RESUMEN
Acute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2-like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease-like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígeno CD47 , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológicoRESUMEN
Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a "don't eat me" signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N-terminal pyro-glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell-mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab-mediated direct growth inhibition, complement-dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα-Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose-dependent manner, suggesting that pyro-glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab-dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte-mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell-mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.
Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Antígenos de Diferenciación/química , Antineoplásicos Inmunológicos/administración & dosificación , Antígeno CD47/metabolismo , Neoplasias/tratamiento farmacológico , Receptores Inmunológicos/química , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Animales , Antígenos de Diferenciación/metabolismo , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cetuximab/administración & dosificación , Cetuximab/farmacología , Sinergismo Farmacológico , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Neoplasias/metabolismo , Panitumumab/administración & dosificación , Panitumumab/farmacología , Unión Proteica/efectos de los fármacos , Receptores Inmunológicos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite several therapeutic advances, patients with multiple myeloma (MM) require additional treatment options since no curative therapy exists yet. In search of a novel therapeutic antibody, we previously applied phage display with myeloma cell screening and developed TP15, a scFv targeting intercellular adhesion molecule 1 (ICAM-1/CD54). To more precisely evaluate the antibody's modes of action, fully human IgG1 antibody variants were generated bearing wild-type (MSH-TP15) or mutated Fc to either enhance (MSH-TP15 Fc-eng.) or prevent (MSH-TP15 Fc k.o.) Fc gamma receptor binding. Especially MSH-TP15 Fc-eng. induced potent antibody-dependent cell-mediated cytotoxicity (ADCC) against malignant plasma cells by efficiently recruiting NK cells and engaged macrophages for antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Binding studies with truncated ICAM-1 demonstrated MSH-TP15 binding to ICAM-1 domain 1-2. Importantly, MSH-TP15 and MSH-TP15 Fc-eng. both prevented myeloma cell engraftment and significantly prolonged survival of mice in an intraperitoneal xenograft model. In the subcutaneous model MSH-TP15 Fc-eng. was superior to MSH-TP15, whereas MSH-TP15 Fc k.o. was not effective in both models - reflecting the importance of Fc-dependent mechanisms of action also in vivo. The efficient recruitment of immune cells and the potent anti-tumor activity of the Fc-engineered MSH-TP15 antibody hold significant potential for myeloma immunotherapy.
Asunto(s)
Mieloma Múltiple , Animales , Humanos , Ratones , Citotoxicidad Celular Dependiente de Anticuerpos , Línea Celular Tumoral , Inmunoglobulina G , Molécula 1 de Adhesión Intercelular/genética , Mieloma Múltiple/tratamiento farmacológico , Receptores de IgG/genéticaRESUMEN
Triggering of the complement cascade induces tumor cell lysis via complement-dependent cytotoxicity (CDC) and attracts and activates cytotoxic cells. It therefore represents an attractive mechanism for mAb in cancer immunotherapy development. The classical complement pathway is initiated by IgG molecules that have assembled into ordered hexamers after binding their Ag on the tumor cell surface. The requirements for CDC are further impacted by factors such as Ab epitope, valency, and affinity. Thus, mAb against well-validated solid tumor targets, such as the epidermal growth factor receptor (EGFR) that effectively induces complement activation and CDC, are highly sought after. The potency of complement activation by IgG Abs can be increased via several strategies. We identified single-point mutations in the Fc domain (e.g., E345K or E430G) enhancing Fc:Fc interactions, hexamer formation, and CDC after Ab binds cell-surface Ag. We show that EGFR Abs directed against clinically relevant epitopes can be converted into mAb with unprecedented CDC activity. Alternative strategies rely on increasing the affinity of monomeric IgG for C1q by introduction of a quadruple mutation at the C1q binding site or via generation of an IgG1/IgG3 chimera. In this study we show that selective enhancement of C1q binding via avidity modulation is superior to the unattended increase in C1q binding via affinity approaches, particularly for target cells with reduced EGFR expression levels. Improving Fc:Fc interactions of Ag-bound IgG therefore represents a highly promising and novel approach for potentiating the anti-tumor activity of therapeutic mAb against EGFR and potentially other tumor targets.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Activación de Complemento , Receptores ErbB/inmunología , Inmunoglobulina G/inmunología , Anticuerpos Monoclonales/genética , Sitios de Unión , Línea Celular Tumoral , Complemento C1q/inmunología , Complemento C1q/metabolismo , Receptores ErbB/genética , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Inmunoterapia/métodos , Mutación , Mutación PuntualRESUMEN
Complement-dependent cytotoxicity (CDC) has been suggested to be an important mechanism of action of tumor-targeting Abs. However, single unmodified epidermal growth factor receptor (EGFR)-targeting IgG1 Abs fail to trigger efficient CDC. For the current study, we generated a CDC-optimized variant of the EGFR Ab matuzumab (H425 wt) by introducing amino acid substitutions K326A/E333A (H425 mt). This Ab was then used to elucidate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert Ab-dependent cell-mediated cytotoxicity (ADCC). H425 mt, but not H425 wt, significantly induced complement deposition, release of anaphylatoxins, and CDC against distinct tumor cell lines, whereas no differences in ADCC by MNC or PMN were detected. Notably, stronger cytotoxicity was induced by H425 mt than by H425 wt in whole blood assays and in experiments in which MNC or PMN were combined with serum. Although MNC-ADCC was not affected by C5 cleavage, the cytotoxic activity of PMN in the presence of serum strongly depended on C5 cleavage, pointing to a direct interaction between complement and PMN. Strong cell surface expression of C5a receptors was detected on PMN, whereas NK cells completely lacked expression. Stimulation of PMN with C5a led to upregulation of activated complement receptor 3, resulting in enhanced complement receptor 3-dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR Abs may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in Ab-based tumor therapy.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Antineoplásicos/farmacología , Complemento C5a/inmunología , Receptores ErbB/antagonistas & inhibidores , Leucocitos/inmunología , Neoplasias/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/inmunología , Humanos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Receptor de Anafilatoxina C5a/inmunologíaRESUMEN
Compared to the evolutionary diversity of antibody isotypes, the spectrum of currently approved therapeutic antibodies is biased to the human IgG1 isotype. Detailed studies into the different structures and functions of human isotypes have suggested that other isotypes than IgG1 may be advantageous for specific indications - depending on the complex interplay between the targeted antigen or epitope, the desired mode of action, the pharmacokinetic properties, and the biopharmaceutical considerations. Thus, it may be speculated that with the increasing number of antibodies becoming available against a broadening spectrum of target antigens, identification of the optimal antibody isotype for particular therapeutic applications may become critical for the therapeutic success of individual antibodies. Thus, investments into this rather unexplored area of antibody immunotherapy may provide opportunities for distinction in the increasingly busy 'antibody space'. Therefore, IgG, IgA, IgE as well as IgM isotypes will be discussed in this review.
RESUMEN
BACKGROUND: Engineering of the antibody's fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies. METHODS AND RESULTS: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody. CONCLUSION: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.
RESUMEN
Binding of C1q to target-bound IgG initiates complement-mediated lysis (CML) of pathogens, as well as of malignant or apoptotic cells, and thus constitutes an integral part of the innate immune system. Despite its prominent molecular flexibility and higher C1q binding affinity compared with human IgG1, IgG3 does not consistently promote superior CML. Hence the aim of this study was to investigate underlying molecular mechanisms of IgG1- and IgG3-driven complement activation using isotype variants of the therapeutic epidermal growth factor receptor (EGFR) Ab cetuximab. Both IgG1 and IgG3 Abs demonstrated similar EGFR binding and similar efficiency in Fab-mediated effector mechanisms. Whereas anti-EGFR-IgG1 did not promote CML of investigated target cells, anti-EGFR-IgG3 triggered significant CML of some, but not all tested cell lines. CML triggered by anti-EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement regulatory proteins CD55 and CD59, but not CD46. Notably, anti-EGFR-IgG3 promoted strong C1q and C3b, but relatively low C4b and C5b-9 deposition on analyzed cell lines. Furthermore, anti-EGFR-IgG3 triggered C4a release on all cells but failed to induce C3a and C5a release on CD55/CD59 highly expressing cells. RNA interference-induced knockdown or overexpression of membrane-bound complement regulatory proteins revealed CD55 expression to be a pivotal determinant of anti-EGFR-IgG3-triggered CML and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification. Together, these data suggest human anti-EGFR-IgG3, although highly reactive with C1q, to weakly promote assembly of the classical C3 convertase that is further suppressed in the presence of CD55, forcing human IgG3 to act mainly through the alternative pathway.
Asunto(s)
Antígenos CD55/genética , Vía Alternativa del Complemento/genética , Vía Alternativa del Complemento/inmunología , Citotoxicidad Inmunológica/genética , Receptores ErbB/genética , Inmunoglobulina G/genética , Animales , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/uso terapéutico , Antígenos CD55/biosíntesis , Antígenos CD55/química , Células CHO , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Convertasas de Complemento C3-C5/antagonistas & inhibidores , Convertasas de Complemento C3-C5/genética , Cricetinae , Cricetulus , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Femenino , Marcación de Gen/métodos , Humanos , Inmunoglobulina G/uso terapéutico , Técnicas de Amplificación de Ácido Nucleico/métodos , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunologíaRESUMEN
Monoclonal antibodies constitute a major treatment option for many tumor patients. Due to their specific recognition sites in their constant Fc regions, antibodies are able to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). While the contribution of ADCC to clinical efficacy has been strengthened by observations that patients with favorable Fcγ receptor polymorphisms display better response rates to therapeutic antibodies, the contribution of CDC to their clinical efficacy remains controversial. In the background of high expression of complement-regulatory proteins on tumor cells as well as of the fact that some therapeutic antibodies lack the capacity to trigger efficient CDC, strategies have been implemented to improve either the capacity of antibodies to initiate the complement cascade or to interfere with tumor cells' resistance mechanisms. Although both strategies have demonstrated therapeutic benefit in vitro and in murine models, CDC-enhanced antibodies-to the best of our knowledge-have not been clinically tested, and evidence for the potential of CDC-optimizing approaches has yet to be generated in humans. Hence, the potency of complement activation and its impact on the clinical efficacy of therapeutic antibodies still remains to be elucidated in clinical trials encompassing novel complement-enhancing molecules.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Proteínas del Sistema Complemento/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Activación de Complemento/efectos de los fármacos , Activación de Complemento/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Terapia Molecular DirigidaAsunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos CD20/inmunología , Antineoplásicos Inmunológicos/farmacología , Proteínas del Sistema Complemento/inmunología , Inmunoglobulina A/farmacología , Leucemia Prolinfocítica Tipo Células B/inmunología , Células Mieloides/inmunología , Animales , Antineoplásicos Inmunológicos/inmunología , Células CHO , Cricetulus , Humanos , Inmunoglobulina A/inmunología , Leucemia Prolinfocítica Tipo Células B/tratamiento farmacológico , Leucemia Prolinfocítica Tipo Células B/patología , Células Mieloides/patologíaRESUMEN
Antibody-based immunotherapy is increasingly employed to treat acute lymphoblastic leukemia (ALL) patients. Many T-ALL cells express CD38 on their surface, which can be targeted by the CD38 antibody daratumumab (DARA), approved for the treatment of multiple myeloma. Tumor cell killing by myeloid cells is relevant for the efficacy of many therapeutic antibodies and can be more efficacious with human IgA than with IgG antibodies. This is demonstrated here by investigating antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear (PMN) cells using DARA (human IgG1) and an IgA2 isotype switch variant (DARA-IgA2) against T-ALL cell lines and primary patient-derived tumor cells. ADCP and ADCC are negatively regulated by interactions between CD47 on tumor cells and signal regulatory protein alpha (SIRPα) on effector cells. In order to investigate the impact of this myeloid checkpoint on T-ALL cell killing, CD47 and glutaminyl-peptide cyclotransferase like (QPCTL) knock-out T-ALL cells were employed. QPTCL is an enzymatic posttranslational modifier of CD47 activity, which can be targeted by small molecule inhibitors. Additionally, we used an IgG2σ variant of the CD47 blocking antibody magrolimab, which is in advanced clinical development. Moreover, treatment of T-ALL cells with all-trans retinoic acid (ATRA) increased CD38 expression leading to further enhanced ADCP and ADCC, particularly when DARA-IgA2 was applied. These studies demonstrate that myeloid checkpoint blockade in combination with IgA2 variants of CD38 antibodies deserves further evaluation for T-ALL immunotherapy.
Asunto(s)
Antígeno CD47 , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Humanos , Inmunoglobulina ARESUMEN
Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcγ receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies' effector functions.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Receptores de IgG , Aminoácidos , Antígenos CD19 , Proteínas del Sistema Complemento , Fragmentos Fc de Inmunoglobulinas , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMEN
Antibody-dependent cellular phagocytosis (ADCP) by macrophages, an important effector function of tumor targeting antibodies, is hampered by 'Don´t Eat Me!' signals such as CD47 expressed by cancer cells. Yet, human leukocyte antigen (HLA) class I expression may also impair ADCP by engaging leukocyte immunoglobulin-like receptor subfamily B (LILRB) member 1 (LILRB1) or LILRB2. Analysis of different lymphoma cell lines revealed that the ratio of CD20 to HLA class I cell surface molecules determined the sensitivity to ADCP by the combination of rituximab and an Fc-silent variant of the CD47 antibody magrolimab (CD47-IgGσ). To boost ADCP, Fc-silent antibodies against LILRB1 and LILRB2 were generated (LILRB1-IgGσ and LILRB2-IgGσ, respectively). While LILRB2-IgGσ was not effective, LILRB1-IgGσ significantly enhanced ADCP of lymphoma cell lines when combined with both rituximab and CD47-IgGσ. LILRB1-IgGσ promoted serial engulfment of lymphoma cells and potentiated ADCP by non-polarized M0 as well as polarized M1 and M2 macrophages, but required CD47 co-blockade and the presence of the CD20 antibody. Importantly, complementing rituximab and CD47-IgGσ, LILRB1-IgGσ increased ADCP of chronic lymphocytic leukemia (CLL) or lymphoma cells isolated from patients. Thus, dual checkpoint blockade of CD47 and LILRB1 may be promising to improve antibody therapy of CLL and lymphomas through enhancing ADCP by macrophages.
Asunto(s)
Antígeno CD47 , Leucemia Linfocítica Crónica de Células B , Humanos , Antígeno CD47/metabolismo , Receptor Leucocitario Tipo Inmunoglobulina B1/metabolismo , Rituximab/farmacología , Rituximab/uso terapéutico , Rituximab/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Línea Celular Tumoral , Fagocitosis , Macrófagos , Anticuerpos/metabolismo , Antígenos CD/metabolismoRESUMEN
Immunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with "double-hit" diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies.
Asunto(s)
Citofagocitosis , Linfoma de Células B Grandes Difuso , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Niño , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2 , SulfonamidasRESUMEN
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent malignancy in children and also occurs in adulthood. Despite high cure rates, BCP-ALL chemotherapy can be highly toxic. This type of toxicity can most likely be reduced by antibody-based immunotherapy targeting the CD19 antigen which is commonly expressed on BCP-ALL cells. In this study, we generated a novel Fc-engineered CD19-targeting IgG1 antibody fused to a single chain tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) domain (CD19-TRAIL). As TRAIL induces apoptosis in tumor cells but not in healthy cells, we hypothesized that CD19-TRAIL would show efficient killing of BCP-ALL cells. CD19-TRAIL showed selective binding capacity and pronounced apoptosis induction in CD19-positive (CD19+) BCP-ALL cell lines in vitro and in vivo. Additionally, CD19-TRAIL significantly prolonged survival of mice transplanted with BCP-ALL patient-derived xenograft (PDX) cells of different cytogenetic backgrounds. Moreover, simultaneous treatment with CD19-TRAIL and Venetoclax (VTX), an inhibitor of the anti-apoptotic protein BCL-2, promoted synergistic apoptosis induction in CD19+ BCP-ALL cells in vitro and prolonged survival of NSG-mice bearing the BCP-ALL cell line REH. Therefore, IgG1-based CD19-TRAIL fusion proteins represent a new potential immunotherapeutic agent against BCP-ALL.
RESUMEN
Blockade of the CD47-SIRPα axis improves lymphoma cell killing by myeloid effector cells, which is an important effector mechanism for CD20 antibodies in vivo. The approved CD20 antibodies rituximab, ofatumumab, and obinutuzumab are of human immunoglobulin G1 (IgG1) isotype. We investigated the impact of the variable regions of these 3 CD20 antibodies when expressed as human IgA2 isotype variants. All 3 IgA2 antibodies mediated antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cellular cytotoxicity (ADCC) by polymorphonuclear cells. Both effector mechanisms were significantly enhanced in the presence of a CD47-blocking antibody or by glutaminyl cyclase inhibition to interfere with CD47-SIRPα interactions. Interestingly, an IgA2 variant of obinutuzumab (OBI-IgA2) was consistently more potent than an IgA2 variant of rituximab (RTX-IgA2) or an IgA2 variant of ofatumumab (OFA-IgA2) in triggering ADCC. Furthermore, we observed more effective direct tumor cell killing by OBI-IgA2 compared with RTX-IgA2 and OFA-IgA2, which was caspase independent and required a functional cytoskeleton. IgA2 variants of all 3 antibodies triggered complement-dependent cytotoxicity, with OBI-IgA2 being less effective than RTX-IgA2 and OFA-IgA2. When we investigated the therapeutic efficacy of the CD20 IgA2 antibodies in different in vivo models, OBI-IgA2 was therapeutically more effective than RTX-IgA2 or OFA-IgA2. In vivo efficacy required the presence of a functional IgA receptor on effector cells and was independent of complement activation or direct lymphoma cell killing. These data characterize the functional activities of human IgA2 antibodies against CD20, which were affected by the selection of the respective variable regions. OBI-IgA2 proved particularly effective in vitro and in vivo, which may be relevant in the context of CD47-SIRPα blockade.
Asunto(s)
Antígenos CD20 , Inmunoglobulina A , Citotoxicidad Celular Dependiente de Anticuerpos , Humanos , Inmunoglobulina G , RituximabRESUMEN
Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos Inmunológicos/farmacología , Neutrófilos/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Línea Celular Tumoral , Cetuximab/farmacología , Femenino , Células HCT116 , Humanos , Inmunoglobulina G/inmunología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Inmunológicos , Receptores Fc/inmunologíaRESUMEN
The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Especificidad de Anticuerpos , Antineoplásicos Inmunológicos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Tiburones/inmunología , Animales , Anticuerpos Antiidiotipos/genética , Especificidad de Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Línea Celular Tumoral , Expresión Génica , Biblioteca de Genes , Humanos , Inmunoconjugados/genética , Inmunoconjugados/farmacología , Inmunofenotipificación , Linfoma/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores de Antígenos de Linfocitos B/sangre , Receptores de Antígenos de Linfocitos B/genética , Proteínas Recombinantes de Fusión/genética , Tiburones/genéticaRESUMEN
The complement system represents a powerful part of the innate immune system capable of removing pathogens and damaged host cells. Nevertheless, only a subset of therapeutic antibodies are capable of inducing complement dependent cytotoxicity, which has fuelled the search for new strategies to potentiate complement activation. Properdin (FP) functions as a positive complement regulator by stabilizing the alternative pathway C3 convertase. Here, we explore a novel strategy for direct activation of the alternative pathway of complement using bi-specific single domain antibodies (nanobodies) that recruit endogenous FP to a cell surface. As a proof-of-principle, we generated bi-specific nanobodies with specificity toward FP and the validated cancer antigen epidermal growth factor receptor (EGFR) and tested their ability to activate complement onto cancer cell lines expressing EGFR. Treatment led to recruitment of FP, complement activation and significant deposition of C3 fragments on the cells in a manner sensitive to the geometry of FP recruitment. The bi-specific nanobodies induced complement dependent lysis of baby hamster kidney cells expressing human EGFR but were unable to lyse human tumour cells due to the presence of complement regulators. Our results confirm that FP can function as a surface bound focal point for initiation of complement activation independent of prior C3b deposition. However, recruitment of FP by bi-specific nanobodies appears insufficient for overcoming the inhibitory action of the negative complement regulators overexpressed by many human tumour cell lines. Our data provide general information on the efficacy of properdin as an initiator of complement but suggest that properdin recruitment on its own may have limited utility as a platform for potent complement activation on regulated cell surfaces.