Tomas Mrkvan

New HPMA copolymer-based drug carriers with covalently bound hydrophobic substituents for solid tumour targeting

Various conjugates of anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond to the drug carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesised. Structure of the conjugates differed in the type and the content of hydrophobic substituent (dodecyl, oleic acid and cholesterol moieties) introduced into the polymer structure. In aqueous solutions the conjugates self-assembled into high-molecular-weight supramolecular structures, such as polymeric micelles or stable hydrophilic nanoparticles 13-37 nm in diameter, depending on the type and the content of hydrophobic substituents. Treatment of mice bearing EL-4 T cell lymphoma with the conjugates in the therapeutic regime of drug administration (i.v.) resulted in significant tumour regression with up to 100% of long-term survivors, depending on the dose and the detailed structure of the carrier. The nanoparticles formed by the conjugate bearing cholesterol moiety exhibited prolonged blood circulation and enhanced tumour accumulation indicating an important role of the EPR effect in excellent anticancer activity of the conjugate.

Conjugates of doxorubicin with graft HPMA copolymers for passive tumor targeting

Synthesis, physicochemical behavior, tumor accumulation and preliminary anticancer activity of a new biodegradable graft copolymer-doxorubicin (DOX) conjugates designed for passive tumor targeting were investigated. In the graft high-molecular-weight conjugates the multivalent N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer was grafted with a similar but semitelechelic HPMA copolymer; both types of polymer chains were bearing doxorubicin attached by hydrazone bonds enabling intracellular pH-controlled drug release. The polymer grafts were attached to the main chain through spacers, degradable enzymatically or reductively, facilitating, after the drug release, intracellular degradation of the graft polymer carrier to short fragments excretable from the organism by glomerular filtration. The graft polymer-DOX conjugate exhibited prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice indicating the important role of the EPR effect in the anticancer activity. The graft polymer-DOX conjugates showed a significantly higher antitumor activity in vivo than DOX.HCl or the linear polymer conjugate when tested in mice bearing 38C13 B-cell or EL4 T-cell lymphoma, with a significant number of long-term-surviving (LTS) mice with EL4 T-cell lymphoma treated with a single dose 15 mg DOX equiv./kg on day 10.

Cytostatic and immunomobilizing activities of polymer-bound drugs: experimental and first clinical data

An N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing doxorubicin and human immunoglobulin as an actively/passively targeting moiety was used in four patients with generalized breast cancer resistant to standard cytotoxic chemotherapy. The dose and time schedule were deduced from a Phase I clinical trial in which doxorubicin bound to HPMA copolymer carrier (PK1) was tested. It was confirmed that the Dox-HPMA-HuIg conjugate is stable and doxorubicin remains in the peripheral blood with a small amount also in the urine, mostly in its polymer-bound form. More than 116 biochemical, immunological and hematological parameters were determined for blood samples taken from patients 24 h, 48 h, 72 h and 1 to 11 weeks after treatment. Depending on the patient, some parameters decreased permanently or temporarily to the normal level (CRP, C3, CA 72-4, beta(2)-microglobulin, ferritin, CEA, CA 125, CD4, CD8, CE19, CD16(+)56(+), leu, ery) and some moved markedly towards physiological values (AST, ALT, ALP, GMT, CA 15-3, NSE, AFP). While the number of peripheral blood reticulocytes was significantly decreased after treatment with the classical free drug, their number was not affected or was even elevated after treatment with Dox-HPMA-HuIg. Increased absolute numbers of CD16(+)56(+) and CD4(+) cells in the peripheral blood and activation of NK and LAK cells in all patients support data obtained in experimental animals, pointing to a dual, i.e. cytostatic and immunomobilizing character of Dox-HPMA conjugates containing a targeting immunoglobulin moiety.

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.

HPMA copolymer-bound doxorubicin targeted to tumor-specific antigen of BCL1 mouse B cell leukemia.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin and targeted with B1 monoclonal antibody (mAb) to BCL1 leukemia cells was synthesised and tested in vitro and in vivo. BCL1 leukemia growing in syngenic Balb/c mice was selected as a tumor model system. B1 mAb recognising the idiotype of surface IgM on BCL1 cells was used as a targeting moiety. Both B1 mAb and doxorubicin were conjugated to HPMA copolymer carrier by aminolysis through a tetrapeptidic Gly-Phe(D,L)-Leu-Gly spacer to ensure the intracellular delivery and controlled release of the drug. B1 mAb-targeted conjugate was shown to possess strictly tumor-specific binding capacity to target BCL1 cells in vitro. A similar conjugate, but containing human nonspecific Ig (HuIg) instead of B1 mAb, failed to bind to BCL1 cells. In vitro, B1 mAb-targeted conjugate demonstrated 40-fold higher cytotoxic effect than nontargeted or human nonspecific Ig-containing HPMA copolymer-bound doxorubicin. Conjugate targeted with B1 mAb was also shown to bind to target BCL1 cells in vivo. B1 mAb-targeted conjugate was shown to be more efficient in the treatment of established BCL1 leukemia than free doxorubicin, nontargeted and human nonspecific Ig-containing conjugate. Antibody-targeted polymeric drugs are thus promising conjugates for cancer treatment.

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.

Preclinical Evaluation of Linear HPMA-Doxorubicin Conjugates with pH-Sensitive Drug Release: Efficacy, Safety, and Immunomodulating Activity in Murine Model

ABSTRACTPurposeIn vivo efficacy and safety of HPMA-based copolymers armed with doxorubicin via a spacer containing pH-sensitive linkage that can be prepared within a broad range of attached drug contents (1) was tested in murine tumor models.MethodsMice bearing T cell lymphoma EL4 or B cell lymphoma 38C13 were treated with a single dose of the conjugate (15, 25, and 75 mg Dox eq./kg i.v.) in a therapeutic regime. Anti-tumor resistance of the cured animals was proved by a second challenge with a lethal dose of tumor cells without additional treatment.ResultsThe content of drug bound to the polymer is an important parameter in relation to the conjugate therapeutic efficacy. The best anti-tumor effects were produced by conjugates with 10 – 13 wt% of bound doxorubicin. Free doxorubicin up to 4.6% relative to total drug content had no impact on the treatment efficacy and acute toxicity. The conjugates induced a complete cure of mice and regular treatment-dependent development of specific anti-tumor resistance. No myelosuppression or organ damage was observed.ConclusionsA well-defined HPMA copolymer-doxorubicin conjugate with pH-sensitive drug release is a good candidate for clinical trials as it has remarkable anti-tumor efficacy and a favorable safety profile.

Drug-HPMA-HuIg Conjugates Effective Against Human Solid Cancer

N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA)-bound doxorubicin conjugated with human Ig as a targeting moiety was used for the first time in the setting of metastatic breast cancer (patients E. G., J. K., K. R. and K.H.) and angiosarcoma (patient D. H) resistant to conventional cytotoxic chemotherapy. It was confirmed that Dox-PHPMA-Hulg conjugate is stable and doxorubicin remains in the peripheral blood mostly in its polymer-bound form. In patients E. G., J. K., K.R. and K.H. more than 116 biochemical and immunological parameters were tested in blood samples taken from the patients 24 h, 48 h, 72 h, 7 d, 14 d and 21, 28 and eight weeks after the treatment. In the patient E. G., 15 parameters had pathological values before the treatment. During the treatment, seven parameters dropped permanently or temporarily to a normal level and seven moved markedly towards the physiological value. While the number of peripheral blood reticulocytes was significantly decreased after the treatment with free doxorubicin, their number was stable or elevated after the treatment with Dox-PHPMA-Hulg conjugate. Increased absolute number of CD16+56+ and CD4+ cells in the peripheral blood and activation of NK and LAX cells in a human patients support the data previously obtained in experimental animals pointing to a dual role, i.e. the cytotoxic and immunomobilizing character of doxorubicin-PHPMA conjugates containing a targeting immunoglobulin moiety.

Overcoming Immunoescape Mechanisms of BCL1 Leukemia and Induction of CD8+ T-Cell–Mediated BCL1-Specific Resistance in Mice Cured by Targeted Polymer-Bound Doxorubicin

BALB/c mice bearing syngeneic BCL1 leukemia, a mouse model of human chronic lymphocytic leukemia, were treated with polymer-bound doxorubicin conjugate targeted with BCL1-specific monoclonal antibody. Such treatment can cure up to 100% of mice and the cured mice show long-lasting resistance to BCL1 leukemia. We show that both CD4+ and CD8+ T cells are required for establishment of the resistance, but only CD8+ T cells are necessary for its maintenance. BCL1 cells express MHC class I and II and also costimulatory molecules CD80 and CD86, which can aid eliciting of antitumor response. On the other hand, BCL1 cells also use several immunoescape mechanisms, such as expression of PD-L1, PD-L2, and interleukin-10. BCL1 cells thus can be recognized by BCL1-specific T cells, but instead of effective priming, such T cells are anergized or deleted by apoptosis. Moreover, BCL1 leukemia progression is accompanied by robust expansion of CD4+CD25+Foxp3+ regulatory T (Treg) cells. Although it has been shown that depletion of Treg cells in tumor-bearing mice can retard tumor growth, direct evidence that expansion of Treg cells can promote tumor growth was lacking. In this study, we provide first direct evidence that expanded Treg cells can indeed promote tumor progression by using mice with selectively expanded Treg cells before inoculation of BCL1 leukemia. Finally, we have also shown that elimination of some immunoescape mechanism (e.g., deletion of Treg) can significantly improve the therapeutic outcome of chemotherapy.

HPMA-Anticancer Drug Conjugates

Homopolymer poly(HPMA) was originally developed as a blood expander “Duxon.” Later on, linear nondegradable HPMA copolymer of a molecular weight ∼25 kDa was used as the backbone to which drugs, mostly doxorubicin, were attached through different side chains and different covalent bonds. Homopolymer and copolymers are non-toxic, biocompatible, and non-immunogenic molecules. To increase their accumulation in solid tumors and achieve maximal EPR effect, branched and grafted high molecular weight derivatives were designed containing oligopeptidic cross-links which can be degraded by lysosomal enzymes. In addition, linear HPMA copolymers were synthesized to form high molecular weight supramolecular structures. To fulfill the requirements for active targeting, poly- and monoclonal antibodies, carbohydrates, lectins, growth hormones, cell-surface active proteins and peptides have been employed. Non-targeted and targeted polymer–drug derivatives based on HPMA have both cytostatic and immunostimulating activity. Their impressive anti-tumor effects most likely result from the combination of strong direct cytotoxicity and a systemic anticancer resistance regularly induced during the treatment.