Fusion protein for local activation of T cells

Abstract

The present invention provides oncolytic virus particles comprising a fusion protein and a nucleic acid construct encoding the fusion protein, preferably a nucleic acid construct encoding the fusion protein, wherein the fusion protein comprises or comprises, from the N-terminus to the C-terminus, a secretion leader peptide-anti-CD3 antibody section-hinge-HIV-Tat.

Description

[Technical Field]

[0001] The present invention relates to a viral particle containing a fusion protein for local activation of cytotoxic T cells, particularly for use in the treatment of solid tumors, and a nucleic acid construct encoding the fusion protein. The presence of the fusion protein in a tumor has the advantage of activating the immune response of cytotoxic T cells against the tumor, particularly T cells that are specifically targeted to tumor antigens. When the fusion protein of the present invention is expressed, for example, from a nucleic acid construct contained in a viral particle, particularly when expressed in tumor tissue, it has the advantage of activating T cells at the site of expression of the fusion protein with little or no activity to activate T cells outside the tumor where the fusion protein is expressed. The fusion protein of the present invention further has the advantage of binding to tissue, such as tumor tissue, and activating T cells at its site, independently of the presence of specific tumor-specific antigens in the tissue. Therefore, the fusion protein can be used in the treatment of tumors without determining tumor-specific antigens, and can be used in the treatment of tumors lacking tumor-specific antigens.

[0002] Preferably, the fusion protein of the present invention is for use in activating T cells in tissue, for example, for use in the treatment of tumors, and the fusion protein is present in the tissue (e.g., tumor tissue), expressed in the tissue, or introduced into the tissue, in order to activate T cells in the tissue.

[0003] The fusion protein is preferably encoded by a nucleic acid construct contained in an oncolytic virus particle, and as a result, administration of the virus particle to a human results in the dominant presence of the virus particle in or near tumor tissue, and the expression of the fusion protein in or near tumor tissue. Generally, the fusion protein is preferably encoded by a nucleic acid construct packaged in the virus particle for transient expression only. The virus particle is preferably an oncolytic virus. [Background technology]

[0004] Liao et al., Cancer Gene Therapy (2003) 10, 779-790 (Non-Patent Literature 1) describes the expression of a fusion protein containing anti-CD3-scFv, a linker, and a transmembrane domain in cells for the purpose of activating T cells in cells expressing the fusion protein, for use in tumor therapy. The fusion protein was produced in cultured cells by transient transfection with lipofectamine in vitro. This publication concludes that reducing the efflux of such fusion protein from cells expressing this fusion protein is important for activating T cells.

[0005] Liao et Roffler, Gene Therapy (2000) 7, 339-347 (Non-Patent Literature 2) describes the activation of cytotoxic T cells by expressing a fusion protein of the hinge region-transmembrane domain of anti-CD3-scFv-IgG and the cytoplasmic domain of CD80 as a surface-bound protein after transfection of tumor cells cultured in vitro.

[0006] WO2021 / 239586A1 (Patent Document 1) describes oncolytic viruses having inactivated E4 orf3 and mutated E4 orf4.

[0007] Kulkarni et al., Biochemical and Biophysical Research Communications 70-712 (2005) (Non-Patent Literature 3) describes the extracellular expression of a fusion protein consisting of Tat-LAMP (CD107b, a lysosome-associated membrane protein). The ability of surface-bound whole Tat to activate PBMCs was tested by expressing a Tat-LAMP fusion on HEK293 cells, and the addition of anti-CD3 increased PBMC activation.

[0008] Lapidot and Litovchick, Drug Development Research 502-515 (2000) (Non-Patent Literature 4) provides an overview of HIV-Tat antagonists for the treatment of AIDS.

[0009] Zauli et al., J Immunol 2216-2224 (1996) (Non-patent Literature 5) describes that co-immobilization of anti-CD3 antibody and total HIV-1 Tat on plastic induces the proliferation of CD4+ T cells.

[0010] In Chirmule et al., J Virol. 492-498 (1995) (Non-patent Literature 6), T cells pretreated with LiCl were brought into contact with total tat, and then tetanus antigen pulsed chemotherapeutic (APC) was added to analyze the HIV infection pathway.

[0011] Lim et al., PLOS ONE (2013) (Non-Patent Literature 7) describes the endocytosis of a cell membrane-permeable peptide fused to anti-Ras scFv and, for comparison, a Tat-scFv fusion.

[0012] A linker for use in fusion proteins is described in Chen et al., Adv Drug Deliv. Rev. 1357-1369 (2013) (Non-Patent Literature 8).

[0013] Urba et al., Cancer Res 2394-2401 (1992)(Non-Patent Document 9) relates to anti-CD3 antibodies for use in tumor treatment.

[0014] US2015 / 0098960A1 (Patent Document 2) describes a fusion protein for treating cancer, which contains the transcription factor domain of Tat, the cysteine-rich domain, and the C-terminal domain of Tat.

[0015] Wang et al., BMC Biotechnology (2017) 17:57 (Non-Patent Document 10) describes that the C-terminal fusion to the foldon domain results in a trimer and improves the catalytic activities of lichenase and xylanase.

Prior Art Documents

Patent Documents

[0016]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0017]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0018] Object of the invention The object of the present invention is to provide alternative, preferably improved, fusion proteins for use in the treatment of tumors, particularly for activating cytotoxic T cells, and carriers for fusion proteins for use in the treatment of tumors. [Means for solving the problem]

[0019] Description of the Invention The present invention achieves the above objective by, in particular, a fusion protein and a nucleic acid construct encoding the fusion protein, preferably an oncolytic virus particle comprising the nucleic acid construct encoding the fusion protein, wherein the fusion protein comprises a secretion leader peptide - anti-CD3 antibody section - hinge - from the N-terminus to the C-terminus. The anti-CD3 antibody section comprises or consists of HIV-Tat, optionally having a tag domain between the scFv and the hinge, and the anti-CD3 antibody section is preferably a single-chain antibody of the anti-CD3-scFv domain, for example, a variable light chain (VL), a linker, and a variable heavy chain (VH), such as VL-linker-VH or VH-linker-VL, where the linker is, for example, a Gly-Ser-linker, and the tag domain is, for example, a mycHis-tag containing a His oligomer for affinity purification, which can be detected by an anti-Myc antibody, and the HIV-Tat is a positively charged section of the HIV-Tat protein derived from HIV, and therefore here the protein transduction domain of this HIV-Tat is also referred to as the HIV-Tat domain, and is also simply referred to as HIV-Tat as a preferred representative of the protein transduction domain. In general, in this specification, the anti-CD3 antibody section is represented by the anti-CD3 scFv domain.

[0020] It has been found that the anti-CD3-scFv domain of the fusion protein leads to the activation of cytotoxic T cells within solid tumors and / or the infiltration of activated cytotoxic T cells into solid tumors, and that this activation is not limited to tumor cells expressing the fusion protein, but also occurs in the vicinity of tumor tissue where the tumor cells expressing the fusion protein are located. While the secretion of the fusion protein, caused by its secreted leader peptide and the absence of the transmembrane and intracellular domains, also leads to the diffusion of the fusion protein within tumor tissue, the HIV-Tat domain is thought to prevent the unrestricted distribution of the fusion protein and preferentially lead to the gradual diffusion of the fusion protein into tissues in the direct vicinity of the cells expressing the fusion protein. Currently, it is thought that the HIV-Tat domain leads to the fusion protein adhering to tissues, such as tumor tissue, after secretion from the expressing cells, and that the presence of the fusion protein activates cytotoxic T cells. Therefore, the fusion protein has the advantage of activating cytotoxic T cells even in tumor cells that are not transduction by viral particles to express the fusion protein and are only present in the vicinity of the cells expressing the fusion protein. The cell-binding properties of the fusion protein containing the HIV-Tat domain do not depend on the presence of a specific antigen in the tissue; that is, the cell-binding properties of the fusion protein are not limited to a specific tumor antigen; therefore, the expression of the fusion protein does not limit the broad applicability of the carrier viral particle encoding the fusion protein. Accordingly, the viral particle encoding the fusion protein, preferably an oncolytic virus, is suitable for use in activating T cells in tissues adjacent to cells infected with the viral particle, for example, in the treatment of tumors. Here, the localization of T cell activation is determined by the specificity of the viral particle, for example, specificity to the target tissue, for example, tumor tissue.

[0021] Since the fusion protein is secreted from cells infected with the virus encoding the fusion protein, the activity of the fusion protein in activating T cells, particularly in tissues near the infected cells, is independent of the type of virus.

[0022] Viral particles, preferably oncolytic viral particles, containing a nucleic acid construct encoding a fusion protein are used as carriers, resulting in the expression of the fusion protein by recipient human cells, preferably tumor cells. Alternatively, liposomes can be used as carriers, the liposomes containing the nucleic acid encoding the fusion protein. In one embodiment, the nucleic acid construct encoding the fusion protein is contained within the liposomes, the liposomes preferably containing a tumor-specific protein, for example, the liposomes may have tumor-specific ligands bound to the liposome membrane, such as antibodies specific to tumor-specific antigens. Alternatively or additionally, the liposomes may be intended for use in direct intratumoral injection.

[0023] In general, the protein sequences and domain arrangements of the fusion proteins described herein are described from the N-terminus to the C-terminus.

[0024] An exemplary fusion protein consisting of secretory leader peptide - anti-CD3-scFv - tag - hinge - HIV-Tat is, MALLLCFVLLCGVVDFARSLSASDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGG GSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSEQ KLISEEDLNMHTGHHHHHPRGPTIKPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVN NKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRTPGKGRKKRRQRRR (Sequence ID 1) contains anti-CD3 scFV as a VL-hinge-VH specific to human CD3, and has a 6xHis tag as an optional tag; therefore, the fusion protein may consist of amino acids 1-278 fused to amino acids 285-530 of Sequence ID 1. and MALLLCFVLLCGVVDFARSLSASDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYW GQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKEQ KLISEEDLNMHTGHHHHHPRGPTIKPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVN NKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRTPGKGRKKRRQRRR (SEQ ID NO: 3) contains anti-CD3 scFV as VH-hinge-VL specific to human CD3, and has a 6xHis tag as an optional tag, and therefore the fusion protein may consist of amino acids 1-273 fused to amino acids 285-566 of SEQ ID NO: 2. The fusion protein of the present invention may have an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with one or both of the sequences of SEQ ID NO: 1 and SEQ ID NO: 3, and may optionally have an additional oligomerized domain positioned between the hinge and HIV-Tat, preferably in combination with a hydrophilic spacer, for example, the fusion protein may have an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with one or both of the sequences of SEQ ID NO: 2 and SEQ ID NO: 4.

[0025] In one embodiment, the fusion protein may consist of a secretory leader peptide-anti-CD3 antibody section-hinge-PTD, for example, a secretory leader peptide-anti-CD3 antibody section-hinge-HIV-Tat.

[0026] In a preferred embodiment, the fusion protein contains an oligomerization domain between its hinge and HIV-Tat, which results in, for example, dimerization or trimerization of the secreted fusion protein. A preferred oligomerization domain is a fibrin domain. In this embodiment, the fusion protein comprises or consists of a secreted leader peptide-anti-CD3 antibody section-hinge-oligomerized domain-HIV-Tat, arranged from the N-terminus to the C-terminus, optionally having a tag domain between the scFv and the hinge, wherein the anti-CD3 antibody section is anti-CD3-scFv, which is a single-chain antibody of a variable light chain (VL), linker, and variable heavy chain (VH) in a VL-linker-VH or VH-linker-VL configuration, the linker being, for example, a Gly-Ser-linker, the tag domain being, for example, a MycHis-tag containing a His oligomer for affinity purification by metal ion affinity, which can be detected by an anti-Myc antibody, and the HIV-Tat is a positively charged section of the HIV-Tat domain derived from HIV.

[0027] An exemplary fusion protein consisting of a secretory leader peptide - anti-CD3-scFv - tag - hinge - oligomerized domain - HIV-Tat is, MALLLCFVLLCGVVDFARSLS ASDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSDIKLQQSGAELARP GASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSEQKLISEEDLNMHTGHHHHHHPRGPTIKPCP PCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQ LSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRTPGKGYIPEAPRDGQAYVRKDGEWVLLSTFLSPAGGGGSGGRKKRRQRRR (SEQ ID NO: 2) contains anti-CD3 scFV as a VH-hinge-VL specific to human CD3, has a 6xHis tag as an optional tag, has a fibrin domain as an oligomerization domain, and contains a hydrophilic spacer section between the oligomerization domain and HIV-Tat. and MALLLCFVLLCGVVDFARSLSASDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVS SGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKEQKLISEEDLNMHTGHHHHH HPRGPTIKPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYIL PPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRTPGKGYIPEAPRDGQAYVRKDGEWVLLSTFLSPAGGGGSGGRKKRRQRRR (SEQ ID NO: 4) contains anti-CD3 scFV as a VH-hinge-VL specific to human CD3, has a 6xHis tag as an optional tag, has a fibrin domain as an oligomerization domain, and contains a hydrophilic spacer section between the oligomerization domain and HIV-Tat. Generally, the arrangement of the hydrophilic spacer between the oligomerization domain and HIV-Tat is optional and preferred. The fibrin domain results in trimerization of the secreted fusion protein. It has been found that oligomerization of the fusion protein results in higher efficacy of T cell activation compared to fusion proteins without an oligomerization domain. As an alternative to the fibrin domain, GCN4, in particular its human homolog activating transcription factor 4 (ATF4), can be included in the fusion protein.

[0028] Generally, the aforementioned fusion proteins may lack a tag because the tag is only available as an epitope for analytical antibodies, or, in the case of a 6xHis tag, is only available to enable Ni-affinity purification of the fusion protein.

[0029] Generally, the hinge preferably has an amino acid sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with at least one of the amino acids 285...520 of SEQ ID NO: 1, 285...520 of SEQ ID NO: 2, 285...520 of SEQ ID NO: 3, and 285...520 of SEQ ID NO: 4.

[0030] Generally, the fibrin domain preferably has an amino acid sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with at least one of the amino acids 521-550 of SEQ ID NO: 2 and at least 90%, or at least 95%, of amino acids 521-550 of SEQ ID NO: 4.

[0031] Preferably, the HIV-Tat domain (also referred to here as the protein transduction domain) has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology with at least one of the amino acids 521-530 of SEQ ID NO: 1, 557-566 of SEQ ID NO: 2, 521-530 of SEQ ID NO: 3, and 557-566 of SEQ ID NO: 4. For the purposes of the present invention, the HIV-Tat domain also includes a peptide section of at least 5 amino acids or at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, for example, up to 20 amino acids in each case, for example, up to 19 amino acids, up to 18 amino acids, up to 17 amino acids, up to 16 amino acids, up to 15 amino acids, up to 14 amino acids, up to 13 amino acids, up to 12 amino acids, up to 11 amino acids, up to 10 amino acids, preferably 5 to 20 amino acids or 9 to 20 amino acids.

[0032] With respect to the object of the present invention, the HIV-Tat domain has at least 40%, preferably at least 50%, more preferably at least 60%, at least 70%, or at least 80% of amino acids, and / or at least 4, at least 5, or preferably at least 6 amino acids, each independently selected from the group Arg, Lys, and His, for example, arginine, lysine, or both arginine and lysine (optionally including histidine). For example, with respect to the object of the present invention, the HIV-Tat domain has 6 to 9 arginine, lysine, or combinations of arginine and lysine, and each optionally further comprises histidine, for example, 1 to 5 histidines. Furthermore, with respect to the object of the present invention, the HIV-Tat domain has the herpes simplex virus protein VP22 (SEQ ID NO: 81) and the protein transduction domain of Antennapedia homeodomain protein (AntP) (SEQ ID NO: 82). The HIV-Tat domain preferably has SEQ ID NO: 20.

[0033] For the purposes of the present invention, these peptide sections are included in the term HIV-Tat domain because, after secretion, these peptide sections in the fusion protein have the function of binding the fusion protein to cells in the vicinity of the secreting cell. Accordingly, for the purposes of the present invention, the HIV-Tat domain as defined herein may also be called a transduction domain.

[0034] Anti-CD3 scFv generally preferably has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology to one of the variable light chain sections of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or homology to one of the variable heavy chain sections of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0035] Selectively, the fusion protein is encoded on a nucleic acid construct under the translational control of the IRES (Internal Ribosome Entry Site), which is a combination of the IRES and the nucleic acid construct encoding the fusion protein, positioned at 3' of a viral protein, e.g., E1B. As a result, the transcription of the mRNA encoding the fusion protein is under the control of the viral promoter, e.g., the E1B promoter, and the translation of the fusion protein is IRES-dependent.

[0036] Preferably, the nucleic acid construct encoding the fusion protein of the present invention is placed in an expression unit that avoids potent expression leading to overexpression of the fusion protein. Preferred weak expression of the fusion protein can be achieved by an expression unit that combines a viral (e.g., E1B) promoter or a physiological promoter with an IRES motif for controlling the expression of the fusion protein of the present invention, resulting in a reduced expression level of the gene located at 3' of the IRES motif. In the combination of the promoter and the IRES that controls the translation of the nucleic acid encoding the fusion protein, the promoter can be any promoter, e.g., a potent, moderate, or weak promoter, e.g., a physiological or viral promoter. An exemplary potent promoter is the EF1α promoter. Alternatively, the fusion protein of the present invention can be expressed, preferably in human cells, preferably in human tumor cells, under the direct control of a weak promoter having lower activity than a potent physiological phosphoglycerate kinase (PGK) promoter, e.g., a promoter with the strength of a glycerol aldehyde-3-phosphate dehydrogenase promoter, or a tumor-specific promoter. Therefore, the nucleic acid section encoding the fusion protein is preferably under the direct control of a weak promoter, such as a phosphoenolpyruvate carboxykinase promoter, a glycerolaldehyde-3-phosphate dehydrogenase promoter, or a tumor-specific promoter, each preferably of human origin.

[0037] The sequence listing shows the amino acid sequence from the N-terminus to the C-terminus and the coding nucleic acid sequence from 5' to 3' of the fusion protein of the present invention, and in detail, Sequence ID 1 is a fusion protein (anti-CD3h-TAT) that has a secretion leader, a variable light chain-linker-variable heavy chain configuration, is directional to human CD3, has a Myc tag, a His tag, a hinge domain and HIV-Tat. Sequence ID 2 is a fusion protein (anti-CD3h-TAT-trimer) having a secretion leader, a variable light chain-linker-variable heavy chain configuration, an anti-CD3 scFv (anti-human CD3 scFv) that is directional to human CD3, a Myc tag, a His tag, a hinge domain, a fibrinin domain, a hydrophilic spacer, and HIV-Tat. Sequence ID 3 is a fusion protein (anti-CD3h-TAT) that has a secretion leader, a variable heavy chain-linker-variable light chain configuration, is directional to human CD3, has a Myc tag, a His tag, a hinge domain and HIV-Tat. Sequence ID 4 is a fusion protein (anti-CD3h-TAT-trimer) containing a secretion leader, a variable heavy chain-linker-variable light chain arrangement, an anti-CD3 scFv (anti-human CD3 scFv) that is directional to human CD3, a Myc tag, a His tag, a hinge domain, a fibrinin domain, a hydrophilic spacer, and HIV-Tat. Sequence ID 5 contains the coding DNA for the fusion protein (anti-CD3h-TAT) of Sequence ID 1. Sequence ID 6 contains the coding DNA for the fusion protein (anti-CD3h-TAT trimer) of Sequence ID 2. Sequence ID 7 contains the coding DNA for the fusion protein (anti-CD3h-TAT) of Sequence ID 3. Sequence ID 8 contains the coding DNA for the fusion protein of Sequence ID 4 (anti-CD3h-TAT trimer). Sequence ID 9 is a fusion protein (anti-CD3m-TAT) that has a secretion leader, a variable light chain-linker-variable heavy chain configuration, is directional to mouse CD3, has a Myc tag, a His tag, a hinge domain and HIV-Tat. Sequence ID No. 10 is a fusion protein (anti-CD3m-TAT-trimer) having a secretion leader, a variable light chain-linker-variable heavy chain configuration, an anti-CD3 scFv (anti-mouse CD3 scFv) that is directional to mouse CD3, a Myc tag, a His tag, a hinge domain, a fibrinin domain, a hydrophilic spacer, and HIV-Tat. Sequence ID 11 contains the coding DNA for the fusion protein (anti-CD3m-TAT) of Sequence ID 9. Sequence ID 12 contains the coding DNA for the fusion protein (anti-CD3m-TAT) of Sequence ID 10. Sequence ID 13 describes the coding DNA of a preferred viral vector encoding the fusion protein of the present invention at nucleotide numbers 4339...5919, and the coding sequence of the fusion protein can be replaced by the coding sequence of any fusion protein of the present invention. Sequence ID 71 describes the coding DNA of a preferred viral vector encoding the fusion protein of the present invention at nucleotide numbers 4339...6027 in an embodiment containing a fibrin domain as a trimerizing domain, wherein the coding sequence of the fusion protein can be replaced with the coding sequence of any fusion protein of the present invention.

[0038] Oncolytic viral particles are, for example, herpes simplex virus, reovirus, Newcastle disease virus, varicella-stomatitis virus, parvovirus H1, measles virus, vaccinia virus, maraba virus, poliovirus, coxsackievirus, preferably adenovirus, in particular adenovirus having a deletion or inactivation of gene E1B5k and / or gene E1B19k, wherein such inactivation is introduced, for example, by mutation, particularly adenovirus serotype 5, adenovirus having a deletion in the Rb-binding domain of E1A (also referred to as delta-24 adenovirus or delta-922-947 adenovirus), and adenovirus containing a tumor-specific promoter that controls the expression of E1A (this is, for example, a telomerase promoter, AFP promoter, PSA promoter, DF1 / Muc promoter, tyrosinase promoter, HIF1α-dependent promoter or E2F / C-myc-dependent promoter).

[0039] A preferred oncolytic virus is the viral particle described in WO2021 / 239586A1 (the contents of which are incorporated herein by reference). A preferred embodiment of the oncolytic viral particle encoding the fusion protein of the present invention is an adenovirus, which preferably contains the nucleic acid sequence of Sequence ID No. 13, where the sequence encoding the fusion protein with anti-mouse CD3 scFv nucleotide numbers 4405-5985 can be optionally replaced by one embodiment of a fusion protein encoding an anti-human CD3 scFv, for example, a fusion protein containing anti-human CD3-scFv-linker-HIV-Tat (optionally including an oligomerized domain and / or hydrophilic spacer between the linker and the HIV-Tat domain). Preferably, the DNA-coding adenovirus comprises, from 5' to 3', a left-inverted end repeat (L-ITR), a first promoter, an E1A coding sequence, an E1B promoter controlling the E1B coding sequence, an expression cassette for at least one effector molecule, preferably an expression cassette encoding inhibitory RNA (RNAi) specific to RNAi target sites within the E1A coding sequence and / or the E1B promoter and / or the E1B coding sequence, a vector backbone sequence, and on the opposite DNA strand, an expression cassette for E4 containing orf4, an expression cassette for E4 containing a promoter driving the expression of E4 in a functional relationship, and a right-inverted end repeat (R-ITR). The arrangement of the L-ITR, promoters controlling the expression of E1A and E1B, preferably the arrangement of the expression cassettes for at least one effector molecule on one DNA strand, and Preferably, the arrangement of the coding sequence for inhibitory RNA under the control of a promoter activated by the presence of p53 on one DNA strand, such as the prMinRGC promoter, and the arrangement of the expression cassette for E4 on the opposite DNA strand correspond to the arrangement of the R-ITR, the E4-promoter, and the arrangement of orf constituting the coding sequence of E4 from 5' to 3' on the opposite strand. The expression cassette for the E4-orf4 coding protein is arranged on the DNA strand opposite to the strand encoding the L-ITR, the first promoter, the E1A coding sequence, and the E1B promoter and the E1B coding sequence. The L-ITR is located at one end of the DNA, and the R-ITR is located at the opposite end of the DNA from the L-ITR.

[0040] The first promoter is preferably CMV (nucleotide numbers 380-625 of SEQ ID NO: 13), which has the advantage of enabling effective inhibition of the E1A-coding sequence, the E1B promoter, and the E1B-coding sequence by an inhibitory RNA molecule that is activated with a time delay following cell infection and expressed under the control of prMinRGC.

[0041] Preferably, the coding sequence of E1A, the E1B promoter, and the coding sequence of E1B contain an RNAi target site for binding inhibitory RNA, such as shRNA, and the adenovirus encoding DNA contains an expression cassette for the inhibitory RNA, such as shRNA, that binds to the RNAi target site, and the expression cassette is preferably under the control of a promoter activated by the presence of p53, such as the promoter prMinRGC. The coding sequences of inhibitory RNAs that inhibit the expression of adenovirus genes, such as E1A, E1B, E4, pTP, and Pol, are preferably expressed under the control of the prMinRGC promoter. The prMinRGC promoter is preferred as a promoter activated by the presence of p53.

[0042] Preferably, the virus particle is encoded by the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 71 and includes it.

[0043] As an alternative to viral particles, nucleic acid constructs encoding fusion proteins can be contained within liposomes used as carriers, or the nucleic acid constructs can be associated with synthetic carrier particles. Generally, carriers containing nucleic acid constructs encoding fusion proteins are provided in formulations suitable for direct injection into tumor tissue, and such carriers are preferably used for direct injection into tumor tissue.

[0044] Generally, the fusion protein is a soluble protein, meaning it lacks a transmembrane domain, and it is secreted from a cell containing a nucleic acid construct that encodes the fusion protein.

[0045] The present invention will be described by example with reference to the drawings. [Brief explanation of the drawing]

[0046] - Figures 1A-1D show the results of flow cytometry (FACS) for the activation markers CD25 and CD69 on mouse CD4+ T cells and CD8+ T cells in the presence of the fusion protein of the present invention for CD90.2+ leukocytes.

[0047] - Figure 2 shows the rate of mouse cancer cell death by T cells in the presence of the fusion protein of the present invention.

[0048] - Figures 3A to 3D show data regarding the binding of the fusion protein of the present invention to human CD4+ T cells.

[0049] - Figures 4A to 4E show data regarding the binding of the fusion protein of the present invention to human CD8+ T cells.

[0050] - Figures 5A-5B show data on the activation of both human CD4+ T cells and CD8+ T cells in relation to anti-CD69 antibody staining, based on embodiments of the fusion protein.

[0051] - Figures 6A-6B show intracellular cytokine staining related to TNFα cytokine production by human CD4+ and CD8+ T cells induced by fusion proteins.

[0052] - Figure 7A shows a schematic of a nucleic acid sequence encoding a preferred viral particle having the fusion protein of the present invention. - Figure 7B shows data regarding increased tumor regression in mice caused by the expression of the fusion protein of the present invention. - Figure 7C shows data regarding the improvement in survival of tumor-bearing mice caused by the expression of the fusion protein of the present invention.

[0053] - Figure 8A shows a schematic of the nucleic acid construct for the expression of the fusion protein of the present invention. - Figure 8B is a Western blot of the culture supernatant of 293T cells expressing the fusion protein of the present invention under the control of a weak or strong promoter. - Figure 8C shows data regarding the improvement in survival of tumor-bearing mice caused by the expression of the fusion protein of the present invention.

[0054] - Figure 9A shows data regarding increased tumor regression in mice caused by the expression of the fusion protein of the present invention. - Figure 9B shows data regarding the improvement in survival of tumor-bearing mice caused by the expression of the fusion protein of the present invention.

[0055] - Figure 10A shows data regarding the activation markers in mouse splenocytes after the addition of the fusion protein of the present invention, or the activation markers when a comparative protein lacking the TAT protein transduction domain is used. - Figure 10B shows data regarding the activation markers in mouse splenocytes after the addition of the fusion protein of the present invention, or the activation markers when a comparative protein lacking the TAT protein transduction domain is used. - Figure 10C shows data regarding the activation markers in mouse splenocytes after the addition of the fusion protein of the present invention, or the activation markers when a comparative protein lacking the TAT protein transduction domain is used. - Figure 10D shows data regarding the activation markers in mouse splenocytes after the addition of the fusion protein of the present invention, or the activation markers when a comparative protein lacking the TAT protein transduction domain is used.

[0056] - Figure 11A shows data regarding the activation marker of spleen cells after the addition of the fusion protein of the present invention, or the activation marker when a comparative protein lacking the anti-CD3 domain is used. - Figure 11B shows data regarding the activation marker of spleen cells after the addition of the fusion protein of the present invention, or the activation marker when a comparative protein lacking the anti-CD3 domain is used. - Figure 11C shows data regarding the activation markers of splenocytes after the addition of the fusion protein of the present invention, or the activation markers when a comparative protein lacking the anti-CD3 domain is used. - Figure 11D shows data regarding the activation marker of spleen cells after the addition of the fusion protein of the present invention, or the activation marker when a comparative protein lacking the anti-CD3 domain is used.

[0057] - Figure 12A shows data regarding the functional activation marker of spleen cells after the addition of the fusion protein of the present invention, or the functional activation marker when a comparative protein lacking the anti-CD3 domain is used. - Figure 12B shows data regarding the activation marker of spleen cells after the addition of the fusion protein of the present invention, or the activation marker when a comparative protein lacking the anti-CD3 domain is used.

[0058] - Figure 13A shows a schematic of in vivo tumor therapy using viral particles expressing the fusion protein of the present invention, followed by the administration of an anti-PD1 antibody. - Figures 13B, 13C, and 13D show individual tumor sizes for comparative therapies, including therapy using a virus expressing the fusion protein of the present invention. - Figure 13E shows the survival rate, and Figure 13F shows the average tumor size of these animals.

[0059] - Figure 14A is a micrograph of tumor tissue after treatment with αPD1 alone. - Figure 14B is a micrograph of tumor tissue after treatment with αPD1 and empty oncolytic viruses. - Figure 14C is a micrograph of tumor tissue after treatment with αPD1 and an oncolytic virus expressing the fusion protein of the present invention. - Figures 14D, 14E, and 14F show the results of spatial analysis of CD8+ T cells against tumor cells (PC+). - Figures 14G, 14H, and 14I show the results of spatial analysis of CD4+ T cells against tumor cells (PC+). - Figure 14J shows the median distance from tumor cells to CD8+ T cells. - Figure 14K shows the median distance from tumor cells to CD4+ T cells. - Figure 14L is a micrograph of tumor tissue after treatment with αPD1 antibody alone. - Figure 14M is a micrograph of tumor tissue after treatment with αPD1 antibody and empty oncolytic virus. - Figure 14N is a micrograph of tumor tissue after treatment with an αPD1 antibody combined with an oncolytic virus encoding the fusion protein of the present invention. - Figure 14O shows the percentage of CD8+ T cells that have been in direct contact with other CD8+ T cells. - Figure 14P shows the percentage of CD8+ T cells that are in close proximity to other CD8+ T cells. - Figure 14Q shows the proportion of CD8+ T cells that are similar to other CD8+ T cells. - Figure 14R shows the percentage of CD4+ T cells in direct contact with CD8+ T cells. - Figure 14S shows the proportion of CD4+ T cells located in close proximity to CD8+ T cells. - Figure 14T represents the proportion of CD4+ T cells that are similar to CD8+ T cells. - Figure 14U shows the median distance from CD8+ T cells to CD4+ T cells. - Figure 14V shows the median distance between CD8+ T cells.

[0060] In the figure, * indicates significant, ** indicates more significant, *** indicates even more significant, and ns indicates not significant. In the example, the viral particle is based on oncolytic adenovirus Ad5 / 11 having a ΔE1A deletion and p53-dependent expression of RNAi, essentially resulting in viral protein expression in p53-deficient tumor cells and limited levels of viral protein in p53-normal cells. This preferred viral particle is encoded by or derived from Sequence ID No. 13, and the coding sequence for the fusion protein from nucleotides 4405 to 5985 can be replaced by a nucleotide sequence encoding an embodiment of the fusion protein of the present invention, for example, one of Sequence ID No. 1, Sequence ID No. 2, Sequence ID No. 3, and Sequence ID No. 4. [Examples]

[0061] Example 1: Activation of T cells in vitro due to the presence of fusion proteins As an example of a fusion protein without an oligomerization domain, a fusion protein consisting of a secretory leader peptide-mouse anti-CD3-scFv-hinge-HIV-Tat was produced in HEK-293 cells. Separately, as an example of a fusion protein containing an oligomerization domain, a fusion protein consisting of a secretory leader peptide-mouse anti-CD3-scFv-tag-hinge-oligomerization domain-HIV-Tat was produced in HEK-293 cells. These fusion proteins each contained an amino acid sequence corresponding to SEQ ID NO: 9 (e.g., encoded by SEQ ID NO: 11) or an amino acid sequence corresponding to SEQ ID NO: 10 (e.g., encoded by SEQ ID NO: 12), respectively, with anti-mouse CD3 scFv instead of anti-human CD3 scFv in SEQ ID NOs: 1 and 3.

[0062] Briefly, HEK-293 cells were cultured and transfected with polyethyleneimine (PEI) using a nucleic acid construct containing SEQ ID NO: 11 or SEQ ID NO: 12 encoding one of the fusion proteins, and then cultured for 72 hours. The fusion proteins were isolated from the cell culture supernatant by Ni-affinity chromatography using a Ni-NTA column. The purified fusion proteins were added to separate aliquots of newly isolated mouse splenocytes and incubated under cell culture conditions for 24 hours. Briefly, splenocytes were isolated from mouse spleens, and one of the fusion proteins was added to 1 mL of splenocyte culture medium at amounts of 0.5 μg, 1 μg, 5 μg, or 10 μg, respectively.

[0063] After incubating splenocytes with the fusion protein, the splenocytes were analyzed by flow cytometry (FACS) for the activation markers CD25 and CD69 on CD4+ T cells and CD8+ T cells in CD90.2+ leukocytes. The fusion protein (αCD3) does not contain the oligomerized domain. TAT ), and a fusion protein containing a fibrin oligomerization domain (αCD3 TATThe results for the trimer (CD4+ T cells) are shown in Figures 1A and 1B, and for CD8+ T cells in Figures 1C and 1D. The results show that the percentage of both CD4+ and CD8+ T cells with activated CD25+ and CD69+ increased with increasing concentration of the added fusion protein. Furthermore, it is shown that the trimerized form of the fusion protein containing the fibrintin oligomerization domain induced increased activation in both CD4+ and CD8+ T cells at the same protein concentration.

[0064] Furthermore, one of the fusion proteins consisting of secretory leader peptide-anti-mouse CD3-scFv-hinge-HIV-Tat, or secretory leader peptide-anti-mouse CD3-scFv-tag-hinge-oligomerized domain-HIV-Tat, was added to 1 × 10⁻¹⁰ 6 One newly isolated mouse splenocyte was added in a total amount of 1 μg in 1 mL of culture medium, resulting in 1 × 10⁶ cells. 5 In addition to individual mouse colon cancer cells (MC38), the effector:target cell ratio was set to 10:1. After 24 hours of co-incubation under cell culture conditions, the number of surviving cancer cells was determined by flow cytometry. Figure 2 shows that both fusion proteins resulted in increased cancer cell death, and that the fusion protein containing the oligomerized domain resulted in increased tumor cell death. Regarding MC38 tumor cells, the fusion protein containing the fibrin oligomerized domain (Figure 2: MC38 + αCD3) TAT -Trimer) is an embodiment that does not contain the oligomerized domain (secretionary leader peptide-anti-mouse CD3-scFv-hinge-HIV-TAT, Figure 2: MC38+αCD3 TAT The same protein concentration was used as in the previous example. Therefore, this effective activation of T cells showed a significantly higher molar activation effect with respect to embodiments containing the oligomerized domain.

[0065] Example 2: Activation of human T cells by fusion proteins from PBMCs Human T cells were isolated from human whole blood as PBMCs by pancoll gradient centrifugation as the buffy coat layer. Secreted leader peptide - anti - human CD3 - scFv - hinge - HIV - Tat (SEQ ID NO: 1, αCD3h TAT , or alternatively SEQ ID NO: 3), or secreted leader peptide - anti - human CD3 - scFv - tag - hinge - oligomerization domain - HIV - Tat (SEQ ID NO: 2, αCD3h TAT - trimer, or alternatively SEQ ID NO: 4). After incubating PBMCs for 24 hours in 1 mL of cell culture medium containing 1 μg of the fusion protein, activation of human T cells was analyzed by measuring CD69 as an activation marker by FACS on CD4+ and CD8+ T cells. During FACS, in addition to the labeled anti - CD69 antibody, labeled anti - CD4 antibody, anti - CD8 antibody, anti - CCR7 antibody and anti - CD45RA antibody were used, TNFα was determined by intracellular immunocytokine staining (ICS), and anti - c - myc antibody was used for specific staining of the fusion protein.

[0066] The FACS results shown in FIGS. 3A - D indicate that subtypes of human CD4+ T cells, specifically naive T cells, T effectors and central memory T cells, associated with the fusion protein as shown by the binding of the anti - c - myc antibody.

[0067] The FACS results shown in FIGS. 4A - E indicate that subtypes of human CD8+ T cells, specifically naive T cells, T effectors, T EMRA and central memory T cells, also associated with the fusion protein as shown by the binding of the anti - c - myc antibody.

[0068] The FACS results shown in FIGS. 5A - B regarding anti - CD69 antibody staining indicate that both CD4+ and CD8+ T cells were secreted leader peptide - anti - human CD3 - scFv - hinge - HIV - Tat (SEQ ID NO: 1, hMATE TAT), or secretory leader peptide-mouse anti-CD3-scFv-hinge-HIV-Tat, or secretory leader peptide-anti-human CD3-scFv-tag-hinge-oligomerized domain-HIV-Tat (SEQ ID NO: 2, hMATE TAT This indicates that each embodiment of the fusion protein (consisting of a trimer) was activated.

[0069] The FACS results shown in Figures 6A-B, concerning intracellular cytokine staining of TNFα (TNFa), indicate cytokine production by CD4+ and CD8+ T cells activated by one of the aforementioned fusion proteins.

[0070] In summary, these results demonstrate that the fusion protein of the present invention effectively binds to CD4+ and CD8+ T cells, including several subtypes of T cells, and that the binding of the fusion protein potently activates both CD4+ and CD8+ T cells (including activation of cytotoxic cytokine production).

[0071] Example 3: Treatment of solid tumors with oncolytic adenovirus expressing fusion proteins As a generally preferred example of an oncolytic virus, also called an oncolytic viral particle, we used adenovirus serotype 5 (Ad5 / 11-p53), which, in normal non-cancerous cells, expresses shRNA that is directional to a binding site in its nucleic acid construct, positioned to avoid the expression of the viral gene in normal cells, depending on the presence of p53, while in tumor cells with lower p53 expression, it allows the expression and replication of the encoded gene. Furthermore, this oncolytic virus had an inactivated E4 orf3 locus and a mutant E4 orf4. A preferred embodiment of this oncolytic virus is encoded by SEQ ID NO: 13, which encodes the fusion protein of the present invention in an embodiment that does not include an oligomerization domain. This oncolytic virus was found to be specific to tumor cells, particularly for use in the treatment of tumors. The preferred oncolytic viral particle is adenovirus Ad5 / 11, which contains a nucleic acid sequence encoding a fusion protein, in this case encoding a fusion protein containing anti-mouse CD3 scFv to bind to T cells of experimental mice. In this example, the fusion protein consisted of a secreted leader peptide-anti-mouse CD3-scFv-hinge-HIV-Tat, as an exemplary embodiment. A schematic of the nucleic acid sequence encoding the viral particle is shown in Figure 7A. It is shown that the sequence encoding the fusion protein is under the translational control of IRES and is transcriptionally regulated by the E1B promoter. Generally favorably, this viral particle, which depends on the presence of p53, generates shRNA that inhibits the replication of the viral particle itself, for example, in healthy tissue expressing p53.

[0072] As an example of treatment for solid tumors, 1 × 10⁶ mice were given to immunonormal C57BL / 6 mice. 7 Individual syngeneic colon adenocarcinoma cells (MC38) were subcutaneously injected into the right flank, and the mouse was then subjected to, for example, a 200 mm slab. 3 The patient was kept under surveillance for 6 days until tumor growth was detected.

[0073] Virus particle (Ad5 / 11p53-αCD3) according to Sequence ID No. 13 TATFor comparison, physiological saline (NaCl) or virus particles without the fusion protein (Ad5 / 11p53) were used as negative controls. This corresponds to SEQ ID NO: 13 but does not contain the coding sequence of the fusion protein (i.e., it does not contain nucleotide numbers 4405-5985 of SEQ ID NO: 13). Adenoviruses containing the DNA of SEQ ID NO: 13 and comparative adenoviruses were produced separately, for example, in cultured HEK293 cells. Virus particles were isolated from the culture supernatant and 1 × 10⁶ 9 It was injected into the tumor as a single dose.

[0074] Figure 7B shows tumor volume in mice, demonstrating that the comparative adenovirus (Ad5 / 11p53) resulted in reduced tumor growth compared to physiological saline (NaCl), as well as the viral particles encoding the fusion protein of the present invention (Ad5 / 11p53-αCD3 TAT This indicates that it resulted in a significant reduction in tumor growth compared to comparative adenoviruses.

[0075] Figure 7C shows the treatment of mice with tumors with physiological saline (control), a comparative adenovirus (Ad5 / 11p53) that does not encode a fusion protein, and a viral particle (SEQ ID NO: 13, Ad5 / 11p53-αCD3) that encodes a fusion protein but is otherwise identical. TAT This shows the survival rate after treatment at ).

[0076] Comparison of the antitumor effect of viral particles encoding the fusion protein with that of adenoviruses shows that the fusion protein significantly increases the antitumor activity of viral particles.

[0077] Example 4: Treatment of solid tumors with oncolytic adenovirus expressing fusion proteins under the control of a weak promoter. A generally preferred example of an oncolytic virus, also called an oncolytic virus particle, is adenovirus serotype 5 (Ad5 / 11p53-αCD3), which encodes the fusion protein of the present invention, represented by SEQ ID NO: 13. TATThe following was used to test the effect of promoter strength on the transcription of the nucleic acid sequence encoding the fusion protein. The CMV promoter was placed to control the transcription of the DNA section encoding the fusion protein as a representative of a strong promoter, by replacing the IRES in SEQ ID NO: 13, which is positioned at 5' to the DNA section encoding the fusion protein. Using an expression unit containing the IRES-motif positioned at 5' to the nucleic acid section encoding the fusion protein, weak expression similar to that achieved when a weak promoter controlling the transcription of the DNA section encoding the fusion protein, as shown in SEQ ID NO: 13, was achieved. As a further example, the DNA section encoding the fusion protein in SEQ ID NO: 13 was replaced with a section encoding the fusion protein containing an oligomerized domain, according to amino acid SEQ ID NO: 2 or SEQ ID NO: 4. These are under the control of IRES in each embodiment.

[0078] Figure 8A shows a fusion protein that does not contain an oligomerized domain, namely a fusion protein consisting of secretion leader-anti-human CD3 scFv-MycTag-HisTag-hinge-HIV-Tat (αCD3 TAT For example, to control the transcription of SEQ ID NO: 1 or SEQ ID NO: 3, a nucleic acid construct containing the CMV promoter (CMV, hTertAd) for strong expression, or a nucleic acid construct containing IRES (Ad5 / 11-p53) for weak expression, or a fusion protein containing an oligomeric domain, i.e., secretion leader-anti-human CD3 scFv-MycTag-HisTag-hinge-fibrintin domain-hydrophilic spacer-HIV-Tat(αCD3) TAT This schematically represents a nucleic acid construct under the control of a weak promoter that controls the transcription of a fusion protein consisting of a trimer (e.g., SEQ ID NO: 2 or SEQ ID NO: 4) (where the fibrin domain represents the oligomerization domain and the hydrophilic spacer is optional). A preferred embodiment of this oncolytic virus is encoded by SEQ ID NO: 71, which encodes the fusion protein of the present invention in an embodiment in which the fibrin domain is included as the oligomerization domain.

[0079] The upper photograph in Figure 8B shows a Western blot of the culture supernatant of HEK293 cells 48 hours after infection with viral particles encoding the fusion protein of the present invention, using immunodetection with labeled anti-Myc antibody. For each 50 μL protein sample of supernatant, the Western blot shows in lane 1 the viral particles encoding the fusion protein (Ad5 / 11p53-αCD3) under the control of the E1B promoter and IRES-motif for the expression of the fusion protein of the present invention. TAT ), in lane 2, as a control, a similar viral particle (Ad5 / 11p53) that does not encode the fusion protein of the present invention, and in lane 3, a viral particle (hTertAd-αCD3) that encodes the fusion protein under the direct control of a strong promoter represented by the CMV promoter. TAT ), and in lane 4, a similar viral particle (hTertAd) containing a strong promoter (CMV promoter) that does not encode the fusion protein of the present invention is shown as a control. This indicates that while viral particles encoding the fusion protein under the control of a strong promoter result in high expression of the fusion protein, in this experiment, expression of the fusion protein under the control of the E1B promoter and IRES motif is not sufficient for direct detection in the supernatant and shows low expression levels.

[0080] The lower photograph in Figure 8B shows a Western blot of a protein sample obtained from the cell culture supernatant after protein purification by metal ion affinity chromatography for targeted enrichment of the fusion protein. Supernatant was obtained from 293T cells 48 hours after infection with viral particles encoding the fusion protein of the present invention, and immunodetection was performed using labeled anti-Myc antibody. For a 50 μL sample of purified protein, lane 1 (unlabeled) shows purified protein from an empty virus (Ad5 / 11p53) lacking expression of the fusion protein, and lane 2 shows viral particles (Ad5 / 11p53-αCD3) encoding the fusion protein under the control of the E1B promoter / IRES of the present invention. TATLane 3 encodes a fusion protein containing an oligomerized domain in a viral particle (Ad5 / 11p53-αCD3) after infection with the purified protein in the E1B promoter / IRES of the present invention. TAT - Indicates a trimer.

[0081] This positive detection after enriching the fusion protein of the present invention demonstrates that the fusion protein of the present invention, when expressed under the control of a weak expression unit (E1B promoter linked to IRES), is present in a soluble form in the supernatant of infected cells, for both embodiments without the oligomerization domain and embodiments including the oligomerization domain.

[0082] Figure 8C shows the survival rates of C57BL / 6 mice with the MC38 tumor described in Example 3 that received intratumoral injection of these viral particles. The results demonstrate that expression of the fusion protein of the present invention under the control of a weak promoter results in dramatically improved survival rates, i.e., tumor therapy, compared to treatment with oncolytic viral particles encoding the same fusion protein under the control of a strong promoter, and compared to oncolytic viral particles without the fusion protein (SEQ ID NO: 13, which lacks the nucleic acid sequence encoding the fusion protein).

[0083] Example 5: Treatment of solid tumors with oncolytic adenovirus expressing fusion proteins A generally preferred example of an oncolytic virus, also called an oncolytic virus particle, is adenovirus serotype 5 encoding the fusion protein of the present invention, represented by SEQ ID NO: 13, which does not contain the oligomerized domain of the fusion protein of the present invention (αCD3 TAT Embodiments encoding (αCD3), and a fusion protein (αCD3) containing an oligomerized domain. TAT -In the trimer embodiment, C57BL / 6 mice with MC38 tumors were used for testing, as described in Example 3.

[0084] The viral particles are based on the oncolytic adenovirus Ad5 / 11, which has a ΔE1A deletion and p53-dependent expression of RNAi according to SEQ ID NO: 13, and encode a fusion protein that does not contain an oligomerized domain (for example, according to SEQ ID NO: 9 (Ad5 / 11p53ΔE1A-αCD3) TAT )), or encoding a fusion protein containing an oligomerized domain (for example, by SEQ ID NO: 10 (Ad5 / 11p53ΔE1A-αCD3 TAT -Trimer). As negative controls, 0.9% physiological saline (NaCl) and viral particles (Ad5 / 11p53ΔE1A) that did not contain the coding sequence of the fusion protein were used.

[0085] Figure 9A shows that tumor volume was significantly lower with respect to viral particles expressing one of the fusion proteins of the present invention, and Figure 9B shows that survival rate, i.e., tumor treatment, was significantly improved with respect to viral particles expressing one of the fusion proteins of the present invention, and that fusion proteins containing oligomerized domains showed even better efficacy.

[0086] Example 6: Activation of spleen cells in vitro As a representative example, a fusion protein consisting of the secretory leader peptide of SEQ ID NO: 1, mouse anti-CD3 scFv-hinge, and HIV-Tat (the HIV-Tat domain is the protein transduction domain (Tat-PTD) of HIV-Tat in SEQ ID NO: 20) was used. For comparison, a fusion protein consisting of the secretory leader peptide, mouse anti-CD3 scFv-hinge, corresponding to amino acid numbers 1...520 of SEQ ID NO: 1, i.e., lacking the protein transduction domain, was used. For further comparison, trimerized variants of the fusion protein of the present invention were tested against trimerized scFv.

[0087] In short, newly isolated mouse splenocytes were incubated with 1 μg of purified fusion protein (MATE) consisting of secretory leader peptide-mouse anti-CD3 scFv-hinge-HIV-Tat, or with a comparative fusion protein lacking a protein transduction domain (PTD). After 24 hours of incubation, T cells were immunostained for the activation markers CD25 and CD69 and measured by flow cytometry, and IFNγ expression was assayed using ICS.

[0088] The results are shown in Figure 10. These show that while spleen cells without protein supplementation (control) were not activated, i.e., only background activity was observed, the fusion protein of the present invention (αCD3TAT) containing the HIV-Tat PTD showed significantly higher activation activity compared to αCD3 scFv alone (αCD3) (i.e., without PTD), which showed very slight activation. Correspondingly, the trimerized fusion protein of the present invention (αCD3TAT-trimer) containing HIV-Tat as the PTD also showed significantly higher activation activity compared to trimerized αCD3 scFv alone (αCD3) (i.e., without PTD). Activation is shown in Figure 10A as the proportion of CD4+ cells relative to CD25+ and CD69+ cells, in Figure 10B as the proportion of CD4+ cells relative to IFNγ-positive cells, in Figure 10C as the proportion of CD8+ cells relative to CD25+ and CD69+ cells, and in Figure 10D as the proportion of CD4+ cells relative to IFNγ-positive cells.

[0089] This demonstrates that the PTD domain of the fusion protein of the present invention contributes significantly to the activation of splenic cells, while the activity of αCD3 scFv alone in activating splenic cells is considerably small.

[0090] As a further control, the fusion protein (αCD3-TAT) or its trimerized variant (αCD3-TAT-trimer) was tested for activation of mouse splenocytes compared to a protein lacking the αCD3-scFv (Hinge-TAT) (which corresponds to SEQ ID NO: 1 lacking amino acids 1...21, i.e., amino acids 22...278, directly linked to amino acids 279...530 in SEQ ID NO: 1). The results are shown in Figure 11, demonstrating that proteins lacking the CD3-binding scFv domain, i.e., the PTD domain and Hinge, do not result in T cell activation. Figure 11A shows the percentage of CD4+ cells among CD25+ cells, indicating that background activation by a negative assay (control) without protein addition was achieved by comparative Hinge-TAT, while the fusion protein (αCD3TAT) and trimerized fusion protein (αCD3TAT-trimer) of the present invention resulted in effective activation. Figure 11B shows the results for CD8+ cells compared to CD69+ cells, Figure 11C shows the results for CD25+ cells, and Figure 11D shows the results for CD69+ cells.

[0091] Figure 12A shows the percentage of CD4+ cells among IFNγ-positive cells, and Figure 12B shows the percentage of CD8+ cells among IFNγ-positive cells.

[0092] The results in Figure 10, as well as Figures 11 and 12, confirm that only the fusion protein of the present invention results in effective activation of CD4+ and CD8+ cells, while the comparative scFv alone or comparative PTD alone results in only very slight or background activation.

[0093] Example 7: Use of the fusion protein of the present invention in combination with an αPD1 antibody in tumor therapy As shown in Figure 13A, MC38 subcutaneous tumors were implanted in mice on day 0, and 10 9Individual adenovirus serotype 5 (Ad5 / 11-p53) virus particles or virus particles expressing the fusion protein of the present invention used in Example 3 were administered intratumorally (it) on day 5, followed by administration of 75 μg of anti-PD1 antibody (αPD1) on day 6, repeated twice a week on the indicated days. Survival statistics were calculated using the log-rank (Mantel-Cox) test. Significance of tumor volume measurements was examined using a two-sided, unpaired t-test. * The evaluation was performed using p ≤ 0.05. The growth of individual tumors is shown on the right (Figures 13B, 13C, 13D).

[0094] Figure 13B shows the growth of individual tumors in different mice treated with control therapy using 0.9% NaCl and αPD1 alone, Figure 13C shows the individual tumor volumes of mice treated with αPD1 in addition to empty viral particles (Ad5 / 11) for comparison, and Figure 13D shows the tumor volumes of mice treated with viral particles expressing the fusion protein of the present invention (Ad5 / 11-αCD3-TAT-trimer) in combination with αPD1. Except for one of the five animals, tumor growth was effectively suppressed by exemplary oncolytic viruses expressing the fusion protein of the present invention in combination with subsequent administration of αPD1, while treatment with αPD1 alone (NaCl 0.9% it) slowed tumor growth, and αPD1 treatment in combination with pre-administration of empty viral particles (Ad5 / 11) had only a slight positive effect on tumor growth.

[0095] Figure 13E shows the survival rate, and Figure 13F shows the average tumor volume for oncology using anti-PD1 antibody alone (αPD1 + 0.9% NaCl) or anti-PD1 combined with empty virus particles (αPD1 + Ad5 / 11) for comparison, as well as for a combination of anti-PD1 antibody and the fusion protein of the present invention expressed therefrom by oncolytic virus particles (αPD1 + Ad5 / 11-αCD3TAT-trimer).

[0096] The results demonstrate that tumor growth and survival rates in αPD1 therapy for solid tumors were significantly improved by viral particles expressing the fusion protein of the present invention. Anti-PD1 antibodies, representative compounds that block the PD-1 / PD-L1 immune checkpoint, demonstrate that oncolytic viruses containing the fusion protein, particularly the nucleic acid construct encoding the fusion protein, are suitable for use in oncology therapies involving the administration of compounds that block the PD-1 / PD-L1 immune checkpoint.

[0097] Figure 14 shows that expression of the fusion protein of the present invention by oncolytic virus during αPD1 checkpoint blockade improves proximity between T cells and tumor cells, as well as the interaction between CD4+ and CD8+ T cells.

[0098] Subcutaneous MC38 tumors immobilized in mice were treated with single intratumor doses of Ad5 / 11-αCD3TAT-trimer, Ad5 / 11, and physiological saline, each in conjunction with two applications of the αPD1 antibody described above. The spatial phenotype of intratumor immune cells was evaluated by multiplex immunohistochemical staining of CD8+ T cells (yellow), CD4+ T cells (green), CD161+ NK cells (magenta), F4 / 80+ macrophages (white), pancadherin+ (PC+) tumor cells (red), and nuclei (DAPI / blue). Figures 14A, 14B, and 14C show spatial immunohistochemical analysis of the proximity between T cell subpopulations and tumor cells. Representative composite images from each group are shown in Figures 14A, 14B, and 14C. Cells within a maximum distance of 15 μm represent direct contact, close proximity is defined by a radius of up to 30 μm, and nearby cells are found within a radius of 50 μm. PC+ cells (tumor cells identified as pancadherin-positive) closest to each CD8+ T cell are marked with a line. Quantitative analysis of proximity between tumor cells and T cells is shown in Figures 14D, 14E, and 14F for CD8+ cells, and in Figures 14G, 14H, and 14I for CD4+ cells. These results indicate that, in combination with αPD1 antibody, the fusion protein of the present invention is more effective in bringing both CD8+ and CD4+ T cells closer to tumor cells and localizing them than αPD1 antibody alone or αPD1 antibody combined with empty viral particles (Ad5 / 11).

[0099] Figures 14J and 14K show the median distance of PC+ tumor cells to CD8+ T cells or CD4+ T cells. Spatial immunophenotypic analysis was used to examine T cell clustering within the tumor after treatment. Representative synthetic images are shown in Figures 14L, 14M, and 14N. Distance analysis between CD8+ cells is shown in Figures 14O, 14P, and 14Q. Figures 14R, 14S, and 14T show proximity analysis of CD8+ and CD4+ T cells. Figures 14U and 14V show the results of median distance measurements between CD8+ T cells and between CD8+ T cells and CD4+ T cells, respectively.

[0100] These data demonstrate that intratumoral expression of the fusion protein of the present invention promotes cluster formation of CD4+ and CD8+ T cells associated with the mutual stimulation of these immune cell types when expressed by oncolytic viruses in tumors.

Claims

1. A fusion protein comprising an anti-CD3 antibody section, characterized in that it comprises or consists of an anti-CD3 antibody section, a hinge, and a protein transduction domain (HIV-Tat domain) from the N-terminus to the C-terminus.

2. The fusion protein according to claim 1, characterized in that the protein transduction domain is a peptide section of at least 5 to 20 amino acids, for example, up to 19 amino acids, and the peptide section has at least 40% and / or at least 4 amino acids independently selected from the group of arginine, lysine, and histidine.

3. The fusion protein according to claim 1 or 2, characterized in that it preferably includes an oligomerized domain positioned between the hinge and the protein transduction domain in combination with a hydrophilic spacer.

4. The fusion protein according to any one of claims 1 to 3, characterized in that the oligomerized domain is a fibrin domain or a GCN4 domain.

5. The fusion protein according to claim 4, characterized in that the fibrin domain has an amino acid sequence that is at least 80% homologous or identical to at least one of amino acids 521-550 of SEQ ID NO: 2 and amino acids 521-550 of SEQ ID NO:

4.

6. The fusion protein according to any one of claims 1 to 5, characterized in that the hinge has an amino acid sequence that is at least 80% homologous or identical to at least one of the amino acids 285 to 520 of SEQ ID NO: 1, amino acids 285 to 520 of SEQ ID NO: 2, amino acids 285 to 520 of SEQ ID NO: 3, and amino acids 285 to 520 of SEQ ID NO:

4.

7. The fusion protein according to any one of claims 1 to 6, characterized in that the protein transduction domain has at least 80% homology or identity with at least one of the amino acids 521 to 530 of SEQ ID NO: 1, amino acids 557 to 566 of SEQ ID NO: 2, amino acids 521 to 530 of SEQ ID NO: 3, amino acids 557 to 566 of SEQ ID NO: 4, and SEQ ID NO: 20, or the PTD is SEQ ID NO: 81 or SEQ ID NO:

82.

8. The fusion protein according to any one of claims 1 to 7, characterized in that the anti-CD3 antibody section is an anti-CD3 scFv specific to human CD3.

9. A fusion protein according to any one of claims 1 to 8, for use in the treatment of tumors, particularly for use in the treatment of solid tumors.

10. A nucleic acid construct for use in tumor treatment, comprising an anti-CD3 antibody section, characterized in that it comprises a fusion protein according to any one of claims 1 to 9.

11. The nucleic acid construct according to claim 10, characterized in that it is contained in a virus particle.

12. The nucleic acid construct according to claim 10, characterized in that it is contained in liposomes.

13. A nucleic acid construct according to any one of claims 10 to 12, characterized in that it is contained in adenovirus particles.

14. A nucleic acid construct according to any one of claims 10 to 13, comprising a nucleotide section of nucleotides 1 to 3706 and a nucleotide section of nucleotides 5986 to 37135 of SEQ ID NO: 13, wherein a coding sequence encoding the fusion protein is included between these nucleotide sections under the control of a promoter, or comprising a nucleotide section of nucleotides 1 to 3706 and a nucleotide section of nucleotides 6094 to 37243 of SEQ ID NO: 71, wherein a coding sequence encoding the fusion protein is included between these nucleotide sections under the control of a promoter.

15. The nucleic acid construct according to any one of claims 10 to 14, characterized in that the coding sequence encoding the fusion protein is placed under the control of a weak promoter.

16. The nucleic acid construct according to any one of claims 10 to 14, characterized in that the coding sequence encoding the fusion protein is located at 3' of IRES and the promoter is located at 5' of IRES.

17. An oncolytic virus for use in the treatment of solid tumors, comprising a nucleic acid construct according to any one of claims 10 to 16.

18. An oncolytic virus for use in the treatment of a solid tumor according to claim 17, wherein the administration of the oncolytic virus is combined with the administration of a compound that blocks the PD-1 / PD-L1 immune checkpoint.

Images