Keyhole Limpet Hemocyanin Conjugate Research Articles

Production of monoclonal antibodies for sandwich immunoassay detection of ciguatoxin 51-hydroxyCTX3C

Every year, more than 50,000 people in subtropical and tropical regions suffer from ciguatera seafood poisoning. The extremely low level of the causative neurotoxins (ciguatoxins) in fish has hampered the preparation of antibodies for detection of the toxins. In this study, we produced a monoclonal antibody (8H4) against the right end of ciguatoxin CTX1B ( 1) and 51-hydroxyCTX3C ( 3) by immunizing mice with the keyhole limpet hemocyanin-conjugate of the synthetic HIJKLM ring fragment ( 10). We used 8H4 and another previously reported monoclonal antibody (10C9) that recognizes the left end of 3 to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect 3. The assay could detect 3 down to the ppb level and lacked cross-reactivity with other related marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

A Phage Display Screen and Binding Studies with Acetylated Low Density Lipoprotein Provide Evidence for the Importance of the Scavenger Receptor Cysteine-rich (SRCR) Domain in the Ligand-binding Function of MARCO

MARCO is a class A scavenger receptor capable of binding both gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.

Synthesis and immunological properties of N-modified GM3 antigens as therapeutic cancer vaccines.

The problem of immunotolerance to GM3, an important tumor-associated trisaccharide antigen, seriously hinders its usage in cancer vaccine development. To solve this problem, the keyhole limpet hemocyanin (KLH) conjugates of a series of GM3 derivatives were synthesized and screened as therapeutic cancer vaccines. First, the beta-linked anomeric azides of differently N-acylated GM3 analogues were prepared by a highly convergent procedure. Next, a pentenoyl group was linked to the reducing end of the carbohydrate antigens following selective reduction of the azido group. The linker was thereafter ozonolyzed to give an aldehyde functionality permitting the conjugation of the antigens to KLH via reductive amination. Finally, the immunological properties of the resultant glycoconjugates were studied in C57BL/6 mice by assessing the titers of specific antibodies induced by the GM3 analogues. While KLH-GM3 elicited low levels of immune response, the KLH conjugates of N-propionyl, N-butanoyl, N-iso-butanoyl, and N-phenylacetyl GM3s induced robust immune reactions with antibodies of multiple isotypes, indicating significantly improved and T-cell dependent immune responses that lead to isotype switching, affinity maturation, and the induction of immunological "memory". It was suggested that GM3PhAc-KLH is a promising vaccine candidate for glycoengineered immunotherapy of cancer with GM3 as the primary target.

Modification of lupus-associated 60-kDa Ro protein with the lipid oxidation product 4-hydroxy-2-nonenal increases antigenicity and facilitates epitope spreading

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. The Ro ribonucleoprotein particle, composed of a 60-kDa protein noncovalently associated with human cytoplasmic RNA, is the target of antibodies in 25–40% of lupus patients. Purified human 60-kDa Ro was found to be oxidatively modified. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore we hypothesized that oxidation by-products, such as 4-hydroxy-2-nonenal (HNE), could lead to neoantigens like HNE-modified 60-kDa Ro, which could in turn initiate autoimmunity or drive epitope spreading. To test this hypothesis we immunized rabbits with either HNE-modified 60-kDa Ro or the unmodified Ro. Intramolecular epitope spreading within the Ro molecule and intermolecular epitope spreading to La, double-stranded DNA, nRNP, and Sm occurred preferentially in HNE–Ro-immunized animals. Nonspecific anti-HNE antibody, generated by immunization with HNE–keyhole limpet hemocyanin conjugate, did not significantly bind to these autoantigens. These data may suggest a hitherto unappreciated mechanism by which oxidative stress facilitates epitope spreading in SLE.

Blending of a conventional Mycoplasma hyopneumoniae vaccine with a positive marker: tracking of immunised pigs by peptide-specific antibodies raised to the marker component

Highly immunodominant marker antigens simply blended to existing veterinary vaccines may represent a smart approach for addressing the still open issue of vaccination compliance. This approach was evaluated by blending a widely deployed Mycoplasma hyopneumoniae vaccine with a peptide–KLH (Keyhole Limpet Hemocyanin) conjugate as marker. Piglets were vaccinated twice with: (i) a combination of the M. hyopneumoniae-specific vaccine and the marker, (ii) M. hyopneumoniae-specific vaccine, (iii) marker alone or (iv) placebo dose only. All piglets which received the M. hyopneumoniae-specific vaccine/marker formulation or, as control, the marker blended with Montanide ® IMS1313 adjuvant responded to the respective immunisation from day 21 to 77 post vaccination as seropositive for the appropriate peptide and KLH. However, the responder rate to M. hyopneumoniae of piglets administered with M. hyopneumoniae-specific vaccine/marker was slightly reduced at day 35 and 49 post immunisation in comparison with piglets vaccinated with M. hyopneumoniae-specific vaccine alone. Accordingly, we conclude that this marker technology could be successfully applied to label a whole set of vaccines prevented that the blending process will be optimised.

258 A phase I trial in patients with solid tumours using autologous dendritic cells loaded with mannan-conjugated recombinant MUC1 protein

Antigens such as ganglioside GD3, neutral glycolipid Lewisy (Ley) and mucins MUC1 and MUC2 are over-expressed on the cell surface of many tumors. We have shown previously that conjugation of antigens such as these to keyhole limpet hemocyanin (KLH) and the use of immunological adjuvant QS-21 is the optimal approach for inducing high titer IgM and IgG antibodies. These antibodies are able to bind with natural antigens on the tumor cell surface and mediate complement dependent cytotoxicity and/or antibody dependent cell mediated cytotoxicity. Immunization of patients with monovalent vaccines containing these and a variety of other antigens have demonstrated both the consistent immunogenicity and the safety of these vaccines. Now, in preparation for the use of polyvalent conjugate vaccines in the clinic, we have addressed for the first time with conjugate vaccines against cancer antigens several questions in the pre-clinical setting, including whether immunogenicity of the individual components is decreased in the polyvalent vaccine and issues relating to vaccine formulation and administration. We have immunized groups of mice with GD3–KLH, Ley–KLH, MUC1–KLH and MUC2–KLH conjugates and QS-21 separately or mixed and administered at one or four sites. High titer IgM and IgG antibodies were induced against each of the four antigens whether administered singly in separate mice, at separate sites in the same mice, or mixed and administered at a single site or at four sites, or administered subcutaneously (s.c.) or intraperitoneally (i.p.). These antibodies reacted specifically with the respective antigens and tumor cells expressing these antigens. There was no evidence of suppression of the antibody response against any one of the antigens by the presence of the other conjugates in the vaccine. Immunogenicity of the four individual antigens conjugated to KLH and QS-21 is not affected by mixing the four together and administering them at a single subcutaneous site.

Vaccine therapy for small cell lung cancer

One novel approach to the treatment of lethal residual disease in patients with small cell lung cancer (SCLC) relies on the induction of a host-immune response to attack chemoresistant tumor cells. Because of its neuroectodermal origin, SCLC has a number of specific antigens that could be capitalized on as immune targets. This article reviews two vaccine strategies currently in clinical study. The anti-idiotype vaccine to the GD3 ganglioside, BEC-2, has recently been tested in a phase III trial. In this trial, patients with SCLC who had completed initial chemotherapy were randomized to observation or vaccination with BEC-2 plus bacillus Calmette-Guerin adjuvant. A series of other trials have established the immunogenicity of several keyhole limpet hemocyanin conjugate vaccines relevant to SCLC, including GM2, Globo H, fucosyl GM1, and polysialic acid. To optimize an immune response against a broad range of tumor phenotypes, these components will be combined into a polyvalent vaccine. A randomized phase II trial of this polyvalent vaccine is planned to start in 2004.

Development of H1e histone linker-specific antibodies by means of synthetic peptides.

A large body of data suggests that the linker histones family (H1) affects gene expression. Investigation of the linker histones role is then of a major interest in cell cycle studies with implications in gene therapy. Indeed, it has been shown that in most tissues a switch of histone subtypes occurs when the cells cease to divide. To investigate linker histone role in gene or transgene expression, an antibody against subtypes of H1 would be useful for immunoprecipitation experiments and further assays measuring H1subtypes-DNA interactions in living cells. In order to produce an antibody against the H1e subtype of linker histones, two synthetic peptides derived from two regions of the H1e mouse histone protein were examined for their potential, [as keyhole limpet hemocyanin (KLH) conjugates] to elicit polyclonal anti-H1e antibodies in New Zealand white rabbits. Selection of the peptide sequences was based on amino acid differences within the different classes of histones and between mice and rabbit histones as well. The evaluation of their potential immunogenic properties was based on examination of peptide hydropathy using predicting algorithms. Immunoglobulins (IgG) obtained from immunized and nonimmunized rabbits were tested using enzyme-linked immunosorbent assay (ELISA) procedures, Western immunoblot, and immunofluorescence experiments. Results showed that the selected synthetic peptides gave rise to a high-titer polyclonal antibody able to recognize the H1e histone under various conditions. This polyclonal antibody did not cross-react with other histones. To our knowledge, this is the first antibody produced against the mouse H1e linker histone.

Development of monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic assay for lasalocid and semduramicin

Monoclonal antibodies (MAbs) against lasalocid and semduramicin were prepared using keyhole limpet hemocyanin conjugates for the immunization of mice. With these MAbs, we developed quantitative enzyme-linked immunosorbent assay (ELISA) methods for lasalocid and semduramicin. The ELISAs were quantitative in the ranges of 0.1-50 ng/mL for lasalocid and 0.05-12.5 ng/mL for semduramicin, and showed 50% inhibition concentrations of 1.2 ng/mL for lasalocid and 0.5 ng/mL for semduramicin. The coefficient of variations (CV%) of lasalocid were 0.3-4.4% for intra-assay and 0.5-5.1% for inter-assay and those of semduramicin were 0.1-4.6% for intra-assay and 0.3-5.2% for inter-assay. The detection limits for lasalocid and semduramicin were 10 ng/g and 5 ng/g in chicken liver and muscle, respectively. Based on the immunochromatographic method, rapid test kits for lasalocid and semduramicin were also developed. With these kits, the detection limits of lasalocid were 50 ng/mL for standard solution and 125 ng/g for chicken muscle, and those of semduramicin were 10 ng/mL for standard solution and 100 ng/g for chicken muscle.

Efficacy of the 26-kilodalton outer membrane protein and two P5 fimbrin-derived immunogens to induce clearance of nontypeable Haemophilus influenzae from the rat middle ear and lungs as well as from the chinchilla middle ear and nasopharynx.

The rat middle ear and lung clearance model has been used to show that the nontypeable Haemophilus influenzae 26-kDa outer membrane protein OMP26 is highly efficacious as a mucosal immunogen, inducing significantly enhanced clearance in immunized rats upon direct challenge of these two anatomic sites. Similarly, the chinchilla model of middle ear and nasopharyngeal clearance has been used to show that two P5 fimbrin adhesin-derived immunogens, LB1 and lipoprotein D (LPD)-LB1(f)(2,1,3), are highly efficacious as parenteral immunogens. Both induced significantly augmented clearance of nontypeable H. influenzae upon challenge of these sites. Here, these three nontypeable H. influenzae immunogens in addition to six bovine serum albumin and keyhole limpet hemocyanin conjugates of the synthetic peptide LB1(f) were assayed for relative efficacy in the reciprocal rodent model system. OMP26 was assayed in the chinchilla host by a parenteral immunization route, with clearance of the middle ear and nasopharynx used as outcome measures. Both LB1 and LPD-LB1(f)(2,1,3) were assayed in the rat host with a mucosal immunization route and clearance of nontypeable H. influenzae from the lungs and middle ears as outcome measures. Both of the immunogens were found to induce a high-titered and specific immune responses in the heterologous host system. Moreover, each was found to be highly efficacious in the reciprocal host system, providing strong support for the continued development and inclusion of both OMP26 and P5 fimbrin-derived peptides as candidate vaccine antigens directed at otitis media caused by nontypeable H. influenzae.

Development of Polyclonal Antibodies Against Domoic Acid for Their Use in Electrochemical Biosensors

The current work describes the raising of polyclonal antibodies (pAbs) against domoic acid (DA), an algal toxin produced by the diatom Pseudonitzschia pungens. They were screened for sensitivity and selectivity using a competitive enzyme-linked immunosorbent assay (ELISA). The antiserum produced against a keyhole limpet haemocyanin (DA-KLH) conjugate displayed a high affinity for free DA. The optimized horseradish peroxidase (HRP) ELISA had a detection limit of 0.6 ng mL−1 (ppb) and a working range of 0.8–300 ppb DA applying a streptavidin-biotin amplification system (ABC system). Furthermore this antiserum did not cross-react with similar chemical structures and algal toxins such as kainic acid, aspartic acid, glutamic acid, geranic acid and 2-methyl-3-butenoic acid. When the ELISA was compared using an alkaline phosphatase (AP) label we found the system to behave in a similar manner to the optimized HRP system but the linear range was smaller in the high DA concentration range. These pAbs were then used in the optimization of a screen-printed electrode (SPE) system for measurement of DA. A disposable screen-printed carbon electrode coupled with amperometric detection of p-aminophenol at +300 mV vs. Ag/AgCl, produced by the enzyme AP, was used for signal measurement. The sensor incorporates a relevant range for toxin detection, by which humans become ill (Iverson, F.; Truelove, J. Toxicology and seafood toxins: domoic acid. Natural Toxins 1994, 2, 334–339.) with detection limits achieved at SPE to the order of ppb. The SPE system is simple and cost-effective due to its disposable nature, and analysis time is complete in 30 min. In addition, recovery experiments on DA for both ELISA and SPE highlighted the functionality of these systems yielding a ±12% deviation for the true value for the ELISA using AP and ±25% for the sensor.

Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for dihydrostreptomycin in milk

Monoclonal antibody (MAb) against streptomycin was prepared by using a streptomycin–keyhole limpet hemocyanin conjugate for the immunization of mice. Splenocytes from BALB/c immunized mice were fused with P3X63Ag8U.1 myeloma cells. This resulted in one hybridoma cell line. Using this MAb, we developed quantitative assays for dihydrostreptomycin by means of an enzyme-linked immunosorbent assay (ELISA). Fifty percent inhibition concentration (IC 50) for the MAb was 2.0 ng ml −1. The detection limit was 0.1 ng ml −1 and the standard deviations were 0.5–4.7% for intra-assay and 0.9–5.9% for inter-assay, respectively. The detection limit using peroxidase was 10 ng ml −1 in milk. Using the MAb produced, a rapid test kit based on the immunochromatographic method was developed. The detection limit using the kit was 100 ng ml −1 in milk.

Vaccination with CD20 peptides induces a biologically active, specific immune response in mice

CD20 is a 33-kD B-cell antigen that is expressed from the early pre-B-cell stage of development and is lost on differentiation of B cells into plasma cells. Because CD20 is expressed strictly by B cells, it is an attractive target for B-cell lymphoma therapy. Monoclonal antibodies to CD20 have been used successfully in the treatment of B-cell lymphomas. We hypothesized that a vaccine consisting of CD20 peptide sequences might be capable of inducing an active, specific, humoral immune response to the protein. Vaccine therapy would have the advantage of generating a polyclonal response to the antigen in contrast to the monoclonal response of an infused antibody. Balb/c mice were vaccinated with prototype vaccine constructs that consisted of peptides representing the human or mouse CD20 extracellular sequences conjugated to carrier proteins and mixed with QS21 adjuvant. Sera from the vaccinated mice demonstrated high-titer, specific antibodies to various epitopes on the immunizing peptides in enzyme-linked immunosorbent assay, weaker antibody binding to native CD20 on cells by flow cytometry, and antibody-mediated complement killing of CD20(+) cells in some cases. Specific proliferation and secretion of interleukin 4 and interferon gamma by mouse spleen cells in response to the immunizing peptides were also demonstrated. Mice vaccinated with the CD20 peptide keyhole limpet hemocyanin conjugates had a 25% decrease in CD19(+) splenic B cells relative to control mice. These data indicate that a biologically active, specific immune response to CD20 can be elicited in mice vaccinated with CD20 peptide conjugates.

Enhancement of the protective efficacy of an oprF DNA vaccine against Pseudomonas aeruginosa

The outer membrane protein F gene ( oprF) of Pseudomonas aeruginosa was recently shown by us to protect mice from P. aeruginosa chronic pulmonary infection when used as a DNA vaccine administered by three biolistic (gene gun) intradermal inoculations given at 2-week intervals. In the present study, we used two different strategies to improve the protective efficacy of the DNA vaccine. In the first strategy, mice were primed with two biolistic intradermal inoculations with the oprF vaccine and then were given a final intramuscular booster immunization containing either a synthetic peptide–keyhole limpet hemocyanin (KLH) conjugate or a chimeric influenza virus. Both the synthetic peptide conjugate and the chimeric virus contained peptide 10, a previously identified immunoprotective epitope of protein F. The second strategy involved the addition of a second outer membrane protein to the vaccine. DNA encoding a fusion protein comprised of the C-terminal half of protein F fused to OprI was administered by three biolistic intradermal inoculations. Challenge with P. aeruginosa in a chronic pulmonary infection model demonstrated that boosting with the chimeric virus (but not with peptide–KLH) or adding oprI to the DNA vaccine significantly enhanced protection as compared to that afforded by the oprF vaccine given alone. Thus, both strategies appear to augment the protection afforded by an oprF-only DNA vaccine.

15-Deoxy-Δ12,14-prostaglandin J2: A PROSTAGLANDIN D2 METABOLITE GENERATED DURING INFLAMMATORY PROCESSES

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J(2) series, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), which have been suggested to exert anti-inflammatory effects in vivo. Meanwhile, the mechanism of these effects is not well understood and the natural site and the extent of its production in vivo remain unclear. In the present study, we raised a monoclonal antibody specific to 15d-PGJ(2) and determined its production in inflammation-related events. The monoclonal antibody (mAb11G2) was raised against the 15d-PGJ(2)-keyhole limpet hemocyanin conjugate and was found to recognize free 15d-PGJ(2) specifically. The presence of 15d-PGJ(2) in vivo was immunohistochemically verified in the cytoplasm of most of the foamy macrophages in human atherosclerotic plaques. In addition, the immunostaining of lipopolysaccharide-stimulated RAW264.7 macrophages with mAb11G2 demonstrated an enhanced intracellular accumulation of 15d-PGJ(2), suggesting that the PGD(2) metabolic pathway, generating the anti-inflammatory PGs, is indeed utilized in the cells during inflammation. The activation of macrophages also resulted in the extracellular production of PGD(2), which was associated with a significant increase in the extracellular 15d-PGJ(2) levels, and the extracellular 15d-PGJ(2) production was reproduced by incubating PGD(2) in a cell-free medium and in phosphate-buffered saline. Moreover, using a chiral high performance liquid chromatography method for separation of PGD(2) metabolites, we established a novel metabolic pathway, in which PGD(2) is converted to 15d-PGJ(2) via an albumin-independent mechanism.

Dendritic cells pulsed with a tumor-specific peptide induce long-lasting immunity and are effective against murine intracerebral melanoma.

Dendritic cells (DCs) are specialized cells of the immune system that are capable of generating potent immune responses that are active even within the "immunologically privileged" central nervous system. However, immune responses generated by DCs have also been demonstrated to produce clinically significant autoimmunity. Targeting the epidermal growth factor receptor variant III (EGFRvIII), which is a mutation specific to tumor tissue, could eliminate this risk. The purpose of this study was to demonstrate that DC-based immunizations directed solely against this tumor-specific antigen, which is commonly found on tumors that originate within or metastasize to the brain, could be efficacious. C3H mice were vaccinated with DCs mixed with a keyhole limpet hemocyanin conjugate of the tumor-specific peptide, PEP-3, which spans the EGFRvIII mutation, or the random-sequence peptide, PEP-1, and were intracerebrally challenged with a syngeneic melanoma expressing a murine homologue of EGFRvIII. Systemic immunization with DCs mixed with PEP-3-keyhole limpet hemocyanin generated antigen-specific immunity. Among mice challenged with intracerebral tumors, this resulted in an approximately 600% increase in the median survival time (>300 d, P < 0.0016), relative to control values. Sixty-three percent of mice treated with DCs mixed with the tumor-specific peptide survived in the long term and 100% survived rechallenge with tumor, indicating that antitumor immunological memory was also induced. In a murine melanoma model, immunization with DCs mixed with tumor-specific peptide results in an antigen-specific immunological response that recognizes the EGFRvIII mutation, has potent antitumor efficacy against intracerebral tumors that express EGFRvIII, and results in long-lasting antitumor immunity.

Comparison of antibody titers after immunization with monovalent or tetravalent KLH conjugate vaccines

Antigens such as ganglioside GD3, neutral glycolipid Lewis y (Le y) and mucins MUC1 and MUC2 are over-expressed on the cell surface of many tumors. We have shown previously that conjugation of antigens such as these to keyhole limpet hemocyanin (KLH) and the use of immunological adjuvant QS-21 is the optimal approach for inducing high titer IgM and IgG antibodies. These antibodies are able to bind with natural antigens on the tumor cell surface and mediate complement dependent cytotoxicity and/or antibody dependent cell mediated cytotoxicity. Immunization of patients with monovalent vaccines containing these and a variety of other antigens have demonstrated both the consistent immunogenicity and the safety of these vaccines. Now, in preparation for the use of polyvalent conjugate vaccines in the clinic, we have addressed for the first time with conjugate vaccines against cancer antigens several questions in the pre-clinical setting, including whether immunogenicity of the individual components is decreased in the polyvalent vaccine and issues relating to vaccine formulation and administration. We have immunized groups of mice with GD3–KLH, Le y–KLH, MUC1–KLH and MUC2–KLH conjugates and QS-21 separately or mixed and administered at one or four sites. High titer IgM and IgG antibodies were induced against each of the four antigens whether administered singly in separate mice, at separate sites in the same mice, or mixed and administered at a single site or at four sites, or administered subcutaneously (s.c.) or intraperitoneally (i.p.). These antibodies reacted specifically with the respective antigens and tumor cells expressing these antigens. There was no evidence of suppression of the antibody response against any one of the antigens by the presence of the other conjugates in the vaccine. Immunogenicity of the four individual antigens conjugated to KLH and QS-21 is not affected by mixing the four together and administering them at a single subcutaneous site.

Synthesis of LJP 993, a multivalent conjugate of the N-terminal domain of beta2GPI and suppression of an anti-beta2GPI immune response.

LJP 993, a tetravalent conjugate of the amino-terminal domain (domain 1) of beta2GPI, was synthesized, and studies were carried out to explore the ability of LJP 993 to bind anti-beta2GPI antibodies and to function as a B cell toleragen. Domain 1 was expressed in Pichia pastoris, and the N-terminus was site-specifically modified by a transamination reaction converting the N-terminal glycine to a glyoxyl group. A tetravalent platform was synthesized with linkers that terminate in aminooxy groups. This was accomplished by preparing an ethylene glycol-based heterobifunctional linker that contains both a Boc-protected aminooxy group and a free primary amine. The linker was used to modify a tetravalent platform molecule by reacting the amino groups on the linker with 4-nitrophenyl carbonate esters on the platform to provide a linker-modified platform, and the Boc protecting groups were removed to provide a tetravalent aminooxy platform. Glyoxylated domain 1 was attached to the platform to provide LJP 993 by formation of oxime bonds. The protein domains of LJP 993 retain activity as evidenced by the ability of LJP 993 to bind to anti-beta2GPI antibodies. Dissociation constants (Kd) for domain 1 and LJP 993 bound to immobilized affinity-purified anti-beta2GPI antibodies from autoimmune thrombosis patients were determined using surface plasmon resonance. An immunized mouse model was developed to test the ability of LJP 993 to act as a toleragen. A thiol containing domain 1 analogue was expressed in insect cells using the baculovirus expression system, and it was used to prepare an immunogenic conjugate of domain 1 and maleimide-derivatized keyhole limpet hemocyanin (KLH). Mice were immunized with the KLH conjugate, and spleen cells were harvested from the immunized mice. The cells were incubated with various concentrations of LJP 993 and transferred to mice whose immune systems had been compromised by irradiation. The hosts were then boosted with the KLH-domain 1 conjugate, and after 7 days their antibody levels were measured. Host mice receiving cells that were treated with LJP 993 produced significantly lower amounts of anti-domain 1 antibodies than controls which received untreated cells, indicative of B cell tolerance.

Keyhole limpet hemocyanin conjugate vaccines against cancer: the Memorial Sloan Kettering experience.

Passively administered and actively induced antibodies have been associated with the eradication of circulating tumor cells and micrometastases in mice and humans. We have identified a series of cell surface carbohydrate and peptide antigens on melanomas, sarcomas, and cancer of the breast, prostate. ovary, and lung tissues. We found that breaking tolerance toward these tumor antigens was best achieved using vaccines containing antigens chemically conjugated to keyhole limpet hemocyanin (KLH) plus a potent immunological adjuvant (QS-21). To date, by using this approach to vaccination. antibodies have been induced in patients against glycolipid antigens GM2, GD2, GD3, FucosylGM1, Globo H, and Lewis Y, and glycoprotein (mucin) antigens Tn, sialyl Tn. TF, and MUC1. More recently, in a comparative study we investigated the T cell response induced by MUCI-KLH conjugates. Although a MUC1-specific T cell response was not consistently detected in any patient, the role of KLH in orienting the cytokine environment was crucial. We were able to confirm that KLH in these conjugate vaccines induces a Th1 T cell response as demonstrated by the high anti-KLH INF-gamma secretion and the IgGI and IgG3 subclasses of this high titer IgG antibodies induced. Clinical trials using KLH conjugated glycolipid and glycoprotein vaccines, are currently ongoing. These range from phase I/II single antigens trials with glycosilated MUC1, polysialic acid, synthetic Fucosyl GMI and GD2, to phase II trials with a polyvalent vaccine containing six or seven antigens. Randomized phase II trials with polyvalent vaccines are planned for initiation in 2001-2002 in patients with ovarian, breast, and prostate cancer.

Effects of Active Immunization against Somatostatin or Somatostatin Analogues on Male Rat Growth

Effects of active immunization against somatostatin (SRIF) or various forms of somatostatin analogues on growth were investigated in male rats. In experiment 1, SRIF14, SRIF28, octreotide, Tyr 3 -octreotide or MK 608 were conjugated to human serum globulin and used as immunogens. Thirty-five Sprague-Dawley male rats were divided into seven groups and immunized against phosphate-buffered saline (PBS) (control 1), or one of the conjugated immunogens at five weeks of age. Booster immunizations were performed at 7, 9 and 12 weeks of age. Only SRIF14 induced the production of anti-somatostatin antibodies. There were no differences in blood GH concentration and growth performance between the SRIF14 group (with anti-SRIF antibodies) and other groups (without the antibodies). Although growth performance for the whole period was not affected by immunization with SRIF14 conjugate, daily weight gain and feed efficiency for 2 weeks after the third booster immunization were significantly improved, suggesting that the production of antibody against somatostatin can reach the minimal level for improving growth performance only after the third booster immunization in male rats. Experiment II was designed as a 2 2 2 2 2 factorial arrangement in which SRIF14 or Tyr11-SRIF14 was conjugated onto thyroglobulin or keyhole limpet haemocyanin (KLH) by glutaraldehyde or m-maleimidobenzoyl-N-hydroxysuccinimide coupling methods, and the immunization schedule was the same as experiment I. Anti-SRIF antibody titre values of the rats that received the conjugated SRIF14 or Tyr 11 -SRIF14 were higher than those injected with PBS. The levels of anti-SRIF antibody were not affected by the different coupling methods or by the different carrier proteins. In the Ag3 group treated with SRIF14 + KLH conjugate, total weight gain for the whole period and feed efficiency for 2 weeks after the third booster immunization were significantly different from other groups. Although GH concentration in blood was not changed by the production of antibody, our results show the possibility that active immunization against SRIF can improve growth performance in the male rat and SRIF14 peptide may be a potential candidate for active immunization.