Recent Advances in Cancer Research and Therapy

Editors

1

Cancer Biotherapy

Progress in China

Zhen-Yu Ding and Yu-Quan Wei*

Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, People’s Republic of China

1.1 Introduction

Public health problems in China have never been so seriously considered as they are today. Acquired immune deficiency syndrome (AIDS), SARS, avian flu, and swine flu (H1N1) have all posed great threats to the nation. However, the most critical public health problem still comes from chronic (noncontagious) diseases, including cancer, cardiovascular disease, and diabetes mellitus. A report from the World Health Organization (WHO) warned that chronic diseases were becoming the most lethal killer of people worldwide. Due to the deterioration of the environment and widely prevalent but uncontrolled smoking, the morbidity of cancer is still on a rapid rise. A retrospective survey that investigated the cause of death in citizens from both urban and rural areas was conducted by the Ministry of Health in 2008. This survey reported that the mortality associated with malignancy increased eightfold over the past three decades. Cancer moved ahead of respiratory disease to become the most deadly disease for rural citizens, while it is the number two killer of people in urban districts (http://61.49.18.102/newshtml/21698.htm).

Cancer has emerged as a great burden for the Chinese health care system. Liver cancer (hepatocellular carcinoma, HCC), gastric cancer, and esophageal cancer still remain the most common causes of death in patients with cancer. However, the prevalence of lung cancer is increasing dramatically.

The past century has witnessed rapid progress in the field of cancer treatment. Surgery and radiotherapy have become mainstays for the treatment of locoregional disease, followed by increasingly effective chemotherapy for disseminated disease. However, despite the great effort that has been devoted, many believe the war against cancer is being lost, and cancer continues to exert a great threat to humankind. In clinical practice, the prognosis is still discouraging for most cancer patients. In addition, radiotherapy and chemotherapy kill tumor cells in a nonspecific way and inevitably cause toxicities while reducing patient tolerance. Therefore, there is a high but unmet need to develop and implement innovative approaches to cure cancer. Fortunately, in recent years, our treatment armamentarium has expanded beyond conventional treatment modalities to include biotherapy, which acts against cancer in a more specific way.

Chinese scientists in oncology, immunology, biochemistry, chemistry, biology, and pharmacology have conducted significant levels of both basic and translational research, advancing our knowledge in these fields. The focus of this chapter will be on selected topics in which substantial progress has been made by Chinese scientists, especially tumor biotherapy.

The discovery of biotherapy dates back one century and was possibly due to chance. The observation of a relationship between infection and cancer regression led Dr. Coley to establish the so-called Coley toxin, which was a mixture of bacterial toxins for the treatment of patients with cancer. Biotherapy, sometimes referred to as biological therapy, biological response modifier (BRM), or immunotherapy, is a treatment used to boost or restore the ability of the host body to fight cancer, infections, and other diseases (http://www.cancer.gov/templates/db_alpha.aspx?CdrID=44483). It is also used to lessen certain side effects that may be caused by other cancer treatments. Agents used in biotherapy include monoclonal antibodies, growth factors, and vaccines. In our opinion, biotherapy also includes gene therapy and targeted therapy, as well as immunotherapy. Cancer biotherapy can be mainly categorized into the following: immunotherapy, gene therapy, antiangiogenesis therapy, and targeted therapy. In this chapter, we will discuss the progress of biotherapy with respect to these categories.

1.2 Immunotherapy

1.2.1 Cancer Vaccine

A vaccine is an active immunogen that can induce protective immunity. Effective vaccines have drastically reduced the incidence of many infectious diseases such as smallpox, poliomyelitis, and diphtheria. Researchers are working hard to make use of this modality to treat cancer, hoping to translate the success of antimicrobial therapy to cancer therapy. A cancer vaccine is a therapeutic vaccine that is usually administered to patients already suffering from cancer. The dream of developing a cancer vaccine has been partially realized only recently. In this area, Chinese researchers are able to remain current with the progress of those abroad who are at the frontiers of investigation.

The identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is very important for the development of peptide-based, cancer-specific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumor-associated antigen. Although several epitopes of the heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. Chinese researchers conducted a study to predict and identify the CTL epitopes in the mouse heparanase protein. The results showed that of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398–405 (LSLLFKKL; mHpa398) and 519–526 (FSYGFFVI; mHpa519) lysed three types of carcinoma cells that expressed both heparanase and H-2K(b) (B16 melanoma cells, EL4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also successfully treating tumor-bearing hosts. The authors suggested that mHpa398 and mHpa519 peptides are novel H-2K(b)-restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo.¹

Gastrin-releasing peptide (GRP), a bombesin-like peptide, is an autocrine growth factor that can stimulate the growth of various cancer cells. A novel protein vaccine HSP65-(GRP-10)(6) (HG6) that consists of six copies of a 10-amino acid residue epitope of GRP C-terminal fragment carried by mycobacterial 65 kDa HSP65 was constructed and then used to immunize mice via subcutaneous injection. Strong humoral and cell-mediated immune responses were induced. High titers of anti-GRP antibodies were detected in the immunized mice sera by ELISA and verified by Western blot analysis. The activity of CD4+ T lymphocytes, especially the high levels of interferon (INF)-gamma, was observed in mice immunized with HG6 when compared with HSP65 or PBS. Immunogenic tumor therapy with a vaccine based on GRP was effective in both protective and therapeutic antitumor immunity in breast tumor models in mice. The purified GRP monoclonal antibody (McAb) was proven to be potent in inhibiting EMT-6 tumor cell proliferation in vitro. The attenuation induced by active immune responses on tumor-induced angiogenesis was observed within an intradermal tumor model in mice. This result showed that immune responses elicited by a novel chimeric protein vaccine targeting GRP can suppress the proliferation of the EMT-6 breast tumor cell line in mice, and that it may be of importance in the further exploration of the applications of other autocrine growth factors identified in humans and in other animals in cancer therapy.²

1.2.2 Cell Therapy

The field of cancer immunotherapy has been recently invigorated by the discovery that vaccination with dendritic cells (DCs) pulsed with tumor antigens is a potent strategy to elicit protective and therapeutic immunity in tumor-bearing hosts. DCs are considered to be the most potent and efficient professional antigen-presenting cells (APCs) identified to date and are capable of activating both resting and naïve T-cells. In fact, most active immunotherapeutic strategies do not stimulate a direct immune response, but rather, they recruit or improve the delivery of antigens to the APCs, mostly DCs. It is well known that DCs express high levels of both class I and II MHC molecules on their cell surfaces in addition to CD28 and intercellular adhesion molecules, which are two of the most critical co-stimulatory molecules for T-cell activation. Their extraordinary capacity to capture, process, and present exogenous antigens also makes DCs the most powerful APCs for the generation of antitumor immunity. The ability to isolate DCs from patients’ peripheral blood and expand them in vitro can help overcome the initial obstacles associated with production. Chinese scientists have made great contributions to the exploitation and application of DCs for cancer biotherapy, gaining recognition from peers abroad.

Tumor-derived exosomes have been proposed as a new type of cancer vaccine. Heat shock proteins (HSPs) are potent Th1 adjuvants. Heat stress can induce HSP and MHC-I expression in tumor cells, leading to the increased immunogenicity of these cells. To improve the immunogenicity of exosomes as a cancer vaccine, Chinese researchers have induced more efficient HLA-A*0201-restricted and carcinoembryonic antigen (CEA)-specific CTL responses by immunization with exosomes prepared from heat-stressed CEA-positive tumor cells. In this study, the researchers identified the composition of CEA+/HS-Exo and observed their effects on human DC maturation. CEA+/HS-Exo contained CEA as well as more HSP70 and MHC-I, while significantly inducing DC maturation. They later evaluated the DCs’ ability to induce a CEA-specific immune response in vivo in HLA-A2.1/Kb transgenic mice, as well as a CEA-specific CTL response in vitro in HLA-A*0201+ healthy donors and HLA-A*0201+CEA+ cancer patients. The immunization of HLA-A2.1/Kb transgenic mice with CEA+/HS-Exo was more efficient in priming a CEA-specific CTL, while the CTL showed antitumor effects when adoptively transferred to SW480-bearing nude mice. Moreover, the in vitro incubation of lymphocytes from HLA-A*0201+ healthy donors and HLA-A*0201+CEA+ cancer patients with CEA+/HS-Exo-pulsed autologous DCs induced HLA-A*0201-restricted and CEA-specific CTL responses. This study showed that CEA+/HS-Exo had a superior immunogenicity than did CEA+/Exo in inducing CEA-specific CTL responses and suggested that exosomes derived from heat-stressed tumor cells be used as efficient vaccines for cancer immunotherapy.³

In another report, researchers from the same group used Hsp70-like protein-1 fusion protein to enhance the induction of a CEA-specific CD8+ CTL response via a DC vaccine. HSPs have been shown to interact with APCs and have a potent adjuvant capability to induce antigen-specific CD8+ CTL and Th1 responses. Hsp70-like protein-1 (Hsp70L1), a new member of the Hsp70 subfamily, acts as a potent Th1 adjuvant. In this report, a tumor antigen-specific immune response was induced by DCs pulsed with a recombinant fusion protein of Hsp70L1 and the CEA(576–669) fragment of CEA containing CAP-1 (an HLA-A2-restricted CTL epitope). The fusion protein CEA(576–669)-Hsp70L1 can promote DC maturation and activate DCs to produce cytokines such as interleukin-12 (IL-12), IL-1beta, and tumor necrosis factor-alpha (TNF-alpha), as well as chemokines such as macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta, indicating the adjuvant ability of Hsp70L1 in the fusion protein. CEA-specific HLA-A2.1-restricted CD8+ CTLs, either from patients with CEA+/HLA-A2.1+ colon carcinoma or from splenocytes of immunized HLA-A2.1/Kb transgenic mice, can be generated more efficiently after stimulations or immunizations with DCs pulsed by CEA(576–669)-Hsp70L1 than with DCs pulsed by CEA(576–669) alone, resulting in increased secretions of the Th1 cytokine IFN-gamma and the more potent killing of target cells in an antigen-specific and HLA-A2.1-restricted manner. The adoptive transfer of splenocytes from transgenic mice immunized with CEA(576–669)-Hsp70L1-pulsed DCs can more markedly inhibit tumor growth and prolong survival in nude mice bearing CEA+/HLA-A2.1+ human colon carcinoma. Therefore, Hsp70L1 has a potent adjuvant effect in the form of a fusion protein, indicating that Hsp70L1 may be widely used as a Th1 adjuvant to prepare antigenic fusion proteins for treatment of cancer or infectious diseases.⁴

It is noteworthy that the efficacy of DC therapy was confirmed not only in preclinical studies but also in pivotal clinical trials. A phase II trial has just been completed, which confirmed the efficacy of antigen-pulsed DC (APDC) for the treatment of metastatic colon and rectal cancer (mCRC). APDC has entered into a phase III trial, and once it passes the phase III trial, the agent will soon enter into clinical testing.

The tumoricidal efficacy of chemotherapy agents for a panel of solid and hematological tumors has been established, although recently accumulating evidence has shown that an immunological effect was also present for at least some of the agents.⁵ Chinese scientists have also played a role in this area. In a recently published paper, they provided the proof-of-principle that low-dose paclitaxel is able to change the tumor microenvironment and improve the outcome of treatment with an intratumoral DC vaccine in a murine lung cancer model.

Their data showed that low-dose paclitaxel, which induced apoptosis in approximately 10% of tumor cells, was not toxic to bone marrow cells or DCs while it stimulated DC maturation and function in vitro. Although the tumor cells inhibited DC differentiation in vitro, this immunosuppressive effect was abrogated by the pretreatment of tumor cells with low-dose paclitaxel. They tested whether the pretreatment of tumor-bearing mice with low-dose paclitaxel in vivo would improve the antitumor potential of a DC vaccine administered intratumorally, and they found significant inhibition of tumor growth in mice treated with low-dose paclitaxel plus intratumoral administration of the DC vaccine, as observed by increased tumor infiltration by CD4+ and CD8+ T-cells and elevated tumor-specific IFN-gamma production by the draining of lymph node cells. These data indicated that low-dose chemotherapy before intratumoral delivery of DCs might be associated with beneficial alterations of the intratumoral microenvironment, thus providing support for antitumor immunity.⁶

Researchers from China have also focused on defining the mechanism and modifying the therapeutic strategy associated with DC therapy. It is known that certain immunosuppressive mechanisms triggered by tumor cells or their derivatives are a major obstacle. In one study, researchers studied the role of DC-derived IL-10 and its negative impact on vaccine efficacy in mouse models. Liver tumor cells were injected via the portal vein, giving rise to disseminated intrahepatic tumors, or were delivered subcutaneously to form solid but extrahepatic tumors. Bone-marrow-derived DCs were generated from normal or IL-10-deficient mice and used as a vector to deliver tumor antigens (Ags). This study demonstrated that DCs devoid of IL-10, a potent immunosuppressive cytokine, are superior to conventional DCs in triggering antitumor immunity. The IL-10(−/−)DCs were highly immunogenic, expressed enhanced levels of surface class II MHC molecules, and secreted increased amounts of Th1-related cytokines. By inducing tumor-specific killing and through the establishment of immunological memory, the vaccines delivered by IL-10(−/−)DCs could evoke strong therapeutic and protective immunity against HCC in mouse models. These findings will have great clinical impact once they are translated into a treatment for malignant, and potentially infectious, diseases in humans.⁷

To improve the therapeutic effect of DC therapy, a proposal involving genetically modified targeted DCs in vitro was tested by Chinese researchers. In this study, researchers created a chimeric CD40 molecule that incorporates a single chain Fv (scFv) molecule specific for the human ErbB2 antigen and fused it to the membrane and cytosolic domains of murine CD40. After adenoviral transfer to bone-marrow-derived DCs, this chimeric receptor (CR) induced nuclear factor-kappaB (NF-kappaB)-dependent DC activation and effector function when cultured with immobilized ErbB2 protein or ErbB2-positive tumor cells in vitro. In vivo migration assays showed that approximately 40% of the injected CR-modified DCs (scFv-CD40-DC) effectively migrated to ErbB2-positive tumors, where they were activated after ErbB2 antigen stimulation, and sequentially homed into the draining lymph nodes. In murine ErbB2-positive D2F2/E2 breast tumor (BALB/c) and EL4/E2 thymoma (C57BL/6) models, intravenous (i.v.) injection of 1×10(6) scFv-CD40-DCs significantly inhibited tumor growth and cured other established tumors. Importantly, the treatment of mice via injection of scFv-CD40-DCs was effective in preventing both ErbB2-positive and parental ErbB2-negative tumor rechallenge. An analysis of the underlying mechanism revealed that i.v. infusion of scFv-CD40-DCs elicited tumor-specific CTL responses, while the transfer of CTLs from scFv-CD40-DC-treated mice protected the naive mice against a subsequent tumor challenge. These results support the concept that the genetic modification of DCs with a tumor-associated antigen-specific CD40 CR might be a useful strategy for the treatment of human cancers.⁸

Chinese scientists have begun to dissect the role of DCs in tumor immune escape and have proposed novel strategies to overcome this problem. Although DC-based cancer vaccines can initiate antitumor immune responses, regulatory DC subsets involved in the tolerance induction attracted much attention recently. The stromal microenvironment of the spleen, lung, and liver can program the generation of CD11clowCD11bhighIalow DCs with regulatory function (CD11bhighIalow regulatory DCs). In one study, researchers used freshly isolated tumor cells to mimic a tumor microenvironment to coculture DCs; they found that freshly isolated tumor cells could drive DCs to differentiate into regulatory DCs with a CD11clowCD11bhighIalow phenotype and display high expressions of IL-10, NO, vascular endothelial growth factor (VEGF), and arginase I. Tumor-educated CD11bhighIalow regulatory DCs inhibited CD4+ T-cell proliferation, both in vitro and in vivo. 3LL lung-cancer-derived TGF-beta and PGE2 were responsible for the generation of regulatory DCs. PGE2 was the main inducer of arginase I in regulatory DCs. Arginase I played a major role in the suppression of T-cell response via regulatory DCs induced by 3LL lung cancer. A natural counterpart of CD11bhighIalow DCs was identified in tumor tissue, and CD11bhighIalow DCs sorted from 3LL lung cancer tissue expressed arginase I and inhibited T-cell response. These data demonstrated that tumors can educate DCs to differentiate into a regulatory DC subset, which contributes to the constitution of an immunosuppressive tumor microenvironment and promotes tumor immune escape.⁹

In addition to DCs, natural killer (NK) cells also play critical roles in antitumor immunity. Myeloid-derived suppressor cells (MDSCs), a population of CD11b(+)Gr-1(+) myeloid cells that expand dramatically during tumor progression, can inhibit T-cells and DCs, contributing to tumor immune escape. In one study, a group of researchers investigated the regulation of the innate function of NK cells by MDSCs in a tumor-bearing host. This study found that the function of NK cells in the liver and spleen was significantly impaired in all tumor-bearing models, indicating that the impairment of hepatic NK cell function by a tumor is a universal phenomenon. Orthotopic liver cancer-bearing mice were established as a tumor model to investigate how hepatic NK cells are impaired. This study showed that the downregulation of NK cell function is inversely correlated with a marked increase of MDSCs in the liver and spleen. MDSCs inhibit cytotoxicity, NKG2D expression, and IFN-gamma production of NK cells both in vitro and in vivo. After incubation with MDSCs, NK cells could not be activated to produce IFN-gamma. Furthermore, membrane-bound TGF-beta1 on MDSCs was responsible for the MDSC-mediated suppression of NK cells. The impaired function of hepatic NK cells in orthotopic liver cancer-bearing mice could be restored by the depletion of MDSCs, but not by regulatory T-cells. Therefore, cancer-expanded MDSCs can induce the anergy of NK cells via membrane-bound TGF-beta1. MDSCs, but not regulatory T-cells, are the main negative regulators of hepatic NK cell function in a tumor-bearing host. This study provides new mechanistic explanations for tumor immune escape.¹⁰

Stem cells are gaining increased attention for their potential application in cancer biotherapy. In a study, mesenchymal stem cells (MSCs) were adenovirally engineered to secrete IL-12 (AdIL-12-MSCs) and evaluated for their anticarcinogenic efficacy against three types of unestablished tumor models, including B16 melanoma, LLC Lewis lung cancer, and HCC hepatoma. The injection of AdIL-12-MSCs into protected mice before tumor inoculation prevented all 12 of the mice in the B16 preventive groups, 10 out of 12 in the LLC lung cancer model and 11 out of 12 mice in the HCC hepatoma model from developing tumors. In contrast, the control groups that were pretreated with PBS exhibited 100% carcinogenesis; the tumor formation rates in the free-AdIL-12 and vacant MSC groups were between approximately 83% and 100%, even with plentiful angiogenesis and newborn lymphatic vessels, as well as distant metastases. As a novel approach, AdIL-12-MSC has shown expected preventive effects on carcinogenesis (P<0.01) with superior low-toxic, broad-spectrum, and long-range qualities. These data indicate that AdIL-12-MSCs possess the potential for tropism to preclinical tumor lesions. They deprive the survival or hibernation of tumor cells that have escaped from conventional treatments, preventing revival and recurrence.¹¹

1.2.3 Antibody Therapy

The production of the monoclonal antibody (McAb) by Kohler and Milstein was a great contribution to health care during the last century. The idea of capitalizing on the power of a McAb to fight malignancy has partly become a reality in daily oncological practice. Currently, there are over 100 McAb under clinical research. Eighteen therapeutic McAbs were approved by the FDA, with most of them for use in oncology. McAb therapy has become the mainstay of tumor immunotherapy.

A naked antibody recognizes and attaches to critical receptors in tumor cells. In this way, they may abrogate the interaction between endogenous ligands and their receptors, inhibit the phosphorylation of receptor tyrosine kinase (RTK), and block downstream signaling. This cascade of events eventually leads to cell cycle stoppage, cessation of proliferation, or apoptosis.

The Fc fragment of an antibody may be very important in inducing the glomerization of a series of complements. The complex formed by these complements has a direct tumoricidal effect (i.e., by attacking cell membranes) or can release a signal for other effector cells to kill the cell (e.g., opsonizing). The Fc fragment also binds specific receptors on the surfaces of effector cells such as NK cells or T-cells, and this antibody-dependent cytotoxicity (ADCC) plays an important role in the antitumor effect of an antibody. Equally promising is the use of a conjugate antibody (Figure 1.1), which is composed of two parts, the conjugates and the antibody. The antibody itself does not possess antitumor activity, but rather carries the conjugate (magic bullet) such as the isotope (e.g., Tuxuetan), toxin, or chemotherapy agent to the tumor site. Currently, there is an intense interest in developing more conjugated antibodies for clinical use.

Figure 1.1 A naked antibody can eradicate cancer in several ways: blockade of a growth signal, ADCC, or CDC.

Because of its great therapeutic and economical potential, China has invested heavily into the research and design of novel antibody agents. One result has been the development and commercial availability of an antibody-¹³¹I-labeled metuximab injection (Licartin) for the treatment of HCC.

A pilot study recruited 24 HCC patients and randomly divided them into three groups to receive 18.5, 27.75, or 37 MBq/kg of Licartin per kilogram of body weight, respectively. Licartin was injected by hepatic artery intubation. The positive imaging result of MAb scanning in 24 patients showed that Licartin accumulated more significantly in hepatomas. These data showed that ¹³¹I-labeled metuximab could deliver relatively selective radiation to tumor tissues.¹² Researchers also carried out clinical trials to show that Licartin was safe and effective in HCC patients. In a phase I trial, 28 patients were randomly assigned to receive the injection in 9.25-, 18.5-, 27.75-, or 37-MBq/kg doses by hepatic artery infusion. In a multicenter phase II trial, 106 patients received the injection (27.75 MBq/kg) on day 1 of a 28-day cycle. The response rate and survival rate were the endpoints. No life-threatening toxic effects were found. The safe dosage was 27.75 MBq/kg. Blood clearance displayed a biphasic model, and its half-life was 90.56−63.93 h. In the phase II trial, the injection was found to be targeted and concentrated in the tumor tissues. Of the 73 patients completing two cycles, 6 (8.22%) had a partial response, 14 (19.18%) had a minor response, and 43 (58.90%) had stable disease (SD). The 21-month survival rate was 44.54%. The survival rate of progression-free patients was significantly higher than that of patients with progressive disease after either one or two cycles (P<0.0001 or P=0.0019).¹³

The Licartin antibody was also shown to be effective in preventing hepatoma recurrence after liver transplantation in a randomized controlled trial. This trial sought to assess the antirecurrence efficacy of Licartin after orthotopic liver transplantation (OLT) in advanced HCC patients. Sixty post-OLT patients with HCC who were at tumor stage 3 or 4 and outside the Milan criteria before OLT were randomized into two groups. Three weeks after OLT, the treatment group received 15.4 MBq/kg of Licartin, while the control group received a placebo intravenously 3 times at an interval of 28 days. At the 1-year follow-up, the recurrence rate decreased by 30.4% (P=0.0174) while the survival rate increased by 20.6% (P=0.0289) in the treatment group compared with the control group. In comparing the control group and the treatment group, the hazard ratio for recurrence was 3.60 (95% confidence interval (CI), 1.50–8.60), and the ratio for death was 3.87 (95% CI, 1.23–12.21). Licartin treatment also resulted in an earlier decreased alpha-fetoprotein (AFP) level and a longer time with a normal AFP level than seen with the placebo (P=0.0016). No Licartin-related toxic effects were observed. The authors concluded that Licartin is a promising drug for preventing post-OLT tumor recurrence in advanced HCC patients excluded by the currently strict criteria for OLT.¹⁴ However, human antimouse antibody (HAMA) response in some patients after administration limited the clinical use of Licartin. To counter this, scientists have tried to develop a more effective antibody fragment with less immunogenicity that can reduce the immunogenicity of the murine antibody. Specifically, they have attempted to humanize HAb18 by variable domain resurfacing based on the three-dimensional structure of the Fv fragment. They fabricated a humanized version of HAb18scFv and HAb18-huscFv based on the human IgG1Fc fragment to form (HAb18-huscFv)(2)-Fc. The reactivity of (HAb18-huscFv)(2)-Fc to the serum of patients with HAMA responses decreased while its specificity and similar binding activity was retained.¹⁵

This group is now developing an ¹³¹I-labeled-CAb1 F(ab′)2 antibody for the re-treatment of colon cancer.¹⁶ A new chimeric IgG1 antibody, hCAb, which could be specifically directed against a cell-surface-associated glycoprotein in colorectal cancer cells, was prepared by genetic engineering technology. In this study, a standard 51Cr release assay showed that like many other clinically validated IgG1 monoclonal antibodies, hCAb primarily acts by ADCC. The maximal cell lysis of ADCC induced by hCAb was over 50% in the presence of peripheral blood mononuclear cells (PBMCs). Moreover, in vivo studies showed potent antitumor effects in nude mice with SW480 and Hce-8693 tumor xenografts. Treatment with hCAb induced a dramatic reduction (over 70%) in tumor volume in comparison to the untreated control group. Furthermore, during the period of treatment, the animals treated with hCAb did not show signs of wasting or other visible signs of toxicity. No obvious tissue damage was detected in any of the vital organs. The chimeric antibody hCAb may be a promising candidate in the treatment of human colorectal cancer. This study can provide a reference for the potential application of hCAb in future clinical trials.

Therapeutic efficacy, suitable dose, and administration times of ¹³¹I-CAb1 F(ab′)2 were investigated in another report. In human colon cancer xenografts, ¹³¹I-CAb1 F(ab′)2 at doses of 125, 375, and 1125 muCi were administrated intraperitoneally on days 6 and 18 after implantation of HR8348 cells with CAb1 high reactivity. The treatment of 125, 375, and 1125 muCi ¹³¹I-CAb1 F(ab′)2 samples did not significantly decrease the mean survival time of nude mice when compared with nontreated groups (P=0.276, 0.865, and 0.582, respectively). Moreover, the mean survival times of nude mice receiving 375 and 1125 muCi of ¹³¹I-CAb1 F(ab′)2 were significantly longer than that of the 5-fluorouracil-treated groups (P=0.018 and 0.042). Tumor growth inhibition rates of the first therapy were 35.67% and 41.37%, with corresponding ¹³¹I-labeled antibody dosages of 375 and 1125 muCi. After a single attack dosage, a second reinforcement therapy may significantly increase the efficacy. The tumor growth inhibition rates with 125, 375, and 1125 muCi ¹³¹I-labeled antibody on day 20 post-therapy were 42.65%, 56.56%, and 84.41%, respectively. Histopathological examination revealed that tissue necrosis of varying degrees was found in the ¹³¹I-CAb1 F(ab′)2-treated groups. This study showed that ¹³¹I-CAb1 F(ab′)2 is safe and effective for colon cancer treatment and may be a novel and potentially adjuvant therapeutic option for colon cancer.¹⁷

This group attempted to generate a reconstituted human-mouse chimeric Fab (cFab) of CAb-1 in vitro to reduce its antigenicity and increase its penetration capacity. First, the genes of the heavy- and light-chain variable regions (VH, VL) of CAb-1 were cloned. Then, the chimeric light chains (cL) and Fd (cFd) were constructed and expressed in Escherichia coli. Finally, the reconstituted cFab was obtained by gradient dialysis of the mixture of cFd and cL. SDS-PAGE and Western blot analysis showed that the reconstituted cFab had a recovery rate of 70.2% when the initial total concentration of cL and cFd proteins was 100 μg/ml. The reconstituted cFab maintained its affinity and specificity to colon cancer cells more than did its parental antibody, as determined by immunostaining analysis, FACS, and ELISA. These results established a foundation for further application of cFab in the diagnosis and treatment of colon cancer.¹⁸

Despite the effectiveness of the anti-CD20 monoclonal antibody (mAb) Rituximab (C2B8) in the treatment of B-cell lymphoma, its efficacy remains variable and often modest. Chinese scientists are working to modify the antibody. In a study, they developed two genetically engineered tetravalent antibodies (TetraMcAb) derived from the anti-CD20 mAbs C2B8 and 2F2, respectively. TetraMcAbs, with a molecular mass that is only 25 kDa higher than native divalent antibodies (DiMcAb), were shown not only to be as effective in mediating complement-dependent cytotoxicity (CDC) and ADCC against B-cell lymphomas as are DiMcAbs, but also to have antiproliferative and apoptosis-inducing activities that are markedly superior to those of DiMcAbs. Interestingly, whereas 2F2 and C2B8 were equally effective in inducing cell growth arrest and apoptosis, the functions of their tetravalent versions, 2F2(scFvHL)(4)-Fc and C2B8(scFvHL)(4)-Fc, were significantly different. 2F2(scFvHL)(4)-Fc exhibited considerably more potent antiproliferative and apoptosis-inducing activity than that of C2B8(scFvHL)(4)-Fc. Immunotherapeutic studies further showed that 2F2(scFvHL)(4)-Fc was far more effective in prolonging the survival of severe combined immunodeficient mice bearing systemic Daudi or Raji tumors than C2B8, 2F2, and C2B8(scFvHL)(4)-Fc, suggesting that it might be a promising therapeutic agent for B-cell lymphoma.¹⁹

Tumor necrosis treatment (TNT) uses degenerating tumor cells and necrotic regions of tumors as targets for radioimmunotherapy. When linked to the therapeutic radionuclide ¹³¹I, a recombinant chimeric TNT antibody (¹³¹I-chTNT) can deliver therapeutic doses to tumors regardless of the location or type of malignancy. The therapeutic efficacy and toxicity of ¹³¹I-chTNT in advanced lung cancer patients were studied in a pivotal registered trial. All 107 patients who were entered into the study and who completed the therapy had experienced treatment failures in prior radiotherapy or chemotherapy an average of three times. The results showed an ORR of 34.6% (complete response, 3.7%; partial response, 30.8%; no change, 55.1%; and progressive disease, 10.3%) in all patients and 33% in 97 nonsmall-cell lung cancer (NSCLC) patients. A biodistribution study demonstrated the excellent localization of radioactivity in tumors in both systemically and intratumorally injected patients. The most obvious adverse side effect was mild and reversible bone marrow suppression. Radioimmunotherapy with ¹³¹I-chTNT was well tolerated and may be used systemically or locally to treat refractory tumors of the lung.²⁰

1.3 Gene Therapy

China occupies a prominent position in the field of cancer gene therapy. Years ago, the first gene therapy product, Adp53, gained approval from the SFDA. Later, another product of the oncolytic adenovirus H101 received qualification to become the second therapeutic drug for cancer treatment in China.

The use of gene therapy and virotherapy has contributed significantly to the treatment of cancer, though neither technology has proven to be very successful until recently. However, a research group had combined the benefits of both technologies to develop a new method called cancer targeting gene–virotherapy (briefly CTGVT), as initiated by Prof. Liu in 2001.²¹ An antitumor gene was inserted into an oncolytic virus (OV) such as ZD55, which is similar to ONYX-015 in the deletion of a 55 kDa gene of Ad·E1B but with the addition of a clone site in ZD55 for the insertion of foreign genes. CTGVT is actually an OV-gene therapy that is also referred to as gene armed oncolytic virus therapy (GAOVT).²² The antitumor effect of the OV-gene (CTGVT or GAOVT) strategy is much better than that of Ad-gene (the gene therapy), which was proven in approximately 60 peer-reviewed SCI papers for different genes and different OVs.²³–³⁰ This effectiveness is because OV can replicate several hundred times and because the inserted gene will be replicated with the same magnitude,³¹ thereby increasing its antitumor effect. Currently, the most effective antitumor drug comes from the CTGVT (OV-gene) technology, including OncoHSV-GM-CSF (OV from the herpes simplex virus with GM-CSF) from BioVex,³² OncoPox-GM-CSF from Jennerex,³² OncoAd-GM-CSF, and OncoAd-IL-24. The biotechnology giant Amgen has spent 1 billion USD to purchase OncoHSV-GM-CSF from BioVex, proving the importance of the CTGVT strategy.

CTGVT is only the basis upon which many modifications have been carried out. The first modification of CTGVT was the combined use of two genes, ZD55-gene1 plus ZD55-gene2, which was called cancer targeting dual gene–virotherapy (CTGVT-DG). Two genes may have a compensative or synergetic effect, thereby increasing their antitumor effects. By using ZD55-TRAIL plus Ad-K5, ZD55-TRAIL plus ZD55-Smac, ZD55-TRAIL plus ZD55-MnSOD, ZD55-TRAIL plus ZD55-hSST2R, ZD55-TRAIL plus ZD55-Cyld, and ZD55-TRAIL plus ZD55-IL-24, all of the xenografts in the liver, colorectal, pancreatic, and gastric cancers of nude mice were completely eradicated.²³–²⁸

To enhance safety and more accurately target specific cancers, many modifications were made in the E1 region, especially the E1A region (e.g., by replacing the E1A native promoter by AFP promoter).³³ As such, this adenovirus may be an ideal way to target liver cancer. We can also make additional modifications of the adenovirus in order to target cancer stem cells and kill them, which equates to the killing of cancer from the root. Doing so enables the complete eradication of the cancer from its origin.

The bright prospects associated with the conditional replicative oncolytic adenovirus have attracted increasing numbers of researchers. In another study, an oncolytic adenovirus was constructed to selectively silence Polo-like kinase 1 (plk1) in tumor cells. plk1 is a serine/threonine protein kinase that is important in many mitotic processes. Previous observations have validated plk1 as a promising therapeutic target. Two artificial features were engineered into one wild-type adenovirus type 5 (wt-Adv5) genome to generate a new oncolytic adenovirus (M1). First, M1 contains a 27-bp deletion in the E1A region, which confers potent oncolytic efficacy. Second, M1 is armed with a fragment of the antisense plk1 cDNA that substitutes the E3 region encoding 6.7K and gp19K. In this design, the tumor-selective replication of M1 would activate the promoters of the native adenovirus E3 to express the antisense plk1 cDNA preferentially in tumor cells and silence the tumor-associated plk1 protein. By virtue of combining oncolysis and plk1 targeting, M1 exhibited potent antitumoral efficacy both in vitro and in vivo. The systemic administration of M1 plus cisplatin induced complete tumor regression in 80% of orthotopic hepatic carcinomas in mouse models.³⁴

Epidermal growth factor receptor (ErbB1, EGFR) is overexpressed in a variety of human cancer cells and has been considered to be a logical target for drug delivery. Recently, groups from China have developed a novel-targeted delivery system strategy for cancer therapy. In their studies, scientists attempted to identify novel ligands with specific binding capabilities to EGFR by screening a phage display peptide library, and they identified an enriched phage clone encoding the amino acid sequence YHWYGYTPQNVI (designated as GE11). Competitive binding assay and Scatchard analysis revealed that the GE11 peptide binds specifically and efficiently to EGFR with a dissociation constant of approximately 22 nM, although with much lower mitogenic activity than with EGF. The peptides were internalized preferentially into cells that highly expressed EGFR, and they accumulated in tumor xenografts that overexpressed EGFR after i.v. delivery in vivo. In gene delivery studies, GE11-conjugated polyethylenimine (PEI) vectors were less mitogenic, but still quite efficient at transfecting genes into cells and tumor xenografts that expressed high levels of EGFR. Therefore, they argued that GE11 is a potentially safe and efficient targeting moiety for selective drug delivery systems mediated through EGFR.³⁵ Similarly, they were able to select a short peptide sequence (WTIIQRREDGSVDFQRTWKEYK, GA3 in name) that was specific to Tie2, an endothelium-specific RTK known to play an important role in tumor angiogenesis. Their study showed that GA3 could not only specifically bind to SMMC7721-Tie2 but also compete with angiopoietin-2 in binding. An in vivo experiment demonstrated that 125I-labeled GA3 favorably accumulates in SPC-A1 xenograft tumor tissues that positively express Tie2.³⁶

Later, scientists attempted to apply the nonviral gene delivery system GE7 for cancer gene therapy. In this study, the human cyclin-dependent kinase inhibitor gene p21(WAF1) and murine cytokine gene granulocyte-macrophage colony-stimulating factor (GM-CSF) were used simultaneously for in vivo gene therapy through systemic injections of the EGFR-targeted GE7/DNA complex into murine hepatoma-bearing mice. The combined administration of p21(WAF1) and GM-CSF remarkably inhibited the growth of subcutaneously transplanted hepatoma Hepa cells and significantly increased the survival rate of the tumor-bearing mice. The activities of NK cells and specific CTLs were clearly enhanced after the combined gene therapy.³⁷ The researchers also tested the targeted delivery system in the ovarian cancer model in rats. The researchers investigated the antitumor effects of the HSV1-tk/GCV strategy with the delivery system (GE7) when administered through the ovarian artery and tail vein. Their findings indicated that the administration of the GE7/HSV1-tk complex via the ovarian artery could be a promising avenue for future human ovarian cancer treatment.

Telomerase reverse transcriptase (TERT) is the key determinant of telomerase activity that plays a crucial role in cellular immortalization and oncogenesis. In a recently published report, a tumor-selective replication-competent adenovirus (RCAd), SG300, was constructed by using a modified promoter of human telomerase reverse transcriptase (hTERT). The antitumor efficacy of SG300 in HCC was assessed both in vitro and in vivo. The results showed that SG300 had a better cancer-selective replication-competent ability and more potential in targeted therapy for HCC.³⁸

In addition, the same group constructed a novel replicative adenovirus CNHK300 in which the hTERT promoter with three extra E-boxes downstream of the promoter was introduced and used to regulate the adenoviral E1a gene; they also studied its properties of selective replication in cancer cells and its antitumoral activity. Experiments in vitro and in vivo demonstrated that CNHK300 can selectively target to hTERT-positive cancer cells and replicate in them, resulting in an oncolytic or antitumoral effect. CNHK300 is superior to ONYX-015 in terms of selective replication and oncolytic or antitumoral effect. The toxicity assay showed no signs of toxicity to liver cells even at the higher dosage of CNHK300 in vivo.³⁹

This group then investigated the antitumor effects of the gene–viral therapeutic system, CNHK300-murine endostatin (CNHK300-mE), in HCC. CNHK300-mE was constructed by employing the hTERT promoter to drive the expression of the adenovirus E1a gene and by cloning the therapeutic gene murine endostatin (mE) into the adenovirus genome. Hepatocellular cells of the HepGII and Hep3B lines were used as liver cancer models to test its efficacy. The results showed that CNHK300-mE was capable of specifically replicating in the telomerase-positive HCC cells and mediating the effective expression of the therapeutic gene in vitro and in vivo.⁴⁰

Therapeutic monoclonal antibodies are increasingly applied in many clinical applications, although complicated technologies and a high cost still limit their wide use. To obtain sustained serum antibody concentrations with a single injection and to lower the costs of antibody protein therapy, the same group has developed an adenovirus-mediated full-length antibody gene therapy. In this study, full-length antibody light- and heavy-chain sequences were linked with an internal ribosome entry site (IRES) and inserted into an adenoviral vector under the control of a cytomegalovirus promoter. The expression of antibodies both in vitro and in vivo were evaluated with an ELISA, and their antitumor efficacy was evaluated in SKOV-3-inoculated nude mice. A single i.v. injection of 2×10⁹ plaque-forming units of Ad5-TAb per mouse resulted in not only a sustained level of over 40 µg/ml serum antibody for at least 4 weeks but also a significant tumor elimination in ovarian cancer in SKOV-3-inoculated nude mice. It seems that an in vivo full-length antibody gene delivery system results in the continuous production of a full-length antibody at high concentrations even after a single administration.⁴¹

One research direction in gene therapy is combinatory (multi-gene) delivery. A research group from China has exploited this strategy using the cotransfers of wild-type p53, GM-CSF, and co-stimulating factor B7-1. In its first study, the group evaluated the in vitro effects of the combination of these three genes via a recombinant adenovirus on the growth and immunogenicity of Hep-2 or primary laryngeal cancer cells. By introducing the wild-type p53 gene, Hep-2 cell growth was inhibited via enhanced apoptosis. By introducing GM-CSF and B7-1 genes, the immunogenicity of the cancer cells was enhanced. Significant proliferation of tumor-infiltrating lymphocytes (TILs) and tumor-specific cytotoxicity of CTLs were induced in vitro. Furthermore, the combined effect of GM-CSF and B7-1 was even more evident than that of any individual agent. These results suggest that the cotransfer of human wild-type p53, GM-CSF, and B7-1 genes into tumor cells via recombinant adenovirus technology can be further developed into a potential combination gene therapy strategy for cancer.⁴²

In the following experiment, a model of multiple myeloma (MM), which remains an incurable disease despite the use of high-dose chemotherapy and stem cell transplantation, was used to test the efficacy of the abovementioned combination. In this study, scientists evaluated the effects of an Ad-p53/GM-CSF/B7-1-modified myeloma cell vaccine strategy aimed to induce apoptosis in order to augment the immunogenicity of MM cells. Both the MM cell lines and purified primary myeloma cells were infected with Ad-p53/GM-CSF/B7-1. The combined effect of these three genes on inducing CTLs was more evident than that of p53 or any combinations of only two agents (i.e., p53 plus GM-CSF or p53 plus B7-1). Furthermore, the significant proliferation of autologous peripheral blood lymphocytes (PBLs) and a specific cytotoxicity against autologous primary MM cells were induced in vitro.⁴³

Combinatory gene therapy could be used to simultaneously correct multiple genetic defects in cancer. The overexpression of Bcl2 and mutations of p53 represent two of the most common molecular defects in tumors. In the nucleus, p53 induces cell cycle arrest, while it interacts with Bcl2 outside the nucleus to regulate signal pathways involved in apoptosis. To potentiate antitumor activity, a double target approach has been proposed that combines H101, a recombinant oncolytic adenovirus that targets the inactive p53 in tumors, with a small interfering RNA (siRNA) (siBCL2) that targets Bcl2. In cell culture, this combined treatment significantly enhanced apoptosis and cytotoxicity more than that shown by treatment with either H101 or siBCL2 alone. In animals carrying tumor xenografts, the combined treatment of H101 and siBCL2 significantly inhibited tumor growth and prolonged survival. At the end of the study, all animals in the combined therapy group survived, with two of the five animals showing a complete eradication of their tumors. Interestingly, siBCL2 treatment increased H101 viral replication in both the treated cells and tumor tissues. The simultaneously targeting of two tumor-specific gene abnormalities using an oncolytic adenovirus and siRNA potentiates total antitumor activity.⁴⁴

The antitumor effect of a genetically modified myeloma cell vaccine was tested in a nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice myeloma xenograft model. A human immune system was established in NOD/SCID mice by intraperitoneal injection of human peripheral blood lymphocytes (huPBLs). After being inoculated subcutaneously with the irradiated myeloma cell line sko-007 with adenovirally transferred GFP or p53, as well as GM-CSF and B7-1 genes, huPBL-NOD/SCID mice were challenged by subcutaneous injection of nontransferred sko-007 cells. The results indicated that the Ad-p53/GM-CSF/B7-1-infected sko-007 cell vaccination significantly reduced local tumor growth compared with controls.⁴⁵ In addition, this strategy was also confirmed to be effective in a nasopharyngeal carcinoma mouse model of NOD/SCID.⁴⁶

siRNA has been widely accepted as a valuable tool for the analysis of gene function. Its potential as a gene therapy agent has been intensively studied as well. A report has been published highlighting the delivery of Stat3-specific siRNA with an attenuated Salmonella typhimurium for the treatment of tumor burden in mice models. The constitutively activated transcription factor signal transducer and activator of transcription 3 (STAT3) promote survival of a number of human tumors. In this study, the relative efficacies of the attenuated S. typhimurium alone or combined with Stat3-specific siRNA, in terms of tumor growth and metastasis, were investigated. The bacteria preferentially homed into tumors over normal liver and spleen tissues in vivo. S. typhimurium that express plasmid-based Stat3-specific siRNAs significantly inhibited tumor growth, reduced the number of metastastic organs, and extended the lifetime for C57BL6 mice bearing an implanted prostate tumor when it was compared against bacterial treatment alone. These results suggest that attenuated S. typhimurium combined with an RNA interference approach might be more effective for the treatment of primary as well as metastatic cancers.⁴⁷

In another study, an siRNA vector specific to human phosphatidylethanolamine-binding protein 4 (hPEBP4) was tested for its efficacy in cancer therapy. hPEBP4 is a newly discovered antiapoptotic factor that was found to be expressed in up to 50% of breast cancers but in only <4% of normal breast tissues. The silencing of hPEBP4 potentiated TNF-alpha-induced apoptosis and cell cycle arrest in MCF-7 cells, which was due to the increased mitogen-activated protein kinase activation and the enhanced phosphatidylethanolamine externalization. Further investigation showed that the silencing of hPEBP4 in MCF-7 cells promoted the TNF-alpha-induced stability of p53; upregulation of phospho-p53ser15, p21waf/cip, and Bax; and downregulation of Bcl-2 and Bcl-xL, which were shown to depend on extracellular signal-regulated kinase 1/2 and c-jun NH2-terminal kinase activation by hPEBP4 silencing. Moreover, an increased proportion of cells in the G0–G1 phase of the cell cycle were observed in hPEBP4-silenced MCF-7 cells with TNF-alpha treatment, while the expression of cyclin A and cyclin E was downregulated more significantly. The authors inferred that the silencing of hPEBP4 expression may be a promising approach for the treatment of breast carcinoma.⁴⁸

siRNA for another oncogene and a nononcogene have been reported frequently during the past few years. Octamer 4 (Oct4), a member of the POU family of transcription factors, plays a key role in the maintenance of pluripotency and proliferation potential of embryonic stem cells. Cancer-stem-cell-like cells (CSCLCs) are reported to comprise a minor population in tumors or even in tumor cell lines that also express Oct4. The role of Oct4 in CSCLCs still remains to be defined. In one study, researchers showed that almost all murine Lewis lung carcinoma 3LL cells and human breast cancer MCF-7 cells express Oct4 at high levels in vitro. This expression of Oct4 is successfully reduced by siRNA, which eventually results in cell apoptosis. The Oct4/Tcl1/Akt1 signal pathway has been observed to be involved in this event. The repression of Oct4 reduces Tcl1 expression and further downregulates the level of p-Ser.473-Akt1. In vivo, only approximately 5% of tumor cells expressed Oct4 in established 3LL and MCF-7 tumor models. siRNA against Oct4 successfully decreases the CSCLCs and markedly inhibits tumor growth. In summary, Oct4 might maintain the survival of CSCLCs partly through Oct4/Tcl1/Akt1 by inhibiting apoptosis, which strongly indicates that targeting Oct4 may have important clinical applications in cancer therapy.⁴⁹

Suicide gene therapy has been an area of interest among researchers for many years. Chinese scientists are currently working to improve outcomes using this method. A report has been presented in which the suicide gene cytosine deaminase (CD) was combined with radiotherapy for local recurrent rectal cancer. The efficiency of liposome-mediated CD gene transfection can be improved by radiation. With radiation of 2, 4, 6, and 8 Gy, the efficiency of liposome-mediated transfection increased from 21.3% to 62.2%, 78.0%, 83.2%, and 87.8%, respectively. CD expression was enhanced as well. A cancer cell inhibition experiment showed that the combined liposome-mediated CD gene therapy with radiation had a much stronger antitumor effect. With the HR8348 tumor xenograft model, the suppression of the tumor xenograft was observed. Compared with the control group, tumor volume was inhibited by 81.5%, 48.5%, and 37.4%, respectively, in the combined CD/5-fluorocytosine (CD/5-FC) with radiation group, CD/5-FC group, and radiation group. The wet weight of the tumor decreased by 80%, 41.7%, and 37.7%, respectively, across the same groups. These findings suggested that the combination of liposome-mediated CD gene therapy with radiation is a safe and efficient anticancer method. Its therapeutic efficacy may satisfy clinical treatment needs associated with local recurrent rectal cancer.⁵⁰

Human alpha-defensin-1 (HNP1), a small antimicrobial peptide, showed cytotoxicity to tumor cells in vitro as well as inhibitory activity for pathological neovascularization in vivo. A recently published paper reported on gene therapy with a plasmid that expressed a secretable form of HNP1 for assaying its antitumor activity. The expression and secretion of HNP1 were determined by reverse transcription-PCR and ELISA in vitro. The expression of HNP1 in the A549 tumor cells caused significant growth inhibition. This effect is most likely cell autonomous, as a significant number of recombinant HNP1 proteins were found to be accumulated in the cytoplasm according to immunohistochemical staining using an anti-HNP1 antibody. The supernatant containing the secreted HNP1 also failed to produce any noticeable antitumor activity. Flow cytometry and Hoechst 33258 staining showed that the number of apoptotic cells among the A549 cells expressing the recombinant HNP1 proteins was significantly greater than that of the nontransfected control cultures, suggesting that this growth-inhibitory activity was the result of an apoptotic mechanism triggered by the intracellular HNP1. The antitumor activity of intracellularly expressed HNP1 was also shown in vivo. A decreased microvessel density and increased lymphocyte infiltration were observed in tumor tissues from HNP1-treated mice through histological analysis. These results indicated that intracellularly expressed HNP1 induces tumor cell apoptosis, which inhibits tumor growth. The antiangiogenesis effect of HNP1 may contribute to its inhibitory activity in vivo, and HNP1 might induce the host immune response to the tumor. These findings provide a rationale for developing an HNP1-based gene therapy for cancer.⁵¹

This group also achieved progress in virus-mediated gene therapy. For example, scientists in this group have tried to manipulate the selectivity of adenoviruses by modifying them with mannan. The mannose receptor was presented on APCs such as DCs; mannan modification was proposed to mask the natural selectivity of the adenovirus and target the virus to DCs, while loading the antigen therein (Figure 1.2). They used TERT as a model antigen to test the efficacy of this strategy. Their results supported the notion that a mannan-modified adenovirus is a useful tumor vaccine.⁵² Vesicular stomatitis virus (VSV), which replicates selectively in tumor cells, has become a promising agent for tumor biotherapy. Previously, VSV was combined with gemcitabine (Gemzar) for the treatment of lung cancer.⁵³ In further studies, they explored the possibility of gene therapy based on the M protein of VSV. The cDNA for the M protein was amplified and cloned into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid or the control empty plasmid pcDNA3.1(+) was mixed with a cationic liposome and introduced into various tumor cell lines in vitro, including the lung cancer cell line LLC, A549 cells, CT26 colon cancer cells, and MethA fibrosarcoma cells. The data showed that the M protein induced remarkable apoptosis of cancer cells in vitro compared with controls. It was found that the tumors treated with the M protein plasmid grew much more slowly, while the survival of the mice was significantly prolonged compared with mice treated with the control plasmid. In MethA fibrosarcoma, the tumors treated with the M protein plasmid completely regressed in certain cases, while the mice acquired long-term protection against the same tumor cell in rechallenge experiments. These results showed that the M protein of VSV can act both as an apoptosis inducer and immune response initiator, which may account for its extraordinary antitumor effects and warrant its further development in cancer gene therapy.⁵⁴

Figure 1.2 Schematic presentation of the modification of an adenovirus with mannan to abrogate its natural selectivity, conferring its specificity to DCs.

Chinese scientists are working hard to innovate traditional gene therapies by expanding upon new technologies. Targeted gene modification mediated by single-stranded oligonucleotides (SSOs) holds great potential for widespread use in a number of biological and biomedical fields, including functional genomics and gene therapy. Using this approach, scientists have created specific genetic changes in a number of prokaryotic and eukaryotic systems. In mammalian cells, the precise mechanism of SSO-mediated chromosome alteration remains to be established, and there have been problems in obtaining reproducible targeting efficiencies. It has previously been suggested that the chromatin structure, which changes throughout the cell cycle, may be a key factor underlying these variations in efficiency. Researchers have systematically investigated SSO-mediated gene repair at various phases of the cell cycle in a mammalian cell line. It was found that the efficiency of SSO-mediated gene repair was elevated by approximately 10-fold in thymidine-treated S-phase cells. The increase in repair frequency correlated positively with the duration of SSO/thymidine coincubation with host cells after transfection. This evidence suggests that these increased repair frequencies arise from a thymidine-induced slowdown of the replication fork progression. Thus, this evidence provides fresh insights into the mechanism of SSO-mediated gene repair in mammalian cells and demonstrates how its efficiency may be reliably and substantially increased.⁵⁵

1.4 Antiangiogenesis Therapy

Several lines of direct and indirect evidence have indicated that the growth and persistence of solid tumors and their metastases are dependent on angiogenesis. As a strategy for cancer therapy, antiangiogenic therapy attempts to stop new vessels forming around a tumor and to break up the existing network of abnormal capillaries that feeds the cancerous mass (Figure 1.3). Angiogenesis has become a very promising target for both experimental and clinical therapies in cancer. Currently, the idea of antiangiogenic therapy in cancer has led to a burgeoning field of research. In this field, China has obtained achievements comparable to those abroad. Only 2 years after the approval of the first antiangiogenic drug, bevacizumab (Avastin), China now possesses her first antiangiogenic drug, recombinant endostatin (Endostar). Some recent progress in the research and development of antiangiogenesis will be introduced below.

Figure 1.3 Tumor growth is due to an imbalance between angiogenesis and antiangiogenesis. Tumors induce angiogenesis, which favors tumor growth, while the therapeutic goal is to tip the balance toward