RESUMO
Immuno-oncology therapies engage the immune system to treat cancer. BiTE (bispecific T-cell engager) technology is a targeted immuno-oncology platform that connects patients' own T cells to malignant cells. The modular nature of BiTE technology facilitates the generation of molecules against tumor-specific antigens, allowing off-the-shelf immuno-oncotherapy. Blinatumomab was the first approved canonical BiTE molecule and targets CD19 surface antigens on B cells, making blinatumomab largely independent of genetic alterations or intracellular escape mechanisms. Additional BiTE molecules in development target other hematologic malignancies (eg, multiple myeloma, acute myeloid leukemia, and B-cell non-Hodgkin lymphoma) and solid tumors (eg, prostate cancer, glioblastoma, gastric cancer, and small-cell lung cancer). BiTE molecules with an extended half-life relative to the canonical BiTE molecules are also being developed. Advances in immuno-oncology made with BiTE technology could substantially improve the treatment of hematologic and solid tumors and offer enhanced activity in combination with other treatments.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Hematológicas/terapia , Imunoterapia Adotiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Antígenos CD19/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires strict adherence by patients and lifelong medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency but cannot cure patients. The bispecific T cell-engaging (BiTE) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. Here, we generated BiTE antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1 + 2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ε scFv. These engineered human BiTE antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs) as well as in macrophages cocultured with autologous CD8+ T cells, the most potent being the human CD4(1 + 2) BiTE [termed CD(1 + 2) h BiTE] antibody construct and the CD4(1 + 2)L17b BiTE antibody construct. The CD4(1 + 2) h BiTE antibody construct promoted HIV infection of human CD4-/CD8+ T cells. In contrast, the neutralizing B12 and the VRC01 BiTE antibody constructs, as well as the CD4(1 + 2)L17b BiTE antibody construct, did not. Thus, BiTE antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential.IMPORTANCE HIV is a chronic infection well controlled with the current cART. However, we lack a cure for HIV, and the HIV pandemic goes on. Here, we showed in vitro and ex vivo that a BiTE antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE antibody constructs display efficient killing of gp120-expressing cells and inhibited replication in ex vivo HIV-infected PBMCs or macrophages. We believe that BiTE antibody constructs recognizing HIV gp120 could be a very valuable strategy for a cure of HIV in combination with cART and compounds which reverse latency.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antivirais/uso terapêutico , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Células CHO , Cricetinae , Cricetulus , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Imunoterapia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Ligação Proteica , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologiaRESUMO
Antibody-based immunotherapy represents a promising strategy to target and eliminate chemoresistant leukemic cells. Here, we evaluated the CD33/CD3-bispecific T cell engaging (BiTE) antibody (AMG 330) for its suitability as a therapeutic agent in acute myeloid leukemia (AML). We first assessed CD33 expression levels by flow cytometry and found expression in >99% of patient samples (n = 621). CD33 was highest expressed in AMLs with NPM1 mutations (P < .001) and lower in AMLs with complex karyotypes and t(8;21) translocations (P < .001). Furthermore, leukemic stem cells within the CD34(+)/CD38(-) compartment displayed CD33 at higher levels than healthy donor stem cells (P = .047). In MS-5 feeder cell-based long-term cultures that supported the growth of primary AML blasts for up to 36 days, AMG 330 efficiently recruited and expanded residual CD3(+)/CD45RA(-)/CCR7(+) memory T cells within the patient sample. Even at low effector to target ratios, the recruited T cells lysed autologous blasts completely in the majority of samples and substantially in the remaining samples in a time-dependent manner. This study provides the first correlation of CD33 expression levels with AML genotype in a comprehensive analysis of adult patients. Targeting CD33 ex vivo using AMG 330 in primary AML samples led to T cell recruitment and expansion and remarkable antibody-mediated cytotoxicity, suggesting efficient therapeutic potential in vivo.
Assuntos
Anticorpos Biespecíficos/imunologia , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Biespecíficos/uso terapêutico , Técnicas de Cultura de Células , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Genótipo , Humanos , Cariotipagem , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Fatores de TempoRESUMO
T cell-engaging CD19/CD3-bispecific BiTE Ab blinatumomab has shown an 80% complete molecular response rate and prolonged leukemia-free survival in patients with minimal residual B-lineage acute lymphoblastic leukemia (MRD(+) B-ALL). Here, we report that lymphocytes in all patients of a phase 2 study responded to continuous infusion of blinatumomab in a strikingly similar fashion. After start of infusion, B-cell counts dropped to < 1 B cell/µL within an average of 2 days and remained essentially undetectable for the entire treatment period. By contrast, T-cell counts in all patients declined to a nadir within < 1 day and recovered to baseline within a few days. T cells then expanded and on average more than doubled over baseline within 2-3 weeks under continued infusion of blinatumomab. A significant percentage of reappearing CD8(+) and CD4(+) T cells newly expressed activation marker CD69. Shortly after start of infusion, a transient release of cytokines dominated by IL-10, IL-6, and IFN-γ was observed, which no longer occurred on start of a second treatment cycle. The response of lymphocytes in leukemic patients to continuous infusion of blinatumomab helps to better understand the mode of action of this and other globally T cell-engaging Abs. The trial is registered with www.clinicaltrials.gov identifier NCT00560794.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Antígenos CD19/imunologia , Complexo CD3/imunologia , Sistema Imunitário/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Adulto , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/farmacocinética , Soro Antilinfocitário/administração & dosagem , Soro Antilinfocitário/efeitos adversos , Soro Antilinfocitário/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/farmacocinética , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Bombas de Infusão , Ativação Linfocitária/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
Persistence or recurrence of minimal residual disease (MRD) after chemotherapy results in clinical relapse in patients with acute lymphoblastic leukemia (ALL). In a phase 2 trial of B-lineage ALL patients with persistent or relapsed MRD, a T cell-engaging bispecific Ab construct induced an 80% MRD response rate. In the present study, we show that after a median follow-up of 33 months, the hematologic relapse-free survival of the entire evaluable study cohort of 20 patients was 61% (Kaplan-Meier estimate). The hema-tologic relapse-free survival rate of a subgroup of 9 patients who received allogeneic hematopoietic stem cell transplantation after blinatumomab treatment was 65% (Kaplan-Meier estimate). Of the subgroup of 6 Philadelphia chromosome-negative MRD responders with no further therapy after blinatumomab, 4 are in ongoing hematologic and molecular remission. We conclude that blinatumomab can induce long-lasting complete remission in B-lineage ALL patients with persistent or recurrent MRD. The original study and this follow-up study are registered at www.clinicaltrials.gov as NCT00198991 and NCT00198978, respectively.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Adulto , Antineoplásicos/uso terapêutico , Terapia Combinada , Intervalo Livre de Doença , Seguimentos , Transplante de Células-Tronco Hematopoéticas , Humanos , Estimativa de Kaplan-Meier , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras B/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Recidiva , Análise de Sobrevida , Transplante HomólogoRESUMO
Epidermal growth factor receptor (EGFR)-specific monoclonal antibodies predominantly inhibit colorectal cancer (CRC) growth by interfering with receptor signaling. Recent analyses have shown that patients with CRC with mutated KRAS and BRAF oncogenes do not profit from treatment with such antibodies. Here we have used the binding domains of cetuximab and pantitumumab for constructing T cell-engaging BiTE antibodies. Both EGFR-specific BiTE antibodies mediated potent redirected lysis of KRAS- and BRAF-mutated CRC lines by human T cells at subpicomolar concentrations. The cetuximab-based BiTE antibody also prevented at very low doses growth of tumors from KRAS- and BRAF-mutated human CRC xenografts, whereas cetuximab was not effective. In nonhuman primates, no significant adverse events were observed during treatment for 3 wk at BiTE serum concentrations inducing, within 1 d, complete lysis of EGFR-overexpressing cancer cells. EGFR-specific BiTE antibodies may have potential to treat CRC that does not respond to conventional antibodies.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Receptores ErbB/antagonistas & inibidores , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados , Cetuximab , Neoplasias Colorretais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes ras/efeitos dos fármacos , Humanos , Mutação/efeitos dos fármacos , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf , Fatores de RiscoRESUMO
Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the "Tn antigen." Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen.
Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Animais , Anticorpos Monoclonais , Afinidade de Anticorpos , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Cristalografia por Raios X , Epitopos/química , Glicopeptídeos/química , Glicopeptídeos/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Técnicas In Vitro , Camundongos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Eletricidade Estática , Ressonância de Plasmônio de SuperfícieAssuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Adulto , Idoso , Seguimentos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Neoplasia Residual , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Recidiva , Análise de Sobrevida , Transplante Homólogo , Resultado do TratamentoRESUMO
Blinatumomab is a first-in-class immunotherapy based on the bispecific T-cell engager (BiTE®) immune-oncology platform, which redirects CD3+ T cells to kill CD19+ target cells. The objective of this analysis was to describe the correlation between B- and T-cell kinetics and response to blinatumomab in patients with relapsed or refractory (r/r) non-Hodgkin lymphoma (NHL). The clinical efficacy of treatment with blinatumomab in patients with r/r NHL was recently investigated in a phase 1 dose-escalation and expansion trial (NCT00274742) wherein 76 patients received blinatumomab by continuous intravenous infusion at various doses (0.5-90 µg/m2/day). B-Cell depletion and expansion of CD3+, CD4+, and CD8+ T cells was analyzed in patients stratified per clinical response (complete response [CR], n = 16; partial response [PR], stable disease [SD], or progressive disease [PD], n = 54) for at least 4 weeks (additional 4 weeks after clinical benefit) from the date of administration of blinatumomab until dose-limiting toxicity or PD. B-cell depletion kinetics were faster in patients who had a CR than in patients who did not have a complete response (PR, SD, or PD). T-cell expansion (T-cell counts exceeding the baseline level on day 22) was more pronounced in patients with CR than in patients without CR. T-cell expansion in patients with CR correlated with increased T-cell counts of both CD4+ and CD8+ T cells compared with patients without CR. Patients with r/r NHL who achieved a CR had faster B-cell depletion and increased expansion of CD3+, CD4+, and CD8+ T cells than patients who did not achieve a CR.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Linfócitos B/efeitos dos fármacos , Linfoma não Hodgkin/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Linfócitos B/imunologia , Humanos , Linfoma não Hodgkin/imunologia , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/imunologia , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) remains a disease with high unmet medical need, as most patients do not achieve durable response with available treatments. Prostate-specific membrane antigen (PSMA) is a compelling target for mCRPC. It is highly expressed by primary and metastatic prostate cancer cells, with increased expression after progression on androgen deprivation therapy. EXPERIMENTAL DESIGN: We developed AMG 160, a half-life extended, bispecific T-cell engager immuno-oncology therapy that binds PSMA on prostate cancer cells and cluster of differentiation 3 on T cells for treatment of mCRPC. AMG 160 was evaluated in vitro and in mCRPC xenograft models. AMG 160 tolerability was assessed in nonhuman primates (NHP). AMG 160 activity as monotherapy and in combination with a PSMA-imaging agent, novel hormonal therapy, and immune checkpoint blockade was evaluated. RESULTS: AMG 160 induces potent, specific killing of PSMA-expressing prostate cancer cell lines in vitro, with half-maximal lysis of 6-42 pmol/L. In vivo, AMG 160 administered weekly at 0.2 mg/kg engages T cells administered systemically and promotes regression of established 22Rv-1 mCRPC xenograft tumors. AMG 160 is compatible with the imaging agent gallium 68-labeled PSMA-11, and shows enhanced cytotoxic activity when combined with enzalutamide or an anti-programmed death-1 antibody. AMG 160 exhibits an extended half-life and has an acceptable safety profile in NHPs. CONCLUSIONS: The preclinical characterization of AMG 160 highlights its potent antitumor activity in vitro and in vivo, and its potential for use with known diagnostic or therapeutic agents in mCRPC. These data support the ongoing clinical evaluation of AMG 160 in patients with mCRPC.See related commentary by Kamat et al., p. 2675.
Assuntos
Transferência Adotiva/métodos , Antígenos de Superfície/imunologia , Glutamato Carboxipeptidase II/imunologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linfócitos T/imunologia , Animais , Complexo CD3/antagonistas & inibidores , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Glutamato Carboxipeptidase II/antagonistas & inibidores , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Neoplasias de Próstata Resistentes à Castração/patologia , Linfócitos T/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aim: We report results of a first-in-human study of pasotuxizumab, a PSMA bispecific T-cell engager (BiTE®) immune therapy mediating T-cell killing of tumor cells in patients with advanced castration-resistant prostate cancer. Patients & methods: We assessed once-daily subcutaneous (SC) pasotuxizumab. All SC patients developed antidrug antibodies; therefore, continuous intravenous (cIV) infusion was assessed. Results: A total of 47 patients received pasotuxizumab (SC: n = 31, 0.5-172 µg/d; cIV: n = 16, 5-80 µg/d). The SC maximum tolerated dose was 172.0 µg/d. A sponsor change stopped the cIV cohort early; maximum tolerated dose was not determined. PSA responders occurred (>50% PSA decline: SC, n = 9; cIV, n = 3), including two long-term responders. Conclusion: Data support pasotuxizumab safety in advanced castration-resistant prostate cancer and represent evidence of BiTE monotherapy efficacy in solid tumors. Clinical trial registration: NCT01723475 (ClinicalTrials.gov).
Assuntos
Anticorpos Biespecíficos , Antineoplásicos Imunológicos , Neoplasias de Próstata Resistentes à Castração , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-Idade , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/uso terapêutico , Antígenos de Superfície/imunologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/sangue , Complexo CD3/imunologia , Glutamato Carboxipeptidase II/imunologia , Imunoterapia , Infusões Intravenosas , Injeções Subcutâneas , Dose Máxima Tolerável , Neoplasias de Próstata Resistentes à Castração/sangue , Neoplasias de Próstata Resistentes à Castração/imunologia , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/terapia , Resultado do TratamentoRESUMO
Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.
Assuntos
Anticorpos Biespecíficos/metabolismo , Vacinas Anticâncer , Melanoma/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Complexo CD3/imunologia , Células CHO , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/imunologia , Cricetinae , Cricetulus , Citotoxicidade Imunológica , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Humanos , Melanoma/patologia , Melanoma/terapia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety. METHODS: We recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation. RESULTS: ING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells. CONCLUSION: A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.
RESUMO
Blinatumomab, a CD19/CD3-bispecific T-cell engager (BiTE) immuno-oncology therapy for the treatment of B-cell malignancies, is associated with neurologic adverse events in a subgroup of patients. Here, we provide evidence for a two-step process for the development of neurologic adverse events in response to blinatumomab: (i) blinatumomab induced B-cell-independent redistribution of peripheral T cells, including T-cell adhesion to blood vessel endothelium, endothelial activation, and T-cell transmigration into the perivascular space, where (ii) blinatumomab induced B-cell-dependent T-cell activation and cytokine release to potentially trigger neurologic adverse events. Evidence for this process includes (i) the coincidence of T-cell redistribution and the early occurrence of most neurologic adverse events, (ii) T-cell transmigration through brain microvascular endothelium, (iii) detection of T cells, B cells, and blinatumomab in cerebrospinal fluid, (iv) blinatumomab-induced T-cell rolling and adhesion to vascular endothelial cells in vitro, and (v) the ability of antiadhesive agents to interfere with blinatumomab-induced interactions between T cells and vascular endothelial cells in vitro and in patients. On the basis of these observations, we propose a model that could be the basis of mitigation strategies for neurologic adverse events associated with blinatumomab treatment and other T-cell therapies. SIGNIFICANCE: This study proposes T-cell adhesion to endothelial cells as a necessary but insufficient first step for development of blinatumomab-associated neurologic adverse events and suggests interfering with adhesion as a mitigation approach.
Assuntos
Anticorpos Biespecíficos/efeitos adversos , Adesão Celular/efeitos dos fármacos , Células Endoteliais/imunologia , Síndromes Neurotóxicas/imunologia , Linfócitos T/imunologia , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Encéfalo/irrigação sanguínea , Encéfalo/imunologia , Encéfalo/patologia , Adesão Celular/imunologia , Linhagem Celular , Criança , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Feminino , Humanos , Incidência , Linfoma não Hodgkin/sangue , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/imunologia , Masculino , Microvasos/citologia , Microvasos/imunologia , Microvasos/patologia , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/imunologia , Síndromes Neurotóxicas/epidemiologia , Síndromes Neurotóxicas/patologia , Síndromes Neurotóxicas/prevenção & controle , Leucemia-Linfoma Linfoblástico de Células Precursoras B/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Linfócitos T/efeitos dos fármacosRESUMO
MuS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. A recent study has shown that microS110 has significant anti tumor activity at well-tolerated doses as low as 5 microg/kg in orthotopic breast and lung cancer models (Amann et al. in Cancer Res 68:143-151, 2008). Here, we have explored the safety profile of microS110 at higher doses. Escalation to 50 microg/kg microS110 caused in mice transient loss of body weight, and transient piloerection, hypomotility, hypothermia and diarrhoea. These clinical signs coincided with serum peaks of TNF-alpha, IL-6, IL-2, IFN-gamma and IL-4, and an increase of surface markers for T cell activation. Because activation of T cells in response to BiTE antibodies is typically dependent on target cells, we analyzed mouse blood for the presence of EpCAM(+) cells. Various mouse strains presented with a subpopulation of 2-3% EpCAM(+) blood cells, mostly B and T lymphocytes, which was not detected in human blood samples. In vitro experiments in which the number of EpCAM(+) cells in blood samples was either reduced or increased suggested that both T cell activation and cytokine release in response to microS110 was dependent on the number of target-expressing cells. In support for a role of EpCAM(+) lymphocytes in the observed side effects, reduction of EpCAM(+) blood cells in mice via a low-dose pre treatment with microS110 dramatically increased the tolerability of animals up to at least 500 microg/kg of the BiTE antibody. This high tolerability to microS110 occurred in the presence of non-compromised T cells. No damage to EpCAM(+) epithelial tissues was evident from histopathological examination of animals daily injected with 100 microg/kg microS110 for 28 days. In summary, these observations suggest that side effects of microS110 in mice were largely caused by an acute T cell activation that was triggered by a subpopulation of EpCAM(+) lymphocytes. Because humans have extremely low numbers of EpCAM(+) cells in blood, this toxicity of an EpCAM-specific BiTE may be specific for mice.
Assuntos
Antígenos de Neoplasias/sangue , Moléculas de Adesão Celular/sangue , Epitopos de Linfócito T/sangue , Subpopulações de Linfócitos , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Células CHO , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Ativação Linfocitária , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/genética , Especificidade da EspécieRESUMO
The EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in malignant cells and thus provides opportunities for selective targeting of tumor cells. We describe here the development of a novel, bispecific single-chain antibody (bscAb) referred to as bscEphA2xCD3. This molecule simultaneously targets EphA2 on tumor cells and the T-cell receptor/CD3 complex on T cells and possesses structural and functional characteristics of the recently developed BiTE technology. An EphA2-specific single-chain antibody was selected for recognition of an epitope that is preferentially exposed on malignant cells based on the concept of epitope exclusion; this was fused to a CD3-specific single-chain antibody to generate bscEphA2xCD3. The resultant bscAb redirected unstimulated human T cells to lyse EphA2-expressing tumor cells both in vitro and in vivo. In separate experiments, efficient tumor cell lysis was achieved in vitro at drug concentrations Assuntos
Anticorpos Biespecíficos/farmacologia
, Complexo CD3/imunologia
, Imunização Passiva/métodos
, Neoplasias/terapia
, Receptor EphA2/imunologia
, Animais
, Anticorpos Biespecíficos/imunologia
, Especificidade de Anticorpos
, Células CHO
, Processos de Crescimento Celular/imunologia
, Linhagem Celular Tumoral
, Cricetinae
, Cricetulus
, Epitopos/imunologia
, Humanos
, Junções Intercelulares/imunologia
, Ativação Linfocitária
, Camundongos
, Camundongos Endogâmicos NOD
, Camundongos SCID
, Neoplasias/imunologia
, Neoplasias/patologia
, Linfócitos T/imunologia
, Ensaios Antitumorais Modelo de Xenoenxerto
RESUMO
Many kinds of bispecific antibodies recruiting T cells for cancer therapy have been developed. Side-by-side comparison has shown that CD19-/CD3-bispecific antibodies of the diabody, tandem diabody (Tandab) and quadroma format had similar cytotoxic activity, with Tandab being the most active format. Tandab has also been claimed to be superior to single-chain (sc) Fv-based bispecific constructs although data from a side-by-side comparison are not available. In this study, we compared side-by-side MT103 (bscCD19xCD3), a single-chain bispecific antibody of the BiTE class, with a CD19-/CD3-bispecific representative of the Tandab class. Based on literature data, we have constructed, produced and characterized the LL linker version of Tandab, which was reported to be the most active version of Tandab proteins. A dimeric protein of 114kDa was obtained that showed proper bispecific binding to CD3- and CD19-positive cells and could redirect both pre-stimulated and unstimulated human T cells for lysis of human B lymphoma lines Raji, MEC-1 and Nalm-6. Raji cells were lysed at a half-maximal concentration (EC50) of 10 nM Tandab using pre-stimulated T cells, which closely matched the published activity of LL-Tandab with this particular cell line. MT103 had between 700- and 8000-fold higher efficacy than Tandab for redirected lysis of the three human B lymphoma lines. These data demonstrate that under identical experimental conditions, the BiTE format has far superior activity compared to the Tandab format and is also superior to conventional diabody and quadroma formats. The extraordinary potency of the BiTE class and its representative MT103 may translate into improved anti-tumor activity, lower dosing and lower costs of production compared to other bispecific antibody formats.
Assuntos
Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD19/imunologia , Antineoplásicos/imunologia , Complexo CD3/imunologia , Linfoma/imunologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Linfoma/tratamento farmacológico , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêuticoRESUMO
BACKGROUND: Blinatumomab is a bispecific T-cell engager (BiTE®) antibody construct targeting CD3ε on T cells and CD19 on B cells. We describe the relationship between pharmacokinetics (PK) of blinatumomab and pharmacodynamic (PD) changes in peripheral lymphocytes, serum cytokines, and tumor size in patients with non-Hodgkin lymphoma (NHL). METHODS: In a phase 1 study, 76 patients with relapsed/refractory NHL received blinatumomab by continuous intravenous infusion at various doses (0.5 to 90 µg/m2/day). PD changes were analyzed with respect to dose, blinatumomab concentration at steady state (Css), and cumulative area under the concentration-versus-time curve (AUCcum). RESULTS: B-cell depletion occurred within 48 hours at doses ≥5 µg/m2/day, followed first-order kinetics, and was blinatumomab exposure-dependent. Change in tumor size depended on systemic blinatumomab exposure and treatment duration and could be fitted to an Emax model, which predicted a 50% reduction in tumor size at AUCcum of ≥1,340 h×µg/L and Css of ≥1,830 pg/mL, corresponding to a blinatumomab dose of 47 µg/m2/day for 28 days. The magnitude of transient cytokine elevation, observed within 1-2 days of infusion start, was dose-dependent, with less pronounced elevation at low starting doses. CONCLUSION: B-lymphocyte depletion following blinatumomab infusion was exposure-dependent. Transient cytokine elevation increased with dose; it was less pronounced at low starting doses. Tumor response was a function of exposure, suggesting utility for the PK/PD relationship in dose selection for future studies, including NHL and other malignant settings.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Antineoplásicos/administração & dosagem , Linfócitos B/metabolismo , Linfoma não Hodgkin/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Área Sob a Curva , Citocinas/sangue , Relação Dose-Resposta a Droga , Feminino , Humanos , Infusões Intravenosas , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
INTRODUCTION: Distant metastases of solid tumors are usually associated with fatal outcome. Disseminated cancer cells are considered early indicators of metastasis. Their sensitive detection and quantification would be a valuable tool for staging of disease and as guidance for therapeutic decisions. EXPERIMENTAL DESIGN: We established a highly sensitive and quantitative multimarker real-time RT-PCR assay for amplification of cancer-related genes MAGE-A1, -A2, -A3/6, -A4, -A10 and -A12 using SYBR green I to detect one single tumor cell in 2 mL of blood or bone marrow. The feasibility of the assay was tested in a large cohort of 177 patients with locally confined prostate carcinoma. RESULTS: Analysis revealed frequent MAGE expression in venous blood and bilateral bone marrow samples (25.5% of all cases) and yielded the first quantitative profile of MAGE expression with a broad range of transcript concentrations for individual markers in the minimal systemic tumor load of patients with localized cancer. CONCLUSIONS: Rare transcripts of different MAGE-A genes can be quantified in clinical samples of cancer patients by a sensitive multimarker real-time RT-PCR. Because of frequent expression of MAGE genes in various types of cancer the multimarker MAGE real-time RT-PCR may be generally useful for detection, quantification and characterization of the individual disseminated tumor load in cancer patients.
Assuntos
Biomarcadores Tumorais/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Carga Tumoral , Idoso , Antígenos de Neoplasias/genética , Expressão Gênica , Humanos , Masculino , Antígenos Específicos de Melanoma , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/análise , RNA Neoplásico/análise , Sensibilidade e Especificidade , Transcrição GênicaRESUMO
Certain bispecific single-chain antibody constructs exhibit an extraordinary potency for polyclonal T cell engagement and target cell lysis. Here we studied the structural basis for this potency, using laser scanning confocal microscopy. Cytolytic human T cell synapses could be triggered either by addition of a specific peptide antigen or an Ep-CAM-/CD3-bispecific T cell engager (BiTE). Both kinds of synapses showed a comparable distribution of all protein markers investigated. Two other BiTEs constructed from different Ep-CAM-specific antibodies gave similar results. BiTEs could also induce lytic synapses between human T cells and a MHC class I-negative, Ep-CAM cDNA-transfected cell line resulting in potent target cell lysis. This shows that certain T cell recognition molecules on target cells are dispensable for synapse formation and BiTE activity, and suggests that BiTE-activated polyclonal T cells may ignore major immune evasion mechanisms of tumor cells in vivo, such as loss of MHC class I expression.