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1.
Recent Results Cancer Res ; 214: 71-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31473849

RESUMO

Bispecific T cell engagers are antibody constructs directed to a tumor-specific target on the one hand and to CD3-positive T cells on the other hand. Blinatumomab is a compound with specificity for the pan-B cell marker CD19. Clinical activity was tested in relapsed and refractory (R/R) non-Hodgkin's Lymphoma (NHL), R/R acute lymphoblastic leukemia (ALL), and ALL patients with minimal residual disease. Trials have already been started in de novo ALL. The most clinically relevant toxicities are neurologic events and cytokine release syndrome as with other T cell-activating therapies. The mechanisms of resistance are not fully understood. Higher leukemia load and later stage disease represent unfavorable factors. Besides, an upregulation of regulatory T cells and inhibitory molecules like PD-1/PD-L1 may have a role as the loss of target by several mechanisms. The future will show whether the use of bispecifics in ALL can change the standard treatment algorithms and whether bispecific T cell engagers will also be successfully used in other malignant entities.


Assuntos
Anticorpos Biespecíficos/farmacologia , Linfoma não Hodgkin/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos T/citologia , Antígenos CD19 , Ensaios Clínicos como Assunto , Humanos , Neoplasia Residual/terapia
3.
Proc Natl Acad Sci U S A ; 116(15): 7533-7542, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30898885

RESUMO

Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Cancer Res Clin Oncol ; 145(5): 1179-1190, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30778749

RESUMO

PURPOSE: Bi-specific antibody (BsAb) is an emerging novel format of antibody. We aimed to develop the natural killer (NK) cell receptor NK group 2, member D (NKG2D)-mediated, immune surveillance system. In this system, the NKG2D ligand MHC class I-related chain A (MICA) was fused with BsAb, which targeted a cluster of differentiation 24 (CD24), a tumor-initiating cell marker that is over-expressed on hepatocellular carcinoma (HCC). METHODS: The Homo MICA extracellular domains (hMICA) were fused to the end of the heavy chain of cG7 with the flexible pentapeptide (Gly-Gly-Gly-Gly-Ser; G4S), which formed the cG7-MICA that was further identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB). The targeting specificity was characterized using the Surface Plasmon Resonance (SPR) technology and a flow cytometry assay. Furthermore, the design of BsAb cG7-MICA that targeted CD24 and NKG2D was proven to enhance antibody-dependent, cell-mediated cytotoxicity (ADCC) in vitro by the CytoTox 96 Nonradioactive Cytotoxicity assay. Degranulation and a cytokine production assay of NK cells demonstrated that NK cells were activated effectively by cG7-MICA. Further, in HCC-bearing nude mice, the anti-tumor effects of cG7-MICA combined with sorafenib were verified again. RESULTS: We purified cG7-MICA successfully, and it has a high affinity. In vivo, cG7-MICA recruited NK cells to the tumor site and improved the anti-tumor efficacy of sorafenib. cG7-MICA also activated NK cells to release interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), and it increased the CD107a expression on the surface of the NK cells in vitro. CONCLUSION: NK cells play a major role in the natural, innate immune system, and they have the function of identifying and killing target cells. cG7-MICA remodels the function of MICA molecules to activate NK cells, which provides a possible strategy for HCC-targeting immunotherapy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antígeno CD24/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/agonistas , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Anticorpos Biespecíficos/genética , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoterapia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Receptores Fc/metabolismo , Transdução de Sinais , Sorafenibe/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Adv Clin Chem ; 88: 151-167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30612605

RESUMO

Congenital hemophilia A, a relatively common and sometimes life-threatening bleeding disorder, is caused by inherited deficiency of clotting factor (F) VIII. The adoption of an appropriate medical and environmental prophylaxis is critical for long-term management of hemophilia because it will considerably reduce the number of both mild and severe bleeding episodes. Among the many therapeutic options that have become available over the past decades, ACE910 (also known as emicizumab) is a bispecific immunoglobulin G antibody characterized by its unique ability to bind FIX or FIXa on one arm and FX on the other, thus abrogating FVIII activity in vivo. Several phase I to III clinical trials have now been published, confirming the clinical efficacy and relative safety of this new agent for long-term prophylaxis of hemophilia A, especially those patients having FVIII inhibitors. The recent regulatory clearance of ACE910 in many countries will hence impose additional challenges to clinical laboratories because the panel of available tests will need to address the emerging issue of monitoring patients treated with this novel anti-hemophilic agent by using conventional as well as innovative approaches. Therefore, this article is aimed to provide an update on clinical background and challenges of laboratory assessment in hemophilia A patients undergoing ACE910 administration.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Hemofilia A/tratamento farmacológico , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Testes de Coagulação Sanguínea/métodos , Ensaios Clínicos como Assunto , Monitoramento de Medicamentos/métodos , Fator VIII/metabolismo , Hemofilia A/metabolismo , Hemofilia A/prevenção & controle , Humanos
8.
J Biochem ; 165(6): 487-495, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30597085

RESUMO

The human fibroblast growth factor-2 (FGF-2) highly expressed in tumours is an important factor to promote tumour angiogenesis and lymphangiogenesis. A disulphide-stabilized diabody (ds-Diabody) could specifically target FGF-2 and show its advantages in inhibition of tumour angiogenesis and growth. It is very important for antibody drugs to confirm the fine epitope. Here, theoretical structure models of FGF-2 and antibody were built by homology modelling. The amino acid residues in the interaction interface of antigen and antibody were analysed by molecular docking. The potential epitope was predicted by homology modelling and molecular docking of antigen-antibody and site-directed mutation assays of alanine scanning. The predicted epitope was verified by antigen mutagenesis and enzyme-linked immunosorbent assay (ELISA). The epitope mapping assay showed that the epitope of ds-Diabody against FGF-2 was defined by the discontinuous sites including six amino acid residues (P23, Q65, R69, G70, Y82 and R118). The results showed that the epitope was localized in the interaction interface of FGF-2 and ds-Diabody. The fine epitope mapping provided the important information for understanding the inhibition activity of ds-Diabody against FGF-2 and helping in the further development of ds-Diabody against FGF-2 as a potentially promising antibody drug for future cancer therapy.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Dissulfetos/química , Mapeamento de Epitopos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/imunologia , Neoplasias/tratamento farmacológico , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/uso terapêutico , Reações Antígeno-Anticorpo , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Fator 2 de Crescimento de Fibroblastos/genética , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neoplasias/imunologia , Neoplasias/patologia
9.
Expert Opin Drug Saf ; 18(2): 75-85, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30681006

RESUMO

INTRODUCTION: During the last decade, new FVIII/IX concentrates have been developed for the treatment of patients affected by hemophilia A/B. Significant progress has been achieved regarding their half-life, but the old issue of immunogenicity and new concerns about safety need to be addressed. AREAS COVERED: After the implementation of virucidal methods, both plasma-derived and recombinant clotting factor concentrates achieved a very safe profile. The development of anti-FVIII antibodies is the major adverse event of replacement therapy with both FVIII concentrates. Furthermore, the new extended half-life concentrates, protein fused or pegylated, raised some concerns about their side effects. EXPERT OPINION: The treatment of hemophilia A with inhibitors by induction of immunotolerance and using by-passing concentrates, improved the quality of life of patients but did not allow them to have a life expectancy like that of patients without inhibitors. The new humanized monoclonal antibody (MAb) ACE910, mimicking FVIII function, seems to be able to reduce the bleedings of hemophilia A patients with inhibitors. The post-marketing surveillance will clarify if the adverse events observed during the phase III clinical trials and compassionate use were due to the association with a Prothrombin activated complex concentrate or to the prothrombotic effect of the drug itself.


Assuntos
Coagulantes/administração & dosagem , Hemofilia A/tratamento farmacológico , Hemofilia B/tratamento farmacológico , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacologia , Coagulantes/efeitos adversos , Coagulantes/farmacocinética , Desenvolvimento de Medicamentos , Fator IX/administração & dosagem , Fator IX/efeitos adversos , Fator IX/farmacocinética , Fator VIII/administração & dosagem , Fator VIII/efeitos adversos , Fator VIII/farmacocinética , Meia-Vida , Hemofilia B/fisiopatologia , Humanos , Qualidade de Vida , Proteínas Recombinantes
10.
Autoimmun Rev ; 18(2): 123-128, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30572136

RESUMO

Targeting various disease pathways using monoclonal antibodies (mAbs) has transformed the treatment paradigm for inflammatory bowel disease (IBD), with these agents exhibiting improved efficacy over corticosteroids or immunosuppressive therapies. Antibodies targeting tumor necrosis factor α (TNF-α) were the first approved biologics for IBD, followed by the more recent approval of antibodies targeting the α4ß7 integrin heterodimer and ustekinumab, which targets the p40 subunit of interleukin-23. Current efforts are focused on the development of additional biologics targeting these known and other newly discovered pathways. Still significant unmet needs remain, as a large proportion of patients either fail to achieve remission or fail to respond altogether. Both Crohn's disease and ulcerative colitis are complex and heterogeneous diseases with several molecular pathways involved in disease pathophysiology. We propose an additional therapeutic approach to the treatment of IBD, bispecific antibodies (BsAbs), which combine two distinct binding specificities within a single biologic to allow the simultaneous targeting of multiple disease-causing cytokines or pathways. Although primarily used in oncology thus far, the unique combinatorial mechanism of action of BsAbs may provide new therapeutic options for a broad range of clinical applications, including autoimmune and inflammatory diseases. This review will discuss the current status of BsAb development in general and potentially therapeutic application in IBD.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Doenças Inflamatórias Intestinais/terapia , Anticorpos Biespecíficos/farmacologia , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Humanos , Doenças Inflamatórias Intestinais/patologia
11.
Methods Mol Biol ; 1904: 11-51, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539465

RESUMO

Since the approval of the first monoclonal antibody (mAb), rituximab, for hematological malignancies, almost 30 additional mAbs have been approved in oncology. Despite remarkable advances, relatively weak responses and resistance to antibody monotherapy remain major open issue. Overcoming resistance might require combinations of drugs blocking both the major target and the emerging secondary target. We review clinically approved combinations of antibodies and either cytotoxic regimens (chemotherapy and irradiation) or kinase inhibitors. Thereafter, we focus on the most promising and currently very active arena that combines mAbs inhibiting immune checkpoints or growth factor receptors. Clinically approved and experimental oligoclonal mixtures of mAbs targeting different antigens (hetero-combinations) or different epitopes of the same antigen (homo-combinations) are described. Effective oligoclonal mixtures of antibodies that mimic the polyclonal immune response will likely become a mainstay of cancer therapy.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Humanos , Imunomodulação/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Biologia de Sistemas/métodos , Resultado do Tratamento
12.
Methods Mol Biol ; 1884: 335-347, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465214

RESUMO

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Bioensaio/métodos , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Biespecíficos/uso terapêutico , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antineoplásicos/uso terapêutico , Bioensaio/instrumentação , Antígeno CD47/genética , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Células CHO , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Separação Celular/instrumentação , Separação Celular/métodos , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Cricetulus , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/patologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
13.
MAbs ; 11(2): 322-334, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30569825

RESUMO

CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Antígeno CD47/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Ativação Linfocitária/efeitos dos fármacos
14.
Anticancer Res ; 38(7): 4273-4279, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29970561

RESUMO

BACKGROUND/AIM: Epithelial cell adhesion molecule (EpCAM) is expressed in various types of cancer, including breast cancer, and is correlated with metastasis, invasion, therapeutic resistance and prognosis. Moreover, several cell surface markers, such as CD44 and EpCAM, are molecular targets on cancer stem-like cells of breast cancer. The aim of this study was to investigate whether catumaxomab, a clinical-grade bispecific antibody that binds to both EpCAM on tumor cells and CD3 on T-cells, combined with activated T-cells can eliminate chemoresistant triple-negative breast cancer (TNBC) cells in vitro. MATERIALS AND METHODS: First, a cell line (MUK-BC1) was established from human breast carcinoma cells derived from a patient with chemoresistant and disseminated breast cancer. These EpCAM-positive TNBC cells were almost completely resistant to various drug-mediated cytotoxicities up to a concentration of 10 µg/ml. RESULTS: Pre-treatment with catumaxomab and subsequent addition of interleukin-2/OKT3-activated autologous T-cells eliminated EpCAM-positive TNBC cells. CONCLUSION: Catumaxomab combined with activated T-cells may be a potent therapeutic modality to overcome chemoresistant EpCAM-positive TNBC cells.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Imunoterapia/métodos , Linfócitos T/imunologia , Neoplasias de Mama Triplo Negativas , Adulto , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Feminino , Humanos , Ativação Linfocitária
15.
Int J Nanomedicine ; 13: 3189-3201, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29881272

RESUMO

Introduction: Bispecific antibodies that engage immune cells to kill cancer cells are actively pursued in cancer immunotherapy. Different types of bispecific antibodies, including single-chain fragments, Fab fragments, nanobodies, and immunoglobulin Gs (IgGs), have been studied. However, the low molecular weight of bispecific antibodies with single-chain or Fab fragments generally leads to their rapid clearance in vivo, which limits the therapeutic potential of these bispecific antibodies. Materials and methods: In this study, we used a site-specific PEGylation strategy to modify the bispecific single-domain antibody-linked Fab (S-Fab), which was designed by linking an anticarcinoembryonic antigen (anti-CEA) nanobody with an anti-CD3 Fab. Results: The half-life (t1/2) of PEGylated S-Fab (polyethylene glycol-S-Fab) was increased 12-fold in vivo with a slightly decreased tumor cell cytotoxicity in vitro as well as more potent tumor growth inhibition in vivo compared to S-Fab. Conclusion: This study demonstrated that PEGylation is an effective approach to enhance the antitumor efficacy of bispecific antibodies.


Assuntos
Anticorpos Biespecíficos/farmacologia , Complexo CD3/imunologia , Antígeno Carcinoembrionário/imunologia , Imunoterapia/métodos , Polietilenoglicóis/química , Animais , Anticorpos Biespecíficos/química , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Meia-Vida , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Masculino , Camundongos SCID , Neoplasias/terapia , Ratos Sprague-Dawley , Anticorpos de Domínio Único/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Drug Deliv ; 25(1): 1066-1079, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29718725

RESUMO

Anti-mPEG/anti-human epidermal growth factor receptor 2 (HER2) bispecific antibodies (BsAbs) non-covalently bound to a docetaxel (DTX)-loaded mPEGylated lecithin-stabilized micellar drug delivery system (LsbMDDs) were endowed with active targetability to improve the chemotherapeutic efficacy of DTX. DTX-loaded mPEGylated LsbMDDs formulations were prepared using lecithin/DSPE-PEG(2K or 5K) nanosuspensions to hydrate the thin film, and then they were subjected to ultrasonication. Two BsAbs (anti-mPEG/anti-DNS or anti-HER2) were simply mixed with the LsbMDDs to form BsAbs-LsbMDDs formulations, respectively, referred as the DNS-LsbMDDs and HER2-LsbMDDs. Results demonstrated that the physical characteristics of the BsAbs-LsbMDDs were similar to those of the plain LsbMDDs but more slowly released DTX than that from the LsbMDDs. Results also showed that the HER2-LsbMDDs suppressed the growth of HER2-expressing MCF-7/HER2 tumors, increasing the amount taken up via an endocytosis pathway leading to high drug accumulation and longer retention in the tumor. In conclusion, the BsAbs-LsbMDDs preserved the physical properties of the LsbMDDs and actively targeted tumors with a drug cargo to enhance drug accumulation in tumors leading to greater antitumor activity against antigen-positive tumors.


Assuntos
Anticorpos Biespecíficos/química , Antineoplásicos/química , Portadores de Fármacos/química , Nanopartículas/química , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Receptor ErbB-2/antagonistas & inibidores , Taxoides/química , Animais , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Docetaxel , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Lecitinas/química , Células MCF-7 , Masculino , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Taxoides/farmacologia
17.
PLoS One ; 13(4): e0195823, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29672587

RESUMO

An increasing number of monoclonal antibodies and derivatives such as antibody-drug conjugates (ADC) are of the IgG1 and IgG4 isotype with distinct structural and functional properties. In cases where antibody-mediated cytotoxicity is not desired, IgG4 is often used, as its Fc region is relatively poor at inducing antibody-dependent cell-mediated or complement-dependent cytotoxicity. IgG4 ADCs with highly cytotoxic drugs against proliferating target cells but which lack or have diminished antibody effector functions against quiescent cells may have a favorable safety profile compared to IgG1. Another unique property of the IgG4 subclass is the capability to exchange half antibodies in vivo creating randomly bispecific antibodies. To investigate the functional properties of process-derived antibody species, and determine the influence of shuffling on the therapeutic efficacy, several model antibodies on the basis of the anti-CD138 antibody-drug conjugate BT062 (Indatuximab ravtansine) were generated: (I) A wild type nBT062, (II) a stable nBT062 comprising mutations to prevent half-antibody exchange, (III) a half nBT062 lacking covalent binding between two heavy chains and (IV) a stabilized, bispecific nBT062-natalizumab antibody with a second, monovalent specificity against CD49d. All nBT062 model variants were capable of CD138-specific binding and antigen-mediated internalization into cells. Furthermore, all nBT062 models inhibited tumor growth in vitro after conjugation with the maytansinoid DM4. The in vivo effects of the different molecular variants were assessed in the MAXF1322 xenograft model. The bispecific nBT062-natalizumab-DM4 demonstrated the least efficacy and was only moderately active even without the co-administration of a human IgG preparation. Wild type, stable and half nBT062-DM4 models demonstrated great anti-tumor activities. The efficacy of wild type and half nBT062-DM4 was reduced in the presence of IgG, while stable nBT062-DM4 was only marginally influenced. These pre-clinical data demonstrate the advantage of introducing half-antibody exchange-preventing mutations into therapeutic IgG4-based antibody drug-conjugates.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Imunoconjugados/farmacologia , Imunoglobulina G/farmacologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Variação Genética , Humanos , Imunoconjugados/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Mutação , Ligação Proteica , Ensaios Antitumorais Modelo de Xenoenxerto
19.
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
20.
Biotechnol Lett ; 40(5): 789-795, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29600425

RESUMO

OBJECTIVE: To promote targeting specificity of anti-CD47 agents, we have constructed a novel bispecific antibody fusion protein against EGFR and CD47, which may minimize the "off-target" effects caused by CD47 expression on red blood cells. RESULTS: The novel bispecific antibody fusion protein, denoted as Bi-SP could simultaneously bind to EGFR and CD47 and exhibited potent phagocytosis-stimulation effects in vitro. Bi-SP treatment with a low dose more effectively inhibited tumor growth than either EGFR-targeting antibody, Pan or the SIRPα variant-Fc (SIRPαV-Fc) in the A431 xenograft tumor model. In addition, the treatment with Bi-SP produced less red blood cell (RBC) losses than the SIRPαV-Fc treatment, suggesting its potential use for minimizing RBC toxicity in therapy. CONCLUSIONS: Bi-SP with improved therapeutic index has the potential to treat CD47+ and EGFR+ cancers in clinics.


Assuntos
Anticorpos Biespecíficos/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Antígeno CD47/antagonistas & inibidores , Carcinoma/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Camundongos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Índice Terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
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