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1.
Nat Cancer ; 3(4): 418-436, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35469014

RESUMO

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.


Assuntos
Anticorpos Biespecíficos , Neoplasias Epiteliais e Glandulares , Anticorpos Biespecíficos/farmacologia , Receptores ErbB/metabolismo , Humanos , Imidazóis , Neoplasias Epiteliais e Glandulares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides , Pirazinas , Receptores Acoplados a Proteínas G/metabolismo
2.
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
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.
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
5.
Cancer Cell ; 33(5): 922-936.e10, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29763625

RESUMO

HER2-driven cancers require phosphatidylinositide-3 kinase (PI3K)/Akt signaling through HER3 to promote tumor growth and survival. The therapeutic benefit of HER2-targeting agents, which depend on PI3K/Akt inhibition, can be overcome by hyperactivation of the heregulin (HRG)/HER3 pathway. Here we describe an unbiased phenotypic combinatorial screening approach to identify a bispecific immunoglobulin G1 (IgG1) antibody against HER2 and HER3. In tumor models resistant to HER2-targeting agents, the bispecific IgG1 potently inhibits the HRG/HER3 pathway and downstream PI3K/Akt signaling via a "dock & block" mechanism. This bispecific IgG1 is a potentially effective therapy for breast cancer and other tumors with hyperactivated HRG/HER3 signaling.


Assuntos
Anticorpos Biespecíficos/administração & dosagem , Imunoglobulina G/administração & dosagem , Neoplasias/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoglobulina G/farmacologia , Células MCF-7 , Camundongos , Modelos Moleculares , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/química , Receptor ErbB-3/química , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Trends Biotechnol ; 25(9): 390-4, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17681390

RESUMO

The therapeutic and commercial success of monoclonal antibodies (mAbs) has inspired innovative approaches aimed at increasing their potency and broadening their applicability. Among these, cocktails of recombinant human mAbs are a logical next step because they combine the technological advances made in the field of antibody engineering with the notion that the ingredients of polyclonal-antibody preparations act in concert to optimally exert and recruit effector functions. Cocktails of mAbs have entered clinical trials, and new technology platforms are being developed for their generation. On the basis of preclinical and early clinical results, the question is not whether cocktails of mAbs have a bright future as therapeutics, but rather what platform is able to reproducibly and cost effectively generate efficacious concoctions that are approvable by the regulatory authorities.


Assuntos
Animais Geneticamente Modificados/imunologia , Anticorpos Monoclonais/biossíntese , Proteínas Recombinantes/imunologia , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Biofarmácia/tendências , Ensaios Clínicos como Assunto , Quimioterapia Combinada , Humanos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese
7.
Nucleic Acids Res ; 31(11): e59, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12771223

RESUMO

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.


Assuntos
Bacteriófagos/genética , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão/genética , Proteínas do Capsídeo , Proteínas de Ligação a DNA/genética , Humanos , Região Variável de Imunoglobulina/genética , Mutação , Técnicas de Amplificação de Ácido Nucleico , Células U937 , Proteínas Virais de Fusão/genética
9.
Biotechnol Prog ; 19(1): 163-8, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12573020

RESUMO

The number of therapeutic monoclonal antibodies in production is expected to rise rapidly in the next few years. As a result, there is much focus on the optimization of antibody expression platforms. Several issues are important including the speed of transition from bench to manufacturing, yield of IgG, and quality (particularly of the glycan structures present on immunoglobulins). We have characterized the human cell line PER.C6 for its ability to produce recombinant IgG. Production yields are still being optimized, but in nonfed batch culture, PER.C6 is able to grow to a cell density of 5 x 10(6) cells/mL and produce 300-500 mg/L IgG; this is likely to increase significantly in fed batch cultures. The generation of antibody-producing cell lines is fast, as rounds of amplification of inserted genes are not required for high production yields. The gene copy number of inserted genes is in the region of 1-10 copies per genome. In addition, PER.C6 is a human cell line, and so does not add glycans, which are immunogenic in humans. A core fucose molecule is essentially always present, and galactose residues are present at a physiological level (0, 1, and 2 galactose residues per glycan are present at a ratio of 1:2:1). No hybrid or high-mannose structures are seen.


Assuntos
Regulação da Expressão Gênica/genética , Imunoglobulina G/biossíntese , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Retina/metabolismo , Reatores Biológicos , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Clonagem Molecular/métodos , Humanos , Imunoglobulina G/genética , Polissacarídeos/metabolismo , Controle de Qualidade , Proteínas Recombinantes/genética , Retina/citologia , Retina/embriologia , Retina/fisiologia , Transfecção/métodos
10.
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
11.
Br J Haematol ; 117(1): 226-37, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11918560

RESUMO

Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34+CD38- cells from purified CD34+ cells upon stimulation with Flt3-ligand, stem cell factor and thrombopoietin. Over a 100-fold (range 80 to 128-fold) expansion of CD34+CD38- cells was observed with bone marrow and cord blood (CB). The expanded CD34+CD38- cells remained negative for lineage-specific markers and could be induced to differentiate into granulocytes, monocytes, megakaryocytes, erythrocytes, and T and B-lymphocytes in vitro. Lineage differentiation assays with single CD34+CD38- cells showed no loss of multilineage potential of expanded cells after ex vivo culture. We also demonstrated that the increase in frequency of CD34+CD38- cells was not as a result of the downregulation of CD38 expression during the culture. Quantitative analysis showed that the number of 6 week cobblestone area forming cells (CAFCwk6), a measure of proliferating HSC, in cytokine-stimulated CD34+ cells were increased by 20-fold. Expanded CD34+CD38- cells could be transduced efficiently with retroviruses encoding the low affinity nerve growth factor receptor (LNGFR) marker gene (17% to 44%, mean 27%), resulting in long-lasting expression of retroviral-encoded genes in progeny HSC and differentiated progenitors. We conclude that the combination Flt3-ligand (FL), stem cell factor and thrombopoietin (TPO) induced strong ex vivo proliferation of CD34+CD38- cells and that the absolute number of expanded cells with stem cell activity increased substantially in this population.


Assuntos
Antígenos CD34 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Adenoviridae/genética , Células da Medula Óssea/imunologia , Contagem de Células , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Sangue Fetal , Vetores Genéticos/administração & dosagem , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Proteínas de Membrana/farmacologia , Receptor de Fator de Crescimento Neural/genética , Fator de Células-Tronco/farmacologia , Estimulação Química , Trombopoetina/farmacologia , Transdução Genética
12.
Blood ; 104(4): 1034-41, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15069014

RESUMO

When a blood clot is formed, vitronectin (VN) is incorporated. Here we studied the consequence of VN incorporation for platelet interactions under flow. Perfusion of whole blood over a fibrin network, formed from purified fibrinogen, resulted in approximately 20% surface coverage with blood platelets. Incorporation of purified multimeric VN into the fibrin network resulted in a 2-fold increase in surface coverage with platelets and in enhancement of platelet aggregate formation. A human monoclonal antibody (huMab VN18), directed against the multimeric form of VN, inhibited platelet adhesion to the combined fibrin/VN matrix to the level of adhesion on fibrin alone. This inhibition was also shown when whole blood was perfused over a plasma-derived clot. Surprisingly, the inhibitory action of the antibody was not directed toward VN incorporated into the fibrin network but toward VN released from the platelets. We conclude that VN-potentiated platelet-clot interaction requires VN in the clot and multimeric VN bound to the platelet surface. Our results provide evidence that homotypic VN interactions contribute to platelet adhesion and aggregation to a blood clot. This report demonstrates for the first time that self-assembly of VN may provide a physiologically relevant contribution to platelet aggregation on a blood clot.


Assuntos
Fibrina/metabolismo , Adesividade Plaquetária , Trombose/etiologia , Vitronectina/metabolismo , Vitronectina/fisiologia , Anticorpos Monoclonais/efeitos dos fármacos , Plaquetas/química , Plaquetas/metabolismo , Plaquetas/fisiologia , Dimerização , Humanos , Perfusão , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Vitronectina/imunologia
13.
Cancer Immunol Immunother ; 53(9): 799-808, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15197494

RESUMO

The formation of a provisional extracellular matrix represents an important step during tumor growth and angiogenesis. Proteins that participate in this process become activated and undergo conformational changes that expose biologically active cryptic sites. Activated matrix proteins express epitopes not found on their native counterparts. We hypothesized that these epitopes may have a restricted tissue distribution, rendering them suitable targets for therapeutic human monoclonal antibodies (huMabs). In this study, we exploited phage antibody display technology and subtractive phage selection to generate human monoclonal antibody fragments that discriminate between the activated and native conformation of the extracellular matrix protein vitronectin. One of the selected antibody fragments, scFv VN18, was used to construct a fully human IgG/kappa monoclonal antibody with an affinity of 9.3 nM. In immunohistochemical analysis, scFv and huMab VN18 recognized activated vitronectin in tumor tissues, whereas hardly any activated vitronectin was detectable in normal tissues. Iodine 123-radiolabeled huMabVN18 was shown to target to Rous sarcoma virus-induced tumors in chickens, an animal model in which the epitope for huMab VN18 is exposed during tumor development. Our results establish activated vitronectin as a potential target for tumor therapy in humans.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Vírus do Sarcoma Aviário/patogenicidade , Doenças das Aves Domésticas/terapia , Sarcoma Aviário/terapia , Vitronectina/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos , Galinhas , Neoplasias do Colo/imunologia , Neoplasias do Colo/terapia , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Humanos , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina , Radioisótopos do Iodo , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Doenças das Aves Domésticas/diagnóstico por imagem , Doenças das Aves Domésticas/imunologia , Conformação Proteica , Radioimunodetecção , Sarcoma Aviário/diagnóstico por imagem , Sarcoma Aviário/imunologia
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