Linkage of doxorubicin (Dox) to a water-soluble synthetic N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) eliminates most of the systemic toxicity of the free drug. In EL-4 lymphoma-bearing C57BL/6 mice, a complete regression of pre-established tumours has been achieved upon treatment with Dox-PHPMA-HuIg conjugate. The treatment was effective using a range of regimens and dosages, ranging from 62.5 to 100% cured mice treated with a single dose of 10-20 mg of Dox eq./kg, respectively. Fractionated dosages producing lower levels of the conjugate for a prolonged time period had substantial curative capacity as well. The cured mice developed anti-tumour protection as they rejected subsequently re-transplanted original tumour. The proportion of tumour-protected mice inversely reflected the effectiveness of the primary treatment. The treatment protocol leading to 50% of cured mice produced only protected mice, while no mice treated with early treatment regimen (i.e. starting on day 1 after tumour transplantation) rejected the re-transplanted tumour. Exposure of the host to the cancer cells was a prerequisite for developing protection. The anti-tumour memory was long lasting and specific against the original tumour, as the cured mice did not reject another syngeneic tumour, melanoma B16-F10. The immunity was transferable to naïve recipients in in vivo neutralization assay by spleen cells or CD8(+) lymphocytes derived from cured animals. We propose an effective treatment strategy which eradicates tumours without harming the protective immune anti-cancer responses.

Division of Immunology and Gnotobiology Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Ehrlich P. Studies in immunity. New York: Plenum; 1906.

Ringsdorf H. Structure and properties of pharmacologically active polymers. J Poly Sci Polym Symp. 1975;101:135–153.

Maeda H, Matsumura Y. Tumoritropic and lymphotropic principles of macromolecular drugs. Crit Rev Ther Drug Carrier Syst. 1989;6:193–210. PubMed

Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release. 2000;65:271–284. doi: 10.1016/S0168-3659(99)00248-5. PubMed DOI

Noguchi Y, Wu J, Duncan R, Strohalm J, Ulbrich K, Akaike T, Maeda H. Early phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissues. Jpn J Cancer Res. 1998;89:307–314. PubMed PMC

Wu J, Akaike T, Hayashida K, Okamoto T, Okuyama A, Maeda H. Enhanced vascular permeability in solid tumor involving peroxynitrite and matrix metalloproteinases. Jpn J Cancer Res. 2001;92:439–451. PubMed PMC

Hortobagyi GN. Anthracyclines in the treatment of cancer. An overview. Drugs. 1997;54(Suppl 4):1–7. PubMed

Rihova B, Kopeckova P, Strohalm J, Rossmann P, Vetvicka V, Kopecek J. Antibody-directed affinity therapy applied to the immune system: in vivo effectiveness and limited toxicity of daunomycin conjugated to HPMA copolymers and targeting antibody. Clin Immunol Immunopathol. 1988;46:100–114. doi: 10.1016/0090-1229(88)90010-4. PubMed DOI

Rossmann P, Rihova B, Strohalm J, Ulbrich K. Morphology of rat kidney and thymus after native and antibody-coupled cyclosporin A application (reduced toxicity of targeted drug) Folia Microbiol (Praha) 1997;42:277–287. PubMed

St’astny M, Ulbrich K, Strohalm J, Rossmann P, Rihova B. Abnormal differentiation of thymocytes induced by free cyclosporine is avoided when cyclosporine bound to N-(2-hydroxypropyl)methacrylamide copolymer carrier is used. Transplantation. 1997;63:1818–1827. PubMed

St’astny M, Strohalm J, Plocova D, Ulbrich K, Rihova B. A possibility to overcome P-glycoprotein (PGP)-mediated multidrug resistance by antibody-targeted drugs conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier. Eur J Cancer. 1999;35:459–466. doi: 10.1016/S0959-8049(98)00373-6. PubMed DOI

Kopecek J, Kopeckova P, Minko T, Lu Z. HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action. Eur J Pharm Biopharm. 2000;50:61–81. doi: 10.1016/S0939-6411(00)00075-8. PubMed DOI

Jelinkova M, Strohalm J, Plocova D, Subr V, St’astny M, Ulbrich K, Rihova B. Targeting of human and mouse T-lymphocytes by monoclonal antibody-HPMA copolymer–doxorubicin conjugates directed against different T-cell surface antigens. J Control Release. 1998;52:253–270. doi: 10.1016/S0168-3659(97)00210-1. PubMed DOI

Kovar M, Strohalm J, Ulbrich K, Rihova B. In vitro and in vivo effect of HPMA copolymer-bound doxorubicin targeted to transferrin receptor of B-cell lymphoma 38C13. J Drug Target. 2002;10:23–30. doi: 10.1080/10611860290007496. PubMed DOI

Kanwar J, Berg R, Lehnert K, Krissansen G. Taking lessons from dendritic cells: multiple xenogeneic ligands for leukocyte integrins have the potential to stimulate anti-tumor immunity. Gene Ther. 1999;6:1835–1844. doi: 10.1038/sj.gt.3301016. PubMed DOI

Hersey P. Impediments to successful immunotherapy. Pharmacol Ther. 1999;81:111–119. doi: 10.1016/S0163-7258(98)00038-2. PubMed DOI

Griffioen AW, Damen CA., Mayo KH, Barendsz-Janson AF, Martinotti S, Blijham GH, Groenewegen G. Angiogenesis inhibitors overcome tumor induced endothelial cell anergy. Int J Cancer. 1999;80:315–319. doi: 10.1002/(SICI)1097-0215(19990118)80:2<315::AID-IJC23>3.0.CO;2-L. PubMed DOI

Rihova B, Strohalm J, Prausova J, Kubackova K, Jelinkova M, Rozprimova L, Sirova M, Plocova D, Etrych T, Subr V, Mrkvan T, Kovar M, Ulbrich K. Cytostatic and immunomobilizing activities of polymer-bound drugs: experimental and first clinical data. J Control Release. 2003;91:1–16. doi: 10.1016/S0168-3659(03)00235-9. PubMed DOI

Rihova B, Kubackova K. Clinical implications of N-(2-hydroxypropyl)methacrylamide copolymers. Curr Pharm Biotechnol. 2003;4:311–322. doi: 10.2174/1389201033489711. PubMed DOI

Fidler IJ. Selections of successive tumour lines for metastasis. Nat New Biol. 1973;242:148–149. PubMed

Ulbrich K, Subr V, Strohalm J, Plocova D, Jelinkova M, Rihova B. Polymeric drugs based on conjugates of synthetic and natural macromolecules. I. Synthesis and physico-chemical characterisation. J Control Release. 2000;64:63–79. doi: 10.1016/S0168-3659(99)00141-8. PubMed DOI

Rihova B, Bilej M, Vetvicka V, Ulbrich K, Strohalm J, Kopecek J, Duncan R. 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. Biomaterials. 1989;10:335–342. doi: 10.1016/0142-9612(89)90075-6. PubMed DOI

Winn HJ. Immune mechanisms in homotransplantation. II. Quantitative assay of the immunologic activity of lymphoid cells stimulated by tumor homografts. J Immunol. 1961;86:228–239. PubMed

Parajuli P, Pisarev V, Sublet J, Steffel A, Varney M, Singh R, LaFace D, Talmadge JE. Immunization with wild-type p53 gene sequences coadministered with Flt3 ligand induces an antigen-specific type 1 T-cell response. Cancer Res. 2001;61:8227–8234. PubMed

Matzinger P. The JAM test. A simple assay for DNA fragmentation and cell death. J Immunol Methods. 1991;145:185–192. doi: 10.1016/0022-1759(91)90325-A. PubMed DOI

Rihova B, Strohalm J, Kubackova K, Jelinkova M, Hovorka O, Kovar M, Plocova D, Sirova M, St’astny M, Rozprimova L, Ulbrich K. Acquired and specific immunological mechanisms co-responsible for efficacy of polymer-bound drugs. J Control Release. 2002;78:97–114. doi: 10.1016/S0168-3659(01)00489-8. PubMed DOI

Kawakami Y, Fujita T, Matsuzaki Y, Sakurai T, Tsukamoto M, Toda M, Tsumimoto H. Identification of human tumor antigens and its implications for diagnosis and treatment of cancer. Cancer Sci. 2004;95:784–791. doi: 10.1111/j.1349-7006.2004.tb02182.x. PubMed DOI PMC

Sapir T, Shoenfeld Y. Facing the enigma of immunomodulatory effects of intravenous immunoglobulin. Clin Rev Allergy Immunol. 2005;29:185–199. doi: 10.1385/CRIAI:29:3:185. PubMed DOI

Shoenfeld Y, Fishman P. Gamma-globulin inhibits tumor spread in mice. Int Immunol. 1999;11:1247–1252. doi: 10.1093/intimm/11.8.1247. PubMed DOI

Shapiro S, Shoenfeld Y, Gilburd B, Sobel E, Lahat N. Intravenous gamma globulin inhibits the production of matrix metalloproteinase-9 in macrophages. Cancer. 2002;95:2032–2037. doi: 10.1002/cncr.10905. PubMed DOI

Prasad NK, Papoff G, Zeuner A, Bonnin E, Kazatchkine MD, Ruberti G, Kaveri SV. Therapeutic preparations of normal polyspecific IgG (IVIg) induce apoptosis in human lymphocytes and monocytes: a novel mechanism of action of IVIg involving the Fas apoptotic pathway. J Immunol. 1998;161:3781–3790. PubMed

Ekberg C, Nordstrom E, Skansen-Saphir U, Mansouri M, Raqib R, Sundqvist VA, Fernandez C. Human polyspecific immunoglobulin for therapeutic use induces p21/WAF-1 and Bcl-2, which may be responsible for G1 arrest and long-term survival. Hum Immunol. 2001;62:215–227. doi: 10.1016/S0198-8859(00)00250-0. PubMed DOI

Mrkvan T, Sirova M, Etrych T, Chytil P, Strohalm J, Plocova D, Ulbrich K, Rihova B. Chemotherapy based on HPMA copolymer conjugates with pH-controlled release of doxorubicin triggers anti-tumor immunity. J Control Release. 2005;110:119–129. doi: 10.1016/j.jconrel.2005.09.028. PubMed DOI

Krawczyk CM, Verstovsek S, Ujhazy P, Maccubbin D, Ehrke MJ. Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor alpha combination treatment of EL4-lymphoma-bearing C57BL/6 mice. Cancer Immunol Immunother. 1995;40:347–357. PubMed PMC

Ehrke MJ, Verstovsek S, Pocchiari SK, Krawczyk CM, Ujhazy P, Zaleskis G, Maccubbin D, Meer JM, Mihich E. Thymic anti-tumor effectors in mice cured of lymphoma by cyclophosphamide plus TNF-alpha therapy: phenotypic and functional characterization up to 20 months after initial tumor inoculation. Int J Cancer. 1998;76:579–586. doi: 10.1002/(SICI)1097-0215(19980518)76:4<579::AID-IJC22>3.0.CO;2-1. PubMed DOI

Ehrke MJ, Verstovsek S, Krawczyk CM, Ujhazy P, Zaleskis G, Maccubbin DL, Mihich E. Cyclophosphamide plus tumor necrosis factor-alpha chemoimmunotherapy cured mice: life-long immunity and rejection of re-implanted primary lymphoma. Int J Cancer. 1995;63:463–471. PubMed

Ehrke MJ, Verstovsek S, Maccubbin DL, Ujhazy P, Zaleskis G, Berleth E, Mihich E. Protective specific immunity induced by doxorubicin plus TNF-alpha combination treatment of EL4 lymphoma-bearing C57BL/6 mice. Int J Cancer. 2000;87:101–109. doi: 10.1002/1097-0215(20000701)87:1<101::AID-IJC15>3.0.CO;2-B. PubMed DOI

Ehrke MJ, Verstovsek S, Ujhazy P, Meer JM, Eppolito C, Maccubbin DL, Mihich E. Doxorubicin plus tumor necrosis factor alpha combination treatments in EL4-lymphoma-bearing C57BL/6 mice. Cancer Immunol Immunother. 1998;45:287–298. doi: 10.1007/s002620050445. PubMed DOI PMC

Ehrke MJ, Verstovsek S, Zaleskis G, Ho RL, Ujhazy P, Maccubbin DL, Mihich E. Specific anti-EL4-lymphoma immunity in mice cured 2 years earlier with doxorubicin and interleukin-2. Cancer Immunol Immunother. 1996;42:221–230. doi: 10.1007/s002620050274. PubMed DOI PMC

Ho RL, Maccubbin D., Ujhazy P, Zaleskis G, Eppolito C, Mihich E, Ehrke MJ. Immunological responses critical to the therapeutic effects of adriamycin plus interleukin 2 in C57BL/6 mice bearing syngeneic EL4 lymphoma. Oncol Res. 1993;5:363–372. PubMed

Gautam SC, Chikkala NF, Ganapathi R, Hamilton TA. Combination therapy with adriamycin and interleukin 2 augments immunity against murine renal cell carcinoma. Cancer Res. 1991;51:6133–6137. PubMed

Vasey PA, Kaye SB, Morrison R, Wilson P, Twelves C, Duncan R, Thomson AH, Murray LS, Hilditch TE, Murray T, Burtles S, Fraier D, Frigerio E, Cassidy J. Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: first member of a new class of chemotherapeutic agents–drug–polymer conjugates. Cancer Research Campaign Phase I/II Committee. Clin Cancer Res. 1999;5:83–94. PubMed

Julyan PJ, Seymour LW, Ferry DR, Daryani S, Boivin CM, Doran J, David M, Anderson D, Christodoulou C, Young AM, Hesslewood S, Kerr DJ. Preliminary clinical study of the distribution of HPMA copolymers bearing doxorubicin and galactosamine. J Control Release. 1999;57:281–290. doi: 10.1016/S0168-3659(98)00124-2. PubMed DOI

Jelinkova M, Strohalm J, Etrych T, Ulbrich K, Rihova B. Starlike vs. classic macromolecular prodrugs: two different antibody-targeted HPMA copolymers of doxorubicin studied in vitro and in vivo as potential anticancer drugs. Pharm Res. 2003;20:1558–1564. doi: 10.1023/A:1026170830782. PubMed DOI

Kovar M, Mrkvan T, Strohalm J, Etrych T, Ulbrich K, St’astny M, Rihova B. HPMA copolymer-bound doxorubicin targeted to tumor-specific antigen of BCL1 mouse B cell leukemia. J Control Release. 2003;92:315–330. doi: 10.1016/S0168-3659(03)00340-7. PubMed DOI

Kovar M, Mrkvan T, Sirova M, Sťastny M, Etrych T, Strohalm J, Plocova D, Ulbrich K, Rihova B (2004) HPMA copolymer-bound doxorubicin targeted with monoclonal antibody to tumor-specific antigen of BCL1 leukemia cells can completely cure tumor-bearing mice and establish long-term immunological memory to the original tumor. 31st annual meeting and exposition of the Controlled Release Society. Honolulu, HI, USA

Rihova B, Strohalm J, Kovar M, Mrkvan T, Subr V, Hovorka O, Sirova M, Rozprimova L, Kubackova K, Ulbrich K. Induction of systemic antitumour resistance with targeted polymers. Scand J Immunol. 2005;62(Suppl 1):100–105. doi: 10.1111/j.1365-3083.2005.01617.x. PubMed DOI

Rihova B, Strohalm J, Hoste K. Immunoprotective therapy with targeted anticancer drugs. Macromol Symp. 2001;172:21–28. doi: 10.1002/1521-3900(200107)172:1<21::AID-MASY21>3.0.CO;2-C. DOI

Hanson HL, Donermeyer DL, Ikeda H, White JM, Shankaran V, Old LJ, Shiku H, Schreibe RD, Allen PM. Eradication of established tumors by CD8+ T cell adoptive immunotherapy. Immunity. 2000;13:265–276. doi: 10.1016/S1074-7613(00)00026-1. PubMed DOI

Miki S, Ksander B, Streilein JW. Studies on the minimum requirements for in vitro “cure” of tumor cells by cytotoxic T lymphocytes. Reg Immunol. 1992;4:352–362. PubMed

Ksander BR, Acevedo J, Streilein JW. Local T helper cell signals by lymphocytes infiltrating intraocular tumors. J Immunol. 1992;148:1955–1963. PubMed

Guerder S, Carding SR, Flavell RA. B7 costimulation is necessary for the activation of the lytic function in cytotoxic T lymphocyte precursors. J Immunol. 1992;155:5167–5174. PubMed

Seymour LW, Ulbrich K, Steyger PS, Brereton M, Subr V, Strohalm J, Duncan R. Tumour tropism and anti-cancer efficacy of polymer-based doxorubicin prodrugs in the treatment of subcutaneous murine B16F10 melanoma. Br J Cancer. 1994;70:636–641. PubMed PMC

Eager Nemunaitis R. J. GM-CSF gene-transduced tumor vaccines. Mol Ther. 2005;12:18–27. doi: 10.1016/j.ymthe.2005.02.012. PubMed DOI

Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother. 2005;54:721–728. doi: 10.1007/s00262-004-0653-2. PubMed DOI PMC

Evaluation of linear versus star-like polymer anti-cancer nanomedicines in mouse models

HPMA-based star polymer biomaterials with tuneable structure and biodegradability tailored for advanced drug delivery to solid tumours

Doxorubicin attached to HPMA copolymer via amide bond modifies the glycosylation pattern of EL4 cells

Preclinical evaluation of linear HPMA-doxorubicin conjugates with pH-sensitive drug release: efficacy, safety, and immunomodulating activity in murine model