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Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.
Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Claudinas/imunologia , Imunoconjugados/uso terapêutico , Neoplasias Pancreáticas/terapia , Neoplasias Gástricas/terapia , Adenocarcinoma/terapia , Animais , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Humanos , Imunoconjugados/sangue , Camundongos , Neoplasias Pancreáticas/sangue , Ratos , Neoplasias Gástricas/sangueRESUMO
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 XenoenxertoRESUMO
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/químicaRESUMO
Interleukin 15 (IL-15) and IL-2 have distinct immunological functions even though both signal through the receptor subunit IL-2Rß and the common γ-chain (γ(c)). Here we found that in the structure of the IL-15-IL-15Rα-IL-2Rß-γ(c) quaternary complex, IL-15 binds to IL-2Rß and γ(c) in a heterodimer nearly indistinguishable from that of the IL-2-IL-2Rα-IL-2Rß-γ(c) complex, despite their different receptor-binding chemistries. IL-15Rα substantially increased the affinity of IL-15 for IL-2Rß, and this allostery was required for IL-15 trans signaling. Consistent with their identical IL-2Rß-γ(c) dimer geometries, IL-2 and IL-15 showed similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induced similar signals, and the cytokine specificity of IL-2Rα versus IL-15Rα determined cellular responsiveness. Our results provide new insights for the development of specific immunotherapeutics based on IL-15 or IL-2.
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Interleucina-15/imunologia , Interleucina-2/imunologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Interleucina-15/química , Interleucina-15/metabolismo , Interleucina-2/química , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/metabolismo , Ligantes , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Transdução de SinaisRESUMO
Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.
Assuntos
Anticorpos Biespecíficos/imunologia , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Especificidade de Anticorpos , Antígenos CD20/imunologia , Linfócitos B/imunologia , Complexo CD3/imunologia , Cetuximab , Citotoxicidade Imunológica , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação Puntual , Ratos , Ratos Sprague-Dawley , Receptores Fc/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Linfócitos T/imunologiaRESUMO
Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.
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Antiparasitários/química , Cisteína Endopeptidases/química , Plasmodium falciparum/enzimologia , Inibidores de Proteases/química , Proteínas de Protozoários/química , Sulfonas/química , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologia , Animais , Antiparasitários/uso terapêutico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/enzimologia , Cristalografia por Raios X , Desenho de Fármacos , Cinética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/enzimologia , Inibidores de Proteases/uso terapêutico , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Sulfonas/uso terapêutico , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/enzimologiaRESUMO
Interleukin-2 (IL-2) is an immunoregulatory cytokine that binds sequentially to the alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gammac) receptor subunits. Here we present the 2.8 angstrom crystal structure of a complex between human IL-2 and IL-2Ralpha, which interact in a docking mode distinct from that of other cytokine receptor complexes. IL-2Ralpha is composed of strand-swapped "sushi-like" domains, unlike the classical cytokine receptor fold. As a result of this domain swap, IL-2Ralpha uses a composite surface to dock into a groove on IL-2 that also serves as a binding site for antagonist drugs. With this complex, we now have representative structures for each class of hematopoietic cytokine receptor-docking modules.
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Interleucina-2/química , Receptores de Interleucina/química , Cristalografia por Raios X , Humanos , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2 , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores de Interleucina/metabolismoRESUMO
Stiff-man syndrome (SMS) is a rare autoimmune disorder of the central nervous system associated with autoantibodies to glutamate decarboxylase (GAD). We isolated five brain-reactive human monoclonal antibodies, with reactivity distinct from GAD, from peripheral blood of a patient newly diagnosed with SMS. Two antibodies reacted with both Purkinje cells and ependymal cells, and precipitated an 80-kDa protein from rat neuronal primary cultures, which was also recognized by 12% (3/25) of SMS sera and 13% (2/15) of SMS cerebrospinal fluid (CSF) samples. The corresponding antigen was identified as 17 beta-hydroxysteroid dehydrogenase type 4 and may represent a possible novel target of autoimmunity in SMS.