RESUMEN
Introduction: Despite advances in treating high-risk neuroblastoma, 50-60% of patients still suffer relapse, necessitating new treatment options. Bispecific trifunctional antibodies (trAbs) are a promising new class of immunotherapy. TrAbs are heterodimeric IgG-like molecules that bind CD3 and a tumor-associated antigen simultaneously, whereby inducing a TCR-independent anti-cancer T cell response. Moreover, via their functional Fc region they recruit and activate cells of the innate immune system like antigen-presenting cells potentially enhancing induction of adaptive tumor-specific immune responses. Methods: We used the SUREK trAb, which is bispecific for GD2 and murine Cd3. Tumor-blind trAb and the monoclonal ch14.18 antibody were used as controls. A co-culture model of murine dendritic cells (DCs), T cells and a neuroblastoma cell line was established to evaluate the cytotoxic effect and the T cell effector function in vitro. Expression of immune checkpoint molecules on tumor-infiltrating T cells and the induction of an anti-neuroblastoma immune response using a combination of whole cell vaccination and trAb therapy was investigated in a syngeneic immunocompetent neuroblastoma mouse model (NXS2 in A/J background). Finally, vaccinated mice were assessed for the presence of neuroblastoma-directed antibodies. We show that SUREK trAb-mediated effective killing of NXS2 cells in vitro was strictly dependent on the combined presence of DCs and T cells. Results: Using a syngeneic neuroblastoma mouse model, we showed that vaccination with irradiated tumor cells combined with SUREK trAb treatment significantly prolonged survival of tumor challenged mice and partially prevent tumor outgrowth compared to tumor vaccination alone. Treatment led to upregulation of programmed cell death protein 1 (Pd-1) on tumor infiltrating T cells and combination with anti-Pd-1 checkpoint inhibition enhanced the NXS2-directed humoral immune response. Conclusion: Here, we provide first preclinical evidence that a tumor vaccination combined with SUREK trAb therapy induces an endogenous anti-neuroblastoma immune response reducing tumor recurrence. Furthermore, a combination with anti-Pd-1 immune checkpoint blockade might even further improve this promising immunotherapeutic concept in order to prevent relapse in high-risk neuroblastoma patients.
Asunto(s)
Anticuerpos Biespecíficos , Antineoplásicos , Neuroblastoma , Animales , Ratones , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Linfocitos T , Antineoplásicos/uso terapéutico , Neuroblastoma/patologíaRESUMEN
BACKGROUND: Intraoperative blood salvage (IBS) is regarded as an alternative to allogeneic blood transfusion excluding the risks associated with allogeneic blood. Currently, IBS is generally avoided in tumor surgeries due to concern for potential metastasis caused by residual tumor cells in the erythrocyte concentrate. METHODS: The feasibility, efficacy and safety aspects of the new developed Catuvab procedure using the bispecific trifunctional antibody Catumaxomab was investigated in an ex-vivo pilot study in order to remove residual EpCAM positive tumor cells from the autologous erythrocyte concentrates (EC) from various cancer patients, generated by a IBS device. RESULTS: Tumor cells in intraoperative blood were detected in 10 of 16 patient samples in the range of 69-2.6 × 105 but no residual malignant cells in the final erythrocyte concentrates after Catuvab procedure. IL-6 and IL-8 as pro-inflammatory cytokines released during surgery, were lowered in mean 28-fold and 52-fold during the Catuvab procedure, respectively, whereas Catumaxomab antibody was detected in 8 of 16 of the final EC products at a considerable decreased and uncritical residual amount (37 ng in mean). CONCLUSION: The preliminary study results indicate efficacy and feasibility of the new medical device Catuvab allowing potentially the reinfusion of autologous erythrocyte concentrates (EC) produced by IBS device during oncological high blood loss surgery. An open-label, multicenter clinical study on the removal of EpCAM-positive tumor cells from blood collected during tumor surgery using the Catuvab device is initiated to validate these encouraging results.
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Anticuerpos Biespecíficos/farmacología , Molécula de Adhesión Celular Epitelial/metabolismo , Células Neoplásicas Circulantes/metabolismo , Recuperación de Sangre Operatoria/instrumentación , Anciano , Eritrocitos/metabolismo , Estudios de Factibilidad , Humanos , Neoplasias/cirugía , Recuperación de Sangre Operatoria/métodos , Proyectos PilotoRESUMEN
BACKGROUND: Neuroblastoma is the most common extracranial solid tumor of childhood. Patients with high-risk disease undergo extremely aggressive therapy and nonetheless have cure rates below 50%. Treatment with the ch14.18 monoclonal antibody (dinutuximab beta), directed against the GD2 disialoganglioside, improved 5-year event-free survival in high-risk patients when administered in postconsolidation therapy and was recently implemented in standard therapy. Relapse still occurred in 57% of these patients, necessitating new therapeutic options. Bispecific trifunctional antibodies (trAbs) are IgG-like molecules directed against T cells and cancer surface antigens, redirecting T cells (via their CD3 specificity) and accessory immune cells (via their functioning Fc-fragment) toward tumor cells. We sought proof-of-concept for GD2/CD3-directed trAb efficacy against neuroblastoma. METHODS: We used two GD2-specific trAbs differing only in their CD3-binding specificity: EKTOMUN (GD2/human CD3) and SUREK (GD2/mouse Cd3). This allowed trAb evaluation in human and murine experimental settings. Tumor-blind trAb and the ch14.18 antibody were used as controls. A coculture model of human peripheral blood mononuclear cells (PBMCs) and neuroblastoma cell lines was established to evaluate trAb antitumor efficacy by assessing expression of T-cell surface markers for activation, proinflammatory cytokine release and cytotoxicity assays. Characteristics of tumor-infiltrating T cells and response of neuroblastoma metastases to SUREK treatment were investigated in a syngeneic immunocompetent neuroblastoma mouse model mimicking minimal residual disease. RESULTS: We show that EKTOMUN treatment caused effector cell activation and release of proinflammatory cytokines in coculture with neuroblastoma cell lines. Furthermore, EKTOMUN mediated GD2-dependent cytotoxic effects in human neuroblastoma cell lines in coculture with PBMCs, irrespective of the level of target antigen expression. This effect was dependent on the presence of accessory immune cells. Treatment with SUREK reduced the intratumor Cd4/Cd8 ratio and activated tumor infiltrating T cells in vivo. In a minimal residual disease model for neuroblastoma, we demonstrated that single-agent treatment with SUREK strongly reduced or eliminated neuroblastoma metastases in vivo. SUREK as well as EKTOMUN demonstrated superior tumor control compared with the anti-GD2 antibody, ch14.18. CONCLUSIONS: Here we provide proof-of-concept for EKTOMUN preclinical efficacy against neuroblastoma, presenting this bispecific trAb as a promising new agent to fight neuroblastoma.
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Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Inmunoterapia/métodos , Neuroblastoma/tratamiento farmacológico , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Metástasis de la NeoplasiaRESUMEN
Transurethral resection of the tumor (TUR-B) followed by adjuvant intravesical treatment with cytostatic drugs or Bacillus Calmette-Guérin (BCG) as standard therapy of non-muscle-invasive bladder cancer (NMIBC) is associated with a high recurrence rate of about 60-70%, considerable side effects and requires close monitoring. Alternative treatment options are warranted. Two patients with epithelial cell adhesion molecule (EpCAM)-positive recurrent non-muscle invasive bladder cancer were treated the first time by an intravesical administration of the trifunctional bispecific EpCAM targeting antibody catumaxomab (total dosage of 470 and 1120 µg, respectively). The binding and killing activity of catumaxomab in urine milieu was evaluated in vitro. In contrast to its previous systemic application catumaxomab was well tolerated without any obvious signs of toxicity. Relevant cytokine plasma levels were not detected and no significant systemic drug release was observed. The induction of a human anti-mouse-antibody (HAMA) reaction was either absent or untypically weak contrary to the high immunogenicity of intraperitoneal applied catumaxomab. Tumor cells that were detectable in urine patient samples disappeared after catumaxomab therapy. Endoscopically confirmed recurrence-free intervals were 32 and 25 months. Our data suggest that intravesical administration of catumaxomab in NMIBC is feasible, safe and efficacious, thus arguing for further clinical development of catumaxomab in this indication.
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Anticuerpos Biespecíficos/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Microambiente Tumoral/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Anciano , Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cistoscopía , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Estadificación de Neoplasias , Unión Proteica , Retratamiento , Resultado del Tratamiento , Microambiente Tumoral/inmunología , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/inmunologíaAsunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trastornos Linfoproliferativos/etiología , Trastornos Linfoproliferativos/terapia , Translocación Genética , Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/farmacología , Complejo CD3/antagonistas & inhibidores , Niño , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 8 , Monitoreo de Drogas , Humanos , Inmunoterapia , Trastornos Linfoproliferativos/mortalidad , Trastornos Linfoproliferativos/patología , Masculino , Estadificación de Neoplasias , Recurrencia , Resultado del TratamientoRESUMEN
Combinatorial approaches of immunotherapy hold great promise for the treatment of malignant disease. Here, we examined the potential of combining an immune checkpoint inhibitor and trifunctional bispecific antibodies (trAbs) in a preclinical melanoma mouse model using surrogate antibodies of Ipilimumab and Catumaxomab, both of which have already been approved for clinical use. The specific binding arms of trAbs redirect T cells to tumor cells and trigger direct cytotoxicity, while the Fc region activates accessory cells eventually giving rise to a long-lasting immunologic memory. We show here that T cells redirected to tumor cells by trAbs strongly upregulate CTLA-4 expression in vitro and in vivo. This suggested that blocking of CTLA-4 in combination with trAb treatment enhances T-cell activation in a tumor-selective manner. However, when mice were challenged with melanoma cells and subsequently treated with antibodies, there was only a moderate beneficial effect of the combinatorial approach in vivo with regard to direct tumor destruction in comparison to trAb therapy alone. By contrast, a significantly improved vaccination effect was obtained by CTLA-4 blocking during trAb-dependent immunization. This resulted in enhanced rejection of melanoma cells given after pre-immunization. The improved immunologic memory induced by the combinatorial approach correlated with an increased humoral antitumor response as measured in the sera and an expansion of CD4+ memory T cells found in the spleens.
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Anticuerpos Biespecíficos/administración & dosificación , Linfocitos T CD4-Positivos/trasplante , Antígeno CTLA-4/genética , Inmunoterapia/métodos , Melanoma Experimental/terapia , Animales , Anticuerpos Biespecíficos/farmacología , Linfocitos T CD4-Positivos/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Memoria Inmunológica , Ipilimumab/administración & dosificación , Ipilimumab/farmacología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Bazo/citología , Bazo/inmunología , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: EpCAM is overexpressed in many neoplasms including ovarian cancer. We screened the EpCAM-coding gene TACSTD1 for single nucleotide polymorphisms (SNPs), which could alter ovarian cancer risk, impact upon disease progression, or alter binding of the therapeutic EpCAM-binding antibody, catumaxomab. METHODS: DNA fragments of 10 healthy volunteers were analyzed to identify SNPs. Subsequently, DNA of ovarian cancer patients (n = 117) and age-matched healthy controls (n = 115) was genotyped by restriction fragment length polymorphism and pyrosequencing. TACSTD1 genotypes 4461T>C were cloned into a gene expression vector; Hek293 cells were subsequently used for stable transfection. FACS analysis of the transfected Hek293 cells was conducted with HO-3-the EpCAM binding site of catumaxomab-to determine antibody binding. RESULTS: One SNP was detected in exon 3 (4461T>C; rs1126497), resulting in an amino acid exchange at position 115 (Met115Thr). Another polymorphism was found in the 3'UTR (17225A>G; rs1421). Genotyping of patients and controls for these SNPs did not reveal significant differences in genotype distribution. Regarding 17225A>G, the homozygous AA-genotype was associated with diminished progression-free survival (PFS; p = 0.032). Overall survival, FIGO-stage, grading, and age did not differ significantly between genotypes. FACS analysis of transfected Hek293 cells overexpressing EpCAM 115Met/Thr showed binding of HO-3 to both proteins. CONCLUSIONS: The AA-genotype of 17225A>G seems to be associated with diminished PFS in ovarian cancer patients. The amino acid exchange resulting from 4461T>C does not appear to alter binding of HO-3, suggesting that treatment with catumaxomab can be offered to patients regardless of their TACSTD1-genotype.
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Anticuerpos Biespecíficos/genética , Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Neoplasias Ováricas/genética , Adulto , Anciano , Estudios de Casos y Controles , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/uso terapéutico , Molécula de Adhesión Celular Epitelial , Femenino , Genotipo , Células HEK293 , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Riesgo , TransfecciónRESUMEN
Trifunctional bispecific antibodies (trAb) are novel anticancer drugs that recruit and activate different types of immune effector cells at the targeted tumor. Thus, tumor cells are effectively eliminated and a long-lasting tumor-specific T-cell memory is induced. The trAb Ektomab is directed against human CD3 on T cells and the tumor-associated ganglioside GD2, which is an attractive target for immunotherapy of melanoma in humans. To optimize clinical applicability, we studied different application routes with respect to therapeutic efficacy and tolerability by using the surrogate trAb Surek (anti-GD2 × anti-murine CD3) and a murine melanoma engineered to express GD2. We show that subcutaneous injection of the trAb is superior to the intravenous delivery pathway, which is the standard application route for therapeutic antibodies. Despite lower plasma levels after subcutaneous administration, the same tumor-protective potential was observed in vivo compared with intravenous administration of Surek. However, subcutaneously delivered Surek showed better tolerability. This could be explained by a continuous release of the antibody leading to constant plasma levels and a delayed induction of proinflammatory cytokines. Importantly, the induction of counter-regulatory mechanisms was reduced after subcutaneous application. These findings are relevant for the clinical application of trifunctional bispecific antibodies and, possibly, also other immunoglobulin constructs. Mol Cancer Ther; 14(8); 1877-83. ©2015 AACR.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antineoplásicos/farmacología , Gangliósidos/antagonistas & inhibidores , Administración Intravenosa , Animales , Anticuerpos Biespecíficos/administración & dosificación , Antineoplásicos/administración & dosificación , Disponibilidad Biológica , Línea Celular Tumoral , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Inyecciones Subcutáneas , Activación de Linfocitos/inmunología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/mortalidad , Melanoma Experimental/patología , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Trifunctional bispecific antibodies (trAbs) used in tumor immunotherapy have the unique ability to recruit T cells toward antigens on the tumor cell surface and, moreover, to activate accessory cells through their immunoglobulin Fc region interacting with activating Fcγ receptors. This scenario gives rise to additional costimulatory signals required for T cell-mediated tumor cell destruction and induction of an immunologic memory. Here we show in an in vitro system that most effective trAb-dependent T-cell activation and tumor cell elimination are achieved in the presence of dendritic cells (DCs). On the basis of these findings, we devise a novel approach of cancer immunotherapy that combines the specific advantages of trAbs with those of DC-based vaccination. Simultaneous delivery of trAbs and in vitro differentiated DCs resulted in a markedly improved tumor rejection in a murine melanoma model compared with monotherapy.
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Anticuerpos Biespecíficos/farmacología , Células Dendríticas/inmunología , Inmunoterapia , Melanoma/terapia , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Femenino , Melanoma/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Trifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model. METHODS: We generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated. RESULTS: Despite its low affinity of approximately 10(7) M(-1) for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC(50) of 70 ng/ml [0.47 nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4(+) and CD8(+) T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced. CONCLUSION: Our data suggest that Surek revealed strong tumor elimination and anti-tumor immunization capabilities. The results warrant further clinical development of the human therapeutic equivalent antibody Ektomab.
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Anticuerpos Biespecíficos/uso terapéutico , Especificidad de Anticuerpos/inmunología , Gangliósidos/inmunología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Traslado Adoptivo , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/farmacología , Anticuerpos Antineoplásicos/uso terapéutico , Citotoxicidad Inmunológica/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Humanos , Sueros Inmunes , Inmunidad Humoral/efectos de los fármacos , Inmunidad Humoral/inmunología , Inmunización , Inmunoglobulina G/inmunología , Melanoma/sangre , Ratones , Neoplasias Cutáneas/sangre , Análisis de Supervivencia , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factores de Tiempo , Resultado del TratamientoRESUMEN
A major goal of tumor immunotherapy is the induction of long-lasting systemic T-cell immunity. Bispecific antibodies (bsAbs) that lack the immunoglobulin Fc region confer T-cell-mediated killing of tumor cells but do not induce long-term memory. In contrast, trifunctional bsAbs comprise an appropriate Fc region and, therefore, not only recruit T cells but also accessory cells that bear activating Fcγ receptors (FcγR), providing additional T-cell-activating signals and securing presentation of tumor-derived antigens to T cells. In this study, we show that trifunctional bsAbs induce a polyvalent T-cell response and, therefore, a vaccination effect. Mice were treated with melanoma cells and with a trifunctional bsAb directed against the melanoma target antigen ganglioside GD2 in addition to murine CD3. The trifunctional bsAb activated dendritic cells and induced a systemic immune response that was not replicated by treatment with the F(ab')2-counterpart lacking the Fc region. Restimulation of spleen and lymph node cells in vitro yielded T-cell lines that specifically produced interferon-γ in response to tumor. In addition, trifunctional bsAb-induced T cells recognized various specific peptides derived from melanoma-associated antigens. Moreover, these polyvalent responses proved to be tumor-suppressive and could not be induced by the corresponding bsF(ab')2-fragment. Taken together, our findings provide preclinical proof of concept that trifunctional bsAbs can induce tumor-specific T cells with defined antigen specificity.
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Anticuerpos Biespecíficos/farmacología , Vacunas contra el Cáncer/farmacología , Epítopos de Linfocito T/inmunología , Melanoma Experimental/terapia , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Complejo CD3/inmunología , Vacunas contra el Cáncer/inmunología , Femenino , Gangliósidos/inmunología , Activación de Linfocitos , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Trifunctional antibodies (trAbs) are promising novel anticancer biologics with a particular mode of action capable of linking innate with adaptive immunity. Based on their unique structure, trifunctional IgG-like heterodimeric antibodies, consisting of nonhuman mouse and rat immunoglobulin halves are able to redirect T lymphocytes, as well as accessory cells, to the tumor site. This recruitment of immune cells is accompanied by cellular activation events elicited by anti-CD3, as well as Fcγ-receptor engagement of trAbs supported by a proinflammatory Th1-biased cytokine milieu. All necessary immunological factors required for long-term vaccination-like effects are stimulated along trAb-mediated therapeutic interventions. Thus, the concerted interplay of antibody-dependent cellular cytotoxicity plus the polyclonal T-cell cytotoxicity and Fcγ-receptor-driven induction of long-lasting immune responses after the initial tumor cell elimination represent the major hallmarks of trAb-mediated treatment of malignant diseases.
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Inmunidad Adaptativa , Anticuerpos/uso terapéutico , Antineoplásicos/uso terapéutico , Inmunidad Innata , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos/inmunología , Antineoplásicos/inmunología , Biofarmacia , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Ensayos Clínicos como Asunto , Humanos , Neoplasias/inmunología , Linfocitos T/inmunologíaRESUMEN
Patients with malignant ascites secondary to primary carcinomas benefit from intraperitoneal therapy with the trifunctional antibody catumaxomab (anti-EpCAM × anti-CD3). Here, we report the analysis of peritoneal fluid samples from 258 patients with malignant ascites randomized to catumaxomab or control groups to investigate the molecular effects of catumaxomab treatment. In the catumaxomab group, tumor cell numbers and peritoneal levels of VEGF decreased, whereas the activation status of CD4(+) and CD8(+) T-cell populations increased more than two-fold after treatment. Notably, CD133(+)/EpCAM(+) cancer stem cells vanished from the catumaxomab samples but not from the control samples. In vitro investigations indicated that catumaxomab eliminated tumor cells in a manner associated with release of proinflammatory Th1 cytokines. Together, our findings show that catumaxomab therapy activates peritoneal T cells and eliminates EpCAM(+) tumor cells, establishing a molecular and cellular basis to understand in vivo efficacy within the immunosuppressed malignant ascites tissue microenvironment.
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Anticuerpos Biespecíficos/uso terapéutico , Ascitis/tratamiento farmacológico , Ascitis/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Humanos , Activación de Linfocitos , Monitoreo Fisiológico/métodosRESUMEN
AIMS: Catumaxomab is the first EMEA approved trifunctional anti-EpCAMxanti-CD3 antibody for the treatment of cancer patients with malignant ascites. A phase II pharmacokinetic study was conducted to determine local and systemic antibody concentrations and anti-drug antibody (ADA) development. METHODS: Thirteen cancer patients with symptomatic malignant ascites were treated with four ascending doses of 10, 20, 50, and 150 microg catumaxomab intraperitoneally (i.p.) infused on days 0, 3, 6 or 7 and 10. The pharmacokinetics of catumaxomab were studied by implementation of supportive data from a non clinical mouse tumour model. Additionally, ADA development was monitored. RESULTS: Ten out of 13 patients were evaluable for pharmacokinetic analysis. Catumaxomab became increasingly concentrated in ascites during the course of treatment, attaining effective concentrations in the ng ml(-1) range. Catumaxomab remained immunologically active even after several days in the circulation. The observed systemic catumaxomab exposure was low (<1%), with a maximal median plasma concentration (C(max)) of 403 pg ml(-1). The mean elimination half-life in the plasma was 2.13 days. All patients developed ADA, but not before the last infusion. High observed inter-individual variability and low systemic exposure may be explained by the inverse correlation between tumour burden, effector cell numbers and systemic antibody bioavailability as demonstrated in a defined mouse tumour model. CONCLUSIONS: Based on the high and effective local concentrations, low systemic exposure and acceptable safety profile, we confirmed that the i.p. application scheme of catumaxomab for the treatment of malignant ascites is appropriate.
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Anticuerpos Biespecíficos/farmacocinética , Factores Inmunológicos/farmacocinética , Neoplasias/tratamiento farmacológico , Anticuerpos Biespecíficos/sangre , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/sangre , Ascitis/metabolismo , Disponibilidad Biológica , Citocinas/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Factores Inmunológicos/sangre , Factores Inmunológicos/inmunología , Inyecciones IntraperitonealesRESUMEN
The Triomab family of trifunctional, bispecific antibodies that maintain an IgG-like shape are novel tumor targeting agents. These chimeras consist of two half antibodies, each with one light and one heavy chain, that originate from parental mouse IgG2a and rat IgG2b isotypes. This combination allows cost-effective biopharmaceutical manufacturing at an industrial scale since this specific mouse/rat isotype combination favors matching of corresponding antibody halves during production by means of quadroma technology. Whereas every Triomab family member is composed of an anti-CD3 rat IgG2b half antibody for T cell recognition, the antigen binding site presented by the mouse IgG2a isotype is exchangeable. Several Triomab antibodies have been generated that bind to tumor-associated antigens, e.g., EpCAM (catumaxomab), HER2/neu (ertumaxomab), CD20 (FBTA05), gangliosides GD2/GD3 (Ektomun), on appropriate tumor target cells associated with carcinomas, lymphomas or melanomas. Catumaxomab (Removab) was launched in Europe for treatment of malignant ascites in April 2009. Here, we report the structural and functional characterization of this product. Mass spectrometry revealed an intact mass of 150511 Dalton (Da) and 23717 Da, 24716 Da, 51957 Da and 52019 Da of the reduced and alkylated rat light chain, mouse light chain, rat heavy chain, mouse heavy chain chains, respectively. The observed masses were in agreement with the expected masses based on the amino acid sequence obtained from cDNA sequencing. The glycosylation profile was similar to other human IgG consisting of biantennary oligosaccharides with different numbers of terminal galactose. CD spectroscopy showed mainly beta-sheets secondary structure that is typical for IgG antibodies. Binding measurement revealed the unique trifunctional features of catumaxomab. Other analytical tools were used to evaluate characteristics of catumaxomab preparations, including the presence of isoforms and aggregates.
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Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Ratones , Peso Molecular , RatasRESUMEN
Human epidermal growth factor receptor 2 (HER2/neu) is an important target for the treatment of the breast cancers in which it is overexpressed. However, no approved anti-HER2/neu therapy is available for the majority of breast cancer patients, who express HER2/neu at low levels (with scores of 1+ or 2+/fluorescence in situ hybridization-negative). The trifunctional antibody ertumaxomab targets HER2/neu, CD3, and activating Fcgamma receptors. In presence of ertumaxomab, tri-cell complexes consisting of tumor cells, T cells, and accessory cells form to cause tumor cell lysis. In a phase I trial with metastatic breast cancer patients, ertumaxomab could be applied safely and resulted in radiographically confirmed clinical responses. In this study, we compare ertumaxomab- and trastuzumab-mediated killing of cancer cell lines that express HER2/neu at low and high levels. Under optimal conditions for trastuzumab-mediated destruction of HER2/neu-overexpressing cells, only ertumaxomab was able to mediate the elimination of tumor cell lines that express HER2/neu at low levels (1+). Ertumaxomab-mediated activity was accompanied by a Th1-based cytokine release, a unique mode of action of trifunctional antibodies. Competitive binding studies with trastuzumab and 520C9 mapped the binding site of ertumaxomab to the extracellular regions II and III of the HER2/neu ectodomain. This site is distinct from the binding site of trastuzumab, so that HER2/neu-expressing tumor cells can be eliminated by ertumaxomab in the presence of high amounts of trastuzumab. The ability of ertumaxomab to induce cytotoxicity against various tumor cell lines, including those with low HER2/neu antigen density, may provide a novel therapeutic option for breast cancer patients who are not eligible for trastuzumab treatment.
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Anticuerpos Biespecíficos/toxicidad , Anticuerpos Monoclonales/toxicidad , Neoplasias de la Mama/patología , Receptor ErbB-2/genética , Adenocarcinoma/patología , Antineoplásicos/toxicidad , Neoplasias del Ciego/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias del Íleon/patología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Neoplasias Pulmonares/patología , Receptor ErbB-2/efectos de los fármacosRESUMEN
Trifunctional bispecific antibodies can efficiently mediate tumor cell killing by redirecting T cells and immune accessory cells to the tumor cell. Here, we describe the new trifunctional antibody, Bi20 (FBTA05, anti-CD20 x anti-CD3), that connects B cells and T cells via its variable regions and recruits FcgammaRI(+) accessory immune cells via its Fc region. Bi20 mediated efficient and specific lysis of B-cell lines and of B cells with low CD20 expression levels that were derived from CLL patients. Remarkably, T-cell activation and tumor cell killing occurred in an entirely autologous setting without additional effector cells in 5 of 8 samples. In comparison, rituximab, a chimeric monoclonal CD20 antibody, demonstrated a significantly lower B-cell eradication rate. Additionally, Bi20, but not rituximab, upregulated the activation markers CD25 and CD69 on both CD4(+) and CD8(+) T cells in the presence of accessory immune cells. CD14(+) accessory cells and the monocyte cell line THP-1 were activated via binding of the Fc region of Bi20, given that T cells were simultaneously engaged by the antibody. Bi20 induced a strong Th1 cytokine pattern characterized by high IFN-gamma and very low IL-4 secretion. In conclusion, Bi20 may offer new immunotherapeutic options for the treatment of B-cell lymphomas.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos CD20/inmunología , Antineoplásicos/farmacología , Complejo CD3/inmunología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Animales , Anticuerpos Biespecíficos/farmacología , Linfoma de Burkitt/tratamiento farmacológico , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Focalización Isoeléctrica , Leucemia Linfocítica Crónica de Células B/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Depleción Linfocítica , Linfoma de Células B/inmunología , RatonesRESUMEN
OBJECTIVE: Use of a trifunctional bispecific antibody (trAb) given concomitantly with allogeneic cell therapy to achieve an anti tumor effect without graft-vs-host disease (GVHD). MATERIALS AND METHODS: A trAb-directed against murine CD3 and human epithelial cell adhesion molecule (EpCAM) (BiLu), was given alone or concomitantly with interleukin (IL)-2-activated (LAK) H-2(b) donor splenocytes to H-2(d/b) mice inoculated with murine melanoma cells transfected with human EpCAM. RESULTS: A total of 32/38 mice treated with BiLu and LAK splenocytes, were tumor-free survivors without GVHD for >200 days following inoculation of a 100% lethal tumor dose (5 x 10(4)). Of 28 disease-free surviving mice previously treated with LAK splenocytes and BiLu, 24 mice proved resistance to a second tumor challenge of 10(4) cells given >210 days following the first tumor inoculation with no evidence of disease for >150 days. In contrast, only 4 of 13 disease-free survivor mice treated with naïve splenocytes and BiLu, and 5 of 10 disease-free survivor controls treated with BiLu only, resisted the second tumor challenge. Induction of antitumor immunity was more efficient and long-lasting (>150 days) in mice previously injected with a lethal tumor cell dose of 5 x 10(4) cells than in mice previously inoculated with 5 x 10(3) tumor cells. CONCLUSION: Concomitantly treatment of allogeneic LAK cells and trAb-induced an efficient long-lasting antitumor immunity. Considering the documented efficacy of anti-EpCAM bispecific antibody in various metastatic cancers, clinical application of our approach may be justified in patients with minimal residual disease at high risk for tumor recurrence.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Enfermedad Injerto contra Huésped/prevención & control , Inmunoterapia Adoptiva , Linfocitos/inmunología , Melanoma Experimental/terapia , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Complejo CD3/inmunología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Línea Celular Tumoral , Cruzamientos Genéticos , Supervivencia sin Enfermedad , Molécula de Adhesión Celular Epitelial , Femenino , Enfermedad Injerto contra Huésped/inmunología , Humanos , Interleucina-2/farmacología , Transfusión de Linfocitos , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Factores de Tiempo , Transgenes , Trasplante HomólogoRESUMEN
Trifunctional antibodies are able to redirect T cells and Fcgamma receptor(+) accessory immune cells to tumor targets. The simultaneous activation of these different classes of effector cells results in efficient killing of the tumor cells by different mechanisms such as phagocytosis and perforin-mediated cytotoxicity. Here, we introduce 2 new trifunctional antibodies specific for human melanoma. These trifunctional antibodies recognize with one binding arm CD3 on human T cells. The other binding arm is directed against melanoma-associated proteoglycans or melanoma-associated gangliosides (GD2 as well as GD3). They mediate specific lysis of various melanoma cell lines in correlation with the level of antigen expression in short-term cytotoxicity experiments. A combination of the 2 trifunctional antibodies was equally or even more efficient. Moreover, they induced a strong Th1 cytokine pattern with high amounts of IFN-gamma and low or no IL-4. Accordingly, CD4(+) and especially CD8(+) T cells expanded, whereas B cells, NK cells and monocytes decreased. The cytokine response was up to 16-fold higher when tumor cells were present. IFN-gamma reached cytotoxic concentrations for SK-MEL-23 melanoma cells. The induction of a T-cell-activatory and melanoma cell-inhibitory cytokine milieu together with the redirection of T-cell- and accessory cell-mediated cytotoxicity are interesting features of these trifunctional antibodies. They may be a new option for the therapy of human malignant melanoma.