Monoclonal antibodies play an increasingly important role for the development of new drugs across multiple therapy areas. The term 'developability' encompasses the feasibility of molecules to successfully progress from discovery to development via evaluation of their physicochemical properties. These properties include the tendency for self-interaction and aggregation, thermal stability, colloidal stability, and optimization of their properties through sequence engineering. Selection of the best antibody molecule based on biological function, efficacy, safety, and developability allows for a streamlined and successful CMC phase. An efficient and practical high-throughput developability workflow (100 s-1,000 s of molecules) implemented during early antibody generation and screening is crucial to select the best lead candidates. This involves careful assessment of critical developability parameters, combined with binding affinity and biological properties evaluation using small amounts of purified material (<1 mg), as well as an efficient data management and database system. Herein, a panel of 152 various human or humanized monoclonal antibodies was analyzed in biophysical property assays. Correlations between assays for different sets of properties were established. We demonstrated in two case studies that physicochemical properties and key assay endpoints correlate with key downstream process parameters. The workflow allows the elimination of antibodies with suboptimal properties and a rank ordering of molecules for further evaluation early in the candidate selection process. This enables any further engineering for problematic sequence attributes without affecting program timelines.

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IntroductionAnterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer.MethodsTo investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-α (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER).ResultsAGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown.ConclusionsTransiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.

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-Z with F and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. The affinity of the PD-L1-binding Affibody ligand Z was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of F-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. NOTA-Z 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 ( = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. 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.

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.