ABSTRACT
Interleukin 15 (IL-15) and IL-2 have distinct immunological functions even though both signal through the receptor subunit IL-2Rß and the common γ-chain (γ(c)). Here we found that in the structure of the IL-15-IL-15Rα-IL-2Rß-γ(c) quaternary complex, IL-15 binds to IL-2Rß and γ(c) in a heterodimer nearly indistinguishable from that of the IL-2-IL-2Rα-IL-2Rß-γ(c) complex, despite their different receptor-binding chemistries. IL-15Rα substantially increased the affinity of IL-15 for IL-2Rß, and this allostery was required for IL-15 trans signaling. Consistent with their identical IL-2Rß-γ(c) dimer geometries, IL-2 and IL-15 showed similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induced similar signals, and the cytokine specificity of IL-2Rα versus IL-15Rα determined cellular responsiveness. Our results provide new insights for the development of specific immunotherapeutics based on IL-15 or IL-2.
Subject(s)
Interleukin-15/immunology , Interleukin-2/immunology , Animals , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Humans , Interleukin-15/chemistry , Interleukin-15/metabolism , Interleukin-2/chemistry , Interleukin-2/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-2 Receptor beta Subunit/metabolism , Ligands , Lymphocytes/immunology , Lymphocytes/metabolism , Mice , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Signal TransductionABSTRACT
The prominent role of voltage-gated sodium channel 1.7 (Nav1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtype-selective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, because they naturally evolved to be potent ion channel blockers. Here, we report the engineering of highly potent and selective inhibitors of the Nav1.7 channel based on tarantula ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to the Nav1.7 channel. The resulting microproteins are highly potent (IC50 to Nav1.7 of 2.5 nm) and selective. We achieved 80- and 20-fold selectivity over the closely related Nav1.2 and Nav1.6 channels, respectively, and the IC50 on skeletal (Nav1.4) and cardiac (Nav1.5) sodium channels is above 3000 nm The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.
Subject(s)
NAV1.7 Voltage-Gated Sodium Channel/chemistry , Pain Management/methods , Protein Engineering , Voltage-Gated Sodium Channel Blockers/pharmacology , HEK293 Cells , Humans , NAV1.7 Voltage-Gated Sodium Channel/drug effects , Patch-Clamp Techniques , Phylogeny , Spider Venoms/chemistryABSTRACT
The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.
Subject(s)
Aminobenzoates/chemistry , Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Immunoconjugates/chemistry , Oligopeptides/chemistry , Pancreatic Neoplasms/drug therapy , Aminobenzoates/blood , Aminobenzoates/pharmacokinetics , Aminobenzoates/pharmacology , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Carbamates/chemistry , Cathepsin B/chemistry , Cathepsin B/metabolism , Cell Line, Tumor , Dipeptides/chemistry , Drug Delivery Systems/methods , Drug Stability , Female , Humans , Immunoconjugates/blood , Immunoconjugates/pharmacokinetics , Immunoconjugates/pharmacology , Mice , Mice, Nude , Models, Molecular , Oligopeptides/blood , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/pathology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.
Subject(s)
Antiparasitic Agents/chemistry , Cysteine Endopeptidases/chemistry , Plasmodium falciparum/enzymology , Protease Inhibitors/chemistry , Protozoan Proteins/chemistry , Sulfones/chemistry , Trypanosoma brucei brucei/enzymology , Trypanosoma cruzi/enzymology , Animals , Antiparasitic Agents/therapeutic use , Chagas Disease/drug therapy , Chagas Disease/enzymology , Crystallography, X-Ray , Drug Design , Kinetics , Malaria, Falciparum/drug therapy , Malaria, Falciparum/enzymology , Protease Inhibitors/therapeutic use , Protein Binding , Protein Structure, Tertiary , Protozoan Proteins/antagonists & inhibitors , Sulfones/therapeutic use , Trypanosomiasis, African/drug therapy , Trypanosomiasis, African/enzymologyABSTRACT
Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.
Subject(s)
Antibodies, Bispecific/immunology , CD3 Complex/immunology , Claudins/immunology , Immunoconjugates/therapeutic use , Pancreatic Neoplasms/therapy , Stomach Neoplasms/therapy , Adenocarcinoma/therapy , Animals , Carcinoma, Pancreatic Ductal/therapy , Cell Line, Tumor , Humans , Immunoconjugates/blood , Mice , Pancreatic Neoplasms/blood , Rats , Stomach Neoplasms/bloodABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
Subject(s)
Escherichia coli/genetics , Immunoconjugates/metabolism , Protein Engineering , Streptomyces/enzymology , Transglutaminases/genetics , Transglutaminases/metabolism , Amino Acid Sequence , Base Sequence , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Enzyme Precursors/isolation & purification , Enzyme Precursors/metabolism , Gene Expression , Industrial Microbiology , Models, Molecular , Molecular Sequence Data , Mutagenesis , Mutation , Plasmids/genetics , Protein Structure, Tertiary , Solubility , Streptomyces/chemistry , Streptomyces/genetics , Transglutaminases/chemistry , Transglutaminases/isolation & purificationABSTRACT
Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the alpha, beta and the "common gamma" (gamma(c)) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor-ligand, and receptor-receptor interactions that are necessary for formation of the high-affinity IL-2/alphabetagamma(c) signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its alpha receptor, a moderately entropically favorable low affinity interaction between IL-2 and its beta receptor, and no interaction between IL-2 and the shared receptor, gamma(c). Formation of the stable intermediate trimolecular complexes of IL-2 with alpha and beta receptors, as well as IL-2 with beta and gamma(c) receptors proceeds through enthalpy-entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor alpha and beta chains, suggesting that a preformed alphabeta complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Ralphabetagamma(c) high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by gamma(c) contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.
Subject(s)
Interleukin-2/metabolism , Protein Isoforms/metabolism , Receptors, Interleukin-2/metabolism , Signal Transduction/physiology , Calorimetry , Interleukin-2/chemistry , Macromolecular Substances , Models, Molecular , Protein Binding , Protein Isoforms/chemistry , Protein Structure, Quaternary , Receptors, Interleukin-2/chemistry , ThermodynamicsABSTRACT
The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency in vitro and in vivo. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.
Subject(s)
Aminobenzoates/pharmacokinetics , Drug Carriers/pharmacokinetics , Oligopeptides/pharmacokinetics , Aminobenzoates/administration & dosage , Aminobenzoates/chemistry , Animals , Antibodies/administration & dosage , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Stability , Female , HEK293 Cells , Humans , Macaca fascicularis , Mice, SCID , Oligopeptides/administration & dosage , Oligopeptides/chemistry , RatsABSTRACT
Stiff-man syndrome (SMS) is a rare autoimmune disorder of the central nervous system associated with autoantibodies to glutamate decarboxylase (GAD). We isolated five brain-reactive human monoclonal antibodies, with reactivity distinct from GAD, from peripheral blood of a patient newly diagnosed with SMS. Two antibodies reacted with both Purkinje cells and ependymal cells, and precipitated an 80-kDa protein from rat neuronal primary cultures, which was also recognized by 12% (3/25) of SMS sera and 13% (2/15) of SMS cerebrospinal fluid (CSF) samples. The corresponding antigen was identified as 17 beta-hydroxysteroid dehydrogenase type 4 and may represent a possible novel target of autoimmunity in SMS.
Subject(s)
17-Hydroxysteroid Dehydrogenases/immunology , Autoantibodies/immunology , Central Nervous System/immunology , Enoyl-CoA Hydratase , Multienzyme Complexes , Neurons/immunology , Stiff-Person Syndrome/enzymology , Stiff-Person Syndrome/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Autoantibodies/blood , Autoantibodies/isolation & purification , Cells, Cultured , Central Nervous System/physiopathology , Estradiol/metabolism , Female , Fetus , Humans , Hydro-Lyases , Mice , Middle Aged , Molecular Sequence Data , Neurons/enzymology , Peroxisomal Multifunctional Protein-2 , Purkinje Cells/enzymology , Purkinje Cells/immunology , Rats , Rats, Wistar , Recombinant Fusion Proteins/immunology , Stiff-Person Syndrome/bloodABSTRACT
Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymatic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compounds at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chemical instability, as was observed with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.
Subject(s)
Antibodies/chemistry , Antineoplastic Agents/chemistry , Immunoconjugates/chemistry , Animals , Antibodies/immunology , Half-Life , Humans , Immunoconjugates/pharmacokinetics , Immunoconjugates/therapeutic use , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/immunology , Mice , Neoplasms/drug therapy , Rats , Transglutaminases/metabolism , Tubulin Modulators/chemistryABSTRACT
Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.
Subject(s)
Antibodies, Bispecific/immunology , Immunoglobulin G/metabolism , Protein Engineering/methods , Animals , Antibodies, Bispecific/genetics , Antibodies, Bispecific/isolation & purification , Antibodies, Bispecific/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Antibody Specificity , Antigens, CD20/immunology , B-Lymphocytes/immunology , CD3 Complex/immunology , Cetuximab , Cytotoxicity, Immunologic , Female , Histocompatibility Antigens Class I/metabolism , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Mice , Mice, Inbred C57BL , Point Mutation , Rats , Rats, Sprague-Dawley , Receptors, Fc/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , T-Lymphocytes/immunologyABSTRACT
The interleukin-7 receptor α chain (IL-7Rα) gene was identified as a top non-major histocompatibility complex-linked risk locus for multiple sclerosis (MS). Recently, we showed that a T helper 1 (T(H)1)-driven, but not a T(H)17-driven, form of MS exhibited a good clinical response to interferon-ß (IFN-ß) therapy. We now demonstrate that high serum levels of IL-7, particularly when paired with low levels of IL-17F, predict responsiveness to IFN-ß and hence a T(H)1-driven subtype of MS. We also show that although IL-7 signaling is neither necessary nor sufficient for the induction or expansion of T(H)17 cells, IL-7 can greatly enhance both human and mouse T(H)1 cell differentiation. IL-7 alone is sufficient to induce human T(H)1 differentiation in the absence of IL-12 or other cytokines. Furthermore, targeting IL-7/IL-7Rα is beneficial in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Mice treated with IL-7Rα-blocking antibodies before or after onset of paralysis exhibited reduced clinical signs of EAE, with reduction in peripheral naïve and activated T cells, whereas central memory T, regulatory T, B, and natural killer cell populations were largely spared. IL-7Rα antibody treatment markedly reduced lymphocyte infiltration into the central nervous system in mice with EAE. Thus, a serum profile of high IL-7 may signify a T(H)1-driven form of MS and may predict outcome in MS patients undergoing IFN-ß therapy. Blockade of IL-7 and the IL-7Rα pathway may have therapeutic potential in MS and other autoimmune diseases.
Subject(s)
Interferon-beta/therapeutic use , Interleukin-7/blood , Interleukin-7/immunology , Multiple Sclerosis/blood , Multiple Sclerosis/drug therapy , Th1 Cells/cytology , Th1 Cells/immunology , Animals , Antibodies/pharmacology , Antibody Specificity/drug effects , Cell Differentiation/drug effects , Encephalomyelitis, Autoimmune, Experimental/blood , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Humans , Immunologic Memory/drug effects , Interferon-beta/immunology , Mice , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Receptors, Interleukin-7/antagonists & inhibitors , Th1 Cells/drug effects , Treatment OutcomeABSTRACT
The cytokine interleukin-15 (IL-15) signals through the formation of a quaternary receptor complex composed of an IL-15-specific alpha receptor, together with beta and gammac receptors that are shared with interleukin-2 (IL-2). The initiating step in the formation of this signaling complex is the interaction between IL-15 and IL-15Ralpha, which is a single sushi domain bearing strong structural homology to one of the two sushi domains of IL-2Ralpha. The crystal structure of the IL2-Ralpha/IL-2 complex has been determined, however little is known about the analogous IL-15Ralpha/IL-15 binding interaction. Here we show that recombinant IL-15 can be overexpressed as a stable complex in the presence of its high affinity receptor, IL-15Ralpha. We find that this complex is 10-fold more active than IL-15 alone in stimulating proliferation and survival of memory phenotype CD8 T cells. To probe the ligand/receptor interface, we used solution NMR to map chemical shifts on 15N-labeled IL-15Ralpha in complex with unlabeled IL-15. Our results predict that the binding surface on IL-15Ralpha involves strands C and D, similar to IL-2Ralpha. The interface, as predicted here, leaves open the possibility of trans-presentation of IL-15 by IL-15Ralpha on an opposing cell.
Subject(s)
Interleukin-15 Receptor alpha Subunit/chemistry , Interleukin-15/chemistry , Binding Sites , CD8-Positive T-Lymphocytes/immunology , Humans , Interleukin-15/genetics , Interleukin-15/metabolism , Interleukin-15 Receptor alpha Subunit/genetics , Interleukin-15 Receptor alpha Subunit/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Conformation , Protein Interaction Mapping , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Recently, we have identified proinsulin (P-Ins)(73-90) as an immunodominant T cell epitope of HLA-DRB1*0401 (DR4) subjects with beta-islet cell autoimmunity and of HLA-DR4/CD4 double-transgenic mice immunized with human P-Ins. We have compared the fine specificities of one human CD4 T cell clone and two mouse T cell hybridoma clones recognizing this epitope, and, although these three clones all recognized the same core region (LALEGSLQK), there were major differences in how they interacted with the peptide (p)/HLA complex, reflecting the fact that human P-Ins is a foreign antigen in the mouse and an autoantigen in the type 1 diabetes patient. The human T cell clone was forkhead transcription factor 3 (Foxp3)-positive, a marker for regulatory T cell lineages, and secreted predominantly IL-5, IL-10, and low levels of IFNgamma in response to P-Ins(73-90). This finding is compatible with the previously detected regulatory cytokine pattern in subjects with beta-cell autoimmunity. However, added N- or C-terminal amino acids drastically changed HLA and tetramer binding capacity as well as T cell reactivity and the cytokine phenotype of the P-Ins(73-90)-specific human CD4 T cell clone, suggesting a potential for this P-Ins epitope as a target for therapeutic intervention in HLA-DR4-positive humans with beta-islet cell autoimmunity or recent-onset type 1 diabetes.
Subject(s)
Epitopes , HLA-DR Antigens/immunology , Peptide Fragments/metabolism , Proinsulin/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Amino Acids/metabolism , Animals , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , HLA-DRB1 Chains , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , PhenotypeABSTRACT
Interleukin-2 (IL-2) is an immunoregulatory cytokine that acts through a quaternary receptor signaling complex containing alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gc) receptors. In the structure of the quaternary ectodomain complex as visualized at a resolution of 2.3 angstroms, the binding of IL-2Ralpha to IL-2 stabilizes a secondary binding site for presentation to IL-2Rbeta. gammac is then recruited to the composite surface formed by the IL-2/IL-2Rbeta complex. Consistent with its role as a shared receptor for IL-4, IL-7, IL-9, IL-15, and IL-21, gammac forms degenerate contacts with IL-2. The structure of gammac provides a rationale for loss-of-function mutations found in patients with X-linked severe combined immunodeficiency diseases (X-SCID). This complex structure provides a framework for other gammac-dependent cytokine-receptor interactions and for the engineering of improved IL-2 therapeutics.
Subject(s)
Interleukin-2/chemistry , Receptors, Interleukin-2/chemistry , Receptors, Interleukin/chemistry , Crystallography, X-Ray , Humans , Interleukin Receptor Common gamma Subunit , Interleukin-2/metabolism , Interleukin-2/therapeutic use , Interleukin-2 Receptor alpha Subunit , Interleukin-2 Receptor beta Subunit , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Receptors, Interleukin/metabolism , Receptors, Interleukin-2/genetics , Receptors, Interleukin-2/metabolism , Recombinant Proteins/therapeutic use , Severe Combined Immunodeficiency/geneticsABSTRACT
Interleukin-2 (IL-2) is an immunoregulatory cytokine that binds sequentially to the alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gammac) receptor subunits. Here we present the 2.8 angstrom crystal structure of a complex between human IL-2 and IL-2Ralpha, which interact in a docking mode distinct from that of other cytokine receptor complexes. IL-2Ralpha is composed of strand-swapped "sushi-like" domains, unlike the classical cytokine receptor fold. As a result of this domain swap, IL-2Ralpha uses a composite surface to dock into a groove on IL-2 that also serves as a binding site for antagonist drugs. With this complex, we now have representative structures for each class of hematopoietic cytokine receptor-docking modules.