RESUMO
Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.
Assuntos
Vacinas Anticâncer , Imunoconjugados , Humanos , Imunoconjugados/farmacologia , Anticorpos , Endossomos , Receptores ErbBRESUMO
Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Animais , Linfócitos T CD8-Positivos , Citocinas/uso terapêutico , Humanos , Linfócitos do Interstício Tumoral , Macaca fascicularis , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Microambiente Tumoral , ZircônioRESUMO
BACKGROUND: Programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) blocking antibodies including cemiplimab have generated profound clinical activity across diverse cancer types. Tumorous PD-L1 expression, as assessed by immunohistochemistry (IHC), is an accepted predictive marker of response to therapy in some cancers. However, expression is often dynamic and heterogeneous, and therefore not reliably captured by IHC from tumor biopsies or archival samples. Thus, there is significant need for accurate whole-body quantification of PD-L1 levels. METHODS: We radiolabeled the novel human anti-PD-L1 antibody REGN3504 with zirconium-89 (89Zr) using the chelator p-SCN-Bn-Deferoxamine to enable non-invasive immuno-positron emission tomography (immuno-PET) of PD-L1 expression. PET imaging assessed the localization of 89Zr-REGN3504 to multiple human tumor xenografts. Mice genetically humanized for PD-1 and PD-L1 were used to assess the biodistribution of 89Zr-REGN3504 to normal tissues and the estimated human radiation dosimetry of 89Zr-REGN3504 was also determined. Pharmacokinetics of REGN3504 was assessed in monkeys. RESULTS: Clear localization of 89Zr-REGN3504 to human tumor xenografts was observed via PET imaging and ex vivo biodistribution studies demonstrated high (fourfold to sixfold) tumor:blood ratios. 89Zr-REGN3504 specifically localized to spleen and lymph nodes in the PD-1/PD-L1 humanized mice. 89Zr-REGN3504 immuno-PET accurately detected a significant reduction in splenic PD-L1 positive cells following systemic treatment with clodronate liposomes. Radiation dosimetry suggested absorbed doses would be within guidelines for other 89Zr radiolabeled, clinically used antibodies. Pharmacokinetics of REGN3504 was linear. CONCLUSION: This work supports the clinical translation of 89Zr-REGN3504 immuno-PET for the assessment of PD-L1 expression. Future clinical studies will aim to investigate the utility of 89Zr-REGN3504 immuno-PET for predicting and monitoring response to anti-PD-1 therapy.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígeno B7-H1/metabolismo , Neoplasias/diagnóstico por imagem , Radioisótopos/química , Zircônio/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Haplorrinos , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias/imunologia , Tomografia por Emissão de Pósitrons , Distribuição TecidualRESUMO
The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo-acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Imunoconjugados/farmacologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Neoplasias Gástricas/patologiaRESUMO
A modeling and simulation approach was used for quantitative comparison of a new generation HER2 antibody drug conjugate (ADC, PF-06804103) with trastuzumab-DM1 (T-DM1). To compare preclinical efficacy, the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of PF-06804103 and T-DM1 was determined across a range of mouse tumor xenograft models, using a tumor growth inhibition model. The tumor static concentration was assigned as the minimal efficacious concentration. PF-06804103 was concluded to be more potent than T-DM1 across cell lines studied. TSCs ranged from 1.0 to 9.8 µg/mL (n = 7) for PF-06804103 and from 4.7 to 29 µg/mL (n = 5) for T-DM1. Two experimental models which were resistant to T-DM1, responded to PF-06804103 treatment. A mechanism-based target mediated drug disposition (TMDD) model was used to predict the human PK of PF-06804103. This model was constructed and validated based on T-DM1 which has non-linear PK at doses administered in the clinic, driven by binding to shed HER2. Non-linear PK is predicted for PF-06804103 in the clinic and is dependent upon circulating HER2 extracellular domain (ECD) concentrations. The models were translated to human and suggested greater efficacy for PF-06804103 compared to T-DM1. In conclusion, a fit-for-purpose translational PK/PD strategy for ADCs is presented and used to compare a new generation HER2 ADC with T-DM1.
Assuntos
Ado-Trastuzumab Emtansina/farmacocinética , Antineoplásicos Imunológicos/farmacocinética , Imunoconjugados/farmacocinética , Neoplasias/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Administração Intravenosa , Ado-Trastuzumab Emtansina/administração & dosagem , Animais , Antineoplásicos Imunológicos/administração & dosagem , Linhagem Celular Tumoral , Simulação por Computador , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoconjugados/administração & dosagem , Macaca fascicularis , Masculino , Camundongos , Modelos Biológicos , Neoplasias/patologia , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with hematologic cancers have improved outcomes after treatment with bispecific antibodies that bind to CD3 on T cells and that redirect T cells toward cancer cells. However, clinical benefit against solid tumors remains to be shown. We made a bispecific antibody that targets both the common prostate tumor-specific antigen PSMA and CD3 (PMSAxCD3) and provide evidence for tumor inhibition in several preclinical solid tumor models. Mice expressing the human extracellular regions of CD3 and PSMA were generated to examine antitumor efficacy in the presence of an intact immune system and PSMA expression in normal tissues. PSMAxCD3 accumulated in PSMA-expressing tissues and tumors as detected by immuno-PET imaging. Although PSMAxCD3 induced T-cell activation and showed antitumor efficacy in mice with low tumor burden, PSMAxCD3 lost efficacy against larger solid tumors, mirroring the difficulty of treating solid tumors in the clinic. Costimulatory receptors can enhance T-cell responses. We show here that costimulation can enhance the antitumor efficacy of PSMAxCD3. In particular, 4-1BB stimulation in combination with PSMAxCD3 enhanced T-cell activation and proliferation, boosted efficacy against larger tumors, and induced T-cell memory, leading to durable antitumor responses. The combination of CD3 bispecific antibodies and anti-4-1BB costimulation represents a therapeutic approach for the treatment of solid tumors.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glutamato Carboxipeptidase II/imunologia , Glutamato Carboxipeptidase II/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Zircônio/farmacocinéticaRESUMO
The immune status of tumors critically influences their responsiveness to PD1 blockades and other immune-based therapies. Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is a clinically validated predictive biomarker of response to checkpoint-inhibitor therapy in a limited number of clinical settings but is poorly predictive in most. With emerging evidence that multiple pathways and immune-checkpoint proteins may coordinately contribute to the adaptive immune resistance, the identification and quantitation of multiple immune markers in tumor tissue could help identify the controlling pathways in a given patient, guide the selection of optimal therapy, and monitor response to treatment. We developed and validated a sensitive and robust immuno-liquid chromatography-parallel reaction monitoring assay to simultaneously quantify the expression levels of six immune markers (CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2) using as little as 1-2 mg of fresh frozen tissue. The lower limit of quantitation ranged from 0.07 ng/mg protein for PD1 to 1.0 ng/mg protein for CD4. The intrabatch accuracy was within -16.6% to 15.0% for all proteins at all concentrations, and the variation ranged from 0.8% to 14.7%, while interbatch accuracy was within -6.3% to 8.6%, and the variation ranged from 1.3% to 12.8%. The validated assay was then applied to quantify all six biomarkers in different tissues and was confirmed to have sufficient sensitivity (0.07-1.00 ng/mg protein) and reproducibility (variation ranged from 4.3 to 12.0%). In an analysis of 26 cervical tumors, CD8A and CD4 were detected in all tumors, followed by PD-L1 in 85%, LAG-3 in 65%, PD1 in 50%, and PD-L2 in 35%. The strongest correlations were observed between CD8A and CD4 ( r = 0.88) and CD8A and LAG-3 ( r = 0.86). PD1 was not significantly correlated with any of the other proteins tested. This method can be applied to survey the immune signatures across tumor types and tailored to incorporate additional markers as needed.
Assuntos
Biomarcadores Tumorais/genética , Cromatografia de Afinidade/normas , Cromatografia Líquida/normas , Peptídeos/análise , Espectrometria de Massas em Tandem/normas , Neoplasias do Colo do Útero/diagnóstico , Sequência de Aminoácidos , Antígenos CD/genética , Antígenos CD/imunologia , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Antígenos CD4/genética , Antígenos CD4/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Criopreservação/métodos , Feminino , Expressão Gênica , Humanos , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Espectrometria de Massas em Tandem/métodos , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17-34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.
RESUMO
There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.
Assuntos
Produtos Biológicos/química , Imunoconjugados/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Ácidos Carboxílicos/química , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Cisteína/química , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Lisina/química , Maleimidas/química , Camundongos , Piranos/química , Distribuição TecidualRESUMO
The stability of the connection between the antibody and the toxin can have a profound impact on ADC safety and efficacy. There has been increasing evidence in recent years that maleimide-based ADCs are prone to payload loss via a retro-Michael type reaction. Herein, we report a mild method for the hydrolysis of the succinimide-thioether ring which results in a "ring-opened" linker. ADCs containing this hydrolyzed succinimide linker show equivalent cytotoxicity, improved in vitro stability, improved PK exposure, and improved efficacy as compared to their nonhydrolyzed counterparts. This method offers a simple way to improve the stability, exposure, and efficacy of maleimide-based ADCs.
Assuntos
Imunotoxinas/química , Succinimidas/química , Sulfetos/química , Animais , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Hidrólise , Imunotoxinas/sangue , Imunotoxinas/uso terapêutico , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Estabilidade ProteicaRESUMO
Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low-antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.
Assuntos
Anticorpos/metabolismo , Imunoconjugados/metabolismo , Preparações Farmacêuticas/síntese química , Engenharia de Proteínas/métodos , Animais , Anticorpos/sangue , Anticorpos/química , Anticorpos/toxicidade , Técnicas de Cultura Celular por Lotes , Células CHO , Morte Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Cricetulus , Cisteína/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Imunoconjugados/toxicidade , Preparações Farmacêuticas/sangue , Preparações Farmacêuticas/química , Estabilidade Proteica/efeitos dos fármacos , RatosRESUMO
Objectives of the present investigation were: (1) to compare three literature reported tumor growth inhibition (TGI) pharmacodynamic (PD) models and propose an optimal new model that best describes the xenograft TGI data for antibody drug conjugates (ADC), (2) to translate efficacy of the ADC Trastuzumab-emtansine (T-DM1) from mice to patients using the optimized PD model, and (3) to apply the translational strategy to predict clinically efficacious concentrations of a novel in-house anti-5T4 ADC, A1mcMMAF. First, the performance of all four of the PD models (i.e. 3 literature reported + 1 proposed) was evaluated using TGI data of T-DM1 obtained from four different xenografts. Based on the estimates of the pharmacodynamic/pharmacokinetic (PK/PD) modeling, a secondary parameter representing the efficacy index of the drug was calculated, which is termed as the tumor static concentration (TSC). TSC values derived from all four of the models were compared with each other, and with literature reported values, to assess the performance of these models. Subsequently, using the optimized PK/PD model, PD parameters obtained from different cell lines, human PK, and the proposed translational strategy, clinically efficacious doses of T-DM1 were projected. The accuracy of projected efficacious dose range for T-DM1 was verified by comparison with the clinical doses. Aforementioned strategy was then applied to A1mcMMAF for projecting its efficacious concentrations in clinic. TSC values for A1mcMMAF, obtained by fitting TGI data from 4 different xenografts with the proposed PK/PD model, were estimated to range from 0.6 to 11.5 µg mL⻹. Accordingly, the clinically efficacious doses for A1mcMMAF were projected retrospectively. All in all, the improved PD model and proposed translational strategy presented here suggest that appropriate correction for the clinical exposure and employing the TSC criterion can help translate mouse TGI data to predict first in human doses of ADCs.
Assuntos
Anticorpos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Imunoconjugados/farmacologia , Imunoconjugados/farmacocinética , Neoplasias Experimentais/tratamento farmacológico , Ado-Trastuzumab Emtansina , Animais , Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Maitansina/análogos & derivados , Maitansina/farmacocinética , Maitansina/farmacologia , Camundongos , Camundongos Nus , Trastuzumab , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for therapy. PSMA ADC is an antibody-drug conjugate (ADC) that consists of a fully human anti-PSMA monoclonal antibody conjugated to monomethylauristatin E through a valine-citrulline linker. In this study, the antitumor activity of PSMA ADC was evaluated against a panel of prostate cancer cell lines in vitro and in a novel in vivo model of taxane-refractory human prostate cancer. In vitro cell killing was efficient for cells with abundant PSMA expression (>10(5) molecules/cell; IC(50) ≤ 0.022 nmol/L) and 1,000-fold less efficient for cells with undetectable PSMA (IC(50) > 30 nmol/L). Intermediate potency (IC(50) = 0.80 nmol/L) was observed for cells with approximately 10(4) molecules of PSMA per cell, indicating a threshold PSMA level for selective cell killing. Similar in vitro activity was observed against androgen-dependent and -independent cells that had abundant PSMA expression. In vitro activity of PSMA ADC was also dependent on internalization and proper N-glycosylation/folding of PSMA. In contrast, less potent and nonselective cytotoxic activity was observed for a control ADC, free monomethylauristatin E, and other microtubule inhibitors. PSMA ADC showed high in vivo activity in treating xenograft tumors that had progressed following an initial course of docetaxel therapy, including tumors that were large (>700 mm(3)) before treatment with PSMA ADC. This study defines determinants of antitumor activity of a novel ADC. The findings here support the clinical evaluation of this agent in advanced prostate cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Imunoconjugados/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Aminobenzoatos/química , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/toxicidade , Anticorpos Monoclonais Humanizados , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glutamato Carboxipeptidase II/imunologia , Glutamato Carboxipeptidase II/metabolismo , Humanos , Imunoconjugados/uso terapêutico , Imunoconjugados/toxicidade , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Nus , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/metabolismo , Estadiamento de Neoplasias , Oligopeptídeos/química , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prostate-specific membrane antigen (PSMA) is the prototypic cell-surface marker of prostate cancer and provides an attractive target for monoclonal antibody (mAb) targeted therapies. In this study, a novel antibody-drug conjugate (ADC) was generated by linking a fully human PSMA mAb to monomethylauristatin E (MMAE), a potent inhibitor of tubulin polymerization. The PSMA ADC was evaluated for antitumor activity in vitro and in a mouse xenograft model of androgen-independent human prostate cancer. The PSMA ADC eliminated PSMA-expressing cells with picomolar potency and >700-fold selectivity in culture. When used to treat mice with established human C4-2 tumors, the PSMA ADC significantly improved median survival 9-fold relative to vehicle or isotype-matched ADC (P = 0.0018) without toxicity. Treatment effects were also manifest as significant (P = 0.0068) reduction in serum levels of prostate-specific antigen (PSA). Importantly, 40% of treated animals had no detectable tumor or measurable PSA at day 500 and could be considered cured. The findings support development of PSMA antibody-auristatin conjugates for therapy of prostate cancer.
Assuntos
Anticorpos Monoclonais/química , Antígenos de Superfície/imunologia , Glutamato Carboxipeptidase II/imunologia , Imunoconjugados/farmacologia , Oligopeptídeos/química , Neoplasias da Próstata/tratamento farmacológico , Células 3T3 , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutamato Carboxipeptidase II/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
These studies demonstrate the feasibility of targeted therapy for the treatment of disseminated peritoneal disease using (212)Pb-labeled Herceptin as an in vivo generator of (212)Bi. In vitro studies compare the potential of the bismuth radioisotopes, (213)Bi and (212)Bi, to that of (212)Pb. Overall, (212)Pb results in a higher therapeutic index than either bismuth radioisotope, requiring lower radioactivity (microCi) for effective cytotoxic response. A pilot radioimmunotherapy (RIT) experiment treating mice bearing 5 d LS-174T intraperitoneally (i.p.) xenografts determined a maximum tolerated dose (MTD) of 20-40 microCi with i.p. administration. A specific dose response was observed and 10 microCi was selected as the effective operating dose for future experiments. Median survival of tumor-bearing mice receiving 10 microCi increased from 19 to 56 days (p = 0.008). The efficacy of (212)Pb-Herceptin was also assessed in a human pancreatic carcinoma xenograft (Shaw; i.p.) animal model previously reported as unresponsive to 213Bi-Herceptin (p = 0.002). Multiple dosing of (212)Pb-Herceptin was evaluated in both animal models. The median survival of mice bearing 3 d LS-174T i.p. xenografts increased to 110 days, with up to 3 doses of (212)Pb-Herceptin given at approximately monthly intervals; however, there was no evidence of a correlation with the second and third doses (p = 0.98). No improvement in median survival was noted with a similar regimen in the Shaw xenograft model.
Assuntos
Doenças Peritoneais/terapia , Radioimunoterapia/métodos , Receptor ErbB-2/metabolismo , Partículas alfa , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Citometria de Fluxo , Genes erbB-2 , Humanos , Imunoconjugados/química , Radioisótopos de Chumbo , Dose Máxima Tolerável , Camundongos , Transplante de Neoplasias , Radioimunoensaio , Receptor ErbB-2/imunologia , Fatores de Tempo , TrastuzumabRESUMO
The studies reported herein demonstrate the efficacy of alpha-particle-targeted radiation therapy of peritoneal disease with Herceptin as the targeting vehicle. Using the CHX-A-DTPA linker, Herceptin was radiolabeled with indium-111 and bismuth-213 with high efficiency without compromising immunoreactivity. A pilot radioimmunotherapy study treating mice bearing 5-day LS-174T (i.p.) xenografts, a low but uniform HER2 expressing, human colon carcinoma, with a single dose of (213)Bi-CHX-A"-Herceptin, proved disappointing. This defined the effect of tumor burden/size on tumor response to radioimmunotherapy with alpha-radiation. A more successful experiment with a lower tumor burden (3 days) in mice followed. A specific dose-response (P = 0.009) was observed, and although a maximum-tolerated dose was not determined, a dose of 500 to 750 muCi was selected as the operating dose for future experiments based on changes in animal weight. Median survival was increased from 20.5 days for the mock-treated mice to 43 and 59 days with 500 and 750 muCi, respectively. The therapeutic effectiveness of (213)Bi-CHX-A"-Herceptin was also evaluated in a second animal model for peritoneal disease with a human pancreatic carcinoma (Shaw). The results of this study were not as dramatic as with the former model, and higher doses were required to obtain an increase in survival of the mice (P = 0.001).
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias do Colo/radioterapia , Radioisótopos de Índio/uso terapêutico , Ácido Pentético/análogos & derivados , Doenças Peritoneais/radioterapia , Radioimunoterapia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Neoplasias do Colo/metabolismo , Neoplasias do Colo/secundário , Feminino , Humanos , Radioisótopos de Índio/farmacocinética , Isotiocianatos/química , Isotiocianatos/metabolismo , Camundongos , Camundongos Nus , Ácido Pentético/química , Ácido Pentético/metabolismo , Doenças Peritoneais/metabolismo , Projetos Piloto , Receptor ErbB-2/metabolismo , Estereoisomerismo , Taxa de Sobrevida , Distribuição Tecidual , Transplante Heterólogo , TrastuzumabRESUMO
Prostate-specific membrane antigen (PSMA) is a type 2 integral membrane glycoprotein that serves as an attractive target for cancer immunotherapy by virtue of its abundant and restricted expression on the surface of prostate carcinomas and the neovasculature of most other solid tumors. However, relatively little is known about the molecular structure of this target. Here, we report that PSMA is expressed on tumor cells as a noncovalent homodimer. A truncated PSMA protein, lacking transmembrane and cytoplasmic domains, also formed homodimers, indicating that the extracellular domain is sufficient for dimerization. PSMA dimers but not monomers displayed a native conformation and possessed high-level carboxypeptidase activity. A unique dimer-specific epitope was identified by using one of a panel of novel mAbs. When used to immunize animals, dimer but not monomer elicited antibodies that efficiently recognized PSMA-expressing tumor cells. These findings on PSMA structure and biology may have important implications for active and passive immunotherapy of prostate and other cancers.
Assuntos
Antígenos de Superfície/química , Antineoplásicos/toxicidade , Glutamato Carboxipeptidase II/química , Células 3T3 , Animais , Anticorpos Monoclonais , Antígenos de Superfície/genética , Antígenos de Superfície/isolamento & purificação , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Cricetinae , Dimerização , Glutamato Carboxipeptidase II/genética , Glutamato Carboxipeptidase II/isolamento & purificação , Humanos , Masculino , Camundongos , Neoplasias da Próstata/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transfecção , Células Tumorais CultivadasRESUMO
The alpha-particle-emitting radionuclides 213Bi, 211At, 224Ra are under investigation for the treatment of leukemias, gliomas, and ankylosing spondylitis, respectively. 213Bi and 211At were attached to monoclonal antibodies and used as targeted immunotherapeutic agents while unconjugated 224Ra chloride selectively seeks bone. 225Ac possesses favorable physical properties for radioimmunotherapy (10d half-life and 4 net alpha particles), but has a history of unfavorable radiolabeling chemistry and poor metal-chelate stability. We selected functionalized derivatives of DOTA as the most promising to pursue from out of a group of potential 225Ac chelate compounds. A two-step synthetic process employing either MeO-DOTA-NCS or 2B-DOTA-NCS as the chelating moiety was developed to attach 225Ac to monoclonal antibodies. This method was tested using several different IgG systems. The chelation reaction yield in the first step was 93+/-8% radiochemically pure (n=26). The second step yielded 225Ac-DOTA-IgG constructs that were 95+/-5% radiochemically pure (n=27) and the mean percent immunoreactivity ranged from 25% to 81%, depending on the antibody used. This process has yielded several potential novel targeted 225Ac-labeled immunotherapeutic agents that may now be evaluated in appropriate model systems and ultimately in humans.
Assuntos
Actínio/química , Compostos Radiofarmacêuticos/química , Anticorpos Monoclonais , Radioimunoterapia , Compostos Radiofarmacêuticos/síntese químicaRESUMO
Unlike beta particle-emitting isotopes, alpha emitters can selectively kill individual cancer cells with a single atomic decay. HuM195, a humanized anti-CD33 monoclonal antibody, specifically targets myeloid leukemia cells and has activity against minimal disease. When labeled with the beta-emitters (131)I and (90)Y, HuM195 can eliminate large leukemic burdens in patients, but it produces prolonged myelosuppression requiring hematopoietic stem cell transplantation at high doses. To enhance the potency of native HuM195 yet avoid the nonspecific cytotoxicity of beta-emitting constructs, the alpha-emitting isotope (213)Bi was conjugated to HuM195. Eighteen patients with relapsed and refractory acute myelogenous leukemia or chronic myelomonocytic leukemia were treated with 10.36 to 37.0 MBq/kg (213)Bi-HuM195. No significant extramedullary toxicity was seen. All 17 evaluable patients developed myelosuppression, with a median time to recovery of 22 days. Nearly all the (213)Bi-HuM195 rapidly localized to and was retained in areas of leukemic involvement, including the bone marrow, liver, and spleen. Absorbed dose ratios between these sites and the whole body were 1000-fold greater than those seen with beta-emitting constructs in this antigen system and patient population. Fourteen (93%) of 15 evaluable patients had reductions in circulating blasts, and 14 (78%) of 18 patients had reductions in the percentage of bone marrow blasts. This study demonstrates the safety, feasibility, and antileukemic effects of (213)Bi-HuM195, and it is the first proof-of-concept for systemic targeted alpha particle immunotherapy in humans.
Assuntos
Partículas alfa/uso terapêutico , Imunotoxinas/uso terapêutico , Leucemia Mieloide Aguda/radioterapia , Leucemia Mielomonocítica Crônica/radioterapia , Partículas alfa/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Antígenos CD34/imunologia , Bismuto/administração & dosagem , Bismuto/uso terapêutico , Crise Blástica/patologia , Crise Blástica/radioterapia , Humanos , Leucemia Mieloide Aguda/patologia , Leucemia Mielomonocítica Crônica/patologia , Leucopenia/etiologia , Radioisótopos/administração & dosagem , Radioisótopos/uso terapêuticoRESUMO
Novel 1,4,7-triazacyclononane-N,N',N' '-triacetic acid (NOTA) based octadentate ligands [2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-ylethyl)carbonylmethylamino]acetic acid tetrahydrochloride (1) and [3-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-propyl)carbonylmethylamino]acetic acid tetrahydrochloride (2) with pendent donor groups as potential yttrium chelators for radioimmunotherapy (RIT) have been prepared via a convenient and high-yield cyclization route. The complexation kinetics of the novel chelates with Y(III) was investigated and compared to that of 1,4,7,10-tetraazacyclododecane-N,N',N' ',N" '-tetraacetic acid (DOTA), a macrocyclic chelating agent well recognized as forming very stable complexes with yttrium but also limited in usage because of slow Y(III) complex formation rates. The in vitro stability of the corresponding (88)Y-labeled complexes in human serum was assessed by measuring the release of (88)Y from the complexes over 14 days. The in vivo biodistribution of (86)Y-labeled 1 in mice was evaluated and compared to that of the (86)Y-DOTA complex. Formation of the Y complex of 1 was significantly more rapid than that of either 2 or DOTA. Serum stability of the (88)Y complex formed with 1 was equivalent to the DOTA complex, while the complex formed with 2 proved to be significantly unstable. The results obtained from a biodistribution study indicate that the (86)Y-1 complex possesses in vivo stability comparable to the analogous DOTA complex.