RESUMO
A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Sítios de Ligação de Anticorpos , Receptores de Antígenos/química , Receptores de Antígenos/imunologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Humanos , ProteóliseRESUMO
Bispecific antibodies (bsAbs) have emerged as promising therapeutics. A bispecific diabody (bsDb) is a small bsAb consisting of two distinct chimeric single-chain components, with two possible arrangements of the domains. We previously reported the effect of domain order on the function of a humanized bsDb targeting the epidermal growth factor receptor (EGFR) on cancer cells, and CD3 on T cells. Notably, the co-localization of a T-cell receptor (TCR) with CD3 is bulky, potentially affecting the cross-linking ability of bsDbs, due to steric hindrance. Here, we constructed and evaluated humanized bsDbs, with different domain orders, targeting EGFR and CD16 on natural killer (NK) cells (hEx16-Dbs). We predicted minimal effects due to steric hindrance, as CD16 lacks accessory molecules. Interestingly, one domain arrangement displayed superior cytotoxicity in growth inhibition assays, despite similar cross-linking abilities for both domain orders tested. In hEx16-Dbs specifically, domain order might affect the agonistic activity of the anti-CD16 portion, which was supported by a cytokine production test, and likely contributed to the superiority of one of the hEx16-Dbs. Our results indicate that both the target antigen and mode of action of an antibody must be considered in the construction of highly functional bsAbs.
Assuntos
Anticorpos Biespecíficos/imunologia , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Receptores de IgG/imunologia , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Humanos , Imunoterapia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Receptores de IgG/antagonistas & inibidores , Linfócitos T/imunologiaRESUMO
Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues-which can be chemical conjugation sites-were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Fusão Gênica , Neoplasias/tratamento farmacológico , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Biologia Computacional , Escherichia coli/genética , Humanos , Modelos Moleculares , Muromonab-CD3/química , Muromonab-CD3/genética , Mutação , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Recent advances in biotechnology have enabled the generation of antibodies with high affinity for the surfaces of specific inorganic materials. Herein, we report the synthesis of functional materials from multiple nanomaterials by using a small bispecific antibody recombinantly constructed from gold-binding and ZnO-binding antibody fragments. The bispecific antibody-mediated spontaneous linkage of gold and ZnO nanoparticles forms a binary gold-ZnO nanoparticle composite membrane. The relatively low melting point of the gold nanoparticles and the solubility of ZnO in dilute acidic solution then allowed for the bottom-up synthesis of a nanoporous gold membrane by means of a low-energy, low-environmental-load protocol. The nanoporous gold membrane showed high catalytic activity for the reduction of p-nitrophenol to p-aminophenol by sodium borohydride. Here, we show the potential utility of nanoparticle pairing mediated by bispecific antibodies for the bottom-up construction of nanostructured materials from multiple nanomaterials.
Assuntos
Anticorpos Biespecíficos/química , Desenho de Fármacos , Nanopartículas/química , Nanotecnologia , Catálise , Dimerização , Ouro/química , Fragmentos de Imunoglobulinas/química , Membranas Artificiais , Porosidade , Óxido de Zinco/químicaRESUMO
In nanotechnological devices, mass transport can be initiated by pressure driven flow, diffusion or by employing molecular motors. As the scale decreases, molecular motors can be helpful as they are not limited by increased viscous resistance. Moreover, molecular motors can move against diffusion gradients and are naturally fitted for nanoscale transportation. Among motor proteins, kinesin has particular potential for lab-on-a-chip applications. It can be used for sorting, concentrating or as a mechanical sensor. When bound to a surface, kinesin motors propel microtubules in random directions, depending on their landing orientation. In order to circumvent this complication, the microtubule motion should be confined or guided. To this end, dielectrophoretically aligned multi-walled-carbon nanotubes (MWCNT) can be employed as nanotracks. In order to control more precisely the spatial repartition of the MWCNTs, a screening method has been implemented and tested. Polygonal patterns have been fabricated with the aim of studying the guiding and the microtubule displacement between MWCNT segments. Microtubules are observed to transfer between MWCNT segments, a prerequisite for the guiding of microtubules in MWCNT circuit-based biodevices. The effect of the MWCNT organization (crenellated or hexagonal) on the MT travel distance has been investigated as well.
Assuntos
Microtúbulos/química , Nanotubos de Carbono/química , Desenho de Equipamento , Cinesinas/metabolismo , Microscopia de Fluorescência , Peso MolecularRESUMO
As a complementary tool to nanofluidics, biomolecular-based transport is envisioned for nanotechnological devices. We report a new method for guiding microtubule shuttles on multi-walled carbon nanotube tracks, aligned by dielectrophoresis on a functionalized surface. In the absence of electric field and in fluid flow, alignment is maintained. The directed translocation of kinesin propelled microtubules has been investigated using fluorescence microscopy. To our knowledge, this is the first demonstration of microtubules gliding along carbon nanotubes.
RESUMO
Gliding of microtubule filaments on surfaces coated with the motor protein kinesin has potential applications for nano-scale devices. The ability to guide the gliding direction in three dimensions allows the fabrication of tracks of arbitrary geometry in space. Here, we achieve this by using kinesin-coated glass wires of micrometer diameter range. Unlike previous methods in which the guiding tracks are fixed on flat two-dimensional surfaces, the flexibility of glass wires in shape and size facilitates building in-vitro devices that have deformable tracks.
Assuntos
Vidro/química , Cinesinas/química , Microtúbulos/química , Microscopia de FluorescênciaRESUMO
Glioblastoma multiforme (GBM) remains an untreatable human brain malignancy. Despite promising preclinical studies using oncolytic herpes simplex virus (oHSV) vectors, efficacy in patients has been limited by inefficient virus replication in tumor cells. This disappointing outcome can be attributed in part to attenuating mutations engineered into these viruses to prevent replication in normal cells. Alternatively, retargeting of fully replication-competent HSV to tumor-associated receptors has the potential to achieve tumor specificity without impairment of oncolytic activity. Here, we report the establishment of an HSV retargeting system that relies on the combination of two engineered viral glycoproteins, gD and gB, to mediate highly efficient HSV infection exclusively through recognition of the abundantly expressed epidermal growth factor receptor (EGFR) on glioblastoma cells. We demonstrate efficacy in vitro and in a heterotopic tumor model in mice. Evidence for systemically administered virus homing to the tumor mass is presented. Treatment of orthotopic primary human GBM xenografts demonstrated prolonged survival with up to 73% of animals showing a complete response as confirmed by magnetic resonance imaging. Our study describes an approach to HSV retargeting that is effective in a glioma model and may be applicable to the treatment of a broad range of tumor types.
Assuntos
Receptores ErbB/metabolismo , Glioblastoma/terapia , Terapia Viral Oncolítica/métodos , Simplexvirus/genética , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Feminino , Vetores Genéticos , Células HT29 , Humanos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Plasmídeos , Recombinação Genética , Simplexvirus/fisiologia , Resultado do Tratamento , Células Vero , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The treatment strategy for iatrogenic intracranial vasospasm has not been established. We reported a rare case of stent-retriever use for the treatment of iatrogenic vasospasm after mechanical thrombectomy. The patient presented acute ischemic stroke due to the occlusion of the left middle cerebral artery (MCA). The patient underwent a successfully mechanical thrombectomy, however, severe stenosis was visualized in MCA, which was considered to be an iatrogenic mechanical vasospasm. This vasospasm was successfully treated with the Solitaire stent device, and the patient recovered from ischemic symptoms. A stent-retriever is a safe and effective treatment for iatrogenic vasospasm after mechanical thrombectomy.
Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Trombectomia/efeitos adversos , Resultado do Tratamento , Stents/efeitos adversos , Doença IatrogênicaRESUMO
T cells and natural killer (NK) cells are major effector cells recruited by cancer therapeutic bispecific antibodies; however, differences in the populations of these cells in individual tumors limit the general use of these antibodies. In the present study, trispecific antibodies were created, namely T cell and NK cell engagers (TaKEs), that recruit both T cells and NK cells. Notably, three Fcfused TaKEs were designed, TaKE1Fc, TaKE2Fc and TaKE3Fc, using variable fragments targeting the epidermal growth factor receptor on tumor cells, CD3 on T cells, and CD16 on NK cells. Among them, TaKE1Fc was predicted to form a circular tetrabodylike configuration and exhibited the highest production and greatest cancer growth inhibitory effects. TaKE1 was prepared from TaKE1Fc by digesting the Fc region for further functional evaluation. The resulting TaKE1 exhibited trispecificity via its ability to bind cancer cells, T cells and NK cells, as well as comparable or greater cancer growth inhibitory effects to those of two bispecific antibodies that recruit T cells and NK cells, respectively. A functional trispecific antibody with the potential to exert strong therapeutic effects independent of T cell and NK cell populations was developed.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Humanos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Células Matadoras Naturais , Neoplasias/terapia , Linfócitos TRESUMO
A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart's chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG-Fab2-type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG-Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG-Fab2 type bsAb will expand the bsAb production method.
Assuntos
Anticorpos Biespecíficos , Inteínas , Trans-Splicing , Processamento de Proteína , Imunoglobulina G/genéticaRESUMO
Prodrug design is a promising approach for reducing the off-target effects of therapeutic antibodies, particularly bispecific antibodies (bsAbs) that recruit T cells for activation; this design uses masking sequences that inhibit antibody binding until they reach the tumor microenvironment, where they are removed. In this study, we propose PAS, a polypeptide sequence composed of repeated Pro, Ala, and Ser residues, as a universal masking sequence. PAS has no specificity, but can inhibit antibody binding through steric hindrance caused by its large fluid dynamic radius and disordered structure; additionally, its length can be adjusted. We fused PAS to the N-terminus of an anti-CD3 single-chain variable fragment (scFv) and a bsAb, that targets both the epidermal growth factor receptor and CD3, via a recognition sequence cleaved by cancer-related proteases. PAS integration inhibited anti-CD3 scFv binding with higher efficacy than the epitope sequence, and the extent of inhibition was proportional to the length of the PAS sequence. For masked bsAbs, T cell-binding ability, cancer growth inhibition effects, and T cell activation effects were also reduced depending on the length of PAS and were fully restored upon removing PAS sequences using protease. The masking procedure using PAS was successfully applied to another scFv. The provision to adjust the masking effects of PAS by tuning its length, makes PAS fusion a valuable tool for the universal design of prodrug antibodies.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Pró-Fármacos , Anticorpos de Cadeia Única , Humanos , Linfócitos T , Pró-Fármacos/uso terapêutico , Neoplasias/tratamento farmacológico , Microambiente TumoralRESUMO
Although the development of small therapeutic antibodies is important, the affinity tags used for their purification often result in heterogeneous production and immunogenicity. In this study, we integrated Staphylococcus aureus protein A (SpA) binding ability into antibody fragments for convenient and tag-free purification. SpA affinity chromatography is used as a global standard purification method for conventional antibodies owing to its high binding affinity to the Fc region. SpA also has a binding affinity for some variable heavy domains (VH) classified in the VH3 subfamily. Through mutagenesis based on alignment and structural modeling results using the SpA-VH3 cocrystal structure, we integrated the SpA-binding ability into the anti-CD3 single-chain Fv. Furthermore, we applied this mutagenesis approach to more complicated small bispecific antibodies and successfully purified the antibodies using SpA affinity chromatography. The antibodies retained their biological function after purification. Integration of SpA-binding ability into conventional antibody fragments simplifies the purification and monitoring of the production processes and, thus, is an ideal strategy for accelerating the development of small therapeutic antibodies. Furthermore, because of its immunoactivity, the anti-CD3 variable region with SpA-binding ability is an effective building block for developing engineered cancer therapeutic antibodies without the Fc region.
Assuntos
Anticorpos Biespecíficos , Anticorpos de Cadeia Única , Anticorpos Biespecíficos/genética , MutagêneseRESUMO
Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.
Assuntos
Anticorpos Biespecíficos/imunologia , Antineoplásicos/metabolismo , Complexo CD3/imunologia , Receptores ErbB/imunologia , Anticorpos de Cadeia Única/imunologia , Linfócitos T/imunologia , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Citotoxinas/química , Citotoxinas/genética , Citotoxinas/imunologia , Citotoxinas/farmacologia , Humanos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacologiaRESUMO
Antibodies, with their high affinity and specificity, are widely utilized in the field of protein engineering, medicinal chemistry, and nanotechnology applications, and our recent studies have demonstrated the recognition and binding of antibody for the surface on inorganic material. In this study, we generated a high-affinity gold-binding antibody fragment by a combination of peptide-grafting and phage-display techniques and showed the availability of the material-binding fragment for one-pot functionalization of nanoparticles as interface molecules. After a gold-binding peptide sequence was grafted into one of the complementarity determining regions of a single variable domain of a heavy-chain camel antibody, a combinatorial library approach raised by 20 times the affinity of the peptide-grafted fragment. The high-affinity gold-binding fragment (E32) spontaneously adsorbed on gold nanoparticles, and consequently the nanoparticles formed a stable dispersion in a high-ionic-strength solution. Multivalent and bispecific antibodies constructed on the E32 platform by means of fusion technology functionalized gold nanoparticles in one pot, and these functionalized nanoparticles could be used to obtain surface plasmon resonance scattering images of cancer cells and to spontaneously link two different nanomaterials. Here, we propose the bispecific antibodies as convenient interface molecules in the nanosized world.
Assuntos
Anticorpos/imunologia , Camelus/imunologia , Ouro , Nanopartículas Metálicas , Sequência de Aminoácidos , Animais , Anticorpos/química , Dados de Sequência Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.
Assuntos
Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais Humanizados/metabolismo , Expressão Gênica , Imunoglobulina G/metabolismo , Ovário/enzimologia , Sialiltransferases/genética , Animais , Anticorpos Biespecíficos/genética , Anticorpos Monoclonais Humanizados/genética , Células CHO , Cricetinae/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Imunoglobulina G/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sialiltransferases/metabolismo , Regulação para Cima , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.
Assuntos
Complexo Antígeno-Anticorpo/química , Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Muramidase/química , Conformação Proteica , Animais , Asparagina , Galinhas , Cristalografia por Raios X , Ligação de Hidrogênio , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Modelos Moleculares , Estrutura Molecular , Muramidase/antagonistas & inibidores , Muramidase/genética , Mutagênese Sítio-Dirigida , TermodinâmicaRESUMO
Recent advances in molecular evolution technology enabled us to identify peptides and antibodies with affinity for inorganic materials. In the field of nanotechnology, the use of the functional peptides and antibodies should aid the construction of interface molecules designed to spontaneously link different nanomaterials; however, few material-binding antibodies, which have much higher affinity than short peptides, have been identified. Here, we generated high affinity antibodies from material-binding peptides by integrating peptide-grafting and phage-display techniques. A material-binding peptide sequence was first grafted into an appropriate loop of the complementarity determining region (CDR) of a camel-type single variable antibody fragment to create a low affinity material-binding antibody. Application of a combinatorial library approach to another CDR loop in the low affinity antibody then clearly and steadily promoted affinity for a specific material surface. Thermodynamic analysis demonstrated that the enthalpy synergistic effect from grafted and selected CDR loops drastically increased the affinity for material surface, indicating the potential of antibody scaffold for creating high affinity small interface units. We show the availability of the construction of antibodies by integrating graft and evolution technology for various inorganic materials and the potential of high affinity material-binding antibodies in biointerface applications.
Assuntos
Anticorpos , Afinidade de Anticorpos , Peptídeos/imunologia , Engenharia de Proteínas/métodos , Adsorção , Óxido de Alumínio/química , Óxido de Alumínio/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/imunologia , Cobalto/química , Cobalto/imunologia , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Nanotecnologia , Óxidos/química , Óxidos/imunologia , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Conformação Proteica , Propriedades de Superfície , Termodinâmica , Óxido de Zinco/química , Óxido de Zinco/imunologiaRESUMO
We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy.
Assuntos
Anticorpos Biespecíficos/toxicidade , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Antígenos/imunologia , Complexo CD3/imunologia , Divisão Celular/efeitos dos fármacos , Cromatografia em Gel , Dimerização , Receptores ErbB/imunologia , Humanos , Células Matadoras Ativadas por Linfocina/efeitos dos fármacos , Células Matadoras Ativadas por Linfocina/imunologia , Cinética , Luz , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Dobramento de Proteína , Proteínas Recombinantes/uso terapêutico , Espalhamento de Radiação , Software , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
A plasmonic biosensor chip of silver-coated PMMA grating with a zinc oxide (ZnO) overlayer is fabricated for surface plasmon field-enhanced fluorescence (SPF) detection of Cy5-labeled green fluorescent protein (GFP). A bispecific antibody (anti-GFP x anti-ZnO antibody) prepared in our lab is densely immobilized on the sensor chip for GFP detection. The sensitivity of the plasmonic biosensors is improved due to densely packed antibodies and ZnO-coating that suppresses nonspecific protein adsorption and fluorescent quenching. With the ZnO-coated plasmonic chip, Cy5-labeled GFP of 10 pM can be detected through SPF. This sensitivity is 100 higher compared with the normal fluorescent detection on a ZnO-coated glass slide.