MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 34, Number 3, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2014.0072
Production of a High-affinity Monoclonal Antibody Reactive with Folate Receptors Alpha and Beta Taku Nagai,1 Yuko Furusho,1 Hua Li,1 Kazuhisa Hasui,1 Sumika Matsukita,2 Kazunobu Sueyoshi,3 Masakazu Yanagi,4 Masaki Hatae,5 Sonshin Takao,6 and Takami Matsuyama 6
Folate receptors a (FRa) and b (FRb) are two isoforms of the cell surface glycoprotein that binds folate. The expression of FRa is rare in normal cells and elevated in cancer cells. Thus, FRa-based tumor-targeted therapy has been a focus area of laboratory research and clinical trials. Recently, it was shown that a significant fraction of tumor-associated macrophages expresses FRb and that these cells can enhance tumor growth. Although FRa and FRb share 70% identity in their deduced amino acid sequence, a monoclonal antibody (MAb) reactive with both receptors has not been developed. A MAb that can target both FRa-expressing cancer cells and FRbexpressing tumor-associated macrophages may provide a more potent therapeutic tool for cancer than individual anti-FRa or anti-FRb MAbs. In this study, we developed a MAb that recognizes both FRa and FRb (antiFRab). The anti-FRab specifically stained trophoblasts and macrophages from human placenta, synovial macrophages from rheumatoid arthritis patient, liver macrophages from cynomolgus monkey and common marmoset, and cancer cells and tumor-associated macrophages from ovary and lung carcinomas. Surface plasmon resonance showed that the anti-FRab bound to soluble forms of the FRa and FRb proteins with high affinity (KD = 6.26 · 10 -9 M and 4.33 · 10 -9 M, respectively). In vitro functional analysis of the anti-FRab showed that this MAb mediates complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis of FRa-expressing and FRb-expressing cell lines. The anti-FRab MAb is a promising therapeutic candidate for cancers in which macrophages promote tumor progression.
Introduction
F
olate is an essential vitamin that is used in singlecarbon-donor reactions in the biosynthesis of purines and thymidylates and is needed for methionine synthesis.(1) Folate can enter the cell through three different routes: reduced folate carrier (RFC) with low affinity for folate, proton-coupled folate transporter (PCFT), and folate receptors (FRs).(2–4) RFC and PCFT are ubiquitously expressed in most normal tissues. There are three isoforms of the folate receptor in humans. The expression of FRa and FRb isoforms is restricted to the surface of epithelial cells such as trophoblasts, the choroid plexus and proximal kidney tubules, and activated macrophages, respectively. The g isoform is a soluble protein secreted by myeloid cells that has not been well characterized. Interestingly, many cancer types including ovary, lung, and colorectal carcinomas overexpress FRa, while tumor-associated macrophages (TAMs) express FRb.(5–8) Regardless of the physiological role for FRs in cancer cells, the disparate expression levels of FRa between cancerous and normal cells make FRa an attractive
candidate for targeted biological therapy of cancers. FRabased tumor-targeted therapy using folate derivatives, antiFRa monoclonal antibodies (MAbs), and folate-conjugated drugs have been an active area of laboratory research and clinical trials.(9–11) We previously showed that the depletion of FRb-expressing TAMs using an anti-FRb immunotoxin reduced tumor growth.(7) Although FRa and b share 70% identity in their deduced amino acid sequences,(12) a MAb reactive with both FRa and b has not been reported. A MAb that can target both cancer cells and tumor-associated macrophages may provide a more potent therapeutic tool for cancer than individual anti-FRa or anti-FRb MAbs. In this study, we developed a MAb reactive with FRa and FRb and examined its in vitro effects on FRa- and FRb-expressing cell lines. Materials and Methods Cell lines
B300-19 (murine pre-B cell), KB (human nasopharynx cell), NS1 (murine myeloma cell), RBL2H3 (rat basophilic
1
Department of Immunology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. Department of Clinical Pathology, Kagoshima Kousei-Ren Hospital, Kagoshima, Japan. Department of Clinical Pathology, 4Department of Surgery, 5Department of Obstetrics, Kagoshima City Hospital, Kagoshima, Japan. 6 The Center for Advanced Biomedical Sciences and Swine Research, Kagoshima University, Kagoshima, Japan. 2 3
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leukemia cell), and SKOV-3 (human ovarian carcinoma cell) cell lines were obtained from ATCC (Manassas, VA). JFP-J1 hybridoma cells producing anti-GFP rat IgG2a were obtained from Riken BioResource Center (Tsukuba, Japan). The human ovarian carcinoma cell line IGROV-1 was a kind gift from Morphotech (Exton, PA). Both SKOV-3 and IGROV-1 were cultured in RPMI-1640 medium (Life Technologies, Carlsbad CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Nacalai Tesque Inc., Kyoto, Japan). The other cell lines were cultured in IMDM medium (Life Technologies) supplemented with 10% heatinactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin, except for B300-19 cells, which were maintained in the same medium containing 50 mM 2-mercaptoethanol. Human placenta, rheumatoid arthritis synovium, liver from cynomolgus monkey and common marmoset were obtained from Kagoshima University. Surgical specimens were obtained from 12 patients with ovarian carcinomas and 19 patients with lung cancer at Kagoshima City Hospital and Kagoshima Kousei-Ren Hospital. The diagnoses were made both clinically and pathologically. Tissue samples contained 10 serous and 2 clear cell carcinomas of the ovary, 13 adenocarcinomas, and 6 squamous cell carcinomas of the lung. This study was approved by the ethics committee of Kagoshima University; written informed consent was obtained from all of the subjects. Production of MAb reactive with FRa and FRb
FRb-expressing RBL2H3 and B300-19 were prepared by transfecting human FRb in the pEF-BOS vector, as previously described.(7,13) Wistar Kyoto rats (Charles River Laboratories, Yokohama, Japan) were injected via the hind footpads with 100 mL of an emulsion containing 1 · 107 of FRb-expressing RBL2H3 and Freund’s complete adjuvant. After 16 days, the lymphocytes from iliac lymph nodes were fused with NS1 myeloma cells at a ratio of 4:1 in 50% polyethyleneglycol (PEG 1500, Roche, Mannheim, Germany). The hybridoma cells were placed on 96-well plates and cultured IMDM medium supplemented with 10% heatinactivated FBS and HAT (Life Technologies). After 7 days post-fusion, the hybridoma supernatants were screened by means of immunofluorescence assay against FRb-B300-19 and KB. One MAb, named clone 5 (rat IgG2a) was selected for its reactivity with both KB and FRb-B300-19. The hybridoma culture supernatants were purified using goat anti-rat IgG-agarose affinity chromatography (Rockland, Gilbertsville, PA) and used for further experiments. The F(ab’)2 fragment was prepared using pepsin-agarose (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. All animal studies were performed in accordance with the ethical guidelines for animal experiments at Kagoshima University. Human peripheral blood mononuclear cells (PBMCs), monocytes, and M-CSF-differentiated macrophages were prepared from the peripheral blood of four healthy donors, as previously described.(7,13) Briefly, PBMCs were isolated using a Ficoll-hypaque density gradient, and monocytes were separated from PBMCs by adhesion to culture dishes. To obtain macrophages, monocytes were cultured in IMDM
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containing 10% FBS and 25 ng/mL recombinant human MCSF (PeproTech, Rocky Hill, NJ) for 7 days. Flow cytometric analysis
After blocking non-specific binding with 10% human-type AB serum, the cells were incubated with 1 mg of anti-human FRa (Mov 18, murine IgG1, which was a gift from Dr. S. Canevari, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy); anti-human FRb (94b, murine IgG1, which was produced in our laboratory)(7,11); clone 5; or one of the following two isotype-matched MAbs: murine IgG1 (MOPC-21, BD Pharmingen, San Jose, CA) or rat IgG2a (R35-95, also from BD Pharmingen) in PBS containing 1% bovine serum albumin and 0.1% NaN3. To examine the effect of soluble FRg in human serum, KB and FRb-expressing B300-19 were incubated with clone 5 or rat IgG2a in the presence of 50% of human-type AB serum. After washing with PBS containing 1% bovine serum albumin and 0.1% NaN3, cells were stained with fluorescein isothiocyanate (FITC)-labeled goat F(ab’)2 anti-rat IgG (H + L) (Leino Technologies, St. Louis, MO) or FITC-labeled goat F(ab’)2 anti-mouse IgG (H + L) (Southern Biotech, Birmingham, AL). For dual-color staining of PBMCs and macrophages, cells were incubated with clone 5, FITC-labeled murine anti-human CD3 (UCHT1, Bio-Rad Laboratories, Hercules, CA), Alexa Fluor 488-labeled murine anti-human CD14 (HCD14, BioLegend, San Diego, CA), FITC-labeled murine murine anti-human CD20 (2H7, Bio-Rad), rat IgG2a, or Alexa Fluor 488-labeled isotype control MAbs (BD Pharmingen). The reactivities of clone 5 and rat IgG2a were further detected with APC-labeled goat F(ab’)2 anti-rat IgG (sc-3832, Santa Cruz Biotechnology, Dallas, TX). The stained cells were analyzed using a CyAnTM ADP flow cytometer (Beckman Coulter, Brea, CA). Immunohistochemical analysis
Immunohistochemical analysis was performed as previously described.(7,14) Briefly, acetone-fixed frozen sections (6 mm) were incubated with anti-FRa, anti-FRb, clone 5, anti-CD68 (EBM11, Dako, Copenhagen, Denmark) for human tissues, anti-CD68 (Y1/82A, BD Pharmingen) for cynomolgus monkey and common marmoset tissues, or isotype-matched MAbs (murine IgG1, murine IgG2b or rat IgG2a). The staining was visualized with a horseradish peroxidase-conjugated MAX-PO secondary antibody kit (Nichirei Co., Tokyo, Japan) and NovaRed substrate (Vector Laboratories, Burlingame, CA), according to the manufacturer’s instructions. Slides were archived using a Digital Sight CCD camera (DS-Fi1, Nikon, Tokyo, Japan) and computer-aided image analyzer (NIS-Elements, Nikon). The staining intensity was assessed in 10 randomly selected 400x fields in each section by two investigators who were blinded to all clinical details. Positive staining in tumor tissues was defined as staining of >1% of the total cell number. Production of recombinant soluble FRa and FRb proteins
Recombinant soluble FRa-Fc protein was prepared as described previously.(15) Briefly, human FRa cDNA (amino acid residues 23-234) without signal peptide and glycophosphatidylinositol anchor peptide was prepared from RT-PCR product derived from KB cells. The primer sequences were
MAb REACTIVE WITH FOLATE RECEPTOR ALPHA AND BETA
5¢-tccaggttccactggtgaccagacaaggattgcatgggccaggactg-3¢ (upstream) and 5¢-gctagcggatccacgcggaaccagactcatggctgcagca tagaacc-3¢ (downstream). The murine Igk leader sequence at the N-terminus was inserted by overlap extension PCR using the following upstream primers: 5¢-tcctgctatgggtactgctgctc tgggttccaggttccactggtgac-3¢ and 5¢-cgtacgagccaccatggagaca gacacactcctgctatgggtactgctg-3¢. These primers were introduced restriction enzyme sites (underlined) for subcloning into the BsiWI and NheI sites in the human Fc fusion protein expression vector pUCOE.(16) CHO-S cells (Life Technologies) were transfected with pUCOE-FRa using a FreeStyle Max
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Cho Expression System (Life Technologies) according to the manufacturer’s instructions. FRa-Fc protein was purified from culture medium supernatants by Protein A affinity column chromatography (GE Healthcare, Tokyo, Japan). The human FRb cDNA (amino acid residues 1-230 containing the Kozak consensus sequence) without the glycophosphatidylinositol anchor peptide was amplified from human FRb in the pEF-BOS vector by the following primer sets: 5¢-agaaagacatggtctggaaatggatg3¢ (upstream) and 5¢-tcaattcacatgcatggctgcagcatagaacc-3¢ (downstream). The PCR product was ligated into the pCR2.1-
FIG. 1. Reactivity of clone 5 with FRaexpressing and FRb-expressing cells. (A) FRa-expressing cell lines IGROV-1 and KB, FRa-negative SKOV-1, FRb-transfected B30019, and B300-19 were incubated with clone 5, anti-FRa (Mov18), anti-FRb (94b), or isotypematched control MAb (murine IgG1 or rat IgG2a) and the cells were analyzed by flow cytometry after incubating with fluorescentlabeled secondary antibodies. White and hatched histograms show fluorescence signals with the isotype-matched control MAb and anti-FR MAb, respectively. (B) PBMCs and M-CSFdifferentiated macrophages were incubated with FITC-labeled MAbs (anti-CD3, antiCD14, anti-CD16, or anti-CD20), or Alexa 488-labeled anti-CD14 MAb and clone 5. Incubation with clone 5 was followed by APClabeled goat anti-rat antibody. The fluorescence signals were analyzed by flow cytometry. Double-positive cells are indicated as percentages. Data are representative of independent experiments from four donors.
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TOPO vector (Life Technologies), and the KpnI/XhoIdigested insert was then ligated into a pBacPAK9 baculovirus transfer vector (Clontech Laboratories, Mountain View, CA). The recombinant baculovirus was produced by transfecting Sf21 cells (Clontech) with pBacPAK9-FRb and BacPAK6 viral DNA (Clontech) using the BacPAKTM Baculovirus Expression System (Clontech) according to the manufacturer’s instructions. Suspension cultures of Sf21 cells (2 · 106 cells/mL) were infected with the recombinant virus stock at a multiplicity of infection of 10 for 2 days. FRb was purified from culture medium supernatants by affinity chromatography using clone 5 immobilized on NHS-activated Sepharose 4 Fast Flow (GE Healthcare). The purity of these proteins was evaluated by 7.5–15% gradient SDS-PAGE. Surface plasmon resonance
Clone 5 was diluted in 10 mM sodium acetate buffer (pH 4.5) to a final concentration of 18 mg/mL, and coupled to a general layer medium (GLM) chip (Bio-Rad) using a ProteOn Amine Coupling Kit (Bio-Rad). Remaining activated carboxylic groups were quenched with ethanolamine. The immobilization level was approximately 13,000 RU. The blank channel remained empty to serve as a reference. The purified analyte (FRa-Fc or FRb) was injected into the ProteOn XPR36 system (Bio-Rad) at four different concentrations (25, 12.5, 3.16, and 1.5 mg/mL). Regeneration was performed with 10 mM glycine–HCl (pH 2.5). All binding sensorgrams were collected, processed, and analyzed using the integrated ProteOn Manager software (Bio-Rad). Binding curves were fitted using the Langmuir model describing a 1:1 binding stoichiometry. Each individually captured antibody interacting with the four concentrations of antigen was fitted using ka, kd, and KD. ka is the association rate constant for the antibody–antigen binding, kd is the dissociation rate constant for the pair, and KD is the equilibrium dissociation constant (kd/ka). Complement-dependent cytotoxicity (CDC) activity
CDC activity was performed as described in the literature with slight modification.(17) Briefly, target cells (KB or FRbB300-19)were seeded at 5 · 104 cells/well in 96-well flatbottom plates in quadruplicate. In preliminary experiments using concentrations of 0.1, 1, 10, and 25 mg/mL of clone 5, the optimal concentration for CDC was determined to be 10 or 25 mg/mL. One hundred microliters of clone 5 or control rat IgG2a at 10 mg/mL was added to each well and incubated for 15 min at room temperature. Then, 10% baby rabbit
‰ FIG. 2. Presence of FR-expressing cells in placenta, rheumatoid arthritis synovium, and liver from cynomolgus monkey and common marmoset. (A) Placenta and rheumatoid arthritis synovium were incubated with anti-FRa (Mov18), isotypematched control MAb (mouse IgG1 or rat IgG2a), and the staining patterns were evaluated by immunohistochemistry as described in Materials and Methods. Original magnifications, 400· (placenta) and 200· (rheumatoid arthritis synovium). (B) Liver from cynomolgus monkey and common marmoset was incubated with anti-FRa (Mov18), anti-FRb (94b), anti-FRab (clone 5), anti-CD68 (Y1/82A), or isotype-matched control MAb (mouse IgG1 or rat IgG2b), and the staining patterns were evaluated by immunohistochemistry as described in Materials and Methods. Original magnification, 200 · .
MAb REACTIVE WITH FOLATE RECEPTOR ALPHA AND BETA
complement (CL3441, Cedarlane Laboratories, Ontario, Canada) was added to each well and incubated for 2 h at 37C and 5% CO2. For spontaneous or maximum controls, target cells without the MAb and complement were added to medium alone or 1% Triton X-100, respectively. The cytotoxicity was evaluated by colorimetric assay using a Cell Counting Kit-8, according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). CDC activity was calculated as cytotoxicity (%) = (experimental colorimetric assay of target cells – spontaneous colorimetric assay of target cells)/ (maximum colorimetric assay of target cells – spontaneous colorimetric assay of target cells) · 100. Antibody-dependent cellular cytotoxicity (ADCC) activity
Spleens from Wistar rats were minced, homogenized in IMDM, and centrifuged after passing through a cell strainer
FIG. 3. Presence of FR-expressing cells in carcinomas. Ovarian and lung carcinomas were incubated with anti-FRa (Mov18), anti-FRb (94b), anti-FRab (clone 5), anti-CD68 (EBM11), or isotype-matched control MAb (mouse IgG1 or rat IgG2a), and the staining patterns were evaluated by immunohistochemistry as described in Materials and Methods. This figure is representative of six positively stained serous lung carcinomas and six positively stained lung adenocarcinomas. Original magnification, 200 · .
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Table 1. Reactivity of Anti-FRa, Anti-FRb, and Anti-FRab MAbs in Carcinoma Tissues Tumor AntiAntiAntihistology FRa FRb FRab Carcinoma (patient numbers) positive positive positive Ovarian
Lung
Serous carcinoma (n = 10) Clear cell carcinoma (n = 2) Adenocarcinoma (n = 13) Squamous cell carcinoma (n = 6)
6
4
6
0
0
0
6
4
6
0
0
0
(BD Pharmingen). The cell pellet was suspended in RBC lysis buffer (Sigma-Aldrich), and these splenocytes were used as effector cells. Target cells (KB or FRb-B300-19) were seeded at 5 · 104 cells/well in 96-well U-bottom plates in quadruplicate. In preliminary experiments using concentrations of 0.1, 1, 10, and 25 mg/mL of clone 5, the optimal concentration for ADCC was 10 or 25 mg/mL. One hundred microliters of clone 5 or rat IgG2a at 10 mg/mL and effector cells (PBMCs or rat splenocytes) at 5 · 106 cells were added to each well at an effector cell/target cell ratio of 100:1, and further incubated for 4 h at 37C and 5% CO2. The cytotoxicity was measured by lactate dehydrogenase release in culture supernatants (Cytotoxicity Detection Kit plus, Roche Applied Science, Penzberg, Germany), according to the manufacturer’s instructions. ADCC activity was calculated as cytotoxicity (%) = (experimental release of target cells – spontaneous release of effector cells – spontaneous release of target cells)/(maximal release of target cells – spontaneous release of target cells) · 100.
FIG. 4. Effect of human serum on reactivity of clone 5 with FRa-expressing and FRb-expressing cells. FRa-expressing KB and FRb-transfected B300-19 were incubated with clone 5 or isotype-matched rat IgG2a in the presence or absence of human-type AB serum. The cells were analyzed by flow cytometry as described in Materials and Methods. White and hatched histograms show fluorescence signals with the IgG2a and clone 5, respectively.
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Antibody-dependent cellular phagocytosis (ADCP) activity
Results
KB were labeled with CellVue Claret Far Red (SigmaAldrich) and seeded at 2 · 104 cells/well in 96-well U-bottom plates in quadruplicate. In preliminary experiments using concentrations of 0.1, 1, 10, and 25 mg/mL of clone 5, the optimal concentration for ADCP was 10 or 25 mg/mL. One hundred microliters of clone 5, its F(ab’)2 fragment or rat IgG2a at 10 mg/ mL, and macrophages (1 · 105) at an effector cell/target cell ratio of 5:1 were added to each well, and further incubated for 4 h at 37C and 5% CO2. After a 4-h incubation, cells were harvested and blocked with human-type AB serum, and subsequently stained with a mixture of Alexa Fluor 488-labeled murine antihuman CD11b (ICRF44, BD Pharmingen) and Alexa Fluor 488labeled murine anti-human CD14 to identify macrophages. The cell-associated fluorescence of Alexa 488/FITC (FL1, macrophages) and Far Red (FL4, target cells) was measured by flow cytometry. ADCP activity was calculated as ratios (%) of KB counts phagocytosed by macrophages to total KB counts.
Clone 5 MAb reacts with FRa- and FRb-expressing cells
Statistical analysis
Statistical analyses were performed using the nonparametric Mann–Whitney U-test. A value of p < 0.05 was considered to be statistically significant.
Clone 5 MAb reacted with the FRa-expressing cell lines IGROV-1, KB, and FRb-transfected B300-19, but not with FRa-negative SKOV3 and B300-19 (Fig. 1A). In addition, clone 5 reacted with a small fraction of monocytes (CD14+) and with most macrophages cultured with M-CSF, but not T cells (CD3+), B cells (CD20+), and natural killer cells (CD16+) (Fig. 1B). In immunohistochemistry experiments, clone 5 stained FRa-expressing trophoblasts and FRb-expressing macrophages from human placenta, FRb-expressing macrophages from rheumatoid arthritis synovium, and liver macrophages from cynomolgus monkey and common marmoset (Fig. 2A, B). In addition, clone 5 stained a large fraction of cancer cells and a significant fraction of TAMs in ovarian and lung carcinomas (Fig. 3). In greater detail, clone 5 stained cancer cells and/or macrophages in six out of 10 samples of serous carcinoma of the ovary (60%) and six out of 13 samples of adenocarcinomas of the lung (46%), but not in clear cell carcinoma of the ovary and squamous cell carcinoma of the lung (Table 1). The previous report suggested the presence of soluble FRg in human serum.(4) We next examined whether human serum
FIG. 5. Determination of apparent steady-state dissociation constants for the interaction of clone 5 with FRa and FRb. (A) Recombinant FRa-Fc and FRb proteins were purified by Protein A and clone 5 affinity column chromatography, respectively, and their relative purities were evaluated by SDS-PAGE. Molecular weight markers are shown on the right. (B) Rate constants and steady-state dissociation constants were measured using immobilized clone 5 and FRa-Fc and FRb as analytes. For each experiment, four concentrations of analyte were assayed in triplicate. ka is the association rate constant for the antibody–antigen binding, kd is the dissociation rate constant for the pair, and KD is the equilibrium dissociation constant (kd/ka). (C) Kinetics of FRa-Fc and FRb binding to immobilized clone 5 are shown in sensorgrams. Protein concentrations are indicated next to the curves.
MAb REACTIVE WITH FOLATE RECEPTOR ALPHA AND BETA
inhibits the binding of clone 5 to FRa- or FRb-expressing cells in order to know the reactivity of clone 5 with FRg. As shown in Figure 4, the binding was not inhibited in the presence of human serum. Clone 5 MAb has high affinity for soluble FRa and FRb
Soluble FRa, containing the human IgG1-Fc portion, and soluble FRb were produced as described in Materials and Methods. The purified soluble FRa and FRb proteins migrated as single bands of 65 kDa and 22 kDa, respectively, in SDS-PAGE, consistent with their estimated molecular weights (Fig. 5A). In the surface plasmon resonance analysis, soluble FRa-Fc and FRb reacted with immobilized clone 5 on sensor chips with high affinity (KD = 6.26 · 10-9 M and 4.33 · 10-9 M, respectively) (Fig. 5B). In vitro functional analysis of clone 5 with FRa- and FRb-expressing cell lines
We examined whether clone 5 mediates CDC and ADCC in FRa-expressing KB and FRb-transfected B300-19. Clone 5 significantly mediated CDC in these cells in the presence of rabbit complement relative to the control MAb (Fig. 6A). A previous report indicated that rat IgG2a could mediate ADCC by human natural killer cells.(18) Clone 5 MAb mediated considerable ADCC in FRa-expressing KB and FRb-transfected
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B300-19 by human blood mononuclear cells; however, this level of ADCC was not statistically significant compared to that mediated by the control MAb. In contrast, clone 5 significantly mediated ADCC in FRa-expressing and FRbexpressing cells by rat spleen cells compared to the control MAb (Fig. 6B). Next, ADCP was examined using FRaexpressing KB and macrophages. In Figure 7A, regions 1 and 4 show Far Red-labeled KB cells and macrophages stained with the mixture of Alexa 488-labeled anti-CD11b and anti-CD14 MAbs, respectively, while KB phagocytosed by macrophages are shown as double-positive cells in region 3. Clone 5 significantly mediated the phagocytosis of KB cells by macrophages compared to the control MAb, whereas the F(ab’)2 form was inactive, indicating the relevance of the Fc gamma receptor to this ADCP (Fig. 7). Discussion
We obtained many anti-FRb-producing hybridoma clones by immunizing human FRb-transfected rat cell line into rats. Unexpectedly, we found that one clone reacts with FRa in addition to FRb. Clone 5 (hereafter referred to as anti-FRab) reacted with FRa- and FRb-expressing cell lines, a small fraction of peripheral blood monocytes, and most macrophages cultured with M-CSF. The immunohistochemical analysis showed that an anti-FRab stained cancer cells and a significant
FIG. 6. CDC and ADCC activity of clone 5 in FRa- and FRb-expressing cells. (A) FRa-expressing KB or FRb-expressing B300-19 were incubated in the presence of 10 mg/mL of clone 5 (-) or control rat IgG2a (,) with 10% baby rabbit complement. Cytotoxicity was evaluated by colorimetric assay. CDC was calculated as cytotoxicity (%), as described in Materials and Methods. Percentages are mean – SEM of four independent experiments. *p < 0.05 versus control rat IgG2a. (B) FRa-expressing KB or FRb-expressing B300-19 were incubated with PBMCs (left) or rat splenocytes (right) at an effector-target ratio of 100:1 in the presence of 10 mg/mL of clone 5 (-) or control rat IgG2a (,). Cytotoxicity was measured by lactate dehydrogenase release in culture supernatants. ADCC activity was calculated as cytotoxicity (%) as described in Materials and Methods. Percentages are mean – SEM of independent experiments from four different donors. *p < 0.05 versus control rat IgG2a.
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FIG. 7. ADCP activity of macrophages against FRa-expressing KB cells mediated by clone 5. (A) KB cells were labeled with CellVue Claret Far Red, and human macrophages were labeled with a mixture of Alexa Fluor 488-labeled CD11b and CD14. These cells were combined at an effector-target ratio of 5:1 and incubated in the presence of 10 mg/mL of rat IgG2a, clone 5, or the F(ab’)2 fragment, as described in Materials and Methods. ADCP activity of macrophages against FRa-expressing KB cells was detected by flow cytometry. Percentages in region 3 are the ratios of KB counts phagocytosed by macrophages to total KB counts. Data are representative of independent experiments from four different donors. (B) Percentages are the ratios of KB counts phagocytosed by macrophages to total KB counts in the presence of rat IgG2a, clone 5, or its F(ab’)2 fragment. Percentages are mean – SEM of independent experiments from four different donors. **p < 0.01 versus rat IgG2a and the F(ab’)2 fragment.
fraction of TAMs. The percentages of FRa positive cells in the serous type of ovarian cancer were lower compared to previous reports.(19,20) The small sample size in our study might have caused a slight bias. An anti-FRab also reacted with activated macrophages from the cynomolgus monkey and common marmoset. Further studies in nonhuman primates may provide useful information about the pharmacodynamics and toxicity of the anti-FRab MAb as a prelude to clinical trials. An anti-FRab showed similar binding with high affinity to soluble FRa and FRb. It has been reported that there is a significant extent of amino acid identity between FRa, FRb, and FRg and that FRg may be present as the soluble form in serum but not on the plasma membrane of myeloid cells.(4) The binding of an anti-FRab to FRa- and FRb-expressing cells was not inhibited in the presence of human serum. Although whether an anti-FRab reacts with FRg has not been determined, the above finding supports that the MAb could target FRa- and FRb-expressing cells in vivo, even in the presence of soluble FRg. Fc gamma-mediated effector functions such as CDC and ADCC have been suggested to be important for the in vivo activity of therapeutic antibodies.(21) An anti-FRab mediated CDC and ADCC in FRa- and FRb-expressing cells, suggesting that this MAb could induce cytotoxicity in FRa-
expressing cancer cells and FRb-expressing TAMs in vivo. In addition, anti-FRab mediated ADCP of FRa-expressing KB cells by macrophages via the Fc gamma receptor. The cytotoxic or phagocytotic potential of a rat-derived anti-FRab in ADCC and ADCP using human cells may be increased by changing the rat Fc portion to a humanized form. Furthermore, a fully humanized anti-FRab will be required in clinical application for cancer. It has been reported that TAMs can stimulate angiogenesis and enhance tumor cell invasion, motility, and intravasation. TAMs are also immunosuppressive, preventing tumor cell attack by natural killer and T cells during tumor progression.(22,23) There is strong evidence for specific subsets of TAMs as prognostic factors.(24–26) We reported that FRbexpressing macrophages were prominent in the perivascular area of the tumor-invasive front and produced VEGF.(14,27) In fact, the increased number of FRb-expressing TAMs was correlated with a poor prognosis in pancreatic cancer. Thus, an anti-FRab MAb that targets cancer cells and a subset of TAMs may become a more potent tool to inhibit cancer growth compared to either an anti-FRa or an anti-FRb MAb. Folateconjugated drugs delivered via FR-mediated cellular uptake have been shown to be promising anti-cancer therapeutics in preclinical animal models and early clinical trials.(28,29)
MAb REACTIVE WITH FOLATE RECEPTOR ALPHA AND BETA
Interestingly, a folate-conjugated mouse polyclonal immunoglobulin induced ADCC in FRa-expressing cells, inhibited tumor growth, and improved the survival rate in an animal model.(30) However, these folate-conjugated agents have not been well characterized with regard to their ability to target TAMs. Relative to anti-FR MAbs, FR-mediated cellular uptake of the folate-conjugated drugs may be inhibited by high folate serum concentrations. Furthermore, in addition to FRexpressing cells, folate-conjugated drugs may target RFC- and PFCT-expressing normal cells and cause detrimental side effects. One disadvantage using MAbs is their reduced penetration into tumors due to their high molecular weight compared to low molecular weight drugs. It is anticipated that administering a smaller anti-FRab MAb-derived polypeptide such as a single-chain or a single-domain MAb would result in an increase in tumor penetration. In conclusion, this study suggests that the anti-FRab MAb described here is a promising therapeutic candidate for cancers in which macrophages promote tumor progression. Author Disclosure Statement
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15.
The authors have no financial interests to disclose. References
1. McNulty H, Pentieva K, Hoey L, Strain J, and Ward M: Nutrition throughout life: folate. Int J Vitam Nutr Res 2012;82:348–354. 2. Desmoulin SK, Hou Z, Gangjee A, and Matherly LH: The human proton-coupled folate transporter: biology and therapeutic applications to cancer. Cancer Biol Ther 2012;13:1355–1373. 3. Low PS, and Antony AC: Folate receptor-targeted drugs for cancer and inflammatory diseases. Adv Drug Deliv Rev 2004;56:1055–1058. 4. Elnakat H, and Ratnam M: Distribution, functionality and gene regulation of folate receptor isoforms: implications in targeted therapy. Adv Drug Deliv Rev 2004;6:1067–1084. 5. Leamon CP, and Jackman AL: Exploitation of the folate receptor in the management of cancer and inflammatory disease. Vitam Horm 2008;79:203–233. 6. Teng L, Xie J, Teng L, and Lee RJ: Clinical translation of folate receptor-targeted therapeutics. Expert Opin Drug Deliv 2012;9:901–908. 7. Nagai T, Tanaka M, Tsuneyoshi Y, Xu B, Michie SA, Hasui K, Hirano H, Arita K, and Matsuyama T: Targeting tumor-associated macrophages in an experimental glioma model with a recombinant immunotoxin to folate receptor beta. Cancer Immunol Immunother 2009;58:1577–1586. 8. Puig-Kro¨ger A, Sierra-Filardi E, Domı´nguez-Soto A, Samaniego R, Corcuera MT, Go´mez-Aguado F, Ratnam M, Sa´nchez-Mateos P, and Corbı´ AL: Folate receptor beta is expressed by tumor-associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages. Cancer Res 2009;69:9395–6403. 9. Gibbs DD, Theti DS, Wood N, Green M, Raynaud F, Valenti M, Forster MD, Mitchell F, Bavetsias V, Henderson E, and Jackman AL: BGC 945, a novel tumor-selective thymidylate synthase inhibitor targeted to alpha-folate receptoroverexpressing tumors. Cancer Res 2005;65:11721–11728. 10. Armstrong DK, White AJ, Weil SC, Phillips M, and Coleman RL: FarletuzuMAb (a monoclonal antibody against
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folate receptor alpha) in relapsed platinum-sensitive ovarian cancer. Gynecol Oncol 2013;129:452–458. Reddy JA1, Dorton R, Bloomfield A, Nelson M, Vetzel M, Guan J, and Leamon CP: Rational combination therapy of vintafolide (EC145) with commonly used chemotherapeutic drugs. Clin Cancer Res 2014;20:2104–2114. Shen F, Ross JF, Wang X, and Ratnam M: Identification of a novel folate receptor, a truncated receptor, and receptor type beta in hematopoietic cells: cDNA cloning, expression, immunoreactivity, and tissue specificity. Biochemistry 1994;33:1209–1215. Nagayoshi R1, Nagai T, Matsushita K, Sato K, Sunahara N, Matsuda T, Nakamura T, Komiya S, Onda M, and Matsuyama T: Effectiveness of anti-folate receptor beta antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages. Arthritis Rheum 2005;52:2666–2675. Kurahara H, Takao S, Kuwahata T, Nagai T, Ding Q, Maeda K, Shinchi H, Mataki Y, Maemura K, Matsuyama T, and Natsugoe S: Clinical significance of folate receptor b-expressing tumor-associated macrophages in pancreatic cancer. Ann Surg Oncol 2012;19:2264–2271. Chen C1, Ke J, Zhou XE, Yi W, Brunzelle JS, Li J, Yong EL, Xu HE, and Melcher K: Structural basis for molecular recognition of folic acid by folate receptors. Nature 2013;500:486–489. Boscolo S1, Mion F, Licciulli M, Macor P, De Maso L, Brce M, Antoniou MN, Marzari R, Santoro C, and Sblattero D: Simple scale-up of recombinant antibody production using an UCOE containing vector. N Biotechnol 2012;29:477–484. Gazzano-Santoro H, Ralph P, Ryskamp TC, Chen AB, and Mukku VR: A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody. J Immunol Methods 1997;202:163–171. Song ES, Young K, and Sears DW: Rat and human natural killers exhibit contrasting immunoglobulin G subclass specificities in antibody-dependent cellular cytotoxicity reflecting differences in their Fc receptors (Fc gamma R). J Leukoc Biol 1990;48:524–530. Ko¨bel M, Madore J, Ramus SJ, Clarke BA, Pharoah PD, Deen S, Bowtell DD, Odunsi K, Menon U, Morrison C, Lele S, Bshara W, Sucheston L, Beckmann MW, Hein A, Thiel FC, Hartmann A, Wachter DL, Anglesio MS, Høgdall E, Jensen A, Høgdall C, Kalli KR, Fridley BL, Keeney GL, Fogarty ZC, Vierkant RA, Liu S, Cho S, Nelson G, Ghatage P, Gentry-Maharaj A, Gayther SA, Benjamin E, Widschwendter M, Intermaggio MP, Rosen B, Bernardini MQ, Mackay H, Oza A, Shaw P, Jimenez-Linan M, Driver KE, Alsop J, Mack M, Koziak JM, Steed H, Ewanowich C, DeFazio A, Chenevix-Trench G, Fereday S, Gao B, Johnatty SE, George J, Galletta L; AOCS Study Group, Goode EL, Kjær SK, Huntsman DG, Fasching PA, Moysich KB, Brenton JD, and Kelemen LE: Evidence for a timedependent association between FOLR1 expression and survival from ovarian carcinoma: implications for clinical testing. An Ovarian Tumour Tissue Analysis consortium study. Br J Cancer 2014;111:2297–307. O’Shannessy DJ, Somers EB, Smale R, and Fu YS: Expression of folate receptor-a (FRA) in gynecologic malignancies and its relationship to the tumor type. Int J Gynecol Pathol 2013;32:258–68. Mitoma H, Horiuchi T, Tsukamoto H, Tamimoto Y, Kimoto Y, Uchino A, To K, Harashima S, Hatta N, and Harada M: Mechanisms for cytotoxic effects of anti-tumor necrosis
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factor agents on transmembrane tumor necrosis factor alphaexpressing cells: comparison among inflixiMAb, etanercept, and adalimuMAb. Arthritis Rheum 2008;58:1248–1257. Noy R, and Pollard JW: Tumor-associated macrophages: from mechanisms to therapy. Immunity 2014;41:49–61. De Palma M, and Lewis CE: Macrophage regulation of tumor responses to anticancer therapies. Cancer Cell 2013;23:277–286. Shigeoka M, Urakawa N, Nakamura T, Nishio M, Watajima T, Kuroda D, Komori T, Kakeji Y, Semba S, and Yokozaki H: Tumor associated macrophage expressing CD204 is associated with tumor aggressiveness of esophageal squamous cell carcinoma. Cancer Sci 2013;104:1112–1119. Prosniak M, Harshyne LA, Andrews DW, Kenyon LC, Bedelbaeva K, Apanasovich TV, Heber-Katz E, Curtis MT, Cotzia P, and Hooper DC: Glioma grade is associated with the accumulation and activity of cells bearing M2 monocyte markers. Clin Cancer Res 2013;19:3776–3786. Laoui D, Movahedi K, Van Overmeire E, Van den Bossche J, Schouppe E, Mommer C, Nikolaou A, Morias Y, De Baetselier P, and Van Ginderachter JA: Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions. Int J Dev Biol 2011;55:861–867. Escribese MM, Sierra-Filardi E, Nieto C, Samaniego R, Sa´nchez-Torres C, Matsuyama T, Calderon-Go´mez E, Vega MA, Salas A, Sa´nchez-Mateos P, and Corbı´ AL: The prolyl hydroxylase PHD3 identifies proinflammatory mac-
rophages and its expression is regulated by activin A. J Immunol 2012;189:1946–1954. 28. Reddy JA, Dorton R, Westrick E, Dawson A, Smith T, Xu LC, Vetzel M, Kleindl P, Vlahov IR, and Leamon CP: Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate. Cancer Res 2007;67:4434–4442. 29. Luyckx M, Votino R, Squifflet JL, and Baurain JF: Profile of vintafolide (EC145) and its use in the treatment of platinum-resistant ovarian cancer. Int J Women Health 2014;6:351–358. 30. Li H, Lu Y, Piao L, Wu J, Yang X, Kondadasula SV, Carson WE, and Lee RJ: Folate-immunoglobulin G as an anticancer therapeutic antibody. Bioconjug Chem 2010;21:961–968.
Address correspondence to: Takami Matsuyama The Center for Advanced Biomedical Sciences and Swine Research Kagoshima University 8-35-1 Sakuragaoka Kagoshima 890-8544 Japan E-mail: [email protected] Received: September 19, 2014 Accepted: February 27, 2015
Production of a High-affinity Monoclonal Antibody Reactive with Folate Receptors Alpha and Beta Taku Nagai,1 Yuko Furusho,1 Hua Li,1 Kazuhisa Hasui,1 Sumika Matsukita,2 Kazunobu Sueyoshi,3 Masakazu Yanagi,4 Masaki Hatae,5 Sonshin Takao,6 and Takami Matsuyama 6
Folate receptors a (FRa) and b (FRb) are two isoforms of the cell surface glycoprotein that binds folate. The expression of FRa is rare in normal cells and elevated in cancer cells. Thus, FRa-based tumor-targeted therapy has been a focus area of laboratory research and clinical trials. Recently, it was shown that a significant fraction of tumor-associated macrophages expresses FRb and that these cells can enhance tumor growth. Although FRa and FRb share 70% identity in their deduced amino acid sequence, a monoclonal antibody (MAb) reactive with both receptors has not been developed. A MAb that can target both FRa-expressing cancer cells and FRbexpressing tumor-associated macrophages may provide a more potent therapeutic tool for cancer than individual anti-FRa or anti-FRb MAbs. In this study, we developed a MAb that recognizes both FRa and FRb (antiFRab). The anti-FRab specifically stained trophoblasts and macrophages from human placenta, synovial macrophages from rheumatoid arthritis patient, liver macrophages from cynomolgus monkey and common marmoset, and cancer cells and tumor-associated macrophages from ovary and lung carcinomas. Surface plasmon resonance showed that the anti-FRab bound to soluble forms of the FRa and FRb proteins with high affinity (KD = 6.26 · 10 -9 M and 4.33 · 10 -9 M, respectively). In vitro functional analysis of the anti-FRab showed that this MAb mediates complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis of FRa-expressing and FRb-expressing cell lines. The anti-FRab MAb is a promising therapeutic candidate for cancers in which macrophages promote tumor progression.
Introduction
F
olate is an essential vitamin that is used in singlecarbon-donor reactions in the biosynthesis of purines and thymidylates and is needed for methionine synthesis.(1) Folate can enter the cell through three different routes: reduced folate carrier (RFC) with low affinity for folate, proton-coupled folate transporter (PCFT), and folate receptors (FRs).(2–4) RFC and PCFT are ubiquitously expressed in most normal tissues. There are three isoforms of the folate receptor in humans. The expression of FRa and FRb isoforms is restricted to the surface of epithelial cells such as trophoblasts, the choroid plexus and proximal kidney tubules, and activated macrophages, respectively. The g isoform is a soluble protein secreted by myeloid cells that has not been well characterized. Interestingly, many cancer types including ovary, lung, and colorectal carcinomas overexpress FRa, while tumor-associated macrophages (TAMs) express FRb.(5–8) Regardless of the physiological role for FRs in cancer cells, the disparate expression levels of FRa between cancerous and normal cells make FRa an attractive
candidate for targeted biological therapy of cancers. FRabased tumor-targeted therapy using folate derivatives, antiFRa monoclonal antibodies (MAbs), and folate-conjugated drugs have been an active area of laboratory research and clinical trials.(9–11) We previously showed that the depletion of FRb-expressing TAMs using an anti-FRb immunotoxin reduced tumor growth.(7) Although FRa and b share 70% identity in their deduced amino acid sequences,(12) a MAb reactive with both FRa and b has not been reported. A MAb that can target both cancer cells and tumor-associated macrophages may provide a more potent therapeutic tool for cancer than individual anti-FRa or anti-FRb MAbs. In this study, we developed a MAb reactive with FRa and FRb and examined its in vitro effects on FRa- and FRb-expressing cell lines. Materials and Methods Cell lines
B300-19 (murine pre-B cell), KB (human nasopharynx cell), NS1 (murine myeloma cell), RBL2H3 (rat basophilic
1
Department of Immunology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. Department of Clinical Pathology, Kagoshima Kousei-Ren Hospital, Kagoshima, Japan. Department of Clinical Pathology, 4Department of Surgery, 5Department of Obstetrics, Kagoshima City Hospital, Kagoshima, Japan. 6 The Center for Advanced Biomedical Sciences and Swine Research, Kagoshima University, Kagoshima, Japan. 2 3
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leukemia cell), and SKOV-3 (human ovarian carcinoma cell) cell lines were obtained from ATCC (Manassas, VA). JFP-J1 hybridoma cells producing anti-GFP rat IgG2a were obtained from Riken BioResource Center (Tsukuba, Japan). The human ovarian carcinoma cell line IGROV-1 was a kind gift from Morphotech (Exton, PA). Both SKOV-3 and IGROV-1 were cultured in RPMI-1640 medium (Life Technologies, Carlsbad CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Nacalai Tesque Inc., Kyoto, Japan). The other cell lines were cultured in IMDM medium (Life Technologies) supplemented with 10% heatinactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin, except for B300-19 cells, which were maintained in the same medium containing 50 mM 2-mercaptoethanol. Human placenta, rheumatoid arthritis synovium, liver from cynomolgus monkey and common marmoset were obtained from Kagoshima University. Surgical specimens were obtained from 12 patients with ovarian carcinomas and 19 patients with lung cancer at Kagoshima City Hospital and Kagoshima Kousei-Ren Hospital. The diagnoses were made both clinically and pathologically. Tissue samples contained 10 serous and 2 clear cell carcinomas of the ovary, 13 adenocarcinomas, and 6 squamous cell carcinomas of the lung. This study was approved by the ethics committee of Kagoshima University; written informed consent was obtained from all of the subjects. Production of MAb reactive with FRa and FRb
FRb-expressing RBL2H3 and B300-19 were prepared by transfecting human FRb in the pEF-BOS vector, as previously described.(7,13) Wistar Kyoto rats (Charles River Laboratories, Yokohama, Japan) were injected via the hind footpads with 100 mL of an emulsion containing 1 · 107 of FRb-expressing RBL2H3 and Freund’s complete adjuvant. After 16 days, the lymphocytes from iliac lymph nodes were fused with NS1 myeloma cells at a ratio of 4:1 in 50% polyethyleneglycol (PEG 1500, Roche, Mannheim, Germany). The hybridoma cells were placed on 96-well plates and cultured IMDM medium supplemented with 10% heatinactivated FBS and HAT (Life Technologies). After 7 days post-fusion, the hybridoma supernatants were screened by means of immunofluorescence assay against FRb-B300-19 and KB. One MAb, named clone 5 (rat IgG2a) was selected for its reactivity with both KB and FRb-B300-19. The hybridoma culture supernatants were purified using goat anti-rat IgG-agarose affinity chromatography (Rockland, Gilbertsville, PA) and used for further experiments. The F(ab’)2 fragment was prepared using pepsin-agarose (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. All animal studies were performed in accordance with the ethical guidelines for animal experiments at Kagoshima University. Human peripheral blood mononuclear cells (PBMCs), monocytes, and M-CSF-differentiated macrophages were prepared from the peripheral blood of four healthy donors, as previously described.(7,13) Briefly, PBMCs were isolated using a Ficoll-hypaque density gradient, and monocytes were separated from PBMCs by adhesion to culture dishes. To obtain macrophages, monocytes were cultured in IMDM
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containing 10% FBS and 25 ng/mL recombinant human MCSF (PeproTech, Rocky Hill, NJ) for 7 days. Flow cytometric analysis
After blocking non-specific binding with 10% human-type AB serum, the cells were incubated with 1 mg of anti-human FRa (Mov 18, murine IgG1, which was a gift from Dr. S. Canevari, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy); anti-human FRb (94b, murine IgG1, which was produced in our laboratory)(7,11); clone 5; or one of the following two isotype-matched MAbs: murine IgG1 (MOPC-21, BD Pharmingen, San Jose, CA) or rat IgG2a (R35-95, also from BD Pharmingen) in PBS containing 1% bovine serum albumin and 0.1% NaN3. To examine the effect of soluble FRg in human serum, KB and FRb-expressing B300-19 were incubated with clone 5 or rat IgG2a in the presence of 50% of human-type AB serum. After washing with PBS containing 1% bovine serum albumin and 0.1% NaN3, cells were stained with fluorescein isothiocyanate (FITC)-labeled goat F(ab’)2 anti-rat IgG (H + L) (Leino Technologies, St. Louis, MO) or FITC-labeled goat F(ab’)2 anti-mouse IgG (H + L) (Southern Biotech, Birmingham, AL). For dual-color staining of PBMCs and macrophages, cells were incubated with clone 5, FITC-labeled murine anti-human CD3 (UCHT1, Bio-Rad Laboratories, Hercules, CA), Alexa Fluor 488-labeled murine anti-human CD14 (HCD14, BioLegend, San Diego, CA), FITC-labeled murine murine anti-human CD20 (2H7, Bio-Rad), rat IgG2a, or Alexa Fluor 488-labeled isotype control MAbs (BD Pharmingen). The reactivities of clone 5 and rat IgG2a were further detected with APC-labeled goat F(ab’)2 anti-rat IgG (sc-3832, Santa Cruz Biotechnology, Dallas, TX). The stained cells were analyzed using a CyAnTM ADP flow cytometer (Beckman Coulter, Brea, CA). Immunohistochemical analysis
Immunohistochemical analysis was performed as previously described.(7,14) Briefly, acetone-fixed frozen sections (6 mm) were incubated with anti-FRa, anti-FRb, clone 5, anti-CD68 (EBM11, Dako, Copenhagen, Denmark) for human tissues, anti-CD68 (Y1/82A, BD Pharmingen) for cynomolgus monkey and common marmoset tissues, or isotype-matched MAbs (murine IgG1, murine IgG2b or rat IgG2a). The staining was visualized with a horseradish peroxidase-conjugated MAX-PO secondary antibody kit (Nichirei Co., Tokyo, Japan) and NovaRed substrate (Vector Laboratories, Burlingame, CA), according to the manufacturer’s instructions. Slides were archived using a Digital Sight CCD camera (DS-Fi1, Nikon, Tokyo, Japan) and computer-aided image analyzer (NIS-Elements, Nikon). The staining intensity was assessed in 10 randomly selected 400x fields in each section by two investigators who were blinded to all clinical details. Positive staining in tumor tissues was defined as staining of >1% of the total cell number. Production of recombinant soluble FRa and FRb proteins
Recombinant soluble FRa-Fc protein was prepared as described previously.(15) Briefly, human FRa cDNA (amino acid residues 23-234) without signal peptide and glycophosphatidylinositol anchor peptide was prepared from RT-PCR product derived from KB cells. The primer sequences were
MAb REACTIVE WITH FOLATE RECEPTOR ALPHA AND BETA
5¢-tccaggttccactggtgaccagacaaggattgcatgggccaggactg-3¢ (upstream) and 5¢-gctagcggatccacgcggaaccagactcatggctgcagca tagaacc-3¢ (downstream). The murine Igk leader sequence at the N-terminus was inserted by overlap extension PCR using the following upstream primers: 5¢-tcctgctatgggtactgctgctc tgggttccaggttccactggtgac-3¢ and 5¢-cgtacgagccaccatggagaca gacacactcctgctatgggtactgctg-3¢. These primers were introduced restriction enzyme sites (underlined) for subcloning into the BsiWI and NheI sites in the human Fc fusion protein expression vector pUCOE.(16) CHO-S cells (Life Technologies) were transfected with pUCOE-FRa using a FreeStyle Max
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Cho Expression System (Life Technologies) according to the manufacturer’s instructions. FRa-Fc protein was purified from culture medium supernatants by Protein A affinity column chromatography (GE Healthcare, Tokyo, Japan). The human FRb cDNA (amino acid residues 1-230 containing the Kozak consensus sequence) without the glycophosphatidylinositol anchor peptide was amplified from human FRb in the pEF-BOS vector by the following primer sets: 5¢-agaaagacatggtctggaaatggatg3¢ (upstream) and 5¢-tcaattcacatgcatggctgcagcatagaacc-3¢ (downstream). The PCR product was ligated into the pCR2.1-
FIG. 1. Reactivity of clone 5 with FRaexpressing and FRb-expressing cells. (A) FRa-expressing cell lines IGROV-1 and KB, FRa-negative SKOV-1, FRb-transfected B30019, and B300-19 were incubated with clone 5, anti-FRa (Mov18), anti-FRb (94b), or isotypematched control MAb (murine IgG1 or rat IgG2a) and the cells were analyzed by flow cytometry after incubating with fluorescentlabeled secondary antibodies. White and hatched histograms show fluorescence signals with the isotype-matched control MAb and anti-FR MAb, respectively. (B) PBMCs and M-CSFdifferentiated macrophages were incubated with FITC-labeled MAbs (anti-CD3, antiCD14, anti-CD16, or anti-CD20), or Alexa 488-labeled anti-CD14 MAb and clone 5. Incubation with clone 5 was followed by APClabeled goat anti-rat antibody. The fluorescence signals were analyzed by flow cytometry. Double-positive cells are indicated as percentages. Data are representative of independent experiments from four donors.
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TOPO vector (Life Technologies), and the KpnI/XhoIdigested insert was then ligated into a pBacPAK9 baculovirus transfer vector (Clontech Laboratories, Mountain View, CA). The recombinant baculovirus was produced by transfecting Sf21 cells (Clontech) with pBacPAK9-FRb and BacPAK6 viral DNA (Clontech) using the BacPAKTM Baculovirus Expression System (Clontech) according to the manufacturer’s instructions. Suspension cultures of Sf21 cells (2 · 106 cells/mL) were infected with the recombinant virus stock at a multiplicity of infection of 10 for 2 days. FRb was purified from culture medium supernatants by affinity chromatography using clone 5 immobilized on NHS-activated Sepharose 4 Fast Flow (GE Healthcare). The purity of these proteins was evaluated by 7.5–15% gradient SDS-PAGE. Surface plasmon resonance
Clone 5 was diluted in 10 mM sodium acetate buffer (pH 4.5) to a final concentration of 18 mg/mL, and coupled to a general layer medium (GLM) chip (Bio-Rad) using a ProteOn Amine Coupling Kit (Bio-Rad). Remaining activated carboxylic groups were quenched with ethanolamine. The immobilization level was approximately 13,000 RU. The blank channel remained empty to serve as a reference. The purified analyte (FRa-Fc or FRb) was injected into the ProteOn XPR36 system (Bio-Rad) at four different concentrations (25, 12.5, 3.16, and 1.5 mg/mL). Regeneration was performed with 10 mM glycine–HCl (pH 2.5). All binding sensorgrams were collected, processed, and analyzed using the integrated ProteOn Manager software (Bio-Rad). Binding curves were fitted using the Langmuir model describing a 1:1 binding stoichiometry. Each individually captured antibody interacting with the four concentrations of antigen was fitted using ka, kd, and KD. ka is the association rate constant for the antibody–antigen binding, kd is the dissociation rate constant for the pair, and KD is the equilibrium dissociation constant (kd/ka). Complement-dependent cytotoxicity (CDC) activity
CDC activity was performed as described in the literature with slight modification.(17) Briefly, target cells (KB or FRbB300-19)were seeded at 5 · 104 cells/well in 96-well flatbottom plates in quadruplicate. In preliminary experiments using concentrations of 0.1, 1, 10, and 25 mg/mL of clone 5, the optimal concentration for CDC was determined to be 10 or 25 mg/mL. One hundred microliters of clone 5 or control rat IgG2a at 10 mg/mL was added to each well and incubated for 15 min at room temperature. Then, 10% baby rabbit
‰ FIG. 2. Presence of FR-expressing cells in placenta, rheumatoid arthritis synovium, and liver from cynomolgus monkey and common marmoset. (A) Placenta and rheumatoid arthritis synovium were incubated with anti-FRa (Mov18), isotypematched control MAb (mouse IgG1 or rat IgG2a), and the staining patterns were evaluated by immunohistochemistry as described in Materials and Methods. Original magnifications, 400· (placenta) and 200· (rheumatoid arthritis synovium). (B) Liver from cynomolgus monkey and common marmoset was incubated with anti-FRa (Mov18), anti-FRb (94b), anti-FRab (clone 5), anti-CD68 (Y1/82A), or isotype-matched control MAb (mouse IgG1 or rat IgG2b), and the staining patterns were evaluated by immunohistochemistry as described in Materials and Methods. Original magnification, 200 · .
MAb REACTIVE WITH FOLATE RECEPTOR ALPHA AND BETA
complement (CL3441, Cedarlane Laboratories, Ontario, Canada) was added to each well and incubated for 2 h at 37C and 5% CO2. For spontaneous or maximum controls, target cells without the MAb and complement were added to medium alone or 1% Triton X-100, respectively. The cytotoxicity was evaluated by colorimetric assay using a Cell Counting Kit-8, according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). CDC activity was calculated as cytotoxicity (%) = (experimental colorimetric assay of target cells – spontaneous colorimetric assay of target cells)/ (maximum colorimetric assay of target cells – spontaneous colorimetric assay of target cells) · 100. Antibody-dependent cellular cytotoxicity (ADCC) activity
Spleens from Wistar rats were minced, homogenized in IMDM, and centrifuged after passing through a cell strainer
FIG. 3. Presence of FR-expressing cells in carcinomas. Ovarian and lung carcinomas were incubated with anti-FRa (Mov18), anti-FRb (94b), anti-FRab (clone 5), anti-CD68 (EBM11), or isotype-matched control MAb (mouse IgG1 or rat IgG2a), and the staining patterns were evaluated by immunohistochemistry as described in Materials and Methods. This figure is representative of six positively stained serous lung carcinomas and six positively stained lung adenocarcinomas. Original magnification, 200 · .
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Table 1. Reactivity of Anti-FRa, Anti-FRb, and Anti-FRab MAbs in Carcinoma Tissues Tumor AntiAntiAntihistology FRa FRb FRab Carcinoma (patient numbers) positive positive positive Ovarian
Lung
Serous carcinoma (n = 10) Clear cell carcinoma (n = 2) Adenocarcinoma (n = 13) Squamous cell carcinoma (n = 6)
6
4
6
0
0
0
6
4
6
0
0
0
(BD Pharmingen). The cell pellet was suspended in RBC lysis buffer (Sigma-Aldrich), and these splenocytes were used as effector cells. Target cells (KB or FRb-B300-19) were seeded at 5 · 104 cells/well in 96-well U-bottom plates in quadruplicate. In preliminary experiments using concentrations of 0.1, 1, 10, and 25 mg/mL of clone 5, the optimal concentration for ADCC was 10 or 25 mg/mL. One hundred microliters of clone 5 or rat IgG2a at 10 mg/mL and effector cells (PBMCs or rat splenocytes) at 5 · 106 cells were added to each well at an effector cell/target cell ratio of 100:1, and further incubated for 4 h at 37C and 5% CO2. The cytotoxicity was measured by lactate dehydrogenase release in culture supernatants (Cytotoxicity Detection Kit plus, Roche Applied Science, Penzberg, Germany), according to the manufacturer’s instructions. ADCC activity was calculated as cytotoxicity (%) = (experimental release of target cells – spontaneous release of effector cells – spontaneous release of target cells)/(maximal release of target cells – spontaneous release of target cells) · 100.
FIG. 4. Effect of human serum on reactivity of clone 5 with FRa-expressing and FRb-expressing cells. FRa-expressing KB and FRb-transfected B300-19 were incubated with clone 5 or isotype-matched rat IgG2a in the presence or absence of human-type AB serum. The cells were analyzed by flow cytometry as described in Materials and Methods. White and hatched histograms show fluorescence signals with the IgG2a and clone 5, respectively.
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Antibody-dependent cellular phagocytosis (ADCP) activity
Results
KB were labeled with CellVue Claret Far Red (SigmaAldrich) and seeded at 2 · 104 cells/well in 96-well U-bottom plates in quadruplicate. In preliminary experiments using concentrations of 0.1, 1, 10, and 25 mg/mL of clone 5, the optimal concentration for ADCP was 10 or 25 mg/mL. One hundred microliters of clone 5, its F(ab’)2 fragment or rat IgG2a at 10 mg/ mL, and macrophages (1 · 105) at an effector cell/target cell ratio of 5:1 were added to each well, and further incubated for 4 h at 37C and 5% CO2. After a 4-h incubation, cells were harvested and blocked with human-type AB serum, and subsequently stained with a mixture of Alexa Fluor 488-labeled murine antihuman CD11b (ICRF44, BD Pharmingen) and Alexa Fluor 488labeled murine anti-human CD14 to identify macrophages. The cell-associated fluorescence of Alexa 488/FITC (FL1, macrophages) and Far Red (FL4, target cells) was measured by flow cytometry. ADCP activity was calculated as ratios (%) of KB counts phagocytosed by macrophages to total KB counts.
Clone 5 MAb reacts with FRa- and FRb-expressing cells
Statistical analysis
Statistical analyses were performed using the nonparametric Mann–Whitney U-test. A value of p < 0.05 was considered to be statistically significant.
Clone 5 MAb reacted with the FRa-expressing cell lines IGROV-1, KB, and FRb-transfected B300-19, but not with FRa-negative SKOV3 and B300-19 (Fig. 1A). In addition, clone 5 reacted with a small fraction of monocytes (CD14+) and with most macrophages cultured with M-CSF, but not T cells (CD3+), B cells (CD20+), and natural killer cells (CD16+) (Fig. 1B). In immunohistochemistry experiments, clone 5 stained FRa-expressing trophoblasts and FRb-expressing macrophages from human placenta, FRb-expressing macrophages from rheumatoid arthritis synovium, and liver macrophages from cynomolgus monkey and common marmoset (Fig. 2A, B). In addition, clone 5 stained a large fraction of cancer cells and a significant fraction of TAMs in ovarian and lung carcinomas (Fig. 3). In greater detail, clone 5 stained cancer cells and/or macrophages in six out of 10 samples of serous carcinoma of the ovary (60%) and six out of 13 samples of adenocarcinomas of the lung (46%), but not in clear cell carcinoma of the ovary and squamous cell carcinoma of the lung (Table 1). The previous report suggested the presence of soluble FRg in human serum.(4) We next examined whether human serum
FIG. 5. Determination of apparent steady-state dissociation constants for the interaction of clone 5 with FRa and FRb. (A) Recombinant FRa-Fc and FRb proteins were purified by Protein A and clone 5 affinity column chromatography, respectively, and their relative purities were evaluated by SDS-PAGE. Molecular weight markers are shown on the right. (B) Rate constants and steady-state dissociation constants were measured using immobilized clone 5 and FRa-Fc and FRb as analytes. For each experiment, four concentrations of analyte were assayed in triplicate. ka is the association rate constant for the antibody–antigen binding, kd is the dissociation rate constant for the pair, and KD is the equilibrium dissociation constant (kd/ka). (C) Kinetics of FRa-Fc and FRb binding to immobilized clone 5 are shown in sensorgrams. Protein concentrations are indicated next to the curves.
MAb REACTIVE WITH FOLATE RECEPTOR ALPHA AND BETA
inhibits the binding of clone 5 to FRa- or FRb-expressing cells in order to know the reactivity of clone 5 with FRg. As shown in Figure 4, the binding was not inhibited in the presence of human serum. Clone 5 MAb has high affinity for soluble FRa and FRb
Soluble FRa, containing the human IgG1-Fc portion, and soluble FRb were produced as described in Materials and Methods. The purified soluble FRa and FRb proteins migrated as single bands of 65 kDa and 22 kDa, respectively, in SDS-PAGE, consistent with their estimated molecular weights (Fig. 5A). In the surface plasmon resonance analysis, soluble FRa-Fc and FRb reacted with immobilized clone 5 on sensor chips with high affinity (KD = 6.26 · 10-9 M and 4.33 · 10-9 M, respectively) (Fig. 5B). In vitro functional analysis of clone 5 with FRa- and FRb-expressing cell lines
We examined whether clone 5 mediates CDC and ADCC in FRa-expressing KB and FRb-transfected B300-19. Clone 5 significantly mediated CDC in these cells in the presence of rabbit complement relative to the control MAb (Fig. 6A). A previous report indicated that rat IgG2a could mediate ADCC by human natural killer cells.(18) Clone 5 MAb mediated considerable ADCC in FRa-expressing KB and FRb-transfected
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B300-19 by human blood mononuclear cells; however, this level of ADCC was not statistically significant compared to that mediated by the control MAb. In contrast, clone 5 significantly mediated ADCC in FRa-expressing and FRbexpressing cells by rat spleen cells compared to the control MAb (Fig. 6B). Next, ADCP was examined using FRaexpressing KB and macrophages. In Figure 7A, regions 1 and 4 show Far Red-labeled KB cells and macrophages stained with the mixture of Alexa 488-labeled anti-CD11b and anti-CD14 MAbs, respectively, while KB phagocytosed by macrophages are shown as double-positive cells in region 3. Clone 5 significantly mediated the phagocytosis of KB cells by macrophages compared to the control MAb, whereas the F(ab’)2 form was inactive, indicating the relevance of the Fc gamma receptor to this ADCP (Fig. 7). Discussion
We obtained many anti-FRb-producing hybridoma clones by immunizing human FRb-transfected rat cell line into rats. Unexpectedly, we found that one clone reacts with FRa in addition to FRb. Clone 5 (hereafter referred to as anti-FRab) reacted with FRa- and FRb-expressing cell lines, a small fraction of peripheral blood monocytes, and most macrophages cultured with M-CSF. The immunohistochemical analysis showed that an anti-FRab stained cancer cells and a significant
FIG. 6. CDC and ADCC activity of clone 5 in FRa- and FRb-expressing cells. (A) FRa-expressing KB or FRb-expressing B300-19 were incubated in the presence of 10 mg/mL of clone 5 (-) or control rat IgG2a (,) with 10% baby rabbit complement. Cytotoxicity was evaluated by colorimetric assay. CDC was calculated as cytotoxicity (%), as described in Materials and Methods. Percentages are mean – SEM of four independent experiments. *p < 0.05 versus control rat IgG2a. (B) FRa-expressing KB or FRb-expressing B300-19 were incubated with PBMCs (left) or rat splenocytes (right) at an effector-target ratio of 100:1 in the presence of 10 mg/mL of clone 5 (-) or control rat IgG2a (,). Cytotoxicity was measured by lactate dehydrogenase release in culture supernatants. ADCC activity was calculated as cytotoxicity (%) as described in Materials and Methods. Percentages are mean – SEM of independent experiments from four different donors. *p < 0.05 versus control rat IgG2a.
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FIG. 7. ADCP activity of macrophages against FRa-expressing KB cells mediated by clone 5. (A) KB cells were labeled with CellVue Claret Far Red, and human macrophages were labeled with a mixture of Alexa Fluor 488-labeled CD11b and CD14. These cells were combined at an effector-target ratio of 5:1 and incubated in the presence of 10 mg/mL of rat IgG2a, clone 5, or the F(ab’)2 fragment, as described in Materials and Methods. ADCP activity of macrophages against FRa-expressing KB cells was detected by flow cytometry. Percentages in region 3 are the ratios of KB counts phagocytosed by macrophages to total KB counts. Data are representative of independent experiments from four different donors. (B) Percentages are the ratios of KB counts phagocytosed by macrophages to total KB counts in the presence of rat IgG2a, clone 5, or its F(ab’)2 fragment. Percentages are mean – SEM of independent experiments from four different donors. **p < 0.01 versus rat IgG2a and the F(ab’)2 fragment.
fraction of TAMs. The percentages of FRa positive cells in the serous type of ovarian cancer were lower compared to previous reports.(19,20) The small sample size in our study might have caused a slight bias. An anti-FRab also reacted with activated macrophages from the cynomolgus monkey and common marmoset. Further studies in nonhuman primates may provide useful information about the pharmacodynamics and toxicity of the anti-FRab MAb as a prelude to clinical trials. An anti-FRab showed similar binding with high affinity to soluble FRa and FRb. It has been reported that there is a significant extent of amino acid identity between FRa, FRb, and FRg and that FRg may be present as the soluble form in serum but not on the plasma membrane of myeloid cells.(4) The binding of an anti-FRab to FRa- and FRb-expressing cells was not inhibited in the presence of human serum. Although whether an anti-FRab reacts with FRg has not been determined, the above finding supports that the MAb could target FRa- and FRb-expressing cells in vivo, even in the presence of soluble FRg. Fc gamma-mediated effector functions such as CDC and ADCC have been suggested to be important for the in vivo activity of therapeutic antibodies.(21) An anti-FRab mediated CDC and ADCC in FRa- and FRb-expressing cells, suggesting that this MAb could induce cytotoxicity in FRa-
expressing cancer cells and FRb-expressing TAMs in vivo. In addition, anti-FRab mediated ADCP of FRa-expressing KB cells by macrophages via the Fc gamma receptor. The cytotoxic or phagocytotic potential of a rat-derived anti-FRab in ADCC and ADCP using human cells may be increased by changing the rat Fc portion to a humanized form. Furthermore, a fully humanized anti-FRab will be required in clinical application for cancer. It has been reported that TAMs can stimulate angiogenesis and enhance tumor cell invasion, motility, and intravasation. TAMs are also immunosuppressive, preventing tumor cell attack by natural killer and T cells during tumor progression.(22,23) There is strong evidence for specific subsets of TAMs as prognostic factors.(24–26) We reported that FRbexpressing macrophages were prominent in the perivascular area of the tumor-invasive front and produced VEGF.(14,27) In fact, the increased number of FRb-expressing TAMs was correlated with a poor prognosis in pancreatic cancer. Thus, an anti-FRab MAb that targets cancer cells and a subset of TAMs may become a more potent tool to inhibit cancer growth compared to either an anti-FRa or an anti-FRb MAb. Folateconjugated drugs delivered via FR-mediated cellular uptake have been shown to be promising anti-cancer therapeutics in preclinical animal models and early clinical trials.(28,29)
MAb REACTIVE WITH FOLATE RECEPTOR ALPHA AND BETA
Interestingly, a folate-conjugated mouse polyclonal immunoglobulin induced ADCC in FRa-expressing cells, inhibited tumor growth, and improved the survival rate in an animal model.(30) However, these folate-conjugated agents have not been well characterized with regard to their ability to target TAMs. Relative to anti-FR MAbs, FR-mediated cellular uptake of the folate-conjugated drugs may be inhibited by high folate serum concentrations. Furthermore, in addition to FRexpressing cells, folate-conjugated drugs may target RFC- and PFCT-expressing normal cells and cause detrimental side effects. One disadvantage using MAbs is their reduced penetration into tumors due to their high molecular weight compared to low molecular weight drugs. It is anticipated that administering a smaller anti-FRab MAb-derived polypeptide such as a single-chain or a single-domain MAb would result in an increase in tumor penetration. In conclusion, this study suggests that the anti-FRab MAb described here is a promising therapeutic candidate for cancers in which macrophages promote tumor progression. Author Disclosure Statement
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The authors have no financial interests to disclose. References
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Address correspondence to: Takami Matsuyama The Center for Advanced Biomedical Sciences and Swine Research Kagoshima University 8-35-1 Sakuragaoka Kagoshima 890-8544 Japan E-mail: [email protected] Received: September 19, 2014 Accepted: February 27, 2015
