RESUMO
Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.
Assuntos
Anticorpos , Região Variável de Imunoglobulina , Região Variável de Imunoglobulina/química , Fragmentos de ImunoglobulinasRESUMO
Generation of bispecific antibodies (BsAbs) having two unique Fab domains requires heterodimerization of the two heavy chains and pairing of each heavy chain with its cognate light chain. An alternative bispecific scaffold (Bipod) comprising an scFv and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. However, unpredictable levels of chain expression and scFv-induced aggregation can complicate purification and reduce the yield of desired Bipod. Here, we describe a high-throughput method for generation of Bipods based on protein A and CH1 domain affinity capture. This method exploits over-expression of the scFv chain to maximize heterodimer yield. Bipods purified by this method have purity suitable for cell-based functional assays and in vivo studies.
Assuntos
Anticorpos Biespecíficos/química , Fragmentos Fab das Imunoglobulinas/química , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/química , Animais , Produtos Biológicos/uso terapêutico , Células CHO , Cricetulus , DNA/química , Dimerização , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Epitopos/química , Humanos , Imunoglobulina G/genética , Imunossupressores/uso terapêutico , Mutação , Neoplasias/terapia , Plasmídeos , Domínios ProteicosRESUMO
CD33 is expressed in 90% of patients with acute myeloid leukemia (AML), and its extracellular portion consists of a V domain and a C2 domain. A recent study showed that a single nucleotide polymorphism (SNP), rs12459419 (C > T), results in the reduced expression of V domain-containing CD33 and limited efficacy of V domain-binding anti-CD33 antibodies. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33 and to CD3, to induce T-cell recruitment and CD33+ tumor cell cytotoxicity independently of their SNP genotype status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro along with T-cell activation and cytokine release. JNJ-67571244 also exhibited statistically significant antitumor activity in vivo in established disseminated and subcutaneous mouse models of human AML. Furthermore, this antibody depletes CD33+ blasts in AML patient blood samples with concurrent T-cell activation. JNJ-67571244 also cross-reacts with cynomolgus monkey CD33 and CD3, and dosing of JNJ-67571244 in cynomolgus monkeys resulted in T-cell activation, transient cytokine release, and sustained reduction in CD33+ leukocyte populations. JNJ-67571244 was well tolerated in cynomolgus monkeys up to 30 mg/kg. Lastly, JNJ-67571244 mediated efficient cytotoxicity of cell lines and primary samples regardless of their SNP genotype status, suggesting a potential therapeutic benefit over other V-binding antibodies. JNJ-67571244 is currently in phase 1 clinical trials in patients with relapsed/refractory AML and high-risk myelodysplastic syndrome.
Assuntos
Leucemia Mieloide Aguda , Linfócitos T , Animais , Domínios C2 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Macaca fascicularis , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Linfócitos T/metabolismoRESUMO
Alternative scaffold molecules represent a class of proteins important to the study of protein design and mechanisms of protein-protein interactions, as well as for the development of therapeutic proteins. Here, we describe the generation of a library built upon the framework of a consensus FN3 domain sequence resulting in binding proteins we call Centyrins. This new library employs diversified positions within the C-strand, CD-loop, F-strand and FG-loop of the FN3 domain. CIS display was used to select high-affinity Centyrin variants against three targets; c-MET, murine IL-17A and rat TNFα and scanning mutagenesis studies were used to define the positions of the library most important for target binding. Contributions from both the strand and loop positions were noted, although the pattern was different for each molecule. In addition, an affinity maturation scheme is described that resulted in a significant improvement in the affinity of one selected Centyrin variant. Together, this work provides important data contributing to our understanding of potential FN3 binding interfaces and a new tool for generating high-affinity scaffold molecules.
Assuntos
Biblioteca Gênica , Ligação Proteica , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Interleucina-17 , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ratos , Alinhamento de Sequência , Fator de Necrose Tumoral alfaRESUMO
The use of consensus design to produce stable proteins has been applied to numerous structures and classes of proteins. Here, we describe the engineering of novel FN3 domains from two different proteins, namely human fibronectin and human tenascin-C, as potential alternative scaffold biotherapeutics. The resulting FN3 domains were found to be robustly expressed in Escherichia coli, soluble and highly stable, with melting temperatures of 89 and 78°C, respectively. X-ray crystallography was used to confirm that the consensus approach led to a structure consistent with the FN3 design despite having only low-sequence identity to natural FN3 domains. The ability of the Tenascin consensus domain to withstand mutations in the loop regions connecting the ß-strands was investigated using alanine scanning mutagenesis demonstrating the potential for randomization in these regions. Finally, rational design was used to produce point mutations that significantly increase the stability of one of the consensus domains. Together our data suggest that consensus FN3 domains have potential utility as alternative scaffold therapeutics.
Assuntos
Fibronectinas/química , Tenascina/química , Sequência de Aminoácidos , Sequência Consenso , Cristalografia por Raios X , Escherichia coli , Fibronectinas/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tenascina/genéticaRESUMO
Despite intensive research, there are very few reagents with which to modulate and dissect the mRNA splicing pathway. Here, we describe a novel approach to identify such tools, based on detection of the exon junction complex (EJC), a unique molecular signature that splicing leaves on mRNAs. We developed a high-throughput, splicing-dependent EJC immunoprecipitation (EJIPT) assay to quantitate mRNAs spliced from biotin-tagged pre-mRNAs in cell extracts, using antibodies to EJC components Y14 and eukaryotic translation initiation factor 4aIII (eIF4AIII). Deploying EJIPT we performed high-throughput screening (HTS) in conjunction with secondary assays to identify splicing inhibitors. We describe the identification of 1,4-naphthoquinones and 1,4-heterocyclic quinones with known anticancer activity as potent and selective splicing inhibitors. Interestingly, and unlike previously described small molecules, most of which target early steps, our inhibitors represented by the benzothiazole-4,7-dione, BN82685, block the second of two trans-esterification reactions in splicing, preventing the release of intron lariat and ligation of exons. We show that BN82685 inhibits activated spliceosomes' elaborate structural rearrangements that are required for second-step catalysis, allowing definition of spliceosomes stalled in midcatalysis. EJIPT provides a platform for characterization and discovery of splicing and EJC modulators.
Assuntos
Benzoquinonas/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Naftoquinonas/farmacologia , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Spliceossomos/efeitos dos fármacos , Tiazóis/farmacologia , Biotina/química , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação/métodos , Precursores de RNA/química , Precursores de RNA/metabolismo , Spliceossomos/metabolismoRESUMO
Pre-mRNA splicing deposits multi-protein complexes, termed exon junction complexes (EJCs), on mRNAs near exon-exon junctions. The core of EJC consists of four proteins, eIF4AIII, MLN51, Y14 and Magoh. Y14 is a nuclear protein that can shuttle between the nucleus and the cytoplasm, and binds specifically to Magoh. Here we delineate a Y14 nuclear localization signal that also confers its nuclear export, which we name YNS. We further identified a 12-amino-acid peptide near Y14's carboxyl terminus that is required for its association with spliced mRNAs, as well as for Magoh binding. Furthermore, the Y14 mutants, which are deficient in binding to Magoh, could still be localized to the nucleus, suggesting the existence of both the nuclear import pathway and function for Y14 unaccompanied by Magoh.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Éxons , Células HeLa , Humanos , Dados de Sequência Molecular , Sinais de Exportação Nuclear , Transporte Proteico , Proteínas de Ligação a RNA/químicaRESUMO
Messenger RNAs produced by splicing are translated more efficiently than those produced from similar intronless precursor mRNAs (pre-mRNAs). The exon-junction complex (EJC) probably mediates this enhancement; however, the specific link between the EJC and the translation machinery has not been identified. The EJC proteins Y14 and magoh remain bound to spliced mRNAs after their export from the nucleus to the cytoplasm and are removed only when these mRNAs are translated. Here we show that PYM, a 29-kDa protein that binds the Y14-magoh complex in the cytoplasm, also binds, via a separate domain, to the small (40S) ribosomal subunit and the 48S preinitiation complex. Furthermore, PYM knockdown reduces the translation efficiency of a reporter protein produced from intron-containing, but not intronless, pre-mRNA. We suggest that PYM functions as a bridge between EJC-bearing spliced mRNAs and the translation machinery to enhance translation of the mRNAs.
Assuntos
Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Éxons , Biossíntese de Proteínas , Splicing de RNA , RNA Mensageiro/genética , Ribossomos/metabolismo , Linhagem Celular , Humanos , Imunoprecipitação , Ligação ProteicaRESUMO
The exon junction complex (EJC) is a protein complex that assembles near exon-exon junctions of mRNAs as a result of splicing. EJC proteins play important roles in postsplicing events including mRNA export, cytoplasmic localization, and nonsense-mediated decay. Recent evidence suggests that mRNA translation is also influenced by the splicing history of the transcript. Here we identify eIF4A3, a DEAD-box RNA helicase and a member of the eIF4A family of translation initiation factors, as a novel component of the EJC. We show that eIF4A3 associates preferentially with nuclear complexes containing the EJC proteins magoh and Y14. Furthermore, eIF4A3, but not the highly related eIF4A1 or eIF4A2, preferentially associates with spliced mRNA. In vitro splicing and mapping experiments demonstrate that eIF4A3 binds mRNAs at the position of the EJC. Using monoclonal antibodies, we show that eIF4A3 is found in the nucleus whereas eIF4A1 and eIF4A2 are found in the cytoplasm. Thus, eIF4A3 likely provides a splicing-dependent influence on the translation of mRNAs.
Assuntos
Núcleo Celular/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Proteínas de Transporte Nucleocitoplasmático , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Núcleo Celular/imunologia , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/imunologia , Éxons/fisiologia , Humanos , Substâncias Macromoleculares , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Splicing of pre-mRNA in eukaryotes imprints the resulting mRNA with a specific multiprotein complex, the exon-exon junction complex (EJC), at the sites of intron removal. The proteins of the EJC, Y14, Magoh, Aly/REF, RNPS1, Srm160, and Upf3, play critical roles in postsplicing processing, including nuclear export and cytoplasmic localization of the mRNA, and the nonsense-mediated mRNA decay (NMD) surveillance process. Y14 and Magoh are of particular interest because they remain associated with the mRNA in the same position after its export to the cytoplasm and require translation of the mRNA for removal. This tenacious, persistent, splicing-dependent, yet RNA sequence-independent, association suggests an important signaling function and must require distinct structural features for these proteins. RESULTS: We describe the high-resolution structure and biochemical properties of the highly conserved human Y14 and Magoh proteins. Magoh has an unusual structure comprised of an extremely flat, six-stranded anti-parallel beta sheet packed against two helices. Surprisingly, Magoh binds with high affinity to the RNP motif RNA binding domain (RBD) of Y14 and completely masks its RNA binding surface. CONCLUSIONS: The structure and properties of the Y14-Magoh complex suggest how the pre-mRNA splicing machinery might control the formation of a stable EJC-mRNA complex at splice junctions.