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1.
Proc Natl Acad Sci U S A ; 121(22): e2319029121, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38781214

RESUMO

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.


Assuntos
Peptídeos , Humanos , Peptídeos/imunologia , Peptídeos/química , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Complexo Principal de Histocompatibilidade/imunologia , Haptenos/imunologia , Ligação Proteica , Microscopia Crioeletrônica
2.
Cell Rep ; 42(11): 113374, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37938973

RESUMO

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of ß-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.


Assuntos
Glioblastoma , Humanos , Glioblastoma/patologia , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
3.
Proc Natl Acad Sci U S A ; 120(28): e2302485120, 2023 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-37399416

RESUMO

The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of KRAS Asp12, a feature that closely resembles that of a small-molecule inhibitor, MTRX1133. The monobody also directly interacted with H95, a residue not conserved in RAS isoforms. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a monobody generated hundreds of functional and nonfunctional single-point mutants, which identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results further illustrate the plasticity of the S-II pocket, which may be exploited for the design of next-generation KRAS(G12D)-selective inhibitors.


Assuntos
Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Mutação , Transformação Celular Neoplásica/genética , Carcinogênese , Neoplasias Pancreáticas/genética
4.
bioRxiv ; 2023 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-37131623

RESUMO

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD + degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD + . Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)-key downstream effectors of LKB1- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCE: Loss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

5.
Cancer Discov ; 13(1): 132-145, 2023 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-36250888

RESUMO

Intracellular oncoproteins can be inhibited with targeted therapy, but responses are not durable. Immune therapies can be curative, but most oncogene-driven tumors are unresponsive to these agents. Fragments of intracellular oncoproteins can act as neoantigens presented by the major histocompatibility complex (MHC), but recognizing minimal differences between oncoproteins and their normal counterparts is challenging. We have established a platform technology that exploits hapten-peptide conjugates generated by covalent inhibitors to create distinct neoantigens that selectively mark cancer cells. Using the FDA-approved covalent inhibitors sotorasib and osimertinib, we developed "HapImmune" antibodies that bind to drug-peptide conjugate/MHC complexes but not to the free drugs. A HapImmune-based bispecific T-cell engager selectively and potently kills sotorasib-resistant lung cancer cells upon sotorasib treatment. Notably, it is effective against KRASG12C-mutant cells with different HLA supertypes, HLA-A*02 and A*03/11, suggesting loosening of MHC restriction. Our strategy creates targetable neoantigens by design, unifying targeted and immune therapies. SIGNIFICANCE: Targeted therapies against oncoproteins often have dramatic initial efficacy but lack durability. Immunotherapies can be curative, yet most tumors fail to respond. We developed a generalizable technology platform that exploits hapten-peptides generated by covalent inhibitors as neoantigens presented on MHC to enable engineered antibodies to selectively kill drug-resistant cancer cells. See related commentary by Cox et al., p. 19. This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Neoplasias Pulmonares , Complexo Principal de Histocompatibilidade , Humanos , Neoplasias Pulmonares/patologia , Antígenos de Histocompatibilidade Classe I , Antígenos de Neoplasias , Peptídeos/farmacologia , Antígenos de Histocompatibilidade , Haptenos
6.
J Mol Biol ; 434(5): 167402, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34958778

RESUMO

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain of human CD99, and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.


Assuntos
Antígeno 12E7/imunologia , Anticorpos , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Antígeno 12E7/antagonistas & inibidores , Adulto , Anticorpos/química , Anticorpos/imunologia , Anticorpos/uso terapêutico , Criança , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia
7.
Nature ; 595(7867): 404-408, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34163073

RESUMO

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.


Assuntos
Proteínas Musculares/genética , Mutação , Síndromes Miastênicas Congênitas/tratamento farmacológico , Síndromes Miastênicas Congênitas/genética , Envelhecimento , Agrina/genética , Agrina/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/imunologia , Modelos Animais de Doenças , Feminino , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Masculino , Camundongos , Terapia de Alvo Molecular , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Síndromes Miastênicas Congênitas/imunologia , Fosforilação , Fosfotirosina/genética , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Receptores Proteína Tirosina Quinases/agonistas , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Recidiva , Sinapses/metabolismo
8.
Nat Commun ; 12(1): 2656, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976200

RESUMO

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.


Assuntos
Anticorpos Monoclonais/farmacologia , Proteínas Mutantes/antagonistas & inibidores , Mutação , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Camundongos Nus , Proteínas Mutantes/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
9.
J Exp Med ; 218(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045063

RESUMO

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Ductal Pancreático/imunologia , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/imunologia , Mutação de Sentido Incorreto , Neoplasias Pancreáticas/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Microambiente Tumoral/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
10.
J Biosci Bioeng ; 126(2): 153-161, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29548844

RESUMO

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies.


Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/metabolismo , Testes Imunológicos de Citotoxicidade/métodos , Ensaios de Triagem em Larga Escala/métodos , Anticorpos Biespecíficos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Vacinas Anticâncer/análise , Vacinas Anticâncer/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoterapia , Células Tumorais Cultivadas
11.
Proc Natl Acad Sci U S A ; 111(40): 14631-6, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-25246589

RESUMO

Investigations into the genomic landscape of histone modifications in heterochromatic regions have revealed histone H3 lysine 9 dimethylation (H3K9me2) to be important for differentiation and maintaining cell identity. H3K9me2 is associated with gene silencing and is organized into large repressive domains that exist in close proximity to active genes, indicating the importance of maintenance of proper domain structure. Here we show that nickel, a nonmutagenic environmental carcinogen, disrupted H3K9me2 domains, resulting in the spreading of H3K9me2 into active regions, which was associated with gene silencing. We found weak CCCTC-binding factor (CTCF)-binding sites and reduced CTCF binding at the Ni-disrupted H3K9me2 domain boundaries, suggesting a loss of CTCF-mediated insulation function as a potential reason for domain disruption and spreading. We furthermore show that euchromatin islands, local regions of active chromatin within large H3K9me2 domains, can protect genes from H3K9me2-spreading-associated gene silencing. These results have major implications in understanding H3K9me2 dynamics and the consequences of chromatin domain disruption during pathogenesis.


Assuntos
Cromatina/metabolismo , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Níquel/farmacologia , Acetilação , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Brônquios/citologia , Fator de Ligação a CCCTC , Linhagem Celular , Cromatina/genética , Epigênese Genética/genética , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Genoma Humano/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
12.
ACS Appl Mater Interfaces ; 5(17): 8628-32, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-23945148

RESUMO

The grating substrate covered with a metal layer, a plasmonic chip, and a bispecific antibody can play a key role in the sensitive detection of a marker protein with an immunosensor, because of the provision of an enhanced fluorescence signal and the preparation of a sensor surface densely modified with capture antibody, respectively. In this study, one of the tumor markers, a soluble epidermal growth factor receptor (sEGFR), was selected as the target to be detected. The ZnO- and silver-coated plasmonic chip with precise regularity and the appropriate duty ratio in the periodic structure further enhanced the fluorescence intensity. As for sensor surface modification with capture antibody, a bispecific antibody (anti-sEGFR and anti-ZnO antibody), the concentrated bispecific antibody solution was found to nonlinearly form a surface densely immobilized with antibody, because the binding process of a bispecific antibody to the ZnO surface can be a competitive process with adsorption of phosphate. As a result, the interface on the plasmonic chip provided a 300× enhanced fluorescence signal compared with that on a ZnO-coated glass slide, and therefore sEGFR was found to be quantitatively detected in a wide concentration range from 10 nM to 700 fM on our plasmonic surface.


Assuntos
Anticorpos/imunologia , Corantes Fluorescentes/química , Imunoensaio , Técnicas Analíticas Microfluídicas , Carbocianinas/química , Receptores ErbB/imunologia , Prata/química , Ressonância de Plasmônio de Superfície , Óxido de Zinco/química
13.
Bioconjug Chem ; 23(9): 1934-44, 2012 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-22873669

RESUMO

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 Superfície
14.
J Biol Chem ; 285(10): 7784-93, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20044483

RESUMO

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/imunologia
15.
J Phys Chem B ; 114(1): 480-6, 2010 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-20000396

RESUMO

Using an artificial peptide library, we have identified a peptide with affinity for ZnO materials that could be used to selectively accumulate ZnO particles on polypropylene-gold plates. In this study, we fused recombinant green fluorescent protein (GFP) with this ZnO-binding peptide (ZnOBP) and then selectively immobilized the fused protein on ZnO particles. We determined an appropriate condition for selective immobilization of recombinant GFP, and the ZnO-binding function of ZnOBP-fused GFP was examined by elongating the ZnOBP tag from a single amino acid to the intact sequence. The fusion of ZnOBP with GFP enabled specific adsorption of GFP on ZnO substrates in an appropriate solution, and thermodynamic studies showed a predominantly enthalpy-dependent electrostatic interaction between ZnOBP and the ZnO surface. The ZnOBP's binding affinity for the ZnO surface increased first in terms of material selectivity and then in terms of high affinity as the GFP-fused peptide was elongated from a single amino acid to intact ZnOBP. We concluded that the enthalpy-dependent interaction between ZnOBP and ZnO was influenced by the presence of not only charged amino acids but also their surrounding residues in the ZnOBP sequence.


Assuntos
Proteínas Imobilizadas/química , Peptídeos/química , Óxido de Zinco/química , Sequência de Aminoácidos , Ouro/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas Imobilizadas/metabolismo , Polipropilenos/química , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Termodinâmica
16.
Biochem Biophys Res Commun ; 365(4): 751-7, 2008 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-18039464

RESUMO

Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Peptídeos/química , Peptídeos/imunologia , Engenharia de Proteínas/métodos , Óxido de Zinco/química , Óxido de Zinco/imunologia , Sítios de Ligação , Ligação Proteica
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