RESUMO
This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm -19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.
Assuntos
Proteínas de Bactérias/química , Domínio de Fibronectina Tipo III , Cloreto de Sódio/química , Thermoanaerobacter/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Temperatura Alta , Simulação de Dinâmica Molecular , Domínios Proteicos , Estabilidade Proteica , Thermoanaerobacter/genéticaRESUMO
Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure-function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indications. In this review, our basic understanding of the antibody structure is described along with how that knowledge has leveraged the engineering of antibody and antibody-related therapeutics having the appropriate antigen affinity, effector function, and biophysical properties. The platforms examined include the development of antibodies, antibody fragments, bispecific antibody, and antibody fusion products, whose efficacy and manufacturability can be improved via humanization, affinity modulation, and stability enhancement. We also review the design and selection of binding arms, and avidity modulation. Different strategies of preparing bispecific and multispecific molecules for an array of therapeutic applications are included.
RESUMO
JAK3-activating mutations are commonly seen in chronic or acute hematologic malignancies affecting the myeloid, megakaryocytic, lymphoid, and natural killer (NK) cell compartment. Overexpression models of mutant JAK3 or pharmacologic inhibition of its kinase activity have highlighted the role that these constitutively activated mutants play in the T-cell, NK cell, and megakaryocytic lineages, but to date, the functional impact of JAK3 mutations at an endogenous level remains unknown. Here, we report a JAK3A572V knockin mouse model and demonstrate that activated JAK3 leads to a progressive and dose-dependent expansion of CD8+ T cells in the periphery before colonization of the bone marrow. This phenotype is dependent on the γc chain of cytokine receptors and presents several features of the human leukemic form of cutaneous T-cell lymphoma (L-CTCL), including skin involvements. We also showed that the JAK3A572V-positive malignant cells are transplantable and phenotypically heterogeneous in bone marrow transplantation assays. Interestingly, we revealed that activated JAK3 functionally cooperates with partial trisomy 21 in vivo to enhance the L-CTCL phenotype, ultimately leading to a lethal and fully penetrant disorder. Finally, we assessed the efficacy of JAK3 inhibition and showed that CTCL JAK3A572V-positive T cells are sensitive to tofacitinib, which provides additional preclinical insights into the use of JAK3 inhibitors in these disorders. Altogether, this JAK3A572V knockin model is a relevant new tool for testing the efficacy of JAK inhibitors in JAK3-related hematopoietic malignancies.
Assuntos
Cromossomos de Mamíferos/metabolismo , Neoplasias Hematológicas/metabolismo , Janus Quinase 3/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Mutação de Sentido Incorreto , Neoplasias Experimentais/metabolismo , Trissomia , Substituição de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Cromossomos de Mamíferos/genética , Técnicas de Introdução de Genes , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Janus Quinase 3/genética , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/patologia , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologiaRESUMO
CD19 is a transmembrane protein expressed on malignant B cells, but not in other lineages or other tissues, which makes it an attractive target for monoclonal antibody-mediated immunotherapy. Anti-CD19 antibody B43 was utilized in a bispecific T-cell engager (BiTE) blinatumomab that demonstrated potency for the treatment of relapsed acute lymphoblastic leukemia. To gain insight into the mechanism of action of the antibody, the crystal structure of B43 Fab was determined in complex with CD19 and in the unbound form. The structure revealed the binding epitope, explained the lack of cross-reactivity toward non-human species, and suggested the key-and-lock mechanism of antigen recognition. Most unexpectedly, the structure revealed a unique molecular topology of CD19. Rather than a tandem of c-type immunoglobulin folds predicted from the amino acid sequence, the extracellular domain of CD19 exhibits an elongated ß-sandwich formed by two immunoglobulin folds by swapping their C-terminal halves. This is the first structure of CD19, which has no sequence homologs.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD19/química , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
In therapeutic antibody discovery and early development, mice and cynomolgus monkey are used as animal models to assess toxicity, efficacy and other properties of candidate molecules. As more candidate antibodies are based on human immunoglobulin (IgG) subclasses, many strategies are pursued to simulate the human system in the test animal. However, translation rate from a successful preclinical trial to an approved drug is extremely low. This may partly be due to differences in interaction of human IgG based candidate molecules to endogenous Fcγ receptors of model animals in comparison to those of human Fcγ receptors. In this study, we compare binding characteristics of human IgG subclasses commonly used in drug development (IgG1, IgG2, IgG4) and their respective Fc silent versions (IgG1σ, IgG2σ, IgG4 PAA) to human, mouse, and cynomolgus monkey Fcγ receptors. To control interactions between Fab and Fc domains, the test IgGs all have the same variable region sequences. We found distinct variations of interaction of human IgG subclasses to model animal Fcγ receptors in comparison to their human counterparts. Particularly, cynomolgus monkey Fcγ receptors showed consistently tighter binding to human IgGs than human Fcγ receptors. Moreover, the presumably Fc silent human IgG4 PAA framework bound to cynomolgus monkey FcγRI with nanomolar affinity while only very weak binding was observed for the human FcγRI. Our results highlighted the need for a thorough in vitro affinity characterization of candidate IgGs against model animal Fcγ receptors and careful design of preclinical studies.
Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Isotipos de Imunoglobulinas/metabolismo , Imunoterapia/métodos , Receptores de IgG/metabolismo , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Região Variável de Imunoglobulina/genética , Macaca fascicularis , Camundongos , Ligação Proteica , Pesquisa Translacional BiomédicaRESUMO
The homeostatic chemokine CCL17, also known as thymus and activation regulated chemokine (TARC), has been associated with various diseases such as asthma, idiopathic pulmonary fibrosis, atopic dermatitis and ulcerative colitis. Neutralization of CCL17 by antibody treatment ameliorates the impact of disease by blocking influx of T cells. Monoclonal antibody M116 derived from a combinatorial library shows potency in neutralizing CCL17-induced signaling. To gain insight into the structural determinants of antigen recognition, the crystal structure of M116 Fab was determined in complex with CCL17 and in the unbound form. Comparison of the structures revealed an unusual induced-fit mechanism of antigen recognition that involves cis-trans isomerization in two CDRs. The structure of the CCL17-M116 complex revealed the antibody binding epitope, which does not overlap with the putative receptor epitope, suggesting that the current model of chemokine-receptor interactions, as observed in the CXCR4-vMIP-II system, may not be universal.
RESUMO
The Protein Data Bank (PDB) is the global archive for structural information on macromolecules, and a popular resource for researchers, teachers, and students, amassing more than one million unique users each year. Crystallographic structure models in the PDB (more than 100,000 entries) are optimized against the crystal diffraction data and geometrical restraints. This process of crystallographic refinement typically ignored hydrogen bond (H-bond) distances as a source of information. However, H-bond restraints can improve structures at low resolution where diffraction data are limited. To improve low-resolution structure refinement, we present methods for deriving H-bond information either globally from well-refined high-resolution structures from the PDB-REDO databank, or specifically from on-the-fly constructed sets of homologous high-resolution structures. Refinement incorporating HOmology DErived Restraints (HODER), improves geometrical quality and the fit to the diffraction data for many low-resolution structures. To make these improvements readily available to the general public, we applied our new algorithms to all crystallographic structures in the PDB: using massively parallel computing, we constructed a new instance of the PDB-REDO databank (https://pdb-redo.eu). This resource is useful for researchers to gain insight on individual structures, on specific protein families (as we demonstrate with examples), and on general features of protein structure using data mining approaches on a uniformly treated dataset.
Assuntos
Biologia Computacional/métodos , Proteínas/química , Algoritmos , Cristalografia por Raios X , Mineração de Dados , Bases de Dados de Proteínas , Ligação de Hidrogênio , Modelos Moleculares , Conformação ProteicaRESUMO
Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.
Assuntos
Anticorpos Monoclonais Humanizados/química , Afinidade de Anticorpos/fisiologia , Tromboplastina/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Regiões Determinantes de Complementaridade/química , Humanos , Camundongos , Modelos Moleculares , Engenharia de Proteínas/métodosRESUMO
The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Fc/imunologia , Proteína Estafilocócica A/imunologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/imunologia , Proteínas Mutantes/metabolismo , Mutação , Ligação Proteica/imunologia , Domínios Proteicos , Receptores Fc/metabolismo , Proteína Estafilocócica A/metabolismoRESUMO
Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signaling pathways. One engineering strategy is to identify Fc mutations that facilitate antibody multimerization on the cell surface directly. From the analyses of the crystal packing of IgG1 structures, we identified a novel set of Fc mutations, T437R and K248E, that facilitated antibody multimerization upon binding to antigens on cell surface. In a NF-κB reporter assay, the engineered T437R/K248E mutations could facilitate enhanced agonism of an anti-OX40 antibody without the dependence on FcγRIIB crosslinking. Nonetheless, the presence of cells expressing FcγRIIB could facilitate a boost of the agonism of the engineered antibody with mutations on IgG1 Fc, but not on the silent IgG2σ Fc. The Fc engineered antibody also showed enhanced effector functions, including antibody-dependent cell-meditated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity, depending on the IgG subtypes. Also, the engineered antibodies showed normal FcRn binding and pharmacokinetic profiles in mice. In summary, this study elucidated a novel Fc engineering approach to promote antibody multimerization on a cell surface, which could enhance agonism and improve effector function for anti-TNFR antibodies as well as other therapeutic antibodies.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Imunoterapia/métodos , Engenharia de Proteínas/métodos , Receptores OX40/agonistas , Animais , Citotoxicidade Celular Dependente de Anticorpos , Humanos , Camundongos , MutaçãoRESUMO
BACKGROUND: ß-Amyloid (Aß) peptide is believed to play a pivotal role in the development of Alzheimer's disease. Passive immunization with anti-Aß monoclonal antibodies may facilitate the clearance of Aß in the brain and may thus prevent the downstream pathology. Antibodies targeting the immunodominant N-terminal epitope of Aß and capable of binding both the plaques and soluble species have been most efficacious in animal models. Structural studies of such antibodies with bound Aß peptides provided the basis for understanding the mechanisms of action and the differences in potency. To gain further insight into the structural determinants of antigen recognition and the preferential Aß conformations, we determined the crystal structure of murine antibody C706 in complex with the N-terminal Aß 1-16 peptide sequence. METHODS: The antigen-binding fragment of C706 was expressed in HEK293 cells and was crystallized in complex with the Aß peptide. The X-ray structure was determined at 1.9-Å resolution. RESULTS: The binding epitope of C706 is centered on residues Arg5 and His6, which provide the majority of interactions. Unlike most antibodies, C706 recognizes a coiled rather than extended conformation of Aß. CONCLUSIONS: Comparison with other antibodies targeting the N-terminal section of Aß suggests that the conformation of the bound peptide may be linked to the immunization protocol and may reflect the preference for the extended conformation in the context of a longer Aß peptide as opposed to the coiled conformation in the isolated short peptide.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Animais , Especificidade de Anticorpos , Cristalografia por Raios X , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Camundongos , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes de Fusão/químicaRESUMO
Tissue factor (TF) initiates the extrinsic pathway of blood coagulation through sequential binding and activation of coagulation factors VII (FVII) and X (FX). In addition, through activation of G-protein-coupled protease activated receptors (PARs) TF induces cell signaling that is related to cancer, angiogenesis and inflammation. Monoclonal antibodies (mAbs) proved to be a useful tool for studying the interplay between TF signaling and coagulation. MAb 10H10 is unique in that it blocks the signaling pathway and thus inhibits angiogenesis and tumor growth without interfering with coagulation. It was also presumed that mAb 10H10 recognizes the cryptic pool of TF devoid of procoagulant activity. The crystal structure of the 10H10 Fab was determined in the absence and in the presence of the TF extracellular domain (ECD). The structures show that the antibody operates by the key-and-lock mechanism causing no conformational changes in either Fab or TF. The TF:10H10 interface is extensive and includes five segments of TF in both the N-terminal and C-terminal domains of the ECD. Neither the known epitope of FVII, nor the putative epitope of FX overlaps with the 10H10 binding site. The 10H10 epitope points to the likely location of the PAR2 exosite. It is also the hypothetical site of TF interaction with integrins that may play a major role in the encryption-decryption process.
Assuntos
Anticorpos Monoclonais/metabolismo , Epitopos/metabolismo , Transdução de Sinais , Tromboplastina/química , Tromboplastina/metabolismo , Animais , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
CD27 is a T-cell and B-cell co-stimulatory glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is dependent on the availability of the TNF-like ligand CD70. Therapeutic approaches to treating autoimmune diseases and cancers with antagonistic and agonistic anti-CD27 monoclonal antibodies (mAbs), respectively, have recently been developed. Mouse anti-human CD27 mAb 2177 shows potency in neutralizing CD70-induced signaling; however, it does not block the binding of soluble CD70. To provide insight into the mechanism of action of the mAb, the crystal structure of the CD27 extracellular domain in complex with the Fab fragment of mAb 2177 was determined at 1.8â Å resolution. CD27 exhibits the assembly of cysteine-rich domains characteristic of the TNF receptor superfamily. The structure reveals a unique binding site of mAb 2177 at the edge of the receptor molecule, which allows the mAb to sterically block the cell-bound form of CD70 from reaching CD27 while leaving the ligand epitope clear. This mode of action suggests a potential dual use of mAb 2177 either as an antagonist or as an agonist.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Complexo Antígeno-Anticorpo/química , Ligante CD27/química , Fragmentos Fab das Imunoglobulinas/química , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/química , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Complexo Antígeno-Anticorpo/genética , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Ligante CD27/genética , Ligante CD27/imunologia , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Células Sf9 , Spodoptera , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding affinity and ability to block NF-κB signaling, however their clearance rates in monkeys are very different. The pharmacokinetics profiles could be epitope dependent. To identify the epitopes, we determined the crystal structure of the ternary complex between CD27 and the Fab fragments of these non-competing antibodies. The structure reveals the binding modes of the antibodies suggesting that their mechanisms of action are distinctly different and provides a possible explanation of the in vivo data.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos , Ligante CD27/química , Ligante CD27/imunologia , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Meia-Vida , Humanos , Macaca fascicularis , CamundongosRESUMO
Designed ankyrin repeat proteins (DARPin®) are artificial non-immunoglobulin binding proteins with potential applications as therapeutic molecules. DARPin 6G9 binds interleukin-13 with high affinity and blocks the signaling pathway and as such is promising for the treatment of asthma and other atopic diseases. The crystal structures of DARPin 6G9 in the unbound form and in complex with IL-13 were determined at high resolution. The DARPin competes for the same epitope as the IL-13 receptor chain 13Rα1 but does not interfere with the binding of the other receptor chain, IL-4Rα. Analysis of multiple copies of the DARPin molecule in the crystal indicates the conformational instability in the N-terminal cap that was predicted from molecular dynamics simulations. Comparison of the DARPin structures in the free state and in complex with IL-13 reveals a concerted movement of the ankyrin repeats upon binding resulted in the opening of the binding site. The induced-fit mode of binding employed by DARPin 6G9 is very unusual for DARPins since they were designed as particularly stable and rigid molecules. This finding shows that DARPins can operate by various binding mechanisms and suggests that some flexibility in the scaffold may be an advantage.
Assuntos
Repetição de Anquirina , Anticorpos/química , Anticorpos/imunologia , Interleucina-13/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/genética , Cristalografia por Raios X , Humanos , Macaca fascicularis , Modelos Moleculares , Engenharia de Proteínas , Estrutura Secundária de ProteínaRESUMO
Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically "silent".
RESUMO
Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload. Overall, 26 of the 94 positions were identified as ideal for cysteine modification, conjugation and drug delivery. Conjugation-tolerant positions were mapped onto a crystal structure of the Centyrin, providing a structural context for interpretation of the mutagenesis experiment and providing a foundation for a Centyrin-targeted delivery platform.
Assuntos
Portadores de Fármacos/química , Fibronectinas/química , Engenharia de Proteínas , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cristalografia por Raios X , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacologia , Receptores ErbB/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Humanos , Maleimidas/química , Modelos Moleculares , Conformação Proteica em Folha beta , Domínios ProteicosRESUMO
The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment.
Assuntos
Anticorpos/genética , Anticorpos/uso terapêutico , Engenharia de Proteínas/métodos , Animais , Anticorpos/química , Anticorpos/metabolismo , Humanos , Distribuição TecidualRESUMO
To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths. The longer CDRs with tandem glycines or serines have more conformational diversity than the others. CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. The stem regions of 14 of the variant pairs are in the 'kinked' conformation, and only 2 are in the extended conformation. The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts.