Photoimmunotechnology as a powerful biological tool for molecular-based elimination of target cells and microbes, including bacteria, fungi and viruses.

Microbial pathogens, including bacteria, fungi and viruses, can develop resistance to clinically used drugs; therefore, finding new therapeutic agents is an ongoing challenge. Recently, we reported the photoimmuno-antimicrobial strategy (PIAS), a type of photoimmunotechnology, that enables molecularly targeted elimination of a wide range of microbes, including the viral pathogen severe acute respiratory syndrome coronavirus 2 and the multidrug-resistant bacterial pathogen methicillin-resistant Staphylococcus aureus (MRSA). PIAS works in the same way as photoimmunotherapy (PIT), which has been used to treat recurrent head and neck cancer in Japan since 2020. Both PIAS and PIT use a monoclonal antibody conjugated to a phthalocyanine derivative dye that undergoes a shape change when photoactivated. This shape change induces a structural change in the antibody-dye conjugate, resulting in physical stress within the binding sites of the conjugate and disrupting them. Therefore, targeting accuracy and flexibility can be determined based on the specificity of the antibody used. In this protocol, we describe how to design a treatment strategy, label monoclonal antibodies with the dye and characterize the products. We provide detailed examples of how to set up and perform PIAS and PIT applications in vitro and in vivo. These examples are PIAS against microbes using MRSA as a representative subject, PIAS against viruses using severe acute respiratory syndrome coronavirus 2 in VeroE6/TMPRSS2 cells, PIAS against MRSA-infected animals, and in vitro and in vivo PIT against cancer cells. The in vitro and in vivo protocols can be completed in ~3 h and 2 weeks, respectively.

Development of a Monoclonal Antibody Targeting HTLV-1 Envelope gp46 Glycoprotein and Its Application to Near-Infrared Photoimmuno-Antimicrobial Strategy.

Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, causes adult T-cell leukemia-lymphoma, HTLV-1 associated myelopathy/tropical spastic paraparesis, and HTLV-1 uveitis. Currently, no antiretroviral therapies or vaccines are available for HTLV-1 infection. This study aimed to develop an antibody against the HTLV-1 envelope protein (Env) and apply it to a near-infrared photoimmuno-antimicrobial strategy (NIR-PIAS) to eliminate HTLV-1 infected cells. We established mouse monoclonal antibodies (mAbs) against HTLV-1 Env by immunization with a complex of liposome and the recombinant protein. Detailed epitope mapping revealed that one of the mAbs bound to the proline-rich region of gp46 and exhibited no obvious neutralizing activity to inhibit viral infection. Instead, the mAb was rarely internalized intracellularly and remained on the cell surface of HTLV-1-infected cells. The antibody conjugated to the photosensitive dye IRDye700Dx recognized HTLV-1 infected cells and killed them following NIR irradiation. These results suggest that the novel mAb and NIR-PIAS could be developed as a new targeted therapeutic tool against HTLV-1 infected cells.

Antimicrobial strategy for targeted elimination of different microbes, including bacterial, fungal and viral pathogens.

The continuous emergence of microbial pathogens for which there are no effective antimicrobials threatens global health, necessitating novel antimicrobial approaches. Here, we present a targeted antimicrobial strategy that can be applied to various microbial pathogens. A photoimmuno-conjugate composed of an antibody against the target pathogen and a photoplastic phthalocyanine-derivative probe that generates photo-induced mechanical stress was developed based on photoimmuno-technology. This strategy, named as photoimmuno-antimicrobial strategy (PIAS), eliminates targeted pathogens, regardless of the target species or drug-resistance status. Specifically, PIAS acts on a broad range of microbes, including the bacterial pathogen Staphylococcus aureus, fungal pathogen Candida albicans, including their drug-resistant strains, and viral pathogen SARS-CoV-2, the causative agent of COVID-19. Furthermore, PIAS protects mice from fatal infections without damaging the non-targeted host microbiota and tissues. This study may contribute to the development of next-generation anti-infective therapies.

Cancer neovasculature-targeted near-infrared photoimmunotherapy (NIR-PIT) for gastric cancer: different mechanisms of phototoxicity compared to cell membrane-targeted NIR-PIT.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) constitutes a new class of molecular-targeted theranostics utilizing monoclonal antibody (mAb)-photosensitizer conjugates and NIR light. In this study, we developed a new type of NIR-PIT targeting vascular endothelial growth factor receptor 2 (VEGFR-2) expressed on vascular endothelium in an experimental gastric cancer model and evaluated the feasibility by comparing conventional NIR-PIT targeting cancer cell membrane in vitro and in vivo. METHODS: HER2-positive human gastric cancer cells, NCI-N87, were used for the experiments. Anti-HER2 mAb, trastuzumab and anti-VEGFR-2 mAb, DC101 were conjugated to photosensitizer, IR700. Phototoxicity in response to NIR-PIT were investigated in vitro and in vivo. Microvessel densities, as an indicator of angiogenesis, were counted in harvested xenografts after NIR-PIT to elucidate the mechanism. RESULTS: DC101-IR700 did not induce phototoxic effect in vitro because of the absence of expression of VEGFR-2 in NCI-N87 cancer cells. However, it induced an antitumor effect in NCI-N87 xenograft tumors accompanied with damage in tumor neovasculature as determined by decreasing tumor microvessel density, which represents a different mechanism than that of conventional NIR-PIT targeting antigens expressed on the tumor cell membrane. CONCLUSION: We demonstrated a new approach of NIR-PIT utilizing a target on vascular endothelium, such as VEGFR-2, and this treatment might lead to the development of a new therapeutic strategy for human gastric cancer.

Photoimmunotherapy targeting biliary-pancreatic cancer with humanized anti-TROP2 antibody.

Photoimmunotherapy (PIT) is a new type of tumor-specific treatment utilizing monoclonal antibody (mAb)-photosensitizer conjugates and near-infrared (NIR) light irradiation. One potential PIT target, the type I transmembrane protein TROP2, is expressed at high levels in many cancers, including pancreatic carcinoma (PC) and cholangiocarcinoma (CC), in which its expression is correlated with poor prognosis and tumor aggressiveness. In this study, we assessed the efficacy of PIT utilizing newly developed humanized anti-TROP2 mAb conjugated to the photosensitizer IR700 (TROP2-IR700) for PC and CC. Immunohistochemistry on PC and CC tissue microarrays confirmed that TROP2 is overexpressed in about half of PC and CC specimens. Using cultured PC and CC cells, TROP2-IR700 localized TROP2-specific and target-specific cell killing was observed after NIR light irradiation. In addition, TROP2-IR700 was localized to mouse xenograft tumors expressing TROP2 after intravenous injection. PC and CC xenograft tumor growth was significantly inhibited by TROP2-targeted PIT relative to controls. The efficacy of TROP2-targeted PIT in vitro and against xenografted tumors in vivo suggests promise as a therapy for human PC and CC, both of which currently have dismal prognoses and limited therapeutic options.

Near-Infrared Photochemoimmunotherapy by Photoactivatable Bifunctional Antibody-Drug Conjugates Targeting Human Epidermal Growth Factor Receptor 2 Positive Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a new class of molecular targeted cancer therapy based on antibody-photoabsorber conjugates and NIR light irradiation. Recent studies have shown effective tumor control, including that of human epidermal growth factor receptor 2 (HER2)-positive cancer, by selective molecular targeting with NIR-PIT. However, the depth of NIR light penetration limits its use. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent maytansinoid DM1. Here, we developed bifunctional antibody-drug-photoabsorber conjugates, T-DM1-IR700, that can work as both NIR-PIT and chemoimmunotherapy agents. We evaluated the feasibility of T-DM1-IR700-mediated NIR light irradiation by comparing the in vitro and in vivo cytotoxic efficacy of trastuzumab-IR700 (T-IR700)-mediated NIR light irradiation in HER2-expressing cells. T-IR700 and T-DM1-IR700 showed almost identical binding to HER2 in vitro and in vivo. Owing to the presence of internalized DM1 in the target cells, NIR-PIT using T-DM1-IR700 tended to induce greater cytotoxicity than that of NIR-PIT using T-IR700 in vitro. In vivo NIR-PIT using T-DM1-IR700 did not show a superior antitumor effect to NIR-PIT using T-IR700 in subcutaneous small-tumor models, which could receive sufficient NIR light. In contrast, NIR-PIT using T-DM1-IR700 tended to reduce the tumor volume and showed significant prolonged survival compared to NIR-PIT using T-IR700 in large-tumor models that could not receive sufficient NIR light. We successfully developed a T-DM1-IR700 conjugate that has a similar immunoreactivity to the parental antibody with increased cytotoxicity due to DM1 and potential as a new NIR-PIT agent for targeting tumors that are large and inaccessible to sufficient NIR light irradiation to activate the photoabsorber IR700.

Combination photoimmunotherapy with monoclonal antibodies recognizing different epitopes of human epidermal growth factor receptor 2: an assessment of phototherapeutic effect based on fluorescence molecular imaging.

Photoimmunotherapy is a new class of molecular targeted cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer and irradiation with near-infrared (NIR) light for both imaging and therapy. Here, we sought to determine the feasibility of combining photoimmunotherapy using conjugates of human epidermal growth factor receptor 2 (HER2)-specific mAb-photosensitizer IR700, trastuzumab-IR700 and pertuzumab-IR700. HER2-expressing and non-expressing cells were treated with mAb-IR700 conjugates and irradiated with NIR light. Fluorescence imaging and cytotoxic effects were examined in cultured HER2-expressng cancer cell lines and in a mouse tumor xenograft model. Trastuzumab-IR700 and pertuzumab-IR700 could specifically bind to HER2 without competing, and the combination treatment of both agents yielded stronger HER2-specific IR700 fluorescence signals than with the treatment with either agent singly. A cytotoxicity assay showed that the combination treatment of both trastuzumab-IR700 and pertuzumab-IR700 followed by NIR light irradiation induced stronger cytotoxic effect than with treatment of either agent plus NIR light irradiation. Furthermore, the phototoxic and cytotoxic effects of mAb depended on HER2-specific IR700 signal intensities. Consistent with in vitro studies, in xenograft tumor models also, IR700 fluorescence imaging-guided NIR light irradiation after the combination treatment of trastuzumab-IR700 and pertuzumab-IR700 led to stronger antitumor effects than by treatment with either agent followed by NIR light irradiation. In conclusion, fluorescence molecular imaging can facilitate the assessment of treatment outcomes of molecular targeted photoimmunotherapy, which holds great potential in facilitating better outcomes in cancer patients.

Molecular targeted photoimmunotherapy for HER2-positive human gastric cancer in combination with chemotherapy results in improved treatment outcomes through different cytotoxic mechanisms.

BACKGROUND: Photoimmunotherapy (PIT) is a novel type of molecular optical imaging-guided cancer phototherapy based on a monoclonal antibody conjugated to a photosensitizer, IR700, in combination with near-infrared (NIR) light. PIT rapidly causes target-specific cell death by inducing cell membrane damages and appears to be highly effective; however, we have previously demonstrated that tumor recurrences were eventually seen in PIT-treated mice, likely owing to inhomogeneous mAb-IR700 conjugate distribution in the tumor, thus limiting the effectiveness of PIT as a monotherapy. Here, we examined the effects of human epidermal growth factor-2 (HER2)-targeted PIT in combination with 5-fluorouracil (5-FU) compared to PIT alone for HER2-expressing human gastric cancer cells. METHODS: NCI-N87 cells, HER2-positive human gastric cancer cells, were used for the experiments. Trastuzumab, a monoclonal antibody directed against HER2, was conjugated to IR700. To assess the short-term cytotoxicity and examine the apoptotic effects upon addition of 5-FU in vitro, we performed LIVE/DEAD and caspase-3 activity assays. Additionally, to explore the effects on long-term growth inhibition, trypan blue dye exclusion assay was performed. NCI-N87 tumor xenograft models were prepared for in vivo treatment studies and the tumor-bearing mice were randomized into various treatment groups. RESULTS: Compared to PIT alone, the combination of HER2-targeted PIT and 5-FU rapidly induced significant cytotoxicity in both the short-term and long-term cytotoxicity assays. While both 5-FU and/or trastuzumab-IR700 conjugate treatment induced an increase in caspase-3 activity, there was no additional increase in caspase-3 activity upon NIR light irradiation after incubation with 5-FU and/or trastuzumab-IR700. The combination of HER2-targeted PIT and 5-FU resulted in greater and longer tumor growth inhibition than PIT monotherapy in vivo. This combined effect of PIT and 5-FU is likely owing to their different mechanisms of inducing tumor cell death, namely necrotic membrane damage by PIT and apoptotic cell death by 5-FU and trastuzumab. CONCLUSIONS: PIT in combination with 5-FU resulted in enhanced antitumor effects compared to PIT alone for HER2-expressing human gastric cancer in vitro and in vivo. This combination photoimmunochemotherapy represents a practical method for treating human gastric cancer and should be investigated further in the clinical setting.

Near infrared photoimmunotherapy for lung metastases.

Lung metastases are a leading cause of cancer related deaths; nonetheless current treatments are limited. Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of intravenously injected antibodies that target tumors with the toxicity induced by photosensitizers activated by NIR-light. Herein, we demonstrate the efficacy of NIR-PIT in a mouse model of lung metastases. Experiments were conducted with a HER2, luciferase and GFP expressing cell line (3T3/HER2-luc-GFP). An antibody-photosensitizer conjugate (APC) consisting of trastuzumab and a phthalocyanine dye, IRDye700DX, was synthesized. In vitro NIR-PIT-induced cytotoxicity was light dose dependent. With 3D culture, repeated NIR-PIT could eradicate entire spheroids. In vivo anti-tumor effects of NIR-PIT included significant reductions in both tumor volume (p = 0.0141 vs. APC) and bioluminescence image (BLI) (p = 0.0086 vs. APC) in the flank model, and prolonged survival (p < 0.0001). BLI demonstrated a significant reduction in lung metastases volume (p = 0.0117 vs. APC). Multiple NIR-PIT doses significantly prolonged survival in the lung metastasis model (p < 0.0001). These results suggested that NIR-PIT is a potential new therapy for the local control of lung metastases.

Photoimmunotherapy targeting prostate-specific membrane antigen: are antibody fragments as effective as antibodies?

UNLABELLED: Photoimmunotherapy is a highly cell-selective cancer therapy based on an armed antibody conjugate with a phthalocyanine-based photosensitizer, IR700. Photoimmunotherapy induces rapid and highly specific necrosis in targeted cancer cells after exposure to near-infrared (NIR) light. Cells not expressing the antigen are not affected. To date, photoimmunotherapy has been demonstrated only with full antibody-IR700 conjugates. In this study, small and bivalent antibody fragments, including anti-prostate-specific membrane antigen (PSMA) diabody (Db) and minibody (Mb), were compared with intact IgG for their effectiveness as photoimmunotherapy agents. METHODS: Radioiodinated antibody and antibody fragments with (125)I were used to determine the timing of maximum binding of each anti-PSMA antibody fragment on the cell surface in vivo in mice bearing either PSMA-positive or -negative PC3 tumors. Then therapeutic efficacy of photoimmunotherapy was examined by exposing mice to NIR light at 2 time points based on the time of maximum cell surface binding at 6 h after injection for Db-IR700 and 24 h after injection for Mb-IR700 and IgG-IR700 as well as 24 h after the peak uptake times. RESULTS: Photoimmunotherapy with the same molar concentration of PSMA-Db-IR700, PSMA-Mb-IR700, and PSMA-IgG-IR700 conjugate showed similar therapeutic effects in vitro and in vivo on PSMA-positive PC3 tumor xenografts in cytotoxicity and survival curves (P > 0.05). CONCLUSION: The use of PSMA-Db-IR700 conjugate results in the shortest time interval between injection and NIR exposure without compromising therapeutic effects of photoimmunotherapy.

Prostaglandin E-major urinary metabolite as a reliable surrogate marker for mucosal inflammation in ulcerative colitis.

BACKGROUND: C-reactive protein (CRP) is used as a biomarker of ulcerative colitis (UC) activity, but CRP levels are sometimes insufficient to reflect UC activity. Therefore, a simple noninvasive biomarker assay with sufficient sensitivity and specificity to accurately reflect UC activity is desired. Since prostaglandin E2 production and colonic inflammation are associated, we evaluated whether prostaglandin E-major urinary metabolite (PGE-MUM) can be used as such a biomarker. METHODS: Patients with UC (n = 99) were enrolled from March 2011 to February 2012. UC activity was evaluated using the simple clinical colitis activity index in 99 patients, Mayo endoscopic scoring (Mayo) in 79 patients, and Matts' grading (Matts) in 64 patients. PGE-MUM levels were measured by radioimmunoassay kit and compared against CRP levels as a control. RESULTS: Both PGE-MUM and CRP levels correlated with UC activity (P < 0.01). Areas under the receiver operating characteristic curves of simple clinical colitis activity index, Mayo, and Matts for PEG-MUM were each higher than for CRP (0.93 > 0.73, 0.90 > 0.77, and 0.89 > 0.75, respectively). In multivariate logistic regression models, PGE-MUM was a significant independent predictor of histologic remission (sensitivity/specificity, 0.82/0.82) when the cutoff value was set to 17.0 µg/g creatinine, but CRP was not (0.69/0.69) (P < 0.01). CONCLUSIONS: Compared with CRP level, PGE-MUM level demonstrated better sensitivity for reflecting UC activity, especially in cases of histologic inflammation, and thus seems to be a better evaluator of mucosal healing. Because this method is simple, quick, and noninvasive, PGE-MUM seems to be a useful biomarker of UC.

Acute cytotoxic effects of photoimmunotherapy assessed by 18F-FDG PET.

UNLABELLED: We have recently developed a cancer-specific therapy, photoimmunotherapy, which uses an antibody-IR700 (phototoxic phthalocyanine dye) conjugate to bind to the cell membrane and near-infrared light to induce immediate and highly specific tumor killing in vivo. For monitoring the acute cytotoxic effects of photoimmunotherapy before the tumor begins to shrink, we used (18)F-FDG PET before and after this intervention in mice. METHODS: Photoimmunotherapy was performed by binding panitumumab (anti-HER1)-IR700 to HER1-positive tumor cells (A431), followed by near-infrared light irradiation in vitro and in vivo. The uptake of (18)F-FDG in the tumor after photoimmunotherapy was evaluated in cellular uptake studies and PET imaging studies. Serial histologic analyses were conducted after photoimmunotherapy. RESULTS: The in vitro cellular uptake of (18)F-FDG was reduced as the dose of light increased, and at high light dose (2 J/cm(2)) the uptake was reduced by more than 99% within 1 h after photoimmunotherapy. In vivo (18)F-FDG PET imaging showed that the accumulation of radioactivity in the treated tumors decreased 76% at 75 min after photoimmunotherapy and did not change for 24 h. In contrast, no significant changes were demonstrated in nontreated tumors. None of tumors changed size within 24 h after photoimmunotherapy, although diffuse necrosis was observed in photoimmunotherapy-treated tumors. CONCLUSION: Immediate cytotoxic effects induced by photoimmunotherapy were clearly detected by decreased glucose uptake using (18)F-FDG PET even before changes in tumor size became evident. (18)F-FDG allows the clinical assessment of the therapeutic effects of photoimmunotherapy earlier than anatomic methods that rely on tumor size.

Fluorescence endoscopic detection of murine colitis-associated colon cancer by topically applied enzymatically rapid-activatable probe.

OBJECTIVES: Screening colonoscopy to monitor for early colitis-associated colon cancer (CAC) is difficult due to the aberrant mucosal patterns associated with long-standing colitis. The aim of this study was to develop a rapid fluorescent detection method for use during colonoscopy for improving the detection of CAC utilising a topically applied enzymatically activatable probe (gGlu-HMRG) which fluoresces in the presence of γ-glutamyltranspeptidase (GGT), an enzyme associated with cancer. METHODS: Expression of GGT in colon cell lines was examined with fluorescence microscopy and flow cytometry. A mouse model (azoxymethane/dextran sulphate sodium) of CAC was used and mice were examined with white light and fluorescence colonoscopy before and after topical gGlu-HMRG administration. RESULTS: Expression of GGT, although variable, was higher in human colon cancer cells than normal human colon cells. Using fluorescence colonoscopy in mice, gGlu-HMRG fluorescent lesions were detected 5 min after topical administration and fluorescence persisted for at least 30 min. Fluorescence guided biopsy revealed all fluorescent lesions that contained cancer or dysplasia (n=16), whereas three out of 12 non-fluorescent lesions contained low grade dysplasia and others did not contain neoplastic histology. Microscopic inflammatory infiltration also had variable fluorescence but in general was much lower (∼10-fold) in signal than cancer. Repeat fluorescence endoscopy allowed individual tumours to be monitored. CONCLUSION: These results suggest that gGlu-HMRG can improve endoscopic detection of CAC with a higher target to background ratio than conventional white light colonoscopy. This could be of benefit to patients with long-standing colitis who must undergo repeated screening colonoscopies.

In vivo real-time lymphatic draining using quantum-dot optical imaging in mice.

The lymphatic system is essential for fluid regulation and for the maintenance of host immunity. However, in vivo lymph flow is difficult to track in real time, because of the lack of an appropriate imaging method. In this study, we combined macro-zoom fluorescence microscopy with quantum-dot (Qdot) optical lymphatic imaging to develop an in vivo real-time optical lymphatic imaging method that allows the tracking of lymph through lymphatic channels and into lymph nodes. After interstitial injection of Qdots in a mouse, rapid visualization of the cervical lymphatics and cervical lymph nodes was achieved. Real-time monitoring of the injected Qdots revealed that the cortex of the node enhanced first followed by a net-like pattern in the central portion of the node. Histology revealed that the rim and net-like enhancing regions corresponded to the subcapsular sinuses and medullary sinuses respectively. Additionally, multiplexed two-color real-time lymphatic tracking was performed with two different Qdots. With this real-time imaging system, we successfully tracked microscopic lymphatic flow in vivo. This method could have a potential impact for lymphatic research in visualizing normal or abnormal functional lymphatic flows.

Immediate in vivo target-specific cancer cell death after near infrared photoimmunotherapy.

BACKGROUND: Near infrared (NIR) photoimmunotherapy (PIT) is a new type of cancer treatment based on a monoclonal antibody (mAb)-NIR phthalocyanine dye, (IR700) conjugate. In vitro cancer-specific cell death occurs during NIR light exposure in cells previously incubated with mAb-IR700 conjugates. However, documenting rapid cell death in vivo is more difficult. METHODS: A luciferase-transfected breast cancer cell (epidermal growth factor receptor+, MDA-MB-468luc cells) was produced and used for both in vitro and in vivo experiments for monitoring the cell killing effect of PIT. After validation of cytotoxicity with NIR exposure up to 8 J/cm2in vitro, we employed an orthotopic breast cancer model of bilateral MDA-MB-468luc tumors in female athymic mice, which subsequently received a panitumumab-IR700 conjugate in vivo. One side was used as a control, while the other was treated with NIR light of dose ranging from 50 to 150 J/cm2. Bioluminescence imaging (BLI) was performed before and after PIT. RESULTS: Dose-dependent cell killing and regrowth was successfully monitored by the BLI signal in vitro. Although tumor sizes were unchanged, BLI signals decreased by >95% immediately after PIT in vivo when light intensity was high (>100 J/cm2), however, in mice receiving lower intensity NIR (50 J/cm2), tumors recurred with gradually increasing BLI signal. CONCLUSION: PIT induced massive cell death of targeted tumor cells immediately after exposure of NIR light that was demonstrated with BLI in vivo.

Real-time monitoring of in vivo acute necrotic cancer cell death induced by near infrared photoimmunotherapy using fluorescence lifetime imaging.

A new type of monoclonal antibody (mAb)-based, highly specific phototherapy (photoimmunotherapy; PIT) that uses a near infrared (NIR) phthalocyanine dye, IRDye700DX (IR700) conjugated with a mAb, has recently been described. NIR light exposure leads to immediate, target-selective necrotic cell death in vitro. Detecting immediate in vivo cell death is more difficult because it takes at least 3 days for the tumor to begin to shrink in size. In this study, fluorescence lifetime (FLT) was evaluated before and after PIT for monitoring the immediate cytotoxic effects of NIR mediated mAb-IR700 PIT. Anti-epidermal growth factor receptor (EGFR) panitumumab-IR700 was used for targeting EGFR-expressing A431 tumor cells. PIT with various doses of NIR light was conducted in cell pellets in vitro and in subcutaneously xenografted tumors in mice in vivo. FLT measurements were obtained before and 0, 6, 24, and 48 hours after PIT. In vitro, PIT at higher doses of NIR light immediately led to FLT shortening in A431 cells. In vivo PIT induced immediate shortening of FLT in treated tumors after a threshold NIR dose of 30 J/cm(2) or greater. In contrast, lower levels of NIR light (10 J/cm(2) or smaller) did not induce shortening of FLT. Prolongation of FLT in tissue surrounding the tumor site was noted 6 hours after PIT, likely reflecting phagocytosis by macrophages. In conclusion, FLT imaging can be used to monitor the acute cytotoxic effects of mAb-IR700-induced PIT even before morphological changes can be seen in the targeted tumors.

In vivo breast cancer characterization imaging using two monoclonal antibodies activatably labeled with near infrared fluorophores.

INTRODUCTION: The gene expression profiles of cancer cells are closely related to their aggressiveness and metastatic potential. Antibody-based immunohistochemistry (IHC) of tissue specimens is a common method of identifying expressed proteins in cancer cells and increasingly inform treatment decisions. Molecular imaging is a potential method of performing similar IHC studies in vivo without the requirement for biopsy or tumor excision. To date, antibody-based imaging has been limited by high background levels related to slow clearance, making such imaging practical. However, optically activatable imaging agents, which are only fluorescent when bound to their cognate receptor, open the possibility of doing in vivo multi-color IHC. METHODS: We describe the use of activatable, near infrared fluorescence-labeled AlexaFluor680 (Alexa680) conjugated panitumumab (Pan) targeted against human epidermal growth factor receptor (EGFR) (Pan-Alexa680) and Indocyanine Green (ICG) conjugated trastuzumab (Tra) targeted against human epidermal growth factor receptor type 2 (HER2) (Tra-ICG) were synthesized and evaluated in cells in vitro and in an orthotopic breast cancer mouse model in vivo. RESULTS: Pan-Alexa680 (self-quenched; SQ) and Tra-ICG were initially quenched but demonstrated a 5.2- and 50- fold dequenching capacity under detergent treatment, respectively. In vitro microscopy and flow cytometry using MDA-MB-468 (EGFR+/HER2-) and 3T3/HER2 cells (EGFR-/HER2+), demonstrated specific fluorescence signal for each cell type based on binding to Pan-Alexa680(SQ) or Tra-ICG. An in vivo imaging study employing a cocktail of Pan- Alexa680(SQ) and Tra-ICG (each 50 µg) was injected into mice with orthotopic MDA-MB-468 and 3T3/HER2 tumors in the breast. Each probe visualized only the target-specific breast tumor. CONCLUSIONS: Multi-color target-specific fluorescence breast cancer imaging can be achieved in vivo by employing two activatable fluorescent probes administered as a cocktail. The images allowed us to see a specific receptor expression in each breast tumor without post-image processing.

MR and optical imaging of early micrometastases in lymph nodes: triple labeling with nano-sized agents yielding distinct signals.

Few imaging methods are available for depicting in vivo cancer cell migration within the lymphatic system. Detection of such early micrometastases requires extremely high target to background. In this study, we dual-labeled human breast cancer cells (MDA-MB468) with a small particle of iron oxide (SPIO) and a quantum dot (QD), and tracked these cells in the lymphatic system in mice using in vivo MRI and optical imaging. A generation-6 gadolinium-dendrimer-based MRI contrast agent (Gd-G6) was employed for visualizing regional lymphatic channels and nodes. Since Gd-G6 shortened T(1) leading to high signal, whereas SPIO-labeled cancer cells greatly lowered signal, a small number of cells were simultaneously visualized within the draining lymphatic basins. One million dual-labeled cancer cells were subcutaneously injected into the paws of mice 24 h prior to imaging. Then whole body images were acquired pre- and post-intracutaneous injection of Gd-G6 with 3D-T(1) w-FFE and balanced-FFE sequences for cancer cell tracking and MR lymphangiography. In vivo MRI clearly visualized labeled cancer cells migrating from the paw to the axillary lymph nodes using draining lymphatics. In vivo optical imaging using a fluorescence surgical microscope demonstrated tiny cancer cell clusters in the axillary lymph node with high spatial resolution. Thus, using a combination of MRI and optical imaging, it is possible to depict macro- and early micrometastases within the lymphatic system. This platform offers a versatile research tool for investigating and treating lymphatic metastases in animal models.

Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate.

Armed antibody-based targeted molecular therapies offer the possibility of effective tumor control with a minimum of side effects. Photoimmunotherapy (PIT) employs a monoclonal antibody-phototoxic phthalocyanine dye, IR700 conjugate, that is activated by focal near-infrared (NIR) light irradiation after antibody binding to the targeted tumor cell surface leading to rapid necrotic cell death. Therapy by single NIR light irradiation was effective without significant side effects; however, recurrences were seen in most treated mice probably because of inhomogeneous distribution of panitumumab-IR700 immunoconjugate in the tumor, leading to ineffective PIT. We describe here an optimized regimen of effective PIT method for the same HER1-overexpressing tumor model (A431) with fractionated administration of panitumumab-IR700 conjugate followed by systematic repeated NIR light irradiation to the tumor based on timing of antibody redistribution into the remnant tumor under the guidance of IR700 fluorescence signal. Eighty percent of the A431 tumors were eradicated with repeated PIT without apparent side effects and survived tumor-free for more than 120 days even after stopping therapy at day 30. Therapeutic effects were monitored using IR700 fluorescent signal. PIT is a promising highly selective and clinically feasible theranostic method for treatment of mAb-binding tumors with minimal off-target effects.