Cancer therapy

Abstract

The present invention relates to a cancer therapy. More specifically, it relates to a method of treating cancer using a modified oncolytic adenovirus and / or a pharmaceutical composition comprising the same.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980033607.0 and the invention name “Cancer Therapy”, which entered the Chinese national phase of PCT international patent application PCT / EP2019 / 056770 with an application date of March 19, 2019. Technical Field

[0002] The present invention relates to a modified oncolytic adenovirus; a pharmaceutical composition comprising the same; and a method for treating cancer using the same. Background Art

[0003] Over the past decade, as immunotherapy and stimulation of the patient's own immune system to target and attack cancer have gained popularity, the understanding of the role of oncolytic viruses in cancer treatment has changed significantly. At the beginning of this century, oncolytic viruses were considered to be active agents in cancer treatment solely through their inherent ability to lyse tumor cells through oncolysis. Recently, their use as cancer vaccines has received attention, and their ability to release tumor antigens from cancer cells to activate the immune system when oncolytic is considered to be an important feature in the design of the ultimate immunotherapy against cancer.

[0004] Adenoviruses are highly immunogenic viruses that are commonly used as vectors in various vaccine approaches to combat infectious diseases. Importantly, they have an excellent ability to elicit and enhance immune responses. In addition, the presence of oncolytic adenovirus within tumors and the immunogenic cell death it causes may lead to clinically relevant antitumor immunity by causing the expression of TH1-type immunomodulatory factors such as IFNγ to develop an unfavorable tumor microenvironment toward a more susceptible state. However, the immunogenicity of oncolytic adenoviruses is a double-edged sword; antiviral immunity is often so overwhelming that it overrides the much weaker immune response elicited against self-antigens expressed by the tumor.

[0005] Peptide vaccines have been an attractive concept in the immunotherapy of cancer, but the clinical efficacy of traditional peptide vaccine approaches is generally considered to be poor. Cancer vaccines deliver a combination of cancer antigens and adjuvants, which should provide the necessary inflammatory signals to initiate an anti-tumor immune response. However, central and peripheral tolerance and the process of immune editing upon tumor progression often lead to a loss of T cell reactivity, which has been observed in vitro when selecting candidate peptides for peptide vaccines. Even if the tumor expresses the selected antigen, current vaccine adjuvants are simply not potent enough to break tolerance in immunosuppressive tumors. In addition, many peptide vaccine approaches are based on short peptides that match the exact minimal sequence of CD8+ T cell epitopes that bind to MHC class I. Despite encouraging results in preclinical tumor models, short peptides have been shown to cause systemic peripheral tolerance in patients, presumably due to the presentation of the peptides by non-professional APCs or immature DCs that lack important co-stimulatory signals.

[0006] The present invention combines the above two vaccine methods in such a way that the method takes advantage of the advantages of both. The physical entity of combining oncolytic adenovirus and cancer antigen solves the following problems: a) for immune cells, the virus-specific target is overwhelmingly strong without sufficient tumor-specific targets; b) the problem of weak adjuvant; and c) the problem of short peptide vaccine inducing central tolerance. Therefore, the present invention relates to a peptide-coated conditional replicating adenovirus (PeptiCRAd), which is a combination of the following two clinically proven cancer immunotherapies in an innovative and unique way: oncolytic adenovirus and peptide vaccine. PeptiCRAd uses immunogenic viruses as active carriers of tumor-specific peptides to guide the immune system to specifically target and kill cancer cells. In addition, in contrast to oncolytic virus vaccines that genetically encode tumor antigens, coating adenoviruses with immunogenic peptides makes PeptiCRAd technology highly adaptable to all cancers; the same carrier virus can be used to treat all cancers and can be adapted simply by coating the virus with different polylysine extension peptides. This unique technology takes advantage of the significant immunogenicity of adenoviruses and simultaneously guides CD8+T cell responses to tumor tissues. Summary of the invention

[0007] According to a first aspect of the present invention, there is provided a modified adenovirus or vector comprising:

[0008] At least one of the following polypeptides, which is covalently or non-covalently linked to the viral capsid and is not genetically encoded by the adenoviral vector:

[0009] i)VFGIELMEVDPIGHLYIFAT[SEQ ID NO:1]MAGE-A3 161-180;

[0010] ii)YLAMPFATPMEEAELARRSLA[SEQ ID NO:2]NY-ESO-1 91-110;

[0011] iii)RGPESRLLEFYLAMPFATPM[SEQ ID NO:3]NY-ESO-1 81-100; or

[0012] iv) a polypeptide that is at least 60% identical thereto.

[0013] In a preferred embodiment of the present invention, the adenovirus is capable of replicating in target cells and has lytic activity.

[0014] Still more preferably, the polypeptide of part iv) has 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 the polypeptide of part 1), or part ii), or part iii).

[0015] Advantageously, the polypeptides can stimulate a peptide-specific immune response in a subject, and still more advantageously, since the polypeptides are not genetically encoded by the adenoviral vector, but are already covalently or non-covalently linked to the capsid, such linkage can be performed quickly and efficiently, i.e., without waiting for viral replication to occur in the host cell. Typically, to facilitate linkage of at least one or two or three polypeptides, the one or more polypeptides are polylysine extended using at least 4, ideally 5, 6, 7, 8 or 9 lysines. Most typically, 6 or 9 lysines are used and most preferably linked at the amino terminus of the polypeptide.

[0016] Therefore, the peptide attached to the adenovirus is:

[0017] KKKK(KKKKK)-VFGIELMEVDPIGHLYIFAT[SEQ ID NO:7]; and / or

[0018] KKKK(KKKKK)-YLAMPFATPMEAELARRSLA[SEQ ID NO:8]; and / or

[0019] KKKK (KKKKK) -RGPESRLLEFYLAMPFATPM [SEQ ID NO: 9]; or:

[0020] KKKKKK-VFGIELMEVDPIGHLYIFAT[SEQ ID NO:4]; and / or

[0021] KKKKKK-YLAMPFATPMEAELARRSLA [SEQ ID NO: 5]; and / or

[0022] KKKKKK-RGPESRLLEFYLAMPFATPM [SEQ ID NO:6].

[0023] In another preferred embodiment of the present invention, the adenovirus can be any type and species in the family Adenoviridae, such as but not limited to human adenovirus, but most often human adenovirus. Most advantageously, the adenovirus is capable of replicating and killing cancer cells while redirecting the antiviral immune response to the tumor, so that the modified adenovirus or vector is replicative and has an oncolytic effect.

[0024] The adenovirus or vector used in the present invention can be made tumor-specific in replication, for example, the adenovirus vector can contain modifications in the E1, E3, E4 and / or L3 genes, such as insertion of a tumor-specific promoter, gene deletion and insertion of a transgene.

[0025] Ideally, the adenovirus or vector is oncolytic, ie, is able to infect and kill cancer cells by replicating selectively in tumor cells relative to normal cells.

[0026] The backbone of the adenovirus or vector may be of any serotype. In one embodiment of the present invention, the serotype of the adenovirus or vector backbone is selected from serotype 3 or serotype 5. As used herein, "adenovirus serotype 5 (Ad5) nucleic acid backbone" refers to the genome of Ad5 and "adenovirus serotype 3 (Ad3) nucleic acid backbone" refers to the genome of Ad3.

[0027] In addition, the virus or vector can be chimeric, such as an Ad5 / 3, Ad3 / 5 or Ad5 / 35 vector. For example, an "Ad5 / 3 vector" refers to a chimeric virus or vector that has parts of both an Ad5 vector and an Ad3 vector.

[0028] The "capsid" of an adenovirus or vector refers to the protein shell of the virus. The capsid is composed of several oligomeric structural subunits made of proteins called protomers.

[0029] Most preferably, the adenovirus or vector is serotype 5, which is selected because it is optimal for eliciting and boosting T cell responses; it triggers the CD8+ T cells required for tumor killing; any pre-existing immunity to oncolytic viruses can enhance the efficacy of intratumoral treatment and it has a good clinical safety record.

[0030] In yet another preferred embodiment of the present invention, the adenovirus or vector is further modified to include any one or more of the following features, including any and all combinations thereof.

[0031] The Ad5 / 3 chimeric substitution replaces the adenovirus serotype 5 fiber knob region with the adenovirus serotype 3 fiber knob region; this allows the virus or vector to circumvent the Ad5 natural receptor, the coxsackievirus-adenovirus receptor (CAR), and instead use the Ad3 natural receptor, desmoglein 2 (DSG2), for internalization. DSG2 is present in large quantities in cancer cells.

[0032] Adenovirus infection is initiated by recognition of host cell receptors with the aid of specialized proteins on the surface of the virus, namely the adenovirus spike protein, and in particular the globular carboxyl-terminal domain of the adenovirus spike protein, referred to as the carboxyl-terminal knob domain. Therefore, when referring to the knob of the adenovirus spike protein herein, it is referring to the globular carboxyl-terminal domain of the adenovirus spike protein.

[0033] The E1A gene is deleted, wherein the deletion is the nucleotides encoding amino acids 122-129 (LTCHEACF); this deletion is a safety measure because the viral or vector E1A protein cannot bind to the retinoblastoma (Rb) molecule and release the transcription factor E2F from Rb for viral gene transcription. Therefore, the adenovirus or vector depends on the presence of free E2F in the host cell and can replicate its genome in dividing normal cells or cancer cells, in which free E2F is continuously available. Therefore, the modification relatively protects non-dividing cells and targets dividing cells or cancer cells.

[0034] Partial deletion of the E3 gene would enhance immunogenicity given the immunosuppressive effects of this gene product. Ideally, the 14.7K gene would be partially or completely deleted.

[0035] In a preferred embodiment, the modified adenovirus or vector comprises the insertion of at least one transgene encoding a co-stimulatory molecule, and ideally, comprises the insertion of two transgenes, wherein one of the genes causes activation of the innate immune system and the other causes activation of the adaptive immune system. Preferred transgenes include CD40L, which activates the innate immune system by driving CD8+ T cell responses using APCs, and OX40L, which activates the adaptive immune system by increasing clonal expansion, survival, and formation of large memory T cell pools of CD8+ T cells.

[0036] Many transgenes can be placed at different positions in the adenovirus or vector. For example, a transgene can be placed in a partially or completely deleted E3 region, under an E3 promoter or an exogenous promoter; or placed in a partially or completely deleted E1 region, under an E1 promoter or an exogenous promoter; or placed in a partially or completely deleted L3 region, under an L3 promoter or an exogenous promoter.

[0037] Most preferably, the OX40L, ideally human OX40L, is located in the E3B region, replacing the 14.7K deletion of the gene.

[0038] Also most preferably, CD40L, ideally human CD40L, is inserted into the late region of the virus, particularly the late region 3 (L3), ideally downstream of the 23K gene.

[0039] In a preferred embodiment, both transgenes are inserted into the virus, OX40L and CD40L.

[0040] In an alternative approach, the DNA encoding OX40L and the DNA encoding CD40L can be linked and inserted as a fusion molecule using known genetic engineering techniques, such as using an internal ribosome entry site (IRES) or more preferably by using a self-cleaving 2A peptide, which has the advantages of being small in size and having a high cleavage efficiency between genes upstream and downstream of the 2A peptide. Typically, CD40L is inserted immediately downstream of OX40L, but it is possible to practice the invention using the opposite configuration.

[0041] The viruses or vectors utilized in the present invention may also contain other modifications in addition to those described above. Any additional components or modifications may be optionally used but are not essential to the present invention.

[0042] As can be seen above, the adenoviruses or vectors of the invention have been engineered to stimulate an immune response against cancer, particularly in the tumor environment where, typically, the immune system is compromised by evasion mechanisms exploited by cancer cells.

[0043] Surprisingly, our data indicate that the addition of the immunostimulatory transgenes human OX40L and human CD40L into the 14.7K locus does not impair the oncolytic efficacy of the virus of the invention when compared to the backbone virus Ad5 / 3D24 or a virus with an immunostimulatory transgene such as human GM-CSF that replaces the gp19K / 7.1K gene that is deleted. This is surprising because the transgenes may significantly affect viral replication due to their size and direct effects on infected cells. In addition, the deletion of the 14.7K gene has not been as extensively studied as the deletion of the gp19K / 7.1K gene, and therefore may have unexpected consequences on the replication machinery of the virus, particularly if the transgene is incorporated into the 14.7K deletion site.

[0044] We have also demonstrated that the viruses of the present application are capable of producing functional human transgenes from the 14.7K locus. This was unexpected because we somewhat unusually used a transcription cassette with a viral 2A processing site between the two transgenes.

[0045] Furthermore, we demonstrated that the viruses of the present invention are capable of eliciting MAGE-A3 and NY-ESO-1 specific immune responses.

[0046] Therefore, the present invention extends to a pharmaceutical composition comprising at least one modified adenovirus or vector of the present invention and a suitable carrier. In a preferred embodiment of the present invention, the pharmaceutical composition is formulated for intratumoral, intramuscular, intraarterial, intravenous, intrapleural, intracapsular, intracavitary or peritoneal injection or oral administration.

[0047] Therefore, in a further aspect, the present invention relates to a method for treating cancer in an individual, said method comprising administering to said individual an effective amount of a pharmaceutical composition comprising at least one adenovirus or vector according to the present invention.

[0048] Additionally or alternatively, the present invention relates to at least one adenovirus or vector or composition according to the present invention for use in treating cancer.

[0049] Additionally or alternatively, the present invention relates to the use of at least one adenovirus or vector or composition according to the present invention for treating cancer.

[0050] Additionally or alternatively, the present invention relates to the use of at least one adenovirus or vector according to the present invention for the manufacture of a medicament for the treatment of cancer.

[0051] Given that tumors have evolved several immunosuppressive mechanisms to counteract the body's immune cells, the therapy of the present invention is also implemented in combination with the use of checkpoint molecules. The best characterized checkpoint pathways are the cytotoxic T lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 pathways (PD-1 / PD-L1). Therefore, the adenovirus or vector of the present invention can be used in combination with checkpoint regulators such as anti-PD1 molecules, anti-PD-L1 molecules, or anti-CTLA-4 molecules to counteract the immunosuppressive tumor environment and induce a strong anti-immune response.

[0052] The modified adenovirus or vector acts as an active adjuvant because it provides the danger signal required for an optimal immune response to the target peptide, and in the case of a replicating adenovirus or vector, it retains its ability to lyse and replicate its genome in the cancer cells it infects. Oncolytic cell killing is immunogenic in nature, which causes changes in the tumor microenvironment that may enhance the immune response to the peptide / tumor. Therefore, the use of our modified adenovirus physically complexed with long peptides (20 amino acids, without a poly-lysine tail) produces a superior anti-tumor immune response when compared to peptide vaccines alone or oncolytic vaccines.

[0053] Most preferably, the cancer referred to herein includes any one or more of the following cancers: nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, kidney cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, laryngeal 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 site, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, disease), cervical cancer, cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease disease), non-Hodgkin's 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, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum 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.

[0054] In the appended claims and the preceding description of the invention, unless the context requires otherwise due to express language or necessary implication, the word "comprises" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. for specifying the presence of stated features, but not excluding the presence or addition of further features in various embodiments of the invention.

[0055] All references cited in this specification, including any patents or patent applications, are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Furthermore, no admission is made that any prior art constitutes part of the common general knowledge in the art.

[0056] Preferred features of each aspect of the invention may be described in conjunction with any other aspect.

[0057] Other features of the invention will become apparent from the following examples. In general, the invention extends to any novel feature or any novel combination of features disclosed in this specification (including the appended claims and drawings). Therefore, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention should be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

[0058] Furthermore, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or similar purpose.

[0059] Throughout the description and claims of this specification, unless the context requires otherwise, the singular includes the plural. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity unless the context requires otherwise. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Embodiments of the present invention will now be described by way of example only with reference to the following, in which:

[0061] Figure 1 A schematic diagram of a modified adenovirus according to the present invention is shown. Adenovirus seraphate type 5, which is optimal in eliciting and enhancing T cell responses, primarily triggers CD8+ T cell responses: required for killing tumor cells, pre-existing immunity against oncolytic viruses enhances the efficacy of intratumoral therapy and has a good clinical safety record. It has the following genetic modifications: (1) a chimeric Ad5 / 3 capsid for increased tumor cell transduction; (2) a D24 deletion in E1A for tumor-specific viral replication; (3) a partial deletion of E3 for increased immunogenicity. Two co-stimulatory molecules as transgenes are used to optimally activate both the innate and adaptive branches of the immune system: innate branch: CD40, for licensing APCs to drive CD8+ T cell responses; adaptive branch: OX40L, for increasing the clonal expansion and survival of CD8+ T cells and forming a larger pool of memory T cells. Specifically, the Ad5 adenovirus is modified to include the serotype 3 fiber knot protein, i.e., the Ad3 fiber knot, and at least one polypeptide of the invention, and ideally two or three polypeptides, are attached to the viral capsid using a polylysine tail. These polypeptides do not form part of the viral genome, but are covalently or non-covalently attached to the capsid. This innovation is referred to herein as PeptiCRAd-1, and thus, it exploits all major arms of the immune system to attack cancer.

[0062] Figure 2Two of the selected peptides are shown. They do not bind directly to MHC class I molecules, but need to be presented by APCs for antigen processing. Therefore, the peptides cannot bind to MHC class I molecules on non-professional APCs, and therefore do not cause transient activation and subsequent anergy of CD8+T cells. Only activated professional APCs (which can accept further costimulation from the virally encoded CD40L present in the further modified adenovirus) can present these peptides to T cells, thereby causing robust activation and proliferation of peptide-specific CD8+T cells (further costimulation and enhanced memory T cell responses are provided by the virally encoded OX40L present in the further modified adenovirus).

[0063] Figure 3 It is shown that when compared to a clinically used unmodified peptide with known immune activation capabilities in cancer patients, Figure 2 The poly-lysine tail at the N-terminus of the peptide does not change the immunological properties of the peptide.

[0064] Figure 4 Shown are the average hydrodynamic diameters of two NY-ESO-1 peptide-virus complexes in water and physiological salt at different peptide concentrations.

[0065] Figure 5 The frequency of T cells in treated tumors and contralateral untreated tumors when treated with modified viruses coated with NYESO-1 and MAGE-A3 peptides according to the present invention (PeptiCRAd-1) containing viruses expressing OX40L and CD40L containing 5 / 3 capsids or the same virus without NYESO-1 and MAGE-A3 peptide coatings (VALO-C1) or peptides alone are shown. The number of CD3+ T cells (A), CD4+ T cells (B) and CD8+ T cells (C) in each treatment group is depicted as the number of cells per gram of tumor tissue. Compared with the sham treatment group, treatment in all groups resulted in higher T cell frequencies. The highest numbers were seen in tumors treated with VALO-C1 or PeptiCRAd.

[0066] Figure 6 The frequency of all immune cells (CD45+ cells) in treated tumors and contralateral untreated tumors when treated with PeptiCRAd-1 or the same virus without peptide coating (VALO-C1) or peptide alone is shown. The frequencies were similar in all groups, with slightly lower numbers in sham-treated animals.

[0067] Figure 7 It was shown that PeptiCRAd and VALO-C1 treatment reduced the percentage of regulatory T cells among all TILs in treated tumors.

[0068] Figure 8 The percentages of NY-ESO-1-specific CD8+ T cells (A) and MAGE-A3-specific CD8+ T cells (B) in blood tumors when treated with PeptiCRAd-1 or the same virus without a peptide coating (VALO-C1) or peptide alone are shown. The percentages are calculated from all CD8+ T cells per 1 ml of blood. When compared to sham-treated or peptide-treated animals, PeptiCRAd treatment produced the highest percentage of NY-ESO-1-specific CD8+ T cells, and both VALO-C1 and PeptiCRAd treatment produced higher percentages of MAGE-A3-specific CD8+ T cells.

[0069] Fig. 9 The percentages of NY-ESO-1-specific CD8+ T cells or MAGE-A3-specific CD8+ T cells in treated tumors or contralateral untreated tumors when treated with PeptiCRAd-1 or the same virus without a peptide coating (VALO-C1) or peptide alone are shown. The percentages are calculated from all CD8+ T cells per gram of tumor tissue. VALO-C1 and PeptiCRAd are equally effective in recruiting peptide-specific CD8+ TILs to tumors.

[0070] Fig.10 The results showed that PeptiCRAd coated with NY-ESO-1 and MAGE-A3 proteins could still prevent tumor growth in a humanized mouse melanoma model even when treatment was initiated against large, well-established tumors. 6 SK-MEL-2 cells were implanted subcutaneously (one tumor per animal) into the flank of NOD / Shi-scid / IL-2Rγ knockout immunodeficient mice. On day 13, 5×10 6 On day 16, 5×10 PBMCs were injected intratumorally 4 1×10 plasmacytoid and myeloid dendritic cells were given on days 16, 17, 18 (priming) and 25 (boosting). 9VP dose of intratumoral PeptiCRAd treatment. The first PeptiCRad dose was given immediately after DC injection. Tumor growth was tracked. Animals were sacrificed on day 32. PeptiCRAd = Ad5 / 3-D24-OX40L-CD40L, an oncolytic adenovirus with a 24 bp deletion in E1A, a 5 / 3 chimeric capsid, and CD40L and OX40L transgenes expressed from the 14.7K locus, coated with NY-ESO-1 peptide and MAGE-A3 peptide; OX40L PeptiCRAd = Ad5 / 3-D24-OX40L, an oncolytic adenovirus with a 24 bp deletion in E1A, a 5 / 3 chimeric capsid, and an OX40L transgene expressed from the 14.7K locus, coated with NY-ESO-1 peptide and MAGE-A3 peptide.

[0071] Fig.11 It is shown that OX40L-PeptiCRAd increased the number of MAGE-A3-specific CD8+T cells in peripheral blood compared to sham-treated animals. Two animals treated with PeptiCRAd also showed an increase in the number of MAGE-A3-specific CD8+T cells in the blood. Anti-MAGE-A3 T cells were assessed by flow cytometry (pentamer analysis) at the end of the tumor growth study mentioned above on day 32.

[0072] Fig.12 (A) Three separate SPR responses of AdV preparations diluted in sterile water and during immobilization on the APTES sensor using sterile water as running buffer. Concentration C = approximately 1.93 × 10 11 VP / ml. (B) SPR responses with the following three different peptide coatings: PEP1455 = KKKKKK-VFGIELMEVDPIGHLYIFAT (SEQ ID NO: 4); PEP1456 = KKKKKKKKK-VFGIELMEVDPIGHLYIFAT (SEQ ID NO: 7); PEP1508 = KKKKKK-YLAMPFATPMEAELARRSLA (SEQ ID NO: 5). Stock solutions with increasing concentrations (5 mg / ml in sterile water) were allowed to interact with the immobilized AdV particles. Sterile water was used as running buffer. Arrows indicate the time points of injection of the corresponding concentrations of peptide samples and asterisks indicate the time points when flushing with running buffer was started. The Δ 最大 Value and Δ 最小 The values ​​correspond to the SPR response used to calculate the number of peptides for each virion interaction in each case (100 μM of peptide in solution (Δ 最大 ) and the absence of peptide in solution (Δ 最小 )).

[0073] Fig.13 SPR response when increasing concentrations of peptide-coated samples PEP1455, PEP1456 and PEP1508 diluted in A195 buffer were allowed to interact with immobilized AdV particles. Arrows indicate the time point of injection of the corresponding concentration of peptide samples and asterisks indicate the time point of starting to wash with running buffer (A195 medium). 最大 Value and Δ 最小 The values ​​correspond to the SPR response used to calculate the number of peptides interacting with each virion in each case (100 μM of peptide in solution (Δ 最大 ) and the absence of peptide in solution (Δ 最小 )). The subscripts "minimum, median" and "maximum, median" refer to the median of two individual measured values ​​calculated from the corresponding measurements. DETAILED DESCRIPTION

[0074] This application relates to the following specific implementation methods.

[0075] 1. A modified adenovirus or vector comprising:

[0076] At least one of the following polypeptides, covalently or non-covalently attached to the viral capsid,

[0077] Not genetically encoded by the adenoviral vector:

[0078] i)VFGIELMEVDPIGHLYIFAT[SEQ ID NO:1];

[0079] ii) YLAMPFATPMEAELARRSLA [SEQ ID NO: 2];

[0080] iii)RGPESRLLEFYLAMPFATPM [SEQ ID NO:3]; or

[0081] iv) a polypeptide that is at least 60% identical thereto.

[0082] 2. The modified adenovirus or vector according to item 1, wherein the polypeptide is polylysine-extended using at least 3, 4, 5, 6, 7, 8 or 9 lysines.

[0083] 3. The modified adenovirus or vector according to item 2, wherein the lysine is linked to the amino terminus of the polypeptide.

[0084] 4. The modified adenovirus or vector according to any one of the preceding numbers, wherein the polypeptide is:

[0085] KKKKKK(KKK)-VFGIELMEVDPIGHLYIFAT[SEQ ID NO:7]; and / or

[0086] KKKKKK(KKK)-YLAMPFATPMEAELARRSLA[SEQ ID NO:8]; and /

[0087] or

[0088] KKKKKK(KKK)-RGPESRLLEFYLAMPFATPM[SEQ ID NO:9]; or

[0089] yes:

[0090] KKKKKK-VFGIELMEVDPIGHLYIFAT[SEQ ID NO:4]; and / or

[0091] KKKKKK-YLAMPFATPMEAELARRSLA [SEQ ID NO: 5]; and / or

[0092] KKKKKK-RGPESRLLEFYLAMPFATPM [SEQ ID NO:6].

[0093] 5. The modified adenovirus or vector according to any one of the preceding numbers, wherein the adenovirus is a human adenovirus.

[0094] 6. A modified adenovirus or vector according to any one of the preceding numbers, wherein the adenovirus comprises modifications in the E1 and / or E3 and / or E4 and / or L3 genes, the modifications comprising the insertion of a tumor-specific promoter, at least one gene deletion and the insertion of at least one transgene.

[0095] 7. A modified adenovirus or vector according to any one of the preceding numbers, wherein the adenovirus is of serotype 5.

[0096] 8. A modified adenovirus or vector according to any of the preceding numbers, wherein the adenovirus is further modified to include an Ad5 / 3 chimeric substitution, i.e., replacing the fiber knob region of adenovirus serotype 5 with the fiber knob region of adenovirus serotype 3.

[0097] 9. A modified adenovirus or vector according to any one of the preceding numbers, wherein the adenovirus is further modified to include a deletion of the E1A gene, wherein the deletion is at least those nucleotides encoding amino acids 122-129.

[0098] 10. The modified adenovirus or vector according to any one of the preceding numbers, wherein the adenovirus is further modified to include a partial or complete deletion of the 14.7k gene.

[0099] 11. A modified adenovirus or vector according to any one of the preceding numbers, wherein the adenovirus is further modified to include the insertion of at least one transgene encoding a co-stimulatory molecule.

[0100] 12. The modified adenovirus or vector according to item 11, wherein the virus comprises at least two transgenes, wherein one of the transgenes causes activation of the innate immune system and the other causes activation of the adaptive immune system.

[0101] 13. The modified adenovirus or vector according to item 12, wherein the virus comprises a transgene encoding CD40L and / or OX40L.

[0102] 14. The modified adenovirus or vector according to item 13, wherein the transgene is human.

[0103] 15. The modified adenovirus or vector according to number 13 or 14, wherein the OX40L is located in the E3B region, replacing the gene 14.7K deletion.

[0104] 16. The modified adenovirus or vector according to any one of numbers 13-14, wherein the CD40L molecule is inserted immediately downstream of OX40L using a 2A processing site.

[0105] 17. A modified adenovirus or vector according to any one of the preceding numbers, wherein the ratio of peptide to virus is in the range of 1 ug to 5 ug per 3E+9 virus particles.

[0106] 18. A pharmaceutical composition comprising at least one modified adenovirus or vector according to any one of numbers 1 to 17 in combination with a suitable carrier.

[0107] 19. The pharmaceutical composition of number 18, wherein the composition is formulated for intratumoral, intramuscular, intraarterial, intravenous, intrapleural, intracapsular, intradermal, intracavitary or peritoneal injection or oral administration.

[0108] 20. At least one adenovirus or vector according to any one of numbers 1 to 17 or a pharmaceutical composition according to any one of numbers 18 to 19 for use in treating cancer.

[0109] 21. Use of at least one adenovirus or vector according to any one of numbers 1 to 17 in the manufacture of a medicament for treating cancer.

[0110] 22. A method for treating cancer in a patient, the method comprising administering to the patient an effective amount of at least one adenovirus or vector according to any one of numbers 1-17 or a composition according to number 18 or 19.

[0111] 23. The method for treating cancer according to number 22, wherein at least one adenovirus or vector according to any one of numbers 1-17 or the composition according to number 18 or 19 is administered together with a cellular checkpoint regulator.

[0112] 24. The method for treating cancer according to No. 23, wherein the checkpoint regulator is an anti-PD1 molecule, an anti-PD-L1 molecule or an anti-CTLA-4 molecule.

[0113] 25. The adenovirus or vector or use according to number 20 or 21 or the method according to number 22-24, wherein the cancer comprises any one or more of the following cancers: nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, kidney cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, Schilling's disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, fibroblastic fibrosis, Sarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's 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, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum 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.

[0114] Methods and Materials

[0115] Peptides:

[0116] The peptides were selected to have acetate counterions, which is a suitable salt form for human patients. The peptides were manufactured by PepScan.

[0117] The specific peptides in PeptiCRAd intended for clinical use are derived from:

[0118] - MAGE-A3 protein (amino acids 161-180, sequence KKKKKK(KKK)-VFGIELMEVDPIGHLYIFAT), a 20-mer peptide with a 6 (SEQ ID NO: 4) or 9 (SEQ ID NO: 7) lysine tail extending from the amino terminus;

[0119] - NY-ESO-1 protein (amino acids 91-110, sequence KKKKKK(KKK)-YLAMPFATPMEAELARRSLA), a 20-mer peptide with a 6 (SEQ ID NO: 5) or 9 (SEQ ID NO: 8) lysine tail extending from the amino terminus;

[0120] - NY-ESO-1 protein (amino acids 81-100, sequence KKKKKK(KKK)-RGPESRLLEFYLAMPFATPM), a 20-mer peptide with a 6 (SEQ ID NO: 6) or 9 (SEQ ID NO: 9) lysine tail extending from the amino terminus.

[0121] Adenovirus used in PeptiCRAd formulations:

[0122] Adenovirus delta 24 serotype 5 virus (AdV 5 / 3) with a modified fiber knob from adenovirus serotype 3. This model virus is not exactly the virus intended for clinical use, but it has the same viral capsid. The difference between this model virus and the clinical virus is that the clinical virus will ideally include gene inserts encoding CD40L and OX40L, which have no effect on the viral capsid and the properties studied herein.

[0123] PeptiCRAd complex formation:

[0124] Individual peptides were diluted in water or 0.5% saline to achieve a peptide stock concentration of 5ug / μl. Virus stock (1,45E+12 VP / ml) in A195 buffer was diluted in water to achieve a concentration of 1E+9 VP / μl. Virus-peptide complexes were prepared by mixing 1 μl or 3 μl of virus dilution (equivalent to 1E+9 VP or 3E+9 VP) with variable volumes of peptide to achieve the target peptide to virus ratio.

[0125] (PeptiCRAd, single peptide); two separate virus aliquots are coated with a single peptide each. The two separate single peptide coated PeptiCRAds (NY-ESO-1 complex and MAGE-A3 complex) are then combined just before tumor injection. The separately coated viruses can also be given as separate injections. Alternatively, the NY-ESO-1 peptide and the MAGE-A3 peptide are first mixed together and then the peptide mixture is used to form the PeptiCRAd complex.

[0126] Zetasizer measurements of size and charge of the complexes:

[0127] Zeta potential particle size analyzer measurements were performed immediately after complex formation, 15 minutes after the components were mixed, or after the complex was left to stand for about 1.5 hours at room temperature (RT). Virus-peptide complexes were first diluted by adding 700 μl of water to the sample, which was then transferred to the measuring cuvette. The hydrodynamic diameter (nm) was measured, followed by the zeta potential (mV). These parameters were measured three times and the average size and zeta potential were recorded.

[0128] Immunological efficacy of PeptiCRAd-1 peptide:

[0129] CD8+ T cells from melanoma patients with known NY-ESO-1 and MAGE-A3 T cell activity were pre-stimulated with unmodified peptides (SEQ ID NO: 2NY-ESO-1 91-110: YLAMPFATPMEAELARRSLA; or SEQ ID NO: 1MAGE-A3 161-180: VFGIELMEVDPIGHLYIFAT). Recognition of polylysine-extended peptides (SEQ ID NO: 5NY-ESO-191-110: KKKKKKYLAMPFATPMEAELARRSLA; or SEQ ID NO: 4MAGE-A3 161-180: KKKKKKVFGIELMEVDPIGHLYIFAT) was investigated by standard IFN-γ ELISPOT methods. The following ELISPOT protocol was used in the experiment: CD4+ / CD8+T cells purified by cell separation columns (Miltenyi Biotech, Lund, Sweden) were pre-sensitized with irradiated autologous PBMCs (25,000 cells / well) pulsed with peptides (10 ug / ml) to remove CD4+ and CD8+T cells. The recognition of pre-sensitized CD4+ / CD8+T cells to peptide-pulsed (1 ug / ml) autologous antigen-presenting cells (EBV-transformed B cells or DCs) was tested in IFNγELISPOT assays on days 10-12. After incubation (37°C) for 16 hours, the number of antigen-specific T cells producing cytokines was evaluated using AID EliSpot Reader Classic ELR 07 (Autoimmun Diagnostika GmbH, Strassberg, Germany, AID Co., Ltd., Strassberg, Germany).

[0130] In vivo immunization

[0131] NOD / Shi-scid / IL-2Rγ knockout immunodeficient mice were humanized using hematopoietic stem cells (CD34+, HLA-B35+) isolated from human umbilical cord blood. A375 human melanoma tumors (2×10 per 100 μl) were implanted subcutaneously. 6 Animals were randomized based on humanization rate and tumor size. Animals were treated with PeptiCRAd-1 or the same virus (VALO-C1) without peptide coating (the viral dose for both groups was 1×10 8 ; For PeptiCRAd, 1×10 7 Peptide vaccines (0.12ug or 30ug) were administered intradermally with polyinosinic acid (Poly-IC) as an adjuvant.

[0132] Treatment was initiated 25 days after randomization (day 0) with a high dose of cyclophosphamide (1 mg / mouse, intravenously). Treatment was given intratumorally (sham, virus, and PeptiCRAd groups) or intradermally (peptide control group) on days 1, 2, 3, and 12. Secondary tumors were implanted in the contralateral flank 2 days after the third treatment (day 5). No treatment was given for secondary tumors.

[0133] Using dextramer analysis, peripheral blood mononuclear cells (PBMC) and tumor infiltrating CD8+ lymphocytes (TIL) were analyzed by flow cytometry for NY-ESO-1YLAMPFATPMEAELARRSLA SEQID NO:2 and MAGE-A3VFGIELMEVDPIGHLYIFAT SEQ ID NO:1 specific CD8+T cells. Different immune cell subsets in PBMC and TIL were evaluated. Flow cytometric analysis was performed on an Attune NxT flow cytometer (Life Technologies).

[0134] PBMC mouse model immunization

[0135] 2.10 6 SKMEL-2 tumor cells (HLA-B35+) were implanted into the right flank of 35 8-week-old NOD-Prkdcem26Cd52 / IL-2Rγem26Cd22 / NjuCrl immunodeficient mice (NCG) (day 0). On day 13, 5×10 6 HLA-B35+ human peripheral blood mononuclear cells (PBMCs) from two different donors. On day 16, 1×10 9 Intratumoral treatment was initiated with a viral dose of VP of 50 VP with either a virus expressing OX40L containing 5 / 3 capsid complexed with NYESO-1 (SEQ ID NO: 2) and MAGE-A3 (SEQ ID NO: 1) ("OX40L PeptiCRAd") or a virus expressing OX40L and CD40L containing 5 / 3 capsid complexed with NYESO-1 (SEQ ID NO: 2) and MAGE-A3 (SEQ ID NO: 1) ("PeptiCRAd"). Simultaneously with the first PeptiCRAd treatment, 50'000 autologous plasmacytoid and myeloid dendritic cells were injected intratumorally. Tumors were treated with intratumoral PeptiCRAd injections without the addition of dendritic cells on days 17, 18 (priming by first treatment), and 25 (boost). The treatment schedule is shown in Fig.10 Tumor growth was followed. Animals were sacrificed on day 32. OX40L-PeptiCRAd and PeptiCRad contain a 24 bp deletion in E1A, a deleted gp19k / 7.1K region, a human OX40L transgene expressed from the 14.7K locus, and 5 / 3 chimeric fibers.

[0136] Virus-peptide complex

[0137] Immobilization of viral particles and the interaction between peptide coatings and viral particles immobilized on the APTES functionalized silica sensor surface were measured using a multi-parameter surface plasmon resonance (SPR) instrument equipped with a 2-channel polydimethylsiloxane (PDMS) flow channel and a 96-well plate autosampler assembly (MP-SPR 220A). The peptide coatings were as follows: PEP1455 = KKKKKK-VFGIELMEVDPIGHLYIFAT (SEQ ID NO: 4); PEP1456 = KKKKKKKKK-VFGIELMEVDPIGHLYIFAT (SEQ ID NO: 7); PEP1508 = KKKKKK-YLAMPFATPMEAELARRSLA (SEQ ID NO: 5). The temperature, flow rate and laser wavelength used for all SPR measurements were +20°C, 20 μL / min and 680 nm, respectively.

[0138] For measurements in water, adenovirus with 5 / 3 chimeric spikes (approximately 1.93 × 10 11 VP / ml) were fixed on the APTES sensor for 12 minutes, and three replicates were measured for each sample. Sterile water was also used as running buffer. For measurements in A195 buffer, adenovirus was first diluted in PBS and fixed on the APTES sensor for 12 minutes. After fixation, A195 was used instead of PBS as running buffer, and measurements were performed in triplicate.

[0139] For measurements in sterile water, peptide stocks were prepared by dissolving 5 mg of peptide in 1 ml of water (5 mg / ml) to yield corresponding concentrations of 1.65 mM 6K-MAGE-A3 (PEP1455), 1.46 mM 9K-MAGE-A3 (PEP1456), and 1.66 mM 6K-NY-ESO (PEP1508). Peptide samples were injected sequentially at increasing concentrations for 6 minutes, followed by an 8-minute run buffer washout period between each sample concentration. The peptide concentrations used were 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM.

[0140] For measurements in A195 buffer, peptide stock solutions were prepared by dissolving 340 ug of the corresponding peptide in A195 medium to form a 100 μM sample solution, which was then used as a stock solution for preparing peptide sample solutions with lower concentrations. Peptide samples were injected sequentially at increasing concentrations for 10 minutes, followed by a 15-minute run buffer washout period between each sample concentration. The peptide concentrations used were 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM.

[0141] The calculation used to estimate the number of peptides bound per virion was based on a geometric calculation using the estimated number of virions immobilized on the SPR sensor surface and the maximum number of adsorbed peptides determined by SPR measurements.

[0142] Results and Discussion

[0143] Effect of the net charge of peptides on complex formation

[0144] The MAGE-A3 peptide has a net charge of -3,9 in the absence of a lysine tail. The three acidic amino acids presenting a negative charge are located near the amino terminus (amino acid 5, amino acid 8, and amino acid 10). We studied whether it is necessary to compensate for the negative net charge of the peptide with a longer positively charged lysine tail (9 lysines instead of 6 lysines) to achieve the proper binding of the peptide on the viral surface. The zeta potential and average complex size of the peptide-coated virus sample containing 1E+9 virus particles compounded with any of 20ug or 40ug of peptide were determined to evaluate the effect of the lysine tail length on the complex formation and stability of the complex. Immediately after the complex is formed, 15 minutes after the components are mixed, and after the complex is left to stand for about 1.5 hours at room temperature (RT), the zeta potential particle size analyzer is measured. The results are shown in Table 1.

[0145] The size and zeta potential results show that complexes with the MAGE-A3 peptide are formed regardless of the length of the lysine tail, indicating that negatively charged amino acids at least at specific positions of the peptide chain do not impair the formation of the complex. Longer lysine tails give the complexes a size that is about 10% larger and a slightly lower zeta potential than the corresponding peptides with shorter lysine tails. Based on the size of the freshly prepared samples and the complexes after the samples were kept at room temperature for 1.5 hours, the complexes with either lysine tail were stable. In the freshly prepared complexes, the size was not affected by the amount of peptide used for complex formation, but in the samples kept for 1.5 hours, the results were a bit irrelevant. In summary, it was decided to use six lysine tail peptides in subsequent zeta potential particle size analyzer studies.

[0146] Effect of the ratio of 6K-MAGE-A3 peptide to virus on the size and zeta potential of the complex

[0147] Peptide to virion ratios of 15ug, 30ug, 45ug and 60ug peptide per 3E+9 virions were prepared to evaluate the effect of ratio on complex formation and stability based on determination of size and zeta potential of the complexes (freshly prepared complexes vs. complexes kept at room temperature for 2 hours).

[0148] The results in Table 2 show the following trend, that is, the lower the ratio of peptide to virus in the tested range, the smaller the size of the complex, which indicates that an optimal ratio can be determined, which produces peptide-virus complexes composed of a single virus or small aggregates formed by several viruses. The complex is also quite stable for at least 2 hours, with a zeta potential of about +30mV.

[0149] An attempt was made to determine the optimal peptide to virus ratio using 6K-MAGE-A3 peptide, as shown in Table 3. The complex size results showed that as low as 1 ug-5 ug of 6K-MAGE-A3 peptide per 3E+9 virus particles (equivalent to a molar ratio range of 6.6E+4 to 3.3E+5) produced stable complexes. However, at lower ratios, the complexes tended to aggregate.

[0150] Effect of virus coating with 6K-MAGE-A3 peptide on infectivity

[0151] The ability of the complexed virus to infect tumor cells is an important aspect of the mechanism of action of PeptiCRAd. This is why the effect of peptide coating on viral infectivity was evaluated by preparing complexes with different peptide to virus ratios and determining the infectivity of the complexes on A549 cells based on an immunocytochemistry assay (ICC). The results in Table 4 show that using a ratio of about 1 ug of the relevant peptide to virus, 62% of the infectivity of the virus-only sample was retained, indicating that viral coating does not significantly affect infectivity.

[0152] Stability of 6K-MAGE-A3-PeptiCRAd at different temperatures

[0153] To evaluate the stability of 6K-MAGE-A3-PeptiCRAd at different temperatures, complexes were prepared by mixing 15ug or 30ug of 6K-MAGE-A3 peptide per 3E+9 VP and storing the samples under different conditions. Stability was tested by a zeta potential particle size analyzer from samples stored for 18 hours to 20 hours at room temperature, +5°C, -20°C, and -80°C. The results are shown in Table 5. The average hydrodynamic diameter remained fairly stable in the range of 165nm to 276nm during storage at different temperatures, indicating that no significant aggregation occurred at any temperature tested. The zeta potential was above the level of +30mV in each sample, indicating good stability. The smallest particle size was obtained after storage at -20°C, but storage at +4°C also seemed to help prevent aggregation.

[0154] Effect of the ratio of 6K-NY-ESO-1 peptide (p81-100) to virus on the size and zeta potential of complexes prepared in water

[0155] Another clinically significant peptide, the NY-ESO-1 peptide, was studied in terms of complex formation properties (size and zeta potential) determined by zeta potential particle size analyzer measurements. The results are shown in Table 6a. With respect to the size of the complex, this particular peptide appears to follow a sinusoidal curve, starting with a small complex size at a small peptide to virus ratio, then increasing in size as the ratio becomes larger, reaching a local maximum at 1 ug peptide, after which the complex size begins to decrease until 60 ug, at which point the complex size reaches a minimum of 180 nm. With respect to the stability of the complex, the zeta potential results indicate that a higher peptide to virus ratio is favorable compared to 6K-MAGE-A3. The NY-ESO-1 peptide has a native charge of -1, with two negatively charged amino acids and two positively charged amino acids near the amino terminus (acidic amino acids are located at positions 4 and 9 of the peptide sequence and basic amino acids are located at positions 1 and 6 of the peptide sequence). The remainder of the sequence of NY-ESO-1 is fairly hydrophobic, as is the MAGE-A3 peptide.

[0156] Effect of the ratio of 6K-NY-ESO-1 peptide (p91-110) to virus on the size and zeta potential of complexes prepared in water

[0157] The 6K-NY-ESO-1 peptide (p91-110) was also studied in terms of complex formation properties (size and zeta potential) determined by zeta potential particle size analyzer measurements. The results are shown in Table 6b. Regarding the size of the complex, the peptide range of 0.5 ug to 1 ug produced only moderately aggregated complexes, indicating that the optimal ratio with this peptide exists somewhere between 1 ug and 5 ug per 3E+9 VPs. The corresponding zeta potential values ​​are still close to zero, indicating that the stability of the complex may not be optimal and there is a tendency to aggregate.

[0158] The NY-ESO-1 peptide (p91-110) has a natural charge of -1, with two negatively charged amino acids and two positively charged amino acids near the carboxyl terminus (the acidic amino acids are located at positions 11 and 13 of the peptide sequence and the basic amino acids are located at positions 16 and 17 of the peptide sequence). The remainder of the sequence of NY-ESO-1 is quite hydrophobic. The location of the positive charge near the C-terminus may also enable the C-terminus to bind to the virus surface.

[0159] PeptiCRAd coated with both 6K-MAGE-A3 peptide and 6K-NY-ESO-1 peptide

[0160] Two clinically significant peptides, NY-ESO-1 peptide and MAGE-A3 peptide, were studied in the form of mixtures for complex formation properties based on zeta potential particle size analyzer measurements. Complexes were prepared in both water and physiological NaCl (0.9%). The results are shown in Table 7a and Table 7b. The average size of the complexes prepared by mixing equal amounts of each peptide with the virus was larger than the average size of the complexes prepared from the 6K-MAGE-A3 peptide alone and smaller than the size of the complexes prepared from the 6K-NY-ESO-1 peptide (p81-100) alone. The average size of less than 300 nm indicates that only moderate aggregation occurred during complex formation. The complexes can be prepared in water and 0.9% NaCl with comparable complex sizes.

[0161] The average size of the complexes prepared by mixing equal amounts of 6K-MAGE-A3 and 6K-NY-ESO-1 peptide (p91-110) with the virus was larger than that of the complexes prepared by 6K-MAGE-A3 and 6K-NY-ESO-1 peptide (p81-100). The average size difference of the complexes was about 1.8 times in water, but decreased to 1.3 times in physiological NaCl. The average size of 400 nm-500 nm indicated that only moderate aggregation occurred during complex formation.

[0162] Effect of the ratio of NY-ESO-1 peptide to virus on the size and zeta potential of complexes prepared in physiological NaCl

[0163] Since the zeta potential particle size analyzer size results of the complexes formed with the NY-ESO-1 peptide (p81-100) in water showed a sinusoidal-like behavior as a function of peptide concentration, we investigated whether performing the complex formation step in physiological salt solution would prevent possible non-specific interactions. The results are shown in Table 8a. The results show that the complex size as a function of increasing peptide concentration follows a trend very similar to that of the complexes prepared in water. At a peptide to virus ratio in the range of 5 ug to 60 ug, the complex size is larger, but nevertheless fairly consistent. The results suggest that the optimal peptide to virus ratio can be found in the range of 1 ug to 5 ug per 3E+9 VP.

[0164] The zeta potential particle size analyzer average size results of complexes formed with NY-ESO-1 peptide (p91-110) in water indicated that at peptide to virus ratios above 1 ug, aggregation increased, resulting in complex sizes greater than 3000 nm. We investigated whether performing the complex formation step in physiological saline solution would prevent aggregation. The results are shown in Table 8b and show that the complex size is fairly constant in the range of 200 nm-400 nm with increasing peptide concentration. Thus, physiological NaCl in the liquid phase during complex formation can prevent aggregation quite significantly, as can be seen in Table 8b. Figure 1 As can be seen in Figure 1 Data for two NY-ESO-1 peptide-virus complexes in water and physiological salt are shown. The complex size remained within 217nm-245nm at the relevant peptide to virus ratio of 1ug-5ug per 3E+9 VP.

[0165] Immunological efficacy of PeptiCRAd-1 peptide

[0166] Upon in vitro stimulation, poly-lysine-extended NY-ESO-1 and MAGE-A3 peptides triggered IFN-γ production in cancer patient-derived antigen-specific CD8+ T cells as effectively as unmodified peptides ( Figure 3 ), indicating that poly-L-lysine modification does not affect function.

[0167] PeptiCRAd elicits peptide-specific immune responses in a humanized mouse model

[0168] All active treatments (peptide alone, virus without peptide coating [VALO-C1], and virus with NY-ESO-1 and MAGE-A3 peptide coating [PeptiCRAd]) increased the number of immune cells in primary tumors compared with sham-treated animals. Both VALO-C1- and PeptiCRAd-1-treated animals showed more T cells (CD3, CD4, CD8) in primary tumors compared with peptide vaccine or sham-treated animals after treatment, while the number of total infiltrating immune cells (CD45) was similar in all groups (45 and 50%, respectively). Figure 5 and Figure 6 ).

[0169] In addition, the number of T regulatory cells (CD3+ / CD4+ / FoxP3+) was reduced in primary tumors treated with VALO-C1 and PeptiCRAd-1 compared with primary tumors from peptide vaccine or sham-treated animals ( Figure 7 This suggests that intratumorally administered immunogenic adenovirus (VALO-C1 or PeptiCRAd-1) modulates the tumor microenvironment by reducing local immunosuppression.

[0170] Unlike VALO-C1 treated animals, PeptiCRAd-1 treated animals showed more CD4+ and CD8+ T cells in untreated secondary tumors than in treated primary tumors, suggesting that tumor targeting by the viral peptide coating is critical for inducing effects in distant untreated tumors (Table 9). In addition, PeptiCRAd-1 treated animals had more NYESO-specific CD8+ T cells in the blood after priming (mean = 4.3% of total CD8+ cells) compared to OV treated (mean = 0.6%) or peptide vaccine treated (mean = 0.6%) animals. Figure 8 In both treated and untreated tumors, the frequencies of MAGE-specific CD8+ TILs in PeptiCRAd- and VALOC1-treated animals were higher than the frequencies of MAGE-specific CD8+ T cells in peptide-treated animals ( Fig. 9 Interestingly, a higher frequency of MAGE-specific CD8+ T cells was seen in VALOC1- and PeptiCRAd-treated tumors compared with NY-ESO-1-specific CD8+ T cells, whereas the opposite was true in the blood.

[0171] PeptiCRAd elicits peptide-specific immune responses in a PBMC mouse model

[0172] Even when treatment was initiated against large, well-established tumors, treatment with PeptiCRAd complexed with NY-ESO-1 and MAGE-A3 resulted in tumor growth arrest in a humanized mouse melanoma model ( Fig.10 ). Compared with sham-treated mice, mice treated with OX40L-PeptiCRAd showed significantly more MAGE-A3-specific CD8+ T cells among all CD8+ T cells in PBMCs, indicating that PeptiCRAd treatment can elicit peptide-specific responses in humanized mice ( Fig.11 ).

[0173] Adenovirus with 5 / 3 chimeric capsid can complex with NY-ESO-I peptide and MAGE-A3 peptide under optimal complexation conditions

[0174] The interaction between peptide coatings and adenoviral particles (AdV) was studied using surface plasmon resonance (SPR) technology. The main objectives were 1) to determine the binding kinetics of NY-ESO-1 peptide and MAGE-A3 peptide to the virus in different media (sterile water and A195 medium (a common medium for virus preparation)); 2) to estimate how many peptide molecules can be bound to one virus particle in each medium; and 3) to evaluate the stability of the peptide-virus complex. The peptide coatings are as follows: PEP1455 = KKKKKK-VFGIELMEVDPIGHLYIFAT (SEQ ID NO: 4); PEP1456 = KKKKKKKKK-VFGIELMEVDPIGHLYIFAT (SEQ ID NO: 7); PEP1508 = KKKKKK-YLAMPFATPMEAELARRSLA (SEQ ID NO: 5).

[0175] The SPR response of AdV particles in sterile water was about 0.7°, which corresponds to 50% coverage of the virus in the detection zone (1.4° corresponds to 100% coverage) (Table 10), giving a total of 5×10 7 When AdV particles were run in A195 buffer, the SPR response was approximately 1.2°, which corresponds to 86% coverage of the detection surface ( Fig.12 ). These results give that 8.6×10 7 These results may indicate that the structure of the virus is better preserved in A195 buffer than in sterile water or that A195 buffer facilitates the adsorption of the virus to the APTES surface.

[0176] Peptide 1455 and peptide 1508 appeared to adsorb better to immobilized virions in sterile water than in A195 medium ( Fig.12 (B) and Fig.13 ). Peptide 1456, on the contrary, was significantly better adsorbed on the immobilized virus particles in A195 medium than in water. The number of peptides adsorbed per virus is calculated in Table 10.

[0177] Based on the results, PEP1455 is not completely soluble in A195 buffer and PEP1456 appears to be a more promising modification in terms of solubility and coating efficiency as judged by the ratio of peptide to virus in A195 buffer. The results indicate that peptide-virus complexes can be prepared using adenovirus constructs with 5 / 3 chimeric capsids and 6 / 9 K-peptides in an appropriate buffer environment. As shown by the SPR results, the complexes are quite stable without a free peptide environment. In addition, the ratio of peptide to virus particles is a peptide sequence-specific factor and also depends on the presence of optimal conditions for complex formation.

[0178] Summarize

[0179] For the three peptides tested (one 6K-MAGE-A3 and two 6K-NY-ESO-1), the complex size seemed to depend on the peptide to virus ratio and appeared to be a peptide-specific property that had to be determined for each peptide and each combination of peptide and virus. In the case of 6K-NY-ESO-1(p91-110), aggregation and possible nonspecific interactions during complex formation could be prevented to some extent by preparing the complexes in physiological salt solution instead of water, but this was not the case for 6K-NY-ESO-1(p81-100), indicating a peptide-specific behavior.

[0180] When relevant peptide to virus ratios were used, complex formation with the 6K-MAGE-A3 peptide did not significantly reduce viral infectivity and 62% of infectivity was retained.

[0181] Based on the stability results of the MAGE-A3 complex, the optimal storage conditions for the complex appear to be +4°C or -20°C.

[0182] PeptiCRAd with peptide coating was superior to naked oncolytic adenovirus (VALO-C1) or standard peptide vaccination in triggering systemic tumor-targeted CD8+ T cell responses and CD8+ TIL infiltration into untreated distant tumors. The data suggest that PeptiCRAd improves the tumor targeting specificity of standard oncolytic viruses.

[0183] Table 1: Average diameter and zeta potential of peptide-virus complexes prepared by mixing virus and peptide at a ratio of 1E+9 virus particles to 20ug or 40ug of MAGE-A3 peptide with 6 or 9 lysine tails.

[0184]

[0185] Table 2: Average hydrodynamic diameter and zeta potential of peptide-virus complexes measured immediately or 2 hours after preparation by mixing 3E+9 virus particles with different amounts of 6K-MAGE-A3 peptide as indicated in the table.

[0186]

[0187]

[0188] Table 3: Average hydrodynamic diameter and zeta potential results of peptide-virus complexes prepared by mixing 3E+9 virus particles with different amounts of 6K-MAGE-A3 peptide.

[0189]

[0190] Table 4: Infectivity of PeptiCRAd complexes prepared using different ratios of MAGE-A3 peptide to virus (3E+9 vp).

[0191]

[0192] Table 5: Average hydrodynamic diameter and zeta potential results of peptide-virus complexes prepared by mixing 3E+9 virus particles with 15ug or 30ug of 6K-MAGE-A3 peptide after being kept at different temperatures for 18-20 hours.

[0193]

[0194]

[0195] Table 6a: Average hydrodynamic diameter and zeta potential of peptide-virus complexes prepared by mixing 3E+9 virus particles with different amounts of 6K-NY-ESO-1 peptide (p81-100).

[0196]

[0197] Table 6b: Average hydrodynamic diameter and zeta potential of peptide-virus complexes prepared by mixing 3E+9 virus particles with different amounts of 6K-NY-ESO-1 peptide (p91-110).

[0198]

[0199]

[0200] Table 7a: Average hydrodynamic diameter and zeta potential of peptide-virus complexes prepared by mixing 3E+9 virus particles with 1 ug of 6K-NY-ESO-1 peptide (p81-100) and MAGE-A3 peptide in water or physiological NaCl (0.9%).

[0201]

[0202] Table 7b: Average hydrodynamic diameter and zeta potential of peptide-virus complexes prepared by mixing 3E+9 virus particles with 1 ug of both 6K-NY-ESO-1 peptide (p91-110) and 6K-MAGE-A3 peptide in water or physiological NaCl (0.9%).

[0203]

[0204] Table 8a: Average hydrodynamic diameter and zeta potential of peptide-virus complexes prepared by mixing 3E+9 virus particles with different amounts of 6K-NY-ESO-1 peptide (p81-100) in physiological NaCl (0.9%).

[0205]

[0206] Table 8b: Average hydrodynamic diameter and zeta potential of peptide-virus complexes prepared by mixing 3E+9 virus particles with different amounts of 6K-NY-ESO-1 peptide (p91-110) in physiological NaCl (0.9%).

[0207]

[0208] Table 9: Animals treated with PeptiCRAd showed more CD8+ and CD4+ T cells in untreated distant tumors than in treated primary tumors, indicating that PeptiCRAd treatment achieved more efficient antigen presentation and subsequent T cell homing to distant tumors compared with OV. Results are described as the ratio of the number of tumor-infiltrating lymphocytes in untreated tumors to treated tumors ± SEM.

[0209]

[0210] Table 10: Results of SPR-based measurements of viruses and virus-peptide complexes.

[0211]

Claims

1. Use of at least one modified adenovirus or a pharmaceutical composition comprising said at least one modified adenovirus in combination with a suitable carrier in the preparation of a medicament for treating cancer, wherein said modified adenovirus comprises: At least one of the following polypeptides, covalently or non-covalently attached to the viral capsid, which polypeptide is not genetically encoded by the adenovirus: i)VFGIELMEVDPIGHLYIFAT[SEQ ID NO:1]; or ii) YLAMPFATPMEAELARRSLA [SEQ ID NO: 2]; wherein the adenovirus comprises a transgene encoding OX40L, or CD40L and OX40L; and wherein the cancer is selected from the group consisting of nasopharyngeal carcinoma, hepatocellular carcinoma, kidney cancer, connective tissue cancer, intestinal cancer, colon cancer, rectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, Schilling's 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 site, carcinoid, gastrointestinal carcinoid, fibrosarcoma, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer , Wilms tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's 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, stomach cancer, thymus cancer, thyroid cancer, trophoblastic carcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum 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.

2. The use according to claim 1, wherein the polypeptide is polylysine-extended using 3, 4, 5, 6, 7, 8 or 9 lysines.

3. The use according to claim 2, wherein the lysine is linked to the amino terminus of the polypeptide.

4. The use according to any one of claims 1 to 3, wherein the polypeptide is: KKKKKKKKKK-VFGIELMEVDPIGHLYIFAT[SEQ ID NO:7]; and / or KKKKKKKKK-YLAMPFATPMEAELARRSLA [SEQ ID NO:8]; or: KKKKKK-VFGIELMEVDPIGHLYIFAT[SEQ ID NO:4]; and / or KKKKKK-YLAMPFATPMEAELARRSLA [SEQ ID NO:5].

5. The use according to any one of claims 1 to 3, wherein the adenovirus is a human adenovirus.

6. The use according to any one of claims 1 to 3, wherein the adenovirus comprises modifications in the E1 and / or E3 and / or E4 and / or L3 genes, the modifications comprising the insertion of a tumor-specific promoter, at least one gene deletion and the insertion of at least one transgene.

7. The use according to any one of claims 1 to 3, wherein the adenovirus is serotype 5.

8. The use according to any one of claims 1-3, wherein the adenovirus is further modified to include an Ad5 / 3 chimeric substitution, i.e., replacing the fiber knob region of adenovirus serotype 5 with the fiber knob region of adenovirus serotype 3.

9. The use according to any one of claims 1 to 3, wherein the adenovirus is further modified to include a deletion of the E1A gene, wherein the deletion is at least those nucleotides encoding amino acids 122-129.

10. The use according to any one of claims 1 to 3, wherein the adenovirus is further modified to include a partial or complete deletion of the 14.7k gene.

11. The use according to any one of claims 1 to 3, wherein the adenovirus is further modified to include the insertion of at least one transgene encoding a co-stimulatory molecule.

12. The use according to claim 11, wherein the virus comprises at least two transgenes, wherein one of the transgenes encodes a co-stimulatory molecule that activates the innate immune system and the other encodes a co-stimulatory molecule that activates the adaptive immune system.

13. The use according to any one of claims 1-3, wherein the transgene is human.

14. The use according to any one of claims 1-3, wherein the OX40L is located in the E3B region, replacing the gene 14.7K deletion.

15. The use according to claim 14, wherein the CD40L molecule is inserted immediately downstream of OX40L using the 2A processing site.

16. The use according to any one of claims 1-3, wherein the ratio of peptide to virus is in the range of 1 ug to 5 ug per 3E+9 virus particles.

17. The use according to any one of claims 1 to 16, wherein the at least one adenovirus or the pharmaceutical composition is formulated for administration together with a cellular checkpoint regulator.

18. The use according to claim 17, wherein the checkpoint regulator is an anti-PD1 molecule, an anti-PD-L1 molecule or an anti-CTLA-4 molecule.

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