Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3–10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3–10 and HSC-3 was 1.4 × 10−9 and 3.2 × 10−9 M, respectively. C44Mab-34 could detect CD44v3–10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.


Introduction
Head and neck cancers mainly arise from the oral cavity, pharynx, larynx, and nasal cavity. These tumors exhibit strong associations with smoking tobacco products, alcohol, and infection with human papillomavirus (HPV) types 16 and 18 [1]. The estimated number of new cases in the oral cavity and pharynx in the United States increased from 35,310 in 2008 to 54,540 in 2023 due to rising HPV-positive cases [2][3][4]. Mortality rates continue to increase for the oral cavity cancers associated with HPV infection (cancers of the tongue, tonsil, and oropharynx) by about 2% per year in men and 1% per year in women [2].
Although many different histologies exist in head and neck cancers, head and neck squamous cell carcinoma (HNSCC) is the common type. The treatment options for HNSCC include surgery, chemo-radiation, molecular targeted therapy, immunotherapy, or a combination of these modalities [5]. Despite the development in cancer treatment, metastasis and drug resistance remain the main causes of death [6]. Although survival can be improved, the impairment due to surgery and the toxicities of treatments deteriorate the patient's quality of life. Thus, the 5-year survival rate remains stagnant at approximately 50% [1].
Cancer stem cells (CSCs) play critical roles in tumor development through their important properties, including self-renewal, resistance to therapy, and tumor metastasis [7][8][9].
All the cells were grown in a humidified incubator at 37 • C with 5% CO 2 .

Purification of CD44ec
The purification of CD44ec from the culture supernatant of LN229/CD44ec was performed using an anti-RAP tag mAb (clone PMab-2) and a RAP peptide (GDDMVNPGLEDRIE) [54,55]. The culture supernatant (5 L) was passed through a 2 mL bed volume of PMab-2-sepharose, and the process was repeated three times. After washing the beads with 100 mL of phosphate-buffered saline (PBS, Nacalai Tesque, Inc.), CD44ec was eluted with 0.1 mg/mL of a RAP peptide in a step-wise manner (2 mL × 10). The purity of CD44ec was determined by Coomassie Brilliant Blue (CBB) staining using the Bio-Safe CBB G-250 Stain (Bio-Rad Laboratories, Inc., Berkeley, CA, USA) (Supplemental Figure S1A).

Hybridomas
Female BALB/c mouse was purchased from CLEA Japan (Tokyo, Japan). The Animal Care and Use Committee of Tohoku University approved the animal experiments (permit number: 2019NiA-001). The immunization of CD44ec was performed as described previously [29].
The splenic cells were fused with P3U1 cells using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA). The culture supernatants of hybridomas were screened using an enzyme-linked immunosorbent assay (ELISA) against CD44ec. The supernatants were further screened using CHO/CD44v3-10 and parental CHO-K1 cells by flow cytometry using SA3800 Cell Analyzers (Sony Corp. Tokyo, Japan).

Dissociation Constant (K D ) Determination by Flow Cytometry
Serially diluted C 44 Mab-34 was treated with CHO/CD44v3-10 and HSC-3 cells. Then, the cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:200). BD FACSLyric and BD FACSuite software version 1.3 (BD Biosciences) were used for fluorescence data collection and analysis, respectively. The GeoMean of each histogram, including primary mAb (C 44 Mab-34) + secondary Ab (Alexa Fluor 488-conjugated anti-mouse IgG) and only secondary Ab (for background), was determined. We further withdrew the background from each data and determined the dissociation constant (K D ) by GraphPad Prism 8 (the fitting binding isotherms to built-in one-site binding models; GraphPad Software, Inc., La Jolla, CA, USA).

Immunohistochemical Analysis
Formalin-fixed paraffin-embedded (FFPE) sections of the OSCC tissue array (OR601c) were purchased from US Biomax Inc. (Rockville, MD, USA). The OSCC tissue array was 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-34 (10 µg/mL) and C 44 Mab-46 (1 µg/mL) for 1 h at room temperature and then treated with the EnVision+ Kit for mouse (Agilent Technologies Inc.) for 30 min. The chromogenic reaction was conducted using 3,3 -diaminobenzidine tetrahy-drochloride (DAB; Agilent Technologies Inc.). The counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation). To examine the sections and obtain images, we used a Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany).

Development of an Anti-CD44v7/8 mAb, C 44 Mab-34
In this study, we purified human CD44ec as an immunogen ( Figure 1). One mouse was immunized with CD44ec, and hybridomas were seeded into 96-well plates. The supernatants were first screened by the reactivity to CD44ec by ELISA. Subsequently, the supernatants, which were positive for CHO/CD44v3-10 cells and negative for CHO-K1 cells, were further selected using flow cytometry. Finally, anti-CD44 mAb-producing clones were established by limiting dilution. Among them, C 44 Mab-34 (IgG 1 , kappa) was shown to recognize CD44p421-440 (GHQAGRRMDMDSSHSTTLQP), which corresponds to the variant 7-and variant 8-encoded sequence (Supplementary Table S1). In contrast, C 44 Mab-34 never recognized other CD44v3-10 extracellular regions. These results indicate that C 44 Mab-34 specifically recognizes the border region between variants 7 and 8. Furthermore, the binding epitopes were determined by ELISA using peptides, which cover the extracellular domain of CD44v3-10.

Immunohistochemical Analysis against Tumor Tissues Using C 44 Mab-34
We next examined whether C 44 Mab-34 could be used for immunohistochemical analyses using FFPE sections. We used sequential sections of an OSCC tissue microarray. In a well-differentiated OSCC section, the clear membranous staining in OSCC was observed by C 44 Mab-34 and C 44 Mab-46 ( Figure 5A,B). In an OSCC section with the stromal-invaded phenotype, C 44 Mab-34 strongly stained stromal-invaded OSCC and could clearly distinguish tumor cells from stromal tissues ( Figure 5C). In contrast, C 44 Mab-46 stained both invaded tumor cells and surrounding stroma cells ( Figure 5D). In Figure 5E,F, C 44 Mab-34 and C 44 Mab-46 never stained tumor tissue, but clear stromal staining was observed by C 44 Mab-46 ( Figure 5F). We have summarized the data of the immunohistochemical analysis of CD44 expression in tumor cells in Table 1  . Western blot analysis using C44Mab-34. The cell lysates from CHO-K1, CHO/CD44s, and CHO/CD44v3-10 (10 µg) were electrophoresed and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 10 µg/mL of C44Mab-46 (A), 10 µg/mL of C44Mab-34 (B), and 1 µg/mL of an anti-β-actin mAb (C). Then, the membranes were incubated with anti-mouse immunoglobulins conjugated with peroxidase for C44Mab-46, C44Mab-34, and an anti-βactin mAb. The red arrows indicate the CD44s (75~100 kDa). The black arrows indicate the CD44v3-10.

Immunohistochemical Analysis against Tumor Tissues Using C44Mab-34
We next examined whether C44Mab-34 could be used for immunohistochemical analyses using FFPE sections. We used sequential sections of an OSCC tissue microarray. In a well-differentiated OSCC section, the clear membranous staining in OSCC was observed by C44Mab-34 and C44Mab-46 ( Figure 5A,B). In an OSCC section with the stromal-invaded phenotype, C44Mab-34 strongly stained stromal-invaded OSCC and could clearly distinguish tumor cells from stromal tissues ( Figure 5C). In contrast, C44Mab-46 stained both invaded tumor cells and surrounding stroma cells ( Figure 5D). In Figure 5E,F, C44Mab-34 and C44Mab-46 never stained tumor tissue, but clear stromal staining was observed by C44Mab-46 ( Figure 5F). We have summarized the data of the immunohistochemical analysis of CD44 expression in tumor cells in Table 1; C44Mab-34 stained 42 out of 49 (86%) cases of OSCC. These results indicate that C44Mab-34 is useful for the immunohistochemical analysis of FFPE tumor sections.  Mab-46 (B, D, F), followed by treatment with the Envision+ kit. The chromogenic reaction was conducted using 3,3 -diaminobenzidine tetrahydrochloride (DAB). The counterstaining was performed using hematoxylin. Scale bar = 100 µm.

Discussion
Head and neck cancer is the seventh most common type of cancer worldwide, and it exhibits aggressive development in clinical settings [58]. Head and neck cancer remains a complex disease with a profound impact on patients and their quality of life after surgical ablation and therapies. Knowledge of the disease has been accumulated with regard to tumor biology and prevention, and therapeutic options have been simultaneously developed [58]. HNSCC is the most common type of head and neck cancer, and it has been revealed as the second-highest CD44-expressing cancer type in the Pan-Cancer Atlas [59]. CD44 overexpression is associated with poor prognosis and resistance to therapy [60][61][62]. Reduced CD44 expression leads to the growth suppression of tumor cells [17,63]. Therefore, CD44 is considered an important target for mAb therapies. In this study, we developed a novel anti-CD44v7/8 mAb, C 44 Mab-34, and showed multiple applications to flow cytometry (Figures 2 and 3), Western blotting (Figure 4), and the immunohistochemistry of OSCC ( Figure 5).
An anti-CD44v7/8 mAb (clone VFF-17) was previously developed, and it has been mainly used for the immunohistochemistry of normal tissue and tumors [64,65]. The epitope of VFF-17 mAb was determined by binding studies with fusion proteins encoding v7 or v8 exons, either alone or in combination [66]. However, a detailed amino acid sequence of the epitope has not been determined. As shown in Supplementary Table S1, C 44 in v8)). These results suggest that C 44 Mab-34 recognizes the border sequence between v7 and v8. In addition, CD44 is known to be heavily glycosylated [67], and the glycosylation pattern is thought to depend on the host cells. Since the epitope of C 44 Mab-34 contains predicted and confirmed O-glycan sites [67], further studies are needed on whether the recognition of C 44 Mab-34 is affected by glycosylation.
Among many CD44v types, CD44v8-10, CD44v6-10, CD44v4-10, and CD44v3-10 were mainly detected in SCC cells by semi-quantitative RT-PCR analysis (manuscript submitted). Since C 44 Mab-34 recognizes the border sequence between v7 and v8 (Supplemental Table S1), C 44 Mab-34 can distinguish CD44v8-10 and the longer CD44v (v6-10, v4-10, and v3-10). Furthermore, the inclusion of these variants (from v8-10 to the longer variants) is promoted by EGF signaling [68,69]. If the expression of CD44v8-10 and the longer variants are differently regulated in normal and tumor cells, C 44 Mab-34 could contribute to tumor diagnosis and therapy. We are now investigating the C 44 Mab-34 reactivity against other tumor tissues together with the epitope analyses.
∆Np63 is known as a marker of basal cells of stratified epithelium and SCC [70]. ∆Np63 mediates HA metabolism and signaling [71]. Specifically, ∆Np63 directly binds to the p63-binding sequence on the promoter/enhancer region of the CD44 gene [71]. In whole-exome sequencing data analysis from 74 HNSCC-normal pairs, the ∆Np63-encoded gene, TP63, was identified as a significantly mutated gene that results in the activation of the ∆Np63 pathway [72]. The relationship between ∆Np63 activation and CD44 transcription should be investigated in future studies. Furthermore, the mechanism of the variant 7/8 inclusion by alternative splicing remains to be determined.
An anti-pan CD44 mAb, RG7356, demonstrated some efficacy and an acceptable safety profile in the phase I study. However, the study was terminated due to no evidence of a clinical and dose-response relationship with RG7356 [73]. Furthermore, a variant 6-specific CD44 mAb-drug conjugate (bivatuzumab-mertansine) was also evaluated in clinical trials. However, lethal epidermal necrolysis halted further development. The efficient accumulation of mertansine in the skin was most likely responsible for the high toxicity [74,75]. Therefore, the therapeutic effects of CD44 mAbs have been disappointing until now.
Near-infrared photoimmunotherapy (NIR-PIT) is a novel tumor therapy that uses a targeted mAb-photoabsorber conjugate (APC) [76]. The mAb binds to the targeted cell surface antigen, and the photoactivatable dye IRDye700DX (IR700) induces the disruption of the cellular membrane after NIR-light exposure. Since NIR-light exposure can be performed at tumor sites locally, APC can exert antitumor effect selectivity while minimizing damage to the surrounding tissue [77,78]. Preclinical studies indicate that NIR-PIT induces tumor necrosis and immunogenic cell death through the induction of innate and adaptive immunity [79]. A first-in-human phase I and II trial of NIR-PIT with RM-1929 (an anti-epidermal growth factor receptor mAb, cetuximab-IR700 conjugate) in patients with inoperable HNSCC was conducted and exhibited the efficacy [80].
A preclinical study of anti-CD44 mAb-based NIR-PIT has been reported [81]. The study used an anti-mouse/human pan-CD44 mAb, IM7, conjugated with IR700 (CD44-IR700) in a syngeneic mouse model of OSCC. The CD44-IR700 can induce significant antitumor responses after a single injection of the conjugate and NIR-light exposure in CD44-expressing OSCC tumors [81]. As shown in Figure 5D,F, a pan-CD44 mAb, C 44 Mab-46, recognized not only tumor cells but also stromal tissue and probably immune cells, which are important for antitumor immunity. Therefore, CD44v is a promising tumor antigen for NIR-PIT, which could be a new modality for OSCC with locoregional recurrence.

Data Availability Statement:
The data presented in this study are available in the article and supplementary material.

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