Cancer Biotherapy and Radiopharmaceuticals

Antibodies are highly versatile proteins with the ability to be used to target diverse compounds, such as radionuclides for imaging and therapy, or drugs and toxins for therapy, but also can be used unconjugated to elicit therapeutically beneficial responses, usually with minimal toxicity. This update describes a new procedure for forming multivalent and/or multispecific proteins, known as the dock-and-lock (DNL) technique. Developed as a procedure for preparing bispecific antibodies capable of binding divalently to a tumor antigen and monovalently to a radiolabeled hapten-peptide for pretargeted imaging and therapy, this methodology has the flexibility to create a number of other biologic agents of therapeutic interest. A variety of constructs, based on anti-CD20 and CD22 antibodies, have been made, with results showing that multispecific antibodies have very different properties from the respective parental monospecific antibodies. The technique is not restricted to antibody combination, but other biologics, such as interferon-α2b, have been prepared. These types of constructs not only allow small biologics to be sustained in the blood longer, but also to be selectively targeted. Thus, DNL technology is a highly flexible platform that can be used to prepare many different types of agents that could further improve cancer detection and therapy.

Exosomes (EXOs) derived from tumor cells have been used to stimulate antitumor immune responses. It has been demonstrated that EXO released by tumor cells engineered to express cytokines are of enhanced stimulatory effect on CD8⁺ cytotoxic T-lymphocyte (CTL) responses and antitumor immunity. J558 is a mouse myeloma cell line expressing tumor antigen P1A. In this study, we purified EXO_TNF-a, EXO_IL-2, and EXO_IFN-γ released by three cytokine-gene (TNF-α, IL-2 and IFN-γ)-engineered J558 (J558_TNF-a, J558_IL-2 and J558_IFN-γ) tumor cell lines from their culture supernatants, respectively, by differential ultracentrifugation. These EXOs showed a “saucer” or round shape with a diameter between 50 and 90 nm by electron microscopy and contained EXO-associated proteins, such as LAMP-1 and AIP1, but not lysate-associated protein galectin, by Western blot analysis. EXO displayed expression of molecules (H-2K^d, CD54, and P1A) similarly to, but to a lesser extent to, J558 tumor cells. We then compared the stimulatory effect of these EXOs on P1A-specific CD8⁺ CTL responses and antitumor immunity 6 days subsequent to intravenous (i.v.) EXO immunization (30 μg/each BALB/c mouse). We demonstrated that EXO_TNF-α immunization was able to induce more efficient P1A-specific CD8⁺ T-cell response accounting for 0.62% of the total CD8⁺ T-cell population, using PE-H-2K^d/P1A peptide and FITC-CD8 staining by flow cytometric analysis then EXO_IL-2 (0.31%) and EXO_IFN-γ (0.22%) immunization (P < 0.05), respectively, at day 6 after immunization. EXO_IL-2 and EXO_IFN-γ vaccine (i.v. 30 μg/each mouse) only protected 3 of 8 (38%) and 2 of 8 (25%) mice from tumor growth after subcutaneous (s.c.) challenging of immunized mice with J558 tumor cells (0.5 × 10⁶ cells/each mouse), whereas EXO_TNF-α immunization protected all 8 of 8 (100%) mice from tumor growth (P < 0.05). Taken together, we demonstrate that EXO_TNF-a released by engineered J558_TNF-a tumor cells more efficiently stimulate tumor antigen P1A-specific CD8⁺ CTL responses and antitumor immunity than EXO_IL-2 and EXO_IFN-γ released by engineered J558_IL-2 and J558_IFN-γ tumor cells. Therefore, TNF-α-expressing tumor cell-released EXO may represent a more effective EXO-based vaccine in the induction of antitumor immunity.

Interleukin-24 (IL-24)/melanoma differentiation associated gene-7 (mda-7) as a novel tumor-suppressor gene has potent antitumor activities in a broad spectrum of human cancers through the activation of various signaling pathways. However, the suppressive effect of adenovirus-mediated IL-24 (Ad-IL-24) expression on human laryngeal cancers is still elusive. In this study, we explored the therapeutic effect of Ad-IL-24 on human laryngeal cancers in vitro and in vivo in an athymic nude mouse model, using a Hep-2 human laryngocarcinoma cell line, and a WI-38 human diploid cell line served as a normal cell control. We demonstrated that Ad-IL-24 induced significant growth inhibition and apoptosis, upregulated the expression of P21, P27, and Bax, downregulated Bcl-2 expression, and activated caspase-3 in Hep-2 laryngeal tumor cells, while it exerted no direct effect on the in vitro proliferation of WI-38 normal diploid cells. Moreover, intratumoral injections of Ad-IL-24 in nude mice bearing Hep-2 tumors significantly suppressed the laryngeal xengrafted tumor growth and reduced microvessel density (MVD) and VEGF expression in tumors. This retarded tumor growth in vitro and in vivo elicited by Ad-IL-24 was closely associated with the upregulation of proliferation-related molecules P21 and P27, decrease in the ratio of anti- to proapoptotic molecules Bcl-2/Bax, followed by the activation of caspase-3, leading to apoptosis via intrinsic apoptotic pathways, and the reduced expression of proangiogenic factor VEGF involved in the inhibition of tumor angiogenesis. Thus, our results indicate that the potent, selective killing activity of Ad-IL-24 in laryngeal cancer cells, but not in normal cells, makes this vector a potential candidate for laryngeal cancer gene therapy.

We investigated whether natural killer (NK) cells in the tumor microenvironment have a radiosensitization effect. The radiosensitization effect of combined CpG and Herceptin^® (Genentech, Inc., South San Francisco, CA) (CpG/Herceptin), given before or after radiation, was evaluated by using a murine colon cancer cell line overexpressing human HER2/neu, CT26HER2/neu. In vitro radiosensitization effects were investigated by coculture of CT26HER2/neu with splenocytes, CpG, and Herceptin before applying radiation. Tumor cells, cocultured with CpG-pretreated splenocytes and Herceptin, were more vulnerable to radiation damage. In BALB/c mice injected with CT26HER2/neu, CpG/Herceptin administered before radiotherapy was associated with a better retardation of tumor growth than when administered after radiotherapy. The radiosensitization effect was significantly abrogated by NK-cell depletion, indicating that NK cells play an essential role in it. Further, surviving mice treated with CpG or CpG/Herceptin and reverse transcriptase were resistant to renewed tumor challenge, suggesting the presence of an induced immune response to the tumor. Neoadjuvant immunotherapy with CpG/Herceptin may improve response to radiotherapy of HER2/neu-expressing tumors.

The aim of this study was to develop a ¹⁸⁸Re-labeled porphyrin-based tumor-specific agent and to evaluate its biologic behavior, including tumor-regressing effectiveness, in mouse tumor models for possible use in achieving targeted cancer radiotherapy. ¹⁸⁸Re was obtained from an alumina-column–based ¹⁸⁸W-¹⁸⁸Re generator constructed in-house. The compound, 5,10,15,20-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin, was synthesized and labeled with ¹⁸⁸ReO₄ ⁻. ¹⁸⁸Re-labeled porphyrin complex was produced with a radiochemical purity of ∼98% with reasonably good in vitro stability (>24 hours at 4°C). Swiss mice bearing thymic lymphoma and fibrosarcoma were used as tumor models. The biodistribution studies revealed satisfactory tumor retention (2.07% ± 0.80% injected activity per g) with insignificant activities in blood (0.53%), liver (0.26%) and kidney (0.04%) at 24 hours. The radiolabeled conjugate treatment increased the average tumor-doubling time and decreased the average specific growth rate substantially in thymic lymphoma, compared to fibrosarcoma tumor. ¹⁸⁸Re-labeled 5,10,15,20-tetrakis[3,4 bis(carboxymethyleneoxy)phenyl] porphyrin has specific affinity toward the fibrosarcoma and thymic lymphoma tumors in mice. Thymic lymphoma was found to be more sensitive to the radionuclide complex, compared to fibrosarcoma. The ¹⁸⁸Re-labeled porphyrin complex showed promising results and warrants further investigations.

Vascular endothelial growth factor (VEGF) plays an important role in the growth and metastasis of non-small-cell lung cancer (NSCLC). The aim of this study was to develop an RNA-interference approach that targets VEGF, using a recombinant plasmid, and to explore its antitumor efficacy in NSCLC in vivo. shRNA-targeting VEGF was cloned into pGenesil-2 plasmid vector and then transfected into A549 human lung cancer cells, using cationic liposome. Reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analysis were used to evaluate the silencing effects of VEGF-shRNA on A549 cells in vitro. Further, the growth-inhibition capacity of VEGF-shRNA on A549 lung carcinoma xenografts was tested in nude mice. Proliferation, apoptosis, and angiogenesis in tumor tissues were measured by PCNA, TUNEL, and CD31 immunohistochemistry, respectively. shRNA-targeting VEGF significantly silenced VEGF expression in A549 lung cancer cells, as confirmed by RT-PCR and ELISA assay (P < 0.01). In vivo, the VEGF-shRNA delayed tumor growth and reduced tumor weight by ∼61.96%, compared with control groups (P < 0.05), accompanied with angiogenesis inhibition (P < 0.01) and apoptosis induction (P < 0.01). Our data showed that the knockdown of VEGF by shRNA might be a potential therapeutic approach against human NSCLC.

Activation of autophagy is a hallmark in tumor cells treated with chemotherapy, but the role of autophagy in acquired resistance of lung adenocarcinoma to cisplatin-based chemotherapy remains to be clarified. Our aim was to address that question by surveying the autophagic activity in parental lung adenocarcinoma cell line A549 and its 8-fold, more resistant subcell line, A549/DDP, which was obtained by treating cisplatin with increasing concentrations. A549/DDP and A549 cells were exposed to serum-free culture medium or ionizing radiation. To measure the stress-induced autophagy, LC3-II, as an autophagosome marker, was measured by immunofluorescence and Western blotting. To determine the effect of 3-MA, a known inhibitor of autophagy, on overcoming acquired cisplatin resistance, the MTT assay and flow cytometry were performed. Western blotting analysis demonstrated that LC3-II was increased in A549/DDP cells, compared with those of parental A549 cells, under stress conditions. Meanwhile, immunofluorescence staining showed that LC3-II protein was located mainly in the cytoplasm of A549/DDP. We also found that 3-MA can enhance the growth inhibition and apoptotic effect of cisplatin in acquired resistant cells (A549/DDP). Collectively, our results provide evidence that the upregulation of autophagy plays a major role in cisplatin resistance of A549/DDP cells.

Radiolabeled neuropeptides are widely explored for targeting tumours for either imaging or radiotherapeutic purposes. After binding to their receptors, these peptides are rapidly internalized into lysosomes, where they are degraded by proteolytic enzymes, such as cathepsins. The aim of this study was to investigate the effect of the inclusion of specific cleavage sites for cathepsin B into the peptide sequence. The cleavage site, GFLG, together with a series of dipeptides for pharmacokinetic modification of radiometabolites, were, therefore, inserted into a peptide that binds to the gastrin/CCK2 receptor. The receptor binding of the peptides was explored in AR42J cells, rates of internalization, and externalization of the radionuclide were measured and the nature of the radiometabolites explored. The effects of the modifications on biodistribution in tumor-bearing mice was explored by high-resolution single-photon emission computed tomography imaging. Differences in rates of externalization from tumor cells in vitro and in the rates of washout from tumor and kidney in vivo were observed. These results indicate that insertion of an enzymatic cleavage site, such as that for cathepsin B, into a neuropeptide appears to have an influence on the intracellular processing, which results in a change in the rate of egress of radioactivity from target and nontarget tissues.

Nonviral delivery systems are relatively easy to produce in the large scale, are safe, and elicit a negligible immune response. Nanoparticles (NPs) offer promise as nonviral vectors as biocompatible and -degradable carriers of drugs with targeting to specific sites by surface receptors of monoclonal antibodies (mAbs). We investigated the effect of four PEG-PLGA (polyethylene glycol-polylactic-co-glycolic acid) NP systems on drug-resistant B-chronic lymphocytic leukemia (B-CLL) cells in vitro, three of them encapsulating the drug, hydroxylchloroquine (HDQ), two with NP surface coatings of mAbs (NP1) CD20, (NP2) CD19, and CD20, and one (NP3) with no mAb, but tagged with the fluorescent marker, fluorescein isothiocyanate. The fourth NP system (NP4) was coated with anti-CD19/FITC and anti-CD20/Alexa-Fluor^® antibodies, but did not contain the active drug, HCQ. Our data indicate that PEG-PLGA nanoparticles with surface mAbs are suitable for selective drug delivery to B-CLL cells and produce a strong apoptotic effect when loaded with the lysosomotropic agent, HDQ.

Fibroblast growth factor 18 (FGF18) is one of the genes downstream of Wnt, one of the most important signaling pathways activated in colon cancer. An FGF18 promoter containing a single T-cell factor/lymphocyte enhancing factor 1 (TCF/LEF1) binding site was inserted upstream of a thymidine kinase (TK) suicide gene module, while a bacterial β-Gal (LacZ) element served as the reporter gene. Following transient transfection with pUCFGF18LacZ, β-Gal staining showed that 5% of SW480, 10% of HCT116, 0% of human umbilical vein endothelial cells (HUVECs) and 0% of normal colon cells (NCCs) had expressed LacZ. β-Gal enzyme-linked immunosorbent assay revealed that the ratio of pUCFGF18LacZ activity to that of positive control was 0.09 and 0.25 in SW480 and HCT116, respectively (significantly higher than mock plasmid), while there were no significant changes in the β-Gal expression in HUVEC and NCC cells transfected with pUCFGF18LacZ or mock plasmid. Following transfection with pUCFGF18TK and pUCCMVTK (positive control), cytotoxicity analysis of transfected cells showed that treatment with ganciclovir (GCV) significantly decreased SW480 and HCT116 cell survival at GCV concentrations above 20 μg/mL. An inverse correlation between GCV concentration and cell viability was evident in both colon cancer cell lines following transfection with these suicide plasmids. pUCFGF18TK and pUCCMVTK induced apoptosis after the administration of GCV in HCT116, but not in SW480, as demonstrated by M30 cytodeath antibody. This discrepancy may stem from differences in the mechanisms of TK/GCV-induced apoptosis in p53-proficient (HCT116) and -deficient (SW480) cells. The specific activity of the FGF18 promoter in HCT116 and SW480 may reflect the advantage of this promoter over artificial promoters containing artificial TCF/LEF binding sites.

Copper(II) bis(thiosemicarbazone) are very useful for blood flow and hypoxic imaging. The aim of this study was to identify structure-activity relationships (SARs) within a series of analogues with different substitution patterns in the ligands, in order to design improved hypoxia imaging agents and elucidate hypoxia selectivity mechanisms. Genetic algorithms (GAs) were used to develop specific copper metal-ligand force field parameters for the MM3 force-field calculations. These new parameters produced results in good agreement with experiment and previously reported copper metal-ligand parameters. A successful quantitative SAR (QSAR) for predicting the several classes of Cu(II)-chelating ligands was built using a training set of 21 Cu(II) complexes. The QSAR exhibited a correlation between the predicted and experimental test set. The QSAR preformed with great accuracy; r ² = 0.95 and q ² = 0.90 utilizing a leave-one-out cross-validation with multiple linear regression analysis to find correlation between different calculated molecular descriptors of these complexes. The final QSAR mathematical models were found as the following: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}&\hbox{QSAR model for copper(II) bis(thiosemicarbazone) }\\&\hbox{Log P} \ = \{ 3.01698 \ (\pm 0.0590)\} - \hbox{LUMO} \ \{ 0.1248 \ (\pm 0.068)\} + \hbox{MR} \ \{ 0.3219 \ (\pm 0.086)\} \\& n = 21 \quad |r| = 0.972 \quad s = 0.188 \quad F = 98.102 \end{align*} \end{document}

The resulting models could act as an efficient strategy for estimating the hypoxic conditions through imaging and provide some insights into the structural features related to the biological activity of these compounds.