Metabolic activation of 1,2-dibromo-3-chloropropane: evidence for the formation of reactive episulfonium ion intermediates

The nematocide and soil fumigant 1,2-dibromo-3-chloropropane (DBCP) is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidney. It has been proposed that both cytochrome P-450 mediated activation and glutathione (GSH) conjugation pathways are operative in DNA damage and organotropy induced by DBCP. To determine the chemical mechanisms involved in the bioactivation of DBCP and to assess a role for an episulfonium ion intermediate, the mechanism of formation of GSH conjugate metabolites of DBCP was investigated. Five biliary GSH conjugates of DBCP were isolated from rats and identified by fast atom bombardment tandem mass spectrometry: S-(2,3-dihydroxy-propyl)glutathione (I), S-(2-hydroxypropyl)glutathione (IIA), S-(3-chloro-2-hydroxypropyl)glutathione (III), 1,3-di(S-glutathionyl)propan-2-ol (IV), and 1-(glycyl-S-cysteinyl)-3- (S-glutathionyl)propan-2-ol (V). The mechanisms of conjugate formation were addressed by assessing deuterium retention in conjugates derived from [1,1,2,3,3-2H5] DBCP (D5-DBCP). GSH conjugates I, III, IV, and V displayed quantitative retention of deuterium, an observation consistent with the formation of an episulfonium ion intermediate. GSH conjugate IIA, however, retained three atoms of deuterium, thus invoking a P-450 mechanism in its genesis. The involvement of glutathione transferase (GST) and sequential episulfonium ion intermediates in the formation of metabolites I, III, and IV was demonstrated in vitro. Upon incubation of DBCP with GST, metabolites I, III, and IV were identified by tandem mass spectrometry and were found to arise with quantitative retention of deuterium when D5-DBCP was employed as a substrate. An additional GSH conjugate, 1,2,3-tri(S-glutathionyl)propane (VI), was observed as the major metabolite in incubations of GST with DBCP. When the incubations of DBCP with GST were performed in H2(18)O, metabolite I incorporated two atoms of 18O, and metabolites III and IV incorporated one atom of 18O. The ability of GST to catalyze the formation of the four GSH conjugates observed in vivo, with quantitative retention of deuterium and incorporation of 18O from H2(18)O, may be rationalized by a mechanism invoking the initial formation of S-(2-bromo-3-chloropropyl)glutathione. Rearrangement of this unstable conjugate via several reactive episulfonium ions, with either hydrolysis by water or alkylation of GSH at various stages, would account for the pattern of metabolites and their status of isotopic enrichment observed under various incubation conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and evaluation of novel Tc-99m labeled probestin conjugates for imaging APN/CD13 expression in vivo

The enzyme aminopeptidase N (APN, also known as CD13) is known to play an important role in tumor proliferation, attachment, angiogenesis, and tumor invasion. In this study, we hypothesized that a radiolabeled high affinity APN inhibitor could be potentially useful for imaging APN expression in vivo. Here, we report synthesis, radiolabeling, and biological evaluation of new probestin conjugates containing a tripeptide, N,N-dimethylglycyl-l-lysinyl-l-cysteinylamide (N(3)S), chelator. New probestin conjugates were synthesized by solid-phase peptide synthesis method, purified by reversed-phase HPLC, and characterized by electrospray mass spectrometry. The conjugates were complexed with Re(V) and (99m)Tc(V) by transmetalation using corresponding Re(V) or (99m)Tc(V) gluconate synthon. The mass spectral analyses of ReO-N(3)S-Probestin conjugates were consistent with the formation of neutral Re(V)O-N(3)S complexes. Initial biological activity of ReO-N(3)S-Probestin conjugates determined by performing an in vitro APN enzyme assay using intact HT-1080 cells demonstrated higher inhibition of APN enzyme activity than bestatin. In vivo biodistribution and whole body planar imaging studies of (99m)TcO-N(3)S-PEG(2)-Probestin performed in nude mice xenografted with human fibrosarcoma tumors derived from HT-1080 cells demonstrated a tumor uptake value of 2.88 ± 0.64%ID/g with tumor-to-blood and tumor-to-muscle ratios of 4.8 and 5.3, respectively, at 1 h postinjection (p.i.). Tumors were clearly visible in whole body planar image obtained at 1 h p.i., but not when the APN was competitively blocked with a coinjection of excess nonradioactive ReO-N(3)S-PEG(2)-Probestin conjugate. These results demonstrate the feasibility of using high affinity APN inhibitor conjugates as targeting vectors for in vivo targeting of APN.     

Evaluation of synthetic schemes to prepare immunogenic conjugates of Vibrio cholerae O139 capsular polysaccharide with chicken serum albumin

Vibrio cholerae serotype O139 is a new etiologic agent of epidemic cholera. There is no vaccine available against cholera caused by this serotype. V. cholerae O139 is an encapsulated bacterium, and its polysaccharide capsule is an essential virulent factor and likely protective antigen.This study evaluated several synthetic schemes for preparation of conjugates of V. cholerae O139 capsular polysaccharide (CPS) with chicken serum albumin as the carrier protein (CSA) using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents. Four conjugates described here as representative of many experiments were synthesized in 2 steps: 1) preparation of adipic acid hydrazide derivative of CPS (CPS_AH) or of CSA (CSA_AH), and 2) binding of CPS_AH to CSA or of CPS to CSA_AH. Although all conjugates induced CPS antibodies, the conjugate prepared by EDC-mediated binding of CPS and CSA_AH (EDC:CPS-CSA_AH) was statistically significantly less immunogenic than the other three conjugates. Representative sera from mice injected with these three conjugates contained antibodies that mediated the lysis of V. cholerae O139 inoculum.Evaluation of the different synthetic schemes and reaction conditions in relation to the immunogenicity of the resultant conjugates provided the basis for the preparation of a V. cholerae O139 conjugate vaccine with a medically useful carrier protein such as diphtheria toxin mutant.     

Enzymatic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments

CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains four extracellular sequences homologous to Ig VL domains. The first of these (V1) is sufficient for binding to HIV; however, the structural basis for this binding has yet to be elucidated. While several models for the structure of Ig-like domains in CD4 have been proposed on the basis of crystal structures of Ig VL domains, direct evidence that CD4 and VL domains fold similarly has not been obtained. To produce individual domains of CD4 for structural studies, we used molecular fusions of such domains with Ig heavy chain (CD4 immunoadhesins), which are very efficiently expressed and secreted in mammalian cells and can be easily isolated in single-step purification with protein A. Since these fusion molecules are antibody-like homodimeric proteins, we investigated the possibility that they might be cleaved enzymatically to produce Fd-like and Fc fragments. We found that cleavage with papain releases an Fd-like fragment containing the V1 and V2 CD4 domains; this fragment fully retains the ability to bind to the HIV-1 envelope glycoprotein gp120 and to block HIV infection in vitro. Moreover, folding of the CD4 domains in the Fd-like fragment and in the parent immunoadhesin is indistinguishable, as indicated by circular dichroism. Spectral analysis of the Fd-like fragment suggests that secondary structure content is identical with that predicted from the known structure of Ig VL domains; this directly supports the hypothesis that the V1 and V2 domains of CD4 fold similarly to Ig VL domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of glutathione conjugates of troglitazone in human hepatocytes

Troglitazone (TGZ) is an orally active antihyperglycemic agent used in the treatment of noninsulin-dependent diabetes mellitus. Several cases of liver failure following TGZ administration led to its withdrawal from the market. The mechanism of toxicity is still not understood. The formation of toxic metabolites is believed to play an important role. Herein, we report the biotransformation of TGZ in human hepatocytes. TGZ at 50 microM concentration was incubated with cryopreserved human hepatocytes. Four metabolites were found-glucuronide, sulfate, and two glutathione (GSH) conjugates of TGZ. The two GSH metabolites could be conjugation at the 6-hydroxychromane nucleus and the thiazolidinedione ring. Alternatively, the conjugation could be one of the two rings, with the two GSH metabolites are diastereomers. The sulfate conjugate was the major metabolite found. The cytochrome P450 (CYP) inhibitors furafylline (CYP1A1/2), omeprazole (CYP2C19), ketoconazole (CYP3A4), and sulfaphenazole (CYP2C9) had no inhibitory effect on the TGZ metabolism suggesting that several P450s may play a role in the TGZ metabolic pathway. Previous studies in our laboratory have shown a large interindividual variation between different donors in cytotoxicity after dosing with TGZ. Based on EC(50) values, donors were classified as sensitive or resistant. The sensitive human donors were found to form significantly less troglitazone GSH conjugates and glucuronides than the resistant donors.     

Phenotypic differences in expression of cytochrome P-450g but not its mRNA in outbred male Sprague-Dawley rats

Cytochrome P-450g was isolated from livers of adult male Sprague-Dawley (CD) rats. Antibody to P-450g cross-reacted with several proteins in Western blots of liver microsomes from male CD rats. An immunospecific antibody was prepared by adsorption over immunoaffinity columns of Sepharose-bound solubilized rat liver microsomes from female CD and male Fischer 344 rats containing little or no P-450g. The immunopurified antibody recognized a single protein on Western blots of liver microsomes from male CD rats with an electrophoretic mobility identical to that of P-450g. Using this antibody, P-450g was shown to be male specific in the CD rat and expressed at maturity. Adult male CD rats were shown to fall into two distinct populations, those expressing high levels of P-450g (+g) and those expressing low levels of P-450g (-g). The P-450g content of the two populations differed 10- to 20-fold. P-450g was low or absent in liver microsomes of both sexes of adult Fischer rats. Purified P-450g catalyzed the hydroxylation of testosterone and androstenedione principally at the 6 beta-position and progesterone at the 16 alpha- and 6 beta-positions in reconstituted systems. However, the hydroxylation of these steroids by liver microsomes from the (+g) phenotype did not differ from that of the (-g) phenotype. Translatable mRNA for P-450g could be detected in livers of adult male CD rats but not female rats. However, the level of P-450g mRNA in livers of adult male CD rats with the (+g) phenotype did not differ from that of (-g) phenotype. These data suggest that phenotypic differences in the expression of P-450g do not depend on differences in mRNA content. This study provides a clear example of a P-450 isozyme which is markedly variable in an outbred strain of rat and absent in an inbred strain. Such a marked variability in an enzyme involved in metabolism of endogenous and exogenous substrates could account for some of the strain differences in susceptibility to toxic chemicals.     

H2O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H₂O₂ at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H₂O₂ to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H₂O₂ (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.     

Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

Sequence specific association of tryptic peptides with multiwalled carbon nanotubes: effect of localization of hydrophobic residues

A model-free approach has been used to study the association of peptides onto multiwalled carbon nanotubes (MWCNT) in aqueous solution at ambient pH to understand the molecular basis of interaction of the peptides with MWCNT. The peptides obtained by tryptic digestion of cytochrome P450cam from P. putida were allowed to interact with MWCNT, and several peptides were found to bind to the nanotube leading to formation of stable homogeneous dispersion of the bionano conjugates of MWCNT. The peptides bound to the MWCNT were separated from the unbound peptides and sequence analyses by tandem MS/MS technique identified the strongly bound peptides as well as the unbound and the weakly bound peptides. The peptide-MWCNT conjugate was further characterized by TEM as well as Raman, FTIR, vis-NIR absorption, and circular dichroism spectroscopy. A model based on the hydrophobicity of residues in the peptides suggested that the amphiphilic peptides with localized hydrophobic residues at the center or at one end of the sequence form stable dispersions of the peptide-MWCNT conjugates.     

The accessory molecules CD5 and CD6 associate on the membrane of lymphoid T cells

CD5 and CD6 are closely related lymphocyte surface receptors of the scavenger receptor cysteine-rich superfamily, which show highly homologous extracellular regions but little conserved cytoplasmic tails. Both molecules are expressed on the same lymphocyte populations (thymocytes, mature T cells, and B1a cells) and share similar co-stimulatory properties on mature T cells. Although several works have been reported on the molecular associations and the signaling pathway mediated by CD5, very limited information is available for CD6 in this regard. Here we show the physical association of CD5 and CD6 at the cell membrane of lymphocytes, as well as their localization at the immunological synapse. CD5 and CD6 co-immunoprecipitate from Brij 96 but not Nonidet P-40 cell lysates, independently of both the co-expression of other lymphocyte surface receptors and the integrity of CD5 cytoplasmic region. Fluorescence resonance energy transfer analysis, co-capping, and co-modulation experiments demonstrate the physical in vivo association of CD5 and CD6. Analysis of T cell/antigen-presenting cells conjugates shows the accumulation of both molecules at the immunological synapse. These results indicate that CD5 and CD6 are structurally and physically related receptors, which may be functionally linked to provide either similar or complementary accessory signals during T cell activation and/or differentiation.     

Enhancement of immunotoxin efficacy by acid-cleavable cross-linking agents utilizing diphtheria toxin and toxin mutants

We have utilized a new class of acid-cleavable protein cross-linking reagents in the construction of antibody-diphtheria toxin conjugates (Srinivaschar, K., and Neville, D. M., Jr. (1989) Biochemistry 28, 2501-2509). The potency of anti-CD5 conjugates assayed by inhibition of protein synthesis on CD5 bearing cells (Jurkat) is correlated with cross-linker hydrolytic rates. The maximum increase in potency of the cleavable conjugates over non-cleavable conventional conjugates is 50-fold and is specific for the CD5 uptake route as judged by competition with excess anti-CD5. The potency of conjugates made from diphtheria toxin and the anti-high molecular weight melanoma-associated antigen (HMW-MAA) is enhanced 3-10-fold by a cleavable cross-linker. However the potency of transferrin or anti-CD3 diphtheria toxin conjugates is only minimally enhanced (2-3-fold). Mutant diphtheria toxins, CRM103 and CRM9, previously shown to express less than 1/100 of the wild type in binding affinity were substituted into these conjugates as probes for possible intracellular toxin receptor interactions. Both mutants were equally as toxic to Jurkat target cells exhibiting 1/700 the wild-type potency. CRM9 non-cleavable conjugates were equally as potent as wild-type conjugates for transferrin and anti-CD3-mediated uptake but not for anti-CD5-mediated uptake where toxicity was reduced 60-fold over the wild-type analog. The cleavable cross-linker enhanced the toxicity of anti-CD5-CRM103 and anti-CD5-CRM9 conjugates, but potency was only 1/10 that of the analogous wild-type cleavable conjugate. These data are consistent with a model in which potentiation of toxicity of the anti-CD5 and anti-high molecular weight melanoma-associated antigen conjugates by the cleavable cross-linker occurs from an enhanced intracellular toxin-toxin receptor interaction that ultimately results in increased toxin translocation to the cytosol compartment. In contrast, these data indicate that the anti-CD3 and transferrin uptake systems do not require this interaction in agreement with previous work (Johnson, V.G., Wilson, D., Greenfield, L., and Youle, R. J. (1988) J. Biol. Chem. 263, 1295-1300).     

Effect of alcohols on binding of camphor to cytochrome P450cam: spectroscopic and stopped flow transient kinetic studies

Addition of alcohols to cytochrome P450cam (CYP101) was shown to release the substrate camphor from the heme pocket of the enzyme. The release of the substrate was found to be caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme. The far-UV CD and near-UV CD spectra reveal that addition of alcohols to cytochrome P450cam cause a small change in the secondary structural elements but a significant change in the tertiary structural organization of this enzyme. The CD spectra at the heme region at various concentrations of alcohols indicate a substantial change in the tertiary structural organization around the heme moiety too. The equilibrium constant associated with the binding of camphor to Cyt P450cam is strongly dependent on the concentration of alcohols and the corresponding free energy associated with the binding is found to scale linearly with the concentration of alcohols. Kinetic experiments on binding of camphor to Cyt P450cam show that both k(on) and k(off) rate constants are strongly affected by addition of alcohols suggesting that alcohol expel camphor out of the heme cavity of Cyt P450cam by affecting tertiary structure of Cyt P450cam as well as by modifying the solubility properties of camphor in aqueous medium.     

Circular dichroism studies on the binding of type I substrates and reverse type I compounds to rabbit liver microsomal cytochrome P-450.

Liver microsomes from control, 3-methylcholanthrene-treated, and phenobarbital-treated New Zealand White rabbits were examined for differences detectable by circular dichroism (CD) spectroscopy. Addition of the Type I substrate cyclohexane to phenobarbital microsomes decreases the negative ellipticity at about 418 nm and concomitantly increases the negative ellipticity at about 395 nm. Cyclohexane added to microsomes from control or 3-methylcholanthrene-treated animals shows little or no CD changes in these wavelength regions. The effect by cyclohexane is completely reversed by the subsequent addition of butanol-1. Addition of benzo[a]pyrene to phenobarbital microsomes also decreases the negative ellipticity at about 418 nm, and this effect can be completely reversed with the subsequent addition of butanol-1. The ellipticity at about 395 nm is reversed in sign and is markedly increased by benzo[a]pyrene, however, and this effect is not changed with the subsequent addition of butanol-1. Restoring the cyclohexane- or benzo[a]pyrene-induced changes by the subsequent addition of alcohol is proportional to the aliphatic chain length, with 4 or more carbon atoms being maximally effective. Primary alcohols inhibit aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) activity of phenobarbital microsomes, and the inhibitory effect is enhanced with increasing chain length of the alcohols; 4 or more carbon atoms being maximally effective. Stimulation of monooxygenase metabolism of cyclohexane or benzo[a]pyrene by NADPH results in restoration of the negative ellipticity band at about 418 nm, whereas the ellipticity peak at about 395 nm remains unchanged. More negative ellipticity at about 210 and 222 nm is found in phenobarbital microsomes than in control or 3-methylcholanthrene microsomes and cyclohexane addition in vitro increases these negative ellipticity peaks in phenobarbital microsomes but not in control or 3-methylcholanthrene microsomes.These results show that with CD studies one can detect directly both high spin (negative ellipticity peak at 385–395 nm) and low spin (negative ellipticity peak at about 418 nm) P-450 iron in liver microsomes from control, 3-methylcholanthrene-treated, or phenobarbital-treated rabbits. These data are consistent with a weak ligand such as oxygen, rather than a stronger ligand such as nitrogen, in the sixth position of 6-coordinated (low spin) ferric iron in P-450 in vivo. Type I substrates such as cyclohexane or benzo[a]pyrene, when bound to P-450, change low spin P-450 iron to the high spin state. Cyclohexane-bound high spin P-450 iron in vitro is more easily converted to low spin iron by butanol-1 than is benzo[a]pyrene-bound high spin P-450 iron. Liver microsomal proteins from phenobarbital-treated rabbits have a higher helical content than those from either control or 3-methylcholanthrene-treated rabbits. Cyclohexane addition in vitro increases this helical character only in phenobarbital microsomes, indicating that one or more forms of phenobarbital-induced P-450 apoproteins is (are) more specific for cyclohexane binding and metabolism than control or 3-methylcholanthrene-induced forms of P-450.     

CD4 is involved in a post-binding event in the cytolytic reaction mediated by human CD4+ cytotoxic T lymphocyte clones

CTL lyse their target cells in discrete phases. First, the CTL bind to the target cell in a Mg2+-dependent manner followed by a Ca2+-dependent cytolytic phase. In the present study, we investigated the role of CD4 in the different phases of the cytolytic reaction mediated by human CD4+ class II MHC-specific CTL clones by using a single cell assay. It was found that the anti-CD4+ mAb OKT4A, which blocks cytotoxic reactions by CD4+ CTL clones as measured with a 51Cr release assay, only marginally affects the formation of conjugates. It appeared that OKT4A more strongly blocked the post-binding phase of the cytolytic reaction. In contrast, anti-leukocyte function-associated mAb strongly blocked the formation of conjugates but not the subsequent lytic phase. As was found previously with CD8+ CTL clones, anti-TCR mAb generally did not affect the formation of conjugates. One exception was noted. The activity of a CD4+ CTL clone, HY-640, could not be blocked by OKT4A, but was affected by an anti-TCR mAb. This anti-TCR mAb could partly reduce the formation of conjugates between HY-640 cells and their specific target cells. These results suggest that this clone has a high affinity TCR, which can contribute to the formation of conjugates. Although preincubation of the CTL clones with OKT4A only marginally affects the number of conjugates upon subsequent mixture with target cells, it was observed that incubation at 37 degrees C of preformed conjugates with OKT4A markedly reduced the number of conjugates. This dissociation of preformed conjugates was optimal only after 2 h of incubation. In contrast, an anti-leukocyte function-associated mAb induced a much more rapid dissociation of preformed conjugates.     

Individually specified drug immunoconjugates in cancer treatment

Forty-three patients with disseminated refractory malignancies each received an individually-specified combination of either Adriamycin (24 patients) or mitomycin-C (19 patients) conjugated murine monoclonal antibodies. Tumors were typed using a panel of antibodies with both immunohistochemistry and flow cytometry. Cocktails of up to six antibodies were selected based on binding greater than 80% of the malignant cells in the biopsy specimen. These monoclonal antibody cocktails were drug conjugated and administered intravenously. Seventeen out of twenty-four patients had reactions to the administration of Adriamycin immunoconjugates, but these were tolerable in all but two patients. Fever, chills, pruritus and skin rash were by far the most common transitory reactions. All were well controlled with premedication. In several patients it was demonstrated that there was limited antigenic drift among various biopsies within the same patient over time. Up to 1 gram of Adriamycin and up to 5 grams of monoclonal antibody were administered. The limiting factor appeared to be a variable dissociation of active Adriamycin from the antibody which unpredictably caused hemopoietic depression. Similar findings were noted in 19 patients with mitomycin-C conjugates. Thrombocytopenia at a 60mg dose of mitomycin-C in this schedule was dose limiting. Preliminary serological evidence suggests that the development of an IgM antibody which is specific against the mouse monoclonal antibody has the specificity and sensitivity to predict clinical reactions. These antibodies were quantitatively less in mitomycin-C patients. Selected patients were re-treated. One patient with chronic lymphocytic leukemia had re-treatment on three occasions and demonstrated regression of peripheral lymph nodes. Two patients with breast carcinoma had definite improvement in ulcerating skin lesions and two patients with tongue carcinoma had shrinkage of their lesions. No responses were seen with mitomycin-C conjugates but binding was noted to tumors and colon with likely drug induced colitis seen after colon binding. This study demonstrates the feasibility and illustrates technical considerations in preparing drug immunoconjugate cocktails for patients with refractory malignancies. Cocktail formulation and antibody delivery was accomplished. The major technical hurdle appears to be the selection of effective conjugation methods that can be used to optimally bind drugs to monoclonal antibodies for targeted cancer therapy.     

Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2)

Repifermin, a truncated form of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 °C). Electrostatic interactions between polyanions and several FGFs have been reported to enhance the thermal stability of these proteins. Polyethylene glycol (PEG) was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS) as an alternative means to stabilize and noncovalently PEGylate KGF-2. Physical characteristics of KGF-2:polyanion-PEG complexes were examined using a variety of methods including circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning calorimetry, and dynamic light scattering. When compared to KGF-2 alone, subtle changes in CD spectra and fluorescence emission maxima were found when KGF-2 was formulated with the synthetic PEG-polyanions. Highly PEGylated polyanions (DS-PEG5) did not bind KGF-2 as well as conjugates with fewer PEG chains. The molecular weight of PEG did not have a noticeable effect on KGF-2 binding to the various PEG-polyanion conjugates. At optimal molar ratios, PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature by approximately 9-17 °C. Thus, polyanion-PEG conjugates improved the stability of KGF-2 and also offered a new electrostatic PEGylation scheme that may be extrapolated to other heparin-binding proteins.     

Synthesis of glycocluster-tumor antigenic peptide conjugates for dendritic cell targeting

The use of dendritic cells (DC) for the development of therapeutic cancer vaccines is attractive because of their unique ability to present tumor epitopes via the MHC class I pathway to induce cytotoxic CD8+ T lymphocyte responses. C-Type membrane lectins, DC-SIGN and the mannose receptor (MR), present on the DC surface, recognize oligosaccharides containing mannose and/or fucose and mediate sugar-specific endocytosis of synthetic oligolysine-based glycoclusters. We therefore asked whether a glycotargeting approach could be used to induce uptake and presentation of tumor antigens by DC. To this end, we designed and synthesized glycocluster conjugates containing a CD8+ epitope of the Melan-A/Mart-1 melanoma antigen. These glycocluster-Melan-A conjugates were obtained by coupling glycosynthons: oligosaccharyl-pyroglutamyl-beta-alanine derivatives containing either disaccharides, a dimannoside (Manalpha-6Man) or lactoside, or a Lewis oligosaccharide, to Melan-A 16-40 peptide comprising the 26-35 HLA-A2 restricted T cell epitope, extended with an oligolysine stretch at the C-terminal end. We showed by confocal microscopy and flow cytometry that fluorescent-labeled Melan-A glycoclusters containing either dimannoside or Lewis oligosaccharide were taken up by DC and concentrated in acidic vesicles; conversely lactoside glycopeptides were not at all taken up. Furthermore, using surface plasmon resonance, we showed that dimannoside and Lewis-Melan-A conjugates bind MR and DC-SIGN with high affinity. DC loaded with these conjugates, but not with the lactose-Melan-A conjugate, led to an efficient presentation of the Melan-A epitope eliciting a CD8+ T-lymphocyte response. These data suggest that synthetically designed glycocluster-tumor antigen conjugates may induce antigen cross-presentation by DC and represent a promising tool for the development of tumor vaccines.     

Custom-tailored drug immunoconjugates in cancer therapy.

Forty-three patients with disseminated refractory malignancies each received an individually specified combination of either Adriamycin (n = 24) or mitomycin-C (n = 19) conjugated to a cocktail of murine monoclonal antibodies (mAb). Cancers were typed with both immunohistochemistry and flow cytometry using a panel of antibodies. Cocktails of up to six antibodies were selected based on total binding of greater than 80% of the malignant cells in the biopsy specimen. These mAb cocktails were then drug conjugated, safety tested, and administered intravenously. The Adriamycin immunoconjugates were well tolerated in 22/24 patients, with 17/24 having significant side effects. Fever, chills, pruritis, and skin rash were by far the most common transitory reactions. All were well controlled with premedication. A total of up to 1 g Adriamycin and 5 g mAb were administered to each patient. The limiting factor appeared to be a variable dissociation of active Adriamycin from the antibody that unpredictably caused hemopoietic depression. Similar findings were noted among 19 patients treated with mitomycin-C conjugates. Thrombocytopenia at a 60-mg dose of mitomycin-C in this schedule was dose limiting. Serological evidence suggested that the development of an immunoglobulin M antibody specific against the mouse mAb had the specificity and sensitivity to predict clinical reactions. These antibodies were quantitatively less in mitomycin-C-treated patients. Selected patients were retreated. One patient with chronic lymphocytic leukemia was treated on three occasions with regression of peripheral lymph nodes. Two patients with breast carcinoma had definite improvement in ulcerating skin lesions, and two patients with tongue carcinoma had shrinkage of their lesions. No responses were seen with mitomycin-C conjugates but binding was noted to tumors. Drug-induced colitis was seen at higher doses with some binding of these conjugates to normal colon epithelium. This study demonstrated the feasibility of preparing individually specified drug immunoconjugate cocktails for patients with refractory malignancies. Cocktail formulation and antibody delivery to the tumor in vivo was accomplished. There was limited antigenic drift among various biopsies within the same patient over time. The major technical hurdle continues to be the selection of effective drug conjugation methods to optimally bind drugs to mAbs for targeted cancer therapy.     

Potential for tumor therapy with iodine-125 labeled immunoglobulins

Because of the high intranuclear toxicity of Auger-electron emitters, the use of radioiodinated (¹²³I, ¹²⁵I) 5-iodo-2′-deoxyuridine (IUdR)-antibody conjugates for cancer therapy has been examined. The results have demonstrated that all the conditions necessary for labeling DNA in vivo are present: uptake of the radiolabeled immunoglobulin by target cells, its subsequent internalization, the degradation of the IUdR-protein conjugate by lysosomal enzymes, and the incorporation of the radionucleoside into DNA.