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1.
J Biol Chem ; 291(23): 12254-70, 2016 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-27129281

RESUMO

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.


Assuntos
Anticorpos Monoclonais/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X2/imunologia , Receptores Purinérgicos P2X3/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Adjuvante de Freund , Células HEK293 , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/prevenção & controle , Canais Iônicos/química , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos BALB C , Microscopia Confocal , Dor/induzido quimicamente , Dor/metabolismo , Dor/prevenção & controle , Multimerização Proteica/imunologia , Ratos , Receptores Purinérgicos P2X2/química , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X3/química , Receptores Purinérgicos P2X3/metabolismo , Ácido Trinitrobenzenossulfônico , Dor Visceral/induzido quimicamente , Dor Visceral/metabolismo , Dor Visceral/prevenção & controle
2.
Bioconjug Chem ; 26(4): 650-9, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25643134

RESUMO

The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.


Assuntos
Aminobenzoatos/química , Anticorpos Monoclonais/química , Antineoplásicos/química , Imunoconjugados/química , Oligopeptídeos/química , Neoplasias Pancreáticas/tratamento farmacológico , Aminobenzoatos/sangue , Aminobenzoatos/farmacocinética , Aminobenzoatos/farmacologia , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Carbamatos/química , Catepsina B/química , Catepsina B/metabolismo , Linhagem Celular Tumoral , Dipeptídeos/química , Sistemas de Liberação de Medicamentos/métodos , Estabilidade de Medicamentos , Feminino , Humanos , Imunoconjugados/sangue , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Camundongos , Camundongos Nus , Modelos Moleculares , Oligopeptídeos/sangue , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/patologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Mol Cancer Ther ; 22(2): 155-167, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36459691

RESUMO

STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody-drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug-antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non-small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).


Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias do Endométrio , Imunoconjugados , Neoplasias Pulmonares , Feminino , Humanos , Animais , Camundongos , Imunoconjugados/química , Tubulina (Proteína)/metabolismo , Receptor 1 de Folato , Antineoplásicos/farmacologia , Neoplasias do Endométrio/tratamento farmacológico , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Oncotarget ; 9(102): 37700-37714, 2018 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-30701025

RESUMO

STRO-001 is a site-specific, predominantly single-species, fully human, aglycosylated anti-CD74 antibody-drug conjugate incorporating a non-cleavable linker-maytansinoid warhead with a drug-antibody ratio of 2 which was produced by a novel cell-free antibody synthesis platform. We examined the potential pharmacodynamics and anti-tumor effects of STRO-001 in multiple myeloma (MM). CD74 expression was assessed in MM cell lines and primary bone marrow (BM) MM biopsies. CD74 mRNA was detectable in CD138+ enriched plasma cells from 100% (892/892) of patients with newly diagnosed MM. Immunohistochemistry confirmed CD74 expression in 35/36 BM biopsies from patients with newly diagnosed and relapsed/refractory MM. Cytotoxicity assays demonstrated nanomolar STRO-001 potency in 4/6 MM cell lines. In ARP-1 and MM.1S tumor-bearing mice, repeat STRO-001 dosing provided significant antitumor activity with eradication of malignant hCD138+ BM plasma cells and prolonged survival. In a luciferase-expressing MM.1S xenograft model, dose-dependent STRO-001 efficacy was confirmed using bioluminescent imaging and BM tumor burden quantification. Consistent with the intended pharmacodynamic effect, STRO-001 induced dose-responsive, reversible B-cell and monocyte depletion in cynomolgus monkeys, up to a maximum tolerated 10 mg/kg, with no evidence of off-target toxicity. Collectively, these data suggest that STRO-001 is a promising therapeutic agent for the treatment of MM.

5.
Chem Biol ; 20(2): 161-7, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23438745

RESUMO

Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymatic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compounds at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chemical instability, as was observed with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.


Assuntos
Anticorpos/química , Antineoplásicos/química , Imunoconjugados/química , Animais , Anticorpos/imunologia , Meia-Vida , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/imunologia , Camundongos , Neoplasias/tratamento farmacológico , Ratos , Transglutaminases/metabolismo , Moduladores de Tubulina/química
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