Photochemical Internalization Enhanced Vaccination Is Safe, and Gives Promising Cellular Immune Responses to an HPV Peptide-Based Vaccine in a Phase I Clinical Study in Healthy Volunteers.

Background and Aims: Photochemical internalization (PCI) is a technology for inducing release of endocytosed antigens into the cell cytosol via a light-induced process. Preclinical experiments have shown that PCI improves MHC class I antigen presentation, resulting in strongly enhanced CD8+ T-cell responses to polypeptide antigens. In PCI vaccination a mixture of the photosensitizing compound fimaporfin, vaccine antigens, and an adjuvant is administered intradermally followed by illumination of the vaccination site. This work describes an open label, phase I study in healthy volunteers, to assess the safety, tolerability, and immune response to PCI vaccination in combination with the adjuvant poly-ICLC (Hiltonol) (ClinicalTrials.gov Identifier: NCT02947854). Methods: The primary objective of the study was to assess the safety and local tolerance of PCI mediated vaccination, and to identify a safe fimaporfin dose for later clinical studies. A secondary objective was to analyze the immunological responses to the vaccination. Each subject received 3 doses of HPV16 E7 peptide antigens and two doses of Keyhole Limpet Hemocyanin (KLH) protein. A control group received Hiltonol and vaccine antigens only, whereas the PCI groups in addition received fimaporfin + light. Local and systemic adverse effects were assessed by standard criteria, and cellular and humoral immune responses were analyzed by ELISpot, flow cytometry, and ELISA assays. Results: 96 healthy volunteers were vaccinated with fimaporfin doses of 0.75-50 µg. Doses below 17.5 µg were safe and tolerable, higher doses exhibited local tolerability issues in some study subjects, mainly erythema, and pain during illumination. There were few, and only mild and expected systemic adverse events. The employment of PCI increased the number of subjects exhibiting a T-cell response to the HPV peptide vaccine about 10-fold over what was achieved with the antigen/Hiltonol combination without PCI. Moreover, the use of PCI seemed to result in a more consistent and multifunctional CD8+ T-cell response. An enhancement of the humoral immune response to KLH vaccination was also observed. Conclusions: Using PCI in combination with Hiltonol for intradermal vaccination is safe at fimaporfin doses below 17.5 µg, and gives encouraging immune responses to peptide and protein based vaccination.

Targeting immune checkpoint B7-H3 antibody-chlorin e6 bioconjugates for spectroscopic photoacoustic imaging and photodynamic therapy.

In this study, we constructed bioconjugates of targeting immune checkpoint B7-H3 antibody and chlorin e6 to treat non-small cell lung cancer under the guidance of spectroscopic photoacoustic and fluorescence imaging. The B7-H3-Ce6 conjugates could display effective tumor diagnosis and therapy and provide a novel approach for immunotherapy.

Combined Photosensitization and Vaccination Enable CD8 T-Cell Immunity and Tumor Suppression Independent of CD4 T-Cell Help.

Cytotoxic T lymphocytes (CTLs) are key players in fighting cancer, and their induction is a major focus in the design of therapeutic vaccines. Yet, therapeutic vaccine efficacy is limited, in part due to the suboptimal vaccine processing by antigen-presenting cells (APCs). Such processing typically takes place via the MHC class II pathway for CD4 T-cell activation and MHC class I pathway for activation of CD8 CTLs. We show that a combination of skin photochemical treatment and immunization, so-called photochemical internalization (PCI) facilitated CTL activation due to the photochemical adjuvant effect induced by photosensitizer, oxygen, and light. Mice were immunized intradermally with antigen and photosensitizer, followed by controlled light exposure. PCI-treated mice showed strong activation of CD8 T cells, with improved IFN-γ production and cytotoxicity, as compared to mice immunized without parallel PCI treatment. Surprisingly, the CD8 T-cell effector functions were not impaired in MHC class II- or CD4 T-cell-deficient mice. Moreover, PCI-based vaccination caused tumor regression independent of MHC class II or CD4 T cells presence in melanoma bearing mice. Together, the data demonstrate that PCI can act as a powerful adjuvant in cancer vaccines, even in hosts with impaired T-helper functions.

Immunological effects of photodynamic therapy in the treatment of actinic keratosis and squamous cell carcinoma.

The use of photodynamic therapy is extensive, due to its antitumoral, antibacterial and photorejuvenation effects. It destroys tumor via direct cell destruction and indirectly via vascular shutdown, induction of acute local inflammatory response and activation of the immune system. Both innate and adaptive immune cells are involved in the immunological effects of photodynamic therapy. In addition to UV-induced DNA damage, inflammation and immunosuppression are also essential elements in the pathogenesis of actinic keratosis. Both immunosuppression induced by UV and defective immune response to dysplastic keratinocytes may be the target of photodynamic therapy to eliminate actinic keratosis. These elements are discussed in the present review, highlighting the possible mechanism of photodynamic therapy to effectively treat actinic keratosis.

Sensitization of immune cells following hexylaminolevulinate photodynamic therapy of cervical intraepithelial neoplasia.

BACKGROUND: Effects of photodynamic therapy (PDT) were tested with respect to immune cell stimulation in cervical intraepithelial neoplasia (CIN). METHODS: A patient with CIN received hexaminolevulinate (HAL) and subsequent PDT. These data were compared to a placebo PDT patient and a healthy HPV16-vaccinated donor. Isolation of peripheral blood mononuclear cells (PBMC) was performed before PDT and at 4 different time points after PDT. The proliferation of these PBMC was tested by [3H]thymidine incorporation following stimulation with control or HPV16-L1 peptides. Potential effects on the CD4+ and CD8+ cell subsets were analysed. RESULTS: The data revealed an unchanged or decreased proliferation of HPV16-L1 peptide-stimulated PBMC as compared to placebo patient. HPV16-L1 peptide incubation of PBMC from the HAL patient demonstrated significant proliferative capacity after PDT. The CD4+ and CD8+ T cell populations in placebo patient were slightly increased after HPV16-L1 peptide administration at each time point. Mixed results were obtained for CD4+ cells as compared to controls and nearly unchanged amounts of CD8+ cells were detectable following HPV16-L1 peptide exposure of PBMC from the HAL/PDT-treated patient. CONCLUSIONS: These findings suggest a T cell reaction from CIN patients during repeated HAL/PDT treatment. However, further immune cell populations might be involved during PDT.

Photodynamic therapy induces an immune response against a bacterial pathogen.

Photodynamic therapy (PDT) employs the triple combination of photosensitizers, visible light and ambient oxygen. When PDT is used for cancer, it has been observed that both arms of the host immune system (innate and adaptive) are activated. When PDT is used for infectious disease, however, it has been assumed that the direct antimicrobial PDT effect dominates. Murine arthritis caused by methicillin-resistant Staphylococcus aureus in the knee failed to respond to PDT with intravenously injected Photofrin(®). PDT with intra-articular Photofrin produced a biphasic dose response that killed bacteria without destroying host neutrophils. Methylene blue was the optimum photosensitizer to kill bacteria while preserving neutrophils. We used bioluminescence imaging to noninvasively monitor murine bacterial arthritis and found that PDT with intra-articular methylene blue was not only effective, but when used before infection, could protect the mice against a subsequent bacterial challenge. The data emphasize the importance of considering the host immune response in PDT for infectious disease.

IL-6 potentiates tumor resistance to photodynamic therapy (PDT).

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) is an anticancer modality approved for the treatment of early disease and palliation of late stage disease. PDT of tumors results in the generation of an acute inflammatory response. The extent and duration of the inflammatory response is dependent upon the PDT regimen employed and is characterized by rapid induction of proinflammatory cytokines, such as IL-6, and activation and mobilization of innate immune cells. The importance of innate immune cells in long-term PDT control of tumor growth has been well defined. In contrast the role of IL-6 in long-term tumor control by PDT is unclear. Previous studies have shown that IL-6 can diminish or have no effect on PDT antitumor efficacy. STUDY DESIGN/MATERIALS AND METHODS: In the current study we used mice deficient for IL-6, Il6(-/-) , to examine the role of IL-6 in activation of antitumor immunity and PDT efficacy by PDT regimens known to enhance antitumor immunity. RESULTS: Our studies have shown that elimination of IL-6 had no effect on innate cell mobilization into the treated tumor bed or tumor draining lymph node (TDLN) and did not affect primary antitumor T-cell activation by PDT. However, IL-6 does appear to negatively regulate the generation of antitumor immune memory and PDT efficacy against murine colon and mammary carcinoma models. The inhibition of PDT efficacy by IL-6 appears also to be related to regulation of Bax protein expression. Increased apoptosis was observed following treatment of tumors in Il6(-/-) mice 24 hours following PDT. CONCLUSIONS: The development of PDT regimens that enhance antitumor immunity has led to proposals for the use of PDT as an adjuvant treatment. However, our results show that the potential for PDT induced expression of IL-6 to enhance tumor survival following PDT must be considered.

Introduction.

In this book you will find chapters describing the most up-to-date information concerning the important clinical applications and methodology of photodynamic therapy. This introduction will provide some background on general mechanisms, cellular targets, and note a few significant new findings relevant to photodynamic action in clinical practice. A short description of the development of photodynamic therapy at Roswell Park Cancer Institute is included.

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein.

Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185(HER-2-ECD) antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185(HER-2-ECD)-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.

A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus.

Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.

Photodynamic therapy-generated vaccines: relevance of tumour cell death expression.

Recent investigations have established that tumour cells treated in vitro by photodynamic therapy (PDT) can be used for generating potent vaccines against cancers of the same origin. In the present study, cancer vaccines were prepared by treating mouse SCCVII squamous cell carcinoma cells with photosensitiser chlorin e6-based PDT and used against poorly immunogenic SCCVII tumours growing in syngeneic immunocompetent mice. The vaccine potency increased when cells were post-incubated in culture after PDT treatment for 16 h before they were injected into tumour-bearing mice. Interfering with surface expression of phosphatidylserine (annexin V treatment) and apoptosis (caspase inhibitor treatment) demonstrated that this post-incubation effect is affiliated with the expression of changes associated with vaccine cell death. The cured mice acquired resistance to re-challenge with the same tumour, while the engagement of cytotoxic T lymphocytes was demonstrated by detection of high numbers of degranulating CD8+ cells in vaccinated tumours. The vaccines prepared from ex vivo PDT-treated SCCVII tumour tissue were also highly effective, implying that surgically removed tumour tissue can be directly used for PDT vaccines. This opens attractive prospects for employing PDT vaccines tailored for individual patients targeting specific antigens of the patient's tumour.

The development and characterisation of porphyrin isothiocyanate-monoclonal antibody conjugates for photoimmunotherapy.

A promising approach to increase the specificity of photosensitizers used in photodynamic therapy has been through conjugation to monoclonal antibodies (MAb) directed against tumour-associated antigens. Many of the conjugations performed to date have relied on the activated ester method, which can lead to impure conjugate preparations and antibody crosslinking. Here, we report the development of photosensitizer-MAb conjugates utilising two porphyrin isothiocyanates. The presence of a single reactive isothiocyanate allowed facile conjugation to MAb FSP 77 and 17.1A directed against internalizing antigens, and MAb 35A7 that binds to a non-internalizing antigen. The photosensitizer-MAb conjugates substituted with 1-3 mol of photosensitizer were characterised in vitro. No appreciable loss of immunoreactivity was observed and binding specificity was comparable to that of the unconjugated MAb. Substitution with photosensitizer had a minimal effect on antibody biodistribution in vivo for the majority of the conjugates, although a decreased serum half-life was observed using a cationic photosensitizer at the higher loading ratios. Tumour-to-normal tissue ratios as high as 33.5 were observed using MAb 35A7 conjugates. The internalizing conjugate showed a higher level of phototoxicity as compared with the non-internalizing reagent, using a cell line engineered to express both target antigens. These data demonstrate the applicability of the isothiocyanate group for the development of high-quality conjugates, and the use of internalizing MAb to significantly increase the photodynamic efficiency of conjugates during photoimmunotherapy.

Photosensitizer-antibody conjugates for detection and therapy of cancer.

Photodynamic therapy (PDT) is a promising approach for the treatment of superficially localized tumors. A limitation, however, is the lack of selectivity of the photosensitizers, which can result in severe toxicity. In this overview, the possibilities for using monoclonal antibodies (MAbs) for selective delivery of photosensitizers to tumors, are discussed. This approach is called photoimmunotherapy (PIT). For PIT to be successful, sufficient amounts of sensitizer should be coupled to the MAb without altering its biological properties. A challenging aspect herein is the hydrophobicity of therapeutic photosensitizers. Options for direct and indirect coupling of photosensitizers to MAbs are evaluated, while pros and cons are indicated. Special attention is paid to the quality testing of photoimmunoconjugates, as this information is important for further optimization of PIT. Results obtained thus far with PIT in in vitro and in vivo model systems are discussed. Despite the encouraging progress made, showing the high selectivity of photoimmunoconjugates, PIT still awaits initial clinical evaluation.

Polymorphous light eruption. A case report and consideration of the hardening mechanism.

BACKGROUND: Polymorphous light eruption (PLE) is a common form of photodermatosis. The pathogenesis of the disease and the mechanism of the hardening phenomenon, however, have not been clarified. OBSERVATION: We report a 62-year-old Japanese woman with PLE. Provocation and hardening induction testing revealed that hardening was induced at the sites irradiated by a sufficient dose of UV for reproduction of the lesion. Topical steroid could prevent a positive reaction but not the hardening induction. The expression of adhesion molecules and CD1a was decreased at the hardening site. CONCLUSION: From these results, the hardening phenomenon in our case might be due to immunosuppression by UV exposure supported by the down-regulation of cell adhesion molecule expression and partially due to the depletion of endogenous antigens which cause a delayed-type hypersensitivity reaction.

Singlet oxygen producing photosensitizers in photophoresis.

In this study, the immunosuppressive properties of two photosensitizers (benzoporphyrin derivative monoacid ring A (BPD) and Photofrin (HPD)), used for the photodynamic therapy of cancer, were investigated. The investigations were performed in our rat model for photophoresis. The validity of this model has been amply demonstrated. It enables a distinction to be made between antigen-specific and antigen non-specific immune suppression. With this model, the immune response which can be specifically suppressed is the contact hypersensitivity (CHS). CHS is induced by 2,4-dinitrofluorobenzene (DNFB). Both BPD and HPD are able to suppress CHS induced by DNFB. Furthermore, this generated suppression is transferable by the spleen cells of treated animals and is antigen non-specific.

Antigenicity of poly(dA-dT) . poly(dA-dT) photocrosslinked with 8-methoxypsoralen.

We report the formation of photocrosslink (diad-duct) between duplex poly(dA-dT) and 8-methoxypsoralen inferred on the basis of UV and fluorescence characteristics, Tm, nuclease S1 digestibility, and hydroxyapatite column chromatography. The photocrosslink was highly immunogenic inducing high titer antibodies in goat. The affinity constant of antigen-antibody interaction quantitated by precipitin titration was found to be 2.71 x 10(9) M-1, calculated by Langmuir isotherm plot. These antibodies were highly specific showing recognition of almost exclusively of nucleic acid-furocoumarin photoadducts and could be used as a probe to detect and quantitate DNA modified with furocoumarins in health and disease. Antibodies against poly(dA-dT)-furocoumarin photocrosslink might be a better probe because of repeated A-T sequences on the immunogen.

Antigen binding specificity of antibodies against 8-methoxypsoralen photoadducted DNA.

The photoadduct between native DNA and 8-methoxypsoralen was characterized on the basis of UV and fluorescence characteristics, Tm, nuclease S 1 digestibility and hydroxyapatite column chromatography. Approximately 80% of photoadduct has at least one diadduct (crosslink) per DNA molecule and only thymine was modified to the extent of 69%. The photo-crosslink was highly immunogenic inducing antibodies having apparent association constant of 1.3 x 10(-9)M. The immune IgG was highly specific towards nucleic acid-furocoumarin crosslink site without appreciable recognition of B-conformation or nucleic acid bases. The antibodies could be used as probe to detect and quantitate nucleic acid modified with furocoumarins in health and disease.

Specific targeting of phototoxic haptenated liposomes to a hapten-specific B cell lymphoma.

A method is reported to eliminate B lymphocytes specific for a haptenated lipid by using the lipid hapten to target a photosensitive drug to them. The photosensitizer eosin was coupled to a phospholipid and incorporated into trinitrophenol (TNP)-bearing small unilamellar vesicles of egg phosphatidylcholine (PC) and cholesterol in order to target the photosensitizer to B lymphoma cells (A20-HL) with TNP-specific membrane IgM receptors in vitro. Exposure of the treated cells to visible light led to an antigen-specific toxic effect indicated by inhibition of cell proliferation. A significantly higher concentration of liposomal eosin was required to inhibit control B cells. These were genetically identical B lymphoma cells (A20-2J) which lack only the DNA for the surface antigen receptor. Furthermore, pretreatment with TNP-conjugated keyhole limpet hemocyanin or anti-IgM antibody abolished the antigen-specific toxic effect, confirming that the TNP-targeted liposomal eosin mediates its effect by binding to the Ig antigen receptors on TNP-specific B cells. Incubation of cells with the TNP-bearing phototoxic liposomes at 4 degrees C instead of 37 degrees C did not alter the antigen-specific targeting effect, suggesting that uptake of the liposomal drug into the cells is not necessary for its toxic effect. Replacement of the liposomal phospholipid (egg PC) with saturated species of PC having higher phase transition temperatures or with sphingomyelin caused a decrease of the antigen-specific effect. These results demonstrate the potential use of antigen-bearing liposomal phototoxic drugs for the purpose of targeting and eliminating B cells with antigen-specific surface Ig receptors.