RESUMO
Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated. Four rituximab scFv variants in the V(L)-V(H) orientation with different linker lengths between the V domains (scFv-1, scFv-3, scFv-5, scFv-8), plus the scFv-5 with a C-terminal cysteine (Cys-Db) for site-specific modification were generated. The scFv-8 and Cys-Db were radioiodinated with (124)I for PET imaging, and biodistribution of (131)I-Cys-Db was carried out at 2, 4 10 and 20 h. The five anti-CD20 scFv variants were expressed as fully functional dimers. Shortening the linker to three or one residue did not produce higher order of multimers. Both (124)I-labeled scFv-8 and Cys-Db exhibited similar tumor targeting at 8 h post injection, with significantly higher uptakes than in control tumors (P < 0.05). At 20 h, less than 1% ID/g of (131)I-labeled Cys-Db was present in tumors and tissues. Specific tumor targeting and high contrast images were achieved with the anti-CD20 diabodies. These agents extend the repertoire of reagents that can potentially be used to improve detection of low-grade lymphomas.
Assuntos
Anticorpos Monoclonais/química , Linfoma de Células B/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Anticorpos de Cadeia Única/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos , Dimerização , Feminino , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Radioisótopos do Iodo , Linfoma de Células B/imunologia , Camundongos , Camundongos Endogâmicos , Rituximab , Anticorpos de Cadeia Única/imunologiaRESUMO
We have previously demonstrated preclinical in vivo targeting of prostate stem cell antigen (PSCA) using a humanized anti-PSCA 2B3 monoclonal antibody (mAb). However, humanization resulted in 5-fold loss of apparent affinity relative to the parental mAb (1 nM). In this study, diabodies (scFv dimers of 55 kDa) were generated from 2B3 including variants with different linker lengths as well as back-mutations to original murine residues to improve affinity. Parental 2B3 (p2B3) and back-mutated 2B3 (bm2B3) diabodies (Dbs) with five- or eight-amino acid linkers (p2B3-Db5, p2B3-Db8, bm2B3-Db5 and bm2B3-Db8) were evaluated for binding to PSCA by flow cytometry and affinities were determined by surface plasmon resonance. Back-mutation restored the affinity from 5.4 to 1.9 nM. Stability, evaluated by size exclusion, revealed that diabodies with eight-residue linkers existed as a mixture of dimeric and monomeric species at low concentrations (Assuntos
Anticorpos Biespecíficos
, Região Variável de Imunoglobulina
, Glicoproteínas de Membrana/imunologia
, Proteínas de Neoplasias/imunologia
, Neoplasias da Próstata/diagnóstico por imagem
, Sequência de Aminoácidos
, Animais
, Anticorpos Monoclonais/genética
, Anticorpos Monoclonais/imunologia
, Anticorpos Monoclonais/metabolismo
, Afinidade de Anticorpos
, Antígenos de Neoplasias
, Linhagem Celular
, Escherichia coli/genética
, Citometria de Fluxo
, Proteínas Ligadas por GPI
, Humanos
, Região Variável de Imunoglobulina/genética
, Região Variável de Imunoglobulina/imunologia
, Região Variável de Imunoglobulina/metabolismo
, Masculino
, Glicoproteínas de Membrana/análise
, Camundongos
, Camundongos SCID
, Modelos Moleculares
, Dados de Sequência Molecular
, Mutação
, Proteínas de Neoplasias/análise
, Transplante de Neoplasias
, Tomografia por Emissão de Pósitrons
, Neoplasias da Próstata/imunologia
, Multimerização Proteica
, Estabilidade Proteica
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/imunologia
, Proteínas Recombinantes/metabolismo
, Transplante Heterólogo
RESUMO
Small, engineered antibody fragments such as diabodies (50 kDa noncovalent dimers of single-chain Fv fragments) are useful alternatives to their larger antibody counterparts. However, due to their size, they are more susceptible to disruption of their antigen binding sites when modified using random conjugation techniques. Previous work has demonstrated the utility of a C-terminal cysteine modification for site-specific radiolabeling of an anti-CEA diabody, resulting in the creation of a cys-diabody (CysDb). In the present work, the adaptability of the CysDb system was explored by creating two additional CysDbs: one specific for CD20 and one for HER2. Purified CysDbs of both specificities demonstrated behavior consistent with stable, covalent dimers harboring a readily reducible disulfide bond. Each CysDb was site-specifically conjugated to three different fluorophores for optical detection: the large fluorescent proteins phycoerythrin (PE) and allophycocyanin (APC), and the small fluorescent molecule Alexa Fluor488. Fluorophore-conjugated CysDbs bound specifically to their targets in both antigen systems and with each different fluorescent tag as determined by flow cytometry. In vitro specific antigen binding was observed in the presence of a mixture of specific and nonspecifically conjugated CysDbs. Conjugates retained both specificity and fluorescence, demonstrating the successful expansion of the CysDb repertoire to new targets and to new site-specific conjugation possibilities.
Assuntos
Antígenos CD20/imunologia , Cisteína/metabolismo , Corantes Fluorescentes/química , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Receptor ErbB-2/imunologia , Compostos de Sulfidrila/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Sensibilidade e EspecificidadeRESUMO
PURPOSE: Prostate stem cell antigen (PSCA) is a cell surface glycoprotein that is overexpressed in prostate cancer, including hormone refractory disease. Previous preclinical studies showed the intact anti-PSCA antibodies, 1G8 and hu1G8, localized specifically to PSCA-expressing xenografts. Optimal micro positron emission tomography (microPET) imaging using hu1G8, however, required a delay of 168 hours postinjection. In this study, the 2B3 minibody (an 80-kDa engineered antibody fragment) has been produced for rapid targeting and imaging. EXPERIMENTAL DESIGN: A gene encoding a PSCA-specific minibody, V(L)-linker-V(H)-hinge-huIgG1 C(H)3, was assembled. The minibody was expressed by secretion from mammalian cells and purified by cation exchange chromatography. Relative affinity and specificity were determined by competition ELISA and flow cytometry. Serial microPET imaging using a 124I-labeled minibody was conducted at 4 and 21 hours in mice bearing LAPC-9 AD, LAPC-9 AI, PC-3, and LNCaP-PSCA human prostate cancer xenografts. Tumor and tissue biodistribution was determined, and region of interest analysis of the images was conducted. RESULTS: Yields of 20 mg/L purified 2B3 minibody were obtained that showed specific binding to LNCaP-PSCA cells. Purified 2B3 minibody showed specific binding to LNCaP-PSCA cells with an apparent affinity of 46 nmol/L. Radioiodinated 2B3 minibody showed rapid nontarget tissue and blood clearance kinetics (t1/2beta = 11.2 hours). MicroPET scanning using the 124I-2B3 minibody showed both androgen-dependent and -independent tumors as early as 4 hours and excellent high contrast images at 21 hours postinjection. CONCLUSIONS: Imaging PSCA-positive prostate cancer is feasible using an intermediate size antibody fragment at 21 hours.