pHLIP®-Mediated Delivery of STING Agonists

Abstract

Disclosed herein are compositions comprising pHLIP® peptides linked to STING agonists, pharmaceutical compositions thereof, and methods of treatment using such compositions. TIFF2025527495000068.tif75128

Description

[Technical Field]

[0001] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. R01 GM073857 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0002] FIELD OF THE INVENTION Disclosed herein are compositions comprising pHLIP® peptides linked to STING agonists, pharmaceutical compositions thereof, and methods of treatment using such compositions. [Background technology]

[0003] background Conventional cancer treatments use toxic agents that do not differentiate well between normal and tumor tissue, limiting their use due to adverse side effects. Recent research in cancer immunotherapy has demonstrated that enhancing cancer patients' innate immune systems, alone or in combination with conventional treatments, may have a decisive impact on cancer treatment outcomes. Stimulator of interferon genes (STING) agonists target tumor cells within the cytosol, such as cancer cells and activated myeloid cells within the tumor microenvironment. STING agonists (STINGa) stimulate immune responses, but specific and efficient targeting of tumor cells is required to avoid adverse side effects. Furthermore, there are significant advantages to delivering STING agonists to activated immune cells within tumors, leading to stronger activation of the STING pathway and inflammatory responses.

[0004] However, one obstacle to the use of small molecule STINGα is their poor pharmacokinetics. Another limitation is that some small molecule STINGα molecules are polar molecules and tend not to efficiently cross cell membranes. For example, STING agonists such as cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) are hydrophilic, negatively charged molecules. Because the cell membrane is a nonpolar, hydrophobic environment, such molecules are associated with low membrane permeability.

[0005] Therefore, an ongoing challenge in the field of immunotherapy is to find ways to improve the pharmacokinetics of the small molecule STINGa, delivering STING agonists specifically into the cytosol of tumor cells, specifically activated immune cells and potentially stromal cells, while avoiding delivery to healthy cells. Summary of the Invention

[0006] As used herein, the following structure: Peptide-Linker-STING A composition is described comprising: Peptide is pHLIP® peptide; Linker is a cleavable linker; "STING" is diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, S NZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, TTI-10001, CRD 5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511, and derivatives thereof; and Each "-" is a covalent bond.

[0007] In some situations, the pHLIP® peptide has the following sequence: X n Y m ; Y m X n ; X n Y m X j ; Y m X n Y i ; Y m X n [[ID= 26]]Y i X j ; X n Y m X j Y i ; Y m X n Y i X j Y l ; X n Y m X j Y i X l ; Y m X n Y i X j Y l X h ; X n Y m X j Y i X h Y g ; Y m X n Y i X j Y l X h Y g ; X n Y m X j Y i X h Y g X f ; (XY) n ; (YX) n ; (XY) n Y m ; (YX) n Y m ; (XY) n X m ; (YX) n Xm ; Y m (XY) n ; Y m (YX) n ; X n (XY) m ; X n (YX) m ; (XY) n Y m (XY) i ; (YX) n Y m (YX) i ; (XY) n X m (XY) i ; (YX) n X m (YX) i ; Y m (XY) n ; Y m (YX) n ; X n (XY) m ; or X n (YX) m and (i) Each Y is independently a solvation energy DG X cor a nonpolar amino acid with a RI >+0.50, or glycine (Gly); (ii) each X is independently a protonatable amino acid; (iii) n, m, i, j, l, h, g, and f each independently represent an integer of 1 to 8.

[0008] In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000002.tif4128, where "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), an azide-containing amino acid, or other modified amino acid. In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000003.tif4128, where "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids.

[0009] In some aspects, the pHLIP® peptide comprises a sequence selected from the group consisting of any one of SEQ ID NOs. 1-219. In some aspects, the pHLIP® peptide has a sequence selected from the group consisting of any one of SEQ ID NOs. 1-219. In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000004.tif4128. In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000005.tif4128. In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000006.tif4128. In some aspects, the pHLIP® peptide has the sequence: It has a sequence consisting of TIFF2025527495000007.tif4128.

[0010] A composition comprising the following structure: Peptide-Linker-STING where: The peptide has the sequence: pHLIP® peptide containing TIFF2025527495000008.tif12128; Linker is a cleavable linker; STING is a STING agonist; and Each "-" is a covalent bond.

[0011] In some aspects, the pHLIP® peptide has the sequence: In some aspects, the pHLIP® peptide has the sequence: In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000011.tif12128. In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000012.tif4128. In some aspects, the pHLIP® peptide has the sequence: It consists of TIFF2025527495000013.tif4128.

[0012] In some aspects, the STING agonist comprises a cyclic dinucleotide (CDN). In other aspects, the STING agonist comprises a cyclic purine dinucleotide. In some aspects, the STING agonist is a non-nucleotide small molecule. In some aspects, the STING agonist is selected from the group consisting of diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, SNZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, TTI-10 001, CRD5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511, and derivatives thereof. In some aspects, the STING agonist is diABZI. In some aspects, the STING agonist is MK-1454. In some aspects, the STING agonist is MK-2118. In some aspects, the STING agonist is TAK-676. In other aspects, the STING agonist is BMS-986301. In some aspects, the STING agonist is BI1387446.

[0013] In some aspects, all of the amino acids in the pHLIP® peptides of the compositions disclosed herein are D-amino acids.

[0014] In some aspects, the Linker of the compositions disclosed herein comprises a disulfide bond or an acid-labile bond. In some aspects, the Linker is self-immolative.

[0015] In some aspects, the composition has the following structure: TIFF2025527495000014.tif81128.

[0016] In some aspects, pharmaceutical compositions are provided that include at least one of the compositions disclosed herein and one or more pharmaceutically acceptable excipients.

[0017] In some aspects, a method for treating cancer in a subject is provided, comprising administering a composition or pharmaceutical composition disclosed herein to a subject in need thereof. In some aspects, the cancer is a solid tumor. In some aspects, the composition or pharmaceutical composition disclosed herein is injected directly into the tumor mass. In some aspects, the composition or pharmaceutical composition disclosed herein is administered systemically. In some aspects, the subject is a human.

[0018] definition pHLIP® peptide (pH-low insertion peptide) is a linear, water-soluble membrane peptide that, at neutral pH, weakly interacts with cell membranes and does not insert into lipid bilayers; however, at slightly acidic pH (<7.0), pHLIP® inserts into cell membranes to form stable transmembrane α-helices. In addition to tumor cells, which are characterized by a low pH (<7.0), immune cells within the tumor mass are also characterized by a low pH (<7.0). Cells within the tumor mass environment, such as macrophages, are also characterized by a low pH. By conjugating pHLIP® to a STING agonist, the composition and method specifically deliver drugs directly to cancerous cells and immune cells (macrophages) and into their cytosol due to their acidic cell surface. Delivering a STING agonist using pHLIP® peptide can strengthen the immune system, e.g., by stimulating the production of interferon-γ, and initiate and / or establish a tumor-specific immune response to combat cancer.

[0019] As used herein, "effective," when referring to an amount of a composition, refers to a quantity of the composition sufficient to produce a desired response. For example, the amount of cargo, such as a STING agonist, when used in the methods of the present disclosure produces a desired response, e.g., an enhanced antitumor effect, without undue adverse side effects (such as toxicity, irritation, or allergic response), commensurate with a reasonable benefit / risk ratio.

[0020] In some embodiments, the subject is a mammal. In particular embodiments, the mammal is a rodent (e.g., a mouse or rat), a primate (e.g., a chimpanzee, a gorilla, a monkey, a gibbon, a baboon), a cow, a camel, a dog, a cat, a horse, a llama, a sheep, a goat, or a pig. In some embodiments, the subject is a human.

[0021] The transitional term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes elements, steps, or ingredients not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those "that do not materially affect the basic and novel characteristics" of the claimed invention.

[0022] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a disease," "a disease state," or "a nucleic acid" is a reference to one or more such embodiments and includes equivalents thereof known to those skilled in the art, and so forth.

[0023] As used herein, "treatment" includes, for example, suppressing, alleviating, or halting the progression of a disorder. Treatment also includes preventing or ameliorating any one or more symptoms of a disorder. As used herein, "suppressing" the progression of a disease or complications in a subject means preventing or alleviating the progression and / or complications of a disease in a subject.

[0024] As used herein, a "pharmaceutically acceptable" carrier or excipient refers to a carrier or excipient that is suitable for use in humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio. It may be, for example, a pharmaceutically acceptable solvent, suspending agent, or vehicle for delivering the compound to a subject.

[0025] Each aspect disclosed herein is believed to be applicable to each of the other disclosed aspects, and thus, any combination of the various elements described herein is within the scope of the invention.

[0026] Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. [Brief explanation of the drawings]

[0027] [Figure 1-1]Characterization of pHLIP-STINGa. a) Schematic of the interaction of pHLIP-STINGa with membrane lipid bilayers (pHLIP polymers are shown in dark blue, and STINGa is shown as a red circle). In state I, pHLIP-STINGa forms an unstructured polymer in solution at normal pH. State II illustrates the interaction of pHLIP-STINGa with membranes at normal pH. State III represents the transmembrane helical orientation of pHLIP induced by low pH, which leads to the translocation of STINGa across the lipid bilayer and its release in the cytoplasm. These three states were monitored by signal changes in the fluorescence (b) and CD (c) spectra of pHLIP-STINGa interacting with POPC liposomes. d) Kinetics of the fluorescence change induced by a decrease in pH in the presence of POPC liposomes are shown. pH transitions monitored by changes in fluorescence intensity (e) and CD (f) spectral signals are shown (experimental points and fitting curves are red, 95% confidence intervals are pink). g) OCD spectra of pHLIP-STINGa recorded immediately after deposition onto the supported bilayer and 12 hours after pHLIP-STINGa insertion into the bilayer was complete. h) IFN signaling pathway activation induced by pHLIP-STINGa in THP1-Blue-ISG cells polarized to M2 macrophages by PMA, IL-4, and IL-13 is shown. These results were normalized to the activity of STINGa alone at the highest concentration tested (considered to be 100%). [Figure 1-2] See description of Figure 1-1. [Figure 2A]Tumor targeting, PK, biodistribution, tumor and serum cytokines, tumor stroma, and immune cell uptake. a) Images of CT26 tumor targeting in mice 40 hours after a single IP injection of ICG-pHLIP-STINGa (100 μM, 300 μl). Tumor sites before and after shaving are shown. b) Normalized fluorescence recorded in blood collected at different time points after a single IV injection of ICG-pHLIP-STINGa (200 μM, 100 μl) is shown (mean and standard error). Data were fitted with an exponential function (red line). c) Kinetics of CT26 tumor targeting by ICG-pHLIP-STINGa and clearance of the agent from major organs are shown. Mean fluorescence per area was calculated for each organ and tissue collected at different time points after a single IV injection of ICG-pHLIP-STINGa (200 μM, 100 μl). d) IL-6 and TNF-α cytokine levels in tumors and serum determined by ELISA at different time points after a single IV injection of STINGa or pHLIP-STINGa (200 μM, 150 μl) are shown compared to control mice (all points, mean and SE are shown; p levels were calculated using the Kolmogorov-Smirnov two-tailed nonparametric test). e) The proportion of tumor stromal and immune cell populations within the TME targeted by Al647-pHLIP-STINGa was determined by FACS analysis of CT26 tumors harvested 24 hours after a single IP injection of Al647-pHLIP-STINGa (100 μM, 300 μl). (All points, mean and standard deviation are shown in the graph; values ​​are shown in the table.) f) Colocalization of Al647-pHLIP-STINGa with Al488-CD140b-Antb-stained CAFs, Al594-CD206-Antb-stained TAMs, and DAPI-stained cell nuclei within the TME is shown on images taken at different magnifications using 10x, 20x, and 40x objectives. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F]See legend to Figure 2A. [Figure 3A] CT26 tumor eradication and development of immune memory. a) Experimental design. b) Shown are CT26 tumor growth curves in Balb / c mice after a single administration of different agents at dose levels of 100 μM in 300 μl for IP injection or 200 μM in 150 μl for IV injection on day 1, when tumor volume reached approximately 100 mm3. The asterisk (*) indicates that this mouse in the IV group received a second dose of pHLIP-STINGa on day 41, when tumors began to regrow. c) Kaplan-Meier survival plot obtained for the data shown in panel b. d) CT26 tumor growth curves obtained for tumor-free mice in the groups that received a single IP or IV injection of pHLIP-STINGa on day 1. On day 61, these mice were re-injected with CT26 cancer cells into the left flank. e) Growth curve of large CT26 tumors after a single IP injection of pHLIP-STINGa (100 μM 300 μl) on day 1, when tumor volumes reached 400–700 mm3. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4-1]Clearance of tumor stroma and increase in pH within the TME. a-d) Cell numbers within the TME in control (mice receiving a single IP injection of vehicle) and treatment (mice receiving a single IP injection of Al647-pHLIP-STINGa (100 μM, 300 μl)) mice were determined by FACS analysis. The percentage of total viable cells within the TME (a), the number of CAFs (CD3-, CD45-, CD140b+) quantified as a percent of total viable cells (b), the number of TAMs (CD45+, CD11b+, F4 / 80+, CD206+) (c), and mMDSCs (CD45+, CD3-, CD11b+, F4 / 80-, Ly6Chi, Ly6G-) (d), both quantified as a percent of CD45+ immune cells, are shown (all points, mean and standard deviation, p-level <0.002 for all graphs). e-g) pH imaging and signal quantification using ICG-pHLIP are shown. Mice bearing CT26 tumors (approximately 100 mm3) on the right flank were divided into two groups (5 mice per group). One group (treatment) was treated with a single IP injection of pHLIP-STINGa (100 μM, 300 μl) on day 1, while the other group (control) received no treatment. On day 3, the ICG-pHLIP pH imaging probe was administered as a single IP injection (50 μM in 100 μl) to both the control (untreated) and pHLIP-STINGa-treated groups. 24 hours later (day 4), the animals were euthanized, and tumors were harvested, cut in half, and imaged. Representative images are shown in panels e-f, and the normalized mean fluorescence signal calculated for all tumor pieces (5 animals per group) is shown in panel g (all points, means and standard deviations are shown; p levels were calculated using the Kolmogorov-Smirnov two-tailed nonparametric test). [Figure 4-2] See description of Figure 4-1. [Figure 5]Self-immolation kinetics. HPLC chromatograms recorded at 280 nm and 320 nm for pHLIP-STINGa treated with DTT in 50 mM phosphate buffer, pH 7.4, containing 150 mM NaCl, at different time points (0, 30, and 60 min) after DTT addition are shown. Retention times are as follows: pHLIP-STINGa: 16.78 min, pHLIP: 17.02 min (no signal at 320 nm), SH-PAB-STINGa (modified diABZI cleaved from pHLIP): 12.16 min, STINGa (original diABZI): 10.27 min, and sacrificial product: 6.95 min. [Figure 6] Fluorescence of diABZI. Fluorescence spectra of STINGa(diABZI) in phosphate buffer excited at 295 nm and 322 nm are shown. [Figure 7] OCD spectra. OCD spectra were recorded 12 hours after deposition of solutions of pHLIP-STINGa and POPC onto the substrate at pH 5 and pH 3.3. [Figure 8] Cell viability. THP1 cells were activated and polarized into M2 macrophages by treatment with PMA for 6 hours, followed by IL-4 / IL-13 for an additional 18 hours. After activation and polarization, all factors were removed, and cells were treated with pHLIP-STINGα in FBS-free DMEM at pH 6.4 for 3 hours. FBS was then added to 10%, the pH was raised to pH 7.4, and cells were incubated for 48 hours before performing an MTS colorimetric assay. [Figure 9] Biodistribution of ICG-pHLIP-STINGa in organs and tissues. After a single IV injection of ICG-pHLIP-STINGa (200 μM, 100 μl), the mean fluorescence per area was calculated for each tissue and organ at 2, 4, 24, 48, 72, and 96 hours post-injection. All points, means, and standard deviations are shown. [Figure 10]Mean levels of cytokines (IL-6, TNF-α, IFN-β) in tumor supernatants and serum measured by ELISA 4 and 16 hours after a single IV (200 μM 150 μl) or IP (100 μM 300 μl) injection of pHLIP(Laa)-STINGa and pHLIP(Daa)-STINGa. [Figure 11] Immunological memory was not developed with 4T1 cancer cells. 4T1 tumor growth curves are shown for the "control" group (a) and the "second reinjection of 4T1 cancer cells" group (b). Mice in the control group were injected with 10 4T1 cancer cells into the right flank. Mice in the second reinjection group were initially inoculated with 5 × 10 CT26 cancer cells. When tumor volume reached approximately 100 mm, a single IP injection of pHLIP-STINGa (100 μM, 300 μl) on day 1 eradicated the tumors. Subsequently, 5 × 10 CT26 cancer cells were inoculated into the left flank on day 61, but tumor growth did not occur. Finally, 10 4T1 cancer cells were inoculated into the right flank on day 101, resulting in 4T1 tumor growth. c) Kaplan-Meier survival plots obtained for the data shown in panels a and b. [Figure 12] Treatment of CT26 tumors in athymic nude mice lacking T cells. a-b) CT26 tumor growth curves in nude athymic mice are shown after a single IP injection of pHLIP-STINGa (100 μM, 300 μl) on day 1, when tumor volumes reached approximately 100-200 mm3. c) Kaplan-Meier survival plots obtained for the data shown in panels a-b. [Figure 13]Treatment of 4T1 tumors. 105 4T1 breast cancer cells were inoculated into the right flank of Balb / C mice. When tumor volume reached approximately 100 mm3 (day 1), a single IP injection of pHLIP-STINGa (300 μl of 100 μM) was administered to the pHLIP-STINGa and pHLIP-STINGa+PD-1 groups (indicated by red arrows). Three IP injections of PD-1 antibody (250 μg per injection) were administered to the PD-1 and pHLIP-STINGa+PD-1 groups on days 4, 9, and 14 (indicated by blue arrows). The control group received no treatment. Tumor growth is shown for control (a), pHLIP-STINGa (b), PD-1 (c), and pHLIP-STINGa+PD-1 (d). e) Kaplan-Meier survival plots based on the data shown in panels a–d are presented. The statistical significance of the p-level for improved survival for pHLIP-STINGa vs. control and PD-1 vs. control was determined to be p<0.02 and p<0.004 for pHLIP-STINGa+PD-1 vs. control, calculated using log-rank (all time points weighted equally), Breslow (all time points weighted by the number of cases at risk at each time point), and Talone-Ware (all time points weighted by the square root of the number of cases at risk at each time point). [Figure 14] Body weight. Shown is the change in body weight after a single IP injection of pHLIP-STINGα (100 μM, 300 μl) on day 1. Similar body weight curves were obtained in Balb / c mice and athymic nude mice with other administration routes. [Figure 15] Intratumoral bleeding was observed within 2–4 days after injection of pHLIP-STINGa. Shown is an image of a Balb / c mouse bearing a CT26 tumor on the right flank the day after receiving a single IP injection of pHLIP-STINGa (300 μM, 100 μl). DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description Described herein are compositions comprising a pHLIP® peptide linked to a STING agonist.

[0029] STING agonists Cyclic dinucleotides are exemplary STING agonists, although other small molecules can be used as well. Exemplary STING agonists include diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, SNZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, TT In some aspects, the STING agonist includes I-10001, CRD5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511, and derivatives thereof. (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-6 76, SNZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, TTI-10 001, CRD5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511, and derivatives thereof.In some aspects, the STING agonist is selected from the group consisting of diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, SNZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, The STING agonist is selected from the group consisting of TTI-10001, CRD5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, and CF511. In one aspect, the STING agonist is diABZI. In some aspects, the STING agonist is MK-1454. In some aspects, the STING agonist is MK-2218.

[0030] pHLIP® Peptides In some aspects, the pHLIP® peptide has the following sequence: n Y m ; Y m X n ; X n Y m X j ; Y m X n Y i ; Y m X n Y i X j ; X n Y m X j Y i ; Y m X n Y i X j Y l ; X n Y m X j Y i X l ; Y m X n Y i X j Y l Xh ; X n Y m X j Y i X h Y g ; Y m X n Y i X j Y l X h Y g ; X n Y m X j Y i X h Y g X f ; (XY) n ; (YX) n ; (XY) n Y m ; (YX) n Y m ; (XY) n X m ; (YX) n X m ; Y m (XY) n ; Y m (YX) n ; X n (XY) m ; X n (YX) m ; (XY) n Y m (XY) i ; (YX) n Y m (YX) i ; (XY) n X m (XY) i ; (YX) n X m (YX) i ; Y m (XY) n ; Y m (YX) n ; X n (XY) m ; or X n (YX) m and has (i) Each Y is independently the solvation energy DG X cora nonpolar amino acid with a RI >+0.50, or glycine (Gly); (ii) each X is independently a protonatable amino acid; (iii) n, m, i, j, l, h, g, and f each independently represent an integer of 1 to 8.

[0031] In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000015.tif4128, where "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), an azide-containing amino acid, or other modified amino acid. In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000016.tif4128, where "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids.

[0032] An example of a pHLIP® peptide is: TIFF2025527495000017.tif4128, and AEQNPIY (SEQ ID NO: 4) represents the flanking sequence. TIFF2025527495000018.tif4128 represents the membrane insertion sequence and DADEGT (SEQ ID NO: 6) represents the flanking sequence.

[0033] Other exemplary pHLIP® peptides are shown in the table below, with sequences provided from N- to C-terminus.

[0034] Table 1: Non-limiting examples of pHLIP® sequences TIFF2025527495000019.tif27166TIFF2025527495000020.tif238166TIFF2025527495000021.tif232166TIFF2025527495000022.tif43166

[0035] Table 2: Non-limiting examples of pHLIP® sequences TIFF2025527495000023.tif145165* Ac means acetylated N-terminus and Am means amidated C-terminus.

[0036] Table 3: Coded amino acids and exemplary non-coded amino acids, including L-isomers, D-isomers, α-isomers, β-isomers, glycol modifications, and methyl modifications TIFF2025527495000024.tif225148TIFF2025527495000025.tif235148TIFF2025527495000026.tif40148

[0037] Table 4: Non-limiting examples of protonatable residues and their substitutions, including L-, D-, α-, and β-isomers TIFF2025527495000027.tif20128

[0038] Table 5: Examples of substitutions of encoded amino acids TIFF2025527495000028.tif112146

[0039] Table 6: Non-limiting examples of membrane insert sequences belonging to different groups of pHLIP® peptides Each protonatable residue (shown in bold) can be replaced by its substitution from Table 4. Each non-polar residue can be replaced by its coded amino acid substitution from Table 5 and / or a non-coded amino acid substitution from Table 3. TIFF2025527495000029.tif195144

[0040] Table 7: Non-limiting examples of pHLIP® sequences Cysteine, lysine, azide-modified amino acids, or alkynyl-modified amino acids can be incorporated into the N-terminal (first 6 residues) or C-terminal (last 6 residues) portions of the peptide for attachment to cargo and linker. TIFF2025527495000030.tif208163TIFF2025527495000031.tif199163

[0041] In some aspects, the pHLIP® peptide comprises a sequence selected from the group consisting of any one of SEQ ID NOs. 1-219. In some aspects, the pHLIP® peptide is from the group consisting of any one of SEQ ID NOs. 1-219. In other aspects, the pHLIP® peptide has the sequence: TIFF2025527495000032.tif4128. In one aspect, the pHLIP® peptide has the sequence: TIFF2025527495000033.tif4128. In one aspect, the pHLIP® peptide has the sequence: TIFF2025527495000034.tif4128. In one aspect, the pHLIP® peptide has the sequence: It has a sequence consisting of TIFF2025527495000035.tif4128.

[0042] Linker Linkers can be relatively small, such as just a few atoms, or quite large polymers of 4-5 kDa. The Linker can react with free thiols at one end to spontaneously form disulfide bonds using a thiopyridine as a leaving group, and with activated amine or hydroxyl groups at the other end to form carbamates or carbonates, respectively, in the presence of DIPEA and, optionally, DMAP or other activator-bases. This material can be used when pHLIP® (A) is protected at its amino terminus, e.g., by N-acetylation. This material can also be reacted with pHLIP® having a cysteine ​​residue or with a thiol-containing linker for subsequent conjugation with pHLIP®, forming a conjugate by disulfide exchange using a thiopyridine as a leaving group. This material can be used to form conjugates with pHLIP® having a lysine residue when pHLIP® is protected at its amino terminus, e.g., by N-acetylation.

[0043] In some aspects, succinimidyl 3-(2-pyridyldithio)propionate (SPDP) crosslinker is used. SPDP is a short-chain crosslinker for amine-sulfhydryl linkages via NHS ester and pyridyldithiol reactive groups, which form a cleavable (reducible) disulfide bond with the sulfhydryl of cysteine. SPDP is used to activate and purify the NH derivative of cCDN (e.g., c[3'-AHC-G(2',5')pA(3',5')p] or c[G(2',5')p-2'-AHC-A(3',5')p]) and exchange the disulfide with the SH of the single cysteine ​​(Cys) at the C-terminus of the pHLIP® peptide to generate pHLIP®-SS-cCDN.

[0044] In some aspects, the following crosslinkers may be used: LC-SPDP (succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate); Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate); PEG4-SPDP (PEGylated long-chain SPDP crosslinker); PEG12-SPDP (PEGylated long-chain SPDP crosslinker); SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate); Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate); SMPT (4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene); DTME (dithiobismaleimidoethane).

[0045] In some aspects, the Linker is attached to the C-terminus of the pHLIP® peptide.

[0046] composition In one aspect, the compositions disclosed herein have the following structure: Peptide-Linker-STING Including, Peptide is pHLIP® peptide; Linker is a cleavable linker; "STING" is diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, S NZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, TTI-10001, CRD 5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511, and derivatives thereof; and Each "-" is a covalent bond.

[0047] In some aspects, the pHLIP® peptide has the following sequence: n Y m ; Y m X n ; X n Y m X j ; Y m X n Y i ; Y m X n Y i X j ; X n Y m X j Y i ; Y m X n Y i X j Y l ; X n Y m X j Y i X l ; Y m X n Y i X j Y l Xh ; X n Y m X j Y i X h Y g ; Y m X n Y i X j Y l X h Y g ; X n Y m X j Y i X h Y g X f ; (XY) n ; (YX) n ; (XY) n Y m ; (YX) n Y m ; (XY) n X m ; (YX) n X m ; Y m (XY) n ; Y m (YX) n ; X n (XY) m ; X n (YX) m ; (XY) n Y m (XY) i ; (YX) n Y m (YX) i ; (XY) n X m (XY) i ; (YX) n X m (YX) i ; Y m (XY) n ; Y m (YX) n ; X n (XY) m ; またはX n (YX) m を有し、 (i) 各Yは、独立して、溶媒和エネルギーDG X cora nonpolar amino acid with a RI >+0.50, or glycine (Gly); (ii) each X is independently a protonatable amino acid; (iii) n, m, i, j, l, h, g, and f each independently represent an integer of 1 to 8.

[0048] In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000036.tif4128, where "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids, or other modified amino acids. In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000037.tif4128, where "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids.

[0049] In some aspects, the pHLIP® peptide comprises a sequence selected from the group consisting of any one of SEQ ID NOs. 1-219. In some aspects, the pHLIP® peptide has a sequence selected from the group consisting of any one of SEQ ID NOs. 1-219. In other aspects, the pHLIP® peptide has the sequence: TIFF2025527495000038.tif4128. In one aspect, the pHLIP® peptide has the sequence: TIFF2025527495000039.tif4128. In one aspect, the pHLIP® peptide has the sequence: TIFF2025527495000040.tif4128. In one aspect, the pHLIP® peptide has the sequence: It has a sequence consisting of TIFF2025527495000041.tif4128.

[0050] In one aspect, the STING agonist is diABZI. In another aspect, the STING agonist is MK-1454. In yet another aspect, the STING agonist is MK-2118. In another aspect, the STING agonist is TAK-676. In another aspect, the STING agonist is BMS-986301. In yet another aspect, the STING agonist is BI1387446.

[0051] In some aspects, the compositions disclosed herein have the following structure: Peptide-Linker-STING Including, The peptide has the sequence: pHLIP® peptide containing TIFF2025527495000042.tif12128; Linker is a cleavable linker; STING is a STING agonist; and Each "-" is a covalent bond, These are also disclosed herein.

[0052] In some aspects, the pHLIP® peptide has the sequence: In some aspects, the pHLIP® peptide has the sequence: In some aspects, the pHLIP® peptide has the sequence: It consists of TIFF2025527495000045.tif12128.

[0053] In some aspects, the pHLIP® peptide has the sequence: TIFF2025527495000046.tif4128. In some aspects, the pHLIP® peptide has the sequence: It consists of TIFF2025527495000047.tif4128.

[0054] In some aspects, the STING agonist comprises a cyclic dinucleotide (CDN). In some aspects, the STING agonist comprises a cyclic purine dinucleotide. In some aspects, the STING agonist is a non-nucleotide small molecule. In some aspects, the STING agonist is selected from the group consisting of diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, SNZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, TTI-10 001, CRD5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511, and derivatives thereof. In some aspects, the STING agonist is diABZI. In some aspects, the STING agonist is MK-1454. In some aspects, the STING agonist is MK-2118. In some aspects, the STING agonist is TAK-676. In other aspects, the STING agonist is BMS-986301. In some aspects, the STING agonist is BI1387446.

[0055] In some aspects, all of the amino acids in the pHLIP® peptides of the compositions disclosed herein are D-amino acids.

[0056] In some aspects, the Linker according to the present disclosure comprises a disulfide bond or an acid-labile bond. In some aspects, the Linker is self-immolative. In some aspects, the composition has the following structure: TIFF2025527495000048.tif80128.

[0057] Treatment method Also provided is a method of using the compositions described herein to treat cancer in a subject in need thereof, comprising administering the compositions or pharmaceutical compositions disclosed herein. In some aspects, the cancer is a solid tumor. In some aspects, the compositions or pharmaceutical compositions disclosed herein are injected directly into the tumor mass. In some aspects, the subject is a human.

[0058] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes for making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, and alternative methods may be used to achieve similar results. [Example]

[0059] Example 1: Synthesis of pHLIP®-STINGa The diABZI STING agonist was modified with a linker to prepare o-pyridyl-dithioethyl-carbamoyl-PAB-STING (Pys-PAB-STINGα). This agent was synthesized and purified by Iris Biotech GmbH. All pHLIP peptides used in this study were synthesized and purified by CSBio. The following pHLIP sequence, with a single Cys residue at the membrane-inserting end of the peptide, was used for conjugation to Pys-PAB-STINGα: pHLIP(Laa): TIFF2025527495000049.tif4128 (all amino acids are in the L configuration) and pHLIP(Daa): TIFF2025527495000050.tif4128 (all amino acids are in the D configuration).

[0060] The pHLIP® peptide and Pys-PAB-STINGa were mixed in a 1:1 molar ratio in dimethyl sulfoxide (DMSO). To this reaction mixture (1 / 10 of the total volume), argon-saturated sodium phosphate buffer, pH 7.4 (100 mM) containing 150 mM NaCl, was added, and the reaction mixture was kept at room temperature (RT) for 2 hours. The pHLIP®-STINGa construct was purified by reverse-phase high-performance liquid chromatography (HPLC) using a Zorbax SB-C18, 9.4 x 250 mm, 5 μm column (Agilent Technologies) with a gradient of 10% to 75% acetonitrile in water containing 0.05% trifluoroacetic acid (TFA). To prepare fluorescent versions of this agent, Al647-pHLIP®-STINGa and ICG-pHLIP-STINGa, the pHLIP peptide: The N-acetylated version of TIFF2025527495000051.tif4128 (all amino acids in the L-configuration) was used. pHLIP® was first conjugated to Pys-PAB-STING and then purified. Next, ICG-NHS ester (Iris Biotech GmbH) or Alexa647-NHS ester (Life Technologies) was conjugated to the N-terminal lysine residue of pHLIP in DMSO at a molar ratio of 1:1.5. Sodium bicarbonate buffer, pH 8.3 (100 mM), was added to this reaction mixture (1 / 10 of the total volume), and the reaction mixture was kept at RT until conjugation was complete. Final purification was performed as described above. The product was lyophilized and characterized by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry and analytical HPLC. The concentration of pHLIP-STINGα conjugates was determined by absorbance using the following molar extinction coefficients: ε for pHLIP-STINGα; 322 =46,200M -1 cm -1 ε in ICG-pHLIP-STINGα 800 =137,000M -1 cm -1 and ε in Al647-pHLIP-STINGa 651=270,000M -1 cm -1 .

[0061] Example 2: Stability in mouse and human plasma To establish the stability of pHLIP(Laa)-STINGa and pHLIP(Daa)-STINGa in plasma, pHLIP-STINGa was mixed with single-donor human or BALB / c mouse plasma (Innovative Research) at a concentration of 200 μM and incubated in plasma at 37°C for 0, 2, 4, or 24 hours. Plasma proteins were precipitated with methanol (1:5 plasma to methanol volume ratio) and centrifuged at 13.4 rpm for 10 minutes. The supernatant was collected and analyzed by HPLC using a Zorbax SB-C18 4.6 x 250 mm, 5 μm column with a gradient of 10% to 75% acetonitrile in water containing 0.05% TFA. Chromatograms were recorded at 220 nm, 280 nm, and 320 nm. As controls, pHLIP(Laa), pHLIP(Daa), Pys-PAB-STINGa, SH-PAB-STINGa (Pys-PAB-STINGa conjugated to a Cys residue), and diABZI (Invivogen) were analyzed for stability in plasma under the same conditions, and the results are provided in Table 8 below.

[0062] (Table 8) TIFF2025527495000052.tif47128

[0063] Example 3: Dynamics of self-sacrifice To induce linker self-immolation, a solution of pHLIP(Laa)-STINGa was treated with dithiothreitol (DTT). At different time points (from 30 minutes to 2 hours after treatment), samples were analyzed by HPLC using a Zorbax SB-C18 4.6 x 250 mm, 5 μm column with a gradient of 10% to 75% acetonitrile in water containing 0.05% TFA. Chromatograms were recorded at 220 nm, 280 nm, and 320 nm. diABZI and pHLIP were used as controls under the same HPLC conditions.

[0064] The results of this study are provided in Figure 5.

[0065] Example 4: Biophysical studies The interaction of pHLIP(Laa)-STINGa with liposomes was investigated by recording the fluorescence and circular dichroism (CD) of the constructs using a PC1 spectrofluorometer (ISS) and a MOS-450 spectrometer (Bio-Logic Science Instruments), respectively, with the temperature control set at 25°C. Liposomes composed of large unilamellar vesicles were prepared by extrusion. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid (Avanti Polar Lipids) in chloroform was desolvated on a rotary evaporator and dried under vacuum for a minimum of 2 hours. The phospholipid film was rehydrated in 2 mM citrate phosphate buffer, pH 7.3, vortexed, and passed 21 times through an extruder (using a 50 nm pore size).

[0066] Fluorescence spectra were recorded from 310 nm to 550 nm with an excitation wavelength of 295 nm and a slit size of 1.0 mm. The excitation polarizer was set at 54.7 degrees (the "magic angle"), while the emission polarizer was set at 0 degrees to reduce Wood's anomalous diffraction. CD spectra were recorded from 190 nm to 260 nm with a step size of 1 nm. The concentrations of pHLIP-STINGa and POPC were 7 μM and 1.4 mM, respectively. STINGa(diABZI) fluorescence was also recorded when excited at wavelengths of 295 nm and 350 nm.

[0067] The pH-dependent insertion of pHLIP-STINGa into the lipid bilayer of POPC liposomes was investigated by monitoring either the change in fluorescence intensity at 400 nm or the change in molar ellipticity at 230 nm as a function of pH. After adding an aliquot of citric acid, the pH of the solution containing pHLIP-STINGa and POPC liposomes was measured using an Orion PerHecT ROSS combination pH microelectrode and an Orion Dual Star pH / ISE benchtop meter. The normalized fluorescence intensity or millidegree ellipticity values ​​were plotted as a function of pH. The pH dependence was fitted to the Henderson-Hasselbalch equation to determine the cooperation (n) and transition midpoint (pK). The fitting equation used was as follows: For a single transition TIFF2025527495000053.tif7128 Two transitions TIFF2025527495000054.tif8128Here, SII and SIII represent the spectral signals of states II and III, respectively, and SII' represents the CD signal of the intermediate state between II and III.

[0068] Fluorescence kinetics was measured using an SFM-300 mixing system (Bio-Logic Science Instruments) coupled with a MOS-450 spectrometer, with the temperature control set at 25°C. To minimize air bubbles in the samples, all samples were degassed before measurement. pHLIP-STINGa and POPC samples were incubated overnight to equilibrate; at this point, the majority of the agent was bound to the liposomal lipid bilayer. To track pHLIP-STINGa membrane insertion, a solution containing 14 μM pHLIP-STINGa and 2.8 mM POPC was mixed with citric acid to lower the pH from 8 to 3.5. To monitor the change in fluorescence intensity during pHLIP-STINGa insertion into POPC liposomes induced by the pH decrease, the emission signal was observed through a filter with an excitation wavelength of 295 nm and a cutoff wavelength of 320 nm.

[0069] For orientational circular dichroism (OCD) measurements, bilayers were prepared on quartz slides (Starna) specially polished for far-UV measurements. The slide cleaning procedure included the following steps: 1) immersion in cuvette cleaning solution for 24 hours, 2) rinsing with deionized distilled water, 3) ultrasonic cleaning in 2-propanol for 10 minutes, 4) ultrasonic cleaning in acetone, 5) another ultrasonic cleaning in 2-propanol, 6) rinsing with deionized water, 7) immersion in a piranha solution consisting of 25% hydrogen peroxide and 75% sulfuric acid, and 8) rinsing with Milli-Q purified water. POPC lipid monolayers were deposited on the quartz substrate by the Langmuir-Blodgett (LB) method using a KSV mini-trough. For LB deposition, a small amount of POPC lipid in chloroform was spread on the surface of the subphase, and the solvent was allowed to evaporate for approximately 10 minutes. The monolayer was then compressed to 32 mN / m. Once the surface pressure stabilized, the first slide was inserted into the trough and held there for 60 seconds to allow the surface pressure to stabilize again. It was then withdrawn from the subphase at a rate of 10 mm / min. The second layer was created by fusion with POPC vesicles. Approximately 80 μl of either a sample containing 7 μM pHLIP-STINGα and 0.7 mM POPC in 2 mM pH 5.0 or 2 mM pH 3.3 citrate-phosphate buffer, or approximately 80 μl of a POPC blank containing 0.7 mM POPC in citrate-phosphate buffer (without pHLIP-STINGα), was spread onto the slide. This process was repeated for eight more slides, which were then stacked on top of each other. Spacers were placed between the slides to prevent them from sticking together. "Time 0" OCD spectra were measured for both pH samples and the POPC blank. The slides were then stored at 4°C for 6 hours at 100% humidity. After 6 hours, excess solution was shaken off each slide and replaced with 80 μL of the corresponding pH buffer. The slides were re-stacked together, while filling with buffer, to form a complete set of 8 slides (16 bilayers) and stored at 100% humidity for an additional 6 hours at 4° C. At the end of the 12-hour incubation period, the "12-hour" OCD spectrum was measured.The OCD spectrum of the POPC blank was subtracted from the OCD spectrum of the sample.

[0070] All data were fitted to appropriate equations by nonlinear least-squares curve fitting employing the Levenberg-Marquardt algorithm using Origin 8.5.

[0071] Characterization of pHLIP-STINGa is provided in Figures 1, 5, and 6.

[0072] Example 5: Activation of IFN in cells THP-1-Blue™-ISG cells (Invivogen) expressing an interferon (IFN) regulatory factor (IRF)-inducible secreted embryonic alkaline phosphatase (SEAP) reporter construct were used. Cells were maintained in RPMI growth medium supplemented with L-glutamine, sodium pyruvate, 10% fetal bovine serum (FBS), normasin, and ciprofloxacin hydrochloride at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cells were seeded at a density of 75,000 cells / well in 96-well plates. To generate M2-polarized macrophages, cells were first treated with 185 ng / mL phorbol 12-myristate 13-acetate (PMA) for 6 hours, followed by 20 ng / mL interleukin-4 (IL-4) and 20 ng / mL interleukin-13 (IL-13) (both from PeproTech) for an additional 16 hours. Upon completion of polarization, the growth medium was replaced with FBS-free Dulbecco's Modified Eagle's Medium (DMEM) pH 6.9 containing increasing amounts of pHLIP(Laa)-STINGa or STINGa (up to 10.0 μM). After 2 h of incubation, an equal volume of RPMI supplemented with 20% heat-inactivated FBS was added, and the cells were incubated for an additional 48 h. SEAP activity was assessed using QUANTI-Blue™ solution (Invivogen) to assess type I interferon protein levels: 150 μl of colorimetric reagent was added to 50 μl of cell supernatant for 30 min at 37°C, followed by absorbance measurements at 655 nm.

[0073] Example 6: Cell viability THP1 cells (ATCC, TIB-202) were maintained in RPMI growth medium supplemented with 2-mercaptoethanol, 10% FBS, and ciprofloxacin hydrochloride at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cells were seeded at a density of 30,000 cells / well in 96-well plates. To generate M2-polarized macrophages, cells were first treated with 185 ng / mL PMA for 6 hours, followed by 20 ng / mL IL-4 and IL-13 for an additional 16 hours. Upon completion of polarization, the growth medium was replaced with FBS-free DMEM pH 6.4 containing increasing amounts of pHLIP(Laa)-STINGa. After 3 hours of incubation, an equal volume of RPMI supplemented with 20% FBS was added. Cell viability was assessed using CellTiter 96 AQ. ueous The proliferation was assessed after 48 hours using the One Solution Cell Proliferation Assay (Promega); colorimetric reagent was added to the cells for 1 hour, followed by absorbance measurements at 490 nm.

[0074] These results are provided in FIG.

[0075] Example 7: Treatment of mice For the treatment of CT26 tumors, 5 × 10 4 CT26 mouse colon cancer cells (ATCC, CRL-2638) were injected subcutaneously (SQ) in 100 μl of growth medium into the right flank of 7- to 9-week-old female Balb / c mice or athymic female nude mice (Hsd Athymic Nude-Foxn1nu strain) (both from Envigo RMS, Inc.). Tumor size was 100 mm. 3 ("small tumor") or 400-700 mm 3On day 1, when tumors reached a large size ("large tumor"), mice were randomized into groups, weighed, and administered a single intraperitoneal (IP) or intravenous (IV) injection of pHLIP(Laa)-STINGa, pHLIP(Daa)-STINGa, pHLIP(Laa), STINGa(diABZI), or vehicle. Vehicle, pHLIP(Laa), STINGa(diABZI), and pHLIP(Laa)-STINGa were administered as a single IP injection of 300 μl at 100 μM. pHLIP(Daa)-STINGa was administered as a single IV injection of 150 μl at 200 μM. These compounds were dissolved in DMSO to form stock solutions and transferred to 20% PEG400 in saline containing 0.9% sodium chloride (vehicle). Residual DMSO in the final solution injected into animals was less than 2%. Tumor volume and body weight were measured three times a week throughout the study. Tumor measurements were performed using calipers, and tumor volume (V) was calculated using the following formula: V=0.52 L W 2 where L is the measured tumor length and W is the tumor width. 3 At this time, mice were removed from the study and euthanized.

[0076] Mice in the pHLIP-STINGa treatment group that remained tumor-free were re-challenged with tumor cells injected into the opposite flank on day 61 after the single pHLIP-STINGa injection. Tumor-free mice were maintained for an additional 40 days (a total of 100 days after treatment with pHLIP-STINGa), and most were euthanized. On day 101, 5 tumor-free mice were re-injected into the 10 5 A control group of female Balb / c mice received another SQ injection of 10 4T1 mouse breast cancer cells (ATCC, CRL-2539) into the right flank. 5 4T1 cancer cells were injected into the right flank, and tumor growth was compared between groups.

[0077] For the treatment of 4T1 triple-negative breast cancer, 10 54T1 mouse breast cancer cells (ATTC, CRL-2539) were injected SQ in 100 μl of growth medium into the right flank of 7-9 week-old Balb / c female mice (Envigo). Tumors were grown to a volume of 100 mm. 3 On day 1, when tumor volume reached 1500 mm, body weight was measured and the mice were randomly assigned to four groups. On day 1, mice in groups #2 and #4 received a single IP injection of pHLIP(Laa)-STINGa (100 μM, 300 μl). On days 4, 9, and 14, mice in groups #3 and #4 received three IP injections of anti-mouse PD-1 antibody (BioCell, CD279) (250 μg / mouse per injection). Mice in the control group (group #1) did not receive any treatment. Tumor volume and body weight were measured three times a week. When tumor volume reached 1500 mm, tumor volume was measured three times a week. 3 Mice were euthanized when the

[0078] These results are provided in Figures 11-15.

[0079] Example 8: ELISA for blood and tumor samples To determine cytokine levels in blood and tumor samples, 5 × 10 4 CT26 cancer cells were injected SQ into the right flank of female Balb / c mice in 100 μl of growth medium. Tumors with a volume of 150–250 mm were grown. 3At this time, mice received a single IV or IP injection of pHLIP(Laa)-STINGa, pHLIP(Daa)-STINGa, STINGa, or no injection. At 4, 16, and 24 h postinjection, animals were euthanized, and blood and tumor samples were collected. Blood samples were held at room temperature for 40 min and then centrifuged at 5,000 g for 20 min at +4°C to collect the supernatant (serum). Tumors were frozen in liquid nitrogen. Both serum and tumor tissue samples were stored at -80°C until further processing and analysis. Tumor samples were processed on ice using a bullet blender (Next Advance) equipped with 1 mm diameter zirconium silicate beads. The processed tumor supernatant was used for enzyme-linked immunosorbent assay (ELISA) assays. A matched antibody pair kit for mouse tissue necrosis factor α (TNF-α) (Sino Biological), a matched antibody pair kit for mouse interleukin 6 (IL-6) (Abcam), and precoated plates for mouse IFN-β (PBL Assay Science) were used. ELISA assays were performed using serum and tumor samples. For TNF-α, the capture antibody was diluted in phosphate-buffered saline (PBS) (Sigma-Aldrich), and for IL-6, the capture antibody was diluted in coating buffer (Abcam). The diluted capture antibody was incubated in the plate overnight at +4°C and washed the next day with PBS / Tween wash buffer (Sigma-Aldrich). The TNF-α plate was blocked with 2% bovine serum albumin (BSA) in wash buffer (Thermo Scientific), while the IL-6 and IFN-β plates were blocked with the dilution buffer from the corresponding kit. Blocking was performed for 2 h at RT on an orbital shaker at 200 rpm. After blocking, the plates were washed and then incubated with diluted tumor and serum samples and the corresponding standard solutions from each ELISA kit. The samples were incubated for 2 hours at room temperature on an orbital shaker at 200 rpm.TNF-α and IFN-β plates were incubated with diluted horseradish peroxidase (HRP)-conjugated detection antibodies for 1 hour at room temperature. IL-6 plates were incubated with diluted biotin-conjugated detection antibodies for 1 hour at room temperature, followed by diluted HRP-streptavidin conjugate (Abcam) for 1 hour at room temperature. All plates were washed and incubated with a 1:1 mixture of 3,3',5,5'-tetramethylbenzidine (TMB) (Invitrogen) and peroxide solution (Thermo Scientific) for up to 20 minutes, after which stop solution (10% H2SO4) was added to the plates. Signals from the wells were quantified by absorbance measured at 450 nm using a Bio-Rad iMark microplate reader. Different dilution schemes were tested in duplicate, and a calibration curve was generated using antibody standards.

[0080] These results are provided in FIG.

[0081] Example 9: Biodistribution, PK and Imaging 5 × 10 for pharmacokinetic (PK), biodistribution, and pH imaging studies 4 CT26 cancer cells were injected SQ into the right flank of female Balb / c mice in 100 μl of growth medium, resulting in a volume of 150–200 mm 3Tumors were allowed to grow until they reached a mass of 100 μL. For PK and biodistribution studies, a single tail vein injection of 100 μL of 200 μM ICG-pHLIP(Laa)-STINGa was administered. Animals were euthanized at 2, 4, 24, 48, 72, and 96 hours post-injection, and blood was collected into K2EDTA vacutainer tubes (BD). Necropsies were performed immediately after euthanasia. Blood, tumors, and major organs (kidneys, liver, spleen, pancreas, lungs, heart, large intestine, small intestine, bone, muscle, and brain) were collected and imaged ex vivo immediately after necropsy. Blood (150 μL) was imaged in a 96-well plate with black bottom and walls. The zero time point (0 min) was obtained by imaging ICG-pHLIP-STINGa diluted with blood from uninjected control mice (dilutions were performed assuming a mouse contained 80 ml / kg of blood). The fluorescence at time zero was set to 100%, and the fluorescence recorded at 2, 4, 24, and 48 hours post-injection (pi) was calculated as a percentage of the signal at time zero. To determine half-lives, points were fitted with a single exponential decay function.

[0082] For in vivo imaging, Balb / c mice and athymic nude mice were cultured with tumors measuring 150–250 mm 3 At this time, a single IP (200 μM 100 μl) or IP (300 μM 150 μl) injection of ICG-pHLIP(Laa)-STINGa was administered, and in vivo imaging was performed at 1, 2, 4, 24, 40-48, 74, 100, 170, and 195 h post-injection.

[0083] For the pH imaging study, mice were divided into two groups. On day 1, mice in group 1 received a single IP injection of pHLIP(Laa)-STINGa (100 μM, 300 μl). Control mice in group 2 received no treatment. On day 3, mice in groups 1 and 2 received a single IP injection of 50 μM, 100 μl of the acidity imaging probe ICG-pHLIP (Iris Biotech, GmbH). On day 4 (or 24 h after ICG-pHLIP injection), all animals were euthanized, and tumors were harvested, cut in half, and imaged.

[0084] In vivo and ex vivo bright-field and near-infrared fluorescence imaging was performed using a Stryker 1588 AIM endoscope system equipped with an L10 AIM light source (808 nm excitation and approximately 815–850 nm collection) and a 1588 AIM camera using a 10 mm scope. The lens was held at a fixed distance from the organ surface within a closed (light-tight) area. Imaging was performed with three different settings. Digital images of the organs were saved in the green channel, transferred to 8-bit files, and processed using the ImageJ program. A threshold was set from pixel intensities ranging from 1 to 255, excluding background with a pixel intensity of 0. Organ and tumor boundaries were defined using bright-field images. Mean organ fluorescence was calculated using the calculated total fluorescence intensity and the total area of ​​each organ.

[0085] The results from these studies are provided in Figures 2, 3, and 9.

[0086] Example 10: FACS analysis The uptake of Al647-pHLIP(Laa)-STINGa by tumor cells in CT26 tumors was analyzed by fluorescence-activated cell sorting (FACS) analysis. 5 CT26 cancer cells were injected SQ into the flank of female Balb / c mice (8-12 weeks old) in 100 μl of 0% Matrigel. Tumor volumes were 150-250 mm. 3 At the time of tumor growth, mice were divided into two groups. Treatment group #1 (8 mice) received a single IP injection of Al647-pHLIP(Laa)-STINGa (300 μM, 100 μl), while control group #2 (5 mice) received a single IP injection of vehicle (1% DMSO in PBS). After 24 hours, all animals were euthanized, and tumors were harvested for processing. Tumor stroma and immune cells were identified using the following markers: CD4 T cells: CD45 + , CD3 + , CD11b - , CD4 + , CD8 - CD8 T cells: CD45 + , CD3 + , CD11b - , CD4 - , CD8 + Tregs: CD45 + , CD3 + , CD11b - , CD4 + , CD25 + , FoxP3 + mMDSCs: CD45 + , CD3 - , CD11b + , F4 / 80 - , Ly6C high , Ly6G - gMDSCs: CD45 + , CD3 - , CD11b + , F4 / 80 - , Ly6C low , Ly6G + M1: CD45 + , CD11b + , F4 / 80 + , CD206 - M2: CD45 + , CD11b + , F4 / 80 + , CD206 + DCs: CD45 + , CD11b + , CD11c + , MHCII + , F4 / 80 - CAFs: CD3 - , CD45 - , CD140b +

[0087] Staining was performed on tumor samples, Fluorescence Minus One (FMO) controls, and Single Color Controls (SCC) as shown below. Tumor: Live / Dead, CD45, CD3, CD4, CD8, CD25, FoxP3, CD11b, F4 / 80, Ly6C, Ly6G, CD206, CD11c, MHCII, CD140b Tumor-FMO: CD3, CD25, FoxP3, F4 / 80, Ly6C, Ly6G, CD206, CD11c, MHCII, CD140b SCC: Unstained, CD45, CD3, CD4, CD8, CD25, FoxP3, CD11b, F4 / 80, Ly6C, Ly6G, CD206, CD11c, MHCII, CD140b, Live / Dead

[0088] The following anti-mouse antibodies were used: CD45-APC-Fire750 clone 30-F11; CD8a-BV650 clone 53-6.7; CD25-BV605 clone PC61; F4 / 80-PE-Dazzle-594 clone BM8; Ly-6C-FITC clone HK1.4; Ly-6G-BV785 clone 1A8; CD206-BV421 clone C068C2; CD140b-PE clone APB5; CD11c-BV711 clone N418; IA / IE-PE / Cy7 clone M5 / 114.15.2 (BioLegend); CD3e-BUV496 clone 145-2C11; CD4-BUV395 clone GK1.5; CD11b-BUV737 clone M1 / 70 (BD Biosciences); and FoxP3. PerCP-Cy5.5 clone FJK-16s (Thermo Fisher); and Live / Dead Aqua-V500 (Life Technologies). The tumor processing procedure was as follows: tumor samples were dissociated using the gentleMACS™ protocol "Tumor Dissociation Kit" according to the manufacturer's instructions. The samples were filtered through a 70 μm cell strainer, rinsed twice in PBS / 2.5% FBS buffer, and the total volume of the sample was measured. A single cell suspension was prepared at 1 × 10 in PBS pH 7.4. 7The cells / mL were prepared in each well of a 96-well plate and stored on ice. All incubation steps were protected from light. Plates were washed by spinning at 300 × g (or 400 × g) for 3 minutes and discarding the supernatant. Live / Dead reagent was added to each sample, incubated for 15 minutes at 4°C, and then washed. Fc block (Mu TruStain FcX / anti-FcγRIV, Biolegend) diluted in staining buffer (BD) was added to the sample and incubated for 10 minutes at 4°C. Cell surface antibodies diluted in staining buffer supplemented with Brilliant Stain Buffer Plus (BD) were then added for 30 minutes at 4°C, followed by washing. Cells were fixed in FoxP3 Fix / Perm solution, incubated at room temperature for 30 minutes, and then washed. For single-color controls, one drop of Ultra Comp Beads (Thermo Fisher) was added to each single-color control well. For live / dead controls, one drop of ArC Amine Reactive Compensation Beads (Life Technologies) was added, followed by the addition of each antibody to the appropriate well. Al647-pHLIP-STINGa was imaged using the isotype control Al647 clone MOPC-21 (BioLegend) in the Alexa47 channel. Following the incubation step, a wash step was performed. Cell counts in the control and treatment groups, as well as the cellular uptake of Al647-pHLIP-STINGa, were determined.

[0089] These results are provided in FIG.

[0090] Example 11: Immunohistochemistry and Imaging For immunohistochemical analysis, 5 x 10 4 CT26 cancer cells were injected SQ into the right flank of female BALB / c mice in 100 μl of growth medium. Tumor volumes were 150–250 mm. 3At 100 μM, mice received a single IP injection of Al647-pHLIP(Laa)-STINGa (100 μl). Tumors were frozen and sectioned at 10–20 μm using a ThermoFisher HM525 NX microscope. Sections were stained with fluorescent antibodies CD206-AL594 (BioLegend), CD68-AL594 (BioLegend), CD140b-AL488 (Invitrogen), and 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich), or hematoxylin-eosin (H&E) staining using hematoxylin 7211 (ThermoFisher) and eosin Y (Poly Scientific). Antibody-stained sections were air-dried for 10 minutes, washed in distilled water for 2 minutes, and then fixed in 4% paraformaldehyde 37% (Sigma-Aldrich) for 12 minutes. They were then washed in Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich) for 5 minutes and air-dried for 10 minutes. A cover slide was placed on top of a layer of petrolatum (Equate) applied to the slide around the tissue. The sections were incubated with blocking buffer containing 5% 10% BSA (ThermoFisher) for 2 hours at room temperature and then washed. The sections were treated with antibodies in blocking buffer for 2 hours at room temperature and then washed. A coverslip was placed over the tissue using an organo / limonene mount. Tissue sections were imaged on an EVOS Fl Auto 2 inverted fluorescence microscope using 10x, 20x, and 40x objectives with appropriate filters in brightfield and fluorescence modes.

[0091] Example 12: Statistical Analysis The Kolmogorov-Smirnov two-sided nonparametric test was used to determine p-levels. The log-rank (all time points are weighted equally), Breslow (all time points are weighted by the number of cases at risk at each time point), and Talone-Ware (all time points are weighted by the square root of the number of cases at risk at each time point) methods were used to determine p-levels for survival plots.

[0092] The patents and scientific literature referred to herein establish knowledge available to those skilled in the art.All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference.All published foreign patents and patent applications cited herein are incorporated herein by reference.The Genbank and NCBI deposits cited herein and identified by accession number are incorporated herein by reference.All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference.

[0093] While the present invention has been particularly shown and described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. The following structure: Peptide-Linker-STING A composition comprising: Peptide is pHLIP® peptide; Linker is a cleavable linker; "STING" is diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, S NZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, TTI-10001, CRD 5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511, and derivatives thereof; and Each "-" is a covalent bond; composition.

2. The pHLIP® peptide has the following sequence: X h Y m ; Y m X n ; X n Y m X j ; Y m X n Y i ; Y m X n Y i X j ; X n Y m X j Y i ; Y m X n Y i X j Y l ; X n Y m X j Y i X l ; Y<00oo029>X n Y i X j Y l X h ; X n Y m X j Y i X h Y g ; Y m X n Y i X j Y l X<000oo46>Y g ; X n Y m X j Y i X h Y g X f ; (XY) n ; (YX) n ; (XY) n Y m ; (YX) n Y m ; (XY) n X m ; (YX) n X m ; Y m (XY) It should be noted that there may be some inaccuracies in the original text, especially in the repeated tags and the unclear "XY" and "YX" notations. The translation is done as accurately as possible based on the provided text. n ; Y m (YX) n ; X n (XY) m ; X n (YX) m ; (XY) n Y m (XY) i ; (YX) n Y m (YX) i ; (XY) n X m (XY) i ; (YX) n X m (YX) i ; Y m (XY) n ; Y m (YX) n ; X n (XY) m ; or X n (YX) m and has (i) Each Y is individually calculated as the solvation energy DG X cor a nonpolar amino acid with a nucleotide sequence >+0.50, or Gly; (ii) each X is individually a protonatable amino acid; (iii) n, m, i, j, l, h, g, and f are each independently an integer from 1 to 8; 10. The composition of claim 1.

3. The pHLIP® peptide has the sequence: a sequence comprising "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids; 3. The composition of claim 1 or 2.

4. The pHLIP® peptide has the sequence: a sequence consisting of "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids; 3. The composition of claim 1 or 2.

5. The composition of claim 1 or 2, wherein the pHLIP® peptide comprises a sequence selected from the group consisting of any one of SEQ ID NOs. 1-219.

6. The composition of claim 1 or 2, wherein the pHLIP® peptide has a sequence selected from the group consisting of any one of SEQ ID NOs. 1-219.

7. The pHLIP® peptide has the sequence:

3. The composition of claim 1 or 2, having a sequence comprising:

8. The pHLIP® peptide has the sequence:

3. The composition of claim 1 or 2, having a sequence comprising:

9. The pHLIP® peptide has the sequence:

3. The composition of claim 1 or 2, having a sequence consisting of:

10. The pHLIP® peptide has the sequence:

3. The composition of claim 1 or 2, having a sequence consisting of:

11. 11. The composition of any one of claims 1 to 10, wherein all amino acids in the pHLIP® peptide are D-amino acids.

12. The composition of any one of claims 1 to 11, wherein the STING agonist is diABZI.

13. The composition of any one of claims 1 to 11, wherein the STING agonist is MK-1454.

14. The composition of any one of claims 1 to 11, wherein the STING agonist is MK-2118.

15. The composition of any one of claims 1 to 11, wherein the STING agonist is TAK-676.

16. 12. The composition of any one of claims 1 to 11, wherein the STING agonist is BMS-986301.

17. 12. The composition of any one of claims 1 to 11, wherein the STING agonist is BI1387446.

18. The composition according to any one of claims 1 to 17, wherein the Linker comprises a disulfide bond or an acid-labile bond.

19. 19. The composition of claim 18, wherein the Linker is self-immolative.

20. A pharmaceutical composition comprising the composition of any one of claims 1 to 19 and one or more pharmaceutically acceptable excipients.

21. 20. A method of treating cancer in a subject, comprising administering to a subject in need thereof a composition according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 20.

22. 22. The method of claim 21, wherein the cancer is a solid tumor.

23. The method according to claim 21 or 22, wherein the composition according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 20 is injected directly into the tumor mass.

24. The method according to claim 21 or 22, wherein the composition according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 20 is administered systemically.

25. The method of any one of claims 21 to 24, wherein the subject is a human.

26. The following structure: Peptide-Linker-STING A composition comprising: Peptide is a pHLIP® peptide comprising the sequence; Linker is a cleavable linker; STING is a STING agonist; and Each "-" is a covalent bond; composition.

27. The pHLIP® peptide has the sequence:

27. The composition of claim 26, comprising:

28. The pHLIP® peptide has the sequence:

27. The composition of claim 26, comprising:

29. The pHLIP® peptide has the sequence: or 27. The composition of claim 26, consisting of:

30. The pHLIP® peptide has the sequence:

30. The composition of claim 29, consisting of:

31. The pHLIP® peptide has the sequence:

30. The composition of claim 29, consisting of:

32. 32. The composition of any one of claims 26 to 31, wherein all amino acids in the pHLIP® peptide are D-amino acids.

33. 33. The composition of any one of claims 26-32, wherein the STING agonist comprises a cyclic dinucleotide (CDN).

34. 34. The composition of claim 33, wherein the STING agonist comprises a cyclic purine dinucleotide.

35. The composition of any one of claims 26 to 32, wherein the STING agonist is a non-nucleotide small molecule.

36. STING agonists include diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, SNZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JNJ-6196, BI-STING, GSK532, TTI-10001, and CRD5500. , LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511, and derivatives thereof.

37. 37. The composition of claim 36, wherein the STING agonist is diABZI.

38. 37. The composition of claim 36, wherein the STING agonist is MK-1454.

39. 37. The composition of claim 36, wherein the STING agonist is MK-2118.

40. 37. The composition of claim 36, wherein the STING agonist is TAK-676.

41. 37. The composition of claim 36, wherein the STING agonist is BMS-986301.

42. 37. The composition of claim 36, wherein the STING agonist is BI1387446.

43. The composition of any one of claims 26 to 42, wherein the Linker comprises a disulfide bond or an acid-labile bond.

44. 44. The composition of claim 43, wherein the Linker is self-immolative.

45. The following structure:

27. The composition of claim 26, having:

46. A pharmaceutical composition comprising the composition of any one of claims 26 to 45 and one or more pharmaceutically acceptable excipients.

47. 46. ​​A method of treating cancer in a subject, comprising administering to a subject in need thereof the composition of any one of claims 26 to 45, or the pharmaceutical composition of claim 46.

48. 48. The method of claim 47, wherein the cancer is a solid tumor.

49. The method of claim 47 or 48, wherein the composition of any one of claims 26 to 45 or the pharmaceutical composition of claim 46 is injected directly into the tumor mass.

50. The method of claim 47 or 48, wherein the composition of any one of claims 26 to 45 or the pharmaceutical composition of claim 46 is administered systemically.

51. The method of any one of claims 47 to 50, wherein the subject is a human.

Images