Reporter gene-based optoacoustic imaging of E. coli targeted colon cancer in vivo.

Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.

Membrane-associated human tyrosinase is an enzymatically active monomeric glycoprotein.

Human tyrosinase (hTyr) is a Type 1 membrane bound glycoenzyme that catalyzes the initial and rate-limiting steps of melanin production in the melanosome. Mutations in the Tyr gene are linked to oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder. Currently, the application of enzyme replacement therapy for a treatment of OCA1 is hampered by the absence of pure hTyr. Here, full-length hTyr (residues 1-529) was overexpressed in Trichoplusia ni larvae infected with a baculovirus, solubilized with detergent and purified using chromatography. Michaelis-Menten kinetics, enzymatic specific activity, and analytical ultracentrifugation were used to compare the hTyr in detergent with the soluble recombinant intra-melanosomal domain, hTyrCtr (residues 19-469). Active hTyr is monomeric in detergent micelles suggesting no stable interactions between protein molecules. Both, hTyr and hTyrCtr, exhibited similar enzymatic activity and ligand affinity in L-DOPA and L-Tyrosine reactions. In addition, expression in larvae is a scalable process that will allow high yield protein production. Thus, larval production of enzymatically active human tyrosinase potentially could be a useful tool in developing a cure for OCA1.

Safety and immunogenicity of vaccination with MART-1 (26-35, 27L), gp100 (209-217, 210M), and tyrosinase (368-376, 370D) in adjuvant with PF-3512676 and GM-CSF in metastatic melanoma.

The effectivenes of cancer vaccines in inducing CD8(+) T-cell responses remains a challenge, resulting in a need for testing more potent adjuvants. Our objective was to determine the safety and immunogenicity of vaccination against melanoma-related antigens employing MART-1, gp100, and tysosinase paptides combined with the TLR9 agonist PF-3512676 and local granulocyte macrophage-colony stimulating factor in oil emulsion. Using continuous monitoring of safety and a 2-stage design for immunologic efficacy, 20 immune response evaluable patients were targetted. Vaccinations were given subcutaneously on days 1 and 15 per cycle (1cycle=28 d) for up to 13 cycles. Interferon-γ enzyme-linked immunosorbent spot was used as the primary assay measuring the frequency of peripheral antigen-specific CD8(+) T cells at days 50 and 90 compared with baseline (target ≥ 9/20 immunologic responses). Clinical responses were measured by Response Evaluation Criteria In Solid Tumors every 8 weeks. Twenty-two (including 20 immune response evaluable) melanoma patients were enrolled. All had American Joint Committe on Cancer stage IV (5M1a, 6M1b, 11M1c) and most had previously received therapy. Eight had previously treated brain metastases. An average of 3.5 cycles of vaccination per patient was administered. Clinical response data were available for 21 patients. There were 2 partial response and 8 stable disease lasting 2-7 months. One patient with ongoing partial response continued on treatment. At a median follow-up of 7.39 months (range, 3.22-20.47 mo), median progression-free survival was 1.9 months (90% confidence interval, 1.84-3.68) and median overall survival was 13.4 months (90% confidence interval,11.3-∞). No regimen-related grade 3/4/5 toxicities were observed. There were 9/20 patients with positive enzyme-linked immunosorbent spot at day 50 and/or day 90. Our adjuvant regimen combining PF-3512676 and granulocyte macrophage-colony stimulating factor was safe and is worthy of further testing with these or alternative peptides, potentially in combination with antibodies that target immunoregulatory checkpoints.

Safety and efficacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor.

OBJECTIVE: To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs. ANIMALS: 111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved. PROCEDURES: 58 dogs received an initial series of 4 injections of huTyr vaccine (102 µg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death. RESULTS: Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM. IMPACT FOR HUMAN MEDICINE: Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

Extracts from Amazonian plants have inhibitory activity against tyrosinase: an in vitro evaluation

Dermatological disorders related to pigmentation result in tenuous hyper or hypopigmentation Cosmetic and pharmaceutical products containing depigmenting substances are used in the treatment of patients who have high pigmentation disorders, such as melasma or chloasma, post-inflammatory hyperpigmentation, senile lentigo and ephelides. Skin lightening agents are not yet totally effective or safe and therefore intensive research for the discovery of new agents is continuous. Enzyme inhibitors involved in melanogenesis, such as tyrosinase, have been discovered in Asian countries, including those isolated from plant extracts. The Brazilian flora has the highest species diversity in the world, and the chemical, pharmacological and cosmetic potential for the discovery of new skin whitening agents is in proportion with this biodiversity. For these reasons, 25 aqueous and 24 organic extracts obtained from 19 plants native to the Amazon rain forest and to the Atlantic forest, belonging to 11 different families, were evaluated as tyrosinase inhibitors. Nine out of 49 extracts showed inhibitory activity in the screening process. The 50 percent inhibitory activity (IA50) was calculated, revealing that the most active extracts were the organic extracts from the leaves and stem of Ruprechtia sp. (IA50 33.76 mg.mL-1) and the organic extract from the aerial organs of Rapanea parviflora (IA50 64.19 mg.mL-1).

Problemas dermatológicos relacionados com a pigmentação resultam em hiperpigmentações ou hipopigmentação cutâneas. Produtos cosméticos e farmacêuticos com atividade despigmentante são utilizados para o tratamento de pacientes que apresentam distúrbios de hiperpigmentação, tais como melasma ou cloasma, hiperpigmentação pós-inflamatória, lentigem senil e efélides. Os despigmentantes atualmente utilizados não são totalmente eficazes ou seguros, razão pela qual há intensa pesquisa, principalmente em países asiáticos, com a finalidade de se obter novos agentes com esta ação, em especial inibidores de enzimas envolvidas na melanogênese, como a tirosinase. Considerando-se que algumas substâncias obtidas de plantas apresentam essa atividade, a flora brasileira constitui-se uma fonte potencial de obtenção de novos despigmentantes. Por essa razão, 25 extratos aquosos e 24 orgânicos obtidos de 19 plantas da Floresta Amazônica e Mata Atlântica, provenientes de 11 diferentes famílias, foram avaliados quanto à atividade de inibição da tirosinase. Do total de 49 extratos testados, 9 mostraram atividade. Os valores de concentração da atividade inibitória 50 por cento (AI 50 por cento), foram calculados e o mais ativo foi o extrato orgânico das folhas e caule de Ruprechtia sp. (AI50 33,76 mg.mL-1) seguido do extrato orgânico dos órgãos aéreos de Rapanea parviflora (AI50 64,19 mg.mL-1).

Polyhemoglobin-tyrosinase, an oxygen carrier with murine B16F10 melanoma suppression properties: a preliminary report.

Melanoma now represents the fifth most common cancer in North America and it has increased dramatically in the past decade. One of the approaches shows that lowering of tyrosine level can inhibit the growth of melanoma in cell culture and in mice bearing B16BL6 melanoma. However, human cannot tolerate the tyrosine restricted diets for lowering tyrosine due to nausea, vomiting, and severe body weight loss. We therefore prepare a novel soluble polyhemoglobin-tyrosinase complex. Our studies show that this preparation can lower systemic tyrosine level in normal animals. This preparation also prevents the native tyrosinase from having adverse effects and from rapid removal after injection. In cell culture study, we find that this preparation inhibits the growth of murine B16F10 melanoma culture. Furthermore, in animal studies we observe that daily intravenous injection of this polyhemoglobin-tyrosinase preparation significantly delays the growth of B16F10 melanoma in mice, without causing adverse effects or changes in the growth of the treated animals.

Phase I/II study of treatment with dendritic cell vaccines in patients with disseminated melanoma.

Previous studies have suggested that immunotherapy with dendritic cell (DC) vaccines may be effective in treatment of patients with AJCC stage IV melanoma. We examined this treatment in phase I/II studies in 33 patients with good performance status and low volume disease. Nineteen patients received DCs plus autologous lysates and 14 patients DCs plus peptides from the melanoma antigens MAGE-3.A2, tyrosinase, gp100, and MART-1. Keyhole limpet hemocyanin (KLH) was used as a helper protein and influenza peptide was given as a positive control. DCs were produced from adherent cells in blood lymphocytes (monocytic DCs), grown in IL-4 and GM-CSF without a maturation step. The DCs were injected into inguinal lymph nodes at weekly intervals (x4), 2 weeks (x1), and 4-weekly intervals (x2). There were 3 responses (3 partial responses) and 1 mixed response in the 19 patients treated with DCs plus autologous lysates. No responses were seen in the group treated with DCs plus peptides. Stable disease (defined as no progression over a period of 3 months) was seen in 4 patients in group 1 and 5 patients in group 2. Treatment was not associated with significant side effects. We examined whether DTH skin tests or assays of IFN-gamma cytokine production may be useful predictors of clinical responses. Twenty-two of 30 patients had DTH responses to KLH and 12 of 13 patients had DTH responses to the influenza peptide. Five of 15 DTH responses were seen against autologous lysates. This was strongly correlated with clinical responses. Approximately half the patients had responses to MART-1 peptide and a third to the other melanoma peptides. Similarly, cytokine production assays showed responses to influenza in 7 of 13 patients, and approximately one third of patients had responses to the other peptides. No IFN-gamma responses were seen in 5 patients against their autologous lysates. There was no correlation between assays of IFN-gamma production and clinical responses. The present studies suggest that autologous lysates may be more effective than the melanoma peptides used in the study as the source of antigen for DC vaccines. DTH responses to autologous lysates appear useful predictors of clinical responses, but further work is needed to identify other measures associated with clinical responses.

Synthesis and analysis of urea and carbamate prodrugs as candidates for melanocyte-directed enzyme prodrug therapy (MDEPT).

The suitability of 4-di(2-chloroethyl)aminoanilino-4-hydroxyphenethylaminomethanone 2 to act as a prodrug for melanocyte-directed enzyme prodrug therapy (MDEPT) is assessed. Thus its synthesis, ability to generate a cytotoxic agent upon exposure to tyrosinase, and stability within different sera are reported. A comparison is made to illustrate that the new urea prodrug 2 is a more suitable candidate for MDEPT than the corresponding carbamate prodrug 1.

Long-term freedom from recurrence in 2 stage IV melanoma patients following vaccination with tyrosinase peptides.

We report here on 2 patients who received adjuvant vaccination with an HLA-A2- or HLA-A24-restricted tyrosinase peptide, respectively, and GM-CSF for frequently relapsing stage IV melanoma. Following resection of metastases and irradiation of brain metastases in 1 patient, both patients were without evidence of disease when receiving the first vaccination. While the patients had had 9 and 12, respectively, mostly s.c., relapses during the 3 years before vaccination, they experienced freedom from relapse for more than 2 years after vaccination. We found a T-cell response to the vaccine peptide in both patients in the peripheral blood by ex vivo IFN-gamma ELISPOT assay. The T-cell population could be further characterized by 4-color flow cytometry in 1 patient, showing that the majority of the peptide-specific CD3(+)CD8(+)IFN-gamma(+) T cells were granzyme B-positive and CCR-7-negative, characterizing them as effector T cells with the ability to mediate cytotoxicity and migrate to inflamed tissues. In this patient also, augmentation of the T-cell response to autologous tumor cells by vaccination could be detected. A single-site postvaccination relapse occurred in both patients, showing downregulation of tyrosinase expression in 1 patient, while normal expression levels for tyrosinase, MHC class I antigens and components of the antigen-processing machinery were found in the other patient. These results suggest that peptide vaccination resulted in a prolonged relapse-free interval in these high-risk patients.

Pre-existing immunity to tyrosinase-related protein (TRP)-2, a new TRP-2 isoform, and the NY-ESO-1 melanoma antigen in a patient with a dramatic response to immunotherapy.

We have performed a detailed analysis of the recognition of melanoma Ags by the tumor-infiltrating lymphocytes (TIL) 1790, isolated from a patient who experienced a dramatic tumor regression following immunization with peptides from the gp100, MART-1, and tyrosinase Ags. This TIL was found to recognize HLA-A2-restricted CTL epitopes in tyrosinase-related protein (TRP)-2 (clone MR7) and NY-ESO-1 (clone M8). These epitopes were the same as the previously identified nonapeptide TRP-2: 180-188, and the overlapping NY-ESO-1 peptides, obtained by using lymphocytes from in vitro stimulation. We also cloned a previously unknown TRP-2 mRNA isoform (TRP-2-6b) that contained two novel exons alternatively spliced from the sixth intron between exons 6 and 7 of TRP-2 mRNA. The isoform encoded an HLA-A2-restricted antigenic epitope recognized by TIL clone MB4. An immunologic analysis of the patient's PBMC obtained before treatment showed the presence of high reactivity against NY-ESO-1 and both TRP-2 Ags, but not the Ags used for immunization. Because immune response against these Ags was less pronounced, it is possible that NY-ESO-1, TRP-2, and TRP-2-6b may be of importance in the generation of CTL-mediated tumor destruction and may have played a role in the dramatic tumor regression seen in this patient.

Depletion of CD25+ regulatory cells results in suppression of melanoma growth and induction of autoreactivity in mice.

Treatment with monoclonal antibodies (mAbs) specific for CD25 (anti-CD25 mAb) has been shown to suppress growth of a variety of different tumours in mice. These studies did not however determine whether or not anti-CD25 mAbs facilitate tumour rejection by depletion of regulatory T cells or by binding to tumour-specific effector cells. Using a murine model of melanoma we have found that treatment of mice with anti-CD25 mAb facilitates long-term CD4+ T cell-mediated tumour immunity through depletion of CD25+ regulatory cells. We further show that the effector CD4+ T cells confer long-term tumour immunity even in the presence of CD25+ regulatory cells and do not require CD8+ T cells for tumour rejection. The inhibitory impact of anti-CD25 mAb treatment on tumour growth may be the result of depleting CD25+ regulatory cells that normally inhibit the generation of immune responses to self-antigens that are shared by the tumour. We have performed experiments to determine whether or not immune responses to melanocyte antigens are generated in anti-CD25 mAb-treated, melanoma-immune mice. The results of the experiments indicate that a T cell response to the melanocyte antigen tyrosinase accompanies suppression of tumour growth in mice lacking CD25+ regulatory cells.

Identification of a new shared HLA-A2.1 restricted epitope from the melanoma antigen tyrosinase.

Tyrosinase has many advantages as a target antigen for the immunotherapy of patients with melanoma because it is expressed in nearly all melanoma specimens with a high degree of cellular homogeneity, and its distribution in normal tissues is limited to melanocytes. To broaden our ability to direct cellular immune responses against this protein, we pursued an investigation to identify new shared human leukocyte antigen (HLA)-A2.1 restricted epitopes from tyrosinase. Peptides were synthesized that fit a permissive HLA-A2.1 binding motif and did not span common sites of polymorphism. The binding affinity of each peptide to HLA-A2.1 relative to a standard peptide with intermediate binding affinity was evaluated in a competitive inhibition assay. Twelve peptides were selected that had binding affinities within 80% of that of the standard peptide, and these were used to stimulate peripheral blood mononuclear cells (PBMC) in vitro from three HLA-A2.1+ patients with metastatic melanoma. Cytotoxic T lymphocytes that specifically recognized peptide-pulsed target cells as well as HLA-A2.1+ tyrosinase+ melanoma cells were raised from one patient with tyrosinase:8-17 (CLLWSFQTSA). To evaluate further the immunogenicity of this peptide, PBMC from 23 HLA-A2.1+ patients were stimulated in vitro with tyrosinase:8-17. Eleven bulk T-cell cultures demonstrated specific peptide recognition, and six of these also recognized HLA-A2.1+ tyrosinase+ melanoma cells. These data suggest that tyrosinase:8-17 may be clinically useful for the treatment of patients with melanoma.

Inhibition of experimental autoimmune vitiligo by oral administration of mushroom tyrosinase.

Experimental autoimmune vitiligo was induced by intradermal injection of mushroom tyrosinase emulsified in complete Freund's adjuvant in female C57BL/6 mice. The onset of vitiligo was characterized by hair hypopigmentation and total melanocyte depletion in the basal layer of the epidermis. Oral administration of mushroom tyrosinase prevented the expression of mushroom tyrosinase induced experimental autoimmune vitiligo. Based on the results it is likely that oral administration of mushroom tyrosinase may have practical implications in vitiligo.

Phase 2 trial of vaccination with tyrosinase peptides and granulocyte-macrophage colony-stimulating factor in patients with metastatic melanoma.

This phase II study was performed to determine the induction of a specific T-cell response, the clinical response rate, and toxicity of vaccination with different HLA class I-binding peptide epitopes derived from the melanocyte differentiation antigen tyrosinase in patients with stage IV melanoma. The study population consisted of 16 patients with metastatic disease and two patients who were macroscopically free of disease at study entry after resection of recurrent skin lesions. Patients received intradermal injections of 200 microgram [corrected] peptide corresponding to their HLA type on day 3, and 75 or 150 microg granulocyte-macrophage colony-stimulating factor on days 1 to 4. Vaccinations were repeated at weeks 2, 4, 6, 10, and 14. Monitoring of peptide-specific T-cell frequencies in the peripheral blood was performed using an interferon gamma ELISPOT assay. Eleven of the 16 patients with metastatic disease went off the protocol within the first 10 weeks because of tumor progression. Of the five patients with metastatic disease who received all six vaccinations, one patient showed a mixed response with regression of some lung metastases; two patients with progressive disease before vaccination had stable disease for 6 and 18+ months; and two patients had progression of their disease. The two patients who had all their metastases resected before vaccination did not have relapses for 6 and 12+ months after vaccination. Induction of tyrosinase-reactive T cells was found in these two patients and in two others with metastatic disease, including the one who achieved a mixed response and one with stable disease. This study shows limited clinical and immunologic activity of HLA class 1-peptide vaccination in combination with granulocyte-macrophage colony-stimulating factor in stage IV melanoma patients.

Related age changes in the elastase and lipid contents of normal and hemiplegic subjects of various ages.

Elastase and lipid levels of the plasmas of subjects and of those who have suffered a stroke have been measured, and it has been shown that the normal age related fall in elastase content is mirrored more closely by the fall in high density lipoprotein cholesterol than by changes in total cholesterol. After a stroke a peak level of the elastase concentration is reached later than the peak of the total cholesterol, but before the peak for the HDL-cholesterol is attained. Highest levels of the ratio of these two (a major risk factor in vascular disease) appear to precede both the stroke and the elevation of the elastase level at ages below 76 whereas at higher ages than this the elevation of elastase precedes the lipid changes. It is therefore deduced that the whole question of elastolytic activity in the plasma is intimately bound up with the lipid content of the plasma and with the age of the subject as mirrored in the status of the elastic tissue in the vessel wall.

Tyrosinase immobilized within artificial cells for detoxification in liver failure: I. preparation and in vitro studies.

The in vitro removal of tyrosine by means of perfusion over immobilized tyrosinase within artificial cells was studied. The assayed activity of the microencapsulated tyrosinase was about half the value of the same amount of enzyme in free solution. Further studies were conducted to determine the stability, kinetics, and clearance of this system. The results obtained suggest that immobilized tyrosinase within artificial cells is efficient in removing tyrosine in vitro and further study in an animal model is feasible.

Hydroxyanisole-induced regression of the Harding-Passey melanoma in mice.

The drug p-hydroxyanisole (OHA) was found to inhibit the incorporation of 3H-thymidine in the Harding-Passey melanoma cells in culture. Because the cultured cells had lost some of their pigment-forming capacity, the enzyme tyrosinase was added to the culture. This greatly increased the sensitivity of the cells to OHA, strongly suggesting that cells producing the enzyme would be preferentially killed by the drug. An in-vivo study of the effect of OHA injected into tumour-bearing mice showed a beneficial effect, including increased survival time, reduction in tumour size and in many cases complete loss of tumour and no recurrence. An experiment with animals immunologically suppressed by radiation suggests that the effect is not an immunological one.