PHLIP-mediated STING agonist delivery

Abstract

Disclosed herein are compositions comprising # imgabs0 # peptides linked to STING agonists, pharmaceutical compositions thereof, and methods of treatment using such compositions.

Description

[0001] Statement Regarding Federally Funded Research

[0002] This invention was made with government support under Grant No. R01 GM073857 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0003] Disclosed herein are compositions comprising a STING agonist linked to Peptide compositions, pharmaceutical compositions thereof, and therapeutic methods using such compositions. Background Art

[0004] Conventional cancer treatments use toxic agents that do not discriminate well between normal and tumor tissues, and therefore their application is limited due to their harmful side effects. Recent studies in cancer immunotherapy have shown that enhancing the innate immune system of cancer patients can have a crucial impact on the outcome of cancer treatment, and are effective when used alone or in combination with conventional treatments. The target of stimulator of interferon genes (STING) agonists is located in the cytoplasm of tumor cells, including cancer cells and activated myeloid cells in the tumor microenvironment. Although STING agonists (STINGa) stimulate immune responses, they must be specifically and effectively targeted to tumor cells to avoid adverse side effects. There are also significant advantages in delivering them to activated immune cells within tumors, thereby more effectively activating the STING pathway and triggering an inflammatory response.

[0005] However, one obstacle to using small molecule STINGa is its poor pharmacokinetics. Another limitation is that some small molecule STINGa tend to be polar molecules and cannot effectively cross cell membranes. STING agonists, such as cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), are hydrophilic and negatively charged molecules. Since the cell membrane is a non-polar, hydrophobic environment, the membrane permeability of such molecules is poor.

[0006] Therefore, the current challenge in the field of immunotherapy is to find ways to deliver STING agonists specifically to the cytoplasm of tumor cells (especially activated immune cells and potentially stromal cells) while avoiding delivery into healthy cells, as well as to improve the pharmacokinetics of the small molecule STINGa. Summary of the invention

[0007] A composition comprising the following structure:

[0008] Peptide-Linker-STING

[0009] The peptide is Peptides,

[0010] wherein the linker is a cleavable linker;

[0011] Wherein "STING" is a STING agonist 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-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 their derivatives; and

[0012] As described herein, each "-" is a covalent bond.

[0013] In some respects, The 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 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 Yg , 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 ,in

[0014] (i) Each Y is independently a having a solvation energy (DG X cor >+0.50) non-polar amino acids or glycine (Gly);

[0015] (ii) each X is independently a protonatable amino acid,

[0016] (iii) n, m, i, j, l, h, g, and f are each independently an integer of 1 to 8.

[0017] In some respects, The peptide comprises a sequence containing the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWXG (SEQ ID NO: 220), wherein "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), an azide-containing amino acid or other modified amino acids. In some aspects, The peptide comprises a sequence consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWXG (SEQ ID NO: 220), wherein "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids.

[0018] In some respects, The peptide comprises a sequence selected from any one of SEQ ID NOs: 1-219. In some aspects, The peptide has a sequence selected from any one of SEQ ID NOs: 1-219. In some aspects, The peptide has a sequence comprising the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide has a sequence comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219). In some aspects, The peptide has a sequence consisting of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide has a sequence consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

[0019] A composition comprising the following structure:

[0020] Peptide-Linker-STING

[0021] Wherein the peptide is a peptide comprising the following sequence Peptides

[0022] ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218) or

[0023] ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 219);

[0024] wherein the linker is a cleavable linker

[0025] wherein STING is a STING agonist; and

[0026] Each "-" is a covalent bond.

[0027] In some respects, The peptide comprises the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide comprises the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219). In some aspects, The peptide consists of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218) or ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219). In some aspects, The peptide consists of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide consists of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

[0028] 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: 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-61 96, BI-STING, GSK532, TTI-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. 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.

[0029] In some aspects, the compositions disclosed herein All amino acids in peptides are D-amino acids.

[0030] 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-destructive.

[0031] In some aspects, the composition has the following structure:

[0032]

[0033] In some aspects, a pharmaceutical composition is provided, comprising at least one composition disclosed herein and one or more pharmaceutically acceptable excipients.

[0034] In some aspects, a method for treating cancer in a subject in need thereof is provided, comprising administering a composition or pharmaceutical composition disclosed herein to the subject. In some aspects, the cancer is a solid tumor. In some aspects, the composition or pharmaceutical composition disclosed herein is injected directly into a tumor mass. In some aspects, the composition or pharmaceutical composition disclosed herein is systemically administered. In some aspects, the subject is a human.

[0035] definition

[0036] Low pH insertion peptide (pH Low Insertion Peptide) is a linear, water-soluble membrane peptide that interacts weakly with the cell membrane under neutral pH conditions and does not insert into the lipid bilayer; however, under weakly acidic pH (<7.0), Inserted into the cell membrane and forms a stable transmembrane α-helix. In addition to tumor cells characterized by low pH (<7.0), immune cells within the tumor mass are also characterized by low pH (<7.0). For example, cells in the tumor mass environment (such as macrophages) are also characterized by low pH. In combination with STING agonists, the compositions and methods deliver drugs specifically and directly to cancer cells and immune cells (macrophages), from their acidic cell surfaces into their cytoplasm. Peptide-delivered STING agonists enhance the immune system, for example by stimulating interferon-γ production to mount and / or establish a tumor-specific immune response to fight cancer.

[0037] As used herein, "effective" when referring to the amount of a composition refers to an amount of the composition sufficient to produce a desired response. For example, when used in the manner disclosed herein, the amount of cargo (e.g., STING agonist) produces a desired response (e.g., enhanced anti-tumor effect) without undue adverse side effects (e.g., toxicity, irritation, or allergic reaction) at a reasonable benefit / risk ratio.

[0038] In some embodiments, the subject is a mammal. In certain embodiments, the mammal is a rodent (e.g., mouse or rat), a primate (e.g., chimpanzee, gorilla, monkey, gibbon, baboon), cattle, camel, dog, cat, horse, llama, sheep, goat or pig. In some embodiments, the subject is a human.

[0039] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the scope of a claim to the specified materials or steps of the claimed invention "and those materials or steps that do not materially affect the basic and novel characteristics" of the claimed invention.

[0040] 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 "disease", "disease state", or "nucleic acid" is a reference to one or more such embodiments, including equivalents thereof known to those skilled in the art, etc.

[0041] As used herein, "treatment" includes, for example, inhibiting the progression of a disease, regression, or stasis. Treatment also includes preventing or ameliorating any symptom or symptom group of the disease. As used herein, "inhibiting" disease progression or disease complications in a subject refers to preventing or reducing disease progression and / or disease complications in a subject.

[0042] As used herein, "pharmaceutically acceptable" carriers or excipients refer to carriers or excipients suitable for use in humans and / or animals, which have no excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions) and have a reasonable benefit / risk ratio. It can be, for example, a pharmaceutically acceptable solvent, suspending agent, or carrier for delivering the compounds of the invention to a subject.

[0043] Each embodiment disclosed herein is applicable to every other disclosed embodiment. Therefore, all combinations of the various elements described herein are within the scope of the present invention.

[0044] Other features and advantages of the present invention will be apparent from the following description of its preferred embodiments and the claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1.Characterization of pHLIP-STINGaa) Schematic diagram of the interaction of pHLIP-STINGa with the membrane lipid bilayer (dark blue represents pHLIP polymers and red circles represent STINGa). In state I, pHLIP-STINGa forms unstructured polymers in normal pH solution. State II represents pHLIP-STINGa interacting with the membrane at normal pH. State III represents that low pH triggers pHLIP to the transmembrane helical orientation, resulting in the translocation of STINGa across the lipid bilayer and its release into the cytoplasm. These three states are monitored by changes in fluorescence (b) and CD (c) spectral signals of pHLIP-STINGa interacting with POPC liposomes. d) shows the kinetics of fluorescence changes triggered by a decrease in pH in the presence of POPC liposomes. The figure shows that the pH transition is monitored by changes in fluorescence intensity (e) and CD (f) spectral signals (experimental points and fitting curves, red, 95% confidence intervals in pink). g) The figure shows the OCD spectra of pHLIP-STINGa recorded immediately after deposition on the support bilayer, and 12 hours later, when pHLIP-STINGa was fully inserted into the bilayer. h) pHLIP-STINGa-induced activation of the IFN signaling pathway in THP1-Blue-ISG cells polarized into M2 macrophages under PMA, IL-4 / IL-13 induction is shown. The results are normalized to the activity at the maximum concentration under the action of STINGa alone, and the activity at the maximum concentration is set as 100%.

[0046] Figure 2.Tumor targeting, PK, biodistribution, tumor and serum cytokines, tumor stroma and immune cell uptake. a) CT26 tumor targeting imaging performed 40 hours after a single intraperitoneal injection of ICG-pHLIP-STINGa (100μM, 300μl) in mice. Tumor sites before and after shaving are shown. b) Normalized fluorescence (mean and SE) recorded in blood collected at different time points after a single intravenous injection of ICG-pHLIP-STINGa (200μM, 100μl) is shown. The data were fitted by an exponential function (red line). c) The kinetics of ICG-pHLIP-STINGa targeting to CT26 tumors and the clearance of the agent from major organs are shown. The mean fluorescence intensity of the area was calculated in each organ and tissue collected at different time points after a single intravenous injection of ICG-pHLIP-STINGa (200μM, 100μl). d) Shows the levels of IL-6 and TNFα cytokines in tumors and serum determined by ELISA at different time points after a single intravenous injection of STINGa or pHLIP-STINGa (200 μM, 150 μl) compared to control mice (all points, means and SE are shown, and p levels were calculated using the Kolmogorov-Smirnov two-tailed nonparametric test). e) The percentage of tumor stromal and immune cell populations within the TME targeted by Al647-pHLIP-STINGa was determined by FACS analysis on CT26 tumors harvested 24 hours after a single intraperitoneal injection of Al647-pHLIP-STINGa (100 μM, 300 μl) (all points, means, standard deviations are shown on the graphs, and data are presented in table form). f) shows that Al647-pHLIP-STINGa co-localizes with Al488-CD140b-antibody-stained CAFs, Al594-CD206-antibody-stained TAMs, and DAPI-stained cell nuclei in the TME, and the images were obtained using objective lenses of different magnifications (10x, 20x, and 40x).

[0047] Figure 3 Eradication of CT26 tumors and generation of immune memory. a) Experimental design. b) Shown is the effect of a single administration of different agents on the growth curve of CT26 tumors in Balb / c mice, injected on day 1, when the tumor volume is approximately 100 mm 3, dose levels were either intraperitoneally injected (100 μM, 300 μl) or intravenously injected (200 μM, 150 μl). The asterisk (*) indicates that this mouse in the intravenous group received a second dose of pHLIP-STINGa on day 41, at which time the tumor had begun to regrow. c) Kaplan-Meier survival curve obtained based on the data in panel b. d) CT26 tumor growth curve obtained from tumor-free mice in the group that received a single intraperitoneal or intravenous injection of pHLIP-STINGa on day 1. On day 61, these mice were re-injected with CT26 cancer cells in the left flank. e) Shown on day 1, when the tumor volume had reached 400-700mm 3 Figure 3 Growth curve of CT26 tumors in mice after a single intraperitoneal injection of pHLIP-STINGa (100 μM, 300 μl).

[0048] Figure 4 .Tumor stroma disappears and pH increases in the TME. ad) FACS analysis was performed to determine the number of cells in the TME in the control group (mice that received a single intraperitoneal injection of vehicle) and the treated group (mice that received a single intraperitoneal injection of Al647-pHLIP-STINGa (100 μM, 300 μl)). The percentage of all viable cells in the TME (a), CAFs quantified as a percentage of all viable cells (CD3 - 、CD45 - 、CD140b + ) number (b), quantified as CD45 + The percentage of immune cells TAM (CD45 + 、CD11b + 、F4 / 80 + 、CD206 + ) (c) and quantified as CD45 + The percentage of mMDSC (CD45 + 、CD3 - 、CD11b + 、F4 / 80 - ,Ly6C hi ,Ly6G - ) (d) (all points, mean and standard deviation, p level < 0.002 for all graphs). eg) shows pH imaging and signal quantification using ICG-pHLIP. Mice bearing CT26 tumors (approximately 100 mm) on the right flank 3) were divided into 2 groups (5 animals per group). One group (treated group) was treated by a single intraperitoneal injection of pHLIP-STINGa (100 μM, 300 μl) on day 1, while the other group (control group) did not receive any treatment. On day 3, the control group (untreated group) and the pHLIP-STINGa treated group were provided with ICG-pHLIP pH imaging probe by a single intraperitoneal injection (50 μM, 100 μl), and the animals were euthanized 24 hours later (day 4), and the tumors were harvested, cut in half and imaged. Representative images are shown in panels ef, and the normalized mean fluorescence signal calculated for all tumor fragments (5 animals per group) is shown in panel g (all points, means and standard deviations are shown, and p levels are calculated using the Kolmogorov-Smirnov two-tailed nonparametric test).

[0049] Figure 5 . Self-immolation kinetics. HPLC chromatograms recorded at 280 nm and 320 nm of pHLIP-STINGa treated with DTT dissolved in 50 mM phosphate buffer (containing 150 mM NaCl, pH 7.4) at different time points (0, 30, 60 minutes) after the addition of DTT are shown. The retention times are as follows: pHLIP-STINGa: 16.78 minutes, pHLIP: 17.02 minutes (no signal at 320 nm), SH-PAB-STINGa (modified diABZI cleaved from pHLIP): 12.16 minutes, STINGa (original diABZI): 10.27 minutes, destruction product: 6.95 minutes.

[0050] Figure 6 .DiABZI fluorescence. Shown are the fluorescence spectra of STINGa (diABZI) excited at 295 nm and 322 nm in phosphate buffer.

[0051] Figure 7 OCD spectra. OCD spectra were recorded after 12 h of deposition of pHLIP-STINGa and POPC solutions in the support layer at pH 5 and pH 3.3.

[0052] Figure 8 .Cell viability. THP1 cells were activated and polarized to M2 macrophages by treating them with PMA for 6 hours and then with IL-4 / IL-13 for another 18 hours. After activation and polarization, all factors were removed and cells were treated with pHLIP-STINGa (pH 6.4) in DMEM without FBS for 3 hours. Then, FBS was added to 10%, the pH was increased to pH 7.4, and the cells were incubated for 48 hours before MTS calorimetry.

[0053] Fig. 9 .Biodistribution of ICG-pHLIP-STINGa in organs and tissues. After a single intravenous injection of ICG-pHLIP-STINGa (200 μM, 100 μl), the mean fluorescence per unit area of ​​each tissue and organ was calculated at 2, 4, 24, 48, 72 and 96 hours after injection. All points, mean values ​​and standard deviations are shown.

[0054] Fig.10 Mean levels of cytokines (IL-6, TNF-α, IFN-β) in tumor supernatants and serum were measured by ELISA 4 and 16 h after a single intravenous (200 μM, 150 μl) or intraperitoneal injection (100 μM, 300 μl) of pHLIP(Laa)-STINGa and pHLIP(Daa)-STINGa.

[0055] Fig.11 .4T1 cancer cells did not generate immune memory. Shown are the 4T1 tumor growth curves of the “control” group (a) and the “second reinjection of 4T1 cancer cells” group (b). The control group mice were implanted with 10 5 The mice in the second re-injection group were first inoculated with 5x10 4 CT26 cancer cells. When the tumor volume reaches about 100mm 3 At day 1, mice received a single intraperitoneal injection of pHLIP-STINGa (100 μM, 300 μl) to eradicate tumors, and then on day 61, mice were inoculated with 5×10 4 Ct26 cancer cells, tumors did not develop, and finally on day 101, 10 5 4T1 cancer cells develop into 4T1 tumors. c) Kaplan-Meier survival curves obtained for the data shown in panels a and b.

[0056] Fig.12 . Treatment of CT26 tumors in athymic nude mice lacking T cells. ab) When the tumor volume of athymic nude mice reached approximately 100-200mm 3 CT26 tumor growth curves in athymic nude mice after a single intraperitoneal injection of pHLIP-STINGa (100 μM, 300 μl) on day 1. c) Kaplan-Meier survival curves obtained for the data shown in panels a and b.

[0057] Fig.13 .4 Treatment of T1 tumors. 10 5 4T1 breast cancer cells were inoculated into the right flank of Balb / C mice. When the tumor volume reached about 100 mm 3On day 1 (day 1), the pHLIP-STINGa group and pHLIP-STINGa+PD-1 group received a single intraperitoneal injection of pHLIP-STINGa (100 μM, 300 μl) (indicated by the red arrow), and the PD-1 group and pHLIP-STINGa+PD-1 group received three intraperitoneal injections of PD-1 antibody (250 μg each injection) on days 4, 9, and 14, respectively (indicated by the blue arrow). The control group did not receive treatment. Tumor growth of the control (a), pHLIP-STINGa (b), PD-1 (c), and pHLIP-STINGa+PD-1 (d) is shown. e) Kaplan-Meier survival curves were drawn based on the data shown in panels ad. Statistical significance for improved survival compared with pHLIP-STINGa versus control and PD-1 versus control was set at a p level of <0.02 and p<0.004 for pHLIP-STINGa+PD-1 versus control, calculated using the logistic regression (by weighting all time points equally), the Breslow method (by weighting all time points by the number of cases at risk at each time point), and the Tarone-Ware method (by weighting all time points by the square root of the number of cases at risk at each time point).

[0058] Fig.14 Body weight. Shown are changes in body weight after a single intraperitoneal injection of pHLIP-STINGa (100 μM, 300 μl) on day 1. Similar body weight curves were obtained in Balb / c and athymic nude mice by other administration routes.

[0059] Fig.15 Intratumoral hemorrhage was observed within 2-4 days after pHLIP-STINGa injection. Images of Balb / c mice bearing CT26 tumors in the right flank are shown the day after a single intraperitoneal injection of pHLIP-STINGa (300 μM, 100 μl). DETAILED DESCRIPTION

[0060] Described herein are compounds comprising a STING agonist linked to Peptide compositions.

[0061] STING agonists

[0062] Cyclic dinucleotides are typical STING agonists, but other small molecules can also be used. 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-619 6. BI-STING, GSK532, TTI-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 their derivatives. In some aspects, the STING agonist is selected from 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-61 96. BI-STING, GSK532, TTI-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 their derivatives. In some aspects, the STING agonist is selected from 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, CRD5500, LB-061, ALG-031048, RVU312-4787, TAK-500, C L656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, 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.

[0063] Peptides

[0064] In some respects, The 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 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 Ym 、(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 ,in

[0065] (i) Each Y is independently a having a solvation energy (DG X cor >+0.50) non-polar amino acids or glycine (Gly);

[0066] (ii) each X is independently a protonatable amino acid,

[0067] (iii) n, m, i, j, l, h, g, and f are each independently an integer of 1 to 8.

[0068] In some respects, The peptide comprises a sequence containing the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWXG (SEQ ID NO: 220), wherein "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), an azide-containing amino acid or other modified amino acids. In some aspects, The peptide comprises a sequence consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWXG (SEQ ID NO: 220), wherein "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids.

[0069] An example of a peptide is AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT (SEQ ID NO: 3), wherein AEQNPIY (SEQ ID NO: 4) represents the flanking sequence, WARYADWLFTTPLLLLDLALLV (SEQ ID NO: 5) represents the membrane insertion sequence, and DADEGT (SEQ ID NO: 6) represents the flanking sequence.

[0070] Other examples The peptides are listed in the table below. The sequences are provided from N-terminus to C-terminus.

[0071] Table 1: Non-limiting examples of sequences.

[0072]

[0073]

[0074]

[0075]

[0076] Table 2: Non-limiting examples of sequences.

[0077] name sequence SEQ ID NO: Var14-Rev Ac-TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA-Am SEQ.ID NO.64 Sh AEQNPIYWARYADWLFTTPL SEQ.ID NO.65 Sh-Cys AEQNPIYWARYADWLFTTPCL SEQ.ID NO.66 Cvs-Sh ACEQNPIYWARYADWLFTT PL SEQ.ID NO.67 Sh-1Trp AEQNPIYFARYADWLFTTPL SEQ.ID NO.68 Sh-W2 AEQNPIYFARYADLLFPTTLAW SEQ ID NO.69 Sh-W1 AEQNPIYWARYADLLFPTTLAF SEQ ID NO.70 Sh-2W AEQNPIYWARYADLLFPTTLAW SEQ ID NO.71 Sh-1D KEDQNPWARYADLLFPTTLAW SEQ.ID NO.72 Sh-1Db KEDQNPWARYADLLFPTTLW SEQ ID NO73 Var12-ID ACEDQNPWARYADLLFPTTLAW SEQ.ID NO.74 Var10-2D ACEDQNPWARYADWLFPTTLLLLD SEQ.ID NO.75 Var13-1E ACEEQNPWARYAELLFPTTLAW SEQ.ID NO.76 Var1 1-2E ACEEQNPWARYAEWLFPTTLLLLE SEQ.ID NO.77 Var7-3E ACEEQNPWARYLEWLFPTETLLLEL SEQ.ID NO.78 Var7-3Eb ACEEQNPQAEYAEWLFPTTLLLLE SEQ ID NO: 79

[0078] *Ac indicates acetylated N-terminus; Am indicates amidated C-terminus

[0079] Table 3: Encoded and exemplary non-encoded amino acids, including L isomers, D isomers, α isomers, β isomers, Diolization and methylation modifications.

[0080]

[0081]

[0082]

[0083]

[0084] Table 4: Non-limiting examples of protonated residues and their substitutions, including L-isomers, D-isomers, α-isomers and β-isomer.

[0085]

[0086] Table 5: Examples of encoded amino acid substitutions.

[0087]

[0088] Table 6: Belonging to different groups Non-limiting examples of membrane insertion sequences for peptides.

[0089] Each protonatable residue (in bold) can be replaced with its substituent in Table 4. Each non-polar residue can be replaced with its encoded amino acid substituent in Table 5 and / or a non-encoded amino acid substituent in Table 3.

[0090]

[0091]

[0092] Table 7: Non-limiting examples of sequences.

[0093] Cysteine, lysine, azide-modified amino acids, or alkyne-modified amino acids can be incorporated into the N-terminal (first six residues) or C-terminal (last six residues) portion of the peptide for conjugation with cargo and linkers.

[0094]

[0095]

[0096]

[0097] In some respects, The peptide comprises a sequence selected from any one of SEQ ID NOs: 1-219. In some aspects, The peptide consists of any one of SEQ ID NOs: 1-219. In other aspects, The peptide has a sequence comprising the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide has a sequence comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219). In some aspects, The peptide has a sequence consisting of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide has a sequence consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

[0098] Connectors

[0099] Linkers can be relatively small, e.g., only a few atoms, to fairly large polymers of 4-5 kDa. One end of the linker can react with a free thiol to spontaneously form a disulfide bond with thiopyridine as a leaving group; the other end reacts with an activated amine or hydroxyl in the presence of DIPEA and, in some cases, DMAP or other activating bases to form a carbamate or carbonate, respectively. If (A) is protected at its amino terminus, for example by N-acetylation. or with a thiol-bearing linker, followed by conjugated, and the conjugate is formed by disulfide exchange (thiopyridine as leaving group). If the amino terminus is protected, for example by N-acetylation, the material can be used with proteins containing lysine residues. A conjugate is formed.

[0100] In some aspects, a succinimidyl 3-(2-pyridyldithio) propionate (SPDP) crosslinker is used. SPDP is a short chain crosslinker used to form cleavable (reducible) disulfide bonds with cysteine ​​sulfhydryls via NHS-ester and pyridyl dithiol reactive groups to achieve conjugation of amines to sulfhydryls. SPDP is used to activate NH2 derivatives 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]), purified and The SH of a single cysteine ​​(Cys) at the C-terminus of the peptide was exchanged for a disulfide bond to obtain

[0101] In some aspects, the following cross-linking agents can 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 cross-linker); PEG12-SPDP (pegylated, long-chain SPDP cross-linker); 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 (bismaleimidoethane disulfide).

[0102] In some aspects, the linker is connected to C-terminus of the peptide.

[0103] Composition

[0104] In one aspect, the compositions disclosed herein comprise the following structure:

[0105] Peptide-Linker-STING

[0106] The peptide is Peptides,

[0107] wherein the linker is a cleavable linker;

[0108] Wherein, "STING" is a STING agonist selected from the following: diABZI, ADU-S100 (MIW815), MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI1387446, E7766, TAK-676, SNZ281, IMSA-101, IMSA-201, SYNB1891, JNJ-4412, JN J-6196, BI-STING, GSK532, TTI-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; and

[0109] Each "-" is a covalent bond.

[0110] In some respects, The peptide has the following sequence: Xn AND m ,AND m X n 、X n AND m X j ,AND m X n AND i ,AND m X n AND i X j 、X n AND m X j AND i ,AND m X n AND i X j AND l 、X n AND m X j AND i X l ,AND m X n AND i X j AND l X h 、X n AND m X j AND i X h AND g ,AND m X n AND i X j AND l X h AND g 、X n AND m X j AND i X h AND g X f (XY) n (YX) n (XY) n AND m (YX) n AND m (XY) n X m (YX) n X m ,AND m (XY) n ,AND m (YX) n 、Xn (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 ,in

[0111] (i) Each Y is independently a having a solvation energy (DG X cor >+0.50) non-polar amino acids or glycine (Gly);

[0112] (ii) each X is independently a protonatable amino acid,

[0113] (iii) n, m, i, j, l, h, g, and f are each independently an integer of 1 to 8.

[0114] In some respects, The peptide comprises a sequence containing the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWXG (SEQ ID NO: 220), wherein "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), an azide-containing amino acid or other modified amino acids. In some aspects, The peptide comprises a sequence consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWXG (SEQ ID NO: 220), wherein "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids.

[0115] In some respects, The peptide comprises a sequence selected from any one of SEQ ID NOs: 1-219. In some aspects, The peptide has a sequence selected from any one of SEQ ID NOs: 1-219. In other aspects, The peptide has a sequence comprising the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, Peptide The peptide has a sequence comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219). In some aspects, The peptide has a sequence consisting of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide has a sequence consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

[0116] In some aspects, the STING agonist is diABZI. In other aspects, the STING agonist is MK-1454. In another aspect, the STING agonist is MK-2118. In another aspect, the STING agonist is TAK-676. In other aspects, the STING agonist is BMS-986301. In yet another aspect, the STING agonist is BI1387446.

[0117] In one aspect, the compositions disclosed herein comprise the following structure:

[0118] Peptide-Linker-STING

[0119] Wherein the peptide is a peptide comprising the following sequence Peptides

[0120] ADQDNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 218) or

[0121] ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 219),

[0122] wherein the linker is a cleavable linker;

[0123] wherein STING is a STING agonist; and

[0124] Wherein each "-" is a covalent bond, also disclosed herein.

[0125] In some respects, The peptide comprises the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide comprises the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219). In some aspects, The peptides consisted of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218) or ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

[0126] In some respects, The peptide consists of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218). In some aspects, The peptide consists of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

[0127] 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 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-61 96, BI-STING, GSK532, TTI-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. 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.

[0128] In some aspects, the compositions disclosed herein All amino acids in peptides are D-amino acids.

[0129] In some aspects, the linker of the present disclosure comprises a disulfide bond or an acid labile bond. In some aspects, the linker is self-destructive. In some aspects, the composition has the following structure:

[0130]

[0131] Treatment

[0132] Provided are methods of treating cancer in a subject in need thereof using a composition as described herein, comprising administering a composition or pharmaceutical composition disclosed herein. In some aspects, the cancer is a solid tumor. In some aspects, the composition or pharmaceutical composition disclosed herein is injected directly into a tumor mass. In some aspects, the subject is a human.

[0133] Examples are provided below to facilitate a more comprehensive understanding of the present invention. The following examples illustrate exemplary ways of 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 only for illustrative purposes, because other methods can be used to obtain similar results.

[0134] Example

[0135] Embodiment 1: Synthesis

[0136] The diABZI STING agonist was modified with a linker to prepare o-pyridyl-dithioethyl-carbamoyl-PAB-STING (Pys-PAB-STINGa). This reagent was synthesized and purified by Iris Biotech GmbH. All pHLIP peptides used in the study were synthesized and purified at CSBio. For conjugation with Pys-PAB-STINGa, the following pHLIP sequences were used, which have a single Cys residue at the membrane-inserting end of the peptide:

[0137] pHLIP(Laa):ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218), wherein all amino acids are in the L configuration, and

[0138] pHLIP (Daa): ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219), wherein all amino acids are in the D configuration.

[0139] The peptide and Pys-PAB-STINGa were mixed in dimethyl sulfoxide (DMSO) at a molar ratio of 1:1. Sodium phosphate buffer (100 mM) containing 150 mM NaCl (pH 7.4), saturated in argon, was then added to the reaction mixture (1 / 10 of the total volume), and the reaction mixture was kept at room temperature (RT) for 2 hours. Purification was performed by reverse phase high performance liquid chromatography (HPLC) using a Zorbax SB-C18, 9.4×250 mm, 5 μm column (Agilent Technology) Constructs, gradient from 10% to 75% acetonitrile / water (containing 0.05% trifluoroacetic acid (TFA)). To prepare the fluorescent version of the reagent ( and ICG-pHLIP-STINGa), an N-acetylated version of the pHLIP peptide AKDDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 221) was used, in which all amino acids were in the L configuration. Conjugation with Pys-PAB-STING followed by purification. ICG-NHS ester (IrisBiotech GmbH) or Alexa647-NHS ester (Life Technologies) was then conjugated to the N-terminal lysine residue of pHLIP in a molar ratio of 1:1.5 in DMSO. Sodium bicarbonate buffer (100 mM) with a pH value of 8.3 was added to the reaction mixture (1 / 10 of the total volume), and the reaction mixture was kept at room temperature until the 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 the pHLIP-STINGa conjugate was determined by absorbance using the following molar extinction coefficient: for pHLIP-STINGaε 322 =46,200M -1 cm -1 For ICG-pHLIP-STINGaε 800 =137,000M -1 cm -1 and for Al647-pHLIP-STINGaε 651 =270,000M -1 cm -1 .

[0140] Example 2: Stability in mouse and human plasma

[0141] To determine 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 stored in plasma at 37°C for 0, 2, 4, or 24 hours. Plasma proteins were precipitated with methanol (1:5 volume ratio of plasma to methanol) and centrifuged at 13.4 rpm for 10 minutes. The supernatant was collected and analyzed by HPLC using a Zorbax SB-C18 4.6×250 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. pHLIP (Laa) was used as a control, and the stability of pHLIP (Daa), Pys-PAB-STINGa, SH-PAB-STINGa (wherein Pys-PAB-STINGa was conjugated with a Cys residue) and diABZI (Invivogen) in plasma was analyzed under the same conditions. The results are shown in Table 8 below.

[0142] Table 8

[0143]

[0144] Example 3: Self-destruction dynamics

[0145] To trigger the self-destruction of the linker, pHLIP(Laa)-STINGa solutions were 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 4.6×250mm, 5μm column with a gradient of 10% to 75% acetonitrile in water (containing 0.05% TFA). Chromatograms were recorded at 220nm, 280nm and 320nm. DiABZI and pHLIP were used as controls under the same HPLC conditions.

[0146] The results of the study are shown in Figure 5 middle.

[0147] Example 4: Biophysical studies

[0148] The fluorescence and circular dichroism (CD) of the constructs were recorded using a PC1 fluorescence spectrophotometer (ISS) and a MOS-450 spectrometer (Bio-Logic Science Instruments), respectively, to study the interaction of pHLIP(Laa)-STINGa with liposomes, and the temperature control was set to 25°C. Liposomes consisting of large unilamellar vesicles were prepared by extrusion. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids (Avanti Polar Lipids) in chloroform were desolvated on a rotary evaporator and dried under vacuum for at least 2 hours. The phospholipid film was rehydrated in 2 mM citrate phosphate buffer (pH 7.3), vortexed, and passed through an extruder (using a 50 nm membrane pore size) 21 times.

[0149] Fluorescence spectra were recorded from 310 nm to 550 nm at an excitation wavelength of 295 nm and a slit size of 1.0 mm. The excitation polarizer was set to 54.7 degrees (the "magic angle"), while the emission polarizer was set to 0 degrees to reduce Wood's anomaly. CD spectra were recorded from 190 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. In addition, the fluorescence of STINGa (diABZI) was recorded under excitation at 295 nm and 350 nm wavelengths.

[0150] The pH-dependent insertion of pHLIP-STINGa into the lipid bilayer of POPC liposomes was studied by monitoring the fluorescence intensity at 400 nm as a function of pH or the molar ellipticity at 230 nm as a function of pH. After adding an equal portion 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 and ISE benchtop meter. The normalized fluorescence intensity or milli-ellipticity values ​​were plotted as a function of pH. The pH dependence was fitted with the Henderson-Hasselbach equation to determine the cooperativity (n) and midpoint (pK) of the transition. The fitting equation used was

[0151] For a single conversion and

[0152] #imgpt90#For both transformations.

[0153] Where SII and SIII represent the spectral signals in states II and III, respectively, and SII′ represents the CD signal of the intermediate between states II and III.

[0154] Fluorescence kinetics were measured using an SFM-300 mixing system (Bio-Logic Science Instruments) coupled to a MOS-450 spectrometer with temperature control set to 25°C. All samples were degassed prior to measurement to minimize bubbles in the sample. pHLIP-STINGa and POPC samples were incubated overnight to reach equilibrium, at which point most of the reagents were bound to the liposome lipid bilayer. To follow the insertion of pHLIP-STINGa into the membrane, a solution containing 14 μM pHLIP-STINGa and 2.8 mM POPC was mixed with citric acid to lower the pH from pH 8 to 3.5. To monitor the changes in fluorescence intensity during the insertion of pHLIP-STINGa into POPC liposomes induced by the pH drop, the emission signal was observed through a 320 nm cutoff filter under 295 nm excitation.

[0155] For the measurement of oriented circular dichroism (OCD), supported bilayers were prepared on quartz slides that were specially polished for far-UV measurements (Starna). The slide cleaning procedure consisted of the following steps: 1) soaking in cuvette cleaning solution for 24 h, 2) rinsing with deionized distilled water, 3) sonication in 2-propanol for 10 min, 4) sonication in acetone, 5) sonication again in 2-propanol, 6) rinsing with deionized water, 7) soaking in piranha solution consisting of 25% hydrogen peroxide and 75% sulfuric acid, 8) rinsing with Milli-Q purified water. POPC lipid monolayers were deposited on quartz substrates by the Langmuir-Blodgett (LB) method using a (KSV microcell). To deposit LB, a small amount of POPC lipids was dissolved in chloroform, spread on the subphase surface, and the solvent was allowed to evaporate for about 10 min. Next, the monolayer was compressed to 32 mN / m. When the surface pressure stabilized, the first slide was inserted into the groove and held for 60 seconds to allow the surface pressure to stabilize again, and then it was pulled out of the subphase at a speed of 10 mm / min. The second layer was prepared by the method of POPC vesicle fusion. About 80 μl of sample (containing 7 μM pHLIP-STINGa and 0.7 mM POPC in 2 mM pH 5.0 or 2 mM pH 3.3 citrate phosphate buffer) or POPC blank control (containing 0.7 mM POPC (without pHLIP-STINGa) in citrate phosphate buffer) was spread on the slide. The process was repeated for another eight slides, which were then stacked together. Spacers were placed between the slides to prevent them from sticking. The "0 hour" OCD spectra of the samples in the two pHs and the POPC blank control were measured. The slides were then stored at 100% humidity at 4°C for 6 hours. After 6 hours, the excess solution on each slide was removed and replaced with 80 μL of buffer of the corresponding pH. The slides were stacked together again and filled with buffer to obtain a complete set of 8 slides (16 double layers) and stored at 100% humidity at 4°C for another 6 hours. At the end of the 12-hour incubation period, the "12-hour" OCD spectrum was measured. The OCD spectrum of the POPC blank control was subtracted from the OCD spectrum of the sample.

[0156] All data were fit to appropriate equations by a nonlinear least squares curve fitting procedure using Origin 8.5 with the Levenberg-Marquardt algorithm.

[0157] Characterization of pHLIP-STINGa Figure 1 , Figure 5 and Figure 6 shown.

[0158] Example 5: Activation of IFN in cells

[0159] Using THP-1-Blue TM -ISG cells (Invivogen), which express interferon (IFN) regulatory factor (IRF)-induced secretory embryonic alkaline phosphatase (SEAP) reporter constructs. Cells were cultured in RPMI growth medium supplemented with L-glutamine, sodium pyruvate, 10% fetal bovine serum (FBS), normocin and ciprofloxacin hydrochloride in a humidified atmosphere (5% CO2 and 95% air) at 37°C. Cells were seeded in 96-well plates at a density of 75,000 cells / well. In order to generate M2 polarized macrophages, cells were first treated with 185ng / mL phorbol 12-myristate 13-acetate (PMA) for 6 hours, then 20ng / mL of interleukin 4 (IL-4) IL-4 and 20ng / mL interleukin 13 (IL-13) (both from PeproTech) were added and treated for another 16 hours. After polarization was completed, the growth medium was replaced with Dulbecco's modified Eagle's medium (DMEM) without FBS, pH 6.9, containing increasing concentrations of pHLIP(Laa)-STINGa or STINGa (up to 10.0 μM). After two hours of incubation, an equal volume of RPMI supplemented with 20% heat-inactivated FBS was added and the cells were incubated for an additional 48 hours. TM Solution (Invivogen) was used to assess SEAP activity to evaluate type I interferon protein levels: 150 μl of colorimetric reagent was added to 50 μl of cell supernatant, incubated at 37°C for 30 min, and then the absorbance was measured at 655 nm.

[0160] Example 6: Cell Viability

[0161] THP1 cells (ATCC, TIB-202) were cultured in RPMI growth medium supplemented with 2-mercaptoethanol, 10% FBS and ciprofloxacin hydrochloride in a humidified atmosphere (5% CO2 and 95% air) at 37°C. Cells were seeded in 96-well plates at a density of 30,000 cells / well. To generate M2 polarized macrophages, cells were first treated with 185ng / mL PMA alone for 6 hours, followed by the addition of 20ng / mL IL-4 and IL-13 for another 16 hours. After polarization was completed, the medium was replaced with DMEM (pH6.4) without FBS containing increasing concentrations of pHLIP(Laa)-STINGa. After three hours of incubation, an equal volume of RPMI supplemented with 20% FBS was added. After 48 hours, the cells were cultured using CellTiter 96AQ ueousCell viability was assessed using the One Solution Cell Proliferation Assay (Promega); the colorimetric reagent was added to the cells for one hour, and then the absorbance was measured at 490 nm.

[0162] The results are shown in Figure 8 .

[0163] Example 7: Treatment of mice

[0164] To treat CT26 tumors, place 5 × 10 4 CT26 mouse colorectal cancer cells (ATCC, CRL-2638) were injected subcutaneously (SQ) into the right flank of female Balb / c mice or athymic female nude mice (strain HsdAthymic Nude-Foxn1nu) at 7 to 9 weeks of age (all from Envigo RMS, Inc). On day 1, when tumors reached 100 mm 3 ("small tumor") or 400-700mm 3 ("large tumors"), mice were randomized, body weights were measured, and agents, including pHLIP(Laa)-STINGa, pHLIP(Daa)-STINGa, pHLIP(Laa), STINGa(diABZI), or vehicle, were administered by a single intraperitoneal (IP) or intravenous (IV) injection. Vehicle, pHLIP(Laa), STINGa(diABZI), and pHLIP(Laa)-STINGa (100 μM, 300 μl) were administered by a single intraperitoneal injection. pHLIP(Daa)-STINGa (200 μM, 150 μl) was administered by a single intravenous injection. Compounds were dissolved in DMSO as stock solutions and transferred to a solution of 20% PEG400 in saline containing 0.9% sodium chloride (vehicle). Less than 2% DMSO remained in the final solution injected into the animals. Tumor volume and body weight were measured 3 times a week throughout the study. Tumors were measured using calipers and tumor volume (V) was calculated according to the following formula:

[0165] V=0.52·L·W 2

[0166] Where L is the length of the measured tumor and W is the width. When the tumor volume is greater than 2000mm 3 When , mice were removed from the study and euthanized.

[0167] On day 61 after a single injection of pHLIP-STINGa, mice without tumors in the pHLIP-STINGa-treated group were challenged again with tumor cells injected into the other flank. The tumor-free mice were housed for an additional 40 days (a total of 100 days after treatment with pHLIP-STINGa), at which point most mice were euthanized. On day 101, five tumor-free mice were again challenged with 10 subcutaneous injections of pHLIP-STINGa into the right flank. 5 4T1 mouse breast cancer cells (ATCC, CRL-2539). In addition, the control group received 10 5 Female Balb / c mice were infected with 4T1 cancer cells, and tumor growth was compared between the groups.

[0168] For the treatment of 4T1 triple-negative breast tumors, place 10 μl of growth medium in 100 μl of 5 4T1 murine breast cancer cells (ATTC, CRL-2539) were injected subcutaneously into the right flank of 7- to 9-week-old Balb / c female mice (Envigo). 3 The time was counted as day 1, body weight was measured, and the mice were randomly divided into four groups. On day 1, mice in groups 2 and 4 received a single intraperitoneal injection of pHLIP(Laa)-STINGa (100μM, 300μl). On days 4, 9, and 14, mice in groups 3 and 4 received three intraperitoneal injections of anti-mouse PD-1 antibody (BioCell, CD279), each injection of 250μg / mouse. Mice in the control group (Group 1) did not receive any treatment. Tumor volume and body weight were measured three times a week. When the tumor volume was greater than 1500mm 3 When , the mice were euthanized.

[0169] The results are shown in Figure 11-Figure 15 .

[0170] Example 8: ELISA in blood and tumor samples

[0171] To determine cytokine levels in blood and tumor samples, 5 × 10 4 CT26 cancer cells were injected subcutaneously into the right flank of female Balb / c mice. When the tumor volume reached 150-250mm 3Mice received a single intravenous or intraperitoneal injection of pHLIP(Laa)-STINGa or pHLIP(Daa)-STINGa, STINGa, or no injection. Animals were euthanized at 4, 16, and 24 hours after injection, and blood and tumors were collected. Blood samples were stored at room temperature for 40 minutes, centrifuged at 5000g for 20 minutes at +4°C, and the supernatant (serum) was collected. Tumors were frozen in liquid nitrogen. 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) and 1 mm diameter zirconium silicate beads (Next Advance). The processed tumor supernatant was used for enzyme-linked immunosorbent assay (ELISA). Matched antibody pair kits for mouse tissue necrosis factor alpha (TNF-α) (Sino Biological), mouse interleukin 6 (IL-6) (Abcam), and pre-coated plates for mouse IFN-β (PBL Assay Science) were used. ELISA assays were performed using serum and tumor samples. For TNF-α, the captured antibody was diluted in phosphate buffered saline (PBS) (Sigma-Aldrich) and for IL-6, the captured antibody was diluted in coating buffer (Abcam). The diluted captured antibody was incubated in the plate at +4°C overnight and washed with PBS / Tween wash buffer (Sigma-Aldrich) the next day. The TNF-α plate was blocked with a wash buffer of 2% bovine serum albumin (BSA) (Thermo Scientific), and the IL-6 and IFN-β plates were blocked with the dilution buffer in the corresponding kits. Blocking was performed for 2 hours at room temperature 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 of each ELISA kit. The samples were incubated for 2 hours at room temperature on an orbital shaker at 200 rpm. The TNF-α and IFN-β plates were incubated with diluted horseradish peroxidase (HRP)-coupled detection antibodies for 1 hour at room temperature. The IL-6 plates were incubated with diluted biotin-conjugated detection antibody for 1 hour at room temperature, followed by incubation with diluted HRP-streptavidin conjugate (Abcam) for 1 hour at room temperature. All plates were washed and incubated with 3,3',5,5'-tetramethylbenzidine (TMB) (Invitrogen) and peroxide solution (mixed in a 1:1 ratio) (Thermo Scientific) for 20 minutes, and then stop solution (10% H2SO4) was added to the plates. The signal from the wells was quantified by measuring the absorbance at 450nm using a Bio-Rad iMark microplate reader. Different dilution schemes were tested in duplicate, and a calibration curve was constructed using antibody standards.

[0172] The results are shown in Fig.10 .

[0173] Example 9: Biodistribution, PK and Imaging

[0174] For pharmacokinetics (PK), biodistribution, and pH imaging, 5 × 10 4 CT26 cancer cells were injected subcutaneously into the right flank of female Balb / c mice, and tumors were grown to a volume of 150-200 mm. 3 . To study PK and biodistribution, a single tail vein injection of ICG-pHLIP(Laa)-STINGa (200 μM, 100 μl) was given. Animals were euthanized at 2, 4, 24, 48, 72 and 96 hours after injection, and blood was collected in K2 EDTA vacuum blood collection tubes (BD), and autopsy was performed immediately after euthanasia. Blood, tumors and major organs (kidneys, liver, spleen, pancreas, lungs, heart, large and small intestines, bones, muscles, brain) were collected and imaged ex vivo immediately after autopsy. Blood (150 μl) was placed in a 96-well plate with a black bottom and walls for imaging. At time zero (0 minutes), ICG-pHLIP-STINGa diluted in blood collected from control mice (which never received any injections) was imaged (the dilution was based on the assumption that mice contain 80 ml / kg of blood). The fluorescence at time zero was defined as 100%, and then the fluorescence at 2, 4, 24, and 48 hours after injection was recorded and calculated as a percentage of the signal at time zero. A single exponential decay function was fitted to these points to determine the half-life.

[0175] For in vivo imaging, when tumors reach 150-250 mm 3 Balb / c and athymic nude mice were given a single IP (200 μM, 100 μl) or IP (300 μM, 150 μl) injection of ICG-pHLIP(Laa)-STINGa and imaged in vivo at 1, 2, 4, 24, 40-48, 74, 100, 170, and 195 h after injection.

[0176] For pH imaging studies, mice were divided into 2 groups. On day 1, mice in group 1 received a single intraperitoneal injection of pHLIP(Laa)-STINGa (100 μM, 300 μl), while mice in the control group (group 2) did not receive any treatment. On day 3, mice in groups 1 and 2 received a single intraperitoneal injection of the acidity imaging probe ICG-pHLIP (50 μM, 100 μl) (Iris Biotech, GmbH). On day 4 (or 24 hours after injection of ICG-pHLIP), all animals were euthanized, tumors were harvested, cut in half and imaged.

[0177] In vivo and in vitro brightfield and near-infrared fluorescence imaging was performed using a Stryker 1588AIM endoscope system equipped with an L10 AIM light source (808 nm excitation light, light collection range of approximately 815 to 850 nm) and a 1588AIM camera (using a 10 mm lens). The lens was maintained at a fixed distance from the organ surface in an enclosed (light-blocked) area. Imaging was performed under three different settings. Digital images of organs were saved as green channel data, converted to 8-bit files, and processed using ImageJ software. Background with pixel intensity of 0 was excluded by setting a pixel intensity threshold (ranging from 1 to 255). Brightfield images were used to determine the boundaries of organs and tumors. The calculated total fluorescence intensity and total area of ​​each organ were used to calculate the average organ fluorescence.

[0178] The results of the study are shown in Figure 2 , Figure 3 and Fig. 9 middle.

[0179] Example 10: FACS analysis

[0180] Fluorescence activated cell sorting (FACS) was used to analyze the uptake of Al647-pHLIP(Laa)-STINGa by tumor cells in CT26 tumors. 5 CT26 cancer cells were injected subcutaneously into the flank of female Balb / c mice (8 to 12 weeks old). 3 At 1 pm, the mice were divided into two groups. Eight mice in the treatment group (Group 1) received a single intraperitoneal injection of Al647-pHLIP(Laa)-STINGa (300 μM, 100 μl), and five mice in the control group (Group 2) received a single intraperitoneal injection of vehicle (1% DMSO in PBS). After 24 hours, all animals were euthanized and tumors were collected for processing. The following markers were used to identify tumor stroma and immune cells:

[0181] CD4 T cells: CD45 + 、CD3 + 、CD11b - 、CD4 + 、CD8 -

[0182] CD8 T cells: CD45 + 、CD3 + 、CD11b - 、CD4 - 、CD8 +

[0183] Treg:CD45 + 、CD3 + 、CD11b - 、CD4 + 、CD25 + 、FoxP3 +

[0184] mMDSC:CD45 + 、CD3 - 、CD11b + 、F4 / 80 - ,Ly6C 高 ,Ly6G -

[0185] gMDSC:CD45 + 、CD3 - 、CD11b + 、F4 / 80 - ,Ly6C 低 ,Ly6G +

[0186] M1:CD45 + 、CD11b + 、F4 / 80 + 、CD206 -

[0187] M2:CD45 + 、CD11b + 、F4 / 80 + 、CD206 +

[0188] DC:CD45 + 、CD11b + 、CD11c + , MHCII + 、F4 / 80 -

[0189] CAF:CD3 - 、CD45 - 、CD140b +

[0190] Stain tumor samples, fluorescence minus one (FMO) controls, and single color controls (SCC) as follows:

[0191] Tumor: Live / Dead, CD45, CD3, CD4, CD8, CD25, FoxP3, CD11b, F4 / 80, Ly6C, Ly6G, CD206, CD11c, MHCII, CD140b

[0192] Tumor-FMO: CD3, CD25, FoxP3, F4 / 80, Ly6C, Ly6G, CD206, CD11c, MHCII, CD140b

[0193] SCC: Unstained, CD45, CD3, CD4, CD8, CD25, FoxP3, CD11b, F4 / 80, Ly6C, Ly6G, CD206, CD11c, MHCII, CD140b, Live / Dead

[0194] 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 (from BioLegend); CD3e-BUV496 clone 145-2C11, CD4-BUV395 clone GK1.5; CD11b-BUV737 clone M1 / 70 (from BD Biosciences), FoxP3 PerCP-Cy5.5 clone FJK-16s (Thermo Fisher) and Live / Dead Aqua-V500 (Life Technologies). Tumor processing procedures were as follows: GentleMACS TM Tumor samples were dissociated using the protocol "Tumor Dissociation Kit". The samples were filtered through a 70 μm cell strainer, rinsed twice in PBS / 2.5% FBS buffer, and then the total sample volume was measured. 7Single cell suspensions were prepared at a concentration of 10 cells / mL and placed in individual wells of a 96-well plate on ice. All incubation steps were performed in the dark. Washing was performed as follows: the culture plate was spun at 300 × g (or 400 × g) for 3 minutes and the supernatant was discarded. Live / dead reagent was added to each sample and incubated at 4°C for 15 minutes before washing. Fc-blocking agent (Mu TruStain FcX / anti-FcγRIV, Biolegend) diluted in staining buffer (BD) was added to the sample and incubated at 4°C for 10 minutes, followed by the addition of cell surface antibodies diluted in staining buffer supplemented with Brilliant Stain Buffer Plus (BD), incubated at 4°C for 30 minutes, and then washed. Cells were fixed using FoxP3 fixation / permeabilization solution and incubated at room temperature for 30 minutes before washing. For single-color controls, one drop of Ultra Comp beads (Thermo Fisher) was added to each single-color control well. For live / dead controls, a drop of ArC amine-reactive compensation beads (Life Technologies) was added to each corresponding well, and then each antibody was added to the appropriate well. Al647-pHLIP-STINGa was imaged in the Alexa47 channel, and the Al647 clone MOPC-21 (BioLegend) was used as an isotype control in the Alexa47 channel. The incubation step was followed by a wash step. The cell number and cellular uptake of Al647-pHLIP-STINGa were determined for the control and treatment groups.

[0195] The results are as follows Figure 4 shown.

[0196] Example 11: Immunohistochemistry and imaging

[0197] For immunohistochemical analysis, 5 × 10 4 CT26 cancer cells were injected subcutaneously into the right flank of female BALB / c mice. When the tumor volume reached 150-250mm 3Mice received a single intraperitoneal injection of Al647-pHLIP(Laa)-STINGa (300 μM, 100 μl) at 4 °C. Tumors were cryosectioned using a ThermoFisher HM525 Nx to prepare 10-20 μm sections. 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 and eosin (H&E) using hematoxylin 7211 (ThermoFisher) and eosin Y (Poly Scientific). The antibody-stained sections were air dried for 10 minutes, then washed with distilled water for 2 minutes, fixed in 4% paraformaldehyde 37% (Sigma-Aldrich) for 12 minutes, washed with Dulbecco's phosphate buffered saline (DPBS) (Sigma-Aldrich) for 5 minutes, and air dried again for 10 minutes. A layer of vaseline (Equate) was applied to the edge of the slide around the tissue and then covered with a coverslip. The sections were incubated with blocking buffer containing 5% of 10% BSA (Thermo Fisher) at room temperature for 2 hours and then washed. The sections were added with antibodies in blocking buffer, treated at room temperature for 2 hours, and then washed. Coverslips were mounted using organic / limonene on the surface of the tissue. Imaging of tissue sections was performed using an EVOS Fl Auto 2 fluorescent inverted microscope, using 10x, 20x and 40x objectives, through appropriate filters in bright field and fluorescence mode.

[0198] Example 12: Statistical Analysis

[0199] The Kolmogorov-Smirnov two-tailed nonparametric test was used to establish the p-level. For the survival curves, the p-level was established by using the log-rank (all time points were weighted equally), the Breslow method (all time points were weighted by the number of cases at risk at each time point), and the Tarone-Ware method (all time points were weighted by the square root of the number of cases at risk at each time point).

[0200] The patents and scientific literature mentioned herein establish the relevant knowledge possessed by those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are hereby incorporated by reference herein. All published foreign patents and patent applications cited herein are hereby incorporated by reference herein. The Genbank and NCBI submissions cited herein (represented by accession numbers) are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference herein.

[0201] While the present invention has been particularly shown and described with reference 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 defined in the appended claims.

Claims

1. A composition comprising the following structure: Peptide-Linker-STING The peptide is Peptides, wherein the linker is a cleavable linker; in, "STING" is a STING agonist 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-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 their derivatives; and Each "-" is a covalent bond.

2. The composition according to claim 1, wherein The 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 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 X m , Y m (XY) n and Y m (YX) n and X n (XY) m and X n (YX) m and (XY) n Y m (XY) i and (YX) n Y m (YX) i and (XY) n X m (XY) i and (YX) n X m (YX) i and Y m (XY) n and Y m (YX) n and X n (XY) m or X n (YX) m where (i) Each Y is independently a having a solvation energy (DG X cor >+0.50) non-polar amino acids or glycine; (ii) each X is independently a protonatable amino acid, (iii) n, m, i, j, l, h, g, and f are each independently an integer of 1 to 8.

3. The composition according to claim 1 or 2, wherein The peptide comprises a sequence containing the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWXG (SEQ ID NO: 220), wherein "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), an azide-containing amino acid or other modified amino acids.

4. The composition according to claim 1 or 2, wherein The peptide comprises a sequence consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWXG (SEQ ID NO: 220), wherein "X" is a functional group for conjugation purposes selected from lysine (Lys), cysteine ​​(Cys), azide-containing amino acids or other modified amino acids.

5. The composition according to claim 1 or 2, wherein The peptide comprises a sequence selected from any one of SEQ ID NOs: 1-219.

6. The composition according to claim 1 or 2, wherein The peptide has a sequence selected from any one of SEQ ID NOs: 1-219.

7. The composition according to claim 1 or 2, wherein The peptide has a sequence comprising the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218).

8. The composition according to claim 1 or 2, wherein The peptide has a sequence comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

9. The composition according to claim 1 or 2, wherein The peptide has a sequence consisting of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218).

10. The composition according to claim 1 or 2, wherein The peptide has a sequence consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

11. The composition according to any one of claims 1 to 10, wherein the All amino acids in peptides are D-amino acids.

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

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

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

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

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

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

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

19. The composition of claim 18, wherein the linker is self-destructive.

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

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

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

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

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

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

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

27. The composition according to claim 26, wherein the The peptide comprises the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218).

28. The composition according to claim 26, wherein the The peptide comprises the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

29. The composition according to claim 26, wherein the The peptide consists of the following sequence (a) ADQDNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 218) or (b) ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 219).

30. The composition according to claim 29, wherein the The peptide consists of the sequence ADQDNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 218).

31. The composition according to claim 29, wherein the The peptide consists of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 219).

32. according to the composition described in any one of claims 26-31, wherein said All amino acids in peptides are D-amino acids.

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

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

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

36. The composition of any one of claims 26-32, wherein the STING agonist is selected from 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, CRD5500, LB-061, ALG-031048, RVU312-4787, TAK-500, CL656, CDK-002, CF501, CF502, CF503, CF504, CF505, CF506, CF507, CF508, CF509, CF510, CF511 and their derivatives.

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

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

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

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

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

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

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

44. The composition of claim 43, wherein the linker is self-destructive.

45. The composition of claim 26, wherein the composition has the following structure:

46. ​​A pharmaceutical composition comprising the composition of any one of claims 26-45 and one or more pharmaceutical excipients.

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

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 26-45 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 26-45 or the pharmaceutical composition of claim 46 is administered systemically.

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

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