Background
Traditional therapies to treat cancer include chemotherapies and radiotherapies; although they are effective against some tumors, they also bring unpleasant side effects due to their unspecific attacks on normal cells and tissues. In recent years, immunotherapies involving checkpoint blockade have achieved huge success, checkpoint blockade is target-specific with much less side effects, and they unleash the power of immune cells [
1,
2]. A variety of checkpoint inhibitors have been approved for the treatment of tumors [
3‐
10]. Although these treatments are effective in some patients, there is still a large percentage of patients who do not respond well to these treatments and cannot benefit from these existing checkpoint immunotherapies.
A family of inhibitory receptors that interact with nectin and nectin-like family molecules has gained increasing attention [
11‐
13], including TIGIT, CD96, PVRIG and the costimulatory receptor CD226, which share the same ligands CD155 and PVRL2 [
14]. The interaction between TIGIT and CD155/PVRL2 suppresses anti-tumor and anti-viral immune responses in both direct and indirect manners [
15]. High expression of TIGIT leads to the exhaustion of CD8
+ T cells and NK cells [
16,
17], and its expression is associated with the prognosis of tumor patients [
18‐
20]. On the other hand, CD96 plays an important role in mouse lung metastasis and subcutaneous tumor models [
21,
22]. Human CD96
+ NK cells are functionally exhausted with impaired IFN-γ and TNF-α production [
23]. PVRIG, also known as CD112R, has been discovered in the year of 2016 [
24]. Being a recently discovered inhibitory receptor in this family, researches on this receptor are very limited compared with those on TIGIT, CD96 and CD226. PVRIG expresses on T cells and NK cells, and its expression on T cells increases with cell activation [
24]. Upregulation of PVRIG on CD8
+ T cells causes their exhaustion in lymphocytic choriomeningitis virus infection [
25]. CD8
+ T cells from PVRIG-deficient mice show stronger antigen-specific effector functions during acute
Listeria monocytogenes infection [
26]. Furthermore, PVRIG-deficient mice display significantly reduced tumor growth due to enhanced CD8
+ T cell function [
26].
Besides CD8
+ T cells, NK cells are also essential anti-tumor effector cells [
27]. The introduction of the term “cold tumor” leads to the boost emergence of anti-tumor immunotherapies involving NK cells, which are particularly important for treating cytotoxic T lymphocyte (CTL)-insensitive tumors with no or minimum MHC class I expression. Reduced NK cell number or impaired NK cell function has been associated with the progression of various types of cancers [
28,
29]. It has been reported that blocking PVRIG not only promotes cytokine secretion and proliferation of human T cells, but also enhances antibody-dependent cell-mediated cytotoxicity (ADCC) of human NK cells [
24,
30]. In addition, PVRIG blockade enhances NK cell killing of its ligand PVRL2
hi acute myeloid cells [
31]. However, the role of PVRIG in the regulation and immunotherapy of NK cells in the solid tumor microenvironment has not been investigated.
In this study, we generated a rat anti-mouse PVRIG monoclonal antibody (mAb) that specifically blocks the interaction between PVRIG and its ligand PVRL2. Genetic knock-out of PVRIG in mice or treatment with anti-PVRIG mAb (both early and late treatments) significantly inhibited the exhaustion of NK cells and slowed tumor growth in several murine tumor models. We showed that besides CD8+ T cells, the presence of NK cells was also critical for the therapeutic effects of PVRIG blockade. Furthermore, we generated mouse anti-human PVRIG mAb and found that anti-human PVRIG (anti-hPVRIG) slowed tumor growth in both human NK cell- and peripheral blood mononuclear cell (PBMC)-reconstituted xenograft murine models. These findings indicate that blockade of PVRIG not only promotes the anti-tumor immunity of CD8+ T cells, but also unleashes the anti-tumor power of NK cells, therefore making PVRIG a promising immune checkpoint target to treat cancer.
Methods
Mice
C57BL/6J mice were purchased from Shanghai Experimental Animal Center (Shanghai, China) or GemPharmatech Corporation Limited (Nanjing, China). Rag1−/− mice were purchased from GemPharmatech Corporation Limited (Nanjing, China). C57BL/6 Pvrig+/– mice were generated by Beijing Biocytogen Corporation Limited (Beijing, China), and Pvrig−/− mice were bred in-house. B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) were purchased from Beijing Biocytogen Corporation Limited (Beijing, China). All mice were maintained in a specific pathogen-free facility for use. Mice were used between 6 and 8 weeks of age. Animal experiments were approved by the ethics committee of the University of Science and Technology of China.
Cell lines
Lewis Lung Carcinoma (LLC) cell line was purchased from the cell bank of the Chinese Academy of Science (Shanghai, China). MCA205 fibrosarcoma cell line was purchased from BNCC (Beijing, China). MC38 colon adenocarcinoma cell line was kindly provided by Professor Yangxin Fu from University of Texas Southwestern Medical Center (Dallas, USA). Hybridoma cells PK136 (anti-NK1.1; in vivo depletion) were purchased from ATCC (Manassas, USA). Clone 1 (rat IgG1,κ), a blocking monoclonal antibody to mouse PVRIG, was a novel clone generated in-house by our laboratory with no prominent ADCC. Clone 2 (mouse IgG1,κ), a blocking monoclonal antibody to human PVRIG, was a novel clone generated in-house by our laboratory with no prominent ADCC. All cell lines were maintained in DMEM medium containing 10% FBS. SW620 human colon cancer cell line, A375 human melanoma cell line and SK-OV-3 human ovarian cancer cell line were purchased from the cell bank of the Chinese Academy of Science (Shanghai, China). SW620 cell line was maintained in L-15 medium containing 10% FBS. A375 cell line was maintained in DMEM medium containing 10% FBS. SK-OV-3 cell line was maintained in McCoy's 5a medium containing 10% FBS. NKG cell lines were established and maintained as previously described [
32]. All cell lines were tested negative for mycoplasma contamination.
Identification of anti-PVRIG antibodies
For rat anti-mouse PVRIG monoclonal antibody (Clone 1) binding assay, 2 × 105 293T-mouse PVRIG cells were incubated with different concentrations of antibodies (Clone 1), and then the binding frequency was detected using APC-conjugated goat anti-rat IgG antibody (Poly4054, Biolegend, San Diego, USA). For assessing PVRIG-PVRL2 antagonistic activity, 2 × 105 293T-mouse PVRIG cells were incubated with different concentrations of antibodies (Clone 1) and 10 μg/mL mouse PVRL2-hFc fusion protein. APC-conjugated mouse anti-human IgG Fc antibody (HP6017, Biolegend, San Diego, USA) was used to detect the binding frequency of mCD112-hFc fusion protein.
For mouse anti-human PVRIG monoclonal antibody (Clone 2) binding assay, 2 × 105 293T-human PVRIG cells were incubated with different concentrations of antibodies (Clone 2), and then the binding frequency was detected using APC-conjugated goat anti-mouse IgG antibody (Poly4053, Biolegend, San Diego, USA). For assessing PVRIG-PVRL2 antagonistic activity, 2 × 105 293T-human PVRL2 cells were incubated with different concentrations of antibodies (Clone 2) and 10 μg/mL human PVRIG-Fc fusion protein. APC-conjugated mouse anti-human IgG Fc antibody (HP6017, Biolegend, San Diego, USA) was used to detect the binding frequency of human PVRIG-Fc fusion protein.
Surface plasmon resonance (SPR)
SPR measurements were performed on the Biacore 8 K high-throughput molecular interaction detection system (GE Healthcare, Little Chalfont, UK). The mouse PVRIG-Fc fusion protein or human PVRIG-Fc fusion protein was immobilized on a CM5 sensor chip (GE Healthcare, Little Chalfont, UK) under 25 degrees according to the manufacturer’s instructions. Anti-PVRIG antibody was flowed at increasing concentrations in the running buffer at 30 μL/min. The sensor chip was regenerated with 50 mM NaOH for every cycle. Specific binding of anti-PVRIG antibody to antigen was calculated automatically using the response to a blank channel as a reference. All the raw sensogram data were processed and fit using the Biacore 8 K Evaluation software version 1.1. (GE Healthcare, Little Chalfont, UK).
Transplant tumor models
For early antibody treatment experiment, C57BL/6 mice or Rag1−/− mice were inoculated subcutaneously with 5 × 104 MC38 cells. Mice were randomized into treatment groups 3 days later and treated with anti-PVRIG (250 μg; purified in-house from Clone 1 hybridoma cell supernatant), isotype-matched control antibody (250 μg; purified in-house from rat serum) or PBS by intraperitoneal injection for six times (once every 3 days). For late antibody treatment experiment, C57BL/6 mice were inoculated subcutaneously with 2 × 105 MC38 cells. Mice were randomized into treatment groups when tumor size reaches 100–150 mm3 and treated with anti-PVRIG (250 μg; purified in-house from Clone 1 hybridoma cell supernatant) or isotype-matched control antibody (250 μg; purified in-house from rat serum) by intraperitoneal injection for six times (once every 3 days). To evaluate the effect of combined therapy, C57BL/6 mice were treated intraperitoneally with isotype-matched control antibody (250 μg), anti-PD-L1 (100 μg; 10F.9G2, Bio X Cell, Lebanon, USA), anti-PVRIG (250 μg) or anti-PD-L1 (100 μg) combined with anti-PVRIG (250 μg) for six times (once every 3 days) starting on day 3. To evaluate the tumor growth in wild-type and Pvrig−/− mice, mice were inoculated subcutaneously with 5 × 104 MCA205 cells, 2 × 105 MC38 cells or 1 × 106 LLC cells.
For human NK cell-reconstituted xenograft model, female B-NDG mice were inoculated subcutaneously with 1 × 106 SW620 colon cancer cells on day 0. Mice were grouped randomly and received 1 × 107 expanded human NK cell transfer on days 7, 12 and 17, along with control antibody treatment or anti-human PVRIG mAb (250 μg; purified in-house from Clone 2 hybridoma cell supernatant) on days 7, 10, 13, 16 and 19. All mice were injected intraperitoneally with 50,000 IU recombinant human IL-2 every two days starting on day 7. For human PBMC-reconstituted xenograft model, female B-NDG mice were inoculated subcutaneously with 1 × 106 SW620 colon cancer cells on day 0. Mice were grouped randomly and received 1 × 107 human PBMC transfer on day 7. After that, mice were treated with PBS, control antibody or anti-human PVRIG mAb (250 μg) by intraperitoneal injection for five times (once every 3 days). Tumors were measured every two or three days by caliper, and tumor volume was calculated as 0.5 × length × width × width. Mice were euthanized when tumor size reaches 1000 mm3.
Isolation of tumor-infiltrating lymphocytes (TILs)
TILs were isolated by dissociating tumor tissue in the presence of collagenase IV (1 mg/mL, Sigma-Aldrich, St. Louis, USA) and DNase I (15 U/mL, Sigma-Aldrich, St. Louis, USA) for 1 h before centrifugation on a discontinuous Percoll gradient (GE Healthcare, Little Chalfont, UK). Isolated cells were then used in various assays to evaluate the phenotype and function of NK cells and T cells.
Antibodies
Monoclonal antibodies to mouse NK1.1 (PK136), mouse PVRIG (Clone 1) and human PVRIG (Clone 2) were purified in-house from hybridoma cell supernatant. Anti-PD-L1 antibody (10F.9G2) and anti-CD8β antibody (53-5.8) were purchased from Bio X Cell (Lebanon, USA). The isotype-matched control antibodies (rat IgG) were purified in-house from rat serum. The following reagents were used: FITC-conjugated antibodies to mouse CD226 (10E5, BioLegend, San Diego, USA), CD69 (H1.2F3, BD Pharmingen, San Diego, USA) and CD107a (1D4B, BD Pharmingen, San Diego, USA); PE-conjugated antibodies to mouse CD8β (H35-17.2, BD Pharmingen, San Diego, USA), CD96 (3.3, BioLegend, San Diego, USA), NKG2D (CX5, BD Pharmingen, San Diego, USA) and Perforin (eBioOMAK-D, eBioscience, San Diego, USA); PerCP-eFluor 710-conjugated antibody to mouse Granzyme B (NGZB, eBioscience, San Diego, USA); PE-Cy7-conjugated antibodies to mouse TIGIT (GIGD7, eBioscience, San Diego, USA), TRAIL (N2B2, BioLegend, San Diego, USA) and Ki67(SolA15, eBioscience, San Diego, USA); BV421-conjugated antibiodies to mouse Tim-3 (RMT3-23, BioLegend, San Diego, USA), TNF-α (MP6-XT22, BioLegend, San Diego, USA) and FasL (MFL3, eBioscience, San Diego, USA); BV510-conjugated antibody to mouse CD45 (30-F11, BD Pharmingen, San Diego, USA); BV605-conjugated antibody to mouse NK1.1 (PK136, BioLegend, San Diego, USA); Alexa fluor 647-conjugated antibody to mouse NKp46 (29A1.4, BD Pharmingen, San Diego, USA); BV785-conjugated antibody to mouse PD-1 (29F.1A12, BioLegend, San Diego, USA); BV786-conjugated antibody to mouse NKG2A/C/E (20d5, BD Pharmingen, San Diego, USA) and IFN-γ (XMG1.2, BD Pharmingen, San Diego, USA); BUV395-conjugated antibody to mouse CD3ε (145-2C11, BD Pharmingen, San Diego, USA); BUV563-conjugated antibody to mouse CD4 (GK1.5, BD Pharmingen, San Diego, USA); BUV737-conjugated antibody to mouse CD8α (53-6.7, BD Pharmingen, San Diego, USA).
The following antibodies were also used: FITC-conjugated antibody to human IFN-γ (B27, BioLegend, San Diego, USA); PE-conjugated antibody to human TNF-α (Mab11, BioLegend, San Diego, USA); PerCP-Cy5.5-conjugated antibody to human CD3ε (HIT3a, BioLegend, San Diego, USA); PE-Cy7-conjugated antibody to human CD8α (RPA-T8, BD Pharmingen, San Diego, USA); BV421-conjugated antibody to human CD4 (RPA-T4, BD Pharmingen, San Diego, USA); BV510-conjugated antibody to human CD16 (3G8, BD Pharmingen, San Diego, USA) and CD107a (H4A3, BioLegend, San Diego, USA); BV605-conjugated antibody to human CD56 (5.1H11, BioLegend, San Diego, USA); and APC-conjugated antibody to human PVRIG (W16216D, BioLegend, San Diego, USA).
Intracellular cytokine staining
For CD107a and intracellular cytokine staining, splenocytes and TILs cells were stimulated for 4 h with 30 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, USA) and 1 µM ionomycin (Sigma-Aldrich, St. Louis, USA) in the presence of 2.5 μg/mL monensin (eBioscience, San Diego, USA). After stimulation, cells were stained for surface markers, fixed and permeabilized with FoxP3 fixation buffer (eBioscience, San Diego, USA) according to the manufacturer’s instructions. Fixed cells were stained with antibodies to IFN-γ, TNF-α and Granzyme B. All samples were acquired on an LSRFortessa (BD, Franklin Lakes, USA) and were analyzed using FlowJo software (BD, Franklin Lakes, USA).
In vivo cell depletion
For depletion of NK1.1+ cells or CD8+ T cells, mice were given intraperitoneal injection of 200 μg mAb to NK1.1 (PK136; purified in-house from cell supernatant) or 200 μg mAb to CD8β (53-5.8, Bio X Cell, Lebanon, USA) 24 h before challenge, and then the antibodies were injected once every week.
Isolation of human PBMCs and NK cells
Peripheral blood samples from healthy controls were collected from The First Affiliated Hospital of Anhui Medical University (Hefei, China). And peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation according to the manufacturer’s instructions. NK cells were purified by negative selection using human NK Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s instructions. The purity of NK cells was > 90% as determined by flow cytometry. Human PBMCs and purified NK cells were cultured in RPMI1640 medium with 10% FBS and 100 IU/mL recombinant human IL-2.
In vitro co-culturing system
1 × 106 human PBMCs or 1 × 105 NKG cells were co-cultured with 4 × 104 SW620 colon cancer cells for 24 h either in the presence of anti-human PVRIG antibody or isotype-matched control antibody (mouse IgG1). CD107a antibody and monensin were added to the culture 4 h before harvest.
Cytotoxicity assay
Tumor cell lines were labeled with CFSE (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions. Labeled tumor cells were co-cultured with effector cells in 96-well plate for 4 h in the presence of anti-human PVRIG antibody or isotype-matched control antibody (mouse IgG1) at different effector/target ratios. For the spontaneous death control, CFSE-labeled target cells were cultured alone followed by the addition of 7AAD, and lysed cells (CFSE+ 7AAD+) were identified by flow cytometry.
Immunohistochemistry
Paraffin sections of human tumor tissues were purchased from the Shanghai Outdo Biotech Co. Ltd. (Shanghai, China). Paraffin sections were de-waxed, rehydrated, subjected to heat-induced epitope retrieval (HIER) and followed by incubation with primary antibodies to human PVRIG (Clone 2, generated in house) and PVRL2 (AF2229, R&D Systems, Minneapolis, USA) respectively. The signal was detected using the DAB Peroxidase Substrate Kit (SK-4100, Vector Labs, Burlingame, USA).
TCGA data analysis
The Cancer Genome Atlas (TCGA) database was used to analyze the gene expression correlations between
PVRIG and other immune checkpoints, as well as the overall survival of colon adenocarcinoma patients based on their
PVRIG gene expression. The correlations were analyzed by UCSC xena at
https://xena.ucsc.edu. The Kaplan–Meier survival of colon adenocarcinoma patients was analyzed by OncoLnc at
http://www.oncolnc.org.
Statistical analysis
Statistical analyses were performed in GraphPad Prism (La Jolla, USA) using appropriate tests as indicated in the figure legends (unpaired two-tailed t test, paired two-tailed t test, one-way ANOVA followed by Tukey’s multiple comparisons test, two-way ANOVA or the Mantel–Cox test). P < 0.05 was considered significant in all analyses.
Discussion
Chemotherapies and radiotherapies are effective against majority of tumors; however, they also bring many unpleasant side effects. Unlike traditional anti-tumor therapies, immunotherapies involving checkpoint blockade have gained their credit due to specific targeting and less side effects and have revolutionized the ways to treat cancer [
1]. Although a variety of checkpoint inhibitors targeting CLTA-4, PD-1 and PD-L1 have shown impressive potent anti-tumor immunity and durable responses in some patients, there is still a large percentage of patients who do not respond to and therefore cannot benefit from these treatments [
37], emphasizing the importance of finding novel immune checkpoint targets or combinational strategies.
Nectin and nectin-like families are important cell–cell adhesion molecules belonging to the immunoglobulin superfamily [
38]. Some nectin family molecules can interact with receptors on the surface of immune cells to participate in immune regulation [
11]. The co-inhibitory receptors CD96, TIGIT, PVRIG and the costimulatory receptor CD226 comprise a critical regulatory system for lymphocyte activity and anti-tumor immunity, and they share the same ligands CD155 and PVRL2[
14]. Engagement of CD226 with its ligands PVRL2 and CD155 on target cells is essential for enhancing the anti-viral and anti-tumor functions of NK cells and T cells [
39,
40], whereas CD96, TIGIT and PVRIG counterbalance CD226-dependent lymphocyte activation. Blockade of CD96 reduces the experimental and spontaneous metastases dependent of CD226 and INF-γ [
21,
41]. Anti-TIGIT antibody mediates the rescue of anti-tumor responses of effector T cells that requires the costimulatory signal of CD226 [
16,
42]. TIGIT preferentially binds to CD155, whereas PVRIG is the main inhibitory receptor for PVRL2 [
24,
43]. PVRIG binds to PVRL2 with a higher affinity than CD226 [
24], and blockade of PVRIG/PVRL2 interaction is very likely to result in the dimmed inhibitory effect of PVRIG and enhanced activating effect of CD226.
The latest research has shown that PVRIG is highly expressed on the terminally exhausted CD8
+ T cells, suggesting that PVRIG is important in the exhaustion of CD8
+ T cells [
25]. Indeed, blockade of PVRIG restores the proliferation and cytokine production of T cells [
24,
36], and genetic deletion of PVRIG enhances IFN-γ production of tumor-infiltrating CD8
+ T cell and slows the tumor growth [
26]. Consistent with their study, we showed that both genetic deficiency and blockade of PVRIG could enhance the cytotoxicity and IFN-γ production of tumor-infiltrating CD8
+ T cells in MC38 tumor-bearing mice.
Besides CD8
+ T cells, NK cells are also essential in anti-tumor immunity. The role of NK cells should not be underestimated. Previous studies have reported that blocking PVRIG enhances the ADCC function of human NK cells [
30] and cytotoxicity against PVRL2
hiPVR
lo acute myeloid leukemia target cells [
31]. However, the role of PVRIG on NK cells in tumor microenvironment has not been investigated. In our study, we used three in vivo murine tumor models to show that tumor-infiltrating PVRIG
+ NK cells are more exhausted (higher CD96, TIGIT, Tim-3, PD-1 and NKG2A expression) compared with PVRIG
− tumor-infiltrating NK cells (with the exception of NKG2A on MCA205 tumor model-derived PVRIG
+ NK cells). CD96, TIGIT, Tim-3, PD-1 and NKG2A are typical NK cell exhaustion-related markers [
44], and their upregulation (both in percentage and MFI) indicated the potential exhaustion or exhausted phenotype of PVRIG
+ NK cells. Genetic knock-out of PVRIG or pharmacologic blockade of PVRIG/PVRL2 interaction significantly inhibited the exhaustion of NK cells and enhanced their cytotoxicity and IFN-γ production and therefore limited the subcutaneous tumor growth and prolonged the survival of tumor-bearing mice. By depletion of NK cells or/and CD8
+ T cells, respectively, we showed that NK cells and CD8
+ T cells both contributed to the therapeutic effects of PVRIG blockade in MC38 tumor model. Concurrent depletion of NK cells and CD8
+ T cells resulted in larger tumor size and shorter survival time in tumor-bearing mice treated with anti-PVRIG mAb. Furthermore, by constructing MC38 tumor model using
Rag1−/− mice (which lack T cells and B cells), we showed that anti-PVRIG mAb was effective even in the absence of adaptive immunity, suggesting the importance of NK cells in PVRIG-targeted treatments.
It was reported previously that treatment with anti-PVRIG mAb alone shows no inhibitory effect on tumor growth in murine CT26 colon cancer model [
26], consistent with their study, we observed no influence of anti-PVRIG mAb on CT26 tumor-bearing mice (data not shown). However, both early (when tumor size is around 10 mm
3) and late (when tumor size is around 100–150 mm
3) treatments with our anti-PVRIG mAb alone were effective against MC38 tumors in vivo, resulting in significant inhibition of tumor growth and prolonged survival of tumor-bearing mice. The potential effectiveness of PVRIG blockade in CT26 colon cancer can be boosted to significantly reduce tumor burden by combined blockade with anti-PD-L1[
26], suggesting that combinational therapies can be more effective [
45]. Indeed, our study showed that combined blockade of PVRIG and PD-L1 significantly reduced tumor size in MC38 tumor-bearing mice and resulted in better therapeutic effects than using either mAb alone. Given the importance of CD226 in the lysis of tumor cells, combinational therapies based on the blockade of inhibitory receptors in this family could be very attractive. As reported, blocking CD96 in
Tigit−/− mice is more effective than in wild-type mice in experimental and spontaneous lung metastasis models [
21]. Blockade of PVRIG with TIGIT effectively enhances the cytokine production and cytotoxicity of human CD8
+ T cells in vitro [
36]. In addition, combined blockade of PVRIG and TIGIT shows a better performance in promoting human NK cell ADCC function triggered by trastuzumab than blocking either one alone [
30]. A recent study has also proved that combined blockade of PVRIG and TIGIT further improves the activation and cytotoxicity of NK cells [
31]. These results indicate the potential of PVRIG in combined blockade, and triple combination involving PVRIG (with anti-TIGIT and anti-PD-1) will also launch soon.
COM701, a first-in-class therapeutic antibody targeting human PVRIG generated by Compugen, shows early signs of efficacy, both as a monotherapy and in combination with the PD-1 inhibitor nivolumab in patients with a variety of advanced solid tumors in a Phase I clinical trial [
46]. Here, the mouse anti-human PVRIG mAb generated in our study could entirely block the interaction between PVRIG and PVRL2 with high binding affinity (4 pM) to human PVRIG. The blockade of PVRIG using this mAb enhanced the cytokine secretion and cytotoxicity of human NK cells against various tumor cell lines in vitro. Furthermore, we used human NK cell- or PBMC-reconstituted xenograft model to verify the anti-tumor efficacy of anti-hPVRIG in vivo. As expected, blocking human PVRIG significantly reduced the tumor size in NK cell-reconstituted xenograft mice, suggesting that PVRIG blockade could be effective by acting only on NK cells. The PBMC-reconstituted xenograft model showed similar results, in which the use of anti-hPVRIG significantly reduced tumor size in PBMC-reconstituted xenograft mice, again, proving the anti-tumor efficacy of anti-hPVRIG mAb generated in our laboratory.
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