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1.
Nat Commun ; 12(1): 4445, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290245

RESUMO

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.


Assuntos
Ligante 4-1BB/agonistas , Anticorpos Biespecíficos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Ligante 4-1BB/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos , Humanos , Inibidores de Checkpoint Imunológico/imunologia , Tolerância Imunológica/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Imunoterapia , Ativação Linfocitária/efeitos dos fármacos
2.
Expert Opin Biol Ther ; 19(7): 721-733, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31286786

RESUMO

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/metabolismo , Anticorpos Biespecíficos/farmacocinética , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Citocinas/análise , Citocinas/metabolismo , Células HL-60 , Meia-Vida , Humanos , Lectinas Tipo C/imunologia , Leucemia Mieloide Aguda/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Receptores Mitogênicos/imunologia , Linfócitos T/metabolismo
3.
Biotechnol Bioeng ; 106(5): 741-50, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20564612

RESUMO

Therapeutic monoclonal antibodies, a highly successful class of biological drugs, are conventionally manufactured in mammalian cell lines. A recent approach to increase the therapeutic effectiveness of monoclonal antibodies has been to combine two or more of them; however this increases the complexity of development and manufacture. To address this issue a method to efficiently express multiple monoclonal antibodies from a single cell has been developed and we describe here the generation of stable cell clones that express high levels of a human monoclonal antibody mixture. PER.C6 cells were transfected with a combination of plasmids containing genes encoding three different antibodies. Clones that express the three corresponding antibody specificities were identified, subcloned, and passaged in the absence of antibiotic selection pressure. At several time points, batch production runs were analyzed for stable growth and IgG production characteristics. The majority (11/12) of subclones analyzed expressed all three antibody specificities in constant ratios with total IgG productivity ranging between 15 and 20 pg/cell/day under suboptimal culture conditions after up to 67 population doublings. The growth and IgG production characteristics of the stable clones reported here resemble those of single monoclonal antibody cell lines from conventional clone generation programs. We conclude that the methodology described here is applicable to the generation of stable PER.C6(R) clones for industrial scale production of mixtures of antibodies.


Assuntos
Anticorpos Monoclonais/biossíntese , Biotecnologia/métodos , Expressão Gênica , Anticorpos Monoclonais/genética , Técnicas de Cultura de Células , Linhagem Celular , Vetores Genéticos , Humanos , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
4.
J Mol Biol ; 387(3): 548-58, 2009 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-19361421

RESUMO

To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (V(H)) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (V(L)) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the V(H) regions in this V(L)-restricted panel with a previously published repertoire of anti-TT V(H) regions with cognate V(H)-V(L) pairing showed a very similar distribution of V(H), D(H) and J(H) gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-V(L) anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-V(L) anti-TT V(H) repertoire was combined with a collection of naïve V(L) regions and again selected for binding to TT, many of the V(H) genes were recovered in combination with a diversity of V(L) regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT V(H) combined with 15 diverse V(L) chains were determined and found to be identical to each other and to the original isolate restricted to a single-V(L) chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding V(H) regions from an immunized repertoire have promiscuous features. These V(H) regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, V(H) region diversity and affinity of a natural repertoire.


Assuntos
Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/imunologia , Toxoide Tetânico/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Epitopos/química , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Dados de Sequência Molecular , Biblioteca de Peptídeos , Toxoide Tetânico/química
5.
Eur Biophys J ; 34(1): 28-41, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15241571

RESUMO

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein-lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.


Assuntos
Membrana Celular/metabolismo , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/metabolismo , Fluidez de Membrana/fisiologia , Glicoproteínas de Membrana/metabolismo , Lipídeos de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Serina Endopeptidases/metabolismo , Animais , Células CHO , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/química , Fluidez de Membrana/efeitos dos fármacos , Lipídeos de Membrana/química , Ligação Proteica , Serina Endopeptidases/química , Serina Endopeptidases/farmacologia , Receptores Toll-Like
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