Multiple myeloma is a type of cancer characterized by the uncontrolled clonal expansion of malignant plasma cells within the bone marrow (Palumbo and Anderson, 2011). Standard therapeutic regimens with FDA-approved substances, such as immunomodulatory drugs (lenalidomide and pomalidomide) and proteasome inhibitors (bortezomib) (Richardson
Programmed death-ligand 1 (PD-L1), also known as B7 homolog 1 (B7-H1) or CD274, is an immune checkpoint molecule that transmits an inhibitory signal to counterpart cells via the binding of PD-1 and CD80 (Zou and Chen, 2008). PD-L1 is expressed in various cells, including antigen-presenting cells (dendritic cells and macrophages), activated B cells, and other nonlymphoid tissue cells, including the heart, lung, liver, and kidney (Iwai
The blockade of PD-L1 also mitigated immunosuppression mediated by the myeloid-derived suppressor cells (MDSCs), which are a heterogeneous population of immature myeloid cells that have different PD-1/PD-L1 expression levels depending on the tumor type (Deng
In this study, we developed a new anti-PD-L1 antibody (Ab) that binds to mouse and human PD-L1. The Ab significantly inhibited syngeneic myeloma cell growth in mice. There were no significant changes in the MDSC composition of tumor-bearing mice following anti-PD-L1 treatment, but the anti-PD-L1 antibody induced antibody-dependent cellular cytotoxicity (ADCC)-associated myeloma cell death. We sought to validate the synergistic efficacy of anti-PD-L1 Ab treatment with lenalidomide in the murine model of multiple myeloma and observed only PD-L1-driven inhibition of tumor growth (lenalidomide itself was not effective in the mouse model). Collectively, our results show that the newly developed anti-PD-L1 Ab may be a therapeutic candidate for multiple myeloma, though further studies are required to differentiate it from other anti-PD-L1 therapeutic Abs.
NS-1 and MOPC-315 murine myeloma cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The NS-1 cells were maintained in RPMI 1640 medium, and the MOPC-315 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% of fetal bovine serum and 1% anti-anti solution. Both cell lines were sub-cultured every 2-3 days. Lenalidomide (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO, and the stock solution was stored at –70°C. Phycoerythrin- (PE) conjugated anti-mouse PD-L1 antibody was purchased from BD Bioscience (San Jose, CA, USA) and used as a control antibody for the mouse PD-L1 expression assays.
A novel anti-PD-L1 antibody with human antibody residues (Choi
The study specimens were obtained from multiple myeloma patients who underwent bone marrow aspiration at the Korea University Anam Hospital (Seoul, Korea) . The study protocol was approved by the Institutional Review Board of the Korea University Medical Center (Seoul, Korea), and the patients provided written informed consent. All methods were performed according to the relevant guidelines and regulations (IRB No. 2018AN0150). Aspirated bone marrow and peripheral blood samples were diluted to a ratio of 1:1 with phosphate-buffered saline (PBS) and layered over the same original blood volume of Ficoll (Histopaque-1077; Sigma-Aldrich) in a 50 mL conical tube. The specimens were centrifuged at 2,000 rpm for 30 min at room temperature, and the upper layer of the opaque interface (containing the mononuclear cells) was aspirated and transferred to a new conical tube. The collected cells were washed with 5 mL of PBS and centrifuged at 1,200 rpm for 10 min at room temperature.
Six-week-old female Balb/c mice were purchased from KOATECH (Pyeongtaek, Korea). The mice were maintained under specific pathogen-free conditions for 1 week in the experimental facilities at Kangwon National University (Chuncheon, Korea), where they received sterilized food and water ad libitum and were housed at 20-22°C on a 12 h light/dark cycle. All of the animal experiments were performed according to the approved guidelines of the Institutional Animal Care and Use Committee of Kangwon National University (KW-140811-2). To establish a mouse myeloma model, 7- or 8-week-old mice were challenged with 5×106 NS-1 cells (subcutaneously or intraperitoneally) or 107 of NS-1 or MOPC-315 cells (intravenously). Tumor length, height, and width were measured with calipers, and the tumor volume was calculated as 1/6π×length (mm)×height (mm)×width (mm). To evaluate the anti-myeloma efficacy of lenalidomide and the anti-human/murine PD-L1 antibody, 10 mg/kg of Lenalidomide (every day) and 5 mg/kg of PD-L1 antibody (every 2-3 days) was intraperitoneally injected. Mouse body condition was scored as 0 (normal), 1 (rough or harsh hair), 2 (partial paralysis of the hind leg or emergence of a cancer nodule near the skin), 3 (complete paralysis of the hind leg or a larger tumor mass), 4 (complete paralysis of the hind leg and reduction of motion), or 5 (death).
To analyze PD-L1 expression on the cell surface, 5×105 of NS-1 and MOPC-315 cells were stained with PE-conjugated anti-mouse PD-L1 (BD Bioscience) and Alexa Flour 488-conjugated anti-human/mouse-PD-L1 antibodies for 15 min 4°C in flow cytometry staining buffer (FACS; PBS supplemented with 1% fetal bovine serum (FBS), 2 mM EDTA). After staining, the cells were washed with 1 ml of FACS buffer and analyzed on a FACSVerse instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). For analysis of the human PBMC and bone marrow cells, frozen cell stock vials were thawed at 37°C, and approximately 105 PBMC or bone marrow cells were stained with PE-Cy7-conjugated anti-CD3 antibody, brilliant violet 421 (BV421)-conjugated anti-CD138 antibody, APC-conjugated anti-human PD-L1 antibody (29E.2A3), or unconjugated anti-human/murine PD-L1 antibody. To detect the anti-human/murine PD-L1 antibody, a second staining was conducted with Alexa Flour 647-conjugated anti-mouse IgG antibody. After staining, the cells were washed with 1 mL of FACS buffer and analyzed on the FACSVerse instrument. Flow cytometry experiment to detect MDSCs were conducted as reported previously and some parts of procedures were revised (Song
The formalin-fixed paraffin-embedded specimens were sectioned (4-5 µm) and placed on slides, which were deparaffinized and then rehydrated. Antigen retrieval was performed by boiling in a pressure cooker for 10 min with a sodium citrate buffer (pH 6.0), and permeabilization was performed with 0.5% Triton X-100. The specimens were blocked using 5% normal donkey serum for 1 h at room temperature and then incubated overnight at 4°C with the primary antibodies targeting CD138 (1:100; R&D System, Minneapolis, MN, USA) and PD-L1 (ABM4E54, 1:100; Abcam, Cambridge, UK). The samples were then incubated with the fluorochrome-conjugated secondary antibodies for the CD138 (Alexa Fluor 488, 1:200; Invitrogen, Carlsbad, CA, USA) and PD-L1 tests (Alexa Fluor 647, 1:200; Invitrogen) at room temperature for 1 h. Isotype-matched antibodies were used as the negative controls, and the nuclei were highlighted using DAPI (4′,6-diamidino-2-phenylindole) mounting medium (ProLongTM Diamond Antifade Mountant with DAPI; Invitrogen). The samples were imaged on an automated fluorescence microscope (200× magnification; EVOS FL Auto; Life Technologies) and processed in CellesteTM image analysis software (Invitrogen).
MOPC-315 cells and splenocytes were seeded in a 96-well plate (104 cells/well); the cells were treated with 1, 5, 50, 100, and 200 μg/ml drugs (lenalidomide and anti-PD-L1 antibody) and incubated for 4 h in an incubator at 37°C and 5% carbon dioxide (CO2). After incubation, 10 μL of cell counting kit-8 (Cell Counting Kit-8, Dojindo Co., Kumamoto, Japan) were added to each well and incubated for an additional 2 h. The absorbance at 450 nm was measured by a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA).
To isolate mouse peripheral blood mononuclear cells, mouse blood was collected by heart puncture in tubes containing EDTA. Spleen, mesenteric, inguinal, axillary, and cervical lymph node samples were obtained from naïve C57Bl/6 mice and mechanically strained through a 100 μm nylon strainer. The blood and strained cells were mixed, layered on 3 ml of Histopaque®-1077 (Sigma-Aldrich), and gently centrifuged for 30 min at room temperature with minimum acceleration and deceleration. Interphase cells were collected and washed 3 times with cold PBS, and the red blood cells (RBCs) were lysed with RBC lysis buffer. PBMCs (2-3×106) were cultured in complete RPMI medium supplemented with 20 ng/mL of murine IL-2 for 48 h.
The MOPC-315 cells were equally divided and stained with 0.5 μM and 5 μM of cell trace violet (CTV, Invitrogen) for 10 mins at 37°C. The MOPC-315 cells stained with 5 uM of CTV were reacted with 0.1 μg/mL of anti-PD-L1 Ab for 30 min at 4°C; the 0.5 uM CTV-stained cells were an internal control (no antibody added). The cells were washed 3 times with cold PBS and mixed equally, and the mixed MOPC-315 cells (5×104) were co-cultured with IL-2 primed mouse PBMC for 4 h at 37°C. After incubation, the cells were analyzed on the FACSVerse instrument. The specific lysis ratio was calculated as
Statistical analyses were performed in Graphpad Prism 5 (GraphPad Software, LLC, San Diego, CA, USA). Unpaired two-tailed Student’s
To validate a novel therapeutic approach to treat multiple myeloma, we constructed a chimeric, mouse-compatible version of a newly generated, fully human anti-PD-L1 Ab that maintained the human VH and VL domains and fused with the mouse IgG2a and kappa constant domains to avoid mouse anti-human IgG1 immune responses. The Ab retained the effector functions, such as the antibody-dependent cellular cytotoxicity (ADCC) and the complement-dependent cytotoxicity (CDC) in mice (Fig. 1A). The original anti-PD-L1 Ab was isolated through phage display screening using a naïve human antibody library and had cross-species reactivity to both murine and human PD-L1. It also showed strong
PD-L1 was expressed on the plasma membrane of CD138+ cells from multiple myeloma patients, and analysis of the bone marrow cells of multiple myeloma patients revealed CD138+ cells with high PD-L1 expression (Fig. 2A). The expression of PD-L1 on CD138+ cells from multiple myeloma patients was detected with flow cytometry, and the mean fluorescence index of PD-L1 was compared to isotype control Ab staining (Fig. 2B). The expression of PD-L1 on CD138+ cells has been associated with bad patient prognosis (Yousef
To assess the therapeutic effects of the newly developed anti-PD-L1 Ab
Multiple myeloma causes cancer cell accumulation in the bone marrow, so we adopted intravenous injection models of multiple myeloma cells. Intravenous injection of the NS-1 cells into BALB/c mice (1×107 cells per mouse) induced lethality within 80 days after tumor challenge, whereas anti-PD-L1 Ab treatment (5 times, 2-day intervals) extended the survival (Fig. 4A). Likewise, intraperitoneal injection of anti-PD-L1 Ab increased mouse survival compared to the MOPC-315 cells-injected mice treated with isotype control Ab (Fig. 4C). We also visually scored the body condition of the mice, based on the general appearance, occurrence of paralysis or solid tumors, and morbidity (as described in the materials and methods). Interestingly, treatment with anti-PD-L1 Ab significantly ameliorated the condition of the NS-1- and MOPC-1-inoculated mice (Fig. 4B, 4D). Together, these results suggest that the newly generated anti-PD-L1 Ab successfully inhibited the growth of multiple myeloma in syngeneic murine models.
Lenalidomide is an immunomodulatory drug used to treat multiple myeloma. We investigated if the combination of lenalidomide and anti-PD-L1 Ab has synergistic effects in a murine myeloma model. Groups of mice were subcutaneously injected with MOPC-315 cells (5×106 cells per mouse) and treated with lenalidomide (10 mg/kg) and anti-PD-L1 Ab (100 µg/mouse). Starting from day 14, lenalidomide was administered every day, and anti-PD-L1 Ab was injected every other day. The anti-PD-L1 Ab treatment significantly inhibited tumor growth, but we did not observe any decrease in tumor size in the mice treated with lenalidomide (compared to the PBS-treated control group; Fig. 5A). Furthermore, there were no additive antitumor effects when lenalidomide was combined with anti-PD-L1 Ab (Fig. 5A). These results highlight the antitumor effects of anti-PD-L1 Ab on the multiple myeloma in murine models but do not reveal any synergistic effects of anti-PD-L1 and lenalidomide.
Myeloid-derived suppressor cells (MDSCs) are a mixed population of immature myeloid cells, containing neutrophils, monocytes, and immature dendritic cells (Lee
As another antitumor mechanism of anti-PD-L1 Ab, we assessed if the Ab can mediate ADCC on multiple myeloma cells. ADCC plays an important role in the antitumor activities of several monoclonal antibodies targeting cancer (Giles
Multiple myeloma is a hematological malignancy with clonal plasma cell disorder and represents about one percent of all reported cancers. Recently established standardized therapies with immunomodulatory drugs such as lenalidomide and pomalidomide with proteasome inhibitors (bortezomib, carfilzomib, and ixazomib) markedly improved the outcomes for multiple myeloma patients (Jelinek
Contrary to expectations, however, recent clinical reports have shown that the blockade of PD-1 is not effective in multiple myeloma patients (Tremblay-LeMay
Targeting PD-L1 has several advantages over a PD-1 inhibitor (Tremblay-LeMay
The blockade of PD-L1 on tumor cells may be beneficial via intrinsic and extrinsic effects through the inhibition of PD-1/PD-L1 binding. The latter helps T and NK cells respond to cancer cells by overcoming the tumor-associated immune evasion mechanisms (Ray
We found that the expression of PD-L1 in multiple myeloma cells can be detected on the surface or in the nucleus. Others have indicated that the soluble PD-L1 levels in the serum of patients are associated with disease prognosis (Wang
In this study, we did not observe synergistic antitumor effects of lenalidomide and PD-L1 combined treatments in the murine model of myeloma (lenalidomide did not show antitumor effects against the mouse myeloma cells). This may be due to the amino acid differences between the human and mouse cereblon (CRBN) protein; isoleucine is replaced by valine at position 391 in the murine CRBN protein. Few studies have reported marginal antitumor effects of lenalidomide in syngeneic mouse models (Vo
Finally, we developed a novel anti-PD-L1 antibody and this antibody can be applied to treat multiple myeloma. The newly developed anti-PD-L1 antibody showed significant antitumor effects against multiple myeloma in mice. This antibody can activate anti-cancer immune response by blocking immunocheck point molecules, and also directly eliminate cancer cells expressing PD-L1 molecule, suggesting that this anti-PD-L1 Ab is a promising candidate to treat multiple myeloma.
This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (NRF-2017M3A9C8060387, NRF-2017M3A9C8060390).