Immunization of breast cancer patients using a synthetic sialyl-Tn glycoconjugate plus Detox adjuvant

We have synthesized various formulations that have potential for active specific immunotherapy (ASI) of human cancers. Sialyl-Tn (STn) is a potentially important target structure for ASI because its expression on mucins is a strong, independent predictor of poor prognosis, suggesting that it may have functional significance in the metastatic process. In this first pilot study of synthetic sialyl-Tn hapten conjugated to keyhole limpet hemocyanin (STn-KLH), with Detox. adjuvant, toxicity and humoral immunogenicity were assessed in 12 patients with metastatic breast cancer. Toxicity was minimal, restricted to local cutaneous reactions (apart from transient nausea and vomiting following single low-dose cyclophosphamide treatment). Using STn-conjugated human serum albumin in a solid-phase enzyme-linked immunosorbent assay, it was shown that all patients developed IgM and IgG specific for the synthetic STn hapten. Following immunization, most patients were shown to develop increased titres of complement-mediated cytotoxic antibodies, partially inhibited by synthetic STn hapten, but not by the related TF hapten. We also detected IgM and IgG antibodies reactive with natural STn determinants expressed on ovine submaxillary mucin, the STn specificity of this reactivity being confirmed by hapten inhibition. Evaluation of clinical efficacy in a small pilot study is difficult. Five patients are alive 12 or more months after entry, and another 4 patients are alive 6 or more months after entry into the study. All 3 patients with known widespread bulky disease progressed despite ASI, 2 having died from widespread cancer. Two patients had partial responses, each lasting 6 months. While several patients had disease stability for 3–10 months, 1 patient with pulmonary metastases remains stable 15 months after entry into the program.     

A novel and efficient method for synthetic carbohydrate conjugate vaccine preparation: synthesis of sialyl Tn-KLH conjugate using a 4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide (MMCCH) linker arm

STn (NeuAc26GalNAc-O-Ser/Thr) is a carbohydrate epitope overexpressed in various human carcinomas. Clinical trials are underway using synthetic STn or STn trimeric glycopeptides [STn, cluster; STn(c) conjugated with keyhole limpet hemocyanin (KLH) as active specific immunotherapy for these cancers. These vaccines have been prepared by conjugating a crotyl ethyl amide derivative of STn or STn(c) to KLH by direct reductive amination after ozonolysis. In the case of STn(c) the conjugation efficiency and the resulting epitope ratios were low. This may be due to steric hinderance of the short spacer arm. To overcome these difficulties, without resynthesis, the STn(c) glycopeptide was modified by attachment of an MMCCH (4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide) spacer arm to the aldehyde derivative, and then conjugated with thiolated KLH. This method gave a higher epitope ratio and yield than the direct method. The STn(c)-MMCCH-KLH conjugate induced high titer antibodies in mice against STn(c). This method may be generally applicable for large synthetic oligosaccharides.     

Specificities of anti-sialyl-Tn and anti-Tn monoclonal antibodies generated using novel clustered synthetic glycopeptide epitopes

The fine specificities of MAbs generated using novel synthetic clustered STn and Tn glycopeptides as immunogens were compared with the anti-TAG-72 antibodies B72.3 and CC49. Hapten inhibition experiments demonstrated the specificity of several of the MAbs for STn and Tn expressed on ovine submaxillary mucin and tumor derived MUC-1 mucin. Amongst the STn specific MAbs only the B195.3 MAb shows absolute dependence on the presence of sialic acid and specificity to the simple disaccharide NANAA α2-6-GalNAc. Identification of tumor associated carbohydrate epitopes in cluster and monomer configurations are possible using MAbs detecting the defined structure specificities described herein. This revised version was published online in November 2006 with corrections to the Cover Date.     

Immunogenicity of synthetic TF-KLH (keyhole limpet hemocyanin) and sTn-KLH conjugates in colorectal carcinoma patients

Mucins of colorectal carcinomas overexpress the cancer-associated disaccharides Thomsen-Friedenreich antigen (TF) and sialyl-Tn antigen (sTn), making these antigens suitable for active specific immunotherapy. Patients at high risk for recurrent colon cancer, but free from disease after surgical resection, were immunized with synthetic TF and sTn covalently attached by a two-carbon crotyl linker to keyhole limpet hemocyanin (KLH). Four groups of patients were treated with TF-KLH without adjuvant, TF-KLH plus the immunological adjuvant Detox, sTn-KLH plus Detox, or sTn-KLH plus the immunological adjuvant QS-21, and the serological response was monitored. Enzyme-linked immunosorbent assay (ELISA), do-blot immunostains, and inhibition assays were used to identify antibody responses against synthetic TF and sTn epitopes and against natural antigens, including asialoglycophorin expressing TF antigen, and ovine submaxillary mucin and the human colon cancer line LS-C expressing sTn antigen. Our results demonstrate that vaccines containing TF or sTn-KLH conjugates plus immunological adjuvants Detox and especially QS-21 induced high IgM and IgG antibody titers against the respective synthetic disaccharide epitopes. However, when tested against natural antigens expressing these disaccharide epitopes, IgM antibodies showed weak to moderate reactivity, while IgG antibodies were almost totally unreactive. On the basis of these results we are continuing to test modifications of synthetic TF and sTn epitopes to identify those that induce IgM and IgG antibodies that are more reactive with these antigens as they are expressed on tumor mucins.This work was supported by grants from the National Institutes of Health (CA33049, CA52491, CA08748) and the Chemotherapy Foundation     

Redefined substrate specificity of ST6GalNAc II: a second candidate sialyl-Tn synthase

The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-linked oligosaccharides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11). It has been suggested that only ST6GalNAc I can synthesize carbohydrate structures of sialyl-Tn-antigen; i.e., NeuAc alpha2-6GalNAc-O-Thr/Ser [Kurosawa et al., J. Biol. Chem. 269, 19048-19053 (1994)] based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity toward asialoagalacto-fetuin. In this study, we present evidence that both ST6GalNAc I and II exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GalNAc I but also II are candidates for sialyl-Tn synthases.     

An O-acetylated sialyl-Tn is involved in ovarian cancer-associated antigenicity

We reported previously that a monoclonal antibody, 6G9, raised against bovine submaxillary mucin (BSM) reacted with mucinous ovarian cancer and recognized tumor-associated sialylated carbohydrate antigens. To obtain structural information on the carbohydrate antigens recognized by 6G9, the reactivity of several mucins and carbohydrates with the antibody was determined by ELISA. Exoglycosidase digestion of BSM showed that 6G9 recognized Sia as a nonreducing monosaccharide but neither Gal nor GlcNAc. Reactivity of BSM with 6G9 decreased markedly on de-O-acetylation of BSM in mild alkali, and O-acetyl Sia obtained from BSM reacted with the antibody, indicating the presence of O-acetyl groups on Sia in the epitope. A sialyl-Tn structure located in the epitope was also demonstrated by the findings that de-O-acetylated BSM retained weak but significant reactivity with 6G9 and that ovine submaxillary mucin, major sugar chains of which are sialyl-Tn, reacted with 6G9 stronger than de-O-acetylated BSM. Furthermore, weak reactivity of NeuAcalpha2 --> 6GalNAc prepared from BSM demonstrated that 6G9 recognized the sialyl-Tn structure, but the modification of Sia with O-acetyl groups was essential for the recognition. The failure of 9-O-acetyl NeuAc, synthesized chemically, to react with the antibody implied that 6G9 recognized sialyl-Tn with O-acetylation on Sia distinct from C-9 O-acetylation.     

SialylTn-mAb17-1A carbohydrate-protein conjugate vaccine: effect of coupling density and presentation of SialylTn

Carbohydrate antigens resulting from aberrant glycosylation of tumor cells, such as SialylTn, represent attractive targets for cancer vaccination. However, T-cell-independent carbohydrate antigens are poorly immunogenic and fail to induce memory and IgG class switch. Clustered expression patterns of some carbohydrates on the cell surface add further complexity to the design of carbohydrate-based vaccines. We describe here a vaccine consisting of SialylTn carbohydrate epitopes coupled to a highly immunogenic carrier molecule, mAb17-1A, adsorbed on alhydrogel and coformulated with a strong adjuvant, QS-21. The SialylTn-mAb17-1A conjugate vaccine was administered in Rhesus monkeys, and the immune responses against mAb17-1A, SialylTn, ovine submaxillary mucin, and tumor cells were analyzed. The data demonstrate that the density of carbohydrate epitopes on the carrier is an essential parameter for induction of anti-carbohydrate specific memory IgG immune responses. Furthermore, the influence of different types of presentation of SialylTn (monomeric vs trimers vs clustered via a branched polyethylenimine linker) on antibody titers and specificity was studied. High-density coupling of SialylTn epitopes to mAb17-1A induced the strongest immune response against synthetic SialylTn and showed also the highest reactivity against natural targets, such as OSM and tumor cells.     

A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers

Previously using a series of monovalent vaccines, we demonstrated that the optimal method for inducing an antibody response against cancer cell-surface antigens is covalent conjugation of the antigens to keyhole limpet hemocyanin (KLH) and the use of a saponin adjuvant. We have prepared a heptavalent-KLH conjugate vaccine containing the seven epithelial cancer antigens GM2, Globo H, Lewis(y), TF(c), Tn(c), STn(c), and glycosylated MUC1. In preparation for testing this vaccine in the clinic, we tested the impact on antibody induction of administering the individual conjugates plus adjuvant compared with a mixture of the seven conjugates plus adjuvant, and of several variables thought to augment immunogenicity. These include approaches for decreasing suppressor cell activity or increasing helper T-lymphocyte activity (low dose cyclophosphamide or anti-CTLA-4 MAb), different saponin adjuvants at various doses (QS-21 and GPI-0100), and different methods of formulation (lyophilization and use of polysorbate 80). We find that: (1). Immunization with the heptavalent-KLH conjugate plus GPI-0100 vaccine induces antibodies against the seven antigens of comparable titer to those induced by the individual-KLH conjugate vaccines, high titers of antibodies against Tn (median ELISA titer IgM/IgG 320/10240), STn (640/5120), TF (320/10240), MUC1 (80/20480), and globo H (640/40); while lower titers of antibodies against Lewis(y)()(160/0) and only occasional antibodies against GM2 are induced. (2). These antibodies reacted with the purified synthetic antigens by ELISA, and with naturally expressed antigens on the cancer cell surface by FACS. (3). None of the approaches for further altering the suppressor cell/helper T-cell balance nor changes to the standard formulation by lyophilization or use of polysorbate 80 had any impact on antibody titers. (4). An optimal dose of saponin adjuvant, QS-21 (50 microg) or GPI-0100 (1000 microg), is required for optimal antibody titers. This heptavalent vaccine is sufficiently optimized for testing in the clinic.     

Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin‐I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies. Copyright © 2003 John Wiley & Sons, Ltd.     

Immunization of mice with fucosyl-GM1 conjugated with keyhole limpet hemocyanin results in antibodies against human small-cell lung cancer cells

Fucosyl-GM1 (Fuc-GM1) [Fucα1 → 2Galβ1 → 3GalNAcβ1 → 4(NeuAcα2-3)Galβ1 → 4Glcβ1 → O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein keyhole limpet hemocyanin (KLH) or covalently linked with KLH, plus immunological adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC. Received: 25 March 1999 / Accepted: 5 August 1999     

Altered GalNAc-alpha-2,6-sialylation compartments for mucin-associated sialyl-Tn antigen in colorectal adenoma and adenocarcinoma.

Sialyl-Tn (STn), a mucin-associated disaccharide antigen carried by apomucins such as MUC2, plays an important role in tumor biology. However, little is known about the subcellular localization and compartments involved in STn synthesis. In this study we used immunoelectron microscopy to localize STn and MUC2 apomucin in human colorectal tissues. MUC2 apomucin was localized predominantly in the rough endoplasmic reticulum (RER) in normal colorectal mucosa (n=6), colorectal adenoma (n=8), and colorectal adenocarcinoma (n=10). STn, recognized by monoclonal antibody TKH2, was not readily detectable in normal colorectal mucosa but becomes manifest in both trans-Golgi apparatus and mucin droplets in colorectal adenoma. In colorectal adenocarcinoma, STn was localized not only in late but also in early Golgi compartments, and particularly in some RER lumens. Furthermore, electron microscopic in situ hybridization revealed that gold particles representing MUC2 mRNA are primarily localized over the RER. Our findings indicate that in colorectal adenoma STn sialylation takes place in the trans-Golgi apparatus, whereas in colorectal cancer STn sialylation occurs in all the Golgi compartments and in the RER.     

Design,Synthesis, and Docking Study of Quinine-9-Triazolyl Conjugates

To develop a better chemotherapeutically potential candidate for lung cancer treatment and cure with repurposed motifs, quinine has been linked with biocompatible CuAAC-inspired regioselective 1,2,3-triazole linker and a series of ten novel 1,2,3-triazolyl-9-quinine conjugates have been developed by utilizing click conjugation of glycosyl ether alkynes with 9-epi-9-azido-9-deoxy-quinine under standard click conditions. In parallel, the docking study indicated that the resulting conjugates have an overall appreciable interaction with ALK-5 macromolecules. Moreover, the mannose-triazolyl conjugate exhibited the highest binding interactions of −7.6 kcal/mol with H-bond interaction with the targeted macromolecular system and indicate the hope for future trials for anti-lung cancer candidates.     

The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids. Chromosomal localization of the BSM1 gene and relations to ovine and porcine counterparts.

We previously elucidated five distinct protein domains (I-V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of approximately 55 tandem repeats of 329 amino acids and that domains III-V are encoded by a 58.4-kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2. 2-q2.3. The amino-acid sequence of six tandem repeats (two full and four partial) were found to have only 92-94% identities. We propose that the variability in the amino-acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68-94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81-amino-acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N-terminal and C-terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes.     

A lectin from the Asian horseshoe crabTachypleus tridentatus: purification,specificity and interaction with tumour cells

A lectin from the haemolymph of the Asian horseshoe crabTachypleus tridentatus was purified to homogeneity by affinity chromatography on Sepharose 4B-boundN-acetylneuraminic acid. The specificity of this lectin was studied by haemagglutination inhibition with sialic acid analogues,N-acetylhexosamines and glycoproteins. For the interaction with the agglutinin theN-acetyl group and the glyceryl side chain ofN-acetylneuraminic acid are important, while presence of an aglycon, specially an -glycosidically linked lactose increases affinity to the lectin. The strongest glycoprotein inhibitors were ovine as well as bovine submaxillary mucin andCollocalia mucin, all beingO-chain glycoproteins but carrying completely different carbohydrate chains. The majority ofN-chain proteins were inactive. As the lectin agglutinates human erythrocytes, but not the murine lymphoma lines Eb and ESb or the human colon carcinoma HT 29, these cancer cells apparently lack the Tachypleus tridentatus agglutinin-receptor which is present on red cells andO-chain glycoproteins.Abbreviations TTA Tachypleus tridentatus agglutinin - SDS sodium dodecyl sulfate - BSM bovine sub-maxillary mucin - VCS Vibrio cholerae sialidase - OSM ovine submaxillary mucin - WGA Wheat germ agglutinin - NeuAc N-acetylneuraminic acid.     

Different modes of sialyl-Tn expression during malignant transformation of human colonic mucosa

Monoclonal antibodies TKH2 and B72.3, which react with the mucin-associated sialyl-Tn(STn) antigen, preferentially bind to cancerous but not normal colonic tissues. If O-acetyl groups are removed by saponification of tissues, MAb TKH2 will react with normal colonocytes, whereas MAb B72.3 remains non-reactive. To explain this difference in binding specificity, we tested both MAbs against synthetic constructs of single (monomeric) or clustered (trimeric) STn epitopes by enzyme immunoassay. Both MAb TKH2 and MAb B72.3 reacted with trimeric STn, but MAb TKH2 demonstrated greater binding than MAb B72.3 to monomeric STn. This suggests that normal colonic mucosa expresses monomeric STn epitopes, but that with transformation to malignancy, clustered STn epitopes appear. The appearance of clustered STn epitopes during colonic carcinogenesis represents a novel pattern of carbohydrate antigen expression and implicates alterations at the level of apomucins and/or glycosyltransferases responsible for cluster epitope formation.     

A structurally diversified linker enhances the immune response to a small carbohydrate hapten

The tether employed to covalently attach β-mannan disaccharide glycoconjugates influences the specificity of rabbit antibodies that protect against Candida albicans. Two glycoconjugates containing (1?→?2)-β-mannan disaccharides linked to chicken serum albumin (CSA) either via a structurally uniform or via a stereodiversified spacer were prepared and evaluated in immunization trials in mice and rabbits. Immunization with conjugate vaccine possessing a structurally diversified linker induced higher IgG titers against Candida albicans cell wall phosphomannan than a conjugate with a structurally uniform linker. These results suggest that affinity maturation and the specific antibody response can be shifted towards recognition of the desired hapten by employing a linker with diversified configuration.     

In vivo andin vitro antitumor activity of mitomycin C conjugates at 7-N position through a linker containing thiocarbamate bond with CD10 monoclonal antibody

Through a linker containing thiocarbomate bound to the 7-N position of mitomycin C (MMC), conjugates with a monoclonal antibody to CD10 (NL-1) were prepared, and their antitumor activities were examined. All five conjugates, except one, showedin vitro cytotoxity to two CD10⁺ lymphoid cell lines superior to MMC. The conjugate displaying the highest cytotoxicity was selected and further tested against three CD10⁺ and two CD10 lymphoid cell linesin vitro. The conjugate with NL-1 antibody demonstrated higher cytotoxic activity against CD10+ tumor cells than the control conjugate with normal immunoglobulin, while there was no significant difference, when tested against CD10^– tumors. The cytotoxic activity of the NL-1 conjugate to CD10+ tumors was significantly blocked by NL-1 antibody. In vivo antitumor activity of the NL-1 conjugate was then tested against a CD10⁺ tumor transplanted to nude mice, and side effects were recorded. The NL-1 conjugate (4 mg/kg) showed anin vivo antitumor effect similar to MMC (2 mg/kg), which is at nearly maximal tolerable dose; the latter induced decreases in numbers of leukocytes and platelets, while the former did not, suggesting less side effect by the NL-1 conjugate. Since MMC demonstrates a broad spectrum of antitumor activity, the conjugate, as such, may be applicable for the treatment of cancer patients.     

Specificity of submaxillary gland sialyltransferases

A method has been developed to determine the activities of specific sialyltransferases by analysis of the products of the reaction. This method, which utilizes high performance liquid chromatography, distinguishes addition of sialic acid to the N-acetylgalactosamine vs. galactose residues of the mucin disaccharide Galβ(1→3)GalNac, and can be used to distinguish formation of the 3′- and 6′-isomers of sialyllactose. For the bovine, ovine, and porcine submaxillary extracts, more than 95% of the activity with asialo ovine submaxillary mucin is due to formation of NeuAc α(2→6)GalNAc. With lactose as the acceptor, more than 95% of the α(2→3) isomer is produced. Activity with asialofetuin is due solely to the O-linked chain, with relative activity toward the galactose vs. GalNAc residues of 0.32, 1.5, and 0.10 for bovine, ovine, and porcine, respectively. The rat submaxillary gland extract showed equal formation of 3′- and 6′-sialyllactose, and very low activity with asialo ovine submaxillary mucin. However, at least 40% of the activity toward the Galβ(1→3)GalNAc disaccharide of asialofetuin was directed toward the GalNAc residue. The relative preference of the N-acetylgalactosaminide α(2→6) sialyltransferase for a monosaccharide vs. a substituted GalNAc may play a role in regulation of chain length during mucin synthesis.     

Pre-immunotherapy serum CA27.29 (MUC-1) mucin level and CD69+ lymphocytes correlate with effects of Theratope® sialyl-Tn-KLH cancer vaccine in active specific immunotherapy

Patients with metastatic breast, colorectal or ovarian cancers received active specific immunotherapy (ASI) with Theratope® sialyl-Tn-KLH (keyhole limpet hemocyanin) cancer vaccine emulsified in Detox? adjuvant. The median log₂ anti-STn IgG titer generated by ASI, estimated by enzyme-linked immunosorbent assay with solid-phase ovine submaxillary mucin, was 5.322 (range = 0?–?9.322). Following ASI, 51 patients who generated titers higher than the median value for anti-STn⁺ mucin IgG survived longer than 46 patients who generated lower titers below the median. 38 of the patients were phenotyped for CD69 prior to ASI. The patients with lower numbers of CD69⁺ peripheral blood lymphocytes prior to immunotherapy (pre-ASI) also had low serum CA27.29 cancer antigen (MUC-1) levels, and had longer times to disease progression and improved survival following ASI. Elevated pre-ASI serum CA27.29 tumor antigen levels were associated with higher numbers of CD69⁺ PBL, with decreased anti-STn antibody production and decreased survival following ASI. The data are compatible with the hypothesis that elevated serum MUC-1 mucin is specifically immunosuppressive.     

Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates

Two Zn-Pc-peptide conjugates bearing either a short linker or a long PEG-linker between the macrocycle and a bifunctional peptide containing the nucleoplasmin and HIV-1 Tat 48-60 sequences have been synthesized in order to increase the Pc cell-targeting ability and to evaluate the effect of the linker. The presence of the peptide chain increased the water solubility of the Pc macrocycle and, consequently, its fluorescence in aqueous solutions. The highest fluorescence quantum yields were observed at low pH (5.0) for both conjugates and were always higher for the conjugate bearing the short linker. Both conjugates were found to have low dark cytotoxicity toward human HEp2 cells (IC50 > 77 microM) but were highly phototoxic (IC50 < 2 microM at 1 J cm-2). The conjugate bearing the long PEG-linker accumulated the most within cells (26 times more than the unconjugated Zn-Pc), followed by the short linker conjugate (17 times more than the unconjugated Zn-Pc). Both conjugates were found to localized preferentially within the cell lysosomes.