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

Yuma Kudo; Hiroyuki Suzuki; Tomohiro Tanaka; Mika K. Kaneko; Yukinari Kato

doi:10.3390/antib12020031

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

Kudo, Yuma;Suzuki, Hiroyuki;Tanaka, Tomohiro;Kaneko, Mika K.;Kato, Yukinari 2023-04-28 00:00:00 antibodies Article Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody C Mab-3 for Multiple Applications against Pancreatic Carcinomas 1 1 , 2 , 1 , 2 1 , 2 1 , 2 , Yuma Kudo , Hiroyuki Suzuki * , Tomohiro Tanaka , Mika K. Kaneko and Yukinari Kato * Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan * Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.); Tel.: +81-29-853-3944 (H.S. & Y.K.) Abstract: 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 (C Mab-3; IgG , kappa) recognized peptides of the variant-5-encoded 44 1 region, indicating that C Mab-3 is a speciﬁc mAb for CD44v5. Moreover, C Mab-3 reacted with 44 44 CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by ﬂow cytometry. The 9 9 apparent K of C Mab-3 for CHO/CD44v3–10 and PK-1 was 1.3 10 M and 2.6 10 M, D 44 respectively. C Mab-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 C Mab-3 is useful for Citation: Kudo, Y.; Suzuki, H.; detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic Tanaka, T.; Kaneko, M.K.; Kato, Y. cancer diagnosis and therapy. Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody Keywords: CD44; CD44 variant 5; monoclonal antibody; ﬂow cytometry; immunohistochemistry C Mab-3 for Multiple Applications against Pancreatic Carcinomas. Antibodies 2023, 12, 31. https:// doi.org/10.3390/antib12020031 1. Introduction Academic Editor: Christian Kellner Pancreatic cancer has become the third leading cause of death in men and women Received: 30 January 2023 combined in the United States in 2023 [1]. The development of pancreatic cancer has Revised: 24 March 2023 been explained by four common oncogenic events, including KRAS, CDKN2A, SMAD4, Accepted: 10 April 2023 and TP53 [2,3]. However, pancreatic cancer shows a heterogeneity in drug response and Published: 28 April 2023 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, Copyright: © 2023 by the authors. immunogenic, and aberrantly differentiated endocrine exocrine (ADEX), which correspond Licensee MDPI, Basel, Switzerland. to the histopathological characteristics [5]. Additionally, various marker proteins have This article is an open access article been investigated for the early diagnostic and drug responses of pancreatic cancers [6]. distributed under the terms and Studies have suggested that CD44 plays important roles in malignant progression of tumors conditions of the Creative Commons through its cancer stemness and metastasis-promoting properties [7,8]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ CD44 is a type I transmembrane glycoprotein that is expressed as a wide variety of 4.0/). isoforms in various types of cells. [9]. The variety of isoforms is produced by the alternative Antibodies 2023, 12, 31. https://doi.org/10.3390/antib12020031 https://www.mdpi.com/journal/antibodies Antibodies 2023, 12, 31 2 of 16 splicing of CD44 mRNA. The CD44 standard isoform (CD44s) is the smallest isoform of CD44 (85–95 kDa); it is presented on the membrane of most vertebrate cells. CD44s mRNA is assembled by the ﬁrst ﬁve and the last ﬁve constant region exons [10]. The CD44 variant isoforms (CD44v) are produced by the alternative splicing of middle variant exons (v1–v10) and the standard exons of CD44s [11]. CD44v is heavily glycosylated, leading to various molecular weights (~250 kDa) owing to N-glycosylation and O-glycosylation [12]. Both CD44s and CD44v (pan-CD44) are known as hyaluronic acid (HA) receptors that mediate cellular homing, migration, adhesion, and proliferation [13]. CD44v is overexpressed in carcinomas and induce metastatic properties [14,15]. A growing body of evidence suggests that CD44v plays critical roles in the promotion of tumor invasion, metastasis, cancer-initiating properties [16], and resistance to chemo- and radiotherapy [7,17]. Reports indicated the important functions of each variant’s exon- encoded region. The v3-encoded region functions as a co-receptor for receptor tyrosine kinases [18]. Since the v3-encoded region possesses heparan sulfate moieties, it can recruit to heparin-binding epidermal growth factor-like growth factor (HB-EGF) and ﬁbroblast growth factors (FGFs). Furthermore, the v6-encoded region forms a ternary complex with HGF and its receptor c-MET, which is essential for its activation [19]. Additionally, oxidative stress resistance is mediated by the v8–10-encoded region through binding with a cystine– glutamate transporter (xCT) subunit [20]. Therefore, establishment and characterization of mAbs that recognize each CD44v is thought to be essential for understanding each variant’s function and development of CD44-targeting tumor diagnosis and therapy. However, the function and distribution of the variant-5-encoded region in tumors has not been fully understood. Our group established the novel anti-pan-CD44 mAbs, C Mab-5 (IgG , kappa) [21] 44 1 and C Mab-46 (IgG , kappa) [22] using the Cell-Based Immunization and Screening 44 1 (CBIS) method and immunization with the CD44v3–10 ectodomain, respectively. Both C Mab-5 and C Mab-46 have epitopes within the standard exon (1 to 5)-encoding 44 44 sequences [23–25]. Furthermore, we showed that both C Mab-5 and C Mab-46 are 44 44 applicable to ﬂow cytometry and immunohistochemistry in oral [21] and esophageal squamous cell carcinomas (SCC) [22]. We have also investigated the antitumor effects of core-fucose-deﬁcient C Mab-5 in mouse xenograft models of oral SCC [26]. In this study, we developed a novel anti-CD44v5 mAb, C Mab-3 (IgG , kappa), by the CBIS 44 1 method and evaluated its applications, including ﬂow cytometry, Western blotting, and immunohistochemical analyses. 2. Materials and Methods 2.1. Cell Lines 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 g/mL streptomycin, 0.25 g/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 . 2.2. Plasmid Construction and Establishment of Stable Transfectants CD44v3–10 open reading frame was obtained from the RIKEN BRC through the Na- tional Bio-Resource Project of the MEXT, Japan. CD44s cDNA was ampliﬁed using the HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany) and LN229 (a glioblas- toma cell line) cDNA as a template. CD44v3–10 and CD44 cDNAs were subcloned into pCAG-Ble-ssPA16 vectors with a signal sequence and N-terminal PA16 tag of 16 amino acids (GLEGGVAMPGAEDDVV) [21,27–30]; this can be detected by NZ-1, which was orig- Antibodies 2023, 12, 31 3 of 16 inally developed as an anti-human podoplanin mAb [31–46]. The pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3–10 vectors were transfected into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientiﬁc, Inc.), which offers an innovative electropo- ration method that utilizes a proprietary biologically compatible pipette tip chamber to generate a more uniform electric ﬁeld for a signiﬁcant increase in transfection efﬁciency and cell viability. By the limiting dilution method, CHO/CD44s and CHO/CD44v3–10 clones were ﬁnally established. 2.3. 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 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). 2.4. Enzyme-Linked Immunosorbent Assay (ELISA) Fifty-eight synthesized peptides, covering the CD44v3–10 extracellular domain [23], were synthesized by Sigma-Aldrich Corp. (St. Louis, MO, USA). The peptides (1 g/mL) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientiﬁc Inc.). Plate washing was performed with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.). After blocking with 1% (w/v) bovine serum albumin (BSA) in PBST, C Mab-3 (10 g/mL) was added to each well. Then, the wells were further incubated with peroxidase-conjugated anti-mouse immunoglobulins (1:2000 dilution; Agilent Technologies Inc., Santa Clara, CA, USA). One-Step Ultra TMB (Thermo Fisher Scientiﬁc Inc.) was used for enzymatic reactions. An iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA) was used to mesure the optical density at 655 nm. 2.5. Flow Cytometry CHO-K1, CHO/CD44v3–10, PK-1, and PK-8 were obtained using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were incubated with C Mab-3, C Mab-46, or blocking buffer (control) (0.1% BSA in PBS) for 44 44 30 min at 4 C. Then, the cells were treated with Alexa Fluor 488-conjugated secondary antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4 C. The data were analyzed using the SA3800 Cell Analyzer and SA3800 software ver. 2.05 (Sony Corp.). 2.6. Determination of Dissociation Constant (K ) via Flow Cytometry CHO/CD44v3–10 and PK-1 cells were treated with serially diluted C Mab-3 (0.01–10 g/mL). Then, the cells were incubated with Alexa Fluor 488-conjugated sec- ondary antibody. Fluorescence data were analyzed using BD FACSLyric and BD FACSuite software version 1.3 (BD Biosciences, Franklin Lakes, NJ, USA). The K 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). Antibodies 2023, 12, 31 4 of 16 2.7. Determination of K via Surface Plasmon Resonance (SPR) Measurement of K between C Mab-3 and the epitope peptide was performed using D 44 SPR. C Mab-3 was immobilized on the sensor chip CM5 according to the manufacturer ’s protocol by Cytiva (Marlborough, MA, USA). C Mab-3 (10 g/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 Mab-3. The K between 44 D C 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 using Biacore X100 evaluation software (Cytiva). 2.8. Western Blot Analysis The total cell lysates (10 g of protein) were separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The separated proteins were transferred onto polyvinylidene diﬂuoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The blocking was performed with 4% skim milk (Nacalai Tesque, Inc.) in PBST. The membranes were incubated with 10 g/mL of C Mab-3, 10 g/mL of C Mab-46, 44 44 0.5 g/mL of NZ-1, or 1 g/mL of an anti- -actin mAb (clone AC-15; Sigma-Aldrich Corp.) and then incubated with peroxidase-conjugated anti-mouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.) for C Mab-3, C Mab-46, and anti- -actin. Anti-rat 44 44 immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.) conjugated to peroxidase was used for NZ-1. The chemiluminescence signals were obtained with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) and detected using a Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan). 2.9. 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 ar- rays (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 nor- mal 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 carci- noma 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 Mab-3 (1 g/mL) and C Mab-46 44 44 (1 g/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. 3. Results 3.1. Development of an Anti-CD44v5 mAb, C Mab-3 In the CBIS method, we used a stable transfectant (CHO/CD44v3–10 cells) as an immunogen (Figure 1). Mice were immunized with CHO/CD44v3–10 cells, and hybrido- mas were seeded into 96-well plates. The supernatants, which are negative for CHO-K1 cells and positive for CHO/CD44v3–10 cells, were selected using ﬂow-cytometry-based high throughput screening. By limiting dilution, anti-CD44-mAb-producing clones were ﬁnally established. Among them, C Mab-3 (IgG , kappa) was shown to recognize both 44 1 CD44p311–330 (AYEGNWNPEAHPPLIHHEHH) and CD44p321–340 peptides (HPPLI- HHEHHEEEETPHSTS), which correspond to the variant-5-encoded sequence (Table 1 Antibodies 2023, 12, x FOR PEER REVIEW 5 of 17 In the CBIS method, we used a stable transfectant (CHO/CD44v3–10 cells) as an im- munogen (Figure 1). Mice were immunized with CHO/CD44v3–10 cells, and hybridomas were seeded into 96-well plates. The supernatants, which are negative for CHO-K1 cells and positive for CHO/CD44v3–10 cells, were selected using flow -cytometry-based high throughput screening. By limiting dilution, anti-CD44-mAb-producing clones were fi- nally established. Among them, C44Mab-3 (IgG1, kappa) was shown to recognize both Antibodies 2023, 12, 31 5 of 16 CD44p311–330 (AYEGNWNPEAHPPLIHHEHH) and CD44p321–340 peptides (HPPLIH- HEHHEEEETPHSTS), which correspond to the variant-5-encoded sequence (Table 1 and Supplementary Figure S1). In contrast, C44Mab-3 did not recognize other CD44v3–10 ex- and Supplementary Figure S1). In contrast, C Mab-3 did not recognize other CD44v3–10 tracellular regions. These results indicated that C44Mab-3 specifically recognizes the CD44 extracellular regions. These results indicated that C Mab-3 speciﬁcally recognizes the variant-5-encoded sequence. CD44 variant-5-encoded sequence. Figure 1. A schematic illustration of anti-human CD44 mAbs production. (A) Structure of CD44. Figure 1. A schematic illustration of anti-human CD44 mAbs production. (A) Structure of CD44. CD44s mRNA is assembled by the ﬁrst ﬁve (1 to 5) and the last ﬁve (16 to 20) exons and translates CD44s mRNA is assembled by the first five (1 to 5) and the last five (16 to 20) exons and translates CD44s. The mRNAs of CD44 variants are produced by the alternative splicing of middle variant CD44s. The mRNAs of CD44 variants are produced by the alternative splicing of middle variant exons exons and and translate translate multiple multiple CD44v CD44v such such as as CD CD44v3–10, 44v3–10, CD CD44v4–10, 44v4–10, CD CD44v6–10, 44v6–10, and and C CD44v8–10. D44v8–10. (B) CHO/CD44v3–10 cells were intraperitoneally injected into BALB/c mice. (C) The splenocytes and P3U1 cells were fused and the hybridomas were produced. (D) The screening was conducted by ﬂow cytometry using parental CHO-K1 and CHO/CD44v3–10 cells. (E) After cloning and additional screening, a clone (C Mab-3 (IgG , kappa)) was established. Furthermore, the binding epitope 44 1 was determined by enzyme-linked immunosorbent assay (ELISA) using peptides that cover the extracellular domain of CD44v3–10. Antibodies 2023, 12, 31 6 of 16 Table 1. Determination of the binding epitope of C Mab-3 by ELISA. Peptide Coding Exon * Sequence C Mab-3 CD44p21–40 2 QIDLNITCRFAGVFHVEKNG CD44p31–50 2 AGVFHVEKNGRYSISRTEAA CD44p41–60 2 RYSISRTEAADLCKAFNSTL CD44p51–70 2 DLCKAFNSTLPTMAQMEKAL CD44p61–80 2/3 PTMAQMEKALSIGFETCRYG CD44p71–90 2/3 SIGFETCRYGFIEGHVVIPR CD44p81–100 3 FIEGHVVIPRIHPNSICAAN CD44p91–110 3 IHPNSICAANNTGVYILTSN CD44p101–120 3 NTGVYILTSNTSQYDTYCFN CD44p111–130 3/4 TSQYDTYCFNASAPPEEDCT CD44p121–140 3/4 ASAPPEEDCTSVTDLPNAFD CD44p131–150 4/5 SVTDLPNAFDGPITITIVNR CD44p141–160 4/5 GPITITIVNRDGTRYVQKGE CD44p151–170 5 DGTRYVQKGEYRTNPEDIYP CD44p161–180 5 YRTNPEDIYPSNPTDDDVSS CD44p171–190 5 SNPTDDDVSSGSSSERSSTS CD44p181–200 5 GSSSERSSTSGGYIFYTFST CD44p191–210 5 GGYIFYTFSTVHPIPDEDSP CD44p201–220 5 VHPIPDEDSPWITDSTDRIP CD44p211–230 5/v3 WITDSTDRIPATSTSSNTIS CD44p221–240 5/v3 ATSTSSNTISAGWEPNEENE CD44p231–250 v3 AGWEPNEENEDERDRHLSFS CD44p241–260 v3 DERDRHLSFSGSGIDDDEDF CD44p251–270 v3/v4 GSGIDDDEDFISSTISTTPR CD44p261–280 v3/v4 ISSTISTTPRAFDHTKQNQD CD44p271–290 v4 AFDHTKQNQDWTQWNPSHSN CD44p281–300 v4 WTQWNPSHSNPEVLLQTTTR CD44p291–310 v4/v5 PEVLLQTTTRMTDVDRNGTT CD44p301–320 v4/v5 MTDVDRNGTTAYEGNWNPEA CD44p311–330 v5 AYEGNWNPEAHPPLIHHEHH + CD44p321–340 v5 HPPLIHHEHHEEEETPHSTS + CD44p331–350 v5/v6 EEEETPHSTSTIQATPSSTT CD44p341–360 v5/v6 TIQATPSSTTEETATQKEQW CD44p351–370 v6 EETATQKEQWFGNRWHEGYR CD44p361–380 v6 FGNRWHEGYRQTPREDSHST CD44p371–390 v6/v7 QTPREDSHSTTGTAAASAHT CD44p381–400 v6/v7 TGTAAASAHTSHPMQGRTTP CD44p391–410 v7 SHPMQGRTTPSPEDSSWTDF CD44p401–420 v7 SPEDSSWTDFFNPISHPMGR CD44p411–430 v7/v8 FNPISHPMGRGHQAGRRMDM CD44p421–440 v7/v8 GHQAGRRMDMDSSHSTTLQP CD44p431–450 v8 DSSHSTTLQPTANPNTGLVE CD44p441–460 v8 TANPNTGLVEDLDRTGPLSM CD44p451–470 v8/v9 DLDRTGPLSMTTQQSNSQSF CD44p461–480 v8/v9 TTQQSNSQSFSTSHEGLEED CD44p471–490 v9 STSHEGLEEDKDHPTTSTLT CD44p481–500 v9/v10 KDHPTTSTLTSSNRNDVTGG CD44p491–510 v9/v10 SSNRNDVTGGRRDPNHSEGS CD44p501–520 v10 RRDPNHSEGSTTLLEGYTSH CD44p511–530 v10 TTLLEGYTSHYPHTKESRTF CD44p521–540 v10 YPHTKESRTFIPVTSAKTGS CD44p531–550 v10 IPVTSAKTGSFGVTAVTVGD CD44p541–560 v10 FGVTAVTVGDSNSNVNRSLS CD44p551–570 v10/16 SNSNVNRSLSGDQDTFHPSG CD44p561–580 v10/16 GDQDTFHPSGGSHTTHGSES CD44p571–590 16/17 GSHTTHGSESDGHSHGSQEG CD44p581–600 16/17 DGHSHGSQEGGANTTSGPIR CD44p591–606 17 GANTTSGPIRTPQIPEAAAA +, OD655 0.3; , OD655 < 0.1. * The CD44 exon-encoded regions are illustrated in Figure 1. Antibodies 2023, 12, 31 7 of 16 3.2. Flow Cytometric Analysis of C Mab-3 to CD44-Expressing Cells We next investigated the reactivity of C Mab-3 against CHO/CD44v3–10 and CHO/CD44s cells by ﬂow cytometry. C Mab-3 recognized CHO/CD44v3–10 cells in a dose-dependent manner (Figure 2A) but do not recognize either CHO/CD44s (Figure 2B) or CHO-K1 (Figure 2C) cells. An anti-pan-CD44 mAb, C Mab-46 [22], rec- ognized CHO/CD44s cells (Supplementary Figure S2). Furthermore, C Mab-3 also rec- Antibodies 2023, 12, x FOR PEER REVIEW 8 of 17 ognized pancreatic cancer cell lines, such as PK-1 (Figure 2D) and PK-8 (Figure 2E), in a dose-dependent manner. Figure Figure 2. 2. FloFlow w cytometry cytometry using using C44Mab C -3 Mab-3 against against CD44-expr CD44-expr essing ce essing lls. CHO/ cells. CD44v CHO/CD44v3–10 3–10 (A), (A), CHO/CD44s (B), CHO-K1 (C), PK-1 (D), and PK-8 (E) cells were treated with 0.01–10 μg/mL of CHO/CD44s (B), CHO-K1 (C), PK-1 (D), and PK-8 (E) cells were treated with 0.01–10 g/mL of C44Mab-3, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (Red line). The C Mab-3, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (Red line). The black line represents the negative control (blocking buffer ) . black line represents the negative control (blocking buffer). 3.3. Determination of the Binding Affinity of C44Mab-3 by Flow Cytometry to CD44-Expressing Cells and SPR with the Epitope Peptide Antibodies 2023, 12, x FOR PEER REVIEW 9 of 17 Next, we determined the binding affinity of C 44Mab-3 to CHO/CD44v3–10 and PK-1 Antibodies 2023, 12, 31 8 of 16 using flow cytometry. As shown in Figure 3, the KD of CHO/CD44v3–10 and PK-1 was 1.3 −9 −9 × 10 M and 2.6 × 10 M, respectively, indicating that C44Mab-3 possesses high affinity for CD44v3–10 and endogenous CD44v5-expressing cells. 3.3. Determination of the Binding Afﬁnity of C Mab-3 by Flow Cytometry to CD44-Expressing We also measured the KD of C44Mab-3 with the epitope peptide (CD44p311–330) using Cells and SPR with the Epitope Peptide Biacore X100. The binding kinetics and measured values are summarized in Supplemen- Next, we determined the binding afﬁnity of C Mab-3 to CHO/CD44v3–10 and PK-1 −6 tary Figure S3. The KD of CD44p311–330 was 5.5 × 10 M. using ﬂow cytometry. As shown in Figure 3, the K of CHO/CD44v3–10 and PK-1 was 9 9 1.3 10 M and 2.6 10 M, respectively, indicating that C Mab-3 possesses high afﬁnity for CD44v3–10 and endogenous CD44v5-expressing cells. Figure 3. The binding affinity of C44Mab-3 to CD44-expressing cells. CHO/CD44v3–10 (A) and PK- Figure 3. The binding afﬁnity of C Mab-3 to CD44-expressing cells. CHO/CD44v3–10 (A) and 1 (B) cells were suspended in 100 μL of serially diluted C44Mab-3 at the indicated concentrations. PK-1 (B) cells were suspended in 100 L of serially diluted C Mab-3 at the indicated concentrations. Then, cells were treated with Alexa Fluor 488-conjugated secondary antibody. Fluorescence data Then, cells were treated with Alexa Fluor 488-conjugated secondary antibody. Fluorescence data were collected and the apparent dissociation constant (KD) was calculated using GraphPad PRISM were collected and the apparent dissociation constant (K ) was calculated using GraphPad PRISM 8. 8. Error bars represent means ± SDs. Error bars represent means SDs. 3.4. Western Blot Analysis We also measured the K of C Mab-3 with the epitope peptide (CD44p311–330) using D 44 We next performed Western blot analysis to investigate the sensitivity of C44Mab-3. Biacore X100. The binding kinetics and measured values are summarized in Supplementary Total cell lysates from CHO-K1, CHO/CD44s, CHO/ 6 CD44v3–10, PK-1, and PK-8 were Figure S3. The K of CD44p311–330 was 5.5 10 M. Antibodies 2023, 12, 31 9 of 16 Antibodies 2023, 12, x FOR PEER REVIEW 10 of 17 3.4. Western Blot Analysis We next performed Western blot analysis to investigate the sensitivity of C Mab-3. Total cell lysates from CHO-K1, CHO/CD44s, CHO/CD44v3–10, PK-1, and PK-8 were analyzed. As shown in Figure 4A, an anti-pan-CD44 mAb, C44Mab-46, recognized the ly- analyzed. As shown in Figure 4A, an anti-pan-CD44 mAb, C Mab-46, recognized the sates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa). C44Mab-3 de- lysates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa). C Mab-3 tected CD44v3–10 as bands of more than 180-kDa. Furthermore, C44Mab-3 detected en- detected CD44v3–10 as bands of more than 180-kDa. Furthermore, C Mab-3 detected dogenous CD44v5-containing CD44v in PK-1 and PK-8 cells. However, C44Mab-3 did not endogenous CD44v5-containing CD44v in PK-1 and PK-8 cells. However, C Mab-3 did detect any bands from lysates of CHO-K1 and CHO/CD44s cells (Figure 4B). An anti-PA16 not detect any bands from lysates of CHO-K1 and CHO/CD44s cells (Figure 4B). An tag mAb (NZ-1) recognized the lysates from both CHO/CD44s (~75 kDa) and anti-PA16 tag mAb (NZ-1) recognized the lysates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa) (Figure 4C). These results indicated that C44Mab-3 specifi- CHO/CD44v3–10 (>180 kDa) (Figure 4C). These results indicated that C Mab-3 speciﬁcally cally detects exogenous CD44v3–10 and endogenous CD44v5-containing CD44v. detects exogenous CD44v3–10 and endogenous CD44v5-containing CD44v. Figure 4. Western blot analysis using C44Mab-3. The cell lysates of CHO-K1, CHO/CD44s, Figure 4. Western blot analysis using C Mab-3. The cell lysates of CHO-K1, CHO/CD44s, CHO/CD44v3–10, PK-1, and PK-8 (10 μg) were electrophoresed and transferred onto polyvinyli- CHO/CD44v3–10, PK-1, and PK-8 (10 g) were electrophoresed and transferred onto polyvinylidene dene fluoride (PVDF) membranes. The membranes were incubated with 10 μg/mL of C44Mab-46 ﬂuoride (PVDF) membranes. The membranes were incubated with 10 g/mL of C Mab-46 (A), (A), 10 μg/mL of C44Mab-3 (B), 0.5 μg/mL of an anti-PA16 tag mAb (NZ-1) (C), and 1 μg/mL of an 10 g/mL of C Mab-3 (B), 0.5 g/mL of an anti-PA16 tag mAb (NZ-1) (C), and 1 g/mL of an anti-β-actin mAb (D). Then, the membranes were incubated with anti-mouse immunoglobulins con- anti- -actin mAb (D). Then, the membranes were incubated with anti-mouse immunoglobulins jugated with peroxidase for C44Mab-46, C44Mab-3, and anti-β-actin. Anti-rat immunoglobulins con- conjugated with peroxidase for C Mab-46, C Mab-3, and anti- -actin. Anti-rat immunoglobulins 44 44 jugated with peroxidase were used for NZ-1. The red arrows indicate CD44s (~75 kDa). The black conjugated with peroxidase were used for NZ-1. The red arrows indicate CD44s (~75 kDa). The black arrows indicate CD44v3–10 or CD44v5 (>180 kDa). arrows indicate CD44v3–10 or CD44v5 (>180 kDa). 3.5. Immunohistochemical Analysis Using C44Mab-3 against Tumor Tissues Antibodies 2023, 12, 31 10 of 16 3.5. Immunohistochemical Analysis Using C Mab-3 against Tumor Tissues We next examined whether C Mab-3 could be used for immunohistochemical anal- yses using FFPE sections. We ﬁrst examined the reactivity of C Mab-3 and C Mab-46 44 44 in an oral SCC tissue. As shown in Supplementary Figure S4, C Mab-3 exhibited a clear membranous staining and could clearly distinguish tumor cells from stromal tissues. In contrast, C Mab-46 stained both. We then investigated the reactivity of C Mab-3 and 44 44 C 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 Mab-3 showed clear membranous staining in pancreatic carcinoma cells with a rela- tively larger cytoplasm (Figure 5A). C Mab-46 also stained the same type of pancreatic carcinoma cells (Figure 5B). The staining intensity of C Mab-3 was much stronger than that of C Mab-46 (Figure 5A,B). Furthermore, diffusely spread tumor cells in the stroma were stained by both C Mab-3 and C Mab-46 (Figure 5C,D). In contrast, both C Mab-3 44 44 44 and C Mab-46 did not stain the typical ductal structure of PDAC (Figure 5E,F). In addition, stromal staining using C Mab-46 was observed in several tissues (Figure 5F). Importantly, normal pancreatic epithelial cells were not stained by C 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 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 Mab-3 could be useful for immunohistochemical analysis of FFPE tumor sections and could recognize a speciﬁc type of pancreatic carcinoma. Table 2. Immunohistochemical analysis using C Mab-3 against pancreatic carcinoma tissue arrays. Tissue Array Age Sex Organ Pathology Diagnosis TNM Grade Stage Type C Mab-3 PA241c 66 F Pancreas Adenocarcinoma T2N0M0 1 I malignant + 66 F Pancreas Adjacent normal pancreas tissue – 54 F Pancreas Adenocarcinoma T3N0M0 2 II malignant – 54 F Pancreas Adjacent normal pancreas tissue – 44 M Pancreas Adenocarcinoma T3N0M0 2 II malignant – 44 M Pancreas Adjacent normal pancreas tissue – 59 M Pancreas Adenocarcinoma T2N0M0 3 I malignant – 59 M Pancreas Adjacent normal pancreas tissue – 63 F Pancreas Adenocarcinoma T2N0M0 3 I malignant + 63 F Pancreas Adjacent normal pancreas tissue – 53 F Pancreas Adenocarcinoma T3N0M0 3 II malignant – 53 F Pancreas Adjacent normal pancreas tissue – PA484 35 M Pancreas Normal pancreas tissue - - - normal – 38 F Pancreas Normal pancreas tissue - - - normal – 38 M Pancreas Normal pancreas tissue - - - normal – 60 M Pancreas Adenocarcinoma T3N0M0 2 II malignant – 68 F Pancreas Adenocarcinoma T2N0M0 2 I malignant + 54 F Pancreas Adenocarcinoma T3N0M0 2 II malignant – 42 F Pancreas Adenocarcinoma T3N0M0 2 II malignant – 65 M Pancreas Adenocarcinoma T3N0M0 2 II malignant – 75 F Pancreas Adenocarcinoma T3N0M1 2 IV malignant – 57 M Pancreas Adenocarcinoma T3N0M0 3 II malignant + 44 M Pancreas Adenocarcinoma T3N0M0 3 II malignant – 47 M Pancreas Adenocarcinoma T3N0M0 - II malignant – 41 M Pancreas Adenocarcinoma T4N1M0 2 III malignant – 64 F Pancreas Adenocarcinoma T3N0M0 2 II malignant – 58 F Pancreas Adenocarcinoma T3N0M0 3 II malignant – 47 F Pancreas Adenocarcinoma T3N1M0 3 III malignant + 78 M Pancreas Adenocarcinoma T2N0M0 3 I malignant + 49 M Pancreas Adenocarcinoma T3N0M0 2 II malignant + 53 F Pancreas Adenocarcinoma T3N0M0 3 II malignant + 60 M Pancreas Adenocarcinoma T2N0M0 3 I malignant + 57 F Pancreas Adenocarcinoma T2N0M0 3 I malignant – 61 M Pancreas Mucinous adenocarcinoma T3N0M1 2 IV malignant – 69 M Pancreas Undifferentiated carcinoma T2N0M0 - I malignant + +, OD655 0.3; , OD655 < 0.1. Antibodies 2023, 12, x FOR PEER REVIEW 12 of 17 49 M Pancreas Adenocarcinoma T3N0M0 2 II malignant + 53 F Pancreas Adenocarcinoma T3N0M0 3 II malignant + 60 M Pancreas Adenocarcinoma T2N0M0 3 I malignant + 57 F Pancreas Adenocarcinoma T2N0M0 3 I malignant – Antibodies 2023, 12, 31 11 of 16 61 M Pancreas Mucinous adenocarcinoma T3N0M1 2 IV malignant – 69 M Pancreas Undifferentiated carcinoma T2N0M0 - I malignant + Figure 5. Immunohistochemical analysis using C Mab-3 and C Mab-46 against pancreatic ade- 44 44 nocarcinomas and normal pancreatic tissues. After antigen retrieval, serial sections of pancreatic carcinoma tissue arrays (Catalog number: PA484) were incubated with 1 g/mL of C Mab-3 or C Mab-46, followed by treatment with the Envision+ kit. The color was developed using 3,3 - diaminobenzidine tetrahydrochloride (DAB), and the sections were counterstained with hematoxylin. Scale bar = 100 m. (A–F) pancreatic adenocarcinomas; (G,H) normal pancreas tissues. Antibodies 2023, 12, 31 12 of 16 4. 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 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 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 Mab-3 recognized both CD44p311–330 and CD44p321–340 peptides, which commonly possess the HPPLIHHEHH sequence. The epitope of C 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 Mab-3 does not contain serine or threonine, the recognition of C Mab-3 is thought to be independent of the glycosylation. Immunohistochemistry using VFF-8 and conventional RT-PCR analyses were per- formed against PDAC [49]. VFF-8 recognized PDAC but not normal pancreas cells. Fur- thermore, 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 ﬁnd- ing (Figure 5A,C,G). Furthermore, we found that C Mab-3 could detect atypical types of PDAC, including metaplastic and diffusely invaded tumor cells (Figure 5A,C). In contrast, C Mab-3 did not stain a typical ductal structure of PDAC (Figure 5E) and normal pan- creatic 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) pan- creatic 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 DNp63 transcriptional network, hypermethylation of pancreatic endodermal determinant genes, and a poor prog- nosis [5]. DNp63 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, DNp63 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 DNp63-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 ad- vanced 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 bivatuzumabmertansine drug conjugate was con- ducted. 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 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 Antibodies 2023, 12, 31 13 of 16 development of the therapeutic use of C Mab-3, further investigations are required to reduce the toxicity to the above tissues. We previously converted a mouse IgG subclass of mAbs into IgG mAb and pro- 1 2a duced defucosylated mAbs using fucosyltransferase-8-deﬁcient CHO-K1 cells. The defu- cosylated IgG mAbs showed potent antibody-dependent cellular cytotoxicity in vitro 2a and suppressed tumor xenograft growth [26,57–63]. Therefore, the production of a class- switched and defucosylated version of C Mab-3 is required to evaluate the antitumor activity in vivo. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antib12020031/s1, Figure S1, Determination of the binding epitope of C Mab-3 by ELISA. Figure S2, Recognition of CHO/CD44s and CHO/CD44v3–10 by C Mab-46 44 44 using ﬂow cytometry. Figure S3, Measurement of dissociation constants (K ) between C Mab-3 D 44 and the epitope peptide using SPR. Figure S4, Immunohistochemical analysis using C Mab-3 and C Mab-46 against oral squamous cell carcinoma tissue. Figure S5, Immunohistochemical analysis using C Mab-3 and C Mab-46 against pancreatic adenocarcinomas and normal pancreatic tissues. 44 44 Author Contributions: Y.K. (Yuma Kudo), H.S. and T.T. performed the experiments. M.K.K. and Y.K. (Yukinari Kato) designed the experiments. H.S. and Y.K. (Yuma Kudo) analyzed the data. Y.K. (Yuma Kudo), H.S. and Y.K. (Yukinari Kato) wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported in part by Japan Agency for Medical Research and De- velopment (AMED) under Grant Numbers: JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.) and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientiﬁc Research (KAKENHI) grant nos. 21K20789 (to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.). Institutional Review Board Statement: The animal study protocol was approved by the Ani- mal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies involving animals. Informed Consent Statement: Not applicable. 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. References 1. Siegel, R.L.; Miller, K.D.; Wagle, N.S.; Jemal, A. Cancer statistics, 2023. CA Cancer J. Clin. 2023, 73, 17–48. [CrossRef] 2. Jones, S.; Zhang, X.; Parsons, D.W.; Lin, J.C.; Leary, R.J.; Angenendt, P.; Mankoo, P.; Carter, H.; Kamiyama, H.; Jimeno, A.; et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 2008, 321, 1801–1806. [CrossRef] 3. Waddell, N.; Pajic, M.; Patch, A.M.; Chang, D.K.; Kassahn, K.S.; Bailey, P.; Johns, A.L.; Miller, D.; Nones, K.; Quek, K.; et al. 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Abstract

antibodies Article Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody C Mab-3 for Multiple Applications against Pancreatic Carcinomas 1 1 , 2 , 1 , 2 1 , 2 1 , 2 , Yuma Kudo , Hiroyuki Suzuki * , Tomohiro Tanaka , Mika K. Kaneko and Yukinari Kato * Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan * Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.); Tel.: +81-29-853-3944 (H.S. & Y.K.) Abstract: 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 (C Mab-3; IgG , kappa) recognized peptides of the variant-5-encoded 44 1 region, indicating that C Mab-3 is a speciﬁc mAb for CD44v5. Moreover, C Mab-3 reacted with 44 44 CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by ﬂow cytometry. The 9 9 apparent K of C Mab-3 for CHO/CD44v3–10 and PK-1 was 1.3 10 M and 2.6 10 M, D 44 respectively. C Mab-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 C Mab-3 is useful for Citation: Kudo, Y.; Suzuki, H.; detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic Tanaka, T.; Kaneko, M.K.; Kato, Y. cancer diagnosis and therapy. Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody Keywords: CD44; CD44 variant 5; monoclonal antibody; ﬂow cytometry; immunohistochemistry C Mab-3 for Multiple Applications against Pancreatic Carcinomas. Antibodies 2023, 12, 31. https:// doi.org/10.3390/antib12020031 1. Introduction Academic Editor: Christian Kellner Pancreatic cancer has become the third leading cause of death in men and women Received: 30 January 2023 combined in the United States in 2023 [1]. The development of pancreatic cancer has Revised: 24 March 2023 been explained by four common oncogenic events, including KRAS, CDKN2A, SMAD4, Accepted: 10 April 2023 and TP53 [2,3]. However, pancreatic cancer shows a heterogeneity in drug response and Published: 28 April 2023 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, Copyright: © 2023 by the authors. immunogenic, and aberrantly differentiated endocrine exocrine (ADEX), which correspond Licensee MDPI, Basel, Switzerland. to the histopathological characteristics [5]. Additionally, various marker proteins have This article is an open access article been investigated for the early diagnostic and drug responses of pancreatic cancers [6]. distributed under the terms and Studies have suggested that CD44 plays important roles in malignant progression of tumors conditions of the Creative Commons through its cancer stemness and metastasis-promoting properties [7,8]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ CD44 is a type I transmembrane glycoprotein that is expressed as a wide variety of 4.0/). isoforms in various types of cells. [9]. The variety of isoforms is produced by the alternative Antibodies 2023, 12, 31. https://doi.org/10.3390/antib12020031 https://www.mdpi.com/journal/antibodies Antibodies 2023, 12, 31 2 of 16 splicing of CD44 mRNA. The CD44 standard isoform (CD44s) is the smallest isoform of CD44 (85–95 kDa); it is presented on the membrane of most vertebrate cells. CD44s mRNA is assembled by the ﬁrst ﬁve and the last ﬁve constant region exons [10]. The CD44 variant isoforms (CD44v) are produced by the alternative splicing of middle variant exons (v1–v10) and the standard exons of CD44s [11]. CD44v is heavily glycosylated, leading to various molecular weights (~250 kDa) owing to N-glycosylation and O-glycosylation [12]. Both CD44s and CD44v (pan-CD44) are known as hyaluronic acid (HA) receptors that mediate cellular homing, migration, adhesion, and proliferation [13]. CD44v is overexpressed in carcinomas and induce metastatic properties [14,15]. A growing body of evidence suggests that CD44v plays critical roles in the promotion of tumor invasion, metastasis, cancer-initiating properties [16], and resistance to chemo- and radiotherapy [7,17]. Reports indicated the important functions of each variant’s exon- encoded region. The v3-encoded region functions as a co-receptor for receptor tyrosine kinases [18]. Since the v3-encoded region possesses heparan sulfate moieties, it can recruit to heparin-binding epidermal growth factor-like growth factor (HB-EGF) and ﬁbroblast growth factors (FGFs). Furthermore, the v6-encoded region forms a ternary complex with HGF and its receptor c-MET, which is essential for its activation [19]. Additionally, oxidative stress resistance is mediated by the v8–10-encoded region through binding with a cystine– glutamate transporter (xCT) subunit [20]. Therefore, establishment and characterization of mAbs that recognize each CD44v is thought to be essential for understanding each variant’s function and development of CD44-targeting tumor diagnosis and therapy. However, the function and distribution of the variant-5-encoded region in tumors has not been fully understood. Our group established the novel anti-pan-CD44 mAbs, C Mab-5 (IgG , kappa) [21] 44 1 and C Mab-46 (IgG , kappa) [22] using the Cell-Based Immunization and Screening 44 1 (CBIS) method and immunization with the CD44v3–10 ectodomain, respectively. Both C Mab-5 and C Mab-46 have epitopes within the standard exon (1 to 5)-encoding 44 44 sequences [23–25]. Furthermore, we showed that both C Mab-5 and C Mab-46 are 44 44 applicable to ﬂow cytometry and immunohistochemistry in oral [21] and esophageal squamous cell carcinomas (SCC) [22]. We have also investigated the antitumor effects of core-fucose-deﬁcient C Mab-5 in mouse xenograft models of oral SCC [26]. In this study, we developed a novel anti-CD44v5 mAb, C Mab-3 (IgG , kappa), by the CBIS 44 1 method and evaluated its applications, including ﬂow cytometry, Western blotting, and immunohistochemical analyses. 2. Materials and Methods 2.1. Cell Lines 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 g/mL streptomycin, 0.25 g/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 . 2.2. Plasmid Construction and Establishment of Stable Transfectants CD44v3–10 open reading frame was obtained from the RIKEN BRC through the Na- tional Bio-Resource Project of the MEXT, Japan. CD44s cDNA was ampliﬁed using the HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany) and LN229 (a glioblas- toma cell line) cDNA as a template. CD44v3–10 and CD44 cDNAs were subcloned into pCAG-Ble-ssPA16 vectors with a signal sequence and N-terminal PA16 tag of 16 amino acids (GLEGGVAMPGAEDDVV) [21,27–30]; this can be detected by NZ-1, which was orig- Antibodies 2023, 12, 31 3 of 16 inally developed as an anti-human podoplanin mAb [31–46]. The pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3–10 vectors were transfected into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientiﬁc, Inc.), which offers an innovative electropo- ration method that utilizes a proprietary biologically compatible pipette tip chamber to generate a more uniform electric ﬁeld for a signiﬁcant increase in transfection efﬁciency and cell viability. By the limiting dilution method, CHO/CD44s and CHO/CD44v3–10 clones were ﬁnally established. 2.3. 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 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). 2.4. Enzyme-Linked Immunosorbent Assay (ELISA) Fifty-eight synthesized peptides, covering the CD44v3–10 extracellular domain [23], were synthesized by Sigma-Aldrich Corp. (St. Louis, MO, USA). The peptides (1 g/mL) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientiﬁc Inc.). Plate washing was performed with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.). After blocking with 1% (w/v) bovine serum albumin (BSA) in PBST, C Mab-3 (10 g/mL) was added to each well. Then, the wells were further incubated with peroxidase-conjugated anti-mouse immunoglobulins (1:2000 dilution; Agilent Technologies Inc., Santa Clara, CA, USA). One-Step Ultra TMB (Thermo Fisher Scientiﬁc Inc.) was used for enzymatic reactions. An iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA) was used to mesure the optical density at 655 nm. 2.5. Flow Cytometry CHO-K1, CHO/CD44v3–10, PK-1, and PK-8 were obtained using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were incubated with C Mab-3, C Mab-46, or blocking buffer (control) (0.1% BSA in PBS) for 44 44 30 min at 4 C. Then, the cells were treated with Alexa Fluor 488-conjugated secondary antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4 C. The data were analyzed using the SA3800 Cell Analyzer and SA3800 software ver. 2.05 (Sony Corp.). 2.6. Determination of Dissociation Constant (K ) via Flow Cytometry CHO/CD44v3–10 and PK-1 cells were treated with serially diluted C Mab-3 (0.01–10 g/mL). Then, the cells were incubated with Alexa Fluor 488-conjugated sec- ondary antibody. Fluorescence data were analyzed using BD FACSLyric and BD FACSuite software version 1.3 (BD Biosciences, Franklin Lakes, NJ, USA). The K 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). Antibodies 2023, 12, 31 4 of 16 2.7. Determination of K via Surface Plasmon Resonance (SPR) Measurement of K between C Mab-3 and the epitope peptide was performed using D 44 SPR. C Mab-3 was immobilized on the sensor chip CM5 according to the manufacturer ’s protocol by Cytiva (Marlborough, MA, USA). C Mab-3 (10 g/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 Mab-3. The K between 44 D C 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 using Biacore X100 evaluation software (Cytiva). 2.8. Western Blot Analysis The total cell lysates (10 g of protein) were separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The separated proteins were transferred onto polyvinylidene diﬂuoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The blocking was performed with 4% skim milk (Nacalai Tesque, Inc.) in PBST. The membranes were incubated with 10 g/mL of C Mab-3, 10 g/mL of C Mab-46, 44 44 0.5 g/mL of NZ-1, or 1 g/mL of an anti- -actin mAb (clone AC-15; Sigma-Aldrich Corp.) and then incubated with peroxidase-conjugated anti-mouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.) for C Mab-3, C Mab-46, and anti- -actin. Anti-rat 44 44 immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.) conjugated to peroxidase was used for NZ-1. The chemiluminescence signals were obtained with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) and detected using a Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan). 2.9. 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 ar- rays (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 nor- mal 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 carci- noma 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 Mab-3 (1 g/mL) and C Mab-46 44 44 (1 g/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. 3. Results 3.1. Development of an Anti-CD44v5 mAb, C Mab-3 In the CBIS method, we used a stable transfectant (CHO/CD44v3–10 cells) as an immunogen (Figure 1). Mice were immunized with CHO/CD44v3–10 cells, and hybrido- mas were seeded into 96-well plates. The supernatants, which are negative for CHO-K1 cells and positive for CHO/CD44v3–10 cells, were selected using ﬂow-cytometry-based high throughput screening. By limiting dilution, anti-CD44-mAb-producing clones were ﬁnally established. Among them, C Mab-3 (IgG , kappa) was shown to recognize both 44 1 CD44p311–330 (AYEGNWNPEAHPPLIHHEHH) and CD44p321–340 peptides (HPPLI- HHEHHEEEETPHSTS), which correspond to the variant-5-encoded sequence (Table 1 Antibodies 2023, 12, x FOR PEER REVIEW 5 of 17 In the CBIS method, we used a stable transfectant (CHO/CD44v3–10 cells) as an im- munogen (Figure 1). Mice were immunized with CHO/CD44v3–10 cells, and hybridomas were seeded into 96-well plates. The supernatants, which are negative for CHO-K1 cells and positive for CHO/CD44v3–10 cells, were selected using flow -cytometry-based high throughput screening. By limiting dilution, anti-CD44-mAb-producing clones were fi- nally established. Among them, C44Mab-3 (IgG1, kappa) was shown to recognize both Antibodies 2023, 12, 31 5 of 16 CD44p311–330 (AYEGNWNPEAHPPLIHHEHH) and CD44p321–340 peptides (HPPLIH- HEHHEEEETPHSTS), which correspond to the variant-5-encoded sequence (Table 1 and Supplementary Figure S1). In contrast, C44Mab-3 did not recognize other CD44v3–10 ex- and Supplementary Figure S1). In contrast, C Mab-3 did not recognize other CD44v3–10 tracellular regions. These results indicated that C44Mab-3 specifically recognizes the CD44 extracellular regions. These results indicated that C Mab-3 speciﬁcally recognizes the variant-5-encoded sequence. CD44 variant-5-encoded sequence. Figure 1. A schematic illustration of anti-human CD44 mAbs production. (A) Structure of CD44. Figure 1. A schematic illustration of anti-human CD44 mAbs production. (A) Structure of CD44. CD44s mRNA is assembled by the ﬁrst ﬁve (1 to 5) and the last ﬁve (16 to 20) exons and translates CD44s mRNA is assembled by the first five (1 to 5) and the last five (16 to 20) exons and translates CD44s. The mRNAs of CD44 variants are produced by the alternative splicing of middle variant CD44s. The mRNAs of CD44 variants are produced by the alternative splicing of middle variant exons exons and and translate translate multiple multiple CD44v CD44v such such as as CD CD44v3–10, 44v3–10, CD CD44v4–10, 44v4–10, CD CD44v6–10, 44v6–10, and and C CD44v8–10. D44v8–10. (B) CHO/CD44v3–10 cells were intraperitoneally injected into BALB/c mice. (C) The splenocytes and P3U1 cells were fused and the hybridomas were produced. (D) The screening was conducted by ﬂow cytometry using parental CHO-K1 and CHO/CD44v3–10 cells. (E) After cloning and additional screening, a clone (C Mab-3 (IgG , kappa)) was established. Furthermore, the binding epitope 44 1 was determined by enzyme-linked immunosorbent assay (ELISA) using peptides that cover the extracellular domain of CD44v3–10. Antibodies 2023, 12, 31 6 of 16 Table 1. Determination of the binding epitope of C Mab-3 by ELISA. Peptide Coding Exon * Sequence C Mab-3 CD44p21–40 2 QIDLNITCRFAGVFHVEKNG CD44p31–50 2 AGVFHVEKNGRYSISRTEAA CD44p41–60 2 RYSISRTEAADLCKAFNSTL CD44p51–70 2 DLCKAFNSTLPTMAQMEKAL CD44p61–80 2/3 PTMAQMEKALSIGFETCRYG CD44p71–90 2/3 SIGFETCRYGFIEGHVVIPR CD44p81–100 3 FIEGHVVIPRIHPNSICAAN CD44p91–110 3 IHPNSICAANNTGVYILTSN CD44p101–120 3 NTGVYILTSNTSQYDTYCFN CD44p111–130 3/4 TSQYDTYCFNASAPPEEDCT CD44p121–140 3/4 ASAPPEEDCTSVTDLPNAFD CD44p131–150 4/5 SVTDLPNAFDGPITITIVNR CD44p141–160 4/5 GPITITIVNRDGTRYVQKGE CD44p151–170 5 DGTRYVQKGEYRTNPEDIYP CD44p161–180 5 YRTNPEDIYPSNPTDDDVSS CD44p171–190 5 SNPTDDDVSSGSSSERSSTS CD44p181–200 5 GSSSERSSTSGGYIFYTFST CD44p191–210 5 GGYIFYTFSTVHPIPDEDSP CD44p201–220 5 VHPIPDEDSPWITDSTDRIP CD44p211–230 5/v3 WITDSTDRIPATSTSSNTIS CD44p221–240 5/v3 ATSTSSNTISAGWEPNEENE CD44p231–250 v3 AGWEPNEENEDERDRHLSFS CD44p241–260 v3 DERDRHLSFSGSGIDDDEDF CD44p251–270 v3/v4 GSGIDDDEDFISSTISTTPR CD44p261–280 v3/v4 ISSTISTTPRAFDHTKQNQD CD44p271–290 v4 AFDHTKQNQDWTQWNPSHSN CD44p281–300 v4 WTQWNPSHSNPEVLLQTTTR CD44p291–310 v4/v5 PEVLLQTTTRMTDVDRNGTT CD44p301–320 v4/v5 MTDVDRNGTTAYEGNWNPEA CD44p311–330 v5 AYEGNWNPEAHPPLIHHEHH + CD44p321–340 v5 HPPLIHHEHHEEEETPHSTS + CD44p331–350 v5/v6 EEEETPHSTSTIQATPSSTT CD44p341–360 v5/v6 TIQATPSSTTEETATQKEQW CD44p351–370 v6 EETATQKEQWFGNRWHEGYR CD44p361–380 v6 FGNRWHEGYRQTPREDSHST CD44p371–390 v6/v7 QTPREDSHSTTGTAAASAHT CD44p381–400 v6/v7 TGTAAASAHTSHPMQGRTTP CD44p391–410 v7 SHPMQGRTTPSPEDSSWTDF CD44p401–420 v7 SPEDSSWTDFFNPISHPMGR CD44p411–430 v7/v8 FNPISHPMGRGHQAGRRMDM CD44p421–440 v7/v8 GHQAGRRMDMDSSHSTTLQP CD44p431–450 v8 DSSHSTTLQPTANPNTGLVE CD44p441–460 v8 TANPNTGLVEDLDRTGPLSM CD44p451–470 v8/v9 DLDRTGPLSMTTQQSNSQSF CD44p461–480 v8/v9 TTQQSNSQSFSTSHEGLEED CD44p471–490 v9 STSHEGLEEDKDHPTTSTLT CD44p481–500 v9/v10 KDHPTTSTLTSSNRNDVTGG CD44p491–510 v9/v10 SSNRNDVTGGRRDPNHSEGS CD44p501–520 v10 RRDPNHSEGSTTLLEGYTSH CD44p511–530 v10 TTLLEGYTSHYPHTKESRTF CD44p521–540 v10 YPHTKESRTFIPVTSAKTGS CD44p531–550 v10 IPVTSAKTGSFGVTAVTVGD CD44p541–560 v10 FGVTAVTVGDSNSNVNRSLS CD44p551–570 v10/16 SNSNVNRSLSGDQDTFHPSG CD44p561–580 v10/16 GDQDTFHPSGGSHTTHGSES CD44p571–590 16/17 GSHTTHGSESDGHSHGSQEG CD44p581–600 16/17 DGHSHGSQEGGANTTSGPIR CD44p591–606 17 GANTTSGPIRTPQIPEAAAA +, OD655 0.3; , OD655 < 0.1. * The CD44 exon-encoded regions are illustrated in Figure 1. Antibodies 2023, 12, 31 7 of 16 3.2. Flow Cytometric Analysis of C Mab-3 to CD44-Expressing Cells We next investigated the reactivity of C Mab-3 against CHO/CD44v3–10 and CHO/CD44s cells by ﬂow cytometry. C Mab-3 recognized CHO/CD44v3–10 cells in a dose-dependent manner (Figure 2A) but do not recognize either CHO/CD44s (Figure 2B) or CHO-K1 (Figure 2C) cells. An anti-pan-CD44 mAb, C Mab-46 [22], rec- ognized CHO/CD44s cells (Supplementary Figure S2). Furthermore, C Mab-3 also rec- Antibodies 2023, 12, x FOR PEER REVIEW 8 of 17 ognized pancreatic cancer cell lines, such as PK-1 (Figure 2D) and PK-8 (Figure 2E), in a dose-dependent manner. Figure Figure 2. 2. FloFlow w cytometry cytometry using using C44Mab C -3 Mab-3 against against CD44-expr CD44-expr essing ce essing lls. CHO/ cells. CD44v CHO/CD44v3–10 3–10 (A), (A), CHO/CD44s (B), CHO-K1 (C), PK-1 (D), and PK-8 (E) cells were treated with 0.01–10 μg/mL of CHO/CD44s (B), CHO-K1 (C), PK-1 (D), and PK-8 (E) cells were treated with 0.01–10 g/mL of C44Mab-3, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (Red line). The C Mab-3, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (Red line). The black line represents the negative control (blocking buffer ) . black line represents the negative control (blocking buffer). 3.3. Determination of the Binding Affinity of C44Mab-3 by Flow Cytometry to CD44-Expressing Cells and SPR with the Epitope Peptide Antibodies 2023, 12, x FOR PEER REVIEW 9 of 17 Next, we determined the binding affinity of C 44Mab-3 to CHO/CD44v3–10 and PK-1 Antibodies 2023, 12, 31 8 of 16 using flow cytometry. As shown in Figure 3, the KD of CHO/CD44v3–10 and PK-1 was 1.3 −9 −9 × 10 M and 2.6 × 10 M, respectively, indicating that C44Mab-3 possesses high affinity for CD44v3–10 and endogenous CD44v5-expressing cells. 3.3. Determination of the Binding Afﬁnity of C Mab-3 by Flow Cytometry to CD44-Expressing We also measured the KD of C44Mab-3 with the epitope peptide (CD44p311–330) using Cells and SPR with the Epitope Peptide Biacore X100. The binding kinetics and measured values are summarized in Supplemen- Next, we determined the binding afﬁnity of C Mab-3 to CHO/CD44v3–10 and PK-1 −6 tary Figure S3. The KD of CD44p311–330 was 5.5 × 10 M. using ﬂow cytometry. As shown in Figure 3, the K of CHO/CD44v3–10 and PK-1 was 9 9 1.3 10 M and 2.6 10 M, respectively, indicating that C Mab-3 possesses high afﬁnity for CD44v3–10 and endogenous CD44v5-expressing cells. Figure 3. The binding affinity of C44Mab-3 to CD44-expressing cells. CHO/CD44v3–10 (A) and PK- Figure 3. The binding afﬁnity of C Mab-3 to CD44-expressing cells. CHO/CD44v3–10 (A) and 1 (B) cells were suspended in 100 μL of serially diluted C44Mab-3 at the indicated concentrations. PK-1 (B) cells were suspended in 100 L of serially diluted C Mab-3 at the indicated concentrations. Then, cells were treated with Alexa Fluor 488-conjugated secondary antibody. Fluorescence data Then, cells were treated with Alexa Fluor 488-conjugated secondary antibody. Fluorescence data were collected and the apparent dissociation constant (KD) was calculated using GraphPad PRISM were collected and the apparent dissociation constant (K ) was calculated using GraphPad PRISM 8. 8. Error bars represent means ± SDs. Error bars represent means SDs. 3.4. Western Blot Analysis We also measured the K of C Mab-3 with the epitope peptide (CD44p311–330) using D 44 We next performed Western blot analysis to investigate the sensitivity of C44Mab-3. Biacore X100. The binding kinetics and measured values are summarized in Supplementary Total cell lysates from CHO-K1, CHO/CD44s, CHO/ 6 CD44v3–10, PK-1, and PK-8 were Figure S3. The K of CD44p311–330 was 5.5 10 M. Antibodies 2023, 12, 31 9 of 16 Antibodies 2023, 12, x FOR PEER REVIEW 10 of 17 3.4. Western Blot Analysis We next performed Western blot analysis to investigate the sensitivity of C Mab-3. Total cell lysates from CHO-K1, CHO/CD44s, CHO/CD44v3–10, PK-1, and PK-8 were analyzed. As shown in Figure 4A, an anti-pan-CD44 mAb, C44Mab-46, recognized the ly- analyzed. As shown in Figure 4A, an anti-pan-CD44 mAb, C Mab-46, recognized the sates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa). C44Mab-3 de- lysates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa). C Mab-3 tected CD44v3–10 as bands of more than 180-kDa. Furthermore, C44Mab-3 detected en- detected CD44v3–10 as bands of more than 180-kDa. Furthermore, C Mab-3 detected dogenous CD44v5-containing CD44v in PK-1 and PK-8 cells. However, C44Mab-3 did not endogenous CD44v5-containing CD44v in PK-1 and PK-8 cells. However, C Mab-3 did detect any bands from lysates of CHO-K1 and CHO/CD44s cells (Figure 4B). An anti-PA16 not detect any bands from lysates of CHO-K1 and CHO/CD44s cells (Figure 4B). An tag mAb (NZ-1) recognized the lysates from both CHO/CD44s (~75 kDa) and anti-PA16 tag mAb (NZ-1) recognized the lysates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa) (Figure 4C). These results indicated that C44Mab-3 specifi- CHO/CD44v3–10 (>180 kDa) (Figure 4C). These results indicated that C Mab-3 speciﬁcally cally detects exogenous CD44v3–10 and endogenous CD44v5-containing CD44v. detects exogenous CD44v3–10 and endogenous CD44v5-containing CD44v. Figure 4. Western blot analysis using C44Mab-3. The cell lysates of CHO-K1, CHO/CD44s, Figure 4. Western blot analysis using C Mab-3. The cell lysates of CHO-K1, CHO/CD44s, CHO/CD44v3–10, PK-1, and PK-8 (10 μg) were electrophoresed and transferred onto polyvinyli- CHO/CD44v3–10, PK-1, and PK-8 (10 g) were electrophoresed and transferred onto polyvinylidene dene fluoride (PVDF) membranes. The membranes were incubated with 10 μg/mL of C44Mab-46 ﬂuoride (PVDF) membranes. The membranes were incubated with 10 g/mL of C Mab-46 (A), (A), 10 μg/mL of C44Mab-3 (B), 0.5 μg/mL of an anti-PA16 tag mAb (NZ-1) (C), and 1 μg/mL of an 10 g/mL of C Mab-3 (B), 0.5 g/mL of an anti-PA16 tag mAb (NZ-1) (C), and 1 g/mL of an anti-β-actin mAb (D). Then, the membranes were incubated with anti-mouse immunoglobulins con- anti- -actin mAb (D). Then, the membranes were incubated with anti-mouse immunoglobulins jugated with peroxidase for C44Mab-46, C44Mab-3, and anti-β-actin. Anti-rat immunoglobulins con- conjugated with peroxidase for C Mab-46, C Mab-3, and anti- -actin. Anti-rat immunoglobulins 44 44 jugated with peroxidase were used for NZ-1. The red arrows indicate CD44s (~75 kDa). The black conjugated with peroxidase were used for NZ-1. The red arrows indicate CD44s (~75 kDa). The black arrows indicate CD44v3–10 or CD44v5 (>180 kDa). arrows indicate CD44v3–10 or CD44v5 (>180 kDa). 3.5. Immunohistochemical Analysis Using C44Mab-3 against Tumor Tissues Antibodies 2023, 12, 31 10 of 16 3.5. Immunohistochemical Analysis Using C Mab-3 against Tumor Tissues We next examined whether C Mab-3 could be used for immunohistochemical anal- yses using FFPE sections. We ﬁrst examined the reactivity of C Mab-3 and C Mab-46 44 44 in an oral SCC tissue. As shown in Supplementary Figure S4, C Mab-3 exhibited a clear membranous staining and could clearly distinguish tumor cells from stromal tissues. In contrast, C Mab-46 stained both. We then investigated the reactivity of C Mab-3 and 44 44 C 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 Mab-3 showed clear membranous staining in pancreatic carcinoma cells with a rela- tively larger cytoplasm (Figure 5A). C Mab-46 also stained the same type of pancreatic carcinoma cells (Figure 5B). The staining intensity of C Mab-3 was much stronger than that of C Mab-46 (Figure 5A,B). Furthermore, diffusely spread tumor cells in the stroma were stained by both C Mab-3 and C Mab-46 (Figure 5C,D). In contrast, both C Mab-3 44 44 44 and C Mab-46 did not stain the typical ductal structure of PDAC (Figure 5E,F). In addition, stromal staining using C Mab-46 was observed in several tissues (Figure 5F). Importantly, normal pancreatic epithelial cells were not stained by C 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 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 Mab-3 could be useful for immunohistochemical analysis of FFPE tumor sections and could recognize a speciﬁc type of pancreatic carcinoma. Table 2. Immunohistochemical analysis using C Mab-3 against pancreatic carcinoma tissue arrays. Tissue Array Age Sex Organ Pathology Diagnosis TNM Grade Stage Type C Mab-3 PA241c 66 F Pancreas Adenocarcinoma T2N0M0 1 I malignant + 66 F Pancreas Adjacent normal pancreas tissue – 54 F Pancreas Adenocarcinoma T3N0M0 2 II malignant – 54 F Pancreas Adjacent normal pancreas tissue – 44 M Pancreas Adenocarcinoma T3N0M0 2 II malignant – 44 M Pancreas Adjacent normal pancreas tissue – 59 M Pancreas Adenocarcinoma T2N0M0 3 I malignant – 59 M Pancreas Adjacent normal pancreas tissue – 63 F Pancreas Adenocarcinoma T2N0M0 3 I malignant + 63 F Pancreas Adjacent normal pancreas tissue – 53 F Pancreas Adenocarcinoma T3N0M0 3 II malignant – 53 F Pancreas Adjacent normal pancreas tissue – PA484 35 M Pancreas Normal pancreas tissue - - - normal – 38 F Pancreas Normal pancreas tissue - - - normal – 38 M Pancreas Normal pancreas tissue - - - normal – 60 M Pancreas Adenocarcinoma T3N0M0 2 II malignant – 68 F Pancreas Adenocarcinoma T2N0M0 2 I malignant + 54 F Pancreas Adenocarcinoma T3N0M0 2 II malignant – 42 F Pancreas Adenocarcinoma T3N0M0 2 II malignant – 65 M Pancreas Adenocarcinoma T3N0M0 2 II malignant – 75 F Pancreas Adenocarcinoma T3N0M1 2 IV malignant – 57 M Pancreas Adenocarcinoma T3N0M0 3 II malignant + 44 M Pancreas Adenocarcinoma T3N0M0 3 II malignant – 47 M Pancreas Adenocarcinoma T3N0M0 - II malignant – 41 M Pancreas Adenocarcinoma T4N1M0 2 III malignant – 64 F Pancreas Adenocarcinoma T3N0M0 2 II malignant – 58 F Pancreas Adenocarcinoma T3N0M0 3 II malignant – 47 F Pancreas Adenocarcinoma T3N1M0 3 III malignant + 78 M Pancreas Adenocarcinoma T2N0M0 3 I malignant + 49 M Pancreas Adenocarcinoma T3N0M0 2 II malignant + 53 F Pancreas Adenocarcinoma T3N0M0 3 II malignant + 60 M Pancreas Adenocarcinoma T2N0M0 3 I malignant + 57 F Pancreas Adenocarcinoma T2N0M0 3 I malignant – 61 M Pancreas Mucinous adenocarcinoma T3N0M1 2 IV malignant – 69 M Pancreas Undifferentiated carcinoma T2N0M0 - I malignant + +, OD655 0.3; , OD655 < 0.1. Antibodies 2023, 12, x FOR PEER REVIEW 12 of 17 49 M Pancreas Adenocarcinoma T3N0M0 2 II malignant + 53 F Pancreas Adenocarcinoma T3N0M0 3 II malignant + 60 M Pancreas Adenocarcinoma T2N0M0 3 I malignant + 57 F Pancreas Adenocarcinoma T2N0M0 3 I malignant – Antibodies 2023, 12, 31 11 of 16 61 M Pancreas Mucinous adenocarcinoma T3N0M1 2 IV malignant – 69 M Pancreas Undifferentiated carcinoma T2N0M0 - I malignant + Figure 5. Immunohistochemical analysis using C Mab-3 and C Mab-46 against pancreatic ade- 44 44 nocarcinomas and normal pancreatic tissues. After antigen retrieval, serial sections of pancreatic carcinoma tissue arrays (Catalog number: PA484) were incubated with 1 g/mL of C Mab-3 or C Mab-46, followed by treatment with the Envision+ kit. The color was developed using 3,3 - diaminobenzidine tetrahydrochloride (DAB), and the sections were counterstained with hematoxylin. Scale bar = 100 m. (A–F) pancreatic adenocarcinomas; (G,H) normal pancreas tissues. Antibodies 2023, 12, 31 12 of 16 4. 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 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 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 Mab-3 recognized both CD44p311–330 and CD44p321–340 peptides, which commonly possess the HPPLIHHEHH sequence. The epitope of C 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 Mab-3 does not contain serine or threonine, the recognition of C Mab-3 is thought to be independent of the glycosylation. Immunohistochemistry using VFF-8 and conventional RT-PCR analyses were per- formed against PDAC [49]. VFF-8 recognized PDAC but not normal pancreas cells. Fur- thermore, 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 ﬁnd- ing (Figure 5A,C,G). Furthermore, we found that C Mab-3 could detect atypical types of PDAC, including metaplastic and diffusely invaded tumor cells (Figure 5A,C). In contrast, C Mab-3 did not stain a typical ductal structure of PDAC (Figure 5E) and normal pan- creatic 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) pan- creatic 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 DNp63 transcriptional network, hypermethylation of pancreatic endodermal determinant genes, and a poor prog- nosis [5]. DNp63 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, DNp63 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 DNp63-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 ad- vanced 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 bivatuzumabmertansine drug conjugate was con- ducted. 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 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 Antibodies 2023, 12, 31 13 of 16 development of the therapeutic use of C Mab-3, further investigations are required to reduce the toxicity to the above tissues. We previously converted a mouse IgG subclass of mAbs into IgG mAb and pro- 1 2a duced defucosylated mAbs using fucosyltransferase-8-deﬁcient CHO-K1 cells. The defu- cosylated IgG mAbs showed potent antibody-dependent cellular cytotoxicity in vitro 2a and suppressed tumor xenograft growth [26,57–63]. Therefore, the production of a class- switched and defucosylated version of C Mab-3 is required to evaluate the antitumor activity in vivo. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antib12020031/s1, Figure S1, Determination of the binding epitope of C Mab-3 by ELISA. Figure S2, Recognition of CHO/CD44s and CHO/CD44v3–10 by C Mab-46 44 44 using ﬂow cytometry. Figure S3, Measurement of dissociation constants (K ) between C Mab-3 D 44 and the epitope peptide using SPR. Figure S4, Immunohistochemical analysis using C Mab-3 and C Mab-46 against oral squamous cell carcinoma tissue. Figure S5, Immunohistochemical analysis using C Mab-3 and C Mab-46 against pancreatic adenocarcinomas and normal pancreatic tissues. 44 44 Author Contributions: Y.K. (Yuma Kudo), H.S. and T.T. performed the experiments. M.K.K. and Y.K. (Yukinari Kato) designed the experiments. H.S. and Y.K. (Yuma Kudo) analyzed the data. Y.K. (Yuma Kudo), H.S. and Y.K. (Yukinari Kato) wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported in part by Japan Agency for Medical Research and De- velopment (AMED) under Grant Numbers: JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.) and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientiﬁc Research (KAKENHI) grant nos. 21K20789 (to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.). Institutional Review Board Statement: The animal study protocol was approved by the Ani- mal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies involving animals. Informed Consent Statement: Not applicable. 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. References 1. Siegel, R.L.; Miller, K.D.; Wagle, N.S.; Jemal, A. Cancer statistics, 2023. CA Cancer J. Clin. 2023, 73, 17–48. [CrossRef] 2. Jones, S.; Zhang, X.; Parsons, D.W.; Lin, J.C.; Leary, R.J.; Angenendt, P.; Mankoo, P.; Carter, H.; Kamiyama, H.; Jimeno, A.; et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 2008, 321, 1801–1806. [CrossRef] 3. Waddell, N.; Pajic, M.; Patch, A.M.; Chang, D.K.; Kassahn, K.S.; Bailey, P.; Johns, A.L.; Miller, D.; Nones, K.; Quek, K.; et al. 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Antibodies – Multidisciplinary Digital Publishing Institute

Published: Apr 28, 2023

Keywords: CD44; CD44 variant 5; monoclonal antibody; flow cytometry; immunohistochemistry

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

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

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

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

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