Laurence Fayadat-Dilman

Anti-IL-17A therapy protects against bone erosion in experimental models of rheumatoid arthritis

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine secreted by a subset of memory T cells and other innate immune cells. It is associated with rheumatoid arthritis (RA) due to IL-17A expression in RA synovial fluid. The severe bone erosive rat adjuvant-induced arthritis (rAIA) and mouse collagen-induced arthritis (mCIA) models were used to address the therapeutic efficacy of anti-IL-17A treatment with a focused investigation on bone protection. In the rAIA model, treatment with anti-IL-17A completely alleviated arthritis, lowered the level of receptor activator of NFκB ligand (RANKL), and inhibited structural damage to the bones. In the mCIA model, IL-17A neutralization coincident with arthritis development or in mice with established arthritis diminished joint swelling by inhibiting disease initiation and progression. Intriguingly, even the few joints that became outwardly severely inflamed in the presence of an anti-IL-17A antagonist had diminished joint histopathology scores compared to severely inflamed, control-treated mice. The bone-preserving property correlated with decreased RANKL message in severely inflamed paws of arthritic mice. These data identify IL-17A as a key factor in inflammation-mediated bone destruction and support anti-IL-17A therapy for the treatment of inflammatory bone diseases such as RA.

Novel Technologies for Generation of Bispecific Constructs

Bispecific molecules can be differentiated from traditional monoclonal antibodies as they are able to address multiple targets simultaneously. These modalities may offer additional advantages with respect to target engagement that may not be feasible by traditional combination therapies with single agents. Therefore, design of bispecific constructs requires particular attention to target and drug selection for successful application of this class of therapeutics. In the past, progress in advancing bispecific molecules into the clinical arena was slow, mainly due to challenges associated with generating bispecific molecules in sufficient quality and quantity. However, due to recent progress in rapidly evolving technologies that encompass state-of-the-art engineering, production, and development of recombinant protein scaffolds, development of novel bispecific modalities has witnessed an exponential growth. In this chapter the current landscape for bi- and multi-specific modalities from design, production, and developability aspects is discussed.

Development of Anti-CD74 Antibody–Drug Conjugates to Target Glucocorticoids to Immune Cells

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

In vivo Imaging of the Programmed Death Ligand 1 by 18F Positron Emission Tomography

Programmed death ligand 1 (PD-L1) is an immune regulatory ligand that binds to the T-cell immune check point programmed death 1. Tumor expression of PD-L1 is correlated with immune suppression and poor prognosis. It is also correlated with therapeutic efficacy of programmed death 1 and PD-L1 inhibitors. In vivo imaging may enable real-time follow-up of changing PD-L1 expression and heterogeneity evaluation of PD-L1 expression across tumors in the same subject. We have radiolabeled the PD-L1–binding Affibody molecule NOTA-ZPD-L1_1 with 18F and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. Methods: The affinity of the PD-L1–binding Affibody ligand ZPD-L1_1 was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of 18F-AlF. In vivo studies were performed in PD-L1–positive, PD-L1–negative, and mixed tumor-bearing severe combined immunodeficiency mice. Tracer was injected via the tail vein, and dynamic PET scans were acquired for 90 min, followed by γ-counting biodistribution. Immunohistochemical staining with an antibody specific for anti–PD-L1 (22C3) was used to evaluate the tumor distribution of PD-L1. Immunohistochemistry results were then compared with ex vivo autoradiographic images obtained from adjacent tissue sections. Results: NOTA-ZPD-L1_1 was labeled, with a radiochemical yield of 15.1% ± 5.6%, radiochemical purity of 96.7% ± 2.0%, and specific activity of 14.6 ± 6.5 GBq/μmol. Surface plasmon resonance showed a NOTA-conjugated ligand binding affinity of 1 nM. PET imaging demonstrated rapid uptake of tracer in the PD-L1–positive tumor, whereas the PD-L1–negative control tumor showed little tracer retention. Tracer clearance from most organs and blood was quick, with biodistribution showing prominent kidney retention, low liver uptake, and a significant difference between PD-L1–positive (percentage injected dose per gram [%ID/g] = 2.56 ± 0.33) and –negative (%ID/g = 0.32 ± 0.05) tumors (P = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. Conclusion: Our results show that Affibody ligands can be effective at targeting tumor PD-L1 in vivo, with good specificity and rapid clearance. Future studies will explore methods to reduce kidney activity retention and further increase tumor uptake.

T cell activation and anti-tumor efficacy of anti-LAG-3 antibodies is independent of LAG-3 – MHCII blocking capacity

LAG-3 has been shown to act as an inhibitory molecule involved in the regulation of T cell activation, proliferation and homeostasis. Exhausted T cell populations that evolve in the tumor microenvironment or during chronic viral infections show coordinated expression of LAG-3 and PD-1. LAG-3 is structurally related to CD4 and binds to MHCII. Anti-LAG-3 antibodies have demonstrated preclinical efficacy in several disease models in particular when combined with anti-PD-1 antibodies. Studies have proposed that LAG-3 blockade is efficacious in both CD4+ and CD8+ T cells despite the lack of significant MHCII levels on CD8+ T cell. In the present study, we evaluated if anti-LAG-3 efficacy is dependent on the ability of the antibody to inhibit the binding of LAG-3 to MHCII. We have compared in a series of in vitro assays the biological activity of two distinct anti-mouse LAG-3 antibodies: C9B7W which does not block the LAG-3 – MHCII interaction and an in-house generated antibody 28G10 that strongly blocks the interaction between LAG-3 and MHCII. Biophysical characterization revealed that C9B7W and 28G10 recognize distinct epitopes on LAG-3, therefore explaining the difference in LAG3-MHCII interruption. However, no differences were observed in T cell activation assays between the two antibodies using TCR transgenic CD4+ T cells. In addition, their ability to synergize with an anti-PD-1 antibody was also comparable. To understand if the overall enhancement in CD4+ T cell activation by C9B7W and 28G10 was achieved through different mechanisms, we profiled gene expression in T cells stimulated in the presence of anti-LAG-3 antibodies. No significant difference was found between the two antibodies. Consistent with this observation, we did not see an additive effect of C9B7W and 28G10 when used together in vitro. Neither antibody demonstrated a significant effect on the activity of TCR transgenic CD8+ T cells in in vitro functional assays. Furthermore, the two antibodies demonstrated comparable anti-tumor efficacy in in vivo syngeneic tumor models when dosed in combination with anti-PD-1. In conclusion, our studies demonstrate that the activity of LAG-3-targeting antibodies is not associated with their ability to disrupt LAG-3-MHCII interaction. This would suggest that anti-LAG-3 antibodies should enhance both CD4+ and CD8+ T cell function. Case in contrast, our in vitro data demonstrates that LAG-3 targeting augments the activation of CD4+ T cells significantly more than CD8+ T cells.