Gursaran P. Talwar, Talwar Research Foundation, E-8, Neb Valley, New Delhi 110068, India. E-mail: email@example.com
Citation Talwar GP, Gupta JC, Shankar NV. Immunological approaches against human chorionic gonadotropin for control of fertility and therapy of advanced-stage cancers expressing hCG/subunits. Am J Reprod Immunol 2011; 66: 26–39
The year 2011 marks the 84th year of the discovery of human chorionic gonadotropin (hCG) by Ascheim and Zondek. Originally considered and employed as a reliable diagnostic index for pregnancy, the multiple roles of hCG as an initiator and sustainer of pregnancy are now recognized. Besides pregnancy, the expression of hCG or its subunits is observed in a number of cancers of diverse type, in particular at advanced stage. Cancers expressing hCG/subunits have poor prognosis and adverse survival. Thus, immunological approaches against hCG have applications for control of fertility and for treatment of terminal cancers. Various mechanisms by which hCG exercises its action are discussed. These include its role as autocrine growth promoter, inhibitor of apoptosis, promotor of angiogenesis, invasiveness, and protection against rejection by the immune system. The article reviews various vaccines developed for control of fertility and for therapy of advanced-stage cancers expressing ectopically hCG/subunits. Also reviewed are the recombinant fully humanized and chimeric antibodies usable for emergency contraception, as vacation contraceptive, and as therapeutic antibodies for treatment of cancers.
Human chorionic gonadotropin (hCG) is a unique hormone. Its existence was discovered by Selmar Aschheim and Bernhard Zondek in 1927.1 They reported that the blood and urine of pregnant women contained a gonad-stimulating substance. On injecting this substance subcutaneously in immature female mice, it led to follicular maturation, luteinization, and haemorrhage into the ovarian stroma. This procedure became known as the Ascheim Zondek pregnancy test, the very first of its kind.
hCG is made by a woman soon after conception. Robert Edwards, who got the Nobel Prize in Medicine (for year 2010), and his colleagues were the first to report the presence of hCG in the culture fluid of early embryos from eggs fertilized in-vitro.2 It plays a critical role in implantation of the embryo onto the uterus. Marmoset embryos exposed to antibodies against beta subunit of hCG do not implant, whereas the same embryos exposed to normal globulins implant normally.3 A similar role of hCG in implantation of the embryo in humans is provided by the observation that sexually active women of reproductive age immunized with a vaccine generating antibodies against hCG do not become pregnant and their menstrual cycles remain regular without lengthening of the luteal phase.4 For a long time, hCG was believed to be made and secreted in normal healthy women only in pregnancy. Recent observations by Alexander group5 indicate the expression of hCG by human endometrial cells during luteal phase. It is not unlikely that hCG made during this phase of the cycle prepares the endometrium to receive the fertilized egg.
An unexpected site of expression of hCG and its subunits (α and β) in men and in non-pregnant women is in a variety of cancers such as lung cancer,6 bladder carcinoma,7 colorectal carcinoma,8 pancreatic carcinoma,9 breast cancer,10 cervical carcinoma,11 oral cancers,12 vulva/vaginal cancers,13 prostate cancer,14 and gastric carcinomas.15 Patients harboring such cancers have poor prognosis and adverse survival. It is not yet fully clear why advanced-stage terminal cancers start expressing hCG and or its subunits, the de-differentiation of normal cells back to embryonic stage is a possibility.
Thus, immunological approaches against hCG have potential of use not only for control of fertility, but also as new therapeutic options for advanced stage, invariably drugs refractory, hCG expressing tumors.
hCG in pregnancy
hCG has a role not only in initiation but also in sustenance of pregnancy. It is no doubt critical for implantation even though the precise mechanism is not fully clear. Leukemia inhibitory factor is up-regulated by hCG.16 hCG inhibits IL-2 in peripheral blood mononuclear cells (PBMC) and modulates the immune response during pregnancy.17 hCG exercises an inhibitory effect on blast transformation of lymphocytes to mitogens and allogenic cells.18,19 hCG also elicits immunoregulatory properties by suppressing mitogen-induced responses of T18,20,21 and B lymphocytes.19 Regulatory T cells (Treg) present at the fetal–maternal interface are believed to provide immune tolerance favoring the fetus. Moreover, hCG is reported to attract macrophages to the fetal–maternal interface, which prevent the exposure of maternal immune system to paternal antigens in the placenta.22,23 Human gonadotropins promote the decidualization of stromal cells, noticeable not only by the morphology of the stromal cells to get transformed to the decidual phenotype, but also evident from the expression of prolactin.24 Both in vivo and in vitro evidence point out to the formation of syncytium from cytotrophoblasts by the action of hCG.25 A recombinant chimeric antibody cPiPP against hCG prevents the fusion of cytotrophoblasts into syncytium.26 Administration of hCG causes an increase in endothelial cell proliferation.27 Treatment with hCG increases the levels of vascular endothelial growth factor (VEGF) and metaloproteinase-924 and hence promotes angiogenesis.
hCG in cancers
Many actions of hCG in promoting pregnancy may also be operative in its support to cancers. hCG or its subunits enhance the proliferation of tumor cells. Bladder cancer cell line T24, which does not produce hCG or its subunits, after treatment with βhCG showed a marked increase in proliferation.28 This action may be a result of its counteracting the apoptotic effect of TGFβ-1; TGFβ-1-induced apoptosis is dose dependently inhibited by co-incubation with βhCG.29 hCG also causes the down-regulation of Fas, Fas ligand, and BAX and p53, which are major apoptotic factors.30 Reduction in βhCG subunit expression in cervical cancer cell lines by silencing RNA led to apoptosis of the HeLa cells.31
Another important action of hCG or its subunits is on promotion of angiogenesis by stimulating the migration and capillary sprout formation of uterine endothelial cells. High levels of hCG and its subunits is associated with high microvessel density in hCG expressing cancers.15βhCG has also a profound effect on metastasis and invasion of cancer cells via its regulation of E-Cadherin. E-Cadherin, which forms complex with catenins, plays a crucial role in the epithelial cell–cell adhesion and is also an invasion suppressor.32βhCG down-regulates E-Cadherin and thus promotes migration and invasion of cancer cells.33 Evidences indicate that the sudden transformation of non-trophoblastic benign tumors to the malignant type can be attributed to altered genetic expression of βhCG. Benign non-trophoblastic cancer cells expressing type I CG β genes (β6 and β7), which transcribe βhCG with an alanine residue at the position 117, start expressing type II CG β genes (β8,β5,β3,β9) that transcribe βhCG with aspartate residue at position 117 during malignant transformation.34 A possible molecular mechanism by which hCG can promote neoplasm has been proposed recently, which suggests that hCG up-regulates the cell cycle proteins via the mammalian target of rapamycin complex 1 (mTORC1) signaling network.35
Thus, hCG is involved not only in the onset, progression, and maintenance of pregnancy but also in cancers. Recent observations show the presence of hCG or its subunits in a variety of advanced-stage cancers invariably metastasized, radio-resistant, and refractory to available drugs. Vaccines against cancer are therefore expected to have a dual utility of not only in preventing an unwanted pregnancy but also in therapy of hitherto untreatable terminal cancers expressing ectopically hCG or its subunits.
Passive immunization: use of recombinant antibodies for preventive and therapeutic purposes
Immunological inactivation of hCG can be achieved by both active (vaccination) and passive immunization (use of preformed competent antibodies). Vaccination produces a long-term response, whereas the passive immunization is of finite duration.
Preformed antibodies offer a mode of ready intervention. There is no lag period of action, in contrast to the time period required for generation and build up of antibodies following first time vaccination. Efficacy is assured in all recipients over a finite period based on the biological half-life of about 21 days of humanized/chimeric antibodies in humans. On the other hand, the duration of the antibody response after vaccination varies from individual to individual as also the quantum of antibodies formed. Thus, efficacy cannot be guaranteed in all recipients unless the vaccine produces above protective threshold response in all.
The following applications are feasible by employing anti-hCG antibodies:
hCG plays a critical role in implantation of the embryo, which is believed to take place between 6th and 9th day following ovulation in women. Antibodies competent to inactivate hCG bioactivity intercept implantation, hence prevent the onset of pregnancy.3,4 At present, Levo-Norgesterol is employed for emergency contraception, which has to be taken within 48–72 hr of unprotected sex. This window of emergency contraception can be extended by some precious days by taking anti-hCG antibodies. What is more, even if the administration of antibodies is delayed, pregnancy will be aborted up to 7 weeks, the time period over which hCG support is required for sustenance of ovarian corpus luteum for progesterone production and sustenance of early pregnancy.
A load dose of antibodies injected in homologous species are expected to remain in circulation at fair titers for a period of 4–6 weeks based on the decay rate of biological half-life of antibodies of 3 weeks. Furthermore, the amount required for interception of implantation would be modest at this stage as only a limited number of embryonic cells make hCG. Thus, only a small volume of high titer recombinant antibodies would be needed to ward off pregnancy. Repeated intercourses often occur during holidays. Two- to four-week vacations are given officially to all in France and in many other countries of Europe. Labor hailing from rural areas working in cities go back home for about a month each year. A single injection can take care of worries following planned or unplanned intercourses.
Non-Surgical Termination of Pregnancy
The dependence of early pregnancy on corpus luteum support is reported to be for 7–9 weeks.36 This seemingly banal use of antibodies for a process easily performed in clinics can be useful in societies (and countries) where medical termination of pregnancy (MTP) is not legal. It can be practiced in remote villages where no hospitals exist. Also it offers privacy to affected women, who do not wish to continue with an accidental or unwanted pregnancy. Scientific evidence for termination of early pregnancy by anti-hCG antibodies is available from studies in baboons.37 Interestingly, hCG made by trophoblasts of implanted embryo stimulates the production of progesterone by the ovarian corpus luteum, which is vital for sustenance of gestation. Interruption of this process by anti-hCG antibodies would result in termination of pregnancy.
Recombinant humanized and chimeric antibodies
The remarkable work of Köhler and Milstein38 ushered in the era of monoclonal antibodies, which are homogeneous, and of consistent affinity for binding a given epitope. Cloned hybridoma cells secrete very high titers of pure antibody in culture. Mouse monoclonals are at present precious agents for immunoassays and diagnostic kits. Although approved by regulatory agencies for use in humans for therapeutic purposes in earlier years, such as Orthoclone (anti-CD3), LymphoCide (anti-CD22), and Panorex 17-1A (anti-EpCAM), their repeated use is contra-indicated owing to the formation of anti-mouse antibodies in humans (HAMA). Humanization of mouse monoclonals has been achieved in more than one laboratories. Replacement of mouse constant regions in antibody chains by human IgG and kappa/lambda and its fusion with mouse variable fragments (Fab) preserves the antigen binding region of the mouse monoclonal, giving rise to chimeric (human–mouse) antibodies, which are approved by USFDA and many other Drugs Regulatory Authorities for therapeutic use in humans.
We reported several years ago the development of a monoclonal antibody PiPP, against hCG,39 which had high affinity for binding hCG (Ka = 3 × 1010m−1). It was devoid of reactivity with human FSH and TSH and had <5% cross-reaction with hLH. PiPP was converted into a recombinant chimeric antibody (cPiPP) as described elsewhere.40 Recombinant antibodies for clinical therapeutic use in humans are expressed in low yields in mammalian cells, which accounts for their high cost. To cut costs, cPIPP was expressed as a periplasmic protein in tobacco leaves at a high yield of 20 mg of purified protein per Kg fresh tobacco leaves.41 Being given that it was expressed in endoplasmic reticulum of the leaves, plant-specific fucose and xylose residues were not loaded on the antibody.42 cPiPP had an affinity of 1.9 × 1010m−1 for hCG. It was totally devoid of cross-reaction with hFSH and hTSH and had <5% cross-reaction with hLH. The antibody was fully competent to block hCG-induced gain of uterine weight of immature mice in vivo and hCG-induced testosterone production by Leydig cells in vitro.40,41 Its efficacy was also tested in a human cell system. Placental villi cytotrophoblasts, isolated from placental villi of MTP cases, on culture in a medium containing anti-hCG antibodies failed to fuse into syncytium. Furthermore, the production of progesterone by the placental cells was fully blocked by cPiPP.26 These observations vouch for the suitability of cPiPP for use as a vacation contraceptive and for non-surgical termination of pregnancy.
Imaging and selective delivery of radiations and or drugs to cancer cells
Choriocarcinoma trophoblast cells are known to make and secrete hCG.43,44 The cells carry receptors for hCG, by virtue of which hCG acts as an autocrine growth factor for these cells. Radio-iodinated PiPP bound to these cells in vitro. JEG cells administered to Nude mice form a cancerous implant. Injection of 131I-PiPP to such mice led to selective localization of radioactivity at the tumor site, whereas the radioactivity of a similarly radio-iodinated non-relevant antibody is distributed randomly all over the body45 (Fig. 1a,b). The binding of the radio-iodinated PiPP to tumor cells is further confirmed by histioradiography (Fig. 1c). These studies clearly demonstrate the utility of the recombinant antibody for imaging and selective delivery of radiations to the tumor cells. It could be of particular utility for tracing of metastasis of such cancers.
Immunological inactivation of hCG for control of cancers expressing ectopically hCG subunits
The curious phenomenon of cancer cells expressing hCG or its subunits has been discussed elsewhere in this article. We carried out studies on T-lymphoblastic leukemia MOLT-4 and lymphocytic leukemia U-937 cells, both available from ATCC. Both MOLT-4 and U-937 cells were bound with cPiPP. The binding as studied by flow cytometry was on the membranes and was specifically competed by authentic purified hCG.46 hCG was not picked up from other cells but was indeed synthesized by the cancer cells, as permeabilized MOLT-4 cells enabled the detection of the presence of hCG within the cells, to which the antibody permeating in the cells could bind. Incubation of MOLT-4 cells with anti-hCG antibodies did not however impair the viability and multiplication of these cells. Nor were the cells lysed by cPiPP in the presence of complement. In contrast, killing of androgen-independent prostate carcinoma DU145 cells is clearly caused by the complement with another monoclonal 730 which binds to these cells on the membrane.47 What could be the reason for such tumor cells to resist complement-mediated cytotoxicity? This issue is not fully understood, although degradation of complement or interference in its activation by such tumor cells have been hinted.48
Selective delivery of cytotoxic compounds to tumor cells
Being given that cPiPP binds with hCG expressed on membranes of T-lymphoblastic leukemia MOLT-4 cells, the antibody could be employed as a vehicle for selective delivery of cytotoxic compounds to the tumor cells without affecting the normal healthy cells. Diferuloylmethane (curcumin) was used for this purpose. Curcumin is a remarkably safe compound; doses upto 8 g/day show neither side effects nor toxicity in humans.49 Curcumin blocks the cancer pathway by down-regulating the NFKB activation pathway,50 and suppression of IKBα kinase and Akt activation.51
cPiPP was conjugated to curcumin using synthetic chemical reactions. A glycine residue was generated on curcumin using BOC-Glycine. Trifluoroacetic acid was used to remove BOC group from the intermediate BOC-glycine-curcumin. Coupling of curcumin-glycine to exposed acidic amino acids (glutamic and aspartic acid) on the antibody was carried out by carbodiimide. The conjugate of curcumin-cPIPP killed effectively MOLT-4 T-lymphoblastic leukemia cells (Fig. 2). The killing was confirmed by both trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.52
Therapeutic effect of Anti-hCG subunit antibodies on human cancer cells expressing hCG subunits
Many years ago, our colleagues at Harvard Medical School brought to our notice human lung cancer (Chago) cells that expressed ectopically either hCG-α or hCG-β subunits. Antibodies directed at these subunits inhibited the multiplication of these cells in vitro. They also prevented, in a dose-dependent manner, the establishment of the cells as tumor in nude mouse (Fig. 3). In case antibodies were given after establishment of the tumor, they caused the necrosis of the tumor.53
Revival and making of a highly immunogenic vaccine against hCG for control of fertility and advanced-stage cancers
A semisynthetic vaccine was developed previously against hCG.54,55 It consisted of a hetero-species dimer (HSD), the alpha subunit of ovine LH annealed non-covalently to beta subunit of hCG. HSD was conjugated to either tetanus toxoid (TT) or diphtheria toxoid (DT). The reason for using two different carriers was the experience that repeated immunization with hCGβ-TT caused a carrier-induced immune suppression to attached ligand, a phenomenon originally reported by Herzenberg et al.56 Immunization with an alternate carrier overcame such suppression of antibody response.57 The reason for replacing the previous hCGβ with the HSD in the vaccine was its superior immunogenicity.54 Furthermore, the antibodies formed had better neutralization capacity of the hCG bioactivity.58
The HSD-TT/DT vaccine went through multicentre phase I safety trials. It was well tolerated, and no side effects of significance were recorded. Nor were there any notable changes in blood chemistry and hematology.59 The menstrual regularity was maintained and women continued to have ovulatory cycles.60 No change in bleeding profile was observed.
With the approval of the Drugs Controller General of India and Institutional Ethics Committees, phase II efficacy trials were carried out with this vaccine in three major institutions: the All India Institute of Medical Sciences (AIIMS, New Delhi), Postgraduate Institute of Medical Education and Research (PGIMER, Chandigarh), and Safdarjung Hospital, New Delhi. A total of 148 sexually active women of proven fertility with two living children (of which one below 1 year to confirm their contemporary fertility) were enrolled with their informed consent. Many of them had come to clinics earlier for medical termination of unwanted pregnancy. The available contraceptives in the family planning basket either did not suit these women or were not used consistently. Their husbands were reluctant to use condoms. Primary immunization was given by three intramuscular injections of the HSD-TT/DT vaccine adsorbed on alum at monthly interval. Sodium phthalyl lipopolysaccharide (SPLPS), a non-pyrogenic derivative of LPS, was used at 1 mg in the first injection only. Vaccine with the TT or DT as carrier was given alternatively, so as to avoid carrier-induced suppression of antibody response to HSD.
All women made antibodies reactive with hCG.4 However, 110 of the 148 immunized women had hCG bioneutralization titers above 50 ng/mL (a threshold fixed for testing protection against pregnancy) for 3 months or longer. All women continued to ovulate and had regular menstrual cycles. The antibody titers declined with time but booster injections raised the titers (Fig. 4). Eight women completed more than 30 cycles by voluntary intake of booster injections as and when required without becoming pregnant. Nine completed 24–29 cycles, 12 completed 18–23 cycles, 15 completed 12–17 cycles, and 21 women completed 6–11 cycles. The personal diary of women indicated without doubt that they were sexually active with a minimum of two sexual intercourses per week. The semen parameters of husbands were good with high counts of motile sperms. The fact that the women were prone to become pregnant is supported by the record of 26 pregnancies taking place in women at titers falling below 35 ng/mL bioneutralization capacity. Fig. 5 is an illustrative example of a 30-year-old subject with two living children and one MTP. After three primary injections of the vaccine, she took two boosters and remained protected against pregnancy for 13 cycles. In the immediate cycle, when her antibody titers had fallen below 20 ng/mL, she conceived and had a positive pregnancy test.
Although most conceptions occurring at or below protective threshold were terminated at the behest of the subjects (Medical termination of pregnancy is legal in India), four women decided to continue with their pregnancy. They carried their pregnancies normally to term and the progeny born to them had normal developmental landmarks and cognitive abilities as compared to their siblings.61
The efficacy of the vaccine to prevent pregnancy was high with only one pregnancy recorded in 1224 cycles above 50 ng/mL.4,62 These historic phase II trials, the first carried out on a potential birth control vaccine in the world, demonstrated the ability of a vaccine engendering antibodies that are competent to inactivate the bioactivity of hCG, to prevent pregnancy in sexually active women, without impairment of ovulation and derangement of menstrual regularity and bleeding profiles.
The main shortcoming of the HSD vaccine was that it produced above protective threshold of 50 ng/mL antibodies for at least 3 months duration in only 60% women. While 60% protection is acceptable for vaccines against infectious diseases, the requirement for protection against pregnancy is above 80–95%, being given that methods of that order of efficacy are available for family planning.
The Task thus was to enhance the immunogenicity of the next generation of the Anti-hCG vaccine. A lesson from research on malaria vaccine is to employ better adjuvants. Glaxo Smithkline, Merck, Pasteur Sanofi, and many other pharma companies have invested heavily in developing adjuvants. Most of these employ oily emulsions. We developed many years ago an immunotherapeutic vaccine for multibacillary lepromatous leprosy based on a non-pathogenic mycobacteria coded as Mw.63,64 The bacillus is usable in an aqueous suspension and retains immunomodulatory properties in an autoclaved state. This bacillus as vaccine has undergone large-scale field trials in leprosy patients and also in their healthy household contacts. It is approved for human usage by the Drugs Controller General of India and also by USFDA. Besides leprosy, it has been employed as adjunct to standard MDT regime, in category II difficult to treat, tuberculosis patients with good results.65 Dipankar Nandi, at the Indian Institute of Sciences Bangalore, has observed that administration of Mw causes a rise in IL12 and γ-interferon. What is more, it has both preventive and therapeutic action (depending on the stage at which it is given) on development of SP2O myeloma as cancer in mice. The gene sequence of Mw has been determined, and its ancestory studied in the mycobacterium kingdom.66 It was hitherto an unlisted sequence in the Data Bank. To avoid confusion with the Beijing MDR strain of tuberculosis w, it has been named as Mycobacterium indicus pranii (MIP).67,68 MIP has been employed in an autoclaved form in PBS buffer in the revived anti-hCG vaccine described later.
Recombinant anti-hCG vaccine
In light of past experience,69 the carboxy terminal peptide of hCGβ, although specific and free of cross-reaction with hLH, was not employed as it is a poor immunogen, demands use of oily strong adjuvant,70,71 and generates lower affinity antibodies (Ka = 108m−1) than that of hCG for its receptors (Ka = 109m−1). Our previous vaccines employed the carrier TT or DT, which were linked chemically using N-succinimidyl-3-(2 pyridyl dithio) propionate (SPDP), to hCG or HSD purified from pregnant women urine and ovine pituitaries. The vaccines were expensive to make, and despite time controlled reactions, the site of linkage of the carrier to hCGβ or HSD, had inevitable variations. We decided therefore to make a recombinant vaccine in which hCGβ gene was fused at the C-terminal end with B subunit of Escherichia coli heat labile enterotoxin (LTB) (Fig. 6). The choice of LTB as carrier was based on the consideration that it is free of regions causing immune suppression. It is a good mucosal adjuvant75 and generates both IgG and IgA antibody response.76
The complex hCGβ-LTB was cloned and expressed in yeast Pichia pastoris as a secretory protein. The conjugate was purified using ammonium sulfate fractionation followed by ion-exchange chromatography.72 It was absorbed on alhydrogel for immunization. MIP at 5 × 107 autoclaved bacilli was injected as adjuvant intramuscularly. Three primary injections of hCGβ-LTB along with MIP at fortnightly interval generated in every Balb/c mouse bioeffective anti-hCG antibodies. On day 37, the titers were already several fold higher than 50 ng/mL in every mouse. A booster around the 4th month enhanced further the titers to well over 100-folds higher than the protective threshold of 50 ng/mL. The immune response was reversible with antibodies declining with time, but was still well above 50 ng/mL after 8 months. Immunogenicity of the recombinant vaccine was also observed in inbred mice of different genetic background, encompassing haplotypes H-2d, H-2k, H-2b, H-2s, and H-2q. This vaccine has received the approval of the Indian National Review Committee on Genetic Manipulation. It is being produced under GMP conditions for pre-clinical toxicology. If found safe, it is planned to conduct clinical trials with this vaccine for preventing pregnancy, as well as for its possible therapeutic action on cancers expressing hCG or its subunits.
Alternate anti-hCG vaccines
Besides the three vaccines described earlier, namely hCGβ-TT,77 hCGβ carboxy terminal peptide (CTP)-DT70, and HSD-TT,4,62 which went up to the stage of phase I safety and/or phase II efficacy clinical trials for fertility control, the following are the other vaccines devised against hCG, which are primarily being tested against cancers expressing hCG.
hCGβ linked to promiscuous T non-B peptides
In view of the carrier-induced immune suppression brought by the hCGβ vaccine linked to TT as carrier, the carrier was replaced by T non-B peptides peptides, which could communicate across various MHC haplotypes, but not have disadvantage of TT. Gupta et al.78 conjugated hCGβ to three promiscuous Th peptides from the measles virus fusion protein, influenza virus hemagglutinin, and HIV-1 reverse transcriptase. Conjugates were adsorbed on alum and studied for their immunogenicity in mice of different haplotypes. It was observed that while conjugation with each peptide improved the antibody response against hCG, immunization with a combination of these hCGβ-peptide conjugates generated anti-hCG responses higher than that achieved with the individual peptides or TT conjugated hCGβ vaccine.
Avicine: anti-hCGβ CTP vaccine
Pierre Triozzi proposed and tested in cancers the anti-37 CTP of hCGβ vaccine originally developed by Vernon Stevens70. The testing was performed with AVI Biopharma with Avicine as the name of the vaccine. Initially, only 37 carboxy terminal amino acids linked to DT vaccine was employed. Subsequently, a loop peptide from within hCGβ was included to enhance the response. The trial was conducted in 77 patients with metastatic colorectal cancer, which provided evidence of survival benefits comparable to chemotherapy with 5-fluorouracil and Pharmacia-Upjohn’s FDA-approved drug, Camptosar(R). The median survival was 42 weeks for patients responding to Avicine immunologically as compared to 17 weeks in patients that did not respond immunologically to Avicine. On Camptosar and 5-FU alone, the median survival was 39 and 28 weeks (http://www.cancerbacteria.com/trial.html).
With the idea of overcoming the lack of immune response in many patients with Avicine, AVI Biopharma signed an agreement with Abgenix to develop a humanized antibody for passive treatment of patients with cancer.
Another cancer in which Avicine has been tested is the ‘most difficult-to-treat’ cancer of pancreas expressing hCGβ. The trial was conducted in 55 patients. They were treated with either Avicine (AVI Biopharma, WA, USA) or Gemzar (Eli Lilly, IN, USA) or with a combination of the two. One-year survival data for the Avicine alone group is similar to that for Gemzar. However, patients had no significant vaccine-related side effects, as compared to the often severe side effects of chemotherapy with Gemzar. One-year survival of 30% of the patients on both vaccine and Gemzar was better than with either of the treatment alone (http://www.cancerbacteria.com/trial.html).
hCGβ mutant GA68 vaccine
Peter Delves and Ivan Roitt group recognized the merit of using the entire hCGβ instead of the CTP. To get rid of the cross-reaction with hLH, they carried out site-directed mutagenesis to see whether a mutated hCGβ could retain the properties of the entire hCGβ, without cross-reaction with hLH. A single amino acid replacement of arginine at position 68 by glutamic acid resulted in hCGβ generating antibodies devoid of cross-reaction with hLH.79 Along with CellDex Therapeutics Inc. (Needham, MA, USA), a vaccine of hCGβ GA68 linked to a human antibody directed at mannose receptor for delivery of the peptide to human immune cells has been made. Adjuvants employed are GMCSF and two TLR agonists, and poly-ICLC and Resiquimode for TLR3 and TLR8, respectively. The combination is undergoing clinical trials in Middlesex, UK under Prof Ray Iles in patients with bladder cancer expressing ectopically hCGβ. Newspaper report (http://www.dailymail.co.uk/health/article-1293927/Jab-halt-deadly-forms-cancer.html) mentions the remarkable shrinkage of tumors and block of invasiveness of the cancer by the vaccine.
Another vaccine reported in 2004 links hCGβ with a human anti-DC antibody, B-11, at genetic level.80 This vaccine is reported to elicit cell-mediated immune response to tumor-associated antigen(s) in a human in vitro model. Monocytes of a normal human donor were incubated with B-11-hCGβ, activated with CD40 ligand mixed with autologous lymphocytes and tested for their ability to mount hCGβ-specific proliferative and cytotoxic T-lymphocyte response. The procedure led to the generation of tumor-specific HLA class I- and class II-restricted T-cell response (including CTLs) capable of killing human cancer cell lines expressing hCGβ. According to the authors, this is the first time that cellular immune response has been induced by a vaccine in a human in vitro system in contrast to the other vaccines inducing primarily antibody response.
Immunological interventions against hCG, whether by vaccines or by recombinant human/chimeric antibodies, have entered an exciting new phase. They may provide therapeutic options for advanced-stage cancers, which are often metastasized and refractory to available drugs. These would also be useful for the control of fertility for which there is a continuing need of additional more acceptable methods. According to WHO (http://www.who.int/en), more than 120 million couples still have an unmet need for family planning and 45 million pregnancies are terminated each year globally. Two recombinant vaccines have been developed. One employs hCGβ linked to either an antibody homing to Dendrocytes or linked to a mucosal carrier, and the other has β subunit of hCG fused to B subunit of heat labile enterotoxin of E. coli (hCGβ-LTB). The former has been tested in vitro; it induces a cell-mediated immune response against hCG. The second vaccine, hCGβ-LTB, given along with a non-pathogenic human use approved Mycobacterium indicus pranii generates several fold higher antibody response in mice than titers established by previous clinical trials to prevent pregnancy. The third vaccine employs an engineered hCGβ with glutamic acid replacing arginine at position 68, conjugated to a human antibody for delivery to dendrocytes. It is in clinical trials in bladder cancer patients with encouraging results.
Dr G. P. Talwar Talwar Research Foundation, New Delhi, India.