Introduction

Oncology Reports

1021-335X 1791-2431

D.A. Spandidos

10.3892/or.2024.8806

OR-52-5-08806

Articles

Antitumor activities of anti‑CD44 monoclonal antibodies in mouse xenograft models of esophageal cancer

Ishikawa

Kenichiro

1 2 Suzuki

Hiroyuki

1 Ohishi

Tomokazu

3 4 Nakamura

Takuro

1 Yanaka

Miyuki

1 Li

Guanjie

1 Tanaka

Tomohiro

1 Ohkoshi

Akira

2 Kawada

Manabu

4 Kaneko

Mika K.

1 Katori

Yukio

2 Kato

Yukinari

1Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi 980-8575, Japan 2Department of Otolaryngology, Head and Neck Surgery, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi 980-8575, Japan 3Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu, Shizuoka 410-0301, Japan 4Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research Foundation, Shinagawa-ku, Tokyo 141-0021, Japan

Correspondence to: Dr Yukinari Kato, Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan, E-mail: dengfeiwen2012@163.com yukinari.kato.e6@tohoku.ac.jp

11 2024

29 08 2024

52 5

147

18042024 23072024

2024

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

CD44 is a type I transmembrane glycoprotein associated with poor prognosis in various solid tumors. Since CD44 plays a critical role in tumor development by regulating cell adhesion, survival, proliferation and stemness, it has been considered a target for tumor therapy. Anti-CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody-drug conjugates and chimeric antigen receptor-T cell therapy. Anti-pan-CD44 mAbs, C₄₄Mab-5 and C₄₄Mab-46, which recognize both CD44 standard (CD44s) and variant isoforms were previously developed. The present study generated a mouse IgG_2a version of the anti-pan-CD44 mAbs (5-mG_2a and C₄₄Mab-46-mG_2a) to evaluate the antitumor activities against CD44-positive cells. Both 5-mG_2a and C₄₄Mab-46-mG_2a recognized CD44s-overexpressed CHO-K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5-mG_2a and C₄₄Mab-46-mG_2a could activate effector cells in the presence of CHO/CD44s cells and exhibited complement-dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5-mG_2a and C₄₄Mab-46-mG_2a significantly suppressed CHO/CD44s and KYSE770 ×enograft tumor development compared with the control mouse IgG_2a. These results indicate that 5-mG_2a and C₄₄Mab-46-mG_2a could exert antitumor activities against CD44-positive cancers and be a promising therapeutic regimen for tumors.

CD44 mAb ADCC CDC esophageal tumor antitumor activity

Japan Agency for Medical Research and Development (AMED)

JP23ama121008

JP24am0521010

JP23bm1123027

JP23ck0106730

Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI)

22K06995

21K20789

21K07168

22K07224

The present study was supported in part by Japan Agency for Medical Research and Development (AMED) (grant nos. JP23ama121008, JP24am0521010, JP23bm1123027 and JP23ck0106730) and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) (grant nos. 22K06995, 21K20789, 21K07168 and 22K07224).

Introduction

Overexpression of CD44 is observed in various solid tumors, which are involved in the tumor malignant progression through the promotion of cellular proliferation, invasiveness and stemness via specific signaling pathways (1). The diversity of CD44 molecular function is mediated by the alternative splicing (2). CD44 is encoded in 20 exons. The first five (1–5) and the last five (16–20) are constant exons that generate the shortest CD44 standard (CD44s) isoform. The exons 6–15 are alternatively spliced and inserted into the CD44s as variant exons (3). The CD44 splice variants with variant exons are designated CD44 variant (CD44v) isoforms. The inclusion of CD44v exons in various combinations is regulated by receptor tyrosine kinase signaling and splicing activators of pre-mRNA (1).

CD44 possesses an extracellular domain (ectodomain), a transmembrane domain, and an intracellular cytoplasmic domain (4). The ectodomain contains a hyaluronic acid (HA)-binding domain (HABD) that mediates cellular homing, adhesion, migration and proliferation (4). Both CD44s and CD44v isoforms have the HABD, and the HA binding causes conformational changes of the CD44, which results in the promotion of intracellular signaling pathways to regulate cell migration and proliferation (5). In CD44v isoforms, the variant exons-encoding sequences form the stem region, which provides a co-receptor for various growth factors and cytokines. These functions activate specific signaling pathways to promote invasion and stemness (6). Therefore, different isoforms of CD44v exhibit different functions according to the inserted variant exons in the CD44 ectodomain.

The relationship between CD44 expression and prognosis in patients with cancer has been evaluated and showed both poor and favorable outcomes (7). Currently, increased evidence suggests that overexpression of CD44 and its isoforms is an unfavorable indicator in patients with cancer (7). In an analysis of multiple studies, including 583 pancreatic cancer cases, overexpression of CD44 was predictive of poor overall survival, a more advanced stage and more lymph node invasion (8). Furthermore, another meta-analysis investigated the prognostic significance of cancer stem cell markers, including CD44, from 52 studies of ovarian cancer. The study concluded that CD44 overexpression was predictive of worse disease-free survival and resistance to chemotherapy (9).

Several anti-CD44 monoclonal antibodies (mAbs) have been developed for preclinical and clinical research for tumor therapy. An anti-pan-CD44 mAb (H4C4) reduced tumor growth, metastasis and post-radiation recurrence in a human pancreatic tumor xenograft model (10). A humanized anti-pan-CD44 mAb, RG7356, showed the cytotoxicity for B cell leukemia but no cytotoxicity on normal B cells. Administration of RG7356 to immunodeficient mice engrafted with chronic lymphocytic leukemia cells resulted in complete clearance of engrafted leukemia cells (11). Phase I clinical trials with RG7356 were conducted in patients with acute myeloid leukemia (12) and advanced CD44-positive solid tumor patients (13). Although RG7356 showed an acceptable safety profile, the studies were not continued due to the lack of a clinical and/or pharmacodynamic dose-response relationship with RG7356 (13).

Therapies using anti-CD44v6 mAbs were considered since CD44v6 plays critical roles in the malignant progression of tumors (14). Humanized anti-CD44v6 mAbs, including BIWA-4 and BIWA-8, were developed. These mAbs labeled with ¹⁸⁶Re were evaluated in head and neck squamous cell carcinoma xenograft-bearing mice and exhibited therapeutic efficacy (15). Furthermore, the BIWA-4 was developed into a humanized version-drug conjugate, bivatuzumab-mertansine (anti-tubulin agent), which was evaluated in clinical trials (16). However, the clinical trials were terminated because of the severe skin toxicity, such as lethal epidermal necrolysis (17). Since CD44v6 is expressed in normal skin epithelium, the toxicity of mertansine in the skin was most likely responsible for the high toxicity (17,18). Previously, a mutated version of BIWA-4, called BIWA-8, was developed for increasing binding affinity by two amino acid substitutions of the light chain (15). The BIWA-8 was further developed to chimeric antigen receptors (CARs). The BIWA-8 CAR-T showed antitumor activities against multiple myeloma or acute myeloid leukemia engrafted with immunodeficient mice (19). Furthermore, the BIWA-8 CAR-T exhibited efficacy in lung and ovarian carcinomas xenograft models (20), which is expected for an application toward solid tumors.

In our previous studies, an anti-pan-CD44 mAb, C₄₄Mab-5 (IgG₁, kappa) was developed using the Cell-Based Immunization and Screening (CBIS) method (21). Another mAb (C₄₄Mab-46) (22) was created by immunization of the CD44v3-10 ectodomain. Both C₄₄Mab-5 and C₄₄Mab-46 have the epitopes within the constant exon 2- and 5-encoded sequences (23–25) and could be applied to immunohistochemistry in oral squamous cell carcinoma (21) and esophageal squamous cell carcinoma (ESCC) (22), respectively. Furthermore, various anti-CD44v mAbs have been developed, such as C₄₄Mab-6 (an anti-CD44v3 mAb) (26), C₄₄Mab-108 (an anti-CD44v4 mAb) (27), C₄₄Mab-3 (an anti-CD44v5 mAb) (28), C₄₄Mab-9 (an anti-CD44v6 mAb) (29), C₄₄Mab-34 (an anti-CD44v7/8 mAb) (30), C₄₄Mab-1 (an anti-CD44v9 mAb) (31) and C₄₄Mab-18 (an anti-CD44v10 mAb) (32). The combinational use of the anti-CD44 mAbs is essential for comprehensively analyzing human tumors.

In the present study, a mouse IgG_2a type of recombinant C₄₄Mab-5 (5-mG_2a) and C₄₄Mab-46 (C₄₄Mab-46-mG_2a) was produced using fucosyl-transferase 8 (FUT8)-deficient ExpiCHO-S cells and the antitumor activity in xenograft-bearing mice was investigated.

Materials and methods <sec> <title>Cell lines

ESCC cell line KYSE770 was obtained from the Japanese Collection of Research Bioresources. Chinese hamster ovary (CHO)-K1 was obtained from the American Type Culture Collection. CHO/CD44s was previously established by transfecting pCAG-Ble/PA16-CD44s into CHO-K1 cells (22). KYSE770 was cultured in Dulbecco's Modified Eagle's Medium (DMEM; Nacalai Tesque, Inc.) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin, and 100 U/ml penicillin (Nacalai Tesque, Inc.). CHO-K1 was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc.) supplemented with 10% (v/v) FBS, 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin and 100 U/ml penicillin (RPMI-1640 complete medium). CHO/CD44s were cultured in RPMI-1640 complete medium containing 0.5 mg/ml Zeocin (InvivoGen). All cells were cultured in a humidified incubator at 37°C with 5% CO₂.

Antibodies

Anti-pan-CD44 mAbs, C₄₄Mab-5 and C₄₄Mab-46 were previously established (21,22). To generate recombinant mAbs, V_H cDNAs of C₄₄Mab-5 or C₄₄Mab-46 and C_H of mouse IgG_2a were cloned into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation). V_L cDNA of C₄₄Mab-5 or C₄₄Mab-46 and C_L cDNA of mouse kappa light chain were also subcloned into the pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation). The vectors were transduced into BINDS-09 (FUT8-knockout ExpiCHO-S) cells (33). 5-mG_2a and C₄₄Mab-46-mG_2a were purified using Ab-Capcher (ProteNova Co., Ltd.). 281-mG_2a (an anti-hamster podoplanin mAb, control mouse IgG_2a) was previously described (34).

Flow cytometry

The cells, obtained using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.), were treated with 5-mG_2a and C₄₄Mab-46-mG_2a, or control blocking buffer [phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (Nacalai Tesque, Inc.)] for 30 min at 4°C. Subsequently, the cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:2,000; Cell Signaling Technology, Inc.) for 30 min at 4°C. Fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp.) and analyzed using SA3800 software ver. 2.05 (Sony Corp.).

Antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay

An ADCC Reporter Bioassay kit (Promega Corporation) was used for the ADCC reporter bioassay. The human FcγRIIIa receptor and a nuclear factor of activated T-cells (NFAT) response element driving firefly luciferase-expressed Jurkat cells were used as effector cells. The target CHO/CD44s and KYSE770 cells (12,500 cells per well) were seeded into a 96-well white solid plate. The serially diluted 5-mG_2a and C₄₄Mab-46-mG_2a were added to the target cells. The effector cells (75,000 cells in 25 µl) were then added and co-cultured with antibody-treated target cells at 37°C for 6 h. Luminescence was measured with a GloMax luminometer (Promega Corporation).

Complement-dependent cytotoxicity (CDC)

The target CHO/CD44s and KYSE770 cells were labeled using 10 µg/ml Calcein AM (Thermo Fisher Scientific, Inc.). The target cells (1×10⁴ cells/well) were mixed with 100 µg/ml of control 281-mG_2a, 5-mG_2a, or C₄₄Mab-46-mG_2a and rabbit complement (final dilution 1:10, Low-Tox-M Rabbit Complement; Cedarlane Laboratories). The calcein release to the medium was measured after a 4-h incubation. The maximum fluorescence of the medium was also measured after lysing all cells with a buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM EDTA. Cytotoxicity (% of lysis) was calculated as % lysis=(E-S)/(M-S) ×100. E is the fluorescence of combined target and effector cells. S is the spontaneous fluorescence of target cells only. M is the maximum fluorescence measured. Statistical analyses were performed using GraphPad PRISM 6 software (GraphPad Software, Inc.; Dotmatics).

Antitumor activity of 5-mG2a and C44Mab-46-mG2a in xenografts of CHO/CD44s and KYSE770

All animal experiments were performed following regulations and guidelines to minimize animal distress and suffering in the laboratory by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (Numazu, Japan). The animal study protocol was approved (approval nos. 2023-037 and 2023-054) by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (Numazu, Japan). BALB/c nude mice (5-week-old female, a total of 72 mice) were purchased from Jackson Laboratories, Inc. and maintained on an 11-h light/13-h dark cycle at a temperature of 23±2°C and 55±5% humidity in a specific pathogen-free environment, across the experimental period. Food and water were supplied ad libitum. The weight of the mice was monitored twice per week and their health was monitored three times per week. CHO/CD44s and KYSE770 cells (5×10⁶ cells) suspended with BD Matrigel Matrix Growth Factor Reduced (BD Biosciences) were inoculated into the left flank of the mice subcutaneously. On day 7 after the inoculation, 100 µg of 5-mG_2a (n=8), C₄₄Mab-46-mG_2a (n=8), or control 281-mG_2a (n=8) in 100 µl PBS were injected intraperitoneally. Additional antibody injections were performed on days 14 and 21. The tumor volume was measured on days 7, 9, 14, 17, 21 and 23. In the KYSE770 ×enograft experiment (500 µg dosage), 500 µg of 5-mG_2a (n=8), C₄₄Mab-46-mG_2a (n=8), or control 281-mG_2a (n=8) in 100 µl PBS were injected intraperitoneally on days 8 and 13 after the inoculation. The tumor volume was measured on days 8, 12, and 19. The tumor volume was calculated using the formula: Volume=W² × L/2, where W is the short diameter and L is the long diameter. The loss of original body weight was determined to a point >25% (35) and/or a maximum tumor size >3,000 mm³ and/or significant changes in the appearance of tumors as humane endpoints for euthanasia. Cervical dislocation was used for euthanasia. Mice death was confirmed by respiratory arrest and rigor mortis. The xenograft tumors were carefully removed from the sacrificed mice and weighed immediately.

Immunohistochemical analysis

The paraffin-embedded xenograft tumors were autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), sections (4 µm) were incubated with 1 µg/ml of C₄₄Mab-46 and mPMab-1 [a mouse-rat chimeric antibody against mouse podoplanin (36)] for 1 h at room temperature and then treated with the EnVision+ Kit for mouse (Agilent Technologies, Inc.) for 30 min. The color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.). Counterstaining was performed with hematoxylin (Merck KGaA). Hematoxylin and eosin (H&E) staining was performed using hematoxylin and eosin (FUJIFILM Wako Pure Chemical Corporation). Leica DMD108 fluorescence microscope (Leica Microsystems GmbH) was used to examine the sections and obtain images.

Statistical analyses

All data are expressed as the mean ± standard error of the mean (SEM). Two-way ANOVA with Tukey's multiple comparisons test and two-way ANOVA with Sidak's multiple comparisons tests were conducted in CDC and tumor weight measurement, respectively. Two-way ANOVA with Sidak's multiple comparisons test was utilized for tumor volume and mice weight. GraphPad Prism 6 (GraphPad Software, Inc.; Dotmatics) was used for all calculations. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Flow cytometric analysis against CHO/CD44s and KYSE770 cells using 5-mG2a and C44Mab-46-mG2a

In our previous study, anti-pan-CD44 mAbs (C₄₄Mab-5 and C₄₄Mab-46) were established and were shown to be available for flow cytometry and immunohistochemistry (21,22). In the present study, a mouse IgG_2a type of C₄₄Mab-5 and C₄₄Mab-46 (5-mG_2a and C₄₄Mab-46-mG_2a) were produced by combining V_H and V_L of both mAbs with C_H and C_L of mouse IgG_2a, respectively (Fig. 1A). The reactivity of CD44s-overexpressed CHO-K1 cells (CHO/CD44s) and an endogenous CD44-expressing esophageal tumor cell line (KYSE770) was first confirmed. 5-mG_2a and C₄₄Mab-46-mG_2a detected CHO/CD44s in a concentration-dependent manner (Fig. 1B), but did not detect parental CHO-K1 cells (negative control, Fig. S1). 5-mG_2a and C₄₄Mab-46-mG_2a also detected KYSE770 cells in a concentration-dependent manner (Fig. 1C). KYSE770 cells were also recognized by anti-CD44v mAbs including C₄₄Mab-6 (an anti-CD44v3 mAb), C₄₄Mab-3 (an anti-CD44v5 mAb), C₄₄Mab-9 (an anti-CD44v6 mAb), and C₄₄Mab-18 (an anti-CD44v10 mAb) (Fig. S2). These results indicated that 5-mG_2a and C₄₄Mab-46-mG_2a recognize exogenous and endogenous CD44.

5-mG2a and C44Mab-46-mG2a-mediated ADCC pathway activation in the presence of CHO/CD44s and KYSE770 cells

To compare the ADCC pathway activation by 5-mG_2a and C₄₄Mab-46-mG_2a, the ADCC reporter bioassay was performed to measure the biological activity of the FcγRIIIa-mediated pathway activation by mAbs (37). CHO/CD44s cells were treated with diluted mAbs and then incubated with effector cells, which possess the human FcγRIIIa receptor and Firefly luciferase regulated by an NFAT response element. As demonstrated in Fig. 2A, 5-mG_2a and C₄₄Mab-46-mG_2a activated the effector in a concentration-dependent manner (EC₅₀; 6.4×10⁻⁸ g/ml and 5.4×10⁻⁸ g/ml, respectively). In the presence of KYSE770 cells, 5-mG_2a activated the effector (EC₅₀; 2.5×10⁻⁷ g/ml, Fig. 2B). However, C₄₄Mab-46-mG_2a did not (Fig. 2B).

The CDC by 5-mG2a and C44Mab-46-mG2a against CHO/CD44s and KYSE770 cells

The CDC mediated by 5-mG_2a and C₄₄Mab-46-mG_2a against CHO/CD44s and KYSE770 cells was next examined. As shown in Fig. 3, both 5-mG_2a and C₄₄Mab-46-mG_2a significantly exerted CDC against CHO/CD44s cells (67 and 88% cytotoxicity, respectively) and KYSE770 (27 and 31% cytotoxicity, respectively) compared with those induced by control mouse IgG_2a (281-mG_2a). These results demonstrated that 5-mG_2a and C₄₄Mab-46-mG_2a exhibited potent CDC activities against CHO/CD44s and KYSE770 cells.

Antitumor effects of 5-mG2a and C44Mab-46-mG2a against CHO/CD44s xenograft

In the CHO/CD44s xenograft tumors, 100 µg of 5-mG_2a, C₄₄Mab-46-mG_2a, or control mouse IgG_2a were injected into mice intraperitoneally on days 7, 14 and 21, following CHO/CD44s inoculation. On days 7, 9, 14, 17, 21 and 23, the tumor volume was measured. The 5-mG_2a and C₄₄Mab-46-mG_2a administration resulted in a significant reduction in tumor volume on days 17 (P<0.05), 21 (P<0.01) and 23 (P<0.01) compared with that of the control mouse IgG_2a (Fig. 4A). The 5-mG_2a and C₄₄Mab-46-mG_2a administration resulted in 46 and 48% reductions in tumor volume compared with that of the control mouse IgG_2a on day 23, respectively. The histological analyses of tumors are demonstrated in Fig. S3. In the necrotic area of tumors, leukocytes and erythrocytes were infiltrated. Membranous staining of CD44 in tumor and podoplanin-positive macrophage were also detected. However, a significant difference among control, 5-mG_2a and C₄₄Mab-46-mG_2a-treated tumors could not be observed.

The weight of CHO/CD44s tumors treated with 5-mG_2a and C₄₄Mab-46-mG_2a was lower than that of tumors treated with the control mouse IgG_2a (53 and 55% reduction, respectively; P=0.11 and P=0.09; Fig. 4B). Loss of body weight was not observed in the CHO/CD44s tumor-implanted mice during the treatments (Fig. 4C).

Antitumor effects of 5-mG2a and C44Mab-46-mG2a on KYSE770 ×enografts

In the KYSE770 ×enograft models, 100 µg of 5-mG_2a and C₄₄Mab-46-mG_2a and control mouse IgG_2a were injected into mice on days 7, 14 and 21, following KYSE770 inoculation. Although the tendency of the reduction of tumor volume by the treatment with 5-mG_2a and C₄₄Mab-46-mG_2a was observed, significant differences were not obtained (Fig. S4). The dosage (500 µg) was next increased and the antitumor effects were investigated. As revealed in Fig. 5, 500 µg of 5-mG_2a, C₄₄Mab-46-mG_2a and control mouse IgG_2a were injected into mice on days 8 and 13, following KYSE770 inoculation. On days 8, 12 and 19, the tumor volume was measured. The 5-mG_2a and C₄₄Mab-46-mG_2a administration resulted in a significant reduction in tumor volume on day 19 (P<0.01) compared with that of the control mouse IgG_2a (Fig. 5A). The 5-mG_2a and C₄₄Mab-46-mG_2a administration resulted in 51 and 37% reduction of tumor volume compared with that of the control mouse IgG_2a on day 19, respectively. The histological analyses of tumors are demonstrated in Fig. S5. Membranous staining of CD44 in tumors and podoplanin-positive macrophages at the tumor periphery were observed. Infiltrated podoplanin-positive macrophages into tumors were not observed compared with KYSE770 ×enograft.

The weight of KYSE770 tumors treated with 5-mG_2a and C₄₄Mab-46-mG_2a was significantly lower than that of tumors treated with the control mouse IgG_2a [47% (P<0.05) and 40% (P<0.05) reduction, respectively; Fig. 5B]. Loss of body weight was not observed in the KYSE770 tumor-implanted mice during the treatments (Fig. 5C).

Discussion

In the present study, mouse IgG_2a type anti-pan-CD44 mAbs (5-mG_2a and C₄₄Mab-46-mG_2a) was produced and antitumor activity against CHO/CD44s and KYSE770 ×enograft tumors was evaluated. Although 5-mG_2a possesses superior affinity to CD44-expressing cells compared with C₄₄Mab-46-mG_2a (22,38), both 5-mG_2a and C₄₄Mab-46-mG_2a showed comparable ability of effector activation (Fig. 2A), CDC (Fig. 3A) and antitumor activity against CHO/CD44s (Fig. 4). Against KYSE770 cells, 5-mG_2a exhibited superior reactivity in flow cytometry (Fig. 1C) and effector activation (Fig. 2B) compared with C₄₄Mab-46-mG_2a. However, both mAbs showed similar antitumor activity to KYSE770 ×enograft in high dosage (Fig. 5).

After the phase I trial of RG7356 in solid tumors, the developer Roche group reported that the CD44s expression is associated with HA production and predicts response to treatment with RG7356 in both xenograft tumor models and clinical response (39). The group also identified that overexpression of CD44s stimulates the HA production (39). These results suggest the close interplay between CD44s and HA and a potential biomarker to enrich patient responses to anti-CD44 mAb therapy in the clinic. In the present study, CD44s-overexpressed CHO-K1 cells and KYSE770 cells were used, which mainly express CD44v (Fig. S2) (22). Due to the lower levels of total CD44 (Fig. 1) and the CD44v expression in KYSE770 cells, a high dosage (500 µg) of mAbs was needed to suppress the KYSE770 ×enograft.

As revealed in Fig. 2B, C₄₄Mab-46-mG_2a did not activate the effector cells in the presence of KYSE770 but exerted the antitumor effect in vivo (Fig. 5). The CDC is a crucial antitumor mechanism by mAbs (40,41). Some tendency was observed that C₄₄Mab-46-mG_2a showed a more significant CDC effect compared with 5-mG_2a (Fig. 3). The difference could be sufficient to exert similar antitumor effects in vivo by 5-mG_2a and C₄₄Mab-46-mG_2a. There was less infiltrated podoplanin-positive macrophage into KYSE770 ×enograft compared with CHO/CD44 (Figs. S3 and S5). Since the infiltrated effector cells exert ADCC activity (42), this may be one of the reasons why ADCC did not contribute markedly in KYSE770 cells. One more possibility is their epitope. 5-mG_2a recognizes the N-terminal part of CD44 [amino acids 25–36, (25)], and C₄₄Mab-46-mG_2a recognizes the central part of CD44 [amino acids 174–178, (23,24)]. Furthermore, IgG antibodies can form ordered hexamers upon binding to their antigen on cell surfaces. These hexamers efficiently bind the hexavalent complement component C1q, the first step in the classical pathway of complement activation (43,44). The structure of C₄₄Mab-46-mG_2a-CD44 complex may provide the adequate access of complements to exert CDC.

Complement has been considered as an adjunctive component that potentiates the antibody-mediated cytolytic effects. However, complement is currently considered an essential effector of tumor cytotoxic responses of antibody-based immunotherapy, which is guiding new therapeutic options (41). An increasing body of evidence suggests that complement plays critical roles in not only mAb-mediated tumor cytolysis but also several immunomodulatory functions in tumor immunosurveillance and antitumor immunity (45,46). The complicated crosstalk of complement effectors with cellular pathways that drive B cell and T cell responses influences T helper/effector T cell survival, differentiation and B cell activation. Therefore, the involvement of complement in our experimental system and/or immunocompetent mouse models should be investigated in future studies.

Most therapeutic mAbs exhibit adverse effects by recognizing antigens in normal cells (47). Clinical trials of an anti-CD44v6 mAb (BIWA-4) bivatuzumab-mertansine drug conjugate to solid tumors failed because of the skin toxicities (17,18). Therefore, cancer-selective or specific mAbs would reduce the adverse effects. Cancer-specific mAbs (CasMabs) against HER2 [H₂Mab-214 (48) and H₂Mab-250 (49)] have been developed by the authors and the reactivity to cancer and normal cells has been evaluated using flow cytometry. The antitumor effect in mouse xenograft models has been also reported using a mouse IgG_2a or human IgG₁ types recombinant mAbs (50). Some anti-CD44 mAbs which exhibit cancer specificity have been reported (51). Among them, the 4C8 mAb recognizes aberrantly O-glycosylated CD44v6 with Tn (GalNAca1-O-Ser/Thr) antigen. The 4C8 chimeric antigen receptor (CAR)-T cells demonstrated target-specific cytotoxicity in vitro, significant tumor regression, and prolonged survival in vivo (52). In our CasMab development against CD44, already established anti-CD44 mAbs were screened by comparing the reactivity against cancer and normal cells. The CasMabs against CD44 could be applicable for designing modalities such as antibody-drug conjugates and CAR-T cells.

Supplementary Material

Supporting Data

Acknowledgements

The authors thank Mr Shun-ichi Ohba and Ms Akiko Harakawa [Institute of Microbial Chemistry (BIKAKEN), Numazu, the Microbial Chemistry Research Foundation] for technical assistance with animal experiments.

Availability of data and materials

The data generated in the present study are included in the figures and/or tables of this article.

Authors' contributions

KI, HS, TO, TN, MY, GL and TT performed the experiments. MK, MKK and YKato designed the experiments. KI, HS, AO, YKatori and YKato engaged the analysis and interpretation of data. KI, HS, and YKato wrote the manuscript. HS and YKato confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Animal experiments were approved (approval nos. 2023-037 and 2023-054) by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (Numazu, Japan).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Abbreviations

CD44s

CD44 standard

CD44v

CD44 variant

hyaluronic acid

mAb

monoclonal antibody

ADCC

antibody-dependent cellular cytotoxicity

CDC

complement-dependent cytotoxicity

FcγR

Fcγ receptor

CAR

chimeric antigen receptor

FUT8

fucosyl-transferase 8

CHO

Chinese hamster ovary

CBIS

Cell-Based Immunization and Screening

ESCC

esophageal squamous cell carcinoma

PBS

phosphate-buffered saline

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Figure 1.

Flow cytometry using 5-mG_2a and C₄₄Mab-46-mG_2a. (A) Class-switched and defucosylated mouse IgG_2a mAbs, 5-mG_2a and C₄₄Mab-46-mG_2a, were generated from C₄₄Mab-5 and C₄₄Mab-46 (mouse IgG₁), respectively. (B) CHO/CD44s and (C) KYSE770 cells were treated with 10–0.01 µg/ml of 5-mG_2a and C₄₄Mab-46-mG_2a (red) or buffer control (black), followed by Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were analyzed using the SA3800 Cell Analyzer. mAb, monoclonal antibody.

Figure 2.

Evaluation of ADCC activity. The ADCC reporter assay by 5-mG_2a and C₄₄Mab-46-mG_2a in the presence of (A) CHO/CD44s and (B) KYSE770 cells. Values are presented as the mean ± SD. N.D., not determined; ADCC, antibody-dependent cellular cytotoxicity; mAb, monoclonal antibody.

Figure 3.

Evaluation of CDC activity by 5-mG_2a and C₄₄Mab-46-mG_2a against CHO/CD44s and KYSE770 cells. The CDC induced by 5-mG_2a, C₄₄Mab-46-mG_2a, or control mouse IgG_2a (281-mG_2a) against (A) CHO/CD44s and (B) KYSE770 cells. Values are shown as the mean ± SEM. *P<0.05 and **P<0.01; one-way ANOVA with Tukey's multiple comparisons test. CDC, complement-dependent cytotoxicity; mAb, monoclonal antibody.

Figure 4.

Antitumor activity of 5-mG_2a and C₄₄Mab-46-mG_2a against CHO/CD44s xenograft tumor. (A) Measurement of tumor volume in CHO/CD44s xenograft. CHO/CD44s cells (5×10⁶ cells) were injected into mice subcutaneously. On day 7, 100 µg of 5-mG_2a and C₄₄Mab-46-mG_2a or control mouse IgG_2a (281-mG_2a) were injected into mice intraperitoneally. On days 14 and 21, additional antibodies were injected. On days 7, 9, 14, 17, 21 and 23 following the inoculation, the tumor volume was measured. Values are presented as the mean ± SEM. *P<0.05 and **P<0.01; two-way ANOVA with Sidak's multiple comparisons test. (B) The weight (left) and appearance (right) of excised CHO/CD44s xenografts on day 23. Values are presented as the mean ± SEM; one-way ANOVA with Tukey's multiple comparisons test. (C) The body weight (left) and appearance (right) of control mouse IgG2a, 5-mG2a, and C₄₄Mab-46-mG2a-treated mice. Scale bar, 1 cm. mAb, monoclonal antibody.

Figure 5.

Antitumor activity of 5-mG_2a and C₄₄Mab-46-mG_2a against KYSE770 ×enograft tumor. (A) Measurement of tumor volume in KYSE770 ×enograft. KYSE770 cells (5×10⁶ cells) were injected into mice subcutaneously. On day 8, 500 µg of 5-mG_2a and C₄₄Mab-46-mG_2a or control mouse IgG_2a (281-mG_2a) were injected into mice intraperitoneally. On day 13, additional antibodies were injected. On days 8, 12, and 19 following the inoculation, the tumor volume was measured. Values are presented as the mean ± SEM. **P<0.01; two-way ANOVA with Sidak's multiple comparisons test. (B) The weight (left) and appearance (right) of the excised KYSE770 ×enografts on day 19. Values are presented as the mean ± SEM. *P<0.05; one-way ANOVA with Tukey's multiple comparisons test. (C) The body weight (left) and appearance (right) of control mouse IgG_2a, 5-mG_2a and C₄₄Mab-46-mG_2a-treated mice. Scale bar, 1 cm. mAb, monoclonal antibody.