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1.
Haematologica ; 108(7): 1793-1802, 2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-36779592

RESUMO

Cusatuzumab is a high-affinity, anti-CD70 monoclonal antibody under investigation in acute myeloid leukemia (AML). This two-part, open-label, multicenter, phase I/II trial evaluated cusatuzumab plus azacitidine in patients with newly diagnosed AML ineligible for intensive chemotherapy. Patients received a single dose of cusatuzumab at one of four dose levels (1, 3, 10, or 20 mg/kg) 14 days before starting combination therapy. In phase I dose escalation, cusatuzumab was then administered on days 3 and 17, in combination with azacitidine (75 mg/m2) on days 1-7, every 28 days. The primary objective in phase I was to determine the recommended phase II dose (RP2D) of cusatuzumab plus azacitidine. The primary objective in phase II was efficacy at the RP2D (selected as 10 mg/kg). Thirty-eight patients were enrolled: 12 in phase I (three per dose level; four with European LeukemiaNet 2017 adverse risk) and 26 in phase II (21 with adverse risk). An objective response (≥partial remission) was achieved by 19/38 patients (including 8/26 in phase II); 14/38 achieved complete remission. Eleven patients (37.9%) achieved an objective response among the 29 patients in phase I and phase II treated at the RP2D. At a median follow-up of 10.9 months, median duration of first response was 4.5 months and median overall survival was 11.5 months. The most common treatment-emergent adverse events were infections (84.2%) and hematologic toxicities (78.9%). Seven patients (18.4%) reported infusion-related reactions, including two with grade 3 events. Thus, cusatuzumab/azacitidine appears generally well tolerated and shows preliminary efficacy in this setting. Investigation of cusatuzumab combined with current standard-of-care therapy, comprising venetoclax and azacitidine, is ongoing.


Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Azacitidina/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico
2.
Cancer ; 128(5): 1004-1014, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34726773

RESUMO

BACKGROUND: The clinical benefit of cusatuzumab, a CD70-directed monoclonal antibody with enhanced effector functions, was investigated in patients with relapsed/refractory (R/R) cutaneous T-cell lymphoma (CTCL). METHODS: In this cohort expansion of the ARGX-110-1201 study, 27 patients with R/R CTCL received cusatuzumab at 1 (n = 11) or 5 mg/kg (n = 16) once every 3 weeks to investigate its safety, dose, and exploratory efficacy. The pharmacokinetics, immunogenicity, CD70 expression, and CD70/CD27 biology were also assessed. RESULTS: The most common adverse events included infusion-related reactions, pyrexia, and asthenia. Eighteen serious adverse events (grade 1-3) were reported in 11 patients; 1 of these (vasculitis) was considered drug-related. For 8 of the 11 patients receiving 1 mg/kg, anti-drug antibodies (ADAs) affected the minimal concentration, and this resulted in undetectable cusatuzumab concentrations at the end of treatment and, in some cases, a loss of response. This effect was greatly reduced in the patients receiving 5 mg/kg. The overall response rate was 23%; this included 1 complete response and 5 partial responses (PRs) in 26 of the 27 evaluable patients. In addition, 9 patients achieved stable disease. The mean duration on cusatuzumab was 5.2 months, and the median duration was 2.5 months. Patients with Sézary syndrome (SS) achieved a 60% PR rate with a dosage of 5 mg/kg and a 33% PR rate with a dosage of 1 mg/kg; this resulted in an overall response rate of 50% for patients with SS at both doses. CONCLUSIONS: Cusatuzumab was well tolerated, and antitumor activity was observed at both 1 and 5 mg/kg in highly pretreated patients with R/R CTCL. The observed dose-dependent effect on exposure supports the use of 5 mg/kg for future development.


Assuntos
Anticorpos Monoclonais , Antineoplásicos , Linfoma Cutâneo de Células T , Neoplasias Cutâneas , Anticorpos Monoclonais/efeitos adversos , Antineoplásicos/uso terapêutico , Ligante CD27 , Humanos , Linfoma Cutâneo de Células T/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Neoplasias Cutâneas/tratamento farmacológico , Resultado do Tratamento
3.
J Biol Chem ; 291(26): 13846-54, 2016 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-27129274

RESUMO

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229).


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/química , Receptores de Interleucina-6/imunologia , Animais , Camelus , Humanos , Interleucina-6/química , Interleucina-6/imunologia , Camundongos , Estrutura Quaternária de Proteína
4.
Infect Immun ; 84(2): 395-406, 2016 02.
Artigo em Inglês | MEDLINE | ID: mdl-26573738

RESUMO

Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA(-) TcdB(+) strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.


Assuntos
Anticorpos Neutralizantes/imunologia , Antitoxinas/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Clostridioides difficile/imunologia , Enterocolite Pseudomembranosa/prevenção & controle , Lactobacillus/genética , Anticorpos de Domínio Único/imunologia , Administração Oral , Animais , Anticorpos Neutralizantes/genética , Antitoxinas/administração & dosagem , Camelídeos Americanos , Clostridioides difficile/patogenicidade , Cricetinae , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Escherichia coli/genética , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Imunização , Imunização Passiva , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Lactobacillus/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Domínio Único/genética
5.
Lancet Haematol ; 10(11): e902-e912, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37914483

RESUMO

BACKGROUND: Cusatuzumab, a high-affinity anti-CD70 antibody, has shown preliminary activity as a treatment for acute myeloid leukaemia when combined with azacitidine. We aimed to determine the optimum dose for future trials of cusatuzumab in combination with azacitidine in patients with previously untreated acute myeloid leukaemia who are not eligible for intensive chemotherapy. METHODS: In this randomised, phase 2, open-label, dose-optimisation study we enrolled adult patients aged 18 years or older with newly diagnosed acute myeloid leukaemia not eligible for intensive chemotherapy, and with Eastern Cooperative Oncology Group scores of 0-2, from 40 hospitals and centres across seven countries. In part one of the trial, participants were randomly allocated 1:1 to 10 mg/kg or 20 mg/kg intravenous cusatuzumab on days 3 and 17, combined with subcutaneous or intravenous azacitidine 75 mg/m2 on days 1-7 in 28-day cycles. The primary efficacy outcome was the rate of complete remission in the intention-to-treat group. The two dose cohorts were evaluated independently without between-cohort statistical comparison. Safety analyses were performed in all patients who received one dose of study drug. Part two of the trial was planned to be a single-arm expansion to evaluate cusatuzumab plus azacitidine at the cusatuzumab dose level selected in part one (primary hypothesis ≥35% rate of complete remission vs null hypothesis of 20%); however, changes in the acute myeloid leukaemia treatment landscape during this trial made it unlikely that enrolment to part two of the study would be clinically feasible, so the study stopped at the end of part one. The trial was registered at ClinicalTrials.gov, NCT04023526. FINDINGS: 103 patients were enrolled between Aug 30, 2019, and Feb 25, 2020, and randomly assigned to either cusatuzumab 10 mg/kg (n=51) or 20 mg/kg (n=52). Median follow-up was 7·2 months (IQR 10·7 months). 57 of 103 (55%) patients were male and 46 (45%) patients were female, 78 (76%) were White, one (1%) was Asian, and 24 (23%) did not report their race. In the 10 mg/kg group, complete remission rate was 12% (six of 51 patients; 95% CI 6-23) and in the 20 mg/kg group was 27% (14 of 52; 17-40). Grade 3 or worse treatment-emergent adverse events (TEAEs) were similar between the cusatuzumab 10 mg/kg (n=51) and 20 mg/kg (n=51) cohorts and included thrombocytopenia (24 patients [47%] vs 29 [57%]), anaemia (24 [47%] vs 17 [33%]), and neutropenia (20 [39%] in both cohorts). Serious TEAEs were also similar in the two cohorts (44 [86%] vs 40 [78%]). Treatment-related TEAEs leading to death were reported in both groups (three patients [6%] in the 10 mg/kg group vs one patient [2%] in the 20 mg/kg group); the reported causes of death were pneumonia (n=2) and septic shock (n=2). INTERPRETATION: Although part one of this study was not designed to formally compare the two dose cohorts for efficacy, the totality of clinical data for cusatuzumab studies performed to date indicate that cusatuzumab 20 mg/kg plus azacitidine represents the optimal dose for further studies. A phase 1b study investigating the triple combination of cusatuzumab with venetoclax and azacitidine is underway (NCT04150887). FUNDING: Janssen Research & Development and argenx.


Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Adulto , Humanos , Masculino , Feminino , Azacitidina/efeitos adversos , Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/diagnóstico , Indução de Remissão , Esquema de Medicação , Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos
6.
J Infect Dis ; 203(8): 1063-72, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21450996

RESUMO

Influenza A virus infections impose a recurrent and global disease burden. Current antivirals against influenza are not always effective. We assessed the protective potential of monovalent and bivalent Nanobodies (Ablynx) against challenge with this virus. These Nanobodies were derived from llamas and target H5N1 hemagglutinin. Intranasal administration of Nanobodies effectively controlled homologous influenza A virus replication. Administration of Nanobodies before challenge strongly reduced H5N1 virus replication in the lungs and protected mice from morbidity and mortality after a lethal challenge with H5N1 virus. The bivalent Nanobody was at least 60-fold more effective than the monovalent Nanobody in controlling virus replication. In addition, Nanobody therapy after challenge strongly reduced viral replication and significantly delayed time to death. Epitope mapping revealed that the VHH Nanobody binds to antigenic site B in H5 hemagglutinin. Because Nanobodies are small, stable, and simple to produce, they are a promising, novel therapeutic agent against influenza.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Especificidade de Anticorpos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Nanocápsulas , Conformação Proteica , Organismos Livres de Patógenos Específicos , Fatores de Tempo
7.
J Infect Dis ; 204(11): 1692-701, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21998474

RESUMO

Despite the medical importance of respiratory syncytial virus (RSV) infections, there is no vaccine or therapeutic agent available. Prophylactic administration of palivizumab, a humanized monoclonal RSV fusion (F) protein-specific antibody, can protect high-risk children. Previously, we have demonstrated that RSV can be neutralized by picomolar concentrations of a camelid immunoglobulin single-variable domain that binds the RSV protein F (F-VHHb nanobodies). Here, we investigated the mechanism by which these nanobodies neutralize RSV and tested their antiviral activity in vivo. We demonstrate that bivalent RSV F-specific nanobodies neutralize RSV infection by inhibiting fusion without affecting viral attachment. The ability of RSV F-specific nanobodies to protect against RSV infection was investigated in vivo. Intranasal administration of bivalent RSV F-specific nanobodies protected BALB/c mice from RSV infection, and associated pulmonary inflammation. Moreover, therapeutic treatment with these nanobodies after RSV infection could reduce viral replication and reduced pulmonary inflammation. Thus, nanobodies are promising therapeutic molecules for treatment of RSV.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/imunologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antivirais/imunologia , Antivirais/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/imunologia , Fatores de Tempo , Proteínas Virais de Fusão/imunologia , Carga Viral/efeitos dos fármacos
8.
Biomedicines ; 9(6)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200749

RESUMO

Dysregulation of MET signaling has been implicated in tumorigenesis and metastasis. ARGX-111 combines complete blockade of this pathway with enhanced tumor cell killing and was investigated in 24 patients with MET-positive advanced cancers in a phase 1b study at four dose levels (0.3-10 mg/kg). ARGX-111 was well tolerated up to 3 mg/kg (MTD). Anti-tumor activity was observed in nearly half of the patients (46%) with a mean duration of treatment of 12 weeks. NHance® mutations in the Fc of ARGX-111 increased affinity for the neonatal Fc receptor (FcRn) at acidic pH, stimulating transcytosis across FcRn-expressing cells and radiolabeled ARGX-111 accumulated in lymphoid tissues, bone and liver, organs expressing FcRn at high levels in a biodistribution study using human FcRn transgenic mice. In line with this, we observed, in a patient with MET-amplified (>10 copies) gastric cancer, diminished metabolic activity in multiple metastatic lesions in lymphoid and bone tissues by 18F-FDG-PET/CT after two infusions with 0.3 mg/kg ARGX-111. When escalated to 1 mg/kg, a partial response was reached. Furthermore, decreased numbers of CTC (75%) possibly by the enhanced tumor cell killing witnessed the modes of action of the drug, warranting further clinical investigation of ARGX-111.

9.
Nat Med ; 26(9): 1459-1467, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32601337

RESUMO

Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse1,2. Hypomethylating agents (HMAs) are the standard of care in the treatment of older or unfit patients with AML, but responses are modest and not durable3-5. Here we demonstrate that LSCs upregulate the tumor necrosis factor family ligand CD70 in response to HMA treatment resulting in increased CD70/CD27 signaling. Blocking CD70/CD27 signaling and targeting CD70-expressing LSCs with cusatuzumab, a human αCD70 monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity activity, eliminated LSCs in vitro and in xenotransplantation experiments. Based on these preclinical results, we performed a phase 1/2 trial in previously untreated older patients with AML with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine ( NCT03030612 ). We report results from the phase 1 dose escalation part of the clinical trial. Hematological responses in the 12 patients enrolled included 8 complete remission, 2 complete remission with incomplete blood count recovery and 2 partial remission with 4 patients achieving minimal residual disease negativity by flow cytometry at <10-3. Median time to response was 3.3 months. Median progression-free survival was not reached yet at the time of the data cutoff. No dose-limiting toxicities were reported and the maximum tolerated dose of cusatuzumab was not reached. Importantly, cusatuzumab treatment substantially reduced LSCs and triggered gene signatures related to myeloid differentiation and apoptosis.


Assuntos
Antineoplásicos/uso terapêutico , Ligante CD27/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Azacitidina/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Humanos , Leucemia Mieloide Aguda/patologia , Resultado do Tratamento , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
10.
Glycobiology ; 19(2): 182-91, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18997175

RESUMO

The "Le(b) mouse" was established as a model for investigations of the molecular events following Le(b)-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human alpha-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Le(b) glycoproteins in gastric pit and surface mucous cells was obtained in this "Le(b) mouse," as demonstrated by binding of monoclonal anti-Le(b) antibodies. To explore the effects of the human alpha-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic alpha-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc alpha 2Gal beta 3(Fuc alpha 4)GalNAc beta 4 Gal beta 4 Glc beta 1Cer, i.e., a novel Le(b)-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAc beta 3 Gal alpha 3(Fuc alpha 2)Gal beta 4 Glc beta 1Cer, in which the isoglobotetrasaccharide has been combined with Fuc alpha 2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAc beta 3(Fuc alpha 2)Gal beta 3GalNAc beta 4Gal beta 4 Glc beta 1Cer.


Assuntos
Fucosiltransferases/metabolismo , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Helicobacter pylori/metabolismo , Estômago/química , Animais , Sítios de Ligação , Mucosa Gástrica/metabolismo , Glicoproteínas/metabolismo , Humanos , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Estômago/enzimologia
11.
BMC Biotechnol ; 7: 58, 2007 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-17875214

RESUMO

BACKGROUND: Bacteriophages infecting lactic acid bacteria (LAB) are widely acknowledged as the main cause of milk fermentation failures. In this study, we describe the surface-expression as well as the secretion of two functional llama heavy-chain antibody fragments, one binding to the major capsid protein (MCP) and the other to the receptor-binding proteins (RBP) of the lactococcal bacteriophage p2, by lactobacilli in order to neutralise lactococcal phages. RESULTS: The antibody fragment VHH5 that is directed against the RBP, was fused to a c-myc tag and expressed in a secreted form by a Lactobacillus strain. The fragment VHH2 that is binding to the MCP, was fused to an E-tag and anchored on the surface of the lactobacilli. Surface expression of VHH2 was confirmed by flow cytometry using an anti-E-tag antibody. Efficient binding of both the VHH2 and the secreted VHH5 fragment to the phage antigens was shown in ELISA. Scanning electron microscopy showed that lactobacilli expressing VHH2 anchored at their surface were able to bind lactococcal phages. A neutralisation assay also confirmed that the secreted VHH5 and the anchored VHH2 fragments prevented the adsorption of lactococcal phages to their host cells. CONCLUSION: Lactobacilli were able to express functional VHH fragments in both a secreted and a cell surface form and reduced phage infection of lactococcal cells. Lactobacilli expressing llama heavy-chain antibody fragments represent a novel way to limit phage infection.


Assuntos
Bacteriófagos/fisiologia , Camelídeos Americanos/imunologia , Melhoramento Genético/métodos , Fragmentos de Imunoglobulinas/metabolismo , Lactobacillus/fisiologia , Lactobacillus/virologia , Engenharia de Proteínas/métodos , Animais , Sobrevivência Celular , Clonagem Molecular/métodos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Proteínas Recombinantes/metabolismo
12.
Nat Biotechnol ; 20(7): 702-6, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12089555

RESUMO

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo. Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans, were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.


Assuntos
Regulação Bacteriana da Expressão Gênica , Região Variável de Imunoglobulina/biossíntese , Lacticaseibacillus casei/genética , Lacticaseibacillus casei/imunologia , Animais , Cárie Dentária/imunologia , Cárie Dentária/prevenção & controle , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Modelos Genéticos , Ratos , Ratos Sprague-Dawley , Streptococcus mutans/imunologia , Streptococcus mutans/fisiologia
13.
Clin Cancer Res ; 23(21): 6411-6420, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28765328

RESUMO

Purpose: The purpose of this study was to evaluate safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor efficacy of ARGX-110, a glyco-engineered monoclonal antibody, targeting CD70, in patients with CD70 expressing advanced malignancies.Experimental Design: Dose escalation with a sequential 3+3 design was performed in five steps at the 0.1, 1, 2, 5, and 10 mg/kg dose levels (N = 26). ARGX-110 was administered intravenously every 3 weeks until progression or intolerable toxicity. Dose-limiting toxicity was evaluated in the 21 days following the first ARGX-110 administration (Cycle 1). Samples for pharmacokinetics and pharmacodynamics were collected.Results: Dose-limiting toxicity was not observed and the maximum tolerated dose was not reached. ARGX-110 was generally well tolerated, with no dose-related increase in treatment-emergent adverse events (TEAE). The most common TEAE were fatigue and drug related infusion-related reactions (IRR). Of the 20 SAEs reported, five events, all IRRs, were considered related to ARGX-110. ARGX-110 demonstrates dose proportionality over the dose range 1 to 10 mg/kg, but not at 0.1 mg/kg and a terminal half-life of 10 to 13 days. The best overall response was stable disease (14/26) in all 26 evaluable patients with various malignancies and the mean duration of treatment was 15 weeks. No dose-response related antitumor activity was observed, but biomarker readouts provided signs of biological activity, particularly in patients with hematologic malignancies.Conclusions: This dose-escalation phase I trial provides evidence of good tolerability of ARGX-110, pharmacokinetics, and preliminary antitumor activity at all dose levels in generally heavily pretreated patients with advanced CD70-positive malignancies. Clin Cancer Res; 23(21); 6411-20. ©2017 AACR.


Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Ligante CD27/imunologia , Neoplasias/tratamento farmacológico , Adulto , Idoso , Anticorpos Anti-Idiotípicos/efeitos adversos , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Ligante CD27/antagonistas & inibidores , Relação Dose-Resposta a Droga , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/classificação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias/patologia
14.
MAbs ; 8(6): 1126-35, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27211075

RESUMO

The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.


Assuntos
Anticorpos Monoclonais/imunologia , Imunização , Epitopos Imunodominantes/imunologia , Receptores de Glucagon/antagonistas & inibidores , Anticorpos de Cadeia Única/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Peptídeos Catiônicos Antimicrobianos , Células CHO , Camelídeos Americanos/imunologia , Catelicidinas/imunologia , Técnicas de Visualização da Superfície Celular , Células Cultivadas , Cricetulus , Fibroblastos , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas de Membrana , Plasmídeos/genética , Plasmídeos/imunologia , Receptores de Glucagon/genética , Receptores de Glucagon/imunologia , Anticorpos de Cadeia Única/sangue
15.
Sci Rep ; 6: 31621, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27546726

RESUMO

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.


Assuntos
Anticorpos Biespecíficos , Antineoplásicos Imunológicos , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Células A549 , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/isolamento & purificação , Antineoplásicos Imunológicos/farmacologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/farmacologia , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Experimentais/imunologia , Proteínas Proto-Oncogênicas c-met/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Protein Eng Des Sel ; 29(4): 123-33, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26945588

RESUMO

Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.


Assuntos
Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Mutação/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Camelídeos Americanos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Interleucina-6/imunologia , Modelos Imunológicos , Modelos Moleculares , Proteínas Recombinantes/química , Alinhamento de Sequência
17.
Mol Biotechnol ; 31(3): 221-31, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16230772

RESUMO

We compared the anticariogenic effect of lactobacilli expressing a single-chain antibody (scFv) against the SA I/II adhesin of Streptococcus mutans under the control of an inducible or a constitutive promoter. Both lactobacilli expressed equal amounts of scFv and agglutinated S. mutans bacteria expressing SA I/II to a similar level. In a rat caries model, transformed lactobacilli could be detected in the oral cavity throughout the duration of the study. Transformants containing the constitutive promoter were slightly more protective than those containing the inducible promoter suggesting that continuous in situ production of scFv in the oral cavity may be therapeutically superior to intermittent production.


Assuntos
Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Imunização Passiva/métodos , Lactobacillus/genética , Streptococcus mutans/imunologia , Aglutinação , Animais , Anticorpos Antibacterianos/genética , Cárie Dentária/terapia , Citometria de Fluxo , Vetores Genéticos/genética , Ratos , Transformação Bacteriana
18.
MAbs ; 7(4): 693-706, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26018625

RESUMO

Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1ß and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform.


Assuntos
Região Variável de Imunoglobulina , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Animais , Camelídeos Americanos , Camelus , Cristalografia por Raios X , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Estrutura Terciária de Proteína
19.
Cancer Res ; 75(16): 3373-83, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26141862

RESUMO

Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies.


Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/imunologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
20.
PLoS One ; 9(10): e109367, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25347556

RESUMO

Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable candidates for development of alternative post exposure treatment drugs against rabies.


Assuntos
Vírus da Raiva/imunologia , Raiva/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Meia-Vida , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Raiva/prevenção & controle , Raiva/virologia , Vacina Antirrábica/imunologia , Vírus da Raiva/genética , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/genética , Distribuição Tecidual , Carga Viral
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