Blanka Rihova

Biocompatibility of N-(2-hydroxypropyl) methacrylamide copolymers containing adriamycin: Immunogenicity, and effect on haematopoietic stem cells in bone marrow in vivo and mouse splenocytes and human peripheral blood lymphocytes in vitro

N-(2-hydroxypropyl)methacrylamide polymeric prodrugs containing adriamycin bound to polymers via glycylphenylalanylleucylglycine side chains and, in one case, galactosamine bound via the same sequence, were tested for immunogenicity after intravenous, subcutaneous and oral application in two inbred strains of mice. The serum antibody level was determined by enzyme-linked immunoassay on the 3rd and 6th day after the last treatment. It was found that antibodies were only produced in very small amounts. In some experimental groups, the antibody titres measured following administration of copolymer conjugate were comparable with those present in non-treated controls. Attachment of adriamycin to N-(2-hydroxypropyl)methacrylamide copolymer considerably decreased its toxicity against haematopoietic precursors in bone marrow as measured by the in vivo colony-forming unit-spleen assay and its ability to inhibit [3H] thymidine incorporation by mouse splenocytes and human peripheral blood lymphocytes measured in vitro.

Biological evaluation of polymeric micelles with covalently bound doxorubicin.

The main limitation of contemporary anticancer chemotherapy remains to be the insufficient specificity of the drugs for tumor tissue, which decreases the maximum tolerated dose due to severe side effects. Micellar drug delivery systems based on amphiphilic block copolymers with a very narrow size distribution (10 to 100 nm in diameter) is a novel innovative approach. Here, we report biological and pharmacological properties of polymeric micellar conjugate containing doxorubicin (DOX) covalently bound via hydrolytically cleavable hydrazone bonds to the micelle core. The system had a very low systemic toxicity (almost 20 times lower than free DOX) and long circulation in the bloodstream (with half of the dose after 24 h). Significant accumulation of tested micelles within the tumor was confirmed by fluorescent whole body imaging. Our new micellar system showed promising therapeutic activity against established murine EL-4 T-cell lymphoma; it was found that it is able to completely cure about 75% of tumor-bearing mice (with doses of either 1 x 150 mg DOX kg(-1) or 2 x 75 mg DOX kg(-1), administered i.v.). Moreover, treatment with micelles in cured mice induced tumor-specific resistance. Up to 80% of these mice survived rechallenge with original but not with distinct tumor cells.

In vivo expansion of activated naive CD8+ T cells and NK cells driven by complexes of IL-2 and anti-IL-2 monoclonal antibody as novel approach of cancer immunotherapy.

IL-2 is potent imunostimulatory molecule that plays a key role in T and NK cell activation and expansion. IL-2 is approved by the FDA to treat metastatic renal cancer and melanoma, but its extremely short half-life and serious toxicities are significant limitations of its use. It was reported that in vivo biological activity of IL-2 can be increased by association of IL-2 with anti-IL-2 mAb (S4B6). IL-2/S4B6 mAb immunocomplexes were described to be highly stimulatory for NK and memory CD8+ T cells and intermediately also for regulatory T cells. IL-2/JES6-1 mAb immunocomplexes are stimulatory solely for regulatory T cells. In this study we show that although both mentioned IL-2 immunocomplexes are less potent than free IL-2 in vitro, they possess extremely high stimulatory activity to expand activated naive CD8+ T cells in vivo. IL-2 immunocomplexes expand activated naive CD8+ T cells several hundred-fold times after four doses and more than 1000-fold times after six doses (1.5 μg/dose of IL-2), whereas free IL-2 given at the same dosage shows negligible activity. IL-2/S4B6 mAb immunocomplexes also induce massive expansion of NK cells (40% of DX5+NK1.1+ cells in spleen). Importantly, activated naive CD8+ T cells expanded by IL-2 immunocomplexes form robust population of functional memory cells. We also demonstrate in two distinct tumor models that IL-2/S4B6 mAb immunocomplexes possess considerable antitumor activity. Finally, by using radioactively labeled IL-2, we provide for first time direct evidence that IL-2 immunocomplexes have much longer half-life in circulation than free IL-2, being ∼3 h vs <15 min, respectively.

Regulatory T cells and their prognostic value for patients with squamous cell carcinoma of the head and neck

Regulatory T cells (Treg) are important regulators of anti‐cancer immune responses, and an increase in Treg frequency was observed in the blood of cancer patients. Blood samples from 112 patients with head and neck squamous cell carcinoma antigen (HNSCC) were obtained at the time of tumour diagnosis, and lymphocyte subpopulations (CD3+; CD3−CD16+CD56+; CD4+; CD8+; CD19+; CD4+CD45RA+) with emphasis on Treg counts (CD3+CD4+CD25+), complete blood count and tumour markers (squamous cell carcinoma [SCC]; CEA; α‐1‐antitrypsin [AAT]; Cyfra 21–1; C‐reactive protein [CRP]) were analysed. The data were grouped according to TNM classification, and their significance for the course of the disease at an interval of 1 year after the end of the therapy was determined. The percentage of CD8+ cells increased and the CD/D8 ratio decreased with tumour grade. The ratio of B lymphocytes decreased in patients with locoregional metastases (11.25%versus 9.22%). Treg (15.2%) and CD4+ cells (45.3%) increased, while NK cells (11.8%) decreased in HNSCC patients compared to controls (9.0%, 38.1% and 15.8%, respectively). The data obtained at time of diagnosis were used to assess the significance of tumour markers (SCC, Cyfra 21–1 and AAT) for evaluation of prognosis. The erythrocyte counts (4.64 × 1012/l versus 4.45 × 1012/l) and haemoglobin levels (14.58 g/dl versus 14.05 g/dl) decreased, while Treg counts (8.91%versus 15.70%) increased in patients with early recurrence. Our results show that examination of these parameters could be helpful for prognostication in HNSCC patients and aid improvement of treatment strategy.

Immunogenicity of protein-N-(2-hydroxypropyl)methacrylamide copolymer conjugates in A/J and B10 mice

Two proteins (model targeting residues) human immunoglobulin fraction (IgG) and human transferrin have been conjugated to N-(2-hydroxy propyl)methacrylamide (HPMA) copolymer and the antibody titer elicited, after subcutaneous or intraperitoneal administration to A/J and B10 mice of free and conjugated protein, was measured using the ELISA technique. The measured IgG titer against protein-HPMA copolymer conjugates was always higher than the IgM titer. Also, the titer (IgG) measured against native protein was up to 250-fold greater than that raised against protein-HPMA copolymer conjugates. This reduction in antibody titer against conjugate had a limited de pendence on its molecular weight.

Synthesis and evaluation of macromolecular prodrugs of mitomycin C

Abstract Poly- [ N -(2-hydroxyethyl)- l -glutamine] (PHEG) prodrugs of the antitumour antibiotic mitomycin C (MMC) were synthesised using peptidyl spacers, tri- and tetrapeptides, to link the drug to the macromolecular carrier. The relationship between the length and composition of the oligopeptide spacer and the rate of drug release was studied by incubation in buffers, serum and in the presence of enzymes (lysosomal enzymes and collagenase IV). It was observed that tetrapeptide-based conjugates generally release MMC more effectively than tripeptide derivatives. Conjugates having a terminal glycine in the spacer are less stable to hydrolysis than those with a terminal hydrophobic amino acid both in buffer and in serum. The gly-phe-ala-leu conjugate released MMC very rapidly in the presence of both lysosomal enzymes and collagenase IV. Biological experiments indicate that PHEG-MMC conjugates act as prodrugs of MMC: cytotoxicity was observed after hydrolytic release of the active compound in vitro. In vivo studies of P388 solid tumour-bearing mice suggest that conjugates which release MMC slowly may be more effective in inhibiting tumour growth and prolonging animal lifespan. Preliminary in vivo bone marrow toxicity studies indicate that PHEG-MMC prodrugs are less myelosuppressive than free MMC.

Enzymatic degradation and immunogenic properties of derivatized dextrans

Dextran T40 was modified with carboxylic, benzylamide and benzylamine-sulphonated groups (samples 1-10). The polymers were incubated with dextranase and the decrease of molecular weight was determined by high performance size exclusion chromatography. It was shown that the higher the substitution of dextrans, the lower their degradability. Modification of dextran with benzylamine and benzylamine-sulphonated groups appeared to hinder the formation of the enzyme-substrate complex more than the same quantity of carboxylic groups. The immunogenicity of one of the modified dextrans, containing 54% of carboxylic groups and 19.5% of benzylamine-sulphonated groups, was determined after subcutaneous and intravenous administration into Balb/c mice. The antibody titre was very low even if administered in complete Freunds adjuvant and did not depend on the injected dose. On the average, the titres of antibodies were lower by five orders of magnitude compared to bovine gamma globulin.

The Activity of Complement in the Presence of N-(2-hydroxypropyl)methacrylamide Copolymers

This study indicates that N-(2-hydroxypropyl)methacrylamide homopolymers and copolymers containing oligopeptide sequences terminated in carboxylic acid groups, amine groups, aromatic units, or puromycin have no prominent effect on the porcine complement system in vitro. Inhibition of both pathways of the complement system occurred at concentrations highly exceeding the dose suitable for therapeutic purposes.

In vitro and in vivo effect of HPMA copolymer-bound doxorubicin targeted to transferrin receptor of B-cell lymphoma 38C13.

N -(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing the anticancer agent doxorubicin and targeted to the transferrin receptor either with anti-mouse CD71 monoclonal antibody (mAb) or with transferrin were synthesized to evaluate their binding and anti-proliferative activity in vitro and anti-tumor potential against 38C13 B-cell lymphoma in vivo. Both the doxorubicin and the targeting moieties were bound to HPMA copolymer chain by aminolysis via a Gly-Phe (d, l) -Leu-Gly spacer to ensure controlled intracellular release of the conjugated drug. We demonstrated that HPMA copolymer-bound doxorubicin targeted to the transferrin receptor with anti-mouse CD71 mAb strongly retards tumor growth, prolongs the survival and completely cures three out of nine experimental mice with established 38C13 tumors. The conjugate targeted with transferrin was less effective in vitro as well as in vivo. It completely cured only one out of seven experimental mice. Free or non-targeted HPMA copolymer-bound doxorubicin showed only a mild anti-tumor effect within the therapeutic schedule used. In vitro, HPMA copolymer-bound doxorubicin targeted with anti-mouse CD71 mAb shows approximately 4-fold higher cytotoxic effect than HPMA copolymer-bound doxorubicin targeted with transferin and 9-fold higher cytotoxic effect than non-targeted HPMA copolymer-bound doxorubicin.

Selectivity of Antibody-Targeted Anthracycline Antibiotics on T Lymphocytes

Targeted polymeric prodrugs based on N-(2-hydroxypropyl) methacrylamide were tested on human peripheral blood lymphocytes or mouse splenocytes triggered in vitro to proliferation by T cell specific (Con A) or B cell specific (S. aureus Cowan 1) mitogens. Only a selective inhibition of 3H- thymidine incorporation by T lymphocytes was observed after in vitro incuba tion with prodrugs prepared by covalent attachment of daunomycin or adriamycin and antibody (anti-CD3; anti Thy 1.2) to biodegradable oligopeptide side chains of soluble synthetic copolymer HPMA. The in vitro results were confirmed in vivo by inhibition of antibody response to thymus dependent (ARS-BGG) or thymus independent (TNP-E. coli) antigens in normal Balb/c mice and in athymic nu/nu Balb/c mice. After administration of biocompatible, antibody targeted prodrugs only the antibody response against ARS-BGG was considerably reduced.