Laboratory safety evaluation of lokivetmab, a canine anti-interleukin-31 monoclonal antibody, in dogs.

Lokivetmab (Cytopoint®, Zoetis) is a canine monoclonal antibody that specifically binds and neutralizes interleukin (IL)-31. Lokivetmab is approved for use in dogs for the treatment of atopic dermatitis (AD) and allergic dermatitis. The laboratory safety of lokivetmab was evaluated in 2 studies by adapting the science-based, case-by-case approach used for preclinical and early clinical safety evaluation of human biopharmaceuticals. The main objectives were to demonstrate the safety of lokivetmab in healthy laboratory Beagle dogs by using integrated clinical, morphologic, and functional evaluations. In Study 1, dogs were treated s.c. with saline or lokivetmab at 3.3 mg/kg (1X, label dose) or 10 mg/kg (3X intended dose) for 7 consecutive monthly doses, with terminal pathology and histology assessments. In Study 2, the functional immune response was demonstrated in naïve dogs using the T-cell dependent antibody response (TDAR) test with 2 different dose levels of unadjuvanted keyhole limpet hemocyanin (KLH) as the model immunogen. The primary endpoint was anti-KLH IgG antibody titer, and secondary endpoints were ex vivo IL-2 enzyme-linked immunospot (ELISpot) and peripheral blood mononuclear cell lymphoproliferation assays. Both studies included monitoring general health, periodic veterinary clinical evaluations, serial clinical pathology and toxicokinetics, and monitoring for anti-drug antibodies. In both studies, the health of dogs receiving lokivetmab was similar to controls, with no treatment-related changes uncovered. Extensive pathology evaluations of immune tissues (Study 1) revealed no lokivetmab-related morphologic changes, and in dogs treated at 10 mg/kg lokivetmab, immunization with the model antigen KLH did not impair the functional antibody or T-cell recall responses. There were no immunogenicity-related or hypersensitivity-related responses observed in either study. These studies in healthy laboratory dogs showed that lokivetmab was well-tolerated, did not produce any treatment-related effects, and had no effect on immune system morphology or its functional response. These studies also demonstrated the utility of a science-based case-by-case approach to the safety evaluation of a veterinary biopharmaceutical product.

The clinical relevance of humoral immune responses to Globo H-KLH vaccine adagloxad simolenin (OBI-822)/OBI-821 and expression of Globo H in metastatic breast cancer.

An international randomized phase II trial of Globo H (GH) vaccine, adagloxad simolenin/OBI-821 in 349 patients with metastatic breast cancer showed longer progression-free survival (PFS) in vaccinated patients who developed anti-Globo H (anti-GH) IgG than those who did not and the placebo group. The impacts of anti-GH IgM and GH expression on peak anti-GH IgG and clinical outcome were further evaluated. The titers of anti-GH IgG and IgM were determined by ELISA. GH expression in tumor was examined by immunohistochemical staining. Immunophenotyping was conducted by flow cytometry. Adagloxad simolenin elicited anti-GH IgM which peaked at titers ≥1:80 between weeks 5 and 13. The mean anti-GH IgG titer peaked at week 41 and decreased thereafter on the completion of vaccination. One log increase in peak IgM was associated with 10.6% decrease in the HR of disease progression (HR: 0.894, 95% CI: 0.833 to 0.960, p=0.0019). Patients with anti-GH IgM ≥1:320 within first 4 weeks after vaccination had significantly higher maximum anti-GH IgM (p<0.0001) and IgG titers (p<0.0001) than those with <1:320. Moreover, the median PFS appears to be longer for patients with anti-GH IgM ≥1:320 within first 4 weeks than those with anti-GH IgM titer <1:320 (11.1 vs 7.3 months, p=0.164), but not statistically significant. Among patients with H score ≥80 for GH expression by immunohistochemistry, the vaccination group (n=42) seemed to have better PFS than the placebo group (n=23) (HR=0.59; 95% CI: 0.32 to 1.10, p=0.10), but the difference did not reach statistical significance. In addition, peak levels of anti-GH IgM were higher in patients who had lower percentage of activated regulatory T cells (Treg cells; CD4+CD45RA-Foxp3high) at baseline than those who had higher activated Treg cells (p=0.042). This study demonstrates that adagloxad simolenin induced both IgG and IgM antibodies against GH. Anti-GH IgM ≥1:320 within first 4 weeks or low activated Treg cells at baseline may help to select patients who are likely to produce a higher level of GH-specific IgM and IgG in the future.

Intensive therapy with gastropodan hemocyanins increases their antitumor properties in murine model of colon carcinoma.

Various natural compounds have been tested as anticancer therapeutics in clinical trials. Most promising direction for antitumor therapy is the use of substances which enhance the immune system response stimulating tumor-specific lymphocytes. Hemocyanins are large extracellular oxygen transport glycoproteins isolated from different arthropod and mollusk species which exhibit strong anticancer properties. Immunized in mammals they trigger Th1 immune response that promotes unspecific stimulation and adjuvant activity in experimental therapeutic vaccines for cancer and antibody development. In the present study we used two hemocyanins - one isolated from marine snail Rapana thomasiana (RtH) and another one, from the terrestrial snail Helix pomatia (HpH) which have been investigated by using different administration schedules (intensive and mild) in murine model of colon carcinoma. The treatment with RtH and HpH generated high levels of antitumor IgG antibodies, antibody-producing plasma cells and tumor-specific CTLs, stimulated secretion of proinflammatory cytokines, suppressed the manifestation of carcinoma symptoms as tumor growth and size, and prolonged the life span of treated mice. Our results showed a significant anti-cancer effect of RtH and HpH hemocyanins on a murine model of colon carcinoma with promising potential for immunotherapy in various schemes of administration based on cross-reactive tumor-associated epitopes.

The effects of vaccination with keyhole limpet hemocyanin or oral administration of Salmonella enterica serovar Enteritidis on the growth performance of immunoglobulin knockout chickens.

The nutritional cost of activating B cell-mediated immunity is thought to be low in chickens. However, this assumption is incompletely characterized. Immunoglobulin knockout (Ig-KO) chickens lacking B cells and immunoglobulin may potentially be a robust model to investigate the nutritional cost of immunity. Specifically, comparing the growth of Ig-KO chickens to immunocompetent birds (WT) following activation of the immune system should indicate costs associated with B cell-mediated immunity. A total of 3 experiments were conducted to determine if (1) an antibody response to keyhole limpet hemocyanin (KLH), (2) an intra-abdominal injection with Salmonella enterica serovar Enteritidis (SE), or (3) an oral challenge of SE would affect body weight gain (BWG) in homozygous Ig-KO (KO) chickens and their immunocompetent counterparts. In Experiment 1, a significant genotype*vaccination status interaction was observed (P = 0.03) during the period from 0 to 6 D after initial vaccination in which KLH injection reduced BWG in WT birds, but not in KO birds. A genotype*vaccination status interaction was present (P = 0.002) from 0 to 7 D after the first booster in which KLH injection due to reduced BWG in KO birds, but not WT birds. In Experiment 2, both KO and immunocompetent birds lost body weight during the period from 0 to 2 D after the SE injection, with no significant differences due to genotype (P = 0.92). Experiment 3 demonstrated that KO birds gained less weight than immunocompetent birds, with a tendency for less weight gain after an initial challenge (P = 0.07) and significantly so after a secondary challenge (P = 0.03). The results from these experiments collectively demonstrate that B cell-mediated immunity can affect growth performance in chickens. Furthermore, these effects can either preserve or impair growth performance, likely via mechanisms related to the immune response rather than the discrete nutritional cost of B cell-mediated immunity.

Immunotherapeutic Potential of Mollusk Hemocyanins in Combination with Human Vaccine Adjuvants in Murine Models of Oral Cancer.

Mollusk hemocyanins have been used for decades in immunological and clinical applications as natural, nontoxic, nonpathogenic, and nonspecific immunostimulants for the treatment of superficial bladder cancer, as carriers/adjuvants of tumor-associated antigens in cancer vaccine development and as adjuvants to dendritic cell-based immunotherapy, because these glycoproteins induce a bias towards Th1 immunity. Here, we analyzed the preclinical therapeutic potential of the traditional keyhole limpet hemocyanin (KLH) and two new hemocyanins from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH) in mouse models of oral squamous cell carcinoma. Due to the aggressiveness and deadly malignant potential of this cancer, the hemocyanins were applied in combination with adjuvants, such as alum, AddaVax, and QS-21, which have been shown to be safe and effective in human vaccines, to potentiate their antitumor activity. The immunogenic performance of the hemocyanins in combination with the adjuvants was compared, and the best formulation was evaluated for its antitumor effects in two murine models of oral cancer: MOC7 cells implanted in the flank (heterotopic) and bioluminescent AT-84 E7 Luc cells implanted in the floor of the mouth (orthotopic). The results demonstrated that the hemocyanins in combination with QS-21 showed the greatest immunogenicity, as reflected by a robust, specific humoral response predominantly characterized by IgG2a antibodies and a sustained cellular response manifesting as a delayed hypersensitivity reaction. The KLH- and FLH-QS-21 formulations showed reduced tumor development and greater overall survival. Hemocyanins, as opposed to QS-21, had no cytotoxic effect on either oral cancer cell line cultured in vitro, supporting the idea that the antitumor effects of hemocyanins are associated with their modulation of the immune response. Therefore, hemocyanin utilization would allow a lower QS-21 dosage to achieve therapeutic results. Overall, our study opens a new door to further investigation of the use of hemocyanins plus adjuvants for the development of immunotherapies against oral carcinoma.

Anti-cancer properties of gastropodan hemocyanins in murine model of colon carcinoma.

BACKGROUND: Various immunotherapeutic approaches have been used for the treatment of cancer. A number of natural compounds are designed to repair, stimulate, or enhance the immune system response. Among them are the hemocyanins (Hcs) - extracellular copper proteins isolated from different arthropod and mollusc species. Hcs are oxygen transporter molecules and normally are freely dissolved in the hemolymph of these animals. Hemocyanins are very promising class of anti-cancer therapeutics due to their immunogenic properties and the absence of toxicity or side effects. KLH (Megathura crenulata hemocyanin) is the most studied molecule of this group setting a standard for natural carrier protein for small molecules and has been used in anti-tumor clinical trials. RESULTS: The Hcs isolated from marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix pomatia (HpH) express strong in vivo anti-cancer and anti-proliferative effects in the developed by us murine model of colon carcinoma. The immunization with RtH and HpH prolonged the survival of treated animals, improve humoral anti-cancer response and moderate the manifestation of C-26 carcinoma symptoms as tumor growth, splenomegaly and lung metastasis appearance. CONCLUSION: Hemocyanins are used so far for therapy of superficial bladder cancer and murine melanoma models. Our findings demonstrate a potential anti-cancer effect of hemocyanins on a murine model of colon carcinoma suggesting their use for immunotherapy of different types of cancer.

Serologic evidence of autoimmunity in E2696 and E1694 patients with high-risk melanoma treated with adjuvant interferon alfa.

We evaluated Eastern Cooperative Group phase II and III trials E2696 and E1694 to assess the incidence and prognostic significance of autoimmunity induced by adjuvant high-dose interferon-α2b (HDI). In E2696, patients with resectable high-risk melanoma were randomized to receive vaccination with GM2-KLH/QS-1 (GMK) plus concurrent HDI, GMK plus sequential HDI, or GMK alone. E1694 randomized patients to either HDI or GMK. Sera from 103 patients in E2696 and 691 patients in E1694 banked at baseline and up to three subsequent time points were tested by ELISA for the development of five autoantibodies. In E2696, autoantibodies were induced in 16 patients (23.2%; n=69) receiving HDI and GMK and two patients (5.9%; n=34) receiving GMK alone (P=0.031). Of 691 patients in E1694, 67 (19.1%) who received HDI (n=350) developed autoantibodies, but only 16 patients (4.7%) developed autoantibodies in the vaccine group (n=341; P<0.001). Almost all induced autoantibodies were detected at ≥12 weeks after the initiation of therapy. A 1-year landmark analysis among resected stage III patients treated with HDI in E1694 showed a trend toward a survival advantage associated with HDI-induced autoimmunity (hazard ratio=0.80; 95% confidence interval: 0.50-1.98; P=0.33). Therefore, adjuvant HDI therapy is associated with the induction of autoimmunity that should be further investigated prospectively as a surrogate marker of adjuvant therapeutic benefit. This potential biomarker develops over the course of up to 1 year, and cannot be used to alter the course of therapy. Studies of the genetic determinants of this response may better discriminate patients more likely to benefit from HDI immunomodulatory therapy.

Adjuvant ganglioside GM2-KLH/QS-21 vaccination versus observation after resection of primary tumor > 1.5 mm in patients with stage II melanoma: results of the EORTC 18961 randomized phase III trial.

PURPOSE: The GM2 ganglioside is an antigen expressed in the majority of melanomas. The GM2-KLH/QS-21 vaccine induces high immunoglobulin M (IgM) and IgG antibody responses. The EORTC 18961 trial compared the efficacy of GM2-KLH/QS-21 vaccination versus observation. PATIENTS AND METHODS: A total of 1,314 patients with a primary tumor > 1.50 mm in thickness (T3-4N0M0; American Joint Committee on Cancer stage II) were randomly assigned to GM2-KLH/QS-21 vaccination (n = 657) or observation (n = 657). Treatment consisted of subcutaneous injections once per week from week 1 to 4, then every 3 months for the first 2 years and every 6 months during the third year. Primary end point was relapse-free survival (RFS). Secondary end points were distant metastasis-free survival (DMFS) and overall survival (OS). Analyses were by intent to treat. RESULTS: After a median follow-up of 1.8 years, the trial was stopped at the second interim analysis for futility regarding RFS (hazard ratio [HR], 1.00; P = .99) and detrimental outcome regarding OS (HR, 1.66; P = .02). After a median follow-up of 4.2 years, we had recorded 400 relapses, nine deaths without relapse, a total of 236 deaths. At 4 years, the vaccination arm showed a decreased RFS rate of 1.2% (HR, 1.03; 95% CI, 0.84 to 1.25) and OS rate of 2.1% (HR, 1.16; 95% CI, 0.90 to 1.51). Toxicity was acceptable, with 4.6% of patients ending study participation because of toxicity. CONCLUSION: GM2-KLH/QS-21 vaccination does not improve outcome for patients with stage II melanoma.

Oral tolerance induction in humans.

Oral tolerance designates the status of systemic hyporesponsiveness against an antigen that makes contact with the immune system via the mucosa of the gastrointestinal tract. In various animal models of autoimmune disease the feeding of the particular autoantigen has been shown to tolerize the animal, thereby ameliorating the course of disease. In contrast, effectivity has not been found in human trials to induce oral tolerance in patients suffering from autoimmune disease. However, the underlying mechanisms of tolerance in rodents, in particular the induction of anti-inflammatory cytokines, seem to be functional in humans as well. Studies using the human neoantigen keyhole limpet hemocyanin (KLH) offer experimental access to examine cellular and molecular basics of oral tolerance in humans required to raise the efficiency of oral tolerance induction in clinical trials.

Immunization with N-propionyl polysialic acid-KLH conjugate in patients with small cell lung cancer is safe and induces IgM antibodies reactive with SCLC cells and bactericidal against group B meningococci.

PURPOSE: Polysialic acid (polySA) is a polymer side chain bound to the neural cell adhesion molecule that is extensively expressed on the surface of small cell lung cancer (SCLC) cells. In our previous study, a robust antibody response was noted in patients with SCLC after vaccination with 30 µg of keyhole limpet hemocyanin (KLH)-conjugated N-propionylated (NP-) polySA, but peripheral neuropathy and ataxia were detected in several vaccinated patients. The objectives of the current trial were to establish the lowest optimal dose and to confirm the safety of the induction of antibodies against polySA with the NP-polySA vaccine. EXPERIMENTAL DESIGN: Patients with SCLC who completed initial treatment and had no evidence of disease progression were injected with either 10 or 3 µg of NP-polySA conjugated to KLH and mixed with 100 µg of immunologic adjuvant (QS-21) at weeks 1, 2, 3, 4, 8, and 16. RESULTS: Nine patients were enrolled at each of the two dose levels. Prior to vaccination, one patient in each group had low-titer antibodies against polysialic acid. All patients at the 10 µg vaccine dose level responded to vaccination with IgM antibody titers against polysialic acid (median titer 1/1,280 by ELISA), and all but one patient made IgM and IgG antibodies against the artificial vaccine immunogen, NP-polysialic acid (median titer 1/10,240). The antibody responses at the 3 µg vaccine dose level were lower; six of nine patients developed antibodies against polysialic acid (median titer 1/160). Post-vaccination sera from 6/9 and 3/9 patients in the 10 and 3 µg groups reacted strongly with human SCLC cells by fluorescent-activated cell sorting (FACS). Sera from all patients in the 10 µg dose group also had bactericidal activity against group B meningococci with rabbit complement. Self-limited grade 3 ataxia of unclear etiology was seen in 1 of 18 patients. CONCLUSIONS: Vaccination with NP-polySA-KLH resulted in consistent high-titer antibody responses, with the 10 µg dose significantly more immunogenic than the 3 µg dose. This study establishes the lowest optimally immunogenic dose of NP-polysialic acid in this NP-polysialic acid-KLH conjugate vaccine to be at least 10 µg, and it establishes the vaccine's safety. We plan to incorporate NP-polySA into a polyvalent vaccine against SCLC with four glycolipid antigens also widely expressed in SCLC-GD2, GD3, fucosylated GM1, and globo H.

Mucosal immune features to phosphorylcholine by nasal Flt3 ligand cDNA-based vaccination.

Phosphorylcholine (PC) is an immunodominant epitope in some pathogens including Streptococcus pneumoniae and it is well-known that PC-specific antibodies (Abs) play a key role in the induction of protective immunity against pneumococcal infection. In this study, we examined whether nasal administration of DNA plasmid encoding Flt3 ligand gene (pFL) as a mucosal adjuvant plus PC-conjugated keyhole limpet hemocyanin (PC-KLH), would elicit PC-specific immune responses, and characterized mucosal immune responses to PC induced by this nasal vaccination. Nasal immunization with pFL plus PC-KLH enhanced induction of PC-specific IgA and IgM Abs in airway secretions when compared with mice given PC-KLH with or without empty plasmid gene (pORF) as controls; in addition to the mucosal immune responses, PC-specific immune responses in serum were also induced. Furthermore, the mucosal and serum IgA and IgM Abs in mice given pFL plus PC-KLH nasally, exhibited high-specificity for the PC molecule. Of interest, the PC-specific Abs bound dose-dependently to anti-T15 idiotype (AB1-2). Thus, the inhibition of S. pneumoniae colonization on the nasal cavity and lungs after nasal challenge with the live organism was significantly elicited in mice immunized with pFL plus PC-KLH compared to that of mice immunized with antigen with pORF. Taken together, these findings show that nasal administration of pFL with PC-KLH elicited T15-like anti-PC IgA and IgM Abs in the respiratory tracts, and further attenuated S. pneumoniae colonization on the respiratory tracts. Nasal administration of Flt3 ligand cDNA with PC may contribute to the development of nasal vaccination for prevention of S. pneumoniae infection.

Phase III multicenter clinical trial of the sialyl-TN (STn)-keyhole limpet hemocyanin (KLH) vaccine for metastatic breast cancer.

PURPOSE: This double-blind, randomized, phase III clinical trial evaluated time to progression (TTP) and overall survival in women with metastatic breast cancer (MBC) who received sialyl-TN (STn) keyhole limpet hemocyanin (KLH) vaccine. Secondary endpoints included vaccine safety and immune response. EXPERIMENTAL DESIGN: The study population consisted of 1,028 women with MBC across 126 centers who had previously received chemotherapy and had had either a complete or a partial response or no disease progression. All women received one-time i.v. cyclophosphamide (300 mg/m(2)) 3 days before s.c. injection of 100 µg STn-KLH plus adjuvant (treatment group) or 100 µg KLH plus adjuvant (control group) at weeks 0, 2, 5, and 9. Subsequently, STn-KLH without adjuvant or KLH without adjuvant was then administered monthly for 4 months, and then quarterly until disease progression, without cyclophosphamide. RESULTS: STn-KLH vaccine was well tolerated; patients had mild to moderate injection-site reactions and reversible flu-like symptoms. Week-12 antibody testing revealed high specific IgG titers and a high rate of IgM-to-IgG seroconversion; the median IgG titers in STn-KLH recipients were 320 (anti-ovine submaxillary mucin) and 20,480 (anti-STn), with no detectable antimucin antibodies in the control group. The TTP was 3.4 months in the treatment group and 3.0 months in the control group. The median survival times were 23.1 months and 22.3 months, respectively. CONCLUSIONS: Although STn-KLH was well tolerated in this largest to date metastatic breast cancer vaccine trial, no overall benefit in TTP or survival was observed. Lessons were learned for future vaccine study designs.

Antitumor activity of N-acetyl-D-glucosamine-substituted glycoconjugates and combined therapy with keyhole limpet hemocyanin in B16F10 mouse melanoma model.

N-Acetyl-D-glucosamine-substituted glycoconjugates (GCJs) with the polyamidoamine (GN8P) or calix[4]arene (GN4C) scaffold represent ligands for NKR-P1 molecule and induce NK cell-mediated cytotoxicity in vitro. The in vivo effect of these GCJs on mouse melanoma model was determined when administered either alone or in combination with non-specific immunostimulator keyhole limpet hemocyanin (KLH). All types of treatment significantly reduced the tumor growth on day 23, while GN4C as well as KLH were effective continuously (from day 14). The GN4C also induced the longest mean survival time (46.3 ± 11.1 d), followed by KLH+GN4C (36.4 ± 12.1), KLH (35.6 ± 6.5), KLH+GN8P (35.6 ± 6.7), and GN8P (32.4 ± 7.0), compared to controls (29.8 ± 3.6). The B16F10 specific cytotoxicity of peripheral blood cells was significantly elevated by both KLH and GN8P, whereas not by GN4C. KLH increased the effect of the GN4C, but did not influence that of GN8P. GN4C was proved to exert anticancer activity in mouse melanoma model. The combination of KLH with GCJs did not generate synergism.

Unexpected high response rate to traditional therapy after dendritic cell-based vaccine in advanced melanoma: update of clinical outcome and subgroup analysis.

We reviewed the clinical results of a dendritic cell-based phase II clinical vaccine trial in stage IV melanoma and analyzed a patient subgroup treated with standard therapies after stopping vaccination. From 2003 to 2009, 24 metastatic melanoma patients were treated with mature dendritic cells pulsed with autologous tumor lysate and keyhole limpet hemocyanin and low-dose interleukin-2. Overall response (OR) to vaccination was 37.5% with a clinical benefit of 54.1%. All 14 responders showed delayed type hypersensitivity positivity. Median overall survival (OS) was 15 months (95% CI, 8-33). Eleven patients underwent other treatments (3 surgery, 2 biotherapy, 2 radiotherapy, 2 chemotherapy, and 4 biochemotherapy) after stopping vaccination. Of these, 2 patients had a complete response and 5 a partial response, with an OR of 63.6%. Median OS was 34 months (range 16-61). Our results suggest that therapeutic DC vaccination could favor clinical response in patients after more than one line of therapy.

Clinical use of anti-CD25 antibody daclizumab to enhance immune responses to tumor antigen vaccination by targeting regulatory T cells.

CD4(+) regulatory T cells frustrate productive tumor immune surveillance and represent an obstacle for cancer immunotherapy. In mice, anti-CD25 antibody is an effective method of depleting CD25(+) FOXP3(+) T regulatory cells (Tregs) in vivo and enhancing cancer immunity. Here, we propose the use of the anti-CD25 monoclonal antibody daclizumab for the depletion of Tregs in cancer patients. In early results from an ongoing clinical trial, a single intravenous infusion of daclizumab in patients with metastatic breast cancer is associated with a marked and prolonged elimination of CD25(+) FOXP3(+) Tregs in peripheral blood. When a cancer antigen peptide vaccine is administered during the daclizumab-induced Treg nadir, effective generation of cytotoxic T lymphocytes has been observed, including those specific for neo-antigens, such as cytomegalovirus peptide used as an immunological control. If confirmed in additional patients, these observations suggest that daclizumab may be an effective and available therapeutic agent for Treg modulation in patients with cancer.

Immunodominant role of CCHA subunit of Concholepas hemocyanin is associated with unique biochemical properties.

Hemocyanin, the oxygen transporter metallo-glycoprotein from mollusks, shows strong relationship between its notable structural features and intrinsic immunomodulatory effects. Here we investigated the individual contribution of CCHA and CCHB subunits from Concholepas hemocyanin (CCH) to in vivo humoral immune response and their pre-clinical evaluation as immunotherapeutic agent in a mice bladder cancer model, in relation to their biochemical properties. To this end, subunits were purified and well characterized. Homogeneous subunits were obtained by anionic exchange chromatography, and its purity assessed by electrophoretic and immunochemical methods. While each CCH subunit contains eight functional units showing partial cross reaction, the vibrational spectral analysis showed several spectral differences, suggesting structural differences between them. In addition, we demonstrated differences in the carbohydrate content: CCHA had a 3.6% w/w sugar with both N- and O-linked moieties. In turn, CCHB had a 2.5% w/w sugar with N-linked, while O-linked moieties were nearly absent. Considering these differences, it was not possible to predict a priori whether the immunogenic and immunotherapeutic properties of subunits might be similar. Surprisingly, both subunits by itself induced a humoral response, and showed an antitumor effect in the bladder carcinoma cell line MBT-2. However, when immunologic parameters were analyzed, CCHA showed better efficiency than CCHB. No allergic reactions or any toxic effects were observed in mice treated with CCHA, sustaining its potential therapeutic use. Our study supports that CCHA subunit accounts for the most important features involved in the immunogenicity of CCH, such as better hydrophilicity and higher content of carbohydrates.

Detection of humoral response in patients with glioblastoma receiving EGFRvIII-KLH vaccines.

The epidermal growth factor receptor variant III (EGFRvIII) is a consistent tumor-specific mutation that is widely expressed in glioblastoma multiforme (GBM) and other neoplasms. As such it represents a truly tumor-specific target for antitumor immunotherapy. Although endogenous humoral responses to EGFRvIII have been reported in patients with EGFRvIII-expressing breast cancer, it is not known whether de novo responses can be generated or endogenous responses enhanced with an EGFRvIII-specific vaccine. To assess this in clinical trials, we have developed and validated an immunoassay to measure and isolate anti-EGFRvIII and anti-KLH antibodies from the serum of patients vaccinated with an EGFRvIII-specific peptide (PEPvIII) conjugated to keyhole limpet hemocyanin (KLH). Using magnetic beads with immobilized antigen we captured and detected anti-EGFRvIII and anti-KLH antibodies in serum from patients before and after vaccinations. Using this assay, we found that significant levels of antibody for tumor-specific antigen EGFRvIII (>4 microg/mL) and KLH could be induced after vaccination with PEPvIII-KLH.

Keyhole limpet hemocyanin: an effective adjunct against melanoma in vivo.

BACKGROUND: We have previously demonstrated the potent in vitro antiproliferative effects of keyhole limpet hemocyanin (KLH) against melanoma. Our prior studies directed us to hypothesize that KLH would be effective in vivo against melanoma, alone and in combination with conventional immunotherapy. METHODS: Mice were inoculated with 2 x 10(7) HTB68 cells and randomized to 6 groups. Treatment groups consisted of control, KLH 200 microg, alpha interferon (AIFN) 1000 IU, interleukin-2 (IL-2) 5000 IU, KLH + AIFN, and KLH + IL-2. RESULTS: KLH + IL-2 exhibited the greatest reduction in tumor volume (30%) as compared to control (P = .014), followed by KLH + AIFN (28%, P = .031). Singly treated animals had less tumor inhibition: IL-2 (30%, P = .022), KLH (18%, not significant), and AIFN (16%, not significant). CONCLUSIONS: KLH augments the effects of AIFN, one of the standard immunotherapeutic agents against melanoma in vivo. Further in vivo and early clinical studies into the effects of KLH as both a single and combined agent are warranted.

Pilot study of a heptavalent vaccine-keyhole limpet hemocyanin conjugate plus QS21 in patients with epithelial ovarian, fallopian tube, or peritoneal cancer.

PURPOSE: To characterize the safety and immunogenicity of a heptavalent antigen-keyhole limpet hemocyanin (KLH) plus QS21 vaccine construct in patients with epithelial ovarian, fallopian tube, or peritoneal cancer in second or greater complete clinical remission. EXPERIMENTAL DESIGN: Eleven patients in this pilot trial received a heptavalent vaccine s.c. containing GM2 (10 microg), Globo-H (10 microg), Lewis Y (10 microg), Tn(c) (3 microg), STn(c) (3 microg), TF(c) (3 microg), and Tn-MUC1 (3 microg) individually conjugated to KLH and mixed with adjuvant QS21(100 microg). Vaccinations were administered at weeks 1, 2, 3, 7, and 15. Periodic blood and urine samples were obtained to monitor safety (complete blood count, comprehensive panel, amylase, thyroid-stimulating hormone, and urinalysis) and antibody production (ELISA, fluorescence-activated cell sorting, and complement-dependent cytotoxicity). RESULTS: Eleven patients were included in the safety analysis; 9 of 11 patients remained on study for at least 2 weeks past fourth vaccination and were included in the immunologic analysis (two withdrew, disease progression). The vaccine was well tolerated. Self-limited and mild fatigue (maximum grade 2 in two patients), fever, myalgia, and localized injection site reactions were most frequent. No clinically relevant hematologic abnormalities were noted. No clinical or laboratory evidence of autoimmunity was seen. Serologic responses by ELISA were largely IgM against each antigen with the exception of Tn-MUC1 where both IgM and IgG responses were induced. Antibody responses were generally undetectable before immunization. After immunization, median IgM titers were as follows: Tn-MUC1, 1:640 (IgG 1:80); Tn, 1:160; TF, 1:640; Globo-H, 1:40; and STn, 1:80. Only one response was seen against Lewis Y; two were against GM2. Eight of nine patients developed responses against at least three antigens. Antibody titers peaked at weeks 4 to 8 in all patients. Fluorescence-activated cell sorting and complement-dependent cytotoxicity analysis showed substantially increased reactivity against MCF7 cells in seven of nine patients, with some increase seen in all patients. CONCLUSIONS: This heptavalent-KLH conjugate plus QS21 vaccine safely induced antibody responses against five of seven antigens. Investigation in an adequately powered efficacy trial is warranted.

Keyhole limpet hemocyanin potentiates standard immunotherapy for melanoma.

INTRODUCTION: Our hypothesis was that keyhole limpet hemocyanin (KLH) would augment the effects of standard immunotherapies for melanoma including interferon-alpha (AIFN) and interleukin (IL)-2. METHODS: The HTB68 melanoma cell line was treated with KLH, AIFN, and IL-2 as single and combined agents. Cell viability, apoptotic activity, and vascular endothelial growth factor levels were all evaluated. RESULTS: Cell growth was reduced with KLH (28%), AIFN (54%), and IL-2 (29%) (all P < .001). KLH and IL-2 combined exhibited a 47% inhibition of cell growth, whereas KLH and AIFN combined yielded a 67% reduction in cell growth (both P < .001). KLH and AIFN combined significantly increased both early (10%) and late (14%) apoptotic activity compared with controls (5% and 7%, P < .001). CONCLUSIONS: The additive effects exhibited by the combination of KLH with AIFN or IL-2 are encouraging and support combination therapy as an effective treatment for this aggressive disease.