Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging.

Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

Cyanine Phototruncation Enables Spatiotemporal Cell Labeling.

Photoconvertible tracking strategies assess the dynamic migration of cell populations. Here we develop phototruncation-assisted cell tracking (PACT) and apply it to evaluate the migration of immune cells into tumor-draining lymphatics. This method is enabled by a recently discovered cyanine photoconversion reaction that leads to the two-carbon truncation and consequent blue-shift of these commonly used probes. By examining substituent effects on the heptamethine cyanine chromophore, we find that introduction of a single methoxy group increases the yield of the phototruncation reaction in neutral buffer by almost 8-fold. When converted to a membrane-bound cell-tracking variant, this probe can be applied in a series of in vitro and in vivo experiments. These include quantitative, time-dependent measurements of the migration of immune cells from tumors to tumor-draining lymph nodes. Unlike previously reported cellular photoconversion approaches, this method does not require genetic engineering and uses near-infrared (NIR) wavelengths. Overall, PACT provides a straightforward approach to label cell populations with spatiotemporal control.

Antibody-dependent cellular phagocytosis of tropomyosin receptor kinase C (TrkC) expressing cancer cells for targeted immunotherapy.

Conventional cancer therapies such as chemotherapy are non-selective and induce immune system anergy, which lead to serious side effects and tumor relapse. It is a challenge to prime the body's immune system in the cancer-bearing subject to produce cancer antigen-targeting antibodies, as most tumor-associated antigens are expressed abundantly in cancer cells and some of normal cells. This study illustrates how hapten-based pre-immunization (for anti-hapten antibodies production) combined with cancer receptor labeling with hapten antigen constructs can elicit antibody-dependent cellular phagocytosis (ADCP). Thus, the hapten antigen 2,4-dinitrophenol (DNP) was covalently combined with a cancer receptor-binding dipeptide (IYIY) to form a dipeptide-hapten construct (IYIY-DNP, MW = 1322.33) that targets the tropomyosin receptor kinase C (TrkC)-expressed on the surface of metastatic cancer cells. IYIY-DNP facilitated selective association of RAW264.7 macrophages to the TrkC expressing 4T1 cancer cells in vitro, forming cell aggregates in the presence of anti-DNP antibodies, suggesting initiation of anti-DNP antibody-dependent cancer cell recognition of macrophages by the IYIY-DNP. In in vivo, IYIY-DNP at 10 mg/kg suppressed growth of 4T1 tumors in DNP-immunized BALB/c mice by 45% (p < 0.05), when comparing the area under the tumor growth curve to that of the saline-treated DNP-immunized mice. Meanwhile, IYIY-DNP at 10 mg/kg had no effect on TrkC-negative 67NR tumor-bearing mice immunized with DNP. Tumor growth suppression activity of IYIY-DNP in DNP-immunized mice was associated with an increase in the anti-DNP IgG (7.3 × 106 ± 1.6 U/mL) and IgM (0.9 × 106 ± 0.07 U/mL) antibodies after five cycles of DNP treatment, demonstrated potential for hapten-based pre-immunization then treatment with IYIY-DNP to elicit ADCP for improved immunotherapy of TrkC expressing cancers.

Cyanine Masking: A Strategy to Test Functional Group Effects on Antibody Conjugate Targeting.

Conjugates of small molecules and antibodies are broadly employed diagnostic and therapeutic agents. Appending a small molecule to an antibody often significantly impacts the properties of the resulting conjugate. Here, we detail a systematic study investigating the effect of various functional groups on the properties of antibody-fluorophore conjugates. This was done through the preparation and analysis of a series of masked heptamethine cyanines (CyMasks)-bearing amides with varied functional groups. These were designed to exhibit a broad range of physical properties, and include hydrophobic (-NMe2), pegylated (NH-PEG-8 or NH-PEG-24), cationic (NH-(CH2)2NMe3+), anionic (NH-(CH2)2SO3-), and zwitterionic (N-(CH2)2NMe3+)-(CH2)3SO3-) variants. The CyMask series was appended to monoclonal antibodies (mAbs) and analyzed for the effects on tumor targeting, clearance, and non-specific organ uptake. Among the series, zwitterionic and pegylated dye conjugates had the highest tumor-to-background ratio (TBR) and a low liver-to-background ratio. By contrast, the cationic and zwitterionic probes had high tumor signal and high TBR, although the latter also exhibited an elevated liver-to-background ratio (LBR). Overall, these studies provide a strategy to test the functional group effects and suggest that zwitterionic substituents possess an optimal combination of high tumor signal, TBR, and low LBR. These results suggest an appealing strategy to mask hydrophobic payloads, with the potential to improve the properties of bioconjugates in vivo.

Hows and Whys of Tumor-Seeking Dyes.

Active targeting uses molecular fragments that bind receptors overexpressed on cell surfaces to deliver cargoes, and this selective delivery to diseased over healthy tissue is valuable in diagnostic imaging and therapy. For instance, targeted near-infrared (near-IR) dyes can mark tissue to be excised in surgery, and radiologists can use active targeting to concentrate agents for positron emission tomography (PET) in tumor tissue to monitor tumor metastases. Selective delivery to diseased tissue is also valuable in some treatments wherein therapeutic indexes (toxic/effective doses) are key determinants of efficacy. However, active targeting will only work for cells expressing the pivotal cell surface receptor that is targeted. That is a problem because tumors, even ones derived from the same organ, are not homogeneous, patient-to-patient variability is common, and heterogeneity can occur even in the same patient, so monotherapy with one actively targeted agent is unlikely to be uniformly effective. A particular category of fluorescent heptamethine cyanine-7 (Cy-7) dyes, here called tumor seeking dyes, offer a way to circumvent this problem because they selectively accumulate in any solid tumor. Furthermore, they persist in tumor tissue for several days, sometimes longer than 72 h. Consequently, tumor seeking dyes are near-IR fluorescent targeting agents that, unlike mAbs (monoclonal antibodies), accumulate in any solid lesion, thus overcoming tumor heterogeneity, and persist there for long periods, circumventing the rapid clearance problems that bedevil low molecular mass drugs. Small molecule imaging agents and drugs attached to tumor-seeking dyes have high therapeutic indices and long residence times in cancer cells and tumor tissue. All this sounds too good to be true. We believe most of this is true, but the controversy is associated with how and why these characteristics arise. Prior to our studies, the prevailing hypothesis, often repeated, was that tumor seeking dyes are uptaken by organic anion transporting polypeptides (OATPs) overexpressed on cancer cells. This Account summarizes evidence indicating tumor seeking Cy-7 dyes have exceptional accumulation and persistence properties because they covalently bind to albumin in vivo. That adduct formation provides a convenient way to form albumin-bound pharmaceuticals labeled with near-IR fluorophores which can be tracked in vivo. This understanding may facilitate more rapid developments of generally applicable actively targeted reagents.

Fluorescent kinase inhibitors as probes in cancer.

Fluorescent dyes attached to kinase inhibitors (KIs) can be used to probe kinases in vitro, in cells, and in vivo. Ideal characteristics of the dyes vary with their intended applications. Fluorophores used in vitro may inform on kinase active site environments, hence the dyes used should be small and have minimal impact on modes of binding. These probes may have short wavelength emissions since blue fluorophores are perfectly adequate in this context. Thus, for instance, KI fragments that mimic nucleobases may be modified to be fluorescent with minimal perturbation to the kinase inhibitor structure. However, progressively larger dyes, that emit at longer wavelengths, are required for cellular and in vivo work. In cells, it is necessary to have emissions above autofluorescence of biomolecules, and near infrared dyes are needed to enable excitation and observation through tissue in vivo. This review is organized to describe probes intended for applications in vitro, in cells, then in vivo. The readers will observe that the probes featured tend to become larger and responsive to the near infared end of the spectrum as the review progresses. Readers may also be surprised to realize that relatively few dyes have been used for fluorophore-kinase inhibitor conjugates, and the area is open for innovations in the types of fluorophores used.

Norcyanine-Carbamates Are Versatile Near-Infrared Fluorogenic Probes.

Fluorogenic probes in the near-infrared (NIR) region have the potential to provide stimuli-dependent information in living organisms. Here, we describe a new class of fluorogenic probes based on the heptamethine cyanine scaffold, the most broadly used NIR chromophore. These compounds result from modification of heptamethine norcyanines with stimuli-responsive carbamate linkers. The resulting cyanine carbamates (CyBams) exhibit exceptional turn-ON ratios (∼170×) due to dual requirements for NIR emission: carbamate cleavage through 1,6-elimination and chromophore protonation. Illustrating their utility in complex in vivo settings, a γ-glutamate substituted CyBam was applied to imaging γ-glutamyl transpeptidase (GGT) activity in a metastatic model of ovarian cancer. Overall, CyBams have significant potential to extend the reach of fluorogenic strategies to intact tissue and live animal imaging applications.

Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody-Drug Conjugate Linker Chemistry.

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitatively assess the cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. Here we report the development of a near-infrared (NIR) optical imaging approach that measures the site and extent of linker cleavage in mouse models. This approach is enabled by a superior variant of our recently devised cyanine carbamate (CyBam) platform. We identify a novel tertiary amine-containing norcyanine, the product of CyBam cleavage, that exhibits a dramatically increased cellular signal due to an improved cellular permeability and lysosomal accumulation. The resulting cyanine lysosome-targeting carbamates (CyLBams) are ∼50× brighter in cells, and we find this strategy is essential for high-contrast in vivo targeted imaging. Finally, we compare a panel of several common ADC linkers across two antibodies and tumor models. These studies indicate that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or nonhindered disulfides, an observation that is only apparent with in vivo imaging. This strategy enables quantitative comparisons of cleavable linker chemistries in complex tissue settings with implications across the drug delivery landscape.

Role of Albumin in Accumulation and Persistence of Tumor-Seeking Cyanine Dyes.

Some heptamethine cyanine dyes accumulate in solid tumors in vivo and persist there for several days. The reasons why they accumulate and persist in tumors were incompletely defined, but explanations based on uptake into cancer cells via organic anion transporting polypeptides (OATPs) have been widely discussed. All cyanine-based "tumor-seeking dyes" have a chloride centrally placed on the heptamethine bridge (a "meso-chloride"). We were intrigued and perplexed by the correlation between this particular functional group and tumor uptake, so the following study was designed. It features four dyes (1-Cl, 1-Ph, 5-Cl, and 5-Ph) with complementary properties. Dye 1-Cl is otherwise known as MHI-148, and 1-Ph is a close analog wherein the meso-chloride has been replaced by a phenyl group. Data presented here shows that both 1-Cl and 1-Ph form noncovalent adducts with albumin, but only 1-Cl can form a covalent one. Both dyes 5-Cl and 5-Ph have a methylene (CH2) unit replaced by a dimethylammonium functionality (N+Me2). Data presented here shows that both these dyes 5 do not form tight noncovalent adducts with albumin, and only 5-Cl can form a covalent one (though much more slowly than 1-Cl). In tissue culture experiments, uptake of dyes 1 is more impacted by the albumin in the media than by the pan-OATP uptake inhibitor (BSP) that has been used to connect uptake of tumor-seeking dyes in vivo with the OATPs. Uptake of 1-Cl in media containing fluorescein-labeled albumin gave a high degree of colocalization of intracellular fluorescence. No evidence was found for the involvement of OATPs in uptake of the dyes into cells in media containing albumin. In an in vivo tumor model, only the two dyes that can form albumin adducts (1-Cl and 5-Cl) gave intratumor fluorescence that persisted long enough to be clearly discerned over the background (∼4 h); this fluorescence was still observed at 48 h. Tumors could be imaged with a higher contrast if 5-Cl is used instead of 1-Cl, because 5-Cl is cleared more rapidly from healthy tissues. Overall, the evidence is consistent with in vitro and in vivo results and indicates that the two dyes in the test series that accumulate in tumors and persist there (1-Cl and 5-Cl, true tumor-seeking dyes) do so as covalent albumin adducts trapped in tumor tissue via uptake by some cancer cells and via the enhanced permeability and retention (EPR) effect.

A Near-IR Fluorescent Dasatinib Derivative That Localizes in Cancer Cells.

Kinase inhibitors (KIs) have had a huge impact on clinical treatment of various cancers, but they are far from perfect medicines. In particular, their efficacies are limited to certain cancer types and, in many cases, provide only temporary remission. This paper explores the possibility of covalently binding a fluorophore for in vivo optical imaging to the KI dasatinib where the particular fluorophore chosen for this study, a heptamethine cyanine (Cy) derivative, tends to accumulate in tumors. Thus, we hypothesized that the dasatinib-fluorophore conjugate might target tumor cells more effectively than the parent KI, give enhanced suppression of viability, and simultaneously serve as a probe for optical imaging. As far as we are aware, the dasatinib conjugate (1) is the first reported to contain this KI and a probe for near-IR imaging, and it is certainly the first conjugate of a tumor-targeting near-IR dye and a KI of any kind. Conjugate 1 suppressed the viability of liver cancer cells (HepG2) more effectively than dasatinib at the same concentration. In scratch assays, 1 prevented regrowth of the tumor cells. Conjugate 1 is cell permeable, and confocal imaging indicates the fluorescence of those cells is concentrated in the mitochondria than lysosomes. In general, this study suggests there is untapped potential for conjugates of KIs with tumor-targeting near-IR dyes in the development of theranostics for optical imaging and treatment of cancer.

Protein labelling and albumin binding characteristics of the near-IR Cy7 fluorophore, QuatCy.

Free cysteine residues react with QuatCy 1, by simply mixing the protein and dye in aqueous buffer at 37 °C. Another dye, MHI-148, can be used for a similar labelling protocol, but QuatCy reacts faster with all proteins studied, except albumin; it emerges here that this is because MHI-148 instantly forms of a non-covalent complex with albumin, but QuatCy does not. Labelling with QuatCy has advantages insofar as it is over five times brighter, and much more photostable, than MHI-148, and combination labelling with this dye pair will allow multiplexing in the near-IR region.

Productive Manipulation of Cyanine Dye π-Networks.

Polymethine bridges in cyanine dyes may be constrained by setting them into edge-fused ring systems, or extended by conjugation with carefully chosen heterocycles. Recent studies have shown that modifications like these can give significantly brighter dyes with red-shifted absorbance and emission maxima.

On the Mechanisms of Uptake of Tumor-Seeking Cyanine Dyes.

Molecular entities that localize in tumor tissue are clinically important for targeted delivery of diagnostic, imaging, and therapeutic reagents. Often these targeting entities are designed for specific receptors (e.g., EGFR or integrin receptors). However, there is a subset of cyanine-7 dyes that apparently localize in every type of solid tumor tissue (at least, no exceptions have been reported so far), and they persist there for several days. Consequently, these dyes can be used for near-IR optical imaging of tumors in animal studies, they can be conjugated with cytotoxic species to give experimental theranostics, and there is potential for expanding their use into the development of clinically useful derivatives. Data presented in the literature and in this work indicate that the half-lives of these compounds in serum at 37 °C is on the order of minutes to a few hours, so what accounts for the persistent fluorescence of these dyes in tumor tissue over periods of several days? Literature, solely based on tissue culture experiments featuring a particular receptor blocker, indicates that uptake of these dyes is mediated by the organic anion transporter proteins (OATPs). Data presented in this paper agrees with that conclusion for short-term uptake, but significantly expands understanding of the likely reasons for long-term uptake and persistent tumor localization in vivo.

Site-Specific Labeling of Proteins with Near-IR Heptamethine Cyanine Dyes.

Convenient labeling of proteins is important for observing its function under physiological conditions. In tissues particularly, heptamethine cyanine dyes (Cy-7) are valuable because they absorb in the near-infrared (NIR) region (750⁻900 nm) where light penetration is maximal. In this work, we found Cy-7 dyes with a meso-Cl functionality covalently binding to proteins with free Cys residues under physiological conditions (aqueous environments, at near neutral pH, and 37 °C). It transpired that the meso-Cl of the dye was displaced by free thiols in protein, while nucleophilic side-chains from amino acids like Tyr, Lys, and Ser did not react. This finding shows a new possibility for convenient and selective labeling of proteins with NIR fluorescent probes.

Near-infrared duocarmycin photorelease from a Treg-targeted antibody-drug conjugate improves efficacy of PD-1 blockade in syngeneic murine tumor models.

Regulatory T cells (Tregs) play a crucial role in mediating immunosuppression in the tumor microenvironment. Furthermore, Tregs contribute to the lack of efficacy and hyperprogressive disease upon Programmed cell death protein 1 (PD-1) blockade immunotherapy. Thus, Tregs are considered a promising therapeutic target, especially when combined with PD-1 blockade. However, systemic depletion of Tregs causes severe autoimmune adverse events, which poses a serious challenge to Treg-directed therapy. Here, we developed a novel treatment to locally and predominantly damage Tregs by near-infrared duocarmycin photorelease (NIR-DPR). In this technology, we prepared anti-CD25 F(ab')2 conjugates, which site-specifically uncage duocarmycin in CD25-expressing cells upon exposure to NIR light. In vitro, CD25-targeted NIR-DPR significantly increased apoptosis of CD25-expressing HT2-A5E cells. When tumors were irradiated with NIR light in vivo, intratumoral CD25+ Treg populations decreased and Ki-67 and Interleukin-10 expression was suppressed, indicating impaired functioning of intratumoral CD25+ Tregs. CD25-targeted NIR-DPR suppressed tumor growth and improved survival in syngeneic murine tumor models. Of note, CD25-targeted NIR-DPR synergistically enhanced the efficacy of PD-1 blockade, especially in tumors with higher CD8+/Treg PD-1 ratios. Furthermore, the combination therapy induced significant anti-cancer immunity including maturation of dendritic cells, extensive intratumoral infiltration of cytotoxic CD8+ T cells, and increased differentiation into CD8+ memory T cells. Altogether, CD25-targeted NIR-DPR locally and predominantly targets Tregs in the tumor microenvironment and synergistically improves the efficacy of PD-1 blockade, suggesting that this combination therapy can be a rational anti-cancer combination immunotherapy.

QuatCy-I2 and MHI-I2 in Photodynamic Therapy.

MHI-I2 (1) and QuatCy-I2 (2) were compared in terms of properties important for early-stage photodynamic therapy preclinical candidates. Thus, experiments were performed to monitor dark cytotoxicities, light/dark cytotoxicity ratios, selectivity of localization in tumors over other organs, and clearance from the plasma.

Modified norcyanines enable ratiometric pH imaging beyond 1000 nm.

Activatable fluorophores with emission beyond 1000 nm have the potential to enable high contrast imaging in complex in vivo settings. However, there are few scaffolds that can be applied to this challenge. Here we detail the synthesis and evaluation of benzo[c,d]indole-substituted norcyanines that enable pH responsive fluorescence imaging in the long wavelength (>1150 nm) range. A key component of our molecular design is the installation of a hydrophilic substituted quaternary amine in the central dihydropyridine ring system. A compound with a C4'-phenyl substituent, but not the C4'-protio homologue, exhibits absorbance maxima of 740 nm and 1130 nm in basic and acidic media, respectively, with evidence of J-aggregate-like properties. These two distinct absorbances enabled ratiometric imaging of probe internalization in a tumor model. Overall, these studies provide a new class of activatable long-wavelength responsive fluorophores with promising photophysical properties.

Targeted Dual-Modal PET/SPECT-NIR Imaging: From Building Blocks and Construction Strategies to Applications.

Molecular imaging is an emerging non-invasive method to qualitatively and quantitively visualize and characterize biological processes. Among the imaging modalities, PET/SPECT and near-infrared (NIR) imaging provide synergistic properties that result in deep tissue penetration and up to cell-level resolution. Dual-modal PET/SPECT-NIR agents are commonly combined with a targeting ligand (e.g., antibody or small molecule) to engage biomolecules overexpressed in cancer, thereby enabling selective multimodal visualization of primary and metastatic tumors. The use of such agents for (i) preoperative patient selection and surgical planning and (ii) intraoperative FGS could improve surgical workflow and patient outcomes. However, the development of targeted dual-modal agents is a chemical challenge and a topic of ongoing research. In this review, we define key design considerations of targeted dual-modal imaging from a topological perspective, list targeted dual-modal probes disclosed in the last decade, review recent progress in the field of NIR fluorescent probe development, and highlight future directions in this rapidly developing field.

Coumarins to Cyanines: Synthesis of Hemicyanines.

Near-infrared (NIR) emitting fluorophores are powerful tools for optical imaging. However, there are only a handful of broadly employed NIR-emitting scaffolds, and the synthetic methods to prepare these molecules are often problematic. Here, we describe a novel, three-step synthesis of chromene-containing hemicyanine probes exhibiting large Stokes shifts and NIR emissions. We develop a pH-activatable probe for visualizing lysosomal trafficking of mAb conjugates. These studies provide a concise approach to hemicyanines with promising properties.