Shuren Zhang

Enhancement of antitumour immunity by a novel chemotactic antigen DNA vaccine encoding chemokines and multiepitopes of prostate-tumour-associated antigens

DNA vaccines provide an attractive technology against cancer because of their safety record in humans and ease of construction, testing and manufacture. In this study, several DNA fragments encoding multiple cytotoxic T lymphocyte (CTL) and T helper cell epitopes were selected from human prostate‐specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate‐specific antigen (hPSA). These DNA fragments were ligated together to form a novel fusion gene, termed the 3P gene. The secondary lymphoid tissue chemokine (SLC), 3P and human immunoglobulin G Fc genes were inserted into pcDNA3.1 to construct a DNA vaccine, designated pSLC‐3P‐Fc. After vaccination, the DNA is taken up by cells that produce and secrete the SLC‐3P‐Fc fusion proteins, termed chemotactic antigen (chemo‐antigen). The secreted chemo‐antigens, in addition to promoting the co‐localization of naive, non‐polarized memory T cells and dendritic cells, are efficiently captured and processed by dendritic cells via receptor‐mediated endocytosis and then cross‐presented to both major histocompatibility complex class I and class II in a cognate manner. The results of this study demonstrate that vaccination with pSLC‐3P‐Fc by gene gun inoculation induced a strong antitumour response in a mouse tumour model, which significantly inhibited tumour growth and prolonged the survival time of the tumour‐bearing mice. In vitro, the secreted SLC‐3P‐Fc fusion protein can attract lymphocytes from human peripheral blood mononuclear cells (PBMC); when human lymphocytes were stimulated by pSLC‐3P‐Fc‐transfected autologous PBMC, CTLs were induced which could specifically kill hPSM‐, hPAP‐, or hPSA‐expressing tumour cells. These observations provide a new vaccine strategy for cancer therapy through promoting the co‐localization of lymphocytes and the concomitant enhancement of antigen‐specific CD4+ helper and CD8+ cytotoxic T‐cell responses against tumour.

Enhancement of DNA Vaccine Potency by Sandwiching Antigen-Coding Gene Between Secondary Lymphoid Tissue Chemokine (SLC) and IgG Fc Fragment Genes

DNA vaccine has become an attractive approach for generating antigen-specific immunity. Targeting antigens to FcRs for IgG (Fc_Rs) on dendritic cells (DCs) has been demonstrated to enhance antigen presentation. Secondary lymphoid tissue chemokine (SLC) has been shown to increase immune responses not only by promoting co-clustering of T cells and DCs in the lymph nodes and spleen but also by regulating their immunogenic potential for the induction of T cell responses. In this study, using HPV 16 E7 as a model antigen, we constructed a chemotactic-antigen plasmid DNA vaccine (pSLC-E7-Fc) by linking SLC and Fc gene sequences to each end of E7 and evaluate its potency of eliciting specific immune response. We found that immunization with pSLC-E7-Fc generated much stronger E7-specific lymphocyte proliferative and cytotoxic T lymphocyte (CTL) responses than control DNA. All the mice receiving pSLC-E7-Fc prophylactic vaccination remained tumor free upon subcutaneous inoculation of TC-1 cells, while those given control DNA all developed tumors. These tumor-free mice were also protected against TC-1 re-challenge. Complete tumor regression with long-term survival occurred in 72% of mice given pSLC-E7-Fc as therapeutic vaccination. In experimental lung metastasis model wherein TC-1 cells were intravenously injected, therapeutic vaccination with pSLC-E7-Fc significantly reduced the number of tumor nodules in the lung. In vivo depletion with antibodies against CD4+ or CD8+ T cells both resulted in complete abrogation of the pSLC-E7-Fc-induced immunotherapeutic effect. Our data indicate that the DNA vaccine constructed by the fusion of SLC and IgG Fc fragment genes to antigen-coding gene is an effective approach to induce potent anti-tumor immune response via both CD4+ and CD8+ T cells dependent pathways.

Cancer stem cells sustaining the growth of mouse melanoma are not rare.

Cancer stem cell (CSC) is generally believed to be a very small proportion of tumor cells capable of initiating and sustaining growth of the tumor. Its existence is usually demonstrated by xenotransplanting human cancer cells in immunodeficient mice. In this paper, we report that the growth of B16-F10 melanoma cells in syngeneic mice could be maintained by a relatively larger proportion (>10%) of tumor cells. The result of this study does not seem to support the current view that cancer stem cells (CSCs) responsible for the sustainable growth of tumor are rare.

Potent Systemic Antitumor Immunity Induced by Vaccination with Chemotactic-Prostate Tumor Associated Antigen Gene-Modified Tumor Cell and Blockade of B7-H1

We previously reported that several DNA fragments from human prostate-specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate-specific antigen (hPSA) genes were selected and fused to create a novel hPSM-mPAP-hPSA fusion gene (named 3P gene), and human secondary lymphoid tissue chemokine (SLC), 3P, and human IgG Fc genes were inserted into pcDNA3.1 to construct a DNA vaccine, designated pSLC-3P-Fc. In this report, to establish a more efficient treatment for immunotherapy against prostate cancer, the construct was transfected into B16F10 to generate gene-modified tumor cell vaccine (named B16F10-SLC-3P-Fc). In poorly immunogenic B16F10 mouse melanoma model, the immunization with B16F10-SLC-3P-Fc resulted in a strong antitumor response and 50% of tumor-bearing mice achieved long-term survival (>120 days). In vivo depletion of lymphocytes indicated that CD8+ T cells were involved in the direct tumor killing, whereas CD4+ T lymphocytes were required for the induction of CD8+ CTL response in B16F10-SLC-3P-Fc-immunized mice. Splenocytes from B16F10-SLC-3P-Fc-immunized mice specifically recognized and lysed PSM, PAP, PSA, and 3P expressing tumor cells. The combined therapy of B16F10-SLC-3P-Fc plus anti-B7-H1 MAbs further enhanced the immune response. Rechallenge experiment showed that a persistent memory response was successfully induced by the combined therapy. These observations suggest pSLC-3P-Fc-modified tumor cells could serve as a vaccine against prostate cancer, and the therapy combined with anti-B7-H1 MAbs further enhanced the antitumor immune response.

Enhanced antitumor effect against human telomerase reverse transcriptase (hTERT) by vaccination with chemotactic‐hTERT gene‐modified tumor cell and the combination with anti‐4‐1BB monoclonal antibodies

Human telomerase reverse transcriptase (hTERT) represents an attractive target for cancer immunotherapy because hTERT is reactivated in most human tumors. In an attempt to develop an effective vaccine against most human cancers, we constructed chemotactic‐hTERT vaccine. Two hTERT fragments encoding multiple cytotoxic T lymphocyte and T helper cell epitopes were fused as a tumor antigen (named Te). The plasmid based DNA vaccine (pCCL21‐Te‐Fc) was constructed by linking human CCL21 and IgG Fc gene sequences to each end of Te. In poorly immunogenic B16F10 mouse melanoma model, DNA (pCCL21‐Te‐Fc) vaccination significantly inhibited tumor growth and all of the mice were dead by day 52. The immunization with pCCL21‐Te‐Fc‐modified tumor cells (B16/CCL21‐Te‐Fc) resulted in a higher antitumor effect than DNA vaccination and 25% of tumor‐bearing mice achieved long‐term survival (>120 days). The combined therapy of B16/CCL21‐Te‐Fc plus anti‐4‐1BB MAbs further enhanced the immune response, resulting in 75% of tumor‐bearing mice achieved long‐term survival (>120 days) in subcutaneous model and few lung nodules in pulmonary metastasis model. Rechallenge experiment showed that a persistent memory response was successfully induced by the combined therapy. In vivo depletion of lymphocytes indicated that CD8+ T cells were essential in the antitumor activity induced by B16/CCL21‐Te‐Fc plus anti‐4‐1BB MAbs, whereas NK cells and CD4+ T cells played substantial roles. The CTL activity induced by pCCL21‐Te‐Fc‐transfected PBMCs specifically lysed a variety of human leukocyte antigen‐matched and hTERT‐positive human tumor cells, suggesting pCCL21‐Te‐Fc could serve as a vaccine against most human cancers.

A simple and effective method for cancer immunotherapy by inactivated allogeneic leukocytes infusion

Allogeneic mixed leukocytes reaction has been reported to activate vast numbers of T lymphocytes and produce large amounts of type 1 cytokines that are linked to an initiation of antitumor immunity. Using poor immunogeneic B16‐F10 and Lewis lung carcinoma (LLC) tumor model, we evaluated the effects of inactivated allogeneic leukocytes infusion (ALI) on the generation of antitumor immune response, as well as its effect on the primary and metastatic tumor. Allogeneic response promoted the generation of both specific and nonspecific antitumor immunity in an in vitro mixed lymphocytes‐tumor cell culture system. Introveinous infusion of mitotically inactivated allogeneic leukocytes resulted in increased type‐1 cytokines (including IL‐2 and IFN‐γ) release, proliferation of various lymphocyte subsets, and generation of both specific and nonspecific antitumor immune response. As a result of such immune response, ALI caused a delayed tumor growth and a prolonged survival in established B16‐F10 melanoma model. In LLC pulmonary spontaneous metastases model, ALI treatment significantly reduced postoperative tumor metastasis as the lung weights were far smaller than control group (0.16 vs. 0.34 g). Furthermore, after primary tumor resection and ALI treatment, 62.5% mice obtained long‐term survival (>120 days) and there were no tumor growths in these mice when they were rechallenged with the same tumor. These experiments demonstrate that inactivated ALI could activate innate and adoptive antitumor immune response. It would be a simple, effective and secured method for cancer immunotherapy.

Antitumor enhancement by adoptive transfer of tumor antigen primed, inactivated MHC-haploidentical lymphocytes

The present study investigated the antitumor effects by adoptive transfer of tumor antigen primed, inactivated MHC-haploidentical lymphocytes in TC-1 lung cancer mouse model. Our studies revealed that the inactivated MHC-haploidentical effecter cells display the antitumor activity in vitro and target the tumor in vivo. After adoptive transferring these effecter cells, the Th1 cytokines such as IL-2 and IFN-γ are elevated in the serum; the recipient tumor-specific cytotoxic T-cells and natural killer cells are activated; tumor specific memory T cells are induced; tumor growth is inhibited and mouse survival is prolonged. The results indicate that MHC-haploidentical lymphocytes provide both effecter cells which can target the tumor cells through the identical MHC molecules and an adjuvant effects through the unmatched allogeneic MHC molecules which induces endogenous innate and adaptive antitumor immune responses.

Immunotherapy using slow-cycling tumor cells prolonged overall survival of tumor-bearing mice

BackgroundDespite considerable progress in the development of anticancer therapies, there is still a high mortality rate caused by cancer relapse and metastasis. Dormant or slow-cycling residual tumor cells are thought to be a source of tumor relapse and metastasis, and are therefore an obstacle to therapy. In this study, we assessed the drug resistance of tumor cells in mice, and investigated whether vaccination could promote survival.MethodsThe mouse colon carcinoma cell line CT-26 was treated with 5-fluorouracil to assess its sensitivity to drug treatment. Mice with colon tumors were immunized with inactivated slow-cycling CT-26 cells to estimate the efficacy of this vaccine.ResultsWe identified a small population of slow-cycling tumor cells in the mouse colon carcinoma CT-26 cell line, which was resistant to conventional chemotherapy. To inhibit tumor recurrence and metastasis more effectively, treatments that selectively target the slow-cycling tumor cells should be developed to complement conventional therapies. We found that drug-treated, slow-cycling tumor cells induced a more intense immune response in vitro. Moreover, vaccination with inactivated slow-cycling tumor cells caused a reduction in tumor volume and prolonged the overall survival of tumor-bearing mice.ConclusionsThese findings suggest that targeting of slow-cycling tumor cells application using immunotherapy is a possible treatment to complement traditional antitumor therapy.

Enhanced anti-tumor immunity generated by Rituximab-coated tumor cell vaccine

Rituximab is a chimeric monoclonal antibody against CD20, and has been used to treat malignant tumors derived from B cell. We designed a tumor cell vaccine modified by Rituximab, and evaluated anti-tumor effect in human CD20 gene transfected mice tumor model in vivo, and in human CTLs induction by mixed lymphocyte tumor cell culture in vitro. The results demonstrated that the Rituximab-coated tumor cell vaccine had a significant therapeutic effect against tumor pulmonary metastasis formation. Antibody depletion experiments showed CD8+ T Cells were essential for the anti-tumor effect but not NK cells. Capture rate of tumor cells by DCs, which were detected by flow cytometry, was increased by adding Rituximab. The tumor specific cytolysis could be induced by Rituximab-coated tumor cell in human in vitro assay. This therapeutic strategy provides a simple way to potentialize CTLs function to combat cancer and may promote more clinical consideration in immunotherapy for tumors. Rituximab-coated tumor cell vaccine also expanded the clinical Rituximab applications.

LAPTM4B-35 protein is a weak tumor-associated antigen candidate.

Lysosome-associated protein transmembrane 4β (LAPTM4B) is a gene that has been indicated to be involved in cancer. It is located at chromosome 8q22 and is composed of seven exons and six introns. LAPTM4B encodes two protein isoforms: LAPTM4B-35 and LAPTM4B-24. LAPTM4B-35 is markedly upregulated and LAPTM4B-24 is downregulated in several types of cancer. LAPTM4B-35 is 91 amino acids (N91) longer than LAPTM4B-24 at the N-terminus. In the present study, western blotting, enzyme-linked immunosorbent spot analysis and the B16F10-N91 tumor bearing-mice experiments were used to evaluate whether the overexpression of N91 indicates its potential as a candidate tumor-associated antigen. The results revealed that N91 was expressed in a wide range of normal mouse tissues and human peripheral blood mononuclear cells, with varying expression levels. The weak immunogenicity of N91 protein suggested it was a weak candidate antigen; however, the N91 protein was associated with cell proliferation.