Construction And Evaluation Of A Novel Humanized HER2-specific Chimeric Receptor

BACKGROUDGene-engineered T cell for adoptive therapy involves engineering the T cell receptor (TCR) and chimeric angtigen receptor (CAR) for the treatment of patients with cancer. Recognition and responses to tumor cells are mediated by the TCR on engagement with tumor-associated antigen (TAA) that is presented by major histocompatibility complex (MHC) molecules, which are expressed by tumor cells. TCR genes, made up of a-and P-chains, can be derived from tumor-specific T cells. In that case, T cells are incubated with tumor cells from the patient, and TCR genes are clones from T cells that react to the tumor cells. The α-and β-chains associate with the γ-,δ-, ε-andζ-chains of the CD3complex. When the TCR encounters a processed TAA fragment displayed on the MHC of the tumor cell, phosphorylation of immunoreceptor tyrosine-base activation motifs occurs, leading to a cascade of intracellular signaling that results in the release of cytokines and cytotoxic compounds from T cells. The advantage of using TCR genes to endow specificity is that the TAA can be derived from the entire protein composition of tumor cells, including intracellular proteins. In addition, there is the potential for truly tumor-specific responses with the identification of specific mutant proteins that are restricted to tumor cells. However, each TCR gene can only be used in a proportion of patients, owing to the MHC-restricted nature of TCR function. Therefore, to broaden the application of gene-engineered T cells, genes encoding CARs (which operate in a non-MHC-restricted manner) have been generated.CARs are composed of a single-chain antibody variable fragment (scFv) extracellular domain linked through hinge and transmembrane domains to a cytoplasmic signalingregion. Genes encoding scFv are usually derived from a tumor specific antibody, and the transmembrane domains are usually derived from immunoglobulin molecules such as CD8.CD28and IgG.The cytoplasmic signaling region is mainly CD3ζ-chains. At present, there are three generation of CAR, in which co-stimulatory molecules such as CD28and CD137are added to CD3ζ, because it has been proved that the addition of co-stimulatory molecules can lead to increased cytokine production in response to TAA and to an enhanced ability of adoptively transferred T cells to mediated tumor regression.CAR T cells can recognize TAA expressed on tumor cells directly and make T cells active and releasing cytokines such as perforation, granzyme, INF-γ and TNF-α. So CAR T cells can kill the tumor cells by non-MHC-restricted ways.Human epidermal growth factor receptor2(HER2) is a well-recognized mediator of the cancerogenic process. It is dysregulated in a wide range of solid tumors, mainly via protein overexpression and/or gene amplification, thus making HER2an attractive target for tailored treatment. It has been proved that more than30%human cancer tissues, such as breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, prostate cancer, gastric cancer, salivary gland cancers, tongue cancer, head and neck squamous cell cancer, non-small cell lung cancer overexpress HER2protein. Although there are monoclonal antibodies such as trastuzumab using for HER2positive cancer patients, they are too expensive to be used broadly. It is easy to synthesize the CAR gene and CAR T cells can be used in patients in non-MHC-restricted ways. The keypoint of CAR T cells is to find a TAA which is high expressed on tumor cells but is low expressed on normal cells. And HER2protein is a good target for CAR T cells therapy.The nature of the scFv is very important for CAR design. The monoclonal antibodies derived from mice have the potential to cause anti-mouse antibody responses in patients, and so CAR T cells will be cleared within days after injection Humanized monoclonal antibody chA21targeted to HER2protein is produced by Professor Jing Liu in University of Science and Technology of China with a surface epitope masking method (SEM). ChA21can recognize HER2at the domain I which is far away the trastuzumab combined domain and suppress the tumor growth effectively. And the chA21scFv has been a present form Professor Jing Liu.OBJECTIVESTo redirect T cell express the second generation chA21scFv-28AR specific for HER2; to observe the character of CAR T cells; to evaluate the anti-tumor effect of CAR T cells in vitro.METHODS1. Constructing the chA21scFv-28ζ CAR.2. To evaluate the HER2expression on human breast cancer cell lines and ovarian cancer cell lines by flow cytometry.3. To transduce the second generation CAR specific for HER2intohuman T lymphocytes using lentivirus; to evaluate the HER2CAR expression on human T cells by flow cytometry.4. To investigate the function of T cells expressing HER2specific CAR in vitro using IFN-γ and IL-2release assay and cytotoxity assay.RESULTS1. The chA21scFv-28ζCAR is constructed successfully.2. Human breast cancer cell lines including SKBR3、T47D、MCF-7、MDA-MB-231and human ovarian cancer cell lines including SKOV3, OVCAR3, A2780, A1847express HER2protein more or less. SKBR3and SKOV3express HER2protein at a high level. The breast cancer cell line MDA-MB-468and the mouse tumor cell line TC-1are HER2negative cell lines.3. The chA21scFv-28ζCAR specific to HER2is successfully transduced and highly expressed on human T lymphocytes surface determined by flow cytometry. About75%of the active T cells are CD8+T cells, and about80%of these cells are central memory T cells which are CD45RO+CD62L+determined by flow cytometry.4. T cells expressing HER2CAR specifically recognize HER2+tumor cell lines and secret high dose IFN-γ and IL-2. At the same time, T cells expressing HER2CAR also showed specifically cytotoxic against human HER2+tumor cell lines but not HER2negative cell lines in vitro.CONCLUSIONS1. ChA21scFv-28ζ CAR is successfully constructed.2. ChA21scFv-28ζ CAR can be transduced into T cells and be expressed steadily.3. CAR T cells are mostly central memory type and the CAR T cells can proliferate quickly. At the same time, CD8+T cells are the main subtype after expanding in vitro.4. This study demonstrate that direct and specific recognition and killing of human breast cancer and ovarian cancers by gene-engineered T cells redirected by HER2-specific CAR. BACKGROUDAlthough the first-generation CAR T cells had no objective response in clinical studies, the second and the third generation CAR T cells have obvious antitumor effects in tumor therapy, especially in hematological malignancies. Pule et al used CAR T cells to treat8neuroblastoma patients and almost half of these patients responded to the therapy, even1of the patients got completely response.Kochenderfer et al used CAR T cells to treat6lymphoma patients and1of the patients got complete response,2of the patients got partly response. Professor Carl June et al in University of Pennsylvania used CAR T cells to treat3chronic lymphocytic leukemia patients and they found that CAR T cells could expand1000folds and maintain the antitumor effects more than6months. Two of the patients got complete response. So, adoptive CAR T cells therapy have good future for antitumor therapy on clinical. Professor Mauspointed out that CAR T cells occupied the first chair for hematological malignacise therapy。Most adoptive CAR T cells therapies targeted HER2were based on trustuzumab, and it had been proved successful in many cancer models such as breast cancer, ovarian cancer, medulloblastoma, glioblastoma and osteosarcoma. The chA21monoclonal antibody is a new type antibody anti HER2protein, and it recognizes the first domain of HER2protein which is different from trustuzumab and pertuzumab. We have found that chA21scFv-28ζ CAR T cells based on chA21antibody have significant antitumor effect on HER2+cancer cells in vitro, so we need to further clear the antitumor effect of chA21scFv-28ζ CAR T cells to HER2+cancer in vivo.OBJECTIVESTo observe if chA21scFv-28ζ CAR T cells have antitumor effect on HER2+breast cancer in vivo and to find the biological characteristics of chA21scFv-28ζ CAR T cells when they are adoptive transfered in vivo.METHODS1. Use NOD-SCID mice and HER2+breast cancer cell line SKBR3to construct the model.2. Transfer the chA21scFv-28ζCCAR T cells to the mice once a week for two weeks after the tumor volume is about300mm3. Non-transfection T cells are used as control.3. To measure the tumor size by vernier caliper.4. Euthanasia the mice when the tumor volume is about2000mm3. Get the tumor weight and used immunochemistry to detect human CD3+T cells in tumor tissue.RESULTS1. The HER2+cancer models are constructed successfully in mice.2. There is no side effect after the adoptive chA21scFv-28C CAR T cells transfer in mice. Compared to the non-transfected T cells, chA21scFv-28ζ CAR T cells inhibit the tumor growth in mice obviously.3. There are huge amount of CD3+T cells in tumor tissues of the chA21scFv-28ζ CAR T cells therapy group, while there is little CD3+T cells in tumor tissues of the control group.CONCLUSIONS1. ChA21scFv-28ζCCAR T cells could specially inhibit the HER2+tumor growth in vivo.2. ChA21scFv-28ζ CAR T cells could specially home in HER2+cancer tissue.