RESUMEN
Chondroitin sulfate proteoglycan 4 (CSPG4) has been identified as a highly promising target antigen for immunotherapy of triple-negative breast cancer (TNBC). TNBC represents a highly aggressive heterogeneous group of tumors lacking expression of estrogen, progesterone and human epidermal growth factor receptor 2. TNBC is particularly prevalent among young premenopausal women. No suitable targeted therapies are currently available and therefore, novel agents for the targeted elimination of TNBC are urgently needed. Here, we present a novel cytolytic fusion protein (CFP), designated αCSPG4(scFv)-MAP, that consists of a high affinity CSPG4-specific single-chain antibody fragment (scFv) genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. Our data indicate that αCSPG4(scFv)-MAP efficiently targets CSPG4(+) TNBC-derived cell lines MDA-MB-231 and Hs 578T and potently inhibits their growth with IC50 values of â¼200 nM. Treatment with αCSPG(scFv)-MAP resulted in induction of the mitochondrial stress pathway by activation of caspase-9 as well as endonuclease G translocation to the nucleus, while induction of the caspase-3 apoptosis pathway was not detectable. Importantly, in vivo studies in mice bearing human breast cancer xenografts revealed efficient targeting to and accumulation of αCSPG4(scFv)-MAP at tumor sites resulting in prominent tumor regression. Taken together, this preclinical proof of concept study confirms the potential clinical value of αCSPG4(scFv)-MAP as a novel targeted approach for the elimination of CSPG4-positive TNBC.
Asunto(s)
Anticuerpos Monoclonales/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Proteínas tau/metabolismo , Animales , Biomarcadores , Biomarcadores de Tumor , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteoglicanos Tipo Condroitín Sulfato/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Ratones , Terapia Molecular Dirigida , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Tubulina (Proteína)/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas tau/genéticaRESUMEN
Classical immunotoxins compromise a binding component (for example, a ligand, antibody or fragment thereof) and a cytotoxic component, usually derived from bacteria or plants (for example, Pseudomonas exotoxin A or ricin). Despite successful testing in vitro, the clinical development of immunotoxins has been hampered by immunogenicity and unsatisfactory safety profiles. Therefore, research has focused on fully human pro-apoptotic components suitable for the development of cytolytic fusion proteins (CFP). We recently reported that human microtubule-associated protein tau (MAP) can induce apoptosis when delivered to rapidly proliferating cancer cells. Here, we describe a new fully human CFP called H22(scFv)-MAP, which specifically targets CD64(+) cells. We show that H22(scFv)-MAP can efficiently kill proliferating HL-60 pro-monocytic cells in vitro. In addition, the human CFP specifically eliminates polarized M1 macrophages in a transgenic mouse model of cutaneous chronic inflammation. Because M1 macrophages promote the pathogenesis of many chronic inflammatory diseases, targeting this cell population with H22(scFv)-MAP could help to treat diseases such as atopic dermatitis, rheumatoid arthritis and inflammatory bowel disease.
Asunto(s)
Polaridad Celular , Macrófagos/citología , Macrófagos/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas tau/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Polaridad Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Enfermedad Crónica , Humanos , Inflamación/patología , Interferón gamma/farmacología , Macrófagos/efectos de los fármacos , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Unión Proteica/efectos de los fármacos , Anticuerpos de Cadena Única/metabolismo , Piel/patologíaRESUMEN
Antibody-based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64-targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcαRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti-human CD89 single-chain Fragment variable and the well-characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC50 values in range 0.2-3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose-dependent apoptosis-mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89-specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89-ETA' as a potent, novel immunotherapeutic agent.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Neoplasias/inmunología , Leucemia Mieloide/inmunología , Receptores Fc/inmunología , ADP Ribosa Transferasas/inmunología , Anciano , Apoptosis/inmunología , Toxinas Bacterianas/inmunología , Exotoxinas/inmunología , Femenino , Células HL-60 , Humanos , Inmunoterapia/métodos , Interferón gamma/inmunología , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/inmunología , Células U937 , Factores de Virulencia/inmunología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Immunotoxins are fusion proteins that combine a targeting component such as an antibody fragment or ligand with a cytotoxic effector component that induces apoptosis in specific cell populations displaying the corresponding antigen or receptor. Human cytolytic fusion proteins (hCFPs) are less immunogenic than conventional immunotoxins because they contain human pro-apoptotic enzymes as effectors. However, one drawback of hCFPs is that target cells can protect themselves by expressing endogenous inhibitor proteins. Inhibitor-resistant enzyme mutants that maintain their cytotoxic activity are therefore promising effector domain candidates. We recently developed potent variants of the human ribonuclease angiogenin (Ang) that were either more active than the wild-type enzyme or less susceptible to inhibition because of their lower affinity for the ribonuclease inhibitor RNH1. However, combining the mutations was unsuccessful because although the enzyme retained its higher activity, its susceptibility to RNH1 reverted to wild-type levels. We therefore used molecular dynamic simulations to determine, at the atomic level, why the affinity for RNH1 reverted, and we developed strategies based on the introduction of further mutations to once again reduce the affinity of Ang for RNH1 while retaining its enhanced activity. We were able to generate a novel Ang variant with remarkable in vitro cytotoxicity against HL-60 cells and pro-inflammatory macrophages. We also demonstrated the pro-apoptotic potential of Ang-based hCFPs on cells freshly isolated from leukemia patients.
Asunto(s)
Leucemia/patología , Macrófagos/efectos de los fármacos , Ribonucleasa Pancreática/genética , Apoptosis , Proteínas Portadoras/metabolismo , Supervivencia Celular/efectos de los fármacos , Citotoxinas/genética , Citotoxinas/toxicidad , Citometría de Flujo , Células HL-60 , Humanos , Macrófagos/citología , Macrófagos/inmunología , Simulación de Dinámica Molecular , Mutación , Unión Proteica/genética , Ribonucleasa Pancreática/toxicidadRESUMEN
Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL) are rare lymphoproliferative cancer types. Although most HL patients can be cured by chemo- and radio-therapy, 4-50% of patients relapse and have a poor prognosis. The need for improved therapeutic options for patients with relapsed or refractory disease has been addressed by CD30-specific antibody-based immunotherapeutics. However, available CD30-specific monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) or chimeric immunotoxins suffer from the requirement of a functional host immunity, undesirable immune reactions or heterogeneity and instability, respectively. Here, we present a new fusion protein comprised of the CD30-specific antibody single-chain fragment Ki4(scFv) and the human pro-apoptotic effector protein, microtubule-associated protein tau (MAPT). Ki4(scFv)-MAP selectively induced apoptosis in rapidly proliferating L540cy, L428, and Karpas 299 cells in a dose-dependent manner. Tubulin polymerization assays confirmed that Ki4(scFv)-MAP stabilizes microtubules, suggesting a mechanism for its pro-apoptotic action. Dose-finding experiments proved that Ki4(scFv)-MAP is well tolerated in mice compared to the previously reported Ki4(scFv)-ETA'. Ki4(scFv)-MAP significantly inhibited growth of subcutaneous L540cy xenograft tumours in mice. Our data present a novel approach for the treatment of CD30(+) lymphomas, combining the binding specificity of a target-specific antibody fragment with the selective cytotoxicity of MAPT towards proliferating lymphoma cells.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Antígeno Ki-1/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/farmacología , Proteínas tau/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Antígeno Ki-1/inmunología , Ratones , Microtúbulos/metabolismo , Unión Proteica , Estabilidad Proteica , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas tau/inmunologíaAsunto(s)
Antígenos CD/inmunología , Inmunoterapia/métodos , Inmunotoxinas/uso terapéutico , Leucemia Mieloide/terapia , Receptores Fc/inmunología , Apoptosis/efectos de los fármacos , Humanos , Inmunotoxinas/farmacología , Leucemia Mieloide/patología , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Células Tumorales CultivadasRESUMEN
BACKGROUND AND AIMS: Antibodies targeting tumor necrosis factor-alpha [TNF-alpha] are a mainstay in the treatment of inflammatory bowel disease. However, they fail to demonstrate efficacy in a considerable proportion of patients. On the other hand, glycosylation of antibodies might influence not only their immunogenicity but also their structure and function. We investigated whether specific glycosylation patterns of the Fc-fragment would affect the immunogenicity of anti-TNF-alpha antibody in monocyte-derived dendritic cells. METHODS: The effect of a specific Fc-glycosylation pattern on antibody uptake by monocyte-derived dendritic cells [mo-DCs] and how this process shapes the immunologic profile of mo-DCs was investigated. Three N-glycoforms of the anti-TNF-alpha antibody adalimumab, that differed in the content of fucose or sialic acid, were tested: [1] mock treated Humira, abbreviated 'Fuc-G0', where the N-glycan mainly consist of fucose and N-acetylglucosamine [GlcNAc], without sialic acid; [2] 'Fuc-G2S1/G2S2' with fucose and alpha 2,6 linked sialic acid; and [3] 'G2S1/G2S2' with alpha 2,6 linked sialic acid, without fucose. RESULTS: Our data demonstrated that neither fucosylation nor sialylation of anti-TNF-Abs [Fuc-G0, FucG2S1/G2S2, G2S1/G2S2] influence their uptake by mo-DCs. Additionally, none of the differentially glycosylated antibodies altered CD80, CD86, CD273, CD274 levels on mo-DCs stimulated in with lipopolysaccharide in the presence of antibodies. Next, we evaluated the levels of cytokines in the supernatant of mo-DCs stimulated with lipopolysaccharide in the presence of Fuc-G0, Fuc-G2S1/G2S2 or G2S1/G2S2-glycosylated anti-TNF antibodies. Only IL-2 and IL-17 levels were downregulated, and IL-5 production was upregulated by uptake of Fuc-G0 antibodies, as compared to control without antibodies. CONCLUSIONS: The specific modification in the Fc-glycosylation pattern of anti-TNF-alpha Abs does not affect their immunogenicity under the tested conditions. As this study was limited to mo-DCs, further investigation is required to clarify whether Ab uptake into mo-DCs might change the immunological profile of T- and B-cells, in order to ultimately reduce the formation of anti-drug antibodies and to improve the patient care.
Asunto(s)
Adalimumab/farmacología , Células Dendríticas/efectos de los fármacos , Fucosa/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Inhibidores del Factor de Necrosis Tumoral/farmacología , Adalimumab/química , Técnicas de Cultivo de Célula , Glicosilación , Humanos , Monocitos , Inhibidores del Factor de Necrosis Tumoral/químicaRESUMEN
Some of the most promising small molecule toxins used to generate antibody drug conjugates (ADCs) include anti-mitotic agents (e.g., auristatin and its derivatives) which are designed to attack cancerous cells at their most vulnerable state during mitosis. We were interested in identifying a human cystostatic protein eventually showing comparable activities and allowing the generation of corresponding targeted fully human cytolytic fusion proteins. Recently, we identified the human microtubule associated protein tau (MAP tau), which binds specifically to tubulin and modulates the stability of microtubules, thereby blocking mitosis and presumably vesicular transport. By binding and stabilizing polymerized microtubule filaments, MAP tau-based fusion proteins skew microtubule dynamics towards cell cycle arrest and apoptosis. This biological activity makes rapidly proliferating cells (e.g., cancer and inflammatory cells) an excellent target for MAP tau-based targeted treatments. Their superior selectivity for proliferating cells confers additional selectivity towards upregulated tumor-associated antigens at their surface, thereby preventing off-target related toxicity against normal cells bearing tumor-associated antigens at physiologically normal to low levels. In this review, we highlight recent findings on MAP tau-based targeted cytolytic fusion proteins reported in preclinical immunotherapeutic studies.
RESUMEN
BACKGROUND: Targeted imaging and therapy (theranostics) is a promising approach for the simultaneous improvement of cancer diagnosis, prognosis and management. Therapeutic and imaging reagents are coupled to tumor-targeting molecules such as antibodies, providing a basis for truly personalized medicine. However, the development of antibody-drug conjugates with acceptable pharmaceutical properties is a complex process and several parameters must be optimized, such as the controlled conjugation method and the drug-to-antibody ratio. OBJECTIVE: The major aim of this work is to address fundamental key challenges for the development of versatile technology platform for generating homogenous immunotheranostic reagent. METHOD: We conjugated the theranostics reagent IRDye700dx to a recombinant antibody fusion protein containing a self-labeling protein (SNAP-tag) which provides a unique reaction site. RESULTS: The resulting conjugate was suitable for the imaging of cancer cells expressing the epidermal growth factor receptor and demonstrated potent phototherapeutic and imaging activities against them. CONCLUSION: Here, we describe a simple, rapid and robust site-directed labeling method that can be used to generate homogeneous immunoconjugate with defined pharmacological properties.
Asunto(s)
Anticuerpos/uso terapéutico , Neoplasias/tratamiento farmacológico , Nanomedicina Teranóstica , Anticuerpos/química , Relación Dosis-Respuesta a Droga , Receptores ErbB/análisis , Receptores ErbB/biosíntesis , Humanos , Indoles/química , Indoles/uso terapéutico , Estructura Molecular , Compuestos de Organosilicio/química , Compuestos de Organosilicio/uso terapéutico , Fotoquimioterapia , Relación Estructura-ActividadRESUMEN
Triple-negative breast cancer (TNBC) is associated with poor prognosis and high prevalence among young premenopausal women. Unlike in other breast cancer subtypes, no targeted therapy is currently available. Overexpression of epithelial cell adhesion molecule (EpCAM) in 60% of TNBC tumors correlates with poorer prognosis and is associated with cancer stem cell phenotype. Thus, selective elimination of EpCAM(+) TNBC tumor cells is of clinical importance. Therefore, we constructed a fully human targeted cytolytic fusion protein, designated GbR201K-αEpCAM(scFv), in which an EpCAM-selective single-chain antibody fragment (scFv) is genetically fused to a granzyme B (Gb) mutant with reduced sensitivity to its natural inhibitor serpin B9. In vitro studies confirmed its specific binding, internalization and cytotoxicity toward a panel of EpCAM-expressing TNBC cells. Biodistribution kinetics and tumor-targeting efficacy using MDA-MB-468 cells in a human TNBC xenograft model in mice revealed selective accumulation of GbR201K-αEpCAM(scFv) in the tumors after i.v. injection. Moreover, treatment of tumor-bearing mice demonstrated a prominent inhibition of tumor growth of up to 50 % in this proof-of-concept study. Taken together, our results indicate that GbR201K-αEpCAM(scFv) is a promising novel targeted therapeutic for the treatment of TNBC.
Asunto(s)
Apoptosis/efectos de los fármacos , Moléculas de Adhesión Celular/antagonistas & inhibidores , Granzimas/farmacología , Inmunoterapia/métodos , Inmunotoxinas/farmacología , Anticuerpos de Cadena Única/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Carga Tumoral/efectos de los fármacos , Animales , Especificidad de Anticuerpos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Molécula de Adhesión Celular Epitelial , Femenino , Granzimas/genética , Granzimas/farmacocinética , Células HEK293 , Humanos , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Inmunotoxinas/farmacocinética , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/farmacocinética , Distribución Tisular , Transfección , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fc gamma receptor I (FcγRI, CD64) is a well-known target antigen for passive immunotherapy against acute myeloid leukemia and chronic myelomonocytic leukemia. We recently reported the preclinical immunotherapeutic potential of microtubule associated protein tau (MAP) against a variety of cancer types including breast carcinoma and Hodgkin's lymphoma. Here we demonstrate that the CD64-directed human cytolytic fusion protein H22(scFv)-MAP kills ex vivo 15-50% of CD64+ leukemic blasts derived from seven myeloid leukemia patients. Furthermore, in contrast to the nonspecific cytostatic agent paclitaxel, H22(scFv)-MAP showed no cytotoxicity towards healthy CD64+ PBMC-derived cells and macrophages. The targeted delivery of this microtubule stabilizing agent therefore offers a promising new strategy for specific treatment of CD64+ leukemia.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Mieloide Aguda , Proteínas Asociadas a Microtúbulos/farmacología , Terapia Molecular Dirigida/métodos , Receptores de IgG , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Humanos , Inmunotoxinas/farmacología , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Macrophages are key players in controlling the immune response that can adapt to microenvironmental signals. This results in distinct polarization states (classical M1 or alternative M2), that play a differential role in immune regulation. In general, the M1 contribute to onset of inflammation, whereas the M2 orchestrate resolution and repair, whereby failure to switch from predominantly M1 to M2 reinforces a pro-inflammatory environment and chronic inflammation. Here, we show selective elimination of M1 macrophages in vitro by a range of CD64-targeted immunotoxins, including H22(scFv)-ETA'. After re-polarization of already polarized macrophages, still only M1 polarization showed sensitivity toward CD64-directed immunotoxins. The selectivity for M1 was found linked to reduced endosomal protease activity in M1 macrophages as demonstrated by inhibition of endosomal proteases. Using the H22(scFv)-ETA' in a transgenic mouse model for chronic cutaneous inflammation, the M1 specificity was confirmed in vivo and a beneficial effect on inflammation demonstrated. Also ex vivo on skin biopsies from atopic dermatitis and diabetes type II patients with chronically-inflamed skin, a clear M1 specific effect was found. This indicates the potential relevance for human application. Our data show that targeting M1 macrophages through CD64 can be instrumental in developing novel intervention strategies for chronic inflammatory conditions.
Asunto(s)
Dermatitis/inmunología , Macrófagos/inmunología , Receptores de IgG/inmunología , Animales , Diferenciación Celular/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones TransgénicosRESUMEN
The treatment of rhabdomyosarcoma (RMS) is challenging, and the prognosis remains especially poor for high-grade RMS with metastasis. The conventional treatment of RMS is based on multi-agent chemotherapy combined with resection and radiotherapy, which are often marked by low success rate. Alternative therapeutic options include the combination of standard treatments with immunotherapy. We generated a microtubule-associated protein (MAP)-based fully human cytolytic fusion protein (hCFP) targeting the fetal acetylcholine receptor, which is expressed on RMS cells. We were able to express and purify functional scFv35-MAP from Escherichia coli cells. Moreover, we found that scFv35-MAP is rapidly internalized by target cells after binding its receptor, and exhibits specific cytotoxicity toward FL-OH1 and RD cells in vitro. We also confirmed that scFv35-MAP induces apoptosis in FL-OH1 and RD cells. The in vivo potential of scFv35-MAP will need to be considered in further studies.
Asunto(s)
Apoptosis/efectos de los fármacos , Antagonistas Colinérgicos/farmacología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Recombinantes de Fusión/farmacología , Rabdomiosarcoma/tratamiento farmacológico , Camptotecina/farmacología , Línea Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Inmunoterapia , Unión Proteica , Receptores Colinérgicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Células U937RESUMEN
Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays a critical role in many inflammatory diseases. Soluble TNF can be neutralized by monoclonal antibodies (mAbs), and this is a widely-used therapeutic approach. However, some patients do not respond to anti-TNF therapy due to the increased expression of CD64 on monocytes and macrophages. A recent study has shown that CD64 captures anti-TNF mAbs via their Fcγ domain, which induces the transcription of pro-inflammatory genes. Specific blocking of CD64 could therefore be a promising strategy to improve the response to anti-TNF therapy. We used the CD64-specific antibody fragment H22(scFv) and tested its activity against the human CD64(+) cell line HL-60. When stimulated with interferon gamma (IFN-γ), these cells represent a pro-inflammatory phenotype of the monocyte/macrophage lineage. We found that H22(scFv) binds selectively to and blocks CD64, preventing the capture of anti-TNF mAb. Importantly, H22(scFv) itself does not induce CD64 activation. We also found that transmembrane TNF on HL-60 cells stimulated with IFN-γ also contributes to the capture of anti-TNF mAb, although via their Fab domain. In conclusion, the specific blocking of CD64 by H22(scFv) could be used a possible anti-inflammatory mechanism for potentiating the effect of anti-TNF antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores de IgG/inmunología , Anticuerpos de Cadena Única/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos/inmunología , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Células HL-60 , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Interferón gamma/inmunología , Interferón gamma/farmacología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-8/genética , Interleucina-8/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Unión Proteica/inmunología , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/farmacología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The treatment of rhabdomyosarcoma (RMS) remains challenging, with metastatic and alveolar RMS offering a particularly poor prognosis. Therefore, the identification and evaluation of novel antigens, which are suitable targets for immunotherapy, is one attractive possibility to improve the treatment of this disease. Here we show that chondroitin sulfate proteoglycan 4 (CSPG4) is expressed on RMS cell lines and RMS patient material. We evaluated the immunotoxin (IT) αMCSP-ETA', which specifically recognizes CSPG4 on the RMS cell lines RD, FL-OH1, TE-671 and Rh30. It is internalized rapidly, induces apoptosis and thus kills RMS cells selectively. We also demonstrate the specific binding of this IT to RMS primary tumor material from three different patients.
Asunto(s)
ADP Ribosa Transferasas/farmacología , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/farmacología , Proteoglicanos Tipo Condroitín Sulfato/inmunología , Exotoxinas/farmacología , Inmunotoxinas/farmacología , Proteínas de la Membrana/inmunología , Rabdomiosarcoma/patología , Anticuerpos de Cadena Única/farmacología , Factores de Virulencia/farmacología , ADP Ribosa Transferasas/metabolismo , Toxinas Bacterianas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Relación Dosis-Respuesta a Droga , Exotoxinas/metabolismo , Humanos , Inmunotoxinas/inmunología , Inmunotoxinas/metabolismo , Concentración 50 Inhibidora , Proteínas de la Membrana/metabolismo , Unión Proteica , Rabdomiosarcoma/inmunología , Rabdomiosarcoma/metabolismo , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo , Factores de Tiempo , Factores de Virulencia/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMEN
In normal epithelia, the epithelial cell adhesion molecule (EpCAM) expression is relatively low and only present at the basolateral cell surface. In contrast, EpCAM is aberrantly overexpressed in various human carcinomas. Therefore, EpCAM is considered to be a highly promising target for antibody-based cancer immunotherapy. Here, we present a new and fully human cytolytic fusion protein (CFP), designated "anti-EpCAM(scFv)-MAP," that is comprised of an EpCAM-specific antibody fragment (scFv) genetically fused to the microtubule-associated protein tau (MAP). Anti-EpCAM(scFv)-MAP shows potent EpCAM-restricted proapoptotic activity toward rapidly proliferating carcinoma cells. In vitro assays confirmed that treatment with anti-EpCAM(scFv)-MAP resulted in the colocalization and stabilization of microtubules, suggesting that this could be the potential mode of action. Dose-finding experiments indicated that anti-EpCAM(scFv)-MAP is well tolerated in mice. Using noninvasive far-red in vivo imaging in a tumor xenograft mouse model, we further demonstrated that anti-EpCAM(scFv)-MAP inhibited tumor growth in vivo. In conclusion, our data suggest that anti-EpCAM(scFv)-MAP may be of therapeutic value for the targeted elimination of EpCAM(+) carcinomas.
Asunto(s)
Antígenos de Neoplasias/química , Moléculas de Adhesión Celular/química , Neoplasias/terapia , Proteínas tau/química , Animales , Apoptosis , Carcinoma/metabolismo , Línea Celular Tumoral , Proliferación Celular , Molécula de Adhesión Celular Epitelial , Femenino , Células HEK293 , Humanos , Inmunoterapia/métodos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Sistemas de Lectura Abierta , Unión Proteica , Proteínas Recombinantes de Fusión/química , Tubulina (Proteína)/químicaRESUMEN
Immunotoxins are promising targeted therapeutic agents comprising an antibody-based ligand that specifically binds to diseased cells, and a pro-apoptotic protein. Toxic components from bacteria or plants can trigger a neutralizing immune response, so that human effector molecules are more suitable. In this context, the protease granzyme B has been successfully tested in cytotoxicity assays against different cancer cells in vitro and in vivo. Our aim here was to introduce granzyme M as an alternative and novel component of human cytolytic fusion proteins. We fused it to the humanized single-chain antibody fragment (scFv) H22 which specifically binds to CD64, an FcγRI receptor overexpressed on activated myeloid cells and leukemic cells. We show that the humanized cytolytic fusion protein Gm-H22(scFv) specifically targets the acute myeloid leukemia cell line HL60 in vitro and is cytotoxic with an IC50 between 1.2 and 6.4 nM. These findings were confirmed ex vivo using leukemic primary cells from patients, which were killed by granzyme M despite the presence of the granzyme B inhibitor serpin B9. In conclusion, granzyme M is a promising new cell-death inducing component for hCFPs because it specifically and efficiently kills target cells when fused to a targeting component.
Asunto(s)
Granzimas/inmunología , Inmunotoxinas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Granzimas/genética , Granzimas/metabolismo , Células HEK293 , Células HL-60 , Humanos , Inmunotoxinas/farmacología , Células K562 , Leucemia/inmunología , Leucemia/metabolismo , Leucemia/patología , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Células Tumorales CultivadasRESUMEN
Diseases caused by chronic inflammation (e.g., arthritis, multiple sclerosis and diabetic ulcers) are multicausal, thus making treatment difficult and inefficient. Due to the age-associated nature of most of these disorders and the demographic transition towards an overall older population, efficient therapeutic intervention strategies will need to be developed in the near future. Over the past decades, elimination of activated macrophages using CD64-targeting immunotoxins has proven to be a promising way of resolving inflammation in animal models. More recent data have shown that the M1-polarized population of activated macrophages in particular is critically involved in the chronic phase. We recapitulate the latest progress in the development of IT. These have advanced from full-length antibodies, chemically coupled to bacterial toxins, into single chain variants of antibodies, genetically fused with fully human enzymes. These improvements have increased the range of possible target diseases, which now include chronic inflammatory diseases. At present there are no therapeutic strategies focusing on macrophages to treat chronic disorders. In this review, we focus on the role of different polarized macrophages and the potential of CD64-based IT to intervene in the process of chronic inflammation.
Asunto(s)
Inmunotoxinas/uso terapéutico , Inflamación/tratamiento farmacológico , Macrófagos/inmunología , Receptores de IgG/inmunología , Animales , Enfermedad Crónica , HumanosRESUMEN
The asymmetric reduction of ketones is performed by using lyophilized whole cells in neat substrates with defined water activity (a(w)). Ketones and alcohols prone to be unstable in aqueous media can now be converted via biocatalysis.