Modified Mycobacterium bovis vaccine

Abstract

The present invention relates to modified bacteria, pharmaceutical compositions containing same, and methods of using same to prevent or treat disease, particularly, but not limited to, cancer or infection.

Description

[Technical Field]

[0001] The present invention relates to modified bacteria, pharmaceutical compositions containing the same, and methods for preventing or treating diseases, particularly cancer (hereinafter referred to as "cancer") or infections (but not limited to these), using the same. [Background technology]

[0002] The use of modified bacteria as agents for the prevention or treatment of disease is well known, particularly in relation to vaccine therapy, in which case typically, attenuated forms of bacteria are administered to stimulate the immune system, thereby inducing protection against subsequent infections by wild-type bacteria. Such vaccines have been developed, though not limited to, diseases caused by infections with the following bacteria: Haemophilus influenzae, Streptococcus pneumoniae, Bordetella pertussis, Vibrio cholerae, Clostridium tetani, Mycobacterium tuberculosis, Salmonella typhi, and Bacillus anthracis.

[0003] Bacterial vaccines can be classified into various types, namely toxoids, subunit vaccines, dead whole-cell vaccines, and live attenuated vaccines (live attenuated vaccines). Live bacterial vaccines have the advantages of being able to express multiple antigens, being able to be mass-produced, and being able to induce a strong immune response.

[0004] Invasive bacteria such as Salmonella, Listeria, Yersinia, Shigella, and Mycobacterium bovis [Bacillus Calmette-Guerin or BCG] are used as vaccines or vaccine vectors that can induce potent humoral and cellular immune responses. Because these are pathogenic bacteria, they are attenuated to obtain suitable non-pathogenic vaccine strains. Numerous attenuated strains that are non-pathogenic and have limited in vivo growth potential have been reported.

[0005] However, uncertainties remain regarding the effectiveness and mechanism of action of the BCG vaccine. While the protective effect of BCG against disseminated tuberculosis in young children is reasonably well established, the effectiveness of BCG vaccination in adults has varied in clinical trials, and there is no compelling evidence of its protective effect in HIV-infected individuals, a group at high risk of progression of tuberculosis disease after infection.

[0006] BCG is produced from strains of Mycobacterium bovis, which are live, attenuated (less pathogenic) bovine tuberculosis bacteria that have lost the ability to cause disease in humans. These live bacilli become less adaptable to human blood because they make maximum use of available nutrients, and typically can no longer induce disease when introduced into a human host. However, they are similar enough to the wild-type ancestor to provide some degree of immunity to human tuberculosis. BCG vaccine may be 0-80% effective in preventing tuberculosis for 15 years. However, its protective effect appears to vary depending on the laboratory where the vaccine strain is cultured and geographical factors.

[0007] Many different companies manufacture BCG vaccines, sometimes using different genetic substrains of the bacterium. OncoTice is manufactured using the TICE substrain, which was developed by Organon Laboratories (Merck & Co.), Pacis BCG (Dianon Systems), Evans Vaccines (PowderJect Pharmaceuticals), BCG (Statens Serum Institut in Denmark), and BCG (Japan BCG Laboratory).

[0008] There are currently moves to develop viral anti-cancer vaccines, and much of the research to date has involved the use of modified oncolytic viruses.

[0009] At the beginning of this century, oncolytic viruses were recognized as active agents in cancer treatment that act solely through their inherent ability to lyse tumor cells via oncolysis. More recently, they have been investigated because they can release tumor antigens from cancer cells (during oncolysis) and activate the immune system.

[0010] In this context, BCG is known to be an intracellular pathogen that can modulate the tumor microenvironment (TME) through multiple mechanisms, including inducing the massive secretion of chemokines and cytokines that recruit T cells and other immune cells to the TME, as well as through polarization of M2 macrophages towards an M1-like phenotype. It has recently been shown that BCG therapy results in enhanced activation and reduced exhaustion of tumor-specific T cells, leading to enhanced effector function, and that BCG-induced regression of bladder cancer requires tumor-specific CD4+ and CD8+ T cells rather than T cells specific for BCG antigens.

[0011] The present invention relates to novel substances for the prevention and / or treatment of diseases. The present invention can be used for the prevention and / or treatment of infectious diseases (infections), for example any of the foregoing, in particular respiratory infections such as tuberculosis (TB), influenza, SARS, MERS, other coronavirus diseases (e.g. COVID-19) or the common cold. Furthermore, the present invention can be used for the prevention and / or treatment of non-infectious diseases such as cancer or autoimmune diseases. SUMMARY OF THE INVENTION

[0012] Statement of the Invention In a first aspect of the present invention, there is provided live attenuated Mycobacterium bovis (BCG) for use in humans for preventing or treating a disease, wherein said BCG is coated with a plurality of peptide antigens capable of inducing an active immune response against said disease in said human, and said peptides are bound to the bacterium using a polylysine or polyarginine peptide linker.

[0013] The inventors have named this modified BCG according to the present invention PeptiBAC [peptide-coated bacillus Calmette-Guerin].

[0014] In a preferred embodiment of the present invention, said peptide antigen is an antigen associated with said disease and can therefore be used to induce an immune response, whereby a human is protected from said disease, or at least said disease has a lower severity than when this is not used.

[0015] More specifically, when treating infections, particularly respiratory infections, such as coronavirus infections (e.g., SARS-CoV-2), the PeptiBAC platform uses infection-associated antigens (viral antigens), preferably those endogenously expressed by the pathogen, such as those derived from SARS-CoV-2. To coat the modified BCG, various viral MHC class I and / or II epitopes derived from, for example, VME1, AP3A, R1AB, R1A, NS7B, NCAP, and spike proteins may be used. Ideally, PeptiBAC is administered intradermally or intranasally when treating respiratory infections.

[0016] In a preferred embodiment of the present invention, the disease is an infectious disease (infection), and the peptide antigen comprises at least one of the following peptides, ideally, the peptide is covalently or noncovalently bound to the bacterial envelope without being genetically encoded by the BCG bacterial vector.

[0017] i) IAMACLVGLMWLSYFIASFRLFAR (derived from VME1) [Sequence ID 1]; ii) KLIFLWLLWPVTLACFVLAAV (derived from VME1) [SEQ ID NO: 2]; iii) LPKEITVATSRTLSYYKLGA (derived from VME1) [SEQ ID NO: 3]; iv) GLEAPFLYLYALVYFLQSINFV (derived from AP3A) [SEQ ID NO: 4]; v)QMAPISAMVRMYIFFASFYYVWK(derived from R1AB)[SEQ ID NO: 5]; vi)KVTLVFLFVAAIFYLITPVHVMSK(derived from R1AB)[SEQ ID NO: 6]; vii)GLVAEWFLAYILFTRFFYVL(derived from R1AB)[SEQ ID NO: 7]; viii)KRAKVTSAMQTMLFTMLRKL(derived from R1A)[SEQ ID NO: 8]; ix)EIPVAYRKVLLRKNGNKGAG(derived from R1AB)[SEQ ID NO: 9]; x)ELSLIDFYLCFLAFLLFLVLIMLII(derived from NS7B)[Sequence ID 10]; xi)AQFAPSASAFFGMSRIGMEV(derived from NCAP)[SEQ ID NO: 11]; xii)ALALLLLDRLNQLESKMSGK(derived from NCAP)[SEQ ID NO: 12]; xiii)VILLNKHIDAYKTFPPTEPK (derived from NCAP) [SEQ ID NO: 13]; and xiv) A polypeptide that is at least 60% identical to one of the peptides in items i-xiii.

[0018] More preferably, the polypeptide of item xiv) has at least 61, 62, 63, 64, 65, 66, 67, 68, 69, 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% identity with respect to one of the peptides of items i) to xiii).

[0019] More specifically, the PeptiBAC "platform" uses tumor-associated antigens (TAAs), tumor-specific antigens (TSPs), or neogeneic antigens when treating cancer, particularly melanoma or colorectal cancer.

[0020] In preferred embodiments of the present invention, the cancer antigen is derived from tyrosinase-related protein-2 (Trp2) and / or glycoprotein 100 (gp100) endogenously expressed in a specific melanoma (e.g., model B16.F10 melanoma). Alternatively, the cancer antigen is derived from modified tumor rejection antigen AH1, which is derived from the gp70 envelope protein of mouse leukemia virus (MuLV). Ideally, these forms of PeptiBAC are administered intratumorally.

[0021] In a preferred embodiment of the present invention, the disease is cancer, and the peptide antigen comprises at least one of the following peptides, ideally, the peptide is covalently or noncovalently bound to the bacterial envelope without being genetically encoded by the BCG bacterial vector.

[0022] i) SIINFEKL[SEQ ID NO: 14]; ii) SVYDFFVWL (derived from tyrosine-related protein 2) [SEQ ID NO: 15]; iii) KVPRNQDWL (derived from gp100) [SEQ ID NO: 16]; iv) SPSYVYHQF (modified sequence derived from tumor rejection antigen AH1) [SEQ ID NO: 56]; v) A polypeptide that is at least 60% identical to the peptide of item i, ii, iii, or iv.

[0023] More preferably, the polypeptide of item v) has at least 61, 62, 63, 64, 65, 66, 67, 68, 69, 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% identity with respect to one of the peptides of item i), ii), iii) or iv).

[0024] BCG bacteria coated with tumor-specific peptides broaden the immune response to encompass the treatment of tumors associated with peptide antigens coated with modified BCG.

[0025] In yet another preferred embodiment of the present invention, the peptide is a Pan MHC-II molecule, for example, PADRE-AKFVAAWTLKAAA [SEQ ID NO: 17], but the peptide itself is not infection / tumor-related, and PADRE is a universal T helper epitope that can enhance immune responses induced by more specific epitopes, such as infection or tumor-specific epitopes. Therefore, it is preferable to use it in combination with another disease-specific peptide antigen when carrying out the present invention.

[0026] Advantageously, the peptide antigen can stimulate a peptide-specific immune response in a target, and even more advantageously, since the peptide is not genetically encoded by the bacterium and is bound to the bacterium covalently or noncovalently using a peptide linker, this binding can be achieved rapidly and efficiently. Typically, to facilitate the binding of the peptide antigen, the peptide antigen is a polylysine or polyarginine elongated using at least four, ideally five, six, seven, eight, or nine lysine or arginine molecules.

[0027] Most typically, six lysine molecules are used, most preferably bound to the amino terminus of the peptide.

[0028] Therefore, the peptide for binding to bacteria is selected from the group including or consisting of the following:

[0029] KKKKKK(KKK)-IAMACLVGLMWLSYFIASFRLFAR(derived from VME1) [Sequence ID 18 or 48]; KKKKKK(KKK)-KLIFLWLLWPVTLACFVLAAV(derived from VME1) [Sequence ID 19 or 46]; KKKKKK(KKK)-LPKEITVATSRTLSYYKLGA(derived from VME1) [Sequence ID 20 or 47]; KKKKKK(KKK)-GLEAPFLYLYALVYFLQSINFV (derived from AP3A) [SEQ ID NO: 21 or 60]; KKKKKK(KKK)-QMAPISAMVRMYIFFASFYYVWK(derived from R1AB)[SEQ ID NO: 22 or 61]; KKKKKK(KKK)-KVTLVFLFVAAIFYLITPVHVMSK(derived from R1AB) [Sequence ID 23 or 62]; KKKKKK(KKK)-GLVAEWFLAYILFTRFFYVL(derived from R1AB) [Sequence ID 24 or 63]; KKKKKK(KKK)-KRAKVTSAMQTMLFTMLRKL(derived from R1A) [Sequence ID 25 or 64]; KKKKKK(KKK)-EIPVAYRKVLLRKNGNKGAG(derived from R1AB) [Sequence ID 26 or 65]; KKKKKK(KKK)-ELSLIDFYLCFLAFLLFLVLIMLII(derived from NS7B)[Sequence ID 27 or 66]; KKKKKK(KKK)-AQFAPSASAFFGMSRIGMEV(derived from NCAP) [Sequence ID 28 or 67]; KKKKKK(KKK)-ALALLLLDRLNQLESKMSGK (derived from NCAP) [Sequence ID 29 or 59]; KKKKKK(KKK)-VILLNKHIDAYKTFPPTEPK (derived from NCAP) [SEQ ID NO: 30 or 40]; KKKKKK(KKK)-SIINFEKL[SEQ ID NO: 31 or 42]; KKKKKK(KKK)-SVYDFFVWL[Sequence ID 32 or 43]; KKKKKK(KKK)-KVPRNQDWL[Sequence ID 33 or 41]; KKKKKK(KKK)-AKFVAAWTLKAAA[Sequence ID 34 or 39]; KKKKKK(KKK)-SPSYVYHQF[Sequence ID 57 or 58]; [Here, KKKKKK(KKK)- is a 6 KKKKKK or 9 KKKKKK(KKK) amino acid linker, or RRRRRR(RRR)- is a 6 RRRRRR or 9 RRRRRR(RRR) amino acid linker]; and A polypeptide that is at least 60% identical to one of the aforementioned peptides.

[0030] The inventors had also considered carrying out the present invention by using a cell-permeable peptide (CPP) linker to conjugate peptide antigens to BCG, but to their surprise, they found that CPPs are toxic to BCG and therefore reduce its viability, resulting in the loss of the advantages sought when using live attenuated bacteria [for example, generally the expression of multiple antigens, mass production and induction of a potent immune response, and (in the case of cancer treatment) specifically, the induction of massive secretion of chemokines and cytokines that recruit T cells and other immune cells to the tumor microenvironment (TME), the regulation of the TME by multiple mechanisms including the bias of M2 macrophages towards a more M1-like phenotype, and the reduction of depletion and enhancement of activation of tumor-specific T cells, leading to enhanced effector function].

[0031] In another preferred embodiment of the present invention, the modified BCG is coated with multiple different peptide antigens, for example, two different peptide antigens, in which case the modified BCG (PeptiBAC) is bivalent. Alternatively, the modified BCG is coated with three different peptide antigens, in which case the PeptiBAC is trivalent. Alternatively, the modified BCG is coated with four or more different peptide antigens, in which case the PeptiBAC is polyvalent. Ideally, the properties of the different peptide antigens used to coat the bacteria are selected in consideration of the nature of the outcome to be achieved and / or the nature of the disease to be treated. Ideally, antigens expressed on the surface of cancer / disease cells are used as peptide antigens. Alternatively, antigens derived from infectious agents to be targeted are used as peptide antigens. Additionally or alternatively, antigens presented by MHC-I or MHC-II are used. In this way, it is possible to enhance the nature of the immune response to be induced and maximize the effect of PeptiBAC therapy.

[0032] For example, in the case of infection, if only one infectious peptide antigen (which is either a virus-associated antigen or even a T helper epitope effective in treating the infection) is used to coat the modified BCG, the PeptiBAC is referred to as monovalent [e.g., PeptiBAC that targets pan-MHC class I or II molecules (referred to herein as PeptiBAC-P)].

[0033] When two infection peptide antigens that give a total of two different antigens / epitopes (e.g., one or two different infection-associated antigens and / or one or two different T helper epitopes effective in treating infection) are used to coat a modified BCG, PeptiBAC is referred to as bivalent.

[0034] When three infection peptide antigens (e.g., one, two, or three different infection-associated antigens and / or one, two, or three different T helper epitopes) that give a total of three different antigens / epitopes are used to coat a modified BCG, PeptiBAC is referred to as trivalent.

[0035] Similarly, for example, in the case of cancer, if only one cancer peptide antigen (which is either a tumor-associated antigen or even a T helper epitope effective in the treatment of cancer) is used to coat the modified BCG, the PeptiBAC is referred to as monovalent [e.g., PeptiBAC that targets pan-MHC class I or II molecules (referred to herein as PeptiBAC-P)].

[0036] When two cancer peptide antigens that give a total of two different antigens / epitopes (e.g., one or two different tumor-associated / tumor-rejection antigens and / or one or two different T helper epitopes effective in the treatment of cancer) are used to coat a modified BCG, PeptiBAC is referred to as bivalent [e.g., PeptiBAC targeting Trp2 and gp100 (referred to herein as PeptiBAC-TG)].

[0037] When three cancer peptide antigens that provide a total of three different antigens / epitopes (e.g., one, two, or three different tumor-associated / tumor-rejection antigens and / or one, two, or three different T helper epitopes effective in the treatment of cancer) are used to coat modified BCG, PeptiBAC is referred to as trivalent PeptiBAC [e.g., those that target Trp2, gp100, and pan-MHC class I or II molecules (referred to herein as PeptiBAC-TGP)].

[0038] The present invention can be carried out using any strain of BCG, but in preferred embodiments of the present invention, BCG strain BCG-Russia and / or BCG-Denmark strain 1331 and / or BCG-Bulgaria are used. However, those skilled in the art will understand that the modifications between strains are minor and unlikely to affect the implementation of the present invention.

[0039] A distinctive feature of BCG, a bacterium belonging to the genus Mycobacterium, is its complex cell envelope, which contains an inner plasma membrane (IM), a peptidoglycan-arabinogalactan complex, and an outer membrane (OM) covalently bound to arabinogalactan. Peptides bind to this cell envelope.

[0040] In another preferred embodiment of the present invention, the disease is selected from the group including infections, respiratory diseases, influenza, tuberculosis (TB), the common cold, coronavirus infections including SARS and MERS, autoimmune diseases, and cancer.

[0041] In yet another preferred embodiment of the present invention, the bacterium is further modified to include any one or more of the following features (including all combinations thereof):

[0042] In a preferred embodiment, the modified bacterium comprises the insertion of at least one transgene encoding a co-stimulatory molecule, and ideally, the insertion of two transgenes, where one gene results in activation of the innate immune system and the other results in activation of the adaptive immune system. Preferred transgenes include CD40L for activating the innate immune system by the use of antigen-presenting cells (APCs) to induce a CD8+ T cell response, and OX40L for activating the adaptive immune system by enhancing clonal proliferation, CD8+ T cell survival, and the generation of a large pool of memory T cells.

[0043] Alternatively, the DNA encoding OX40L and CD40L may be linked using known genetic engineering techniques and inserted as a fusion molecule. Typically, CD40L is inserted directly downstream of OX40L, but the present invention can also be carried out in the reverse configuration.

[0044] The BCG used in this invention may include modifications other than those described above. Any additional components or modifications may be used as desired, but are not essential to this invention.

[0045] Therefore, the bacteria of the present invention are engineered to stimulate an immune response to diseases such as infectious diseases, thereby acting as a vaccine or therapeutic agent. Furthermore, as stated above, the bacteria of the present invention are engineered to stimulate an immune response to diseases such as cancer, and in particular, to stimulate an immune response to diseases such as cancer in a tumor environment where the immune system is typically impaired by evasion mechanisms used by cancer cells.

[0046] The advantage of this modified BCG platform technology is that it non-genetically introduces disease and immunity-inducing peptides into the BCG vaccine, which makes this approach highly adaptable to, and therefore appropriate for, a personalized immunotherapy approach based on the identification of patient-specific neoantigens.

[0047] Accordingly, the present invention also includes a pharmaceutical composition comprising at least one modified BCG of the present invention and a suitable carrier. In preferred embodiments of the present invention, the pharmaceutical composition is formulated for intradermal, intranasal, subcutaneous, transdermal, intratumoral, intramuscular, intraarterial, intravenous, intrapleural, intravesicular, intracavitary, intraperitoneal injection or oral administration.

[0048] Therefore, in yet another embodiment, the present invention relates to a method for treating a disease in an individual, comprising administering to the individual an effective amount of a modified BCG according to the present invention, or an effective amount of a pharmaceutical composition comprising at least one modified BCG according to the present invention.

[0049] When tumors develop several immunosuppressive mechanisms to counteract the body's immune cells, the treatment of the present invention is also ideally implemented in combination with the use of checkpoint molecules. The most well-characterized checkpoint pathways are cytotoxic T lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 pathway (PD-1 / PD-L1). Therefore, the modified BCG of the present invention can be used in combination with checkpoint modulators or immune checkpoint inhibitors, such as anti-PD1 molecules, anti-PD-L1 molecules, or anti-CTLA-4 molecules, to counteract the immunosuppressive tumor environment and to provoke a potent anti-immune response.

[0050] Modified BCG acts as an active adjuvant because it provides the necessary danger signal for an optimal immune response to the target peptide. Oncolytic cell killing is inherently immunogenic, which causes alterations in the tumor microenvironment that can enhance the immune response to the peptide / tumor. Therefore, using our modified BCG in co-administration with or physically complexed with an immunomodulatory peptide results in a superior antitumor immune response compared to the use of peptide vaccines or BCG vaccines alone.

[0051] In a preferred method of the present invention, the modified BCG is administered before and / or after the administration of a checkpoint modulator molecule or an immune checkpoint inhibitor molecule. Alternatively, the modified BCG is co-administered with a checkpoint modulator molecule or an immune checkpoint inhibitor molecule.

[0052] Therefore, in another aspect of the present invention, a combination therapy is provided comprising modified BCG according to the present invention and at least one checkpoint molecule, such as cytotoxic T lymphocyte protein 4 (CTLA-4) or programmed cell death protein 1 pathway (PD-1 / PD-L1).

[0053] Furthermore, or alternatively, the present invention relates to at least one modified BCG or pharmaceutical composition according to the present invention for use in the treatment of diseases described herein.

[0054] Additionally or alternatively, the present invention relates to the use of at least one modified BCG according to the present invention in the manufacture of a medicament for treating the diseases described herein.

[0055] Most preferably, the cancers referred to herein include one or more of the following: nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary origin, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's cancer. Temporomandibular tumor, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, cervical cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine and pancreatic cancer, glucagonoma, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0056] Most preferably, the infectious diseases (infections) referred to herein include any one or more of the following infectious diseases (infections): respiratory diseases, influenza, tuberculosis (TB), the common cold, or coronavirus infections, including SARS and MERS.

[0057] In the claims described below and the above description of the present invention, unless the context requires a different interpretation due to explicit wording or implicit meaning, the word “includes” or variations such as “includes” or “contains” is used inclusively, that is, it identifies the presence of the features shown, but does not exclude the presence or addition of additional features in the various embodiments of the present invention.

[0058] All references, including any patents or patent applications cited herein, are incorporated herein by reference. None of the references are considered to constitute prior art. Furthermore, none of the prior art is considered to constitute any part of the well-known general knowledge in the art.

[0059] Preferred features of each aspect of the present invention may be as described in relation to any of the other aspects.

[0060] Other features of the present invention will become apparent from the examples described below. Generally speaking, the present invention also includes any novel features or any novel combination of features disclosed herein (including the appended claims and drawings). Accordingly, any features, integers, properties, compounds or chemical parts described in relation to a particular aspect, embodiment or example of the present invention should be understood to be applicable to any other aspect, embodiment or example described herein, insofar as they are not incompatible therewith.

[0061] Furthermore, unless otherwise indicated, any feature disclosed herein may be replaced by an alternative feature that serves the same or similar purpose.

[0062] Throughout this specification and the claims, the singular form includes the plural form unless the context requires otherwise. In particular, where the singular form is used, this specification should be understood to assume not only unity but also plurality, unless the context requires otherwise.

[0063] Next, embodiments of the present invention will be described below as mere examples with reference to the following. [Brief explanation of the drawing]

[0064] [Figure 1]Figure 1 shows schematic diagrams of A) an immunomodulatory peptide containing an N-terminal cell-permeable peptide, B) an immunomodulatory peptide containing an N-terminal polylysine, and C) an immunomodulatory peptide containing polyarginine. The various functional sequences of these substances are color-coded as follows: Blue: cell-permeable peptide sequence (A) or polylysine sequence (B). Green: immunoproteasome processing site. Orange: immunomodulatory peptide or MHC-I-restricted epitope. [Figure 2] Figure 2 shows surface plasmon resonance (SPR) measurements to confirm the high affinity of a cell-permeable peptide (CPP)-containing immunomodulatory peptide [A] and a polylysine (6K) linker-containing immunomodulatory peptide [B] to the surface of BCG bacteria. Immunomodulatory peptides that do not contain either CPP or 6K do not interact with the bacterial surface. Figure 2A shows surface plasmon resonance (SPR) analysis of peptide / BCG interactions. A) Surface plasmon resonance analysis of the interaction between CPP-OVA and BCG. B) Surface plasmon resonance analysis of the interaction between poly-K-Trp2 and BCG. C) Surface plasmon resonance analysis of the interaction between poly-K-AH1 and BCG. Figure 2B shows surface plasmon resonance (SPR) analysis of various binding sites used for coating BCG. Various CPP sequences and cholesterol sites were tested by surface plasmon resonance (SPR) for their efficacy in immobilizing therapeutic peptides on Mycobacterium cell walls. The binding efficacy of the Cady sequence GLWRALWRLLRSLWRLLWRA (SEQ ID NO: 35), the penetratin sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 36), the KLAL sequence KLALKLALKALKAALKLA (SEQ ID NO: 37), the N-terminal cholesterol moiety, and the CPP Tat sequence GRKKRRQRRRPQ (SEQ ID NO: 38) was compared. [Figure 3] Figure 3 shows that the modified BCG of the present invention (hereinafter referred to as PeptiBAC) can efficiently deliver immunomodulatory CPP-containing peptides into dendritic cells (DCs), enabling DCs to present these peptides in the major histocompatibility complex [A]. PeptiBAC can induce DC maturation and activation as measured by the expression of CD40 [B] and CD86 [C] surface markers. [Figure 4] Figure 4 shows the treatment schedule and groups in the upper panel, and the lower panel shows that PeptiBAC therapy reduces tumor growth suppression in a mouse model of melanoma. Panels A-D show the individual growth curves for each treated mouse. Responders in each group are shown in green / light gray. The percentage of responders is shown to the right of the dotted line in each panel. Panel E shows the Kaplan-Meier survival curves for each group. The use of peptiBAC nearly doubles the survival rate. [Figure 5] Figure 5 shows the systemic peptide-specific T cell response induced by the PeptiBAC (PB; BCG complexed with CPP-containing SIINFEKL peptide) platform, as measured by [A] ELISpot assay from the spleen of treated animals and [B] increased influx of CD8+ T cells into the tumor microenvironment, comparing the PB group to the peptide alone (PO) group or the BCG group. [Figure 6] Figure 6 shows [A] the schedule and treatment groups in the animal model of hyperimmunosuppressive mouse melanoma B16.F10, and [B] the individual tumor growth curves of treated animals along with the response rates shown below the blue lines in each group. [Figure 7]Figure 7 shows bacterial plaque formation after formulation of BCG with various amounts of polylysine linker-containing immunomodulatory peptides. BCG was complexed with 10 nm and 40 nm immunomodulatory peptides containing polylysine linkers as bacterial membrane binding sites. Polylysine linker-binding peptides did not affect the viability of BCG bacteria. Figure 7A shows that coating BCG with CPP-containing peptide antigen instead of polylysine-containing peptide antigen reduces BCG viability. BCG was coated with CPP-containing peptide antigen (CPP-OVA) or polylysine-containing antigen (PolyK-OVA), and the complexes were directly plated for colony formation. RAW-Blue cells (100,000 cells / well) were stimulated with BCG or PeptiBAC-OVA (using PolyK-OVA peptide), and NF-κB / AP-1 activation was measured 24 hours after infection. Figure 7B shows that coating Escherichia coli (Gram-negative bacterium) with CPP-containing peptide did not affect bacterial viability. Escherichia coli was coated with 14 nmol of CPP-containing peptide antigen (CPP-Trp2), and the complex was directly plated for colony formation. Figure 7C shows that coating Listeria monocytogenes (Gram-positive bacteria) with CPP-binding peptide does not affect the viability of the microorganisms. Listeria monocytogenes (L. monocytogenes) was coated with 14 nmol of CPP-containing peptide antigen (CPP-Trp2), and the complex was directly plated for colony formation. [Figure 8]Figure 8 shows that macrophages can cross-present antigens delivered by the PeptiBAC platform and may be biased toward an M1-like phenotype. A) Mouse bone marrow-derived macrophages were pulsed with PeptiBAC-OVA, BCG, or polylysine-containing SIINFEKL peptide individually, or left unpulsed (simulation). Cross-presentation was measured by flow cytometry using APC-binding anti-H-2Kb bound to SIINFEKL. B) M2 macrophages were treated with BCG, PeptiBAC-OVA, or LPS (10 μg / ml) for 24 hours, or left untreated (simulation). Expression of MHC-II, CD86, and CD206 was measured by flow cytometry. Each bar represents the mean ± SEM of three technically overlapping experiments. Statistical analysis was performed using one-way ANOVA. **** p<0.0001; *** p<0.001. [Figure 9] Figure 9 shows the mean tumor growth curves for each treatment group in the CT26 colon cancer experiment. PeptiBAC-AH1 (BCG coated with poly-K-containing AH1 epitope peptide), ICI (anti-PD-1 immune checkpoint inhibitor). Statistical analysis was performed using two-way ANOVA. * p<0.05; ** p<0.01. [Figure 10] Figure 10 shows that treatment with PeptiBAC-Trp2 (BCG coated with poly-K-containing Trp2 epitope peptide) increases the response rate to checkpoint inhibitor therapy. Panel [A] shows the individual growth curves of each treated mouse. The percentage of responders (%) is shown to the right of the dotted line in each panel. Panel [B] shows the initial size of the treated tumor in each group. Panel [C] shows the mean tumor growth in each group. Figure 10A shows that PeptiBAC-Trp2 (BCG coated with poly-K-containing Trp2 epitope peptide) combined with anti-PD1 induces strong tumor-specific CD8+ T cell infiltration into tumors in a syngeneic mouse model of B16.F10.9 / K1 melanoma. A) Immunological analysis of tumors in treated mice. B) Immunological analysis of spleens in treated mice. The number of mice in each group was 9-11. Statistical analysis was performed using one-way ANOVA. * p<0.05. [Figure 11] Figure 11 shows that PeptiBAC-AH1 (BCG coated with a poly-K-containing AH1 epitope peptide) combined with anti-PD1 improves tumor growth suppression and induces a systemic tumor-specific CD8+ T cell response and robust tumor-specific CD8+ T cell infiltration into the tumor in a syngeneic mouse model of CT26 colorectal cancer, compared to any single monotherapy. A) Anti-PD-1 immune checkpoint inhibitor alone (100 μg / dose intraperitoneally three times weekly from day 6), BCG alone or in combination with anti-PD-1 immune checkpoint inhibitor, and PeptiBAC-AH1 alone or in combination with anti-PD-1 immune checkpoint inhibitor were administered intratumorally 11, 13, and 25 days after tumor transplantation. Individual tumor growth curves for all treatment groups are shown. A threshold of 450 mm3 was set to determine the proportion of mice responding to these various therapies (dotted line). The percentage of responders in each treatment group is shown to the right of the dotted line. B) Immunological analysis of tumors and spleens of treated mice. The number of mice in each group was 8-10. Statistical analysis was performed using one-way ANOVA. * p<0.05, ** p<0.01. [Figure 12]Figure 12 shows that xenogeneic primary-boost vaccination with the PeptiCRAd platform improves the peptide-specific T cell response induced by the PeptiBAC platform. A) Naive C57BL / 6JOlaHsd immunoqualified mice were subcutaneously inoculated with 1 × 10⁹ VP / dose of PeptiCRAd-Trp2 or 2–8 × 10⁶ CFU / dose of PeptiBAC-Trp2 (BCG coated with poly-K-containing Trp2 epitope peptide) or saline as a simulated treatment group. Primary and booster vaccinations were administered 14 days apart, and mice were euthanized 4 days after booster vaccination, with spleens collected for enzyme-linked immunospot (ELISPOT) assays. There were 4 mice in each vaccination group and 2 mice in the unvaccinated control group. B) In the same manner as in A, mice were vaccinated with PeptiBAC-OVA (BCG coated with poly-K-containing SIINFEKL epitope peptide) or PeptiBAC-OVA, followed by a booster dose with PeptiCRAd-OVA. The number of mice in each vaccination group was 5. [Figure 13] Figure 13 shows surface plasmon resonance (SPR) analysis performed to evaluate peptide binding properties to viral capsids. Electrostatic interactions between SARS-CoV-2 derived peptides (20–24 amino acid lengths) and human adenovirus capsids are shown. These data indicate that all nine peptides selected for the study can electrostatically bind to adenovirus capsids. MAGE-A3 peptide (20aa) was used as a positive control. [Figure 14]Figure 14 shows the interferon-gamma ELISPOT assay to demonstrate a robust T cell response to SARS-CoV-2 derived peptides in peripheral blood mononuclear cells (PBMCs) isolated from COVID-19 convalescent patients. PBMCs were isolated from nine patients treated in the intensive care unit (ICU) for severe COVID-19. PBMCs were collected six months after internalization (prolonged hospitalization). These data demonstrate that all nine selected peptides are clinically relevant because each of them can induce a cytotoxic T cell response in SARS-CoV-2 infected patients. As a control, PBMCs from healthy donors were used to evaluate the T cell response to the same SARS-CoV-2 derived peptides.

[0065] Rationale: Subjects who are hospitalized may be more susceptible. By emphasizing the importance of these individuals' responses, it may be possible to justify the selection of this cohort and this point in time.

[0066] Detailed explanation material and method: Conceptual proof The infectious peptide antigens described herein are biologically adapted to human infections and therefore could not be tested in animal models. Thus, the use of BCG to deliver immunologically effective surface-bound peptide antigens was demonstrated using BCG coated with cancer peptide antigens. This modified BCG was investigated for immunoactivity using both existing mouse cell lines and mouse strains.

[0067] peptide: The peptides used in this study are listed below. These were purchased from PepScan and Ontores.

[0068] CPP peptide: GRKKRRQRRRPQRWEKISIINFEKL[SEQ ID NO: 49] GRKKRRQRRRPQRWEKISVYDFFVWL[Sequence ID 50] GRKKRRQRRRPQRWEKIKVPRNQDWL[Sequence ID 51] GRKKRRQRRRPQRRAKFVAAWTLKAAA[Sequence ID 52] GRKKRRQRRRPQRRAKFVAAWTLKAAAKVPRNQD[Sequence ID 53] GRKKRRQRRRPQRRAKFVAAWTLKAAASVYDFFVWL[Sequence ID 54] GLWRALWRLLRSLWRLLWRA, permeable sequence (SEQ ID NO: 35) RQIKIWFQNRRMKWKK,KLAL sequence (sequence number 36) KLALKLALKALKAALKLA, N-terminal cholesterol portion (SEQ ID NO: 37) and GRKKRRQRRRPQ CPP,Tat sequence (sequence number 38) Polylysine peptide: KKKKKKSIINFEKL (Sequence ID 42) KKKKKKSVYDFFVWL (Sequence No. 43) KKKKKK SPSYVYHQF[Sequence ID 58] Other peptides: RWEKISIINFEKL[SEQ ID NO: 55] SIINFEKL[SEQ ID NO: 14] SPSYVYHQF[Sequence ID 56] SVYDFFVWL[Sequence ID 15] cell line Mouse melanoma cell lines B16.OVA, B16.F10, and B16.F10.K1 were cultured at 37°C / 5% CO2 in DMEM containing 10% fetal bovine serum (FBS) (Life Technologies), 1% L-glutamine, and 1% penicillin / streptomycin. Human triple-negative breast cancer cell line MDMBA436 was cultured at 37°C / 5% CO2 in RPMI containing 10% fetal bovine serum (FBS) (Life Technologies), 1% L-glutamine, and 1% penicillin / streptomycin. Mouse DC strain Jaws II was cultured at 37°C / 5% CO2 in Alpha Minimum Essential Medium containing 20% ​​FBS (Life Technologies), ribonucleoside, deoxyribonucleoside, 4 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate (Life Technologies), and 5 ng / mL mouse GM-CSF (PeproTech, USA).

[0069] The mouse colon cancer CT26.wt cell line was purchased from ATCC and cultured in high-glucose RPMI containing 10% fetal bovine serum (FBS) (Life Technologies), 1% L-glutamine, and 1% penicillin / streptomycin. The B16F10.9 / K1 cell line was kindly provided by Ludovic Martinet (Inserm, France) and cultured in high-glucose DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin. The B16.OVA cell line, a mouse melanoma cell line expressing chicken ovalbumin (OVA), was kindly provided by Professor Richard Vile (Mayo Clinic, Rochester, MN, USA). B16.OVA cells were cultured in DMEM containing 10% FBS (Life Technologies), 1% L-glutamine, 1% penicillin / streptomycin, and 5 mg / mL Geneticin. Mouse dendritic cell line JAWSII was purchased from ATCC and cultured in Alpha Minimum Essential Medium containing 20% ​​FBS (Life Technologies), ribonucleoside, deoxyribonucleoside, 4 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate (Life Technologies), and 5 ng / ml mouse GM-CSF (PeproTech, USA). Mouse macrophage reporter cell line RAW-Blue (InvivoGen) was cultured in DMEM supplemented with 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin, 100 μg / ml normosine (InvivoGen), and 100 μg / ml zeosin (InvivoGen) (as a selective antibiotic). Human lung cancer A549 cell lines were purchased from the NIH and cultured in OptiPRO® SFM supplemented with 10% FBS (Life Technologies), 1% L-glutamine, and 1% penicillin / streptomycin. All cells were cultured at 37°C / 5% CO2 and regularly tested for mycoplasma contamination using a commercially available detection kit (Lonza).

[0070] BCG vaccine preparations: BCG vaccine preparations were purchased from InterVax Ltd (Canada) (BCG vaccine for tuberculosis; BCG-Bulgarian strain), or from AJVaccines (Denmark) (BCG vaccine for tuberculosis; Danish strain 1331), or obtained from the Serum Institute of India (India) (BCG vaccine for tuberculosis, BCG-Russian strain, and ONCO-BCG preparation used for the treatment of bladder cancer). SII BCG (2-8 × 10⁶ colony-forming units [CFU] / vial) and SII-ONCO-BCG vaccine (1-19.2 × 10⁸ CFU / vial) were kindly provided by the Serum Institute of India (Pune, India). BCG vaccine (1.5-6.0 × 10⁶ CFU / vial) was purchased from InterVax (Toronto, Canada). On the other hand, the BCG vaccine AJV (2-8 x 10⁶ CFU / vial) from AJ Vaccines (Copenhagen, Denmark) was a donation from Professor Helen McShane (University of Oxford).

[0071] virus: In heterologous initial and additional experiments, adenovirus (VALO-mD901) expressing mouse OX40L and CD40L was used. The development of VALO-mD901 has already been described (Ylosmaki & Ylosmaki et al., 2021). Briefly, a portion of the E3B region of the pAd5 / 3-D24 backbone plasmid was replaced with the human cytomegalovirus (CMV) promoter region, mouse OX40L, a 2A self-cleaving peptide sequence, the mouse CD40L gene, and a rabbit betaglobin polyadenylation signal. The virus was amplified in A549 cells, purified under a double cesium chloride gradient, and stored in A195 adenovirus storage buffer 16 at -60°C. Viral particle (VP) concentrations were measured at 260 / 280 nm, and infection units (IU) were determined by immunocytochemistry (ICC) by staining hexone proteins in A549-infected cells.

[0072] peptide GRKKRRQRRRPQRWEKISIINFEKL (SEQ ID NO: 49), RWEKISIINFEKL (SEQ ID NO: 55), KKKKKK-SIINFEKL (SEQ ID NO: 42), and SIINFEKL (SEQ ID NO: 14) (containing MHC class I restricted epitopes OVA257-264 from chicken ovalbumin), KKKKKK-SVYDFFVWL (SEQ ID NO: 43), and SVYDFFVWL (SEQ ID NO: 15) (containing MHC class I restricted epitopes Trp2 180-188 from tyrosinase-related protein 2), and KKKKKK-SPSYAYHQF (SEQ ID NO: 44), and SPSYAYHQF (SEQ ID NO: 45) (containing modified MHC class I restricted epitopes [gp70 423-431] from mouse leukemia virus envelope glycoprotein 70, where the original AH1 epitope is modified with V5A to enhance immunogenicity). All peptides were purchased from Zhejiang Ontores Biotechnologies (Zhejiang, China).

[0073] PeptiBAC complex formation 0.75 × 10⁶ resuspended in PBS 5 ~12×10 7 CFU of BCG was complexed with 40-90 nmol of CPP or poly-K (e.g., 6K) peptide resuspended in DMSO and incubated at room temperature for 15 minutes. After complex formation, the PeptiBAC complex was pelleted by centrifugation at 1000 g at room temperature for 10 minutes, and the buffer was changed to remove unbound peptides.

[0074] PeptiCRAd complex formation VALO-mD901 adenovirus (in A195 preservation buffer) and poly(K) elongated Trp2 epitope (in 0.9% physiological saline) were used in a ratio of 1.8 × 10⁶ per virus particle. 5The PeptiCRAd complex was prepared by mixing the peptides in the specified ratios. The mixture was then incubated at room temperature for 15 minutes. For injection into animals, the complex was further diluted with 0.9% saline to obtain the required dose volume.

[0075] Surface plasmon resonance Measurements were performed using a multiparametric SPR Navi220A instrument (Bionavis, Tampere, Finland). PBS (pH 7.4) was used as the running buffer. A constant flow rate of 20 mL / min was maintained throughout the experiment, and the temperature was set to +20°C. A 670 nm laser light was used for surface plasmon excitation. Sensor slides with a silicon dioxide surface were activated by plasma treatment for 5 minutes, and then coated with APTES ((3-aminopropyl)triethoxysilane) by incubation in 50 mM APTES in isopropanol for 4 hours. The sensors were then washed and placed in the SPR instrument, and bacteria were immobilized in situ on the sensor surface of the test channel by injecting a BCG preparation in PBS (pH 7.4) for 12 minutes, followed by washing with PBS. Next, CPP-containing immunomodulatory peptides, polylysine-containing immunomodulatory peptides, or peptides without CPP or polylysine sequences (non-interacting controls) were injected separately into the flow cell's channel.

[0076] To test the interaction between various peptides and the Mycobacterium outer membrane, 100 μM test peptides, either extended with CPP or polylysine sequences, or those without binding moieties (as non-interaction controls), were injected into BCG-coated and uncoated channels of a flow cell.

[0077] The number of peptides per BCG particle was estimated according to the following procedure.

[0078] 1) First, we assumed that the fully coated sensor surface would form a single layer of hexagonally packed BCG particles. This means that (based on geometric calculations) only 74% of the sensor surface is covered with bacteria. For this reason, we assumed the average length (2.36 μm) and width (0.47 μm) of BCG bacteria, and the volume 0.3887 μm. 3 And it was converted into spherical particles with a diameter of 905.5 nm.

[0079] 2) To estimate the thickness of the hexagonal packed layer of BCG particles, optical modeling of the SPR sensor properties was performed for an unmodified sensor without BCG and a sensor completely covered with a layer of BCG particles. However, in the optical modeling of the SPR sensor properties, it was necessary to consider that the model assumes a flat, uniform layer without space, so the volume of a sphere was converted to its corresponding value in a cube using a conversion factor of 0.524 (based on geometric calculations).

[0080] 3) To estimate the theoretical flat and uniform thickness of a completely covered hexagonal packed BCG layer, we first multiplied the average diameter of the BCG by 0.74 (contribution from hexagonal packing), and then by 0.524 (contribution from filling the gaps between spheres within the uniform flat layer).

[0081] 4) In this way, a theoretically flat and uniform thickness of 351.1 nm (assuming an average diameter of 905.5 nm) was obtained for the fully coated hexagonal packed layer of BCG.

[0082] 5) Next, by assuming a refractive index of 1.35 for BCG, the maximum SPR angular response induced by this BCG layer was calculated by optical modeling and obtained 2.28° (see Supplementary Figure 7).

[0083] 6) Next, the measured SPR response during BCG immobilization on the SPR sensor surface was divided by the corresponding maximum SPR angular response (i.e., 2.28°) modeled for a single layer of the hexagonal packed BCG layer. This ratio was then assumed to reflect the proportion of the detection area covered with BCG. For the measurements of the various peptides used in this study, the corresponding ratios were 22.6% (6K-AH1 peptide), 13.6% (6K-TRP2), and 11.0% (CPP-SIINFEKL).

[0084] 7) Since the detection area is determined by the diameter of the laser used in the SPR device, i.e., 1 mm, it was possible to calculate the area covered with BCG by multiplying the detection area by the percentage of the detection area covered with BCG (22.6% for 6K-AH1 peptide, 13.6% for 6K-TRP2 peptide, and 11.0% for CPP-SIINFEKL peptide).

[0085] 8) Next, the footprint area of ​​BCG is calculated based on its assumed diameter of 905.5 nm, resulting in an area of ​​approximately 643,971 nm per BCG particle. 2 I obtained it.

[0086] 9) The number of BCG particles on the sensor surface was obtained by dividing the area of ​​the BCG-coated sensor region (obtained from item 7) by the BCG footprint area (obtained from item 8). In the measurements for various peptides in this study, the corresponding number of BCG particles was 275,269 BCG particles (6K-AH1 peptide), 165,397 BCG particles (6K-TRP2 peptide), and 133,729 BCG particles (CPP-SIINFEKL peptide).

[0087] 10) Next, the number of peptides adsorbed to BCG was calculated from the SPR response measured when a 100 μM peptide was interacted with the BCG layer. The SPR response value of the peptide was converted using a conversion factor of 600 ng / cm². 2By using the ×SPR response (in degrees), the result can be converted into the mass per unit area of the adsorbed peptide. In the present study, the mass / area values determined for various peptides were 35.3 ng / cm 2 (6K-AH1 peptide), 301.6 ng / cm 2 (6K-TRP2 peptide) and 163.0 ng / cm 2 (CPP-SIINFEKL peptide).

[0088] 11) By knowing the detection area, multiplying the detection area by the mass / area of each peptide allows estimation of the absolute mass of the peptide adsorbed to BCG. In the present study, the masses determined for various peptides were approximately 0.277 ng (6K-AH1 peptide), 2.369 ng (6K-TRP2 peptide) and 1.280 ng (CPP-SIINFEKL peptide).

[0089] 12) By knowing the molecular weight of the peptide (1868.21 g / mol for 6K-AH1 peptide, 1944.4 g / mol for 6K-TRP2 peptide, and 3279.9 g / mol for CPP-SIINFEKL peptide), the mass is converted into moles, and finally converted into the number of peptides using Avogadro's constant. In the present study, the numbers of adsorbed peptides determined for various peptides were approximately 8.9×10 10 (6K-AH1 peptide), 7.3×10 11 (6K-TRP2 peptide) and 2.4×10 11 (CPP-SIINFEKL peptide).

[0090] 13) Finally, the number of peptides (obtained from item 12) was divided by the number of BCG particles obtained (from item 9) to estimate the number of adsorbed peptides per BCG particle.

[0091] Cross-presentation and DC activation experiments: For the PeptiBAC cross-presentation experiment, 20 nmol of GRKKRRQRRRPQRWEKISIINFEKL [SEQ ID NO: 49] was complexed with the BCG preparation at 37°C for 15 minutes. The bacteria were then pelletized by centrifugation at 1000×G for 15 minutes, and the unbound peptide was removed by removing the supernatant containing the unbound peptide. 10 6 Jaws II cells were plated in 2 mL of complete alpha minimal essential medium and infected with purified PeptiBAC preparations. After o / n incubation, cells were detached, washed, and stained with SIINFEKL [SEQ ID NO: 14]-conjugated APC-conjugated anti-mouse H-2Kb (141606, BioLegend), APC-conjugated mouse IgGκ isotype control (400119, BioLegend), APC-conjugated anti-mouse CD40 antibody (17-0401-81, Ebioscience), or PerCpCy5.5-conjugated anti-mouse CD86 antibody (105028, BioLegend), PerCP-conjugated anti-mouse CD86 (105025, BioLegend), and FITC-conjugated anti-mouse CD40 (124607, BioLegend) antibodies. Stained samples were analyzed by flow cytometry.

[0092] ELISPOT assay: The amount of peptide-specific, e.g., SIINFEKL-specific, activated interferon-gamma secreting T cells was measured by the ELISPOT assay (CTL, USA) as described by the manufacturer. Briefly, antigen-presenting cells were stimulated using 2 μg of SIINFEKL peptide (SEQ ID NO: 14) (Note: These peptides contained only MHC class I epitopes to allow for the exclusion of nonspecific stimulation that may originate from the CPP Tat sequence or immunoproteasome processing sequence used in the PeptiBAC platform). After 3 days of stimulation, plates were stained and sent to CTL-Europe GmbH for spot counting.

[0093] SIINFEKL((SEQ ID NO: 14)OVA257~264), SVYDFFVWL((SEQ ID NO: 15)TRP2180-188 The amounts of BCG and adenovirus-specific activated interferon-γ secreting T cells were measured by the ELISPOT assay (CTL, Ohio, USA) according to the manufacturer's instructions. Briefly, antigen-presenting cells were stimulated using 2 μg of SIINFEKL or SVYDFFVWL peptide. After 2 or 3 days of stimulation, plates were stained and sent to CTL-Europe GmbH for spot counting.

[0094] The amount of peptide-specific activated interferon-gamma secreting T cells was measured using the ELISPOT assay (ImmunoSpot, Bonn, Germany) according to the manufacturer's instructions. In short, 2.5 × 10⁻⁶ 5 Each PBMC was stimulated with selected peptides containing the conserved region of the coronavirus (2 μg of each peptide) and tested twice in overlapping cycles at 37°C for 72 hours. Spots were counted using the ELISpot reading system (ImmunoSpot, Bonn, Germany) and background (DMSO-only signal) was corrected. T cell response was 1 × 10⁶ 6 This is expressed as a peptide-specific reaction per PBMC.

[0095] Flow cytometry The following antibodies were used in the experiment: TruStain FcX® anti-mouse CD16 / 32 (101320, BioLegend), FITC anti-mouse CD8 (A502-3B-E, ProImmune), phycoerythrin (PE) anti-mouse CD3e (550353, BD Pharmingen), peridinine-chlorophyll-protein (PerCP) anti-mouse CD19 (115531, BioLegend), and PE-cyanine 7 anti-mouse CD4 (25-0041-82 eBioscience). We studied SIINFEKL epitope-specific T cells using APC-labeled H-2Kb / SIINFEKL pentamer (F093-84B-E, ProImmune), SVYDFFVWL (Trp2) epitope-specific T cells using PE-labeled H-2Kb / SVYDFFVWL pentamer (F185-82B-E, Proimmune), and SPSYVYHQF (modified sequence derived from tumor rejection antigen AH1) epitope-specific T cells using PE-labeled H-2Ld / SPSYVYHQF pentamer (F398-82A-E, Proimmune). Flow cytometry analysis was performed using a BD Accuri 6C Plus (BD Biosciences) or BD LSRFortessa™ (BD Biosciences) flow cytometer, and data analysis was performed using FlowJo software v10 (BD Biosciences).

[0096] Bacterial survival rate and macrophage assays To evaluate bacterial viability, CPP-containing peptides or polylysine-containing peptides were complexed with BCG (as described in the PeptiBAC complex formation section), and the complexes were directly plated for colony formation. After 4 weeks of incubation at 37°C, bacterial colonies were counted.

[0097] We evaluated the activation of the NF-κB and AP-1 pathways induced by BCG and PeptiBAC using a mouse RAW-Blue macrophage reporter cell line (InvivoGen) expressing multiple pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs). The presence of PRR agonists expressed by RAW-Blue cells induces NF-κB and AP-1 activation, leading to the secretion of embryonic alkaline phosphatase enzyme (SEAP). In the Quanti-BLUE (InvivoGen) system, substrates change to purple / blue in the presence of SEAP. The relative activating potency of BCG and PeptiBAC was determined by measuring SEAP concentrations using a multi-well plate reader (Varioskan Flash; ThermoLabsystems). For the generation of myeloid-derived macrophages (BMDMs), 10 cells isolated from C57BL / 6JOlaHsd mice were used. 7 Each myeloid cell was seeded in 10 ml of complete medium (RPMI-1640) (Sigma) containing 10 ng / mL recombinant macrophage colony-stimulating factor (ThermoScientific), 10% FBS (Life Technologies), 2 mM L-glutamine, 50 U / mL penicillin, and 50 μg / mL streptomycin (Life Technologies). The cells were cultured at 37°C in a humidified atmosphere of 5% CO2. On day 3, half of the medium was replaced with fresh medium. On day 6, some macrophages were collected and used for cross-presentation experiments. For the remaining macrophages, the medium was gently aspirated and replaced with 10 ml of fresh complete medium containing 20 ng / mL interleukin-4 (IL-4, Life Technologies). After 48 hours of culture, M2-biased macrophages were collected and used for biasing experiments.

[0098] Animal experiments All animal experiments were reviewed and approved by the Experimental Animal Committee of the University of Helsinki and the Provincial Government of Southern Finland. The C57BL / 6JOlaHsd mouse strain was used in all animal experiments. In the B16.OVA animal experiment, 350,000 B16.OVA cells were injected into the right flank of mice, resulting in a tumor size of approximately 50 mm. 3 Once the target period was reached (10-12 days after injection), mice were treated with BCG, PeptiBAC platform, peptide alone, or injection medium alone (simulation). Specifically, they were treated with BCG alone at a dose of 0.75-3 × 10⁵ CFU / dose, PeptiBAC-OVA at a dose of 0.75-3 × 10⁵ CFU / dose, peptide alone, or PBS (as a simulated treatment group). Mice were treated on day 0 and day 2, followed by an additional (booster) treatment on day 9. Tumors were measured every other day until the end of the experiment. In the B16.F10 animal experiment, 150,000 B16.F10 cells were injected into the right flank of mice, and the tumor size was approximately 50 mm. 3 Once the target period was reached (8-10 days after injection), the mice were treated with BCG, various PeptiBAC platforms, or injection media alone (simulation). Mice were treated on day 0 and day 3, followed by an additional treatment on day 9. Tumors were measured every other day until the end of the experiment. On day 27 after tumor transplantation, three mice from each group were euthanized, and the spleen and tumors were collected for ELISPOT and flow cytometry analysis. The survival of the remaining animals was tracked.

[0099] In the B16.F10.K1 animal experiment, 300,000 B16.F10.K1 cells were injected into the right flank of mice along with a 1:1 ratio of Matrigel Basement Membrane Matrix High Concentration (Corning, USA), resulting in a tumor size of approximately 50 mm. 3Once this stage is reached (10-12 days after injection), the mice are treated with BCG, BCG + immune checkpoint inhibitor (anti-PD-1), PeptiBAC, PeptiBAC + immune checkpoint inhibitor, immune checkpoint inhibitor alone, or injection medium only (simulation), specifically 6.25 × 10⁻¹⁴. 6 ~12×10 7 BCG CFU / dose, 6.25 × 10 6 ~12×10 7 The mice were treated with CFU / dose PeptiBAC-Trp2 or PBS (as a simulated treatment group). The group receiving anti-PD-1 (InVivoMab, USA, clone RMP1-14) received 100 μg / dose intraperitoneally three times a week starting from day 16 after tumor transplantation. Mice were treated on days 0 and 2, with an additional treatment on day 14. An immune checkpoint inhibitor was administered intraperitoneally three times a week starting from day 5. Tumors were measured every other day until the end of the experiment.

[0100] For the CT26 colon experiment, 600,000 CT26 cells were injected into the right flank of 8-9 week old immunoqualified female BALB / c mice, and 6.25 × 10⁶ cells were administered on days 11, 13, and 25 post-tumor transplantation. 6 ~12×10 7 BCG CFU / dose, 6.25 × 10 6 ~12×10 7 The mice were treated with CFU / dose PeptiBAC-AH1 or PBS (as a simulated treatment group). The group receiving anti-PD-1 (InVivoMab, USA, clone RMP1-14) received 100 μg / dose intraperitoneally three times a week starting from day 17 post-tumor transplantation. For the initial and booster vaccination experiment, 1 × 10⁶ immunocompetent naive female C57BL / 6JOlaHsd mice aged 8-9 weeks were administered. 9 VP / dose PeptiCRAd VALO-mD901-Trp2, PeptiCRAd VALO-mD901-OVA, 2-8 × 10 6 PeptiBAC-Trp2 CFU / dose, 2-8 × 10 6Mice were treated with CFU / dose PeptiBAC-OVA or saline (as a simulated treatment group). Vaccinations were administered at 14-day intervals, and 4 days after the final injection, the mice were euthanized and their spleens were collected for the ELISPOT assay. All mouse strains were obtained from Envigo (Venray, the Netherlands).

[0101] Results and Discussion: Bacillus Calmette-Guerin vaccines produced from attenuated strains of Mycobacterium bovis can be coated with therapeutic peptides by using cell-permeable peptide sequences or polylysine or polyarginine linker sequences as bacterial membrane-binding anchors.

[0102] The outer membrane of Mycobacterium consists of a lipid-containing bilayer, and the membrane's surface charge is highly negative. Therefore, we hypothesized that immunomodulatory / therapeutic peptides could bind to the outer membrane by using cell-permeable peptide sequences (CPPs) or highly positive amino acid sequences, such as a 6-residue (6K) polylysine or polyarginine extension (see the schematic diagram of the binding method used in Figure 1). To test this, we analyzed anchor affinity by surface plasmon resonance (SPR). SPR analysis showed high and stable affinity to the bacterial outer layer regardless of which anchor portion was used. In this case, binding is entirely dependent on the CPP or 6K portion, because peptides without either portion did not interact with the bacterial outer layer (Figure 2).

[0103] In Figure 2A, various CPP sequences were tested for their efficacy in immobilizing therapeutic peptides within the Mycobacterium cell wall using surface plasmon resonance (SPR) (data not shown). The CPP sequence derived from HIV Tat protein was found to be the most efficient CPP for peptide immobilization (A). In addition to the HIV Tat-derived CPP sequence, positively charged polylysine sequences were found to efficiently immobilize peptides within the cell wall (B and C). Furthermore, the number of peptides bound to BCG bacteria was estimated using these two different binding sites, and for the SIINFEKL antigen containing the N-terminal CPP Tat sequence, the number of peptides bound to BCG was 1.8 × 10⁶. 6 The peptide molecules / bacteria are presumed to be present, and for the Trp2 antigen and the AH1 antigen containing an N-terminal polylysine sequence, the number of peptides bound to BCG is 4.4 × 10⁶, respectively. 6 Peptide molecules / bacteria and 3.2 × 10 5 It was presumed to be a peptide molecule / bacterial.

[0104] In Figure 2B, various CPP sequences were tested for their efficacy in immobilizing therapeutic peptides within the Mycobacterium cell wall using surface plasmon resonance (SPR).

[0105] The CPP sequence derived from the HIV Tat protein was found to be the most efficient CPP for peptide fixation. In addition to the HIV Tat-derived CPP sequence, a positively charged polylysine sequence was found to efficiently fix peptides within the cell wall. Using these two different binding sites, the number of peptides bound to BCG bacteria was estimated. For the SIINFEKL antigen containing the N-terminal CPP Tat sequence, the number of peptides bound to BCG was 1.8 × 10⁶. 6 It was estimated to be a peptide molecule / bacterial. For the Trp2 antigen and the AH1 antigen containing an N-terminal polylysine sequence, the number of peptides bound to BCG was 4.4 × 10⁶, respectively. 6 Peptide molecules / bacteria and 3.2 × 10 5It was presumed to be a peptide molecule / bacterial.

[0106] Antigen-presenting cells can efficiently present immunomodulatory peptides delivered by PeptiBAC.

[0107] Next, we tested whether PeptiBAC, the modified BCG of the present invention, can deliver immunomodulatory peptides into antigen-presenting cells (APCs), and whether these peptides can be readily processed and cross-presented on major histocompatibility complex I (MHC-I) molecules on the surface of antigen-presenting cells. BCG was coated with the CPP-containing immunomodulatory peptide GRKKRRQRRRPQRWEKISIINFEKL [SEQ ID NO: 49] (containing the neoepitope SIINFEKL) to obtain PeptiBAC-OVA. Subsequently, JAWS II dendritic cell (DC) cells were infected with PeptiBAC-OVA, and the cross-presentation efficacy of the neoepitope SIINFEKL was evaluated by flow cytometry (Figure 3).

[0108] SIINFEKL was efficiently cross-presented from PeptiBAC coated with CPP-bound SIINFEKL peptide (SEQ ID NO: 49) because approximately 40% of APCs were shown to cross-present the SIINFEKL epitope. Furthermore, PeptiBAC was shown to enhance DC activation / maturation compared to BCG, as measured by enhanced expression of the activation / maturation markers CD40 and CD86.

[0109] PeptiBAC induces antitumor effects in a syngeneic mouse model of B16.OVA melanoma, and tumor-specific CD8 + It induces strong induction of effector T cells.

[0110] To study the antitumor effects of the PeptiBAC platform, we used B16, a well-established syngeneic mouse melanoma model expressing chicken OVA as a model antigen.

[0111] B16.OVA tumor-bearing mice were treated intratumorally with OVA-targeted PeptiBAC (PeptiBAC-OVA), BCG, CPP-conjugated SIINFEKL peptide alone, or a vehicle-based (mock) treatment. PeptiBAC-OVA-treated animals showed enhanced reduction in tumor growth compared to all other treatment groups. In the peptide-only and mock treatment groups, there was one responder in each group, representing a response rate of 12.5%. In the BCG-treated group, the response rate was 25%, and in the PeptiBAC-OVA-treated group, it was 37.5% (Figure 4). Surprisingly, BCG-treated mice showed the lowest survival rate among all groups. In stark contrast, the survival rate of PeptiBAC-OVA-treated mice was significantly increased compared to the other groups (Figure 4).

[0112] To further validate the PeptiBAC platform, systemic peptide-specific T cell responses induced by various treatment groups were evaluated using the enzyme-linked immunoabsorption spot (ELISpot) assay (Figure 5). Notably, PeptiBAC-OVA treatment was able to induce a large systemic peptide-specific T cell response, as measured by the number of interferon-gamma (INF-G) secreting SIINFEKL-responsive T cells. Other treatments did not induce a SIINFEKL-specific T cell response. + T cell counts were assessed by flow cytometry, and compared to other groups, PeptiBAC-OVA-treated tumors showed increased T cell infiltration into the tumor microenvironment (TME).

[0113] • Trivalent PeptiBAC, which targets neoplastic antigens and helper T cells, exhibits antitumor efficacy in a highly immunosuppressive and invasive mouse model of B16.F10 melanoma.

[0114] To validate the PeptiBAC platform using tumor-associated antigens such as tyrosinase-related protein 2 (Trp2) and glycoprotein 100 (gp100), which are endogenously expressed by B16.F10 melanoma, mice were transplanted with B16.F10 tumors and treated intratumorically with bivalent PeptiBAC targeting Trp2 and gp100 (PeptiBAC-TG), monovalent PeptiBAC targeting pan-MHC class II molecules (PeptiBAC-P), trivalent PeptiBAC targeting Trp2, gp100 and pan-MHC class II molecules (PeptiBAC-TGP), BCG, and vehicle alone (mock-up). PeptiBAC-TGP-treated animals showed enhanced reduction in tumor growth compared to all other treatment groups. In the BCG and mock-up treatment groups, there was one responder in each group, which represented a response rate of 12.5%. No responders were observed in the PeptiBAC-P treatment group. The response rate was 25% in the PeptiBAC-TG treatment group and 50% in the PeptiBAC-TGP treatment group (Figure 6). Similar to previous experiments using B16.OVA tumors, the BCG treatment group showed the lowest survival rate, with only 3 out of 8 mice surviving until the end of the experiment (day 21).

[0115] • Antigen peptides containing polylysine-free CPP reduce BCG survival rates.

[0116] The unexpectedly low potency observed when PeptiBAC was used with CPP-containing OVA antigen prompted testing whether the CPP-containing antigen peptide could be toxic to the bacteria. Indeed, a decrease in BCG survival was observed when coated with the CPP-containing antigen peptide, but not when coated with the polylysine-containing antigen peptide (Figures 7 and 7A). Polylysine-extended SIINFEKL did not inhibit plaque formation, and this was comparable to the control group at both 20 μl and 100 μl (Figure 7). However, when polylysine extension was compared to the use of CPP as an alternative (Figure 7A), it was found that the use of the CPP linker may have a dramatic impact on BCG survival (Figure 7A).

[0117] To test whether this effect on survival rate was BCG-specific, Escherichia coli (Gram-negative bacterium) and Listeria monocytogenes (Gram-positive bacterium) were exposed to CPP, and their survival rates were again measured using plaque or colony counts. It was found that CPP did not affect the survival rates of Escherichia coli (Gram-negative bacterium) and Listeria monocytogenes (Gram-positive bacterium). Colony counts were comparable to the control, and in fact, slightly better for Escherichia coli (Figure 7B). This suggests that the inhibitory effect of CPP on BCG was specific to that bacterium.

[0118] To further verify that polylysine is a suitable binding site, we tested the potential of PeptiBAC coated with a polylysine-containing antigen peptide to activate macrophages. PeptiBAC (containing the polylysine-containing antigen peptide) was as potent as uncoated BCG in activating the NF-κB / AP1 pathway in mouse RAW-blue macrophages (Figure 7A). Since tumor-associated macrophages (TAMs) are important cellular components of TMEs, we also wanted to evaluate the cross-presentation characteristics of macrophages to tumor antigens delivered by PeptiBAC. After infecting bone marrow-derived macrophages (BMDMs) with PeptiBAC-OVA (BCG coated with a polylysine-containing OVA peptide) for 24 hours, we evaluated the cross-presentation efficacy of the epitope (SIINFEKL) by flow cytometry. Notably, SIINFEKL delivered by PeptiBAC was efficiently cross-presented on the surface of BMDMs (Figure 8A). In addition to macrophage presentation, we wanted to confirm whether PeptiBAC possesses the same properties as BCG in terms of macrophage shifting from the M2 state to the M1 state. M2-shifted macrophages were infected with either BCG or PeptiBAC, and the expression of macrophage M2 and M1 markers was analyzed by flow cytometry. Both BCG and PeptiBAC were equally effective in shifting M2 macrophages to the M1 state, as assessed by significant upregulation of both MHC-II and CD86 expression, and significant downregulation of M2 marker CD206 expression (Figure 8B). Based on these data, polylysine was selected as the binding site for use in all subsequent experiments.

[0119] PeptiBAC increases the response rate to checkpoint inhibitor therapy in a treatment-resistant mouse model of B16.F10.K1 melanoma.

[0120] Finally, the synergistic effects of PeptiBAC combined with checkpoint inhibitor therapy were tested using a mouse model of melanoma that is inherently resistant to checkpoint inhibitor therapy. B16.F10.K1-carrying mice were treated with Trp2-targeted PeptiBAC (PeptiBAC-Trp2; where the binding site was a 6K sequence), PeptiBAC-Trp2 combined with an anti-PD-1 antibody (a checkpoint inhibitor against the PD-1 / L-1 axis), BCG, BCG combined with an anti-PD-1 antibody, or an anti-PD-1 antibody alone or vehicle (mock). PeptiBAC-Trp2 treatment alone increased the number of responders to the treatment (response rate 44%), but the synergistic effect of PeptiBAC-Trp2 and the anti-PD-1 antibody further increased the response rate (response rate 71%). In all other groups, the response rate was less than 20%, including anti-PD-1 antibody alone (response rate 20%), which indicates an efficient enhancement of checkpoint inhibitor therapy by using PeptiBAC to mitigate treatment resistance (Figure 10, Panel A, individual tumor growth curves).

[0121] To evaluate the mechanism of tumor growth suppression, we assessed whether there were differences in Trp2-specific T cell responses between treatment groups. Compared to tumors treated with BCG, anti-PD-1 alone, and a combination of BCG and anti-PD-1 ICI, PeptiBAC-Trp2-treated tumors showed a higher tumor-infiltrating CD4 response. + and CD8 + An increase in the number of T cells was observed (Figure 10A, upper panel). Furthermore, compared to tumors treated with BCG, anti-PD-1 alone, and a combination of BCG and anti-PD-1 ICI, PeptiBAC-Trp2-treated tumors showed increased Trp2-specific CD8 + The number of T cells increased (Figure 10A, upper panel). In contrast to other treatment groups, tumors treated with the combination of PeptiBAC-Trp2 and anti-PD-1 showed a significantly higher number of tumor-infiltrating CD4 cells. + and CD8 + T cells and Trp2-specific CD8 +The presence of T cells indicates a synergistic effect on the T cell response resulting from the combination of these two treatment methods (Figure 10A, upper panel A). Furthermore, systemic tumor-specific T cell responses were evaluated by analyzing the spleen of treated mice. CD4 + and CD8 + No significant difference in the number of T cells was observed between the groups.

[0122] Trp2-specific CD8 + The number of T cells increased in the spleen treated with the combination of PeptiBAC-Trp2 and anti-PD-1 ICI compared to other treatment groups, again demonstrating a synergistic effect on the T cell response by combining these two therapeutic approaches (Figure 10A, lower panel B).

[0123] In a syngeneic mouse model of CT26 colorectal cancer, intratumoral treatment with PeptiBAC in the presence of polylysine-containing modified gp70 antigen increased the number of responders to anti-PD-1 therapy, improved tumor suppression, and induced tumor-specific T cell responses.

[0124] To validate the PeptiBAC platform as a more universal cancer vaccine platform, the PeptiBAC platform was tested in a syngeneic mouse model of CT26 colorectal cancer using the modified tumor rejection antigen AH1 combined with anti-PD-1 immune checkpoint inhibitor therapy. AH1 is one of the most characterized tumor rejection antigens in mice, and it is derived from the gp70 envelope protein of mouse leukemia virus (MuLV), which is endogenous in the genomes of most experimental mouse strains, including the BALB / c strain used in these studies. From day 11 post-tumor transplantation, mice were intratumorally treated with BCG, anti-PD-1 alone, PeptiBAC-AH1, a combination of BCG and anti-PD-1, a combination of PeptiBAC-AH1 and anti-PD-1, or saline (as a simulated treatment group). In this case as well, the tumor size threshold was set to 450 mm to determine responders in each treatment group. 3The settings were as follows: The groups treated with the simulated tumor, BCG, anti-PD-1 monotherapy, and a combination of BCG and anti-PD-1 ICI showed similar tumor growth characteristics with response rates of 25%, 22%, 25%, and 10%, respectively. Interestingly, in contrast to the B16.F10.9 / K1 melanoma model, PeptiBAC-AH1 treatment alone did not enhance tumor growth suppression, with a response rate of 25%. Notably, animals treated with the combination of PeptiBAC-AH1 and anti-PD-1 showed highly efficient tumor growth suppression, with the number of responders to anti-PD-1 therapy increasing from 25% to 80%, and a response rate of 80% (Figure 11A; and Figure 9 for mean tumor growth curves). Again, we assessed whether differences in T cell response existed between the treatment groups. Tumor-infiltrating CD4 + and CD8 + Although no significant difference in T cell count was observed between the treatment groups, interestingly, CD8 in PeptiBAC-AH1 treated tumors + The number of T cells was slightly reduced compared to tumors in other treatment groups. AH1-specific CD8 + T cell counts were slightly reduced in tumors treated with BCG and in combination with BCG and anti-PD-1 ICI compared to other treatment groups, but tumors treated with PeptiBAC-AH1 and anti-PD-1 ICI showed a decrease in AH1-specific CD8 + This shows a significant increase in the number of T cells, which suppresses tumor growth in TME and AH1-specific CD8 + This suggests a correlation with the number of T cells (Figure 11B, upper panel). Analysis of systemic tumor-specific T cell responses from the spleen of treated mice showed intergroup CD4 + and CD8 + No significant difference was observed in the number of T cells. However, in the spleens of mice treated with PeptiBAC-AH1 and in combination with PeptiBAC-AH1 and anti-PD-1 ICI, AH1-specific CD8 was higher compared to spleens from other groups. + A significant increase in T cells was observed (Figure 11B, lower panel).

[0125] • A xenogeneic first- and booster vaccination strategy using the PeptiBAC platform in combination with the PeptiCRAd platform improves the T cell response to coated antigens.

[0126] Finally, the PeptiBAC platform was tested in combination with our recently described cancer vaccine platform PeptiCRAd (peptide-coated conditionally replicating adenovirus) using a heterogeneous primary-booster vaccination strategy. By combining two immunologically different platforms coated with the same antigen, we tested whether this heterogeneous primary-booster approach could enhance the T cell-specific immune response in naive mice to MHC-I-constrained epitopes presented by both platforms. For this purpose, two doses of PeptiBAC-Trp2 or PeptiCRAd-Trp2 were administered as allogeneic primary-booster controls, or primary vaccination with PeptiBAC-Trp2 followed by a booster vaccination with PeptiCRAd-Trp2, or primary vaccination with PeptiCRAd-Trp2 followed by a booster vaccination with PeptiBAC-Trp2, were administered at 14-day intervals in naive C57BL / 6JOlaHsd mice. Four days after the booster dose, mice were sacrificially killed, their spleens were harvested, and analyzed for induction of Trp2-specific T cell responses using interferon-gamma ELISPOT. Allogeneic first-instance and booster vaccinations with PeptiCRAd-Trp2, or xenogeneic first-instance and booster vaccinations with PeptiCRAd-Trp2 - PeptiBAC-Trp2, did not induce a significant Trp2-specific T cell response in this vaccination regimen. Allogeneic first-instance and booster vaccinations with PeptiBAC-Trp2 induced a moderate Trp2-specific T cell response, which was significantly enhanced by the xenogeneic first-instance and booster vaccination regimen with PeptiBAC-Trp2 - PeptiCRAd-Trp2 (Figure 12A). The same approach was then tested using an immunodominant epitope of ovalbumin (SIINFEKL), an epitope with higher immunogenicity than Trp2, and the induction of OVA-specific T cell responses was again evaluated using interferon-gamma ELISPOT. In this case as well, the heterologous primary-booster regimen induced a significant enhancement of the OVA-specific T cell response compared to PeptiBAC-OVA vaccination (Figure 12B).

[0127] summary BCG can be coated with a therapeutic peptide (PeptiBAC) using a polylysine or polyarginine linker, which can be done by binding or anchoring the peptide to the bacterial membrane. When administered to humans, this modified BCG is physiologically processed, thereby enabling antigen-presenting cells to efficiently present the immunomodulatory peptide delivered by PeptiBAC.

[0128] PeptiBAC, containing polylysine-containing peptide antigens, induces antitumor effects in melanoma and colon cancer mouse models, and tumor-specific CD8 + It induces strong induction of effector T cells.

[0129] Trivalent PeptiBAC, containing CPP-containing peptide antigens that target neoplastic antigens and helper T cells, exhibits antitumor efficacy in a highly immunosuppressive and invasive melanoma mouse model.

[0130] Furthermore, PeptiBAC, which contains polylysine-containing peptide antigens, increases the response rate to checkpoint inhibitor therapy in known treatment-resistant mouse models of melanoma.

[0131] Notably, treatment with PeptiBAC-Trp2 (a polylysine-containing Trp2 epitope peptide) efficiently sensitized tumors to immune checkpoint inhibitor (ICI) therapy, with this combination therapy group showing a 70% response rate. In addition to enhanced tumor growth suppression, immunological analysis of treated tumors revealed significant infiltration of CD4+, CD8+, and Trp2-specific CD8+ T cells into the TME of PeptiBAC-Trp2+ ICI-treated mice.

[0132] To further evaluate the PeptiBAC platform, we tested it in a syngeneic mouse model of CT26 colorectal cancer using modified tumor rejection antigen AH1 (polylysine-containing AH1 epitope peptide) combined with anti-PD-1 ICI therapy.

[0133] In this model, neither monotherapy showed any effect on tumor growth inhibition, but the combination of PeptiBAC-AH1 and anti-PD-1 ICI showed a remarkable synergistic effect with an 80% response rate. Furthermore, the combination-treated mice showed a significant increase in AH1-specific CD8+ T cell infiltration into the TME. Both PeptiBAC-AH1 monotherapy and the combination of PeptiBAC-AH1 and anti-PD-1 significantly increased AH1-specific CD8+ T cells in the spleen compared to the other treatment groups.

[0134] When heterogeneous primary-booster vaccination was performed using two or more immunologically distinct platforms in succession to deliver antigens, it was shown that the PeptiBAC (containing polylysine-containing antigen peptide) platform, along with another peptide-based cancer vaccine platform (referred to as PeptiCRAd) using, for example, oncolytic adenovirus, can be used as a component in a heterogeneous primary-booster vaccination scheme. Interestingly, when PeptiBAC was used as the primary vaccine and PeptiCRAd as the booster vaccine, an enhancement of the antigen-specific T cell response was observed compared to allogeneic primary-booster vaccination using PeptiBAC alone.

[0135] In summary, these results suggest that PeptiBAC is superior in inducing anti-disease effects, particularly antitumor effects, when the disease is especially invasive, such as in highly immunosuppressive and invasive diseases, or when the disease is known to be treatment-resistant. Furthermore, PeptiBAC is particularly effective when combined with immune checkpoint inhibitor therapy or used in xenolytic first-instance-booster vaccination regimens. [Table 1] [Table 2]

Claims

1. A weakened Mycobacterium bovis (BCG) suitable for use in humans to prevent or treat a disease, wherein the BCG is coated with a plurality of peptide antigens capable of inducing an immune response to the disease in humans, and the peptides are bound to the BCG using a polylysine or polyarginine peptide linker.

2. The attenuated Mycobacterium bovis (BCG) according to claim 1, wherein the polylysine or polyarginine linker each comprises at least 4, 5, 6, 7, 8, or 9 lysine or arginine molecules.

3. The attenuated Mycobacterium bovis (BCG) according to claim 2, wherein the linker consists of six lysine molecules or six arginine molecules.

4. The attenuated Mycobacterium bovis (BCG) according to any one of claims 1 to 3, wherein the BCG is coated with a plurality of different peptide antigens.

5. Attenuated Mycobacterium bovis (BCG) according to any one of claims 1 to 4, wherein at least one of the peptides is MHC-I or MHC-II restrictive.

6. The attenuated Mycobacterium bovis (BCG) according to any one of claims 1 to 5, wherein the disease is an infection.

7. The attenuated Mycobacterium bovis (BCG) according to claim 6, wherein the infection is a respiratory infection.

8. The attenuated Mycobacterium bovis (BCG) according to claim 7, wherein the disease is a viral infection and the peptide antigen is derived from at least one of the following proteins: VME1, AP3A, R1AB, NS7B, NCAP, R1A, and viral spike protein.

9. The disease is an infection, and the peptide antigen is the following peptide: GLVAEWFLAYILFTRFFYVL (derived from R1AB) [Sequence ID 7]; GLEAPFLYLYALVYFLQSINFV (derived from AP3A) [SEQ ID NO: 4]; KVTLVFLFVAAIIFYLITPVHVMSK (derived from R1AB) [SEQ ID NO: 6]; KLIFLWLLWPVTLACFVLAAV (derived from VME1) [SEQ ID NO: 2]; KRAKVTSAMQTMLFTMLRRKL (derived from R1A) [Sequence ID 8]; LPKEITVATSRTLSYYKLGA (derived from VME1) [Sequence ID 3]; AQFAPSASAFFGMSRIGMEV (derived from NCAP) [SEQ ID NO: 11]; VILLNKHIDAYKTFPPTEPK (derived from NCAP) [SEQ ID NO: 13]; ALALLLLLDRLNQLESKMSGK (derived from NCAP) [SEQ ID NO: 12]; IAMACLVGLMWLSYFIASFRLFAR (derived from VME1) [SEQ ID NO: 1]; QMAPISAMVRMYIFFASFYYVWK (derived from R1AB) [SEQ ID NO: 5]; EIPVAYRKVLLRKNGNKGAG (derived from R1AB) [SEQ ID NO: 9]; and ELSLIDFYLCFLAFLLFLVLIMLII (derived from NS7B) [Sequence ID 10]; A weakened Mycobacterium bovis (BCG) according to any one of claims 6 to 8, comprising at least one of the above.

10. The attenuated Mycobacterium bovis (BCG) according to any one of claims 1 to 5, wherein the disease is cancer, and the peptide antigen is selected from the group including tumor-associated antigens (TAAs), tumor-specific antigens (TSPs), or neonatal antigens.

11. The peptide antigen is the following polypeptide: i) SIINFEKL [Sequence ID 14]; ii) SVYDFFVWL [Sequence ID 15]; iii) KVPRNQDWL [Sequence ID 16]; and iv) SPSYVYHQF [Sequence ID 56] The attenuated Mycobacterium bovis (BCG) according to claim 10, comprising at least one of the above.

12. The attenuated Mycobacterium bovis (BCG) according to any one of claims 1 to 11, wherein the peptide antigen comprises AKFVAAWTLKAAA (Padre peptide) [SEQ ID NO: 17].

13. The aforementioned cancers include nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary origin, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, cervical cancer, kidney cancer, Wilms' tumor, and liver cancer. The attenuated Mycobacterium bovis (BCG) according to claim 10 or 11, which is one or more of the following: Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, cardiac cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palatine cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

14. The number of peptides bound to each BCG is 1.8 × 10⁻⁶. ６ A weakened Mycobacterium bovis (BCG) according to any one of claims 1 to 13, exceeding the limits of peptide molecules / bacteria.

15. A pharmaceutical composition comprising attenuated Mycobacterium bovis (BCG) according to any one of claims 1 to 14 and a suitable carrier.

16. The pharmaceutical composition according to claim 15, formulated for intradermal, intranasal, subcutaneous, transdermal, intratumor, intramuscular, intraarterial, intravenous, intrapleural, intravesicular, intracavitary, intracavitary, or peritoneal injection or oral administration.

17. A pharmaceutical composition for combination therapy comprising attenuated Mycobacterium bovis (BCG) according to any one of claims 1 to 14 or a pharmaceutical composition according to any one of claims 15 or 16, and at least one checkpoint modulator or immune checkpoint inhibitor.

18. The combination therapy pharmaceutical according to claim 17, wherein the checkpoint modulator or immune checkpoint inhibitor is cytotoxic T lymphocyte protein 4 (CTLA-4) or programmed cell death protein 1 pathway (PD-1 / PD-L1).

19. A weakened Mycobacterium bovis (BCG) according to any one of claims 1 to 14, or a pharmaceutical composition according to claim 15 or 16, for the manufacture of a pharmaceutical for treating a disease.

20. A weakened Mycobacterium bovis (BCG) according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 or 16 for use in the treatment of cancer, infection, respiratory disease, influenza, TB, influenza, the common cold, or coronavirus infections including SARS and MERS.

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