技術平台

抗體應用於診斷、疾病治療是目前大分子藥物開發的趨勢。要產生針對目標蛋白質的多株或單株抗體，需要純化蛋白質或合成胜肽作為免疫原。然而，蛋白質特性複雜，常在生產純化時出現難以克服的困難。另一方面，如果開發者希望避免蛋白質純化，並決定使用合成胜肽作為免疫原，也存在與胜肽免疫原性相關的問題。大多數時候，合成胜肽由於免疫原性低和半衰期短而無法誘導產生抗體。為了解決這個問題，台灣活力生技新藥股份有限公司採用了一種名為模版重複聚合酶連鎖反應 (LAE) 技術，此技術由黃昭蓮博士和徐嘉澤博士共同開發，該項發明已經在學術刊物(Cancer Res.)發表並獲得專利。通過這項技術，建構了DNA片段，將表位(epitope)編成線性排列。這種設計結合了受體介導、攝取和高效抗原表現，使免疫系統能夠產生任何抗原的抗體，包括對低抗原性的自我抗原。我們技術上的優點是不需要提供任何純化蛋白質或合成胜肽。只需要提供抗體識別表位(epitope)的氨基酸序列（10至12氨基酸）。台灣活力生技新藥股份有限公司應用LAE 技術已有許多不同的表位(epitope)開發的經驗。對DNA topoisomeras II-α (TOP2-α)和DNA topoisomerase II-β (TOP2-β)產生的兩種多株抗體分別對TOP2-α和TOP2-β產生專一性反應。這兩種抗體清楚地區分了這兩種酶，並且不會相互交叉反應。

LAE 技術配合雙組成結構載體設計，其中包含受體結合區（稱為 RBD），得以讓 APC 攝取呈現線性排列串聯之重複表位(epitope)（稱為抗原線性排列聚類域或 ACD）(圖1)。已經證明，含有受體結合區 （RBD） 的載體蛋白質通過受體介導攝取,促使線性排列串聯之重複表位(epitope)進入APC細胞，因此通過APC中的 MHC II 通路呈現抗原(圖2)。研究還證明含有抗原線性排列聚類域（ACD）的蛋白質在誘導B細胞活化方面非常有效(圖2)。事實上，這種雙組結構載體設計能誘導產生IgG抗體，此抗體能有效認得攜帶表位(epitope)的蛋白質且具高親和力及高專一性，這些實証更證明使用這種雙組結構蛋白質於開發治療型疫苗作為疾病治療與預防和抗體做為臨床檢驗之優越性。

Technology Platform

The vision of Taivital Biopharmaceutical Co., LTD. is to serve the biotechnology community by raising specific antibodies that can recognize any desirable protein. We also aim to develop antibody-related products for research, disease diagnosis, and most importantly for therapeutic purposes. To produce polyclonal or monoclonal antibodies against target protein, it needs purified protein or synthetic peptide as immunogen is needed. However, it always associates with difficulties in the purification of the desirable protein. On the other hand, if scientists would like to avoid protein purification and decide to use synthetic peptide as immunogen, there are also problems associated with peptide-based immunization.  Most of the time, synthetic peptide fails to induce antibodies due to its low immunogenicity and short half-life. To solve this problem, Taivital Biopharmaceutical Co., LTD. employs a technique named “Linear Array Epitope” (LAE) developed by Dr. Jaulang Hwang and Dr. Chiatse Hsu, whose findings have been published and patented. With this technique, we construct DNA fragments encoding epitopes in a linear array. This powerful technology that combines the use of receptor-mediated uptake and efficient antigen presentation and allows the induction of immune responses to generate antibodies against any antigen, including self-antigens. The advantage of this technology is that there is no need to supply any purified protein or synthetic peptide.  It only needs to provide the amino acid sequence (10~12 amino acids) of the epitope that they would like the antibody to recognize. The LAE technology has been tested with many different epitopes. Two polyclonal antibodies against DNA topoisomeras II-α (TOP2-α) and DNA topoisomerase II-β (TOP2-β) have been produced with LAE immunogen against TOP2-α and TOP2-β, respectively. These two antibodies clearly distinguish these two enzymes and do not cross-react with each other. LAE technology needs a novel synthetic bipartite protein containing the receptor binding domain (referred to as RBD) for aiding uptake/presentation by APCs and a tandem repeated epitope peptide in linear array (referred to as the antigen clustering domain or ACD), as diagrammatically shown in Fig. 1. It has been demonstrated that bipartite protein containing the receptor binding domain (RBD) facilitate uptake through receptor-mediated endocytosis and hence antigen presentation through the MHC II pathway in APCs (Fig. 2). It has also been demonstrated that carrier proteins containing antigen clustering domain (ACD) are highly effective in inducing strong B cell activation (Fig. 2). Indeed, such a bipartite carrier protein is highly effective in inducing high affinity IgG1 antibody against the protein carrying the epitope, suggesting the general applicability of using this bipartite immunogen for developing therapeutic vaccines for disease treatment and antibodies for clinical diagnosis.

References

• Hsu CT, Ting CY, Ting CJ, Chen TY, Lin CP, Whang-Peng J, and Hwang, J. Vaccination against gonadotropin-releasing hormone (GnRH) using toxin receptor-binding domain-conjugated GnRH repeats. Cancer Res. 2000 60:3701–3705.
• Chiang HL, Lin CY, Jan FD, Lin YS, Hsu CT, Whang-Peng J, Liu LF, Nieh S, Lin CC, Hwang J. A novel synthetic bipartite carrier protein for developing glycotope-based vaccines. Vaccine. 2012; 30:7573-7581
• Lin CY, Ho JY, Hsieh MT, Chiang HL, Chuang JM, WhangPeng J, Chang YC, Tseng YH, Chen SF, Nieh S, Hwang J. Reciprocal relationship of Tn/NF-kappaB and sTn as an indicator of the prognosis of oral squamous cell carcinoma. Histopathology. 2014; 64:713-721.
• Ho CW, Lin CY, Liaw YW, Chiang HL, Chin YT, Huang RL, Lai HC, Hsu YW, Kuo PJ, Chen CE, Lin HY, Whang-Peng J, Nieh S, Fu E, Liu LF, wang J. The cytokine-cosmc signaling axis upregulates the tumorassociated carbohydrate antigen Tn. Oncotarget, 2016; 7: 61930-61944.
• Wang FS, Wu RW, Lain WS, Tsai TC, Chen YS, Sun YC, Ke HJ, Li JC, Hwang J, Ko JY. Sclerostin vaccination mitigates estrogen deficiency induction of bone mass loss and microstructure deterioration. Bone, 2018; 112: 24-34.
• Chen CM, Hwang J, Chou HC, Shiah HS. Tn (N-acetyl-dgalactosamine-O-serine/threonine) immunization protects against hyperoxiainduced lung injury in adult mice through inhibition of the nuclear factor kappa B activity. Int Immunopharmacol., 2018; 59: 261-268.
• Chen CM, Hwang J, Chou HC. Maternal Tn Immunization Attenuates Hyperoxia-Induced Lung Injury in Neonatal Rats Through Suppression of Oxidative Stress and Inflammation. Front Immunol. 2019; 10: 681-.
• Chen CM, Hwang J,Chou HC, Chen Anti-Tn Monoclonal Antibody Attenuates Hyperoxia-Induced Lung Injury by Inhibiting Oxidative Stress and Inflammation in Neonatal Mice. Front Pharmacol. 2020; 11:
• Chen CM, Hwang J, and Chou HC. Immunization with anti-Tn immunogen in maternal rats protects against hyperoxia-induced kidney injury in newborn offspring. Pediatric Research, 2021; 89:476–482
• Chow JC, Chou HC, Hwang J, and Chen CM. Anti-Tn Monoclonal Antibody Ameliorates Hyperoxia-Induced Kidney Injury by Suppressing Oxidative Stress and Inflammation in Neonatal Mice. Mediators of Inflammation 2021; in press.