Generation and characterization of a humanized anti-IL-17A rabbit monoclonal antibody
Introduction
The IL-17 family of cytokines consists of six homologous cytokines, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F [1,2]. IL-17A is the most abundant, potent and well characterized member in the IL-17 family [2]. Biologically active IL-17A exists as a homodimer (A/A) or as a heterodimer (A/F) with IL-17F. Homodimer IL-17A composes of two chains of 155 amino acids connected by disulfide bonds with a molecular weight of 35 kDa. IL-17A works by binding to the complex of IL-17RA and IL-17RC on the surface of various kinds of cells, such as epidermal cells, endothelial cells and fibroblasts [1,3]. For example, IL-17A induced high expression of IL-1, IL-6, TNF and CRP on macrophages and DC cells which will lead to inflammation, and participating in the pathological process of psoriasis and transplant rejection [4]. IL-17A can also cause bone erosion, cartilage damage and participated in the pathological process of rheumatoid arthritis and periodontal disease by inducing RANKL, MMP and osteoclast formation in osteoblasts and chondrocytes [5,6].
So far, its neutralization showed the potential for an enhanced efficacy and an improved safety profile in patients with autoimmune diseases because it is produced and acted locally at the sites of inflammation [7]. For example, neutralizing anti-murine interleukin-17A monoclonal antibody hindered ozone-induced inflammation and glucocorticoids insensitivity in a murine model of asthma [8]. Anti-IL-17A mAb or anti-IL-17RA mAb alleviated the high glucose (HG)-treated rat Müller cell line disorders by blocking IL-17A which will alleviate diabetic retinopathy in rodents [9]. Several monoclonal antibody drugs targeting IL-17A have been approved for the market, including Secukinumab, Ixekizumab and Brodalumab. The Secukinumab act as an IL-17A neutralizing antibody was approved to treat for moderate-to-severe plaque psoriasis, psoriatic arthritis and ankylosing spondylitis [10,11]. Ixekizumab and Brodalumab were approved to treat plaque psoriasis by targeting the IL-17 pathway [[12], [13], [14]]. These findings indicated that the development of antibodies targeting IL-17A has important clinical significance and market prospects which greatly improve the treatment of psoriasis, psoriatic arthritis and other autoimmune diseases.
Rabbits have been a reliable source of polyclonal antibodies for decades, and now rabbit monoclonal antibodies are widely used in various fields, because it is suggested that rabbits may yield a more diversity, high-affinity and high-specificity repertoire of antibodies in comparison to mice [15]. For example, the natural advantage of high signal-to-noise ratio of rabbit monoclonal antibody in IHC detection is favored by clinicians, especially for IHC detection of immune checkpoint target protein used as companion/supplementary diagnosis, such as PD-L1 [16]. Rabbits are a convenient source for obtaining monoclonal anti-idiotype antibodies which are useful for the development of PK and ADA assays [17]. Recently, the first rabbit-derived scFv monoclonal antibody, BEOVU, was approved by the FDA for the treatment of wet age-related macular degeneration (AMD) [18]. In addition, the world's second rabbit antibody drug Vyepti appeared on the market for treatment of migraine in adults [19]. The approval of Beovu and Vyepti indicates that therapeutic antibodies derived from rabbit have entered a new era, greatly enriching the precursor discovery platform for therapeutic antibodies.
In terms of the complexity of IL-17A network, its functions in pro-inflammatory and its cell/tissue protective roles were discovered in different diseases. It has great potential and space to develop new humanized Rabbit-derived IL-17A antibodies which will adapt to treat patients with plaque psoriasis, psoriatic arthritis and ankylosing spondylitis. Therefore, in present study, a new humanized IL-17A antibody HZD37-5 which showed high neutralization and affinity in vivo and in vitro were generated by targeting N78 loci of IL-17A. And HZD37-5 might be better suitable for the treatment of plaque psoriasis, psoriatic arthritis and ankylosing spondylitis, as well as other autoimmune diseases.
Section snippets
Immunization and generation of specific neutralizing B cell clone
Human IL-17A expressed in HEK293 cells were cross-linked to Keyhole limpet hemocyanin (KLH) ( GenScript, Piscataway, NJ, USA) and injected subcutaneously to immunize New Zealand white rabbits. The initial immunization was given 0.5 mg IL-17A with 0.5 ml Freund's complete adjuvant mixture, and the booster immunization was given 0.25 mg IL-17A with 0.5 ml Freund's incomplete adjuvant mixture (V:V = 1:1). Immunized animals with high antigen-binding activity in peripheral serum was selected to
Immunization and selection of candidate antibodies in New Zealand white rabbit
In order to obtain the candidate antibodies, human IL-17A conjugated with KLH was used to immunize New Zealand White Rabbits. After binding and blocking ELISA screening, seven rabbit monoclonal antibodies with the best activity were selected for recombinant expression. The neutralization activity of rabbit antibodies in HFF-1 cells showed that the IC50 for 9#, 19#, 37#, 55# and 58# rabbit antibodies were similar varied from 31.2 to 48.3 ng/ml while the IC50 were 103 and 820 ng/ml for 26# and
Discussion
IL-17A which is mainly derived from Th17 cells has become a promising target for therapeutic development in the treatment of inflammatory and autoimmune diseases [21,22]. IL-17A can form a homodimer IL-17AA or heterodimer IL-17AF which binds to its receptor IL-17RA/IL-17RC leading to nuclear factor κB (nuclear factor kappa B, NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway activation through IL-17R-Act1-TRAF6 signal transduction complex [23]. Increasing numbers of studies
CRediT authorship contribution statement
Wei Chen: Conceptualization, Resources, Methodology, Software, Investigation, Writing – original draft. Yong Kong: Methodology, Software, Investigation. Wang Li: Methodology, Software, Investigation. Yi Zhou: Methodology, Software, Investigation. Meijuan Wu: Methodology, Software, Investigation. Tao Chen: Methodology, Software, Investigation. Yiliang Wu: Methodology, Software, Investigation. Huaiyao Qiao: Formal analysis, Data curation, Visualization. Zhihua Qiu: Formal analysis, Data curation,
Declaration of competing interest
Wei Chen, Yong Kong, Wang Li, Yi Zhou, Meijuan Wu, Tao Chen, Yiliang Wu, Huaiyao Qiao, Zhihua Qiu, and Jiwan Qiu are all employed by Qyuns Therapeutics Co. The intellectual property rights and economic interests related to this project belong to the company, and there is no conflict of interest between myself and the company, and no conflict of interest between each of the authors, hereby declare.
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