RESUMO
The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fragmentos Fc das Imunoglobulinas/imunologia , Peptídeo Hidrolases/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Biomarcadores , Linhagem Celular Tumoral , Galinhas , Humanos , Hidrólise , Metaloproteinase 9 da Matriz/metabolismo , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/metabolismo , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/efeitos adversos , Anticorpos de Cadeia Única/farmacologia , Trastuzumab/administração & dosagem , Trastuzumab/efeitos adversos , Trastuzumab/farmacologiaRESUMO
Through protein engineering and a novel pegylation strategy, a diabody specific to tumor-associated glycoprotein 72 (TAG-72) (PEG-AVP0458) has been created to optimize pharmacokinetics and bioavailability to tumor. We report the preclinical and clinical translation of PEG-AVP0458 to a first-in-human clinical trial of a diabody. Methods: Clinical translation followed characterization of PEG-AVP0458 drug product and preclinical biodistribution and imaging assessments of Iodine-124 trace labeled PEG-AVP0458 (124I-PEG-AVP0458). The primary study objective of the first-in-human study was the safety of a single protein dose of 1.0 or 10 mg/m2 124I-PEG-AVP0458 in patients with TAG-72 positive relapsed/ metastatic prostate or ovarian cancer. Secondary study objectives were evaluation of the biodistribution, tumor uptake, pharmacokinetics and immunogenicity. Patients were infused with a single-dose of 124I labeled PEG-AVP0458 (3-5 mCi (111-185 MBq) for positron emission tomography (PET) imaging, performed sequentially over a one-week period. Safety, pharmacokinetics, biodistribution, and immunogenicity were assessed up to 28 days after infusion. Results: PEG-AVP0458 was radiolabeled with 124I and shown to retain high TAG-72 affinity and excellent targeting of TAG-72 positive xenografts by biodistribution analysis and PET imaging. In the first-in-human trial, no adverse events or toxicity attributable to 124I-PEG-AVP0458 were observed. Imaging was evaluable in 5 patients, with rapid and highly specific targeting of tumor and minimal normal organ uptake, leading to high tumor:blood ratios. Serum concentration values of 124I-PEG-AVP0458 showed consistent values between patients, and there was no significant difference in T½α and T½ß between dose levels with mean (± SD) results of T½α = 5.10 ± 4.58 hours, T½ß = 46.19 ± 13.06 hours. Conclusions: These data demonstrates the safety and feasibility of using pegylated diabodies for selective tumor imaging and potential delivery of therapeutic payloads in cancer patients.
Assuntos
Anticorpos Biespecíficos/efeitos adversos , Antígenos de Neoplasias/metabolismo , Antineoplásicos/efeitos adversos , Neoplasias/tratamento farmacológico , Compostos Radiofarmacêuticos/efeitos adversos , Adulto , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacocinética , Anticorpos Antineoplásicos/genética , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Estudos de Viabilidade , Feminino , Humanos , Infusões Intravenosas , Radioisótopos do Iodo/administração & dosagem , Radioisótopos do Iodo/efeitos adversos , Radioisótopos do Iodo/farmacocinética , Masculino , Camundongos , Neoplasias/diagnóstico , Neoplasias/imunologia , Neoplasias/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/efeitos adversos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacocinética , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: This phase 1 dose-escalation trial studied MM-302, a novel HER2-targeted PEGylated antibody-liposomal doxorubicin conjugate, in HER2-positive locally advanced/metastatic breast cancer. METHODS: Patients were enrolled in four cohorts: MM-302 monotherapy (8, 16, 30, 40, and 50 mg/m2 every 4 weeks [q4w]); MM-302 (30 or 40 mg/m2 q4w) plus trastuzumab (4 mg/kg q2w); MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) q3w; MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) and cyclophosphamide (450 mg/m2) q3w. RESULTS: Sixty-nine patients were treated. The most common adverse events (AEs) were fatigue and nausea. Grade 3/4 AEs of special interest included neutropenia, fatigue, mucosal inflammation, anemia, thrombocytopenia, febrile neutropenia, and palmar-plantar erythrodysesthesia. The MTD was not reached. With MM-302 ≥ 30 mg/m2, overall response rate (ORR) was 13% and median progression-free survival (mPFS) 7.4 months (95% CI: 3·5-10·9) in all arms. In 25 anthracycline-naïve patients, ORR was 28·0% and mPFS 10·9 months (95% CI: 1·8-15·3). Imaging with 64Cu-labeled MM-302 visualized tumor-drug penetrance in tumors throughout the body, including the brain. CONCLUSION: MM-302 monotherapy, in combination with trastuzumab, or trastuzumab plus cyclophosphamide, was well tolerated and showed promising efficacy. The selected phase 2 MM-302 dose was 30 mg/m2 plus 6 mg/kg trastuzumab q3w.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Ciclofosfamida/administração & dosagem , Doxorrubicina/análogos & derivados , Imunoconjugados/administração & dosagem , Receptor ErbB-2/genética , Anticorpos de Cadeia Única/administração & dosagem , Trastuzumab/administração & dosagem , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Encéfalo/diagnóstico por imagem , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Ciclofosfamida/efeitos adversos , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacocinética , Esquema de Medicação , Feminino , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacocinética , Anticorpos de Cadeia Única/efeitos adversos , Anticorpos de Cadeia Única/farmacocinética , Análise de Sobrevida , Trastuzumab/efeitos adversos , Resultado do TratamentoRESUMO
INTRODUCTION: MEDI-565, a bispecific, single-chain antibody targeting human carcinoembryonic antigen on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex, showed antitumor activity in carcinoembryonic antigen-expressing tumors in murine models. PATIENTS AND METHODS: This phase I, multicenter, open-label dose escalation study enrolled adults with gastrointestinal adenocarcinomas. MEDI-565 was given intravenously over 3 hours on days 1 through 5 in 28-day cycles, with 4 single-patient (0.75-20 µg) and 5 standard 3 + 3 escalation (60 µg-3 mg; 1.5-7.5 mg with dexamethasone) cohorts. Primary objective was determining maximum tolerated dose; secondary objectives were evaluating pharmacokinetics, antidrug antibodies, and antitumor activity. RESULTS: Thirty-nine patients were enrolled (mean age, 59 years; 56% male; 72% colorectal cancer). Four patients experienced dose-limiting toxicities (2 at 3 mg; 2 at 7.5 mg + dexamethasone): hypoxia (n = 2), diarrhea, and cytokine release syndrome (CRS). Five patients reported grade 3 treatment-related adverse events: diarrhea, CRS, increased alanine aminotransferase, hypertension (all, n = 1), and hypoxia (n = 2); 6 experienced treatment-related serious adverse events: diarrhea, vomiting, pyrexia, CRS (all, n = 1), and hypoxia (n = 2). MEDI-565 pharmacokinetics was linear and dose-proportional, with fast clearance and short half-life. Nineteen patients (48.7%) had antidrug antibodies; 5 (12.8%) had high titers, 2 with decreased MEDI-565 concentrations. No objective responses occurred; 11 (28%) had stable disease as best response. CONCLUSIONS: The maximum tolerated dose of MEDI-565 in this patient population was 5 mg administered over 3 hours on days 1 through 5 every 28 days, with dexamethasone. Pharmacokinetics were linear. No objective responses were observed.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anticorpos Biespecíficos/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias Gastrointestinais/tratamento farmacológico , Imunoterapia/métodos , Adulto , Idoso , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/farmacocinética , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Complexo CD3/imunologia , Antígeno Carcinoembrionário/imunologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/efeitos adversos , Anticorpos de Cadeia Única/farmacocinética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
RIT has become an attractive strategy in cancer treatment, but still faces important drawbacks due to poor tumor penetration and undesirable pharmacokinetics of the targeting vehicles. Smaller radiolabeled antibody fragments and peptides feature highly specific target accumulation, resulting in low accumulation in healthy tissue, except for the kidneys. Nanobodies are the smallest (MW<15 kDa) functional antigen-binding fragments that are derived from heavy chain-only camelid antibodies. Here, we show that the extend of kidney retention of nanobodies is predominantly dictated by the number of polar residues in the C-terminal amino acid tag. Three nanobodies were produced with different C-terminal amino-acid tag sequences (Myc-His-tagged, His-tagged, and untagged). Dynamic planar imaging of Wistar rats with 111In-DTPA-nanobodies revealed that untagged nanobodies showed a 70% drop in kidney accumulation compared to Myc-His-tagged nanobodies at 50 min p.i.. In addition, coinfusion of untagged nanobodies with the plasma expander Gelofusin led to a final reduction of 90%. Similar findings were obtained with different 177Lu-DTPA-2Rs15d nanobody constructs in HER2pos tumor xenografted mice at 1 h p.i.. Kidney accumulation decreased 88% when comparing Myc-His-tagged to untagged 2Rs15d nanobody, and 95% with a coinfusion of Gelofusin, without affecting the tumor targeting capacity. Consequently, we identified a generic method to reduce kidney retention of radiolabeled nanobodies. Dosimetry calculations of Gelofusin-coinfused, untagged 177Lu-DTPA-2Rs15d revealed a dose of 0.90 Gy/MBq that was delivered to both tumor and kidneys and extremely low doses to healthy tissues. In a comparative study, 177Lu-DTPA-Trastuzumab supplied 6 times more radiation to the tumor than untagged 177Lu-DTPA-2Rs15d, but concomitantly also a 155, 34, 80, 26 and 4180 fold higher radioactivity burden to lung, liver, spleen, bone and blood. Most importantly, nanobody-based targeted radionuclide therapy in mice bearing small estiblashed HER2pos tumors led to an almost complete blockade of tumor growth and a significant difference in event-free survival between the treated and the control groups (P<0.0001). Based on histology analyses, no evidence of renal inflammation, apoptosis or necrosis was obtained. In conclusion, these data highlight the importance of the amino acid composition of the nanobody's C-terminus, as it has a predominant effect on kidney retention. Moreover, we show successful nanobody-based targeted radionuclide therapy in a xenograft model and highlight the potential of radiolabeled nanobodies as a valuable adjuvant therapy candidate for treatment of minimal residual and metastatic disease.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Lutécio/uso terapêutico , Neoplasias Ovarianas/radioterapia , Radioimunoterapia/métodos , Radioisótopos/uso terapêutico , Compostos Radiofarmacêuticos/uso terapêutico , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Linhagem Celular Tumoral , Feminino , Humanos , Lutécio/efeitos adversos , Lutécio/farmacocinética , Masculino , Camundongos , Camundongos Nus , Radioisótopos/efeitos adversos , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/efeitos adversos , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Wistar , Anticorpos de Cadeia Única/efeitos adversos , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/uso terapêutico , TrastuzumabAssuntos
Anticorpos Monoclonais/uso terapêutico , Ponte de Artéria Coronária , Anticorpos de Cadeia Única/uso terapêutico , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Ponte de Artéria Coronária/mortalidade , Humanos , Infarto do Miocárdio/induzido quimicamente , Agregação Plaquetária/efeitos dos fármacos , Anticorpos de Cadeia Única/efeitos adversosRESUMO
Accumulation of amyloid-ß protein (Aß) in the brain is thought to be a causal event in Alzheimer's disease (AD). Immunotherapy targeting Aß holds great promise for reducing Aß in the brain. Here, we evaluated the efficacy and safety of anti-Aß single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aß deposits in an AD mouse model (TgAßPPswe/PS1dE9). First, delivery of scFv59 to the brain was optimized by injecting rAAV serotypes 1, 2, and 5 into the right lateral ventricle. Symmetrical high expression of scFv59 was found throughout the hippocampus and partly in the neocortex in both hemispheres via rAAV1 or rAAV5, while scFv59 expression via rAAV2 was mostly limited to one hemisphere. rAAV1, however, induced apoptosis and microglial activation but rAAV5 did not. Therefore, rAAV5 was selected for therapeutic scFv59 delivery in TgAßPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAßPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aß deposits reduced in the hippocampus. Aß42 levels in cerebrospinal fluid (CSF) tended to increase and the Aß40 : 42 ratio decreased in CSF, suggesting that Aß42 was relocated from the parenchyma to CSF. Hemorrhages associated with a focal increase in blood vessel amyloid were found in the brain. While immunotherapy has great potential for clearing cerebral Aß, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aß immunotherapy is applied.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/imunologia , Proteínas Amiloidogênicas/metabolismo , Hemorragia Cerebral/induzido quimicamente , Anticorpos de Cadeia Única/efeitos adversos , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Precursor de Proteína beta-Amiloide , Análise de Variância , Animais , Antígenos CD , Apoptose/efeitos dos fármacos , Apoptose/genética , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Transformada , Dependovirus/fisiologia , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Ensaio de Imunoadsorção Enzimática/métodos , Proteína Glial Fibrilar Ácida/metabolismo , Imunoterapia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Presenilina-1 , Transdução Genética , TransfecçãoRESUMO
OBJECTIVE: The previous Pexelizumab for Reduction of Infarction and Mortality in Coronary Artery Bypass Graft Surgery I (PRIMO-CABG I) trial (n = 3099) indicated that C5 complement inhibition with pexelizumab might reduce myocardial infarction (MI) and postoperative mortality. PRIMO-CABG II was designed to investigate the safety and efficacy of terminal complement inhibition in reducing perioperative MI and mortality in patients undergoing CABG surgery who have 2 or more predefined preoperative risk factors. METHODS: PRIMO-CABG II, a randomized, double-blind, placebo-controlled trial, enrolled 4254 patients undergoing CABG with or without valve surgery at 249 hospitals in North America and Western Europe from June 2004 to July 2005. The patients were randomly assigned to receive intravenous pexelizumab or placebo. The primary composite endpoint was the incidence of death or MI within 30 days of randomization. RESULTS: The PRIMO-CABG II trial did not meet its prespecified primary endpoint of death or MI at 30 days, the secondary endpoints of death at 30 days, or the development of new or worsening congestive heart failure (relative risk 0.91, 0.82, and 1.01, respectively; P > .05). However, in a combined analysis of both pivotal trials, PRIMO-CABG I and II (n = 7353), death at 30 days was significantly reduced for the greatest risk subset (n = 2156, pexelizumab 5.7% vs placebo 8.1%, P = .024). Furthermore, this mortality reduction persisted throughout the 180-day follow-up period (pexelizumab 11.1% vs placebo 14.4%, P = .036). CONCLUSIONS: Pexelizumab was associated with a nonsignificant 6.7% reduction in the primary composite endpoint of death or MI at postoperative day 30 in CABG patients enrolled in the PRIMO-CABG II trial, despite the suggestion of a more favorable treatment effect in the previous PRIMO-CABG I trial. However, an exploratory analysis of the combined PRIMO I and II data set using an established predictive risk model showed a mortality benefit for high-risk surgical patients.