BACKGROUND: Human interleukin-22 (IL-22) is known as a "dual function" cytokine that acts as a master regulator to maintain homeostasis, structural integrity of the intestinal epithelial barrier, and shielding against bacterial pathogens. On the other hand, the overexpression of IL-22 is associated with hyper-proliferation and recruitment of pathologic effector cells, leading to tissue damage and chronic inflammation in specific diseases including inflammatory bowel disease (IBD). To study a role of IL-22-mediated signaling axis during intestinal inflammation, we generated a set of small protein blockers of IL-22R1 and verified their inhibitory potential on murine model of colitis. METHODS: We used directed evolution of proteins to identify binders of human IL-22 receptor alpha (IL-22R1), designated as ABR ligands. This approach combines the assembly of a highly complex combinatorial protein library derived from small albumin-binding domain scaffold and selection of promising protein variants using ribosome display followed by large-scale ELISA screening. The binding affinity and specificity of ABR variants were analyzed on transfected HEK293T cells by flow cytometry and LigandTracer. Inhibitory function was further verified by competition ELISA, HEK-Blue IL-22 reporter cells, and murine dextran sulfate sodium (DSS)-induced colitis. RESULTS: We demonstrate that ABR specifically recognizes transgenic IL-22R1 expressed on HEK293T cells and IL-22R1 on TNFα/IFNγ-activated HaCaT cells. Moreover, some ABR binders compete with the IL-22 cytokine and function as IL-22R1 antagonists in HEK-Blue IL22 reporter cells. In a murine model of DSS-induced acute intestinal inflammation, daily intraperitoneal administration of the best IL-22R1 antagonist, ABR167, suppressed the development of clinical and histological markers of colitis including prevention of mucosal inflammation and architecture deterioration. In addition, ABR167 reduces the DSS-induced increase in mRNA transcript levels of inflammatory cytokines such as IL-1β, IL-6, IL-10, and IL-17A. CONCLUSIONS: We developed small anti-human IL-22R1 blockers with antagonistic properties that ascertain a substantial role of IL-22-mediated signaling in the development of intestinal inflammation. The developed ABR blockers can be useful as a molecular clue for further IBD drug development.

• Keywords
• Experimental colitis, Immune suppression, Inflammatory bowel disease, Interleukin-22, Protein engineering,
• MeSH
• HEK293 Cells MeSH
• Interleukin-22 MeSH
• Interleukins genetics   metabolism   MeSH
• Colitis * chemically induced   pathology   metabolism   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Receptors, Interleukin * metabolism   genetics   MeSH
• Dextran Sulfate * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Interleukin-22 MeSH
• interleukin-22 receptor MeSH Browser
• Interleukins MeSH
• Receptors, Interleukin * MeSH
• Dextran Sulfate * MeSH

Human immunodeficiency virus type 1 (HIV-1) vaccine immunogens capable of inducing broadly neutralizing antibodies (bNAbs) remain obscure. HIV-1 evades immune responses through enormous diversity and hides its conserved vulnerable epitopes on the envelope glycoprotein (Env) by displaying an extensive immunodominant glycan shield. In elite HIV-1 viremic controllers, glycan-dependent bNAbs targeting conserved Env epitopes have been isolated and are utilized as vaccine design templates. However, immunological tolerance mechanisms limit the development of these antibodies in the general population. The well characterized bNAbs monoclonal variants frequently exhibit extensive levels of somatic hypermutation, a long third heavy chain complementary determining region, or a short third light chain complementarity determining region, and some exhibit poly-reactivity to autoantigens. This review elaborates on the obstacles to engaging and manipulating the Env glycoprotein as an effective immunogen and describes an alternative reverse vaccinology approach to develop a novel category of bNAb-epitope-derived non-cognate immunogens for HIV-1 vaccine design.

• Keywords
• Broadly neutralizing antibodies, Combinatorial protein library, Glycans, HIV-1 vaccine, Non-cognate ligands, Protein mimicry,
• MeSH
• Epitopes immunology   MeSH
• env Gene Products, Human Immunodeficiency Virus immunology   MeSH
• HIV Infections immunology   MeSH
• HIV Antibodies * immunology   MeSH
• HIV-1 * immunology   MeSH
• Humans MeSH
• Ligands MeSH
• Molecular Mimicry immunology   MeSH
• Antibodies, Neutralizing * immunology   MeSH
• Polysaccharides immunology   MeSH
• AIDS Vaccines * immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Epitopes MeSH
• env Gene Products, Human Immunodeficiency Virus MeSH
• HIV Antibodies * MeSH
• Ligands MeSH
• Antibodies, Neutralizing * MeSH
• Polysaccharides MeSH
• AIDS Vaccines * MeSH

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

• Keywords
• Cancer cell migration, GAMG glioblastoma, HEK-Blue IL-6, IL-6, IL-6R blockers, Malignant melanoma, Migrastatics, Pancreatic carcinoma, Protein engineering,
• MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Cell Movement * drug effects   MeSH
• Cell Proliferation * drug effects   MeSH
• Receptors, Interleukin-6 * metabolism   MeSH
• Protein Binding drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IL6R protein, human MeSH Browser
• Receptors, Interleukin-6 * MeSH

One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this "non-cognate ligand" concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.

• Keywords
• Env glycoprotein, HIV vaccine, broadly neutralizing antibody, combinatorial library, protein mimetics, protein scaffold,
• MeSH
• Epitopes MeSH
• env Gene Products, Human Immunodeficiency Virus genetics   MeSH
• HIV Infections * prevention & control   MeSH
• HIV Antibodies MeSH
• HIV-1 * genetics   MeSH
• Mice MeSH
• Antibodies, Neutralizing MeSH
• Viral Pseudotyping MeSH
• Broadly Neutralizing Antibodies MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• env Gene Products, Human Immunodeficiency Virus MeSH
• HIV Antibodies MeSH
• Antibodies, Neutralizing MeSH
• Broadly Neutralizing Antibodies MeSH

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

• Keywords
• computational saturation, directed evolution, interleukin-10, protein engineering, protein scaffold, ribosome display,
• MeSH
• Databases, Protein MeSH
• Interleukin-10 metabolism   MeSH
• Protein Conformation MeSH
• Computer Simulation MeSH
• Protein Engineering MeSH
• Proteins chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Ribosomes metabolism   MeSH
• Directed Molecular Evolution methods   MeSH
• Amino Acid Sequence MeSH
• Protein Stability MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interleukin-10 MeSH
• Proteins MeSH
• Recombinant Proteins MeSH

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

• Keywords
• ABD scaffold, binding protein, combinatorial library, fibrin, fibrinogen Bβ chain, liposome, metallochelation, thrombus imaging, thrombus targeting,
• Publication type
• Journal Article MeSH

BACKGROUND: The development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies. METHODS: We employed a highly complex combinatorial protein library derived from a 5 kDa albumin-binding domain scaffold, fused with support protein of total 38 kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses. FINDINGS: Three most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface. INTERPRETATION: This strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines. FUND: Czech Health Research Council 15-32198A, Ministry of Health, Czech Republic.

• Keywords
• Albumin-binding domain scaffold, Antibody paratope mimetics, Combinatorial protein library, HIV-1 vaccine, Neutralizing antibody, Protein docking,
• MeSH
• Antigens, Viral chemistry   immunology   MeSH
• Epitopes chemistry   immunology   MeSH
• HIV Infections immunology   virology   MeSH
• HIV Envelope Protein gp120 immunology   MeSH
• HIV Antibodies blood   immunology   MeSH
• HIV-1 immunology   MeSH
• Immunoglobulin G blood   immunology   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Models, Molecular MeSH
• Mice MeSH
• Antibodies, Neutralizing blood   immunology   MeSH
• Amino Acid Sequence MeSH
• AIDS Vaccines immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Viral MeSH
• Epitopes MeSH
• HIV Envelope Protein gp120 MeSH
• HIV Antibodies MeSH
• Immunoglobulin G MeSH
• Antibodies, Neutralizing MeSH
• AIDS Vaccines MeSH

Lactococcus lactis, a probiotic bacterium of food origin, has recently been demonstrated as a suitable strain for the production and in vivo delivery of therapeutically important proteins into the gut. We aimed to engineer recombinant L. lactis cells producing/secreting REX binding proteins that have been described as IL-23 receptor (IL-23R) blockers and IL-23R antagonists suppressing the secretion of cytokine IL-17A, a pivotal step in the T-helper Th17-mediated pro-inflammatory cascade, as well as in the development of autoimmune diseases, including inflammatory bowel disease (IBD). To reach this goal, we introduced cDNA sequences coding for REX009, REX115, and REX125 proteins into plasmid vectors carrying a Usp45 secretion signal, a FLAG tag sequence consensus, and a LysM-containing cA surface anchor (AcmA), thus allowing cell-surface peptidoglycan anchoring. These plasmids, or their non-FLAG/non-AcmA versions, were introduced into L. lactis host cells, thus generating unique recombinant L. lactis-REX strains. We demonstrate that all three REX proteins are expressed in L. lactis cells and are efficiently displayed on the bacterial surface, as tested by flow cytometry using an anti-FLAG antibody conjugate. Upon 10-fold concentration of the conditioned media, a REX125 secretory variant can be detected by Western blotting. To confirm that the FLAG/non-FLAG REX proteins displayed by L. lactis retain their binding specificity, cell-surface interactions of REX proteins with an IL-23R-IgG chimera were demonstrated by flow cytometry. In addition, statistically significant binding of secreted REX009 and REX115 proteins to bacterially produced, soluble human IL-23R was confirmed by ELISA. We conclude that REX-secreting L. lactis strains were engineered that might serve as IL-23/IL-23R blockers in an experimentally induced mouse model of colitis.

• Keywords
• IL-23 cytokine, IL-23R, albumin-binding domain, binding protein, lactococcus, surface display,
• Publication type
• Journal Article MeSH

Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists.

• Keywords
• IL-17 receptor, albumin-binding domain, binding protein, combinatorial library, cytokine,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Cell Line MeSH
• Cytokines metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Interleukin-17 metabolism   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Inflammation Mediators metabolism   MeSH
• Models, Molecular MeSH
• Receptors, Interleukin-17 antagonists & inhibitors   chemistry   metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Protein Stability MeSH
• Carrier Proteins metabolism   MeSH
• Protein Binding MeSH
• Inflammation immunology   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Cytokines MeSH
• IgG Fc-binding protein, Streptococcus MeSH Browser
• IL17A protein, human MeSH Browser
• Interleukin-17 MeSH
• Inflammation Mediators MeSH
• Receptors, Interleukin-17 MeSH
• Recombinant Proteins MeSH
• Carrier Proteins MeSH

IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn’s Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis.

• Keywords
• IL-23, albumin-binding domain, binding protein, cytokine, lactococcus, surface display,
• MeSH
• Th17 Cells drug effects   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Interleukin-23 metabolism   MeSH
• Lactococcus lactis metabolism   MeSH
• Humans MeSH
• Proteins genetics   metabolism   pharmacology   MeSH
• Flow Cytometry MeSH
• Recombinant Proteins genetics   metabolism   pharmacology   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Interleukin-23 MeSH
• Proteins MeSH
• Recombinant Proteins MeSH