RESUMO
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.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Linfoma de Células B/terapia , Receptores Fc/imunologia , Tetraspaninas/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Desenvolvimento de Medicamentos , Células HEK293 , Xenoenxertos , Humanos , Imunoglobulina G/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfoma de Células B/imunologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Receptores Fc/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
UNLABELLED: Longitudinal imaging of intratumoral distributions of antibodies in vivo in mouse cancer models is of great importance for developing cancer therapies. In this study, multipinhole SPECT with sub-half-millimeter resolution was tested for exploring intratumoral distributions of radiolabeled antibodies directed toward the epidermal growth factor receptor (EGFr) and compared with full 3-dimensional target expression assessed by immunohistochemistry. METHODS: (111)In-labeled zalutumumab, a human monoclonal human EGFr-targeting antibody, was administered at a nonsaturating dose to 3 mice with xenografted A431 tumors exhibiting high EGFr expression. Total-body and focused in vivo tumor SPECT was performed at 0 and 48 h after injection and compared both visually and quantitatively with full 3-dimensional immunohistochemical staining for EGFr target expression. RESULTS: SPECT at 48 h after injection showed that activity was predominantly concentrated in the tumor (10.5% ± 1.3% of the total-body activity; average concentration, 30.1% ± 4.6% of the injected dose per cubic centimeter). (111)In-labeled EGFr-targeting antibodies were distributed heterogeneously throughout the tumor. Some hot spots were observed near the tumor rim. Immunohistochemistry indicated that the antibody distributions obtained by SPECT were morphologically similar to those obtained for ex vivo EGFr target expression. Regions showing low SPECT activity were necrotic or virtually negative for EGFr target expression. A good correlation (r = 0.86, P < 0.0001) was found between the percentage of regions showing low activity on SPECT and the percentage of necrotic tissue on immunohistochemistry. CONCLUSION: Multipinhole SPECT enables high-resolution visualization and quantification of the heterogeneity of (111)In-zalutumumab concentrations in vivo.
Assuntos
Anticorpos/química , Receptores ErbB/metabolismo , Imageamento Tridimensional , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imunoconjugados/química , Imuno-Histoquímica , Radioisótopos de Índio/química , Camundongos , Camundongos SCID , Transplante de Neoplasias , Reprodutibilidade dos Testes , Tomografia Computadorizada por Raios XRESUMO
Ab-dependent cellular cytotoxicity (ADCC) is recognized as a prominent cytotoxic mechanism for therapeutic mAbs in vitro. However, the contribution of ADCC to in vivo efficacy, particularly for treatment of solid tumors, is still poorly understood. For zalutumumab, a therapeutic epidermal growth factor receptor (EGFR)-specific mAb currently in clinical development, previous studies have indicated signaling inhibition and ADCC induction as important therapeutic mechanisms of action. To investigate the in vivo role of ADCC, a panel of EGFR-specific mAbs lacking specific functionalities was generated. By comparing zalutumumab with mAb 018, an EGFR-specific mAb that induced ADCC with similar potency, but did not inhibit signaling, we observed that ADCC alone was insufficient for efficacy against established A431 xenografts. Interestingly, however, both zalutumumab and mAb 018 prevented tumor formation upon early treatment in this model. Zalutumumab and mAb 018 also completely prevented outgrowth of lung metastases, in A431 and MDA-MB-231-luc-D3H2LN experimental metastasis models, already when given at nonsaturating doses. Finally, tumor growth of mutant KRAS-expressing A431 tumor cells, which were resistant to EGFR signaling inhibition, was completely prevented by early treatment with zalutumumab and mAb 018, whereas ADCC-crippled N297Q-mutated variants of both mAbs did not show any inhibitory effects. In conclusion, ADCC induction by EGFR-specific mAbs represents an important mechanism of action in preventing tumor outgrowth or metastasis in vivo, even of cancers insensitive to EGFR signaling inhibition.
Assuntos
Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/imunologia , Receptores ErbB/antagonistas & inibidores , Neoplasias Experimentais/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Separação Celular , Receptores ErbB/imunologia , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos SCID , Neoplasias Experimentais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We evaluated the dose requirements for sustained in vivo activity of ofatumumab, a human anti-CD20 antibody under development for the treatment of B cell-mediated diseases. In a mouse xenograft model, a single dose, resulting in an initial plasma antibody concentration of 5 microg/ml, which was expected to result in full target saturation, effectively inhibited human B-cell tumour development. Tumour growth resumed when plasma concentrations dropped below levels that are expected to result in half-maximal saturation. Notably, tumour load significantly impacted antibody pharmacokinetics. In monkeys, initial depletion of circulating and tissue residing B cells required relatively high-dose levels. Re-population of B-cell compartments, however, only became detectable when ofatumumab levels dropped below 10 microg/ml. We conclude that, once saturation of CD20 throughout the body has been reached by high initial dosing, plasma concentrations that maintain target saturation on circulating cells (5-10 microg/ml) are probably sufficient for sustained biological activity. These observations may provide a rationale for establishing dosing schedules for maintenance immunotherapy following initial depletion of CD20 positive (tumour) cells.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígenos CD20/imunologia , Linfócitos B/imunologia , Imunoterapia/métodos , Animais , Anticorpos/sangue , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/terapia , Linfoma de Células B/terapia , Macaca fascicularis , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos CD20/imunologia , Antígenos CD20/metabolismo , Sítios de Ligação de Anticorpos , Epitopos/imunologia , Epitopos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Antígenos CD20/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Sítios de Ligação de Anticorpos/genética , Linhagem Celular , Linhagem Celular Tumoral , Via Clássica do Complemento/genética , Via Clássica do Complemento/imunologia , Citotoxicidade Imunológica/genética , Mapeamento de Epitopos , Epitopos/genética , Humanos , Imunoglobulina G/metabolismo , Linfoma/imunologia , Linfoma/patologia , Linfoma/terapia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismoRESUMO
The immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 chains associated with the T cell receptor (TCR) are crucial for TCR signaling. To probe the role of the CD3gamma-ITAM in T cell development, we created knock-in mice in which the CD3gamma chain of the TCR complex is replaced by a mutant signaling-deficient CD3gamma chain, lacking the CD3gamma-ITAM. This mutation results in considerable impairment in positive selection in the polyclonal TCR repertoire. When CD3gamma-deltaITAM mice are crossed to mice expressing transgenic F5 TCRs, their thymocytes are completely unable to perform positive selection in vivo in response to intrathymic ligands. Also, the in vitro positive selection response of double-positive (DP) thymocytes with F5-CD3gamma-deltaITAM mutant receptors to their agonist ligand and many of its variants is severely impaired or abrogated. Yet, the binding and dissociation constants of agonist ligands for the F5 receptor are not affected by the CD3gamma-deltaITAM mutation. Furthermore, DP thymocytes with mutant receptors can respond to agonist ligand with normal antigen sensitivity and to normal levels, as shown by their ability to induce CD69 up-regulation, TCR down-regulation, negative selection, and ZAP70 and c-Jun NH2-terminal kinase activation. In sharp contrast, induction of extracellular signal-regulated kinase (ERK) activation and linker for activation of T cells (LAT) phosphorylation are severely impaired in these cells. Together, these findings underscore that intrinsic properties of the TCR-CD3 complex regulate selection at the DP checkpoint. More importantly, this analysis provides the first direct genetic evidence for a role of the CD3gamma-ITAM in TCR-driven thymocyte selection.