Antibody-cytokine fusion proteins

Cytokines are key players in stimulating and regulating immune responses in physiological and pathophysiological processes. Various cytokines have been approved for therapy of cancer and other diseases and many more are under development. However, therapeutic efficacy is often hampered by severe side effects and poor pharmacokinetic properties. Fusion of cytokines to antibodies or antibody fragments allows for a targeted delivery and should, therefore, improve efficacy and pharmacokinetics. This review provides a comprehensive summary of the developments in the field of targeted cytokine delivery by genetic engineering of antibody-cytokine fusion proteins.     

Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.     

Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression

A recombinant, multifunctional protein has been designed for optimized, cell-targeted DNA delivery and gene expression in mammalian cells. This hybrid construct comprises a viral peptide ligand for integrin alpha(V)beta(3) binding, a DNA-condensing poly-L-lysine domain, and a complete, functional beta-galactosidase protein that serves simultaneously as purification tag and DNA-shielding agent. This recombinant protein is stable; it has been produced successfully in Escherichia coli and can be purified in a single step by affinity chromatography. At optimal molar ratios, mixtures of this vector and a luciferase-reporter plasmid form stable complexes that transfect cultured cells. After exposure to these cell-targeted complexes, steady levels of gene expression are observed for more than 3 days after transfection, representing between 20 and 40% of those achieved with untargeted, lipid-based DNA-condensing agents. The principle to include viral motifs for cell infection in single polypeptide recombinant proteins represents a promising approach towards the design of non-viral modular DNA transfer vectors that conserve the cell-target- ing specificity of native viruses and that do not need further processing after bioproduction in a recombinant host.     

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design

The DVD-Ig™ protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.Key words: dual variable domain immunoglobulin, DVD-Ig, immunotherapy, variable domain, antibody engineering, dual-specific, linker     

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design

The DVD-Ig^TM protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.     

The structure of the interleukin-15 alpha receptor and its implications for ligand binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.     

Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody-targeted interleukin-2

 A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the Cγ1 gene of ch14.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumor load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases. Received: 7 November 1995 / Accepted: 15 December 1995     

Computational design of antibody-affinity improvement beyond in vivo maturation

Antibodies are used extensively in diagnostics and as therapeutic agents. Achieving high-affinity binding is important for expanding detection limits, extending dissociation half-times, decreasing drug dosages and increasing drug efficacy. However, antibody-affinity maturation in vivo often fails to produce antibody drugs of the targeted potency, making further affinity maturation in vitro by directed evolution or computational design necessary. Here we present an iterative computational design procedure that focuses on electrostatic binding contributions and single mutants. By combining multiple designed mutations, a tenfold affinity improvement to 52 pM was engineered into the anti-epidermal growth factor receptor drug cetuximab (Erbitux), and a 140-fold improvement in affinity to 30 pM was obtained for the anti-lysozyme model antibody D44.1. The generality of the methods was further demonstrated through identification of known affinity-enhancing mutations in the therapeutic antibody bevacizumab (Avastin) and the model anti-fluorescein antibody 4-4-20. These results demonstrate computational capabilities for enhancing and accelerating the development of protein reagents and therapeutics.     

Bi-functional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer

Typically, multiple cytokines act in concert to mediate a desired immunological response, and thus more effective therapeutics may be achieved by combining several cytokines with potentially synergistic activities. We have developed a series of bi-functional cytokine fusion proteins which, when additionally linked to an intact antibody (or the Fc portion of an antibody) in a variety of configurations, can be specifically targeted. We focus here mainly on the synergizing cytokine combination interleukin-2/interleukin-12 (IL-2/IL-12), but also demonstrate the utility of this approach with interleukin-4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF). Cytokine activity was retained in constructs where the cytokines were fused in tandem at the carboxyl terminus of the Fc or antibody heavy (H) chain, as well as in constructs where one cytokine was fused at the carboxyl terminus of the H chain while the second cytokine was fused to the amino terminus of either the H or light (L) chain variable region. Even in such constructs, antigen binding of the antibody-cytokine fusion proteins could be maintained. In the context of bi-functional fusion proteins, hetero-dimeric IL-12 could be expressed either in a single-chain form, or maintained as a heterodimer in which the p40 subunit was fused to IL-2. These IL-12/IL-2 bi-functional fusion proteins were shown to induce extremely high levels of interferon-gamma (IFN-gamma), similar to the synergy normally seen with the combined application of the individual cytokines. In addition, these bifunctional molecules were shown to have striking anti-tumor activity as either gene therapy or as an antibody cytokine(s) fusion protein, and may provide a useful approach to the treatment of cancer.     

Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse

Rituximab (RTX), a chimeric IgG1 monoclonal antibody directed against the CD20 antigen, has revolutionized the treatment of B-cell malignancies. Nevertheless, the relapsed/refractory rates are still high. One strategy to increase the clinical effectiveness of RTX is based on antibody-cytokine fusion protein (immunocytokine; ICK) vectorizing together at the tumor site the antibody effector activities and the cytokine co-signal required for the generation of cytotoxic cellular immunity. Such ICKs linking various antibody formats to interleukin (IL)-2 are currently being investigated in clinical trials and have shown promising results in cancer therapies. IL-15, a structurally-related cytokine, is now considered as having a better potential than IL-2 in antitumor immunotherapeutic strategies. We have previously engineered the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. Compared with IL-15, RLI displayed better biological activities in vitro and higher antitumor effects in vivo in murine and human cancer models. In this study, we investigated the advantages of fusing RLI to RTX. Anti-CD20-RLI kept its binding capacity to CD20, CD16 and IL-15 receptor and therefore fully retained both antibody effector functions (ADCC and CDC), and the cytokine potential of RLI. In a severe combined immunodeficiency (SCID) mouse model of disseminated residual lymphoma, anti-CD20-RLI was found to induce long-term survival of 90% of mice up to at least 120 days whereas RLI and RTX, alone or in combination, just delayed the disease onset (100% of death at 28, 40 and 51 days respectively). These findings suggest that such ICK could improve the clinical efficacy of RTX, particularly in patients with refractory B-cell lymphoma.     

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.     

Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse

Rituximab (RTX), a chimeric IgG1 monoclonal antibody directed against the CD20 antigen, has revolutionized the treatment of B-cell malignancies. Nevertheless, the relapsed/refractory rates are still high. One strategy to increase the clinical effectiveness of RTX is based on antibody-cytokine fusion protein (immunocytokine; ICK) vectorizing together at the tumor site the antibody effector activities and the cytokine co-signal required for the generation of cytotoxic cellular immunity. Such ICKs linking various antibody formats to interleukin (IL)-2 are currently being investigated in clinical trials and have shown promising results in cancer therapies. IL-15, a structurally-related cytokine, is now considered as having a better potential than IL-2 in antitumor immunotherapeutic strategies. We have previously engineered the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. Compared with IL-15, RLI displayed better biological activities in vitro and higher antitumor effects in vivo in murine and human cancer models. In this study, we investigated the advantages of fusing RLI to RTX. Anti-CD20-RLI kept its binding capacity to CD20, CD16 and IL-15 receptor and therefore fully retained both antibody effector functions (ADCC and CDC), and the cytokine potential of RLI. In a severe combined immunodeficiency (SCID) mouse model of disseminated residual lymphoma, anti-CD20-RLI was found to induce long-term survival of 90% of mice up to at least 120 days whereas RLI and RTX, alone or in combination, just delayed the disease onset (100% of death at 28, 40 and 51 days respectively). These findings suggest that such ICK could improve the clinical efficacy of RTX, particularly in patients with refractory B-cell lymphoma.     

PEI-g-PEG-RGD/small interference RNA polyplex-mediated silencing of vascular endothelial growth factor receptor and its potential as an anti-angiogenic tumor therapeutic strategy

Tumor angiogenesis appears to be achieved by the expression of vascular endothelial growth factor (VEGF) within solid tumors that stimulate host vascular endothelial cell mitogenesis and possibly chemotaxis. VEGF's angiogenic actions are mediated through its high-affinity binding to 2 endothelium-specific receptor tyrosine kinase, Flt-1 (VEGFR1), and Flk-1/KDR (VEGFR2). RNA interference-mediated knockdown of protein expression at the messenger RNA level provides a new therapeutic strategy to overcome various diseases. To achieve high efficacy in RNA interference-mediated therapy, it is critical to develop an efficient delivering system to deliver small interference RNA (siRNA) into tissues or cells site-specifically. We previously reported an angiogenic endothelial cell-targeted polymeric gene carrier, PEI-g-PEG-RGD. This targeted carrier was developed by the conjugation of the ανβ3/ανβ5 integrin-binding RGD peptide (ACDCRGDCFC) to the cationic polymer, branched polyethylenimine, with a hydrophilic polyethylene glycol (PEG) spacer. In this study, we used PEI-g-PEG-RGD to deliver siRNA against VEGFR1 into tumor site. The physicochemical properties of PEI-g-PEG-RGD/siRNA complexes was evaluated. Further, tumor growth profile was also investigated after systemic administration of PEI-g-PEG-RGD/siRNA complexes.     

Parsing the effects of binding,signaling, and trafficking on the mitogenic potencies of granulocyte colony-stimulating factor analogues

The pharmacodynamic potency of a therapeutic cytokine interacting with a cell-surface receptor can be attributed primarily to three central properties: [1] cytokine/receptor binding affinity, [2] cytokine/receptor endocytic trafficking dynamics, and [3] cytokine/receptor signaling. Thus, engineering novel or second-generation cytokines requires an understanding of the contribution of each of these to the overall cell response. We describe here an efficient method toward this goal in demonstrated application to the clinically important cytokine granulocyte colony-stimulating factor (GCSF) with a chemical analogue and a number of genetic mutants. Using a combination of simple receptor-binding and dose-response proliferation assays we construct an appropriately scaled plot of relative mitogenic potency versus ligand concentration normalized by binding affinity. Analysis of binding and proliferation data in this manner conveniently indicates which of the cytokine properties-binding, trafficking, and/or signaling-are contributing substantially to altered potency effects. For the GCSF analogues studied here, two point mutations as well as a poly(ethylene glycol) chemical conjugate were found to have increased potencies despite comparable or slightly lower affinities, and trafficking was predicted to be the responsible mechanism. A third point mutant exhibiting comparable binding affinity but reduced potency was predicted to have largely unchanged trafficking properties. Surprisingly, another mutant possessing an order-of-magnitude weaker binding affinity displayed enhanced potency, and increased ligand half-life was predicted to be responsible for this net beneficial effect. Each of these predictions was successfully demonstrated by subsequent measurements of depletion of these five analogues from cell culture medium. Thus, for the GCSF system we find that ligand trafficking dynamics can play a major role in regulating mitogenic potency. Our results demonstrate that cytokine analogues can exhibit pharmacodynamic behaviors across a diverse spectrum of "binding-potency space" and that our analysis through normalization can efficiently elucidate hypotheses for the underlying mechanisms for further dedicated testing. We have also extended the Black-Leff model of pharmacological agonism to include trafficking effects along with binding and signaling, and this model provides a framework for parsing the effects of these factors on pharmacodynamic potency.     

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs.     

Novel trastuzumab-DM1 conjugate: Synthesis and bio-evaluation

Antibody-drug conjugates are now of considerable interest and are recommended for the treatment of cancers. Linkers are having a crucial role in potency and efficacy of these drugs. Herein, for the first time, we have used a water-soluble poly-ethylene glycol based linker (succinimidyl-[(N-maleimido propionamido)-diethyleneglycol] [SM(PEG)2]) for lysine amide coupling of DM1 drug to trastuzumab considering evaluation of the effect of using a hydrophilic linker on physicochemical and biological properties of the resulting conjugate in comparison to the conjugate containing succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, which has a relative hydrophobic nature. The physicochemical properties of synthesized conjugates were investigated in terms of drug to antibody ratio, size variants and free drug quantities. In vitro biological activity of trastuzumab-DM1 conjugates was assessed on breast cancer cell lines expressing different levels of HER2 using binding affinity, antiproliferative, apoptosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Synthesized conjugate containing hydrophilic linker, showed higher drug to antibody ratio, no aggregated form and higher cellular toxicity in comparison to SMCC bearing conjugate. Binding affinity and ADCC potential of conjugates was not affected upon the usage of hydrophilic linker. In conclusion, application of SM(PEG)2 for coupling of DM1 to trastuzumab enhance desirable characteristics of the resulting conjugate.     

Conformational Heterogeneity in Antibody-Protein Antigen Recognition: IMPLICATIONS FOR HIGH AFFINITY PROTEIN COMPLEX FORMATION

Specific, high affinity protein-protein interactions lie at the heart of many essential biological processes, including the recognition of an apparently limitless range of foreign proteins by natural antibodies, which has been exploited to develop therapeutic antibodies. To mediate biological processes, high affinity protein complexes need to form on appropriate, relatively rapid timescales, which presents a challenge for the productive engagement of complexes with large and complex contact surfaces (∼600–1800 Ų). We have obtained comprehensive backbone NMR assignments for two distinct, high affinity antibody fragments (single chain variable and antigen-binding (Fab) fragments), which recognize the structurally diverse cytokines interleukin-1β (IL-1β, β-sheet) and interleukin-6 (IL-6, α-helical). NMR studies have revealed that the hearts of the antigen binding sites in both free anti-IL-1β Fab and anti-IL-6 single chain variable exist in multiple conformations, which interconvert on a timescale comparable with the rates of antibody-antigen complex formation. In addition, we have identified a conserved antigen binding-induced change in the orientation of the two variable domains. The observed conformational heterogeneity and slow dynamics at protein antigen binding sites appears to be a conserved feature of many high affinity protein-protein interfaces structurally characterized by NMR, suggesting an essential role in protein complex formation. We propose that this behavior may reflect a soft capture, protein-protein docking mechanism, facilitating formation of high affinity protein complexes on a timescale consistent with biological processes.     

The amino-terminal helix of GM-CSF and IL-5 governs high affinity binding to their receptors.

Transduction of the biological effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) requires the interaction of each cytokine with at least two cell surface receptor components, one of which is shared between these two cytokines. A strategy is presented that allowed us to identify receptor binding determinants in GM-CSF and IL-5. Mixed species (human and mouse) receptors were used to locate unique receptor binding domains on a series of human-mouse hybrid GM-CSF and IL-5 cytokines. Results show that the interaction of these two cytokines with the shared subunit of their high affinity receptor complexes is governed by a very small part of their peptide chains. The presence of a few key residues in the amino-terminal alpha-helix of each ligand is sufficient to confer specificity to the interaction. Comparison with other cytokines suggests that the amino-terminal helix of many of these proteins may contain the recognition element for the formation of high affinity binding sites with the alpha subunit of their multi-component receptors.     

Liver cell-targeted delivery of therapeutic molecules

The liver is the largest internal organ in mammals and is involved in metabolism, detoxification, synthesis of proteins and lipids, secretion of cytokines and growth factors and immune/inflammatory responses. Hepatitis, alcoholic or non-alcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. Safe and efficient delivery of therapeutic molecules (drugs, genes or proteins) into the liver is very important to increase the clinical efficacy of these molecules and to reduce their side effects in other organs. Several liver cell-targeted delivery systems have been developed and tested in vivo or ex vivo/in vitro. In this review, we discuss the literature concerning liver cell-targeted delivery systems, with a particular emphasis on the results of in vivo studies.     

A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy

The pro-inflammatory cytokine interleukin (IL)-1β is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1β have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1β with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a >30-fold increased affinity to human IL-1β compared with its parent antibody. This anti-human IL-1β IgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1β pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1β monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal.