Sprayable, Immune Cell Targeting, and Squaraine NIR Fluorophores for Image-guided Cancer Surgery

Executive summary

Fluorescence-guided surgery (FGS) aids surgeons with real-time visualization of small cancer foci and borders, which improves surgical and prognostic efficacy of cancer. A fluorescent molecule for cancer imaging must:

• Accumulate in cancer cells via active and/or passive targeting mechanisms, or
• Be fluorescent only in the cancerous tissue via activating targeting mechanism.

Here, there are three different fluorophores that not only meet these conditions but show excellence in imaging cancer cells.

1. First, pH-sensitive near-infrared (NIR) fluorophores that exhibit rapid signal changes in acidic tumor microenvironments (TME) and mitochondrial and lysosomal retention. These fluorophores are sensitive to low pH, which allows them to target the acidic tumor microenvironment and activate.
   
   
2. Second, there are immune cell-targeting fluorophores that infiltrate immune cells, including inflammatory monocytes that are recruited from the circulation, and differentiate into macrophages as they migrate into the inflammatory tissues, such as cancerous area.
   
   Therefore, immune cells, including bone marrow-derived and/or tissue-resident/tumor-associated immune cells (TRICs/TAICs), can be a principal target for cancer detection due to the abundance of immune cells in tumoral tissues (e.g., tumor-associated macrophages comprise approximately 50% of tumor mass).
   
   
3. Thirdly, squaraine fluorophores, zwitterionic, and planar structured squaraines, are water-soluble and stable in aqueous solutions, resulting in reduced serum binding and superb optical properties in the NIR wavelengths, allowing surgeons to more precisely perform surgical tumor resection. These squaraines allow for fast and efficient targeting of cancers during fluorescence image-guided surgery.

Unmet need

Despite the steady advances in imaging devices, there is a scarcity of fluorophores available to achieve optimal FGS. FDA-approved small molecule NIR probes are available in the clinic; however, they show no specific targeting capability and have significant limitations in image-guided cancer surgery due to their short blood half-life and nonspecific uptake. In addition, they are not ideal for cancer imaging since they display little on-target binding. 

Value proposition

As the FGS market continues to grow at a CAGR of 6% over the next six years, the need for more effective fluorophores is also growing. Existing FDA-approved fluorophores have proven to not be effective during FGS because of their half-lives and lack of specificity for tumors. These three fluorophores for use in FGS have their own unique properties and tumor-targeting abilities.

Team

Rationale of disease

A common treatment for tumorous cancer is surgery to resect the tumors. Surgery can be a cure for some patients; but oftentimes, it serves as a crucial treatment in preventing metastasis. Fluorescence-guided surgery is used in several surgical applications, including lymphography, angiography, and the identification of solid tumors. This technology capitalizes on FGS for tumor resection.

Background

A common treatment for tumorous cancer is surgery to resect the tumors. Surgery can be a cure for some patients, but oftentimes, it serves as a crucial treatment in preventing metastasis. Fluorescence-guided surgery is used in several surgical applications, including lymphography, angiography, and the identification of solid tumors. This technology capitalizes on FGS for tumor resection.

Unmet needs

Surgical resection of tumors not only involves risks common with surgery, but it does not guarantee that all of the tumor is resected. Tumor foci and borders exist on the microscopic level, making the probability of complete resection low. With FGS, however, surgeons are able to see the smallest parts of tumors and improve the likelihood of success and the patient’s prognosis. The fluorophores presented here would fulfill the need for rapid illumination and retention of fluorescent signals during surgery to resect the smallest areas of tumors.

Technology

Background and proof of concept

Over 50 near-infrared (NIR) fluorescent targeted agents have been synthesized and tested through intravenous injections and topical administration in various cancer-bearing mouse models.  

1. PH compounds are pH-sensitive activatable agents, enabling rapid fluorescence “turn-on” within 10 minutes post-topical administration, which stays over 4 hours in the tumoral tissue. This enables the visualization of submillimeter tumors, tumor foci, and borders, enabling real-time image-guided surgery.
   
   
2. OCTLs are pan tumor-targeted small molecules. After systemic administration in ovarian cancer bearing mice, OCTLs rapidly diffuse across tumor vasculature, show cellular uptake via organic cation transporters (OCTs), and are retained in the lysosome.
   
   
3. Compounds are designed for immune call-specific targeting. After intravenous injection in a pancreatic ductal adenocarcinoma mouse model, SH agents accumulate in immune cells in the bone marrow, followed by infiltration of those bone marrow-derived immune cells to the cancerous region, resulting in fluorescence signal intensity boosting over time on the tumoral site, while reducing signals in the bone marrow.

Advantages and progress

The sprayable NIR fluorophores not only have a significant impact on surgical and diagnostic applications but also provide an effective and scalable strategy to design therapeutic agents for a wide array of cancers. There is a lack of immune cell-targeted small molecular fluorophores reported, making this fluorophore novel. Moreover, the squaraine fluorophores are water-soluble and stable in aqueous solutions, resulting in reduced serum binding and superb optical properties in the NIR wavelengths, allowing surgeons to more precisely perform surgical tumor resection. The squaraine fluorophores display ultrabright optical properties and optimal pharmacokinetics, allowing high-contrast and durable near-infrared imaging for fluorescence-guided surgery of ovarian cancer in mice.

This project impacts the diagnosis and surgery of cancer by providing a highly sensitive and specific imaging modality. The translatability of this study is facilitated by the fact that this platform is based on the FDA-approved fluorophore analogs that are confirmed to be safe repeatedly in preclinical models with minimal invasiveness and adverse effects. These features would support rapid approval by the FDA and open a major pathway toward the clinical development of this technology. 

Once milestones are achieved in current studies, multimodal imaging probes will enter a formal preclinical development process toward an investigational new drug application. To this end, all deliverables are being synthesized to be compatible with cGMP manufacturing to lay a strong foundation for future clinical translation. 

Several promising NIR fluorescent probes currently are undergoing FGS clinical trials, and—for the first time—targeted NIR probe, OTL-38 received an FDA approval for ovarian cancer surgery in 2021.

Mechanism

1. Sprayable fluorophores: Since the tumor microenvironment is acidic, activatable NIR fluorophores detect mildly low pH based on photon electron transfer (PeT).

2. Pan tumor-targeted agents: With their rapid diffusion across tumor vasculature, cellular uptake via organic cation transporters and retention in the lysosome, squaraines are optimal for rapid fluorescence recovery, durable retention, and excellent targetability upon intravenous administration, allowing for intraoperative imaging for a sufficiently long time and detection of small peritoneal dissemination of ovarian cancer.

3. Immune cell-targeting fluorophores: SITT10 is an indocyanine-based fluorophore lacking targeting moieties that have occasionally shown tumor targetability, called structure-inherent tumor targeting. SITT10 can directly target tumor-associated immune cells, as well as myeloblasts and lymphoblasts, which migrate through blood vessels followed by infiltration to the cancerous region.

Competitive advantages