Datopotamab Deruxtecan, a Novel TROP2-directed Antibody-drug Conjugate, Demonstrates Potent Antitumor Activity by Efficient Drug Delivery to Tumor Cells.

Trophoblast cell surface antigen 2 (TROP2) is highly expressed on various epithelial tumors and correlates with poor prognosis. We developed the novel TROP2-directed antibody-drug conjugate (ADC), datopotamab deruxtecan (Dato-DXd, DS-1062a), with a potent DNA topoisomerase I inhibitor (DXd), and evaluated its antitumor activity and safety profiles in preclinical models.The pharmacologic activity and mechanism of action of Dato-DXd were investigated in several human cancer cell lines and xenograft mouse models including patient-derived xenograft (PDX) models. Safety profiles were also assessed in rats and cynomolgus monkeys.Dato-DXd bound specifically to TROP2 and was internalized into tumor cells followed by intracellular trafficking to lysosome and DXd release, which induced DNA damage and apoptosis in TROP2-expressing tumor cells in vitro. Dato-DXd exhibited in vivo antitumor activity with DNA damage induced by the accumulated DXd in TROP2-expressing xenograft tumors, but neither isotype control IgG-ADC nor anti-TROP2 antibody had this effect. Dato-DXd also showed potent antitumor activity with tumor regression in several TROP2-expressing xenograft tumors including NSCLC PDX models. Safety profiles of Dato-DXd in rats and cynomolgus monkeys were acceptable.Dato-DXd demonstrated potent antitumor activity against TROP2-expressing tumors by efficient payload delivery into tumors and acceptable safety profiles in preclinical models. These results suggest Dato-DXd could be a valuable treatment option for patients with TROP2-expressing tumors in the clinical setting.

Antibody Screening System Using a Herpes Simplex Virus (HSV)-Based Probe To Identify a Novel Target for Receptor-Retargeted Oncolytic HSVs.

Herpes simplex virus (HSV) is a promising tool for developing oncolytic virotherapy. We recently reported a platform for receptor-retargeted oncolytic HSVs that incorporates single-chain antibodies (scFvs) into envelope glycoprotein D (gD) to mediate virus entry via tumor-associated antigens. Therefore, it would be useful to develop an efficient system that can screen antibodies that might mediate HSV entry when they are incorporated as scFvs into gD. We created an HSV-based screening probe by the genetic fusion of a gD mutant with ablated binding capability to the authentic HSV entry receptors and the antibody-binding C domain of streptococcal protein G. This engineered virus failed to enter cells through authentic receptors. In contrast, when this virus was conjugated with an antibody specific to an antigen on the cell membrane, it specifically entered cells expressing the cognate antigen. This virus was used as a probe to identify antibodies that mediate virus entry via recognition of certain molecules on the cell membrane other than authentic receptors. Using this method, we identified an antibody specific to epiregulin (EREG), which has been investigated mainly as a secreted growth factor and not necessarily for its precursor that is expressed in a transmembrane form. We constructed an scFv from the anti-EREG antibody for insertion into the retargeted HSV platform and found that the recombinant virus entered cells specifically through EREG expressed by the cells. This novel antibody-screening system may contribute to the discovery of unique and unexpected molecules that might be used for the entry of receptor-retargeted oncolytic HSVs.IMPORTANCE The tropism of the cellular entry of HSV is dependent on the binding of the envelope gD to one of its authentic receptors. This can be fully retargeted to other receptors by inserting scFvs into gD with appropriate modifications. In theory, upon binding to the engineered gD, receptors other than authentic receptors should induce a conformational change in the gD, which activates downstream mechanisms required for viral entry. However, prerequisite factors for receptors to be used as targets of a retargeted virus remain poorly understood, and it is difficult to predict which molecules might be suitable for our retargeted HSV construct. Our HSV-based probe will allow unbiased screening of antibody-antigen pairs that mediate virus entry and might be a useful tool to identify suitable pairs for our construct and to enhance our understanding of virus-cell interactions during infection by HSV and possibly other viruses.

Interleukin-13 receptor α2 is a novel marker and potential therapeutic target for human melanoma.

Malignant melanoma is one of the untreatable cancers in which conventional therapeutic strategies, including chemotherapy, are hardly effective. Therefore, identification of novel therapeutic targets involved in melanoma progression is urgently needed for developing effective therapeutic methods. Overexpression of interleukin-13 receptor α2 (IL13Rα2) is observed in several cancer types including glioma and pancreatic cancer. Although IL13Rα2 is implicated in the progression of various types of cancer, its expression and roles in the malignant melanoma have not yet been elucidated. In the present study, we showed that IL13Rα2 was expressed in approximately 7.5% melanoma patients. While IL13Rα2 expression in human melanoma cells decreased their proliferation in vitro, it promoted in vivo tumour growth and angiogenesis in melanoma xenograft mouse model. We also found that the expression of amphiregulin, a member of the epidermal growth factor (EGF) family, was correlated with IL13Rα2 expression in cultured melanoma cells, xenograft tumour tissues and melanoma clinical samples. Furthermore, expression of amphiregulin promoted tumour growth, implicating causal relationship between the expression of IL13Rα2 and amphiregulin. These results suggest that IL13Rα2 enhances tumorigenicity by inducing angiogenesis in malignant melanoma, and serves as a potential therapeutic target of malignant melanoma.

Oncolytic Herpes Simplex Virus Vectors Fully Retargeted to Tumor- Associated Antigens.

Oncolytic virotherapy is a novel therapeutic modality for malignant diseases that exploits selective viral replication in cancer cells. Herpes simplex virus (HSV) is a promising agent for oncolytic virotherapy due to its broad cell tropism and the identification of mutations that favor its replication in tumor over normal cells. However, these attenuating mutations also tend to limit the potency of current oncolytic HSV vectors that have entered clinical studies. As an alternative, vector retargeting to novel entry receptors has the potential to achieve tumor specificity at the stage of virus entry, eliminating the need for replication-attenuating mutations. Here, we summarize the molecular mechanism of HSV entry and recent advances in the development of fully retargeted HSV vectors for oncolytic virotherapy. Retargeted HSV vectors offer an attractive platform for the creation of a new generation of oncolytic HSV with improved efficacy and specificity.

Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus.

Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors. IMPORTANCE: Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

Cell Surface Antibody Retention Influences In Vivo Antitumor Activity Mediated by Antibody-dependent Cellular Cytotoxicity.

BACKGROUND: Multiple factors affect the in vivo antitumor activity of antibody-based therapeutics; however, the influence of cell surface retention on antibody-dependent cellular cytotoxicity (ADCC) is not fully understood. Here we evaluated the importance of cell surface antibody retention in antitumor activity mediated by ADCC in vivo. MATERIALS AND METHODS: Two mAbs against tumor-associated calcium signal transducer 2 (TACSTD2/TROP2), AR47A6.4.2 and Pr1E11, were used. Antitumor activities against BxPC3 and Colo205 cells were investigated through in vitro and in vivo assays. RESULTS: Pr1E11 showed better cell surface retention than AR47A6.4.2 in vitro although Pr1E11 and AR47A6.4.2 showed equivalent ADCC activity. Complement-dependent cytotoxicity and antiproliferative activity were not observed for either antibody. Pr1E11 exhibited higher antitumor activity than AR47A6.4.2 in vivo. CONCLUSION: Our results suggest that high cell surface retention can result in potent ADCC activity in vivo. This observation could provide novel insight into how effectively screen for antibodies with strong in vivo antitumor activity.

Pr1E11, a novel anti-TROP-2 antibody isolated by adenovirus-based antibody screening, recognizes a unique epitope.

TROP-2 is a type Ⅰ transmembrane glycoprotein that is highly expressed in various epithelial cancer cells, and its increased expression correlates with poor prognosis. Although several anti-TROP-2 antibodies have been described, they were found unsuitable for antitumor therapy use in vivo as naked antibodies. In this study, we established a novel anti-TROP-2 antibody, designated Pr1E11, from mice immunized with primary prostate cancer cells. Antibody screening was based on the infection activity of Adv-LacZ-FZ33, which displays an immunoglobulin G binding domain in the adenoviral fiber protein. We found that Pr1E11 specifically binds to TROP-2 with high affinity and recognizes diverse epithelial cancer cell lines and primary pancreatic cancer tissues. Epitope analysis using TROP-2 deletion mutants revealed that binding site of Pr1E11 is a cysteine-rich domain, a unique epitope compared with other available anti-TROP-2 antibodies. In addition, Pr1E11 exhibited low internalization activity, which may make it suitable for naked antibody therapeutics. Our results suggest that Pr1E11 may stimulate different biological activities from other anti-TROP-2 antibodies based on its unique binding epitope, and is a potential candidate for naked antibody therapeutics for various epithelial cancer treatments.

Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells.

Antibody-drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb-DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.

Short-fiber protein of ad40 confers enteric tropism and protection against acidic gastrointestinal conditions.

The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral-fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short-fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter ß-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases.

Cyclophosphamide enhances antitumor efficacy of oncolytic adenovirus expressing uracil phosphoribosyltransferase (UPRT) in immunocompetent Syrian hamsters.

Oncolytic viruses (OVs) are novel cancer therapeutics with great promise, but host antiviral immunity represents the hurdle for their efficacy. Immunosuppression by cyclophosphamide (CP) has thus been shown to enhance the oncolytic efficacy of many OVs, but its effects on OVs armed with therapeutic genes remain unknown. We have previously reported on the efficacy of AxE1CAUP, an oncolytic adenovirus (OAd) expressing uracil phosphoribosyltransferase (UPRT), an enzyme that markedly enhanced the toxicity of 5-fluorouracil (5-FU), in immunodeficient, Ad-nonpermissive nude mice. Here we explored the efficacy and safety of intratumoral (i.t.) AxE1CAUP/5-FU therapy and of its combination with CP for syngenic HaP-T1 pancreatic cancers in immunocompetent, Ad-permissive Syrian hamsters. AxE1CAUP infected, replicated, expressed UPRT, and increased the sensitivity to 5-FU in HaP-T1 cells in vitro. I.t. AxE1CAUP/5-FU treatment inhibited the growth of subcutaneous HaP-T1 allografts. The combination with high-dose CP inhibited serum Ad-neutralizing antibody formation, increased intratumoral AxE1CAUP replication and UPRT expression, and resulted in further enhanced therapeutic effects with 5-FU. Neither body weight nor histology of the liver and lung changed during these treatments. A clinically-approved, intermediate-dose CP also enhanced the efficacy of i.t. AxE1CAUP/5-FU treatment in these hamsters, which was not affected by preexisting immunity to the vector. These data demonstrate the excellent antitumor efficacy and safety of an OAd armed with a suicide gene in combination with CP for treating syngenic tumors in immunocompetent, Ad-permissive animals, indicating the efficacy of CP in overcoming the hurdle of antiviral immunity for effective OV-mediated gene therapy.

Manipulation of human early T lymphopoiesis by coculture on human bone marrow stromal cells: potential utility for adoptive immunotherapy.

T cell precursors are an attractive target for adoptive immunotherapy. We examined the regulation of human early T lymphopoiesis by human bone marrow stromal cells to explore in vitro manipulation of human T cell precursors in a human-only coculture system. The generation of CD7(+)CD56(-)cyCD3(-) proT cells from human hematopoietic progenitors on telomerized human bone marrow stromal cells was enhanced by stem cell factor, flt3 ligand, and thrombopoietin, but these stimulatory effects were suppressed by interleukin 3. Expression of Notch ligands Delta-1 and -4 on stromal cells additively promoted T cell differentiation into the CD7(+)cyCD3(+) pre-T cell stage, while cell growth was strongly inhibited. By combining these coculture systems, we found that initial coculture with telomerized stromal cells in the presence of stem cell factor, flt3 ligand, and thrombopoietin, followed by coculture on Delta-1- and -4-coexpressing stromal cells led to a higher percentage and number of pre-T cells. Adoptive immunotherapy using peripheral blood T cells transduced with a tumor antigen-specific T cell receptor (TCR) is a promising strategy but has several limitations, such as the risk of forming a chimeric TCR with the endogenous TCR. We demonstrated that incubation of TCR-transduced hematopoietic progenitors with the combination of coculture systems gave rise to CD7(+)TCR(+)CD3(+)CD1a(-) T cell precursors that rapidly proliferated and differentiated under the culture condition to induce mature T cell differentiation. These data show the regulatory mechanism of early T lymphopoiesis on human stromal cells and the potential utility of engineered human stromal cells to manipulate early T cell development for clinical application.

Pancreatic stellate cells enhance stem cell-like phenotypes in pancreatic cancer cells.

The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Recent studies have identified that a portion of cancer cells, called "cancer stem cells", within the entire cancer tissue harbor highly tumorigenic and chemo-resistant phenotypes, which lead to the recurrence after surgery or re-growth of the tumor. The mechanisms that maintain the "stemness" of these cells remain largely unknown. We hypothesized that PSCs might enhance the cancer stem cell-like phenotypes in pancreatic cancer cells. Indirect co-culture of pancreatic cancer cells with PSCs enhanced the spheroid-forming ability of cancer cells and induced the expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. These results suggested a novel role of PSCs as a part of the cancer stem cell niche.

Human bone marrow stromal cells simultaneously support B and T/NK lineage development from human haematopoietic progenitors: a principal role for flt3 ligand in lymphopoiesis.

The regulation of human early lymphopoiesis remains unclear. B- and T-lineage cells cannot develop simultaneously with conventional stromal cultures. Here we show that telomerized human bone marrow stromal cells supported simultaneous generation of CD19(+) CD34(lo/-) CD10(+) cyCD79a(+) CD20(+/-) VpreB(-) pro-B cells and CD7(+) CD34(+) CD45RA(+) CD56(-) cyCD3(-) early T/Natural Killer (NK) cell precursors from human haematopoietic progenitors, and the generation of both lymphoid precursors was promoted by flt3 ligand (flt3L). On the other hand, stem cell factor or thrombopoietin had little or no effect when used alone. However, both acted synergistically with flt3L to augment the generation of both lymphoid precursors. Characteristics of these lymphoid precursors were evaluated by gene expression profiles, rearrangements of IgH genes, or replating assays. Similar findings were observed with primary human bone marrow stromal cells. Notably, these two lymphoid-lineage precursors were generated without direct contact with stromal cells, indicating that early B and T/NK development can occur, at least in part, by stromal cell-derived humoral factors. In serum-free cultures, flt3L elicited similar effects and appeared particularly important for B cell development. The findings of this study identified the potential of human bone marrow stromal cells to support human early B and T lymphopoiesis and a principal role for flt3L during early lymphopoiesis.

Evaluation of selective tumor detection by clinical magnetic resonance imaging using antibody-conjugated superparamagnetic iron oxide.

Active targeting by monoclonal antibodies (mAbs) combined with nanosize superparamagnetic iron oxide (SPIO) is a promising technology for magnetic resonance imaging (MRI) diagnosis. However, the clinical applicability of this technology has not been investigated using appropriate controls. It is important to evaluate the targeting technology using widely used clinical 1.5-Tesla MRI in addition to the high-Tesla experimental MRI. In this study, we measured mAb-conjugated dextran-coated SPIO nanoparticles (CMDM) in vivo using clinical 1.5-Tesla MRI. MRI of tumor-bearing mice was performed using a simple comparison between positive and negative tumors derived from the same genetic background in each mouse. The system provided significant tumor-targeting specificity of the target tumor. To the best of our knowledge, this is the first report on the specific detection of target tumors by mAb-conjugated SPIO using clinical 1.5-Tesla MRI. Our observations provide clues for reliable active targeting using mAb-conjugated SPIO in clinical applications.

IKAROS isoform 6 enhances BCR-ABL1-mediated proliferation of human CD34+ hematopoietic cells on stromal cells.

The BCR-ABL1 induces chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Recent studies revealed high ratios of loss of the IKZF1 gene which encodes IKAROS in BCR-ABL1+ ALL and lymphoblastic crisis (LBC) of CML. However, little is known about the cooperativity between the aberrant IKAROS and BCR-ABL1 in primary human hematopoietic cells. We investigated the effects of expression of BCR-ABL1 and/or IK6, a natural dominant negative isoform of IKAROS, on proliferation and differentiation of human CD34+ cord blood cells with or without human bone marrow-derived stromal cells which support early B cell differentiation. Cell proliferation was remarkably enhanced by co-expression of BCR-ABL1 and IK6, with reduced expression of glycophorin A and increased expression of CD41, especially on stromal cells, compared with expression of BCR-ABL1 alone that resulted in expansion of erythroid progenitors. Interestingly, p190BCR-ABL1 showed higher dependency on stromal cells to stimulate cell growth with IK6, than p210BCR-ABL1. Furthermore, the cooperation was found to depend on direct cell adhesive interaction of hematopoietic progenitors with stromal cells. These findings suggest that IK6 and BCR-ABL1 synergistically contribute to leukemogenesis in human bone marrow stromal microenvironment, and may provide a clue to elucidate the mechanisms of leukemogenesis of Ph+ ALL and CML-LBC.

EpCAM- and EGFR-targeted selective gene therapy for biliary cancers using Z33-fiber-modified adenovirus.

A critical issue in adenovirus (Ad)-based cancer gene therapy is to improve the specificity of gene delivery to cancer cells for better efficacy and safety. We explored methods of retargeting Ad vectors for selective gene therapy of human biliary cancers using the Ad incorporating an IgG Fc-binding motif (Z33) from the Staphylococcus protein A (Ad-FZ33) combined with tumor-specific antibodies. Flow cytometry analysis revealed high-expression levels of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) on human biliary cancer cells. Ad-FZ33 expressing LacZ combined with antibodies against EpCAM or EGFR, followed by ß-gal assay, demonstrated highly efficient gene transduction in these biliary cancer cells, compared to the treatment with control antibody or without antibody. Ad-FZ33 expressing uracil phosphoribosyl transferase (UPRT), an enzyme which greatly enhances the toxicity of 5-fluorouracil (FU), combined with antibodies against EpCAM or EGFR, remarkably enhanced the sensitivity of biliary cancer cells to 5-FU. By contrast, the treatment did not affect the 5-FU sensitivity of the cells not expressing EpCAM or EGFR including normal hepatocytes. Finally, treatments with the UPRT-expressing Ad-FZ33 with antibodies against EpCAM or EGFR, followed by 5-FU administration, significantly suppressed the growth of biliary cancer xenografts in nude mice. These results indicate that the gene therapy mediated by the Z33 fiber modified Ad with anti-EpCAM or anti-EGFR antibodies offers a potentially effective therapeutic modality against biliary cancers.

E1A, E1B double-restricted adenovirus enhances the cytotoxicity and antitumor activity of gemcitabine to renal cell carcinoma.

BACKGROUND: Our previous studies have demonstrated potent oncolysis efficacy of the E1A, E1B double-restricted replication-competent oncolytic adenovirus AxdAdB-3 for treatment of bladder cancer. Here, we reported the feasibility and efficacy of AxdAdB-3 alone, or in combination with gemcitabine for treating renal cell carcinoma. METHODS: Cytopathic effects of AxdAdB-3 were evaluated in human renal cell carcinoma cell lines TOS-1, TOS-2, TOS-3, TOS-3LN, SMKT-R3, SMKT-R4 and ACHN, and in normal human renal proximal tubule epithelial cells (RPTEC). AxdAdB-3 induced down-regulation of the cell cycle was determined by flow cytometry. Combination therapies of AxdAdB-3 with gemcitabine were evaluated in vitro and in vivo on subcutaneous TOS-3LN tumors in a severe combined immunodeficiency disease (SCID) mouse model. RESULTS: AxdAdB-3 was potently cytopathic against the tested most renal cell carcinoma cell lines including TOS-2, TOS-3, TOS-3LN, SMKT-R3 and SMKT-R4, while normal human RPTEC were not destroyed. AxdAdB-3 effectively induced cell cycle S-phase entry. Combined therapy of AxdAdB-3 with gemcitabine demonstrated stronger antitumor effects in vitro and in vivo compared with either AxdAdB-3 or gemcitabine alone. CONCLUSION: AxdAdB-3 alone, or in combination with gemcitabine may be a promising strategy against renal cell carcinoma.

Effects of 5 minutes of neck-muscle vibration immediately before occupational therapy on unilateral spatial neglect.

PURPOSE: To evaluate the effects of neck-muscle vibration for 5 min before occupational therapy (OT) on unilateral spatial neglect (USN). METHOD: In this multiple-baseline design study for 6 weeks (A(1)-B-A(2) design: A(1), A(2); conventional OT without neck-muscle vibration, B; neck-muscle vibration before OT together with conventional OT), we examined 11 right brain-damaged patients in the post-acute phase of stroke who showed USN. Sessions A(1) and A(2): conventional OT for 40 min once daily for 5 days a week. Session B: the left posterior neck muscles of the patient were subjected to vibration for 5 min, without confirming the appearance of a kinaesthetic illusion, immediately before OT, and then the same OT programme as in sessions A(1) and A(2) was performed. Each session lasted 2 weeks. USN and activities of daily living (ADL) were evaluated at 2-week intervals by the Behavioural Inattention Test (BIT) and Functional Independence Measure (FIM), respectively. RESULTS: Significant increases in the total scores in both the conventional subtest and behavioural subtest of the BIT were only seen during session B. FIM scores increased significantly during both sessions A(1) and B. CONCLUSIONS: The application of neck-muscle vibration before OT may have positive effects on USN, but the specific effect on the improvement of ADL is not clear.

Epigenetic modulation enhances the therapeutic effect of anti-IL-13R(alpha)2 antibody in human mesothelioma xenografts.

PURPOSE: The interleukin-13 receptor α2 (IL-13Rα2) is expressed by a variety of human malignant cells. Here, we have examined the constitutive surface expression and the epigenetic regulation of IL-13Rα2 by human mesothelioma. We have also investigated the therapeutic effect of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and anti-IL-13Rα2 monoclonal antibody on mesothelioma xenografts. EXPERIMENTAL DESIGN: Cell surface expression of IL-13Rα2 by various lung carcinomas was analyzed using flow cytometry. Therapeutic effects of anti-IL-13Rα2 and 5-aza-dC were investigated using antibody-dependent cellular cytotoxicity and proliferation assays and by monitoring the survival of mesothelioma-bearing mice. RESULTS: We found that human malignant mesotheliomas expressed surface IL-13Rα2 on their surface and that it was upregulated by treatment with 5-aza-dC. This augmented expression of IL-13Rα2 resulted in growth inhibition of the mesothelioma cells when cocultured with anti-IL-13Rα2 and effector cells, such as splenocytes and peritoneal exudate cells. The growth inhibition of mesothelioma cells was mediated by IFN-γ that was only detected in the supernatant when effector cells were exposed to 5-aza-dC-treated tumors in the presence of anti-IL-13Rα2. Compared with the control or either regimen alone, in vivo administration of anti-IL-13Rα2 in combination with 5-aza-dC significantly prolonged the survival of mice with mesothelioma xenografts. CONCLUSIONS: These observations indicate a promising role for IL-13Rα2 as a target for antibody treatment in malignant mesothelioma, and, in combination with epigenetic regulation by a DNA methylation inhibitor, suggest the potential for a novel strategy to enhance therapeutic potency.

Neural cell adhesion molecule 2 as a target molecule for prostate and breast cancer gene therapy.

In adenovirus-derived gene therapy, one of the problems is the difficulty in specific targeting. We have recently demonstrated that monoclonal antibody (mAb) libraries screened by fiber-modified adenovirus vector (Adv-FZ33), which is capable of binding to immunoglobulin-G (IgG), provide a powerful approach for the identification of suitable target antigens for prostate cancer therapy. Hybridoma libraries from mice immunized with androgen-dependent prostate cancer cell line LNCaP were screened and mAb were selected. Through this screening, we obtained one mAb, designated LNI-29, that recognizes a glycoprotein with an apparent molecular mass of 100 kD. It was identified as neural cell adhesion molecule 2 (NCAM2). Some prostate and breast cancer cell lines highly expressed NCAM2 whereas normal prostate cell lines expressed NCAM2 at low levels. In contrast to the low efficiency of gene transduction by Adv-FZ33 with a control antibody, LNI-29-mediated Adv-FZ33 infection induces high rates of gene delivery in NCAM2-positive cancers. NCAM2-mediated therapeutic gene transduction of uracil phosphoribosyltransferase (UPRT) had a highly effective cytotoxic effect on NCAM2-positive cancer cells, whereas it had less of an effect in cases with a control antibody. In conclusion, NCAM2 should be a novel gene therapy target for the treatment of prostate and breast cancer.