Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients.
Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients.
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DOI：
10.1038/s41598-021-00325-3
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发表时间：
2021-10-21
影响因子：
4.6
通讯作者：
Sandey M
中科院分区：
文献类型：
--
作者：
Marable J;Ruiz D;Jaiswal AK;Bhattacharya R;Pantazes R;Agarwal P;Suryawanshi AS;Bedi D;Mishra A;Smith BF;Sandey M
癌症是导致老年犬只死亡的首要原因。目前，免疫检查点抑制物(ICIS)如抗CTLA4抗体的使用已显著改善了几种晚期癌症的预后。然而，针对CTLA4阻断治疗犬癌患者的ICIS尚未定义。在这项研究中，我们试图开发、表征和评估抗CTLA4的嵌合重链抗体(CHcAbbs)是否为治疗犬癌的可行候选治疗方案。利用磁性辅助细胞分选(MACS)和流式细胞术从酵母纳米体库中鉴定了抗CTLA4纳米体(NBS)。CHcAbbs是通过将编码抗CTLA4 NBS的DNA序列与犬免疫球蛋白B亚类的Fc结构域基因融合而成的。从ExpiCHO-S细胞中纯化了重组cHcAb。用稳定表达犬CTLA4和FcγRI的细胞系研究cHcAbs的结合能力和特异性。从健康犬分离的PBMCs用于检测cHcAbs对犬PBMCs的激活能力。利用犬CTLA4蛋白的胞外区筛选完全合成的酵母纳米体库，鉴定了新的NBS。纯化的NBs与幼犬CTLA4特异性结合。我们报道了由抗CTLA4 NBS和犬免疫球蛋白B亚类Fc区融合而成的嵌合型HcAbs，其大小仅为常规单抗的一半，并形成了二聚体。该嵌合抗体可与犬CTLA4和Fcγ受体特异性结合。由于NBS的结合与犬CTLA4的MYPPPY基序重叠，这些NBS有望立体干扰犬CTLA4与B-7S的相互作用。与人类的CTLA4一样，狗的CTLA4表达在辅助T细胞和一小部分细胞毒性T细胞上。犬Tregs也结构性表达CTLA4，经PMA/Ionomycin刺激后，CTLA4在细胞表面的表达显著增加。CPBMC在激动型抗CD3Ab和cHcAb6刺激下，干扰素-γ的表达显著高于同型对照。这项研究确定了一种新的基于纳米体的CTLA4抑制剂，用于治疗犬癌患者。
Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers. Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs (cPBMCs). Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and Fcγ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of cPBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control. This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients.
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影响因子：
45.3
作者：
Massard, Christophe;Gordon, Michael S.;Segal, Neil H.
通讯作者：
Segal, Neil H.
影响因子：
56.9
作者：
Ostrov, DA;Shi, WX;Nathenson, SG
通讯作者：
Nathenson, SG
DOI：
10.1016/s1470-2045(16)30364-3
发表时间：
2016-10
期刊：
The Lancet. Oncology
影响因子：
--
作者：
Kaufman HL;Russell J;Hamid O;Bhatia S;Terheyden P;D'Angelo SP;Shih KC;Lebbé C;Linette GP;Milella M;Brownell I;Lewis KD;Lorch JH;Chin K;Mahnke L;von Heydebreck A;Cuillerot JM;Nghiem P
通讯作者：
Nghiem P
DOI：
10.1084/jem.20081811
发表时间：
2009-02-16
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Friedline RH;Brown DS;Nguyen H;Kornfeld H;Lee J;Zhang Y;Appleby M;Der SD;Kang J;Chambers CA
通讯作者：
Chambers CA
影响因子：
16.8
作者：
McMahon C;Baier AS;Pascolutti R;Wegrecki M;Zheng S;Ong JX;Erlandson SC;Hilger D;Rasmussen SGF;Ring AM;Manglik A;Kruse AC
通讯作者：
Kruse AC