A phase I, single-center, open-label study of RM-1929 photoimmunotherapy in Japanese patients with recurrent head and neck squamous cell carcinoma.

BACKGROUND: To determine the safety, preliminary efficacy, pharmacokinetics, and immunogenicity of a single cycle of RM-1929 photoimmunotherapy, an anti-EGFR antibody cetuximab conjugated with a light-activatable dye (IRDye®700DX), in Japanese patients with recurrent head and neck squamous cell carcinoma (rHNSCC). METHODS: Patients received a single fixed dose (640 mg/m2) of RM-1929 and a fixed light treatment dose (50 J/cm2 for superficial illumination; 100 J/cm fiber diffuser length for interstitial illumination). Safety, tumor response (modified RECIST v1.1 by central radiology review), pharmacokinetics, and immunogenicity were evaluated. RESULTS: Three Japanese patients were enrolled who had failed ≥ 3 prior lines of therapy including radiation, chemotherapy, cetuximab, and immunotherapy. Target lesions were: submental lesion; right superficial cervical node lesion and oropharynx lesion; and external auditory canal lesion. All patients experienced ≥ 1 treatment-emergent adverse event (TEAE), but none were considered dose-limiting. TEAEs were mild to moderate in severity except for one grade 3 application-site pain, which was transient, resolved without sequelae within 24 h, and did not affect study treatment administration. Thirteen of 17 TEAEs reported were possibly or probably related to study treatment. Three patient reports of application-site pain and localized edema were deemed probably related to study treatment. Objective response was observed in two patients (both partial responses). The third patient had disease progression. RM-1929 concentrations and pharmacokinetic parameters were similar in all patients. No patients tested positive for anti-drug antibodies. CONCLUSIONS: RM-1929 photoimmunotherapy showed a manageable safety profile in rHNSCC. Tumor response in these heavily pre-treated patients was clinically meaningful and warrants further investigation. CLINICAL TRIAL REGISTRATION: The trial was registered with the Japanese registry of clinical trials as jRCT2031200133.

Preclinical imaging of epidermal growth factor receptor with ABY-029 in soft-tissue sarcoma for fluorescence-guided surgery and tumor detection.

BACKGROUND AND OBJECTIVES: Fluorescence-guided surgery using epidermal growth factor receptor (EGFR) targeting has been performed successfully in clinical trials using a variety of fluorescent agents. We investigate ABY-029 (anti-EGFR Affibody® molecule labeled with IRDye 800CW) compared with a small-molecule perfusion agent, IRDye 700DX carboxylate, in a panel of soft-tissue sarcomas with varying levels of EGFR expression and vascularization. METHODS: Five xenograft soft-tissue sarcoma cell lines were implanted into immunosuppressed mice. ABY-029 and IRDye 700DX were each administered at 4.98 µM. Fluorescence from in vivo and ex vivo (fresh and formalin-fixed) fixed tissues were compared. The performance of three fluorescence imaging systems was assessed for ex vivo tissues. RESULTS: ABY-029 is retained longer within tumor tissue and achieves higher tumor-to-background ratios both in vivo and ex vivo than IRDye 700DX. ABY-029 fluorescence is less susceptible to formalin fixation than IRDye 700DX, but both agents have disproportional signal loss in a variety of tissues. The Pearl Impulse provides the highest contrast-to-noise ratio, but all systems have individual advantages. CONCLUSIONS: ABY-029 demonstrates promise to assist in wide local excision of soft-tissue sarcomas. Further clinical evaluation of in situ or freshly excised ex vivo tissues using fluorescence imaging systems is warranted.

Improved Discrimination of Tumors with Low and Heterogeneous EGFR Expression in Fluorescence-Guided Surgery Through Paired-Agent Protocols.

PURPOSE: The goal of fluorescence-guided surgery (FGS) in oncology is to improve the surgical therapeutic index by enhancing contrast between cancerous and healthy tissues. However, optimal discrimination between these tissues is complicated by the nonspecific uptake and retention of molecular targeted agents and the variance of fluorescence signal. Paired-agent imaging (PAI) employs co-administration of an untargeted imaging agent with a molecular targeted agent, providing a normalization factor to minimize nonspecific and varied signals. The resulting measured binding potential is quantitative and equivalent to in vivo immunohistochemistry of the target protein. This study demonstrates that PAI improves the accuracy of tumor-to-healthy tissue discrimination compared to single-agent imaging for in vivo FGS. PROCEDURES: PAI using a fluorescent anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) with untargeted IRDye 700DX carboxylate was compared to ABY-029 alone in an oral squamous cell carcinoma xenograft mouse model at 3 h after dye administration (n = 30). RESULTS: PAI significantly enhanced tumor discrimination, as compared to ABY-029 alone in low EGFR-expressing tumors and highly heterogeneous populations including multiple cell lines with varying expression (diagnostic accuracy: 0.908 vs. 0.854 and 0.908 vs. 0.822; and ROC curve AUC: 0.963 vs. 0.909 and 0.957 vs. 0.909, respectively) indicating a potential for universal FGS image thresholds to determine surgical margins. In addition, PAI achieved significantly higher diagnostic ability than ABY-029 alone 0.25-5-h post injection and exhibited a stronger correlation to EGFR expression heterogeneity. CONCLUSION: The quantitative receptor delineation of PAI promises to improve the surgical therapeutic index of cancer resection in a clinically relevant timeline.

Trastuzumab-based near-infrared photoimmunotherapy in xenograft mouse of breast cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel form of cancer treatment using conjugates of antibody against overexpressed antigens in cancers and photoabsorber IRDye700DX. HER2 is overexpressed in various cancers, for which molecular targeted therapy such as trastuzumab has been developed. The present study investigated the efficacy potential of HER2-targeted NIR-PIT using trastuzumab-IRDye700DX conjugate (Tra-IR700) in HER2-positive breast cancer. We first examined the reactivity of Tra-IR700 and the cytotoxicity of NIR-PIT in vitro. HER2-positive BT-474 and SK-BR-3 cells and HER2-negative BT-20 cells were used. Tra-IR700 fluorescence was only observed in HER2-positive breast cancer cell lines, and the fluorescence was localized to the cell surface. Furthermore, HER2-positive breast cancer cell lines treated with NIR-PIT showed swelling and blebbing shortly after irradiation, and eventually increased PI-positive dead cells. Next, tumor accumulation of Tra-IR700 and tumor damage by NIR-PIT were examined in vivo. Tra-IR700 was administered intravenously to a xenograft model in which BT-474 cells were implanted subcutaneously in BALB/c nude mice. Tra-IR700 fluorescence was the highest in tumor tissue 1 day after administration, and the fluorescence was localized to the cell membrane of tumor cells. At this time point, NIR-PIT resulted in diffuse necrosis of tumor tissues 1 day after irradiation. These results suggest that NIR-PIT with Tra-IR700 induces a highly selective therapeutic effect in a HER2-positive breast cancer model. NIR-PIT using Tra-IR700 is expected to be a novel treatment for HER2-positive cancers, including breast cancer.

Identification of a Suitable Untargeted Agent for the Clinical Translation of ABY-029 Paired-Agent Imaging in Fluorescence-Guided Surgery.

PURPOSE: Non-specific uptake and retention of molecular targeted agents and heterogeneous tissue optical properties diminish the ability to differentiate between tumor and normal tissues using molecular targeted fluorescent agents. Paired-agent imaging (PAI) can increase the diagnostic ability to detect tumor tissue by mitigating these non-specific effects and providing true molecular contrast by co-administration of an untargeted control imaging agent with a targeted agent. This study evaluates the suitability of available clinically translatable untargeted agents for the translation of PAI in fluorescence-guided surgery using an affibody-based targeted imaging agent (ABY-029). EXPERIMENTAL: DESIGN: Three untargeted agents that fluoresce near 700 nm and exhibit good clinical safety profiles (methylene blue, IRDye 700DX, and IRDye 680LT) were tested in combination with the clinically tested IRDye 800CW-labeled anti-epidermal growth factor receptor (EGFR) affibody molecule, ABY-029 (eIND 122,681). Properties of the untargeted agent important for human use and integrity of PAI were tested: (1) plasma protein binding; (2) fluorescence signal linearity in in vitro whole blood dilution; (3) in vivo pharmacokinetic matching to targeted agent in negative control tissue; and (4) in vivo diagnostic accuracy of PAI vs single agent imaging (SAI) of ABY-029 alone in orthotopic oral head and neck squamous cell carcinomas. RESULTS: IRDye 680LT outperformed IRDye 700DX and methylene blue with the highest signal linearity (R2 = 0.9998 ± 0.0002, 0.9995 ± 0.0004, 0.91 ± 0.02, respectively), the highest fluorescence yield in whole blood at 1 µM (104.42 ± 0.05, 103.68 ± 0.09, 101.9 ± 0.2, respectively), and the most closely matched ABY-029 pharmacokinetics in EGFR-negative tissues (binding potential error percentage = 0.31% ± 0.37%, 10.25% ± 1.30%, and 8.10% ± 5.37%, respectively). The diagnostic ability of PAI with ABY-029 and IRDye 680LT outperformed conventional SAI with an area-under-the-receiver-operating-characteristic curve (AUC) value of 0.964 vs. 0.854, and 0.978 vs. 0.925 in the Odyssey scanning system and Pearl wide field imaging system, respectively. CONCLUSION: PAI is a highly promising methodology for increasing detection of tumors in fluorescence-guided surgery. Although not yet clinically approved, IRDye 680LT demonstrates promise as an untargeted agent when paired with ABY-029. The clinical translation of PAI to maximize tumor excision, while minimizing normal tissue removal, could improve both patient survival and life quality.

Near-infrared photoimmunotherapy: design and potential applications for cancer treatment and beyond.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment modality based on a target-specific photosensitizer conjugate (TSPC) composed of an NIR phthalocyanine photosensitizer and an antigen-specific recognition system. NIR-PIT has predominantly been used for targeted therapy of tumors via local irradiation with NIR light, following binding of TSPC to antigen-expressing cells. Physical stress-induced membrane damage is thought to be a major mechanism underlying NIR-PIT-triggered photokilling. Notably, NIR-PIT can rapidly induce immunogenic cell death and activate the adaptive immune response, thereby enabling its combination with immune checkpoint inhibitors. Furthermore, NIR-PIT-triggered "super-enhanced permeability and retention" effects can enhance drug delivery into tumors. Supported by its potential efficacy and safety, NIR-PIT is a rapidly developing therapeutic option for various cancers. Hence, this review seeks to provide an update on the (i) broad range of target molecules suitable for NIR-PIT, (ii) various types of receptor-selective ligands for designing the TSPC "magic bullet," (iii) NIR light parameters, and (iv) strategies for enhancing the efficacy of NIR-PIT. Moreover, we review the potential application of NIR-PIT, including the specific design and efficacy in 19 different cancer types, and its clinical studies. Finally, we summarize possible NIR-PIT applications in noncancerous conditions, including infection, pain, itching, metabolic disease, autoimmune disease, and tissue engineering.

Hurdles for the wide implementation of photoimmunotherapy.

Near infrared photoimmunotherapy (NIR-PIT) is a molecularly targeted treatment for cancers achieved by injecting a conjugate of IRDye700DX® (IR700), a water-soluble silicon phthalocyanine derivative in the near infrared, and a monoclonal antibody that targets cancer cell antigens. NIR-PIT is a highly specific treatment with few side effects that results in rapid immunogenic cell death. Despite it being a very effective and innovative therapy, there are a few challenges preventing full implementation in clinical practice. These include the limits of near infrared light penetration, selection of targets, concerns about tumor lysis syndrome and drug costs. However, NIR-PIT has been approved by the regulatory authorities in Japan, allowing for exploration of how to mitigate challenges while maximizing the benefits of this treatment modality.

Phase 1/2a, open-label, multicenter study of RM-1929 photoimmunotherapy in patients with locoregional, recurrent head and neck squamous cell carcinoma.

BACKGROUND: Recurrent head and neck squamous cell carcinoma (rHNSCC) represents a significant global health burden with an unmet medical need. In this study we determined the safety and efficacy of RM-1929 photoimmunotherapy in patients with heavily pretreated rHNSCC. METHODS: RM-1929 (anti-EGFR-IR700 dye conjugate) was infused, followed by tumor illumination. We evaluated safety, tumor response, and pharmacokinetics. RESULTS: Nine patients were enrolled in Part 1 (dose-finding) and 30 patients in Part 2 (safety and efficacy). No dose-limiting toxicities were experienced in Part 1; 640 mg/m2 with fixed light dose (50 J/cm2 or 100 J/cm) was recommended for Part 2. Adverse events (AEs) in Part 2 were mostly mild to moderate but 19 (63.3%) patients had AE ≥Grade 3, including 3 (10.0%) with serious AEs leading to death (not treatment related). Efficacy in Part 2: unconfirmed objective response rate (ORR) 43.3% (95% CI 25.46%-62.57%); confirmed ORR 26.7% (95% CI 12.28%-45.89%); median overall survival 9.30 months (95% CI 5.16-16.92 months). CONCLUSIONS: Treatment was well tolerated. Responses and survival following RM-1929 photoimmunotherapy in heavily pretreated patients with rHNSCC were clinically meaningful and warrant further investigation. CLINICAL TRIAL INFORMATION: NCT02422979.

The feasibility of Miltuximab®-IRDye700DX-mediated photoimmunotherapy of solid tumors.

BACKGROUND: Photoimmunotherapy (PIT) is an emerging method of cancer treatment based on the use of a photosensitizer near-infrared dye IRDye700DX (IR700) conjugated to a monoclonal antibody. The antibody selectively delivers IR700 to cancer cells, which can then be killed after photoexcitation. Glypican-1 (GPC-1) is a novel target expressed specifically in malignant tumors. We aimed to investigate whether anti-GPC-1 antibody Miltuximab® (Glytherix Ltd., Sydney, Australia) can be conjugated with IR700 for PIT of solid tumors. METHODS: The dye IR700 was conjugated with Miltuximab® and characterized by spectrophotometry and flow cytometry. Miltuximab®-IR700-mediated PIT was tested in prostate (DU-145), bladder (C3 and T-24), brain (U-87 and U-251) and ovarian (SKOV-3) cancer cell lines. After 1 h incubation with Miltuximab®-IR700, the cells were washed by PBS and illuminated using a 690-nm light-emitting diode. The viability of the cells was assessed by a CCK-8 viability kit 24 h later. RESULTS: Miltuximab®-IR700-mediated PIT caused 67.3-92.3% reduction in viability of cells with medium-high GPC-1 expression and did not affect the viability of GPC-1-low cells. Cytotoxicity was attributed to the targeted binding of the conjugate with subsequent photoactivation, as the conjugate or light exposure alone had no effect on the cell viability. Miltuximab®-IR700 did not induce cytotoxicity in cells blocked by unconjugated Miltuximab®. CONCLUSIONS: PIT with Miltuximab®-IR700 appears to be highly specific and effective against GPC-1-expressing cancer cells, indicating that it holds promise for an effective and safe treatment of early stage solid tumors or as adjuvant therapy following surgical resection. These findings necessitate further investigation of PIT with Miltuximab®-IR700 in other GPC-1-expressing cancer cell lines in vitro and in vivo in xenograft tumor models.

Carcinoembryonic antigen-targeted photodynamic therapy in colorectal cancer models.

BACKGROUND: In colorectal cancer, survival of patients is drastically reduced when complete resection is hampered by involvement of critical structures. Targeted photodynamic therapy (tPDT) is a local and targeted therapy which could play a role in eradicating residual tumor cells after incomplete resection. Since carcinoembryonic antigen (CEA; CEACAM5) is abundantly overexpressed in colorectal cancer, it is a potential target for tPDT of colorectal cancer. METHODS: To address the potential of CEA-targeted PDT, we compared colorectal cancer cell lines with different CEA-expression levels (SW-48, SW-480, SW-620, SW-1222, WiDr, HT-29, DLD-1, LS174T, and LoVo) under identical experimental conditions. We evaluated the susceptibility to tPDT by varying radiant exposure and concentration of our antibody conjugate (DTPA-hMN-14-IRDye700DX). Finally, we assessed the efficacy of tPDT in vivo in 18 mice (BALB/cAnNRj-Foxn1nu/nu) with subcutaneously xenografted LoVo tumors. RESULTS: In vitro, the treatment effect of tPDT varied per cell line and was dependent on both radiant exposure and antibody concentration. Under standardized conditions (94.5 J/cm2 and 0.5 µg/µL antibody conjugate concentration), the effect of tPDT was higher in cells with higher CEA availability: SW-1222, LS174T, LoVo, and SW-48 (22.8%, 52.8%, 49.9%, and 51.9% reduction of viable cells, respectively) compared to cells with lower CEA availability. Compared to control groups (light or antibody conjugate only), tumor growth rate was reduced in mice with s.c. LoVo tumors receiving tPDT. CONCLUSION: Our findings suggest cells (and tumors) have different levels of susceptibility for tPDT even though they all express CEA. Furthermore, tPDT can effectively reduce tumor growth in vivo.

Paired-agent imaging for detection of head and neck cancers.

Head and neck cancers overwhelmingly overexpress epidermal growth factor receptor (EGFR). This overexpression has been utilized for head and neck cancers using molecular targeted agents for therapy and cancer cell detection. Significant progress has been made in using EGFR-targeted fluorescent antibody and Affibody molecule agents for fluorescent guided surgery in head and neck cancers. Although success in achieving tumor-to-background ratio of 3-5 have been achieved, the field is limited by the non-specific fluorescence in normal tissues as well as EGFR specific fluorescence in the oral cavity. We propose that paired-agent imaging (PAI) could improve the contrast between tumor and normal tissue by removing the fluorescent signal arising from non-specific binding. Here, ABY-029 - an anti-EGFR Affibody molecule labeled with IRDye 800CW - and IRDye 680RD conjugated to Affibody Control Imaging Agent molecule (IR680-Affctrl) are used as targeted and untargeted control agents, respectively, in a panel of head and neck squamous cell carcinomas (HNSCC) to test the ability of PAI to increase tumor detection. Initial results demonstrate that binding potential, a value proportional to receptor concentration, correlates well to EGFR expression but experimental limitations prevented pixel-by-pixel analysis that was desired. Although promising, a more rigorous and well-defined experimental protocol is required to align ex vivo EGFR immunohistochemistry with in vivo binding potential and fluorescence intensity. Additionally, a new set of paired-agents, ABY-029 and IRDye 700DX, are successfully tested in naïve mice and will be carried forward for clinical translation.

Biodistribution Study of Intravenously Injected Cetuximab-IRDye700DX in Cynomolgus Macaques.

PURPOSE: The use of receptor-targeted antibodies conjugated to photosensitizers is actively being explored to enhance treatment efficacy. To facilitate clinical testing, we evaluated cetuximab conjugated to IRDye700DX (IR700) in cynomolgus macaques. PROCEDURES: Total IR700 and intact cetuximab-IR700 were measured in 51 tissues at 2 and 14 days after intravenous injection of 40 and 80 mg/kg cetuximab-IR700, respectively, and compared with an unlabeled cetuximab-dosed control group (two each per sex per time point per group). RESULTS: The IR700 retrieved from all tissues at 2 and 14 days after dosing was estimated at 34.9 ± 1.8 and 2.53 ± 0.67% of the total dose, respectively. The tissues with the highest levels of intact cetuximab-IR700 at 2 days after dosing were the blood, lung, and skin. Formalin-fixed paraffin-embedded tissue sections at 2 days after dosing showed the highest IR700 signals in the axillary lymph node, mammary gland, and gall bladder. CONCLUSIONS: Both IR700 and intact cetuximab-IR700 biodistributions were consistent with known epidermal growth factor receptor (EGFR) expression, and changes between 2 and 14 days were consistent with rapid metabolism and excretion of the cetuximab-IR700.

Photoimmunotherapy of residual disease after incomplete surgical resection in head and neck cancer models.

Antibody-based photodynamic therapy, or photoimmunotherapy (PIT), is a novel, targeted cancer therapy, which can serve as both a diagnostic and a therapeutic agent. The primary objective of this study was to evaluate the capacity of panitumumab-IRDye700DX (Pan-IR700) to eliminate microscopic tumor remnants in the postsurgical setting, which was accomplished using novel in vitro and in vivo models of residual disease after incomplete resection. Additionally, PIT was evaluated in fresh human-derived cancer tissue. To determine a threshold for cellular regrowth after PIT, an in vitro assay was performed using a range of cells representing microscopic disease quantities. Long-term growth inhibition was induced after treatment of 5 × 10(3) and 1 × 10(4) cells at 6 J. A novel in vivo mouse model of subtotal tumor resection was used to assess the effectiveness of Pan-IR700 mediated PIT to eliminate residual disease and inhibit recurrence in the post-surgical wound bed. Mice receiving surgical treatment plus adjuvant PIT showed a threefold and fourfold reduction in tumor regrowth at 30 days post PIT in the 50% and 90% subtotal resection groups, respectively (as measured by bioluminescence imaging), demonstrating a significant (P < 0.001) reduction in tumor regrowth. To determine the translatability of epidermal growth factor receptor (EGFR)-targeted PIT, SCCHN human tissues (n = 12) were treated with Pan-IR700. A significant reduction (P < 0.001) in ATP levels was observed after treatment with Pan-IR700 and 100 J cm(-2) (48% ± 5%) and 150 J cm(-2) (49% ± 7%) when compared to baseline. Targeting EGFR with Pan-IR700 has robust potential to provide a tumor-specific mechanism for eliminating residual disease in the surgical setting, thereby increasing therapeutic efficacy, prolonging progression-free survival, and decreasing morbidity.