A Novel Monoclonal Antibody to PL2L60 is Effective For Therapy of Various Types of Cancers in Human and Mice

Background: PL2L60 is a PIWIL2-like (PL2L) protein that is specically and widely expressed in various types of hematopoietic and solid tumors. However, it is unknown whether PL2L60 is an effective target for cancer immunotherapy. The current study aimed to investigate the ecacy of an monoclonal antibody (mAb) to PL2L60 (clone KAO3 (cid:0) IgM isotype) in treatment of the cancers either from human or mouse. Methods: The expression of PL2L60 protein in the cell surface and cytoplasm were determined in a panel of human and mouse tumor cell lines by ow cytometry, immunouorescent staining and Western Blotting. The apoptosis and the cell cycle arrest of the tumor cells treated with mAb KAO3 were evaluated by ow cytometry. The tumorigenesis of the mAb KAO3-pretreated tumor cells was determined by tumor incidence and tumor size, and ecacy of mAb KAO3 treatment on tumor growth in tumors-bearing mice were kinetically evaluated. Complement-dependent cytotoxicity tests were utilized to determine the mechanism of mAb KAO3 killing tumor cells. Results: Treatment of human or mouse tumor cells with mAb KAO3 at the time of inoculation eciently inhibited their tumorigenesis in the immunodecient mice. Moreover, injection of mAb KAO3 into established tumors signicantly inhibited their growth, and prolonged survival of the tumor-bearing mice, including lymphoma, breast cancer, lung cancer and cervical cancer. The inhibitory effects of mAb KAO3 were likely associated with its binding to the PL2L60 expressed on tumor cell surface, which may induce cell apoptosis through either activation of complement or blocking cell cycling. The cell cycling was arrested at G 2 /M phase and DNA synthesis may be inhibited. Conclusion: We have identied the PL2L60 as a novel tumor-specic and broad-spectral biomarker, which is recognized by the mAb KAO3 as an ecient target for immunotherapy of both solid and hematopoietic cancers.


Background
The attention has been increasingly garnered to "cancer immunotherapy" because of its e cacy in several cancer types (1)(2)(3)(4)(5). However, the target for cancer immunotherapy is largely individualized or personalized at present and most of the targets are only distributed in a few of cancer types rather than all cancer types (1). Moreover, these molecular targets are not cancer-speci c rather required for normal cell functions. Therefore, the bottleneck for current immunotherapy is lack of a speci c but broad spectral target or antigen which is widely expressed on both hematopoietic and solid cancers. Recently, we have found that the PIWIL2-like (PL2L) protein 60, a product of alienation-activated PIWIL2 gene by intragenic promoter (6, 7), is widely expressed in various hematopoietic and solid tumors, mediating tumorigenesis through promoting tumor cell proliferation and inhibiting apoptosis as well (8).
The PIWIL2 is usually expressed in testis (9-11), but can be activated in somatic cells upon DNA damages to promote DNA repair through remodeling chromatin (12). It plays crucial roles in self-renewal and maintenance of germline stem cells (13,14). Recent studies have indicated that the ectopic expression of PIWIL2 has been observed in various types of primary tumors and tumor cell lines (8, 15), including breast cancer (16), cervical cancer (17), gastric cancer (18), acute myeloid leukemia (19), colorectal cancer (20), colon cancer (21), ovarian cancer (22) and testicular germ cell tumors (23).
It has been reported that PIWIL2 can promote tumorigenesis through regulating several signal transduction pathways (8, [24][25][26][27][28][29][30][31] and inhibiting apoptotic death of tumor cells via activation of Stat3/Bcl-XL pathway (15,24). However, the function of PIWIL2 in tumor development remains controversial, because most of the commercial available antibodies speci c for PIWIL2 could not distinguish the full length PIWIL2 from its variants (8). In the primary breast and cervical cancers, full length PIWIL2 proteins were detected mainly in apoptotic tumor cells but little in living tumor cells (8). In contrast, PIWIL2 variants PL2L proteins, such as PL2L60, are abundantly detected in various types of tumor tissues and tumor cell lines (8, 23), suggesting that the tumorigenic function of PIWIL2 might be mediated mainly by PIWIL2 variants (31).
We and others have found that PIWIL2 has multiple variants including PL2L80, PL2L80A, PL2L60, PL2L60A, PL2L50 and PL2L40 (8, 23,31). Some of the variants appear to be transcribed by intragenic promoters rather than a canonical promoter (6, 7). While full length PIWIL2 can mediate DNA repair acting as a barrier gene to the initiation of tumorigenesis and promote apoptotic cell death in tumor tissues (8, 32), its variants such as PL2L60 (6, 8) and PL2L60A (23) can promote tumorigenesis. Among the variants mentioned above, PL2L60 is predominantly expressed in precancerous stem cells (pCSCs) as well as in various types of human and murine tumor cell lines with a level much higher than full length PIWIL2 (8, [33][34][35]. PL2L60 can promote tumor cell survival and proliferation in vitro through upregulation of STAT3 and BCL2 genes. It can also coordinate with NF-κB to promote tumorigenesis, probably representing a common pathway for the development of tumors in various types of tissues (8, 36, 37). Importantly, peptides derived from PL2L60 can serve as strong immunogens targeting various types of cancers (8, 38). In addition, PL2L60 is also detected in the testicular cells of mice, suggesting its roles in gametogenesis or development (6, 23).
In this study, we investigated the e cacy of mAb KAO3 to PL2L60 in immunotherapy of cancers, which was generated in our laboratory (8). It demonstrated a unique capability to directly induce apoptosis of cancer cells and to inhibit cell proliferation through arresting cell cycle. It effectively inhibited tumorigenesis of pCSCs in the severe-combined immunode cient (SCID) mice and furthermore suppressed established tumor growth when injected intratumorally into lymphoma, breast cancer, lung cancer and cervical cancer. Collectively, the anti-PL2L60 mAb KAO3 is a potentially useful drug for immunotherapy of various types of cancers.

Materials And Methods
Mice, cell lines, medium and mAb KAO3 Severe-combined immunode ciency (SCID) mice, nude mice and C57BL/6 mice were obtained from SLACCAS (Shanghai Laboratory Animal Center), bred in the pathogen-free animal facility at Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, and maintained under standard conditions according to the institutional guidelines of Ren Ji Hospital animal care and ethics review committee. Mice were used at the age of 8-12 weeks old.
The following human and mouse cell lines were used: human colon cancer cell line HCT116 (ATCC® laboratory. Murine precancerous stem cells (pCSCs) clone 2C4 was cloned from a mouse with dendritic cell-like leukemia, as previously described (33). Murine cancer stem cell (CSC) clone 326T-4 was a subclone of 326T clone,which was generated from a thymomas (8). The cells were maintained in R10F (RPMI 1640 plus 10% fetal calf serum supplemented with 5 mM glutamine, 50 mM 2-mecaptoethonal, 100 U/ml penicillin, and 100 mg/ml streptomycin) at 37°C in a humidi ed incubator containing 5% CO2 Monoclonal antibody (mAb) to PL2L60 (clone KAO3, isotype IgM) was generated in our laboratory, and the culture supernatants was used as described except for where indicated (8).
Flow cytometric analysis of cell surface expression of PL2L60 and apoptosis Cancer cells were dissociated with 0.25% trypsin-EDTA (1 mM; Invitrogen) for 1-3 min. Cells were washed with cell sorting buffer (PBS containing 1% fetal calf serum), and then incubated for 1 hour at 4°C with anti-PL2L60 mAbs. Cells were then incubated with phycoerythrin-conjugated goat anti-mouse IgM (1:250 dilution; BioLegend) for 30 min at 4°C. After a nal wash, the cells were re-suspended with 1% paraformaldehyde in PBS containing 1% FBS and analyzed by ow cytometry (BD, San Jose, CA, USA).
For cell apoptosis assay, the Annexin V-APC Apoptosis Detection kit (Biolegend) was used. Tumor cells were harvested and washed twice with pre-cooled PBS after they were treated with or without mAb KAO3 for 24 h. The cells were incubated with a mixture containing annexin V-APC (BioLegend) and propidium iodide (Sigma) in the binding buffer for 15 min in darkness. Apoptotic cells were detected using Annexin V-APC and PI, and were analyzed using a ow cytometer (BD, C6). Three independent experiments were performed.
Immuno uorescent microscopic analysis of PL2L60 expression in the cell membrane and cytoplasm For cell surface staining, tumor cells were harvested,resuspended tumor cells (5 x 10 6 /ml) in PBS, and then plated 0.2 ml cells (1X10 6 cells/well) in 96 well plates. The cells were spun (1000 rpm for 5min) and the supernatants were discarded. The cells were resuspended in 100 µl PBS containing 5 µl supernatants of anti-PL2L60 mAbs KAO3 and gently vortexed for 5 s. The samples were incubated at 4°C for 30 min, washed twice with PBS and resuspended in 100 µl PBS containing 1% paraformaldehyde to each well to x the cells.
For cytoplasmic staining, tumor cells cultured on cover slips were xed in 1% paraformaldehyde for 20 min before being washed, and subsequently blocked with 1% bovine serum albumin in PBS for 30 min. Cells were incubated at room temperature with anti-PL2L60 mAbs (KAO3, 1:100 dilution) in 1% bovine serum albumin. One hour after incubation, cells were washed and incubated with FITC-conjugated goat anti-mouse antibody (IgM, BioLegend).

Western blot analysis of PL2L60 expression in tumor cells
Cell samples were washed in cold PBS twice before they were harvested with trypsin. Then cells were lysed with protein extraction reagent. The total protein concentrations of whole-cell lysates were determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Then the protein was separated using 12% polyacrylamide gel, and transferred to polyvinyl di uoride membranes. After being blocked with 5% bovine serum albumin (BSA) in TBS/Tween 20 (TBST) and incubated with PL2L60-speci c antibody (mAb KAO3 supernatants) at 4°C overnight, the membranes were washed with TBS-T for 5 min.
The wash was repeated three times. After this, the membranes were incubated with horseradish peroxidase conjugated anti-mouse IgM antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h, followed by a 5 min wash with TBST, which was repeated three times. Then the membranes were analyzed with the ECL chemiluminescent detection system (Bio-Rad), and the images were acquired by a Kodak Imaging Station 2000R (Eastman Kodak, USA).

CCK-8 assay for cell proliferation
The effect of anti-PL2L60 mAb (KAO3) on tumor cell proliferation was assessed using CCK-8 assays

Complement-dependent cytotoxicity assay
For complement-dependent cytotoxicity (CDC) assay, tumor cells were plated out at 5x10 4 cells per well. Test antibodies were added at the indicated nal concentrations together with either active or heat inactivated (60°C for 30 minutes) human serum (25% nal serum concentration; Pathway Diagnostics, Dorking, UK). Plates were incubated for 3 hours at 37°C, following which the cell viability reagent was added. Triton1 X-100 was added to the control cell-only wells to establish the maximum lysis control.

Statistical analysis
All data were derived from at least three independent experiments. Values are expressed as the mean ± SD. Experimental groups were compared with the respective control by Student's t-test. Three or more groups were compared by the one-way analysis of variance (ANOVA). Differences were considered signi cant when p values were less than 0.05. * indicated that p value < 0.05; ** indicated that p value < 0.01; and *** indicated that p value < 0.001. Survival analyses were performed using Kaplan-Meier survival curves, and signi cant differences between groups were tested using the log-rank test.
Correlation coe cients were assayed by Spearman's analysis.

Results
Expression of PL2L60 protein in cancer cells In the human primary breast and cervical cancers, full length PIWIL2 proteins were detected mainly in apoptotic tumor cells but little in living tumor cells by immunohistochemistry. In contrast, PIWIL2 variants PL2L proteins, such as PL2L60, are abundantly detected in various types of tumor tissues and tumor cell lines (8), suggesting that the tumorigenic functions of PIWIL2 might be mainly mediated by PIWIL2 variants (6, 7). Therefore, the wide expression of PL2L proteins in various cancer types renders them an ideal broad spectral target for immunotherapy of solid and hematopoietic caners. To test the hypothesis, we rst investigated whether the PL2L proteins were expressed on the cell surface of various types of tumor cell lines, using a monoclonal antibody (mAb KAO3) to a PIWIL2 peptide homologous for both human and mouse (8). In addition to cytoplasm (8), PL2L proteins were also detected by ow cytometry (Fig. 1A & B) and uorescent microscopy (Fig. 1C) (Fig. 1E & G). The expression of PL2L60 protein is the highest in cytoplasm of pCSCs 2C4 (Fig. 1D-F), consistently with its expression on cell surface (Fig. 1B).
The mAb KAO3 effectively inhibited tumor cell proliferation PL2L60 can promote tumor cell survival and proliferation in vitro through up-regulation of STAT3 and BCL2 genes. It can also coordinate with NF-κB to promote tumorigenesis (8). Therefore, we have developed antibodies to PL2L60, such as mAb KAO3, to examine whether it may inhibit cancer cell proliferation by blocking the function of PL2L60 (8). Cancer cells were cultured for 48 hours in the presence of various concentrations of mAb KAO3 (0, 1, 2, 4, and 8µl per well) and examined for their number and viability. As shown in Fig. 2, KAO3 mAb was able to inhibit proliferation of various types of cancer cells in a dose-dependent manner, including murine hematopoietic pCSC line (2C4) and CSC line (326T-4) and human epithelial cancer cell lines such as MDA-MB-231, A549 and HeLa (Fig. 1A). This was associated with cancer cell apoptosis induced by mAb KAO3. Some epithelial cancer cells became round, karyopyknosis was observed under high power microscope and semi-detached cells were observed in the microwells (Fig. 1A). Flow cytometry analysis revealed that apoptotic cells in the KAO3 mAb-treated wells were increased signi cantly (Fig. 2B & C). Accordingly, the number of viable cells was greatly reduced in a dose-dependent manner, too, as revealed by CCK-8 assay (Fig. 2D). These results suggest that anti-PL2L60 mAb(KAO3)may block the function of surface PL2L60 and induce cancer cell apoptosis.
The KAO3 mAb induced cell cycle arrest at the G 2 /M phase in cancer cells To determine the mechanisms underlying cell apoptosis induced by mAb KAO3, the cell cycle distributions of 2C4, 326T-4, MDA-MB-231, A549 and HeLa after anti-PL2L60 mAb (KAO3) treatment were examined. As shown in Fig. 3A, compared with control cells, cancer cells that were treated with anti-PL2L60 mAb (KAO3) for 48 h had slightly decreased or increased proportion of G 0 /G 1 phase and S phase, except for 2C4, which was increased at G 0 /G 1 phase and almost suppressed at S phase ( Fig. 3B &   C). The number of cells that were in G 2 /M phase increased in all lines to various degrees. 2C4, 326T-4 and A549 cells were arrested at G 2 /M phase with higher percentages than MDA-MB-231 and HeLa cells (Fig. 3D), consistently with the observation for their cell viability shown in Fig. 2D). In short, the cell cycling analysis demonstrated that anti-PL2L60 mAb (KAO3) caused signi cant G 2 /M-phase arrest in ve cancer cell lines, 2C4 was most sensitive to mAb KAO3 treatment.
The mAb KAO3 could effectively inhibit various types of tumorigenesis and tumor growth of both human and mice in the SCID mice Since anti-PL2L60 mAb (KAO3) treatment disrupted cancer cell growth and induced cancer cell apoptosis in vitro, we investigated whether KAO3 mAb could be used to directly inhibit tumor growth in vivo. In order to observe the effects of mAb KAO3 on tumorigenicity of tumor cells, tumor treatment program was divided into two stages: one is the initial stages of tumorigenesis, and another is the tumor growth stage.
Human and mouse cancer cells were suspended in the culture supernatant of KAO3 hybridoma (treatment group) or R10F medium (control group) and then inoculated into severe-combined immunode cient (SCID) mice. After tumor formation, tumor-bearing mice in different treatment groups were then divided into two groups, respectively. A group of tumor-bearing mice were injected with KAO3 supernatants intratumorally every two days for treatment, and the other group was injected with culture medium intratumorally as a control. Tumor growth rate was measured every two days.
At the initial stages of tumorigenesis, tumor incidence in different groups were counted when the tumors become palpable. We found that the tumorigenesis of pCSCs (2C4) in the mice was almost completely inhibited by KAO3 mAb. Within 3 weeks of observation, only 60% of the mice (8/14) developed tumors from day 13 to day 21 after inoculation. In contrast, all the mice (100%, 12/12) in the control group developed tumor within 15 days after inoculation. There was a very signi cant statistical difference between the two groups ( Fig. 4A; p = 0.001). The tumor incidence in mice inoculated with KAO3-pretreated human breast cancer cells (MDA-MB-231) was 50%, whereas it was 83% in control group ( Fig. 4C; p = 0.043). The tumor incidence of other three cell lines were 100%, and there were no differences between KAO3 pretreatment groups and the control groups (Fig. 4B, D & E).
Further analysis of the tumor growth kinetics showed that tumors derived from KAO3-pretreated pCSCs grew signi cantly slower than the tumors derived from control (medium-treated) pCSCs (Fig. 5A). Intratumoral treatment of tumors from control group with 100 µl of KAO3 mAb supernatants (Ctrl→KAO3) signi cantly inhibited tumor growth ( Fig. 5B & P/2C4). The tumors which derived from the pretreated pCSCs were further treated with KAO3 mAb (KAO3→ KAO3). Tumor size was smaller than those in the control treatment group (KAO → ctrl). But there was no statistical difference in the two groups because the tumor volume was both restrained after the KAO3 mAb was pretreated ( Fig. 5C & P/2C4: KAO3 → ctrl vs. KAO3 → KAO3). The results indicate that the mAb KAO3 is effective in preventing tumorigenesis of pCSCs and suppressing the growth of established tumors. The same treatment of murine hematopoietic cancer stem cells (CSCs; clone 326T-4), and 326T-4 also led to various degree of tumorigenic or tumor growth inhibition in the immunode cient mice (Fig. 5D-F & P).
Since mAb KAO3 also recognizes human PL2L60 proteins, we treated human breast cancer cells (MDA-MB-231), lung cancer cells (A549) and cervical cancer cells (HeLa) with the culture supernatants of hybridoma KAO3 as described for pCSC (2C4). Similar results were observed that KAO3 mAb could also effectively inhibit the tumorigenesis of human cancer cells and the growth of established tumors in SCID mice ( Fig. 5G-P). The results suggest that mAb KAO3 can also effectively kill or suppress human cancer cells in the SCID mice. These results verify the in vitro nding that KAO3 mAb has therapeutic effects on tumorigenesis and tumor growth either tumors from human or mice, as compared to controls.

The mAb KAO3 mediates complement-dependent cytotoxicity (CDC) to cancer cells of human and mice
To determine the mechanisms underlying therapeutic effects of mAb KAO3 on the cancers of human and mice, we treated with cancer cell lines 2C4, 326T-4, MDA-MB-231, A549 and HeLa cells with mAb KAO3 plus human complement (Fig. 6). The results showed that mouse pCSC line 2C4 and human breast cancer cell line MDA-MB-231 displayed the strongest oncolytic effect in the CDC experiment, followed by mouse lymphoma cell line 326T-4 and human lung cancer cell line A549. The CDC effect in human cervical cancer cell line HeLa was the weakest (Fig. 6A-B). These results are consistent with the expression level of PL2L60 protein on cancer cell surface. It is further suggest that the target of the anti-PL2L60 mAb KAO3 was the PL2L60 protein on the tumor cell surface, and KAO3 may exert its anti-tumor effect through the CDC-dependent mechanisms.

Discussion
We and others have found that PIWIL2 has multiple variants including PL2L80, PL2L80A, PL2L60, PL2L60A, PL2L50 and PL2L40 (8, 23). Some of the variants appear to be transcribed by intragenic promoters rather than a canonical promoter (6, 7). While full length PIWIL2 can mediate DNA repair acting as a barrier gene to the initiation of tumorigenesis and promote apoptotic cell death in tumor tissues (7,8,31). its variants such as PL2L60 (8) and PL2L60A (23) can promote tumorigenesis. Among the variants mentioned above, PL2L60 is predominantly expressed in precancerous stem cells (pCSCs) as well as in various types of cancer cell lines including hematopoietic and solid cancers of human and mice with a level much higher than full length PIWIL2 (8). PL2L60 can promote tumor cell survival and proliferation in vitro through up-regulation of STAT3 and BCL-2 genes. It can also coordinate with NF-κB to promote tumorigenesis, probably representing a common pathway for the development of tumors in various types of tissues (7). Therefore, the development of therapeutic antibodies, which target PL2L60, has great potential for eradicating tumors. Importantly, peptides derived from PL2L60 may serve as strong immunogens targeting various types of cancer. A PIWIL2 peptide which is located in the sequence of all the PL2L proteins and shared between human and mice is a strong immunogen successfully used for generation of rabbit polyclonal antibody and murine monoclonal antibody (mAb) (8). The mAb clones KAO2 and KAO3 have stronger a nity for PL2L60 than the clone KAO1 in the immunohistochemical staining assay and Western blot analysis (8). The ndings implicate that PL2L proteins, especially PL2L60, might be a common target for cancer immunotherapy.
In this study, we used the mAb KAO3 developed in our laboratory to examine whether PL2L60 proteins are a common target for cancer immunotherapy, because PL2L60 proteins are widely expressed in various types of cancer cells including hematopoietic and solid tumors (8). As expected, mAb KAO3 has strong capacity to inhibit various types of tumorigenesis and tumor growth. The differential capability of mAb KAO3 to inhibit tumorigenesis and tumor growth appeared to be associated with the expression level of surface PL2L60 proteins between the cancer cell lines (Fig. 1A-B), but not with intracellular expressions ( Fig. 1C-G), as the e ciency of KAO3 mAb to inhibit tumorigenesis was associated with surface KAO3 + cells in percentage. HeLa contained less KAO3 + cells than other lines and had less sensitivity to mAb KAO3 treatments (Fig. 5). Therefore, the therapeutic e cacy of mAb KAO3 may be determined by the amount of surface PL2L proteins expression. Availability of data and materials: All data generated or analysed during this study are included in this published article.    proportions of cell cycles in G0/G1, S and G2/M phases were compared between the control and treatment groups. The results shown are from three independent experiments. NS, not signi cant; *, p < 0.05; **, p < 0.01; and ***, p < 0.001.