RESUMO
The tumor necrosis factor (TNF) and IL-23/IL-17 axes are the main therapeutic targets in spondyloarthritis. Despite the clinical efficacy of blocking either pathway, monotherapy does not induce remission in all patients and its effect on new bone formation remains unclear. We aimed to study the effect of TNF and IL-17A dual inhibition on clinical disease and structural damage using the HLA-B27/human ß2-microglobulin transgenic rat model of SpA. Immunized rats were randomized according to arthritis severity, 1 week after arthritis incidence reached 50%, to be treated twice weekly for a period of 5 weeks with either a dual blockade therapy of an anti-TNF antibody and an anti-IL-17A antibody, a single therapy of either antibody, or PBS as vehicle control. Treatment-blinded observers assessed inflammation and structural damage clinically, histologically and by micro-CT imaging. Both single therapies as well as TNF and IL-17A dual blockade therapy reduced clinical spondylitis and peripheral arthritis effectively and similarly. Clinical improvement was confirmed for all treatments by a reduction of histological inflammation and pannus formation (p < 0.05) at the caudal spine. All treatments showed an improvement of structural changes at the axial and peripheral joints on micro-CT imaging, with a significant decrease for roughness (p < 0.05), which reflects both erosion and new bone formation, at the level of the caudal spine. The effect of dual blockade therapy on new bone formation was more prominent at the axial than the peripheral level. Collectively, our study showed that dual blockade therapy significantly reduces inflammation and structural changes, including new bone formation. However, we could not confirm a more pronounced effect of dual inhibition compared to single inhibition.
Assuntos
Interleucina-17/antagonistas & inibidores , Espondilartrite/etiologia , Espondilartrite/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Artrite/tratamento farmacológico , Artrite/etiologia , Artrite/metabolismo , Artrite/patologia , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imageamento Tridimensional , Imuno-Histoquímica , Masculino , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ratos , Ratos Transgênicos , Espondilartrite/diagnóstico , Espondilartrite/tratamento farmacológico , Microtomografia por Raio-XRESUMO
Despite the efficacy of biologics for treatment of rheumatoid arthritis (RA), many patients show inadequate responses and likely require neutralization of multiple mediators. Neutralization of both interleukin (IL)-1ß and IL-17A with monoclonal antibodies showed greater efficacy than either agent alone in a mouse arthritis model with cooperative inhibition of key inflammatory factors, IL-6, granulocyte colony-stimulating factor (G-CSF), and CXC chemokine ligand (CXCL)1. Given the potential clinical benefit in RA, we generated a human dual variable domain antibody Ig, ABBV-615, capable of simultaneous binding and neutralization of IL-1ß and IL-17A. ABBV-615 was characterized and evaluated in cynomolgus monkeys for pharmacokinetics and toxicity to enable clinical development. ABBV-615 exhibited affinities (KD) of 12 and 3 pM on human IL-1ß and IL-17A, respectively, and potencies (IC50) of 3 and 58 pM, respectively, as well as excellent drug-like properties. ABBV-615 pharmacokinetics in cynomolgus monkeys was dose proportional from 20 to 100 mg/kg with a mean half-life of 16 days. However, a 13-week repeat-dose toxicity study in cynomolgus monkeys revealed time-dependent spontaneous infections exclusively in skin at all doses tested and not historically seen with single-agent anti-IL-1α/ß or anti-IL-17A. Consistent with reduced resistance to skin infections, IL-1ß- and IL-17A-stimulated human keratinocytes demonstrate cooperative or compensatory production of key antibacterial and inflammatory mediators such as lipocalin-2, G-CSF, CXCL1, IL-8, tumor necrosis factor, and IL-6, which aid in defense against skin bacterial infections. These results illustrate the skin-specific antimicrobial mechanisms of IL-1ß and IL-17A and highlight the importance of understanding unique combinatorial effects of biologic agents.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/tratamento farmacológico , Interleucina-17/imunologia , Interleucina-1beta/imunologia , Pele/efeitos dos fármacos , Pele/microbiologia , Animais , Artrite Experimental/imunologia , Humanos , Macaca fascicularis , Masculino , CamundongosRESUMO
We describe the discovery of a thioester-containing glucocorticoid receptor modulator (GRM) payload and the corresponding antibody-drug conjugate (ADC). Payload 6 was designed for rapid hepatic inactivation to minimize systemic exposure of nonconjugated GRM. Mouse PK indicated that 6 is cleared 10-fold more rapidly than a first-generation GRM payload, resulting in 10-fold lower exposure and 3-fold decrease in Cmax. The anti-mTNF conjugate ADC5 fully inhibited inflammation in mouse contact hypersensitivity with minimal effects on corticosterone, a biomarker for systemic GRM effects, at doses up to and including 100 mg/kg. Concomitant inhibition of P1NP suggests potential delivery to cells involved in the remodeling of bone, which may be a consequence of TNF-targeting or bystander payload effects. Furthermore, ADC5 fully suppressed inflammation in collagen-induced arthritis mouse model after one 10 mg/kg dose for 21 days. The properties of the anti-hTNF conjugate were suitable for liquid formulation and may enable subcutaneous dosing.
Assuntos
Artrite Experimental , Corticosterona , Imunoconjugados , Fator de Necrose Tumoral alfa , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Camundongos , Imunoconjugados/farmacologia , Imunoconjugados/química , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Corticosterona/sangue , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Glucocorticoides/farmacologia , Humanos , Masculino , Modelos Animais de DoençasRESUMO
Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.
Assuntos
Anticorpos , Artrite Experimental , Imunoconjugados , Esteroides , Humanos , Animais , Camundongos , Preparações Farmacêuticas , Receptores de Glucocorticoides/uso terapêutico , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Modelos Animais de Doenças , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêuticoRESUMO
Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.
Assuntos
Imunoconjugados , Pró-Fármacos , Receptores de Glucocorticoides , Inibidores do Fator de Necrose Tumoral , Anticorpos , Pró-Fármacos/farmacologia , Glucocorticoides , Maleimidas , Imunoconjugados/farmacologiaRESUMO
A painful, chronic condition, Rheumatoid Arthritis, is marked by bone erosion and soft tissue swelling at the joint. As treatments are investigated in pre-clinical models, characterizing disease progression is integral to assessing treatment efficacy. Here, in vivo and ex vivo micro-computed tomography (µCT) are used in parallel with traditional caliper score measurement to quantify physiological changes in the tarsal region in a murine, collagen-induced arthritis model. In vivo imaging methods, which are validated here through comparison to ex vivo and caliper methods, afford longitudinal analysis of both bone and soft tissue through a single image acquisition. This method removes the subjectivity of swelling quantification which is inherently associated with traditional caliper measurements. Histopathology offers an additional assessment of bone erosion and inflammation by providing a microscopic characterization of disease activity. In comparison to untreated animals, daily prednisolone (glucocorticoid) treatment is shown to restore bone volume, as reflected through in vivo and ex vivo µCT images, as well as histopathology. Prednisolone-associated reduction in inflammation is shown through in vivo µCT soft tissue volume measurements, paw caliper measurements, and histopathology. The findings reported here provide a comprehensive validation of in vivo µCT with a sensitivity that enables characterization of pre-clinical disease assessment in response to treatment in a murine, collagen-induced arthritis model.
Assuntos
Artrite Reumatoide/diagnóstico por imagem , Colágeno/efeitos adversos , Monitorização Fisiológica/métodos , Microtomografia por Raio-X/métodos , Animais , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Tecido Conjuntivo/diagnóstico por imagem , Tecido Conjuntivo/patologia , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos DBA , Tamanho do Órgão , Gravidade do Paciente , Prednisolona/uso terapêuticoRESUMO
OBJECTIVES: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. METHODS: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. RESULTS: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. CONCLUSION: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.
Assuntos
Artrite Experimental , Artrite Reumatoide , Animais , Anticorpos Monoclonais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Modelos Animais de Doenças , Epitopos , Fibronectinas , Humanos , Camundongos , Distribuição TecidualRESUMO
Glucocorticoid receptor modulators (GRM) are the first-line treatment for many immune diseases, but unwanted side effects restrict chronic dosing. However, targeted delivery of a GRM payload via an immunology antibody-drug conjugate (iADC) may deliver significant efficacy at doses that do not lead to unwanted side effects. We initiated our α-TNF-GRM ADC project focusing on identifying the optimal payload and a linker that afforded stable attachment to both the payload and antibody, resulting in the identification of the synthetically accessible maleimide-Gly-Ala-Ala linker. DAR 4 purified ADCs were shown to be more efficacious in a mouse contact hypersensitivity model than the parent α-TNF antibody. Analysis of P1NP and corticosterone biomarkers showed there was a sufficient therapeutic window between efficacy and unwanted effects. In a chronic mouse arthritis model, α-TNF-GRM ADCs were more efficacious than both the parent α-TNF mAb and an isotype control bearing the same GRM payload.
Assuntos
Antineoplásicos , Imunoconjugados , Animais , Anticorpos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Camundongos , Receptores de GlucocorticoidesRESUMO
Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.
Assuntos
Glucocorticoides , Imunoconjugados , Animais , Humanos , Camundongos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Receptores de Glucocorticoides , Inibidores do Fator de Necrose TumoralRESUMO
The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-x(L), and Bcl-w protein function. There is evidence that Bcl-2-associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.
Assuntos
Autoimunidade/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Artrite Experimental/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Hemocianinas/imunologia , Humanos , Hipersensibilidade Tardia/imunologia , Interferon-alfa/farmacologia , Nefrite Lúpica/induzido quimicamente , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Especificidade por SubstratoRESUMO
For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.
Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/biossíntese , Artrite Experimental/tratamento farmacológico , Região Variável de Imunoglobulina/biossíntese , Engenharia de Proteínas , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/patologia , Células CHO , Cricetinae , Cricetulus , Modelos Animais de Doenças , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/uso terapêutico , Interleucina-12/antagonistas & inibidores , Interleucina-12/imunologia , Interleucina-18/antagonistas & inibidores , Interleucina-18/imunologia , Camundongos , Estrutura Terciária de Proteína , RatosRESUMO
Mutations in the Interleukin (IL)-23/IL-23 receptor loci are associated with increased inflammatory bowel disease (IBD) susceptibility, and IL-23 neutralization has shown efficacy in early clinical trials. To better understand how an excess of IL-23 affects the gastrointestinal tract, we investigated chronic systemic IL-23 exposure in healthy wildtype mice. As expected, IL-23 exposure resulted in early activation of intestinal type 3 innate lymphoid cells (ILC3), followed by infiltration of activated RORγt+ T helper cells. Surprisingly, however, sustained IL-23 stimulus also dramatically reduced classical ILC3 populations within the proximal small intestine, and a phenotypically distinct T-bet expressing ILC3 population emerged. TNFα neutralization, a widely used IBD therapy, reduced several aspects of the IL-23 driven ILC3 response, suggesting a synergy between IL-23 and TNFα in ILC3 activation. In vitro studies supported these findings, revealing previously unappreciated effects of IL-23 and TNFα within the intestine.
Assuntos
Interleucina-23/metabolismo , Intestino Delgado/imunologia , Linfócitos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/patologia , Interleucina-23/administração & dosagem , Intestino Delgado/patologia , Linfócitos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores CCR6/metabolismo , Receptores de Interleucina-7/metabolismo , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Abstract Objectives: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. Methods: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. Results: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. Conclusion: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.
RESUMO
BACKGROUND AND AIMS: Anti-tumour necrosis factor [TNF] monoclonal antibodies [infliximab, adalimumab] induce complete mucosal healing in a proportion of patients with Crohn's disease whereas a TNF receptor fusion protein [etanercept] is not effective and the anti-TNF F[ab']2 fragment [certolizumab] shows a very low rate of complete mucosal healing. In contrast, all four TNF-neutralising drugs have demonstrated efficacy in the treatment of rheumatoid arthritis. These observations suggest that factors other than neutralisation of TNF may contribute to clinical outcomes in Crohn's disease. Here we tested the hypothesis that Fc receptor [FcR]-mediated effects may contribute to the therapeutic response of anti-TNF antibodies in inflammatory bowel disease. METHODS: We modified an IgG2c mouse anti-TNF antibody that binds the high-affinity FcRs to generate an IgG1 isotype with strongly diminished binding. We examined the therapeutic effects of both antibodies in the T cell transfer model of inflammatory bowel disease and the collagen-induced arthritis model. RESULTS: The IgG2c anti-TNF antibody prevented colonic inflammation in the T cell transfer model of colitis, whereas the IgG1 anti-TNF did not. Conversely, both the IgG2c and IgG1 anti-TNFs were similarly effective in reducing the severity of articular inflammation in mouse collagen-induced arthritis. CONCLUSION: These data support the concept that the mechanism of action for TNF-neutralising drugs may differ across immune-mediated diseases and, potentially, between therapeutics within a particular disease. Our data suggest a specific role of Fc-mediated immune regulation in the resolution of intestinal inflammation by anti-TNF monoclonal antibodies.