Targeted antigen delivery systems and their use

Abstract

An antigen delivery system is disclosed, the nanoparticles being surface-modified with a cancer-specific cell-targeting peptide and comprising an immunogenic HLA class I-restricted peptide, the HLA class I-restricted peptide being a vaccine-dependent immunogenic HLA class I-restricted peptide, the cancer-specific cell-targeting peptide comprising the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1). A method of treating a subject with cancer is disclosed, the method comprising administering one of the antigen delivery systems disclosed herein to the subject with cancer. A method of killing cancer cells is disclosed, the method comprising contacting cancer cells with any of the antigen delivery systems disclosed herein, wherein upon entry of the liposomes into the cancer cells, the cancer cells present the vaccine-dependent immunogenic HLA class I-restricted peptide from the antigen delivery system, and the cancer cells generate an immune response against the vaccine-dependent immunogenic HLA class I-restricted peptide, and the immune response against the vaccine-dependent immunogenic HLA class I-restricted peptide targets and kills the cancer cells presenting the vaccine-dependent immunogenic HLA class I-restricted peptide. Disclosed are methods of generating a non-cancer secondary immune response that targets cancer cells, the method comprising administering to a subject having cancer cells one of the antigen delivery systems disclosed herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 081,178, filed September 21, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under W81XWH - 16 - 1 - 0262 awarded by the USA Medical Research Acquisition Activity (USAMRAA), and under 7R01CA164447 and 5R01CA164447 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Reference to a Sequence Listing The sequence listing filed on September 21, 2021, as a text file named "37794_0097Pl_Sequence_Listing.txt" created on September 21, 2021, and having a size of 3,894 bytes, is incorporated herein by reference in accordance with 37 C.F.R. § 1.52(e)(5) of the United States Patent Laws and Regulations.

Background Art

[0004] Cell - mediated (CM) immunotherapy for cancer treatment is designed to activate the body's adaptive immune response against malignant tumors. Generally, the goal of the CM response is to activate a cytotoxic T - cell response against the tumor to eliminate cancer cells. Although the principles of these treatments are simple, current research studying the complexity of the tumor microenvironment and methods attempting to directly activate T cells against tumor antigens demonstrate the difficulties associated with generating an immune response against tumors.

[0005] Several cancer immunotherapies for comorbidity (CM) exist today, including PD-1 inhibitors, injection of live viruses or viral particles into tumors, and adoptive T-cell therapy. However, concerns regarding efficacy, safety, and / or cost limit the use of many of these therapies. To address these concerns, an immune response to HLA class I peptides can be generated using a procedure based on developing a completely synthetic minimal delivery system that facilitates the presentation of human leukocyte antigen (HLA) class I restriction immunogenic peptides specifically on cancer cells, without using live viruses, viral subunits, or biologically derived materials.

[0006] Liposome-based drugs are being developed that consist of neutral stealth liposomes that encapsulate synthetically produced immunogenic HLA class I restriction peptides. In addition, the liposomes have targeted peptides on their outer surface that both specifically accumulate in cancer cells and promote the presentation of immunogenic peptides in HLA class I molecules. Disclosed herein are targeted peptides that exhibit better efficacy in antigen delivery systems. [Overview of the project]

[0007] An immunotherapy based on an antigen delivery system is disclosed that facilitates the presentation of non-cancerous HLA class 1 restriction immunogenic peptides in cancer cells, thereby inducing a secondary immune response against cancer cells.

[0008] An antigen delivery system comprising nanoparticles is disclosed, wherein the nanoparticles are surface-modified with a cancer-specific cell-targeting peptide and contain an immunogenic HLA class I restriction peptide, wherein the HLA class I restriction peptide is a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide comprises the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1). An example of the disclosed antigen delivery system is an antigen delivery system comprising PEGylated liposomes, wherein the PEGylated liposomes are surface-modified with a cancer-specific cell-targeting peptide and contain an immunogenic HLA class I restriction peptide, wherein the HLA class I restriction peptide is a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide comprises the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1).

[0009] A method for treating a subject having cancer is disclosed, comprising administering to the subject having cancer one of the antigen delivery systems disclosed herein. For example, a method for treating a subject having cancer is disclosed, comprising administering an antigen delivery system comprising a PEGylated liposome, wherein the PEGylated liposome is surface-modified with a cancer-specific cell-targeting peptide and comprises an immunogenic human leukocyte antigen (HLA) class I restriction peptide, wherein the HLA class I restriction peptide is a vaccine-dependent immunogenic HLA class I restriction peptide and the cancer-specific cell-targeting peptide comprises the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1).

[0010] A method for treating a subject having cancer is disclosed, comprising administering to the subject having cancer one of the antigen delivery systems disclosed herein. For example, a method for treating a subject having cancer is disclosed, comprising administering an antigen delivery system comprising a PEGylated liposome, wherein the PEGylated liposome is surface-modified with a cancer-specific cell-targeting peptide and comprises an immunogenic HLA class I restriction peptide, wherein the HLA class I restriction peptide is a vaccine-dependent immunogenic HLA class I restriction peptide and the cancer-specific cell-targeting peptide comprises the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1).

[0011] A method for killing cancer cells is disclosed, comprising contacting the cancer cells with one of the antigen delivery systems disclosed herein, wherein, upon entry of the liposomes into the cancer cells, the cancer cells present a vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system, the cancer cells generate an immune response to the vaccine-dependent immunogenic HLA class I restriction peptide, and the immune response to the vaccine-dependent immunogenic HLA class I restriction peptide targets and kills the cancer cells presenting the vaccine-dependent immunogenic HLA class I restriction peptide.

[0012] A method for generating a non-cancer secondary immune response targeting cancer cells is disclosed, comprising administering one of the antigen delivery systems disclosed herein to a subject having cancer cells, wherein the liposomes enter the cancer cells in the subject, the cancer cells present a vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system, the subject generates a non-cancer secondary immune response to the vaccine-dependent immunogenic HLA class I restriction peptide, and the non-cancer secondary immune response targets and kills the cancer cells presenting the vaccine-dependent immunogenic HLA class I restriction peptide.

[0013] Additional advantages of the disclosed methods and compositions are partly described in the following description, partly understood from the description, or can be learned through the practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and achieved by the elements and combinations specifically indicated in the appended claims. It should be understood that both the above summary and the following modes for carrying out the invention are merely illustrative and descriptive and do not limit the invention as described in the claims. [Brief explanation of the drawing]

[0014] The accompanying drawings incorporated herein and forming part thereof illustrate several embodiments of the disclosed methods and compositions, and together with the descriptions, help to illustrate the principles of the disclosed methods and compositions.

[0015] [Figure 1] The following are exemplary flow cytometry results showing normalized alanine variants (A1, A2, A3, and A4) of SRI_MGS5 on H1993 cells. [Figure 2] Examples of relative binding between acetylated SRI_MGS5 peptide and non-acetylated SRI_MGS5 peptide on H1993 and H1299 cells are shown. SRI_MGS5 is a targeted peptide that contains or derives LQWRRDDNVHNFGVWARYRL (SEQ ID NO: 2). For example, SRI_MGS5_V1 is a dimer containing LQWRRDDNVHNFGVWARYRL (SEQ ID NO: 2), and SRI_MGS5_V2 is a dimer containing LQWRRNFGVWARYRL (SEQ ID NO: 1). [Figure 3] Exemplary flow cytometry results showing increased binding of the SRI_MGS5_V2 peptide on NSCLC strains compared to the parent peptide SRI_MGS5_V1 are presented. [Figure 4] Examples of quantitative uptake of SRI_MGS5_V2 at 1H in three NSCLC cell lines at various concentrations are shown. Uptake is expressed as the average number of peptides per cell. Error bars are smaller than some point symbols. [Figure 5] Exemplary flow cytometry results showing the exact number of peptide molecules internalized by the H358 NSCLC cell line over time when incubated with 100 nM dye-labeled SRI_MGS2_V2 are shown. Due to differences in the Y-axis, H358 cell line data are not included in Figure 6 for clarity. [Figure 6] Exemplary flow cytometry results are shown, illustrating the precise number of peptide molecules internalized over time by various NSCLC cell lines when incubated with 100 nM dye-labeled SRI_MGS5_V2. [Figure 7A-7C] Examples of co-localization of the SRI_MGS5_V2 peptide with autophagocytic vesicles (A) H1993 cells treated with 100 nM SRI_MGS5_V1 peptide (Panel A) and optimized SRI_MGS_V2 conjugated to Alexa Fluor 647 (Panels B and C) are shown (red). Autophagocytic vesicles are stained with LC3B antibody (green). Regions corresponding to co-localization of the peptide with autophagocytic vesicles are shown in yellow. [Figure 8] Exemplary near-infrared (NIR) imaging results showing tumor accumulation of SRI_MGS5_V1 and SRI_MGS5_V2 are shown. The peptides were conjugated to Alexa Fluor 750 dye, and 15 μg was intravenously injected via the tail vein into mice with a subcutaneous H1993 NSCLC tumor in the right flank. Both peptides localized to the tumor site within 24 hours, but SRI_MGS5_V1 showed a significant signal along the dorsal side of the animal at 24H. The signal was retained at 72H. [Figure 9] Figure 6 shows an example of quantifying tumor uptake from in vivo images. Both peptides exhibit similar levels of tumor targeting. [Figure 10] Ex vivo imaging of tumors and other organs at 72H from the experiment shown in Figure 6. [Figure 11] This image shows an exemplary in vivo fluorescence image of C57BL / 6 mice 72 hours after injection with SRI_MGS5_V2 conjugated to Alexa Fluor 750 dye. [Figure 12]Exemplary ex vivo fluorescence images of organs obtained from C57BL / 6 mice 72 hours after injection with SRI_MGS5_V2 conjugated to Alexa Fluor 750 dye are shown. [Figure 13] Ex vivo imaging of tumors and other organs at 72H from the experiments shown in Figures 11 and 12 is shown. [Figure 14] The size distribution of antigen-loaded nanoparticles obtained via dynamic light scattering technology is shown. [Figure 15] Examples of the co-localization of autophagosomes with GFP-loaded nanoparticles conjugated to SRI_MGS5_V1 and SRI_MGS5_V2 peptides are shown. The liposomes are loaded with GFP and appear green. Autophagosomes are stained with an LC3B antibody (red). Regions corresponding to the co-localization of liposomes and autophagosomes are shown in yellow. [Figure 16] Exemplary TNF-α ELISA results showing that the SRI_MGS2_V2 targeting peptide facilitates the presentation of encapsulated immunogenic peptides by HLA class I molecules are shown. H1993 cells treated with TALL were co-cultured with HLA-A2 positive PBMC, and the culture supernatant was analyzed for TNF-a secretion. The concentration of all liposomes used in this assay corresponded to approximately 4.5 mg / ml of phosphatidylcholine. The free H250 peptide control was incubated at 5 μmol / L. [Figure 17] Exemplary IFNγ ELISA results showing that the SRI_MGS2_V2 targeting peptide facilitates the presentation of encapsulated immunogenic peptides by HLA class I molecules are shown. H1993 cells treated with TALL were co-cultured with HLA-A2 positive PBMC, and the culture supernatant was analyzed for IFNγ secretion. The concentration of all liposomes used in this assay corresponded to approximately 4.5 mg / ml of phosphatidylcholine. The free H250 peptide control was incubated at 5 μmol / L. [Figure 18]Exemplary TNF-α ELISA results showing that the SRI_MGS2_V2-targeted peptide facilitates the presentation of encapsulated immunogenic peptides by HLA class I molecules in mouse lung cancer cell lines. TALL-treated LLC1 cells were co-cultured with HLA-A2-positive PBMC, and the culture supernatants were analyzed for TNF-α secretion. Even when using a combination of two antigenic peptides (H250 and H516), IFN-γ production did not substantially increase. When CD8+ T cells were depleted from PBMC in the assay, TNF-α production was significantly reduced across all treatment groups. The concentration of all liposomes used in this assay corresponded to approximately 4.5 mg / ml of phosphatidylcholine. Incubate the free H250 peptide control at 5 μmol / L. [Figure 19] Exemplary IFN-γ ELISA results showing that the SRI_MGS2_V2-targeted peptide facilitates the presentation of encapsulated immunogenic peptides by HLA class I molecules in mouse lung cancer cell lines. TALL-treated LLC1 cells were co-cultured with HLA-A2-positive PBMC, and the culture supernatants were analyzed for IFN-γ secretion. When CD8+ T cells were depleted from PBMC in the assay, IFN-γ production was significantly reduced across all treatment groups. The concentration of all liposomes used in this assay corresponded to approximately 4.5 mg / ml of phosphatidylcholine. Incubate the free H250 peptide control at 5 μmol / L. [Figure 20] Exemplary in vivo fluorescence images of tumor-bearing C57BL / 6 mice 72 hours after injection of either SRI_MGS2_V2-conjugated liposomes (top) or blank liposomes (bottom), clearly showing the ability of the targeted peptide to enhance liposome localization within LLC1 tumors. [Figure 21] In vivo quantification of liposome accumulation in LLC1 tumors of C57BL / 6 mice over time. [Figure 22] Exemplary zeta potential measurements of SRI_MGS5_V2-conjugated liposomes loaded with an equimolar mixture of H250 + H516 antigenic peptides. [Figure 23]This shows an exemplary DLS measurement demonstrating the size distribution of SRI_MGS5_V2-conjugated liposomes containing both H250 and H516 antigen peptides. [Figure 24] This shows exemplary mean flux (photons / second) emission from the lungs of both TALL-treated and untreated animals, as evaluated by IVIS imaging. [Figure 25] This shows an exemplary change in the flux (photons / second) from the lungs from day 0 to day 11, expressed as Log(flux day 0 / flux day 11). Each animal is shown individually. Positive values ​​indicate an increase in tumor size. Negative values ​​indicate a decrease in tumor size. [Figure 26] This shows a typical evaluation of tumor nodule formation in the lungs after TALL treatment. (A) The lungs of the TALL-treated group had very few or no nodules (shown in green). (B) The lungs of untreated animals were found to contain several small nodules or a single large nodule. [Figure 27] This shows exemplary mean flux (photons / second) emission from the lungs of both TALL-treated and untreated animals, as evaluated by IVIS imaging. [Figure 28] This shows that SRI-MGS5_V2 improved binding in a panel of cancer cell lines. [Figures 29A-29B] This study demonstrates that MGS5_V2 targets orthotopic triple-negative breast cancer tumors in a syngeneic mouse model. A) Quantitative flow cytometry data showing concentration-dependent binding and internalization of the MGS5_V2 peptide in 4T1 cells. B) A TNBC model showing Luc-expressing 4T1 cells transplanted into the milk fat pad of BALB / c mice (left), and the nIR dye-labeled MGS5_V2 peptide targets 4T1 cells and localizes within the tumor (right). [Figures 30A-30B]This study demonstrates that MGS5_V2 targets orthotopic pancreatic ductal adenocarcinoma tumors in a syngeneic mouse model. A) Flow cytometry data showing concentration-dependent binding and internalization of MGS5_V2 in Pan02 cells. B) PDAC model showing Luc-expressing Pan02 cells transplanted into C57BL / 6 mice (left), and fluorescently labeled MGS5_V2 targets Pan02 cells and localizes within the tumor (right). [Figure 31A-31B] This study demonstrates that MGS5_V2 targets orthotopic glioblastoma tumors in a syngeneic mouse model. A) Flow cytometry data showing concentration-dependent binding and internalization of MGS5_V2 in GL261 cells. B) GBM model showing Luc-expressing GL261 cells transplanted into the brain of B / 6 mice (left), and fluorescently labeled MGS5_V2 targeting GL261 cells (right). [Figure 32] This study demonstrates that MGS SRI_MGS5_V2 mediates the functional presentation of antigen peptides in multiple different cancer types. Treatment group: A - MGS5_V2-targeted liposomes loaded with H250 antigen; B - Positive control: Cells pulsed with free H250 antigen; C - No MGS on liposomes loaded with H250 antigen; D - Blank liposomes - No H250 antigen. [Figure 33] This study demonstrates that SRI_MGS_V2 TALL reduces tumor growth in a 4T1 breast cancer model. Mice were treated with six doses (2 μg antigen peptide / mouse) every other day. [Modes for carrying out the invention]

[0016] The methods and compositions disclosed may be more readily understood by referring to the following detailed description of specific embodiments, the examples contained herein, and the figures and their preceding and succeeding descriptions.

[0017] Unless otherwise specified, the methods and compositions disclosed are not limited to specific synthesis methods, analytical techniques, or reagents, and should therefore be understood to be subject to variation. It should also be understood that the terminology used herein is solely for the purpose of describing specific embodiments and is not intended to be limiting.

[0018] Materials, compositions, and components that can be used for, in combination with, or in preparation for the methods and compositions disclosed herein, or that are products thereof. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to various individual and collective combinations and substitutions of each of these compounds may not be expressly disclosed, but it is understood that each is specifically intended and described herein. Thus, when classes of molecules A, B, and C and classes of molecules D, E, and F and examples of combined molecules are disclosed, A to D are disclosed, and each is individually and collectively intended, even if each is not individually enumerated. Thus, in this example, each of the combinations A to E, A to F, B to D, B to E, B to F, C to D, C to E, and C to F is specifically intended and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and exemplary combinations A to D. Similarly, any subset or combination of these is also specifically intended and disclosed. Therefore, for example, subgroups A-E, B-F, and C-E should be considered specifically contemplated and disclosed from the disclosures of A, B, and C; D, E, and F; and exemplary combinations A-D. This concept applies to all aspects of this application, including but not limited to steps in methods for producing and using the disclosed compositions. Thus, where there are various executable additional steps, each of these additional steps is executable in any particular embodiment or combination of embodiments of the disclosed method, and such combinations should be considered specifically contemplated and disclosed.

[0019] A.Definition It should be understood that the methods and compositions disclosed are not limited to the specific methodologies, protocols, and reagents described, as they may vary. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the invention, which would be limited only by the appended claims.

[0020] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context explicitly indicates otherwise. For example, a reference to "targeted peptide" includes multiple such targeted peptides, and a reference to "its liposome" refers to one or more liposomes and their equivalents known to those skilled in the art.

[0021] As used herein, the terms “subject” or “patient” may be used interchangeably and may refer to any organism to which the proteins or compositions of the present invention may be administered, for example, for experimental, diagnostic, and / or therapeutic purposes. Typical subjects include animals (e.g., non-human primates and mammals such as humans; birds; domestic animals or livestock such as cats, dogs, sheep, goats, cattle, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; rabbits; fish; reptiles; zoo animals and wild animals). Typically, “subject” refers to animals, including humans and mammals such as primates.

[0022] "Treatment" means administering the antigen delivery system or composition of the present invention to a subject such as a human or other mammal (e.g., an animal model) that has increased susceptibility to developing cancer or has cancer, in order to prevent or delay the worsening of the effects of the disease or condition, or to partially or completely reverse the effects of the disease or condition.

[0023] "Prevention" means minimizing the chances that a person with increased susceptibility to developing cancer will actually develop the disease, or otherwise develop the cause of its symptoms.

[0024] As used herein, the terms “administer” and “dosage” refer to any method of providing the disclosed peptide, composition, or pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, ophthalmic administration, intraotoral administration, intracerebral administration, rectal administration, sublingual administration, oral administration, and parenteral administration, and infusion, e.g., intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Dosage may be continuous or intermittent. In various embodiments, the preparation may be administered therapeutically, i.e., to treat an existing disease or condition. In further various embodiments, the preparation may be administered prophylactically, i.e., to prevent a disease or condition. In some embodiments, those skilled in the art can determine an effective dose, effective schedule, or effective route of administration of the disclosed composition or disclosed protein to treat a subject or induce an immune response. In some embodiments, those skilled in the art can also modify or alter the aspects of the administration process to improve the efficacy of the disclosed antigen delivery system or pharmaceutical preparation.

[0025] The term “percent (%) identity” may be used herein interchangeably with the term “percent (%) homology” and refers to the level of nucleic acid or amino acid sequence identity when aligned with a wild-type sequence using a sequence alignment program. For example, as used herein, 80% homology means the same as 80% sequence identity determined by a defined algorithm, and therefore, homology of a given sequence means having more than 80% sequence identity over a given length of the sequence. Exemplary levels of sequence identity include, but are not limited to, 80%, 85%, 90%, 95%, 98% or more sequence identity of a given sequence to any one coding sequence of any of the proteins of the present invention, as described herein. Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, the BLAST program suite available on the Internet, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul, et al., 1990 and Altschul, et al., 1997. Sequence searches are typically performed using the BLASTN program when evaluating a given nucleic acid sequence against GenBank DNA sequences and nucleic acid sequences in other public databases. The BLASTX program is preferred for searching for nucleic acid sequences that have been translated in all read frames against GenBank protein sequences and amino acid sequences in other public databases. Both BLASTN and BLASTX are run using the BLOSUM-62 matrix with default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0. (See, for example, Altschul, SF, et al., Nucleic Acids Res. 25:3389-3402, 1997).The preferred alignment of selected sequences to determine the "% identity" between two or more sequences is performed using the CLUSTAL-W program in Mac Vector version 13.0.7, operating with default parameters, including, for example, a 10.0 open gap penalty, a 0.1 extended gap penalty, and a BLOSUM 30 similarity matrix.

[0026] Amino acid modifications, such as substitutions, deletions, insertions, or any combination thereof, can be used to arrive at the final derivative, variant, or analogue. Generally, these changes are made on a few nucleotides to minimize molecular modification. However, larger changes may be permissible under certain circumstances.

[0027] Generally, the nucleotide identity between individual mutant sequences may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Therefore, a “mutant sequence” may be one containing one or more amino acid modifications and sharing a biological function with the parent or reference sequence of the present invention (e.g., wild-type sequence), including but not limited to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent sequence, and possessing identified identity to the parent or reference sequence of the present invention. In some embodiments, a mutant-targeted peptide may contain one, two, three, four, or more amino acid base changes compared to the parent or reference sequence of the present invention, and may share or improve upon the biological function, specificity, and / or activity of the parent sequence. Therefore, the mutant-targeted peptide may have identified identity with respect to the parent sequence of the present invention (e.g., SEQ ID NO: 1) and share biological function including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent sequence. The mutant sequence may also share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the reference sequence (e.g., SEQ ID NO: 1).

[0028] The terms “mutant,” “mutant,” or “modified” may be used interchangeably. As used herein, the term “mutant” refers to a modified nucleic acid or protein that exhibits the same characteristics as a reference nucleic acid or protein sequence. A modified targeted peptide may be at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology to the reference sequence. In some embodiments, the reference sequence may be Sequence ID No. 1. A mutant may also include a nucleotide sequence substantially similar to the sequence disclosed herein. “Mutant” or “its variant” may mean any difference from the reference sequence other than a simple deletion of an N-terminal and / or C-terminal amino acid residue or multiple residues. If a mutant includes an amino acid residue substitution, the substitution may be considered conserved or non-conserved. A mutant may include at least one substitution and / or at least one addition, and may also have at least one deletion. A mutant may also include one or more non-native residues.

[0029] As used herein, amino acid “substitution” refers to the replacement of one amino acid residue with a different amino acid residue. The substituted amino acid may be any of the 20 amino acids commonly found in human proteins, as well as atypical or unnatural amino acids. Amino acid residue substitutions can be considered conserved or unconserved. Conservative substitutions are those in the following group: Ser, Thr, and Cys; Leu, ILe, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gln, Asn, Glu, Asp, and His. In some embodiments, the substitution may be an unnatural substitution. For example, the substitution may include selenocysteine ​​at any position (e.g., seleno-L-cysteine), including a substitute for cysteine. Many other “unnatural” amino acid substitutes are known in the art and are available from commercial sources. Examples of unnatural amino acids include D-amino acids, amino acid residues having an acetylaminomethyl group bonded to the sulfur atom of cysteine, pegylated amino acids, and those with the formula NH2(CH2) nExamples of COOH omega amino acids include n, where n is a 2-6 neutral nonpolar amino acid, such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine. Phenylglycine can be substituted for Trp, Tyr, or Phe, citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline can be substituted with hydroxyproline, retaining the conformation that confers the properties of proline.

[0030] "Optional" or "optional" means that the following event, situation, or material may or may not occur, may or may not exist, and that the description includes both the case in which the event, situation, or material occurs or exists, and the case in which it does not occur or does not exist.

[0031] A range may be expressed herein as “about” one particular value and / or “about” another particular value. Where such a range is expressed, unless the context otherwise specifically indicates, the range from one particular value and / or another particular value is also considered to be specifically contemplated and disclosed. Similarly, where a value is expressed as an approximation by the use of the antecedent “about,” the particular value will be understood to form another specifically contemplated embodiment that should be considered disclosed unless the context otherwise specifically indicates. It will also be understood that each endpoint of a range is important with respect to and independently of the other endpoints, unless the context otherwise specifically indicates. Finally, it should be understood that all individual values ​​and subranges of values ​​that fall within an expressly disclosed range should also be considered to be specifically contemplated and disclosed, unless the context otherwise specifically indicates. The foregoing applies in any particular case whether some or all of these embodiments are expressly disclosed.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the disclosed methods and compositions belong. Any methods and materials similar to or equivalent to those described herein may be used in the performance or testing of the methods and compositions, but particularly useful methods, devices, and materials are those described herein. Publications cited herein and the materials from which they are cited are incorporated herein by reference. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such disclosure for the sake of prior art. No reference is recognized as constituting prior art. Discussions of references state the authors' assertions, and the applicant reserves the right to challenge the accuracy and validity of the cited documents. While several publications are mentioned herein, it will be clearly understood that such references do not constitute an acknowledgment that any of these documents form part of the common general knowledge in the art.

[0033] Throughout this specification and the claims, the term “comprise,” and variations thereof such as “comprising” and “comprises,” means “including, but not limited to,” and is not intended to exclude, for example, other additives, components, integers, or processes. In particular, in methods described as including one or more processes or operations, each process is specifically intended to include those listed (unless the process includes a limiting term such as “consisting of”), and each process is not intended to exclude, for example, other additives, components, integers, or processes not listed in the process.

[0034] B. Antigen delivery system An immunotherapy based on an antigen delivery system is disclosed that facilitates the presentation of non-cancerous HLA class 1 restriction immunogenic peptides in cancer cells, thereby inducing a secondary immune response against cancer cells. The disclosed immunotherapy avoids the major hurdle in cancer vaccine development: the need to identify tumor-associated antigens or generate a primary immune response against the tumor. Unlike immunomodulators, the immune response generated by the disclosed antigen delivery system is antigen-specific and does not involve an overall general activation of the immune response. This minimizes the problems of autoimmunity and off-target effects.

[0035] Targeted nanoparticles used to deliver antigens (e.g., viral antigens) for antigen presentation in cells (e.g., cancer cells) are disclosed. A synthetic antigen delivery system suitable for the specific delivery of antigen cargo into cells is also disclosed.

[0036] In some embodiments, the disclosed antigen delivery system comprises a targeted peptide, nanoparticles, and an HLA class I restriction peptide, each of which is described herein.

[0037] In some embodiments, the antigen delivery system does not contain any viral particles, toxins, or biological materials. Therefore, safety concerns in the disclosed antigen delivery system are reduced.

[0038] An antigen delivery system comprising nanoparticles is disclosed, wherein the nanoparticles are surface-modified with a cancer-specific cell-targeting peptide and contain an immunogenic HLA class I restriction peptide, wherein the HLA class I restriction peptide is a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide comprises the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1). An example of the disclosed antigen delivery system is an antigen delivery system comprising PEGylated liposomes, wherein the PEGylated liposomes are surface-modified with a cancer-specific cell-targeting peptide and contain an immunogenic HLA class I restriction peptide, wherein the HLA class I restriction peptide is a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide comprises the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1).

[0039] 1. Targeted peptides An antigen delivery system comprising nanoparticles is disclosed, wherein the nanoparticles are surface-modified with a targeted peptide. For example, an antigen delivery system comprising liposomes is disclosed, wherein the liposomes are surface-modified with a cancer-specific cell-targeting peptide.

[0040] In some embodiments, the targeted peptide is a cell-specific targeted peptide. In some embodiments, the targeted peptide is a cancer cell-specific targeted peptide. Cancer-specific cell-targeted peptides are disclosed. In some non-limiting examples, the cancer-specific cell-targeted peptide targets lung cancer, pancreatic cancer, breast cancer, and / or glioblastoma.

[0041] Targeted peptides are well known and have been used in the past to target nanoparticles to cell types. One particular targeted peptide, LQWRRDDNVHNFGVWARYRL (SEQ ID NO: 2), is a cancer-specific cell-targeting peptide, but it has been found to be less effective for use in disclosed antigen delivery systems due to its hydrophobicity, difficulty in purification, and unstable properties. A targeted peptide is disclosed herein in which amino acids 6-10 of SEQ ID NO: 2 have been removed and the C-terminus and N-terminus have been fused together, resulting in a targeted peptide with increased cell binding and better physiological and chemical properties than the peptide of SEQ ID NO: 2. Thus, in some embodiments, the resulting targeted peptide contains the amino acid sequence of LQWRRNFGVWARYRL (SEQ ID NO: 1). In some embodiments, the reduction in hydrophobicity of SEQ ID NO: 1 dramatically increases the solubility and stability of the peptide, and therefore improves coupling to nanoparticles such as liposomes.

[0042] A targeted peptide comprising the amino acid sequence LQWRRNFGVWARYRL (SEQ ID NO: 1) is disclosed. In some embodiments, the targeted peptide comprising the amino acid sequence SEQ ID NO: 1 is a cancer-specific cell-targeting peptide.

[0043] In some embodiments, the disclosed targeted peptides accumulate specifically in cancer cells and promote the presentation of HLA class I.

[0044] In some embodiments, targeted peptides can be conjugated to the surface of nanoparticles, such as liposomes, using any technique known in the art. For example, targeted peptides can be conjugated to the surface of liposomes by a thiol-ester bond resulting from the Michael addition of a single sulfhydryl group on the targeted peptide to a maleimide present on the liposome. In some embodiments, targeted peptides can be conjugated to the surface of nanoparticles using amide chemistry and / or click chemistry.

[0045] In some embodiments, the targeted peptide may be acetylated at its N-terminus. In some embodiments, the targeted peptide may include a linker at its C-terminus. For example, in some embodiments, the linker may be a PEG linker.

[0046] In some embodiments, the disclosed antigen delivery system may include a dimer of the disclosed targeted peptide, also called a targeted peptide dimer. In some embodiments of the disclosed antigen delivery system, the cancer-specific cell-targeted peptide is a dimer in which at least one of the two targeted peptides contains the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1). In some embodiments, both targeted peptides in the dimer contain the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1). In some embodiments, the targeted peptide dimer is linked via a linker and a branching point, such as PEG. In some embodiments, the branching point may be a lysine residue. For example, in some embodiments, the dimer contains the sequence (CH3CO-LQWRRNFGVWARYRL-PEG11)2K (SEQ ID NO: 3).

[0047] In some embodiments, at least two linkers are present, one at the end of each targeted peptide. In some embodiments, two or more linkers may be used. In some embodiments, the linker may be PEG11. In some embodiments, more or fewer ethylene glycol subunits may be used. For example, PEG may have 2 to 24 ethylene glycols.

[0048] In some embodiments, the targeted peptide dimer may further include a conjugated moiety on the C-terminal side of the branching point, which enables conjugation to liposomes. For example, in some embodiments, the dimer comprises the sequence (CH3CO-LQWRRNFGVWARYRL-PEG11)2K-X (SEQ ID NO: 4), where X is a tag or reactive moiety for conjugation.

[0049] In some embodiments, the disclosed targeted peptides can facilitate the presentation of HLA class I using autophagy. In some embodiments, the disclosed targeted peptides can accumulate in intracellular autophagocytic vesicles. The accumulation of the targeted peptide in the autophagocytic vesicles leads to the accumulation of the entire antigen delivery system within the autophagocytic vesicles, which may result in the presentation of the HLA class I restriction peptide of the antigen delivery system.

[0050] In some embodiments, the targeted peptide may include a sequence having at least 75, 80, 85, 90, 95, or 99% identity with SEQ ID NO: 1. Therefore, variants of SEQ ID NO: 1 are disclosed herein and may be used in the disclosed antigen delivery systems.

[0051] 2. Nanoparticles An antigen delivery system comprising nanoparticles is disclosed, wherein the nanoparticles are surface-modified with a targeted peptide. In some embodiments, the nanoparticles may be any vehicle capable of carrying a cargo, specifically a peptide, without modifying the cargo. In some embodiments, the vehicle needs to have a high payload capacity and shield the unmodified immunogenic peptide cargo because its presentation in HLA class I molecules is limited by the size and position of amino acid residues.

[0052] In some embodiments, nanoparticles may be inorganic, liposomes, virus-like particles, or polymers. A variety of materials exist from which nanoparticles can be synthesized. Examples of such materials include, but are not limited to, lipids (viral envelopes or phospholipids), synthetic polymers such as poly(allylamine hydrochloride) (PAH), poly(acrylic acid) (PAA), and poly(methacrylic acid) (PMA), polypeptides such as poly(lactide-co-glycolide) (PLGA) and poly-L-lysine (PLL), natural polymers such as chitosan, and proteins such as albumin.

[0053] In some embodiments, the disclosed antigen delivery system comprises liposomes. In some embodiments, the liposomes are self-assembled phospholipid bilayers having an aqueous core. In some embodiments, the liposomes can be manufactured in a multilayer structure so that they can enable the encapsulation of both hydrophilic and hydrophobic antigens between different layers. The liposomes can be readily manufactured from synthetic materials, readily loaded with synthetic peptides, and are suitable for modification with targeted peptides. In some embodiments, the liposomes passively accumulate in tumors based on enhanced permeability and retention effects, thus enhancing therapeutic specificity. In some embodiments, the nanoparticles can be metallic, organic, inorganic, and polymeric nanostructures, including dendrimers, micelles, and liposomes.

[0054] In some embodiments, the disclosed antigen delivery system comprises PEGylated liposomes. The PEGylated liposomes comprise the synthetic polymer poly-(ethylene glycol) (PEG). In some embodiments, the presence of PEG on the surface of the liposome can prolong blood circulation time while reducing uptake by mononuclear phagocytic cell systems. Therefore, in some embodiments, PEGylated liposomes are also known as stealth liposomes. In some embodiments, DSPE PEG2000 modified with maleimide can be incorporated into the liposome lipid formulation to enable the coupling of thiol-containing targeted peptides to the liposomes.

[0055] In some embodiments, the liposomes may be 30 nM to 350 nM. In some embodiments, the liposomes may be about 100 nM. 100 nM stealth liposomes for encapsulating synthetically produced immunogenic peptides are disclosed.

[0056] In some embodiments, the nanoparticles may further include a detectable label. In some embodiments, the detectable label may be a chemiluminescent label, a fluorescent label, or an enzymatic label. As used herein, “detectable label” is a nucleic acid, protein, or compound that is detectable or capable of producing a detectable response. The detectable labels according to the present invention may be directly or indirectly conjugated to or encapsulated within the nanoparticles and include radioisotopes, enzymes, haptens, chromophores such as dyes or particles that impart a detectable color (e.g., latex beads or metal particles), luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent moieties), quantum dots, and fluorescent compounds such as fluorescent proteins.

[0057] Suitable fluorescent proteins include green fluorescent protein (GFP) or its variants, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), high-sensitivity GFP (EGFP), high-sensitivity CFP (ECFP), high-sensitivity YFP (EYFP), GFPS65T, emerald, topaz (TYFP), Venus, citrine, m-citrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, cerulean, T-sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer 2, t-dimer 2(12), mRFPl, pocilloporin, and Renilla. Examples of fluorescent proteins include, but are not limited to, GFP, monsterGFP, paGFP, maple protein and kindling protein, phycobiliproteins, and phycobiliprotein conjugates including β-phycoerythrin, R-phycoerythrin, and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, and mPlum (Shaner et al. (2005) Nat. Methods 2:905-909). Any of the various fluorescent and colored proteins derived from anthoan species, such as those described in Matz et al. (1999) Nature Biotechnol. 17:969-973, are suitable for use.

[0058] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO).

[0059] In some embodiments, nanoparticles are conjugated to the targeted peptides described herein using methods known in the art.

[0060] In some embodiments, nanoparticles encapsulate or are loaded with one or more HLA class I restriction peptides described herein. In some embodiments, nanoparticles contain one or more of the same HLA class I restriction peptides. For example, nanoparticles can encapsulate one, two, three, four, five, six, seven, eight, nine, or ten or more of a single HLA class I restriction peptide described herein. In some embodiments, nanoparticles contain one or more of the same HLA class I restriction peptides, but both nanoparticles containing one HLA class I restriction peptide and nanoparticles containing different HLA class I restriction peptides can be used in the disclosed antigen delivery system. For example, multiple HLA class I restriction peptides are used in the antigen delivery system, but each HLA class I restriction peptide is contained within its own nanoparticle.

[0061] 3. HLA class I restriction peptide HLA class I molecules are found on the surface of all nucleated cells in vertebrates. In some embodiments, HLA class I molecules function to present or display peptide fragments to cytotoxic T cells. In some embodiments, HLA class I molecules present peptides from within the cell (i.e., endogenous peptides). In some embodiments, HLA class I molecules contain a class I peptide binding groove that accommodates a processed peptide of 8 to 10 amino acid residues, or possibly 11 to 14 amino acids, known as an HLA class I restriction peptide. Thus, in some embodiments, the disclosed HLA class I restriction peptide is 8 to 14 amino acids long. In some embodiments, the disclosed HLA class I restriction peptide is 8 to 10 amino acids long.

[0062] In some embodiments, the disclosed HLA class I restriction peptide may be a peptide to which the subject has been previously exposed. For example, the peptide may originate from a virus, bacterium, or fungus to which the subject has been previously exposed (infected). In some embodiments, the peptide may originate from a peptide to which the subject has been previously exposed via vaccination. Thus, in some embodiments, the disclosed HLA class I restriction peptide may be a vaccine-dependent immunogenic HLA class I restriction peptide. In some embodiments, a vaccine-dependent immunogenic HLA class I restriction peptide means that the HLA class I restriction peptide originates from a vaccine previously used to immunize a subject receiving one of the disclosed antigen delivery systems. For example, if a subject has been vaccinated with an influenza vaccine, the vaccine-dependent immunogenic HLA class I restriction peptide may be a peptide derived from the influenza vaccine. Vaccine-dependent means that the HLA class I restriction peptide originates from a vaccine containing that peptide, and the subject receiving the antigen delivery system containing the HLA class I restriction peptide has already been vaccinated with that vaccine. Secondary immunogenicity means that an HLA class I restriction peptide induces a secondary immune response to that HLA class I restriction peptide. For example, an HLA class I restriction peptide induces a secondary immune response because it is a vaccine-dependent HLA class I restriction peptide, and therefore the subject has already been vaccinated and produced a primary immune response to the peptide. In some embodiments, the secondary immune response is more rapid and efficient.

[0063] Due to prior exposure to an HLA class I restriction peptide, the peptide may also be called a prior immune response-dependent secondary immunogenic HLA class I restriction peptide. For example, a vaccine-dependent secondary immunogenic HLA class I restriction peptide is disclosed. Prior immune response-dependent or vaccine-dependent may mean that the subject has previously produced an immune response to the HLA class I restriction peptide, and therefore it is secondary immunogenic because the immune response produced to the HLA class I restriction peptide in this delivery system is a secondary immune response.

[0064] In some embodiments, the vaccine-dependent immunogenic HLA class I restriction peptide may be any HLA class I restriction peptide derived from the vaccine containing the peptide. In some embodiments, the vaccine-dependent immunogenic HLA class I restriction peptide may be measles virus hemagglutinin peptide H250. In some embodiments, the vaccine-dependent immunogenic HLA class I restriction peptide may be influenza virus hemagglutinin peptide HA. In some embodiments, the vaccine-dependent immunogenic HLA class I restriction peptide may be smallpox virus H-2Kd restriction vaccine-specific peptide, A5275-83 (VACV-A52). In some embodiments, the vaccine-dependent immunogenic HLA class I restriction peptide may be H250 (SMYRVFEVGV, SEQ ID NO: 5), C166 (SLWGSLLML, SEQ ID NO: 6), H38 (LLAVIFVMFL, SEQ ID NO: 7), H516 (ILPGQDLQYV, SEQ ID NO: 8), H3L (SLSAYIIRV, SEQ ID NO: 9), E2L (KIDYYIPYV, SEQ ID NO: 10), or 0IL (GLNDYLHSV, SEQ ID NO: 11).

[0065] In some embodiments, the HLA class I restriction peptide may be based on the specific HLA type of the target receiving the disclosed antigen delivery system.

[0066] 4. Therapeutic agents In some embodiments, the nanoparticles of the disclosed antigen delivery system may be further loaded with a therapeutic agent. Thus, cells will receive both the HLA class I restriction peptide and the therapeutic agent from the nanoparticles.

[0067] In some embodiments, the therapeutic agent is a cancer treatment agent. In some embodiments, the cancer treatment agent may be any known cancer treatment agent. In some embodiments, the cancer treatment agent may be, but is not limited to, methotrexate, docetaxel-gemcitabine, bevacizumab, cyclophosphamide, erlotinib, gemcitabine, crizotinib, atezolizumab, nivolumab, pembrolizumab, estrogen modulators, hormone-based chemotherapy, fluorouracil, oxaliplatin, irinotecan, leucovorin, carmustine, or temozolomide.

[0068] In some embodiments, the therapeutic agent is a drug that induces autophagy. Since autophagy may be important for antigen presentation, delivering an autophagy-inducing drug in combination with an HLA class I restriction peptide can increase the efficacy of the presented HLA class I restriction peptide. In some embodiments, the autophagy-inducing drug may be, but is not limited to, L-type Ca2+ channel blockers (verapamil, loperamide, amiodarone), calpain inhibitors (calpastatin), ATP-sensitive K+ channel agonists (minoxidil), cAMP reducers (rilmenidine, clonidine), inositol-reducing agents (valproic acid), class I PI3K inhibitors (LY294002), mTOR (rapamycin), AKT (perifosine), and IMPase (Li+).

[0069] C. Composition A composition comprising an antigen delivery system is disclosed. For example, a composition comprising an antigen delivery system is disclosed, wherein the antigen delivery system comprises nanoparticles, the nanoparticles are surface-modified with a cancer-specific cell-targeting peptide and comprises an immunogenic HLA class I restriction peptide, the HLA class I restriction peptide is a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide comprises the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1).

[0070] 1. Pharmaceutical composition In some embodiments, the disclosed compositions may be pharmaceutical compositions. For example, in some embodiments, a pharmaceutical composition is disclosed comprising a composition comprising one or more antigen delivery systems disclosed herein and a pharmaceutically acceptable carrier. "pharmaceutically acceptable" means a material or carrier that would be selected to minimize any degradation of the active ingredient and any adverse side effects in the subject, as is well known to those skilled in the art. Examples of carriers include dimyristoyl phosphatidyl (DMPC), phosphate-buffered saline, or polyvesicular liposomes. For example, PG:PC:cholesterol:peptide or PC:peptide may be used as a carrier in the present invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. ARGennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make it isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution. The pH of the solution may be about 5 to about 8, or about 7 to about 7.5. Further carriers include sustained-release preparations such as a semipermeable matrix of a solid hydrophobic polymer containing the composition, the matrix in the form of a molded article, e.g., a film, a stent (implanted in a blood vessel during angioplasty), liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred, for example, depending on the route of administration and the concentration of the composition being administered. These are most typically standard carriers for drug administration to humans, including solutions such as sterile water, physiological saline, and buffer solutions at physiological pH.

[0071] The pharmaceutical composition may also contain carriers, thickeners, diluents, buffers, preservatives, etc., provided that the intended activity of the polypeptide, peptide, or conjugate of the present invention is not impaired. The pharmaceutical composition may also contain one or more active ingredients (in addition to the composition of the present invention), such as antibacterial agents, anti-inflammatory agents, or anesthetics.

[0072] Pharmaceutical compositions such as those disclosed herein can be prepared for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, vaginal, or rectal), or transdermal (e.g., topical) administration. Aerosol inhalation may also be used to deliver fusion proteins. Accordingly, compositions comprising fusion proteins dissolved or suspended in an acceptable carrier, including but not limited to aqueous carriers such as water, buffered water, saline, or buffered saline (e.g., PBS), can be prepared for parenteral administration. One or more of the excipients included, such as pH adjusters and buffers, tension adjusters, wetting agents, and surfactants, can assist in achieving an approximate physiological state. If the composition contains solid components (which may also be for oral administration), one or more of the excipients may function as binders or fillers (e.g., for formulations such as tablets or capsules). When a composition is formulated for application to the surface of the skin or mucous membrane, one or more of the excipients may be solvents or emulsifiers for formulations such as creams and ointments.

[0073] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions containing physiological saline and buffer media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose, sodium chloride, lactated Ringer's solution, or fixative oils. Intravenous vehicles include fluids and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0074] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous solutions, powders, or oily bases, thickeners, etc., may be necessary or desirable.

[0075] Compositions for oral administration include powders or granules, suspensions or solutions in water or a non-aqueous medium, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions may potentially be administered as pharmaceutically acceptable acid or base-added salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as monalkyl, dialkyl, trialkyl, and arylamines, and substituted ethanolamines.

[0076] The pharmaceutical compositions are sterile and can be sterilized by conventional sterilization techniques, or can be bacterially filtered. Aqueous solutions can be packaged for ready use or lyophilized, and the lyophilized preparations encompassed by this disclosure can be combined with a sterile aqueous carrier before administration. The pH of the pharmaceutical compositions will typically be 3 to 11 (e.g., about 5 to 9) or 6 to 8 (e.g., about 7 to 8). The obtained compositions in solid form can be packaged in multiple single-dose units, each containing a fixed amount of the above-mentioned drug, such as in a sealed package of tablets or capsules. The compositions in solid form can also be packaged in containers for adaptable amounts, such as in a compressible tube designed for topically applicable creams or ointments.

[0077] The pharmaceutical compositions described above can be formulated to contain a therapeutically effective amount of the compositions disclosed herein. In some embodiments, therapeutic administration includes prophylactic administration. Based on genetic testing and other prognostic methods, a physician may, in consultation with the patient, choose prophylactic administration if the patient has a clinically determined predisposition or increased susceptibility (in some cases, significantly increased susceptibility) to one or more autoimmune diseases, or if the patient has a clinically determined predisposition or increased susceptibility (in some cases, significantly increased susceptibility) to cancer.

[0078] The pharmaceutical compositions described herein may be administered to a subject (e.g., a human subject or human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of a clinical disease. Therefore, in some embodiments, the subject is a human subject. In therapeutic use, the composition is administered to a subject (e.g., a human subject) who already has cancer or has been diagnosed with cancer, in an amount sufficient to at least partially improve the condition, its complications, and the signs or symptoms of the consequences, or to inhibit (and preferably halt) the progression of those symptoms. The amount suitable for achieving this is defined as the “therapeutically effective amount.” A therapeutically effective amount of a pharmaceutical composition may be an amount that achieves a cure, but the outcome is only one of several possible outcomes. As stated above, therapeutically effective amounts include amounts that provide a treatment in which the onset or progression of cancer is delayed, inhibited, or prevented, or in which an autoimmune disease or symptoms of an autoimmune disease are improved. One or more of the symptoms may not be very severe. The recovery of the treated individual may be accelerated.

[0079] The total effective amount of conjugates in the pharmaceutical compositions disclosed herein may be administered to mammals as a single dose over a relatively short period, either as a bolus or by infusion, or using fractional therapy protocols in which multiple doses are administered over a longer period (e.g., every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, every 1-2 weeks, or once a month). Alternatively, continuous intravenous infusion sufficient to maintain a therapeutically effective concentration in the blood is also within the scope of this disclosure.

[0080] The pharmaceutical composition may be administered in several ways, depending on whether topical or systemic treatment is desired and the area to be treated.

[0081] D. Treatment methods A method for treating a subject having cancer is disclosed, comprising administering to the subject having cancer one of the antigen delivery systems disclosed herein. In some embodiments, the subject is first diagnosed with cancer and then administered one of the antigen delivery systems disclosed herein.

[0082] A method for treating a subject with cancer is disclosed, comprising administering an antigen delivery system comprising nanoparticles, wherein the nanoparticles are surface-modified with a cancer-specific cell-targeting peptide and comprise an immunogenic HLA class I restriction peptide, the HLA class I restriction peptide being a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide comprising the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1). For example, a method for treating a subject with cancer is disclosed, comprising administering an antigen delivery system comprising PEGylated liposomes, wherein the PEGylated liposomes are surface-modified with a cancer-specific cell-targeting peptide and comprise an immunogenic human leukocyte antigen (HLA) class I restriction peptide, the HLA class I restriction peptide being a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide comprising the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1).

[0083] In some embodiments, an antigen delivery system targets cancer cells in a subject, and when liposomes enter the cancer cells in the subject, the cancer cells present a vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system on its surface in an HLA class I molecule, and the subject generates an immune response to the vaccine-dependent immunogenic HLA class I restriction peptide. In other words, nanoparticles such as liposomes, along with a targeted peptide (e.g., a peptide containing LQWRRNFGVWARYRL (SEQ ID NO: 1)), help to obtain the HLA class I restriction peptide inside the cancer cell, where the cancer cell can then present the HLA class I restriction peptide in a manner that generates an immune response to the HLA class I restriction peptide. In some embodiments of the disclosed method, the immune response to the vaccine-dependent immunogenic antigen targets and kills the cancer cell presenting the vaccine-dependent immunogenic antigen.

[0084] In some aspects of the disclosed method, a cancer-specific cell-targeting peptide targets cancer cells using nanoparticles (e.g., liposomes).

[0085] In some embodiments, the disclosed method may further include administering a therapeutic agent to a subject. In some embodiments, the therapeutic agent is a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent may be a checkpoint inhibitor. In some embodiments, the cancer therapeutic agent may be a chemotherapeutic agent. In some embodiments, the therapeutic agent is an autophagy inducer. In some embodiments, the therapeutic agent is encapsulated in nanoparticles, as is the case with HLA class I restriction peptides. In some embodiments, the therapeutic agent is administered separately from nanoparticles.

[0086] In some embodiments, the disclosed method may further include detecting an immune response in a subject to a vaccine-dependent immunogenic HLA class I restriction peptide. The step of detecting an immune response in a subject to a vaccine-dependent immunogenic HLA class I restriction peptide can confirm the effectiveness of a treatment. In some embodiments, determining the immune response in a subject to a vaccine-dependent immunogenic HLA class I restriction peptide can be an important factor in determining which vaccine-dependent immunogenic HLA class I restriction peptide is best suited for use in an antigen delivery system.

[0087] In some embodiments, subjects are first HLA-typed before treatment. HLA-typed subjects can identify the HLA class I restriction peptide to be used in the antigen delivery system.

[0088] E. Methods to kill cancer cells A method for killing cancer cells is disclosed, comprising contacting the cancer cells with one of the antigen delivery systems disclosed herein, wherein, upon entry of the liposomes into the cancer cells, the cancer cells present a vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system, the cancer cells generate an immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen, and the immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen targets and kills the cancer cells presenting the vaccine-dependent immunogenic HLA class I restriction peptide antigen. In some embodiments, when cancer cells present a vaccine-dependent immunogenic HLA class I restriction peptide, it is intended that this means the vaccine-dependent immunogenic HLA class I restriction peptide is presented on HLA class I molecules on the surface of the cancer cells.

[0089] In some embodiments, the method may further include contacting cancer cells with a therapeutic agent. In some embodiments, the therapeutic agent is a cancer treatment agent. Thus, combination therapies comprising the disclosed antigen delivery system and known cancer treatment agents may be used. In some embodiments, the cancer treatment agent is a checkpoint inhibitor. In some embodiments, the cancer treatment agent may be, but is not limited to, chemotherapy, antibody therapy, immunotherapy, radiotherapy, hormone therapy, stem cell therapy, or targeted therapy. Any known cancer treatment agent may be used in combination with the antigen delivery system described herein.

[0090] In some embodiments, cancer cells are present within the target. Therefore, in some embodiments, contact with the antigen delivery system of cancer cells includes administering the antigen delivery system to the target.

[0091] F. Methods for generating non-cancer secondary immune responses A method for generating a non-cancer secondary immune response targeting cancer cells is disclosed, comprising administering one of the antigen delivery systems disclosed herein to a subject having cancer cells, wherein the liposomes enter the cancer cells in the subject, the cancer cells present a vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system, the subject generates a non-cancer secondary immune response to the vaccine-dependent immunogenic HLA class I restriction peptide, and the non-cancer secondary immune response targets and kills the cancer cells presenting the vaccine-dependent immunogenic HLA class I restriction peptide.

[0092] As described herein, a non-cancer secondary immune response is a secondary immune response to an antigen other than a cancer antigen. In some embodiments, a non-cancer secondary immune response targets an antigen other than a cancer antigen, but can target cancer cells that present a non-cancer antigen on their surface. Thus, as described herein, a non-cancer secondary immune response can target and kill cancer cells that present a non-cancer antigen. For example, cancer cells that present measles peptides (due to an antigen delivery system described herein) can generate a secondary immune response to measles peptides, resulting in the killing of the cancer cells that present measles peptides.

[0093] In some embodiments, the subject is first determined to have received a vaccine containing a vaccine-dependent immunogenic HLA class I restriction peptide.

[0094] In some embodiments, subjects are first HLA-typed before administering an antigen delivery system. HLA-typed subjects can make it possible to identify the HLA class I restriction peptide to be used in the antigen delivery system.

[0095] G. Methods to increase specific localization to cancer cells A method for increasing specific localization to cancer cells is disclosed, comprising administering one of the disclosed antigen delivery systems to a subject having cancer cells, wherein the majority of the nanoparticles (e.g., liposomes) localize to the cancer cells. A method for increasing specific localization to cancer cells is disclosed, comprising administering one of the disclosed antigen delivery systems to a subject having cancer cells, wherein at least 50%, 60%, 70%, 80%, or 90% of the nanoparticles (e.g., liposomes) localize to the cancer cells.

[0096] In some embodiments, there is nonspecific localization of less than 10%, less than 20%, less than 30%, or less than 40% of the nanoparticles.

[0097] H. Kit The materials described above, as well as other materials, can be packaged together in any suitable combination as a kit useful for carrying out or assisting in the carrying out of the disclosed method, or for preparing the disclosed antigen delivery system. It is useful if the kit components within a given kit are designed and adapted to be used together in the disclosed method. For example, a kit is disclosed containing a cancer-specific cell-targeting peptide containing the sequence LQWRRNFGVWARYRL (SEQ ID NO: 1).

[0098] The kit may also contain liposomes or vaccine-dependent immunogenic HLA class I restriction peptides.

[0099] The disclosed kit may also include instructions for constructing or using the disclosed antigen delivery system. Item 1 An antigen delivery system comprising PEGylated liposomes, wherein the PEGylated liposomes are surface-modified with a cancer-specific cell-targeting peptide and contain an immunogenic human leukocyte antigen (HLA) class I restriction peptide, the HLA class I restriction peptide being a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide containing the sequence LQWRRNFGVWARYRL. Section 2 The antigen delivery system according to item 1, wherein the vaccine-dependent immunogenic HLA class I restriction peptide is measles virus hemagglutinin peptide H250. Section 3 The antigen delivery system according to item 1, wherein the vaccine-dependent immunogenic HLA class I restriction peptide is the influenza virus hemagglutinin peptide HA. Section 4 The antigen delivery system according to item 1, wherein the vaccine-dependent immunogenic HLA class I restriction peptide is a smallpox virus H-2Kd restriction vaccinia-specific peptide, A5275-83 (VACV-A52). Section 5 The antigen delivery system according to claim 1, wherein the vaccine-dependent immunogenic HLA class I restriction peptide is H250 (SMYRVFEVGV), C166 (SLWGSLLML), H38 (LLAVIFVMFL), H516 (ILPGQDLQYV), H3L (SLSAYIIRV), E2L (KIDYYIPYV), or 0IL (GLNDYLHSV). Section 6 The antigen delivery system according to item 1, wherein the antigen delivery system does not contain any viral particles, toxins, or biological materials. Section 7 The antigen delivery system according to item 1, wherein the cancer-specific cell-targeting peptide is acetylated at its N-terminus. Section 8 The antigen delivery system according to item 1, wherein the cancer-specific cell-targeting peptide includes a PEG linker at its C-terminus. Section 9 The antigen delivery system according to claim 1, wherein the cancer-specific cell-targeting peptide is a dimer, and at least one of the two targeting peptides comprises the sequence LQWRRNFGVWARYRL. Section 10 The aforementioned dimer has the sequence CH3CO-LQWRRNFGVWARYRL-PEG11. 2 The antigen delivery system described in item 9, including K. Section 11 The antigen delivery system according to claim 9, further comprising the cancer-specific cell-targeting peptide and the lysine branching point of the dimer. Section 12 The antigen delivery system according to paragraph 9, wherein the dimer further comprises a conjugated portion on the C-terminal side of the lysine branching point, the conjugated portion enabling conjugation to the liposome. Section 13 A method for treating a subject with cancer, comprising administering the antigen delivery system described in item 1 to a subject diagnosed with cancer or having cancer. Section 14 The method according to claim 13, wherein the antigen delivery system targets cancer cells in the subject, and when the liposomes enter the cancer cells in the subject, the cancer cells present the vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system, and the subject generates an immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen. Item 15 The method according to claim 14, wherein the immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen targets and kills cancer cells that present the vaccine-dependent immunogenic HLA class I restriction peptide antigen. Section 16 The method according to item 13, wherein the cancer-specific cell-targeting peptide targets the liposomes against the cancer cells. Section 17 The method according to claim 13, further comprising administering a therapeutic agent to the subject. Section 18 The method according to item 17, wherein the therapeutic agent is a cancer treatment agent. Section 19 The method according to claim 13, further comprising detecting the immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen in the subject. Section 20 A method for killing cancer cells, comprising contacting cancer cells with an antigen delivery system described in item 1, wherein when the liposomes enter the cancer cells, the cancer cells present the vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system, the cancer cells generate an immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen, and the immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen targets and kills the cancer cells presenting the vaccine-dependent immunogenic HLA class I restriction peptide antigen. [Examples]

[0100] Utilizing delivery ligands to promote HLA class I presentation has been previously achieved using cholera or Shiga toxins fused to class I restriction immunogenic peptides (17-19). However, these toxins accumulate indiscriminately within cells and are not specifically targeted to cancer cells. The parent peptide, H1299.3, has been found to selectively accumulate in cancer cells and demonstrate limited binding to normal human bronchial epithelial cells, thus potentially offering a wider therapeutic concentration range (20, 21). The disclosed targeted peptide having the amino acid sequence of SEQ ID NO: 1 is based on the parent peptide H1299.3 and possesses the same properties with increased solubility and stability.

[0101] Furthermore, the production of cholera or Shiga toxin requires biosynthesis, leaving concerns regarding the immunogenicity of the toxin carrier. In addition, current research differs from hapten painting or antibody recruitment strategies, in which targeted drugs deliver haptens to the cell surface, leading to antibody recruitment. The disclosed immunotherapy is designed to directly activate T cells through the specific presentation of HLA class I restriction antigens in cancer cells via an internalization mechanism, rather than generating antibody-dependent cytotoxicity from hapten immobilization on the cell surface.

[0102] In some aspects, the disclosed immunotherapies do not use viral products or live viruses, thus differing from current virus-based immunotherapies and reducing the safety concerns associated with these types of therapies.

[0103] Cancer immunotherapy, designed to generate cell-mediated immune responses against tumors, is emerging as a frontline treatment option for cancer, but concerns regarding efficacy, safety, and cost-effectiveness limit the use of these therapies. To address these weaknesses, immunotherapies are being developed that can specifically deliver previously encountered antigen peptides to cancer cells and facilitate their presentation via the MHC class I pathway. One example of immunotherapy utilizes a modular synthetic nanoparticle delivery system containing three components: neutral stealth liposomes, encapsulated synthetic immunogenic HLA class I restriction peptides derived from measles virus (MV), and a tumor-targeting peptide (called H1299.3 or SRI_MGS5_V1) on the outer surface of the liposomes. The targeted peptide leads to specific accumulation of liposomes within cancer cells, facilitating the presentation of MV-derived immunogenic peptides on HLA class I molecules. This system is called TALL (Targeted Antigen Loaded Liposomes). Therefore, TALL can specifically generate a secondary immune response against targeted tumor cells in patients who have previously been vaccinated against MV or infected with MV. In short, the immunotherapy evokes a response in the immune system as if cancer cells were infected with MV, without the use of viral particles. A significant reduction in tumor growth with TALL has been demonstrated in an invasive LLC1 (lung) mouse model. The result is a strong cytotoxic T lymphocyte (CTL) response specifically against the tumor. This approach has advantages over current immunotherapies: 1) it avoids the need to identify tumor-associated antigens (TAAs) or educate the immune system via a primary immune response, and 2) it is TAA-independent and may be effective against tumors with a low mutational burden.

[0104] Unfortunately, optimization experiments are disclosed for the parent peptide LQWRRDDNVHNFGVWARYRL (SEQ ID NO: 2), which has been proven to be hydrophobic, difficult to purify, and unstable. This presented a problem with liposome coupling. Further peptide optimization was necessary for this peptide to become a clinically viable target drug. The result was the identification of LQWRRNFGVWARYRL (SEQ ID NO: 1).

[0105] The current research has three main objectives: Objective 1 - Optimize the immunogenicity peptide payload of TALL using previously identified MHC class I restriction immunogenicity peptides. Objective 2 - Isolate and characterize novel cancer target ligands that specifically internalize in cancer cells and enter the HLA class I pathway using phage display methodologies. Objective 3 - Evaluate the antitumor efficacy of TALL in tumor models.

[0106] The rationale behind these objectives was as follows: A single immunogenic peptide derived from the measles virus has been used in previous studies. The addition of multiple immunogenic peptides can enhance the immune response against tumors by activating additional memory T cells. The original system demonstrated the efficacy of a single targeted peptide. However, it is unlikely that a single peptide will bind to all tumors encountered clinically. Identifying multiple targeted ligands would broaden the range of patients who can benefit from TALL therapeutically. The ultimate goal is to develop clinically useful cancer therapies. Therefore, in vivo antitumor efficacy trials will be conducted using a mouse tumor model.

[0107] H250 has been previously utilized in the TALL system as an immunogenic peptide that is HLA class I restricted and elicits a strong CD8-specific IFNγ response in HLA A*02:01 patients. However, numerous peptides contribute to pathogen-induced immunogenic responses, and expanding the repertoire of antigenic peptides delivered by the disclosed system may be useful.

[0108] One of the goals of the current research is to synthesize additional immunogenic peptides and add them to the existing TALL system.

[0109] Using solid-phase Fmoc chemistry, identified antigenic peptides derived from measles virus (MV) and vaccinia virus (VACV), as shown in Table 1, were synthesized and purified to a purity of over 95%. These antigenic peptides bind to the HLA A*02:01 haplotype. MV and VACV are well-characterized pathogens that induce a Th1 response and have high coverage for vaccine administration against the pathogens in the population. The H250 peptide was also synthesized with the AlexaFluor546 dye attached via a cysteine ​​located at the C-terminus of the peptide. This allows for doping of small amounts of the labeled peptide into liposomes, so that liposome loading efficiency can be tracked and quantified in in vitro and in vivo delivery of the antigenic peptide. [Table 1]

[0110] One objective was to improve the biophysical and biological properties of the parent sequence of (LQWRRDDNVHNFGVWARYRL(SEQ ID NO: 2)). This was achieved by determining the minimal binding motif of LQWRRDDNVHNFGVWARYRL(SEQ ID NO: 2), testing N-terminal and C-terminal modifications, and polymerizing the peptide on a scaffold.

[0111] By determining the minimum binding domain, we can identify locations where peptides can be modified or even cleaved without sacrificing functionality. Reducing peptide length is also expected to lower the cost of synthesizing the targeted portion, while improving synthesis yield and reducing undesirable proteolysis and potential immune responses. Furthermore, key regions of the peptide can be focused on to improve solubility and stability. For each peptide sequence in which amino acids 1-5 (A1), 6-10 (A2), 11-15 (A3), or 16-20 (A4) were replaced with alanine, a set of four peptides was synthesized (Table 2). Previous data showed that the dimer form of the parent peptide, SEQ ID NO: 2, had the optimal valency. Thus, each peptide was ligated to a lysine core containing a biotin tag to create the peptides shown in Figure 1. The peptides were tested for binding to the NSCLC strain using flow cytometry. Once the binding region was determined, a second round of peptides was created in which individual amino acids were replaced with alanine (Table 2). [Table 2]

[0112] Based on the alanine scan results (shown in Figure 1), amino acids 1-5 and 11-20 were determined to contain important binding determinants. A single alanine substitution within amino acids 6-10 did not affect the peptide bond, indicating that this region does not contribute to binding and cell uptake. The peptide consisting only of amino acids 11-20 did not bind to H1993 cells. Surprisingly, the removal of amino acids 6-10 and the fusion of the C-terminus and N-terminus of the parent peptide resulted in a peptide with increased cell binding and better physiological and chemical properties. The SRI_MGS5_V1 peptide was further improved by acetylation and dimerization on a branched lysine core (Figure 2). The finally optimized SRI_MGS5 peptide consists of 15 amino acids, an acetylated N-terminus, and a PEG11 linker on the C-terminus to increase solubility and prevent aggregation, and is dimerized on a core that allows for further modification and attachment to liposomes. ((CH3CO-LQWRRNFGVWARYRL-PEG11)2K-X, where X is a tag or reactive portion for conjugation called SRI_MGS5_V2).

[0113] The final optimized SRI_MGS5_V2 peptide, SRI_MGS5_V1, possesses the following characteristics: It is less hydrophobic compared to the parent peptide, which dramatically increases the peptide's solubility and stability, thus improving coupling to liposomes and making it easier to handle. Flow cytometry data, as shown in Figure 3, demonstrate that SRI_MGS5_V2 has higher binding affinity to several NSCLC strains compared to the parent SRI_MGS5_V1 peptide. Importantly, this data suggests that SRI_MGS5_V2 binds to multiple NSCLC cell lines and has a high potential for broad applicability in NSCLC (Figure 3). Furthermore, SRI_MGS5_V2 has also been shown to bind to several breast cancer strains (MCF-7, MDA-MB-23 Land 4T1), pancreatic cancer strain (Pan02), and glioma cell line (GL-261) (see Figures 28-33). SRI_MGS5_V2 shows minimal binding to normal control human lung epithelial cell lines (HBEC, see Figure 3). This peptide exhibits high discriminatory power between NSCLC cancer cells and normal lung cells.

[0114] A quantitative flow cytometry assay was performed to measure the average number of internalized peptide molecules per cell. SRI_MGS5_V2 was internalized into three human NSCLC cell lines (Figure 4). The number of internalized molecules corresponded to intracellular concentrations of 100–800 nM, resulting in 1 × 10⁶ molecules for H358 cells. 5 From individual molecules / cells, 8 × 10 for H1993 cells 5 This is in the range of individual molecules / cells. The uptake rate at 6 hours is not saturated, and the peptide appears to continue entering cells beyond this time (Figures 5 and 6). This indicates either a high number of receptors that are not yet saturated, or rapid recycling of receptors that enables continuous delivery, leading to high intracellular concentrations of the peptide.

[0115] Uptake of SRI_MGS5_V2 was significantly reduced when experiments were conducted at 4°C, indicating that binding and internalization are receptor-mediated. Uptake of SRI_MGS5_V2 is not toxic to H1993 cells. No significant loss of viability was observed when cells were incubated with a high concentration of 250 μM SRI_MGS5_V2 for 72 hours.

[0116] Confocal microscopy results show that SRI_MGS5_V2 accumulates much more significantly in autophagy vesicles within H1993 NSCLC cells compared to the parent MGS5 V1 (Figure 7). As indicated in the original proposal, SRI_MGS5_V1 promotes MHC class I presentation of the antigen peptide via an autophagy-dependent mechanism, and treatment with an autophagy inhibitor resulted in a significant reduction in IFN-γ secretion. Therefore, accumulation in autophagy vesicles is a crucial element for the success of this immunotherapy. Comparable results are observed in LLC1 cells.

[0117] SRI_MGS5_V2 and its parent peptide, SRI_MGS5 V1, were labeled with the near-infrared dye Alexa Fluor 750 for imaging. Following injection via the tail vein, the peptides accumulated almost equally specifically at the site of subcutaneous H1993 tumors, and the signal was maintained for 72 hours, indicating internalization of the peptide dye conjugate (Figures 8 and 9). Ex vivo imaging at 72H showed that SRI_MGS5_V2 reduced nonspecific accumulation in the lungs and other organs compared to the acetylated parent peptide, SRI_MGS5_V1, which is a desirable quality for NSCLC treatment. For example, SRI_MGS5_V2 showed a 3-fold lower signal in the lungs compared to SRI_MGS5_V1. Similarly, SRI_MGS5_V2 showed 1.6-fold and 2.2-fold lower signals in the liver and kidneys, respectively (Figure 10). Importantly, the corresponding non-acetylated versions of each peptide were also evaluated. No tumor targeting was observed for any of the non-acetylated peptides, highlighting the importance of protecting the N-terminus of the peptide for serum stability.

[0118] SRI_MGS5_V5 also has an LLC1 tumor subcutaneously transplanted into the posterior abdomen of a C57BL / 6 mouse (Figures 11-13). The LLC1 tumor is palpable (approximately 500 mm). 3 The mice were allowed to grow to a certain stage, at which point they were intravenously treated with 15 μg of SRI_MGS5_V2 conjugated to Alexa Fluor 750. Images were taken daily for 72 hours, after which the animals were sacrificed and their organs were evaluated to determine the localization of the targeted peptide. The results showed that SRI_MGS2_V2 was successfully localized within the LLC1 tumor mass. Thus, this peptide can be used for tumor targeting in syngeneic tumor models.

[0119] Liposome optimization was also studied. Liposomes were optimized by coupling with SRI_MGS5_V2, which resulted in greater binding to NSCLC strains and localization within the autophagocytic follicles of these cells. To optimize the TALL system, SRI_MGS5_V2 was coupled to liposomes, which were then characterized and used in co-culture assays to determine inflammatory cytokine production.

[0120] The TALL system was prepared using the stealth liposome formulation used in DOXIL®, but doped with 1.2% DSPE-PEG2000 maleimide to enable SRI_MGS5_V2 conjugation. Dynamic light scattering was used to determine the size of the resulting liposomes to be approximately 100 nm (Figure 14). To determine the loading efficiency, fluorescently labeled antigen peptides were loaded into the liposomes, and they were treated with a surfactant to rupture and release their loads. Fluorescence measurements after surfactant treatment were used to estimate the loading efficiency of the liposome preparation technique. The antigen peptide loading efficiency of the liposomes was determined to be 50%, consistent across batches.

[0121] To ensure that SRI_MGS5_V2-targeted liposomes are still transported to autophagocytic vesicles, green fluorescent protein (GFP) was encapsulated within liposomes modified externally with the SRI_MGS5 variant. Clear co-localization of the liposomes with autophagocytic vesicles is observed, as shown by punctate yellow staining, as shown in Figure 15. Co-localization is observed for both peptides, but again, SRI_MGS5_V2 is superior to the parent peptide.

[0122] Co-culture assays were performed using SRI_MGS5_V2 and the parent peptide to confirm that the optimized peptide mediates the delivery and presentation of the antigen peptide in HLA class I molecules, resulting in equivalent antigen presentation and subsequent immune response. H250 antigen peptides were loaded into liposomes, and SRI_MGS5_V2 or SRI_MGS5 V1 was conjugated to the outer surface of the liposomes via maleimide chemistry. After incubation with H1993 cells, the liposomes were co-cultured with peripheral blood mononuclear cells (PBMCs) from anonymous donors with haplotype HLA A2+ and measles vaccination. ELISA results showed that both the TALL system corresponding to the parent SRI_MGS_V1 and SRI_MGS5_V2 exhibited similar levels of TNF-α production in the co-culture assay (Figure 16). Further experiments were conducted to test different liposome variants and evaluate TNF-α and INF-γ in co-culture. As shown in Figures 17 and 18, complete TALL treatment, containing liposome-encapsulated H250 antigen peptide and targeted Sri_MGS5_V2 peptide on the liposome surface, yielded both IFN-γ and TNF-α in co-culture experiments. Liposomes loaded with H250 peptide but without the H250 targeted peptide or without Sri_MGS5_V2-conjugated liposomes showed a significant decrease in IFN-γ and TNF-α in the same co-culture assay; therefore, both the targeted peptide and the antigen peptide are necessary to observe delivery and presentation that lead to an immune response. Depletion of CD8+ T cells from PBMCs prevented secretion of IFN-γ and TNF-α, indicating that the immune response is CD8+-dependent. The same experiment was completed using mouse LLC1 cells as antigen-presenting cells and lymph node lymphocytes (LNL) from C57B1 / 6 mice vaccinated against HA. Similar data have been observed, and it has been shown that syngeneic tumor models can be used to evaluate the in vivo response to TALL therapy.

[0123] Testing the ability of SRI_MGS5_V2 to actively direct liposomes to tumor mass in an animal model confirms that the targeted peptide can localize liposomes within LLC1 tumor mass in C57BL / 6 mice. To do this, SRI_MGS2_V2 was conjugated to stealth liposomes labeled with the lipophilic NIR dye DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide), which was inserted into the phospholipid membrane of the liposomes and allowed to be imaged in vivo. Liposomes not conjugated to the peptide served as a control. This is particularly important because liposomes are known to accumulate in tumors due to enhanced permeability and retention effect (EPR) effects. Equimolar liposome formulations were intravenously injected via the tail vein into C57B1 / 6 mice with subcutaneous LLC1 tumors. Tumors were imaged in vivo at 24, 48, and 72 hours. As seen in Figures 20 and 21, SRI_MGS5_V2 liposomes localized to a much greater extent within the tumor mass compared to blank liposomes (liposomes without the targeted peptide). Ex vivo imaging of tumors and other organs showed three times the accumulation of SRI_MGS5_V2 targeted liposomes compared to untargeted liposomes in tumors. SRI_MGS5_V2 liposomes showed a 2.5-fold reduction in the lungs compared to untargeted liposomes. Therefore, SRI_MGS5_V2 improves tumor targeting while reducing off-target accumulation in other organs.

[0124] Antigen peptide optimization was also studied. H250 was used as an immunogenic peptide in the TALL system, which is HLA class I restricted and elicits a strong CD8-specific IFNγ response in HLA A*02:01 patients. However, many peptides contribute to pathogen-induced immunogenic responses, and expanding the repertoire of antigen peptides delivered by the disclosed delivery system may be useful.

[0125] One objective was to add additional immunogenic peptides to existing liposomes and test the efficacy of a novel TALL system via co-culture assays. Initially, measles virus-derived H3L and C166 peptides were used as additional antigenic peptides in the TALL system, but unfortunately, they proved to be hydrophobic, difficult to purify, and unstable. Therefore, a different antigenic peptide, H516 (ILPGQDLQYV), identified from patients with the HLA A*02:01 haplotype and highly immunogenic, was used and incorporated into TALL liposomes. The biantigenic peptide (H516+H250) encapsulated liposomes were characterized using DLS and zeta potential measurements (Figures 22 and 23). The size of the resulting liposomes was determined to be approximately 155 nm, and their zeta potential was approximately -25.3 mV.

[0126] Co-culture assays were performed to determine the effectiveness of the dual-antigen liposome system compared to single-antigen H250. SRI_MGS5_V2-targeted liposomes containing both H516 and H250 peptides generated the same levels of IFNγ and TNF-α secretion as liposomes containing only H250 (Figures 17-19). In a similar manner, liposomes encapsulating both H250 and C166 antigen peptides were tested for their ability to stimulate TNF-α secretion in co-culture assays. In this case, dual-antigen peptide liposomes showed a reduction in TNF-α compared to liposomes containing either H250 or C166. Because dual-antigen liposomes showed similar, or in some cases worse, activity, the focus was shifted to the single H250 antigen. However, the results demonstrate that the system is effective with other antigen peptides. Given the modular nature of TALL, different antigens can be used based on patient needs.

[0127] SRI_MGS5_V2 binds to breast, pancreatic, and glioblastoma cell lines and recognizes tumors in syngeneic orthotopic tumor models for each of these tumor types. This indicates that this peptide has broad utility and can recognize many different tumors. Thus, it makes sense to focus on the SRI_MGS2_V2 peptide, as it has the ability to deliver antigen peptides and is known to facilitate the presentation of antigen peptides in MHC class 1.

[0128] Several experiments required the development of a luciferase expressing LLC1 cells. In vivo experiments involved generating subcutaneous LLC1 tumors in mice, followed by TALL treatment. To generate an orthotopic lung tumor model, it was essential to create a suitable luciferase expressing the LLC1 cell line. Lentiviral reporter constructs were used to obtain stable luciferase expression. Stable luciferase expressing the LLC1 cell line was created using lentiviral vector constructs containing the firefly luciferase gene and transduction of LLC1 cells with these constructs.

[0129] The following procedure was used to generate a pseudoviral vector for transduction. The luciferase gene containing the plasmid-pTrip-luc was constructed by replacing the eGFP sequence of the Trip-eGFP plasmid with the firefly luciferase gene between the BamHI and XhoI sites. The coding sequence of the firefly luciferase was PCR amplified from the pGL4-Luc vector using the following primers. 5'-AGAGGATCCACCGGTCGCCACCATGGAAGATGC-CAAAAAC-3'(Sequence No. 12)(Sense) 5'-ATAGCTCGAGTTAGACGTT-GATCCTGGCGC-3'(Sequence ID 13)(Antisense).

[0130] To generate pseudovirus particles, 6*10 5Lenti-X 293T cells were seeded in 4 ml of DMEM medium containing 10% FBS in a 6 cm polystyrene dish 24 hours prior to transfection. The following day, cells were transfected using polyethyleneimine (PEI, MW 25Kd) or Lipofectamine 2000 reagent (Invitrogen). VSV-Gpp pseudoviral particles were packaged by transfection with 0.5 mL of DNA mixture containing 2 μg of pTrip-Luc, 2 μg of CMV-dR8.2 plasmid, and 1 μg of HEF-VSV-G plasmid (expressing VS VG envelope protein). The medium was replaced 16 hours after transfection. The supernatant containing VS V-Gpp pseudoviral particles was collected 36–48 hours after transfection and filtered through a 0.45-μm syringe filter.

[0131] LLC1 cells were transduced with these pseudoviral vectors for 3–4 hours with the addition of polyblen, followed by removal of the vectors and replacement with fresh RPMI medium containing 10% FBS. The transduced LLC1 cells were left in an incubator for 72 hours. Next, the cells were counted using a hemocytometer and diluted to obtain a final density of 100 cells in 1 ml of medium. 100 μL of this cell suspension was added to each well of a 96-well plate, so that 80% of the wells contained single cells. These cells were grown for 7–10 days to obtain a clonal population. Their luciferase expression was tested every other week for a period of 4 weeks using the Promega luciferase assay system to confirm stable luciferase expression.

[0132] Through this process, it was found that luciferase-expressing LLC1 cells drift over time, and meticulous care must be taken to control luciferase expression.

[0133] The antitumor efficacy of the TALL system was evaluated in an orthotopic lung tumor model. The efficacy of optimized TALL therapy was tested in a syngeneic orthotopic lung cancer model. Tests in immune-capable C57BL / 6 mice can provide a measure of the antitumor immune response induced by TALL therapy in vaccinated animals.

[0134] An orthotopic lung tumor model was generated in C57BL / 6 mice using luciferase-expressing LLC1 cells. C57BL / 6 mice were vaccinated using a gene-immunization strategy. A plasmid vector encoding the complete measles hemagglutinin protein containing the CMV promoter sequence was developed, which can generate a strong Th1-like CTL response. Plasmid (10 ng) was administered intramuscularly once a week for a period of 4 weeks. After the vaccination period, lymphocytes were obtained from the spleens of vaccinated mice from the group. Successful vaccination was measured by IFN-Y secretion via a co-culture assay with TALL treatment in peptide-pulsed lymphocytes. After confirming successful vaccination, 1 × 10⁶ cells were collected. 6 Luc-LLC1 cells were delivered to each mouse via tail vein IV injection. Within two weeks, bioluminescence was observed in the animals' lungs upon D-luciferin injection, indicating successful lung tumor formation.

[0135] Animals were treated with TALL-targeted liposomes every other day for a total of six treatments. The control group included animals that received no treatment. Bioluminescence from the lungs of all mice was measured every other day via IP injection of 250 μL of 15 mg / mL D-luciferin solution throughout the entire treatment period, followed by IVIS imaging. Reduced luciferin expression was observed in the lungs of animals in the TALL-treated group (Figure 24), which corresponds to a decrease in tumor size. Increased luciferase expression was observed in untreated mice, through experiments showing an increase in tumor size. Unfortunately, as shown in Figure 24, some animals died in both groups throughout the treatment. Changes in tumor flux in the surviving animals are shown in Figure 25. At the end of the experiment, the lungs of the animals were resected, weighed, and observed for tumor nodule formation. The lungs of TALL-treated animals were found to have little to no nodules compared to untreated animals (Figure 25).

[0136] To evaluate the tumor-reducing effect of TALL, a larger study was conducted using more treatment groups. The treatment groups were as follows: 1) treatment with complete TALL liposomes, 2) TALL liposomes containing an externally targeted peptide but without an antigen peptide, 3) blank liposomes containing neither a targeted peptide nor an antigen peptide, and 4) TALL liposomes without a targeted peptide. In all four groups, increased flux and decreased luciferase expression were observed in the lungs of animals treated with complete TALL compared to all other treatment groups (Figures 26 and 27). The decrease in flux release corresponds to a decrease in total luciferase expression, which indicates a reduction in the total number of tumor cells in the lung, and therefore a reduction in tumor size. A smaller reduction in tumor size was observed in the treatment groups without a targeted peptide. This is expected to be due to the accumulation of several liposomes observed within the tumor mass, due to enhanced permeability and retention (EPR) effects, along with slower internalization into cells. Increased flux release (and therefore tumor size) was observed in both the blank liposome treatment group and the group treated without antigen peptides. This highlights the importance of immunization and the presence of antigen peptides for successful TALL treatment.

[0137] Those skilled in the art will be able to recognize or confirm, through mere habitual experimentation, many equivalents to specific embodiments of the methods and compositions described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. An antigen delivery system comprising PEG-modified liposomes, wherein the PEG-modified liposomes are surface-modified with a cancer-specific cell-targeting peptide and contain an immunogenic human leukocyte antigen (HLA) class I restriction peptide, the HLA class I restriction peptide being a vaccine-dependent immunogenic HLA class I restriction peptide, and the cancer-specific cell-targeting peptide comprising a dimer containing two targeting peptides each having the sequence CH3CO-LQWRRNFGVWARYRL, the two targeting peptides being linked via PEG11 and a lysine branch point to form the sequence (CH3CO-LQWRRNFGVWARYRL-PEG11). ２ A K-forming antigen delivery system.

2. The antigen delivery system according to claim 1, wherein the vaccine-dependent immunogenic HLA class I restriction peptide is measles virus hemagglutinin peptide H250.

3. The antigen delivery system according to claim 1, wherein the vaccine-dependent immunogenic HLA class I restriction peptide is influenza virus hemagglutinin peptide HA.

4. The antigen delivery system according to claim 1, wherein the vaccine-dependent immunogenic HLA class I restriction peptide is a smallpox virus H-2Kd restriction vaccinia-specific peptide, A5275-83 (VACV-A52).

5. The antigen delivery system according to claim 1, wherein the vaccine-dependent immunogenic HLA class I restriction peptide is H250 (SMYRVFEVGV), C166 (SLWGSLLML), H38 (LLAVIFVMFL), H516 (ILPGQDLQYV), H3L (SLSAYIIRV), E2L (KIDYYIPYV), or 0IL (GLNDYLHSV).

6. The antigen delivery system according to claim 1, wherein the antigen delivery system does not contain any viral particles, toxins, or biological materials.

7. The antigen delivery system according to claim 1, wherein the cancer-specific cell-targeting peptide is acetylated at its N-terminus.

8. The antigen delivery system according to claim 1, wherein the cancer-specific cell-targeting peptide includes a PEG linker at its C-terminus.

9. The aforementioned sequence (CH3CO-LQWRRNFGVWARYRL-PEG11) ２ The antigen delivery system according to claim 1, wherein K comprises CH3CO-LQWRRNFGVWARYRL-PEG11-K-PEG11-LRYRAWVGFNRRWQL-CH3CO.

10. The antigen delivery system according to claim 1, wherein the dimer further includes a conjugated portion on the C-terminal side of the lysine branching point, and the conjugated portion enables conjugation to the liposome.

11. A pharmaceutical composition for treating a subject having cancer, comprising the antigen delivery system described in claim 1.

12. The pharmaceutical composition according to claim 11, wherein the antigen delivery system targets cancer cells in the subject, and when the liposomes enter the cancer cells in the subject, the cancer cells present the vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system, and the subject generates an immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen.

13. The pharmaceutical composition according to claim 12, wherein the immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen targets and kills cancer cells that present the vaccine-dependent immunogenic HLA class I restriction peptide antigen.

14. The pharmaceutical composition according to claim 11, wherein the cancer-specific cell-targeting peptide targets the liposomes against the cancer cells.

15. The pharmaceutical composition according to claim 11, further comprising a therapeutic agent.

16. The pharmaceutical composition according to claim 15, wherein the therapeutic agent is a cancer treatment agent.

17. The pharmaceutical composition according to claim 11 for detecting the immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen in the subject.

18. A pharmaceutical composition for killing cancer cells, comprising the antigen delivery system described in claim 1, wherein when the liposomes enter the cancer cells, the cancer cells present the vaccine-dependent immunogenic HLA class I restriction peptide from the antigen delivery system, the cancer cells produce an immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen, and the immune response to the vaccine-dependent immunogenic HLA class I restriction peptide antigen targets and kills the cancer cells presenting the vaccine-dependent immunogenic HLA class I restriction peptide antigen.

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