A Novel Anti-PD-L1 Antibody Exhibits Antitumor Effects on Multiple Myeloma in Murine Models via Antibody-Dependent Cellular Cytotoxicity.

Multiple myeloma is a malignant cancer of plasma cells. Despite recent progress with immunomodulatory drugs and proteasome inhibitors, it remains an incurable disease that requires other strategies to overcome its recurrence and non-response. Based on the high expression levels of programmed death-ligand 1 (PD-L1) in human multiple myeloma isolated from bone marrow and the murine myeloma cell lines, NS-1 and MOPC-315, we propose PD-L1 molecule as a target of anti-multiple myeloma therapy. We developed a novel anti-PD-L1 antibody containing a murine immunoglobulin G subclass 2a (IgG2a) fragment crystallizable (Fc) domain that can induce antibody-dependent cellular cytotoxicity. The newly developed anti-PD-L1 antibody showed significant antitumor effects against multiple myeloma in mice subcutaneously, intraperitoneally, or intravenously inoculated with NS-1 and MOPC-315 cells. The anti-PD-L1 effects on multiple myeloma may be related to a decrease in the immunosuppressive myeloid-derived suppressor cells (MDSCs), but there were no changes in the splenic MDSCs after combined treatment with lenalidomide and the anti-PD-L1 antibody. Interestingly, the newly developed anti-PD-L1 antibody can induce antibody-dependent cellular cytotoxicity in the myeloma cells, which differs from the existing anti-PD-L1 antibodies. Collectively, we have developed a new anti-PD-L1 antibody that binds to mouse and human PD-L1 and demonstrated the antitumor effects of the antibody in several syngeneic murine myeloma models. Thus, PD-L1 is a promising target to treat multiple myeloma, and the novel anti-PD-L1 antibody may be an effective anti-myeloma drug via antibody-dependent cellular cytotoxicity effects.

BLI-Based Functional Assay in Phage Display Benefits the Development of a PD-L1-Targeting Therapeutic Antibody.

The therapeutic functionality of the antibodies from phage display is verified after an initial screening. Several immunological assays such as ELISA, flow cytometry, the western blot, and surface plasmon resonance (SPR) assay are commonly used; the IgG-format antibody is usually preferred to verify the functionality of antibodies, which need elaborative mammalian expression and purification work. Here, we describe a biolayer interferometry (BLI)-based assay that can evaluate the inhibitory functions of antibodies at an earlier stage of screening. To develop a PD-L1-targeting antibody from phage display, we applied the BLI assay to the initial scFv antibody screening, in addition to common ELISA and fluorescence-activated cell sorting (FACS) assays, which showed high advantages and relevance with the in vitro cell-based PD-1/PD-L1 inhibition assay. The same assays for IgG-format antibodies showed high efficiency of the BLI assay in the functional characterization of antibodies, and one candidate selected from the BLI assay resulted in highly efficacious antitumor activity in an in vivo syngeneic mouse study. The BLI assay was also beneficial when searching for antibodies with diverse epitopes. These results demonstrated that the BLI-based inhibition assay is an excellent technique for high-throughput scFv antibody screening in earlier stages and can make phage-display antibody screening more efficient to develop therapeutic candidates.

Novel Antibodies Targeting MUC1-C Showed Anti-Metastasis and Growth-Inhibitory Effects on Human Breast Cancer Cells.

Mucin1 (MUC1) is aberrantly glycosylated and overexpressed in various cancers, and it plays a crucial role in cancerogenesis. MUC1 is a type I membranous protein composed of α and ß subunits. MUC1-α can be cleaved in cancers, exposing MUC1-ß (MUC1-C). MUC1-C is involved with multiple cancer cellular functions, which makes it an attractive target for cancer treatment. However, its multifunctional mechanisms have not been fully elucidated and there has not been a successful therapeutic development against MUC1-C. Through a phage display process, we isolated the specific antibodies for the extracellular domain of MUC1-C. The relevant full IgG antibodies were produced successfully from mammalian cells and validated for their MUC1-C specificities through ELISA, dual FACS analysis, BLI assay, and confocal image analysis. In the comparison with reference antibody, elected antibodies showed characteristic bindings on target antigens. In the functionality assessment of high-ranking antibodies, SKM1-02, -13, and -20 antibodies highly inhibited invasion by triple-negative breast cancer (TNBC) cells and the SKM1-02 showed strong growth inhibition of cancer cells. Our results showed that these MUC1-C specific antibodies will be important tools for the understanding of MUC1 oncogenesis and are also highly effective therapeutic candidates against human breast cancers, especially TNBC cells.

Magnetic Resonance Colonography Enables the Efficacy Assessment of Immune Checkpoint Inhibitors in an Orthotopic Colorectal Cancer Mouse Model.

Immune checkpoint inhibitors (ICIs) have become an effective therapeutic option for colorectal cancer and studies on these drugs have therefore increased greatly. Efficacy assessments of ICIs in preclinical orthotopic colorectal cancer using MRI have not been reported however due to the difficulties in conducting colorectal imaging. The purpose of this present study was to investigate the feasibility of using magnetic resonance colonography (MRC) to evaluate the efficacy of an ICI, an anti-PD-L1 antibody, in an orthotopic colorectal cancer mouse model. The mouse model was generated by the engraftment of colorectal cancer cells into the submucosal layer of the colon. Anti-cancer efficacy was assessed by tumor volume and metastatic tumor number analyses, and these values were significantly lower in the PD-L1 antibody-treated group compared to the controls. Histological analyses using H&E and Ki-67 immunohistochemical staining confirmed a highly efficacious tumor growth inhibition and enhanced infiltration by CD4+ and CD8+ lymphocytes in the PD-L1 antibody-treated group. We conclude that MRC has the potential to be used for ICI efficacy assessments against orthotopic colorectal cancer mouse model.

Generation of a human antibody that inhibits TSPAN8-mediated invasion of metastatic colorectal cancer cells.

Tetraspanin 8 (TSPAN8) is a tumor-associated antigen implicated in tumor progression and metastasis. However, the validation of TSPAN8 as a potential therapeutic target in metastatic colorectal cancer (mCRC) has not yet been studied. In this study, through several in vitro methodologies, we identified a large extracellular loop of TSPAN8 (TSPAN8-LEL) as a key domain for regulating mCRC invasion. Using phage display technology, we developed a novel anti-TSPAN8-LEL human antibody with subnanomolar affinity that specifically recognizes amino acids 140-205 of TSPAN8-LEL in a conformation-dependent manner. Finally, we demonstrated that the antibody specifically reduces invasion in the HCT116 and LoVo mCRC cell lines more potently than in the HCT-8 and SW480 non-mCRC cell lines. Our data suggest that TSPAN8-LEL may play an important role in mCRC cell invasion, and that the antibody we have developed could be a useful tool for inhibiting the invasion of TSPAN8-expressing mCRCs.

Augmentation of natural cytotoxicity by chronic low-dose ionizing radiation in murine natural killer cells primed by IL-2.

The possible beneficial effects of chronic low-dose irradiation (LDR) and its mechanism of action in a variety of pathophysiological processes such as cancer are a subject of intense investigation. While animal studies involving long-term exposure to LDR have yielded encouraging results, the influence of LDR at the cellular level has been less well defined. We reasoned that since natural killer (NK) cells constitute an early responder to exogenous stress, NK cells may reveal sentinel alterations in function upon exposure to LDR. When purified NK cells received LDR at 4.2 mGy/h for a total of 0.2 Gy in vitro, no significant difference in cell viability was observed. Likewise, no functional changes were detected in LDR-exposed NK cells, demonstrating that LDR alone was insufficient to generate changes at the cellular level. Nonetheless, significant augmentation of cytotoxic, but not proliferative, function was detected when NK cells were stimulated with low-dose IL-2 prior to irradiation. This enhancement of NK cytotoxicity was not due to alterations in NK-activating receptors, NK1.1, NKG2D, CD69 and 2B4, or changes in the rate of early or late apoptosis. Therefore, LDR, in the presence of suboptimal cytokine levels, can facilitate anti-tumor cytotoxicity of NK cells without influencing cellular proliferation or apoptosis. Whether these results translate to in vivo consequences remains to be seen; however, our data provide initial evidence that exposure to LDR can lead to subtle immune-enhancing effects on NK cells and may explain, in part, the functional basis underlying, diverse beneficial effects seen in the animals chronically exposed to LDR.

Suppression of human anti-porcine natural killer cell xenogeneic responses by combinations of monoclonal antibodies specific to CD2 and NKG2D and extracellular signal-regulated kinase kinase inhibitor.

Natural killer (NK) cells can destroy xenogeneic tissues by antibody-dependent cell cytotoxicity (ADCC) and direct lysis. Unlike ADCC, activating interactions between human NK receptors and their cognate ligands in pigs are not fully elucidated. We set up this study to identify human NK activating receptors recognizing porcine cells isolated from distinct organs, e.g., aorta, cornea and liver, and to provide a molecular basis for effective immunosuppressive regimens. Among the array of NK receptors tested, NKp46, 2B4, CD49d, CD48, CD2 and NKG2D, only CD2 and NKG2D were shown to be involved in both cytotoxicity and cytokine (interferon-gamma and tumour necrosis factor-alpha) production against porcine targets. Simultaneous blocking of CD2 and NKG2D by combining its monoclonal antibodies further suppressed xenogeneic NK responses. Moreover, addition of a suboptimal dose of PD98059, an extracellular signal-regulated kinase (ERK) kinase inhibitor, to those cells maximally reduced NK cytotoxicity, suggesting that ERK plays an important role in NK-mediated xenoreactivity. These impairments in NK cells were tightly associated with defective intracellular calcium mobilization and the subsequent degranulation process. Therefore, our data demonstrate a distinct role of CD2 and NKG2D on human NK cells in recognizing porcine grafts and further provide a potentially efficacious combinational regimen using anti-CD2 and anti-NKG2D monoclonal antibodies with PD98059 in a pig-to-human transplantation model.