mAb 2C5 Research Articles

Abstract 1997: 2C5 antibody modified dendrimer-based mixed micelles for the treatment of multidrug resistant cancers

Abstract Purpose: A major hurdle in chemotherapy is multidrug resistance (MDR) observed in tumors. Downregulation of efflux proteins such as P-glycoprotein (P-gp) using small interfering RNA (siRNA) can be an effective way to treat resistant tumors. In this study, monoclonal antibody 2C5 (mAb 2C5) - PEG7k-DOPE conjugates were post-inserted into the mixed dendrimer micelles containing generation 4 (G4) polyamidoamine (PAMAM)-PEG2k-DOPE and PEG5k-DOPE. The inherent amphiphilic nature of DOPE conjugates causes the copolymers to self-assemble to form a micelle and encapsulate hydrophobic chemotherapeutic drug in its core. The siRNA electrostatically binds to the cationic charges on the G4 PAMAM dendrimer. The tumor-specific mAb 2C5 on the surface of these nano-preparations resulted in improved tumor targeting. Methods: G4 PAMAM-PEG2k-DOPE was prepared by conjugating G4 PAMAM with para-nitrophenol (pNP)-PEG2k-DOPE. To prepare 2C5-PEG7k-DOPE, mAb 2C5 was mixed with micelles containing pNP-PEG7k-DOPE and PEG5k-DOPE in sodium citrate buffer at pH 5.0 and later adjusted the pH to 8.0. In vitro evaluation of the targeted and non-targeted micelles were performed in both MDA-MB-231 and SKOV-3TR resistant cell lines. Further a xenograft MDA-MB-231 in vivo mice tumor models were performed. Results: The 2C5-conjugated mixed dendrimer micelles have a uniform size distribution with a slightly positive zeta potential. They show a stable size and zeta potential for 20 days. The morphology of the 2C5 modified micelles was confirmed using TEM imaging. A higher cellular association was observed in the micelles with the 2C5 antibody targeting using fluorescence imaging (DOPE-FITC tagged micelle) in both the cell lines. At 30 minutes the 2C5 targeted mixed dendrimer micelles showed high cellular uptake and was analyzed using flow cytometry. We further evaluated the P-gp downregulation using western blot and observed a significant downregulation of P-gp levels in the targeted formulation versus the non-targeted formulation. The in vivo tumor growth inhibition study in nude NCG 572 MDA-MB-231 tumor bearing mice showed that the immuno-micelles loaded with siMDR-1 and chemotherapeutics resulted in significantly increased therapeutic efficacy compared to non-modified mixed dendrimer micelles. Conclusions: We have evaluated the 2C5 antibody-modified mixed dendrimer micelles in different cell models, in addition to the models tested in our previous studies. We have successfully established the stability of the formulation in this study and established a 20-day shelf-life. Cellular association, internalization and P-gp downregulation studies show the superiority of the 2C5 modified dendrimer micelles upon non-targeted preparations or free actives. Citation Format: Satya Siva Kishan Yalamarty, Nina Filipczak, Xiang Li, Tanvi Vinod Pathrikar, Colin Cotter, Vladimir P. Torchilin. 2C5 antibody modified dendrimer-based mixed micelles for the treatment of multidrug resistant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1997.

Co-Delivery of siRNA and Chemotherapeutic Drug Using 2C5 Antibody-Targeted Dendrimer-Based Mixed Micelles for Multidrug Resistant Cancers.

Multidrug resistance (MDR) observed in tumors significantly hinders the efficacy of chemotherapy. Downregulation of efflux proteins, such as P-glycoprotein (P-gp), using small interfering RNA (siRNA) can be an effective way to minimize the resistance in tumors. In this study, monoclonal antibody 2C5 (mAb 2C5)-PEG7k-DOPE conjugates were post-inserted into the mixed dendrimer micelles containing generation 4 (G4) polyamidoamine (PAMAM)-PEG2k-DOPE and PEG5k-DOPE. The inherent amphiphilic nature of DOPE conjugates causes the copolymers to self-assemble to form a micelle, which can encapsulate hydrophobic chemotherapeutic drugs in its core. The siRNA electrostatically binds to the cationic charges on the G4 PAMAM dendrimer. The tumor-specific mAb 2C5 on the surface of these nano-preparations resulted in improved tumor targeting. This active targeting to tumors can cause increase in the drug and siRNA accumulation at the tumor site, and thereby minimizing the off-target effects. The micelles were shown to have higher cellular association and effectiveness in vitro. The immunomicelle preparation was also tested for cytotoxicity in breast (MDA-MB-231) and ovarian (SKOV-3TR) MDR cancer cell lines.

Targeted Delivery of Combination Therapeutics Using Monoclonal Antibody 2C5-Modified Immunoliposomes for Cancer Therapy.

To develop immunoliposomes modified with monoclonal cancer-specific antibody (mAb) 2C5 and co-loaded with a combination of two chemotherapeutics, in order to simultaneously target bulk cancer cells using paclitaxel and cancer stem cells (CSCs) using salinomycin to prevent cancer growth and metastases. Breast cancer cells (MDA-MB-231 and/or SK-BR-3) were chosen as models for all in vitro testing. Liposomes composed of natural phospholipids co-loaded with salinomycin and paclitaxel were prepared and physically characterized. Immunoliposomes modified with mAb 2C5 coupled to polymeric conjugate were prepared and characterized for specific targeting. Wound healing assay was performed using the combination of free drugs in vitro. In vitro studies on cellular interaction and uptake were followed by holographic imaging to study cell-killing, cell-division and proliferation inhibiting effects of the formulation. Ex-vivo study on hemolysis was investigated to check possible toxicity of the formulation. Physical characterization of the liposomes showed stable nanoparticles of consistent and desirable size range (170-220nm), zeta potential (-13mV to - 20mV), polydispersity indices (<0.2) and drug encapsulation efficiencies (~150μg per ml for salinomycin, ~210μg/ml for paclitaxel and 1:1 for combination drug loaded liposomes). Combination therapy strongly affected cancer cell proliferation as shown by significant diminishing of artificial gap closure at the wound site on MDA-MB-231 cells in culture using wound healing assay. Quantitation of changes in wound widths showed ~219μm for drug combination, ~104μm for only paclitaxel, and ~ 7μm for only salinomycin treatments. Statistically significant increase in cellular interaction and specific uptake of the targeted drug co-loaded liposomal nanopreparation (p value ≤ 0.05) by MDA-MB-231 and SK-BR-3 cells confirmed the effectiveness of the approach. Holographic imaging using MDA-MB-231 cells produced visible increase in cell-killing, proliferation and division in vitro. Ex-vivo experimentation showed reduced hemolysis correlating with low toxicity in athymic nude mice model. The results demonstrated the enhanced therapeutic efficacy of a combination of salinomycin and paclitaxel delivered by mAb 2C5-modified liposomal preparation in cancer therapy.

Expression kinetics of β-integrin in Chinese shrimp (Fenneropenaeus chinensis) hemocytes following infection with white spot syndrome virus

Our previous study has demonstrated that an integrin β subunit of Chinese shrimp (Fenneropenaeus chinensis) (FcβInt) involved in WSSV infection. In order to further elucidate the potential role of the FcβInt in the WSSV infection, expression response of FcβInt to WSSV infection in shrimp hemocytes was investigated after intra-muscular injection with the virus. Following time-course hemocytes sampling, the expression variation of FcβInt in hemocytes was examined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linked immunosorbent assay (ELISA) using the monoclonal antibody (Mab) 2C5 against FcβInt, which was successfully produced with recombinant partial FcβInt and exhibited binding to a 120 kDa hemocyte protein. Meanwhile, the dynamic state of FcβInt mRNA level and WSSV copies in hemocytes were determined by quantitative real-time PCR. The result of FCIFA showed that FcβInt was mainly expressed on the semi-granular and granular cells, which was down-regulated at 6 h post infection (p.i.), and significantly increased to the peak level at 12 h p.i., then decreased to the control level by 24 h. However, FcβInt on the hyaline cells was lowly expressed and didn't show active response to the viral infection. The variation of FcβInt concentrations in total hemocytes determined by ELISA was roughly in accordance with the changing tendency of FcβInt expressed on the semi-granular and granular cells. FcβInt mRNA level in total hemocytes was significantly up-regulated to the peak level at 12 h p.i. Moreover, the number of WSSV copies in hemocytes began to exhibit a significant increase at 24 h p.i. The present study demonstrated that WSSV infection would induce a differential regulation of FcβInt expression in different type hemocytes, which provided valuable evidences for the close correlation between FcβInt and WSSV infection.

The effect of dual ligand-targeted micelles on the delivery and efficacy of poorly soluble drug for cancer therapy

We prepared and evaluated transferrin (Tf) and monoclonal antibody (mAb) 2C5-modified dual ligand-targeted poly(ethylene glycol)–phosphatidylethanolamine micelles loaded with a poorly soluble drug, R547 (a selective adenosine triphosphate-competitive cyclin-dependent kinase inhibitor) for enhancement of targeting efficiency and cytotoxicity in vitro and in vivo to A2780 ovarian carcinoma compared to single ligand-targeted micelles. Micellar solubilization significantly improved the solubility of R547 from 1 to 800 μg/mL. The size of modified and non-modified micelles was 13–16 nm. Flow cytometry indicated significantly enhanced cellular association of dual ligand-targeted micelles compared to single ligand-targeted micelles. Confocal microscopy confirmed the Tf receptor-mediated endocytosis of rhodamine-labeled Tf-modified micelles after staining the micelle-treated cells with the endosomal marker Tf–Alexa488. The optimized dual-targeted micelles enhanced cytotoxicity in vitro against A2780 ovarian cancer cells compared to plain and single ligand-targeted micelles. Interestingly, in vivo anti-tumor efficacy was more pronounced for the preparation with a single-targeting ligand (Tf). The specific combination Tf and mAb 2C5 did not yield the expected increase in efficacy as was observed in vitro. This observation suggests that the relationships between targeting ligands in vivo could be more complex than in simplified in vitro systems, and the results of the optimization process should always be verified in vivo.

PH-sensitive poly(histidine)-PEG/DSPE-PEG co-polymer micelles for cytosolic drug delivery

To introduce pH sensitivity into the DSPE-PEG-based micellar system and achieve the quick intracellular drug release in response to the acidity in endosomes, a mixed polymeric micelle was developed based on three grafted copolymers, including 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000(DSPE-PEG2000), antinucleosome antibody (mAb 2C5)-modified DSPE-PEG3400 (DSPE-PEG3400-2C5), and poly(ethylene glycol)-coupled poly(l-histidine) (PHIS-PEG2000). The structure of PHIS-PEG2000 was confirmed by 1H NMR spectroscopy. The mixed micelles with the diameter ranging from 110 to 135 nm were prepared using a dialysis method against pH 7.6 PBS. Paclitaxel (PCT) was used as a model drug, the encapsulation efficiency and loading content of PCT were 88% and 5%, respectively. The mixed micelles composed with 50wt% of PHIS-PEG2000 showed the desired pH-dependent drug release property with much faster drug release than micelles without PHIS-PEG2000. At pH around 5.5, about 75–95% of the loaded drug was released within 2 h. The MTT assay showed PCT-loaded mixed micelles had higher cytotoxicity at pH 5.8 than that at pH 7.4. Further modification of the mixed micelles with anti-cancer nucleosome-specific monoclonal antibody 2C5 significantly increased their cellular uptake efficiency and cytotoxicity. Thus, the low pH in endosomes could trigger the PCT release from the pH-sensitive mixed micelles after 2C5-mediated endocytosis. The results of this study suggest that the mixed micelles (DSPE-PEG2000/DSPE-PEG3400-2C5/PHIS-PEG2000) could enhance the tumor cell-specific internalization and trigger the quick drug release, resulting in the improved anti-cancer efficacy.

PEG-PE-based micelles co-loaded with paclitaxel and cyclosporine A or loaded with paclitaxel and targeted by anticancer antibody overcome drug resistance in cancer cells

The over-expression of the P-glycoprotein (P-gp) in cancer cells is one of the main reasons of the acquired Multidrug Resistance (MDR). Combined treatment of MDR cancer cells with P-gp inhibitors and chemotherapeutic agents could result in reversal of resistance in P-gp-expressing cells. In this study, paclitaxel (PTX) was co-encapsulated in actively targeted (anticancer mAb 2C5-modified) polymeric lipid-core PEG-PE-based micelles with Cyclosporine A (CycA), which is one of the most effective first generation P-gp inhibitors. Cell culture studies performed using MDCKII (parental and MDR1) cell lines to investigate the potential MDR reversal effect of the formulations. The average size of both empty and loaded PEG2000-PE/Vitamin E mixed micelles was found between 10 and 25 nm. Zeta potentials of the formulations were found between −7 and −35 mV. The percentage of PTX in the micelles was found higher than 3% for both formulations and cumulative PTX release of about 70% was demonstrated. P-gp inhibition with CycA caused an increase in the cytotoxicity of PTX. Dual-loaded micelles demonstrated significantly higher cytotoxicity in the resistant MDCKII-MDR1 cells than micelles loaded with PTX alone. Micelle modification with mAb 2C5 results in the highest cytotoxicity against resistant cells, with or without P-gp modulator, probably because of better internalization bypassing the P-gp mechanism. Our results suggest that micelles delivering a combination of P-gp modulator and anticancer drug or micelles loaded with only PTX, but targeted with mAb 2C5 represent a promising approach to overcome drug resistance in cancer cells.

Matrix Metalloprotease 2-Responsive Multifunctional Liposomal Nanocarrier for Enhanced Tumor Targeting

A novel "smart" multifunctional drug delivery system was successfully developed to respond to the up-regulated matrix metalloprotease 2 (MMP2) in the tumor microenvironment and improve cancer cell-specific delivery of loaded drugs. The system represents a surface-functionalized liposomal nanocarrier, for which two functional polyethylene glycol (PEG)-lipid conjugates were synthesized and characterized. The functionalized liposome was further modified with the tumor cell-specific antinucleosome monoclonal antibody (mAb 2C5). In the resulting system, several drug delivery strategies were combined in the same nanocarrier in a simple way and coordinated in an optimal fashion. The functions of the nanocarrier include (i) the hydrophilic and flexible long PEG chains to prevent nanocarrier nonspecific interactions and prolong its circulation time; (ii) a nanoscale size of the system that allows for its passive tumor targeting via the enhanced permeability and retention (EPR) effect; (iii) a mAb 2C5 to allow for the specific targeting of tumor cells; (iv) a matrix metalloprotease 2-sensitive bond between PEG and lipid that undergoes cleavage in the tumor by the highly expressed extracellular MMP2 for the removal of PEG chains; (v) cell-penetrating peptide (TATp) triggering of the enhanced intracellular delivery of the system after long-chain PEG removal and exposure of the previously hidden surface-attached TATp. It is shown that such a design can enhance the targetability and internalization of nanocarriers in cancer cells.

Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody–hapten complex: Rapid and robust IgG response induced by a single subcutaneous injection

Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90μg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens.

Nanobiopolymer for Direct Targeting and Inhibition of EGFR Expression in Triple Negative Breast Cancer

Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.

Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicity

pH-sensitive PEGylated (with PEG-PE) long-circulating liposomes (HSPC:cholesterol and Doxil®), modified with cell-penetrating TAT peptide (TATp) moieties and cancer-specific mAb 2C5 were prepared. A degradable pH-sensitive hydrazone bond between a long shielding PEG chains and PE (PEG2k-Hz-PE) was introduced. TATp was conjugated with a short PEG1k-PE spacer and mAb 2C5 was attached to a long PEG chain (2C5-PEG3.4k-PE). The “shielding” effect of TATp by long PEG chains was investigated using three liposomal models. At normal pH, surface TATp moieties are “hidden” by the long PEG chains. Upon the exposure to lowered pH, this multifunctional carrier exposes TATp moieties after the degradation of the hydrazone bond and removal of the long PEG chains. Enhanced cellular uptake of the TATp-containing immunoliposomes was observed in vitro after pre-treatment at lowered pH (using flow cytometry and fluorescence microscopy techniques). The presence of mAb 2C5 on the liposome surface further enhanced the interaction between the carrier and tumor cells but not normal cells. Furthermore, multifunctional immuno-Doxil® preparation showed increased cellular cytotoxicity of B16-F10, HeLa and MCF-7 cells when pre-incubated at lower pH, indicating TATp exposure and activity. In conclusion, a multifunctional immunoliposomal nanocarrier containing a pH-sensitive PEG-PE component, TATp, and the cancer cell-specific mAb 2C5 promotes enhanced cytotoxicity and carrier internalization by cancer cells and demonstrates the potential for intracellular drug delivery after exposure to lowered pH environment, typical of solid tumors.

Abstract 4433: Nanoconjugate mediated inhibition of EGFR expression of triple negative breast cancer

Abstract Objective: We synthesized novel nanoconjugate based on a polymalic acid (PMLA) nanoplatform for triple negative breast cancer treatment. Aberrant expression of EGFR correlates with poor prognosis of breast cancer and anti-EGFR therapy has been recognized to play an important role for triple negative breast cancer patients. However, conventional small molecule drugs lack tumor specificity and cause adverse effect on normal cells. To improve anti-cancer and tumor specific targeting effects, we synthesized a new nanoconjugate carrying the monoclonal antibody (mAb) 2C5. This antibody has binding specificity for tumor cells, while PMLA harbors a mouse anti-transferrin receptor antibody (TfR) and a antisense oligonucleotide to EGFR (AON) allowed us to inhibit breast cancer growth. Materials and Methods: The nanoconjugates are: (1) PMLA-AON with 2C5 and TfR for tumor endothelial and cancer cell targeting, (2) PMLA-AON with 2C5, and (3) only 2C5 without AON. (4) PBS and (5) PMLA were used as a control. Anti-mouse TfR antibody mediated polymer transcytosis through tumor vascular endothelial cells, and 2C5 targeted tumor cell specifically. EGFR overexpressing (MDA-MB-468, SKBR-3) breast cancer cells were used to evaluate drug effect after treatments in vitro. MDA-MB-468 triple negative breast cancer cells were inoculated subcutaneously in nude mice. Drugs were injected intravenously and tumor sizes were measured. Intravenous injections with a nanoconjugate labeled with Alexa Fluor 680 were done for imaging analysis. Drug accumulation was analyzed by Xenogen IVIS 200 and subsequent confocal microscopy. EGFR, phosphorylated Akt and total Akt expressions were detected by Western blotting using in vivo tumor samples undergoing different drug treatments as described above. Results: In vitro Western blot showed that PMLA-AON with targeting antibody inhibited EGFR synthesis more than naked AON. In vivo imaging and confocal microscopy revealed increased drug accumulation with targeting mAbs. Nanoconjugates carrying both mAb and AON significantly inhibited breast tumor growth in vivo as compared to controls (P&amp;lt;0.03). The highest inhibition of tumor growth was observed in mice treated with the leading nanoconjugate variant (1) PMLA-AON with 2C5 and TfR, (P=0.03 vs. controls; P=0.03 vs. nanoconjugate variant (2)). Leading nanoconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than variant (2) and controls on protein level expression. Summary: The newly designed nanoconjugate with covalently attached monoclonal antibody (2C5 and TfR) delivered EGFR inhibitors specifically into the cancer cells by passing host endothelial system and tumor cell membrane. Systemic treatment of triple negative human breast tumor-bearing nude mice with a nanoconjugate showed significant anti-tumor effects in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2011-4433

Antinuclear Antibodies With nucleosome-restricted Specificity for Targeted Delivery of Chemotherapeutic Agents

Circulating antinuclear autoantibodies (ANAs) are well known to accompany various pathological conditions and can be artificially induced by immunization. Research and clinical data permit us to hypothesize a definite connection between cancer and ANAs. Based on the available data, my group's research suggested that exogenous ANAs may be used as anticancer therapeutics. Among these ANAs, nucleosome-specific ANAs may be particularly useful. Advances in cancer immunotherapy with monoclonal antibodies re-emphasized the role of humoral immunity in neoplasia control. The development of a universal antibody targeting diverse cancers is of clear importance. We showed that certain natural ANAs recognize the surface of numerous tumor cells but not normal cells via cell surface-bound nucleosomes originating from the apoptotically dying neighboring tumor cells, mediate antibody-dependent cellular cytotoxicity of tumor cells in vitro and inhibit the development of murine tumor in syngeneic mice. A single monoclonal antinuclear nucleosome-specific autoantibody, mAb 2C5, specifically recognizes multiple unrelated human tumor cell lines and accumulates at a high tumor-to-normal cell ratio in various human tumors in nude mice. Immunotherapy with mAb 2C5 resulted in significant suppression of the growth of several human tumors. In addition, mAb 2C5, when used in subtherapeutic quantities, can serve as a highly efficient specific ligand to target various drug- or diagnostic agent-loaded pharmaceutical nanocarriers, such as liposomes and polymeric micelles, to various tumors. Here, the data (accumulated predominantly in our laboratory over several years) on mAb 2C5-mediated tumor targeting of chemotherapeutic agents is reviewed.

Production and characterization of agonistic monoclonal antibodies against chicken CD40

CD40 is mainly expressed by professional antigen-presenting cells (APCs). Its ligand, CD40L, is transiently expressed on activated CD4 + T-cells. CD40–CD40L interactions mediate T-cell help to APCs and provide crucial signals for affinity maturation and B-cell class switching. In mammals, agonistic monoclonal anti-CD40 antibodies (mAbs) mimic the effects of CD40L on APCs, leading to enhanced T-cell priming and expansion, increased antibody production and class switching. In this study, we describe agonistic anti-chicken CD40 mAb 2C5. This mAb detected CD40 on primary chicken B-cells and macrophages, DT40 B-cells, and HD11 macrophages, induced NO synthesis in HD11 macrophages, and stimulated DT40 B-cell proliferation. These observations demonstrated at least partial functional equivalence of 2C5 to chicken CD154. This mAb may therefore constitute a new tool to study the role of CD40 in the chicken immune system, and its agonistic effects suggest that it could also be used as an adjuvant.

Tumor-Targeted Nanomedicines: Enhanced Antitumor Efficacy In vivo of Doxorubicin-Loaded, Long-Circulating Liposomes Modified with Cancer-Specific Monoclonal Antibody

The efficacy of drug delivery systems can be enhanced by making them target specific via the attachment of various ligands. We attempted to enhance tumor accumulation and therapeutic effect of doxorubicin-loaded, long-circulating, polyethylene glycol-coated liposomes (Doxil, ALZA Corp.) by coupling to their surface the anticancer monoclonal antibody (mAb) 2C5 with nucleosome-restricted activity that can recognize the surface of various tumors but not normal cells and specifically targets pharmaceutical carriers to tumor cells in vitro and in vivo. Following earlier in vitro results with various cancer cell lines, the mAb 2C5 liposomes were studied in vivo versus plain and nonspecific-IgG liposomes. Antibody coupling to Doxil was done via the "postinsertion" technique. Using (111)In-labeled liposomes, the tissue biodistribution and pharmacokinetic profile were studied, as well as their accumulation in tumors in mice, followed by the whole-body gamma-scintigraphic imaging. Therapeutic efficacy of mAb 2C5-targeted Doxil versus nonspecific IgG-modified and original Doxil controls was followed by registering live tumor growth and determining tumor weights upon mice sacrifice. mAb 2C5-targeted liposomes showed enhanced accumulation in tumors, and the in vivo therapeutic activity of the mAb 2C5-Doxil treatment was found to be significantly superior, resulting in final tumor weights of only 25% to 40% compared with all Doxil control treatments, when tested against the s.c. primary murine tumors of 4T1 and C26 and human PC3 tumor in nude mice. Our results showed the remarkable capability of 2C5-targeted Doxil to specifically deliver its cargo into various tumors, significantly increasing the efficacy of therapy.

Nanosized cancer cell-targeted polymeric immunomicelles loaded with superparamagnetic iron oxide nanoparticles

Stable 30–50 nm polymeric polyethylene glycol–phosphatidylethanolamine (PEG–PE)-based micelles entrapping superparamagnetic iron oxide nanoparticles (SPION) have been prepared. At similar concentrations of SPION, the SPION-micelles had significantly better magnetic resonance imaging (MRI) T2 relaxation signal compared to ‘plain’ SPION. Freeze-fracture electron microscopy confirmed SPION entrapment in the lipid core of the PEG–PE micelles. To enhance the targeting capability of these micelles, their surface was modified with the cancer cell-specific anti-nucleosome monoclonal antibody 2C5 (mAb 2C5). Such mAb 2C5-SPION immunomicelles demonstrated specific binding with cancer cells in vitro and were able to bring more SPION to the cancer cells thus demonstrating the potential to be used as targeted MRI contrast agents for tumor imaging.

Plasma Osteopontin Modulates Chronic Restraint Stress-Induced Thymus Atrophy by Regulating Stress Hormones: Inhibition by an Anti-Osteopontin Monoclonal Antibody

Osteopontin (OPN) is a cytokine implicated in mediating responses to certain stressors, including mechanical, oxidative, and cellular stress. However, the involvement of OPN in responding to other physical and psychological stress is largely unexplored. Our previous research revealed that OPN is critical for hind limb-unloading induced lymphoid organ atrophy through modulation of corticosteroid production. In this study, we demonstrate that OPN(-/-) mice are resistant to chronic restraint stress (CRS)-induced lymphoid (largely thymus) organ atrophy; additionally, the stress-induced up-regulation of corticosterone production is significantly reduced in OPN(-/-) mice. Underlying this observation is the fact that normal adrenocorticotropic hormone levels are substantially reduced in the OPN(-/-) mice. Our data demonstrate both that injection of OPN into OPN-deficient mice enhances the CRS-induced lymphoid organ atrophy and that injection of a specific anti-OPN mAb (2C5) into wild-type mice ameliorates the CRS-induced organ atrophy; changes in corticosterone levels were also partially reversed. These studies reveal that circulating OPN plays a significant role in the regulation of the hypothalamus-pituitary-adrenal axis hormones and that it augments CRS-induced organ atrophy.

Tumor-Specific Anti-Nucleosome Antibody Improves Therapeutic Efficacy of Doxorubicin-Loaded Long-Circulating Liposomes against Primary and Metastatic Tumor in Mice

The efficacy of drug delivery systems can be significantly enhanced by making them target-specific via the attachment of various ligands to their surface. We attempted to enhance tumor accumulation and therapeutic effect of doxorubicin-loaded long-circulating liposomes (Doxil, ALZA Corp.) by coupling to their surface the anticancer monoclonal antibody 2C5 (mAb 2C5) with nuclesome (NS)-restricted activity, that can recognize the surface of various tumor but not normal cells via the surface-bound nucleosomes released from the apoptotically dying neighboring tumor cells and specifically targets pharmaceutical carriers to tumor cells in vitro and in vivo. Antibody coupling to PEGylated doxorubicin-liposomes was performed by the "post-insertion" technique. The pharmacokinetics of plain and immuno-targeted Doxil-mimicking liposomes, as well as their accumulation in primary Lewis lung carcinoma (LLC) tumors in mice was followed by real-time gamma-scintigraphy upon liposomal membrane labeling with (111)In. Therapeutic action of various liposomal formulations was followed by registering primary tumor growth, determining tumor weigh upon mice sacrifice, and by counting the number of metastases in the liver and lungs. 2C5 antibody-targeted liposomes demonstrate significantly enhanced accumulation in LLC tumors. Targeted doxorubicin-loaded PEG-liposomes were significantly more effective in inhibiting tumor growth and metastatic process in the LLC tumor models in mice. Our results clearly show the remarkable capability of 2C5-targeted Doxil to specifically deliver its cargo into various tumor manifestations (solid and metastatic) significantly increasing the efficacy of therapy.

Mixed PEG–PE/vitamin E tumor-targeted immunomicelles as carriers for poorly soluble anti-cancer drugs: Improved drug solubilization and enhanced in vitro cytotoxicity

Two poorly soluble, potent anti-cancer drugs, paclitaxel and camptothecin, were successfully solubilized by mixed micelles of polyethylene glycol–phosphatidyl ethanolamine (PEG–PE) and vitamin E. Drug-containing micelles were additionally modified with anti-nucleosome monoclonal antibody 2C5 (mAb 2C5), which can specifically bring micelles to tumor cells in vitro. The optimized micelles had an average size of about 13–22 nm and the immuno-modification of micelles did not significantly change it. The solubilization of both drugs by the mixed micelles was more efficient than by micelles made of PEG–PE alone. Solubilization of camptothecin in micelles prevented also the hydrolysis of active lactone form of the drug to inactive carboxylate form. Drug-loaded mixed micelles and mAb 2C5-immunomicelles demonstrated significantly higher in vitro cytotoxicity than free drug against various cancer cell lines.

Tumor-Targeted Immuno-liposomes for Delivery of Chemotherapeutics and Diagnostics

The monoclonal antibody, mAb 2C5, recognizing the surface of numerous tumor, but not normal, cells via their surface-bound nucelosomes, was coupled onto sterically protected poly(ethylene glycol) liposomes. In this study, the development of different mAb 2C5-targeted formulations, like radiolabeled liposomes for gamma imaging of tumors and doxorubicin-containing liposomes for cancer chemotherapy, is described. Our data demonstrate the enhanced in vitro anticancer activities and the evident tumor-avid in vivo biodistribution, confirming the superior tumor-selectivity of the mAb 2C5-modified liposomes. This report reveals the eminent potential for this tumor-targeting strategy, with mAb 2C5, in improving the delivery of drugs and imaging agents into various tumors.