Gcmaf: Our Next-Generation Immunotherapy

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GcMAF: our next-generation immunotherapy

Authors Saisei Mirai, based in Osaka, Japan, is a immunotherapy — such as dendritic cells, Yoshihiro Uto1, Hitoshi Hori1, Kentaro medical organization specializing in cancer autologous lymphocyte-activated killer Kubo2, Masamitsu Ichihashi3, Norihiro immunotherapy. The name Saisei Mirai is (LAK) cells, autologous NK cells, mono- Sakamoto3,4, Martin Mette5, Toshio Inui2,3,5 derived from the Japanese words for regen- clonal antibodies, cancer peptide vaccines, eration and future. It is our mission to cytokines, and biological response modifiers 1 Department of Life System, Institute provide cancer patients with the latest, most (BRM) — is rapidly advancing. However of Technology and Science, Graduate effective treatments while also ensuring the in a clinical setting, the results of cancer School, The University of Tokushima, 2-1 best possible quality of life. Cancer immu- immunotherapy are mixed. Minamijosanjima-cho, Tokushima, Japan. notherapy is the use of the immune system We therefore contend that cancer immu- to reject cancer. In particular, it involves notherapy should be customized to each 2 Saisei Mirai Cell Processing Center, stimulating the patient’s immune system individual patient based on their immune Nature21 Building 3F, 3-34-8 Okubo-cho, to locate and eliminate the cells that are status. We propose our next-generation Gc Moriguchi, Osaka, Japan. cancerous. This is achieved with immune protein-derived macrophage activating fac- cells such as Natural Killer cells (NK cells), tor (GcMAF) as a promising candidate for 3 Kobe Saisei Mirai Clinic, Kobe T Lymphocytes, Dendritic Cells (DC), and a patient-friendly cancer immunotherapy. Commerce Industry and Trade Center macrophages, among others. A variety of GcMAF, also known as vitamin D binding Building 23F, 5-1-14, Hamabe-dori, treatments, such as GcMAF, Hyper T/NK protein-macrophage activating factor (DBP- Chuo-ku, Kobe, Japan. cells, Coley vaccine, gene therapy, cancer maf), is a potent endogenous macrophage vaccines, and intravenous high-dose vita- activator found naturally in the blood. Until 4 Division of Food and Drug Evaluation min C or α-lipoic acid are routinely used to recently, GcMAF was not used in cancer Science, Department of Community stimulate the immune system. immunotherapy in spite of it being a key Medicine and Social Healthcare Science, The immune system is the body’s vital player of our innate immune system. Kobe University Graduate School of self defense that protects against disease. It We will review first-generation GcMAF Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, can recognize a wide variety of pathogens developed by Dr Yamamoto and second- Kobe, Japan found in the environment and even tumor generation GcMAF currently under devel- cells borne within the human body. The opment at Saisei Mirai (Fig. 1). We will also 5 Inui Immunotherapy Clinic, 6-14-17 mechanisms of protection fall into two broad present a number of other therapies used in Kindacho, Moriguchi, Osaka, Japan. categories — innate immunity and adaptive combination with GcMAF. immunity. There are several types of cells At Saisei Mirai, we are currently treating in the innate immune system: phagocytic cancer patients with GcMAF-based cancer neutrophils; macrophages; dendritic cells; immunotherapy. We use GcMAF in combi- mast cells; and natural killer (NK) cells. The nation with other immune system-related adaptive immune system is comprised of the therapies, such as regional and whole-body lymphocytes, both T cells and B cells, which hyperthermia, Coley vaccine, which stimu- are able to recognize and remember specific lates fever within the body, and Hyper-T/ pathogens, and their products, including NK-cell therapy. All of these therapies are antibodies. about strengthening the immune system The progression of many diseases, such and take a holistic approach to fighting as cancer and AIDS, follows the collapse cancer rather than a localized approach that of the immune system. We now know is common with conventional therapies such that stimulating the immune system as radiation and surgery. can halt or even reverse such diseases. Immunotherapy has become an attractive Coley vaccine new strategy in the treatment of cancer1. Coley vaccine — also known as Coley’s The laboratory and clinical study of cancer Toxins and Coley Fluid — was one of the

first immunotherapies to enter the clinic. Coley-vaccine therapy is a medical treatment used to induce a fever to improve the immunological competence of the patient. For hundreds of years, doctors have reported cases of tumors that disappeared, apparently

spontaneously after patients developed high SPONSOR FEATURE fevers from infection. In many cases, when fever subsided, tumors had broken down and been absorbed or sloughed off. Infection seemed to be key to these miraculous cures. It was not until 1893 when Dr William Coley began injecting a sterile mixture of Streptococcus pyogenes and Serratia marcescens bacteria into cancer patients to induce fevers, that this observation was put into clinical practice. By using a suspension of killed bacteria, Coley was able to mimic an acute infection, without the risks of an actual pathogen.

Hyperthermia Hyperthermia heat therapy, also called Figure 1: Research map of Gc protein and GcMAF. First-generation GcMAF developed by thermotherapy, is the application of heat to Dr Nobuto Yamamoto. Second-generation GcMAF proposed by Dr Yoshihiro Uto, Dr. Hitoshi the body in order to treat a medical condi- Hori, DrToshio Inui and Dr Kentaro Kubo. tion (Fig 2). It is used to activate a patient’s immunological competence and is often utilized in combination with other medical treatments at Saisei Mirai. At Saisei Mirai a patient can select from a wide range of suitable and compassionate integrative medical treatments in consulta- tion with doctors experienced with cancer immunotherapy. Recently, immunotherapy treatments are being administered to people living with with infections, such as HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV). Saisei Mirai has a rich history in cancer research. It has collaborated with a number of renowned research centers such as Tokushima University, Kagoshima University Figure 2: Whole body hyperthermia. This equipment can heat tumors within the patient’s and Kobe University and has contributed body with a special heat control system. significantly to the clinical application of new cancer therapies. and has been demonstrated to have anti- with these clinical trials: there were no clear angiogenic and anti-tumor activity in vivo5. classifications of patients’ histopathological Discovery and basic study of fi rst- GcMAF also directly inhibits proliferation types, grades, and stages, and the curative generation GcMAF and migration of human prostate cancer judgment was based solely on a patient’s GcMAF, discovered by Dr Nobuto Yamamoto cells or human breast cancer cells independ- N-acetylgalactosaminidase (Nagalase) activ- in 1991, is derived from the group specific ent of its macrophage activation ability6. ity, and neither tumor markers or cytokine component (Gc) protein (vitamin D binding levels were measured, and there was no protein), a member of the albumin super- Clinical study of fi rst-generation control group. family2. GcMAF is an interesting serum gly- GcMAF for cancer and HIV There is also an interesting clinical report coprotein with various biological activities. treatment of HIV treatment using GcMAF10. The It is reported that during an inflammatory Clinical trials using GcMAF in patients with weekly administration of 100 ng of GcMAF response, β-galactosidase of an activated metastatic breast cancer7, prostate cancer8, to 15 non-anemic HIV-infected patients B-cell and sialidase of a T-cell hydrolyze and metastatic colorectal cancer9 have been showed that the number of CD4+ cells the terminal galactose and sialic acid sac- conducted. Cancer did not recur over a four increased to normal levels within 6 weeks, charides of Gc protein to produce GcMAF3. to seven year period in all subjects adminis- and were maintained for the entire 7 years GcMAF has interesting biological activity; it trated weekly doses of 100 ng of GcMAF for after GcMAF therapy, while the number activates macrophages via superoxide radi- 7 to 19 weeks; a result that took everyone by of CD8+ cells decreased to normal levels, cal generation and phagocytic activation4, surprise. However, there are some problems and the amount of HIV-1 RNA and p24

antigen were detectable in these patients blood stream. It is believed that this posi- tive effect resulted from GcMAF-activated macrophages phagocytosizing and destroy- ing the HIV virus. GcMAF was active in the monocytes/macrophages isolated from AIDS 11

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Pharmaceutical development of first-generation GcMAF for immunotherapy There is little or no research being conducted on the pharmaceutical development of GcMAF as a biological drug. The pharmaceutical development of GcMAF with its high homogeneity and purity from human serum is very difficult owing to its parent Gc protein having six major subtypes, namely homodimer or heterodimer of 1f, 1s, and 2, each with a different sugar moiety (Fig. 3). In order to overcome this problem, Dr Yamamoto manufactured the GcMAF mimic glycoprotein using a baculovirus Figure 3: Proposed sugar chain structure of Gc protein subtype. a) Sugar structure proposed expression system. Dr Giovanna Ghirlanda by Dr Nobuto Yamamoto. b) Sugar structure proposed by Dr C.R. Borges with LC-MS/MS and colleagues developed a small molecule analysis. c) Sugar structure proposed by Dr. Yoshihiro Uto and Dr. Hitoshi Hori with lectin GcMAF constituting 23-mer glycopeptides analysis. GalNAc: N-acetylgalactosamine; Gal: galactose; SA: sialic acid; α-Man: α-mannose. with a similar sugar chain of GcMAF with macrophage phagocytic activation abilities comparable to GcMAF12. We also developed A 12-mer GcMAF-mimic glycopeptides having their macrophage phagocytic activation abilities and in-vivo anti-angiogenic activities in the chick embryo’s chorioallantoic membrane (CAM) assay in a joint study with JITSUBO Co., Ltd. We hope that practical pharmaceutical development of GcMAF for clinical use will begin soon.

Pharmaceutical development and B clinical study of second-generation GcMAF for immunotherapy The first-generation GcMAF were prepared by artificial enzymatic treatments from 1f1f- subtype Gc protein. First-generation GcMAF treatment is not personal and does not con- sider each patient’s immune status, possibly caused by differences of Gc protein types, GcMAF level, and immune state of each subject (Fig, 4A). Accordingly, we propose second-generation GcMAF, which considers the patient’s individual immune status. Our second-generation GcMAF-based immuno- Figure 4: The treatment of patients with GcMAF using blood donated by volunteers. therapy (Fig. 4B) is characterized as; 1) the (A) First-generation GcMAF is prepared from the serum of a healthy volunteer having artificial exogeneous preparation of GcMAF 1f1f-subtype Gc protein. This GcMAF is then used to treat unspecified patients. using a sample of the patient’s serum or (B) Second-generation GcMAF is prepared from the serum of the cancer patient or his or her serum from his or her close relatives, 2) close relatives and used to treat that particular patient specifically. examination of patient’s GcMAF level and immunity status before GcMAF treatment, lacking the galactosyl moiety — could be an effective precursor-type macrophage 3) the optimized GcMAF preparation is converted to GcMAF in vivo by processing activator in vivo13. These results make it administered to the cancer patient. with constitutive sialidase. We recently clear that β-galactosidase-treatment is a We hypothesized that the GcMAF precur- found that 1f1f-subtype of preGcMAF, common modification for all subtypes of Gc sor (preGcMAF) — the full Gc protein, only as well as GcMAF itself, could be used as protein to prepare GcMAF in vivo. Morita

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Figure 5: Saisei Mirai Cell Processing Center (CPC). (A) Cell culture room in CPC. Each room has an air lock. To prevent contamination of outside air, dust and microbes, each room is maintained at a set room pressure. The conditions in each room and important equipment is monitored 24 hour a day. (B) Treatment of cell culture in the safety cabinet. The worker wears special clothes and shoes. We treat cells under aseptic conditions and observe the condition of the culture every day under a microscope. (C) The cell-banking system introduced with liquid nitrogen tank. Cells such as T lymphocytes from the patient are kept for long periods for use in the future.

Pharmaceutical Ind., Ltd. is currently devel- and colleagues developed a feasible method to proliferate and maintain their activity for oping preGcMAF for preclinical trials and to obtain large numbers of activated or long periods in vivo19. Therefore, expansion towards clinical application. effective T lymphocytes16,17. Cultivation of of Hyper T cells has the potential to be a suit- At the end of April 2011, Saisei Mirai peripheral blood lymphocytes with immo- able and important factor of adoptive immu- started treating cancer patients using second bilized anti-CD3 monoclonal antibody and notherapy against cancer. NK cells are a generation GcMAF macrophage activation human recombinant interleukin-2 induces a unique subset of lymphocytes, distinct from therapy produced from human serum. As rapid and massive proliferation of T lympho- T lymphocytes. They contribute to essential at March 2012, Saisei Mirai have 137 cancer cytes and greatly augments their cytotoxic immune systems such as host antimicrobial patients registered as using GcMAF. We also activity. Administration of these expanded and antitumor immunity without require- plan to conduct a comparative clinical study TILs demonstrates clinical activity in some ment for prior immune sensitization of the to clarify the efficacy of several combination patients with several types of cancer, making host20. NK cells are promising effector cells therapies of different Gc protein subtypes, it a useful adoptive immunotherapy. for immunotherapy against cancer. Three different content of GcMAF, and different We developed a cultivation method with cell types T lymphocytes, Hyper T cells, macrophage status to find the relationship autologous plasma from patients with spe- and NK cells, are cultured simultaneously. between each combination therapy and the cific antibodies for membrane antigens of Methods to expand one cell type have been curative effect of human serum including natural killer (NK) cells based on Sekine’s developed by several researchers. However, GcMAF. We aim to determine the optimal techniques and our own clinical results. established methods of cultivation are prob- condition of the macrophage activation Briefly, peripheral blood lymphocytes are lematic with respect to cost, complexity and therapy from the results of these clinical and cultivated with immobilized anti-CD3 mon- safety, such as the risks of using biological analytical studies. Furthermore, second- oclonal antibody and human recombinant tissue of animal or human origin includ- generation therapy is now being used for interleukin-2 as per Sekine’s method, adding ing human serum albumin. Therefore, in HIV and also HCV patients at Kobe Saisei several matrix proteins such as fibronectin spite of its possible effectiveness, it has not Mirai Clinic in Kobe, Japan. in plasma or immobilized monoclonal been entirely accepted in general medical antibody such as CD161 for NK cells18. This practice. We developed a simple method Hyper T/NK cell Therapy cultivation method is used not only to obtain of simultaneously culturing — T lympho- In a previous study, tumor-infiltrating activated T lymphocytes, but also ‘Hyper T cytes, Hyper T cells, and NK cells — which lymphocytes (TIL) have been reported cells’ and NK cells. Hyper T cells, a name we together seem to be important for an effec- to be effective in experimental and clini- coined, are unique immature multipotent T tive immune therapy. Another advantage of cal research of advanced cancer14,15. TIL cells with various capabilities. Populations of our method is free of animal and human recognize specific antigens expressed by these cells deplete as we age. In particular, material. Each cell is shown to be effec- autologous tumor cells. However, this a significant decline in number is evidnent tive in cancer, but many problems are also specific antigenicity is too low to achieve a in people over 50 years of age. Hyper T overlooked. For example, it is said that NK high degree of antigenicity in therapeutic cells have a broader specificity for antigens cells have difficulty permeating deep into use. In order to solve this problem, Sekine expressed by autologous tumor cells, are able the tumor tissue. Accordingly, it is necessary

for NK cells to attack a tumor tissue after sialidase of T-cells. J. Immunol. 151, exhibits native-like phagocytic activity. making a tumor is reduced in size by T lym- 2794–2802 (1993). J. Am. Chem. Soc. 128, 7142–7143 phocytes and Hyper T lymphocytes. Such 4. Mohamad, S.B. et al. Preparation (2006). a sensible strategy makes it an effective of Gc protein-derived macrophage 13. Uto, Y. et al. Effect of the Gc-derived immune therapy. Using these cells in combi- activating factor (GcMAF) and its macrophage-activating factor nation allows the advantage of one cell type structural characterization and precursor (preGcMAF) on phagocytic

SPONSOR FEATURE compensate for the disadvantage of using biological activities. Anticancer Res. activation of mouse peritoneal the other alone — their synergistic actions 22, 4297–4300 (2002). macrophages. Anticancer Res. 31, contribute to the eradication of tumor cells. 5. Kisker, O. et al. Vitamin D binding 2489–2492 (2011). Actually, we tested this combination protein-macrophage-activating factor 14. Rosenberg, S.A. et al. A new approach to therapy with systemically administered (DBP-maf) inhibits angiogenesis and the adoptive immunotherapy of cancer T lymphocytes, Hyper T cells and NK cells tumor growth in mice. Neoplasia 5, with tumor-infiltrating lymphocytes. in more than 100 cancer patients. This 32–40 (2003). Science 233, 1318–1321 (1986). method can be of benefit to patients and 6. Pacini, S. et al. Effects of vitamin 15. Rosenberg, S.A. et al. A progress report is a promising immunotherapy. We expect D-binding protein-derived on the treatment of 157 patients with that the described immunotherapy will play macrophage-activating factor on advanced cancer using lymphokine- a central role in future treatments against human breast cancer cells. Anticancer activated killer cells and interleukin-2 certain human cancers, both alone and in Res. 32, 45–52 (2012). or high-dose interleukin-2 alone. N. combination with other therapies such as 7. Yamamoto, N. et al. Immunotherapy Eng. J. Med. 316, 889–897 (1987). GcMAF and/or hyperthermia treatment. of metastatic breast cancer patients 16. Yamazaki, T. et al. Characterization with vitamin D-binding protein- of immobilized anti-CD3 antibody Cell Processing Center derived macrophage activating factor activated T lymphocytes for use in Saisei Mirai Cell Processing Center (CPC) (GcMAF). Int. J. Cancer 122, 461–467 adoptive immunotherapy of patients is a special clean facility for cell culture (2008). with brain tumors. NEUROL Med. and preparation of medicine for the patient 8. Yamamoto, N. et al. Immunotherapy Chir. (Tokyo) 32, 255–261 (1992). (Fig 5). To maintain safety and high quality, for prostate cancer with Gc Protein- 17. Sekine, T. et al. A feasible method only medical specialists with knowledge and derived macrophage-activating factor, for expansion of peripheral blood skills for cell-culture work are employed at GcM AF. Transl. Oncol. 1, 65–72 lymphocytes by culture with the CPC. In our tissue-engineered products, (2008). immobilized anti-CD3 monoclonal such as GcMAF, Hyper T NK cells, and can- 9. Yamamoto, N. et al. Immunotherapy antibody and interleukin-2 for use in cer vaccines are manufactured. of metastatic colorectal cancer with adoptive immunotherapy of cancer vitamin D-binding protein-derived patients. Biomed. and Pharamocother. References macrophage activating factor, GcMAF. 47, 73–78 (1993). 1. Mellman, I. et al. Cancer Cancer Immunol. Immunother. 57, 18. Pozo, D. et al. CD161 (Human NKR- immunotherapy comes of age. Nature 1007–1016 (2008). P1A) signaling in NK cells involves the 480, 480–489 (2011). 10. Yamamoto, N. et al. Immunotherapy activation of acid spingomyelinase. J. 2. Yamamoto, N. et al. Vitamin D3 of HIV-infected patients with Gc Immunol. 176, 2397–2406 (2006). binding protein (group-specific protein-derived macrophage activating 19. Utsuyama, M. et al. Differential component) is a precursor for the factor (GcMAF). J. Med. Virol. 81, age-change in the numbers of macrophage-activating signal factor 16–21 (2009). CD4+CD45RA+ and CD4+CD29+ from lysophosphatidylcholine-treated 11. Yamamoto, N. et al. Structural T cell subsets in human peripheral lymphocytes. Proc. Natl. Acad. Sci. modification of serum vitamin blood. Mech. Ageing. Dev. 63, 57–68 USA 88, 8539–8543 (1991). D3-binding protein and (1992). 3. Yamamoto, N. et al. Conversion of immunosuppression in AIDS patients. 20. Ljunggren, H.G. et al. Prospects for vitamin D3 binding protein (group- AIDS Res. Hum. Retroviruses the use of NK cells in immunotherapy specific component) to a macrophage- 11,1373–1378 (1995). of human cancer. Immunology 7, activating factor by the stepwise action 12. Bogani, F. et al. A designed 329–339 (2007). of β-galactosidase of B-cells and glycoprotein analogue of Gc-MAF

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