Linda Liang

Discovery of Pyrophosphate Diesters as Tunable, Soluble, and Bioorthogonal Linkers for Site-Specific Antibody–Drug Conjugates

As part of an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel pyrophosphate ester linker was discovered to enable the targeted delivery of glucocorticoids. As small molecules, these highly soluble phosphate ester drug linkers were found to have ideal orthogonal properties: robust plasma stability coupled with rapid release of payload in a lysosomal environment. Building upon these findings, site-specific ADCs were made between this drug linker combination and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. Full characterization of these ADCs enabled procession to in vitro proof of concept, wherein ADCs 1-22 and 1-37 were demonstrated to afford potent, targeted delivery of glucocorticoids to a representative cell line, as measured by changes in glucocorticoid receptor-mediated gene mRNA levels. These activities were found to be antibody-, linker-, and payload-dependent. Preliminary mechanistic studies support the notion that lysosomal trafficking and enzymatic linker cleavage are required for activity and that the utility for the pyrophosphate linker may be general for internalizing ADCs as well as other targeted delivery platforms.

Novel Phosphate Modified Cathepsin B Linkers: Improving Aqueous Solubility and Enhancing Payload Scope of ADCs

In an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel phosphate bridged Cathepsin B sensitive linker was developed to enable the targeted delivery of glucocorticoids. Phosphate bridging of the Cathepsin B sensitive linkers allows for payload attachment at an aliphatic alcohol. As small molecule drug-linkers, these aqueous soluble phosphate containing drug-linkers were found to have robust plasma stability coupled with rapid release of payload in a lysosomal environment. Site-specific ADCs were successfully made between these drug-linkers and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. These ADCs demonstrated in vitro targeted delivery of glucocorticoids to a representative cell line as measured by changes in glucocorticoid receptor (GR) mediated gene mRNA levels. This novel linker expands the scope of potential ADC payloads by allowing an aliphatic alcohol to be a stable, yet cleavable attachment site. This phosphate linker may have broad utility for internalizing ADCs as well as other targeted delivery platforms.

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.