Anti-CD44 Variant 10 Monoclonal Antibody Exerts Antitumor Activity in Mouse Xenograft Models of Oral Squamous Cell Carcinomas

CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3–10-overexpressed CHO-K1 (CHO/CD44v3–10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3–10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3–10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3–10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.

Monoclonal antibodies (mAbs) against CD44 have been evaluated in clinical trials [31,32].RG7356, a humanized anti-pan-CD44 mAb, exhibited the antitumor effect for B cell leukemia but no cytotoxicity on normal B cells [33].In a human chronic lymphocytic leukemia-engrafted mouse model, RG7356 administration resulted in complete clearance of engrafted leukemia cells [33].In acute myeloid leukemia [34] and advanced CD44-positive solid tumors [35], phase I clinical trials were conducted.Although RG7356 exhibited an acceptable safety profile, the studies were terminated due to the lack of dose-response relationship with RG7356 in both clinical and pharmacodynamic aspects [35].
Since CD44v expression is restricted in epithelial tissue and carcinomas, anti-CD44v mAbs were developed and evaluated in clinical studies.Humanized anti-CD44v6 mAbs (BIWA-4 and BIWA-8) labeled with 186 Re showed antitumor efficacy in head and neck squamous cell carcinoma (SCC) xenograft-bearing mice [36].Moreover, the antibody-drug conjugate (ADC) of BIWA-4, bivatuzumab-mertansine was developed and evaluated in clinical trials [32].However, the clinical trials were terminated due to severe toxicity in the skin, probably due to the efficient accumulation of mertansine in the skin [32,37].Therefore, anti-CD44 mAbs with more potent efficacy and lower toxicity to normal cells are desired.
The Fc region of therapeutic mAb binds to FcγRs on dendritic cells, macrophages, and neutrophils, which influences the adaptive immune responses through antigen presentation and cytokine production [38].Moreover, the FcγR binding results in the activation of natural killer (NK) cells [39] and macrophages [40], which mediates antibody-dependent cellular cytotoxicity (ADCC).The complement-dependent cellular cytotoxicity (CDC) is also considered as an essential effector function in tumor immunotherapy [41].The Fc region of therapeutic mAbs binds to complement C1q, facilitating the assembly of active C1 complex (C1q, C1r, and C1s).The reaction of the complement cascade finally promotes the assembly of the pore-forming membrane attack complex (MAC or C5b-C9) on the tumor cell membrane, which results in the terminal cell lysis [41].The involvement of CDC in the antitumor effect was first recognized in the treatment of B cell lymphomas by an anti-CD20 mAb, rituximab [42,43].Furthermore, the cytolytic capacity by complement has been shown in anti-CD38 and CD52 immunotherapies for multiple myeloma and chronic lymphocytic leukemia, respectively [43][44][45].Moreover, a growing body of evidence suggests that complement plays critical roles in not only tumor cell lysis but also in several immunologic functions in antitumor immunity [46,47].In the immunotherapy against solid tumors, an anti-HER2 bispecific and biparatopic antibody zanidatamab exerted more potent CDC against HER2-positive breast cancers compared with clinically approved anti-HER2 mAb trastuzumab [48].

Induction
In contrast, C 44 Mab-46-mG 2a significantly exerted CDC against CHO/CD44v3-10 cells in the presence of complements (27% cytotoxicity, p < 0.05) compared with the control (12% cytotoxicity).We did not observe a significant difference in CDC between C 44 Mab-18-mG 2a and the control (Figure 3B) These results indicated that C 44 Mab-18-mG 2a and C 44 Mab-46-mG 2a possess different properties for exerting ADCC and CDC against CHO/CD44v3-10 cells.
OSCC arises from the oral cavity and is a type of head and neck squamous cell carcinoma (HNSCC).HNSCC has been revealed to be associated with alcohol, smoking, and human papillomavirus types 16 and 18 infection [62].In the Pan-Cancer Atlas, HNSCC has been shown as the second-highest CD44-expressing tumor type [63].The overexpression of CD44 is associated with resistance to therapy and unfavorable outcomes [64][65][66].Furthermore, CD44-high cancer stem cells (CSCs) from HNSCC exhibited increased migration, invasiveness, and stemness [67].In immunodeficient mice, the CD44-high CSCs could form more lung metastatic foci than CD44-low cells [68].Therefore, CD44 is considered an important target for mAb therapies.Since C 44 Mab-18 is available to immunohistochemistry [58], C 44 Mab-18 could be used for diagnosis and therapy of OSCC.
In CHO/CD44v3-10 cells, C 44 Mab-18-mG 2a and C 44 Mab-46-mG 2a recognized a common target but mainly exerted ADCC and CDC activity, respectively (Figure 3).The epitope of C 44 Mab-46 was previously determined as the 174-TDDDV -178 sequence in the constant exon 5-encoded region [51], which is relatively apart from transmembrane domain compared to variant exon 10-encoded region recognized by C 44 .To activate the classical pathway of complement, an ordered hexamer formation of IgG mAb is required to bind to the hexavalent complement C1q [69,70].The structure of the C 44 Mab-46-mG 2a -CD44v3-10 complex may provide the appropriate space to form the hexameric structure of the mAb-C1q complex and recruit the pore-forming membrane attack complex to exert CDC.In contrast, C 44 Mab-18-mG 2a showed a higher reactivity to CHO/CD44v3-10 compared with C 44 Mab-46-mG 2a in flow cytometry (Figure 1).The difference in the reactivity and the epitope would influence the ADCC activity.Further studies are required to reveal the relationship among the affinity of mAb, epitope, and ADCC activity.
The limitation of this study is that both C 44 Mab-46-mG 2a and C 44 Mab-18-mG 2a are effective in human xenograft models of nude mice.To apply human tumor therapy, a class switch to human IgG 1 is essential.We previously generated humanized IgG 1 mAbs and evaluated the antitumor activity with injections of human NK cells [71,72].We will produce humanized mAbs from C 44 Mab-46 and C 44 Mab-18 and evaluate the ADCC and CDC in the presence of human NK cells and complement, respectively.Furthermore, the antitumor effects should be investigated with injections of human NK cells.
Near-infrared photoimmunotherapy (NIR-PIT) uses a targeted mAb conjugated with a photoactivatable dye such as IRDye700DX (IR700) [73][74][75][76].When the mAb binds to the antigen-expressed target cells, IR700 induces plasma membrane rupture and immunogenic cell death by NIR-light exposure.Preclinical studies of anti-pan-CD44 mAb-based NIR-PIT (IM7-IR700) were conducted.In the syngeneic mouse model of OSCC, IM7-IR700 administration and the NIR light exposure to OSCC tumors resulted in a significant reduction but failed to induce durable antitumor responses [77].Because IM7 is a pan-CD44 mAb, IM7 might target not only tumor cells but also CD44s-positive immune cells which are involved in the antitumor immunity.The expression of CD44v is low in hematopoietic cells compared with CD44s [2].We previously showed that C 44 Mab-18 can distinguish tumor cells from stromal tissues in immunohistochemistry.In contrast, C 44 Mab-46 stained both tumor and stromal tissue including fibroblasts and leukocytes [58].Therefore, anti-CD44v10 mAbs such as C 44 Mab-18 might be a promising mAb for NIR-PIT without affecting the host immune cells in the tumor microenvironment.
Since both CD44s and CD44v are expressed in normal cells, there is a concern about adverse effects due to the recognition of normal cells by mAbs.In fact, clinical trials of the anti-CD44v6 mAb-ADC to advanced solid tumors were discontinued because of the skin toxicities [32,37].Therefore, cancer-specific antibodies are desired to reduce the adverse effects.A cancer-specific anti-CD44v6 mAb (clone 4C8) recognizes aberrantly O-glycosylated Tn (GalNAcα1-O-Ser/Thr) antigen in the variant exon 6-encoded region.The 4C8 mAb was further developed for chimeric antigen receptor (CAR)-T cells, which exhibited target-specific in vitro cytotoxicity and significant tumor regression in vivo [78].We have developed cancer-specific mAbs (CasMabs) against various tumor antigens, including HER2 (clones H 2 Mab-214 [79] and H 2 Mab-250 [80]), and reported the antitumor effect in mouse xenograft models using recombinant mouse IgG 2a or human IgG 1 mAbs [71,72].These anti-HER2 mAbs were screened by the reactivity to cancer and normal cells in flow cytometry.H 2 Mab-214 was revealed to recognize a locally misfolded structure in the Cys-rich HER2 extracellular domain 4, which usually forms a β-sheet [79].H 2 Mab-250 also shows a specific reactivity against HER2-positive tumor cells, which has been developed as CAR-T-cell therapy.The phase I study has been conducted in the US (NCT06241456).We have developed CasMabs against CD44s or CD44v by comparing the reactivity against tumor and normal cells.The anti-CD44 CasMabs could contribute to developing novel modalities such as ADCs and CAR-T cells.
The tumor volume was calculated using the following formula: Volume = W 2 × L/2, where W is the short diameter and L is the long diameter.All data are expressed as the mean ± standard error of the mean (SEM).In tumor weight measurement, one-way ANOVA with Tukey's multiple comparisons test was conducted.Two-way ANOVA with Tukey's multiple comparisons test was utilized for tumor volume and mice weight.GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) was used for all calculations.p < 0.05 was considered to indicate a statistically significant difference.

2. 5 .
Antitumor Effects of C 44 Mab-18-mG 2a and C 44 Mab-46-mG 2a in the Mouse Xenografts of HSC-2 and SAS In the HSC-2 and SAS xenograft models, C 44 Mab-18-mG 2a , C 44 Mab-46-mG 2a , or control mouse IgG 2a (500 µg/mouse) were intraperitoneally administrated into mice on days 7 and 14, following the inoculation.The tumor volume was measured on days 7, 14, and 17.The C 44 Mab-18-mG 2a and C 44 Mab-46-mG 2a administration resulted in a significant reduction in both tumor volume on day 14 (p < 0.01) and day 17 (p < 0.01) compared with that of the control mouse IgG 2a (Figure 6A,B).The C 44 Mab-18-mG 2a and C 44 Mab-46-mG 2a administration resulted in 43% and 36% reduction in HSC-2 tumor volume, respectively, compared with that treated with the control mouse IgG 2a on day 17.The C 44 Mab-18-mG 2a and C 44 Mab-46-mG 2a administration also resulted in a 33% and 32% reduction in SAS tumor volume, respectively, compared with that treated with the control mouse IgG 2a on day 17.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 9 of 17 46-mG2a administration also resulted in a 33% and 32% reduction in SAS tumor volume, respectively, compared with that treated with the control mouse IgG2a on day 17.The weight of HSC-2 tumors treated with C44Mab-18-mG2a and C44Mab-46-mG2a was significantly lower than that treated with the control mouse IgG2a [Figure 6C, 40% (p < 0.01) and 30% (p < 0.05) reduction, respectively].The weight of SAS tumors treated with C44Mab-18-mG2a and C44Mab-46-mG2a was significantly lower than that treated with the control mouse IgG2a [Figure 6D, 23% (p < 0.01) and 19% (p < 0.05) reduction, respectively].The loss of body weight was not observed in the HSC-2 and SAS tumor-implanted mice during the treatments (Figure 6E,F).