Fig 6 - uploaded by Stefan Gerhardt
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Wall-eyed stereo representation of the CAT-2200 paratope. Residues from the IL-17A homodimer are shown in pale or dark yellow. Residues from the Fab fragments are shown with the light chain in blue and with the heavy chain in green. The seven amino acids that are different between CAT-2200 and the lead antibody TINA-12 have been labeled.
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IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 A resolution in order...
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... changes were identified in CDR3 between the optimized antibody CAT-2200 and the parent TINA12. The crystal structure of the IL-17A/Fab complex allows us to examine the structural context of these changes and to speculate on how they might have improved affinity. Two of the changes, D93P (in VL-CDR3) and W98H (in VH-CDR3), are part of the paratope (Fig. 6). Pro93 in VL-CDR3 forms a stacking interaction with Tyr85 of IL-17A, probably providing a significant improvement in interface surface complementarity. Interestingly, the D93H mutation also appeared in other optimized constructs, consistent with the notion that side-chain stacking may be beneficial. The D93P mutation would restrict ...
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... Apart from two bstrands, in which one was found at N-terminal region and another was at C-terminal region, two more small b-strands were observed between second and third b-strands. However, only b-strands present at the core regions were considered for numbering as per hsIL-17A crystal structure, (pdb id:2vxs; Gerhardt et al., 2009;Fig. 6). ...
Interleukin-17A is a pro-inflammatory cytokine that plays a key role in the immune response to many pathogens and implicated in autoimmune diseases. This molecule is also involved in providing protection to many bacterial and fungal infections of gastro-intestinal tract and respiratory mucosa. Although molecular aspect of IL-17A has been studied in few species, no data are available for buffalo, which is one of the major sources of milk production in India. Therefore, in the present study, IL-17A gene of Indian Murrah Buffalo origin was cloned, expressed, and analyzed using bioinformatic tools. The coding sequence of buffalo IL-17A gene was cloned in prokaryotic expression vector (pET-28a) followed by its expression, purification, and characterization. A computational analysis was performed to understand the sequence, structure, and evolutionary relationship of buIL-17A. It revealed that the length of buIL-17A sequence without signal peptide is 132 amino acids as in cattle. However, sequence identity is found to be 99% due to one amino substitution difference between buffalo and cattle. After analysis, it can be concluded that buIL-17A recombinant protein can be used as a potential immunobiological reagent for diagnostic and therapeutic purpose.
... IL-17 secondary structure. The crystal structures of IL-17AA, IL-17FF and IL-17AF have previously been determined 12,13,42,43 and have similar structural features. Each protomer consists of two b-hairpins (see Fig. 1). ...
Knowledge of protein dynamics is fundamental to the understanding of biological processes, with NMR and 2D-IR spectroscopy being two of the principal methods for studying protein dynamics. Here, we combine these two methods to gain a new understanding of the complex mechanism of a cytokine:receptor interaction. The dynamic nature of many cytokines is now being recognised as a key property in the signalling mechanism. Interleukin-17s (IL-17) are proinflammatory cytokines which, if unregulated, are associated with serious autoimmune diseases such as psoriasis, and although there are several therapeutics on the market for these conditions, small molecule therapeutics remain elusive. Previous studies, exploiting crystallographic methods alone, have been unable to explain the dramatic differences in affinity observed between IL-17 dimers and their receptors, suggesting there are factors that cannot be fully explained by the analysis of static structures alone. Here, we show that the IL-17 family of cytokines have varying degrees of flexibility which directly correlates to their receptor affinities. Small molecule inhibitors of the cytokine:receptor interaction are usually thought to function by either causing steric clashes or structural changes. However, our results, supported by other biophysical methods, provide evidence for an alternate mechanism of inhibition, in which the small molecule rigidifies the protein, causing a reduction in receptor affinity. The results presented here indicate an induced fit model of cytokine:receptor binding, with the more flexible cytokines having a higher affinity. Our approach could be applied to other systems where the inhibition of a protein-protein interaction has proved intractable, for example due to the flat, featureless nature of the interface. Targeting allosteric sites which modulate protein dynamics, opens up new avenues for novel therapeutic development.
... In order to assess the stapling designs, we selected several antibodies to generate in scFv and corresponding spFv formats: two antibodies with kappa light chains (GLk1 and GLk2) from synthetic phage antibody libraries; 37 a lambdacontaining antibody (CAT2200) that binds IL-17A; 38 and two scFv variants, Cris7a and Cris7b, derived from anti-CD3 mAb Cris7, 39 which have potential use in CD3-based T-cell redirecting. We constructed and expressed the scFv and spFv molecules in both LH and HL orientations. ...
Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation (“breathing”) and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.
... The affinity of the CAT-2200 antibody Fab fragment [31] for IL-17A is 20 fold lower compared to the affinities of the secukinumab Fab fragment and the netakimab VHH domain (Table 5). Epitopes in the IL-17A/CAT-2200 and IL-17A/anti-IL-17A-76 complexes partially overlap ( Figure S3a). ...
Interleukin-17 (IL-17) is a cytokine produced by the Th17 cells. It is involved in chronic inflammation in patients with autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and psoriasis. The antibodies targeting IL-17 and/or IL-17R are therapy tools for these diseases. Netakimab is an IL-17A-specific antibody containing a Lama glama VHH derivative domain and a VL variable domain. We have determined the crystal structure of the IL-17A-specific VHH domain in complex with IL-17A at 2.85 Å resolution. Certain amino acid residues of the three complementary-determining regions of the VHH domain form a network of solvent-inaccessible hydrogen bonds with two epitope regions of IL-17A. The β-turn of IL-17A, which forms the so-called epitope-1, appears to be the main region of IL-17A interaction with the antibody. Contacts formed by the IL-17A mobile C-terminal region residues (epitope-2) further stabilize the antibody–antigen complex.
... The IL-17 family of cytokines are homodimeric glycoproteins, or heterodimeric in the case of the IL-17A/F cytokine, and these dimers range from around 35 to 50 kDa in size [4,10,12]. The IL-17 family of cytokines share a conserved C-terminus, which contains four cysteine and two serine residues, which are necessary to form the cysteine knot fold observed in the crystal structures of IL-17A and IL-17F [4,9,10,12,13]. The IL-17 family of cytokines diverge in their N-terminal segments [4,10,12]. What is known about the structural aspects of these molecules has been extensively described in reviews by Zhang et al. (2011) and Liu (2019) [14,15]. ...
Interleukin-17 (IL-17) family cytokines are potent drivers of inflammatory responses. Although IL-17 was originally identified as a cytokine that induces protective effects against bacterial and fungal infections, IL-17 can also promote chronic inflammation in a number of autoimmune diseases. Research in the last decade has also elucidated critical roles of IL-17 during cancer development and treatment. Intriguingly, IL-17 seems to play a role in the risk of cancers that are associated with metabolic disorders. In this review, we summarize our current knowledge on the biochemical basis of IL-17 signaling, IL-17′s involvement in cancers and metabolic disorders, and postulate how IL-17 family cytokines may serve as a bridge between these two types of diseases.
... adalah sebanyak 15 struktur. Ke-15 struktur ini diperoleh dengan metode difraksi sinar X, yaitu berturut turut dari yang terakhir ditemukan, 6WIR [14], 6WIO [14], 5N7W [15], 5VB9 [16], 5N92 [17], 5NAN [17], 5HI3 [18], 5HI5 [18], 5HI4 [18], 5HHV [19], 5HHX [19], 4QHU [20], 4HR9 [21], 4HSA [21] dan 2VXS [22]. Struktur yang pertama kali ditemukan adalah 2VXS pada tahun 2009 berupa kompleks berisi satu dimer IL-7A yang diapit dua fragmen antibodi. ...
Interleukin-17A (IL-17A) merupakan sitokin pro-inflamasi yang terlibat dalam patogenesis beberapa penyakit, antara lain psoriasis, rheumatoid arthritis, kanker, diabetes dan penyakit ginjal stadium akhir. Peningkatan kadar serum IL-17A memberikan petunjuk adanya keterkaitan antara IL-17A pada kejadian nefropati diabetik. Keterkaitan ini juga diperjelas dengan temuan bahwa penghambatan aktivitas IL-17A dapat menurunkan albuminuria, cedera ginjal dan menunda perkembangan nefropati diabetik. Peranan IL-17A ini menjadikannya sebagai pilihan target potensial terapi nefropati diabetik yang sejauh ini bertumpu pada pengendalian optimal sistem renin angiotensin. Penelitian ini bertujuan mengidentifikasi determinan molekul interaksi STK630921 pada IL-17A. Penelitian dilakukan dengan penambatan molekul STK630921 pada protein IL-17A, dilanjutkan dengan simulasi dinamika molekul selama 15 ns. Identifikasi determinan molekul dilakukan menggunakan bantuan perangkat lunak PyPLIF-HIPPOS terhadap hasil simulasi dinamika molekul. Penelitian ini berhasil mengidentifikasikan asam-asam amino yang berperan penting yaitu His29, Trp67, Asn27, Lys114 dan Glu95 dengan interaksi aromatik dan interaksi hidrogen sebagai jenis interaksi yang berperan pada aktivitas STK630921 pada struktur protein 4HR9.
... The binding interface largely overlapped with the IL-17RA binding surface [26]. HB0017 recognised similar regions on IL-17A as that of Fab6785 and a computationally designed antibody, but it demonstrated significantly higher affinity for IL-17A compared with those antibodies [27,28]. Moreover, the epitopes on IL-17A for HB0017 were distinct from those of secukinumab and ixekizumab. ...
The pro-inflammatory cytokine interleukin-17A (IL-17A) is a key driver of multiple inflammatory and immune disorders. Therapeutic antibodies targeting IL-17A have been proven effective in treating patients with these diseases; however, large variations in clinical outcomes have been observed with different antibodies. In this study, we developed HB0017, a novel monoclonal antibody that targets human IL-17A. HB0017 specifically and strongly bound to human, cynomolgus monkey, and mouse IL-17A at the physiological interface with the IL-17A receptor. In human and monkey cells, HB0017 potently antagonized the functions of IL-17A through competitive binding. HB0017 functioned equivalently to that of clinically approved antibodies in terms of therapeutic efficacy for inflammatory disorders and psoriasis in a mouse model. The results indicate that HB0017 may be an alternative biological therapy for treating patients with inflammation and autoimmune diseases.
... The similarity between IL-17 cytokines is higher in the C terminus and in five spatially conserved cysteine residues. N terminus sequences of IL-17B, IL-17C, and IL-17E are substantially different from those found in IL-17A and IL-17F because of a longer extension of the former three proteins (95), suggesting that the N terminus is involved in receptor specificity (96). ...
The interleukin-(IL-)17 family of cytokines is composed of six members named IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17A is the prototype of this family, and it was the first to be discovered and targeted in the clinic. IL-17A is essential for modulating the interplay between commensal microbes and epithelial cells at our borders (i.e., skin and mucosae), and yet, for protecting us from microbial invaders, thus preserving mucosal and skin integrity. Interactions between the microbiota and cells producing IL-17A have also been implicated in the pathogenesis of immune mediated inflammatory diseases and cancer. While interactions between microbiota and IL-17B-to-F have only partially been investigated, they are by no means less relevant. The cellular source of IL-17B-to-F, their main targets, and their function in homeostasis and disease distinguish IL-17B-to-F from IL-17A. Here, we intentionally overlook IL-17A, and we focus instead on the role of the other cytokines of the IL-17 family in the interplay between microbiota and epithelial cells that may contribute to cancer pathogenesis and immune surveillance. We also underscore differences and similarities between IL-17A and IL-17B-to-F in the microbiota-immunity-cancer axis, and we highlight therapeutic strategies that directly or indirectly target IL-17 cytokines in diseases.
... To facilitate the drug discovery process, multiple pharmaceutical companies have been actively engaged in structure-based drug design on IL-17A. To date, several groups have published crystal structures of human IL-17A in apo form and various bound forms [4][5][6][7][8][9]. In their crystallization efforts, one group obtained the recombinant IL-17A through mammalian expression followed by deglycosylation, or through insect expression with truncation and site-directed mutagenesis to suppress glycosylation and eliminate the predicted exposed free thiol group [4,5]. ...
... In their crystallization efforts, one group obtained the recombinant IL-17A through mammalian expression followed by deglycosylation, or through insect expression with truncation and site-directed mutagenesis to suppress glycosylation and eliminate the predicted exposed free thiol group [4,5]. In contrast, other groups chose E. coli expression due to its multiple advantages, such as high expression levels, lack of posttranslational modifications and cost-effectiveness [6,10]. A total of four isoforms of a recombinant human IL-17A variant post-refolding were reported, differing by their interchain disulfide structures [11]. ...
... Two protocols for IL-17A refolding have been published previously. One used the oxido-shuffling pair of cysteine and cystamine at 5 mM and 1 mM respectively [10]; the other used another oxido-shuffling pair, reduced and oxidized glutathione, at 0.3 mM and 0.03 mM respectively [6]. Both protocols applied more of the reducing agent than the oxidizing agent (Table 1), which is in accordance with the commonly established conditions for refolding of disulfide-bonded proteins [12][13][14]. ...
Refolding of human interleukin 17A (IL-17A) has been reported; however, the key refolding protocol was not robust enough to deliver consistent results and to be easily scaled up for crystallization. Here we report an optimized refolding method for IL-17A. Although co-crystal structures of IL-17A with ligands have been obtained with a high-affinity peptide and an anti-IL-17A Fab as stabilizers, neither the production yield nor the characterization of the IL-17A/Fab complex was reported. To facilitate co-crystallization of IL-17A with small-molecule compounds derived from our DNA encoded library, we also describe the method for yield enhancement of anti-IL-17A Fab production and characterize the IL-17A/Fab complex for the first time, providing an essential prerequisite for structure-based drug discovery targeting IL-17A.
... Для поиска молекулярных основ ингибирования этого цито кина определяются кристаллические структуры его комплексов с различными ингибиторами. Получены структуры комплекса IL-17А с Fab-фрагментом нейтрализующего антитела CAT 2200 [64] и тройст венный комплекс с добавлением пептида-ингибитора HAP (Нigh Affinity Peptide) [66]. ...
Cytokines of the IL-17 family play a key role in the host organism defense against bacterial and fungal infections. At the same time, upregulated synthesis of IL-17 cytokines is associated with immunoinflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and others. The members of this family are important therapeutic targets in the treatment of various human chronic inflammatory disorders. Elucidation of signaling pathways involving IL-17 family proteins and analysis of the structure of cytokine complexes with specific antibodies, inhibitors, and receptors are essential for the development of new drugs for the therapy of immunoinflammatory rheumatic diseases.
