Toshiko Harada

Photoimmunotherapy: Comparative effectiveness of two monoclonal antibodies targeting the epidermal growth factor receptor

Photoimmunotherapy (PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by photosensitizers after exposure to near infrared (NIR) light. Herein we compare two commonly available anti‐EGFR monoclonal antibodies, cetuximab and panitumumab, for their effectiveness as PIT agents in EGFR positive tumor models. A photosensitizer, IR‐700, conjugated to either cetuximab (cet‐IR700) or panitumumab (pan‐IR700), was evaluated using EGFR‐expressing A431 and MDAMB468‐luc cells in 2D‐ and 3D‐culture. PIT was conducted with irradiation of NIR light after exposure of the sample or animal to each conjugate. In vivo PIT was performed with fractionated exposure of NIR light after injection of each agent into A431 xenografts or a MDAMB468‐luc orthotopic tumor bearing model.

Glypican-3 Targeted Human Heavy Chain Antibody as a Drug Carrier for Hepatocellular Carcinoma Therapy

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.

Activatable Organic Near-Infrared Fluorescent Probes Based on a Bacteriochlorin Platform: Synthesis and Multicolor in Vivo Imaging with a Single Excitation

Near infrared (NIR) fluorescent probes are ideal for in vivo imaging because they offer deeper tissue penetration and lower background autofluorescence. Although most fluorophores in this range are cyanine-based dyes, several new classes of fluorescent NIR probes have been developed. In this study, we developed organic bacteriochlorin derivatives, NMP4 and NMP5, which are excited with a single green light and emit different narrow, well-resolved bands in the NIR (peak of 739 and 770 nm for NMP4 and NMP5, respectively). When conjugated to galactosyl-human serum albumin (hGSA) or glucosyl-human serum albumin (glu-HSA), both targeting H-type lectins, including the β-d-galactose receptor expressing on ovarian cancer, these agents become targeted, activatable, single excitation, multicolor NIR fluorescence probes. After conjugation to either glu-HSA or hGSA, substantial quenching of fluorescence occurs that is reversed after cell binding and internalization. In vitro studies showed higher cancer cell uptake with NMP4 or NMP5 conjugated to hGSA compared to the same conjugates with glu-HSA. In vivo single excitation two-color imaging was performed after intraperitoneal injection of these agents into mice with disseminated ovarian cancer. Excited with a single green light, distinct NIR emission spectra from each fluorophore were detected and could be distinguished with spectral unmixing. In vivo results using a red fluorescence protein (RFP) labeled tumor model of disseminated ovarian cancer demonstrated high sensitivity and specificity for all probes. The success of single excitation, 2-color NIR fluorescence imaging with a new class of bacteriochlorin-based activatable fluorophores, NMP4 and NMP5, paves the way for further exploration of noncyanine dye-based NIR fluorophores.

Photoimmunotherapy Targeting Prostate-Specific Membrane Antigen: Are Antibody Fragments as Effective as Antibodies?

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 125I 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.

Near Infrared Photoimmunotherapy Targeting EGFR Positive Triple Negative Breast Cancer: Optimizing the Conjugate-Light Regimen

Aim Triple-negative breast cancer (TNBC) is considered one of the most aggressive subtypes of breast cancer. Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs an antibody-photosensitizer conjugate (APC) followed by exposure of NIR light for activating selective cytotoxicity on targeted cancer cells and may have application to TNBC. In order to minimize the dose of APC while maximizing the therapeutic effects, dosing of the APC and NIR light need to be optimized. In this study, we investigate in vitro and in vivo efficacy of cetuximab (cet)-IR700 NIR-PIT on two breast cancer models MDAMB231 (TNBC, EGFR moderate) and MDAMB468 (TNBC, EGFR high) cell lines, and demonstrate a method to optimize the dosing APC and NIR light. Method After validating in vitro cell-specific cytotoxicity, NIR-PIT therapeutic effects were investigated in mouse models using cell lines derived from TNBC tumors. Tumor-bearing mice were separated into 4 groups for the following treatments: (1) no treatment (control); (2) 300 μg of cet-IR700 i.v., (APC i.v. only); (3) NIR light exposure only, NIR light was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day 2 (NIR light only); (4) 300 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 after injection and 100 J/cm2 of light on day 2 after injection (one shot NIR-PIT). To compare different treatment regimens with a fixed dose of APC, we added the following treatments (5) 100 μg of cet-IR700 i.v., NIR light administered at 50 J/cm2 on day 1 and 50 μg of cet-IR700 i.v. immediately after NIR-PIT, then NIR light was administered at 100 J/cm2 on day 2, which were performed two times every week (“two split” NIR-PIT) and (6) 100 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day 2, which were performed three times per week (“three split” NIR-PIT). Result Both specific binding and NIR-PIT effects were greater with MDAMB468 than MDAMB231 cells in vitro. Tumor accumulation of cet-IR700 in MDAMB468 tumors was significantly higher (p < 0.05) than in MDAMB231 tumors in vivo. Tumor growth and survival of MDAMB231 tumor bearing mice was significantly lower in the NIR-PIT treatment group (p < 0.05). In MDAMB468 bearing mice, tumor growth and survival was significantly improved in the NIR-PIT treatment groups in all treatment regimens (one shot NIR-PIT; p < 0.05, “two split” NIR-PIT; p < 0.01, “three split” NIR-PIT; p < 0.001) compared with control groups. Conclusion NIR-PIT for TNBC was effective regardless of expression of EGFR, however, greater cell killing was shown with higher EGFR expression tumor in vitro. In all treatment regimens, NIR-PIT suppressed tumor growth, resulting in significantly prolonged survival that further improved by splitting the APC dose and using repeated light exposures.

Minibody-indocyanine green based activatable optical imaging probes: the role of short polyethylene glycol linkers.

Minibodies show rapider blood clearance than IgGs due to smaller size that improves target-to-background ratio (TBR) in in vivo imaging. Additionally, the ability to activate an optical probe after binding to the target greatly improves the TBR. An optical imaging probe based on a minibody against prostate-specific membrane antigen (PSMA-MB) and conjugated with an activatable fluorophore, indocyanine green (ICG), was designed to fluoresce only after binding to cell-surface PSMA. To further reduce background signal, short polyethylene glycol (PEG) linkers were employed to improve the covalent bonding ratio of ICG. New PSMA-MBs conjugated with bifunctional ICG derivatives specifically visualized PSMA-positive tumor xenografts in mice bearing both PSMA-positive and -negative tumors within 6 h postinjection. The addition of short PEG linkers significantly improved TBRs; however, it did not significantly alter the biodistribution. Thus, minibody-ICG conjugates could be a good alternative to IgG-ICG in the optical cancer imaging for further clinical applications.

Near infrared photoimmunotherapy with avelumab, an anti-programmed death-ligand 1 (PD-L1) antibody.

Near Infrared-Photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate (APC). Programmed cell death protein-1 ligand (PD-L1) is emerging as a molecular target. Here, we describe the efficacy of NIR-PIT, using fully human IgG1 anti-PD-L1 monoclonal antibody (mAb), avelumab, conjugated to the photo-absorber, IR700DX, in a PD-L1 expressing H441 cell line, papillary adenocarcinoma of lung. Avelumab-IR700 showed specific binding and cell-specific killing was observed after exposure of the cells to NIR in vitro. In the in vivo study, avelumab-IR700 showed high tumor accumulation and high tumor-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 μg of avelumab-IR700 i.v.; (3) NIR light exposure only, NIR light was administered; (4) 100 μg of avelumab-IR700 i.v., NIR light was administered. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups (p < 0.001), and significantly prolonged survival was achieved (p < 0.01 vs other groups). In conclusion, the anti-PD-L1 antibody, avelumab, is suitable as an APC for NIR-PIT. Furthermore, NIR-PIT with avelumab-IR700 is a promising candidate of the treatment of PD-L1-expressing tumors that could be readily translated to humans.

Near infrared photoimmunotherapy of B‐cell lymphoma

Near infrared photoimmunotherapy (NIR‐PIT) is a new, highly‐selective cancer theranostics that employs an antibody‐photo absorber conjugate (APC). NIR‐PIT has successfully treated preclinical tumor models with APCs and is now in the first‐in‐human phase 1 clinical trial for head and neck cancer patients against EGFR. CD20 is highly expressed in many B‐cell lymphomas and is emerging as a molecular target for this disease. Here, we describe the use of the anti‐CD20 monoclonal antibody (mAb), rituximab‐IR700 APC for NIR‐PIT of B‐cell lymphoma in two CD20‐expressing lymphoma mouse models. CD20 expressing B‐cell lymphoma cell lines (Daudi and Ramos) were used in this study. Rituximab‐IR700, rituximab conjugated with IRDye700DX, showed specific binding, and cell‐specific killing only after exposure of NIR light to both cells in vitro. To evaluate effects of NIR‐PIT in vivo, tumor‐bearing mice were separated into 4 groups: (1) control; (2) APC i.v. only; (3) NIR light exposure only; (4) APC and NIR light (NIR‐PIT). These were performed every week for up to 3 weeks. Rituximab‐IR700 showed high tumor accumulation and high target‐to‐background ratio in vivo. Tumor growth was significantly inhibited by NIR‐PIT in comparison with the other groups (p < 0.001 for both tumors), and survival was significantly prolonged in both tumors (p < 0.001 for Daudi tumors and p < 0.0001 for Ramos tumors vs other groups). More than half of tumors were cured with this single regimen of NIR‐PIT. In conclusion, anti‐CD20 rituximab‐IR700 works as a highly effective APC for NIR‐PIT against B‐cell lymphoma.

Improved micro-distribution of antibody-photon absorber conjugates after initial near infrared photoimmunotherapy (NIR-PIT).

Near infrared photoimmunotherapy (NIR-PIT), a targeted cancer therapy which uses an antibody-photo absorber conjugate (APC) and near infrared light exposure, dramatically improves nano-drug delivery into treated tumor beds due to enhanced vascular permeability. We investigated the micro-distribution of APCs in a variety of NIR-PIT treated tumors. Either cetuximab (cet) or trastuzumab (tra) conjugated with IR700 (cet-tra-IR700) was administered, as appropriate, to each mouse model of tumor. Tumor-bearing mice implanted with A431-GFP, MDAMB468-GFP, 3T3Her2-GFP or N87-GFP were separated into 5 groups: group 1=no treatment; group 2=cet-tra-IR700 i.v., no light exposure; group 3=cet-tra-IR700 i.v., NIR light exposure; group 4=cet-tra-IR700 i.v. and additional cet-tra-IR700 i.v. at 24h but no light exposure; group 5=cet-tra-IR700 i.v., NIR light exposure and additional cet-tra-IR700 i.v. immediately after NIR but no additional NIR light exposure. In vivo, ex vivo and microscopic fluorescence imaging was performed. Fluorescence from the surface of the tumor (s-tumor) was compared to fluorescence from deeper areas of the tumor (d-tumor). In general, there was no significant difference in the fluorescence intensity of GFP in the tumors among all groups, however the highest IR700 fluorescence intensity was consistently shown in group 5 tumors due to added APC after NIR-PIT. Fluorescence microscopy in all tumor types demonstrated that GFP relative fluorescence intensity (RFI) in s-tumor was significantly lower in group 3 and 5 (NIR-PIT groups) than in group 1, 2, and 4 (no NIR-PIT) yet there was no significant difference in d-tumor RFI among all groups. IR700 fluorescent RFI in the d-tumor was highest in group 5 (NIR-PIT+additional APC) compared to the other groups. Cell killing after NIR-PIT was primarily on the surface, however, APCs administered immediately after NIR-PIT penetrated deeper into tissue resulting in improved cell killing after a 2nd NIR-PIT session. This phenomenon is explained by increased vascular permeability immediately after NIR-PIT.

Photoimmunotherapy of hepatocellular carcinoma-targeting Glypican-3 combined with nanosized albumin-bound paclitaxel

AIM Effectiveness of Glypican-3 (GPC3)-targeted photoimmunotherapy (PIT) combined with the nanoparticle albumin-bound paclitaxel (nab-paclitaxel) for hepatocellular carcinoma was evaluated. MATERIALS & METHODS GPC3 expressing A431/G1 cells were incubated with a phthalocyanine-derivative, IRDye700DX (IR700), conjugated to an anti-GPC3 antibody, IR700-YP7 and exposed to near-infrared light. Therapeutic experiments combining GPC3-targeted PIT with nab-paclitaxel were performed in A431/G1 tumor-bearing mice. RESULTS IR700-YP7 bound to A431/G1 cells and induced rapid target-specific necrotic cell death by near-infrared light exposure in vitro. IR700-YP7 accumulated in A431/G1 tumors. Tumor growth was inhibited by PIT compared with nontreated control. Additionally, PIT dramatically increased nab-paclitaxel delivery and enhanced the therapeutic effect. CONCLUSION PIT targeting GPC3 combined with nab-paclitaxel is a promising method for treating hepatocellular carcinoma.