Development of a Novel Anti-CD44 Variant 8 Monoclonal Antibody C44Mab-94 against Gastric Carcinomas

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeted monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells. These results indicate that C44Mab-94 is useful for detecting CD44v8 in a variety of experimental methods and is expected to become usefully applied to GC diagnosis and therapy.


Introduction
Gastric cancer (GC) is the third leading cause of cancer-related deaths globally [1]. The GC incidence is higher in Eastern Asia than in Western countries [2]. The vast majority of GC are adenocarcinomas, which can be divided into intestinal-type gastric cancer (IGC), diffuse-type gastric cancer (DGC), and mixed histology according to the Lauren classification [3]. The World Health Organization classifies gastric adenocarcinomas into papillary, tubular, mucinous, and poorly cohesive carcinomas [4]. Furthermore, next-generation sequencing defined four molecular subtypes, including Epstein-Barr virus-positive, microsatellite instability, genomically stable, and chromosomally unstable types [5,6]. The analysis also revealed the alterations in the GC genome and provided treatment options with anti-human epidermal growth factor receptor 2 (HER2) therapy [7] or immune checkpoint inhibitor therapy [8]. However, the benefit of those therapies is limited to a small subset of patients. In patients with advanced GC, especially those with DGC, peritoneal metastasis and subsequent development of malignant ascites are the most frequent cause of death [9]. Tanaka et al., therefore, performed a comprehensive multi-omic analysis of malignant ascitic samples and their corresponding tumor cell lines [10]. They

Immunohistochemical Analysis Using C44Mab-94 against Tumor Tissues
We next investigated whether C44Mab-94 could be applied to immunohistochemical analysis using FFPE sections. We first examined the reactivity of C44Mab-94 in the OSCC tissue array because this type was revealed as the second highest CD44-positive cancer type in the Pan-Cancer Atlas [40]. As shown in Supplementary Figure S2A,B, the membranous staining in OSCC was observed by C44Mab-94 and C44Mab-46. In a stromal-invaded OSCC section, C44Mab-94 strongly stained invaded OSCC and could clearly distinguish tumor cells from stromal tissues (Supplementary Figure S2C). In contrast, C44Mab-46 stained both invaded OSCC and surrounding stroma cells (Supplementary Figure S2D). Supplementary Table S1 summarizes the results of OSCC tissue staining.

Immunohistochemical Analysis Using C 44 Mab-94 against Tumor Tissues
We next investigated whether C 44 Mab-94 could be applied to immunohistochemical analysis using FFPE sections. We first examined the reactivity of C 44 Mab-94 in the OSCC tissue array because this type was revealed as the second highest CD44-positive cancer type in the Pan-Cancer Atlas [40]. As shown in Supplementary Figure S2A Table S1 summarizes the results of OSCC tissue staining. We next stained the GC tissue array (BS01011b) using C 44 Mab-94 and C 44 Mab-46. C 44 Mab-94 exhibited membranous staining in IGC ( Figure 6A). C 44 Mab-46 also stained the same type of cancer cells ( Figure 6B). Furthermore, membranous and cytoplasmic staining by C 44 Mab-94 and C 44 Mab-46 was observed in stromal-invaded tumor cells ( Figure 6C,D). In DGC ( Figure 6E,F), diffusely spread tumor cells were strongly stained by both C 44 Mab-94 and C 44 Mab-46. In contrast, neither C 44 Mab-94 nor C 44 Mab-46 stained the ductal epithelial structure of IGC ( Figure 6G,H). Additionally, stromal staining by C 44 Mab-46 was observed in the tissue ( Figure 6H). We next stained the GC tissue array (BS01011b) using C44Mab-94 and C44Mab-46. C44Mab-94 exhibited membranous staining in IGC ( Figure 6A). C44Mab-46 also stained the same type of cancer cells ( Figure 6B). Furthermore, membranous and cytoplasmic staining by C44Mab-94 and C44Mab-46 was observed in stromal-invaded tumor cells ( Figure 6C,D). In DGC ( Figure 6E,F), diffusely spread tumor cells were strongly stained by both C44Mab-94 and C44Mab-46. In contrast, neither C44Mab-94 nor C44Mab-46 stained the ductal epithelial structure of IGC ( Figure 6G,H). Additionally, stromal staining by C44Mab-46 was observed in the tissue ( Figure 6H).
We confirmed that C 44 Mab-94 recognizes a synthetic peptide of the v8-encoded region (DSSHSTTLQPTANPNTGLVE), but not border regions (v7/v8 and v8/v9) by ELISA ( Figure 3). The epitope region possesses multiple confirmed and predicted O-glycosylation sites [50]. C 44 Mab-94 recognized a~75-kDa band in the lysate of CHO/CD44v3-10 ( Figure 5A), which is similar to the predicted molecular size from the amino acids of CD44v3-10. Therefore, C 44 Mab-94 could recognize CD44v3-10 regardless of the glycosylation. A detailed epitope analysis and an investigation of the influence of glycosylation on C 44 Mab-94 recognition are required in future studies.
In a GC cell line, the major transcripts of CD44v, including CD44v3, 8-10, CD44v6-10, CD44v8-10, and CD44v3, 8 were identified [39] ( Figure 1A). C 44 Mab-94 can cover all products of the transcripts and detect the broad CD44v-expressing GC. Since CD44v8-10 plays critical roles in the regulation of ROS defense and GC progression [26], an anti-CD44v9 mAb (clone RV3) has so far mainly been used in immunohistochemistry. Several studies revealed that CD44v9 is a predictive marker for the recurrence of GC [51] and a biomarker for GC patient selection and efficacy of the xCT inhibitor, sulfasalazine [52]. Further investigations are required to reveal the relationship between CD44v8 expression and clinical factors using C 44 Mab-94. Additionally, C 44 Mab-94 recognized both IGC ( Figure 6A) and DGC ( Figure 6E) in immunohistochemistry. It would be worthwhile investigating whether CD44v8 is expressed in a specific molecular subtype of GC [6] in a future study.
A comprehensive analysis of malignant ascites identified the amplifications of cancer driver genes including CD44 [10]. Although the expression pattern of CD44v is not identified, CD44v8 is thought to be an important target for mAb therapy due to the commonly included region in GC [39]. We have shown the antitumor activity using class-switched and defucosylated IgG 2a recombinant mAbs [33]. The defucosylated IgG 2a mAbs were produced by CHO-K1 lacking fucosyltransferases 8; they exhibited potent ADCC activity in vitro, and suppressed the growth of xenograft [53]. Therefore, the production of defucosylated C 44 Mab-94 is one of the strategies used to evaluate the antitumor effect on GC with peritoneal metastasis in the preclinical model.
Clinical applications of a humanized anti-CD44v6 mAb (BIWA-4) bivatuzumabmertansine drug conjugate to solid tumors failed because of skin toxicities [47,48]. The accumulation of the mertansine drug was thought to be a cause of the toxicity [47,48]. Human acute myeloid leukemia (AML) cells also express high levels of CD44 mRNA due to the suppression of methylation of the CpG islands in the promoter [54]. Furthermore, higher expression of CD44v6 was observed in AML patients with FLT3 or DNMT3A mutations. Therefore, a mutated version of BIWA-4-called BIWA-8-was engineered to develop chimeric antigen receptors (CARs) for AML. The CD44v6 CAR-T cells exhibited potent anti-leukemic effects [54,55] indicating that CD44v6 is a rational target of CAR-T therapy for AML harboring FLT3 or DNMT3A mutations. Additionally, the CD44v6 CAR-T also showed an antitumor effect in lung and ovarian cancer xenograft models [56], which is expected for a wider development toward solid tumors.
Since CD44 mRNA is elevated in AML, other CD44 variants might also be transcribed in AML. Furthermore, CD44v8-10 was elevated during chronic myeloid leukemia (CML) progression from chronic phase to blast crisis in a humanized mouse model, which is required for the maintenance of stemness of CML [57]. Therefore, in future, we will investigate the reactivity of C 44 Mab-94 against hematopoietic malignancy. Further studies are required to investigate the selective expression of CD44v8 in leukemia cells, but not in hematopoietic stem cells, to certify its safety as a CAR-T antigen.
In this study, we used tumor cell-expressed CD44v3-10 as an immunogen. This strategy is important for the establishment of cancer-specific mAbs (CasMabs). We previously developed PDPN-targeting CasMabs [58] and podocalyxin-targeting CasMabs [59], which recognize cancer-type aberrant glycosylation of the targets [60]. Anti-PDPN-CasMabs are currently applied to CAR-T therapy in preclinical models [61]. For CasMab development, we should do further screening of our established anti-CD44 mAbs by comparing the reactivity against normal cells. Anti-CD44 CasMabs could be applicable for designing the modalities including ADCs and CAR-T.

Conflicts of Interest:
The authors have no conflict of interest to declare.