Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody C44Mab-3 for Multiple Applications against Pancreatic Carcinomas

Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker and plays tumor promotion and drug resistance roles in various cancers. In particular, the splicing variants are overexpressed in many carcinomas and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant’s (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant-5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3–10 and PK-1 was 1.3 × 10−9 M and 2.6 × 10−9 M, respectively. C44Mab-3 could detect the exogenous CD44v3–10 and endogenous CD44v5 in Western blotting and stained the formalin-fixed paraffin-embedded pancreatic cancer cells but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic cancer diagnosis and therapy.


Introduction
Pancreatic cancer has become the third leading cause of death in men and women combined in the United States in 2023 [1]. The development of pancreatic cancer has been explained by four common oncogenic events, including KRAS, CDKN2A, SMAD4, and TP53 [2,3]. However, pancreatic cancer shows a heterogeneity in drug response and clinical outcomes [4]. Therefore, detailed understanding of pancreatic cancers has been required to improve patient selection for current therapies and to develop novel therapeutic strategies. An integrated genomic analysis of pancreatic ductal adenocarcinomas (PDAC) was performed and defined four subtypes, including squamous, pancreatic progenitor, immunogenic, and aberrantly differentiated endocrine exocrine (ADEX), which correspond to the histopathological characteristics [5]. Additionally, various marker proteins have been investigated for the early diagnostic and drug responses of pancreatic cancers [6]. Studies have suggested that CD44 plays important roles in malignant progression of tumors through its cancer stemness and metastasis-promoting properties [7,8].
CD44 is a type I transmembrane glycoprotein that is expressed as a wide variety of isoforms in various types of cells. [9]. The variety of isoforms is produced by the alternative Chinese hamster ovary (CHO)-K1 and mouse multiple myeloma P3X63Ag8U.1 (P3U1) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The human pancreas cancer cell lines PK-1 and PK-8 were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer at Tohoku University. These cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B (Nacalai Tesque, Inc.), and 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All the cells were grown in a humidified incubator at 37 • C with 5% CO 2 .

Hybridomas
The female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). All animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) and performed according to relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. The mice were intraperitoneally immunized with CHO/CD44v3-10 (1 × 10 8 cells) and Imject Alum (Thermo Fisher Scientific Inc.) as an adjuvant. After the three additional immunizations per week, a booster injection was performed two days before harvesting the spleen cells of immunized mice. The hybridomas were established by the fusion of splenocytes and P3U1 cells using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA). RPMI-1640 supplemented with hypoxanthine, aminopterin, and thymidine (HAT; Thermo Fisher Scientific Inc.) was used for the selection of hybridomas. The supernatants, which are negative for CHO-K1 cells and positive for CHO/CD44v3-10 cells, were selected by flow cytometry using SA3800 Cell Analyzers (Sony Corp. Tokyo, Japan).

Determination of Dissociation Constant (K D ) via Flow Cytometry
CHO/CD44v3-10 and PK-1 cells were treated with serially diluted C 44 Mab-3 (0.01-10 µg/mL). Then, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody. Fluorescence data were analyzed using BD FACSLyric and BD FACSuite software version 1.3 (BD Biosciences, Franklin Lakes, NJ, USA). The K D was determined by the fitting binding isotherms to built-in one-site binding models of GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA).

Determination of K D via Surface Plasmon Resonance (SPR)
Measurement of K D between C 44 Mab-3 and the epitope peptide was performed using SPR. C 44 Mab-3 was immobilized on the sensor chip CM5 according to the manufacturer's protocol by Cytiva (Marlborough, MA, USA). C 44 Mab-3 (10 µg/mL in acetate buffer (pH 4.0; Cytiva)) was immobilized using an amine coupling reaction. The surface of the flow cell 2 of the sensor chip CM5 was treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (NHS), followed by the injection of C 44 Mab-3. The K D between C 44 Mab-3 and the epitope peptide (CD44p311-330) was determined using Biacore X100 (Cytiva). A single cycle kinetics method was used to measure the binding signals. The data were analyzed by 1:1 binding kinetics to determine the association rate constant (ka) and dissociation rate constant (kd) and K D using Biacore X100 evaluation software (Cytiva).

Immunohistochemical Analysis
One formalin-fixed paraffin-embedded (FFPE) oral SCC tissue was obtained from Tokyo Medical and Dental University [47]. FFPE sections of pancreatic carcinoma tissue arrays (Catalog number: PA241c and PA484) were purchased from US Biomax Inc. (Rockville, MD, USA). Pancreas adenocarcinoma tissue microarray with adjacent normal pancreas tissue (PA241c) contains 6 cases of pancreas adenocarcinoma with matched adjacent normal pancreas tissue, with quadruple cores per case. One oral SCC tissue was autoclaved in citrate buffer (pH 6.0; Nichirei biosciences, Inc., Tokyo, Japan), and pancreatic carcinoma tissue arrays were autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C 44 Mab-3 (1 µg/mL) and C 44 Mab-46 (1 µg/mL) for 1 h at room temperature. Then, the sections were incubated with the EnVi-sion+ Kit for mouse (Agilent Technologies Inc.) for 30 min. The color was developed using 3,3 -diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.). Hematoxylin (FUJIFILM Wako Pure Chemical Corporation) was used for the counterstaining. A Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to examine the sections and obtain images.

Determination of the Binding Affinity of C 44 Mab-3 by Flow Cytometry to CD44-Expressing Cells and SPR with the Epitope Peptide
Next, we determined the binding affinity of C 44 Mab-3 to CHO/CD44v3-10 and PK-1 using flow cytometry. As shown in Figure 3, the K D of CHO/CD44v3-10 and PK-1 was 1.3 × 10 −9 M and 2.6 × 10 −9 M, respectively, indicating that C 44 Mab-3 possesses high affinity for CD44v3-10 and endogenous CD44v5-expressing cells.
We also measured the KD of C44Mab-3 with the epitope peptide (CD44p311-330) using Biacore X100. The binding kinetics and measured values are summarized in Supplementary Figure S3. The KD of CD44p311-330 was 5.5 × 10 −6 M.

Western Blot Analysis
We next performed Western blot analysis to investigate the sensitivity of C44Mab-3. Total cell lysates from CHO-K1, CHO/CD44s, CHO/CD44v3-10, PK-1, and PK-8 were We also measured the K D of C 44 Mab-3 with the epitope peptide (CD44p311-330) using Biacore X100. The binding kinetics and measured values are summarized in Supplementary Figure S3. The K D of CD44p311-330 was 5.5 × 10 −6 M.

Immunohistochemical Analysis Using C 44 Mab-3 against Tumor Tissues
We next examined whether C 44 Mab-3 could be used for immunohistochemical analyses using FFPE sections. We first examined the reactivity of C 44 Mab-3 and C 44 Mab-46 in an oral SCC tissue. As shown in Supplementary Figure S4, C 44 Mab-3 exhibited a clear membranous staining and could clearly distinguish tumor cells from stromal tissues. In contrast, C 44 Mab-46 stained both. We then investigated the reactivity of C 44 Mab-3 and C 44 Mab-46 in pancreatic carcinoma tissue arrays. Although we performed the antigen retrieval using citrate buffer (pH 6.0) for pancreatic carcinoma tissue arrays in the same way as with an oral SCC tissue, weak staining was observed. Therefore, we next used EnVision FLEX Target Retrieval Solution High pH for the antigen retrieval procedure; C 44 Mab-3 showed clear membranous staining in pancreatic carcinoma cells with a relatively larger cytoplasm ( Figure 5A). C 44 Mab-46 also stained the same type of pancreatic carcinoma cells ( Figure 5B). The staining intensity of C 44 Mab-3 was much stronger than that of C 44 Mab-46 ( Figure 5A,B). Furthermore, diffusely spread tumor cells in the stroma were stained by both C 44 Mab-3 and C 44 Mab-46 ( Figure 5C,D). In contrast, both C 44 Mab-3 and C 44 Mab-46 did not stain the typical ductal structure of PDAC ( Figure 5E,F). In addition, stromal staining using C 44 Mab-46 was observed in several tissues ( Figure 5F). Importantly, normal pancreatic epithelial cells were not stained by C 44 Mab-3 ( Figure 5G). A similar staining pattern was also observed in another tissue array (Supplementary Figure S5). We summarized the data of immunohistochemical analyses in Table 2; C 44 Mab-3 stained 8 out of 20 cases (40%) (PA484, Figure 5) and 2 out of 6 cases (33%) (PA241c, Supplementary Figure S5) of pancreatic carcinomas. These results indicated that C 44 Mab-3 could be useful for immunohistochemical analysis of FFPE tumor sections and could recognize a specific type of pancreatic carcinoma.

Discussion
PDAC is the most common type of pancreatic cancer and has extremely poor prognosis, with a 5-year survival rate of approximately 10% [48]. Advances in therapy have only achieved incremental improvements in overall outcome but can provide notable benefits for undefined subgroups of patients. PDACs are heterogenous neoplasms with various histology [4] and heterogenous molecular landscapes [5]. Therefore, the identification of early diagnostic markers and therapeutic targets in each group has been desired. In this study, we developed C 44 Mab-3 using the CBIS method ( Figure 1) and determined its epitope as variant-5-encoded region of CD44 (Table 1). Then, we showed the usefulness of C 44 Mab-3 for multiple applications, including flow cytometry (Figures 2 and 3), Western blotting (Figure 4), and immunohistochemistry of PDAC ( Figure 5).
An anti-CD44v5 mAb (clone VFF-8) was previously developed and is mainly used for the immunohistochemical analyses of tumors [49]. The epitope of VFF-8 was determined as IHHEHHEEEETPHSTST in the v5-encoded region by ELISA [50]. As shown in Table 1, C 44 Mab-3 recognized both CD44p311-330 and CD44p321-340 peptides, which commonly possess the HPPLIHHEHH sequence. The epitope of C 44 Mab-3 partially shares that of VFF-8. Further investigation of the detailed epitope mapping is required. In addition, CD44 is known to be heavily glycosylated [12], and the glycosylation pattern is thought to depend on the host cells. Since the epitope of C 44 Mab-3 does not contain serine or threonine, the recognition of C 44 Mab-3 is thought to be independent of the glycosylation.
Immunohistochemistry using VFF-8 and conventional RT-PCR analyses were performed against PDAC [49]. VFF-8 recognized PDAC but not normal pancreas cells. Furthermore, the RT-PCR analysis revealed that the exon v5 appeared in the chain containing at least v4-10 in 80% of PDACs and the cell lines tested. The authors discussed that one of the major differences between normal and PDAC was the linkage of CD44v5 to the CD44v6-containing chain [49]. Our immunohistochemical analysis also support this finding ( Figure 5A,C,G). Furthermore, we found that C 44 Mab-3 could detect atypical types of PDAC, including metaplastic and diffusely invaded tumor cells ( Figure 5A,C). In contrast, C 44 Mab-3 did not stain a typical ductal structure of PDAC ( Figure 5E) and normal pancreatic epithelial cells ( Figure 5G). In addition to conventional PDAC, the World Health Organization has classified nine histological subtypes of PDAC, which further highlight the morphologic heterogeneity of PDAC [4]. It is worthwhile to investigate whether CD44v5 is expressed in a specific subtype of PDAC in a future study.
Large-scale genomic analyses of PDACs defined four subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) ADEX, which correlate with histopathological characteristics [5]. Among them, the squamous subtype is characterized as being enriched for TP53 and KDM6A mutations and having upregulation of the ∆Np63 transcriptional network, hypermethylation of pancreatic endodermal determinant genes, and a poor prognosis [5]. ∆Np63 is known as a marker of basal cells of stratified epithelium and SCC [51]; it is also reported to regulate HA metabolism and signaling [52]. Specifically, ∆Np63 directly regulates the expression of CD44 through p63-binding sites that are located in the promoter region and in the first intron of CD44 gene [52]. Therefore, CD44 transcription could be upregulated in ∆Np63-positive PDAC. However, the mechanism of the variant 5 inclusion during alternative splicing remains to be determined.
Clinical trials of anti-pan-CD44 and variant-specific CD44 mAbs have been conducted [53]. An anti-pan-CD44 mAb, RG7356, exhibited an acceptable safety profile in patients with advanced solid tumors expressing CD44. However, the study was terminated due to no evidence of a clinical and pharmacodynamic dose-response relationship with RG7356 [54]. A clinical trial of a humanized anti-CD44v6 mAb bivatuzumab−mertansine drug conjugate was conducted. However, it failed due to severe skin toxicities [55,56]. The efficient accumulation of mertansine was most likely responsible for the high toxicity [55,56]. Although CD44v5 is not detected in normal pancreatic epithelium by C 44 Mab-3 (this study) and VFF-8 [49], CD44v5 could be detected in normal lung, skin, gastric, and bladder epithelium by VFF-8 [50]. For the development of the therapeutic use of C 44 Mab-3, further investigations are required to reduce the toxicity to the above tissues.

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