RESUMO
A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pre-targeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft tumor model. The in vivo biodistribution studies showed that all the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed that the (111)In-DOTA bispecific antibody had increased liver and spleen uptake, not seen for the (125)I-version due to metabolism and release of the radioiodine from the cells. These data suggest increased clearance of the antibody fusion formats by the mononuclear phagocyte system. Importantly, a pre-targeted study showed specific tumor uptake of (177)Lu-DOTA and a tumor : blood ratio of 199 : 1. This pre-targeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacocinética , Antígeno Carcinoembrionário/imunologia , Compostos Heterocíclicos com 1 Anel/imunologia , Neoplasias/imunologia , Neoplasias/radioterapia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Feminino , Humanos , Radioisótopos do Iodo/química , Radioisótopos do Iodo/farmacocinética , Camundongos , Camundongos Nus , Engenharia de Proteínas , Estabilidade Proteica , Radioimunoterapia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Distribuição TecidualRESUMO
This case study describes early phase purification process development for a recombinant anticancer minibody produced in mammalian cell culture. The minibody did not bind to protein A. Cation-exchange, anion-exchange, hydrophobic-interaction, and hydroxyapatite (eluted by phosphate gradient) chromatographic methods were scouted, but the minibody coeluted with BSA to a substantial degree on each. Hydroxyapatite eluted with a sodium chloride gradient separated BSA and also removed a dimeric contaminant, but BSA consumed so much binding capacity that this proved impractical as a capture tool. Capto MMC media proved capable of supporting adequate capture and significant dimer removal, although both loading and elution selectivity varied dramatically with the amount of supernatant applied to the column. An anion-exchange step was included to fortify overall virus and DNA removal. These results illustrate the value of multimodal chromatography methods when affinity chromatography methods are lacking and conventional alternatives prove inadequate.
RESUMO
BACKGROUND: Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors. METHODS AND FINDINGS: As proof-of-concept, we selected Herceptin (trastuzumab), a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice. CONCLUSIONS: Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Neurônios/citologia , Células-Tronco/metabolismo , Animais , Anticorpos Monoclonais Humanizados , Especificidade de Anticorpos/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Feminino , Imunoglobulina G/imunologia , Camundongos , Camundongos Nus , Neurônios/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Receptor ErbB-2/imunologia , Células-Tronco/efeitos dos fármacos , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Retention mapping of chimeric monoclonal IgG(1), Fc, Fab, F(ab')(2), and aggregated antibody was conducted on hydroxyapatite (HA) by systematically varying phosphate and chloride concentrations during gradient elution in order to characterize the interactions of each solute with calcium and phosphate residues on the solid phase. Lysozyme was used as a control to model cation exchange-dominant interactions. Bovine serum albumin was used as a control for calcium affinity-dominant interactions. Calcium affinity and phosphoryl cation exchange were positively cooperative for IgG-related species. Fc retention was dominated by calcium affinity, while retention of Fab was dominated by cation exchange. F(ab')(2) exhibited a curve shape similar to Fab, but stronger retention. The retention curve for intact IgG incorporated the distinctive elements of its fragments but stronger retention than that predicted by their addition to one another. Aggregate retention paralleled the curve for non-aggregated antibody, with stronger retention by both binding mechanisms. Experimental data revealed evidence of charge repulsion between IgG carboxyls and HA phosphate at low conductivity values. Electrostatic repulsion of amino residues and attraction of carboxyls by HA calcium appeared to be blocked by strong complexation of calcium with mobile phase phosphate.
Assuntos
Durapatita/química , Fragmentos de Imunoglobulinas/química , Animais , Resinas de Troca de Cátion , Bovinos , Cromatografia por Troca Iônica , Soroalbumina Bovina/química , Eletricidade EstáticaRESUMO
This study introduces the application of calcium-derivatized hydroxyapatite for purification of Fab. Fab binds to native hydroxyapatite but fails to bind to the calcium derivatized form. IgG, Fc, and most other protein contaminants bind to the calcium form. This supports Fab purification by a simple flow-through method that achieves greater than 95% purity from papain digests and mammalian cell culture supernatants. Alternatively, Fab can be concentrated on native hydroxyapatite then eluted selectively by conversion to the calcium-derivatized form.
Assuntos
Cálcio/química , Cromatografia de Afinidade/métodos , Durapatita/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Meios de Cultura , Imunoglobulina G/isolamento & purificaçãoRESUMO
Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [125 I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [111 In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [64 Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents.
Assuntos
Albuminas/farmacocinética , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/diagnóstico por imagem , Radioimunodetecção/métodos , Proteínas Recombinantes de Fusão/farmacocinética , Albuminas/genética , Animais , Anticorpos Antineoplásicos/genética , Antígeno Carcinoembrionário/genética , Neoplasias Colorretais/imunologia , Feminino , Humanos , Imunoconjugados/farmacocinética , Região Variável de Imunoglobulina/genética , Radioisótopos de Índio/farmacocinética , Radioisótopos do Iodo/farmacocinética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual/imunologia , Transplante HeterólogoRESUMO
An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.
Assuntos
Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/uso terapêutico , Neoplasias Mamárias Experimentais/terapia , Engenharia de Proteínas , Receptor ErbB-2/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Dados de Sequência MolecularRESUMO
An engineered anti-carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously constructed from the murine anti-CEA T84.66 antibody. Tumor targeting, imaging and biodistribution studies in nude mice bearing LS174T xenografts with radiolabeled anti-CEA diabody demonstrated rapid tumor uptake and fast blood clearance, which are favorable properties for an imaging agent. Current radiolabeling approaches result in random modification of the protein surface, which may impair immunoreactivity especially for smaller antibody fragments. Site-specific conjugation approaches can direct modifications to reactive groups located away from the binding site. Here, cysteine residues were introduced into the anti-CEA diabody at three different locations, to provide specific thiol groups for chemical modification. One version (with a C-terminal Gly-Gly-Cys) existed exclusively as a disulfide-bonded dimer. This cysteine-modified diabody (Cys-diabody) retained high binding to CEA and demonstrated tumor targeting and biodistribution properties identical to the non-covalent diabody. Furthermore, following reduction of the disulfide bond, the Cys-diabody could be chemically modified using a thiol-specific bifunctional chelating agent, for radiometal labeling. Thus, the Cys-diabody provides a covalently linked alternative to conventional diabodies, which can be reduced and modified site-specifically. This format will provide a versatile platform for targeting a variety of agents to CEA-positive tumors.