Human Anti-PD-L1 Peptide Vaccine and Method of Using the Same

Abstract

Compositions related to synthetic PD-L1 peptides, chimeric PD-L1 peptides, and anti-PD-L1 antibodies, and methods for treating cancer, autoimmune diseases and Alzheimer's disease using said peptides or antibodies are disclosed.

Description

Background Technology

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 901,727 filed September 17, 2019, which is incorporated herein by reference in its entirety.

[0003] Cancer is now a leading cause of death in developed countries and globally. The financial burden of this disease, and more importantly, the suffering it causes, is immense. There is a clear and urgent need to accelerate the development and application of new and more effective anticancer therapies. The field of oncology is vast and comprises various indications, including some rare and orphan forms. While oncology is one of the most active fields in terms of drug development, there is still significant unmet demand.

[0004] Recent advances in cancer immunology have documented the importance of T cell-mediated anti-tumor immunity for human cancers, and inhibitory receptors expressed by T cells have become important targets for cancer immunotherapy. Signaling via the immune checkpoint programmed death protein-1 (PD-1) attenuates the anti-tumor immune response, enabling tumor progression. Therapeutics blocking the signaling axis between PD-1 and its ligand, programmed death ligand-1 (PD-L1), with monoclonal antibodies have demonstrated remarkable clinical success in cancer treatment and have shown impressive activity across a wide range of cancer subtypes, even in advanced and metastatic stages of the disease. Therapeutics targeting this pathway are currently in clinical trials. Pembrolizumab and nivolumab are the first class of anti-PD-1 pathway checkpoint inhibitors to receive accelerated approval from the U.S. Food and Drug Administration (FDA) for the treatment of ipilimumab-refractory melanoma.

[0005] Monoclonal antibodies targeting immunological checkpoints, particularly the PD-1 / PD-L1 axis, have delivered remarkable results in cancer treatment over the past few years. Despite their proven utility, antibodies have certain drawbacks as therapeutic agents, including poor tissue / tumor infiltration, which may be particularly relevant when targeting the PD-1:PD-L1 signaling pathway. For example, PD-1-expressing effector T cells are found to infiltrate the solid tissues of PD-L1-expressing tumors. This is problematic for antibodies because their large size hinders their entry into the tumor. Consequently, antibodies may fail to completely antagonize PD-1:PD-L1 signaling at the intended treatment site within the tumor, potentially leading to suboptimal efficacy.

[0006] Checkpoint blocking triggers a new paradigm shift in cancer immunotherapy. However, many cancer patients have not responded to PD-1 / PD-L1 checkpoint blocking. New PD-1 / PD-L1 checkpoint inhibitors are needed for the treatment of cancer, viral infections, autoimmune diseases, and Alzheimer's disease.

[0007] A method and composition related to a synthetic PD-L1 peptide are disclosed.

[0008] In one aspect, a PD-L1 chimeric peptide for stimulating an immune response to a PD-L1 protein is disclosed herein, comprising one or more PD-L1 B cell epitopes, a T helper (Th) epitope (e.g., a measles virus fusion protein peptide such as SEQ ID NO: 6), and a linker (e.g., SEQ ID NO: 7) linking the PD-L1 B cell epitope to the Th epitope, wherein one or more PD-L1 B cell epitopes consist of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. For example, any prior aspect chimeric peptide is disclosed herein, wherein the peptide comprises the amino acid sequence presented in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0009] Also disclosed herein is a synthetic PD-L1 peptide for stimulating an immune response to a PD-L1 protein, comprising one or more of the sequences described in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, comprising a D enantiomer of the disclosed sequence. In one aspect, the synthetic peptide may be acetylated.

[0010] In one aspect, a chimeric peptide is disclosed herein comprising a synthetic peptide of any preceding aspect further comprising a Th epitope (e.g., a measles virus fusion protein peptide such as SEQ ID NO: 6) and a linker (e.g., SEQ ID NO: 7) linking a synthetic PD-L1 peptide to the Th epitope. For example, a chimeric peptide of any preceding aspect is disclosed herein, wherein the peptide comprises the amino acid sequence presented in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0011] In addition, a pharmaceutical composition is disclosed comprising any one or more preceding chimeric or synthetic peptides and a pharmaceutically acceptable vehicle (e.g., a biodegradable vehicle comprising, but not limited to, an adjuvant (e.g., a water-in-oil adjuvant comprising, but not limited to, Montanide).

[0012] In one aspect, any prior aspect pharmaceutical composition is disclosed, further comprising one or more HER-2 B cell epitopes (e.g., SEQ ID NO: 27 or SEQ ID NO: 29), one or more chimeric HER 2 peptides (e.g., SEQ ID NO: 28 or SEQ ID NO: 30), and / or one or more anti-Her-2 antibodies.

[0013] Also disclosed is a HER-2 chimeric peptide for stimulating an immune response to HER-2, comprising one or more HER-2 B cell epitopes, a T helper (Th) epitope (e.g., including but not limited to a measles virus fusion protein such as SEQ ID NO: 6), and a linker (e.g., SEQ ID NO: 7) linking the HER-2 B cell epitope to the Th epitope, wherein one or more HER-2 B cell epitopes consist of a sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 29. For example, the HER-2 chimeric peptide comprises the amino acid sequence presented in SEQ ID NO: 28 or SEQ ID NO: 30.

[0014] In one aspect, an antibody that specifically binds to a chimeric peptide or synthetic peptide of any preceding aspect is disclosed herein.

[0015] Additionally, a method for treating, inhibiting, reducing, decreasing, improving, and / or preventing cancer and / or metastasis (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer), Alzheimer's disease, or autoimmune disease in a subject is disclosed herein, including administering any of the peptides or compositions of any prior aspect to a subject. Brief explanation of the drawing

[0016] The accompanying drawings included in and constituting part of this specification illustrate various embodiments and illustrate the configurations and methods disclosed together with the description. Figure 1 shows an enlarged view of hPD-1 / hPD-L1. The interfaces hPD-1 and hPD-L1 are indicated by blue and green ribbons, respectively. All residues critical to the interaction are highlighted by bars. Residues forming the hydrophobic core are indicated in yellow. Water molecules are indicated by red spheres. Hydrogen bonds are indicated by black dashed lines. (a) Front view (Zak et al., 2015, Structure 23, 2341-2348). (b) PD-1 peptide modeling. Figure 2 shows the predicted B-cell epitopes for human PD-L1 and HER-2 peptides. The amino acid sequences of human PD-L1, peptides 36-53, 50-67, 95-112, and 130-147 were selected for evaluation. Additionally, the existing HER2 (266-296) and HER2 (597-626) were used here as a combination therapy. Figure 3 shows the immunogenicity of the PD-L1 epitope versus the MVF-PD-L1 epitope vaccine in rabbits. Immunogenicity of the MVF-PD-L1 B cell epitope. New Zealand white rabbits were immunized with 1 mg of each MVF-peptide immunogen dissolved in dd H2O (1:1) emulsified in Montanide ISA 720 vehicle (Seppic) along with 333 μg of N-acetylglucosamine-3-acetyl-l-alanyl-d-isoglutamine (nor-MDP). Rabbits were boosted with the same dose at 3-week intervals. Blood was collected via the central auricular artery of the rabbits. Blood was collected weekly (1Y+3, 2Y+1, 2Y+2, 2Y+3, 3Y+1, 3Y+2), and serum was tested for antibody titers by ELISA using the immunogen MVF-PD-L1 peptide. Serum (terminal) from rabbits (3Y+3) immunized with the MVF-PD-1 peptide immunogen was individually tested for the immunogen by ELISA. ELISA plates were coated using a double layer of 200 ng / well peptide. Titer was defined as the highest dilution of serum, and the absorbance value after subtracting the blank was 0.2. Figure 4 shows the antigenicity of PD-L1 epitopes versus recombinant PD-L1 protein. Serum (terminal) from rabbits (3Y+3) immunized with the MVF-PD-L1 peptide immunogen was individually tested for the immunogen rhPD-L1 protein by ELISA. ELISA plates were coated using a double layer of 500 ng / well of rhPD-L1 protein. Figures 5a, 5b, 5c, and 5d illustrate the experimental plan for testing PD-L1 immunization in Balb / c mice. Figure 5a shows Experiment 1: a CT26 WT tumor model for Balb / c mice immunized with MVF-PD-L1 + ISA720. Figure 5b illustrates Plan 1B: Balb / c mouse vaccination and tumor engraftment plan. Balb / c mice aged 6–8 weeks were immunized three times at 3-week intervals with an MVF-peptide immunogen emulsified in ISA 720 containing nor-MDP. Mice were immunized with the MVF-PD-L1 vaccine construct [PD-L1(36–53), PD-L1(50–67), PD-L1(95–112), PD-L1(130–147)] prior to tumor challenge. Blood was collected weekly, and serum was tested for antibody titers using ELISA. Two weeks after the third immunization (3Y), 10 CT26 WT tumor cells per mouse 5 The mice were engrafted. Control mice were treated with PBS twice weekly as a negative control starting 2 days after the tumor challenge, and with anti-mPD-L1 mAb (10F.9G2) twice weekly as a positive control. Tumor growth was observed twice weekly and measured with calipers. Figure 5c shows the Plan 1C antibody control scheme. Figure 5d shows mice immunized with the PD-L1 vaccine challenge 2 weeks after the secondary boost in CT26 WT under Plan 1D. Figure 6 shows the immunogenicity of PD-L1 epitopes versus MVF-PD-L1 immunogens in Balb / c mice. Immunogenicity of MVF-PD-L1 peptides in BALB / c mice immunized with various peptide constructs. Serum collected weekly was titrated against each individual MVF-PD-L1 peptide immunogen. Pink bars represent titers for PD-L1 130 and red bars represent titers for PD-1 (92-110). Figure 7 shows the antigenicity of PD-L1 epitopes versus recombinant human PD-L1 protein in a CT26 tumor model. Serum from mice immunized with the MVF-PD-L1 peptide immunogen was individually tested for the immunogen rhPD-L1 protein by ELISA. ELISA plates were coated using a double layer of 500 ng / well of rhPD-L1 protein. Figure 8 shows individual plots of CT26 WT tumor growth in BALB / c mice immunized with MVF-PD-L1 vaccine components [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112), PD-L1(130-147)], with PBS used as a negative control and anti-mPD-L1mAb(10F.9G2) used as a positive control. Figure 9 shows the mean tumor growth in BALB / c mice immunized with the MVF-PD-L1 vaccine components [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112), PD-L1(130-147)], with PBS used as the negative control and anti-mPD-L1mAb (10F.9G2) as the positive control. The overall curve of tumor growth was analyzed using two-way ANOVA, which showed a significant difference with p<0.01. Figure 10 shows plots of tumor volume LWW at 14 and 16 days for each of the four treatment groups; one-way ANOVA was used to analyze multiple group comparisons, both of which showed p<0.01. Figure 11 shows antibody isotypes in BALB / c mice after immunization with MVF-PD-L1 vaccine components [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112), PD-L1(130-147)], nor-MDP, and ISA 720. Figure 12 shows the PD-1 / PD-L1 biopsy. The assay was performed according to the manufacturer's (Promega) protocol for the PD-1 / PD-L1 blockade biopsy. Only the PD-L1 (130-147) epitope was able to inhibit PD-1 / PD-L1 compared to nivolumab and atezolizumab. The PD-L1 (50, 95, and PD-L1-92) epitopes could not block PD-1 and PD-L1. Figure 13 shows apoptosis (caspase 9 assay). The Caspase-Glo 9 assay kit (Promega, Madison, WI) was used to detect caspases in cells treated in vitro. Figure 14 shows ADCC activity. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action for anti-cancer mAbs. Figures 15a, 15b, 15c, and 15d show the plan for Experiment 2: a CT26 HER-2 tumor model in Balb / c mice immunized with MVF-PD-L1+ISA720. Figure 15a shows Plan 2A, 100 µg / mouse (based on 20 g body weight per mouse = 5 mg / kg) every 3 days during the study period (PMID: 29337305). Figure 15b shows Balb / c mouse vaccination and CT26 HER-2 tumor engraftment. Balb / c mice aged 6–8 weeks were immunized three times at 3-week intervals with an MVF-peptide immunogen emulsified in ISA 720 containing nor-MDP. Mice were immunized with the MVF-PD-L1 vaccine construct [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112), PD-L1(130-147)] prior to the tumor challenge. Blood was collected weekly, and serum was tested for antibody titers using ELISA. Two weeks after the third immunization (3Y), 10 CT26 WT tumor cells per mouse were administered. 5The mice were engrafted. Control mice were treated with PBS twice weekly as a negative control starting 2 days after the tumor challenge, and with anti-mPD-L1 mAb (10F.9G2) twice weekly as a positive control. Tumor growth was observed twice weekly and measured with calipers. Figure 15c shows the plan for antibody control (Plan 2C). Figure 15d shows mice immunized with the PD-L1 vaccine challenge 2 weeks after a secondary boost using CT26 / HER-2. Figure 16 shows the immunogenicity of the MVF-PD-L1 peptide and the combination (MVF-PD-L1-(95) + MVF-PD-1(92-110)) in BALB / c mice immunized with various peptide compositions. Serum was collected weekly and titrated for each individual MVF-PD-L1 or MVF-PD-1 peptide immunogen. Pink bars represent the titers for PD-L1-95; red bars represent the titers for PD-1(92). Figure 17 shows the antigenicity of PD-L1 epitopes versus recombinant human PD-L1 protein in a CT26-HER2 tumor model. Serum from mice immunized with the MVF-PD-L1 peptide immunogen was individually tested for the immunogen rhPD-L1 protein by ELISA. ELISA plates were coated using a double layer of 500 ng / well of rhPD-L1 protein. Figure 18 shows individual plots of CT26 HER2 tumor growth in BALB / c mice immunized with the MVF-PD-L1 vaccine components [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112), PD-L1(130-147)], using PBS as a negative control and anti-mPD-L1 mAb(10F.9G2) as a positive control. Figure 19 shows the mean values ​​of CT26 HER2 tumor growth in BALB / c mice immunized with the MVF-PD-L1 vaccine components [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112), PD-L1(130-147)] and the MVF-PD-L1-(95) + MVF-PD-1(92-110) combination, PBS as a negative control, and anti-mPD-L1 mAb(10F .9G2) as a positive control. The overall curve of tumor growth was analyzed using two-way ANOVA, which showed a significant difference with p<0.01. Figure 20 shows plots of tumor volume LWW at 14 and 16 days for each of the four treatment groups; one-way ANOVA was used to analyze the multiple group comparisons, which showed p<0.05 at 14 days and p<0.01 at 16 days, respectively. Figure 21 shows antibody isotypes in Balb / c mice after immunization with MVF-PD-L1 vaccine components [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112)], PD-L1(130-147)], nor-MDP and ISA 720. Figure 22 shows a comparison of 50% mouse survival times; Balb / c mice immunized with MVF-PD-L1 vaccine constructs [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112), PD-L1(130-147)] with or without MVF-PD-1(92), nor-MDP and ISA 720; 9-10 mice per group, 50% mouse survival times were analyzed using the survival times of 4-5 mice per group after challenging the mice with CT26 HER2 tumor cells. Figures 23a, 23b, and 23c show the plan for Experiment 3: a CT26 HER-2 tumor model on Balb / c mice immunized with combo or triple+ISA720. Figure 23a shows Plan 3A, a CT26 / HER-2 tumor model on Balb / c mice immunized with combo or triple+ISA720. Figure 23b shows Plan 3B, an experiment to test immunization with PD-L1 and HER-2 in Balb / c CT26 / HER-2 mice. Figure 23c shows Plan 3C, Balb / c mouse vaccination and CT26 HER2 tumor engraftment. 6-8 week old Balb / c mice were immunized three times at 3-week intervals with an MVF-peptide immunogen emulsified in ISA 720 containing nor-MDP. Mice were immunized with the MVF-PD-L1 vaccine construct combined with Combo HER2 [PD-L1(36-53), PD-L1(50-67), PD-L1(95-112), PD-L1(130-147)] prior to tumor challenge. Blood was collected weekly, and serum was tested for antibody titers using ELISA. Two weeks after the third immunization (3Y), 10 CT26 WT tumor cells per mouse were administered. 5 The mice were engrafted. Control mice, starting 2 days after the tumor challenge, were treated with PBS twice a week as a negative control and with anti-mPD-L1 mAb (10F.9G2) twice a week as a positive control. Tumor growth was observed twice a week and measured with calipers. Figure 24 shows the immunogenicity of MVF-PD-L1 peptides with combo HER-2 in BALB / c mice immunized with various peptide compositions. Serum collected weekly was titrated against each individual MVF-PD-L1 with each HER2 peptide immunogen. Figure 25 shows the immunogenicity of MVF-PD-L1 peptides with combo HER-2 in BALB / c mice immunized with various peptide compositions. Serum collected weekly was titrated against each individual MVF-PD-L1 with each HER2 peptide immunogen. Figure 26 shows the experimental design for the D2F2 cancer cell challenge. Balb / c mice aged 6–8 weeks were immunized with G1 prior to the tumor challenge, and G2 mice were treated with anti-PD-1 mAb (29F.1A12)(G1) or anti-PD-L1 mAb (10F.9G2)(G2) or MVF-PD-L1 vaccine constructs [PD-L1(92–110)(G3) PD-L1(130–147)(G4)]. Similarly, G10, G11, and G12 were controls and were not treated prior to the tumor cell challenge. G13, G14, and G15 mice were immunized with 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse for G13, 100 µg MVF-PD-1(92-110) + 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse for G14, and 100 µg MVF-PD-L1(130-147) + 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse for G14, and 100 µg MVF-PD-L1(130-147) + 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse for G15. Mice were immunized up to 4 times prior to the tumor challenge. Mice were not subjected to further treatment after challenge with D2F2 / E2 tumor cells. All mice were monitored at least twice per week, and tumors were measured with calipers according to the indicated data. Figure 27 shows a plan for the D2F2 challenge described in Figure 26. Figure 28 shows the results of measuring the immunogenicity of G3 mice immunized with 100 µg MVF-PD-1 (92-110) + ISA720 per mouse using ELISA. Figure 29 shows the results of measuring the immunogenicity of G4 mice immunized with 100 µg MVF-PD-L1 (130-147) + ISA720 per mouse using ELISA. Figure 30 shows the results of measuring the immunogenicity of G13 mice immunized with 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse using ELISA. Figure 31 shows the results of measuring the immunogenicity of G14 mice immunized with 100 µg MVF-PD-1 (92-110) + 100 µg MVF-HER-2 (266-296) + 100 µg MVF-HER-2 (597-626) + ISA720 per mouse using ELISA. Figure 32 shows the results of measuring the immunogenicity of G15 mice immunized with 100 µg MVF-PD-L1 (130-147) + 100 µg MVF-HER-2 (266-296) + 100 µg MVF-HER-2 (597-626) + ISA720 per mouse using ELISA. Figure 33 shows D2F2 WT tumor cell growth in BALB / c mice of the indicated treatment group. Tumor volume was measured with calipers and calculated using the formula: (length x width 2) / 2. The overall curve of tumor growth was analyzed using a full two-way ANOVA, which indicates a significant difference with p < 0.01. All peptide-immunized groups of mice were found to have a lower tumor burden than the PBS group and the mAb-treated groups. Figure 34 shows the log-rank (Mantel-Cox) test for the percentage survival rate of mice treated with the designed group. Figure 35 shows a line plot representing D2F2 / E2 tumor cell growth in BALB / c mice, as shown in the graph. Tumor volume was measured with calipers and calculated using the formula: (length x width 2) / 2. The overall curve of tumor growth was analyzed using a full two-way ANOVA, which showed a significant difference with p < 0.01. Mice treated with all peptides and mAbs were found to have a lower tumor burden than the PBS group. Most importantly, MVF-HER-2 (266-296) + MVF-HER-2 (597-626); Mice immunized with 2XHER2 as MVF-PD-1(92-110) + MVF-HER-2(266-296) + MVF-HER-2(597-626) and MVF-PD-L1(130-147) + MVF-HER-2(266-296) + MVF-HER-2(597-626) showed significant tumor suppression in most tumor-free mice by the end of 28 days after the tumor challenge. Figure 36 shows the log-rank (Mantel-Cox) test representing the percentage survival rate of mice treated with each designed group. Specific details for implementing the invention

[0017] Before the compounds, compositions, articles, devices, and / or methods are disclosed and described, it should be understood that, unless otherwise specified, they are not limited to specific synthetic methods, specific recombinant biotechnological methods, or specific reagents, and that such limitations may naturally vary. Furthermore, it should be understood that the terms used herein are merely for describing specific embodiments and are not intended to be limiting.

[0018] A. Definition

[0019] As used in the specification and appended claims, the singular forms “one” and “it” include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to a “pharmaceutical carrier” includes a mixture of two or more such carriers, etc.

[0020] A range may be expressed in this application as "about" one specific value and / or "about" another specific value. When such a range is expressed, other embodiments include one specific value and / or another specific value. Similarly, when a value is expressed as an approximation, it will be understood that the antecedent "about" is used to indicate that the specific value forms another embodiment. It will be further understood that the endpoint of each range is important both in relation to and independently of other endpoints. Multiple values ​​are disclosed in this application, and each value is also understood to be disclosed in this application as that specific value "about" in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. Furthermore, it is understood that possible ranges between values ​​"less than or equal to," "greater than or equal to," and values ​​are disclosed as appropriately understood by a person skilled in the art. For example, if the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" are disclosed. Additionally, data is provided throughout the application in various formats, and this data indicates a range for a combination of endpoints, starting points, and data elements. For example, when specific data point "10" and specific data point 15 are disclosed, it is considered that not only the range between 10 and 15, but also greater than, equal to, less than, or equal to 10 and 15 are disclosed. Additionally, it is understood that each unit between two specific units is also disclosed. For example, when 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0021] In this specification and the following claims, references will be made to a number of terms defined as having the following meanings:

[0022] "Optional" or "at any time" means that the event or situation described thereafter may or may not occur, and the description includes cases where the event or situation occurs and cases where it does not occur.

[0023] The term "administering" means administration orally, topically, intravenously, subcutaneously, transdermally, via the skin, intramuscularly, intra-articularly, parenterally, intra-arterially, intradermally, intraventricularly, intracranially, intraperitoneally, intralesionally, intranasally, rectalally, vaginally, inhaled, or through an implanted reservoir. The term "parenterally" includes subcutaneous, intravenously, intramuscularly, intra-articularly, synovially, intrasternally, intravertebrally, intrahepatically, intralesionally, and intracranial injection or infusion techniques.

[0024] As used herein, the term “comprising” is intended to mean that the composition and method include the cited elements but do not exclude others. The term “essentially comprising” used to define the composition and method means excluding other elements that are essentially important to the combination. Accordingly, a composition essentially composed of the elements defined herein will not exclude trace amounts of contaminants and pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, etc., in the separation and purification method. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0025] The "effective dose" is an amount sufficient to obtain a beneficial or desired result. The effective dose may be administered as one or more administrations, applications, or dosages.

[0026] As used herein, the terms “treat,” “treating,” “treatment,” and their grammatical variations include partially or completely delaying, alleviating, mitigating, or reducing the intensity of one or more accompanying symptoms of a disorder or condition and / or alleviating, mitigating, or hindering one or more causes of a disorder or condition. Treatment according to the invention may be applied preventively, prophylactically, mitigatingly, or therapeutically. In some examples, the terms “treat,” “treating,” “treatment,” and their grammatical variations include partially or completely reducing the size of a tumor, reducing the number of tumors, and reducing the severity / metastatic potential of a tumor compared to the subject prior to treatment or compared to the incidence of such symptoms in a general or study population.

[0027] References to weight parts of a component in a specific element or composition in the specification and final claims indicate the weight relationship between the element or component of the composition or article in which the weight part is expressed and other elements or components. Thus, in a compound containing 2 weight parts of component X and 5 weight parts of component Y, X and Y are present in a weight ratio of 2:5, and are present in such a ratio regardless of whether additional components are included in the compound. As used herein, “wt.%” or “weight%” or “% by weight” of a component indicates the ratio of the weight of the component to the total weight of the composition containing the component, and is expressed as a percentage, unless specifically stated otherwise.

[0028] Various publications are referenced throughout this application. The disclosures of these publications are incorporated by reference into this application to more completely describe the latest technology related thereto. The disclosed references are also incorporated individually and specifically as references for the materials included in the content discussed in the sentences on which the references depend.

[0029] B. Composition

[0030] The components used to prepare the disclosed composition and the composition itself to be used within the method disclosed herein are disclosed. It is understood that these and other materials are disclosed herein, and that specific references to each of the various individual and collective combinations and permutations may not be explicitly disclosed when combinations, subsets, interactions, groups, etc. of these materials are disclosed, and that each is specifically considered and described herein. For example, where a specific synthetic or chimeric PD-L1 peptide is disclosed and discussed, and where a number of modifications that may be made to a number of molecules comprising the synthetic or chimeric PD-L1 peptide are discussed, all combinations and permutations of the synthetic or chimeric PD-L1 peptide and possible modifications are specifically considered unless otherwise specifically indicated. Thus, if classes of molecules A, B, and C and classes of molecules D, E, and F and examples of combination molecules are disclosed, AD is disclosed, and semantic combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed, even if each is not individually mentioned. Similarly, any subset or combination thereof is disclosed. Thus, for example, a subgroup of AE, BF, and CE is considered to be disclosed. This concept applies to all aspects of the present application, including but not limited to steps of the method for manufacturing and using the disclosed composition. Accordingly, it should be understood that if there are various additional steps that may be performed, each of these additional steps may be performed in any specific embodiment or combination of embodiments of the disclosed method.

[0031] The PD-1 gene, belonging to the immunoglobulin superfamily, codes for a 55 kDa type I transmembrane protein. Both mouse PD-1 and human PD-1 consist of 288 amino acids, with a signaling peptide (20 amino acids) at the N-terminus and a hydrophobic region in the middle, which is the transmembrane region. Human and murine PD-1 proteins share approximately 60%–80% amino acid identity, with the conservation of residues defining four potential N-glycosylation sites and the Ig-V domain. PD-1 is expressed in T cells, B cells, and macrophages. Ligands for PD-1 are the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC). Signaling via the immune checkpoint programmed death protein-1 (PD-1) attenuates the anti-tumor immune response, thereby enabling tumor progression. Therapeutic blocking of the signaling axis between PD-1 and its ligand, programmed cell death ligand-1 (PD-L1), with monoclonal antibodies has demonstrated remarkable clinical success in cancer treatment and impressive activity across a wide range of cancer subtypes. Improvements to traditional PD-1 / PD-L1 blocking are disclosed herein, utilizing smaller non-antibody peptide therapeutics and peptide vaccines capable of stimulating a host immune response to directly block the interaction between PD-1 and PD-L1 or to generate antibodies against PD-L1 that block the PD-1 / PD-L1 interaction.

[0032] Using computer-assisted analysis of PD-L1 B cell epitopes, sequences corresponding to PD-L1 (SEQ No. 1) residues 36-53, 50-67, 95-112, and 130-147 were derived. Accordingly, in one aspect, a synthetic PD-L1 peptide for stimulating an immune response to a PD-L1 protein comprising PD-L1 residues 36-53, 50-67, 95-112, and / or 130-147 is disclosed herein. For example, synthetic PD-L1 peptides for stimulating an immune response to PD-L1 protein, comprising LIVYWEMEDKNIIQFVHG (SEQ No. 2), FVHGEEDLKVQHSSYRQR (SEQ No. 3), YRCMISYGGADYKRITVK (SEQ No. 4), and / or VTSEHELTCQAEGYPKAE (SEQ No. 5), are disclosed herein. In one aspect, the peptide may be acylated and / or amidated. Thus, synthetic PD-L1 peptides for stimulating an immune response to PD-L1 protein, comprising (SEQ No. 2), (SEQ No. 3), (SEQ No. 4), and / or (SEQ No. 5), are disclosed herein; wherein the synthetic peptide is acylated and / or amidated.

[0033] In some cases, the use of analogs of L-amino sequences may have advantages over base sequences, such as resistance to degradation, stability, and ease of synthesis, or may have greater efficacy. In one aspect, it is understood and considered herein that the disclosed synthetic sequences may include L-amino sequences in reverse order from amino to carboxy terminus. For example, the retrosequences of SEQ NO: 2, SEQ NO: 3, SEQ NO: 4, and SEQ NO: 5 are GHVFQIINKDEMEWYVIL (SEQ NO: 12), RQRYSSHQVKLDEEGHVF (SEQ NO: 13), KVTIRKYDAGGYSIMCRY (SEQ NO: 14), and EAKPYGEAQCTLEHESTV (SEQ NO: 15), respectively. These retrosequences may also have mirror images of the base sequences. Accordingly, a synthetic PD-L1 peptide comprising one or more of the sequences described in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and / or SEQ ID NO: 15 is disclosed herein. It has SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and the synthetic peptide comprising SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and / or SEQ ID NO: 15 may be acetylated and / or amidated.

[0034] In addition to the retro-analogous L-amino acid sequences described in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, as presented in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, there are D enantiomer analogs of forward L-amino (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5) and retro-L-amino sequences (SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15) that may have increased resistance to degradation and proteolysis, extended efficacy, and increased ease of synthesis, allowing for better oral administration. Accordingly, in one aspect, a synthetic PD-L1 peptide comprising one or more of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and / or SEQ ID NO: 15 is disclosed herein; wherein the amino acid comprising the sequence is a D amino acid.

[0035] In one aspect, a synthetic PD-L1 peptide initiated by linking the synthetic PD-L1 peptide to a helper T(Th) cell epitope that promotes the release of cytokines that help bypass MHC restriction (i.e., indiscriminate Th cell epitopes) to form a chimeric PD-L1 peptide is understood and considered herein to have increased B cell stimulation. For example, a PD-L1 chimeric peptide for stimulating an immune response to a PD-L1 protein is disclosed herein, comprising one or more PD-L1 B cell epitopes further comprising a T helper (Th) epitope (e.g., a measles virus fusion protein peptide such as SEQ ID NO: 6) in one aspect, wherein one or more PD-L1 B cell epitopes are composed of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and / or SEQ ID NO: 15. It is understood and considered herein that the B cell epitope (i.e., the PD-L1 synthetic peptide) may comprise a D amino acid.

[0036] The Th epitope may be an amino acid of length about 14 to about 22, more preferably about 15 to 21, and most preferably 16. Preferably, the Th cell epitope has one of the following amino acid sequences provided in Table 1.

[0037] Table 1

[0038]

[0039] To link a synthetic PD-L1 peptide and a Th cell epitope, an amino acid linker may be used. Preferably, the linker is a peptide of about 2 to about 15 amino acids, more preferably about 2 to about 10 amino acids, and most preferably about 2 to about 6 amino acids in length. The most preferred linker comprises the amino acid sequence Gly-Pro-Ser-Leu (SEQ: 7). Thus, a chimeric peptide is disclosed herein comprising a synthetic peptide of any one of the prior aspects further comprising, in one aspect, a Th epitope (e.g., a measles virus fusion protein peptide such as SEQ: 6), and a linker (e.g., SEQ: 7) linking the synthetic PD-L1 peptide to the Th epitope. For example, in one aspect, a chimeric PD-L1 peptide for stimulating an immune response to a PD-L1 protein is disclosed, comprising one or more PD-L1 B cell epitopes, a T helper (Th) epitope (e.g., a measles virus fusion protein peptide such as SEQ ID NO: 6), and a linker (e.g., SEQ ID NO: 7) linking the PD-L1 B cell epitope to the Th epitope; wherein the chimeric PD-L1 peptide comprises the amino acid sequence presented below:

[0040] KLLSLIKGVIVHRLEGVEGPSLLIVYWEMEDKNIIQFVHG(SEQ No.: 8), KLLSLIKGVIVHRLEGVEGPSLFVHGEEDLKVQHSSYRQR(SEQ No.: 9), KLLSLIKGVIVHRLEGVEGPSLYRCMISYGGADYKRITVK(SEQ No.: 10), KLLSLIKGVIVHRLEGVEGPSLVTSEHELTCQAEGYPKAE(SEQ No.: 11), KLLSLIKGVIVHRLEGVEGPSLGHVFQIINKDEMEWYVIL(SEQ No.: 16), KLLSLIKGVIVHRLEGVEGPSLRQRYSSHQVKLDEEGHVF(SEQ No.: 17), KLLSLIKGVIVHRLEGVEGPSLKVTIRKYDAGGYSIMCRY(SEQ No.: 18), and / or KLLSLIKGVIVHRLEGVEGPSLEAKPYGEAQCTLEHESTV(Sequence No.: 19).

[0041] As with synthetic peptides, it is understood and considered herein that the amino acids of the synthetic PD-L1 peptide contained within the chimeric PD-L1 peptide may be D amino acid analogs of the L-amino acid within the sequence. Accordingly, in one aspect, a chimeric peptide comprising any of the synthetic PD-L1 peptides disclosed herein is disclosed, further comprising a Th epitope (e.g., a measles virus fusion protein peptide such as SEQ ID NO: 6) and a linker (e.g., SEQ ID NO: 7) linking the synthetic PD-L1 peptide to the Th epitope. For example, in one aspect, a chimeric PD-L1 peptide comprising the amino acid sequences presented in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and / or SEQ ID NO: 19 is disclosed herein; wherein the synthetic PD-L1 peptide sequence (i.e., the B cell epitope) comprises the D amino acid.

[0042] As disclosed herein, the disclosed synthetic and chimeric PD-L1 peptides can be combined with targeted therapies using peptide-based B-cell epitopes and peptide immunotherapies that block cancer growth and spread by inhibiting oncogenic signaling pathways such as HER-2. Human epidermal growth factor receptor 2 (HER-2), also known as HER2, HER-2 / neu, p185neu, ERBB2, or the CD340 receptor family, plays a central role in the pathogenesis of several human cancers, including breast, ovarian, renal, colorectal, and lung cancers, and is associated with more aggressive forms of cancer, increased risk of metastasis, increased tumor invasion, and reduced overall survival. Therefore, HER-2 is a key therapeutic target for several cancers, serving as the binding site for trastuzumab (HERCEPTIN®) and pertuzumab (PERJECTA®). Trastuzumab was the first humanized mAb targeting HER-2 in combination with chemotherapy approved for clinical use in patients with metastatic HER-2 positive breast cancer. When pertuzumab, a humanized mAb that blocks the dimerization of HER-2 with other HER family members, was added to docetaxel and trastuzumab in untreated HER-2 positive breast cancer patients, progression-free survival improved from 12.4 months to 18.5 months and overall survival improved from 40.8 months to 56.5 months. Despite the benefits observed with trastuzumab in about one-third of metastatic patients, HER-2 positive breast cancer responds early, and most responding patients eventually developed acquired resistance within one year of treatment. Targeted therapies using humanized mAbs against HER-2 (trastuzumab, pertuzumab) or chimeric mAbs targeting EGFR (cetuximab, ERBITUX®) significantly improved survival in the adjuvant setting, but still showed toxicity, tolerability, or acquired resistance, so most patients eventually succumbed to the disease with advanced cancer.

[0043] A HER-2 chimeric peptide for stimulating an immune response to HER-2 is also disclosed herein, comprising one or more HER-2 B cell epitopes, a T helper (Th) epitope (e.g., including but not limited to a measles virus fusion protein such as SEQ ID NO: 6), and a linker (e.g., SEQ ID NO: 7) linking the HER-2 B cell epitope to the Th epitope, wherein one or more HER-2 B cell epitopes consist of a sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 29. For example, the HER-2 chimeric peptide comprises the amino acid sequence presented in SEQ ID NO: 28 or SEQ ID NO: 30.

[0044] Any PD-L1 peptide, synthetic PD-L1 peptide, chimeric PD-L1 peptide (e.g., any peptide presented in SEQ ID NOs: 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and / or 19) and / or any HER-2 peptide or chimeric HER-2 peptide (e.g., peptide presented in SEQ ID NOs: 27, 28, 29, or 30) may be formulated into a vaccine or pharmaceutical composition that may be administered therapeutically or prophylactically to subjects who have or are at risk of developing cancer (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer), autoimmune disease, and / or Alzheimer's disease. In one aspect, a vaccine or pharmaceutical composition may comprise any one or more preceding chimeric or synthetic peptides and a pharmaceutically acceptable vehicle (e.g., a biodegradable vehicle comprising, but not limited to, an emulsion containing a pharmaceutically acceptable adjuvant). As used herein, the term “adjuvant” generally refers to a class of substances capable of increasing the magnitude of the immune response induced by the programmed death ligand-1 (PD-L1) chimeric peptide beyond what is expected from the chimeric peptide alone or from the chimeric peptide as described herein in the absence of an adjuvant.

[0045] Suitable adjuvants will be known to those skilled in the art. Non-limiting examples of suitable adjuvants include aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate (also known as alum)), liposomes, virososomes, water-in-oil or oil-in-water emulsions (e.g., Freund adjuvants, Montanide®, MF59®, and AS03), 3- O -Desyl-4'-monophosphoryl lipid A (MPL) and adjuvants containing MPL (e.g., AS01, AS02, and AS04) and saponin-based adjuvants. The saponin-based adjuvants contain saponins or, for example, Quillaja saponaria ( Quillaja saponaria ), Panax Ginseng Panax Noto Ginseng (Panax ginseng Panax notoginseng), Panax Quercus folium (Panax quinquefolium), Platycodon grandiflorum (Platycodon grandiflorum), Polygala senega (Polygala senega), Polygala tenupolia Pfolygala tenuifolia), Quillaja brasiliensis (Quillaja brasiliensis), Astragalus Membraseus ( Axstragalus membranaceus) and Archirantes Bidentata (Achyranthes bidentata)It includes saponin derivatives derived from. Exemplary saponin-based adjuvants include ISCON, ISCON matrix, ISCOMATRIX™ adjuvants, Matrix M™ adjuvants, Matrix C™ adjuvants, Matrix Q™ adjuvants, AbISCO®-100 adjuvants, AbISCO®-300 adjuvants, ISCOPREP™, ISCOPREP™ derivatives, adjuvants containing ISCOPREP™ or ISCOPREP™ derivatives, QS-21, QS-21 derivatives, and adjuvants containing QS-21 or QS21 derivatives. Vaccine compositions as described herein may also be combined with immunomodulators, for example, cytokines, chemokines, and growth factors. Mixtures of two or more adjuvants within the same vaccine composition are also considered herein. In one embodiment, the adjuvant is the water-in-oil adjuvant montanid. For example, a pharmaceutical composition is disclosed herein comprising a PD-L1 peptide, a synthetic PD-L1 peptide, and / or a chimeric PD-L1 peptide (e.g., any peptide as presented in SEQ ID NOs: 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and / or 19), and further comprising one or more HER-2 B cell epitopes (e.g., SEQ ID NO: 27 or SEQ ID NO: 29), one or more chimeric HER 2 peptides (e.g., SEQ ID NO: 28 or SEQ ID NO: 30), and / or one or more anti-Her-2 antibodies.In one aspect, a pharmaceutical composition is disclosed herein that comprises any of PD-L1 peptides, synthetic PD-L1 peptides, and / or chimeric PD-L1 peptides (e.g., any of the peptides presented in SEQ ID NOs: 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and / or 19), with or without a HER-2 B cell epitope, wherein the composition comprises aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, calcium hydroxyphosphate, Freund complete adjuvant, Montanide®, Freund incomplete adjuvant, ISCOM, ISCOM Matrix, ISCOMATRIX™ adjuvant, Matrix M™ adjuvant, Matrix C™ adjuvant, Matrix Q™ adjuvant, AbISCO®-100 adjuvant, AbISCO®-300 It further comprises an adjuvant selected from the group consisting of an adjuvant, ISCOPREP™, an ISCOPREP™ derivative, an adjuvant containing ISCOPREP™ or an ISCOPREP™ derivative, QS-21, a QS-21 derivative, and an adjuvant containing QS-21 or a QS-21 derivative.

[0046] In one aspect, an antibody that specifically binds to any of the PD-L1 chimeric peptide, PD-L1 synthetic peptide, HER-2 chimeric peptide, and HER-2 synthetic peptide disclosed herein is disclosed.

[0047] 1. Sequence similarity

[0048] As discussed herein, the use of the terms homology and identity is understood to mean the same thing as similarity. Therefore, for example, when the word homology is used between two non-natural sequences, it is understood that this does not necessarily indicate an evolutionary relationship between these two sequences, but rather examines the similarity or relationship between their nucleic acid sequences. Many methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity, regardless of whether they are evolutionarily related.

[0049] Generally, it is understood that one method of defining any known variants and derivatives or possible occurrences of the genes and proteins disclosed herein is to define the variants and derivatives in terms of homology to a specific known sequence. Such homology of a specific sequence disclosed herein is also discussed elsewhere in this application. Generally, variants of the genes and proteins disclosed herein have typically at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to the specified sequence or natural sequence. Those skilled in the art readily understand the method of determining the homology of nucleic acids, such as two proteins or genes. For example, homology can be calculated after aligning two sequences so that homology is at the highest level.

[0050] Another method for calculating homology can be performed using a published algorithm. Optimal sequence alignment for comparison is determined by a local homology algorithm (Smith and Waterman, Adv. Appl. Math. 2:482(1981)), by the homology sorting algorithm (Needleman and Wunsch, J. MoL Biol.48:443(1970)), by similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA This can be performed by computer implementations of such algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by testing.

[0051] Any method may generally be used and the results of these various methods may differ in specific cases, but a skilled technician will find that if identity is found in at least one of these methods, the sequence has the specified identity disclosed herein.

[0052] For example, as used herein, a sequence cited as having a specific percentage homology with respect to another sequence refers to a sequence having the cited homology as calculated by any one or more of the calculation methods described above. For example, even if the first sequence does not have 80% homology as calculated by any of the other calculation methods, if the first sequence is calculated to have 80% homology with respect to the second sequence using the Zuker calculation method, the first sequence has 80% homology with respect to the second sequence as defined herein. As another example, even if the first sequence does not have 80% homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation method, or other calculation methods, if the first sequence is calculated to have 80% homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method, the first sequence has 80% homology to the second sequence as defined herein. As yet another example, if the first sequence is calculated to have 80% homology to the second sequence using each respective calculation method, the first sequence has 80% homology to the second sequence as defined herein (in practice, different calculation methods often result in different calculated percentages of homology).

[0053] 2. Peptide

[0054] a) Proteins and peptide variants

[0055] As discussed herein, there are numerous variants of known synthetic PD-L1 peptides and chimeric PD-L1 peptides considered herein. Additionally, known functional PD-L1 strain variants include derivatives of synthetic PD-L1 peptides and chimeric PD-L1 peptides that function in the disclosed methods and compositions. Protein variants and derivatives are well understood by those skilled in the art and may involve amino acid sequence modifications. For example, amino acid sequence modifications generally fall into one or more of the three classes of substitutions, insertions, or deletions. Insertions include amino and / or carboxyl-terminal fusions as well as insertions of single or multiple amino acid residues into the sequence. Insertions are generally smaller than amino or carboxyl-terminal fusions, for example, small insertions of about one to four residues. Immunogenic fusion protein derivatives, such as those described in the Examples, are prepared by fusion of polypeptides large enough to confer immunogenicity to the target sequence by cross-linking in vitro or by culturing recombinant cells transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. Typically, about 2 to 6 or fewer residues are deleted at any site within the protein molecule. These variants are generally produced by site-specific mutagenesis of nucleotides in the DNA encoding the protein, and the DNA encoding the variant is then expressed in recombinant cell cultures. Techniques for creating substitution mutations at predetermined sites of DNA having known sequences, such as M13 primer mutagenesis and PCR mutagenesis, are well known. Amino acid substitutions generally consist of a single residue but can occur at multiple different locations at once; insertions will generally be about 1 to 10 amino acid residues; and deletions will range from about 1 to 30 residues.Deletions or insertions are preferably performed on adjacent pairs, namely the deletion of two residues or the insertion of two residues. Substitutions, deletions, insertions, or combinations thereof may be combined to arrive at the final structure. Mutations must not place the sequence outside the decoding frame and, preferably, will not generate a complementary region capable of generating a secondary mRNA structure. Substitution variants are those in which at least one residue is removed and another residue is inserted in its place. Such substitutions are generally performed according to Tables 2 and 3 below and are referred to as conservative substitutions.

[0056] Table 2: amino acid abbreviations

[0057]

[0058] Table 3: amino acid substitution

[0059] Exemplary conservative substitutions for the original residue, and others, are known in the art.

[0060]

[0061] Substantial changes in functional or immunological identity are achieved by selecting substitutions that are less conservative than those in Table 3, namely, by selecting residues that have a more significantly different effect on (a) the structure of the polypeptide backbone in the substitution region, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the large volume of the side chain. In general, substitutions expected to cause the greatest changes in protein properties are (a) where a hydrophilic residue, e.g., ceryl or threonyl, is substituted with (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) where cysteine ​​or proline is substituted with (or by) another residue; and (c) where a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted with (or by) an electronegative residue, e.g., glutamyl or aspartyl; Or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted with a residue not having a side chain, e.g. glycine, and in this case (e) it will increase the number of sites for sulfation and / or glycosylation.

[0062] For example, replacing one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution is replacing one hydrophobic residue with another or one polar residue with another. Substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included in the mosaic polypeptide provided herein.

[0063] Substitution or deletion mutagenesis may be used to insert a site for N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletion of cysteine ​​or other unstable residues may also be desirable. Deletion or substitution of a potential protein degradation site, e.g., Arg, is achieved, for example, by deleting one of the basic residues or substituting one with a glutaminyl or histidyl residue.

[0064] Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are often deamidated into corresponding glutamyl and asparagyl residues after translation. Alternatively, these residues are deamidated under weakly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of o-amino groups of lysine, arginine, and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco pp 79-86

[1983] ), acetylation of N-terminal amines, and in some cases, amidation of C-terminal carboxyl groups.

[0065] It should be understood that one method of defining variants and derivatives of the proteins disclosed herein is to define the variants and derivatives in terms of homology / identity with respect to a specific known sequence. Variants of these and other proteins disclosed herein are specifically disclosed having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with respect to the mentioned sequence. Those skilled in the art will readily understand how to determine the homology of two proteins. For example, homology can be calculated after aligning the two sequences so that the homology is at the highest level.

[0066] Another method for calculating homology can be performed using a published algorithm. Optimal sequence alignment for comparison is determined by a local homology algorithm (Smith and Waterman, Adv. Appl. Math. 2:482(1981)), by the homology sorting algorithm (Needleman and Wunsch, J. MoL Biol. 48: 443(1970)), by similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA . 85:2444(1988), this can be performed by computer implementations of such algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by testing.

[0067] Homology of the same type is, for example, in the literature (Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol It can be obtained for nucleic acids by the algorithm described in . 183:281-306, 1989).

[0068] The description of conservative mutations and homology is understood to be that variants can be combined in any combination, such as an embodiment having at least 70% homology to a specific sequence that is a conservative mutation.

[0069] As this specification discusses various proteins and protein sequences, it is understood that nucleic acids capable of encoding such protein sequences are also disclosed. This includes all degenerate sequences associated with a specific protein sequence, that is, all nucleic acids having a sequence encoding a specific protein sequence, as well as all nucleic acids including degenerate nucleic acids encoding disclosed variants and derivatives of the protein sequence. Accordingly, it is understood that while each specific nucleic acid sequence may not be described herein, each and all sequences are effectively disclosed and described herein through the disclosed protein sequence. Furthermore, it is understood that while an amino acid sequence does not represent a specific DNA sequence encoding that protein within an organism, and a specific variant of the disclosed protein is disclosed herein, known nucleic acid sequences encoding that peptide or protein are also known, disclosed, and described herein.

[0070] It is understood that there are numerous amino acids and peptide analogs that can be incorporated into the disclosed composition. For example, there are numerous D amino acids or amino acids having functional substituents different from the amino acids presented in Tables 2 and 3. Contrasteric isomers of naturally occurring peptides as well as stereoisomers of peptide analogs are disclosed. These amino acids can be easily incorporated into a polypeptide chain by manipulating a gene construct that charges a tRNA molecule with a selected amino acid and inserts the analog amino acid into the peptide chain in a site-specific manner, for example, using an amber codon.

[0071] Peptide-like molecules may be generated, but they are not linked via natural peptide bonds. For example, linkages to amino acids or amino acid analogs may include CH2NH--, CH2S--, CH2--CH2--, CH=CH--(cis and trans), COCH2--, CH(OH)CH2--, and CHH2SO- (these and other items are referenced in the literature, Spatola, AF in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins , B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A.F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979)(--CH2NH--, CH2CH2--); Spatola et al. Life Sci 38:1243-1249 (1986)(--CH H2--S); Hann J. Chem. Soc Perkin Trans . I 307-314 (1982)(--CH--CH--, cis and trans); Almquist et al. J. Med. Chem . 23:1392-1398 (1980)(--COCH2--); Jennings-White et al. Tetrahedron Lett 23:2533 (1982)(--COCH2--); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982)(--CH(OH)CH2--); Holladay et al. Tetrahedron . Lett 24:4401-4404 (1983)(--C(OH)CH2--); and Hruby Life SciIt can be found in 31:189-199(1982)(--CH2--S--); each of these is incorporated herein by reference). A particularly preferred non-peptide linkage is --CH2NH--. Peptide analogs are understood to have one or more atoms between the bonding atoms, such as β-alanine, γ-aminobutyric acid, etc.

[0072] Amino acid analogs and peptide analogs may have more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad biological activity), reduced antigenicity, etc.

[0073] Since D-amino acids are not recognized by peptidases or the like, D-amino acids can be used to produce more stable peptides. More stable peptides can be produced by systematically substituting one or more amino acids of a common sequence with the same type of D-amino acid (e.g., D-lysine instead of L-lysine). In other words, any inverse of the disclosed sequence (i.e., D-amino acid substitution) is considered herein. Two or more peptides can be cyclized or attached to each other using cysteine ​​residues. This can help restrict the peptides to a specific stereotype. In one aspect, a synthetic PD-L1 peptide comprising one or more of the sequences presented in SEQ NO: 2, SEQ NO: 3, SEQ NO: 4, or SEQ NO: 5 is disclosed herein; wherein the amino acids of the peptide are D enantiomers.

[0074] In one aspect, the disclosed synthetic peptide may be reversed such that the carboxyl terminus at the amino of the peptide is inverted (i.e., retro sequence). In one aspect, the retro sequences of SEQ NO: 2, SEQ NO: 3, SEQ NO: 4, and SEQ NO: 5 are disclosed herein, which each include SEQ NO: 12, SEQ NO: 13, SEQ NO: 14, and SEQ NO: 15. These retro sequences may also have mirror images of the base sequence. In one aspect, the retro sequence may also include a D amino acid substitution (i.e., retro-inverse) sequence. Thus, a synthetic PD-L1 peptide comprising one or more of the sequences presented in SEQ NO: 12, SEQ NO: 13, SEQ NO: 14, and SEQ NO: 15 is disclosed herein; wherein the amino acid of the peptide is a D enantiomer.

[0075] It is understood that any of the D amino acid-substituted synthetic peptides disclosed herein may be used as a PD-L1 epitope in the disclosed PD-L1 chimeric peptide. For example, a chimeric PD-L1 peptide is disclosed herein comprising one or more PD-L1 B cell epitopes, a T helper (Th) epitope, and a linker connecting the PD-L1 B cell epitope to the Th epitope, wherein one or more PD-L1 B cell epitopes are composed of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; wherein the amino acid of the peptide is a D enantiomer. In one aspect, a chimeric PD-L1 peptide is disclosed herein, wherein the peptide comprises the amino acid sequence presented in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; wherein the amino acid of the synthetic PD-L1 peptide is a D enantiomer.

[0076] 3. Transport of pharmaceutical carriers / pharmaceutical products

[0077] As described above, the synthetic PD-L1 peptide, chimeric PD-L1 peptide, synthetic HER-2 peptide, and / or chimeric HER-2 peptide disclosed herein may also be administered in vivo in a pharmaceutically acceptable carrier. Accordingly, in one aspect, a pharmaceutical composition comprising any one or more of the PD-L1 peptides presented in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and / or SEQ ID NO: 30 is disclosed herein.

[0078] "Pharmaceuticalally acceptable" means a substance that is not biologically or otherwise undesirable; that is, the substance may be administered to a subject along with a nucleic acid or vector without causing any undesirable biological effects or interacting in a harmful manner with other components of the pharmaceutical composition containing it. As is well known to those skilled in the art, the carrier will be naturally selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0079] Pharmaceutical compositions comprising the disclosed PD-L1 peptides are understood and considered herein to be particularly useful for the treatment, suppression, reduction, aggravation, improvement, and / or prevention of diseases or conditions in which PD-L1-mediated immunosuppression occurs. Over the past 20 years, another paradigm of cancer treatment has evolved: selective mechanism-based targeted therapies using peptide-based B cell epitopes and peptide-immunotherapies that block the growth and spread of cancer by inhibiting oncogenic signaling pathways; these targeted therapies tend to have fewer side effects than non-specific chemotherapy. Consequently, peptide-based and small molecule inhibitors have garnered attention in drug development strategies in recent years. However, small molecule immune checkpoint inhibitors require significant improvement compared to monoclonal antibodies. Therefore, in one aspect, the disclosed pharmaceutical compositions comprising one or more of the PD-L1 peptides disclosed herein may be combined with disease-specific therapies or vaccines to further increase the efficacy of the PD-L1 peptides. For example, a pharmaceutical composition comprising one or more PD-L1 peptides may be combined with an anti-HER2 antibody, a HER-2 chimeric peptide, and / or a HER-2 B cell epitope for use in the treatment, inhibition, reduction, decrease, improvement, and / or prevention of cancer and / or metastasis (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer).

[0080] In one aspect, a pharmaceutical composition is disclosed herein comprising one or more of the PD-L1 peptide, synthetic peptide, or chimeric peptide disclosed herein (e.g., SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and / or SEQ ID NO: 19), and further comprising one or more HER-2 B cell epitopes (e.g., SEQ ID NO: 27 or 29, or chimeric epitope SEQ ID NO: 28 or 30) and / or an anti-Her-2 antibody. In one aspect, pharmaceutical compositions comprising MVF-PD-L1 (36-53) presented in SEQ ID NO: 8, MVF-PD-L1 (50-67) presented in SEQ ID NO: 9, MVF-PD-L1 (95-112) presented in SEQ ID NO: 10, and / or MVF-PD-L1 (130-147) presented in SEQ ID NO: 11; MVF-HER-2 (266-296) peptides (e.g., as presented in SEQ ID NO: 28), and MVF-HER-2 (597-626) peptides (e.g., as presented in SEQ ID NO: 30) are specifically disclosed herein.

[0081] The composition may be administered orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, transdermally, ex vivo, topically, etc., including local intranasal administration or inhalation. As used herein, "local intranasal administration" means delivery of the composition into the nose and nasal cavity through one or both nostrils and may include delivery by a nebulizer or droplet mechanism, or delivery via aerosolization of nucleic acid or vector. Administration of the composition by inhalation may be made through the nose or mouth via a nebulizer or droplet mechanism. It may also be delivered directly to any part of the respiratory system (e.g., lungs) via intubation. The exact amount of the composition required may vary from subject to subject depending on the subject's species, age, weight and general condition, the severity of the allergic disorder being treated, the specific nucleic acid or vector used, the mode of administration, etc. Therefore, it is not possible to specify an exact amount for every composition. However, an appropriate amount may be determined by a person skilled in the art using only the routine experiments given in the teachings of this invention.

[0082] Parenteral administration of the composition, when used, generally features injection. The injectable formulation may be prepared in conventional forms, such as a liquid solution or suspension, a solid form suitable for a liquid suspension solution prior to injection, or an emulsion. More recently revised parenteral administration approaches involve the use of slow-release or sustained-release systems to maintain a constant dose (e.g., U.S. Patent No. 3,610,795 incorporated herein by reference).

[0083] The material can be a solution or a suspension (e.g., microparticles, liposomes, or incorporated into cells). These can target specific cell types through antibodies, receptors, or receptor ligands. The following reference provides an example of using this technique to target proteins specific to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D.; Br. J. Cancer , 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer , 58:700-703, (1988); Senter, et al., Bioconjugate Chem ., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother ., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews , 129:57-80,(1992); and Roffler, et al., Biochem. Pharmacol Vehicles such as "stealth" and other antibodies are conjugated to liposomes (including lipid-mediated drugs targeting colon carcinoma), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioblastoma cells in vivo. The following reference is an example of using this technique to target proteins specific to tumor tissue (Hughes et al., Cancer Research , 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). Generally, receptors are involved in constitutive or ligand-induced endocytotic pathways. These receptors aggregate in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where receptors are sorted, and then recirculate to the cell surface for intracellular storage or degradation in lysosomes. Internalization pathways perform various functions such as nutrient uptake, removal of activated proteins, macromolecular clearance, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and receptor-level regulation. Many receptors follow one or more intracellular pathways depending on cell type, receptor concentration, ligand type, ligand valence, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409(1991)).

[0084] a) Pharmaceutically acceptable carrier

[0085] A composition containing antibodies can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0086] Suitable carriers and their formulations are described in the literature ( Remington: The Science and Practice of Pharmacy(19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995). Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline solution, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release formulations, such as a semipermeable matrix of a solid hydrophobic polymer containing antibodies, wherein the matrix is ​​in the form of a molded article, e.g., a film, liposome, or microparticle. For example, it will be apparent to those skilled in the art that certain carriers may be more preferred depending on the route of administration and concentration of the composition being administered.

[0087] Pharmaceutical carriers are known to those skilled in the art. These will most commonly be standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffer solutions at physiological pH. The composition may be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0088] In addition to the selected molecule, the pharmaceutical composition may include a carrier, a thickener, a diluent, a buffer, a preservative, a surfactant, etc. The pharmaceutical composition may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc.

[0089] The pharmaceutical composition may be administered in various ways depending on whether local or systemic treatment is required and the site to be treated. Administration may be local (including ophthalmic, vaginal, rectal, and nasal), oral, inhaled, or parenteral, e.g., intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibody may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavernously, or transdermally.

[0090] 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, alcohol / aqueous solutions, saline solutions, and emulsions or suspensions containing buffer media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactate Ringer's, or fixative oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (e.g., 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.

[0091] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oil-based bases, thickeners, etc. may be required or preferred.

[0092] 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, dispersion aids, or binders may be preferred.

[0093] Some of the composition may be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromide, perchloric acid, nitric acid, thiocyanate, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvate, 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 mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0094] b) Therapeutic use

[0095] Effective dosages and schedules for administering the composition may be determined empirically, and making such decisions is within the scope of the art. The dosage range for administering the composition is sufficiently large to produce the desired effect in which the symptoms of the disorder are affected. The dosage must not be large enough to cause adverse effects such as unwanted cross-reactions or anaphylactic reactions. Generally, the dosage will vary depending on age, condition, gender, the severity of the patient's disease, the route of administration, or whether other drugs are included in the regimen, and can be determined by a person skilled in the art. The dosage may be adjusted by the individual physician in the event of adverse indications. Dosage may vary and may be administered as one or more daily doses over a day or several days. Guidelines for appropriate dosages for a given class of medicine can be found in the literature. For example, guidelines for selecting appropriate doses for antibodies can be found in the literature regarding the therapeutic uses of antibodies ( Handbook of Monoclonal Antibodies , Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985)ch. 22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy , Haber et al., eds., Raven Press, New York (1977) pp.365-389). A typical daily dose of an antibody used alone may range from about 1 μg / kg to a maximum of 100 mg / kg body weight or more per day, depending on the factors mentioned above.

[0096] The synthetic PD-L1 peptides, chimeras, and antibodies disclosed herein that inhibit the interaction between PD-1 and PD-L1 may be administered prophylactically to patients or subjects at risk of developing cancer, autoimmune diseases, or Alzheimer's disease, or may be administered therapeutically (i.e., after diagnosis of the disease or onset of symptoms) for the treatment, inhibition, reduction, improvement, and / or prevention of cancer (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer), autoimmune diseases, or Alzheimer's disease.

[0097] Other molecules or antibodies that interact with PD-1 or PD-L1 to inhibit PD-1 / PD-L1 interactions (e.g., pembrolizumab and nivolumab) may be used in combination with the disclosed synthetic PD-L1 peptide, chimeric PD-L1 peptide, or anti-PD-L1 antibody to treat, inhibit, reduce, decrease, improve, and / or prevent cancer, autoimmune disease, or Alzheimer's disease in subjects.

[0098] 4. Antibodies

[0099] (1) Generally, antibodies

[0100] The term “antibody” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to whole immunoglobulin molecules, the term “antibody” includes fragments or polymers of such immunoglobulin molecules, and human or humanized versions or fragments thereof of an immunoglobulin molecule selected for the ability to interact with PD-L1 such that the interaction of PD-1 with PD-L1 is inhibited or the HER-2 receptor is inhibited. Antibodies binding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and / or SEQ ID NO: 19 involved in the interaction between PD-1 and PD-L1 are also disclosed. Additionally, antibodies binding to HER-2 SEQ No. 27, SEQ No. 28, SEQ No. 29, and / or SEQ No. 30 are disclosed herein. Antibodies may be tested for desired activity using the in vitro assay described herein or by similar methods, and then therapeutic and / or prophylactic activity in vivo is tested according to known clinical trial methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize comparable classes for mice. The heavy chain constant domains corresponding to the other classes of immunoglobulins are referred to as alpha, delta, epsilon, gamma, and mu, respectively.

[0101] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies within the population are identical except for possible naturally occurring mutations that may exist in a small subset of antibody molecules. Monoclonal antibodies of the present invention specifically include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining chain(s) are identical or homologous to the corresponding sequence of an antibody derived from a different species or belonging to another antibody class or subclass, as well as insofar as they exhibit the desired antagonistic activity.

[0102] The disclosed monoclonal antibody can be produced using any procedure for generating monoclonal antibodies. For example, the disclosed monoclonal antibody is described in the literature (Kohler and Milstein, Nature It can be prepared using a hybridoma method as described in , 256:495(1975)). In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to induce lymphocytes capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0103] Monoclonal antibodies can also be produced by recombinant DNA methods. The DNA encoding the disclosed monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using an oligonucleotide probe capable of specifically binding to genes encoding the heavy and light chains of a murine antibody). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display technology, as described, for example, in U.S. Patent No. 5,804,440 of Burton et al. and U.S. Patent No. 6,096,441 of Barbas et al.

[0104] In vitro methods are also suitable for producing monovalent antibodies. Digestion of the antibody to produce its fragments, particularly the Fab fragment, can be achieved using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348 published on December 22, 1994, and US Patent No. 4,342,566. Papain digestion of the antibody generally produces two identical antigen-binding fragments, referred to as the Fab fragment, each having a single antigen-binding site and a residual Fc fragment. Pepsin treatment produces a fragment having two antigen-binding sites that is still capable of cross-linking the antigen.

[0105] As used herein, the term “antibody or fragment thereof” includes chimeric antibodies and hybrid antibodies having dual or multiple antigen or epitope specificity, and fragments comprising hybrid fragments, e.g., F(ab')2, Fab', Fab, Fv, sFv, etc. Accordingly, an antibody fragment having the ability to bind to a specific antigen thereof is provided. For example, a fragment of an antibody that maintains PD-L1 binding activity or binds SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and / or SEQ ID NO: 19 is included in the meaning of the term “antibody or fragment thereof”. These antibodies and fragments may be manufactured by techniques known in the art and may be screened for specificity and activity according to the methods described in the examples and general methods for producing and screening antibodies for specificity and activity (Harlow and Lane. Antibodies, A Laboratory Manual.Cold Spring Harbor Publications, New York, (1988).

[0106] In addition, the meaning of "antibody or fragment thereof" includes conjugates of antibody fragments and antigen-binding proteins (single-chain antibodies).

[0107] Regardless of whether they are attached to a different sequence, the fragment may also include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. Such modifications may provide some additional properties, such as the removal or addition of disulfide-bonding-capable amino acids, increased viability, or altered secretory properties. In any case, the antibody or antibody fragment must possess bioactive properties, such as specific binding to cognate antigens. The functional or active region of the antibody or antibody fragment can be identified by mutagenicization of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to those skilled in the art and may include site-specific mutagenicization of the nucleic acid encoding the antibody or antibody fragment (Zoller, MJ Curr. Opin. Biotechnol . 3:348-354, 1992).

[0108] As used herein, the terms "antibody" or "antibodies" may also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., antibodies derived from mice, rats, or rabbits) are naturally antigenic to humans and may therefore cause undesirable immune responses when administered to humans. Accordingly, the use of human or humanized antibodies in the method serves to reduce the likelihood that antibodies administered to humans will cause undesirable immune responses.

[0109] (2) Human antibodies

[0110] The disclosed human antibodies can be produced using any technology. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic mutant mice capable of generating a full repertoire of human antibodies in response to immunization are described (Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., See 7:33(1993)). Specifically, in these chimeric and germline mutant mice, the antibody heavy chain binding region (J (H) Homozygous deletion of the ) gene results in the complete suppression of endogenous antibody production, and successful delivery of the human germline antibody gene array to these germline mutant mice results in the production of human antibodies upon antigen challenge. Antibodies with desired activity are selected using the Env-CD4-co-receptor complex described herein.

[0111] (3) Humanized antibodies

[0112] Antibody humanization technology generally involves the use of recombinant DNA technology to manipulate DNA sequences encoding one or more polypeptide chains of antibody molecules. Thus, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, e.g., sFv, Fv, Fab, Fab', F(ab')2, or other antigen-binding portions of the antibody) containing a portion of an antigen-binding site from a non-human (donor) antibody incorporated into the framework of a human (recipient) antibody.

[0113] To produce a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced with residues from one or more CDRs of a donor (non-human) antibody molecule known to possess desired antigen-binding properties (e.g., specific levels of specificity and affinity for a target antigen). In some cases, Fv framework (FR) residues of the human antibody are replaced with corresponding non-human residues. The humanized antibody may also contain residues not found in the recipient antibody or the imported CDR or framework sequence. Generally, the humanized antibody has one or more amino acid residues introduced from a non-human source. In practice, the humanized antibody is a human antibody in which some CDR residues and possibly some FR residues are substituted with residues from similar sites of a rodent antibody. The humanized antibody generally contains an antibody constant region (Fc), typically at least a portion of the antibody constant region of the human antibody.

[0114] (4) Antibody administration

[0115] The administration of antibodies may be performed as disclosed herein. Nucleic acid approaches for antibody delivery also exist. Broad-spectrum neutralizing anti-PD-L1 antibodies and antibody fragments (including any antibody binding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and / or SEQ ID NO: 19) may also be administered to a patient or subject as a nucleic acid preparation (e.g., DNA or RNA) encoding the antibody or antibody fragment, causing the patient or subject's own cells to absorb the nucleic acid and produce and secrete the encoded antibody or antibody fragment. The delivery of nucleic acids may be accomplished by any means, such as as disclosed herein, for example.

[0116] C. Methods of treating diseases

[0117] It is understood and considered herein that the disclosed compositions, synthetic PD-L1 peptides, and chimeric PD-L1 peptides may be used for the treatment, inhibition, reduction, decrease, improvement, and / or prevention of diseases in which uncontrolled cell proliferation occurs, such as immunosuppression and prevention of programmed cell death, e.g., Alzheimer's disease, autoimmune diseases, or cancer.

[0118] A non-limiting list of different types of autoimmune diseases that can be treated, inhibited, reduced, decreased, improved, and / or prevented using the chimeric or synthetic peptides or pharmaceutical compositions disclosed herein includes, but is not limited to, psoriasis, alopecia areata, primary biliary cirrhosis, autoimmune polyendocrine syndrome, type 1 diabetes mellitus, autoimmune thyroiditis, systemic lupus erythematosus, multiple sclerosis, Guillain-Barré syndrome, Graves disease, Sjögren's syndrome, ulcerative colitis, autoimmune hemolytic anemia, pernicious anemia, psoriatic arthritis, rheumatoid arthritis, recurrent polychondritis, myasthenia gravis, acute disseminated encephalomyelitis, and granulomatosis with polyangiitis.

[0119] A non-limiting list of different types of cancer that can be treated, inhibited, reduced, decreased, improved, and / or prevented using the chimeric or synthetic peptides or pharmaceutical compositions disclosed herein includes lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, endometrial cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer; testicular cancer; Includes, but is not limited to, colorectal and rectal cancer, prostate cancer, ipilimumab-refractory melanoma, or pancreatic cancer.

[0120] Accordingly, in one aspect, a method for treating, inhibiting, reducing, decreasing, improving, and / or preventing cancer, Alzheimer's disease, or autoimmune disease in a subject is disclosed herein, comprising administering a PD-L1 synthetic peptide to a subject, wherein the PD-L1 synthetic peptide comprises one or more of the sequences described in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. It is understood and considered herein that the synthetic peptide may be acetylated, amidated, and / or may comprise a D enantiomer. Accordingly, in one aspect, a method for treating, inhibiting, reducing, decreasing, improving, and / or preventing cancer (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer), Alzheimer's disease, or autoimmune disease in a subject is disclosed herein, comprising administering a PD-L1 synthetic peptide to a subject, wherein the PD-L1 synthetic peptide comprises a D enantiomer and / or D enantiomer retroinverto of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 18, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 as presented in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively.

[0121] In one aspect, the disclosed composition is understood to be capable of being combined with other treatments for a given disease or condition. For example, in one aspect, a method for treating, inhibiting, shrinking, reducing, improving, and / or preventing cancer and / or metastasis is disclosed, comprising administering a PD-L1 peptide, a PD-L1 synthetic peptide, or a PD-L1 chimeric peptide to a subject; wherein the disease or condition is cancer (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer), and the method further comprises the step of administering to a subject one or more HER-2 B cell epitopes (e.g., one or more HER-2 peptides presented in SEQ ID NO. 27 or 29 or a chimeric MVF-HER-2 peptide presented in SEQ ID NO. 28 or 30) and / or one or more anti-HER-2 antibodies. When a HER-2 B cell epitope or anti-HER-2 antibody is administered to a subject, it is understood that the administration may be a separate simultaneous administration of the HER-2 B cell epitope or anti-HER-2 antibody, a pre-administration, or a subsequent administration of the HER-2 B cell epitope or anti-HER-2 antibody that is a component of the same pharmaceutical formulation as the HER-2 B cell epitope or anti-HER-2 antibody, or the PD-L1 peptide, PD-L1 synthetic peptide, or PD-L1 chimeric peptide.For example, a method for treating, inhibiting, shrinking, reducing, improving, and / or preventing cancer and / or metastasis (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer) comprises administering to a subject a pharmaceutical composition comprising one or more of the PD-L1 peptides presented in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; the chimeric PD-L1 peptides presented in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and / or the retro-inverto PD-L1 peptides presented in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; The above method further comprises the step of administering to a subject one or more of the HER-2 B cell epitope HER-2 (266-296) presented in SEQ ID NO: 27 and / or the HER-2 (597-626) presented in SEQ ID NO: 29 and / or the chimeric epitope MVF-HER-2 (266-296) peptide (e.g., as presented in SEQ ID NO: 28) and the MVF-HER-2 (597-626) peptide (e.g., as presented in SEQ ID NO: 30). Accordingly, in one aspect, MVF-PD-L1 (36-53) presented in SEQ ID NO: 8, MVF-PD-L1 (50-67) presented in SEQ ID NO: 9, MVF-PD-L1 (95-112) presented in SEQ ID NO: 10, and / or MVF-PD-L1 presented in SEQ ID NO: 11 A method for treating, inhibiting, shrinking, reducing, improving, and / or preventing cancer and / or metastasis (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer) is disclosed, comprising administering a pharmaceutical composition comprising MVF-PD-L1 (130-147); MVF-HER-2 (266-296) peptides (e.g., as presented in SEQ ID NO: 28), and MVF-HER-2 (597-626) peptides (e.g., as presented in SEQ ID NO: 30) to a subject with cancer.

[0122] It is further understood and considered herein that synthetic peptides for use in treating, inhibiting, reducing, decreasing, improving, and / or preventing cancer, autoimmune disease, or Alzheimer's disease may be components of chimeric peptides. Accordingly, in one aspect, a method for treating, inhibiting, reducing, decreasing, improving, and / or preventing cancer, Alzheimer's disease, or autoimmune disease in a subject is disclosed herein, comprising administering a PD-L1 chimeric peptide to a subject, wherein the chimeric peptide comprises one or more PD-L1 B cell epitopes, T helper (Th) epitopes, and a linker connecting the PD-L1 B cell epitopes to the Th epitopes, wherein one or more PD-L1 B cell epitopes consist of sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. It is understood and considered herein that the synthetic PD-L1 peptide (i.e., PD-L1 B cell epitope) used in the chimeric peptide may be acetylated, amidated, and / or may comprise a D enantiomer. In one aspect, a method for treating, inhibiting, reducing, decreasing, improving, and / or preventing cancer (e.g., breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, and cervical cancer), Alzheimer's disease, or autoimmune disease in a subject is disclosed herein, for example, by administering a PD-L1 chimeric peptide to a subject, wherein the chimeric peptide comprises SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0123] D. Examples

[0124] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are manufactured and evaluated; they are purely illustrative and are not intended to limit the disclosure. While efforts have been made to ensure accuracy with respect to figures (e.g., amounts, temperatures, etc.), some errors and deviations must be taken into account. Unless otherwise stated, parts are by weight, temperatures are in °C or ambient temperatures, and pressures are at or near atmospheric pressure.

[0125] 1. Example 1: Identification of peptide epitopes for huPD-L1

[0126] The selection of candidate B cell epitopes expressed on the surface of PD-L1 was performed in-house (Peptide Companion) using six antigenic correlations described as follows TM , 5x.com) performed by computer-assisted analysis: ( a ) The chain flexibility and mobility profiles of individual sequences were calculated; ( b Hydropathy profiles were generated across 7 residue range settings and then smoothed to a 3-residue range using the Kyte and Doolittle scales; ( c Hydrophilic profiles for the 6-residue window were generated using Hopp and Woods' program; ( d ) Analysis of amino acid residue exposure to water (1.4A probe) was performed by the solvent exposure algorithm; ( e ) Calculated a protrusion index that predicts the accessible and solvent-protruding protein portion; ( f The probability that a 5-residue sequence is antigenic is Welling et al. et al. It was determined by the method of ). Sequences were assigned scores from 1 to 6 and ranked according to their respective index values; the highest-ranked sequences had the highest individual scores for the analyzed results, and consecutive candidates had the next highest scores.

[0127] The highest-scoring epitopes were further ranked by correlation with secondary structural properties; for example, amphiphilic α-helical sequences or β-turned loop regions are preferred over random coil fragments. Chou, Fasman, and Novotny et al. et al. A computer program was used to predict secondary structures (α-helix, β-strand / sheet, β-turn / loop, random coil) and α-helix amphiphilic moments. Finally, individual amino acid sequences were considered. Electrostatic ion pairs and helical dipole interactions in helical segments were also considered (e.g., hydrophobic / hydrophilic balance).

[0128] Four epitopes of PD-L1 were identified using peptide epitope mapping with immunogenicity / antigenicity algorithms, and the analysis of these epitopes was combined with the crystal structure complex of human PD-1 / human PD-L1 (hPD-1 / hPD-L1) as described in the literature (Zak et al. in 2015 (PDB ID: 4ZQK)) to design a chimeric B-cell vaccine based on the extracellular domain of PD-1. Selection was further enhanced by examining the three-dimensional structure of PD-1 (PDB ID codes: 4Z18, 4ZQK, 3BIK). All four epitopes were analyzed using the PyMOL 3-D modeling software DeLano WL (2002). The PyMOL User's Manual It was modeled using [method]. The sequences with the highest scores are shown in Table 4. Using this method, four of the 12 highest-scoring B-cell epitope sequences of human PD-L1, amino acids 36-53, 50-67, 95-112, and 130-147 were selected for evaluation in combination with information obtained from the crystal structure of PD-1:PDL1.

[0129] Table 4. Human PD-L1 Predictor B-cell Epitopes

[0130]

[0131] Although the structures of human PD-1 (PDB 3RRQ) and human PD-L1 (PDB 3BIS, 3FN3, 4Z18, 5C3T) have been determined, they, in turn, failed to explain the significant plasticity for complex formation within human PD-1, which was only very recently demonstrated by the structure of the complete human PD-1 / PD-L1 complex. While the above structures provided a complete description of the interactions, the flat surface of the protein-protein interface still complicates drug design efforts to guide more rational drug development in the absence of structural information regarding small molecule inhibitors in complexes with PD-1 or PD-L1. The crystal structures show that receptor-ligand interactions are mediated in the major portion by residues of the C0CFG strand within both PD-1 and PD-L1 (Fig. 1). Protein-protein contacts involve both hydrophobic and polar interactions, and the total surface area is 1,970 Ų. The interactions are organized around a central hydrophobic core contributed by the two partners and consist of nonpolar residues of the PD-1 front sheet (Val64, Ile126, Leu128, Ala132, Ile134) and the PD-L1 front sheet (LIle54, LTyr56, LMet115, LAla121, LTyr123), including characteristic alkyl-p interactions of the Ile134 and LTyr123 side chains. This hydrophobic region is open to the solvent at the expected antigen-binding site and is adjacent to a buried region of mixed polar / nonpolar interactions on opposite sides of the molecule. These two regions are surrounded by a peripheral network of polar residues (safe in terms of the CDR loop), providing additional hydrogen bond-mediated interactions between the receptor and the ligand.

[0132] 2. Example 2: Synthesis of peptide epitopes for huPD-L1

[0133] Four new peptide sequences identified as targeting human PD-L1 were synthesized using a 9600 Milligen / Biosearch solid-phase peptide synthesizer (Millipore, Bedford, MA, USA) with PyBOP / 6Cl-HOBT coupling reagents for Fmoc / t-butyl chemistry and CLEAR amide resin (Peptides International, Louisville, KY, USA). Some peptide samples were acetylated using 1-acetyimidazole (Sigma-Aldrich St. Lois, MO, USA) prior to cleavage. All peptides were synthesized as chimeric constructs containing a promiscuous T helper epitope derived from the measles virus fusion protein (MVF, amino acids 288-302) using a 4-residue linker (GPSL). Peptides were cleaved from the resin using the cleavage reagent R(TFA) / thioanisole / EDT / anisole (90 / 5 / 3 / 2), and the crude peptides were purified by semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC; Waters, Bedford, MA, USA) using a C-4 Vydac column. RP-HPLC fractions exhibiting identical retention times were aggregated and lyophilized. All peptides demonstrated a purity exceeding 95%. The samples were then characterized by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI) at the Campus Chemical Instrumentation Center (CCIC, The Ohio State University, Columbus, OH, USA) and analyzed using an analytical RP-HPLC system (Waters, Bedford, MA, USA). All peptides possessed accurate molecular weights.

[0134] 3. Example 3: Immunization with peptide epitotype against huPD-L1

[0135] Vaccine antibodies were generated for each peptide using New Zealand white rabbits purchased from Charles River Laboratories (Wilmington, MA, USA). Rabbits were immunized with 1 mg of MVF chimeric peptide emulsified in Montanide ISA 720 (Seppic, Paris, France) and nor-MDP adjuvant (N-acetyl-glucosamine-3-acetyl-alanyl-d-isoglutamine) and boosted twice at 3-week intervals. Antibody titers were monitored by direct ELISA for the peptide immunogen, the peptide B-cell epitope, and the acetylated version of the B-cell epitope. Serum was collected weekly, and animals were sacrificed at week 9. Peptide vaccine antibodies were purified by affinity chromatography using a protein A / G column, and concentrations were measured by the Coomassie protein assay. All experiments were conducted in accordance with the U.S. Public Health Service policy on the humane care and use of laboratory animals, approved by the Ohio State University Institutional Animal Care and Use Committee, and detailed in the accepted protocol.

[0136] 4. Example 4: In vivo study of peptide vaccine PD-L1 epitope: CT-26 tumor model in mice

[0137] The peptide vaccine was dissolved in water and emulsified in Montanide ISA 720 (1:1) and 100 μg of nor-MDP (N-acetylglucosamine-3-acetyl-l-alanyl-d-isoglutamine). 5-6 week old female Balb / c mice (Charles River Laboratories) were vaccinated with 100 μg of the peptide vaccine three times at 3-week intervals, and challenged subcutaneously (sc) with CT-26 tumor cells (100,000 per mouse) 15 days after the third vaccination. Mice immunized with an unrelated MVF peptide chimera were used as a negative control.

[0138] Mice treated twice a week with an anti-mouse PD-L1 MAb (Bio X Cell, West Lebanon, NH) dose of 200 µg / week were used as positive controls. Tumor growth was monitored for up to 18 days after the challenge. During immunization, blood was collected every two weeks and used for ELISA to monitor antibody titers. Mice were euthanized at the end of treatment, and tumor samples were extracted, weighed, and stored for further study and histological examination. Spleens were also collected for further examination.

[0139] 5. Example 5: In vivo study of peptide vaccine PD-L1 epitope: CT-26-HER2 neu tumor model in mouse.

[0140] The vaccine was dissolved in water and emulsified in Montanide ISA 720 (1:1) and 50 μg of nor-MDP (N-acetylglucosamine-3-acetyl-l-alanyl-d-isoglutamine). 5-6 week old female Balb / c mice (Charles River Laboratories) were immunized three times with 100 μg of each peptide vaccine at 3-week intervals, and 2 weeks after the third immunization, the mice were challenged subcutaneously (sc) with CT-26-HER2 neu tumor cells (100,000 per mouse).

[0141] Mice treated twice weekly with anti-mouse PD-L1 MAb 10F.9G2 (Bio X Cell, West Lebanon, NH) at a dose of 200 µg / week were used as positive controls, or mouse IgG purified from mouse serum (Sigma-Aldrich, St. Louis, Mo) was used as a negative control. Tumor growth was monitored for up to 21 days after the challenge. During immunization, blood was collected every two weeks and used for ELISA to monitor antibody titers. Mice were euthanized at the end of treatment, and tumor samples were extracted, weighed, and stored for further study and histological examination.

[0142] Organs (spleen, liver, heart, lung, lymph nodes, brown adipose tissue, kidney, and tumor) were collected from Balb / c mice vaccinated with combination peptides (HER-2, PD-1, and PD-L1) and submitted for analysis to the Comparative Pathology and Mouse Phenotyping Core Facility of the Department of Veterinary Life Sciences and Comprehensive Cancer Center (Pathologist: Krista MD La Perle, DVM, PhD, Dipl. ACVP).

[0143] All experiments were conducted in accordance with the U.S. Public Health Service policy on the humane care and use of laboratory animals, approved by the Ohio State University Institutional Animal Care and Use Committee, and detailed in the accepted protocol.

[0144] 6. Example 6: Cytotoxicity of PD-L1 antibody-dependent cells:

[0145] The human immune system consists of a complex network of immune checkpoint molecules that facilitate the elimination of cells expressing foreign antigens while maintaining tolerance to self-antigens. Immune checkpoint receptors promise novel immunotherapeutic targets for the treatment of various diseases, including cancer and autoimmune-mediated disorders. Programmed cell death protein 1, also known as PD-1 (CD279), is an immunosuppressive receptor expressed on activated T cells and B cells and plays a crucial role in regulating the immune response to tumor antigens. Binding of PD-1 by one of the ligands of PD-L1 (B7-H1) or PD-L2 (B7-DC) on adjacent cells inhibits T-cell receptor (TCR) signaling, TCR-mediated proliferation, transcriptional activation, and cytokine production. Therapeutic antibodies and Fc fusion proteins designed to block PD-1 / PD-L1 interactions have shown promising results in clinical trials for the treatment of various cancers.

[0146] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism of action by which targeted antibodies destroy virus-infected or diseased cells through the destruction of components of the cell-mediated immune system. ADCC is a preferred mechanism for killing targeted cancer cells using antibody-based drugs. Antibodies bind to target antigens on the cell surface. When the Fc effector portion of a target-binding antibody binds to the FcγRIIIa receptor on the cell surface of effector cells (primarily natural killer cells), multiple cross-linking between the two cell types occurs, inducing the activation of the ADCC pathway. The death of target cells is the endpoint of this pathway activation and is used in ADCC biopsies.

[0147] 7. Example 7: PD-L1 Vaccine Immunogenicity and Antitumor Efficacy in a Synonymous Tumor Model of Colon Carcinoma

[0148] A new paradigm of humoral-responsive immunotherapy based on stereomorphic B-cell epitope vaccines is being developed at our institution with the goal of bypassing inherent drug resistance while providing the potential for sustained treatment. Specifically, two novel B-cell epitope-specific vaccines (B-Vaxx) have been developed: a trastuzumab-binding epitope and a pertuzumab-binding epitope, specifically designed using the X-ray structures of the HER-2-trastuzumab and HER-2-pertuzumab complexes. Clinical results from the first human dose-escalation phase of a Phase I study using B-Vaxx were recently reported. The investigational vaccine was safe and well-tolerated, demonstrated antitumor activity, and showed preliminary indications that peptide vaccination can avoid therapeutic resistance and provide a promising alternative to mAb therapy.

[0149] The characterization and development of a novel B-cell peptide epitope targeting human PD-L1, which induces in vivo polyclonal antibodies capable of blocking PD-1 / PD-L1 interactions that potentially mimic the effects of atezolizumab, are presented herein. We demonstrate the preclinical development of a PD-L1 vaccine to design a chimeric B-cell vaccine based on the extracellular domain of PD-L1 using sophisticated peptide mapping and predictive antigenicity algorithms based on the 3D structure of PD-L1, which was linked to the uninhibited T helper cell measles virus fusion protein (MVF).

[0150] The immunogenicity of each peptide epitope was first evaluated in rabbits and Balb / c mice, which induced antibodies recognizing not only the immunogenic synthetic peptides but also the recombinant human PD-L1 protein. A synonymic colorectal CT-26 wt Balb / c mouse tumor model was used to evaluate the efficacy of vaccination with four MVF-PD-L1 peptides as growth inhibitors for CT-26 tumor cells. PD-L1 (130–147), PD-L1 (50–67), and PD-L1 mAb epitopes showed significant inhibition of tumor growth compared to PBS. The experiment was then extended to another synonymic CT26 / HER-2 Balb / c carcinoma model. The antitumor effects of vaccination with all four MVF-PD-L1 epitopes were investigated in a synonymic Balb / c colorectal carcinoma mouse model challenged with CT26 / HER-2 cell lines. Interestingly, PD-L1 (130-147) treatment demonstrated superior tumor growth inhibition, with greater tumor growth inhibition demonstrated compared to PBS and commercially available standard mouse anti-PD-L1 antibodies.

[0151] a) Result

[0152] (1) Design and purification of four novel human PD-L1 (hPD-L1) peptide epitopes

[0153] The tertiary structure of PD-L1 consists of an extracellular domain, a transmembrane domain, and an intracellular region. Since the extracellular domain is responsible for PD-1 binding and can access anti-peptide antibodies, this domain was selected as the immunogenicity region. B-cell epitopes were ranked based on six correlations of antigenicity and correlated with the secondary structure, and the analysis of these epitopes was combined with the crystal structure complex of human PD-1 / human PD-L1 (hPD-1 / hPD-L1). In this analysis, four B-cell epitope sequences of human PD-L1 were identified for further investigation: amino acids: 36-53, 50-67, 95-112, and 130-147 (Fig. 2). Four peptide sequences were synthesized as a chimeric construct containing a "promiscuous" T helper epitope derived from measles virus fusion protein (MVF, amino acids 288-302; KLLSLIKGVIVHRLEGVE) (SEQ No. 6) using a 4-residue linker composed of GPSL.

[0154] (2) In vivo immunization using PD-L1 epitopes results in a strong antibody response in rabbits.

[0155] The immunogenicity of all four peptides was evaluated in inbred rabbits (Fig. 3). Antibody responses were monitored weekly. Titers increased after the first boost, and after the second boost, high titers exceeded 32,000 for PD-L1 36-53 and 50-67, whereas they were 64,000 for PD-L1 95 and 130. Additionally, the antibodies were able to recognize the human recombinant PD-L1 protein (Fig. 4). This thus justifies the selection of the peptide epitopes.

[0156] (3) Efficacy of anti-mouse PD-L1(B7-H1) mAb clone 10F.9G2 against PD-L1 vaccine epitope in inhibiting tumor growth in a synonymous Balb / c model challenged with CT26 colon carcinoma cell line.

[0157] To confirm the efficacy of the in vivo PD-L1 epitope vaccine in CT26 carcinoma cell lines, Balb / c mice (10 mice / group) were immunized with 100 μg each of MVF-PD-L1 (36-53), MVF-PD-L1 (50-67), MVF-PD-L1 (95-112), and MVF-PD-L1 (130-147) emulsified with nor-MDP and Montanide 720 (1:1 ratio) in Plan 1A (Fig. 5a). As described in detail in Plans 1A-D (Figs. 5a, 5b, 5c, and 5d), mice were immunized three times at 3-week intervals. Blood was collected at 1, 2, and 3 weeks after each vaccination and labeled as 1Y+3, 2Y+1, 2Y+2, 2Y+3, 3Y+1, 3Y+2, and 3Y+3, where Y is the immunization number as shown in Tables 5A, 5B, 5C, 5D, and 5E. All PD-L1 epitopes induced high titers of PD-L1 anti-peptide antibodies by ELISA. The data are summarized in Figure 6, and all vaccines showed responsiveness to recombinant PD-L1 protein (Figure 7). Additionally, combinations of PD-1 (92-110) and PD-L1-130-147 were investigated.

[0158] Table 5A: Immunogenicity (Titer) of PD-L1(36-53) Peptide Epitope in Balb / c Mice

[0159]

[0160] Table 5B: Immunogenicity (Titer) of PD-L1 (50-67) Peptide Epitope in Balb / c Mice

[0161]

[0162] Table 5C: Immunogenicity (Titer) of PD-L1(95-112) Peptide Epitope in Balb / c Mice

[0163]

[0164] Table 5D: Immunogenicity (Titer) of PD-L1 (130-147) Peptide Epitope in Balb / c Mice

[0165]

[0166] Table 5E: Immunogenicity (potency) of PD-1 (92-110) and PD-L1 (130-147) peptide epitope combination in Balb / c mice

[0167]

[0168] Two weeks after the third immunization (3Y), CT26 carcinoma cells (1 x 10⁶ per mouse) were injected into mice. 5 Canine cells were inoculated subcutaneously (sc), and tumor formation was monitored daily thereafter. Mice treated with PBS were used as negative controls, and mice treated twice weekly with a 200 μg / dose injection of anti-mouse PD-L1 monoclonal antibody (mAb clone 10F.9G2) were used as positive controls. Tumor growth was monitored daily and measured with calipers. Tumor growth (tumor volume; LWW mm²) for each individual mouse. 3The pattern is presented in Fig. 8. Mice vaccinated with all four MVF-PD-L1 chimeric peptide vaccines showed significant tumor suppression, comparable to that treated with anti-mouse PD-L1 (10F.9G2) mAb monoclonal antibody (+ve control) versus PBS treatment (-ve control). PD-L1 B-cell epitope vaccine treatment showed a similar and significant reduction in tumor growth on day 16 post-challenge compared to the negative control treatment using PBS (Fig. 9). However, most notably, MVF-PD-L1 (130-147), MVF-PD-L1 (50-67), and PD-L1 (36-53) showed the strongest mean tumor growth inhibition at days 14 and 16 in combination with 10F.9G2 PD-L1 mAb, but were not significantly worse than MVF-PD-L1 (36-53), MVF-PD-L1 (95-112), or the MVF-PD-L1 (95) + MVF-PD-L1 (92-110) combinations. These results indicate the potential therapeutic application of the vaccine as a useful inhibitory vaccine. Tumor sizes at various time points are also presented in Figure 10, and one-way ANOVA followed by Tukey's multiple comparison test was used to compare data from multiple groups or between groups compared to a negative control analyzed by the Kaplan-Meier method. Significantly higher rates of tumor growth inhibition, highest survival rates, and lowest levels of lesions were observed in vaccinated PD-L1 (130-147).

[0169] (a) Antibody isotype.

[0170] In addition, various antibody isotypes generated by vaccination were analyzed. The antibody isotypes induced in mice were determined to be the IgG1 class against PD-L1 130-147, which mainly contained IgG 2a (data shown in Figure 11), while IgG 2b was dominant in other epitopes.

[0171] (b) Anti-MVF-PD-L1 130 antibody can block PD-1 / PD-L1 interaction.

[0172] Bioluminescent cell-based PD-1 / PD-L1 blocking bioassays provide a good option for the rapid screening of therapeutic antibodies or other compounds developed to disrupt PD-1 signaling interactions. The function of three anti-PD-L1 antibodies was tested using the PD1 / PD-L1 blocking assay. Purified polyclonal anti-PD-L1 antibodies from rabbits were observed to be capable of blocking PD1 / PD-L1 interactions similarly to commercially available anti-human PD-L1 antibodies (Fig. 12). These results indicate that functional anti-PD-L1 is generated in rabbits immunized with B-cell epitope peptides. Fig. 12 shows the ability of each PD-L1 vaccine to block PD-1:PD-L1 interactions compared to nivolumab and atezolizumab. Surprisingly, only PD-L1 antibodies 130–147 were effective.

[0173] (c) Determination of apoptosis by caspase activity assay.

[0174] Monoclonal antibodies have become attractive therapeutic drug candidates due to their disease-specific targeting potential and low toxicity profiles. Antibody-mediated target cell death is often associated with immune effector mechanisms, such as cytotoxicity of antibody-directed cells, but it can also be induced by the process of apoptosis. Antibody-directed mechanisms, including antigen crosslinking, activation of death receptors, and blockade of ligand-receptor growth or survival pathways, can induce apoptosis in target cells. Depending on the mechanism of action, monoclonal antibodies can induce targeted cell-specific death alone or influence the regulation of anti-apoptotic pathways to enhance target cell sensitivity to chemotherapy or radiotherapy agents. To further investigate this, we evaluated whether anti-PD-L1 peptide vaccine antibodies could induce apoptosis in cancer cells through a caspase activation assay. MC38 (PD-L1-expressing cells) and CT26WT cancer cells were seeded in 96-well plates and cultured overnight at 37°C. The following day, low serum growth medium containing the peptide vaccine antibody, as well as the reference antibody atezolizumab and β-Lapachone as an apoptosis inducer, was added to the wells. The plates were then incubated at 37°C for an additional 24 hours. Next, the Caspase-Glo reagent was added, and caspase-9 release was measured using a irradiometer as a measure of apoptosis induction. Caspase-9 is a member of the cysteine-aspartate-specific protease (caspase) family, which acts as a key initiator in the intrinsic apoptosis pathway of mammalian cells. It was found that the PD-L1 antibody caused a significant increase in the amount of caspase activity in treated cells compared to the negative control (PBS), comparable to that of the mAb atezolizumab. The increased caspase release was more than threefold (Fig. 13), which clearly indicates increased apoptosis.We found that the PD-L1 antibody caused a significant increase in the amount of caspase activity in treated cells compared to the negative control (PBS), comparable to that of the mAb atezolizumab. The increased caspase release was more than threefold (Fig. 13), which clearly indicates increased apoptosis.

[0175] (d) ADCC activity.

[0176] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a critical mechanism of action for anticancer mAbs, through which antibodies recruit FcγR-bearing effector cells to target "diseased" cells for destruction by components of the cell-mediated immune system, such as natural killer cells. However, most PD-1 / PD-L1 antibodies currently in clinical development, such as atezolizumab (MPDL3280A), are known not to mediate antibody-dependent cell-mediated cytotoxicity; this is a humanized monoclonal antibody with an FcγR-binding-deficient IgG1 isotype engineered to eliminate ADCC activity. While ADCC induction can induce apoptosis and potentially enhance tumor death, it can also tame the immune response by leading to the depletion of PD-L1-expressing T cells. To eliminate ADCC and CDC at clinically appropriate doses, the Fc (crystallizable fragment) domain of atezolizumab is engineered to reduce its interaction with FcyR.

[0177] To evaluate the ADCC activity of the purified polyclonal antibody, WIL2-S target cells were used along with MC38 cells and Jurkat effector cells treated with 25 ng / ml IFN-y for 16 hours. Linearity tests for the ADCC response were performed at various antibody concentrations, as shown in Fig. 14, to evaluate the performance of the assay. The antibody induced by the peptide vaccine had an effect similar to that of the mAb atezolizumab and was unable to mediate antibody-dependent cytotoxicity (Fig. 14).

[0178] (4) Efficacy of anti-mouse PD-L1(B7-H1) mAb clone 10F.9G2 against PD-L1 vaccine epitope in tumor growth inhibition in a HER-2-expressing CT26 colon carcinoma cell line challenged synonyms Balb / c model

[0179] CT26 / HER-2 carcinoma cell line To verify the efficacy of the in vivo PD-L1 epitope vaccine, Balb / c mice (10 mice / group) were immunized with 100 μg each of MVF-PD-L1 (36-53), MVF-PD-L1 (50-67), MVF-PD-L1 (95-112), and MVF-PD-L1 (130-147) emulsified with nor-MDP and Montanide 720 (1:1 ratio) in Plan 2A (Fig. 15a). As described in detail in Plans 2A-D (Figs. 15a, 15b, 15c, and 15d), the mice were immunized three times at 3-week intervals. Blood samples were collected 1, 2, and 3 weeks after each vaccination and labeled as 1Y+3, 2Y+1, 2Y+2, 2Y+3, 3Y+1, 3Y+2, and 3Y+3, where Y represents the number of immunizations as shown in Tables 6A, 26B, 6C, 6D, and 6E. All PD-L1 epitopes induced high titers of PD-L1 anti-peptide antibodies by ELISA. The data are summarized in Figure 16, and all vaccines showed responsiveness to recombinant PD-L1 protein (Figure 17). Additionally, combinations of PD-1 (92-110) and PD-L1-95 were investigated.

[0180] Table 6 A: Immunogenicity (Titer) of PD-L1(36-53) Peptide Epitope in Balb / c Mice

[0181]

[0182] Table 6B: Immunogenicity (Titer) of PD-L1 (50-67) Peptide Epitope in Balb / c Mice

[0183]

[0184] Table 6C: Immunogenicity (Titer) of PD-L1(95-112) Peptide Epitope in Balb / c Mice

[0185]

[0186] Table 6D: Immunogenicity (Titer) of PD-L1 (130-147) Peptide Epitope in Balb / c Mice

[0187]

[0188] Table 6E: Immunogenicity (potency) of PD-1 (92) and PD-L1 (95-112) peptide epitope combination in Balb / c mice

[0189]

[0190] 2 weeks after the third immunization (3Y), CT26 carcinoma cells in mice (1 x 10⁶ per mouse) 5 Cells were inoculated subcutaneously (sc), and tumor formation was monitored daily. Mice treated with PBS were used as negative controls, and mice treated twice weekly with a 200 μg / dose injection of anti-mouse PD-L1 monoclonal antibody (mAb clone 10F.9G2) were used as positive controls. Tumor growth was monitored daily and measured with calipers. Tumor growth (tumor volume; LWW mm²) for each individual mouse. 3 The patterns are presented in Fig. 18. Mice vaccinated with all four MVF-PD-L1 chimeric peptide vaccines showed significant tumor suppression, as in the anti-mouse PD-L1 (10F.9G2) mAb monoclonal antibody (+ve control) versus PBS treatment (-ve control). PD-L1 B-cell epitope vaccine treatment showed a similar and significant reduction in tumor growth on day 16 after challenge compared to the negative control treatment using PBS (Fig. 19). However, most notably, the combinations of MVF-PD-L1 (130-147), PD-L1 (36-53), PD-L1 (95-112), PD-L1 (50-67), and MVF-PD-L1 (95) + MVF-PD-L1 (92-110) showed the most potent tumor growth inhibition on day 16, indicating potential therapeutic applicability as useful inhibitory vaccines. Tumor sizes at various time points are also presented in Figure 20, and data from multiple groups or between groups were compared using one-way ANOVA followed by Tukey's multiple comparison test. Compared to negative controls analyzed by the Kaplan-Meier method, significantly higher rates of tumor growth inhibition, the highest survival rates, and the lowest levels of lesions were observed in the vaccinated PD-L1 (130-147).

[0191] In addition, various isotypes of antibodies generated by vaccination were analyzed. The isotypes of antibodies induced in mice were determined to be the IgG1 class against PD-L1 130-147 containing IgG 2a (data presented in Fig. 21), while IgG2b was dominant in other epitopes. Finally, the 50% mouse survival time for each PD-L1 epitope was analyzed, showing that PD-L1 130-147 has a better survival rate compared to other PD-L1 epitopes (Fig. 22).

[0192] (5) Combination treatment with peptide vaccines PD-L1 and HER-2 epitopes results in potent HER-2 and PD-L1 antibody responses and tumor growth inhibition in the Balb / c CT26 / HER-2 model.

[0193] Next, using the CT26 / HER-2 tumor model of Balb / c, we tested the synergistic effects of the combination of anti-PD-L1 and anti-HER-2 immunization therapies to determine whether they could increase immunogenicity, enhance anti-tumor responses, and provide synergistic effects in inhibiting tumor growth. Balb / c mice (10 mice / group) were immunized three times at 3-week intervals with MVF-PD-L1 (36-53, 50-67, 95-112, 130-147) combined with an MVF-HER-2 (266-296) + MVF-HER-2 (597-626) peptide vaccine construct emulsified with ISA 720 (1:1 ratio) as summarized in plans 3A-3C (Figs. 23a, 23b, and 23c). As determined by ELISA, potent HER-2 and PD-L1 antibody responses were induced in all vaccinated mouse populations over a set period (1Y+3, 2Y+1, 2Y+2, 2Y+3, 3Y+1, 3Y+2, 3Y+3, and terminal hemorrhage). Tables 7A, 7B, 7C, 7D, and 7E. These immunogenicity data are summarized in Figures 24 and 25. 1 x 10⁶ from CT26 / HER-2 tumor lines 2 weeks after final boost. 5Tumor cells were transplanted subcutaneously (sc) into mice. Only control mice were treated with PBS to serve as negative controls, or treated with 200 μg / dose anti-mouse PD-L1 mAb (10F.9G2) twice a week during the experiment period to serve as positive controls.

[0194] Table 7A: Immunogenicity (Titer) of PD-L1(36-53)+ Combination HER-2 Peptide Epitope in Balb / c Mice

[0195]

[0196] Table 7B: Immunogenicity (Titer) of PD-L1(50-67)+ Combination HER2 Peptide Epitope in Balb / c Mice

[0197]

[0198] Table 7C: Immunogenicity (Titer) of PD-L1(95-112)+ Combination HER2 Peptide Epitope in Balb / c Mice

[0199]

[0200] Table 7D: Immunogenicity (Titer) of PD-L1(130-147)+ Combination HER2 Peptide Epitope in Balb / c Mice

[0201]

[0202] Table 7E: Combo HER2 immunogenicity (titer) in Balb / c mice

[0203]

[0204] b) Discussion

[0205] The current enthusiasm for cancer immunotherapy stems from the success of certain agents targeting immune checkpoint molecules such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Currently, more than 12 immune checkpoint antibodies targeting CTLA-4 and PD-1 / PD-L1 have received FDA regulatory approval worldwide, and there are numerous ongoing clinical trials for checkpoint inhibitors targeting PD-1 and PD-L1. These therapies have achieved exceptional success in only a small number of patients. The mechanisms underlying innate and acquired resistance are partly responsible for the lack of response to PD-1 / PD-L1 blockade, highlighting the evidence that well-designed combination therapies can expand their use and clinical impact. For example, the combination of ipilimumab and nivolumab received FDA approval due to positive results in patients with unresectable melanoma and promising Phase 1 and 2 trials, but the combination also demonstrated increased toxicity compared to monotherapy.

[0206] Compared to PD-1 inhibitors, PD-L1 inhibitors can reduce the incidence of adverse effects caused by immune disorders. The FDA has approved one humanized mAb (atezolizumab, TECENTRIC®) and two fully human mAbs (avelumab, BAVENCIO® and durvalumab, IMFINZI®) targeting PD-L1. The recently reported avelumab / hPD-L1 complex structure provided clear structural information on how therapeutic mAbs remove the binding of PD-1 / PD-L1. Avelumab, a PD-L1 targeted antibody, is currently undergoing several Phase 3 clinical trials for non-small cell lung cancer (NSCLC) (NCT02395172), advanced renal cell carcinoma (NCT02684006), and gastric cancer (NCT02625610).

[0207] A vaccine targeting PD-L1, composed of the extracellular domain of PD-L1 (PD-L1E) fused to the C-terminus of the diphtheria toxin (DTT) translocation domain, was recently reported. The therapeutic efficacy of the DPDL1E vaccine was evaluated in B16-F10 tumor-bearing C57BL / 6 mice. The authors concluded that the PD-L1-targeting DPDL1E vaccine induces a PD-L1-specific immune response and delays tumor growth in vivo. These results represent a promising path for future research in the exploration of cancer vaccine design.

[0208] Overall, the rational development of diverse immune strategies for managing specific cancers holds substantial potential to transform the therapeutic landscape and improve human disease management. This study also highlights the benefits of using multi-target approaches, as it is increasingly recognized that only the use of drugs affecting multiple signaling pathways yields potent antiproliferative effects and delays the onset of drug resistance. However, this current study demonstrates the feasibility of generating vaccine combinations that can serve as powerful tools for investigating the oncogenic drivers of cancer cells. Currently, there are no commercially available B-cell epitope vaccines targeting the human PD-1 / PD-L1 axis.

[0209] c) Materials and Methods

[0210] (1) Identification and synthesis of peptide epitopes for hPD-L1.

[0211] The selection of candidate B-cell epitopes expressed on the surface of PD-L1 was performed using six antigenic correlations described below in an in-house computer-assisted analysis (Peptide Companion TMThe following was performed by (5z.com): (a) Profiles of chain flexibility and mobility of individual sequences were calculated according to Karplus and Schultz; (b) Hydrophobic profiles were generated across a 7-residue range setting and then smoothed to a 3-residue range using the Kyte and Doolittle scale; (c) Hydrophilic profiles for a 6-residue window were generated using the Hopp and Woods program; (d) Analysis of amino acid residue exposure to water was performed by the solvent exposure algorithm of Rose et al.; (e) The extrusion index was calculated by the method of Thornton et al., which predicts accessible and solvent-extruding protein portions; (f) The probability that a 5-residue sequence is antigenic was determined by the method of Welling et al.; Sequences were assigned scores from 1 to 6 and ranked based on their respective index values: the highest-ranked sequences had the highest individual scores for the analyzed sequences, and consecutive candidates had the next highest scores. The best-scoring epitopes were further ranked according to their correlation with secondary structural properties; for example, amphiphilic α-helical sequences or β-turn loop regions were preferred over random coil fragments. A computer program by Chou, Fasman, and Novotny et al. was used to predict secondary structures (α-helical, β-strand / sheet, β-turn / loop, random coil) and α-helical amphiphilic moments. Finally, individual amino acid sequences were considered. Electrostatic ion pairs and helical dipole interactions in helical segments were also taken into account (e.g., hydrophobic / hydrophilic balance). Peptide epitope mapping was used with an immunogenicity / antigenicity algorithm to identify four epitopes of PD-L1.All four epitopes were modeled using PyMOL 3-D modeling software (DeLano WL 2002, PyMOL User's Manual).

[0212] Four new peptide sequences were synthesized on CLEAR amide resin (Peptides International, Louisville, KY, USA) using a 9600 Milligen / Biosearch solid-phase peptide synthesizer (Millipore, Bedford, MA, USA) with Fmoc / BOP chemistry and PyBOP / HOBT coupling reagents (PB Biosystems, Louisville, KY, USA). Some peptide samples were acetylated using 1-acetyimidazole (Sigma-Aldrich St. Lois, MO, USA) prior to cleavage. All peptides were synthesized as chimeric constructs containing a promiscuous T helper epitope derived from the measles virus fusion protein (MVF, amino acids 288-302) using a 4-residue linker (GPSL). Peptides were cleaved from the resin using the cleavage reagent R(TFA) / thioanisole / EDT / anisole (90 / 5 / 3 / 2), and the unrefined peptides were purified using semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC; Waters, Bedford, MA, USA) with a C-4 Vydac column. RPHPLC fractions exhibiting identical retention times were aggregated and lyophilized. All peptides demonstrated a purity exceeding 95%. The samples were then characterized by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI) at the Campus Chemical Instrumentation Center (CCIC, The Ohio State University, Columbus, OH, USA) and analyzed on an analytical RP-HPLC system (Waters, Bedford, MA, USA). All peptides possessed accurate molecular weights.

[0213] (2) Immunization using hPD-L1 peptide epitope.

[0214] For each peptide, vaccine antibodies were generated in New Zealand white rabbits purchased from Charles River Laboratories (Wilmington, MA, USA). Rabbits were immunized with 1 mg of MVF chimeric peptide emulsified in Montanide ISA 720 (Seppic, Paris, France) and boosted three times at 3-week intervals. For mouse experiments, 6–8-week-old mice were immunized with 0.1 mg of peptide emulsified in ISA 720 (1:1 ratio). Mice were boosted with each dose at 3-week intervals. Blood was then collected, and antibody titers were tested in the serum. Serum was collected weekly, the peptide vaccine antibodies were purified by affinity chromatography using a Protein A / G column, and concentrations were measured using a spectrometer. All experiments were performed in accordance with the U.S. Public Health Service policy on the humane care and use of laboratory animals and were approved by the Ohio State University Institutional Animal Care and Use Committee as detailed in the accepted protocol.

[0215] (3) In vivo study of four peptide vaccine hPD-L1 epitopes: CT-26 and CT26 / HER-2 tumor models in mice.

[0216] 6–8 week old BALB / c mice (Charles River, Wilmington, MA) were used as the standard animal model. Mouse CT26 colon carcinoma cell lines were purchased from American Type Culture Collection Cells and cultured regularly in RPMI1640 medium containing 10% FBS. CT26 / HER2 cells were kindly provided by M. Penichet. The vaccine was dissolved in sterile water and emulsified in Montanide ISA 720 (1:1). 6–8 week old female Balb / c mice (Charles River Laboratories) were immunized three times with 100 μg of each peptide vaccine at 3-week intervals, and 2 weeks after the third immunization, CT26 and / or CT26 / HER-2 tumor cells (1 x 10⁶ per mouse) 5 Mice were challenged with ), which was subcutaneously (sc) implanted in the right flank. 200 μg of rat α-mouse PD-L1 mAb 10F.9G2 (Bio X Cell, West Lebanon, NH) was administered intraperitoneally (ip) twice weekly to the control group following tumor inoculation. Mice were monitored daily, and the formation of palpable tumors was scored; the tumor was challenged when it became necrotic or reached a predetermined 2,000 mm² 3 If it exceeded the size, it was sacrificed. Tumor volume was measured in cubic millimeters with calipers and calculated using the following formula: A×B 2 ×0.5, where A is the largest diameter and B is the widest point perpendicular to the length. During immunization, blood was collected weekly and used in an ELISA to monitor antibody titers.

[0217] (4) Antibody-dependent cell-mediated cytotoxicity (ADCC) reporter biopsy.

[0218] To evaluate ADCC activity, the ADCC Reporter Bioassay (Promega) was used according to the manufacturer's instructions. Briefly, target WIL2-S and MC38 cells were seeded into each well of a 96-well assay plate one day prior. PD-L1 expression was induced in MC38 cells by treating them with IFN-γ (25 ng / ml for 16 hours at 37°C). The following day, serially diluted reference antibody (atezolizumab) or CD20 control antibody, and serially diluted purified PD-L1 130 polyclonal antibody were added to the assay plate. Next, Jurkat cells (immortalized T-lymphocyte cells engineered to stably express the FcγRIIIa receptor as effector cells) were co-cultured with the antibody-treated target cells in a humidified 5% CO2 incubator at 37°C for 6 hours at an effector cell to target cell ratio of 4:1. Once bound to the antibody, engineered Jurkat T-lymphocytes induce the expression of firefly luciferase by activating gene transcription through the activated T-cell nuclear factor (NFAT) pathway. Finally, luciferase activity was quantified using a luciferase assay reagent by a SpectraMax M3 plate reader (Molecular Devices).

[0219] (5) Natural death (Caspase 9 test).

[0220] The Caspase-Glo 9 assay kit (Promega, Madison, WI) was used for caspase detection in cells treated in vitro. The reagent provides a luminescent caspase-9 substrate in combination with luciferase and a cell lysant. Cells are 1 × 10⁶ per well. 4Dog cells were plated in 96-well plates and cultured overnight in complete medium. Subsequently, the cells were treated for 24 hours with an anti-PD-L1 antibody and the reference antibodies atezolizumab and β-Lapachon (10 μM, Sigma Aldrich). Caspase activity was measured by adding 50 μl of CASPASE-GLO® 9 reagent directly to the assay wells to induce cell lysis, followed by caspase cleavage of the substrate and luminescence. Luminescence was detected using a microplate reader (Molecular Devices). The values ​​indicated in the readings are proportional to the amount of caspase activity in the sample.

[0221] (6) PD-1 / PD-L1 biopsy.

[0222] The assay was performed according to the manufacturer's protocol for the PD-1 / PD-L1 blockade biopsy (Promega). Simply put, 4 × 10 5 aAPC / CHO-K1 or PD-L1 aAPC / CHO-K1 cells were seeded into 96-well RPMI-1640 plates containing 10% FBS. After incubation overnight, the medium was aspirated, and sequentially diluted purified PD-L1 or PD-1 antibodies, as well as nivolumab and atezolizumab antibodies (reference antibodies), were added. Next, PD-1 effector cells were co-cultured, and the plates were maintained at 37°C for 5 hours in a humidified 5% CO2 incubator. After mixing with Bio-Glo reagent, luminescence was measured using a SpectraMax M3 plate reader (Molecular Devices).

[0223] (7) Mouse isotyping test.

[0224] Antibody isotypes (i.e., IgA, IgM, IgG1, IgG2a, IgG2b, IgG3) were determined using the Mouse Typer Isotyping Kit (BIO-RAD, Hercules, CA). Briefly, wells of a 96-well assay plate were coated with 200 ng of peptide antigen in ddH2O and incubated overnight at 4°C. The plate was washed with wash buffer (0.05% Tween-20 and 1% horse serum in PBS). The plate was then blocked with 1% BSA in PBS at room temperature for 1 hour. 100 μl of diluted mouse serum was added to each well for 2 hours, and after washing the wells, 100 μl of ready-to-use rabbit anti-mouse subclass antibody was added to each well and incubated at room temperature for an additional 2 hours. The wells were washed again, and 100 μl of a 1 / 3000 dilution of goat anti-rabbit conjugated to HRP antibody (BIO-RAD, Hercules, CA) was added to each well and incubated at room temperature in the dark for 1 hour. Then, the plate was washed, and 50 μl of the prepared substrate solution was added to each well. The reaction was stopped with 25 μl of 5% SDS suspension buffer. Absorbance at 415 nm was determined using an ELISA plate reader. The dilution of each serum sample was determined by the ELISA titer, which indicated an absorbance of 0.4 or greater after subtracting the background.

[0225] (8) Statistical analysis.

[0226] Tumor size was observed daily and measured with calipers. Tumor volume was calculated using the formula: Volume = (Length * Width * Width) / 2. Statistical analysis of the data was performed using GraphPad Prism 8.1.2 (GraphPad Software, Inc.). One-way analysis of variance was followed by Tukey's multiple comparison test to compare data from multiple groups or between groups. p-value or modified to less than 0.05 p-value ...was allowed as a statistically significant difference. * indicates p<0.05, and ** indicates p<0.01.

[0227] 8. Example 8: D2F2 and D2F2E2 Tumor Challenge

[0228] a) Experimental design: This experiment has two parts (10 mice / group, 6-8w):

[0229] (1) Part 1: Four groups of mice from G0 to G4 are challenged with D2F2 WT cancer cells.

[0230] G0, G1, and G2 were the control groups, and mice in these groups were not treated prior to the tumor cell challenge. After the challenge with D2F2 WT tumor cells, G0 mice were treated with PBS (ip) twice weekly starting on day 2 after the challenge, serving as the negative control, while G1 and G2 mice were treated with anti-PD-1 mAb (29F.1A12) or anti-PD-L1 mAb (10F.9G2), respectively. Treatment for the G1 and G2 mouse groups also began on day 2 after the tumor challenge, with 100 µg / mouse administered twice weekly. G1 and G2 were used as positive controls. In contrast, G3 and G4 mice were immunized with 100 µg MVF-PD-1 (92-110) + ISA720 or 100 µg MVF-PD-L1 (130-147) + ISA720 per mouse, respectively. Mice were immunized up to four times prior to tumor challenge. Mice were not subjected to further treatment after challenge with D2F2 WT tumor cells. All mice were monitored at least twice per week, and tumors were measured with calipers as shown in the data (Fig. 26).

[0231] (2) Part 2: Five groups of mice from G10 to G15 were planned to be challenged with D2F2 / E2 cancer cells.

[0232] G10, G11, and G12 served as controls and were not treated prior to the tumor cell challenge. After challenging mice with D2F2 / E2 tumor cells, G10 mice were treated with PBS (ip) twice a week starting on day 2 after the challenge, serving as negative controls, while G11 and G12 mice were treated with anti-PD-1 mAb (29F. 1A12) or anti-PD-L1 mAb (10F. 9G2), respectively. For G1 and G12, treatment began on day 2 after the tumor challenge at 100 µg / mouse twice a week. G11 and G12 were used as positive controls. G13, G14, and G15 mice were immunized with 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse for G13, 100 µg MVF-PD-1(92-110) + 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse for G14, and 100 µg MVF-PD-L1(130-147) + 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse for G15. Mice were immunized up to four times prior to the tumor challenge. After the mice were challenged with D2F2 / E2 tumor cells, they were not subjected to further treatment. All mice were monitored at least twice per week, and tumors were measured with a caliper as shown in the data (Fig. 26).

[0233] b) Experimental plan:

[0234] Mice were immunized with a peptide vaccine designed at 3-week intervals as described in Fig. 26, labeled 1Y, 2Y, 3Y, and 4Y. Blood was collected as indicated in the plan, with numbers representing weeks after immunization; for example, 2Y+2 indicates secondary immunization and blood was collected after 2 weeks (Fig. 27). Mice were challenged with D2F2 WT or D2F2 / E2 at 4Y+5 as shown in Fig. 26. Mice were monitored at least twice per week, and tumors were measured as indicated in the data figures.

[0235] c) Result

[0236] (1) Immunogenicity of G3 mice immunized with 100 µg MVF-PD-1(92-110) + ISA720 per mouse.

[0237] Blood was collected as shown in Fig. 28. For example: 2Y+2 represents secondary immunization and blood was collected after 2 weeks. Antibody titers against MVF-PD-1 (92-110) were measured using an ELISA assay.

[0238] To detect antibodies against MVF-PD-1(92-110) in mouse serum, 100 μl of 2 μg / ml MVF-PD-1(92-110) peptide was coated onto a 96-well assay plate (COSTAR) as the antigen in ddiH2O and incubated overnight at 4°C. The plate was washed with wash buffer (0.05% Tween-20 and 1% horse serum in PBS). Then, the plate was blocked with 200 μl of 1% BSA in PBS at room temperature for 1 hour. After incubation, 100 μl of 2x serially diluted serum was added to each well and incubated at room temperature for 2 hours. The wells were washed again, and 100 μl of a 1 / 500 dilution of goat anti-mouse conjugated with HRP antibody (0.8 mg / ml, Invitrogen) was added to each well and incubated in the dark at room temperature for 1 hour. The plates were washed for the final time, and 50 μl of the prepared ABTS substrate solution was added to each well. The reaction was stopped with 25 μl of 1% SDS in water. Absorbance at 415 nm was determined using an ELISA plate reader. The dilution of each serum sample was determined by the highest antibody ELISA titer observed at an absorbance greater than 0.2.

[0239] (2) Immunogenicity of G4 mice immunized with 100 µg MVF-PD-L1 (130-147) + ISA720 per mouse.

[0240] Blood was collected as shown in Fig. 29: 2Y+2 showed secondary immunity and blood was collected after 2 weeks. Antibody titers against MVF-PD-L1 (130-147) were measured using an ELISA assay.

[0241] To detect antibodies against MVF-PD-L1 (130-147) in mouse serum, 100 μl of 2 μg / ml MVF-PD-L1 (130-147) peptide was coated onto a 96-well assay plate (COSTAR) as the antigen in ddiH2O and incubated overnight at 4°C. The plate was washed with wash buffer (0.05% Tween-20 and 1% horse serum in PBS). Then, the plate was blocked with 200 μl of 1% BSA in PBS at room temperature for 1 hour. After incubation, 100 μl of 2x serially diluted serum was added to each well and incubated at room temperature for 2 hours. The wells were washed again, and 100 μl of a 1 / 500 dilution of goat anti-mouse conjugated with HRP antibody (0.8 mg / ml, Invitrogen) was added to each well and incubated in the dark at room temperature for 1 hour. The plates were washed for the final time, and 50 μl of the prepared ABTS substrate solution was added to each well. The reaction was stopped with 25 μl of 1% SDS in water. Absorbance at 415 nm was determined using an ELISA plate reader. The dilution of each serum sample was determined by the highest antibody ELISA titer observed at an absorbance greater than 0.2.

[0242] (3) Immunogenicity of G13 mice immunized with 100 µg MVF-HER-2(266-296) + 100 µg MVF-HER-2(597-626) + ISA720 per mouse.

[0243] Blood was collected as shown in Fig. 30: for example, 2Y+2 represents secondary immunization, and blood was collected after 2 weeks. Antibody titers against MVF-HER-2 (266-296) or MVF-HER-2 (597-626) were measured individually using an ELISA assay.

[0244] To detect antibodies against MVF-HER-2(266-296) in mouse serum, 100 μl of 2 μg / ml MVF-HER-2(266-296) peptides were coated onto a 96-well assay plate (COSTAR) as antigen in ddiH2O and incubated overnight at 4°C. The plate was washed with wash buffer (0.05% Tween-20 and 1% horse serum in PBS). Then, the plate was blocked with 200 μl of 1% BSA in PBS at room temperature for 1 hour. After incubation, 100 μl of 2x serially diluted serum was added to each well and incubated at room temperature for 2 hours. The wells were washed again, and 100 μl of a 1 / 500 dilution of goat anti-mouse conjugated with HRP antibody (0.8 mg / ml, Invitrogen) was added to each well and incubated in the dark at room temperature for 1 hour. The plates were washed for the final time, and 50 μl of the prepared ABTS substrate solution was added to each well. The reaction was stopped with 25 μl of 1% SDS in ddi water. Absorbance at 415 nm was determined using an ELISA plate reader. The dilution of each serum sample was determined by the highest antibody ELISA titer observed at an absorbance greater than 0.2. The same procedure was performed to detect antibodies against MVF-HER-2 (597-626) in mouse serum. At 4Y+13, the results indicated that antibody titers in CR mice with no tumors or very small tumors were relatively higher than those in mice with large tumors.

[0245] (4) Immunogenicity of G14 mice immunized with 100 µg MVF-PD-1 (92-110) + 100 µg MVF-HER-2 (266-296) + 100 µg MVF-HER-2 (597-626) + ISA720 per mouse.

[0246] Blood was collected as shown in Fig. 31: for example, 2Y+2 indicates secondary immunization and indicates that blood was collected after 2 weeks. Antibody titers for MVF-PD-1 (92-110), MVF-HER-2 (266-296), or MVF-HER-2 (597-626) were individually measured using an ELISA assay.

[0247] To detect antibodies against MVF-PD-1(92-110) in mouse serum, 100 μl of 2 μg / ml MVF-PD-1(92-110) peptide was coated onto a 96-well assay plate (COSTAR) as the antigen in ddiH2O and incubated overnight at 4°C. The plate was washed with wash buffer (0.05% Tween-20 and 1% horse serum in PBS). Then, the plate was blocked with 200 μl of 1% BSA in PBS at room temperature for 1 hour. After incubation, 100 μl of 2x serially diluted serum was added to each well and incubated at room temperature for 2 hours. The wells were washed again, and 100 μl of a 1 / 500 dilution of goat anti-mouse conjugated with HRP antibody (0.8 mg / ml, Invitrogen) was added to each well and incubated in the dark at room temperature for 1 hour. The plates were washed for the final time, and 50 μl of the prepared ABTS substrate solution was added to each well. The reaction was stopped with 25 μl of 1% SDS in ddi water. Absorbance at 415 nm was determined using an ELISA plate reader. The dilution of each serum sample was determined by the highest antibody ELISA titer observed at an absorbance greater than 0.2. The same procedure was performed to detect antibodies against MVF-HER-2 (266-296) and MVF-HER-2 (597-626) in mouse serum. At 4Y+13, the results indicated that antibody titers in CR mice with no tumors or very small tumors were relatively higher than those in mice with large tumors.

[0248] (5) Immunogenicity of G15 mice immunized with 100 µg MVF-PD-L1 (130-147) + 100 µg MVF-HER-2 (266-296) + 100 µg MVF-HER-2 (597-626) + ISA720 per mouse.

[0249] Blood was collected as shown in Fig. 32: for example, 2Y+2 indicates secondary immunization and indicates that blood was collected after 2 weeks. Antibody titers for MVF-PD-L1 (130-147), MVF-HER-2 (266-296), or MVF-HER-2 (597-626) were individually measured using an ELISA assay.

[0250] To detect antibodies against MVF-PD-L1 (130-147) in mouse serum, 100 μl of 2 μg / ml MVF-PD-L1 (130-147) peptides were coated onto a 96-well assay plate (COSTAR) as antigen in ddiH2O and incubated overnight at 4°C. The plate was washed with wash buffer (0.05% Tween-20 and 1% horse serum in PBS). Then, the plate was blocked with 200 μl of 1% BSA in PBS at room temperature for 1 hour. After incubation, 100 μl of 2x serially diluted serum was added to each well and incubated at room temperature for 2 hours. The wells were washed again, and 100 μl of a 1 / 500 dilution of goat anti-mouse conjugated with HRP antibody (0.8 mg / ml, Invitrogen) was added to each well and incubated in the dark at room temperature for 1 hour. The plates were washed for the final time, and 50 μl of the prepared ABTS substrate solution was added to each well. The reaction was stopped with 25 μl of 1% SDS in ddi water. Absorbance at 415 nm was determined using an ELISA plate reader. The dilution of each serum sample was determined by the highest antibody ELISA titer observed at an absorbance greater than 0.2. The same procedure was performed to detect antibodies against MVF-HER-2 (266-296) and MVF-HER-2 (597-626) in mouse serum. At 4Y+13, the results indicated that antibody titers in CR mice with no tumors or very small tumors were relatively higher than those in mice with large tumors.

[0251] (6) Tumor growth and survival

[0252] Figure 33 shows D2F2 WT tumor cell growth in BALB / c mice for each mouse group shown in the graph. Tumor volume was measured with calipers and calculated using the formula: (Length x Width 2) / 2. The overall curve of tumor growth was analyzed using a full two-way ANOVA, which p A significant difference of <0.01 was observed. All peptide-immunized groups of mice were found to have a lower tumor burden than the PBS and mAb-treated groups. One-way ANOVA was used at days 21 and 28 to test all five groups for p-values ​​less than 0.01. Additionally, all peptide-treated groups MVF-PD-1 (92-110) and MVF-PD-L1 (130-147) had smaller tumor volumes than the PBS-negative and mAb-positive controls.

[0253] The log-rank (Mantel-Cox) test was also used to analyze survival rates when mice were treated with the designed groups (Fig. 34). p <0.001 indicates that treatment significantly improved mouse survival. In particular, the MVF-PD-L1(130-147) peptide immunization group showed the best mouse survival rate among all treatment groups, with approximately 60% survival after 45 days of tumor challenge.

[0254] Figure 35 shows D2F2 / E2 tumor cell growth in BALB / c mice for the mouse group as shown in the graph. Tumor volume was measured with calipers and calculated using the formula: (Length x Width 2) / 2. The overall curve of tumor growth was analyzed using a full two-way ANOVA, which pIt showed a significant difference of <0.01. Mice immunized with all peptides and treated with mAbs were found to have a lower tumor burden than the PBS group. Most importantly, MVF-HER-2(266-296) + MVF-HER-2(597-626); MVF-PD-1(92-110) + MVF-HER-2(266-296) + MVF-HER-2(597-626); and MVF-PD-L1(130-147) + MVF-HER-2(266-296) + MVF-HER-2(597-626), all mice immunized with 2XHER2 showed significant tumor suppression, and most mice were tumor-free by the end of day 28 after the tumor challenge.

[0255] On the 21st and 28th, one-way ANOVA was performed on all 6 groups p All groups with values ​​less than 0.01 were tested. All peptide-treated groups—MVF-HER-2 (266-296) + MVF-HER-2 (597-626); MVF-PD-1 (92-110) + MVF-HER-2 (266-296) + MVF-HER-2 (597-626); and MVF-PD-L1 (130-147) + MVF-HER-2 (266-296) + MVF-HER-2 (597-626)—had significantly smaller tumor volumes than both the PBS-negative and mAbs-positive controls. All mice in the peptide-immunized groups had small tumors or were mostly tumor-free on days 21 and 28, respectively.

[0256] In addition, the log-rank (Mantel-Cox) test was used to analyze survival rates when mice were treated with the designed groups (Fig. 36). p<0.001 indicates that treatment significantly improved mouse survival. In particular, the MVF-HER-2(266-296) + MVF-HER-2(597-626) and MVF-PD-L1(130-147) + MVF-HER-2(266-296) + MVF-HER-2(597-626) peptide immunization groups still showed a 100% mouse survival rate 45 days after the tumor challenge, which is the highest among all treatment groups. Mice treated with MVF-PD-1(92-110) + MVF-HER-2(266-296) + MVF-HER-2(597-626) and mAb(PD-1)(29F.1A12) showed a survival rate of approximately 60%.

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[0336] F. Sequence

[0337] Sequence No. 1 Human PD-L1 residue 1-273

[0338] AFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

[0339] Sequence No.: 2 PD-L1(36-53)

[0340] LIVYWEMEDKNIIQFVHG

[0341] Sequence No.: 3 PD-L1(50-67)

[0342] FVHGEEDLKVQHSSYRQR

[0343] Sequence No.: 4 PD-L1(95-112)

[0344] YRCMISYGGADYKRITVK

[0345] Sequence No.: 5 PD-L1(130-147)

[0346] VTSEHELTCQAEGYPKAE

[0347] Sequence No. 6 Measles virus fusion protein (MVF)

[0348] KLLSLIKGVIVHRLEGVE

[0349] Sequence No.: 7 linker

[0350] GPSL

[0351] Sequence No.: 8 MVF- PD-L1(36-53)

[0352] KLLSLIKGVIVHRLEGVEGPSLLIVYWEMEDKNIIQFVHG

[0353] Sequence No.: 9 MVF- PD-L1(50-67)

[0354] KLLSLIKGVIVHRLEGVEGPSLFVHGEEDLKVQHSSYRQR

[0355] Sequence No.: 10 MVF- PD-L1(95-112)

[0356] KLLSLIKGVIVHRLEGVEGPSLYRCMISYGGADYKRITVK

[0357] Sequence No.: 11 MVF- PD-L1(130-147)

[0358] KLLSLIKGVIVHRLEGVEGPSLVTSEHELTCQAEGYPKAE

[0359] Sequence No.: 12 PD-L1(36-53) D peptide retro-inverto

[0360] GHVFQIINKDEMEWYVIL

[0361] Sequence No.: 13 PD-L1 (50-67) D peptide retro-inverto

[0362] RQRYSSHQVKLDEEGHVF

[0363] Sequence No.: 14 PD-L1 (95-112) D peptide retro-inverto

[0364] KVTIRKYDAGGYSIMCRY

[0365] Sequence No.: 15 PD-L1 (130-147) D-peptide retro-inverto

[0366] EAKPYGEAQCTLEHESTV

[0367] Sequence No.: 16 MVF PD-L1 (36-53) D peptide retro-inverto

[0368] KLLSLIKGVIVHRLEGVEGPSLGHVFQIINKDEMEWYVIL

[0369] Sequence No.: 17 MVF PD-L1 (50-67) D peptide retro-inverto

[0370] KLLSLIKGVIVHRLEGVEGPSLRQRYSSHQVKLDEEGHVF

[0371] Sequence No.: 18 MVF PD-L1 (95-112) D peptide retro-inverto

[0372] KLLSLIKGVIVHRLEGVEGPSLKVTIRKYDAGGYSIMCRY

[0373] Sequence No.: 19 MVF PD-L1 (130-147) D peptide retro-inverto

[0374] KLLSLIKGVIVHRLEGVEGPSLEAKPYGEAQCTLEHESTV

[0375] Sequence No.: 20 TT

[0376] NSVDDALINSTIYSYFPSV

[0377] Sequence No.: 21 TT1

[0378] PGINGKAIHLVNNQSSE

[0379] Sequence No.: 22 P2

[0380] QYIKANSKFIGITEL

[0381] Sequence No.: 23 P30

[0382] FNNFTVSFWLRVPKVSASHLE

[0383] Sequence No.: 24 MVF (Natural)

[0384] LSEIKGVIVHRLEGV

[0385] Sequence No.: 25 HBV

[0386] FFLLTRILTIPQSLN

[0387] Sequence No.: 26 CSP

[0388] TCGVGVRVRSRVNAANKKPE

[0389] Sequence No.: 27 HER-2(266-296)

[0390] LHCPALVTYNTDTFESMPNPEGRYTFGASCV

[0391] Sequence No.: 28 MVF HER-2(266-296)

[0392] KLLSLIKGVIVHRLEGVEGPSLLHCPALVTYNTDTFESMPNPEGRYTFGASCV

[0393] Sequence No.: 29 HER-2(597-626)

[0394] VARCPSGVKPDLSYMPIWKFPDEEGACQPL

[0395] Sequence No.: 30 MVF HER-2(597-626)

[0396] KLLSLIKGVIVHRLEGVEGPSLVARCPSGVKPDLSYMPIWKFPDEEGACQPL

Claims

Claim 1 A programmed death ligand-1 (PD-L1) chimeric peptide for stimulating an immune response to a PD-L1 protein comprising one or more PD-L1 B cell epitopes, a T helper (Th) epitope, and a linker connecting the PD-L1 B cell epitope to the Th epitope, wherein the one or more PD-L1 B cell epitopes are sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

5. Claim 2 A chimeric peptide according to claim 1, wherein the Th epitope comprises a measles virus fusion protein peptide. Claim 3 In claim 2, the programmed cell death ligand-1 (PD-L1) chimeric peptide wherein the Th epitope comprises SEQ ID NO:

6. Claim 4 In claim 1, the linker comprises SEQ ID NO: 7, a programmed cell death ligand-1 (PD-L1) chimeric peptide. Claim 5 A programmed cell death ligand-1 (PD-L1) chimeric peptide according to claim 1, wherein the peptide comprises the amino acid sequence presented in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11. Claim 6 A synthetic PD-L1 peptide for stimulating an immune response to a PD-L1 protein, comprising one or more of the sequences presented in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO:

15. Claim 7 In claim 6, a synthetic peptide in which the amino acid comprising the synthetic PD-L1 peptide is a D enantiomer. Claim 8 In claim 6, the synthetic peptide in which the above peptide is acetylated. Claim 9 A chimeric peptide comprising one or more synthetic peptides of claim 6, further comprising a Th epitope and a linker connecting a synthetic PD-L1 peptide to the Th epitope. Claim 10 In claim 9, the chimeric peptide wherein the Th epitope comprises a measles virus fusion protein peptide. Claim 11 In claim 10, the chimeric peptide wherein the Th epitope comprises SEQ ID NO:

6. Claim 12 In claim 9, the chimeric peptide wherein the linker comprises SEQ ID NO:

7. Claim 13 In claim 9, the chimeric peptide comprising the amino acid sequence presented in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO:

19. Claim 14 A pharmaceutical composition for treating cancer, Alzheimer's disease, or autoimmune disease, comprising one or more chimeric peptides or synthetic peptides of any one of claims 1 to 13 and a pharmaceutically acceptable vehicle. Claim 15 A pharmaceutical composition according to claim 14, further comprising one or more HER-2 B cell epitopes. Claim 16 A pharmaceutical composition according to claim 15, wherein the HER-2 B cell epitope comprises one or more of the sequences presented in SEQ ID NO: 27 or 29. Claim 17 A pharmaceutical composition according to claim 16, wherein the HER-2 B cell epitope is a component of one or more chimeric HER-2 peptides presented in SEQ ID NO: 28 or 30. Claim 18 A pharmaceutical composition according to claim 14, wherein the vehicle is a pharmaceutically acceptable adjuvant. Claim 19 A pharmaceutical composition according to claim 18, wherein the said adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, calcium hydroxide phosphate, Freund complete adjuvant, MONTANIDE®, Freund incomplete adjuvant, ISCOM, ISCOM Matrix, ISCOMATRIX™ adjuvant, Matrix M™ adjuvant, Matrix C™ adjuvant, Matrix Q™ adjuvant, AbISCO®-100 adjuvant, AbISCO®-300 adjuvant, ISCOPREP™, ISCOPREP™ derivative, adjuvant containing ISCOPREP™ or ISCOPREP™ derivative, QS-21, QS-21 derivative, and adjuvant containing QS-21 or QS-21 derivative. Claim 20 A pharmaceutical composition according to claim 19, wherein the adjuvant is MONTANIDE®. Claim 21 An antibody that specifically binds to any one of the chimera or synthetic peptide of any one of claims 1 to 13. Claim 22 A pharmaceutical composition according to claim 14, wherein the cancer is selected from the group of cancers consisting of lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the oral cavity, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, endometrial cancer, epithelial carcinoma, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck carcinoma, colorectal cancer, hematopoietic cancer, testicular cancer, prostate cancer, or pancreatic cancer. Claim 23 A pharmaceutical composition according to claim 22, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, colon cancer, non-small cell lung cancer, prostate cancer, or cervical cancer. Claim 24 A pharmaceutical composition according to claim 14, wherein the autoimmune disease is selected from the group consisting of psoriasis, alopecia areata, primary biliary cirrhosis, autoimmune polyendocrine syndrome, type 1 diabetes mellitus, autoimmune thyroiditis, systemic lupus erythematosus, multiple sclerosis, Guillain-Barré syndrome, Graves disease, Sjögren's syndrome, ulcerative colitis, autoimmune hemolytic anemia, pernicious anemia, psoriatic arthritis, rheumatoid arthritis, recurrent polychondritis, myasthenia gravis, acute disseminated encephalomyelitis, and polyangiomatosis with granulomatosis. Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete

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