Synergistic Activation of Innate and Adaptive Immune Mechanisms in the Treatment of Gonadotropin-Sensitive Tumors

Human chorionic gonadotropin (hCG) prolongs the secretion of progesterone from the corpus luteum, providing a critical stimulus for the sustenance of pregnancy. hCG (or individual subunits) is also secreted by a variety of trophoblastic and non-trophoblastic cancers and has been associated with poor prognosis. Early clinical studies have indicated merit in anti-hCG vaccination as potential immunotherapy, but anti-tumor efficacy is believed to be compromised by sub-optimal immunogenecity. In the present study, enhanced tumorigenesis was observed when SP2/O cells were subcutaneously injected in either male or female BALB/c x FVB/J^βhCG/- F1 transgenic mice, establishing the growth-promoting effects of the gonadotropin for implanted tumors in vivo. The utility of Mycobacterium indicus pranii (MIP) was evaluated, as an innate anti-tumor immunomodulator as well as adjuvant in mice. MIP elicited the secretion of the inflammatory cytokines IFNγ, IL-6, IL-12p40, KC and TNFα from murine antigen presenting cells. When MIP was incorporated into an anti-hCG vaccine formulation previously employed in humans (a βhCG-TT conjugate adsorbed on alum), elevated T cell recall proliferative and cytokine responses to hCG, βhCG and TT were observed. MIP increased vaccine immunogenicity in mice of diverse genetic background (including in traditionally low-responder murine strains), leading to enhanced titres of bioneutralizing anti-hCG antibodies which exhibited cytotoxicity towards tumor cells. Individual administration of MIP and βhCG-TT to BALB/c mice subcutaneously implanted with SP2/O cells resulted in anti-tumor effects; significantly, immunization with βhCG-TT supplemented with MIP invoked synergistic benefits in terms of tumor volume, incidence and survival. The development of novel vaccine formulations stimulating both adaptive and innate anti-tumor immunity to induce collaborative beneficial effects may fill a niche in the adjunct treatment of hCG-sensitive tumors that are resistant to conventional therapy.     

Antibodies against human chorionic gonadotropin convert the deglycosylated hormone from an antagonist to an agonist

Chemical deglycosylation of human chorionic gonadotropin (hCG) produced an antagonist (DG-hCG) that specifically bound to hCG receptors but was no longer able to stimulate adenylate cyclase in the murine Leydig tumor cell line, MLTC-1. DG-hCG was restored to an agonist by incubating cells or membranes having the bound analogue with antibodies against hCG (anti-hCG). In the presence of anti-hCG, cyclic AMP accumulation and adenylate cyclase activity were stimulated over DG-hCG alone. There was no accumulation of cyclic AMP when the cells were exposed to anti-hCG alone or DG-hCG and normal serum or anti-hCG first then DG-hCG. Several different batches of anti-hCG were effective but their activity did not correlate with their affinity for DG-hCG or hCG. The effect of anti-hCG on DG-hCG activity was dose- and time-dependent. Maximal stimulation of cyclic AMP was achieved with antisera dilutions of 1:200 or less. When DG-hCG-treated cells were exposed to anti-hCG at 37 degrees C, there was a 10-min lag. The lag was eliminated when the cells were exposed to the antibodies at 4 degrees C for 3 h and then warmed to 37 degrees C. Adenylate cyclase was also activated when Fab fragments prepared by papain digestion of anti-hCG were used, whereas Fc fragments were not effective. Thus, the divalency of the anti-hCG is not the critical factor in the mechanism of antibody action. Our results suggest that anti-hCG converts DG-hCG from an antagonist to an agonist possibly by altering the conformation of the modified hormone.     

Preliminary studies on the indium slide immunoassay for estimation of human chorionic gonadotropin and antihuman chorionic gonadotropin antibody

Human chorionic gonadotropin (hCG) is synthesized and secreted as early as 170 hr after fertilization and has been used as an index for pregnancy. Neutralization of hCG with a beta-subunit hCG vaccine(s) has been proposed as a contraceptive technique. To monitor the duration of effectiveness of the vaccine, it will be necessary to monitor the anti-hCG antibodies, especially those responsible for inhibiting the hCG bioactivity. We report a simple, rapid technique using an indium slide immunoassay for the qualitative estimation of hCG and to monitor a bioeffective anti-hCG antibody. The sensitivity of the indium slide assay to measure hCG ranged from 1 microgram/ml to 1 ng/ml, depending on the format of the assay. The indium slide assay also detected anti-hCG antibodies generated against a specific determinant on hCG recognized by a neutralizing monoclonal antibody (P3W80) in women immunized with a contraceptive vaccine.     

The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG(beta)) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG(beta)-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG(beta)-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG(beta)-specific and anti-hCG(beta)/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG(beta)-R68E was eliminated, mAbs reacting with hCG(beta)-specific epitopes bound to hCG(beta) and hCG(beta)-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG(beta)-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG(beta)-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed.     

Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin

Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.     

Tumoricidal adsorption of Staphylococcus aureus organisms on Ehrlich ascites tumor cells sensitized with rabbit antibody

A tumoricidal effect was observed when protein A-bearing Staphylococcus aureus organisms were adsorbed on Ehrlich ascites tumor cells previously sensitized with antiserum from a rabbit immunized with Ehrlich ascites tumor cells. Electron micrographs showed that staphylococci were firmly attached to the tumor cells, which might explain how effectively the attached cocci killed the tumor cells. The tumoricidal effect was confirmed not only by an in vitro experiment but also by an in vivo one. The possible applications of the tumoricidal adsorption as an indicator for staphylococcal virulence or for selective anti-tumor action was discussed.     

Existence of associated, non-associated, and oligomeric forms of human chorionic gonadotropin subunits in placental extracts

The molecular sizes of human chorionic gonadotropin (hCG) subunits in the native state in normal first trimester placental extracts were determined by gel filtration on Sephacryl S-300, followed by SDS-polyacrylamide gel electrophoresis, protein blotting, and immunobinding analysis using anti-alpha and - beta antibodies. Mature forms of hCG subunits in the extracts were only found in the same fraction as that which contained standard urinary hCG, indicating an alpha beta dimer. On the other hand, immature forms were detected with a wide range of molecular weights, which were higher than that of standard hCG, suggesting oligomerization of associated or non-associated immature subunits. In order to determine the associated state of these subunits, various forms of associated subunits (hCG alpha beta) in placental extracts were immunoprecipitated with anti-hCG antiserum, which only recognized hCG alpha beta, and Protein A-Sepharose. They were then analyzed by SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions, followed by immunobinding assaying. It has been suggested that there are three kinds of hCG alpha beta S (one mature and two immature). To confirm the above results and to clarify the existence of free subunits, placental extracts were subjected to two-dimensional SDS-polyacrylamide gel electrophoresis. With this technique, high molecular weight forms of immature hCG subunits were found to be present in placental cells as an oligomer of not only the alpha beta dimer but of each subunit as well.     

IL-12 enhances efficacy and shortens enrichment time in cytokine-induced killer cell immunotherapy

Cytokine-induced killer (CIK) cells are T cell derived ex vivo expanded cells with both NK and T cell properties. They exhibit potent anti-tumor efficacy against various malignancies in preclinical models and have proven safe and effective in clinical studies. We combined CIK cell adoptive immunotherapy with IL-12 cytokine immunotherapy in an immunocompetent preclinical breast cancer model. Combining CIK cells with IL-12 increased anti-tumor efficacy in vivo compared to either therapy alone. Combination led to full tumor remission and long-term protection in 75% of animals. IL-12 treatment sharply increased the anti-tumor efficacy of short-term cultured CIK cells that exhibited no therapeutic effect alone. Bioluminescence imaging based in vitro cytotoxicity and in vivo homing assays revealed that short-term cultured CIK cells exhibit full cytotoxicity in vitro, but display different tumor homing properties than fully expanded CIK cells in vivo. Our data suggest that short-term cultured CIK cells can be “educated” in vivo, producing fully expanded CIK cells upon IL-12 administration with anti-tumor efficacy in a mouse model. Our findings demonstrate the potential to improve current CIK cell-based immunotherapy by increasing efficacy and shortening ex vivo expansion time. This holds promise for a highly efficacious cancer therapy utilizing synergistic effects of cytokine and cellular immunotherapy.     

Antibody binding to the beta-subunit of deglycosylated chorionic gonadotropin converts the antagonist to an agonist

The murine Leydig tumor cell line, MLTC-1, has a gonadotropin-responsive adenylate cyclase system. Binding of human chorionic gonadotropin (hCG) stimulates the accumulation of cyclic AMP in these cells. Chemically deglycosylated hCG (DG-hCG) is an antagonist that binds with high affinity to the gonadotropin receptor, but fails to stimulate adenylate cyclase. This antagonism can be reversed if the binding of DG-hCG is followed by treatment of the DG-hCG-receptor complex with antibodies against hCG. Polyclonal antibodies against DG-hCG were raised in rabbits. These antibodies were strongly cross-reactive with hCG, bound to both the alpha- and beta-subunits of hCG and DG-hCG, and reversed the antagonism of DG-hCG. The antiserum was divided into two fractions by affinity chromatography on hCG-Sepharose. The fraction that was not retained reacted only with DG-hCG (DG-hCG antibodies) and, on Western blots, bound to both the alpha- and beta-subunits of DG-hCG. DG-hCG antibodies did not reverse the antagonism of DG-hCG. However, using 125I-protein A, we were able to detect binding of these antibodies to the cell surface DG-hCG-receptor complex. The fraction of antibodies retained by the affinity column reacted with both DG-hCG and hCG (DG-hCG/hCG antibodies). On Western blots, DG-hCG/hCG antibodies bound to the beta-subunit, but only weakly to the alpha-subunit of both hCG and DG-hCG. These antibodies also bound to the cell surface DG-hCG-receptor complex. In addition, DG-hCG/hCG antibodies were able reverse the antagonism of DG-hCG. Reversal of DG-hCG antagonism by the whole antiserum was blocked by the beta- but not the alpha-subunit of hCG. Polyclonal antiserum against the beta- but not the alpha-subunit of hCG reversed the antagonism of DG-hCG. From these results, we conclude that antibody binding to specific determinants common to both native and deglycosylated beta-subunit reverses the antagonism of DG-hCG. In addition, antibodies directed against unique determinants on the deglycosylated beta-subunit are not capable of reversing the antagonism of DG-hCG.     

Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice

Human CG is a pregnancy marker secreted by the placenta, and it utilizes the same receptors as does LH. Human CG is a heterodimer, and its subunits are expressed in tissues other than placenta. Similarly, LH/hCG receptors are also expressed in multiple tissues; however, the physiological significance of this expression is unknown. Free hCGbeta is efficiently secreted in vitro in transfected cells and is highly expressed in many human cancers; however, the biological effects of free hCGbeta in vivo are unknown. To study in vivo consequences of elevated levels of free hCGbeta and hCG dimer in both male and female reproductive physiology, we used mouse metallothionein 1 promoter to generate multiple lines of transgenic mice that overexpressed either one or both subunits of hCG. Although mice expressing the glycoprotein hormone alpha subunit are normal and fertile, both male and female transgenic mice overexpressing only the hormone-specific hCGbeta subunit are infertile. The hCGbeta subunit-expressing transgenic female mice progressively develop cystic ovaries, whereas the male transgenic mice are infertile but otherwise are not phenotypically discernible. In contrast, both the male and female transgenic mice coexpressing high levels of the hCG subunits (i.e., the hCG dimer) demonstrate multiple reproductive defects. The male transgenic mice have Leydig cell hyperplasia, very high levels of serum testosterone, reduced testis size, and dramatically enlarged seminal vesicles and are infertile and display overly aggressive behavior when caged with females. The female transgenic mice are also infertile, have elevated levels of serum estradiol, and progressively develop hemorrhagic and cystic ovaries with thecal layer enlargement and stromal cell proliferation and degenerating kidneys. These results suggest that the in vivo biological effects of ectopically expressed free hCGbeta subunit are distinct from those of the hCG dimer and are gender specific. These transgenic mice are useful models for studying the biology of free hCGbeta subunit, for further analyzing the gain of function effects of hCG during early Leydig cell development, and for studying the roles of hCG in ovarian and kidney pathophysiology and function.     

Cellular basis of immunologic interactions in adoptive T cell therapy of established metastases from a syngeneic murine sarcoma

The adoptive transfer of specifically sensitized T lymphocytes can effectively mediate the regression of established local and metastatic tumors. Previous experiments using the weakly immunogenic MCA 105 sarcoma indicated that cellular interactions between transferred L3T4+ helper and Lyt-2+ cytotoxic immune T cells were necessary for mediating tumor regression. In this study, the kinetics of T-T cell interactions were analyzed by in vivo depletion of T cell subsets with mAb. The anti-tumor efficacy of transferred immune cells was abrogated by in vivo administration of either L3T4 or Lyt-2 mAb on the day of cellular therapy. However, if mAb were given 3 days after the transfer of immune cells, depletion of Lyt-2+ but not L3T4+ cells abrogated anti-tumor efficacy. T cell depletion on day 6 after transfer of immune cells had no adverse effect on tumor regression, indicating the period required for T cell reactivity in vivo. Furthermore, depletion of Ia+ cells by in vivo mAb treatment abrogated the anti-tumor efficacy of immune cells. It is thus hypothesized that there are two distinct but sequential phases of in vivo T cell interactions leading to the regression of established tumors after adoptive immunotherapy. An initial "helper/inducer" phase apparently requires the interaction of L3T4+ immune cells and the tumor Ag involving Ia+ cells. The inducement of L3T4+ cell activation is to provide helper function via the secretion of IL-2. The second phase designated as an "effector phase" involves differentiation of immune Lyt-2+ cells under the influence of IL-2 secreted during the helper/inducer phase for generation of mature Lyt-2+ effector cells. To further support the hypothesis of a two-phase process we have examined the phenotype and kinetics of tumor regression mediated by effector cells generated by secondary in vitro sensitization (IVS). Although the IVS cells were generated from fresh MCA 105 immune spleen cells, their anti-tumor efficacy was mediated solely by Lyt-2+ lymphocytes. Kinetic studies revealed that the in vivo requirement of IVS Lyt-2+ effector cells to mediate tumor regression was less than 3 days, and the anti-tumor reactivity of these cells was not affected by in vivo depletion of Ia+ cells. Thus, the IVS reaction is likely representative of the in vivo counterpart of the helper/inducer phase leading to the generation of mature Lyt-2+ immune effector cells. Tumor regression after transfer of Lyt-2+ cells generated in IVS therefore required a relatively shorter period of time than that required after the transfer of fresh noncultured MCA 105 immune spleen cells.     

Protein-secretory patterns of normal and abnormal human placentas with special reference to human chorionic gonadotropin

[35S]Methionine-labeled protein-secretory patterns resolved by two-dimensional polyacrylamide gel electrophoresis in abnormal hydatidiform-mole placentas were compared with those in normal full-term placentas with special reference to human chorionic gonadotropin (hCG) by means of immunoblotting and immunoelectron-microscopic techniques. Although basic protein-secretory patterns of both placentas were similar to each other, four polypeptide spots appeared and one spot disappeared in the hydatidiform-mole samples. Among four newly synthesized and secreted spots, three were immunoreacted with anti-hCG serum by an immunoblotting experiment. Ultrastructural localization of hCG showed that the labeling intensity of anti-hCG serum in hydatidiform-mole placentas was much heavier than that in full-terms ones. Particularly, the Golgi apparatus, middle-sized granules and large bodies were highly immunoreactive. The present study reveals that hydatidiform-mole placentas have different protein-secretory functions especially in hCG synthesis and secretion from those of normal pregnancy.     

Low molecular weight hyaluronan preconditioning of tumor-pulsed dendritic cells increases their migratory ability and induces immunity against murine colorectal carcinoma

We have recently shown that systemic administration of low molecular weight hyaluronan (LMW HA) significantly reduces colorectal carcinoma (CRC) growth in vivo. The elicited response is partially mediated by activated dendritic cells (DC). To potentiate the ability of DC loaded with whole tumor lysate (DC/TL) to induce immunity against CRC in mice, we aimed to study the effects of preconditioning DC with LMW HA for therapeutic vaccination. LMW HA improved maturation of ex vivo generated DC, increased IL-12, decreased IL-10 production, and enhanced a MLR activity in vitro. Although TNF-α showed a similar capacity to mature DC, preconditioning of DC/TL with LMW HA increased their ability to migrate in vitro toward CCL19 and CCL-21 in a CD44- and a TLR4-independent manner; this effect was superior to Poly(I:C), LPS, or TNF-α and partially associated with an increase in the expression of CCR7. Importantly, LMW HA dramatically enhanced the in vivo DC recruitment to tumor-regional lymph nodes. When these LMW HA-treated CRC tumor lysate-pulsed DC (DC/TL/LMW HA) were administered to tumor-bearing mice, a potent antitumor response was observed when compared to DC pulsed with tumor lysate alone and matured with TNF-α. Then, we showed that splenocytes isolated from animals treated with DC/TL/LMW HA presented a higher proliferative capacity, increased IFN-γ production, and secreted lower levels of the immunosuppressive IL-10. Besides, increased specific CTL response was observed in DC/TL/LMW HA-treated animals and induced long-term protection against tumor recurrence. Our data show that LMW HA is superior to other agents at inducing DC migration; therefore, LMW HA could be considered a new adjuvant candidate in the preparation of DC-based anticancer vaccines with potent immunostimulatory properties.     

人绒毛膜促性腺激素β亚基和绵羊垂体促性腺激素共有α亚基组成的单链多...

hCG beta-oLH alpha chimeric cDNA was constructed by using overlapping PCR to contact the codons of C-terminal end of hCG beta with the codons of N-terminal end of oLH alpha, then it was subcloned into nuclear polyhedrosis virus (AcNPV) expression vector pVL1393 to construct expression vector pVL1393-hCG beta-oLH alpha. The insect cells (Sf9) were cotransfected by the expression vector pVL1393-hCG beta-oLH alpha and BaculoGold AcNPV linearized genomic DNA, and recombinant viruses AcNPV-hCG beta-oLH alpha were screened out by plaque assay. Further the insect cells were infected by the recombinant viruses, the recombinant hCG beta-oLH alpha was purified by immunoaffinity chromatography column coupling anti-hCG beta monoclonal antibody from the conditioned media of infected cells. The results of SDS-PAGE silver staining and western blotting showed that hCG beta-oLH alpha single peptide chain had apparent molecular weights of 40.5 kD and 38.0 kD under non-reducing and reducing conditions respectively, indicating the occurrence of disulfide bonds and significant tertiary structure in the single peptide chain. From the results of competitive inhibition of 125I-hCG beta binding we can conclude that the anti-hCG beta antibody-binding activity of hCG beta-oLH alpha chimera is lower than that of native hCG, but higher than that of native hCG beta. Therefore, we assume that the hCG beta-oLH alpha chimera should have potential application as a target antigen of anti-hCG fertility regulatory vaccine.     

Testosterone response of cryptorchid and hypophysectomized rats to human chorionic gonadotrophin (hCG) stimulation

The testosterone responses to a single injection of hCG (100 i.u.) in hypophysectomized (hypox.), cryptorchid or sham-operated rats were followed over a 5-day period. In sham-operated rats, hCG induced a biphasic rise in serum testosterone, peaks being observed at 2 and 72 h. Reduced testis weights, elevated FSH and LH levels and reduced serum testosterone levels were found after 4 weeks of cryptorchidism, but hCG stimulation resulted in a normal 2 h peak in serum testosterone. However, the secondary rise at 72 h in cryptorchid rats was significantly lower than sham-operated rats. Reduced testis weight and undetectable serum FSH and LH levels together with decreased testosterone levels were found 4 weeks after hypophysectomy. Serum testosterone levels rose 2 h after hCG in comparison to hypox. controls but this peak was significantly reduced compared with sham-operated rats. The second rise in serum testosterone began on day 2, peaking on day 4 at levels comparable to that seen in sham-operated rats after hCG. The in vitro basal and hCG stimulated secretion of testosterone by cryptorchid testes was greater than that secreted by normal rat testes (518.0 +/- 45.9 and 3337.6 +/- 304.1 pmol per testis per 4 h compared with 223.6 +/- 24.9 and 1312.9 +/- 141.4 pmol per testis per 4 h for normal rat testes). In cryptorchid animals a single injection of 100 i.u. hCG resulted in a pattern of in vitro refractoriness similar to normal rats, lasting from 12 h to 2 days, during which testosterone secretion was reduced to near basal levels. The in vitro basal and hCG-stimulated secretion of testosterone by hypox. rat testes was severely diminished compared with normal rat testes. The temporal pattern of in vitro secretion of testosterone from hypox. rat testes mimicked the in vivo serum testosterone pattern seen in these animals. This study demonstrates important differences in the in vivo and in vitro testosterone response to hCG after testicular damage.     

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.     

Generation from tumor-bearing mice of lymphocytes with in vivo therapeutic efficacy

Systemic transfer of sensitized lymphocytes can effectively mediate the regression of established tumors. However, virtually all prior experimental applications of this approach have utilized lymphocytes from animals that have been immunized to reject tumor challenge. A similar source of cells is not available in the human. With the use of a weakly immunogenic murine tumor, MCA 105, we demonstrate here that following in vitro sensitization (IVS) with viable tumor cells and interleukin 2, the nontherapeutic lymphoid cells from mice bearing a progressively growing tumor acquired antitumor reactivity capable of mediating the regression of established pulmonary metastases. Although the IVS system induced nonspecific lymphokine-activated killer-like cytotoxic activity from lymphoid cells of normal as well as tumor-bearing mice, therapeutically active cells could only be generated from cultures initiated with lymphoid cells from tumor-bearing animals, indicating that the IVS was a secondary in vitro immune response. Without other treatment, the IVS cells could mediate antitumor effects. However, low doses of exogenous interleukin 2 administration could enhance their therapeutic efficacy. By in vivo T cell subset depletion with monoclonal antibodies, the primary effector cells were identified as belonging to cytotoxic/suppressor T cell lineage expressing the Lyt-2 phenotype. In addition, these therapeutic effector cells could be further expanded in numbers in vitro with continuous stimulation by tumor cells in the presence of interleukin 2. Compared to the number of cells initiating the culture, as many as 126 times the number of cells were obtained after 9 days of IVS followed by in vitro expansion for an additional 5 days. Studies on the kinetics of the occurrence of the pre-effector lymphocytes during tumor growth revealed that they were readily obtained from draining lymph nodes of mice with a broad range of tumor burdens as well as durations of tumor growth. The ability to generate and expand, in vitro, therapeutically active lymphocytes from tumor-bearing hosts has important implications for cellular therapy of human cancers.     

Evaluation of the immunogenicity of a single chain chimeric peptide composed of hCGβ and oLHα for inhibition of the growth of hCGβ-expressing cancer cells

Human chorionic gonadotropin (hCG) is a membrane-associated protein highly expressed in several types of human cancer cells. The expression in the cancer cells indicates that hCG may be a potential target molecule for cancer immunotherapy. The objective of this study was to develop a novel immunogenic molecule, which can efficiently induce the neutralizing antibody against hCG and which is also suitable for mass production. The immunogenicity of the recombinant single chain chimeric protein of hCGβ-oLHα expressed by yeast was examined. Additionally, the inhibitory effects of the anti-hCGβ-oLHα antibody on the growth of hCG-positive cancer cells were determined. It was found that hCGβ-oLHα yielded high titers of anti-hCG rabbit antibody that could effectively neutralize the bioactivity of hCG. The rabbit anti-hCGβ-oLHα IgG inhibited the proliferation of hCG-expressing human colorectal cancer cells (LS-174, HCT-116, HCT-15 and KM-12) in a dose-dependent manner. Furthermore, an intact anti-tumor vaccine was prepared by conjugating hCGβ-oLHα with tetanus toxoid (TT) and this was used to immunize Balb/c mice bearing hCG-expressing SP2/0 tumor cells. The progression of tumors in these immunized mice was remarkably inhibited. These results suggest that hCGβ-oLHα is a new promising immunogenic molecule for the development of an anti-hCG-based cancer vaccine.     

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.     

Monoclonal antibodies from mice bearing polyoma virus-induced tumor

Summary Monoclonal antibodies were produced by fusing NS1/1 myeloma cells with splenocytes from A. BY mice bearing syngeneic polyoma virus-induced SEYF-a tumors.From six separate fusion experiments 514 hybridomas were obtained, 45 of which were found to secrete SEYF-a-binding antibodies. The binding patterns of antibodies secreted by eight hybridomas to a panel of tumor cells and to normal mouse fibroblasts were analyzed by means of an indirect radioimmunoassay. Seven hybridomas were found to secrete antibodies that bound to all cell lines tested. This indicated that certain SEYF-a-associated antigens are widely distributed on a variety of seemingly nonrelated tumor cells.One hybridoma secreted antibodies that exhibited a high binding activity to SEYF-a cells, a low binding activity to two members of the tumor panel, and none at all against most of its constituents, including normal fibroblasts. The results of the binding experiments were further supported by absorption experiments.A subclass analysis of the immunoglobulins secreted by the various hybridomas revealed that three clones secreted IgG1; one clone secreted IgM; and three clones secreted IgG2a. Polyacrylamide gel electrophoresis of two of the secreted antibodies indicated a high degree of homogeneity of the heavy and the light chain of the corresponding antibodies, as would be expected from monoclonal products.The results of this study demonstrate the feasibility of obtaining anti-tumor monoclonal antibodies from tumor bearers, representing the immune response of the tumor bearer against antigens associated with his syngeneic tumor.