RESUMO
BACKGROUND: Infections with Herpes simplex virus (HSV)-1 or -2 usually present as mild chronic recurrent disease, however in rare cases can result in life-threatening conditions with a large spectrum of pathology. Monoclonal antibody therapy has great potential especially to treat infections with virus resistant to standard therapies. HDIT101, a humanized IgG targeting HSV-1/2 gB was previously investigated in phase 2 clinical trials. The aim of this study was to develop a next-generation therapy by combining different antiviral monoclonal antibodies. METHODS: A lymph-node derived phage display library (LYNDAL) was screened against recombinant gB from Herpes simplex virus (HSV) -1 and HDIT102 scFv was selected for its binding characteristics using bio-layer interferometry. HDIT102 was further developed as fully human IgG and tested alone or in combination with HDIT101, a clinically tested humanized anti-HSV IgG, in vitro and in vivo. T-cell stimulating activities by antigen-presenting cells treated with IgG-HSV immune complexes were analyzed using primary human cells. To determine the epitopes, the cryo-EM structures of HDIT101 or HDIT102 Fab bound to HSV-1F as well as HSV-2G gB protein were solved at resolutions < 3.5 Å. RESULTS: HDIT102 Fab showed strong binding to HSV-1F gB with Kd of 8.95 × 10-11 M and to HSV-2G gB with Kd of 3.29 × 10-11 M. Neutralization of cell-free virus and inhibition of cell-to-cell spread were comparable between HDIT101 and HDIT102. Both antibodies induced internalization of gB from the cell surface into acidic endosomes by binding distinct epitopes in domain I of gB and compete for binding. CryoEM analyses revealed the ability to form heterogenic immune complexes consisting of two HDIT102 and one HDIT101 Fab bound to one gB trimeric molecule. Both antibodies mediated antibody-dependent phagocytosis by antigen presenting cells which stimulated autologous T-cell activation. In vivo, the combination of HDIT101 and HDIT102 demonstrated synergistic effects on survival and clinical outcome in immunocompetent BALB/cOlaHsd mice. CONCLUSION: This biochemical and immunological study showcases the potential of an effective combination therapy with two monoclonal anti-gB IgGs for the treatment of HSV-1/2 induced disease conditions.
Assuntos
Herpes Simples , Humanos , Animais , Camundongos , Herpes Simples/imunologia , Herpes Simples/terapia , Herpes Simples/tratamento farmacológico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/imunologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Feminino , Herpesvirus Humano 2/imunologia , Herpesvirus Humano 2/efeitos dos fármacosRESUMO
INTRODUCTION: Photochemical Internalization is a novel drug delivery technology for cancer treatment based on the principle of Photodynamic Treatment. Using a photosensitizer that locates in endocytic vesicles membranes of tumor cells, Photochemical internalization enables cytosolic release of endocytosed antitumor agents in a site-specific manner. The purpose of the present in-vitro study was to explore whether Photochemical Internalization is able to enhance the efficacy of Ranpirnase, a cytotoxic amphibian ribonuclease, for eradication of squamous cell carcinoma of the head and neck. METHODS: Cell viability was measured in 8 primary human cell lines of squamous cell carcinoma of the head and neck after treatment with Ranpirnase and Photochemical Internalization. For Photochemical Internalization the photosensitizer disulfonated tetraphenyl porphine was incubated with tumor cells followed by exposure to blue light (435 nm). RESULTS: Our study demonstrates significant enhancement of antitumor activity of Ranpirnase by Photochemical Internalization. Treatment responses were heterogeneous between the primary cancer cell lines. Combining Photochemical Internalization with Ranpirnase resulted in 4.6 to 1,940-fold increased cytotoxicity when compared with the ribonuclease alone (P < 0.05). CONCLUSION: Cytotoxicity of Ranpirnase can be markedly enhanced by Photochemical Internalization in squamous cell carcinoma of the head and neck.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Fotoquimioterapia/métodos , Ribonucleases/uso terapêutico , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Fotoquímica , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
PURPOSE: Dual-targeted therapy has been shown to be a promising treatment option in recurrent and/or refractory B-cell non-Hodgkin's lymphoma (B-NHL). We generated radioimmunoconjugates (RICs) comprising either a novel humanized anti-CD22 monoclonal antibody, huRFB4, or rituximab, and the low-energy ß-emitter (177)Lu. Both RICs were evaluated as single agents in a human Burkitt's lymphoma xenograft mouse model. To increase the therapeutic efficacy of the anti-CD22 RIC, combination therapy with unlabelled anti-CD20 rituximab was explored. METHODS: The binding activity of CHX-Aâ³-DTPA-conjugated antibodies to target cells was analysed by flow cytometry. To assess tumour targeting of (177)Lu-labelled antibodies, in vivo biodistribution experiments were performed. For radioimmunotherapy (RIT) studies, non-obese diabetic recombination activating gene-1 (NOD-Rag1 (null) ) interleukin-2 receptor common gamma chain (IL2rγ (null) ) null mice (NRG mice) were xenografted subcutaneously with Raji Burkitt's lymphoma cells. (177)Lu-conjugated antibodies were administered at a single dose of 9.5 MBq per mouse. For dual-targeted therapy, rituximab was injected at weekly intervals (0.5 - 1.0 mg). Tumour accumulation of RICs was monitored by planar scintigraphy. RESULTS: Conjugation of CHX-A"-DTPA resulted in highly stable RICs with excellent antigen-binding properties. Biodistribution experiments revealed higher tumour uptake of the (177)Lu-labelled anti-CD22 IgG than of (177)Lu-labelled rituximab. Treatment with (177)Lu-conjugated huRFB4 resulted in increased tumour growth inhibition and significantly longer survival than treatment with (177)Lu-conjugated rituximab. The therapeutic efficacy of the anti-CD22 RIC could be markedly enhanced by combination with unlabelled rituximab. CONCLUSION: These findings suggest that dual targeting with (177)Lu-based CD22-specific RIT in combination with rituximab is a promising new treatment option for refractory B-NHL.
Assuntos
Linfoma de Burkitt/terapia , Imunoconjugados/uso terapêutico , Lutécio/química , Radioimunoterapia/métodos , Rituximab/uso terapêutico , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/química , Animais , Linfoma de Burkitt/radioterapia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoglobulina G/uso terapêutico , Dose Máxima Tolerável , Camundongos , Camundongos Endogâmicos NOD , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the availability of antiviral chemotherapy, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections remain a severe global health problem. Of particular concern is the growing incidence of drug resistance in immunocompromised patients, which stresses the urgency to develop new effective treatment alternatives. We have developed a humanized monoclonal antibody (mAb hu2c) that completely abrogates viral cell-to-cell spread, a key mechanism by which HSV-1/2 escapes humoral immune surveillance. Moreover, mAb hu2c neutralized HSV fully independent of complement and/or immune effector cell recruitment in a highly efficient manner. Prophylactic and therapeutic administration of mAb hu2c completely prevented infection-related mortality of severely immunodeficient mice being challenged with a lethal dose of HSV-1. The high neutralization capacity of mAb hu2c was fully maintained toward clinical HSV isolates being multiresistant to standard antiviral drugs, and infection was fully resolved in 7/8 nonobese diabetic/SCID mice being infected with a multidrug resistant HSV-1 patient isolate. Immunohistochemical studies revealed no significant cross-reactivity of the antibody toward human tissues. These features warrant further clinical development of mAb hu2c as an immunotherapeutic compound for the management of severe and particularly drug-resistant HSV infections.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Herpes Simples/tratamento farmacológico , Simplexvirus/imunologia , Aciclovir , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Farmacorresistência Viral , Herpes Simples/imunologia , Imuno-Histoquímica , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Testes de Neutralização , Simplexvirus/genética , Células VeroRESUMO
Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONC(EGFR)) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC(EGFR) expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONC(EGFR) induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONC(EGFR)-encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC(EGFR) is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC(EGFR)-encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.
Assuntos
Adenoviridae/genética , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Vetores Genéticos/genética , Vírus Oncolíticos/genética , Ribonucleases/administração & dosagem , Ribonucleases/metabolismo , Animais , Anticorpos Antivirais , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Terapia Viral Oncolítica/métodos , RNA/metabolismo , Replicação Viral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab')(2)] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Especificidade de Anticorpos , Células COS , Chlorocebus aethiops , Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/prevenção & controle , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Herpes Genital/imunologia , Herpes Genital/prevenção & controle , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Testes de Neutralização , Mapeamento de Peptídeos , Células Vero , Proteínas do Envelope Viral/químicaRESUMO
HDIT101 is a first-in-class humanized monoclonal antibody recognizing a conserved epitope in glycoprotein B, a target present on the surface of herpes simplex virus 1 (HSV-1) and HSV-2 particles as well as on virus-infected cells. This was a first-in-human, single-center, double-blind, placebo-controlled trial in 24 healthy volunteers, randomized 3:1 (placebo:active) in each of the six dose levels with escalating doses up to 12,150 mg HDIT101. HDIT101 was administered intravenously, to study safety, pharmacokinetics (PKs), and immunogenicity. HDIT101 was well-tolerated in all recipients and no serious or severe adverse events, no infusion-related reactions, and no events suggestive of dose limiting off-target toxicity occurred. The mean serum exposure (area under the curve from zero to infinity [AUC0-∞ ]) of HDIT101 showed a linear increase from 4340 h*µg/ml at a dose of 50 mg to 1,122,247 h*µg/ml at a dose of 12,150 mg. No immunogenic effects following HDIT101 exposure were observed at any of the applied doses. HDIT101 demonstrated the expected PK properties of a monoclonal antibody was well-tolerated, and could be safely administered even at excessively high doses that may be required for treatment of patients with septical HSV spread.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Humanos , Voluntários Saudáveis , Método Duplo-Cego , Anticorpos Monoclonais/efeitos adversos , EpitoposRESUMO
Multiple therapeutic monoclonal antibodies (mAbs) are currently under development or in (pre)clinical study phases to reach regulatory approval. Among these, a new mAb against herpes simplex virus, HDIT101, was recently tested in healthy volunteers during a phase I clinical trial (first-in-human, dose escalation). In the frame of the pharmacokinetic evaluation of this new therapy, a mass spectrometric (MS)-based method was developed for the quantification of HDIT101 in human plasma using liquid chromatography coupled to tandem mass spectrometry. In this work, we describe the development of this bioanalytical assay using the quantification of a HDIT101 surrogate peptide, the assay validation procedure according to the FDA guidelines within the calibration range from 20 to 5000 µg/mL, and its application to plasma samples from the first-in-human clinical trial. This work presents a generic workflow for the development of MS-based quantification assays of new therapeutic antibodies that allows reaching high immunopurification recovery (>98% for HDIT101 over the full calibration range with a precision of 6.9% CV). Surrogate peptide and stable isotopically labeled internal standard were stable, and batch-to-batch accuracies and precisions at the four quality standard levels ranged between -2 and 5% bias and 8 and 11% CV, respectively.
RESUMO
ImmunoRNases represent a highly attractive alternative to conventional immunotoxins for cancer therapy. Quantitative production of immunoRNases in appropriate expression systems, however, remains a major challenge for further clinical development of these novel compounds. Here we describe a method for high-level production and purification of a fully functional immunoRNase fusion protein from supernatants of stably transfected mammalian cells.
Assuntos
Biologia Molecular/métodos , Mieloma Múltiplo/patologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Ribonuclease Pancreático/biossíntese , Ribonuclease Pancreático/imunologia , Transfecção , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Clonais , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Vetores Genéticos/genética , Humanos , Região Variável de Imunoglobulina/imunologia , Camundongos , Proteínas Recombinantes de Fusão/farmacologia , Ribonuclease Pancreático/farmacologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologiaRESUMO
Radioimmunotherapy is considered as treatment option in recurrent and/or refractory B-cell non-Hodgkin lymphoma (B-NHL). To overcome the dose limiting bone marrow toxicity of IgG-based radioimmunoconjugates (RICs), we modified a humanized diabody with 5-, 10-, or 20-kDa polyethylene glycol (PEG) for CD22-targeted radioimmunotherapy using the low-energy ß-emitter lutetium-177 ((177)Lu). A favorable pharmacokinetic profile was observed for the 10-kDa-PEG-diabody in nude mice being xenografted with subcutaneous human Burkitt lymphoma. Even at high doses of 16 MBq this diabody RIC was well tolerated by NOD Rag1(null) IL2rγ(null) (NRG) mice and did not reveal signs of organ long-term toxicity 80 days post injection. Combination therapy of the diabody RIC with unconjugated anti-CD20 Rituximab demonstrated therapeutic efficacy in established disseminated mantle cell lymphoma xenograft models. When compared with the combination of the IgG formatted (177)Lu anti-CD22 antibody and Rituximab, dual targeted therapy with the diabody RIC achieved an improved reduction of disease burden in the first nine days following treatment. The data indicate that the PEGylated anti-CD22 diabody may have potential for extending the repertoire of radiopharmaceuticals for the treatment of patients with B-NHL.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Linfoma de Burkitt/radioterapia , Imunoconjugados/farmacologia , Lutécio/farmacologia , Linfoma de Célula do Manto/radioterapia , Radioimunoterapia/métodos , Radioisótopos/farmacologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/toxicidade , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais Humanizados/toxicidade , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/toxicidade , Imunoglobulinas Intravenosas/farmacologia , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Lutécio/farmacocinética , Lutécio/toxicidade , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Nus , Radioimunoterapia/efeitos adversos , Radioisótopos/farmacocinética , Radioisótopos/toxicidade , Rituximab/farmacologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADCs) have evolved as a new class of potent cancer therapeutics. We here report on the development of ADCs with specificity for the B-cell lineage specific (surface) antigen CD22 being expressed in the majority of hematological malignancies. As targeting moiety a previously generated humanized anti-CD22 single-chain variable fragment (scFv) derivative from the monoclonal antibody RFB4 was reengineered into a humanized IgG1 antibody format (huRFB4). Onconase (ranpirnase), a clinically active pancreatic-type ribonuclease, was employed as cytotoxic payload moiety. Chemical conjugation via thiol-cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the ADC. Development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of Onconase cargos. A minimum of two Onconase molecules per IgG was required for achieving significant in vitro cytotoxicity towards lymphoma and leukemia cell lines. Antibody-drug conjugates with an Onconase to antibody ratio of 3 : 1 exhibited an IC50 of 0.08 nM, corresponding to more than 18,400-fold increased cytotoxicity of the ADC when compared with unconjugated Onconase. These results justify further development of this ADC as a promising first-in-class compound for the treatment of CD22-positive malignancies.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Ribonucleases/farmacologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Antineoplásicos/química , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoglobulina G/imunologia , Ribonucleases/químicaRESUMO
Cytotoxic ribonucleases such as the leopard frog derivative Ranpirnase (Onconase(®)) have emerged as a valuable new class of cancer therapeutics. Clinical trials employing single agent Ranpirnase in cancer patients have demonstrated significant clinical activity and surprisingly low immunogenicity. However, dose-limiting toxicity due to unspecific uptake of the RNase into non-cancerous cells is reached at relatively low concentrations of > 1 mg/m(2). We have in the present study generated a dimeric anti-EGFR Ranpirnase-diabody fusion protein capable to deliver two Ranpirnase moieties per molecule to EGFR-positive tumour cells. We show that this compound mediated far superior efficacy for killing EGFR-positive tumour cells than a monomeric counterpart. Most importantly, cell killing was restricted to EGFR-positive target cells and no dose-limiting toxicity of Ranpirnase-diabody was observed in mice. These data indicate that by targeted delivery of Ranpirnase non-selective toxicity can be abolished and suggests Ranpirnase-diabody as a promising new drug for therapeutic interventions in EGFR-positive cancers.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Ribonucleases/farmacologia , Animais , Carcinoma de Células Escamosas/enzimologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Neoplasias de Cabeça e Pescoço/enzimologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ribonucleases/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.
Assuntos
Afinidade de Anticorpos/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Moléculas de Adesão Celular/metabolismo , Região Variável de Imunoglobulina/química , Lectinas/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Sequência de Aminoácidos , Anticorpos Biespecíficos/metabolismo , Sítios de Ligação de Anticorpos , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Dimerização , Escherichia coli/genética , Citometria de Fluxo , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Células Jurkat , Linfoma de Células B/imunologia , Conformação Proteica , Estrutura Terciária de Proteína , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , TemperaturaRESUMO
Due to its frequent overexpression in a variety of solid tumors the epidermal growth factor receptor (EGFR) is a well-established target for therapeutic interventions in epithelial cancers. In order to target EGFR in head and neck cancer, we have generated a ribonuclease (RNase) fusion protein comprising a humanized anti-EGFR antibody single-chain Fv fragment (scFv) and Ranpirnase, an RNase from Rana pipiens. Fusion of Ranpirnase to the N-terminus of the scFv via a flexible glycine-serine linker (G4S)3 resulted in very poor cytotoxicity of the fusion protein. As endosomal accumulation and lysosomal degradation have been reported to diminish the antitumor efficacy of ribonuclease or toxin-based immunoagents, we explored a fusion peptide from dengue virus that has been reported to be involved in the endosomal escape of the virus. This peptide was introduced as a linker between Ranpirnase and the scFv moiety. The modified immunoRNase exhibited exceptionally high cytotoxicity toward EGFR-expressing head and neck cell lines without affecting specificity. These results indicate that endosomal entrapment needs to be considered for Ranpirnase-based immunoagents and might be overcome by the use of tailored transduction domains from viral proteins.
Assuntos
Antineoplásicos/metabolismo , Vírus da Dengue/genética , Receptores ErbB/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleases/metabolismo , Anticorpos de Cadeia Única/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/química , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Ribonucleases/química , Ribonucleases/genética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genéticaRESUMO
The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected "LYmph Node Derived Antibody Libraries" (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.
Assuntos
Anticorpos Antivirais , Clonagem Molecular , Biblioteca Gênica , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Imunoglobulina G , Região Variável de Imunoglobulina , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Feminino , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Masculino , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaAssuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Antineoplásicos/uso terapêutico , Antígeno Ki-1/imunologia , Neoplasias Experimentais/terapia , Reação em Cadeia da Polimerase/métodos , Receptores de IgG/imunologia , Animais , Anticorpos Biespecíficos/isolamento & purificação , Primers do DNA/química , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfócitos/imunologia , Linfoma/imunologia , Linfoma/terapia , Camundongos , Camundongos SCID , Neoplasias Experimentais/imunologia , Ligação Proteica , Transplante HeterólogoRESUMO
Immunotoxins combining antibody specificity with bacterial or plant toxins are limited by their strong immunogenicity and non-specific toxicity. Ribonucleases of the RNase A protein superfamily provide a solution to address these issues because they show potent antineoplastic activity on cell internalization but do not show appreciable immunogenicity or non-specific toxicity. Their therapeutic value is demonstrated by RNase derived from the frog (Rana pipiens), Onconase (ONC, Alfacell, Inc., New Jersey, USA), the first and only RNase being evaluated in clinical trials at present. Conjugation or fusion of RNases to tumor specific antibodies is a promising approach to further boost tumor cell killing of these compounds. This review focuses on 'targeted RNases/ImmunoRNases' as promising novel anticancer therapeutics.
Assuntos
Antineoplásicos/uso terapêutico , Ribonucleases/uso terapêutico , Animais , Humanos , Imunotoxinas/uso terapêutico , Antígeno Ki-1/imunologia , Pró-Fármacos/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Ribonuclease Pancreático/uso terapêutico , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologiaRESUMO
Ribonucleases (RNases) of the superfamily A exhibit potent antineoplastic activity yet do not mediate appreciable immunogenicity or non-specific toxicity in both animal models and cancer patients. Ranpirnase (Onconase), the first ribonuclease being evaluated as a therapeutic in humans, has progressed to phase III clinical trials in patients with unresectable mesothelioma. Conjugation of RNases to internalizing tumor-targeting monoclonal antibodies was shown to enhance specific cell killing by several orders of magnitude both in vitro and in animal models. In this review we describe the development and current status of genetically engineered 2(nd) generation immunoRNases as promising novel anti-cancer therapeutics.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Ribonuclease Pancreático/uso terapêutico , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/imunologia , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Neoplasias/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/imunologiaRESUMO
To improve selective cytotoxicity and pharmacokinetics of an anti-CD22 antibody single chain Fv (scFv)-ribonuclease fusion protein, a dimeric derivative was generated. Human angiogenin was fused via a (G4S)3 spacer peptide to the carboxy-terminal end of the stable dimeric anti-CD22 VL-VH zero-linker scFv MLT-7. The dimeric fusion protein and a monovalent counterpart were produced as soluble proteins in the periplasm of Escherichia coli. Comparative studies with homogeneously purified fusion proteins revealed that both constructs specifically bound to the target antigen and retained ribonucleolytic activity. However, they exhibited a markedly different capability for killing CD22+ tumor cells. The monomeric construct inhibited protein synthesis of target cells in a dose-dependent manner, but 50% inhibition (IC50) could be achieved only at the highest tested concentration (>350 nM). In contrast, the dimeric fusion protein efficiently killed CD22+ Raji and Daudi tumor cell lines with IC50 values of 74 nM and 118 nM, respectively. These results show that the therapeutic potential of scFv-ANG fusion proteins can be markedly enhanced by engineering dimeric derivatives.