RESUMEN
CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antígenos CD20/inmunología , Antígenos de Neoplasias/inmunología , Proteínas del Sistema Complemento/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Leucemia Linfocítica Crónica de Células B/genética , Tetraspaninas/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Complemento C1q/inmunología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoglobulina G/inmunología , Leucemia Linfocítica Crónica de Células B/sangre , Mutación , Unión Proteica , Rituximab/farmacologíaRESUMEN
Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20+ B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab')2 fragments, as well as C1q-binding-deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted of specific complement components demonstrated that the observed lytic activity, which we termed "accessory CDC," remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR- B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into CD20+ cells: OFA-F(ab')2 fragments were able to induce CDC in the CD20+BCR+ cell population, but not in the CD20+BCR- population. Importantly, OFA-F(ab')2 fragments were able to induce CDC ex vivo in malignant B cells isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD20/metabolismo , Linfocitos B/efectos de los fármacos , Vía Clásica del Complemento , Inmunoterapia Adoptiva/métodos , Linfoma de Células B/terapia , Rituximab/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antígenos CD20/inmunología , Linfocitos B/inmunología , Línea Celular Tumoral , Complemento C1/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inmunoglobulina M/genética , Inmunoglobulina M/metabolismo , Linfoma de Células B/inmunología , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Rituximab/genética , Rituximab/uso terapéuticoRESUMEN
The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3-3 µM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 µM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD20/inmunología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Purinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Quinazolinonas/farmacología , Adenina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis/efectos de los fármacos , Western Blotting , Estudios de Casos y Controles , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Activación de Complemento/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , PiperidinasRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is a solid tumor type that arises in the squamous epithelial cells lining the mucosal surfaces of the upper aerodigestive tract. Long-term survival of patients with advanced disease stage remains disappointing with current treatment options. We show that tissue factor is abundantly expressed on patient-derived HNSCC cell lines, xenograft tumor material, and tumor biopsies from patients with HNSCC. Tisotumab vedotin (TV) is an antibody-drug conjugate (ADC) directed to tissue factor, a protein expressed in many solid tumors. HNSCC cells and xenograft tumors were efficiently eliminated in vitro and in vivo with TV-monotherapy compared with treatment with a control antibody conjugated to monomethyl auristatin E (MMAE). Antitumor activity of TV was also tested in vivo in combination with chemoradiotherapy, standard of care for patients with advanced stage HNSCC tumors outside the oral cavity. Preclinical studies showed that by adding TV to chemoradiotherapy, survival was markedly improved, and TV, not radiotherapy or chemotherapy, was the main driver of antitumor activity. Interestingly, TV-induced cell death in xenograft tumors showed an influx of macrophages indicative of a potential immune-mediated mode-of-action. In conclusion, on the basis of these preclinical data, TV may be a novel treatment modality for patients suffering from head and neck cancer and is hypothesized to improve efficacy of chemoradiotherapy. SIGNIFICANCE: This work shows preclinical in vitro and in vivo antitumor activity of the antibody-drug conjugate Tisotumab vedotin in head and neck cancer models, and enhanced activity in combination with chemoradiotherapy, supporting further clinical development for this cancer type.
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Neoplasias de Cabeza y Cuello , Inmunoconjugados , Humanos , Línea Celular Tumoral , Quimioradioterapia , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Tromboplastina , Ensayos Antitumor por Modelo de Xenoinjerto , AnimalesRESUMEN
BACKGROUND: Elevated tissue factor (TF) expression, although restricted in normal tissue, has been reported in multiple solid cancers, and expression has been associated with poor prognosis. This manuscript compares TF expression across various solid tumor types via immunohistochemistry in a single study, which has not been performed previously. AIMS: To increase insight in the prevalence and cellular localization of TF expression across solid cancer types, we performed a detailed and systematic analysis of TF expression in tumor tissue obtained from patients with ovarian, esophageal, bladder, cervical, endometrial, pancreatic, prostate, colon, breast, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma. The spatial and temporal variation of TF expression was analyzed over time and upon disease progression in patient-matched biopsies taken at different timepoints. In addition, TF expression in patient-matched primary tumor and metastatic lesions was also analyzed. METHODS AND RESULTS: TF expression was detected via immunohistochemistry (IHC) using a validated TF-specific antibody. TF was expressed in all cancer types tested, with highest prevalence in pancreatic cancer, cervical cancer, colon cancer, glioblastoma, HNSCC, and NSCLC, and lowest in breast cancer. Staining was predominantly membranous in pancreatic, cervical, and HNSCC, and cytoplasmic in glioblastoma and bladder cancer. In general, expression was consistent between biopsies obtained from the same patient over time, although variability was observed for individual patients. NSCLC biopsies of primary tumor and matched lymph node metastases showed no clear difference in TF expression overall, although individual patient changes were observed. CONCLUSION: This study shows that TF is expressed across a broad range of solid cancer types, and expression is present upon tumor dissemination and over the course of treatment.
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Carcinoma de Pulmón de Células no Pequeñas , Glioblastoma , Neoplasias de Cabeza y Cuello , Neoplasias Pulmonares , Masculino , Femenino , Humanos , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas de Cabeza y Cuello , Tromboplastina/análisis , Neoplasias Pulmonares/patologíaRESUMEN
Chronic lymphocytic leukemia (CLL) is an immunosuppressive disease characterized by increased infectious morbidity and inferior antitumor activity of immunotherapies. Targeted therapy with Bruton's tyrosine kinase inhibitors (BTKis) or the Bcl-2 inhibitor venetoclax has profoundly improved treatment outcomes in CLL. To overcome or prevent drug resistance and extend the duration of response after a time-limited therapy, combination regimens are tested. Anti-CD20 antibodies that recruit cell- and complement-mediated effector functions are commonly used. Epcoritamab (GEN3013), an anti-CD3×CD20 bispecific antibody that recruits T-cell effector functions, has demonstrated potent clinical activity in patients with relapsed CD20+ B-cell non-Hodgkin lymphoma. Development of CLL therapy is ongoing. To characterize epcoritamab-mediated cytotoxicity against primary CLL cells, peripheral blood mononuclear cells from treatment-naive and BTKi-treated patients, including patients progressing on therapy, were cultured with epcoritamab alone or in combination with venetoclax. Ongoing treatment with BTKi and high effector-to-target ratios were associated with superior in vitro cytotoxicity. Cytotoxic activity was independent of CD20 expression on CLL cells and observed in samples from patients whose condition progressed while receiving BTKi. Epcoritamab induced significant T-cell expansion, activation, and differentiation into Th1 and effector memory cells in all patient samples. In patient-derived xenografts, epcoritamab reduced the blood and spleen disease burden compared with that in mice receiving a nontargeting control. In vitro, the combination of venetoclax with epcoritamab induced superior killing of CLL cells than either agent alone. These data support the investigation of epcoritamab in combination with BTKis or venetoclax to consolidate responses and target emergent drug-resistant subclones.
Asunto(s)
Anticuerpos Biespecíficos , Antineoplásicos , Leucemia Linfocítica Crónica de Células B , Humanos , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Leucocitos Mononucleares , Antineoplásicos/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2 , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéuticoRESUMEN
BACKGROUND: HexaBody®-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC). METHODS: Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38. HexaBody-CD38-induced CDC, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), trogocytosis, and apoptosis were assessed using flow cytometry assays using tumour cell lines, and MM patient samples (CDC). CD38 enzymatic activity was measured using fluorescence spectroscopy. Anti-tumour activity of HexaBody-CD38 was assessed in patient-derived xenograft mouse models in vivo. FINDINGS: HexaBody-CD38 binds a unique epitope on CD38 and induced potent CDC in multiple myeloma (MM), acute myeloid leukaemia (AML), and B-cell non-Hodgkin lymphoma (B-NHL) cells. Anti-tumour activity was confirmed in patient-derived xenograft models in vivo. Sensitivity to HexaBody-CD38 correlated with CD38 expression level and was inversely correlated with expression of complement regulatory proteins. Compared to daratumumab, HexaBody-CD38 showed enhanced CDC in cell lines with lower levels of CD38 expression, without increasing lysis of healthy leukocytes. More effective CDC was also confirmed in primary MM cells. Furthermore, HexaBody-CD38 efficiently induced ADCC, ADCP, trogocytosis, and apoptosis after Fc-crosslinking. Moreover, HexaBody-CD38 strongly inhibited CD38 cyclase activity, which is hypothesized to relieve immune suppression in the tumour microenvironment. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of HexaBody-CD38 in patients with MM. FUNDING: Genmab.
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Antineoplásicos , Mieloma Múltiple , Humanos , Animales , Ratones , ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Citotoxicidad Celular Dependiente de Anticuerpos , Línea Celular Tumoral , Proteínas del Sistema Complemento/metabolismo , Microambiente TumoralRESUMEN
Therapeutic antibodies have broad indications across diverse disease states, such as oncology, autoimmune diseases, and infectious diseases. New research continues to identify antibodies with therapeutic potential as well as methods to improve upon endogenous antibodies and to design antibodies de novo. On April 27-30, 2022, experts in antibody research across academia and industry met for the Keystone symposium "Antibodies as Drugs" to present the state-of-the-art in antibody therapeutics, repertoires and deep learning, bispecific antibodies, and engineering.
Asunto(s)
Anticuerpos Biespecíficos , Humanos , Anticuerpos Biespecíficos/uso terapéutico , InmunoterapiaRESUMEN
CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Humanos , Anticuerpos Biespecíficos/farmacología , Linfocitos T CD8-positivos , Granzimas/farmacología , Complejo CD3/farmacología , Citotoxicidad Inmunológica , Perforina/farmacología , Receptor de Muerte Celular Programada 1 , Neoplasias/tratamiento farmacológico , CitocinasRESUMEN
BACKGROUND: Despite the preclinical promise of CD40 and 4-1BB as immuno-oncology targets, clinical efforts evaluating CD40 and 4-1BB agonists as monotherapy have found limited success. DuoBody-CD40×4-1BB (GEN1042/BNT312) is a novel investigational Fc-inert bispecific antibody for dual targeting and conditional stimulation of CD40 and 4-1BB to enhance priming and reactivation of tumor-specific immunity in patients with cancer. METHODS: Characterization of DuoBody-CD40×4-1BB in vitro was performed in a broad range of functional immune cell assays, including cell-based reporter assays, T-cell proliferation assays, mixed-lymphocyte reactions and tumor-infiltrating lymphocyte assays, as well as live-cell imaging. The in vivo activity of DuoBody-CD40×4-1BB was assessed in blood samples from patients with advanced solid tumors that were treated with DuoBody-CD40×4-1BB in the dose-escalation phase of the first-in-human clinical trial (NCT04083599). RESULTS: DuoBody-CD40×4-1BB exhibited conditional CD40 and 4-1BB agonist activity that was strictly dependent on crosslinking of both targets. Thereby, DuoBody-CD40×4-1BB strengthened the dendritic cell (DC)/T-cell immunological synapse, induced DC maturation, enhanced T-cell proliferation and effector functions in vitro and enhanced expansion of patient-derived tumor-infiltrating lymphocytes ex vivo. The addition of PD-1 blocking antibodies resulted in potentiation of T-cell activation and effector functions in vitro compared with either monotherapy, providing combination rationale. Furthermore, in a first-in-human clinical trial, DuoBody-CD40×4-1BB mediated clear immune modulation of peripheral antigen presenting cells and T cells in patients with advanced solid tumors. CONCLUSION: DuoBody-CD40×4-1BB is capable of enhancing antitumor immunity by modulating DC and T-cell functions and shows biological activity in patients with advanced solid tumors. These findings demonstrate that targeting of these two pathways with an Fc-inert bispecific antibody may be an efficacious approach to (re)activate tumor-specific immunity and support the clinical investigation of DuoBody-CD40×4-1BB for the treatment of cancer.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD40/metabolismo , Ensayos Clínicos como Asunto , Humanos , Activación de Linfocitos , Neoplasias/terapia , Linfocitos TRESUMEN
Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 axis have changed the treatment paradigm for advanced solid tumors; however, many patients experience treatment resistance. In preclinical models 4-1BB co-stimulation synergizes with ICI by activating cytotoxic T- and NK-cell-mediated anti-tumor immunity. Here we characterize the mechanism of action of a mouse-reactive Fc-inert PD-L1×4-1BB bispecific antibody (mbsAb-PD-L1×4-1BB) and provide proof-of-concept for enhanced anti-tumor activity. In reporter assays mbsAb-PD-L1×4-1BB exhibited conditional 4-1BB agonist activity that was dependent on simultaneous binding to PD-L1. mbsAb-PD-L1×4-1BB further blocked the PD-L1/PD-1 interaction independently of 4-1BB binding. By combining both mechanisms, mbsAb-PD-L1×4-1BB strongly enhanced T-cell proliferation, cytokine production and antigen-specific cytotoxicity using primary mouse cells in vitro. Furthermore, mbsAb-PD-L1×4-1BB exhibited potent anti-tumor activity in the CT26 and MC38 models in vivo, leading to the rejection of CT26 tumors that were unresponsive to PD-L1 blockade alone. Anti-tumor activity was associated with increased tumor-specific CD8+ T cells and reduced regulatory T cells within the tumor microenvironment and tumor-draining lymph nodes. In immunocompetent tumor-free mice, mbsAb-PD-L1×4-1BB treatment neither induced T-cell infiltration into the liver nor elevated liver enzymes in the blood. Dual targeting of PD-L1 and 4-1BB with a bispecific antibody may therefore address key limitations of first generation 4-1BB-agonistic antibodies, and may provide a novel approach to improve PD-1/PD-L1 checkpoint blockade.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antígeno B7-H1 , Linfocitos T CD8-positivos , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/uso terapéutico , Microambiente TumoralRESUMEN
Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell-mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell-mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy. SIGNIFICANCE: DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs. See related commentary by Li et al., p. 1184. This article is highlighted in the In This Issue feature, p. 1171.
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Anticuerpos Biespecíficos , Neoplasias , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antígeno B7-H1 , Modelos Animales de Enfermedad , Humanos , Inmunoterapia/métodos , Ratones , Neoplasias/tratamiento farmacológico , Linfocitos TRESUMEN
Apoptosis induction by death receptor (DR)-specific agonistic antibodies is a potentially effective antitumor therapy. Nonetheless, to date, all conventional DR-targeting antibodies that induce apoptosis via FcγR-dependent DR clustering failed to show clinical efficacy. HexaBody-DR5/DR5 (GEN1029) has been developed to overcome full FcγR dependence. HexaBody-DR5/DR5 is a mixture of 2 noncompeting DR5-specific immunoglobulin G1 (IgG1) antibodies, each with an E430G mutation in the Fc domain. This mutation enhances Fc-Fc interactions, resulting in antibody hexamerization, followed by FcγR-independent clustering of DR5 molecules. This unique combination of dual epitope targeting and increased IgG hexamerization resulted in potent preclinical antitumor activity in various solid cancers. In this study, we explored the preclinical activity of HexaBody-DR5/DR5 in multiple myeloma (MM), because MM cells are known to express DR5. In bone marrow samples from 48 MM patients, HexaBody-DR5/DR5 induced potent cytotoxicity of primary MM cells. Importantly, HexaBody-DR5/DR5 mediated the highest cytotoxic activity in samples from relapsed/refractory MM patients, including those who are refractory to daratumumab. This improved cytotoxic activity was observed only in patients who received their last anti-MM treatment <1 month ago, suggesting that anti-MM drugs sensitized MM cells to HexaBody-DR5/DR5. Supporting this, bortezomib combined with HexaBody-DR5/DR5 synergistically increased cytotoxicity in MM cells in 7 of 11 newly diagnosed patients. Lenalidomide also synergized with HexaBody-DR5/DR5, but only via its immunomodulatory effects, presumably by enhancing the antibody-dependent cellular cytotoxicity activity of HexaBody-DR5/DR5. Daratumumab showed additive effects when combined with HexaBody-DR5/DR5. In conclusion, the results of this preclinical study indicate a therapeutic potential for HexaBody-DR5/DR5, especially in recently treated relapsed/refractory MM patients.
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Antineoplásicos , Mieloma Múltiple , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Humanos , Lenalidomida , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genéticaRESUMEN
Epcoritamab (DuoBody-CD3xCD20, GEN3013) is a novel bispecific IgG1 antibody redirecting T-cells toward CD20+ tumor cells. Here, we assessed the preclinical efficacy of epcoritamab against primary tumor cells present in the lymph node biopsies from newly diagnosed (ND) and relapsed/refractory (RR) B-NHL patients. In the presence of T-cells from a healthy donor, epcoritamab demonstrated potent activity against primary tumor cells, irrespective of prior treatments, including CD20 mAbs. Median lysis of 65, 74, and 84% were achieved in diffuse large B-cell lymphoma (n = 16), follicular lymphoma (n = 15), and mantle cell lymphoma (n = 8), respectively. Furthermore, in this allogeneic setting, we discovered that the capacity of B-cell tumors to activate T-cells was heterogeneous and showed an inverse association with their surface expression levels of the immune checkpoint molecule Herpesvirus Entry Mediator (HVEM). In the autologous setting, when lymph node (LN)-residing T-cells were the only source of effector cells, the epcoritamab-dependent cytotoxicity strongly correlated with local effector cell-to-target cell ratios. Further analyses revealed that LN-residing-derived or peripheral blood-derived T-cells of B-NHL patients, as well as heathy donor T-cells equally mediated epcoritamab-dependent cytotoxicity. These results show the promise of epcoritamab for treatment of newly-diagnosed or relapsed/refractory B-NHL patients, including those who became refractory to previous CD20-directed therapies.
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Antineoplásicos Inmunológicos/farmacología , Linfocitos B/efectos de los fármacos , Linfoma Folicular/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células del Manto/tratamiento farmacológico , Anticuerpos Biespecíficos/farmacología , Antígenos CD20/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular Tumoral , Humanos , Activación de Linfocitos/efectos de los fármacos , Linfoma Folicular/inmunología , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células del Manto/inmunología , Linfoma de Células del Manto/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A. METHODS: T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys. FINDINGS: DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies. FUNDING: Genmab.
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Anticuerpos Biespecíficos/inmunología , Antígenos CD20/metabolismo , Complejo CD3/metabolismo , Citotoxicidad Inmunológica , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/farmacología , Especificidad de Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Leucemia de Células B/tratamiento farmacológico , Leucemia de Células B/etiología , Leucemia de Células B/patología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/etiología , Linfoma de Células B/patología , Macaca fascicularis , Ratones , Mutación , Proteínas Recombinantes , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Higher-order death receptor 5 (DR5) clustering can induce tumor cell death; however, therapeutic compounds targeting DR5 have achieved limited clinical efficacy. We describe HexaBody-DR5/DR5, an equimolar mixture of two DR5-specific IgG1 antibodies with an Fc-domain mutation that augments antibody hexamerization after cell surface target binding. The two antibodies do not compete for binding to DR5 as demonstrated using binding competition studies, and binding to distinct epitopes in the DR5 extracellular domain was confirmed by crystallography. The unique combination of dual epitope targeting and increased IgG hexamerization resulted in potent DR5 agonist activity by inducing efficient DR5 outside-in signaling and caspase-mediated cell death. Preclinical studies in vitro and in vivo demonstrated that maximal DR5 agonist activity could be achieved independent of Fc gamma receptor-mediated antibody crosslinking. Most optimal agonism was observed in the presence of complement complex C1, although without inducing complement-dependent cytotoxicity. It is hypothesized that C1 may stabilize IgG hexamers that are formed after binding of HexaBody-DR5/DR5 to DR5 on the plasma membrane, thereby strengthening DR5 clustering and subsequent outside-in signaling. We observed potent antitumor activity in vitro and in vivo in large panels of patient-derived xenograft models representing various solid cancers. The results of our preclinical studies provided the basis for an ongoing clinical trial exploring the activity of HexaBody-DR5/DR5 (GEN1029) in patients with malignant solid tumors.
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Epítopos/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Tetraspanin CD37 has recently received renewed interest as a therapeutic target for B-cell malignancies. Although complement-dependent cytotoxicity (CDC) is a powerful Fc-mediated effector function for killing hematological cancer cells, CD37-specific antibodies are generally poor inducers of CDC. To enhance CDC, the E430G mutation was introduced into humanized CD37 monoclonal IgG1 antibodies to drive more efficient IgG hexamer formation through intermolecular Fc-Fc interactions after cell surface antigen binding. DuoHexaBody-CD37, a bispecific CD37 antibody with the E430G hexamerization-enhancing mutation targeting two non-overlapping epitopes on CD37 (biparatopic), demonstrated potent and superior CDC activity compared to other CD37 antibody variants evaluated, in particular ex vivo in patient-derived chronic lymphocytic leukemia cells. The superior CDC potency was attributed to enhanced IgG hexamerization mediated by the E430G mutation in combination with dual epitope targeting. The mechanism of action of DuoHexaBody-CD37 was shown to be multifaceted, as it was additionally capable of inducing efficient antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vitro. Finally, potent anti-tumor activity in vivo was observed in cell line- and patient-derived xenograft models from different B-cell malignancy subtypes. These encouraging preclinical results suggest that DuoHexaBody-CD37 (GEN3009) may serve as a potential therapeutic antibody for the treatment of human B-cell malignancies.
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Anticuerpos Biespecíficos/farmacología , Antígenos de Neoplasias/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Linfoma de Células B/terapia , Receptores Fc/inmunología , Tetraspaninas/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular Tumoral , Desarrollo de Medicamentos , Células HEK293 , Xenoinjertos , Humanos , Inmunoglobulina G/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Linfoma de Células B/inmunología , Ratones , Ratones SCID , Terapia Molecular Dirigida , Receptores Fc/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
OBJECTIVE: Four different patterns of demyelination have been described in active demyelinating lesions of multiple sclerosis (MS) patients that were biopsied shortly after disease onset. These patterns were suggested to represent heterogeneity of the underlying pathogenesis. The aim of this study was to determine whether lesion heterogeneity also exists in an unselected collection of autopsy material from patients with established MS. METHODS: All MS brain tissue available in the VU Medical Center was assessed for the presence of active demyelinating lesions using magnetic resonance imaging-guided sampling and immunohistochemistry. Tissue blocks containing active demyelinating lesions were evaluated for the presence of complement and antibody deposition, oligodendrocyte apoptosis, differential loss of myelin proteins, and hypoxia-like damage using histology, immunohistochemistry, and confocal microscopy. Blocks with active demyelinating lesions were compared with blocks with active (nondemyelinating) and inactive lesions. RESULTS: Complement and antibodies were consistently associated with macrophages in areas of active demyelination. Preferential loss of myelin proteins, extensive hypoxia-like damage, and oligodendrocyte apoptosis were absent or rare. This pattern was observed in all tissue blocks containing active demyelinating lesions; lesion heterogeneity between patients was not found. INTERPRETATION: The immunopathological appearance of active demyelinating lesions in established MS is uniform. Initial heterogeneity of demyelinating lesions in the earliest phase of MS lesion formation may disappear over time as different pathways converge in one general mechanism of demyelination. Consistent presence of complement, antibodies, and Fcgamma receptors in phagocytic macrophages suggests that antibody- and complement-mediated myelin phagocytosis is the dominant mechanism of demyelination in established MS.
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Sistema Nervioso Central/patología , Esclerosis Múltiple/patología , Fibras Nerviosas Mielínicas/patología , Adulto , Anciano , Apoptosis/inmunología , Autoanticuerpos/análisis , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoinmunidad/inmunología , Autopsia , Biomarcadores/análisis , Biomarcadores/metabolismo , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Activación de Complemento/inmunología , Proteínas del Sistema Complemento/análisis , Proteínas del Sistema Complemento/inmunología , Progresión de la Enfermedad , Femenino , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Macrófagos/inmunología , Macrófagos/patología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Glicoproteína Asociada a Mielina/análisis , Glicoproteína Asociada a Mielina/inmunología , Oligodendroglía/inmunología , Oligodendroglía/patologíaRESUMEN
Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.