Patent Application
20250099625
The current invention relates to diagnostic molecules that can be used in a subject or in vitro to determine whether a subject has a particular disease or whether that disease is susceptible to particular treatments. The invention also relates to methods of making said compounds and the compounds applied to the uses mentioned above.
The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Cancer immunotherapy that trains the immunity to eradicate cancer has revolutionized the landscape of oncology. However, patients often respond differently to the same immunotherapy, and the response rates toward checkpoint blockade therapy remain low in many cancer types (20-40% melanoma, renal cell carcinoma, and colorectal cancer). Tumor-infiltrating leukocytes (TILs) are known to be closely associated with the cancer progression and clinical endpoints of cancer patients. Particularly, clinical data has revealed that high levels of M1 macrophages and cytotoxic T lymphocytes (CTLs) were associated with positive prognosis (Fridman, W. H. et al., Nat. Rev. Clin. Oncol. 2017, 14, 717-734), while increased neutrophil-lymphocyte ratio predicted poor survival of cancer patients (Gentles, A. J. et al., Nat. Med. 2015, 21, 938-945). To assess TILs in tumor immune microenvironment (TIME) for patient stratification and therapeutic predication before and during cancer immunotherapy, flow cytometry, histological staining, and mass cytometry of biopsy tumor tissues have been used. However, single-site biopsy is invasive, static, risky to cause metastasis and is only able to reveal regional information on TIME; whereas, peripheral blood analysis inevitably contains biomarkers secreted from organs other than tumor, reducing the specificity towards TIME.
Molecular imaging offers a real-time and non-invasive way for longitudinal monitoring of overall TIME. However, existing imaging modalities including computed tomography (CT), positron emission tomography (PET) and magnetic resonance imaging (MRI) fail to accurately monitor of TILs in TIME, because they rely on antibody/ligand conjugated contrast agents to target the leukocytes of interest, and their “always-on” signals are inevitably compounded by nonspecific retention in tissues other than tumor. In contrast, activatable molecular optical reporters only trigger their signals against intended biomarkers and thus have minimized background and quantitative signals with the concentration and activity of biomarkers. Recently, activatable molecular reporters have been developed for real-time imaging of leukocytes; however, as their activation mechanism is solely determined by the leukocyte biomarkers, their signals can be nonspecifically triggered by the leukocytes in peripheral blood and inflammatory tissues. Thus, specific real-time non-invasive imaging of TILs remains a great challenge.
Therefore, there exists a need for new molecular fluorescence reports for detection of TILS.
Aspects and embodiments will now be discussed by reference to the following clauses.
1. A compound of formula I:
2. The compound according to Clause 1, or a salt or solvate thereof, wherein A is selected from:
where the point of attachment is denoted by the dotted line.
3. Use of a compound of formula I as defined in Clause 1 or Clause 2, or a salt or solvate thereof in the preparation of an imaging agent for the diagnosis of a condition or disease in a tissue and/or an organ using near-infrared fluorescence.
4. A compound of formula I as defined in Clause 1 or Clause 2, or a salt or solvate thereof for use as an imaging agent for the diagnosis of a condition or disease in a tissue and/or an organ using near-infrared fluorescence.
5. The use according to Clause 3 or the compound for use according to Clause 4, wherein the use is in vivo or in vitro.
6. The use or compound for use according to Clause 5, wherein the in vivo imaging is for the purpose of visualizing the tumour immune microenvironment.
7. A method of diagnosing a condition or disease in a tissue and/or an organ, the method comprising the steps of administering a compound of formula I as defined in Clause 1 or Clause 2, or a salt or solvate thereof to a subject in need of diagnosis and determining the presence or absence of a condition or disease in a tissue and/or an organ using near-infrared fluorescence.
8. A method of determining the susceptibility of a tumour tissue to an immunotherapy, the method comprising the steps of:
9. The method according to Clause 8, wherein the method is conducted in vivo.
10. The method according to Clause 8, wherein the method is conducted in vitro.
11. The method according to any one of Clauses 8 to 10, wherein the method further involves subjecting a subject to a therapeutic treatment regimen based on the results obtained by the method.
12. The method according to any one of Clauses 8 to 11, wherein the always on reference reporter compound is:
In a first aspect of the invention, there is provided a compound of formula I:
In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa.
The phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, and the like.
References herein (in any aspect or embodiment of the invention) to compounds of formula I include references to such compounds perse, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.
Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.
Examples of acid addition salts include acid addition salts formed with acetic, 2,2-dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2-sulphonic, naphthalene-1,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(1S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (−)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g. (+)-L-tartaric), thiocyanic, undecylenic and valeric acids.
Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.
The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.
For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.
Compounds of formula I may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.
Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively, the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ‘chiral pool’ method), by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
In embodiments of the first aspect, A may be selected from:
The reference for (a): J. Leukoc. Biol. 2016, 100, 961; Nat. Med. 2016, 22, 64; J. Biol. Chem. 1997, 272, 9677; and Adv. Mater. 2020, 32, 2000648. The reference for (b): J. Biol. Chem. 2007, 282, 4545; and Cancer Res. 2017, 77, 2318. The reference for (c): J. Biol. Chem. 1979, 254, 4027.
The compounds of formula I may be used to help determine whether a subject is suffering from a particular disease. Thus, in further aspects of the invention, there is provided:
The use of (aa) or the compound for use of (ab) may be conducted in vivo or in vitro. In particular embodiments, the in vivo imaging may be for the purpose of visualizing the tumour immune microenvironment.
The compounds of formula I may also be used to determine whether a particular tumor tissue is susceptible to immunotherapy. Thus, in a further aspect of the invention, there is provided a method of determining the susceptibility of a tumour tissue to an immunotherapy, the method comprising the steps of:
This method may be conducted in vivo or in vitro.
The method above, whether conducted in vitro or in vitro enables the determination of whether a particular tissue can be treated by immunotherapy. Given this, the skilled person can then treat the tumour if it is revealed to be susceptible to the proposed treatment. Thus, in certain embodiments, the method may further involve subjecting a subject to a therapeutic treatment regimen based on the results obtained by the method.
Any suitable always on reference reporter compound may be used herein. For example, the reference reported compound may be:
For the avoidance of doubt, in the context of the present invention, the term “treatment” includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.
The terms “patient” and “patients” include references to mammalian (e.g. human) patients. As used herein the terms “subject” or “patient” are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).
Further embodiments of the invention that may be mentioned include those in which the compound of formula I is isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compound of formula I is not isotopically labelled.
The term “isotopically labelled”, when used herein includes references to compounds of formula I in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to “one or more positions in the compound” will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula I. Thus, the term “isotopically labelled” includes references to compounds of formula I that are isotopically enriched at one or more positions in the compound.
The isotopic labelling or enrichment of the compound of formula I may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine. Particular isotopes that may be mentioned in this respect include 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 35S, 18F, 37Cl, 77Br, 82Br and 125I).
When the compound of formula I is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula I that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or non-radioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.
The compounds of formula I may be prepared for administration to a subject. Thus, in a further aspect of the invention, there is provided a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof as described herein in admixture with one or more of a pharmaceutically acceptable adjuvant, diluent and carrier.
Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.
Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.
Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.
The amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.
For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99% (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90% (w/w) of a release-controlling polymer.
A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50% (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof.
However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic or diagnostic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I.
In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.
All chemicals were purchased from Sigma-Aldrich unless otherwise stated. All amino acid derivatives were bought from GL Biochem. PBr3 and p-aminobenzyl alcohol were purchased from Tokyo Chemical Industry. Anti-mouse PD-L1 (B7-H1) (Clone: 10F.9G2, Cat No. BP0101) was purchased from Bio X Cell. Anti-mouse CD16/32 (Cat No. 101302), Alexa Fluor® 700 anti-mouse CD45 (Cat No. 103128), FITC anti-mouse CD3 (Cat No. 100204), PE anti-mouse CD8a (Cat No. 100708), APC anti-human/mouse GrB recombinant antibody (Cat No. 372204), PerCP anti-mouse/human CD11b (Cat No. 101229), ultra-LEAF™ Purified anti-mouse CD47 antibody (Cat No. 127518), recombinant mouse murine granulocyte macrophage colony stimulating factor (GM-CSF, Cat No. 576304), recombinant rat interferon-gamma (IFN-γ, Cat No. 598802), recombinant rat interleukin-4 (IL-4, Cat No. 776902), intracellular staining perm wash buffer (ISPWB), ACK lysis, PE anti-mouse/human CD11b, and APC anti-mouse Ly-6G were purchased from Biolegend. Neutrophil elastase (NE) polyclonal antibody (PA5-79198), anti-Mo Ly-6G APC (17-9668-82), anti-Mouse NOS2 PE (12-5920-82), Live/Dead™ Fixable Blue Dead Cell Stain (Cat No. L23105), Dynabeads™ FlowComp™ Mouse CD8 kit (Cat No. 11462D) and secondary antibody Alexa Fluor® 488 conjugated goat anti-rabbit IgG (Cat No. 2045215) were purchased from Thermo Fisher Scientific. Caspase-1 (D-3) Alexa Fluor® 647 and F4/80 (C-7) Alexa Fluor® 488 (sc-377009) were purchased from Santa Cruz Biotechnology. GrB was purchased from Novoprotein. Caspase-1 was purchased from BioVision. Cathepsin C (DPPI) and APN were obtained from R&D systems. NE, LPS, bovine serum albumin (BSA), HEPES, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate), PBS, heat-inactivated horse serum, interleukin-2 (IL-2) and DNase I were purchased from Sigma-Aldrich. MTS solution was purchased from PROMEGA PTE LTD. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), Iscove's Modified Dulbecco's Medium (IMDM) and Roswell Park Memorial Institute (RPMI) were purchased from GIBCO. O.C.T. medium was bought from Sakura Fineteck Japan. Heparinized capillary tubes were purchased from Greiner Bio-One GmbH. Type I collagenase and type IV collagenase were purchased from Thermofisher. Dialysis bag was used for dialysis. Dialysis bags were bought from USA Viskase.
Mouse breast cancer cell line 4T1 cells, murine colorectal carcinoma cell line CT26 cells, mouse embryonic fibroblasts 3T3 cells, mouse neutrophils MPRO cells, and mouse macrophage cell line RAW 264.7 cells were purchased from the American Type Culture Collection (ATCC).
UV-Vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer.
Fluorescence spectra were recorded on a Fluorolog 3-TCSPC spectrofluorometer (Horiba Jobin Yvon).
HPLC curves were measured on an Agilent 1260 system equipped with UV detector, G1311B pump, and an Agilent Zorbax SB-C18 RP (9.4×250 mm) column, with CH3OH containing trifluoroacetic acid (TFA, 0.1%) and water containing TFA (0.1%) as the eluent.
1H NMR spectra were recorded on a Bruker 400 MHz NMR.
LCMS spectra were measured on Thermo Finnigan Polaris Q quadrupole ion trap mass spectrometer equipped with a standard electrospray ionization (ESI) source.
Fluorescence imaging of cells was acquired on Laser Scanning Microscope LSM800 (Zeiss).
In vivo animal fluorescence images were taken via an IVIS imaging system (IVIS-CT machine, PerkinElmer), and the region of interest was analyzed via the Living Image 4.3 software.
Tissue sections were obtained on a cryostat (Leica).
HPLC purification was performed on an Agilent 1260 gradient preparative system equipped with a G1361A pump, UV detector and an Agilent Zorbax SB-C18 RP (21.2×150 mm) column, with CH3OH containing TFA (0.1%) and water containing TFA (0.1%) as the eluent (Tables 2 and 3).
All cells were cultured in a humidified environment at 37° C. which contains 5% CO2 and 95% air. 3T3 and RAW 264.7 cells were cultured in DMEM with 10% FBS. 4T1 and CT26 cells were cultured in RPMI 1640 with 10% FBS. MPRO cells were cultured in IMDM with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate containing 10 ng/mL murine GM-CSF, and 20% of heat-inactivated horse serum. CD8 T cells were isolated from spleen of BALB/c mouse by using the Dynabeads® Untouched™ Mouse CD8 Cells kit, and cultured in RPMI 1640 with IL-2. Bone marrow-derived dendritic cells (BMDCs) were isolated from bone marrow of BALB/c mice according to previous protocols (Madaan, A. et al., J. Biol. Methods 2014, 1, e1) and cultured in RPMI 1640 with GM-CSF (20 ng/mL). RAW264.7 cells were polarized to M1 macrophages by incubation with LPS (10 μg/mL) and IFN-γ (20 ng/mL) for 24 h. RAW264.7 cells were polarized to M2 macrophages by incubation with IL-4 (20 ng/mL) for 24 h. Both M1 and M2 macrophages were cultured in DMEM.
All mouse experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC), Nanyang Technological University (NTU). Six-week-old female BALB/c mice were purchased from InVivos, Singapore. For establishment of poorly immunogenic tumors, 4T1 cancer cells in PBS were subcutaneously implanted to the right flank of mice at a density of 1×106 cells/mouse. For establishment of highly immunogenic tumors, CT26 cancer cells in PBS were subcutaneously implanted to the right side of the back of mice at a density of 2×106 cells/mouse.
The in vivo and ex vivo fluorescence intensities were quantified using Living Image 4.3 software for the region of interest analysis. Statistical comparisons between two groups were determined by two-tailed Student's t-test. For statistics analysis, P<0.05 was considered statistically significant; *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.
TAMRs comprise three key units: a tumor-passive targeting moiety, a fluorescent signaling moiety, and a dual-lock TILs responsive moiety that can only be fully cleaved in the presence of both cancer and leukocytes (
IR800-N3 (5 mg, 0.004 mmol) and PVP-alkyne (12 mg, 0.004 mmol) were dissolved in DMSO (1.5 mL). Then, CuSO4·5H2O (1 mg, 0.004 mmol) and sodium ascorbate (1.6 mg, 0.008 mmol) in deionized water (1.5 mL) were added. The reaction mixture was stirred at 25° C. for 12 h. Then, the reaction solution was dialyzed against deionized water for 24 h to remove salts and DMSO, and freeze-dried to get PVP-IR800.
TAMRs were constructed on a near-infrared (NIR) hemicyanine dye (CyOH) (
CyCl was synthesized according to the previous protocol (Huang, J. et al., Nat. Mater. 2019, 18, 1133-1143). K2CO3 (1382 mg, 10 mmol) and resorcinol (1101 mg, 10 mmol) were dissolved in CH3CN (10 mL), followed by stirring at 55° C. for 20 min. Then, a CH3CN solution of CyCl (16.25 mg, 5 mmol) was added to the reaction mixture. The reaction mixture was stirred for another 6 h at 55° C., followed by removing CH3CN. The crude product was purified by silica gel column chromatography using DCM and methanol (CH3OH) (DCM/CH3OH=30/1) to obtain CyOH with a yield of 79%.
MS of CyOH: m/z 467.36. 1H NMR (400 MHz, MeOD): δ (ppm): 8.38 (d, J=12.0 Hz, 1H), 7.59 (s, 1H), 7.49 (d, J=8, 1H), 7.38 (t, 2H), 7.21 (t, 2H), 6.73 (d, J=8, 1H), 6.57 (s, 1H), 6.05 (d, J=16, 1H), 4.07 (t, 2H), 3.45 (t, 2H), 2.74 (t, 2H), 2.68 (t, 2H), 1.91-1.89 (m, 4H), 1.79-1.74 (m, 2H), 1.74 (s, 6H).
N-vinyl pyrrolidinone (1 g, 9 mmol) and AIBN (20 mg) were dissolved in isopropoxyethanol (10 mL), followed by flushing with nitrogen for 10 min. The reaction mixture was stirred at 60° C. for 4 h under the protection of nitrogen. After completion, isopropoxyethanol was concentrated, and the residue in isopropoxyethanol was precipitated into an excess of ethyl ether (200 mL). The white precipitate was centrifuged at 7500 rpm for 10 min and dried at 37° C. under vacuum to get PVP with a yield of 67%.
1H NMR (400 MHz, MeOD): δ (ppm): 3.92-3.74 (m, 32H), 3.29 (m, 54H), 2.38-2.27 (m, 54H), 2.06-2.01 (m, 54H), 1.75-1.46 (m, 60H).
PVP (3 g, 1 mmol) was dissolved in anhydrous THF (40 mL), and NaH (240 mg, 10 mmol) was added. After production of bubbles for 10 min, 3-bromopropyne (1.19 g, 10 mmol) was quickly added to the reaction solution, followed by stirring at 25° C. for 24 h. After completion, THF was concentrated, and the mixture in THF was precipitated into an excess of ethyl ether (200 mL). The white precipitate was centrifuged at 7500 rpm for 10 min. The residue was further dissolved in water and dialyzed against deionized water to remove salts for 4 h (MWCO=1000) and freeze-dried to get PVP-alkyne with a yield of 77%.
1H NMR (400 MHz, MeOD): δ (ppm): 4.17 (s, 2H), 3.92-3.74 (m, 32H), 3.29 (m, 54H), 2.51 (s, 2H), 2.38-2.27 (m, 54H), 2.06-2.01 (m, 54H), 1.75-1.46 (m, 60H).
Peptides Ac-Y(tBu)VAD(OtBu)—OH, Ac—IE(OtBu)FD(OtBu)—OH, and MeOSuc-AAPV-OH were prepared by solid phase peptide synthesis (SPPS, O. A. Musaimi, B. G. de la Torre & F. Albericio, Green Chem. 2020, 22, 996-1018).
1H NMR (400 MHz, MeOD): δ (ppm): 7.18 (d, J=8, 2H), 6.92 (d, J=8, 2H), 4.75 (t, 1H), 4.67-4.63 (m, 1H), 4.44-4.39 (m, 1H), 4.23-4.19 (m, 1H), 3.14-3.09 (m, 1H), 2.88-2.82 (m, 2H), 2.77 (d, J=4, 2H), 1.92 (s, 3H), 1.46 (s, 9H), 1.40 (d, J=8, 3H), 1.33 (s, 9H), 0.98 (t, 6H).
1H NMR (400 MHz, MeOD): δ (ppm): 7.26-7.15 (m, 5H), 4.76 (t, 1H), 4.67-4.63 (m, 1H), 4.32-4.28 (m, 1H), 4.15 (d, J=8, 1H), 3.25 (m, 2H), 3.2 (m, 1H), 2.98-2.92 (m, 1H), 2.79-2.70 (m, 2H), 2.30-2.09 (m, 2H), 2.01 (s, 3H), 1.97-1.90 (m, 1H), 1.86-1.75 (m, 2H), 1.45 (s, 18H), 0.93-0.83 (m, 6H).
1H NMR (400 MHz, MeOD): δ (ppm): 4.64-4.52 (m, 2H), 4.35 (t, 1H), 4.29 (t, 1H), 3.81-3.75 (m, 1H), 3.66 (s, 3H), 3.32-3.30 (m, 1H), 2.69-2.58 (m, 2H), 2.55-2.45 (m, 2H), 2.21-2.18 (m, 2H), 2.04-1.91 (m, 3H), 1.36-1.32 (m, 6H), 0.99 (d, J=8, 6H).
EEDQ (742 mg, 3.0 mmol), PABA (369 mg, 3.0 mmol) and Fmoc-A-OH (311.3 mg, 1.0 mmol) were dissolved in DCM (15 mL), and the reaction mixture was continuously stirred at 25° C. for 6 h. The residues were purified by HPLC and freeze-dried to get Fmoc-A-PABA with a yield of 91%.
1H NMR (400 MHz, MeOD): δ (ppm): 7.82 (d, J=8, 2H), 7.70 (t, 2H), 7.56 (d, J=8, 2H), 7.40 (t, 2H), 7.33 (d, J=8, 4H), 4.58 (s, 2H), 4.41 (d, J=8, 2H), 4.29-4.22 (m, 2H), 1.44 (d, J=8, 3H).
Fmoc-A-PABA (100 mg, 0.24 mmol) was dissolved in anhydrous THF, followed by adding PBr3 (200 mg, 0.72 mmol). The reaction mixture was stirred at 0° C. for 2 h. Then, THF was removed and the residue was dissolved with an excess amount of ethyl acetate (EA, 200 mL). The solution was washed with NaHCO3 aqueous solution for three times. After concentrated and dried, the residue was dissolved by anhydrous CH3CN (50 mL), followed by adding CyOH (37.4 mg, 0.08 mmol) and N,N-diisopropylethylamine (DIPEA, 40 μL). The reaction mixture was stirred at 55° C. for 8 h. Then, CH3CN was removed, and the residue was purified by HPLC and freeze-dried to get Cy-A-Fmoc with a yield of 97%.
1H NMR (400 MHz, CDCl3): δ (ppm): 8.66 (d, J=12, 1H), 7.75 (d, J=8, 2H), 7.67 (d, J=8, 3H), 7.60 (t, 2H), 7.46-7.40 (m, 6H), 7.32 (t, 3H), 7.24 (t, 2H), 7.01-6.98 (m, 2H), 6.44 (d, J=16, 1H), 5.22 (s, 2H), 4.31-4.23 (m, 5H), 4.10 (t, 1H), 3.40 (t, 2H), 2.71 (t, 2H), 2.65 (t, 2H), 1.89-1.86 (m, 4H), 1.75 (s, 6H), 1.74-1.7 (m, 2H), 1.41 (d, J=8, 3H).
Cy-A-Fmoc (10 mg, 0.012 mmol) was dissolved in DMF (2 mL). Piperidine (100 μL) was added to the solution, followed by stirring at 25° C. for 5 min. The mixture was purified by HPLC and freeze-dried to get CyA with a yield of 88%.
MS of CyA: m/z 643.43. 1H NMR (400 MHz, CDCl3): δ (ppm): 8.82 (d, J=16, 1H), 7.91 (d, J=8, 2H), 7.75 (d, J=8, 2H), 7.71-7.68 (m, 2H), 7.57 (d, J=4, 1H), 7.53-7.48 (m, 3H), 7.15 (d, J=4, 1H), 7.09-7.05 (m, 1H), 6.56 (d, J=12, 1H), 5.27 (s, 2H), 4.51 (t, 1H), 4.40 (t, 1H), 4.12-4.07 (m, 1H), 3.17 (t, 2H), 3.0 (t, 2H), 2.81 (t, 1H), 2.75 (t, 1H), 1.86 (s, 6H), 1.83-1.77 (m, 6H), 1.63 (d, J=8, 3H).
CyA (10 mg, 0.016 mmol), Ac-Y(tBu)VAD(OtBu)—OH (29.8 mg, 0.036 mmol), HBTU (12.1 mg, 0.032 mmol), HOBt (4.3 mmg, 0.032 mmol), and DIPEA (4.1 mg, 0.032 mmol) were dissolved in DMF (5 mL), followed by stirring at 25° C. for 2 h. Then, the reaction mixture was purified by HPLC and freeze-dried to get CyA-D(OtBu)AVY(tBu) with a yield of 91%.
1H NMR (400 MHz, CDCl3): δ (ppm): 8.79 (d, J=16, 1H), 8.21 (t, 1H), 8.15 (d, J=8, 1H), 8.00 (d, J=12, 3H), 7.74-7.72 (m, 2H), 7.63-7.57 (m, 2H), 7.50-7.43 (m, 3H), 7.17 (d, J=8, 1H), 7.06-6.98 (m, 3H), 6.55 (d, J=16, 1H), 5.27 (s, 2H), 4.67-4.60 (m, 1H), 4.47-4.37 (m, 2H), 4.31-4.24 (m, 2H), 4.11-4.08 (m, 2H), 3.46 (t, 2H), 3.19 (s, 2H), 3.09-3.05 (m, 1H), 2.81-2.70 (m, 6H), 2.21 (t, 1H), 2.11-2.03 (m, 2H), 2.01-1.97 (m, 2H), 1.93 (s, 3H), 1.83 (s, 6H), 1.80-1.77 (m, 1H), 1.48 (d, J=8, 3H), 1.39 (s, 9H), 1.35 (d, J=8, 3H), 1.31 (s, 9H), 1.02-0.9 (m, 6H).
CyA-D(OtBu)AVY(tBu) (8 mg) was dissolved in TFA (0.95 mL), followed by adding H2O (0.05 mL). The reaction was stirred at 0° C. and monitored by HPLC. After confirming the whole deprotection of tBu and OtBu group, saturated NaHCO3 aqueous solution was added dropwise to neutralize TFA. Then, the mixture was extracted by DCM. After drying with anhydrous Na2SO4, DCM was removed, and the residue was by purified by HPLC and freeze-dried to get the product CyA-DAVY with a yield of 52%.
MS of CyA-DAVY: m/z 1133.56. 1H NMR (400 MHz, CDCl3): δ (ppm): 8.78 (d, J=12, 1H), 8.21 (t, 1H), 7.95-7.91 (m, 1H), 7.76-7.71 (m, 3H), 7.58-7.55 (m, 2H), 7.50-7.43 (m, 4H), 7.39-7.33 (m, 2H), 7.05 (t, 2H), 6.70 (d, J=8, 1H), 6.54 (d, J=12, 1H), 5.27 (s, 2H), 4.65-4.62 (m, 1H), 4.59-4.55 (m, 1H), 4.47-4.44 (m, 1H), 4.39 (t, 2H), 4.26 (t, 1H), 4.10 (t, 1H), 3.26-3.15 (m, 2H), 3.04-2.99 (m, 1H), 2.94-2.92 (m, 1H), 2.91-2.89 (m, 1H), 2.87 (t, 2H), 2.83-2.77 (m, 2H), 2.76-2.72 (m, 2H), 2.23-2.18 (m, 1H), 2.08-2.05 (m, 2H), 2.0-1.97 (m, 2H), 1.93 (s, 3H), 1.83 (s, 6H), 1.71 (m, 1H), 1.48 (d, J=8, 3H), 1.35 (d, J=8, 3H), 1.02-0.9 (m, 6H).
CyA-DAVY (10 mg, 0.009 mmol), OPD (4.9 mg, 0.09 mmol), HBTU (6.8 mg, 0.018 mmol), HOBt (2.4 mg, 0.018 mmol), and DIPEA (2.3 mg, 0.018 mmol) were dissolved in DMF (3 mL). The reaction mixture was stirred at 25° C. for 0.5 h. Then, the reaction mixture was purified by HPLC and freeze-dried to get TASMRM1 with a yield of 41%.
MS of TASMRM1: m/z 612.89. 1H NMR (400 MHz, CDCl3): δ (ppm): 8.76 (d, J=12, 1H), 8.00 (m, 3H), 7.78-7.72 (m, 3H), 7.64-7.56 (m, 3H), 7.50-7.42 (m, 5H), 7.09-6.98 (m, 4H), 6.70 (d, J=16, 2H), 6.55 (d, J=16, 1H), 5.28 (s, 2H), 4.59-4.55 (m, 1H), 4.41-4.35 (m, 3H), 4.33-4.24 (m, 2H), 4.11 (t, 1H), 3.46 (t, 2H), 3.15-3.08 (m, 2H), 2.95-2.91 (m, 1H), 2.83-2.78 (m, 2H), 2.76-2.72 (m, 3H), 2.65-2.59 (m, 1H), 2.23-2.18 (m, 2H), 2.08-2.03 (m, 4H), 1.93 (s, 3H), 1.83 (s, 6H), 1.46 (d, J=8, 3H), 1.37 (d, J=8, 3H), 1.02-0.9 (m, 6H).
CyA (10 mg, 0.016 mmol), Ac-IE(OtBu)FD(OtBu)—OH (21.7 mg, 0.032 mmol), HBTU (12.1 mg, 0.032 mmol), HOBt (4.3 mmg, 0.032 mmol), and DIPEA (4.1 mg, 0.032 mmol) were dissolved in DMF (5 mL). The reaction mixture was stirred at 25° C. for 2 h. Then, the reaction mixture was purified by HPLC and freeze-dried to get CyA-D(OtBu)FE(OtBu)I with a yield of 91%.
1H NMR (400 MHz, CDCl3): δ (ppm): 8.78 (d, J=16, 1H), 8.36-8.0 (m, 2H), 7.75 (t, 2H), 7.57-7.55 (m, 2H), 7.47 (t, 3H), 7.42 (s, 1H), 7.28-7.22 (m, 5H), 7.08-7.05 (m, 2H), 6.54 (d, J=12, 1H), 5.28 (s, 2H), 4.65 (t, 1H), 4.56-4.53 (m, 1H), 4.43-4.37 (m, 3H), 4.23-4.20 (m, 1H), 4.14-4.11 (m, 1H), 3.47 (t, 2H), 3.25-3.20 (m, 2H), 3.06-3.0 (m, 1H), 2.83-2.72 (m, 7H), 2.25-2.14 (m, 2H), 2.07 (s, 3H), 1.97-1.93 (m, 5H), 1.84-1.76 (m, 8H), 1.48 (s, 3H), 1.41 (s, 18H), 1.3-1.19 (m, 2H), 0.96-0.94 (m, 6H).
CyA-D(OtBu)FE(OtBu)I (6.5 mg) was dissolved in TFA (0.95 mL), followed by adding H2O (0.05 mL). The reaction was stirred at 0° C. and monitored by HPLC. After confirming the whole deprotection of OtBu group, saturated NaHCO3 aqueous solution was added dropwise until neutralizing trifluoroacetic acid. Then, the mixture was extracted by DCM. After drying with anhydrous Na2SO4, DCM was removed, and the residue was by purified by HPLC and freeze-dried to get product TASMRCTL with a yield of 58%.
MS of TASMRCTL: m/z 1189.6. 1H NMR (400 MHz, CDCl3): δ (ppm): 8.77 (d, J=16, 1H), 7.98 (s, 1H), 7.73-7.62 (m, 3H), 7.54-7.51 (m, 2H), 7.47-7.25 (m, 4H), 7.24-7.19 (m, 5H), 7.04-6.96 (m, 2H), 6.52 (d, J=12, 1H), 5.26 (s, 2H), 4.63 (t, 1H), 4.54-4.51 (m, 1H), 4.42-4.34 (m, 2H), 4.31-4.22 (m, 2H), 4.11 (d, J=8, 1H), 3.46 (t, 2H), 3.21-3.13 (m, 2H), 3.03-2.97 (m, 3H), 2.86-2.8 (m, 3H), 2.78-2.74 (m, 1H), 2.73-2.71 (m, 1H), 2.35-2.28 (m, 2H), 2.19 (t, 1H), 2.04 (s, 3H), 1.97-1.93 (m, 3H), 1.84-1.76 (s, 6H), 1.46-1.4 (m, 3H), 1.48 (s, 3H), 1.3-1.19 (m, 2H), 0.96-0.94 (m, 6H).
CyA (10 mg, 0.016 mmol), MeOSuc-AAPV-OH (15.0 mg, 0.032 mmol), HBTU (12.1 mg, 0.032 mmol), HOBt (4.3 mmg, 0.032 mmol), and DIPEA (4.1 mg, 0.032 mmol) were dissolved in DMF (5 mL). The reaction mixture was stirred at 25° C. for 2 h. Then, the reaction mixture was purified by HPLC and freeze-dried to get TASMRNE with a yield of 91%.
1H NMR (400 MHz, CDCl3): δ (ppm): 8.78 (d, J=12, 1H), 7.71-7.67 (m, 4H), 7.51-7.42 (m, 6H), 7.1 (t, 2H), 6.53 (d, J=12, 1H), 5.25 (s, 2H), 4.55-4.26 (m, 7H), 3.71-3.57 (m, 2H), 3.5-3.45 (m, 2H), 3.41 (t, 3H), 2.99-2.72 (m, 4H), 2.6-2.5 (m, 4H), 2.22-2.1 (m, 3H), 2.0-1.95 (m, 5H), 1.82 (s, 6H), 1.45-1.4 (m, 2H), 1.35-1.27 (m, 9H), 0.96-0.94 (m, 6H).
Synthesis of TAMRM1, TAMRCTL and TAMRNE
TASMRM1 (12 mg, 0.01 mmol) or TASMRCTL (12 mg, 0.01 mmol) or TASMRNE (11 mg, 0.01 mmol) and PVP-alkyne (30 mg, 0.01 mmol) were dissolved in DMSO (1.5 mL). Then, CuSO4·5H2O (2.5 mg, 0.01 mmol) and sodium ascorbate (4 mg, 0.02 mmol) in deionized water (1.5 mL) were added. The reaction mixture was stirred at 25° C. for 12 h. Then, the reaction solution was dialyzed against deionized water for 24 h to remove salts and DMSO, and freeze-dried to get TAMRM1, TAMRCTL and TAMRNE with yields of 77%, 92% and 88%, respectively.
TAMRM1. 1H NMR (400 MHz, CDCl3): δ (ppm): 8.76 (d, J=12, 1H), 8.00 (m, 3H), 7.82 (s, 1H), 7.78-7.72 (m, 3H), 7.64-7.56 (m, 3H), 7.50-7.42 (m, 5H), 7.09-6.98 (m, 4H), 6.70 (d, J=16, 2H), 6.55 (d, J=16, 1H), 5.28 (s, 2H), 4.59-4.55 (m, 1H), 4.41-4.35 (m, 3H), 4.33-4.24 (m, 2H), 4.15 (s, 2H), 4.11 (t, 1H), 3.92-3.74 (m, 32H), 3.46 (t, 2H), 3.29 (m, 54H), 3.15-3.08 (m, 2H), 2.95-2.91 (m, 1H), 2.83-2.78 (m, 2H), 2.76-2.72 (m, 3H), 2.65-2.59 (m, 1H), 2.38-2.27 (m, 54H), 2.23-2.18 (m, 2H), 2.08-2.03 (m, 58H), 1.93 (s, 3H), 1.83 (s, 6H), 1.75-1.46 (m, 60H), 1.45 (d, J=8, 3H), 1.37 (d, J=8, 3H), 1.02-0.9 (m, 6H).
TAMRCTL. 1H NMR (400 MHz, CDCl3): δ (ppm): 8.77 (d, J=16, 1H), 7.98 (s, 1H), 7.82 (s, 1H), 7.73-7.62 (m, 3H), 7.54-7.51 (m, 2H), 7.47-7.25 (m, 4H), 7.24-7.19 (m, 5H), 7.04-6.96 (m, 2H), 6.52 (d, J=12, 1H), 5.26 (s, 2H), 4.63 (t, 1H), 4.54-4.51 (m, 1H), 4.42-4.34 (m, 2H), 4.31-4.22 (m, 2H), 4.15 (s, 2H), 4.11 (d, J=8, 1H), 3.92-3.74 (m, 32H), 3.46 (t, 2H), 3.29 (m, 54H), 3.21-3.13 (m, 2H), 3.03-2.97 (m, 3H), 2.86-2.8 (m, 3H), 2.78-2.74 (m, 1H), 2.73-2.71 (m, 1H), 2.35-2.28 (m, 56H), 2.19 (t, 1H), 2.06-2.01 (m, 57H), 1.97-1.93 (m, 3H), 1.84-1.76 (s, 6H), 1.75-1.46 (m, 60H), 1.46-1.4 (m, 3H), 1.48 (s, 3H), 1.3-1.19 (m, 2H), 0.96-0.94 (m, 6H).
TAMRNE. 1H NMR (400 MHz, CDCl3): δ (ppm): 8.78 (d, J=12, 1H), 7.82 (s, 1H), 7.71-7.67 (m, 4H), 7.51-7.42 (m, 6H), 7.1 (t, 2H), 6.53 (d, J=12, 1H), 5.25 (s, 2H), 4.55-4.26 (m, 7H), 4.15 (s, 2H), 3.92-3.74 (m, 32H), 3.71-3.57 (m, 2H), 3.5-3.45 (m, 2H), 3.41 (t, 3H), 3.29 (m, 54H), 2.99-2.72 (m, 4H), 2.6-2.5 (m, 4H), 2.38-2.27 (m, 54H), 2.22-2.1 (m, 3H), 2.06-2.01 (m, 54H), 2.0-1.95 (m, 5H), 1.82 (s, 6H), 1.75-1.46 (m, 60H), 1.45-1.4 (m, 2H), 1.35-1.27 (m, 9H), 0.96-0.94 (m, 6H).
CyOHN3 (6 mg, 0.01 mmol) and PEG-alkyne (20 mg, 0.01 mmol) were dissolved in DMSO (1.5 mL). Then, CuSO4·5H2O (2.5 mg, 0.01 mmol) and sodium ascorbate (4 mg, 0.02 mmol) in deionized water (1.5 mL) were added. The reaction mixture was stirred at 25° C. for 12 h. Then the reaction solution was dialyzed against deionized water for 24 h to remove salts and DMSO, and freeze-dried to get PEG-Cy.
CyOHN3 (6 mg, 0.01 mmol) and PVP-alkyne (30 mg, 0.01 mmol) were dissolved in DMSO (1.5 mL). Then, CuSO4·5H2O (2.5 mg, 0.01 mmol) and sodium ascorbate (4 mg, 0.02 mmol) in deionized water (1.5 mL) were added. The reaction mixture was stirred at 25° C. for 12 h. Then the reaction solution was dialyzed against deionized water for 24 h to remove salts and DMSO, and freeze-dried to get PVP-Cy.
Mice were intravenously injected with the uncaged PEG-Cy (5 μmol/kg) and PVP-Cy (5 μmol/kg). Blood samples of PEG-Cy injected mice were collected using heparinized capillary tubes at 1, 4, 7, 11, 16, 25, 35, 55, 75, 95, 120 and 150 min post-injection of PEG-Cy. Blood samples of PVP-Cy injected mice were collected using heparinized capillary tubes at 1, 4, 7, 11, 16, 25, 35, 55, 75, 95, 120, 150 and 180 min post-injection of PVP-Cy. The blood samples in heparinized capillary tubes were then centrifuged at 3500 rpm for 10 min, followed by quantification with HPLC.
PVP was selected as the tumor-passive targeting moiety because we confirmed that it had higher tumor accumulation efficiency (1.5-fold), and longer circulatory half-life (4.4-fold) in comparison to PEG (
TAMRs and TASMRs prepared in Example 1 were characterised.
TAMRs (25 μM) were incubated with their respective leukocyte biomarkers or combination of both tumor and respective leukocyte biomarkers (50 μM DEA NONOate, 1 U Cas-1, 0.5 μg APN, 0.5 μg GrB, 2.5 mU NE) in respective buffers at 37° C. for 2 h. For TAMRM1, the enzymatic experiment was conducted in Cas-1 HEPES buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 0.1% CHAPS, 5% glycerol, 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT)). For TAMRCTL, the enzymatic experiment was conducted in tris buffer (100 mM tris, pH 7.5, 150 mM NaCl). GrB (0.5 μg) was first activated by cathepsin C (0.2 μg) in MES buffer (50 mM MES, pH 5.5, 50 mM NaCl) for 4 h. For TAMRNE, the enzymatic experiment was conducted in NE tris buffer (50 mM Tris, 1 M NaCl, 0.05% (w/v) brij-35, pH 7.5). After completion, UV-Vis absorption and fluorescence spectra of the enzymatic solutions were recorded.
TAMRs or TASMRs (25 μM) were incubated with various enzymes including uPA (0.5 μg), NTR (0.5 μg), GGT (0.5 μg), FAP (0.2 mU), caspase-3 (0.5 μg), CTSS (0.5 μg), GrB (0.5 μg), NE (0.5 mU), APN (0.5 μg), Cas-1 (1 U) and cathepsin C (0.5 μg), and combination of some of these enzymes in their respective buffers at 37° C. for 2 h. For NTR, GGT and caspase-3, the enzymatic experiments were conducted in PBS (10 mM, pH 7.4) buffer. For FAP, the enzymatic experiments were conducted in HEPES buffer (50 mM HEPES, pH 7.4, 0.1% bovine serum albumin (BSA), 5% glycerol). For cathepsin S, the enzymatic experiments were conducted in NaOAc buffer (NaOAc 50 mM, pH 5.5, 5 mM DTT, 250 mM NaCl). For APN, the enzymatic experiments were conducted in tris buffer (50 mM tris, pH 7.0). After completion, the fluorescence intensities of enzymatic solutions were measured by IVIS spectrum imaging system. Excitation: 675 nm. Emission: 720 nm.
Various concentrations of TASMRM1 (2, 4, 8, 20, 40, 80, 160 or 200 μM) were incubated with Cas-1 (0.5 U) at 37° C. for 1 h in HEPES buffer (50 mM HEPES, pH=7.2, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, 10 mM DTT). Various concentrations of TASMRCTL (5, 10, 20, 40, 80, or 120 μM) were incubated with GrB (0.25 μg) at 37° C. for 30 min in tris buffer (100 mM tris, pH 7.5, 150 mM NaCl). Various concentrations of TASMRNE (5, 10, 20, 40, 80, 120, 160 or 200 μM) were incubated with NE (2.5 mU) at 37° C. for 4 min in tris buffer (50 mM Tris, 1 M NaCl, 0.05% (w/v) brij-35, pH 7.5). Various concentrations of CyA (10, 20, 40, 80, 120 or 200 μM) were incubated with APN (0.25 μg) at 37° C. for 20 min in tris buffer (50 mM tris, pH 7.0). After incubation, the mixture was measured by HPLC. The enzymatic reaction velocity (nmol/min or pmol/s) was calculated, plotted as a function of TASMRs or CyA concentrations, and fitted the Michaelis-Menten equation: V=Vmax*[S]/(Km+[S]), where Vmax indicates the maximum theoretical reaction rate, [S] indicates the substrate concentration, and Km indicates the Michaelis constant.
TAMRs(M1, CTL, NE) exhibited similar optical properties with absorption peaks at˜610 and 660 nm, respectively (
HPLC analysis was applied to investigate the structural changes of TASMRs (TAMR precursors) in response to their respective biomarkers. TASMRs instead of TAMRs were used for the study because HPLC traces of TAMRs remained similar before and after enzymatic activation due to the existence of PVP that dominated the elution property. Incubation of TASMRs with both cancer and leukocyte biomarkers resulted in the appearance of the elution peak assigned to CyOH (TR=17.8 min), which was undetectable after incubation with either single biomarker (
The capabilities of TAMRs (prepared in Example 1) to detect TILs were tested against respective leukocytes in the presence or absence of cancer biomarker (APN, Pasqualini, R. et al., Cancer Res. 2000, 60, 722-727).
4T1 cells, CT26 cells, M1 macrophages, CD8 T cells and MPRO cells were placed in 24-well plates with 8000 cells per well, and cultured for 24 h, followed by incubation with TAMRs at a final concentration from 12.5 to 100 μg/mL for 24 h. The cells in each well were collected and centrifuged to remove TAMRs, and resuspended in MTS solution (200 μL, 0.1 mg/mL), and incubated for another 4 h at 37° C. The absorbance of MTS solution was measured via a microplate reader (SpectraMax M5 microplate reader) at 490 nm. Cell viability was calculated by the ratio of the absorbance of the cells incubated with TAMRs to that of the respective control cells.
For cellular imaging of TAMRs, 4T1 cells, CT26 cells, 3T3 cells, M1 macrophages, M2 macrophages, RAW264.7 cells, BMDCs, CD8 T cells and MPRO cells were seeded into confocal cell culture dishes (5×104 cells/dish). After 24 h incubation, the cells were replaced with fresh medium containing TAMRs (10 μM) in the absence or presence of APN (0.5 μg). After incubation for 2 h, cells were washed with PBS for three times, followed by fixation with 4% paraformaldehyde (PFA) for 20 min. Then, the cells were stained with DAPI for the nucleus. Fluorescence images of cells were taken via LSM800 (Zeiss). The fluorescence intensity was quantified by Image J.
Note that all the TAMRs showed negligible cytotoxicity against selective cells in TIME (
AMRs precursors (Cy-DAVY, Cy-DFEI and Cy-VPAA) were synthesized according to previous protocols (He, S. et al., J. Am. Chem. Soc. 2020, 142, 7075-7082). AMRs were synthesized by following the protocol for TAMRs in Example 1.
MS of Cy-DAVY: m/z 1062.61. 1H NMR (400 MHz, CDCl3): δ (ppm): 8.77 (d, J=14, 1H), 7.76-7.71 (m, 3H), 7.58-7.55 (m, 2H), 7.50-7.41 (m, 4H), 7.39-7.33 (m, 1H), 7.06 (d, J=8, 4H), 6.70 (d, J=8, 2H), 6.53 (d, J=12, 1H), 5.25 (s, 2H), 4.76 (t, 1H), 4.59-4.55 (m, 1H), 4.36 (t, 2H), 4.27-4.24 (m, 1H), 4.10 (t, 1H), 3.46 (t, 2H), 3.28-3.10 (m, 1H), 3.05-3.0 (m, 1H), 2.96 (t, 1H), 2.91-2.85 (m, 2H), 2.79-2.72 (m, 2H), 2.74-2.71 (m, 2H), 2.08-2.03 (m, 2H), 2.0-1.97 (m, 3H), 1.91 (s, 3H), 1.83 (s, 6H), 1.71 (m, 1H), 1.36 (d, J=8, 3H), 1.02-0.9 (m, 6H).
MS of Cy-DFEI: m/z 1118.58. 1H NMR (400 MHz, CDCl3) of Cy-DFEI: δ (ppm): 8.76 (d, J=16, 1H), 7.75-7.72 (m, 3H), 7.56 (d, J=4, 2H), 7.50-7.47 (m, 4H), 7.41 (s, 1H), 7.23-7.16 (m, 4H), 7.12-7.07 (m, 3H), 6.53 (d, J=16, 1H), 5.28 (s, 2H), 4.8 (t, 1H), 4.54-4.51 (m, 1H), 4.38 (t, 2H), 4.26-4.23 (m, 1H), 4.13 (d, J=8, 1H), 3.48 (t, 2H), 3.37 (s, 2H), 3.20-3.15 (m, 1H), 3.06-2.96 (m, 2H), 2.79-2.73 (m, 5H), 2.34-2.23 (m, 2H), 2.04 (s, 3H), 1.97-1.93 (m, 4H), 1.84-1.76 (s, 6H), 1.46-1.4 (m, 3H), 1.3-1.19 (m, 2H), 0.90-0.88 (m, 6H).
1H NMR (400 MHz, CDCl3): δ (ppm): 8.80 (d, J=16, 1H), 7.75-7.73 (m, 2H), 7.64-7.62 (m, 2H), 7.57-7.52 (m, 2H), 7.49-7.43 (m, 2H), 7.35-7.27 (m, 2H), 7.13-7.06 (m, 2H), 6.55 (d, J=16, 1H), 5.25 (s, 2H), 4.39 (t, 1), 4.29 (t, 3H), 4.23-4.17 (m, 2H), 3.83-3.81 (m, 2H), 3.67-3.62 (m, 2H), 3.54-3.46 (m, 2H), 3.24-3.15 (m, 1H), 2.82 (s, 2H), 2.75 (t, 1H), 2.72 (s, 2H), 2.68-2.65 (m, 2H), 2.53 (d, J=8, 1H), 2.06-1.94 (m, 4H), 1.66-1.59 (m, 2H), 1.82 (s, 6H), 1.45-1.4 (m, 2H), 1.36-1.34 (m, 6H), 1.22-1.15 (m, 2H), 0.96-0.94 (m, 6H).
The development of imaging agents with high specificity for TILs remains challenging because leukocytes exist in peripheral blood and inflamed tissues (Nourshargh, S. & Alon, R., Immunity 2014, 41, 694-707). The specificity of TAMRs (prepared in Example 1) towards TILs was investigated and compared with their single-lock counterparts (AMRs, prepared in Example 4) (
LPS (5.0 mg/kg) was intraperitoneally injected into living mice, and the LPS-inflamed blood samples were collected 4 h post-injection of LPS. TAMRs and AMRs (10 μM) were respectively incubated with LPS-inflamed and saline-treated blood samples for 30 min. Then, the blood samples were homogenized and centrifuged for 10 min. The supernatants were used for absorption analysis.
For blood incubation experiments, blood samples were first obtained from BALB/c mice. Briefly, saline or LPS (5 mg/kg mice) was intraperitoneally injected into living mice. After 4 h, the blood samples were collected. TAMRs (final concentration: 10 μM) were added into saline-treated and LPS-inflamed blood samples (50 μL). After 30 min, fluorescence images of blood samples were acquired by IVIS spectrum imaging system. Excitation: 675 nm. Emission: 720 nm.
For in vivo specificity detection, living mice bearing LPS-inflamed tissue in the left thigh muscle and subcutaneous CT26 tumor on the right flank were first built. Briefly, CT26 tumors were first inoculated in the right flank. 7 days later, LPS (5 mg/kg) were injected into the left thigh muscle. 24 h later, 10 μL of TAMRs or AMRs (0.1 mM) were locally injected into both LPS-inflamed tissues and CT26 tumors for longitudinal NIRF imaging. NIRF images were acquired by IVIS spectrum imaging system. Excitation: 675 nm. Emission: 720 nm.
The fundamental challenge in molecular imaging of TILs lies in the lack of probe design to distinguish TILs from resident leukocytes in other organs. This challenge is tackled by our dual-locked tandem molecular design that simultaneously incorporate both disease-site and biomarker specificities into signal activation of probes. The dual-locked TAMRs only triggered their fluorescence in the presence of both cancer and leukocyte biomarkers, and thus specifically detected TILs with no false positives from other leukocytes in LPS-induced inflammation; in contrast, their single-lock counterparts failed to do so (
In comparison to their pure forms in PBS solution, TAMRs exhibited almost identical NIRF signals in the saline-treated blood samples, and slightly increased signals in LPS-inflamed blood samples (1.1 to 1.2-fold). In contrast, AMRs showed 1.8 to 3.6-fold, and 2.8 to 4.9-fold higher signals in saline-treated and LPS-inflamed blood samples relative to their NIRF signals in PBS, respectively (
The specificity of TAMRs towards TILs was further verified in living mice bearing LPS-inflamed tissue in the left thigh muscle (Ning, X. et al., Nat. Mater. 2011, 10, 602-607) and subcutaneous CT26 tumor on the right flank (
To determine the clearance pathway and in vivo stability of TAMRs, urine of healthy mice injected with TAMRs (prepared in Example 1) or AMRs (prepared in Example 4) was collected and quantified with HPLC (
Healthy mice were intravenously injected with TAMRM1, TAMRCTL, or TAMRNE (5 μmol/kg) and their single-lock counterparts AMRM1, AMRCTL, or AMRNE (5 μmol/kg), and placed into metabolic cages. Urine samples were collected at 3, 9, and 24 h post-injection of these reporters. The renal clearance of these reporters was examined by HPLC analysis of the urine samples. The urine samples were also centrifuged at 5000 rpm for 15 min, and the UV-Vis absorption spectra of the supernatants were further recorded.
UV-Vis Analysis of Urine Collected from Healthy Mice
Healthy mice were intravenously injected with TAMRs (5 μmol/kg) or AMRs (5 μmol/kg), and placed into metabolic cages. At 12 post-injection timepoint, the urine samples were collected and centrifuged at 5000 rpm for 15 min, and the UV-Vis absorption spectra of the supernatants were further recorded (n=3).
Due to their high-water solubility, the renal clearance efficiency of these reporters at 24 h post-injection was 55-71% of the total injection dosage (
Due to the high renal clearance of TAMRs, they can be applied for fluorescence urinalysis of TIME for evaluation of cancer immunotherapy, showing great potential for clinical translation. We report real-time NIRF imaging of TILs using TAMRs for companion diagnosis and prediction of cancer immunotherapy in the following examples.
The capabilities of TAMRs (prepared in Example 1) for in vivo real-time imaging of TILs were first validated against mice bearing 4T1 tumor, which has been considered as a poorly immunogenic tumor as it shows a lower presence of tumor-suppressive leukocytes (Taylor, M. A. et al., J. Immunother. Cancer 2019, 7, 328). Mice were administrated with aPD-L1, Oxa or the combination of aPD-L1 and Oxa (aPD-L1/Oxa) intraperitoneally (
After tumor inoculation for one week, 4T1 tumor-bearing mice were administrated with aPD-L1 (10 mg/kg), Oxa (6 mg/kg) or the combination of aPD-L1 and Oxa (aPD-L1/Oxa) intraperitoneally every two days for three times. CT26 tumor-bearing mice were administrated with aPD-L1 (10 mg/kg), aCD47 (10 mg/kg) or the combination of aPD-L1 and aCD47 (aPD-L1/aCD47) intraperitoneally every two days for three times. At day 7, tumor penetration reference reporter PVP-IR800 (3 μmol/kg) and TAMRs including TAMRM1, TAMRCTL and TAMRNE (5 μmol/kg) were i.v. injected, and real-time NIRF imaging was monitored for 48 h. For APN inhibition group, bestatin (APN inhibitor, 10 mg/mL, 10 μL) was injected intratumorally 2 h before i.v. injection of TAMRCTL. Fluorescence imaging was taken with excitation at 675 nm and emission at 720 nm for monitoring of activated TAMRs, and excitation at 745 nm and emission at 800 nm for monitoring of IR800.
At 72 h post-injection of PVP-IR800 (3 μmol/kg) and TAMRs (5 μmol/kg), mice were euthanized and major tissues including heart, liver, spleen, lung, kidneys, and tumor were collected and captured by IVIS spectrum imaging system with excitation at 675 nm and emission at 720 nm for monitoring of activated TAMRs, and excitation at 745 nm and emission at 800 nm for monitoring of IR800.
After ex vivo imaging, the major organs were suspended in PBS, homogenized, and centrifuged (10000 rpm, 10 min) to remove insoluble components. The supernatant containing extracted reporters was analyzed by HPLC assay to present the distribution of TAMRs.
At 24 h post-injection of TAMRs (5 μmol/kg), mice were euthanized, and tumors were collected, followed by fixation in 4% PFA. After dehydration with 30% sucrose solution, tumor tissues were embedded in O.C.T. medium for 10 min, followed by cutting into 10-μm sections using a cryostat (Leica, CM1950). Tumor sections were washed with PBS containing 0.1% triton X-100 (PBST), followed by incubation with 3% BSA solution at 25° C. for 2 h to block non-specific binding of antibodies. Tumor sections were stained with Alexa Fluor® 488 anti-F4/80 (C-7), PE anti-mouse CD8a, and NE polyclonal antibody for TAMRM1, TAMRCTL and TAMRNE groups, respectively. For TAMRNE group, the tumor sections were further stained with secondary antibody Alexa Fluor® 488 conjugated goat anti-rabbit IgG. Finally, tumors sections were stained with DAPI for the nucleus. The fluorescence images of tumor sections were captured on LSM800 (Zeiss). Finally, the colocalization of NIRF signals of TAMRs with their respective leukocytes was analyzed using ImageJ software.
After three times of immunotherapy, at day 8, both 4T1 and CT26 tumor-bearing mice in each group were euthanized, and tumors, lymph nodes, and blood cells were harvested to prepare single cell suspension. For evaluation of TILs, tumor tissues were cut into small pieces and digested at 37° C. for 4 h in RPMI 1640 containing type I collagenase (1 mg/mL), type IV collagenase (100 μg/mL), and DNase I (100 μg/mL). Then, the mixture was filtered through a 70 μm cell strainer. For evaluation of leukocytes in lymph node and blood cells, cells of lymph nodes and blood cells were treated with ACK lysis to remove red blood cells. All the single cell suspensions were first blocked with anti-mouse CD16/32, followed by live/dead staining. For CTLs analysis, the cells were stained with Alexa Fluor® 700 anti-mouse CD45, FITC anti-mouse CD3 and PE anti-mouse CD8a for 30 min at 4° C., followed by fixation in 4% PFA in the dark for 20 min at 25° C. Then, cells were resuspended in ISPWB and incubated with APC anti-human/mouse GrB recombinant antibody for 1 h at 25° C., followed by washing with ISPWB for three times. For M1 macrophages analysis, the cells were stained with Alexa Fluor® 700 anti-mouse CD45, PerCP anti-mouse/human CD11 b and Alexa Fluor® 488 anti-F4/80 (C-7) for 30 min at 4° C., followed by fixation in 4% PFA in the dark for 20 min at 25° C. Thereafter, cells were resuspended in ISPWB and incubated with PE anti-mouse NOS2 and Alexa Fluor® 647 anti-Cas-1 (D-3) for 1 h at 25° C., followed by washing with ISPWB for three times. For neutrophils analysis, the cells were stained with Alexa Fluor® 700 anti-mouse CD45, PE anti-mouse/human CD11b and APC anti-mouse Ly-6G for 30 min at 4° C., followed by fixation in 4% PFA in the dark for 20 min at 25° C. Thereafter, cells were resuspended in ISPWB and incubated with NE polyclonal antibody for 1 h at 25° C., followed by washing with ISPWB for three times. The cells were then incubated with secondary antibody Alexa Fluor® 488 conjugated goat anti-rabbit IgG for 1 h at 25° C., followed by washing with ISPWB for three times. The final cells were analyzed with Fortessa X20 (BD Biosciences).
After three times of immunotherapies, TAMRs and the “always-on” reference reporter (PVP-IR800) were intravenously co-injected into 4T1 tumor-bearing mice for longitudinal NIRF imaging (
The immunofluorescence staining showed that greater than 75% of red signals of TAMRs well overlapped with green signals of TILs labeled with FITC-tagged antibodies (
The relationship between TAMRs and TILs was further evaluated with a simple linear regression model. A positive correlation was found between R-NIRFM1 and levels of iNOS+Cas-1+ cells, with a correlation coefficient (R) of 0.75, and a Pearson's r value (p) of 0.82. R-NIRFCTL and R-NIRFNE were positively correlated with the levels of CD8*GrB+ cells and Ly-6G+NE+ cells (RCTL=0.86, ρCTL=0.93, RNE=0.90 and ρNE=0.95), respectively (
The capabilities of TAMRs (prepared in Example 1) for in vivo real-time imaging of TILs were further evaluated against mice bearing CT26 tumor model, which is considered as a highly immunogenic tumor as it shows a higher presence of tumor-suppressive leukocytes (Taylor, M. A. et al., J. Immunother. Cancer 2019, 7, 328). Mice were administrated with aPD-L1, anti-Cluster of Differentiation 47 (aCD47) or the combination of aPD-L1 and aCD47 (aPD-L1/aCD47) intraperitoneally, followed by systemic administration of TAMRs and PVP-IR800 (
After three times of immunotherapies, TAMRs and the “always-on” reference probe (PVP-IR800) were intravenously co-injected into living mice for longitudinal NIRF imaging (
The immunofluorescence staining of tumor sections showed that red signals of TAMRs overlapped well with green signals of TILs labeled with FITC-tagged antibodies (
The relationship between TAMRs and TILs was further evaluated with a simple linear regression model. R-NIRFM1 positively correlated with the levels of iNOS+Cas-1+ cells in the tumor regions (RM1=0.84, ρM1=0.91). R-NIRFCTL and R-NIRFNE were also positively correlated with the levels of CD8+GrB+ cells and Ly-6G+NE+ cells, respectively (RCTL=0.85, ρCTL=0.92, RNE=0.88, ρNE=0.94) (
Therefore, this array of TAMRs enabled real-time multiplex profiling of TILs in TIME, providing a non-invasive way to accurately map out the intertumoral immune contexture. Moreover, TAMRs had high specificity and sensitivity towards TILs, showing over 70% overlap with their respective TILs in immunofluorescence staining of tumor slices (
The therapeutic efficacies of various treatments against both 4T1 and CT26 tumor-bearing mice were evaluated.
For urinalysis, both 4T1 and CT26 tumor-bearing mice treated with different immunotherapeutic were intravenously injected with PVP-IR800 (3 μmol/kg mice) and TAMRs (prepared in Example 1, 5 μmol/kg mice), and placed into metabolic cages. At 12 post-injection timepoint, the urine samples were collected and imaged by IVIS spectrum imaging system with excitation at 675 nm and emission at 720 nm for monitoring of activated TAMRs, and excitation at 745 nm and emission at 800 nm for monitoring of IR800.
The tumor control rate was calculated with the following formulas:
Vt0 means the volume of tumor on day 0, Vt means the volume at certain time, c means the control group (saline, without irradiation), and s means the group needed calculation.
Both 4T1 and CT26 tumor-bearing mice treated with different treatments were intravenously injected with TAMRs (prepared in Example 1, 5 μmol/kg). After 24 h post-injection, mice were euthanized, and tumors were collected, followed by fixation in 4% PFA. After being dehydrated with 30% sucrose, tumor tissues were embedded in O.C.T. medium and cut into 10-μm sections. Tumor sections were washed with PBST, following by staining the nucleus with DAPI. The fluorescence images of the tumor sections were captured on LSM800 (Zeiss) using the tiles mode.
We have inputted in vivo fluorescence imaging data (R-NIRF) at 24 h (n=3) and 48 h (n=3) of each group into GraphPad data table, and plotted it using ROC cure model in Column analyses of GraphPad. Combination of multiple TAMRs was derived from logistic regression of R-NIRFs of TAMRM1, TAMRCTL and TAMRNE.
We have inputted in vivo fluorescence imaging data at 24 h (n=3) and 48 h (n=3), and urinary data at 12 h (n=3) of each group into origin workbook, and created PCA figure using an enhanced version of Principal Component Analysis tool of origin.
For 4T1 tumor-bearing mice, the highest tumor control rate (TCR) (85%) was observed for aPD-L1/Oxa treatment, which was 9.9- and 1.8- and fold higher than that of aPD-L1 (8.6%) and Oxa (48%) treatment, respectively (
The high therapeutic efficacy of aPD-L1/aCD47 treatment was attributed to the synergetic effect: aCD47 inhibited CD47-SIRPa interaction, and promoted phagocytosis of apoptotic cells by macrophages, which further enhanced priming of CTLs. Concurrently, aPD-L1 favored the cytotoxicity of CTLs, and provided an immunostimulatory microenvironment which suppressed the functions of immunosuppressive neutrophils, resulting in an enhanced anticancer efficacy (
High renal clearance efficiency and fluorescence turn-on response of TAMRs provide a convenient way for fluorescence urinalysis of TILs, making TAMRs highly promising for clinical translation. To evaluate the potential of TAMRs for urinalysis, urine samples of mice post-injection of reporters were collected for NIRF measurement. In general, the urinary signals coincided well with the real-time imaging data: urinary R-NIRFM1 and R-NIRFCTL were the highest while R-NIRFNE was the lowest for aPD-L1/Oxa treated 4T1 tumor-bearing mice; urinary R-NIRFM1 and R-NIRFCTL were the highest while R-NIRFNE was the lowest for aPD-L1/aCD47 treated CT26 tumor-bearing mice. (
To determine whether the urinary R-NIRFs at day 7 could predict the therapeutic outcomes at day 20, the correlation analysis between R-NIRFs and the relative tumor volumes was performed. Consistent with the in vivo ratiometric imaging data (
Because urinalysis was performed at day 7 which was two weeks earlier than the treatment endpoint, urinary R-NIRFs of TAMRs served as a non-invasive and accurate way to predict therapeutic outcomes. In addition, comparison of the urinary R-NIRFs for different tumors revealed that positive prognostic TAMRM1 and TAMRCTL exhibited 1.3- and 1.5-fold higher R-NIRFs in CT26 tumors than that in 4T1 tumors, respectively. Thus, TAMRM1 and TAMRCTL clearly distinguished poorly immunogenic tumor (4T1) from highly immunogenic tumor (CT26), showing their stratification capability.
The ability of TAMRs in companion diagnosis and prediction of cancer immunotherapy was further evaluated by PCA. PCA of R-NIRFs of TAMRs distinctly separated untreated 4T1 and CT26 tumors (
TAMRs could also be applied for the microscopic examination of whole-tumor sections, which is one of clinical approaches for evaluation of TILs (
Thus, in addition to urinalysis, TAMRs was able to delineate the spatial distribution of TILs in whole-tumor sections via microscopic examination, serving as another clinical utility for stratification and evaluation of cancer therapy. TAMRs staining revealed the changes in the location and density of TILs after therapy, showing more positive prognostic TILs (M1 macrophages and CTLs) and less negative prognostic TILs (neutrophils) distributed in the center of tumor, and 1.5 to 2.0-fold higher levels of M1 macrophages, 1.6 to 1.9-fold higher levels of CTLs and 3.1 to 4.5-fold lower levels of neutrophils than untreated tumors (
In summary, we developed an unprecedented set of TILs-specific molecular fluorescence reporters (TAMRs) for companion diagnosis and prognosis of cancer immunotherapy. TAMRs have a unique dual-locked sensing mechanism, permitting specific fluorescence correlation with TILs. TAMR-based real-time imaging and urinalysis are non-invasive and dynamic but competent to profile multiple TILs with the sensitivity and specificity at the level equal to static flow cytometry analysis and invasive biopsy. The signal correlation of TAMRs allows for accurate analyses of tumor immunogenicity and longitudinal monitoring of changes in TIME.
Thus, TAMRs not only present a high-throughput, non-invasive, and effective way to screen combinational immunotherapeutic agents in preclinical settings, but also hold the potential in clinical settings to stratify patients for personalized combinational cancer immunotherapy, optimize immunotherapeutic intervention, and predict immunotherapeutic outcome. The modular dual-locked tandem design of TAMRs can be generalized for specific detection of biomarkers from the targeted cell at targeted disease site, advancing the way for precision biomarker profiling using molecular probes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202113855P | Dec 2021 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2022/050903 | 12/13/2022 | WO |
