Tumor-specific targeting of polymer drug delivery systems with recombinant proteins bound via tris(nitrilotriacetic acid).

Antibody-mediated targeting is an efficient strategy to enhance the specificity and selectivity of polymer nanomedicines towards the target site, typically a tumor. However, direct covalent coupling of an antibody with a polymer usually results in a partial damage of the antibody binding site accompanied with a compromised biological activity. Here, an original solution based on well-defined non-covalent interactions between tris-nitrilotriacetic acid (trisNTA) and hexahistidine (His-tag) groups, purposefully introduced to the structure of each macromolecule, is described. Specifically, trisNTA groups were attached along the chains of a hydrophilic statistical copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA), and at the end or along the chains of thermo-responsive di-block copolymers based on N-isopropylmethacrylamide (NIPMAM) and HPMA; His-tag was incorporated to the structure of a recombinant single chain fragment of an anti-GD2 monoclonal antibody (scFv-GD2). Static and dynamic light scattering analyses confirmed that mixing of polymer with scFv-GD2 led to the formation of polymer/scFv-GD2 complexes; those prepared from thermo-responsive polymers formed stable micelles at 37 °C. Flow cytometry and fluorescence microscopy clearly demonstrated antigen-specific binding of the prepared complexes to GD2 positive murine T-cell lymphoma cells EL-4 and human neuroblastoma cells UKF-NB3, while no interaction with GD2 negative murine fibroblast cells NIH-3T3 was observed. These non-covalent polymer protein complexes represent a new generation of highly specific actively targeted polymer therapeutics or diagnostics.

Low levels of vitamin D in population exposed to significant environmental pollution.

In recent years, monitoring of vitamin D levels and possible use of supplementation is gaining attention. Numerous studies showed low levels of vitamin D in winter months followed by improvement during summer. These changes are mostly dependent on the level of sun exposure, but also on geographical location, genetic factors, social-economic status, quality of nutrition and environmental pollution. In this observation we found significant decrease in vitamin D levels in populations exposed to extreme environmental pollution in area of central Europe. This region is known for extreme burden from microparticles originating in chemical industry, surface coal mining and cold-based power stations. Vitamin D levels in all patients was determined by ELISA. Using 540 patients in our department of clinical immunology and allergology we measured the levels of vitamin D in 2016 to 2021 period. In only 4 patients (0.74 %) we found vitamin D levels higher than 30 ng/ml. The curve of observed values does not reflect dependency on sun exposure and does not change during the year. We discuss the effect of environmental contaminants, lifestyle and economic and social factors. From our observations, we propose to directly supplement population with vitamin D, particularly children and seniors. From our observations, we propose to directly supplement population with vitamin D, particularly children and seniors.

Factors, associated with elevated concentration of soluble carbonic anhydrase IX in plasma of women with cervical dysplasia.

Precancerous lesions of human cervix uteri have a tendency for regression or progression. In cervical intraepithelial neoplasia grade 2 (CINII) case there is an uncertainty if a lesion will progress or regress. The carbonic anhydrase IX (CAIX) enzyme is overexpressed in cervical cancer which is more sensitive to radiotherapy. CAIX is associated with poor prognosis in solid hypoxic tumors. The aim of this study was to determine factors related to elevated soluble CAIX (s-CAIX) in high-grade intraepithelial lesion (HSIL) cases. METHODS: Patients diagnosed with HSIL (N = 77) were included into the research group whereas without HSIL (N = 72)-the control group. Concentration of the soluble CAIX (s-CAIX) in plasma was determined by the DIANA ligand-antibody-based method. C. trachomatis was detected from cervical samples by PCR. Primary outcomes were risk factors elevating s-CAIX level in HSIL group. Non-parametric statistical analysis methods were used to calculate correlations. RESULTS: The s-CAIX level in patients with HSIL was elevated among older participants (rs = 0.27, p = 0.04) and with C. trachomatis infection (p = 0.028). Among heavy smokers with HSIL, the concentration of s-CAIX was higher in older women (rs = 0.52, p = 0.005), but was not related to the age of heavy smokers' controls (τ = 0.18 p = 0.40). CONCLUSION: The concentration of s-CAIX was higher among older, heavy smoking and diagnosed with C. trachomatis patients. All these factors increased the risk for HSIL progression.

Immune Response 5-7 Months after Vaccination against SARS-CoV-2 in Elderly Nursing Home Residents in the Czech Republic: Comparison of Three Vaccines.

BACKGROUND AND AIMS: Elderly nursing home residents are especially prone to a severe course of SARS-CoV-2 infection. In this study, we aimed to investigate the complex immune response after vaccination depending on the convalescence status and vaccine. METHODS: Sampling took place in September-October 2021. IgG antibodies against spike protein and nucleocapsid protein, the titer of virus neutralization antibodies against delta and (on a subset of patients) omicron, and cellular immunity (interferon-gamma release assay) were tested in nursing home residents vaccinated with Pfizer, Moderna (both 30-31 weeks after the completion of vaccination), or AstraZeneca (23 weeks) vaccines. The prevalence with 95% confidence intervals (CI) was evaluated in Stata version 17. RESULTS: 95.2% (95% CI: 92.5-97.1%) of the 375 participants had positive results of anti-S IgG, 92.8% (95% CI: 89.7-95.2%) were positive in virus neutralization assay against delta, and 89.0% (95% CI: 84.5-92.5%) in the interferon-gamma-releasing assay detecting cellular immunity. Results of the virus neutralization assay against omicron correlated with those against delta but the neutralization capacity was reduced by about half. As expected, the worst results were found for the AstraZeneca vaccine, although the vaccination-to-test period was the shortest for this vaccine. All immune parameters were significantly higher in convalescent residents than in naive residents after vaccination. No case of COVID-19 occurred during the vaccination-to-test period. CONCLUSIONS: A high immune response, especially among vaccinated convalescents (i.e., residents with hybrid immunity), was found in elderly nursing home residents 5-7 months after vaccination against SARS-CoV-2. In view of this, it appears that such residents are much better protected from COVID-19 than those who are only vaccinated and the matter of individual approach to the booster dose in such individuals should be further discussed.

Tumor Marker B7-H6 Bound to the Coiled Coil Peptide-Polymer Conjugate Enables Targeted Therapy by Activating Human Natural Killer Cells.

Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugated with immunologically active protein B7-H6 via complementary coiled coil VAALEKE (peptide E) and VAALKEK (peptide K) sequences. Receptor B7-H6 was described as a binding partner of NKp30, and its expression has been proven for various tumor cell lines. The binding of B7-H6 to NKp30 activates NK cells and results in Fas ligand or granzyme-mediated apoptosis of target tumor cells. In this work, we optimized the expression of coiled coil tagged B7-H6, its ability to bind activating receptor NKp30 has been confirmed by isothermal titration calorimetry, and the binding stoichiometry of prepared chimeric biopolymer has been characterized by analytical ultracentrifugation. Furthermore, this coiled coil B7-H6-loaded polymer conjugate activates NK cells in vitro and, in combination with coiled coil scFv, enables their targeting towards a model tumor cell line. Prepared chimeric biopolymer represents a promising precursor for targeted cancer immunotherapy by activating the cytotoxic activity of natural killer cells.

Inhibitors of CA IX Enzyme Based on Polyhedral Boron Compounds.

This review describes recent progress in the design and development of inhibitors of human carbonic anhydrase IX (CA IX) based on space-filling carborane and cobalt bis(dicarbollide) clusters. CA IX enzyme is known to play a crucial role in cancer cell proliferation and metastases. The new class of potent and selective CA IX inhibitors combines the structural motif of a bulky inorganic cluster with an alkylsulfamido or alkylsulfonamido anchor group for Zn2+ ion in the enzyme active site. Detailed structure-activity relationship (SAR) studies of a large series containing 50 compounds uncovered structural features of the cluster-containing inhibitors that are important for efficient and selective inhibition of CA IX activity. Preclinical evaluation of selected compounds revealed low toxicity, favorable pharmacokinetics and ability to reduce tumor growth. Cluster-containing inhibitors of CA IX can thus be considered as promising candidates for drug development and/or for combination therapy in boron neutron capture therapy (BNCT).

Cobalt Bis(dicarbollide) Alkylsulfonamides: Potent and Highly Selective Inhibitors of Tumor Specific Carbonic Anhydrase IX.

Carbonic anhydrase IX (CAIX) is an enzyme expressed on the surface of cells in hypoxic tumors. It plays a role in regulation of tumor pH and promotes thus tumor cell survival and occurrence of metastases. Here, derivatives of the cobalt bis(dicarbollide)(1-) anion are reported that are based on substitution at the carbon sites of the polyhedra by two alkylsulfonamide groups differing in the length of the aliphatic connector (from C1 to C4, n=1-4), which were prepared by cobalt insertion into the 7-sulfonamidoalkyl-7,8-dicarba-nido-undecaborate ions. Pure meso- and rac-diastereoisomeric forms were isolated. The series is complemented with monosubstituted species (n=2). Synthesis by a direct method furnished similar derivatives (n=2, 3), which are chlorinated at the B(8,8') boron sites. All compounds inhibited CAIX with subnanomolar inhibition constants and showed high selectivity for CAIX. The best inhibitory properties were observed for the compound with n= 3 and two substituents present in rac-arrangement with Ki =20 pM and a selectivity index of 668. X-ray crystallography was used to study interactions of these compounds with the active site of CAIX on the structural level.

Cobalt Bis(dicarbollide) Alkylsulfonamides: Potent and Highly Selective Inhibitors of Tumor Specific Carbonic Anhydrase IX.

Invited for this month's cover is a collaboration from three institutes from the Czech Academy of Sciences: Institute of Inorganic Chemistry, Institute of Organic Chemistry and Biochemistry, and Institute of Molecular Genetics, and the University of Pardubice. The cover picture shows a family of potent and selective CA IX inhibitors that combines the structural motif of a bulky inorganic cobalt bis(dicarbollide) polyhedral ion with a propylsulfonamido anchor group. Read the full text of the article at 10.1002/cplu.202000574.

The structural basis for the selectivity of sulfonamido dicarbaboranes toward cancer-associated carbonic anhydrase IX.

Human carbonic anhydrase IX (CA IX), a protein specifically expressed on the surface of solid tumour cells, represents a validated target both for anticancer therapy and diagnostics. We recently identified sulfonamide dicarbaboranes as promising inhibitors of CA IX with favourable activities both in vitro and in vivo. To explain their selectivity and potency, we performed detailed X-ray structural analysis of their interactions within the active sites of CA IX and CA II. Series of compounds bearing various aliphatic linkers between the dicarbaborane cluster and sulfonamide group were examined. Preferential binding towards the hydrophobic part of the active site cavity was observed. Selectivity towards CA IX lies in the shape complementarity of the dicarbaborane cluster with a specific CA IX hydrophobic patch containing V131 residue. The bulky side chain of F131 residue in CA II alters the shape of the catalytic cavity, disrupting favourable interactions of the spherical dicarbaborane cluster.

Sulfonamido carboranes as highly selective inhibitors of cancer-specific carbonic anhydrase IX.

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme overexpressed in hypoxic tumors, where it plays an important role in tumor progression. Specific CA IX inhibitors potentially could serve as anti-cancer drugs. We designed a series of sulfonamide inhibitors containing carborane clusters based on prior structural knowledge of carborane binding into the enzyme active site. Two types of carborane clusters, 12-vertex dicarba-closo-dodecaborane and 11-vertex 7,8-dicarba-nido-undecaborate (dicarbollide), were connected to a sulfonamide moiety via aliphatic linkers of varying lengths (1-4 carbon atoms; n = 1-4). In vitro testing of CA inhibitory potencies revealed that the optimal linker length for selective inhibition of CA IX was n = 3. A 1-sulfamidopropyl-1,2-dicarba-closo-dodecaborane (3) emerged as the strongest CA IX inhibitor from this series, with a Ki value of 0.5 nM and roughly 1230-fold selectivity towards CA IX over CA II. X-ray studies of 3 yielded structural insights into their binding modes within the CA IX active site. Compound 3 exhibited moderate cytotoxicity against cancer cell lines and primary cell lines in 2D cultures. Cytotoxicity towards multicellular spheroids was also observed. Moreover, 3 significantly lowered the amount of CA IX on the cell surface both in 2D cultures and spheroids and facilitated penetration of doxorubicin. Although 3 had only a moderate effect on tumor size in mice, we observed favorable ADME properties and pharmacokinetics in mice, and preferential presence in brain over serum.

Identification of Novel Carbonic Anhydrase IX Inhibitors Using High-Throughput Screening of Pooled Compound Libraries by DNA-Linked Inhibitor Antibody Assay (DIANA).

The DNA-linked inhibitor antibody assay (DIANA) has been recently validated for ultrasensitive enzyme detection and for quantitative evaluation of enzyme inhibitor potency. Here we present its adaptation for high-throughput screening of human carbonic anhydrase IX (CAIX), a promising drug and diagnostic target. We tested DIANA's performance by screening a unique compound collection of 2816 compounds consisting of lead-like small molecules synthesized at the Institute of Organic Chemistry and Biochemistry (IOCB) Prague ("IOCB library"). Additionally, to test the robustness of the assay and its potential for upscaling, we screened a pooled version of the IOCB library. The results from the pooled screening were in agreement with the initial nonpooled screen with no lost hits and no false positives, which shows DIANA's potential to screen more than 100,000 compounds per day.All DIANA screens showed a high signal-to-noise ratio with a Z' factor of >0.89. The DIANA screen identified 13 compounds with Ki values equal to or better than 10 µM. All retested hits were active also in an orthogonal enzymatic assay showing zero false positives. However, further biophysical validation of identified hits revealed that the inhibition activity of several hits was caused by a single highly potent CAIX inhibitor, being present as a minor impurity. This finding eventually led us to the identification of three novel CAIX inhibitors from the screen. We confirmed the validity of these compounds by elucidating their mode of binding into the CAIX active site by x-ray crystallography.

Metallacarborane Sulfamides: Unconventional, Specific, and Highly Selective Inhibitors of Carbonic Anhydrase IX.

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme that regulates pH in hypoxic tumors and promotes tumor cell survival. Its expression is associated with the occurrence of metastases and poor prognosis. Here, we present nine derivatives of the cobalt bis(dicarbollide)(1-) anion substituted at the boron or carbon sites by alkysulfamide group(s) as highly specific and selective inhibitors of CAIX. Interactions of these compounds with the active site of CAIX were explored on the atomic level using protein crystallography. Two selected derivatives display subnanomolar or picomolar inhibition constants and high selectivity for the tumor-specific CAIX over cytosolic isoform CAII. Both derivatives had a time-dependent effect on the growth of multicellular spheroids of HT-29 and HCT116 colorectal cancer cells, facilitated penetration and/or accumulation of doxorubicin into spheroids, and displayed low toxicity and showed promising pharmacokinetics and a significant inhibitory effect on tumor growth in syngenic breast 4T1 and colorectal HT-29 cancer xenotransplants.

Microgel Bioreactors for Cancer Cell Targeting by pH-Dependent Generation of Radicals.

The lack of specificity of traditional cytostatics and increasing resistance of cancer cells represent important challenges in cancer therapy. One of the characteristics of cancer cells is their intrinsic oxidative stress caused by higher metabolic activity, mitochondrial malfunction, and oncogene stimulation. This feature can be exploited in the pursuit of more selective cancer therapy, as there is increasing evidence that cancer cells are more sensitive to elevated concentrations of reactive oxygen species than normal cells. In this study, we demonstrate a new concept for cancer cell targeting by in situ production of radicals under physiological conditions. The biologically active radicals are produced in the milieu of cancer cells by enzymatic conversion from an inactive precursor, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt, by using miniature bioreactors represented by cell-sized microgels containing immobilized laccase. We utilize the pH-dependent activity of laccase to generate radicals only at a lower pH (5.7-6.1) that is characteristic of the tumor microenvironment. The composition of the microgels was optimized so as to allow sufficient substrate and radical diffusion, high enzyme activity, and stability under physiological conditions. The functionality of this system was evaluated on three cancer cell lines (HeLa, HT-29, and DLD1) and the cytotoxicity of in situ-produced radicals was successfully proven in all cases. These results demonstrate that cancer cell targeting by in situ-generated radicals using miniature enzymatic reactors may represent an alternative to traditional cytostatics. In particular, the pH-dependence of radical generation and their short-lived nature can ensure localized functionality in the tumor microenvironment and thereby reduce systemic side-effects.

Effects of ß-glucan and Vitamin D Supplementation on Inflammatory Parameters in Patients with Diabetic Retinopathy.

The objective of this article is to evaluate the potential effects of beta-glucan and vitamin D supplementation in patients with diabetic retinopathy. We evaluated the levels of several parameters of inflammatory reactions (C-reactive protein [CRP], serum amyloid A [SAA], and interleukin- [IL-] 6), leptin, and vitamin D. Using a 3-month interval, we divided the patients into three groups: (1) supplemented with beta-glucan and vitamin D, (2) supplemented with vitamin D and placebo, and (3) supplemented with vitamin D alone. By this division, we aim not only to observe whether beta-glucan can increase the effects of vitamin D, but also to eliminate the potential effects of placebo. The doses of vitamin D corresponded to phototype, weight, age, and sex of the individual. Fifty-two diabetic retinopathy patients were selected for our study. We found significant vitamin D deficits in all cases, even after three months of supplementation with vitamin D. Significant changes in levels of CRP were observed in the beta-glucan-supplemented group; levels of SAA and IL-6 were not changed. Leptin levels were significantly lowered in the beta-glucan-supplemented group and increased in the other groups. More detailed studies and/or longer supplementation is necessary.

Radiolabeling of the antibody IgG M75 for epitope of human carbonic anhydrase IX by 61Cu and 64Cu and its biological testing.

Human carbonic anhydrase IX is a membrane enzyme that is significantly expressed in some types of cancer cells, while copper radioisotopes offer wide range of diagnostic, therapeutic and theranostic properties. The work was focused on a new approach to the labelling of antibody IgG M75 for epitope human carbonic anhydrase IX with copper radioisotopes 61Cu and 64Cu and its in vivo testing in mice with inoculated colorectal cancer. Monoclonal antibody IgG M75 for epitope human carbonic anhydrase IX was successfully conjugated with copper-specific chelator "phosphinate" and labelled with 61Cu and 64Cu The obtained molecule has considerable potential as a radioimmuno pharmaceutical suitable for imaging of tumours expressing carbonic anhydrase IX by positron emission tomography (PET).

Polymer Cancerostatics Targeted by Recombinant Antibody Fragments to GD2-Positive Tumor Cells.

A water-soluble polymer cancerostatic actively targeted against cancer cells expressing a disialoganglioside antigen GD2 was designed, synthesized and characterized. A polymer conjugate of an antitumor drug doxorubicin with a N-(2-hydroxypropyl)methacrylamide-based copolymer was specifically targeted against GD2 antigen-positive tumor cells using a recombinant single chain fragment (scFv) of an anti-GD2 monoclonal antibody. The targeting protein ligand was attached to the polymer-drug conjugate either via a covalent bond between the amino groups of the protein using a traditional nonspecific aminolytic reaction with a reactive polymer precursor or via a noncovalent but highly specific interaction between bungarotoxin covalently linked to the polymer and the recombinant scFv modified with a C-terminal bungarotoxin-binding peptide. The GD2 antigen binding activity and GD2-specific cytotoxicity of the targeted noncovalent polymer-scFv complex proved to be superior to the covalent polymer-scFv conjugate.

Effects of Glucan and Vitamin D Supplementation on Obesity and Lipid Metabolism in Diabetic Retinopathy.

BACKGROUND: Diabetes mellitus is a chronic disease manifested by an increase of blood glucose. OBJECTIVE: To evaluate the effects of glucan and vitamin D supplementation in patients with diabetic retinopathy. METHOD: We evaluated the effects of 3-month supplementation with glucan and vitamin D in 54 patients with diabetic retinopathy. We measured levels of vitamin D, cholesterol, HDL cholesterol, and triglycerides. RESULTS: The supplementation strongly decreased the cholesterol levels and improved the levels of HDL cholesterol. In addition, vitamin D levels were strongly improved, but still not at optimal values. CONCLUSION: From our data, we concluded that glucan and vitamin D supplementation strongly influence lipid metabolism and have positive effects on human health.

A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21.

BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. RESULTS: We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. CONCLUSIONS: Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.

Antibody-pHPMA functionalised fluorescent silica nanoparticles for colorectal carcinoma targeting.

The systemic application of highly potent drugs such as cytostatics poses the risks of side effects, which could be reduced by using a carrier system able to specifically deliver the encapsulated drug to the target tissue. Essential components of a nanoparticle-based drug delivery system include the drug carrier itself, a targeting moiety, and a surface coating that minimizes recognition by the immune system. The present work reports on the preparation, in vitro characterization and in vivo testing of a new delivery system consisting of fluorescent silica nanoparticles functionalised with a non-immunogenic stealth polymer poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) and a monoclonal antibody IgG M75 that specifically binds to Carbonic Anhydrase IX (CA IX). CA IX is a promising therapeutic target, as it is a hallmark of several hypoxic tumours including colorectal carcinoma. Uniquely in this work, the monoclonal antibody was covalently coupled to the surface of fluorescently labelled silica nanoparticles via a multivalent amino-reactive co-polymer rather than a traditional bivalent linker. The pHPMA-M75 functionalised SiO2 nanoparticles exhibited excellent colloidal stability in physiological media. Their in vitro characterisation by flow cytometry proved a highly specific interaction with colorectal carcinoma cells HT-29. In vivo study on athymic NU/NU nude mice revealed that the SiO2-pHPMA-M75 nanoparticles are capable of circulating in the blood after intravenous administration and accumulate in the tumour at tenfold higher concentration than nanoparticles without specific targeting, with a considerably longer retention time. Additionally, it was found that by reducing the dose administered in vivo, the selectivity of the nanoparticle biodistribution could be further enhanced in favour of the tumour.

Polymer Cancerostatics Targeted with an Antibody Fragment Bound via a Coiled Coil Motif: In Vivo Therapeutic Efficacy against Murine BCL1 Leukemia.

A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate.