Phage display library derived peptides that bind to human tumor melanin as potential vehicles for targeted radionuclide therapy of metastatic melanoma

Metastatic melanoma remains an incurable disease, and there is a great need for novel therapeutic modalities. We have recently identified melanin as a target for radionuclide therapy of melanoma and demonstrated the feasibility of this approach using a 188-rhenium ( (188)Re)-radiolabeled melanin-binding decapeptide to fungal melanin known as 4B4. Although the results indicated that radiolabeled melanin-binding decapeptide had activity against melanoma, that peptide also manifested high kidney uptake and this might become a concern during clinical trials. We hypothesized that by identifying peptides with different amino acid composition against tumor melanin we might be able to decrease their kidney uptake. Using the Heptapeptide Ph.D.-7 Phage Display Library, we identified three heptapeptides that bind to human tumor melanin. These peptides were radiolabeled with (188)Re via HYNIC ligand, and their comprehensive biodistribution in A2058 human metastatic melanoma tumor-bearing nude mice was compared to that of (188)Re-4B4 decapeptide. While tumor uptake of heptapeptides was quite similar to that of (188)Re-4B4 decapeptide, there was dramatically less uptake in the kidneys at both 3 h (6% ID/g vs 38%) and 24 h (2% ID/g vs 15%) postinjection. Administration of one of the generated heptapeptides, (188)Re-HYNIC-AsnProAsnTrpGlyProArg, to A2058 human metastatic melanoma-bearing nude mice resulted in significant retardation of the tumor growth. Immunofluorescence showed that in spite of their relatively small size heptapeptides were not able to penetrate through the membranes of viable melanoma cells and bound only to extracellular melanin, which provides assurance that they will be safe to healthy melanin-containing tissues during radionuclide therapy. Thus, these heptapeptides appear to have potentially significant advantages for targeted therapy of melanoma relative to existing melanin-binding peptides.     

放射性药物在肿瘤靶向治疗中的应用

放射性药物指供临床诊断或治疗用的放射性核素制剂或其标记化合物。放射性核素靶向治疗是利用对肿瘤细胞具有特异高亲和力的分子载体将核素定向导入特定的肿瘤组织,对肿瘤进行治疗。与传统的放疗和化疗相比,其具有选择性杀伤肿瘤细胞的特点。随着核医学的发展,SPECT/CT、PET/CT的普及,新靶点的发现和新型放射性药物的研发,利用放射性药物进行靶向治疗在肿瘤临床治疗中占据的地位越来越重要。本文简述了放射性药物的分类、组成及特点;综述了针对肿瘤相关抗原的放射免疫药物在非霍奇金淋巴瘤、结直肠癌和前列腺癌中的应用;受体介导的放射性核素药物在治疗神经内分泌肿瘤、前列腺癌和乳腺癌中的临床应用以及基于基因修饰的放射性药物在肿瘤靶向治疗中的实验研究进展。最后总结了放射性药物在肿瘤靶向治疗中的应用前景与面临的挑战,以期为靶向治疗肿瘤的放射性药物的开发和临床应用提供一些参考。     

Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection

Targeted gene transduction to specific tissues and organs through intravenous injection would be the ultimate preferred method of gene delivery. Here, we report successful targeting in a living animal through intravenous injection of a lentiviral vector pseudotyped with a modified chimeric Sindbis virus envelope (termed m168). m168 pseudotypes have high titer and high targeting specificity and, unlike other retroviral pseudotypes, have low nonspecific infectivity in liver and spleen. A mouse cancer model of metastatic melanoma was used to test intravenous targeting with m168. Human P-glycoprotein was ectopically expressed on the surface of melanoma cells and targeted by the m168 pseudotyped lentiviral vector conjugated with antibody specific for P-glycoprotein. m168 pseudotypes successfully targeted metastatic melanoma cells growing in the lung after systemic administration by tail vein injection. Further development of this targeting technology should result in applications not only for cancers but also for genetic, infectious and immune diseases.     

Efficient killing of tumor cells by CAR-T cells requires greater number of engaged CARs than TCRs

Although CAR-T cells are widely used to treat cancer, efficiency of CAR-T cell cytolytic responses has not been carefully examined. We engineered CAR specific for HMW-MAA (high-molecular-weight melanoma-associated antigen) and evaluated potency of CD8+ CAR-T cells to release cytolytic granules and to kill tissue-derived melanoma cells, which express different levels of HMW-MAA. CAR-T cells efficiently killed melanoma cells expressing high level of HMW-MAA, but not melanoma cells with lower levels of HMW-MAA. The same melanoma cells presenting significantly lower level of stimulatory peptide-MHC ligand were readily lysed by T cells transduced with genes encoding α,β-TCR specific for the peptide-MHC ligand. The data suggest that higher level of targeted molecules is required to engage a larger number of CARs than TCRs to induce efficient cytolytic granule release and destruction of melanoma cells. Understanding the difference in molecular mechanisms controlling activation thresholds of CAR- versus TCR-mediated responses will contribute to improving efficiency of CAR T cells required to eliminate solid tumors presenting low levels of targeted molecules.     

Functionalized head-to-head hairpin polyamides: synthesis, double-stranded DNA-binding activity and affinity

A series of 4 functionalized head-to-head-linked hairpin oligo(N-methylpyrrole) carboxamides with different linkers have been synthesized. Their ability to bind double-stranded DNA and sequence specificity were compared and the apparent Kd values of their DNA complexes were determined. These compounds, particularly those with iminodiacetic linkers, revealed a high affinity for DNA (Kd = 4.5-4.8 x 10(-9) M) and sequence specific recognition of 9-10 base pairs.     

Combination of targeted therapy and immunotherapy in melanoma

The treatment of human melanoma has progressed markedly in recent years. Building on the observation that immune recognition is a frequent event in melanoma, a series of immunotherapeutic approaches have been evaluated in clinical trials, culminating in the first phase III study improving overall survival of melanoma patients since 20 years. However, the response rates seen upon immunotherapeutic interventions such as anti-CTLA4 treatment are often low. Furthermore, clinical responses can take several weeks to develop, during which time stage IV melanoma patients often deteriorate. Recent advances in our understanding of the genetic lesions in human melanoma now also allow the specific targeting of the signaling pathway alterations in this disease. Such targeted therapies can lead to high response rates, although the duration of these responses is thus far relatively short. We suggest that the combination of immuno and targeted therapy offers potential for synergy for both conceptual and practical reasons. In this review, we will discuss the potential and possible limitations for such combination therapy, and we describe the most promising combinations of targeted therapy and immunotherapy that can be tested in the clinic in the coming years. The concept of induction therapy by small molecule administration and consolidation by immunotherapeutics also has potential for the treatment of other human cancers.     

Anti-melanoma efficacy of internal radionuclide therapy in relation to melanin target distribution

Targeted internal radionuclide therapy (TRT) could be an efficient, specific way to treat disseminated melanoma. Based on a previous pharmacomodulation study, we selected a quinoxaline-derived molecule (ICF01012) for its melanin specificity and kinetic properties suitable for TRT. Here, we determined the efficacy of [¹³¹I]ICF01012 radiotherapy in vitro and in vivo in relation to melanogenesis using human melanoma models. [¹²⁵I]ICF01012 uptake was first assessed in relation to melanin content. We found that melanin distribution in different models was representative of pathology seen in human tumours: melanin content was high in the extracellular space of SKMel3 tumours, and accumulated primarily in melanophages in M4Beu tumours. Targeted [¹³¹I]ICF01012 radiotherapy had a strong anti-tumoural efficacy in pigmented versus unpigmented tumours, regardless of target distribution and content. This study supports the use of melanin targeting with ¹³¹I-labelled iodoquinoxaline for effective treatment of melanoma.     

Silencing of microRNA-27a facilitates autophagy and apoptosis of melanoma cells through the activation of the SYK-dependent mTOR signaling pathway

Melanoma is considered as an aggressive neoplastic transformation and featured with high metastatic potential. Although some studies have provided targets for novel therapeutic interventions, clinical development of targeted drugs for melanoma still remains obscure. Therefore, this study aims to identify the role of microRNA-27a (miR-27a) in autophagy and apoptosis of melanoma cells in regulating spleen tyrosine kinase (SYK)-mediated the mammalian target of rapamycin (mTOR) signaling pathway. A microarray-based analysis was made to screen differentially expressed genes and predict target miRNA. Melanoma specimens were collected with pigmented nevus as a control. Melanoma cell line Mel-RM was treated with miR-27a inhibitor or pcDNA-SYK to prove their effects on autophagy and apoptosis of melanoma cells. The volume change and tumor mass of nude mice in each group were detected by the tumorigenesis assay. Microarray-based analysis results showed that SYK was lowly expressed in melanoma cells and may be regulated by miR-27a. Besides, miR-27a expression was increased whereas SYK expression was decreased in melanoma tissues. Meanwhile, miR-27a was positively correlated with tumor stage and lymph node metastasis of melanoma tissues. Furthermore, miR-27a targeted SYK and silencing of miR-27a or overexpression of SYK cells promoted autophagy and apoptosis of melanoma cells and reduced their tumorigenic ability in vivo. In conclusion, this study proves that silencing of miR-27a facilitates autophagy and apoptosis of melanoma cells by upregulating SYK expression and activating the mTOR signaling pathway. The finding offers new ideas for the clinical development of melanoma.     

Targeting of Liposomes to Cells Bearing Nerve Growth Factor Receptors Mediated by Biotinylated Nerve Growth Factor

We have used biologically active derivatives of beta-nerve growth factor (NGF), modified by biotinylation via carboxyl groups, to target the specific binding of liposomes to cultured rat and human tumor cells bearing NGF receptors. Liposomes, to be used for targeting, were prepared by conjugating streptavidin to phospholipid amino groups on liposomes prepared by reverse-phase evaporation. Approximately 2,000 streptavidin molecules were incorporated per liposome. Addition of biotinylated NGF, but not of unmodified NGF, could mediate the subsequent binding of radiolabeled streptavidin-liposomes to rat pheochromocytoma PC12 cells in suspension at 4 degrees C. In contrast, incubation with biotinylated NGF did not mediate the binding of hemoglobin-conjugated liposomes. Under optimal incubation conditions, approximately 570 streptavidin-liposomes were specifically bound per cell. Biotinylated NGF was also used to obtain specific binding of streptavidin-liposomes containing encapsulated fluorescein isothiocyanate-labeled dextran to PC12 cells or human melanoma HS294 cells. When HS294 cells were incubated at 37 degrees C following targeted liposome binding at 4 degrees C, the cell-associated fluorescence appeared to become internalized, displaying a perinuclear pattern of fluorescence similar to that observed when lysosomes were stained with acridine orange. Trypsin treatment abolished cell-associated fluorescence when cells were held at 4 degrees C but did not alter the fluorescence pattern in cells following incubation at 37 degrees C. When liposomes containing carboxyfluorescein, a dye capable of diffusing out of acidic compartments, were targeted to HS294 cells, subsequent incubation at 37 degrees C resulted in diffuse cytoplasmic fluorescence, suggesting that internalized liposomes encounter lysosomal or prelysosomal organelles.     

Evaluation of lipophilicity and antitumour activity of parallel carboxamide libraries

Searching for molecules possessing antitumour activity, a parallel molecule library of aromatic carboxamides has been designed and synthesised. This work resulted in a "thiophene" sub-library containing a thiophene core and of a "furoyl" sub-library with a furoyl core, respectively. In both sub-libraries substitutions were carried out with six different groups resulting in six pairs of compounds differing in only the heteroatom of aromatic ring of the cores. To study the importance of the type of cores and the specific substitutions in relation to their lipophilicity and antitumour activity, lipophilicity of carboxamides was determined by chromatographical data (log k') and by software calculated parameters (CLOGP). Pairs of compounds were tested for their ability to inhibit the proliferation of the A431 cells by MTT assay. The isosteric molecule pairs were successfully separated. Our results showed that the experimentally determined (log k') and the calculated (CLOGP) lipophilicity parameters correlated well with each other. Furthermore, lipophilicity values of the thiophene sub-library were always higher than those in the furoyl sub-library. Moreover, compounds of the thiophene sub-library were more active than their respective furoyl pairs in our MTT antiproliferative assay. From these observations we can conclude that the higher the lipophilicity values the higher the antitumour activity of the carboxamides synthesised. Therefore, determination of lipophilicity by measuring the log k' or by calculating the CLOGP values of the carboxamide sub-libraries may help to predict their biological activities.     

Melanoma: surface markers as the first point of targeted delivery of therapeutic genes in multilevel gene therapy

Melanoma is one of the most malignant tumors, aggressively metastasizing by lymphatic and hematogenous routes. Due to the resistance of melanoma cells to many types of chemotherapy, this disease causes high mortality rate. High hopes are pinned on gene therapeutic approaches to melanoma treatment. At present, one of the main problems of the efficient use of the post-genomic generation therapeutic means is the lack of optimal techniques of delivery of foreign genetic material to the patient's target cells. Surface specific markers of melanoma cells can be considered as promising therapeutic targets. This review describes currently known melanoma specific receptors and its stem cells, as well as contains data on melanoma antigens presented on the cell surface by major histocompatibility complex proteins. The ability of surface proteins to internalize might be successfully used for the development of methods of targeted delivery of gene therapeutic constructs. In conclusion, a concept of multilevel gene therapy and the possible role therein of surface determinants as targets of gene systems delivery to the tumor are discussed.     

Molecular modeling study of intercalation complexes of tricyclic carboxamides with d(CCGGCGCCGG)₂ and d(CGCGAATTCGCG)₂

Tricyclic dyes with different mesoatoms such as xanthenes (fluorescein, eosin) anthracenes and acridines (proflavine) approved by the Food and Drug Administration (FDA) for use in foods, pharmaceuticals and cosmetic preparations interact with DNA, and some of them do so through intercalation. Hyperchem 7.5, Spartan 04, Yasara 10.5.14 program packages and molecular modeling, molecular mechanics and dynamics techniques with the oligonucleotides d(CCGGCGCCGG)2 and d(CGCGAATTCGCG)2 were utilized in order to examine the mode of binding to DNA of a range of tricyclic carboxamides bearing N,N-dimethylaminoethyl side chain, i.e., 9-amino-DACA, anthracene, acridine-1-carboxamide, acridine-4-carboxamide (DACA), azacridine, phenazine, pyridoquinoxaline, oxopyridoquinoxaline, phenoxazine and xanthenone or N,N-dimethylaminobutyl moiety, i.e., phenazine and acridine. The bicyclic quinoline-8-carboxamide was also examined for comparison reasons. On the basis of our data, prerequisite for the interaction between protonated N,N-dimethylaminoethyl moiety and guanine is the formation of only one internal hydrogen bond between carboxamide and peri NH + in the case of 9-amino-DACA or peri N in the cases of DACA, azacridine, phenazine and pyridoquinoxaline. The presence of an additional internal hydrogen bond between oxygen carboxamide and protonated N,N-dimethylamino group in the cases of tricyclic systems bearing peri NH (phenoxazine) or O (xanthenone) group, prevents the interaction between side chain and guanine. Also, the formation of one internal hydrogen bond between oxygen carboxamide and protonated N,N-dimethylamino group inhibits the interaction between side chain and guanine in the case of acridine-1-carboxamide. Our findings are in accordance with previously reported results obtained from the kinetic studies of the binding of acridine and related tricyclic carboxamides to DNA.     

Mannose receptor targeting of tumor antigen pmel17 to human dendritic cells directs anti-melanoma T cell responses via multiple HLA molecules

Targeting recycling endocytic receptors with specific Abs provides a means for introducing a variety of tumor-associated Ags into human dendritic cells (DCs), culminating in their efficient presentation to T cells. We have generated a human mAb (B11) against the mannose receptor that is rapidly internalized by DCs through receptor-mediated endocytosis. By genetically linking the melanoma Ag, pmel17, to Ab B11, we obtained the fully human fusion protein, B11-pmel17. Treatment of DCs with B11-pmel17 resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Thus, potent pmel17-specific T cells were cytotoxic toward gp100(+) HLA-matched melanoma targets, but not HLA-mismatched melanoma or gp100(-) nonmelanoma tumor lines. Importantly, competitive inhibition of lysis of an otherwise susceptible melanoma cell line by cold targets pulsed with known gp100 CD8 T cell epitopes as well as a dose-dependent proliferative response to Th epitopes demonstrates that DCs can process targeted Ag for activation of cytotoxic as well as helper arms of the immune response. Thus, the specific targeting of soluble exogenous tumor Ag to the DC mannose receptor directly contributes to the generation of multiple HLA-restricted Ag-specific T cell responses.     

The avidity of tumor‐specific T cells amplified by a plasmacytoid dendritic cell‐based assay can predict the clinical evolution of melanoma patients

The advent of immune checkpoint blockers and targeted therapies has changed the outcome of melanoma. However, many patients experience relapses, emphasizing the need for predictive and prognostic biomarkers. We developed a strategy based on plasmacytoid dendritic cells (pDCs) loaded with melanoma tumor antigens that allows eliciting highly efficient antitumor T‐cell responses. We used it to investigate antitumor T‐cell functionality in peripheral blood mononuclear cells and tumor‐infiltrating lymphocytes from melanoma patients. The pDCs elicited tumor‐specific T cells in different proportions and displaying diverse functional features, dependent upon the stage of the disease, but independent of the histological parameters at diagnosis. Strikingly, the avidity of the MelA‐specific T cells triggered by the pDCs was found to predict patient relapse time and overall survival. Our findings highlighted unexplored aspects of antitumor T‐cell responsiveness in melanoma, and revealed for the first time the structural avidity of tumor‐specific T cells as a crucial feature for predicting clinical evolution.     

Cephalosporin prodrugs of paclitaxel for immunologically specific activation by L-49-sFv-beta-lactamase fusion protein

Paclitaxel conjugates of 7-phenylacetamidocephalosporanic acid were prepared as prodrugs for site specific activation by targeted beta-lactamase. Immunologically specific activation of the prodrug 5 containing 3,3-dimethyl-4-amino-butyric acid as linker in combination with the fusion protein L-49-sFv-beta-lactamase was demonstrated in vitro on 3677 melanoma cells.     

Synthesis and evaluation of acridine- and acridone-based anti-herpes agents with topoisomerase activity

The discovery of new non-nucleoside antiviral compounds is of significant and growing interest for treating herpes virus infections due to the emergence of nucleoside-resistant strains. Using a whole cell virus-induced cytopathogenic assay, we tested a series of substituted triaryl heterocyclic compounds including acridones, xanthones, and acridines. The compounds which showed activity against Herpes Simplex-1 and/or Herpes Simplex-2 were further assayed for inhibition of topoisomerase activity to gain insight into the mechanism of action. The results indicate that the acridine analogs bearing substituted carboxamides and bulky 9-amino functionalities are able to inhibit herpes infections as well as inhibit topoisomerase II relaxation of supercoiled DNA. Given the mechanism of action of amsacrine (a closely related, well-studied 9-amino substituted acridine), the compounds were further tested in a DNA topoisomerase II cleavage assay to determine if the compounds function as poisons. The results show that the acridines synthesized in this study function through a different mechanism to that of amsacrine, most likely by blocking topoisomerase binding to DNA (akin to that of aclarubicin). This not only suggests a unique mechanism of action in treating herpes virus infections, but also may be of great interest in the development of anticancer agents that target topoisomerase II activity.     

Neoadjuvant BRAF‐targeted therapy in regionally advanced and oligometastatic melanoma

Current management of locoregional and oligometastatic melanoma is typically with surgery; however, some patients are unable to undergo resection due to location/size of their tumors and/or the anticipated morbidity of the surgery. While there are currently no established guidelines for neoadjuvant therapy in melanoma, neoadjuvant BRAF‐targeted therapy may make resection more feasible. A retrospective analysis was conducted of 23 patients with BRAFV600‐mutant, stage III/IV melanoma treated with BRAF‐targeted therapy prior to surgery, with no adjuvant treatment. Surgical specimens, preoperative imaging, and clinical outcomes were evaluated. Results: Ten of 23 patients (44%) attained a pathologic complete response (pCR), with no correlation between RECIST response based on preoperative imaging and pathologic response. After a median of 43‐month follow‐up, only 1 patient (10%) with a pCR recurred, while 8 of 13 (62%) patients without a pCR recurred. Patients with a pCR had significantly improved relapse‐free (RFS) and overall survival (OS) compared to patients with residual tumor. Neoadjuvant BRAF‐targeted therapy is associated with a high pCR rate in patients with stage III‐IV melanoma, which may correlate with improved RFS and OS.     

Targeted Gene Delivery by Intravenous Injection of Retroviral Vectors

Specifically and effectively directing a therapeutic gene to its intended site of action is a critical issue for translation of basic genomics to clinical gene therapy. Delivering gene therapy vectors to specific cells or tissues through intravenous injection is the most desirable method for this purpose. In 2001, we reported successful targeted gene transduction in vitro utilizing both oncoretroviral and lentiviral vectors pseudotyped with a chimeric Sindbis virus envelope (ZZ SINDBIS). However, these pseudotypes mediated non-specific gene transduction to liver and spleen in vivo. To address this problem we generated the modified ZZ SINDBIS (termed m168) with significantly less non-specific infectivity. To investigate the ability of m168 pseudotyped lentiviral vector to mediate targeted gene transduction in vivo, we utilized a metastatic tumor model by using mouse melanoma cells engineered to express human P-glycoprotein. We administered the m168 pseudotyped vector conjugated with anti-P-glycoprotein antibody into the mice intravenously to target metastatic melanoma. The m168 pseudotyped vector selectively infected metastatic melanoma cells demonstrating successful targeted gene transduction in vivo. Targeting technology based upon m168 can be further modified for application not only to cancer but also potentially to genetic, neurologic, infectious and immune diseases, thereby expanding the future application of gene therapy.     

Targeting the RAS pathway in melanoma

Metastatic melanoma is a highly lethal type of skin cancer and is often refractory to all traditional chemotherapeutic agents. Key insights into the genetic makeup of melanoma tumors have led to the development of promising targeted agents. An activated RAS pathway, anchored by oncogenic BRAF, appears to be the central motor driving melanoma proliferation. Although recent clinical trials have brought enormous hope to patients with melanoma, adverse effects and novel escape mechanisms of these inhibitors have already emerged. Definition of the limits of the first successful targeted therapies will provide the basis for further advances in management of disseminated melanoma. In this review, the current state of targeted therapy for melanoma is discussed, including the potent BRAF(V600E) inhibitor vemurafenib.     

Recognition of hairpin-containing single-stranded DNA by oligonucleotides containing internal acridine derivatives.

Oligodeoxynucleotides with an internal intercalating agent have been targeted to single-stranded sequences containing hairpin structures. The oligonucleotide binds to nonadjacent single-stranded sequences on both sides of the hairpin structure in such a way as to form a three-way junction. The acridine derivative is inserted at a position that allows it to interact with the three-way junction. The melting temperature (Tm) of complexes formed between the hairpin-containing target and oligonucleotides containing one internal acridine derivative was higher than that obtained with the same target and an unmodified oligonucleotide (DeltaTm = +13 degrees C). The internal acridine provided the oligonucleotide with a higher affinity than covalent attachment to the 5' end. Oligonucleotides could also be designed to recognize a hairpin-containing single-stranded nucleic acid by formation of Watson-Crick hydrogen bonds with a single-stranded part and Hoogsteen hydrogen bonds with the stem of the hairpin. An internal acridine derivative was introduced at the junction between the two domains, the double helix domain with Watson-Crick base pairs and the triple helix domain involving Hoogsteen base triplets in the major groove of the hairpin stem. Oligonucleotides with an internal acridine or an acridine at their 5' end have similar binding affinities for the stem-loop-containing target. The bis-modified oligonucleotide containing two acridines, one at the 5' end and one at an internal site, did not exhibit a higher affinity than the oligonucleotides with only one intercalating agent. The design of oligonucleotides with an internal intercalating agent might be of interest to control gene expression through recognition of secondary structures in single-stranded targets.