Impact of afucosylation strategy on antibody function: a comparative study of glycoengineered anti-CD20 antibodies Obinutuzumab and Obinutuzumab beta

The α1,6-fucosyltransferase (encoded by FUT8 gene) is the key enzyme transferring fucose to the innermost GlcNAc residue on an N-glycan through an α-1,6 linkage in the mammalian cells. The presence of core fucose on antibody Fc region can inhibit antibody-dependent cellular cytotoxicity (ADCC) and reduce antibody therapeutic efficiency in vivo. Chinese hamster ovary (CHO) cells are the predominant production platform in biopharmaceutical manufacturing. Therefore, the generation of FUT8 knock-out (FUT8KO) CHO cell line is favorable and can be applied to produce completely non-fucosylated antibodies. The characterization of monoclonal antibodies as well as host cell glycoprotein impurities are required for quality control purposes under regulation rules. To understand the role of FUT8 in the glycosylation of CHO cells, we generated a FUT8 knock-out CHO cell line and performed a large-scale glycoproteomics to characterize the FUT8KO and wild-type (WT) CHO cells. The glycopeptides were enriched by hydrophilic chromatography and fractionated 25 fractions by bRPLC followed by analysis using high-resolution liquid chromatography mass spectrometry (LC-MS). A total of 7,127 unique N-linked glycosite-containing intact glycopeptides (IGPs), 928 glycosites, and 442 glycoproteins were identified from FUT8KO and WT CHO cells. Moreover, 28.62% in 442 identified glycoproteins and 26.69% in 928 identified glycosites were significantly changed in the FUT8KO CHO compared to wild-type CHO cells. The relative abundance of all the three N-glycan types (high-mannose, hybrid, and complex) was determined in FUT8KO comparing to wild-type CHO cells. Furthermore, a decrease in fucosylation content was observed in FUT8KO cells, in which core-fucosylated glycans almost disappeared as an effect of FUT8 gene knockout. Meantime, a total of 51 glycosylation-related enzymes were also quantified in these two cell types and 16 of them were significantly altered in the FUT8KO cells, in which sialyltransferases and glucosyltransferases were sharply decreased. These glycoproteomic results revealed that the knock-out of FUT8 not only influenced the core-fucosylation of proteins but also altered other glycosylation synthesis processes and changed the relative abundance of protein glycosylation.

4146 Background: Delta-like ligand 3 (DLL3) is an emerging target in multiple neuroendocrine cancers including small cell lung cancer but remains underexplored in GEP NENs. With the ongoing development of multiple classes of therapeutics against DLL3, there is a need to understand the landscape of functional DLL3 expression in GEP NENs. Methods: DLL3 immunohistochemistry (IHC) was completed on available tumor samples from patients (pts) with GEP poorly differentiated neuroendocrine carcinomas (GEP NECs) and grade 3 well differentiated pancreatic NETs (G3 WD PanNETs) treated between 2018-2024 and a tissue microarray of resected G1-G2 WD PanNETs (185 samples total). DLL3 positivity (+) was defined as ≥ 5% weak (1+) IHC staining. H-scores were calculated by combining % of + tumor cells and degree of staining (1, 2, 3+), ranging from 0-300. Correlations between DLL3 status and clinicopathologic features and outcomes were analyzed. Among selected pts with DLL3 IHC+ GEP NENs, DLL3 immunoPET imaging using the diagnostic tracer [ 89 Zr]Zr-DFO-SC16.56 was completed to evaluate functional expression. Results: Among GEP NECs overall, 50/69 were DLL3+ (72%; median H-score 50, interquartile range [IQR] 0-160, range 0-300), including 13/16 esophagogastric (median 45), 11/13 pancreatic (median 60), 7/11 hepatobiliary (median 120), 16/26 colorectal (median 32.5), and 3/3 NECs of other/unknown origin (median 60), with DLL3 expression higher in small cell vs large cell histology (median 120 vs 15, P = 0.011). Among GEP NECs, there was no association between DLL3+ and individual genomic alterations, PFS to 1L platinum-based therapy (median 4.6 mo vs 4.7 mo in DLL3-negative [-], P = 0.435), or OS from diagnosis of advanced disease (median 15.5 vs 12.2 mo in DLL3-, P = 0.629). Among WD PanNETs, DLL3 expression was detected in 3/46 (7%) G1, 1/23 (4%) G2, and 19/47 (40%) G3 tumors, with median Ki67 higher among DLL3+ vs DLL3- tumors overall (42% vs 6%, P < 0.001) and within G3 WD PanNETs alone (48% vs 30%, P = 0.009). Among pts with advanced G3 WD PanNETs, DLL3+ was associated with shorter OS from diagnosis of advanced G3 disease (median OS 23.1 mo vs 43.9 mo in DLL3-, P = 0.012). [ 89 Zr]Zr-DFO-SC16.56 DLL3 PET imaging was completed on 5 pts with DLL3 IHC+ GEP NENs at progression on standard systemic therapy. Notably, a pt with pancreatic NEC and liver metastases (DLL3 IHC H-score 60) demonstrated high tumoral tracer uptake (SUV max 36.7) with 95% of tumor lesions demonstrating DLL3 PET avidity. Among 4 pts with DLL3 IHC+ G3 WD PanNETs, DLL3 PET was positive in 3/4, with SUV max ranging from 14.4-27.5 and % of DLL3 PET+ tumor lesions ranging from 50-100%. Conclusions: DLL3 is expressed on a majority of GEP NECs and on a minority of well differentiated PanNETs marked by high grade disease and poor clinical outcomes. Functional DLL3 PET imaging highly suggests DLL3 as a promising therapeutic target in both GEP NECs and high grade WD PanNETs.

Background: High-grade lung neuroendocrine tumors are a heterogeneous group of cancers with aggressive common features and a lack of effective therapeutic options. Recently, efforts have been made to identify new targets and drugs to improve clinical outcomes in these patients. Because of its high expression in neuroendocrine tumors compared to normal tissue, delta-like ligand 3 (DLL3) has emerged as a new therapeutic target in this setting.1,2 Targeting DLL3 with the half-life extended bispecific T cell engager (HLE BiTE®) immune therapy, tarlatamab, in a phase 1 clinical trial has demonstrated promising anti-tumor activity in small cell lung cancer (SCLC) patients.3 In order to more precisely guide patient selection and clinical trial design, a better understanding and quantification of DLL3 expression is required. Thus, we investigated DLL3 immunohistochemical expression across SCLC, large cell neuroendocrine carcinoma (LCNEC), and neuroendocrine carcinoma with combined histology (CNEC), and its associations with clinicopathological characteristics. Design: Formalin-fixed and paraffin-embedded surgically resected lung neuroendocrine carcinomas (SCLC, n=17; LCNEC, n=20; CNEC, n=8) were selected based on tissue availability at MD Anderson Cancer Center and stained with a commercially available immunohistochemistry assay for DLL3 (clone SP347, Ventana). Total percentage and intensity of DLL3 expression of neuroendocrine carcinoma cells were scored and the results were presented as H-Score (0-300) and percentage (%) of DLL3 positive tumor cells. We correlated DLL3 expression with clinicopathological characteristics. Results: DLL3 expression was observed in 37/45 (82%) of all cases (SCLC: %, median=65%, 0-100%; H-score, median=95, 0-175; LCNEC: %, median=65%, 0-100%; H-score, median=100, 0-180; CNEC: %, median=55%, 0-95%; H-score, median=90, 0-175). In patients with LCNEC, DLL3 % was higher in male patients (p=0.0500) and former smokers (p=0.0298) compared to females and current smokers, respectively. No other clinicopathological associations were found with age, Tumor-Node-Metastasis (TNM) staging system, recurrence, neoadjuvant therapy or overall survival. Conclusion:Our work confirms that most high-grade neuroendocrine tumors express DLL3 across histology types and TNM stage. Our results suggest that a large subset of patients with high-grade lung neuroendocrine tumors may be a target population of interest for DLL3-targeted therapies.

Mammalian expression systems such as Chinese hamster ovary (CHO), mouse myeloma (NS0), and human embryonic kidney (HEK) cells serve a critical role in the biotechnology industry as the production host of choice for recombinant protein therapeutics. Most of the recombinant biologics are glycoproteins that contain complex oligosaccharide or glycan attachments representing a principal component of product quality. Both N-glycans and O-glycans are present in these mammalian cells, but the engineering of N-linked glycosylation is of critical interest in industry and many efforts have been directed to improve this pathway. This is because altering the N-glycan composition can change the product quality of recombinant biotherapeutics in mammalian hosts. In addition, sialylation and fucosylation represent components of the glycosylation pathway that affect circulatory half-life and antibody-dependent cellular cytotoxicity, respectively. In this chapter, we first offer an overview of the glycosylation, sialylation, and fucosylation networks in mammalian cells, specifically CHO cells, which are extensively used in antibody production. Next, genetic engineering technologies used in CHO cells to modulate glycosylation pathways are described. We provide examples of their use in CHO cell engineering approaches to highlight these technologies further. Specifically, we describe efforts to overexpress glycosyltransferases and sialyltransfereases, and efforts to decrease sialidase cleavage and fucosylation. Finally, this chapter covers new strategies and future directions of CHO cell glycoengineering, such as the application of glycoproteomics, glycomics, and the integration of 'omics' approaches to identify, quantify, and characterize the glycosylated proteins in CHO cells. Graphical Abstract.

Fucose depletion from oligosaccharides of human IgG1-type antibodies results in a great enhancement of antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to clarify the effect of fucose removal on effector functions of all human IgG subclasses. A panel of anti-CD20 chimeric antibodies having a matched set of human heavy chain subclasses with different fucose contents in their oligosaccharides was constructed using wild-type and fucosyltransferase-knockout Chinese hamster ovary cells as host cells. As found previously for IgG1, fucose-negative variant of IgG2, IgG3, and IgG4 exhibited enhanced ADCC and FcγRIIIa binding compared with their highly fucosylated counterparts. In contrast, fucose removal did not affect complement-dependent cytotoxicity (CDC) of any IgGs. Consequently, fucose removal from IgG2 and IgG4 resulted in a unique effector function profile; they had potent ADCC and no CDC. In conclusion fucose depletion can provide a panel of IgGs with enhanced ADCC without an impact on other inherent properties specific for each IgG subclass, such as CDC.

Purpose:Delta-like ligand 3 (DLL3) is an emerging target across neuroendocrine cancers, but remains underexplored in gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) including poorly differentiated neuroendocrine carcinomas (GEP NECs) and well differentiated neuroendocrine tumors (NETs). We aimed to define the landscape of DLL3 expression and feasibility of DLL3-targeted imaging in this population.Patients and Methods:We completed DLL3 immunohistochemistry (IHC) on 360 tumor samples from patients with GEP NENs, analyzing associations between DLL3 IHC positivity and clinicopathologic features and outcomes. [89Zr]Zr-DFO-SC16.56 DLL3 immunoPET-CT imaging was performed in six patients with DLL3 IHC-positive advanced GEP NENs as part of a phase II clinical trial.Results:Among GEP NECs, DLL3 expression was identified in 53/75 (71%) samples, was enriched for small cell histology, and did not demonstrate prognostic significance. Among well differentiated pancreatic NETs (PanNETs), DLL3 expression was identified in 22/51 (43%) grade 3 (G3) tumors, with univariate analysis revealing increased mortality risk among patients with DLL3-positive advanced G3 PanNETs (hazard ratio 3.27, 95% confidence interval 1.09–9.78). Between May 28, 2024 and February 10, 2025, six patients with DLL3 IHC-positive GEP NENs were enrolled onto the imaging protocol. [89Zr]Zr-DFO-SC16.56 immunoPET-CT imaging delineated DLL3-avid tumor lesions in five of six patients (two of two GEP NECs, three of four G3 PanNETs). Tumor-specific uptake of [89Zr]Zr-DFO-SC16.56 varied between patients, with maximum standard uptake values ranging from 7.4–36.7, with four of six cases demonstrating DLL3 avidity in ≥ 50% of tumor lesions.Conclusion:DLL3 is expressed on a majority of GEP NECs and on a subset of high grade PanNETs marked by poor outcomes. Functional imaging suggests DLL3 as a promising therapeutic target in both GEP NECs and high grade PanNETs.

It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E- and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAcalpha2-->3Galbeta1-->4(Fucalpha1-->3(sulfo-->6))GlcNAcbeta1--> 3Galbeta1-->3GalNAcalpha1-->Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that beta1,3-N-acetylglucosaminyltransferase-3 (beta3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different beta3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding beta3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 beta1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either beta3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branched O-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of fucose significantly enhances antibody dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) to disrupt the α-1,6-fucosyltranferase (FUT8) gene and subsequently inhibit α-1,6-fucosylation on antibodies expressed in CHO cells.

Small cell lung cancer (SCLC) is known for its high proliferative rate and poor prognosis. Although Delta-like ligand 3 (DLL3) is specifically expressed on the surface of SCLC, the association of DLL3 with prognosis in SCLC remains uncertain. Hence, we aimed to evaluate prognostic role of DLL3 in extensive stage of SCLC treated with first-line chemotherapy. A total of 54 patients with extensive stage of SCLC (ES-SCLC) who were treated with first-line chemotherapy were included for our analysis. In addition, tissue specimen should be available for immuno-histochemical staining for DLL3, and their clinico-pathologic data, including progression-free survival (PFS) and overall survival (OS), were obtained. DLL3 expression and the percentage of tumor cells with DLL3 positive among total cancer cells were analyzed microscopically and DLL3 high and DLL3 low were defined as the percentage of DLL3 positive tumor cells versus total cancer cells ≧ 75% and < 75%, respectively. DLL3 expression was not associated with any of the clinico-pathological characteristics such as age at diagnosis, sex, response to first-line chemotherapy, second-line chemotherapy (Yes or No), and number of metastatic sites. However, response to first-line chemotherapy and number of metastatic sites were correlated to PFS, while DLL3 expression and number of metastatic sites were correlated to OS. DLL3 was highly expressed in SCLC, and not associated with any clinico-pathological characteristics. In survival outcome, DLL3 was correlated with worse OS, which suggests the prognostic role of DLL3 in ES-SCLC.

133 Objectives: Transdifferentiation of prostate adenocarcinoma to Neuroendocrine Prostate Cancer (NEPC) has emerged as one of the leading causes of resistance to androgen deprivation therapy (ADT) [1]. The patients have aggressive disease with visceral metastasis and have short survival time (7-10 months) [2]. Genomic and proteomic characterization of biopsy samples of NEPC lesions indicates loss of androgen receptor (AR) signaling [3]. Therefore, current PET imaging agents such as 18F-FDHT and 68Ga-PSMA11 that rely on functional AR cannot be used. Our goal is to develop a PET based molecular imaging agent that can uniquely identify NEPC lesions. Taking advantage of highly specific expression of Delta-like ligand 3 (DLL3) in NEPC lesions, we have developed zirconium-89 labeled immunoPET agent that can specifically identify NEPC lesions. Methods: We have used well characterized NCI-H660 cell line as representative NEPC model and compared it with AR dependent LNCaP and AR independent PC3 and DU145 cell lines. qPCR was used to measure relative levels of AR-regulated gene transcripts (AR, PSMA, PSA) and NEPC marker DLL3 and normalized to β-actin in the cell lines. Relative protein levels of AR regulated genes and DLL3 were measured by western blot analysis of cellular extracts using β-actin as our control. ImmunoPET agent - 89Zr-SC16 - was developed through the conjugation of the DFO chelator to SC16 (DLL3 specific) mAb and radiolabeled with zirconium-89. Saturation binding assay was performed on the cell line NCI-H660 to determine Bmax and Kd values. For in vivo PET imaging and biodistribution studies, NCI-H660 (DLL3/+) or DU145 (DLL3/-) xenografts were established in 6-8 week old male athymic nude mice. The mice were administered with 89Zr-SC16 and imaged at 24, 48, 72, 96, and 120 h post injection on a PET scanner and at chosen time points mice were euthanized and organs collected for biodistribution studies. Results: Saturation binding assay reveals that Kd =0.35 nM and Bmax = 863 fm/106 cells for 89Zr-SC16. In vitro studies indicated that NCI-H660 cell line was positive for DLL3 and negative for AR, PSA, and PSMA both at transcriptional and translational level. As expected all other cells except PC3 were negative for DLL3. Invivo PET imaging with 89Zr-SC16 showed clear delineation of NCI-H660 (DLL3/+) tumor xenografts. Biodistribution studies showed tumor uptake of 18.4 ± 3.8 %ID/g in the NCI-H660 tumors compared to 5.5 ± 0.5 %ID/g in the DU145 tumors, demonstrating the selective accumulation of the radiotracer in the DLL3-expressing tumors. Conclusions: Our findings demonstrate that only NEPC cells selectively express DLL3 and using DLL3 targeting PET agent 89Zr-SC16 we can non-invasively and uniquely identify NEPC lesions in vivo. Acknowledgement: Partial funding for these studies was provided by 2019 Geoffrey Beene Cancer Research Center Grant.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.

Enhancing the yield and activity of defucosylated antibody produced by CHO-K1 cells using Cas13d-mediated multiplex gene targeting

Recombinant therapeutic proteins (RTPs) are widely used to treat various major diseases. Chinese hamster ovary (CHO) cells are the preferred mammalian cell expression system for the production of RTPs. However, maintaining high productivity while ensuring good-quality RTPs is still challenging. Glycosylation, aggregation, charge variants, and degradation are the main quality attributes of RTPs and can impact their safety, biological activity, stability, and half-life. Modifications of associated genetic factors have been performed to improve the quality of the RTPs. For example, knocking out the α-1,6-fucosyltransferase (FUT8) gene results in the production of fucose-free antibodies, significantly enhancing antibody-dependent cellular cytotoxicity (ADCC). Overexpressing the molecular chaperone GRP78 reduces antibody aggregation rates while improving cell survival rates. Knocking out the carboxypeptidase D (CpD) gene completely eliminates C-terminal lysine heterogeneity, thereby improving the antibody charge uniformity. The deletion of the insulin-degrading enzyme (IDE) gene nearly eliminates insulin degradation. Understanding the genetic factors that influence the quality of therapeutic proteins during CHO cell culture is essential for the production of high-quality therapeutic proteins. This review summarizes the genetic factors contributing to RTP heterogeneity in CHO cells and discusses innovative strategies to address this heterogeneity, such as CRISPR/Cas9-mediated gene knockout, synergistic glycosyltransferase overexpression, and host cell engineering.

Glioma is one of the most common neurological malignancies worldwide. Delta-like ligand 3 (DLL3), an inhibitory ligand-driven activation of the Notch pathway, has been shown to be significantly associated with overall survival in patients with glioma. Therefore, the purpose of this study was to determine whether DLL3 as a biomarker in glioma is associated with patients' clinicopathological features and prognosis. We identified differences in transcriptome and promoter methylation in the Chinese Glioma Genome Atlas (CGGA) in patients with malignant glioma with shorter (less than 1year) and longer (greater than 3years) survival time. Further analysis of The Cancer Genome Atlas (TCGA) revealed that four genes (DLL3, TSPAN15, RTN1, PAK7) are highly associated with patient prognosis and play an indispensable role in evolution. We chose the expression level of DLL3 in glioma patients for our study. Patients were divided into groups with low and high expression of DLL3 according to the cutoff values obtained, and Kaplan-Meier and Cox analysis were used to examine the correlation between DLL3 gene expression and patient survival. We then performed a gene set enrichment analysis (GSEA) to identify significantly enriched signaling pathways. Our results confirmed that the overall survival of patients with low DLL3 expression was significantly shorter than that of patients with high DLL3 expression. GSEA showed that the signaling pathways of the immune process and immune response, among others, were enhanced with the DLL3 low-expression phenotype. Collectively, our findings signify that DLL3 is a potent prognostic factor for glioma, which can provide a viable approach for glioma prognostic assessment and valuable insights for anti-tumor immune-targeted therapies.

Delta-like Ligand 3 (DLL3) targeting therapies are promising in small cell lung cancer (SCLC) treatment. However, DLL3 expression in SCLC and other neuroendocrine neoplasms (NEN) is heterogeneous and not well characterized. We describe the landscape of DLL3 at the mRNA and protein levels across SCLC, large cell neuroendocrine carcinoma (LCNEC), and non-small cell lung cancer. Additionally, we explore its expression in extra-pulmonary NEN (EP-NEN) using a standardized DLL3 IHC assay. DLL3 expression is enriched in SCLC, LCNEC along with combined histology lung cancers. Moreover, we find a wide range of DLL3 expression in high-grade EP-NEN. We describe heterogenous DLL3 expression not only in SCLC but also in different NEN types. This comprehensive characterization of DLL3 can help guide future clinical trial design targeting DLL3 in NEN including LCNEC and EP-NEN that are lacking standard of care treatment options.