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Regulatory T cells (Tregs) are known to inhibit antitumor immunity, yet the specific mechanism by which intratumoral Tregs promote tumor growth remains unclear. To better understand the roles of intratumoral Tregs, we selectively depleted tumor-infiltrating Tregs using anti-CD25-F(ab′)2 near-infrared photoimmunotherapy. Depletion of tumor-infiltrating Tregs induced transient but synchronized IFNγ expression in CD8 T and natural killer (NK) cells. Despite the small fraction of CD8 T and NK cells contained within examined tumors, IFNγ produced by these CD8 T and NK cells led to efficient and rapid tumor vessel regression, intratumoral ischemia, and tumor necrosis/apoptosis and growth suppression. IFNγ receptor expression on vascular endothelial cells was required for these effects. Similar findings were observed in the early phase of systemic Treg depletion in tumor-bearing Foxp3DTR mice; combination with IL15 therapy further inhibited tumor growth and achieved increased complete regression. These results indicate the pivotal roles of intratumoral Tregs in maintaining tumor vessels and tumor growth by suppressing CD8 T and NK cells from producing IFNγ, providing insight into the mechanism of Treg-targeting therapies. Significance: Intratumoral Treg depletion induces synchronized intratumoral CD8 T- and NK-cell activation, IFNγ-dependent tumor vessel regression, and ischemic tumor necrosis/apoptosis, indicating the roles of intratumoral Tregs to support the tumor vasculature.

Near-infrared photoimmunotherapy (NIR-PIT) is a new anti-cancer treatment by NIR light-induced photochemical reactions within antibody-IRdye700DX conjugates, leading to significant immunogenic cell death. The efficacy of NIR-PIT is synergistically enhanced by combining immune activating agents including immune checkpoint inhibitors and cytokines. However, there are still needs for improving the efficacy of NIR-PIT. A recent study showed that cytokines could exert strong anti-cancer effects when administered locally. Interleukin-15 (IL-15) elicits immune responses that include the generation and persistence of cytotoxic T cells and natural killer (NK) cells. Based on these findings, we hypothesized that local IL-15 administration combined with cancer cell-targeted NIR-PIT could be a highly effective therapy. The efficacy of IL-15 was evaluated by intratumorally or intraperitoneally injecting 5 μg of human IL-15 in C57BL/6 mice inoculated s.c. with murine colon cancer MC38 cells. Human IL-15-secreting MC38 (hIL-15-MC38) was constructed by introducing human IL-15-coding plasmids into MC38 cells and the efficacy of CD44-targeted NIR-PIT was compared in vitro and in vivo. The efficacy of intratumoral IL-15 injection combined with CD44-targeted NIR-PIT was also evaluated in MC38 tumor models. Moreover, bilateral tumor models were used to analyze whether local IL-15 administration combined with CD44-targeted NIT-PIT could induce abscopal effects in untreated tumors. Intratumoral IL-15 injection significantly suppressed tumor growth and induced immune activities compared with intraperitoneal injection. Both hIL-15-MC38 and parental MC38 cells had high CD44 expression on their cell surface and in vitro NIR-PIT induced rapid cell death in both two cell lines. When hIL-15-MC38 or MC38 tumor-bearing mice were treated with or without NIR-PIT, hIL-15-MC38 tumor-bearing mice treated with NIR-PIT showed the best tumor growth inhibition and survival. The hIL-15-MC38 tumors treated with NIR-PIT showed significant increases in the cell number/g tumor of CD8+ T cells, NK cells, and natural killer T cells, and significantly higher Granzyme B expression in these cells compared with the control MC38 tumors. Similar results were observed in vivo by intratumoral IL-15 injection combined with NIR-PIT. Moreover, in bilateral tumor models with right hIL-15-MC38 and left MC38 tumors, NIR-PIT against right hIL-15-MC38 tumors significantly suppressed the growth of left untreated MC38 tumors. In conclusion, local IL-15 administration combined with CD44-targeted NIR-PIT promoted anti-cancer immune responses, resulting in the inhibition of growth of treated tumors, longer survival of mice, and abscopal effects in untreated tumors. Thus, locally administered IL-15 is a promising anti-cancer therapy in combination with cancer cell-targeted NIR-PIT. Citation Format: Hiroshi Fukushima, Aki Furusawa, Takuya Kato, Seiichiro Takao, Shuhei Okuyama, Peter L. Choyke, Hisataka Kobayashi. Locally administered Interleukin-15 combined with cancer cell-targeted near-infrared photoimmunotherapy in a syngeneic mouse cancer model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5110.

Introduction: Epithelioid sarcoma (ES) is a rare yet high-grade and highly aggressive soft tissue neoplasm first identified by Enzinger in 1970.1 ES has high reported recurrence and metastasis rates (around 70% and 50%, respectively).2 Unresectable locally advanced and metastatic ES are usually chemoresistant and generally fatal.3 Here, we extended our previous near-infrared photoimmunotherapy (NIR-PIT) study,4 to develop antibody photosensitizer conjugates based on two monoclonal antibodies (mAbs) binding to CD44 or EGFR, both overexpressed on ES tumor cell surface. We conducted cellular studies and established animal models to evaluate the specificity and efficacy of the two mAb conjugates in binding, detecting and eliminating ES. Method: CD44, EGFR mAb or its IgG isotype control was conjugated with a NIR phthalocyanine dye, IR700, to form CD44-IR700, EGFR-IR700 and IgG-IR700, respectively. A bilateral ES tumor model was established by inoculating 1×106 VAESBJ cells in 0.05 ml of Hank's balanced salt solution on either flank of athymic Balb/c (nu/nu) female mouse. Subsequently, 100 μg of CD44-IR700 or EGFR-IR700 or IgG-IR700 was injected intravenously (i.v.), and NIR fluorescence images of IR700 in mice were obtained over a 24-h period (n = 4 per group). For PIT, tumor-bearing mice (n = 4 or 5 per group) received two i.v. injections of 100 μg of CD44-IR700 or EGFR-IR700 at a one-week interval, and tumors were exposed to NIR light exposure at 200 J/cm2 at 24h p.i. Tumor diameters were measured over 3 weeks. IgG-IR700 and PBS-injected mice were used as controls. Results and Discussion: We confirmed CD44 and EGFR overexpression in VAESBJ cells by flow cytometry. Both CD44-IR700 and EGFR-IR700 demonstrated target-specific binding to VAESBJ and cell death when activated by NIR light. Compared to EGFR-IR700, CD44-IR700 exhibited a higher potency in cellular PIT and had a much lower IC50 value. CD44-IR700 and EGFR-IR700 both exhibited preferential accumulation in VAESBJ tumors and showed approximately two to threefold higher retention compared to IgG-IR700. CD44-IR700 or EGFR-IR700 injection combined with NIR light exposure resulted in significant tumor growth delay in VAESBJ tumors compared to the IgG-IR700 or PBS group. Reduction of CD44 and EGFR levels were observed in the corresponding CD44-IR700 or EGFR-IR700-treated tumors as evidenced by flow cytometry and western blotting studies. Our data collectively demonstrate the specificity and efficacy of CD44 or EGFR-based PIT as novel therapeutic approaches that can be integrated into an intraoperative setting for achieving clear tumor margins in localized ES or applied as a standalone therapy for inoperable locally advanced and recurrent ES. References: 1. Enzinger, F. et al. Cancer 1970; 2. Elsamna, S. et al. Acta Oncologica. 2020; 3. Wolf, P et al. Am J Surg. 2008; 4. Jin, J. et al. Sci Rep. 2016. Supported by NIH R35 CA209960 and Emerson Collective Cancer Research Fund. Citation Format: Jiefu Jin, James Barnett, Yelena Mironchik, Hisataka Kobayashi, Adam Levin, Zaver M. Bhujwalla. Phototheranostics of epithelioid sarcoma by targeting CD44 or EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 696.

Introduction: Stromal cells such as cancer associated fibroblasts (CAFs) mediate many of the aggressive characteristics of cancer1 and have an ever-replenishing supply that is largely left intact by our current therapeutic strategies.2 Fibroblast activation protein-α (FAP-α) is a target antigen that been reported to be selectively expressed in tumors by a subset of immunosuppressive stromal fibroblasts. Here, we extended our previous near-infrared photoimmunotherapy (NIR-PIT) study,3 to develop a FAP-α-targeted monoclonal antibody (mAb) photosensitizer conjugate. We developed FAP-α overexpressing MDA-MB-231 and HT-1080 cells to test the feasibility of targeting FAP-α expressing populations in cells and tumors in vivo, in terms of the specificity of the conjugate to bind to, detect, and eliminate FAP-α expressing populations. Method: FAP-α monoclonal antibody (AF3715) and its IgG isotype control were conjugated with a NIR phthalocyanine dye, IR700, to form FAP-α-IR700 and IgG-IR700. FAP-α overexpression was achieved by transducing MDA-MB-231 and HT-1080 cells with lentivirus encoding the gene for human FAP (Accession No. NM_004460.3) that was subcloned into lentiviral vector pMA3211 to obtain 231-FAP and HT-1080-FAP cells. Cell viability was measured by using CCK-8 assay. Bilateral tumor models were established by inoculating 1×106 MDA-MB-231 cells or 1×106 231-FAP cells in 0.05 ml of Hank's balanced salt solution on either side in the mammary fat pads of athymic Balb/c (nu/nu) female mice. A similar number of HT-1080 or HT-1080-FAP cells were inoculated bilaterally in the flank. 50 μg of FAP-α-IR700 or IgG-IR700 was injected i.v., and fluorescence images of IR700 in mice were obtained over a 24-h period (n = 3 per group). At 24 h post injection, mice were euthanized, and tumors were isolated for imaging. For PIT, tumor-bearing mice (n = 4 per group) received two i.v. injections of 100 μg of FAP-α-IR700 at a one-week interval, and tumors were exposed to NIR irradiation at 200 J/cm2 at 24h p.i. Tumor diameters were measured over 2 weeks. IgG-IR700 and PBS-injected mice were used as controls. Results and Discussion: We confirmed FAP-α overexpression in 231-FAP and HT-1080-FAP cells. FAP-α-IR700 was activated by NIR light, causing FAP-α-specific cell death. FAP-α-IR700-mediated phototoxicity was dependent on the conjugate concentration and NIR light dose; and it was inhibited by excess AF3715. We observed the preferential accumulation of FAP-α-IR700 in FAP-α-overexpressing 231-FAP and HT-1080-FAP tumors compared to their wild-type counterparts. The mean fluorescence intensity of FAP-α-IR700 in FAP-α-overexpressing tumors was approximately two to threefold higher than the wild type tumors. FAP-α-IR700 injection together with NIR light exposure resulted in the highest tumor growth delay in 231-FAP tumors and HT-1080-FAP tumors. Our results evaluate and confirm the ability of FAP-α-IR700 to target and eliminate FAP-α-overexpressing cell populations, providing novel opportunities to selectively deplete FAP-α high CAFs in cancers. References: 1. Horimoto, Y. et al. Cell Adh Migr. 2012; 2. Eyden, B. et al. J Cell Mol Med. 2008; 3. Jin, J. et al. Sci.Rep. 2016. Supported by NIH R35 CA209960, P41 EB024495, and Emerson Collective Cancer Research Fund. Citation Format: Jiefu Jin, James D. Barnett, Balaji Krishnamachary, Yelena Mironchik, Hisataka Kobayashi, Zaver Bhujwalla. Phototheranostics of cancer associated fibroblasts by targeting fibroblast activation protein-α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3360.

Regulatory T cells (Tregs) play pivotal roles in inhibiting anti-tumor immunity at different phases and locations (priming phase in lymph node and effector phase in tumor tissue) through their ability to suppress the function of T, NK, and antigen-presenting cells. However, our knowledge about the roles of Tregs in tumor immunity has largely derived from analyses after systemic depletion of Tregs or systemic manipulation of Treg functions in murine tumor models. The specific roles that intra- and extra-tumor Tregs play remain unclear due to technical difficulties to deplete or manipulate Tregs in a spatially specific manner. In order to achieve spatially specific depletion of Tregs in a clinically applicable manner, we have previously developed an anti-CD25 F(ab')2 near-infrared photoimmunotherapy (NIR-PIT), in which an intravenous injection of IR700-conjugated anti-CD25 F(ab')2 followed by irradiation of near-infrared light on tumors achieves selective depletion of intratumor Tregs and suppresses tumor growth. The suppression of tumor growth by the anti-CD25 F(ab')2 NIR-PIT depends on CD8 T and NK cells and their production of IFN-γ; however, the exact target of IFN-γ and the mechanisms of tumor regression remain to be elucidated. In this study, we aimed to dissect the mechanisms of the anti-tumor effect of intratumor Treg depletion by anti-CD25 F(ab')2 NIR-PIT using a series of Ifngr1 knockout mice models including bone-marrow chimera and conditional knockout models. Unexpectedly, we found that the target of IFN-γ in anti-CD25 F(ab')2 NIR-PIT was endothelial cells of tumor vessels. In both MC38 and EO771 tumor models (murine colon and breast cancer, respectively), CD8 T cells, NK cells, and Tregs were mainly located along the tumor vessels, and depletion of intratumor Tregs by anti-CD25 F(ab')2 NIR-PIT induced IFN-γ expression from these perivascular CD8 T and NK cells for up to 6 hours. This transient but synchronized induction of IFN-γ/STAT1 signaling along tumor vessels caused rapid vessel regression, intratumor ischemia, and apoptotic death of tumor cells. Combination with IL-15 treatment further eradicated residual tumor cells to achieve complete remission of MC38 and EO771 tumors. Citation Format: Yutaka Kurebayashi, Peter L. Choyke, Hisataka Kobayashi, Noriko Sato. Intratumor regulatory T cells prevent IFN-gamma-dependent tumor vessel regression and can be selectively targeted by anti-CD25 near-infrared photoimmunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3444.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that employs a targeted monoclonal antibody-photo-absorber conjugate (APC). Following antibody localization of the APC to a tumor cell surface antigen, NIR light is used to induce highly selective cytolysis. Extensive pre-clinical evidence demonstrates that NIR-PIT is effective at inducing tumor cell lysis using a number of different antibody-APC conjugates and NIR-PIT targeting EGFR with a cetuximab-APC conjugate has shown promising results in phase 2 clinical evaluation for the treatment of recurrent head and neck squamous cell carcinoma. NIR-PIT induces rapid, necrotic cell death that yields innate immune ligands that activate dendritic cells, consistent with immunogenic cell death. Yet, NIR-PIT treatment of syngeneic tumors in wild-type mice has mostly failed to induce durable regression of established tumors, suggesting the presence of one or more mechanisms of resistance to formation of meaningful anti-tumor immunity. In this study, we hypothesized that NIR-PIT could induce anti-tumor immunity that was being restricted by the PD-1/PD-L1 signaling axis, and that PD-1 could reverse innate immune resistance to induce durable, effective anti-tumor immune responses. Using a CD44-targeting APC, we demonstrated the ability of PD-1 immune checkpoint blockade to significantly enhance antigen-specific anti-tumor immunity induced by NIR-PIT in multiple syngeneic tumor models (MC38, LLC, and MOC1 cancer cell line). In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade appeared to reverse adaptive immune resistance, resulting in both enhanced pre-existing tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses appeared to correlate with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic anti-tumor immunity. Cured mice resisted tumor challenge, indicating the presence of systemic immune memory. Cumulatively these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in highly antigenic syngeneic models of cancer. Citation Format: Tadanobu Nagaya, Jay Friedman, Yasuhiro Maruoka, Fusa Ogata, Shuhei Okuyama, Paul E. Clavijo, Peter L. Choyke, Clint Allen, Hisataka Kobayashi. Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3206.

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/cm2 or greater. In contrast, lower levels of NIR light (10 J/cm2 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. Cancer Res; 72(18); 4622–8. ©2012 AACR.

Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Despite the recent advances in the treatment of NB, patients with high-risk NB still have poor prognosis. Immunotherapy using anti-GD2 monoclonal antibody (mAb), which binds to disialoganglioside GD2 on the surface of NB cells, has recently become a standard treatment for high-risk NB. However, the therapeutic efficacy of anti-GD2 mAb is insufficient in many clinical trials. Therefore, the enhancement of therapeutic potential of anti-GD2 mAb is needed to improve the clinical outcome of patients with high-risk NB. Near-infrared photoimmunotherapy (NIR-PIT) is a promising antitumor strategy to enhance the therapeutic potential of immunotherapy using an antibody-photoabsorber conjugate (APC). In this study, we investigated the therapeutic efficacy of anti-GD2-based NIR-PIT using anti-GD2 mAb conjugated to the photoabsorber IR700 (anti-GD2-IR700) in human NB cells. Methods: We used 3 human NB cell lines, including IMR-32, CHP-134 and SK-N-SH. The expression of GD2 on the cell surface of NB cells was analyzed by flow cytometric analysis. Anti-GD2-IR700 was synthesized by incubating anti-GD2 mAb and IR700. The mixture was purified with a Sephadex G50 column. The antitumor effect of anti-GD2-IR700 was evaluated using XTT assay. NB cells were incubated with anti-GD2-IR700, anti-GD2 mAb or PBS and NIR light was irradiated at 2.0J/cm2. Results: IMR-32 and CHP-134 cells, but not SK-N-SH cells, expressed GD2 protein on the cell surface. In GD2-positive IMR-32 and CHP-134 cells, administration of anti-GD2-IR700 significantly suppressed the cell viability compared to anti-GD2 mAb or PBS when combined with NIR light irradiation. In contrast, in GD2-negative SK-N-SH cells, anti-GD2-IR700 showed no significant inhibition of cell viability in combination with NIR light irradiation. Conclusion: These results suggest that NIR-PIT using an anti-GD2-IR700 is a promising antitumor strategy to promote the therapeutic efficacy of anti-GD2 immunotherapy for high-risk NB. Citation Format: Hiroshi Nouso, Hiroshi Tazawa, Terutaka Tanimoto, Morimichi Tani, Takanori Oyama, Hiroaki Sato, Kazuhiro Noma, Shunsuke Kagawa, Hisataka Kobayashi, Takuo Noda, Toshiyoshi Fujiwara. Development of near-infrared photoimmunotherapy targeting GD2-positive neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3831.

Background: Esophageal cancer has been reported to express HER family, especially EGFR and HER2, of which expressed cases have been reported to show poor prognosis. However, although there are some clinical trials that target those markers, using cetuximab (Cet), panitumumab (Pan), and trasutuzumab (Tra), the result is not sufficient. On the other hand, cancer-associated fibroblasts (CAFs) are one of the major components of tumor microenvironment and suggested to be associated with those chemoprevention, leading to poor prognosis. Thus, to target both cancer cells and CAFs, we focused on near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT is a new molecular targeted therapy, which is based on a near-infrared (NIR) photosensitizer IR700, conjugated to monoclonal antibody (mAb) targeting particular molecules. We developed the CAFs-targeting PIT targeted on fibroblast activated protein (FAP) and have confirmed the efficacy to FAP-expressed CAFs. In this study, we hypothesized that the effect of cancer-targeting PIT is decreased under CAF-rich condition as like other antibody-based therapy. Here we firstly evaluated the effect of cancer-targeting PIT in esophageal cancer cells and furthermore the possibility of additional effect by “dual-targeting PIT” for CAFs and cancer cells. Materials and Methods: Human esophageal cancer cell lines and human esophageal fibroblasts (FEF3) were used in this study. FEF3 stimulated with conditioned medium (CM) of cancer cells defined as CAFs. We used cetuximab and panitumumab for anti-EGFR antibody and trastuzumab for anti-HER2 antibody. The expression of EGFR, HER2 in esophageal cancer cells was confirmed by Western blot (WB). Cell viability for quantitative evaluation was determined using an XTT Cell Proliferation Kit II. Result: WB demonstrated that HER2 expression was increased in TE4 and OE19, and EGFR was increased in TE8. FEF3 stimulated with CM was increased FAP expression compared with control. XTT assay demonstrated that the effect of cancer-targeting PIT using Pan-IR700, Tra-IR700 is efficient. Additionally, we confirmed that CAFs-targeting PIT worked in co-culture condition with cancer cells as well as monoculture condition. Furthermore, “dual-targeting PIT” showed rapid cell death of both in co-culture condition, and XTT assay showed the additive effects in vitro, as expected. Discussion: In this study, dual-targeting therapy has shown cellular specific cell death by targeting to cell-specific antigen and induced both cells death simultaneously. NIR-PIT can be novel effective therapy for esophageal cancer, and also for CAFs in tumor microenvironment. Thus, “dual-targeting PIT," which is combined with both mAb-IR700 for cancer cells and CAFs, can regulate tumor stroma as well as cancer cells simultaneously and can be highly expected to have strong antitumor effect by remodeling tumor microenvironment. Citation Format: Hiroaki Sato, Kazuhiro Noma, Toru Narusaka, Satoshi Komoto, Yuki Katsura, Takuya Kato, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Yasuhiro Shirakawa, Hisataka Kobayashi, Toshiyoshi Fujiwara. Dual-targeting photoimmunotherapy (NIR-PIT) for esophageal cancer cells and cancer-associated fibroblasts (CAFs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5066.

Optical probes that yield high target-to-background ratios are necessary to detect microfoci of cancer that would otherwise escape detection with white light imaging. Target-specific activation of the optical signal at tumor foci is one mechanism by which high target and low background signal can be achieved. Here, we describe a two-step activation process in which the tumors are first pretargeted with a nonfluorescent biotinylated monoclonal antibody [cetuximab (Erbitux) targeting human epidermal growth factor receptor type 1 (HER1)]. Following this, a second agent, neutravidin-BODIPY-FL fluorescent conjugate, is given and binds to the previously targeted antibody, resulting in an ∼10-fold amplification of the optical fluorescence signal, leading to high tumor-to-background ratios. Spectral fluorescence imaging was done in a mouse model of peritoneal metastasis using a HER1-overexpressing cell line (A431) after pretargeting with biotinylated cetuximab and 3 h after administration of neutravidin-conjugated BODIPY-FL. Both aggregated tumors as well as small cancer implants were clearly visualized in vivo. For lesions ∼0.8 mm or greater in diameter, the spectral fluorescence imaging had a sensitivity of 96% (178 of 185) and a specificity of 98% (188 of 191). This two-step activation paradigm (pretargeting followed by neutravidin-biotin binding with an attached activatable fluorophore) could be useful in tumor-specific molecular imaging of various targets to guide surgical resections. [Cancer Res 2007;67(8):3809–17]

A target cell–specific activation strategy for improved molecular imaging of peritoneal implants has been proposed, in which fluorophores are activated only in living targeted cells. A current example of an activatable fluorophore is one that is normally self-quenched by attachment to a peptide backbone but which can be activated by specific proteases that degrade the peptide resulting in “dequenching.” In this study, an alternate fluorescence activation strategy is proposed whereby self-quenching avidin-rhodamine X, which has affinity for lectin on cancer cells, is activated after endocytosis and degradation within the lysosome. Using this approach in a mouse model of peritoneal ovarian metastases, we document target-specific molecular imaging of submillimeter cancer nodules with minimal contamination by background signal. Cellular internalization of receptor-ligand pairs with subsequent activation of fluorescence via dequenching provides a generalizable and highly sensitive method of detecting cancer microfoci in vivo and has practical implications for assisting surgical and endoscopic procedures. [Cancer Res 2007;67(6):2791–9]

The efficacy of monoclonal antibodies (mAbs) as single agents in targeted cancer therapy has proven to be limited. Arming mAbs with a potent toxic drug could enhance their activity. Here we report that conjugating geldanamycin (GA) to the anti-HER2 mAb Herceptin improved the activity of Herceptin. The IC50s of the immunoconjugate H-GA were 10–200-fold lower than that of Herceptin in antiproliferative assays, depending on the cell line. The H-GA mode of action involved HER2 degradation, which was partially lactacystin sensitive and thus proteasome dependent. The linkage between GA and Herceptin remained stable in the circulation, as suggested by the pharmacokinetics of Herceptin and conjugated GA, which were almost identical and significantly different from that of free GA. Tumor uptake of Herceptin and H-GA were similar (52 ± 7 and 43 ± 7% of the initial injected dose per gram tissue, respectively; P = 0.077), indicating no apparent damage attributable to conjugation. Therapy experiments in xenograft-bearing mice consisted of weekly i.p. doses, 4 mg/kg for 4 months. H-GA showed a greater antitumor effect than Herceptin because it induced tumor regression in 69% of the recipients compared with 7% by Herceptin alone. Median survival time was 145 days as opposed to 78 days, and 31% of the recipients remained tumor free 2 months after therapy was terminated versus 0% in the Herceptin group. Enhancement of Herceptin activity could be of significant clinical value. In addition, the chemical linkage and the considerations in therapeutic regimen described here could be applied to other immunoconjugates for targeted therapy of a broad spectrum of cancers.

Introduction and Purpose: CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to suppress immune responses against cancers. Treg-mediated immunosuppression is a key mechanism for tumor immune-evasion, which could lead cancer immunotherapies to failures. Systemic depletion of Tregs have been tried by using antibodies against them; however, intravenously delivering these antibodies might not sufficiently deplete Tregs in the tumor microenvironment or could substantially deplete effector cells, in case anti-CD25 antibody was used. In addition, development of auto-immune responses could cause potential side effects. To overcome these problems, we exploited near infrared photoimmunotherapy (NIR-PIT) at the tumor to quickly deplete only intratumoral Tregs, aiming to induce anti-tumor immune activation. Experimental Design: F(ab’)2 fragments of an anti-mouse CD25 antibody, PC61.3, were generated and conjugated with a phthalocyanine dye, IRDye-700DX (αnti-CD25-F(ab’)2-IR700). Using the anti-CD25-F(ab’)2-IR700, in vitro NIR-PIT effect was examined against CD25-expressing mouse T lymphocytes, HT-2 clone A5E cells. In vivo CD25-target-NIR-PIT of the tumor was performed after an intravenous injection of the conjugate to mice bearing subcutaneous, luciferase transfected, LL/2 (Lewis lung carcinoma, LL/2-luc) or MC38 (mouse colon cancer, MC38-luc) cancers. Tumor volume, bioluminescence signals (BLI), and Immune responses following a local CD25-target-NIR-PIT were examined. Results: In vitro NIR-PIT-induced cytotoxicity was light dose dependent. CD25-target-NIR-PIT at the tumor quickly and selectively depleted Tregs in the cancers, yet, Tregs in any other organs were not affected. The local CD25-target-NIR-PIT induced a rapid activation and cytotoxic action of CD8 T cells and NK cells within LL/2-luc or MC38-luc tumors. This led to significant reductions of tumor volume (p < 0.0001) and BLI (p < 0.05) in both LL/2-luc and MC38-luc models and prolonged the survival of the mice (p < 0.0001) compared to the non-treated controls. Intriguingly, this local CD25-target-NIR-PIT induced a transient systemic cytokine storm and antitumor-effects on distant non-irradiated specific tumors. Effects of local CD25-target-NIR-PIT were significantly (p < 0.0001) inhibited by a CD8-, NK-, or INFγ−depletion, suggesting the anti-tumor roles of CD8 T cells and NK cells. Conclusions: Quick depletion of intratumoral Tregs by a local CD25-target-NIR-PIT rapidly induced CD8 T- and NK-cell activation, thereby restoring local anti-tumor immunity. Consequently, activated immunity led to regression of not only NIR-PIT-treated tumors but also non-NIR light exposed tumors in separate parts of the body. These observations suggest that using local CD25-target-NIR-PIT may be a promising new strategy for cancer immunotherapy. Citation Format: Kazuhide Sato, Noriko Sato, Biying Xu, Yuko Nakamura, Tadanobu Nagaya, Peter L. Choyke, Hisataka Kobayashi. Cancer treatment by near infrared photoimmunotherapy targeting intratumoral regulatory T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1470.

Near-infrared (NIR) fluorophores have several advantages over visible fluorophores, including improved tissue penetration and lower autofluorescence; however, only indocyanine green (ICG) is clinically approved. Its use in molecular imaging probes is limited because it loses its fluorescence after protein binding. This property can be harnessed to create an activatable NIR probe. After cell binding and internalization, ICG dissociates from the targeting antibody, thus activating fluorescence. ICG was conjugated to the antibodies daclizumab (Dac), trastuzumab (Tra), or panitumumab (Pan). The conjugates had almost no fluorescence in PBS but became fluorescent after SDS and 2-mercaptoethanol, with a quenching capacity of 10-fold for 1:1 conjugates and 40- to 50-fold for 1:5 conjugates. In vitro microscopy showed activation within the endolysosomes in target cells. In vivo imaging in mice showed that CD25-expressing tumors were specifically visualized with Dac-ICG. Furthermore, tumors overexpressing HER1 and HER2 were successfully characterized in vivo by using Pan-ICG(1:5) and Tra-ICG(1:5), respectively. Thus, we have developed an activatable NIR optical probe that “switches on” only in target cells. Because both the antibody and the fluorophore are Food and Drug Administration approved, the likelihood of clinical translation is improved. [Cancer Res 2009;69(4):1268–72]

CD44 transmembrane glycoproteins are cell adhesion molecules that play a role in cancer cell proliferation, migration, and invasion.1 CD44 expression is associated with stem-like breast cancer cells1 and is upregulated under hypoxia2 making it a significant target. Monoclonal antibody (mAb)-linked to photosensitizers have achieved targeted phototherapy.3 We developed a CD44 mAb-targeted photosensitizer complex for combined fluorescent detection and phototherapy (PT) of CD44 expressing triple negative breast cancer cells. The mAb conjugate is activated by near-infrared (NIR) light, and demonstrates phototoxic effects selectively in CD44 expressing breast cancer cells following cell membrane binding. CD44 mAb was conjugated with a NIR phthalocyanine dye, IR700, to form CD44-IR700. MB-MDA-231 (CD44 positive), MCF-7 and BT-474 (CD44 negative) were selected for the cell studies. SDS-PAGE, UV spectroscopy and confocal fluorescence imaging were performed to determine purity, composition and cellular localization of the conjugate. Microscopic observation and cell viability tests were performed to study CD44-specific cell death. Tumor models were established by inoculating 2×106 MB-MDA-231 or BT-474 cells in the upper-right mammary fat pad of athymic female mice. In vivo fluorescence imaging was performed on a Li-Cor Pearl® Impulse scanner. Tumor bearing mice (n = 5 per group) injected with PBS, IR700, anti-CD44 mAb, IgG-IR700 and CD44-IR700 were monitored over a 3 week-period and sacrificed to harvest tumors. NIR light at a dose of 30 J/cm2 was applied on day 1 after injection. CD44-IR700 demonstrated preferential binding to the cell membrane of MB-MDA-231 but not MCF-7 or BT-474 cells. CD44-specific cell death was initiated by CD44-IR700-mediated PT (>90% of MB-MDA-231 cells were killed immediately, while 88% of MCF-7 cells and 90% of BT-474 cells were alive). Irradiation itself was harmless to cells and there was no cytotoxicity associated with IR700 and CD44 antibody or with CD44-IR700 in the absence of irradiation. In vivo fluorescence imaging verified the 3-fold increased retention of CD44-IR700 in MB-MDA-231 tumors compared to BT-474 tumors. CD44-IR700 injection together with a therapeutic dose of NIR light exposure resulted in significant growth delay in MB-MDA-231 tumors, but not BT-474 tumors. We did not observe tumor growth delay in separate groups of mice injected with PBS, IR700, anti-CD44 mAb, or IgG-IR700, irrespective of NIR light application. Histological studies confirmed a significant increase of necrosis in the treated MDA-MB-231 tumors. This novel PT theranostic strategy provides a promising opportunity for treating CD44 expressing primary and metastatic triple negative breast cancer. References: 1. Louderbough et al, Mol Can Res, 2011; 2. Krishnamachary et al, PloS one, 2012; 3. Mitsunaga et al, Nat Med, 2011. Supported by NIH R01 CA136576 and P50 CA103175. Citation Format: Jiefu Jin, Balaji Krishnamachary, Hisataka Kobayashi, Zaver Bhujwalla. IR700-conjugated anti-CD44 monoclonal antibody for phototherapy theranostics of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2484. doi:10.1158/1538-7445.AM2015-2484

Cancer associated fibroblasts (CAFs) are thought to play an essential role in cancer invasion, migration, metastasis and resistance to anticancer drugs. CAFs were originally defined as fibroblasts expressing α-SMA in cancer tissue; however, fibroblast activation protein (FAP) has been recently identified as a more functional and specific surface protein of CAFs. The photoimmunotherapy (PIT) is a new molecular targeted therapy, which is based on a near-infrared (NIR) photosensitizer IR700, conjugated to monoclonal antibody (mAb) targeting particular molecules. PIT induces the selective destruction of targeted cells. The aim of this study is to analyze the influence of CAFs on human esophageal cancer and to develop a novel therapeutic strategy to eliminate CAFs by using FAP-targeting PIT. We produced the IR700-FAP antibody for this study. Human esophageal cancer cell lines (TE-4 and OE-19) and FEF3 human fibroblasts were used in this study. TE-4 and OE-19 cells stimulated with conditioned medium (CM) of CAFs exhibited malignant phenotypes confirmed by invasion assay, migration assay, and colony formation assay. Tumor cells were significantly more malignant when directly co-cultured with CAFs. We further examined whether tumor cells stimulated by CM (CAFs) could acquire the resistance to chemotherapy or radiotherapy. Cell viability assay showed that tumor cells stimulated with CAFs was more resistant to conventional chemotherapy or radiation than unstimulated tumor cells. Western blot analysis demonstrated that E-cadherin expression was decreased and that of vimentin was increased in CAF-stimulated tumor cells, indicating the epithelial mesenchymal transition (EMT) induction. Moreover, OE-19 cells stimulated with CAFs contained more CD133-positive cancer stem-like cells. In vivo experiments demonstrated that subcutaneous TE4 tumors were more refractory to chemotherapy in the presence of co-inoculated CAFs as observed in vitro. Finally, we investigated whether elimination of CAFs by FAP-targeting PIT could affect the resistance to chemotherapy in vitro and in vivo. In vivo PIT following intratumoral anti-FAP mAb-conjugated IR700 suppressed the growth of subcutaneous tumors co-inoculated with CAFs. These results demonstrated that human tumor cells became more malignant and resistant in the presence of CAFs, which could be reversed by using CAF-specific PIT. Targeting fibroblast itself is a unique strategy and can be clinically promising as combination targeting cancer cells and their fundamental microenvironment. Citation Format: Ryoichi Katsube, Kazuhiro Noma, Shinichiro Watanabe, Shinichi Urano, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Hisataka Kobayashi, Toshiyoshi Fujiwara. A novel photoimmunotherapy targeting cancer-associated fibroblasts (CAFs) overcomes therapeutic resistance in human esophageal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 401. doi:10.1158/1538-7445.AM2015-401

Photoimmunotherapy (PIT) of cancer utilizes tumor-specific monoclonal antibodies conjugated to a photosensitizer phthalocyanine dye IR700 which becomes cytotoxic upon irradiation with near infrared light. In this study, we aimed to evaluate the efficacy of PIT on human pancreatic cancer cells in vitro and in vivo in a nude mouse model. An in vitro cytotoxicity assay was used to determine cell death following treatment with PIT for Panc-1, MiaPaCa2, and BxPC-3 human pancreatic cancer cells. For in vivo determination of PIT efficacy, nude mice were implanted with BxPC-3 pancreatic tumors expressing green fluorescent protein (GFP). After tumor engraftment, the mice were divided into two groups: treatment with anti-EGFR-IR700 + 690 nm laser and treatment with 690 nm laser only. Anti-EGFR-IR700 (100 μg) was administered to the treatment group via tail vein injection 24 hours prior to therapy. Tumors were then exposed and treated with phototherapy at an intensity of 150 mW/cm2 for 30 minutes. Whole body imaging was done using an OV-100 small animal imaging system. Anti-EGFR-IR700 antibody bound to the BxPC3 cells to a high degree based on image co-localization. Anti-EGFR-IR700 caused extensive cancer cell killing after light activation compared to control cells in cytotoxicity assays. In the mouse experiments, the treatment group had significantly smaller tumors as measured by GFP than the control after just 96 hours. The present results indicate the high potential of PIT for pancreatic cancer therapy. Citation Format: Nzola de Magalhães, Takashi Murakami, Roger Heim, Lew Makings, Miguel Garcia-Guzman, Hisataka Kobayashi, Robert M. Hoffman, Michael Bouvet. Photoimmunotherapy with an anti-EGFR antibody conjugated to an IRDye700 results in extensive and rapid cell death in vitro and in vivo in human pancreatic cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2019. doi:10.1158/1538-7445.AM2015-2019

Introduction and Purpose Lung metastases are a leading cause of cancer related deaths. Current therapies include surgery, chemotherapy and radiotherapy but cause considerable side effects in the remaining normal lung, chest wall and heart. Therefore, a new-targeted therapy for lung metastases that results in minimal damage to adjacent structures, yet effectively treats lung metastases, is needed. Since one interesting property of the lung is that it transmits light better than other organs, light therapy is a viable alternative therapy. 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. Experimental Design In vitro and in vivo 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, IRDye-700DX, was synthesized. In vitro NIR-PIT cytotoxicity was assessed with dead staining, luciferase activity, and GFP fluorescence intensity. In vivo NIR-PIT was performed in a mouse model of lung metastases as well as a flank model, and assessed by tumor volume and/or bioluminescence (BLI). Results In vitro NIR-PIT-induced cytotoxicity was light dose dependent. In vivo anti-tumor effects of NIR-PIT included significant reductions in both tumor volume (p = 0.0141 vs. APC) and 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 metastases model (p < 0.0001). Conclusions NIR-PIT prolonged survival suggesting that NIR-PIT is a potential new therapy for the local control of lung metastases. Citation Format: Kazuhide Sato, Tadanobu Nagaya, Peter L. Choyke, Hisataka Kobayashi. Near infrared photoimmunotherapy for lung metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4118. doi:10.1158/1538-7445.AM2015-4118

Introduction: Bladder cancer (BCa) is a common cancer and is the most expensive malignancy to treat from diagnosis to death. Although no new therapies have been introduced in the past two decades, data from ‘The Cancer Genome Atlas (TCGA)’ confirms that Epidermal Growth Factor Receptor (EGFR) family and its downstream signaling molecules are altered in almost 72% of muscle invasive bladder cancer (MIBC) cases. However, anti-EGFRs to date have failed to demonstrate any benefit over standard of care chemotherapy. We have applied a technique of employing monoclonal antibodies (mAbs) conjugated with photo-activatable compounds (IRDye 700Dx, a phthalocyanine dye) that are activated by near-infrared light (NIR). When incubated with the conjugate, exposure to NIR destroys only targeted cells. We explored photoimmunotherapy (PIT) in a panel of BCa cell lines. The purpose of this study was to study the efficacy and mechanism of action of PIT using the anti-EGFR panitumumab (Pan)-IR700 immunoconjugate as a selective therapeutic strategy for bladder cancer. Methods: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines. The cytotoxicity of Pan-IR700 was analyzed using LIVE/DEAD and IC50 was measured using the MTS assay. The type of cell death was examined by fluorometric caspase assay, Annexin V-PI staining, and TEM. The mechanism of cell death was elucidated using fluorometric measurement of reactive and singlet oxygen species. Results: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines and cell lines with increasing amounts of surface EGFR (RT4, TCCSUP, 5637 and UMUC-5) were selected for further studies. Pan-IR700 rapidly killed UMUC-5 cells (high EGFR expressions) with an IC50 of 4 nM at 4 J/cm2 NIR. In TCCSUP cells (low surface EGFR), the same IC50 of Pan-IR700 could be achieved at 64 J/cm2 of NIR. No significant cytotoxicity was observed in the presence of IR700 or NIR alone, or in cell lines without any EGFR expression. Absence of any caspases and presence of most cells in the late apoptosis/necrosis quadrant of Annexin V-PI staining suggested that PIT kills cells by necrosis. In TEM, these cells showed classic features of necrotic cell death such as swollen organelles, disintegrated plasma membrane and depleted cytoplasm and nucleoplasm. Cells undergoing PIT showed production of ROS and singlet oxygen species in large amounts immediately after exposure to NIR. However, only the singlet oxygen quencher (NaN3) and not the ROS quencher (Trolox) was able to protect the cells from PIT induced cell death indicating partial involvement of singlet oxygen in PIT-induced necrosis of cells. Conclusions: PIT is a new targeted treatment for bladder cancer. Our data demonstrate that Pan-IR700-induced PIT selectively and efficiently kills EGFR-expressing bladder cancer cells in vitro and therefore warrants further preclinical therapeutic studies in in vivo bladder cancer models. Citation Format: Reema Railkar, Quentin Li, Srinivas Vourganti, Sam J. Brancato, Peter L. Choyke, Hisataka Kobayashi, Piyush K. Agarwal. Molecular targeted photoimmunotherapy as a treatment for bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3311. doi:10.1158/1538-7445.AM2015-3311

The efficacy of monoclonal antibodies (mAbs) as single agents in targeted cancer therapy has proven to be limited. Arming mAbs with a potent toxic drug could enhance their activity. Here we report that conjugating geldanamycin (GA) to the anti-HER2 mAb Herceptin improved the activity of Herceptin. The IC(50)s of the immunoconjugate H-GA were 10-200-fold lower than that of Herceptin in antiproliferative assays, depending on the cell line. The H-GA mode of action involved HER2 degradation, which was partially lactacystin sensitive and thus proteasome dependent. The linkage between GA and Herceptin remained stable in the circulation, as suggested by the pharmacokinetics of Herceptin and conjugated GA, which were almost identical and significantly different from that of free GA. Tumor uptake of Herceptin and H-GA were similar (52 +/- 7 and 43 +/- 7% of the initial injected dose per gram tissue, respectively; P = 0.077), indicating no apparent damage attributable to conjugation. Therapy experiments in xenograft-bearing mice consisted of weekly i.p. doses, 4 mg/kg for 4 months. H-GA showed a greater antitumor effect than Herceptin because it induced tumor regression in 69% of the recipients compared with 7% by Herceptin alone. Median survival time was 145 days as opposed to 78 days, and 31% of the recipients remained tumor free 2 months after therapy was terminated versus 0% in the Herceptin group. Enhancement of Herceptin activity could be of significant clinical value. In addition, the chemical linkage and the considerations in therapeutic regimen described here could be applied to other immunoconjugates for targeted therapy of a broad spectrum of cancers.

Major advances in cancer biology and immunology have been gained using mouse models. However, very few methods are currently available to visualize the deep lymphatic system. A new micro-magnetic resonance lymphangiography (MRL) method in mice, which uses dendrimer-based magnetic resonance imaging contrast agents, was developed. Micro-MRL imaging clearly visualized most of the mouse lymphatic system, including both lymphatics and lymph nodes. This method could detect and distinguish among dilation of lymphatic vessels in a lymphangitis model, proliferative or neoplastic lymph node swellings in a lymphoproliferative model, and inflammatory lymph node swellings in an infection/inflammation model. Changes in the lymphoid system of transgenic mice overexpressing interleukin-15 could be visualized. Abnormal enlarged lymph nodes identified by micro-MRL were selectively removed and analyzed to demonstrate their cell type, receptor expression, and clonality in individual mice. We conclude that the enhanced resolution of this noninvasive micro-MRL can detect and classify lymphatic and lymph node abnormalities in mice, which should have wide applicability to the study of immunology and cancer in both experimental animals and clinical medicine.

The rapid blood flow and perfusion of macromolecules in the inflammatory breast cancer xenograft (WIBC-9), which exhibits a "vasculogenic mimicry" type of angiogenesis without the participation of endothelial cells and expresses high levels of the HER-2/neu antigen, was evaluated in mice using 3D-micro-MR angiography using a novel macromolecular MR contrast agent [G6-(1B4M-Gd)(256)]. Herceptin, which recognizes the HER-2/neu antigen and has similar size (10 nm) to G6-(1B4M-Gd)(256), accumulated and internalized in the WIBC-9 tumors more quickly than in the control MC-5 tumors that progress with normal angiogenesis. Three dimensional micro-MRI with the G6-(1B4M-Gd)(256) macromolecular MRI contrast agent distinguishes between the different types of angiogenesis and is predictive of the rapid accumulation and internalization of Herceptin in the WIBC-9 inflammatory breast cancer xenograft.

In the present study, we examined hemodynamics in vasculogenic mimicry (VM) and angiogenesis of inflammatory breast cancer (IBC) xenografts (WIBC-9), having previously reported on the unique histological features and molecular basis of these processes (K. Shirakawa et al., Cancer Res., 61: 445-451, 2001). Histologically, the WIBC-9 xenografts exhibited invasive ductal carcinoma with a hypervascular structure (angiogenesis) in the tumor margin and VM without endothelial cells, central necrosis, or fibrosis in the tumor center. Results of molecular analysis indicated that WIBC-9 had a vasculogenic phenotype, including expression of Flt-1 and Tie-2. Comparison of WIBC-9 with an established non-IBC xenograft (MC-5), using time-coursed dynamic micromagnetic resonance angiography analysis (with our newly developed intravascular macromolecular magnetic resonance imaging contrast agent), electromicroscopy, and immunohistochemistry, demonstrated blood flow and a VM-angiogenesis junction in the central area of the WIBC-9 tumor. It has previously been considered impossible to prove a connection between VM and angiogenesis using angiography, because there are no intravascular macromolecular magnetic resonance imaging contrast agents that do not exhibit significant leakage through the vascular wall. In the present study, laser-captured microdissection was performed in regions of WIBC-9 tumors that exhibited VM without endothelial cells, central necrosis, or fibrosis, revealing expression of human-Flt-1 and human-Tie2 and the absence of human-CD31, human-endothelin B receptor, and human-thrombin receptor. These facts led us to hypothesize that the VM of WIBC-9 involves hemodynamics that serve to feed WIBC-9 cells, and this in turn suggests a connection between VM and angiogenesis.

A major barrier to cancer treatment is the inability to deliver sufficient concentrations of drug to the interior of tumors without incurring systemic toxicities. Nanomaterials are appealing because they can carry a large drug payload, however, tumor delivery is limited by modest leakage and retention in most tumors. Photoimmunotherapy (PIT) is a newly developed therapy involving the injection of a conjugate composed of an armed monoclonal antibody (mAb) and a near infrared phthalocyanine dye, IR700 [Mitsunaga M. Nature Med 2011;17:1685]. When exposed to NIR light, the conjugate induces a highly-selective necrotic cell death only in mAb-IR700 bound cancer cells. This target-specific necrotic cell death occurs within 10 minutes of exposure to NIR light and results in rapid morphologic changes including cellular swelling, bleb formation, and rupture of vesicles and cell death. Meanwhile, immediately adjacent receptor-negative cells are unharmed. Because PIT initially damages antigen bearing tumor cells immediately adjacent to the vasculature, it causes marked increases in vascular permeability leading to 20-fold enhanced nano-particle delivery into cancer tissue compared with untreated tumor bed. This effect has been termed “super-enhanced permeability and retention (SUPR)” to differentiate it from enhanced permeability and retention (EPR) which is common in tumors. In this study, we used a mixed tumor model containing 99% (EGFR)-positive A431 cells mixed with 1% EGFR-negative Balb3T3/DsRed cells in order to simulate tumor heterogeneity. Mixed tumors were treated with PIT alone or PIT combined with liposomes containing daunorubicin (Daunoxome), utilizing the SUPR effect. PIT completely treated EGFR+ cells, however, EGFR- cells quickly became the dominant cell type leading to death in mice. However, co-injection of Daunoxome could cure tumors following PIT. Therefore, although PIT alone does not eliminate all cancer cells due to heterogeneity of tumor, SUPR allows the delivery of extremely high concentrations of nano-sized anti-cancer reagents (e.g. Daunoxome), resulting in synergistic therapeutic effects of PIT and nano-sized agents. In conclusion, target-selective PIT can induce a SUPR effect leading to the accumulation of dramatically higher concentrations of nano-sized reagents within tumors thus improving the effectiveness of PIT in heterogeneous tumors. Citation Format: Hisataka Kobayashi, Kohei Sano, Takahito Nakajima, Kazuhide Sato, Peter L. Choyke. Photoimmunotherapy (PIT) combined with a nanosized cancer agent successfully treated heterogenous antigen-expressing tumors based on super-enhanced permeability and retention (SUPR) effect. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5400. doi:10.1158/1538-7445.AM2014-5400

A major barrier to cancer treatment is the inability to deliver sufficient concentrations of drug to the interior of tumors without incurring systemic toxicities. Nanomaterials are appealing because they can carry a large drug payload, however, tumor delivery is limited by modest leakage and retention in most tumors. Photo-immunotherapy (PIT) is a newly developed therapy involving the injection of a conjugate composed of an armed monoclonal antibody (mAb) and a near infrared phthalocyanine dye, IR700 [Mitsunaga M. Nature Med 2011;17:1685]. When exposed to NIR light, the conjugate induces a highly-selective necrotic cell death only in mAb-IR700 bound cancer cells. This target-specific necrotic cell death occurs within 10 minutes of exposure to NIR light and results in rapid morphologic changes including cellular swelling, bleb formation, and rupture of vesicles indicating necrotic cell death. Meanwhile, immediately adjacent receptor-negative cells are unharmed. Because it damages cells immediately adjacent to the tumor vasculature, PIT results in marked increases in vascular permeability leading to 12- to 25-fold enhanced nano-particle delivery into cancer tissue, an effect termed “super-enhanced permeability and retention (SUPR)” to differentiate it from inherent enhanced permeability and retention (EPR). Of particular interest is that nano-sized reagents including macromolecules (dendrimers), particles (quantum dots, iron oxide nano-particles), and liposomes up to 300 nm in diameter rapidly enter and are retained within the tumor space following PIT. Therefore, although PIT might not be able to eliminate all cancer cells due to heterogeneity of tumor antigen expression, SUPR allows the delivery of extremely high concentrations of nano-sized anti-cancer reagents such as daunorubicin containing liposome (Daunoxome), resulted in significantly synergic therapeutic effects of PIT and nano-sized agents. In conclusion, target-selective PIT can induce a SUPR effect leading to the accumulation of relatively high concentrations of nano-sized reagents into the tumor bedthus improving effectiveness of PIT.. Citation Format: Hisataka Kobayashi, Kohei Sano, Takahito Nakajima, Peter L. Choyke. Super-enhanced permeability and retention (SUPR) effect induced by photo-immunotherapy (PIT) can accommodate massive nano-sized reagents deep into tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4512. doi:10.1158/1538-7445.AM2013-4512

The prognosis of HER2 positive breast cancer has been improved by Trastuzumab which features high specificity and limited side effects. Numerous randomized trials have confirmed the efficacy and safety of Trastuzumab in HER2-overexpressing cancers. However, many patients who initially respond to Trastuzumab ultimately develop disease progression. Strategies to overcome Trastuzumab-refractory cancer are sought, and drug-conjugation is one of candidates. Trastuzumab-IR700 which is Trastuzumab conjugated with a near-infrared (NIR) photosensitizer, phthalocyanine dye, IR700, has recently been developed as a new type of molecular-target photoimmunotherapy(PIT). This PIT can induce target-selective necrotic cell death only at the place which have Trastuzumab's target part and where exposure of NIR light. Disappointingly, HER2-targeting therapy is applicable to only Her2-expressig tumors which consist of approximately 20-25% of primary breast cancers. In previous study, we developed the adenoviral vector to express HER2-extracellular domain (HER2-ECD), Ad/HER-ECD, and confirmed that it efficiently induced HER2 expression for HER2 negative and HER2-downregulated human cancer cells. So, we hypothesized that Ad/HER-ECD expand the range of tumor entities suitable for Trastuzumab-based PIT. By using Ad/HER-ECD and Trastuzumab-IR700 with PIT we revealed that the direct cell membrane destruction are induced at HER2 negative cancer cells, similarly at HER2 positive cancer cells, in vitro. These results suggest that combination of virally-transduction of target antigen and an antibody-based PIT would expand and potentiate target therapy even for target-negative or attenuated cancer cells. Citation Format: Michihiro Ishida, Shunsuke Kagawa, Kyoko Shimoyama, Shinichiro Watamabe, Kazuhiro Noma, Hiroshi Tazawa, Yuuri Hashimoto, Junji Matsuoka, Hisataka Kobayashi, Toshiyoshi Fujiwara. Neotargeting HER2 negative cancer cells with Trastuzumab-based photoimmunotherapy by viral transduction of HER2-extracellular domain. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3298. doi:10.1158/1538-7445.AM2013-3298

Purpose: There is a need to improve detection of peritoneal ovarian cancer metastases before and during surgery. Previous designs of activatable GSA-fluorophore probes, which target lectin receptors, have used only visible range dyes conjugated to GSA. However, activatable agents emitting fluorescence in the NIR range are advantageous because NIR photons have deeper in vivo tissue penetration and lower background autofluorescence than those emitting in the visible range. An NIR-activatable GSA fluorophore pair can be synthesized using the dye NMP1. This NMP1 dye has two unique absorption peaks (in green and NIR wavelength ranges) with a single NIR emission peak of 780nm. NMP1 thus has two different Stoke's shifts that have the potential to allow separate imaging of tumor nodules both at the surface and at depths below the surface. Experimental Design: GSA was conjugated with 2 NMP1 molecules to create a self-quenching complex (GSA-NMP1). The activation magnitude of GSA-NMP1 was validated by adding 10% SDS. Flow cytometry, fluorescence microscopy, and in vivo spectral fluorescence imaging were carried out to compare GSA-NMP1 with GSA-IR800 (an always on control agent with similar emission to NMP1) in terms of intracellular activation and abilities to allow detection of small ovarian cancer implants in mice models. Sensitivity and specificity of GSA-NMP1 for implant detection were determined by co-localizing NMP1 emission spectra with red fluorescent protein expressed constitutively in SHIN3 tumor implants. Image processing in Java was used to subtract spectral images, with resulting images theorized to show tumor nodules at a depth below the surface. Results: The fluorescence signal of GSA-NMP1 can be activated ∼100-fold and was specifically yielded after internalization into SHIN3 cells 3h after incubation. Submillimeter ovarian cancer implants in the peritoneal cavity were clearly detectable in vivo with spectral fluorescence imaging. Among 555 peritoneal lesions, the sensitivity and specificity for GSA-NMP using NIR and green excitation light, were 75%/92% and 91%/92% for lesions ∼0.8 mm or greater in diameter, respectively. Image processing suggests that nodules at a depth below the small bowel are imaged only with GSA-NMP1 by subtraction of the two excitation images. Conclusions: GSA-NMP1 is useful in imaging peritoneal ovarian cancer metastases, located both superficially and deep in the abdominal cavity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-508. doi:1538-7445.AM2012-LB-508

Targeted molecular therapies of cancer offer the possibility of effective tumor control with a minimum of side effects. However, to date this strategy has had modest results for most tumors. Antibody-based therapies have been among the more successful molecular approaches. The concept of using the antibody as a vector for additional therapy has been attempted with toxins and radio-isotopes with mixed results. Herein we describe an antibody conjugate with a photosensitive near infrared (NIR) phthalocyanine dye, IR700, which can be used as an optical imaging agent at low doses of light but becomes a photoimmunotherapeutic (PIT) at higher doses of light. In this study, we tested the efficacy of PIT with optimized regimen using an EGFR antbody-IR700 conjugate in a xenograft tumor model. For in vitro PIT, EGFR (HER1) expressing A431 cells were incubated with panitumumab-IR700 conjugates (Pan-IR700) which were then irradiated with NIR light. For in vivo PIT, A431 xenograft tumor bearing mice injected with 100 μg of Pan-IR700 every week. Mice were then treated with NIR light on day one (50 J cm−2) and day 2 (100 J cm−2) after Pan-IR700 injection which was administered every week for up to 4 weeks. NIR fluorescence images were obtained in each mouse and the effect of the PIT was analyzed. Pan-IR700 demonstrated specific binding to target receptors on the cell membrane. Target-specific cell death could be induced only in Pan-IR700 binding cancer cells immediately after exposure to NIR light. In vitro microscopy demonstrated that NIR exposure resulted in cellular swelling, bleb formation, and rupture of vesicles indicating necrotic cell death. No phototoxicity was observed in co-cultured HER1 negative Balb3T3 cells, even when MAb-IR700 was not removed from the medium. HER1 target-specific accumulation of MAb-IR700 was observed in mouse xenograft tumors by fluorescence imaging. Repeated administration of Pan-IR700 followed by repeated irradiation with NIR light resulted in effective tumor eradication and prolonged survival compared to control mice with no significant side effects. IR700 fluorescence from the conjugate allowed for image guidance of light delivery as well as monitoring of PIT. In conclusion, Pan-IR700 PIT was effective, only when the antibody conjugates were bound to the cell membrane, but showed no phototoxicity when they were not bound or irradiated with NIR light. Target selective PIT enables selective treatment of cancer with no apparent side effects even with multiple administrations of Pan-IR700 and NIR light irradiation. PIT using MAb-IR700 is a promising theranostics for highly selective treatment of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4830. doi:1538-7445.AM2012-4830

Three modes of cancer therapy, surgery, radiation and chemotherapy, have been central to modern oncologic therapy. Molecular targeted cancer therapies have been introduced to target specific pathways, while minimizing side effects but have had limited success except in several notable cases. The concept of amplifying the effect of molecular targeting by applying physical energy to the bound conjugate is relatively new. In this study, we describe a hydrophilic photosensitizer based on a near infrared (NIR) phthalocyanine dye, IR700, which is covalently conjugated to one of several humanized monoclonal antibodies (MAb) targeting human epidermal growth factor receptor (EGFR) type 1 (HER1) and 2 (HER2). When exposed to light the conjugate induces highly selective cell death in vivo, a process termed “photo-activatable immunotherapy” (PIT). In vitro studies were conducted with Trastuzumab-IR700 bound to HER2-expressing NIH3T3 cells (3T3/HER2) and panitumumab-IR700 bound to HER1-expressing A431 cells which were then irradiated with non harmful NIR light. EGFR-negative Balb3T3 cells were used as controls. In vivo studies were conducted with 3T3/HER2 and A431 xenograft tumors 1 day after intravenous administration of MAb-IR700 followed by non harmful, transcutaneous NIR light. MAb-IR700 demonstrated specific binding to target receptors on the cell membrane, which was followed by gradual internalization into endolysosomal compartments. MAb-IR700 bound target-specific cell death could be induced within 5 minutes of exposure to NIR light and resulted in cellular swelling, bleb formation, and rupture of vesicles indicating necrotic cell death. No phototoxicity was observed in co-cultured receptor-negative Balb3T3 cells after incubation with MAb-IR700, even when MAb-IR700 was not removed from the medium during light exposure. EGFR target-specific accumulation of MAb-IR700 was observed in the mouse xenograft tumors on NIR fluorescence images. Greater than 90% tumor shrinkage within 3 days of the NIR irradiation, was measured, with no apparent side effects, but was only observed in EGFR-expressing tumors. Furthermore, IR700 fluorescence produced by the MAb conjugate permitted guidance of light delivery and allowed for monitoring after therapy. The MAb-IR700 PIT was most effective, when conjugates were bound to the cell membrane, but showed no phototoxicity, when unbound, suggesting a novel mechanism for PIT compared with conventional photodynamic therapies. Target selective PIT based on MAb-IR700 conjugate membrane binding enables selective treatment of cancer with no apparent side effect to normal cells or surrounding tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3618. doi:10.1158/1538-7445.AM2011-3618

Word limit: 2600 characters, including abstract body and title. Optical imaging has the potential to become a robust tool in intraoperative sentinel lymph node mapping because of its portability, high spatial resolution, and absence of radiation. Most preclinical and clinical studies have used conventional fluorescent agents, which need excitation light to emit fluorescence. However, excitation light also induces autofluorescence (background signal) from intrinsic fluorophores in the normal tissue, lowering the target-to-background ratio. We developed two new methods for optical sentinel lymph node mapping to eliminate background signals using two unique nano-particles; an upconverting nano-particle (UCNP) and a self-illuminating bioluminescence resonance energy transfer quantum-dot (BRET-QD) nano-particle. UCNPs are unique nano-sized particles that emit light at shorter wavelengths with excitation in the near infrared (NIR). Since fluorophores typically emit light at longer wavelengths than the excitation light, there is no autofluorescence. Moreover, by changing the composition of the doping metals, UCNPs can be designed to emit at specific wavelengths. When UCNPs were injected into the chin of anesthetized mice, and excited at 950-nm, superficial neck lymph nodes were successfully depicted with no background signals in the visible-range (peak 550 nm) or NIR-range (peak 800 nm) in all mice. Furthermore, with serial injection of the visible and NIR-range UCNPs, simultaneous lymphatic imaging was achieved in two colors. The BRET-QD is self-illuminating and employs the substrate, coelenterazine, which reacts with luciferase conjugated on the BRET-QD. The bioluminescent light in blue efficiently excites the NIR QD, and emits photons in the NIR, therefore, there is no autofluorophore excitation. Moreover, by changing the QDs within BRET-QD, it is possible to alter their emission wavelength. BRET-QD655, (QD655 in its core), was injected at different sites (chin, ear, forepaws, and hind paws) of anesthetized mice. After BRET-QD655 injection, coelenterazine was intravenously injected and imaging was performed without excitation light. In all mice, sentinel lymph nodes in each lymphatic basin were clearly visualized with ultra-low background signals, and lasted at least 30 min after coelenterazine injections. In conclusion, we demonstrate that optical imaging using two unique nano-particles were able to depict sentinel lymph nodes with minimal autofluorescence. This enables the direct, real time sentinel lymph node mapping without extensive post processing of the images. These two novel nano-particles have the potential to be robust tools for sentinel lymph node detection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4332.