Anti-TNF Thioester Glucocorticoid Antibody-Drug Conjugate Fully Inhibits Inflammation with Minimal Effect on Systemic Corticosterone Levels in a Mouse Arthritis Model.

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.

Discovery of ABBV-154, an anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody-Drug Conjugate (iADC).

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.

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

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.

Fibronectin extra domain A as a drug delivery targeting epitope for rheumatoid arthritis.

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.

Design and Development of Glucocorticoid Receptor Modulators as Immunology Antibody-Drug Conjugate Payloads.

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.

Dual Blockade of TNF and IL-17A Inhibits Inflammation and Structural Damage in a Rat Model of Spondyloarthritis.

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.

Fibronectin extra domain A as a drug delivery targeting epitope for rheumatoid arthritis

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.