A novel MICA/B-targeted chimeric antigen receptor augments the cytotoxicity of NK cells against tumor cells.

Chimeric antigen receptor (CAR)-modified immune cells have emerged as a promising approach for cancer treatment, but single-target CAR therapy in solid tumors is limited by immune escape caused by tumor antigen heterogeneity and shedding. Natural killer group 2D (NKG2D) is an activating receptor expressed in human NK cells, and its ligands, such as MICA and MICB (MICA/B), are widely expressed in malignant cells and typically absent from healthy tissue. NKG2D plays an important role in anti-tumor immunity, recognizing tumor cells and initiating an anti-tumor response. Therefore, NKG2D-based CAR is a promising CAR candidate. Nevertheless, the shedding of MICA/B hinders the therapeutic efficacy of NKG2D-CARs. Here, we designed a novel CAR by engineering an anti-MICA/B shedding antibody 1D5 into the CAR construct. The engineered NK cells exhibited significantly enhanced cytotoxicity against various MICA/B-expressing tumor cells and were not inhibited by NKG2D antibody or NKG2D-Fc fusion protein, indicating no interference with NKG2D-MICA/B binding. Therefore, the developed 1D5-CAR could be combined with NKG2D-CAR to further improve the obstacles caused by MICA/B shedding.

The SpyCatcher-SpyTag interaction mediates tunable anti-tumor cytotoxicity of NK cells.

Chimeric antigen receptor (CAR)-modified T and NK cell immunotherapy is a promising approach for cancer treatment. Due to the lack of tunability in anti-tumor activity, conventional CAR therapies have limited efficacy at low tumor antigen densities. To tune the CAR response to tumor cell surface antigens, we have developed a split CAR using the SpyCatcher-SpyTag system. The SpyCatcher serves as the ectodomain to constitute a SpyCatcher-CAR (SpyCAR), while SpyTag is attached to the antibodies that recognize tumor antigens. With dimerization mediated by SpyCatcher and SpyTag, the number and activation level of SpyCARs recruited by tumor antigens depends on the SpyTag number in the "antibody-SpyTag" fusion protein. The results demonstrated that the increasing number of SpyTags effectively enhanced the cytotoxicity of SpyCAR-NK92 cells against target cells. The development of SpyCAR with tunable cytotoxicity provides a novel strategy for CAR-based tumor immunotherapies.

Intrinsic and extrinsic factors determining natural killer cell fate: Phenotype and function.

Natural killer (NK) cells are derived from hematopoietic stem cells. They belong to the innate lymphoid cell family, which is an important part of innate immunity. This family plays a role in the body mainly through the release of perforin, granzyme, and various cytokines and is involved in cytotoxicity and cytokine-mediated immune regulation. NK cells involved in normal immune regulation and the tumor microenvironment (TME) can exhibit completely different states. Here, we discuss the growth, development, and function of NK cells in regard to intrinsic and extrinsic factors. Intrinsic factors are those that influence NK cells to promote cell maturation and exert their effector functions under the control of internal metabolism and self-related genes. Extrinsic factors include the metabolism of the TME and the influence of related proteins on the "fate" of NK cells. This review targets the potential of NK cell metabolism, cellular molecules, regulatory genes, and other mechanisms involved in immune regulation. We further discuss immune-mediated tumor therapy, which is the trend of current research.

Engagement of an optimized lentiviral vector enhances the expression and cytotoxicity of CAR in human NK cells.

Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer NK cells for the efficient expression of CARs. This study reported the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. We constructed a third-generation NKG2D-based CAR that kills cancer cells by targeting up to eight stress-induced ligands (NKG2DLs). Our results demonstrated that the CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered NK cells. This research helps to optimize the production of CAR-NK cells and enhance their functional activity, to provide CAR-NK cell products with better and more uniform quality.

Cytokine Release Syndrome After Modified CAR-NK Therapy in an Advanced Non-small Cell Lung Cancer Patient: A Case Report.

Use of chimeric antigen receptors (CARs), as an immune cell therapy, has generated excellent clinical outcomes against hematologic tumors in recent years. Among them, the CAR-NK (natural killer) therapy has shown better efficacy, and less toxicity, than chimeric antigen receptor T-cell (CAR-T) therapy. In our phase II clinical trials, administering chimeric costimulatory converting receptor (CCCR)-NK92 cells on advanced non-small cell lung cancer patients proved efficacious in cell and animal experiments. However, we observed occurrence of cytokine release syndrome (CRS), a rare and unexpected side effect, never reported before during CAR-NK therapy. Here, we provide a detailed report of the patient's case, emphasize on the need to pay attention to CRS in NK cell therapy, and suggest improvements that will minimize potential toxicity.

A chimeric switch-receptor PD1-DAP10-41BB augments NK92-cell activation and killing for human lung Cancer H1299 Cell.

Engineered natural killer (NK) cell-based therapies have been potentially broadly applicable and exhibited promising results in clinical trials, particularly in the fight against cancers. NK cell immunotherapy however always remains variable. One major obstacle is the inhibitory pathway including PD1/PDL1, providing tumor cells an escape mechanism from immunosurveillance. In this regard, we rationally designed a chimeric switch-receptor (CSR) PD1-DAP10-41BB, which comprising the ectodomain of PD1 fused to the co-stimulatory receptor DAP10 and 41BB. Therefore, by exchanging the transmembrane and cytoplasmic tail of PD1 with positive costimulatory molecules DAP10 and 41BB signaling domains, the negative PD1/PDL1 signal pathway was thus converted into a positive one. This CSR-expressing NK92 cells showed a typical parental NK92 phenotype and improved cytotoxicity against human lung cancer H1299 cells. Besides, the expression of CSR elicited a significant increase of effector molecules such as perforin and granzymes, which can induce apoptosis of H1299 cells. More importantly, in the solid tumor cell H1299-bearing mice model, the CSR-modified NK92 cells significantly inhibited tumor growth. Collectively, we demonstrated that expression of PD1-DAP10-41BB augmented NK92-cell activation and killing in vitro and in vivo, which provides a considerable avenue of using NK-tailored chimeric receptor engineered NK92 cells to treat a wide range of solid tumors.

In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology.

Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies in vitro. Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies in vitro.

Genetical engineering for NK and T cell immunotherapy with CRISPR/Cas9 technology: Implications and challenges.

Immunotherapy has become one of the most promising strategies in cancer therapies. Among the therapeutic alternatives, genetically engineered NK/T cell therapies have emerged as powerful and innovative therapeutic modalities for cancer patients with precise targeting and impressive efficacy. Nonetheless, this approach still faces multiple challenges, such as immunosuppressive tumor microenvironment, exhaustion of immune effector cells in tumors, off-target effects manufacturing complexity, and poor infiltration of effector cells, all of which need to be overcome for further utilization to cancers. Recently, CRISPR/Cas9 genome editing technology, with the goal of enhancing the efficacy and increasing the availability of engineered effector cell therapies, has shown considerable potential in the novel strategies and options to overcome these limitations. Here we review the current progress of the applications of CRISPR in cancer immunotherapy. Furthermore, we discuss issues related to the NK/T cell applications, gene delivery methods, efficiency, challenges, and implications of CRISPR/Cas9.

Chimeric antigen receptor-modified macrophages trigger systemic anti-tumour immunity.

Preliminary results and emerging data have shown that lipid droplet high (LDhi ) immunosuppressive cells accumulate in tumour tissues. By tracking and phenotypic profiling of LDhi cells, we find that LDhi CD19+ , LDhi CD11b+ , and LDhi Ly6G+ immune cell populations appear in the spleen, thymus, and tumour tissues in a syngeneic tumour model. Using a contact-dependent reporter system, we discover a LDhi CCR7hi immunosuppressive cell population that migrates from tumour tissues to the spleen and thymus. Hence, we engineered a family of chimeric antigen receptor-modified macrophages (CAR-Ms) that direct macrophages to CCR7-positive cells and show that the cytosolic domain from Mer receptor tyrosine kinase (MerTK) triggers tumour cell cytotoxicity by the CAR-Ms. In vivo, CCR7-targeted CAR-Ms suppressed tumour growth and prolonged survival by preventing metastasis and by inducing systemic anti-tumour immunity through retarding the migration of LDhi CCR7hi immunosuppressive cells from tumour tissues to distal immune organs, indicating an important role for CCR7 in tumour cell-induced immune tolerance. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

VEGF165b augments NK92 cytolytic activity against human K562 leukemia cells by upregulating the levels of perforin and granzyme B via the VEGR1-PLC pathway.

Pro-angiogenic Vascular endothelial growth factors (VEGFs) exert immunosuppressive functions on some immune cells by interacting with VEGF receptors. Blocking the VEGF/VEGFR pathway could reverse the tumor immunosuppressive microenvironment to some degree. We recently demonstrated that the anti-angiogenic VEGF isoform VEGF165b, similar to other anti-angiogenic agents, inhibit the accumulation immunosuppressive cells such as Tregs and MDSCs. However, whether VEGF165b affects the functions of immune effector cells remain unclear. Here, NK92 cell line was utilized as an immune effector cell model. Our results verified that NK92 cells endogenously express VEGF165 and VEGFR1. Further investigation showed that NK92 treatment with VEGF165b augments its killing ability against human K562 leukemia cells by upregulating perforin and granzyme B through the VEGFR1-PLC pathway, whereas VEGF165b had no impact on the proliferation of NK92 cells in vitro. The results of this study improve our understanding of the immunomodulatory function of VEGF165b, which may help in enhancing the efficacy of NK92-based cancer immunotherapy.

A novel chimeric PD1-NKG2D-41BB receptor enhances antitumor activity of NK92 cells against human lung cancer H1299 cells by triggering pyroptosis.

Chimeric antigen receptor (CAR)-modified adoptive natural killer (NK) cells represent a promising immunotherapeutic modality for cancer treatment but face many challenges in solid tumors. One major obstacle is the immune-suppressive effects induced by inhibitory receptors (IR) including PD1. To interfere with PD1 signaling to augment CAR-NK cells' activity against solid tumors, we rationally designed a novel chimeric costimulatory converting receptor (CCCR), comprising mainly the extracellular domain of PD1, transmembrane and cytoplasmic domains of NKG2D, and the cytoplasmic domain of 41BB. This NK-tailored CCCR was able to switch the negative PD1 signal to an activating signal and hence reversed the immune suppressive effects of PD1. The CCCR-modified NK92 (CCCR-NK92) cells retained typical characteristics of NK cells and exhibited enhanced antitumor activity against human lung cancer H1299 cells in vitro compared with untransduced NK92 cells. The rapid clearance of H1299 cells was caused by CCCR-NK92 cell-induced extensive pyroptosis. In a lung cancer xenograft model, CCCR-NK92 cells significantly inhibited tumor growth. Our results highlight a promising immunotherapeutic potential of using NK-tailored CCCR engineered NK92 cells to treat human lung cancer.

VEGF165b and its mutant demonstrate immunomodulatory, not merely anti-angiogenic functions, in tumor-bearing mice.

A great deal of evidence has shown that anti-angiogenic molecules and antibodies targeting the VEGF-A/VEGFRs signal pathway can also reverse tumor-induced immunosuppression to an extent. VEGF165b, an anti-angiogenic VEGF-A isoform, has demonstrated capacity as an efficacious anti-tumor therapy in mice as an anti-angiogenic agent. However, whether VEGF165b also plays an immunomodulatory role in anti-tumor field remains unclear. mVEGF165b effect on regulatory T cells (Tregs) in vitro were evaluated using flow cytometry and Cell Counting Kit-8 (CCK-8) methods. Its effects on Tregs (or Foxp3 expressing cells) and myeloid-derived suppressor cells (MDSCs) were analyzed in vivo using flow cytometry and immunostaining techniques. In this study, we found VEGF165b and its mutant (its half-life in plasma was extended 10 times while retaining its bioactivity; the VEGF165b mutant is called mVEGF165b for short) inhibited the proliferation of Tregs in vitro. In addition, mVEGF165b dramatically inhibited the accumulation of MDSCs and Tregs (or Foxp3 expressing cells) in the spleen and tumor in tumor-bearing mice. In conclusion, our findings demonstrated for the first time that VEGF165b and its mutant has immunoregulatory functions. It may be used as a potential immunomodulatory agent, beyond its anti-angiogenic capacities, in cancer therapies.

A novel bispecific chimeric PD1-DAP10/NKG2D receptor augments NK92-cell therapy efficacy for human gastric cancer SGC-7901 cell.

Cell-based immunotherapy continues to be a promising avenue for cancers that standard therapy has failed. Although the specificity, avidity, and efficacy of infused cells have improved, immunocytotherapy still faces substantial hurdles. To this end, we developed a structure-based rational design approach and constructed a novel Dual Targeting Chimeric Receptor (DTCR) PD1-DAP10/NKG2D comprising the truncated ectodomain of PD1 fused to a key co-stimulatory receptor DAP10, and subsequently harnessed the activating receptor NKG2D, which evaluated the capacity of solid tumor cell killing. Retroviral transduction of DTCR dramatically increased NK92 cell surface expression of PD1 and NKG2D, which boosted robust cytotoxicity against human gastric cell SGC-7901. Chimeric receptor DTCR stimulation elicited a significant increase of TNF-α and TRAIL, which can trigger apoptosis of SGC-7901 cells. More importantly, DTCR-NK92 cells had considerable antitumor activity in the solid tumor cell SGC-7901-bearing mice model. Collectively, we demonstrated that expression of DTCR markedly augmented the cytotoxic potential of NK92 cells against solid tumor cells, and this potentially promising treatment modality will facilitate clinical translation of potent NK-tailored chimeric receptor strategy for a generalized cellular therapy that may be conducive to treat a wide range of solid tumors.

Aliiroseovarius marinus sp. nov., isolated from seawater.

A novel Gram-stain-negative, rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated A6024T, was isolated from coastal seawater near Rizhao, PR China (119.61° E 35.47° N). The organism grew optimally at 28 °C, in pH 6.0-7.0 and in the presence of 3.0 % (w/v) NaCl. The strain required seawater or artificial seawater for growth and NaCl alone did not support growth. Strain A6024T contained ubiquinone 10 as the sole respiratory quinone and C18 : 1 ω7c (75.2 %) as the most abundant fatty acid. The predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified lipids. The DNA G+C content of strain A6024T was 59.9 mol%. The results of phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Aliiroseovarius halocynthiae MA1-10T, Aliiroseovarius pelagivivens GYSW-22T and Aliiroseovarius crassostreae CV919-312T with 98.3, 97.6 and 97.4 % sequence similarities, respectively. The calculated average nucleotide identity values and DNA-DNA hybridization values between strain A6024T and the phylogenetically related Aliiroseovarius species were in the range 76.0-85.6 % and 19.6-29.4 %, respectively. On the basis of the results of polyphasic analyses, strain A6024T represents a novel species of the genus Aliiroseovarius, for which the name Aliiroseovarius marinus sp. nov. is proposed. The type strain is A6024T (=KCTC 72114T=MCCC 1K03595T).

Leucothrix sargassi sp. nov., isolated from a marine alga [Sargassum natans (L.) Gaillon].

A novel bacterial strain, C3212T, was isolated from a marine alga collected from the sea shore of Yantai, China. The strain was Gram-stain-negative, rod-shaped, aerobic, non-motile, and oxidase- and catalase-positive. Growth was observed at 8-37 °C (optimum, 28 °C), at pH 6.0-9.0 (optimum, pH 7.0) and in the presence of 1.0-7.0 % (w/v) NaCl (optimum, 4.0 %). The major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids of strain C3212T consisted of diphosphatidylglycerol (cardiolipin), phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified polar lipid. The major fatty acids were C16 : 1ω6c and/or C16 : 1ω7c, and C18 : 1ω6c and/or C18 : 1ω7c. The DNA G+C content of strain C3212T was 44.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Leucothrix pacifica XH122T, Leucothrix arctica IMCC 9719T and Leucothrix mucor DSM 2157T with similarities of 98.0, 97.5 and 94.3 %, respectively. Estimated DNA-DNA hybridization values were 14.2, 20.7 and 13.9 % between strain C3212T and L. pacifica XH122T, L. arctica IMCC 9719T and L. mucor DSM 2157T, respectively. Phenotypic, phylogenetic and genomic analyses revealed that strain C3212T represents a novel species of the genus Leucothrix, for which the name Leucothrix sargassi sp. nov. is proposed. The type strain is C3212T (=MCCC 1K03600T=KCTC 72121T).

Structure-based rational design of a novel chimeric PD1-NKG2D receptor for natural killer cells.

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have the potential to provide the potential for the implementation of allogeneic "off-the-shelf" cellular therapy against cancers. Currently, most CARs are not optimized for NK cells, so new NK-tailored CARs are needed. Here, a major activating receptor of NK cells, NKG2D was harnessed to design different chimeric receptors that mediate strong NK cell signaling. In these NKG2D signaling-based chimeric receptors, the extracellular domain of inhibitory receptor PD-1 was employed to reverse the immune escape mediated by PD-1 ligands in the solid tumors. To achieve the rational design of chimeric PD1-NKG2D receptors, we developed a transmembrane protein tertiary structure prediction program (PredMP & I-TASSER) and optimized the conformation of the PD-1 ectodomain by genetically altering the sequences encoding the hinge and intracellular domain. Finally, we identified a chimeric PD1-NKG2D receptor containing NKG2D hinge region and 4-1BB co-stimulatory domain to exhibit stable surface expression and mediate in vitro cytotoxicity of NK92 cells against various tumor cells. This strategy now provides a promising approach for the computer-aided design (CAD) of potent NK cell-tailored chimeric receptors with NKG2D signaling.

Utilizing VEGF165b mutant as an effective immunization adjunct to augment antitumor immune response.

Compelling evidence has shown that blocking VEGF via monoclonal antibodies may be beneficial in that it not only inhibits tumor angiogenesis but also reduces immune suppression and promotes T cell infiltration into tumors. Herein, we determined whether our recently generated VEGF165b mutant could be used as a co-immunization adjunct to augment the peptide cancer-vaccine- induced immune response in a mouse model of breast cancer. When co-immunized mVEGF165b with the peptide-based cancer vaccine (MUC1, a T-cell epitope dominant peptide vaccine from Mucin1), the VEGF antibody titers increased approximately 600,000-fold in mice. Moreover, the anti-VEGF antibody also reduced the frequency of regulatory T cells (Tregs) in both preventive and therapeutic scenarios. Mechanistically, the decrease of the Tregs population was associated with a remarkably increased MUC-1-specific IFN-γ-producing CD8+ T cells and anti-MUC1 humoral response. Finally, this combination co-immunization produced a superior antitumor response and significantly prolonged survival of tumor-bearing mice. In conclusion, our findings suggest that mVEGF165b may be an ideal immunization adjunct to enhance the immune efficacy of peptide-based tumor vaccines by overcoming immune tolerance.

VEGF165b mutant with a prolonged half-life and enhanced anti-tumor potency in a mouse model.

VEGF165b has been shown to be an effective anti-cancer agent; however, its short half-life limits further application in the clinical field. The development of a mutant VEGF165b with a prolonged half-life is urgently needed for its future application. A mutant VEGF165b was generated by inactivation of its plasmin cleavage site. The mutant and native VEGF165b proteins without purification tags were expressed via the Pichia pastoris expression system followed by purification with a HiTrap heparin affinity chromatography column through optimization of the purification conditions. Furthermore, its binding affinity with VEGF Receptors and its functions in vitro and in vivo were examined. Results showed that the half-life of mutant VEGF165b increased to approximately 10 times (Intravenous), 9.1 times (Intraperitoneal) and 5.4 times (Subcutaneous) greater than that of VEGF165b, and the mutation did not cause significant alteration of VEGFR1 and VEGFR2 binding affinity. Mutant VEGF165b inhibited the proliferation and migration of HUVECs in vitro, similar to the native VEGF165b. In a mouse melanoma model, mutant VEGF165b exhibited stronger anti-tumor activity in comparison with its native counterpart. These results indicate that the mutant VEGF165b had a prolonged half-life and retained the anti-angiogenic activity of the native VEGF165b, suggesting that this novel mutant VEGF165b may be a stronger anti-cancer agent.

Thalassotalea atypica sp. nov., isolated from seawater, and emended description of Thalassotalea eurytherma.

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZG4-3-1T, was isolated from coastal seawater of Rizhao, China (119.625° E 35.517° N). The organism grew optimally at 24-28 °C, at pH 7.0 and in the presence of 2.0 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZG4-3-1T contained ubiquinone 8 (Q-8) as the major respiratory quinone and contained C16 : 1ω7c and/or C16 : 1ω6c and C16 : 0 as the dominant fatty acids. The polar lipids of strain RZG4-3-1T were phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminophospholipid. The DNA G+C content of strain RZG4-3-1T was 40.1 mol%. Strain RZG4-3-1T exhibited the highest 16S rRNA gene sequence similarity value (96.0 %) to Thalassotalea eurytherma JCM 18482T. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RZG4-3-1T belonged to the genus Thalassotalea. On the basis of polyphasic analyses, strain RZG4-3-1T represents a novel species of the genus Thalassotalea, for which the name Thalassotalea atypica sp. nov. is proposed. The type strain is RZG4-3-1T (=JCM 31894T=KCTC 52745T=MCCC 1K03276T). An emended description of Thalassotalea eurytherma is also provided.

[Over-expressed HER2 enhances proliferation, migration and invasion of melanoma B16 cells].

Objective To establish a melanoma B16 cell line stably over-expressing the HER2 (ErbB2) gene and study its effect on the proliferation, migration and invasion of melanoma cells. Methods The recombinant plasmid pCMV3-ErbB2 was transfected into B16 melanoma cell line by cationic liposome. The positive clones were screened with hygromycin B. Real-time quantitative PCR (qRT-PCR) was utilized to determine the expression of ErbB2 mRNA. Immunofluorescence was used to evaluate the expression of ErbB2 protein. MTT assay was applied to detect the cell proliferation ability. The cell migration ability was examined by a scratch assay. TranswellTM assay was performed to determine the effect of HER2 over-expression on the capability of B16s invasion. Results The cell line over-expressing HER2 was successfully established as showed by qRT-PCR. Over-expression of HER2 remarkably enhanced the proliferation, invasion and migration of B16 cells. Conclusion Over-expression of HER2 can enhance the growth and invasion of B16 cells.