Enhancing the Anti-tumor Potency of a Novel Siglec-15 Antibody by Engineering its Fc-mediated Effector Functions.

Siglec-15, an inhibitory immune checkpoint, is an emerging target in cancer immunotherapy. Blocking the function of Siglec-15 is an excellent strategy for cancer treatment and antibody blockade has been used to target Siglec-15. However, whether Fc-mediated effector functions contribute to the therapeutic effect of antibodies remains unclear. Herein, we generated a monoclonal antibody, 1-15D1, which had a high binding affinity with Siglec-15 and strongly activated T-cell immune response in vitro. Subsequently, the Fc-mediated effector functions of 1-15D1 were explored in a Siglec-15 humanized mouse model, and further improvement in antitumor efficacy was observed in the mouse IgG2a isotype group. Thus, we demonstrate that the antitumor effects of 1-15D1 were mediated via multiple factors. In addition to the T-cell immune response, 2 novel mechanisms were explored, including the internalization of the cell surface Siglec-15 and Fc-mediated effector functions. In conclusion, our studies not only provide a potential agent for the improvement of cancer immunotherapy but also suggest that a specific role of Fc-mediated immune regulation may improve the therapeutic potency of Siglec-15 monoclonal antibody.

Drifts in N-Linked Glycosylation Result in ADCC Potency Variation of Perjeta® from August 2020 to October 2021 in China.

The proposed biosimilar candidate needs to demonstrate biosimilarity with reference products, and the quality target product profile and biosimilarity assessment criteria are prerequisite, which should be based on extensive characterization of the reference products. In this study, 13 lots of China-sourced pertuzumab (trademark: Perjeta®), with an expiration date from 2020 to 2021, were comprehensively characterized. Despite the consistency of purity, drifts in N-glycan profile were observed, which resulted in the variation of antibody-dependent cellular cytotoxicity (ADCC) activity. In detail, four parametric curves of ADCC activity of the reference product were unparalleled, and the maximum response value was highly related to the content of %afucose than half-maximal effective concentration (EC50). As ADCC is a potential critical quality attribute of Perjeta®, the glycosylation of Perjeta® and its biosimilars should be tightly monitored and controlled.

Selection of Payloads for Antibody-Drug Conjugates Targeting Ubiquitously Expressed Tumor-Associated Antigens: a Case Study.

The main objective of this work was to demonstrate which kind of payload is the suitable choice for antibody-drug conjugates directed to widely expressed tumor-associated antigen. Trop-2 is overexpressed in various solid tumors, but it is also present on the epithelium of several normal tissues. A well-designed anti-Trop-2 ADC demands a good balance of efficacy and toxicity. In this research, MMAE, SN-38, and DXd were selected as candidates for payloads of the anti-Trop-2 mAb SY02. The antitumor activities and safety profiles of these ADCs were investigated to compare the therapeutic windows. Robust in vitro cytotoxicity was observed on human pancreatic cancer cell CFPAC-1 and breast cancer cell MDA-MB-468 with IC50 generally in the subnanomolar range. Consistent with in vitro assay, SY02-DXd and SY02-SN-38 demonstrated superior efficacy in CFPAC-1 xenograft models with TGI rates of 98.2% and 87.3%, respectively. However, SY02-MMAE could hardly inhibit the tumor growth. Subsequently, antitumor activities of these ADCs were further compared in MDA-MB-468 xenograft model. Complete tumor regression was observed in SY02-DXd and SY02-MMAE groups, indicating their potent antitumor activities. In an exploratory safety and pharmacokinetic study, SY02-DXd demonstrated the best safety profile with minimal adverse events in cynomolgus monkeys, while SY02-MMAE exhibited severe on-target skin toxicity which caused death. In conclusion, SY02-DXd demonstrated superior efficacy and safety with the widest therapeutic window. Based on the efficacy and safety results, moderate cytotoxic payloads would be ideal choices for ADCs targeting ubiquitously expressed antigens.

An Innovative Site-Specific Anti-HER2 Antibody-Drug Conjugate with High Homogeneity and Improved Therapeutic Index.

Purpose: Antibody-drug conjugates (ADCs) have emerged as a potent cancer therapeutic option in recent years. DP303c is a HER2-targeting ADC with a cleavable linker-MMAE payload. The current study aimed to evaluate the therapeutic potentials of DP303c in vitro as well as in vivo. Materials and Methods: Size exclusion chromatography (SEC), reverse-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to analyze the physicochemical characterization of DP303c. An enzyme-linked immunosorbent assay (ELISA), a cell-based assay, and bio-layer interferometry (BLI) were used to evaluate DP303c's affinity with HER2 and Fc receptors. A confocal laser scanning microscopy was used to observe the internalization of DP303c. Antibody-dependent cell-mediated cytotoxicity (ADCC) and cytotoxicity assays were used to investigate the activity of DP303c in vitro. The antitumor activity of DP303c was assessed in vivo in the HER2-positive cell-derived xenograft model. Results: DP303c was a site-specific anti-HER2 antibody-drug conjugate with a monomethyl auristatin E (MMAE) with an average drug-to-antibody ratio (DAR) of 2.0. DP303c showed a high affinity with HER2 and could be effectively internalized. In vitro and in vivo, DP303c showed stronger antitumor activity as compared to trastuzumab-DM1 (T-DM1) in a series of HER2-positive cancer cells and cell-derived xenograft (CDX) models, especially in the lower HER2-expressing cells. DP303c also exhibited high serum stability and a good PK profile. Conclusion: DP303c was a steady and homogenous DAR 2 ADC that was predicted to deliver MMAE inhibitor to tumor cells. DP303c demonstrated remarkable anticancer efficacy against T-DM1 in xenograft models. DP303c was a strong candidate for the treatment of patients with HER2-positive cancer.

[Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase].

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

[Expression, purification and characterization of N-glycosylation mutant human IFN-λ1 in Pichia pastoris].

IFN-λ1 is a member of a new family of interferons called type Ⅲ IFNs with similar functions to type ⅠIFNs. Previously we obtained recombinant soluble human rhIFN-λ1 from Pichia pastoris. However, the hyper-glycosylation from P. pastoris brings immunogenicity and low purification recovery rate. To overcome this disadvantage, in this study, we constructed an rhIFN-λ1 mutant (rhIFN-λ1-Nm) devoid of the potential N-glycosylation sites by site-directed mutagenesis. rhIFN-λ1-Nm was successfully expressed and secreted extracellularly in P. pastoris (GS115) using methanol inducible AOX1 promoter with α-mating factor signal sequence. rhIFN-λ1-Nm was purified and characterized. There was no significant difference between rhIFN-λ1-Nm and rhIFN-λ1 in structure and bioactivity. The molecular weight was low after N-glycosylation mutation whereas the glycosylation was much lower. The mutational rhIFN-λ1 (rhIFN-λ1-Nm) could legitimately be developed as substitutes for rhIFN-λ1, and thus it may be developed into a more promising therapeutic reagent in the future.

Suppression of hepatocellular carcinoma proliferation and hepatitis B surface antigen secretion with interferon-λ1 or PEG-interferon-λ1.

Hepatocellular carcinoma (HCC) is a common cancer associated with chronic hepatitis B virus (HBV) infection. Conventional interferon-α (IFN-α) and pegylated IFNs (PEG-IFNs) approved for chronic HBV infection treatment can reduce the risk of HCC but are not suitable for the majority of patients and cause significant side effects. IFN-λ1 is a type III IFN with antiviral, antiproliferative, and immunomodulatory functions similar to type I IFNs but with fewer side effects. However, the tolerability and antitumor activity of PEG-IFN-λ1 in HCC xenograft mice are unknown. In vitro IFN-λ1 treatment of Hep3B and Huh7 human hepatoma cell lines increased MHC class I expression, activated JAK-STAT signaling pathways, induced IFN-stimulated gene expression, and inhibited hepatitis B surface antigen (HBsAg) expression. IFN-λ1 treatment also caused 23.2 and 19.9% growth inhibition of Hep3B and Huh7 cells, respectively, and promoted cellular apoptosis. PEG-IFN-λ1, but not IFN-λ1 treatment, significantly suppressed tumor growth (P=0.002) and induced tumor cell apoptosis in a Hep3B cell xenograft mouse model without significant weight loss or toxicity. PEG-IFN-λ1 also significantly inhibited (P=0.000) serum HBsAg secretion from Hep3B xenograft tumors in vivo. Thus, PEG-IFN-λ1 can suppress Hep3B xenograft tumor growth and inhibit HBsAg production and may be a potential treatment for HBV-related HCC.

Interaction of IFNλR1 with TRAF6 regulates NF-κB activation and IFNλR1 stability.

IFNλR1 is a member of the class II cytokine receptor family, and it associates with IL-10R2 to form a functional receptor complex, IFNλR. This receptor complex transduces signals from IFNλs (IFNλ1, IFNλ2, and IFNλ3), promoting antiviral and antiproliferative activities similar to those of type I IFNs. In an effort to further understand signal transduction through IFNλR1, we used bioinformatics analysis and identified a tumor necrosis factor receptor-associated factor 6 (TRAF6)-binding motif in the intracellular domain of IFNλR1. In subsequent immunoprecipitation and GST pull-down assays, IFNλR1 was shown to immunoprecipitate with TRAF6 and was pulled down by GST-TRAF6. Endogenous IFNλR1 and TRAF-6 interaction implies that these proteins really interact in the cells. This interaction was abrogated upon mutation of the TRAF6-binding motif in IFNλR1. Furthermore, the interaction between IFNλR1 and TRAF6 inhibited TRAF6-induced NF-κB activation, likely due to a reduction in TRAF6 autoubiquitination. Moreover, co-expression of IFNλR1 with TRAF6 significantly increased the stability of IFNλR1, thereby prolonging its half-life and enhancing its steady-state level in cultured cells.

Antitumor activities of recombinant human interferon (IFN)-λ1 in vitro and in xenograft models in vivo for colon cancer.

Interferon (IFN)-λ1, as a newly identified IFN, interacts with the structurally unique IFN-λ1 receptor complex and exhibits antiviral and antiproliferation effects. The major focus of our work was to study the antitumor activities of recombinant human IFN-λ1 (rhIFN-λ1) in vivo and in vitro. The MTT analysis showed that rhIFN-λ1 inhibited the proliferation of HCT116 cells in a dose-dependent manner, but with a less efficacy in HT29 cells than HCT116 cells. IFN-λ1 also activated the STATs and induced apoptosis in both types of cells. In the in vivo study, we found that rhIFN-λ1 suppressed tumor growth in a dose-dependent fashion, with an inhibition rate of 52% of HCT116 (P<0.01) and 56% of HT29 (P<0.01). These results indicate that rhIFN-λ1 could be a new potential IFN reagent for cancer therapy.