体内研究的hGPR 160/ECL 2 mAb克隆。然后我们将测试抗癌细胞活性
我们的抗体的体外研究。在目标2中,前2-3个候选物(对配体的最佳结合亲和力)是
和体外细胞活力测定中的LC 50)将用于(1)体内原位细胞系衍生的异种移植物
(CDX)结肠癌和TNBC的小鼠模型。在目标3中,将对最佳候选药物奥沙利铂进行体外试验
和紫杉醇增强研究。hGPR 160/ECL 2 mAb可能被证明是一个显着的改善,
目前的抗癌治疗对大部分患者来说是不够的。然而,小分子
细胞毒素将继续发挥重要作用,并与hGPR 160/ECL 2抗体联合治疗
可以使它们的细胞毒性作用最大化,同时使用副作用少得多的剂量。
英文摘要
We discovered that a rabbit polyclonal antibody raised against the second extracellular loop of human G-protein-
coupled receptor 160 (hGPR160/ECL2) has two remarkable actions: (1) direct anti-cancer effects on human
colon cancer and triple negative breast cancer (TNBC) cell lines that express the receptor without altering the
viability of a normal colon cancer cells line, and (2) great potentiation of the cytotoxic effects of the small molecule
chemotherapeutic, oxaliplatin, at doses that by themselves have no significant activity. This potentiation indicates
that combination with the hGPR160/ECL2 antibody might yield a considerable improvement in the clinical effect
with cytotoxin doses that are lower and produce fewer side-effects than those in current use. To translate these
observations into a practical therapy, we propose to develop a human monoclonal hGPR160/ECL2 antibody for
further characterization of its anticancer effects and as a step towards developing a therapeutically useful human
monoclonal hGPR160/ECL2 antibody. The current proposal is in response to PAR-22-216 with aims to develop
and test a new biologic agent that both treats cancer and mitigates cancer treatment-related toxicities. We are
unaware of any commercially available small molecule ligands for GPR160. Our recent publication1 and
preliminary data presented here indicate that an hGPR160/ECL2 monoclonal antibody is a viable
chemotherapeutic. However, our preliminary studies used a rabbit polyclonal antibody, which has an irreducible
element of uncertainty concerning the locus of binding. Accordingly, a human monoclonal hGPR160/ECL2
antibody will address this issue and permit unambiguous in vitro experiments on anticancer effects in human
cancer cell lines and in vivo experiments on human cell line-derived xenografts (CDX) in immunodeficient mice.
Success with the work proposed here in three Aims would lead to further work to develop a therapeutic
monoclonal antibody. In Aim 1, we will develop human monoclonal antibodies to hGPR160/ECL2 by immunizing
transgenic mice expressing human heavy and light genes with the hGPR160’s ECL2 amino acid sequence (or
subsequence thereof) conjugated to KLH and developing hybridomas via PEG-fusion. We will screen clonal
antibodies by flow cytometry and down-select using a blocking-of-binding assay to a panel of ~10
hGPR160/ECL2 mAbs clones for further in vitro and in vivo studies. We will then test the anti-cancer cell activity
of our antibodies with in vitro studies. In Aim 2, the top 2-3 candidates (best binding affinity against the ligand
and LC50 in the in vitro cell viability assay) will be advanced for (1) in vivo orthotopic cell line-derived xenograft
(CDX) mouse models of colon cancer and TNBC. In Aim 3, the top candidate will be tested in in vitro oxaliplatin
and paclitaxel potentiation studies. An hGPR160/ECL2 mAb may prove to be a significant improvement to
current anti-cancer treatments that are inadequate for a large percentage of patients. However, small molecule
cytotoxins will continue to have an important role and combination therapy with an hGPR160/ECL2 antibody
may allow their cytotoxic effects to be maximized while using doses with far fewer side effects.
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