Novel HVEM-binding protein and pharmaceutical composition containing same

Abstract

The purpose of the present invention is to provide a novel HVEM-binding protein. The present invention provides a human HVEM-binding protein comprising at least one immunoglobulin single variable region, wherein: the immunoglobulin single variable region includes complementarity determining regions 1-3 which have the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3, respectively; and the immunoglobulin single variable region is a humanized VHH. The protein according to the present invention may further comprise a human immunoglobulin Fc region or a human serum albumin-binding protein, and the immunoglobulin Fc region may be an Fc region of human IgG4.

Description

Novel HVEM-binding protein and pharmaceutical composition containing same REFERENCE TO RELATED APPLICATIONS

[0001] This application benefits from the priority of an earlier Japanese application, Patent Application No. 2023-131057 (filing date: August 10, 2023), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to HVEM binding proteins and pharmaceutical compositions comprising said proteins.

[0003] The incidence of primary brain tumors in Japan is estimated to be approximately 20,000 per year (prevalence rate in 2010: 130.8 per 100,000 people). The five major brain tumors are glioma, meningioma, pituitary adenoma, schwannoma, and craniopharyngioma. Furthermore, it has been reported that the incidence of primary brain tumors, including malignant glioblastoma multiforme, is approximately twice as high in Europe and the United States compared to Japan. While surgical removal is the standard treatment for brain tumors, malignant tumors often require multidisciplinary treatment, including chemotherapy and radiation therapy. Treatment effectiveness depends on how much of the tumor can be removed during the initial surgery. It has been reported that in the case of malignant tumors, failure to remove at least 95-98% of the tumor results in little difference in survival prognosis compared to only removing half of the tumor. Furthermore, malignant brain tumors are said to have a high probability of progressing to more aggressive forms of brain tumors.

[0004] Glioblastoma multiforme (GBM) is the most common and most aggressive form of malignant brain tumor in adults. Despite advances in surgery, radiation therapy, and chemotherapy, the prognosis for glioblastoma patients remains poor, with a median survival time of less than 15 months. Glioblastoma is classified into proneural, neural, classical, and mesenchymal subtypes based on genomic abnormalities from the The Cancer Genome Atlas (TCGA) dataset. The highly proliferative nature of glioblastoma cells is due to alterations in several signaling pathways, including oncogenes and tumor suppressor genes.

[0005] Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily, and members of the BMP subfamily, including BMP-2, BMP-4, and BMP-7, have been reported to induce differentiation, cell cycle arrest, and apoptosis in glioblastoma cells. Many of the BMP target genes identified in glioblastoma to date include intracellular signaling molecules (Non-Patent Document 1). However, it has also been reported that mesenchymal subtype glioblastomas often exhibit resistance to BMPs (Non-Patent Document 2).

[0006] Savary K et al., Oncogene, 32, 5409-5420 (2013)Dalmo et al., Mol. Cancer Res., 18, 981-991 (2020)

[0007] The present invention aims to provide a novel HVEM-binding protein and a pharmaceutical composition containing the protein.

[0008] The present invention provides the following: [1] A human HVEM-binding protein comprising at least one immunoglobulin single variable region, wherein the immunoglobulin single variable region comprises complementarity-determining regions 1 to 3 (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and wherein the immunoglobulin single variable region is a humanized VHH. [2] The HVEM binding protein of [1] above, wherein the immunoglobulin single variable region comprises the amino acid sequence of (v), (vi), or (vii) below: (v) the amino acid sequence of SEQ ID NO: 18, 19, or 20; (vi) an amino acid sequence having at least 80% identity to the sequence of the framework region (excluding amino acids corresponding to the humanized region) in the sequence of (v); (vii) an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added in the sequence of the framework region (excluding amino acids corresponding to the humanized region) in the sequence of (v) or (vi). [3] The HVEM binding protein according to [1] or [2] above, wherein the immunoglobulin single variable region comprises framework regions (FR1, FR2, FR3, and FR4), and the amino acid sequences of FR1, FR2, FR3, and FR4 are (i), (ii), or (iii) below: (i) FR1 having the amino acid sequence represented by SEQ ID NO:5, FR2 having the amino acid sequence represented by SEQ ID NO:8, FR3 having the amino acid sequence represented by SEQ ID NO:11, and FR4 having the amino acid sequence represented by SEQ ID NO:13; (ii) FR1 having the amino acid sequence represented by SEQ ID NO:5, FR2 having the amino acid sequence represented by SEQ ID NO:7, FR3 having the amino acid sequence represented by SEQ ID NO:10, and FR4 having the amino acid sequence represented by SEQ ID NO:13; (iii) FR1 having the amino acid sequence represented by SEQ ID NO:5, FR2 having the amino acid sequence represented by SEQ ID NO:6, FR3 having the amino acid sequence represented by SEQ ID NO:10, and FR4 having the amino acid sequence represented by SEQ ID NO:13.[4] The HVEM binding protein of any of [1] to [3] above, further comprising a human immunoglobulin Fc region or a human serum albumin binding protein. [5] The HVEM binding protein of [4] above, wherein the immunoglobulin Fc region is the Fc region of human IgG4. [6] The HVEM binding protein of [5] above, wherein the amino acid sequence of the Fc region of human IgG4 is the amino acid sequence of SEQ ID NO: 14, 15, or 16. [7] An antibody-drug conjugate comprising the HVEM binding protein of [1] or [4] above and a drug. [8] The antibody-drug conjugate of [7] above, wherein the drug is an anticancer drug. [9] A pharmaceutical composition comprising the HVEM binding protein of any of [1] to [6] above or the antibody-drug conjugate of [7] above.

[10] The pharmaceutical composition of [9] above, for use in inhibiting binding of HVEM to BTLA and / or APRIL.

[11] The pharmaceutical composition according to [9] above, for use in the treatment or prevention of solid cancer or malignant tumor.

[12] The pharmaceutical composition according to any of [9] to

[11] above, for use in the treatment or prevention of glioma.

[13] The pharmaceutical composition according to

[12] above, wherein the glioma is any of oligodendroglioma, oligoastrocytoma, astrocytoma, and glioblastoma multiforme.

[14] The pharmaceutical composition according to

[13] above, wherein the glioblastoma multiforme belongs to any of the proneural subtype, neural subtype, classical subtype, and mesenchymal subtype.

[15] The pharmaceutical composition according to any of [9] to

[14] above, for use in combination with other anticancer drugs.

[16] An inhibitor of the HVEM signaling pathway, comprising the HVEM binding protein according to any of [1] to [6] above.

[17] The inhibitor according to

[16] above, wherein the HVEM signaling pathway is the HVEM-mediated NF-κB signaling pathway.

[18] An anti-cancer immunity inducer comprising the HVEM binding protein according to any one of [1] to [6] above.

[19] A polynucleotide encoding the HVEM binding protein according to any one of [1] to [6] above.

[20] An expression vector comprising the polynucleotide according to

[19] above.

[21] A method for treating or preventing solid cancer, comprising administering to a subject in need thereof the HVEM binding protein of any of [1] to [6] above or a composition comprising same, or the antibody-drug conjugate of [7] above or a composition comprising same.

[22] Use of the HVEM binding protein of any of [1] to [6] above or the antibody-drug conjugate of [7] above for the manufacture of an agent for treating or preventing solid cancer.

[0009] The protein of [1] above may be referred to herein as the "protein of the present invention," and the composition of [9] above may be referred to herein as the "composition of the present invention."

[0010] The present invention is advantageous in that it can provide an HVEM binding protein that has the activity of selectively inhibiting human HVEM and exhibits good pharmacokinetics, as well as a pharmaceutical composition containing the protein.

[0011] Figure 1 shows the degree of humanization of the alpaca anti-human HVEM antibody (VHH-3, SEQ ID NO: 17) (Figure 1a), the single variable region of the humanized anti-human HVEM antibody (HVs80) (SEQ ID NO: 19) (Figure 1b), and the single variable region of the humanized anti-human HVEM antibody (HVR80 and HVR81) (SEQ ID NO: 20) (Figure 1c) (Example 3). Figure 2 shows the effect of alpaca anti-human HVEM antibody gene transfer on the proliferation of mesenchymal subtype cells (Example 5). (a) This figure shows the expression of the alpaca anti-human HVEM antibody (VHH-3) or anti-GFP antibody (negative control) gene transferred into mesenchymal subtype GBM cell line U3054MG. (b) This figure shows that the proliferation of mesenchymal subtype GBM cells is suppressed by gene transfer of the alpaca anti-human HVEM antibody (VHH-3) (n = 3 biological repeats). Figure 3 shows the effects of alpaca anti-human HVEM antibody on the proliferation and cell invasion of mesenchymal subtype cells (Example 6). (a, b) The effect of alpaca anti-human HVEM antibody (VHH-3) on the proliferation of mesenchymal subtype cells. (a) Growth curve and (b) response dose curve (n = 2 biological replicates for each). (c) Spheroids of mesenchymal subtype GBM cells (U3031MG or U3054MG) were cultured on mouse brain slices, and the effect of alpaca anti-human HVEM antibody on the inhibition of cell invasion was shown (n = 9 biological replicates). Representative images (corresponding to the median cell invasion index) for each experimental condition are shown below the graph. Figure 4 shows the effect of intraperitoneal administration of alpaca anti-human HVEM antibody on the in vivo proliferation of mesenchymal subtype GBM cells (n = 5 animals per experimental group, Example 7). (a) Alpaca anti-human HVEM antibody (VHH-3) was intraperitoneally administered to mice at 10 mg / kg once daily. This figure shows the quantification of in vivo proliferation of mesenchymal subtype GBM cells (U3031MG or U3054MG) 14 and 28 days after the start of antibody administration. Antibody administration was initiated 105 days after cell transplantation for mice bearing U3031MG cells, and 91 days after cell transplantation for mice bearing U3054MG cells.(b) Mouse survival curve. The endpoint was the time point when mice showed 20% or more weight loss and / or neurological symptoms. Figure 5 shows that deletion of the HVEM gene by genome editing increases the sensitivity of mesenchymal subtype GBM cells to an anticancer drug (temozolomide) and an EGFR inhibitor (erlotinib) (Example 8). (a) Dose-response curve (n = 3 biological replicates). (b) Quantification of the apoptosis rate (n = 3 biological replicates). (C) Apoptotic cells were stained with Annexin V-FITC and 7-AAD, respectively, and apoptosis of mesenchymal subtype GBM cells was detected by flow cytometry. Cell viability is expressed as a percentage of cell viability in the absence of drug. Figure 6 shows that an alpaca anti-human HVEM antibody (VHH-3) inhibits the interaction between human BTLA and human HVEM (n = 3 independent experiments, Example 9). Recombinant human HVEM protein was immobilized on an immunoassay plate and reacted with alpaca anti-human HVEM antibody (VHH-3) or anti-GFP antibody (negative control) at concentrations ranging from 3 pM to 1 μM. After reacting with recombinant human BTLA-Fc protein, the proportion of recombinant human BTLA-Fc protein that reacted with the recombinant human HVEM protein using anti-Fc antibody was quantified. The binding of HVEM to BTLA was expressed as a ratio to the amount bound in the absence of antibody. Figure 7 shows the pharmacokinetics of anti-human HVEM antibody in cynomolgus monkeys (n = 3 animals per experimental group, Example 10). (a) Blood concentration of humanized anti-human HVEM antibody (HV15) after intravenous injection into cynomolgus monkeys. (b) A graph showing the blood concentration of a humanized anti-human HVEM antibody (HV80) when it was intravenously injected into a cynomolgus monkey. (c) A graph showing the blood concentration of a humanized anti-human HVEM antibody (HV15) when it was subcutaneously injected into a cynomolgus monkey. Figure 8 shows that a humanized anti-human HVEM antibody (HVR81) inhibits the proliferation of mesenchymal subtype GBM cells (U3054MG) in a concentration-dependent manner (Example 12). Each value in the graph represents the average and standard deviation at each antibody concentration. Cell viability is expressed as a ratio to the cell viability in the absence of antibody.Figure 9 shows that humanized anti-human HVEM antibody (HVR81) inhibits the interaction between human BTLA and human HVEM in a concentration-dependent manner (Example 13). Each value in the figure represents the mean and standard deviation at each antibody concentration. The binding of HVEM to BTLA is shown as a ratio to the amount of binding in the absence of antibody. Figure 10 shows that humanized anti-human HVEM antibody (HVR81) inhibits the interaction between human APRIL and human HVEM in a concentration-dependent manner (Example 13). Each value in the figure represents the mean and standard deviation at each antibody concentration. The binding of HVEM to APRIL is shown as a ratio to the amount of binding in the absence of antibody. Figure 11 shows that humanized anti-human HVEM antibody (HVR81) inhibits NF-kB signaling in mesenchymal subtype GBM cells (U3054MG) in a concentration-dependent manner (Example 14). Each value in the figure represents the mean and standard deviation at each antibody concentration. Figure 12 is a photograph (160 μm on each side) showing the intracellular transfer of humanized anti-human HVEM antibody (HVR80) into HVEM-expressing cells (Example 15). Fluorescence emitted by AcidiFluor ORANGE is shown in black, and fluorescence from DAPI, which stains cell nuclei, is shown in light gray (scale bar: 20 μm). Figure 13 shows the inhibition of HEK293T-HVEM cell viability by administration of MMAE-conjugated humanized anti-human HVEM antibody (HVR80) (Example 16). Each value in the figure represents the mean and standard deviation of cell viability at each antibody-drug conjugate concentration. Cell viability is expressed as a percentage of cell viability in the absence of the antibody-drug conjugate. Figure 14 shows the inhibition of HEK293T-HVEM cell viability by administration of PNU-conjugated humanized anti-human HVEM antibody (HVR80) (Example 16). Each value in the figure represents the mean and standard deviation of cell viability at each antibody-drug conjugate concentration. Cell viability is expressed as a percentage of cell viability in the absence of antibody-drug conjugate. Specific Description of the Invention

[0012] The protein of the present invention is characterized by binding to HVEM. Here, "HVEM" is an abbreviation for Herpes virus entry mediator, and refers to a type I transmembrane protein belonging to the TNF / NGF receptor superfamily. HVEM is also known as TNFRSF14 (tumor necrosis factor (TNF) receptor superfamily member 14), CD270, LIGHTR, or ATAR. That is, "HVEM" in the present invention is synonymous with "HVEM / TNFRSF14."

[0013] HVEM is expressed on various tissues and cells, including T cells, B cells, natural killer cells, dendritic cells, hematopoietic cells, and non-hematopoietic cells (parenchymal cells) (Pasero C et al., Curr Opin Pharmacol., 12, 478-485 (2012); Sedy JR and Ramezani-Rad P, Adv. Cancer Res., 42, 145-186 (2019)). Several ligands, including TNF-related cytokines such as LIGHT and LTa, as well as non-TNF-related cytokines such as BTLA, CD160, and SALM5, are known to bind to HVEM.

[0014] In the present invention, the human HVEM gene is based on the nucleotide sequence published by the HUGO Gene Nomenclature Committee HGNC ID: HGNC:11912, and the mouse HVEM gene is based on the nucleotide sequence published by Mouse Genome Informatics (MGI): MGI:2675303. Also in the present invention, the human HVEM protein is based on the amino acid sequence published under GenBank Accession No. NP_003811.2, and the mouse HVEM protein is based on the amino acid sequence published under GenBank Accession No. NP_849262.1. Furthermore, in the present invention, the human HVEM mRNA is based on GenBank Accession No. NM_003820.4, and the mouse HVEM mRNA is based on GenBank Accession No. NM_178931.2.

[0015] The protein of the present invention is a protein that contains at least one immunoglobulin single variable region and is capable of inhibiting the function of HVEM by binding to it.

[0016] As used herein, the term "immunoglobulin variable region" refers to the antigen-binding site of an antibody, and is composed of "complementarity-determining regions" (CDRs) and "framework regions" (FRs). The complementarity-determining regions, also known as hypervariable regions (HVRs), are the most frequently variable regions in the variable region, while the framework regions are relatively highly conserved.

[0017] In the present invention, CDRs can be defined using conventional CDR definitions used in the art, such as Kabat, Chothia, AbM, and contact. The Kabat definition is based on sequence variation and is the most commonly used (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The Chothia definition takes into account the location of structural loops (e.g., Chothia and Lesk, J. Mol. Biol. 196, 901-917 (1987)). The AbM definition is intermediate between the Kabat and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.

[0018] As used herein, the term "immunoglobulin single variable domain" refers to a single immunoglobulin variable domain, rather than a paired immunoglobulin variable domain, that is capable of specifically binding to an epitope of an antigen.

[0019] The single variable region constituting the protein of the present invention can be characterized by complementarity determining regions 1 to 3 (CDR1, CDR2, and CDR3), and the amino acid sequences of CDR1, CDR2, and CDR3 can be identified by the amino acid sequence represented by SEQ ID NO:1, the amino acid sequence represented by SEQ ID NO:2, and the amino acid sequence represented by SEQ ID NO:3, respectively.

[0020] The single variable region constituting the protein of the present invention can also be characterized by framework regions 1 to 4 (FR1, FR2, FR3, and FR4), and FR1, FR2, FR3, and FR4 can be specified by a combination of FR1, FR2, FR3, and FR4 selected from the group consisting of (i), (ii), and (iii) above. The single variable region constituting the protein of the present invention can be specified by CDRs 1 to 3 and FRs 1 to 4, and in this case, the single variable region constituting the protein of the present invention can be formed by linking FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in this order from the N-terminus.

[0021] The single variable region constituting the protein of the present invention can also be identified by the single variable region sequenced in the Examples (the polypeptide (v) above). In addition, in the present invention, polypeptides substantially identical to the polypeptide (v) above are also included in the single variable region constituting the protein of the present invention.

[0022] The polypeptide substantially identical to the single variable region constituting the protein of the present invention can be a polypeptide selected from the above (vi) and (vii).

[0023] In (vi) above, "identity" is used in a sense that includes "homology." Here, "identity" refers to, for example, the degree of identity when the sequences to be compared are appropriately aligned, and means the percentage of exact amino acid matches between the sequences. Identity takes into account, for example, the presence of gaps in the sequences and the properties of the amino acids (Wilbur, Natl. Acad. Sci. USA 80, 726-730 (1983)). The alignment can be performed, for example, using any algorithm. Specifically, homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183, 63-69 (1990)), or Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. Furthermore, identity can be calculated, for example, using known homology search programs such as those described above. For example, identity can be calculated using the default parameters of the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI).

[0024] The identity in (vi) can be at least 80% or more, 85% or more, 90% or more, 91%, 92%, 93%, 94%, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the sequence of the framework region (excluding amino acids corresponding to the humanized region) in the sequence in (v).

[0025] In (vii) above, "one or more amino acids have been deleted, substituted, inserted, and / or added in the sequence of the framework region (excluding the amino acids corresponding to the humanized region)" means that the sequence of the framework region excluding the amino acids corresponding to the humanized region has been altered by deletion, substitution, insertion, and / or addition of a number of amino acids equivalent to those produced by known methods such as site-specific mutagenesis, or a number of amino acids equivalent to those naturally occurring. The alterations in the amino acid sequence may be, for example, continuous or discontinuous.

[0026] The insertion of an amino acid in (vii) above can be, for example, insertion into the interior of the amino acid sequence. Furthermore, the addition of an amino acid in (vii) above can be, for example, addition to either the N-terminus or the C-terminus of the amino acid sequence, or addition to both the N-terminus and the C-terminus.

[0027] The amino acid substitution in (vii) above means that an amino acid residue constituting an amino acid sequence is replaced with another type of amino acid residue. The amino acid substitution in (vii) above may be, for example, a conservative substitution. "Conservative substitution" means substituting one or more amino acids with another amino acid and / or amino acid derivative so as not to substantially alter the function of the protein. In a conservative substitution, the substituted amino acid and the substituted amino acid preferably have similar properties and / or functions, for example. Specifically, chemical properties such as hydrophobicity and hydrophilicity index, polarity, and charge, or physical properties such as secondary structure, are preferably similar. Amino acids or amino acid derivatives with similar properties and / or functions are known in the art. For example, nonpolar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged amino acids (basic amino acids) include arginine, histidine, and lysine, and examples of negatively charged amino acids (acidic amino acids) include aspartic acid and glutamic acid.

[0028] Preferred amino acid modifications in (vii) above include one, two, three, four, five, six, or seven amino acid substitutions, and more preferably, the substitutions are conservative substitutions.

[0029] When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 18, the single variable region constituting the protein of the present invention comprises a CDR1 having the amino acid sequence represented by SEQ ID NO: 1, a CDR2 having the amino acid sequence represented by SEQ ID NO: 2, a CDR3 having the amino acid sequence represented by SEQ ID NO: 3, an FR1 comprising the amino acid sequence represented by SEQ ID NO: 5, an FR2 having the amino acid sequence represented by SEQ ID NO: 6, an FR3 having the amino acid sequence represented by SEQ ID NO: 10, and an FR4 having the amino acid sequence represented by SEQ ID NO: 13. In this case, the single variable region can be formed by linking FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in this order from the N-terminus. When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 18, the sequence of the framework region in (vii) (excluding amino acids corresponding to the humanized region) can be: a sequence excluding glutamic acid (Glu) at position 1 and leucine (Leu) at position 11 in the amino acid sequence of SEQ ID NO: 5, i.e., a sequence corresponding to amino acids at positions 2 to 10 and 12 to 25 in the amino acid sequence of SEQ ID NO: 5; a sequence consisting of amino acids at positions 1 to 14 in the amino acid sequence of SEQ ID NO: 6; a sequence excluding serine (Ser) at position 16 and arginine (Arg) at position 28 in the amino acid sequence of SEQ ID NO: 10, i.e., a sequence corresponding to amino acids at positions 1 to 15, 17 to 27, and 29 to 39 in the amino acid sequence of SEQ ID NO: 10; and a sequence excluding glutamine (Gln) at position 3 and leucine (Leu) at position 6 in the amino acid sequence of SEQ ID NO: 13, i.e., a sequence corresponding to amino acids at positions 1, 2, 4, 5, and 7 to 11 in the amino acid sequence of SEQ ID NO: 13. When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 18, the number of amino acids modified by substitution or the like in (vii) above is, for example, 1 to 16, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0030] When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 19, the single variable region constituting the protein of the present invention comprises a CDR1 having the amino acid sequence represented by SEQ ID NO: 1, a CDR2 having the amino acid sequence represented by SEQ ID NO: 2, a CDR3 having the amino acid sequence represented by SEQ ID NO: 3, an FR1 comprising the amino acid sequence represented by SEQ ID NO: 5, an FR2 having the amino acid sequence represented by SEQ ID NO: 7, an FR3 having the amino acid sequence represented by SEQ ID NO: 10, and an FR4 having the amino acid sequence represented by SEQ ID NO: 13. In this case, the single variable region can be composed of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 linked in this order from the N-terminus. When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 19, the sequence of the framework region in (vii) (excluding amino acids corresponding to the humanized region) is: - a sequence excluding glutamic acid (Glu) at position 1 and leucine (Leu) at position 11 in the amino acid sequence of SEQ ID NO: 5, i.e., a sequence corresponding to amino acids at positions 2 to 10 and 12 to 25 in the amino acid sequence of SEQ ID NO: 5; - a sequence excluding serine (Ser) at position 5 in the amino acid sequence of SEQ ID NO: 7, i.e., a sequence corresponding to amino acids at positions 1 to 4 and 6 to 14 in the amino acid sequence of SEQ ID NO: 7; - a sequence excluding serine (Ser) at position 16 and arginine (Arg) at position 28 in the amino acid sequence of SEQ ID NO: 10, i.e., a sequence corresponding to amino acids at positions 1 to 15, 17 to 27, and 29 to 39 in the amino acid sequence of SEQ ID NO: 10; - a sequence excluding glutamine (Gln) at position 3 and leucine (Leu) at position 6 in the amino acid sequence of SEQ ID NO: 13, i.e., a sequence corresponding to the amino acids at positions 1, 2, 4, 5, and 7 to 11 in the amino acid sequence of SEQ ID NO: 13. When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 19, the number of amino acids modified by substitution or the like in (vii) above is, for example, 1 to 16, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid.

[0031] When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 20, the single variable region constituting the protein of the present invention comprises a CDR1 having the amino acid sequence represented by SEQ ID NO: 1, a CDR2 having the amino acid sequence represented by SEQ ID NO: 2, a CDR3 having the amino acid sequence represented by SEQ ID NO: 3, an FR1 comprising the amino acid sequence represented by SEQ ID NO: 5, an FR2 having the amino acid sequence represented by SEQ ID NO: 8, an FR3 having the amino acid sequence represented by SEQ ID NO: 11, and an FR4 having the amino acid sequence represented by SEQ ID NO: 13. In this case, the single variable region can be composed of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 linked in this order from the N-terminus. When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 20, the sequence of the framework region in (vii) (excluding amino acids corresponding to the humanized region) is: - a sequence excluding glutamic acid (Glu) at position 1 and leucine (Leu) at position 11 in the amino acid sequence of SEQ ID NO: 5, i.e., a sequence corresponding to amino acids at positions 2 to 10 and 12 to 25 in the amino acid sequence of SEQ ID NO: 5; - a sequence excluding glutamine (Gln) at position 4 and alanine (Ala) at position 5 in the amino acid sequence of SEQ ID NO: 8, i.e., a sequence corresponding to amino acids at positions 1 to 3 and 6 to 14 in the amino acid sequence of SEQ ID NO: 8; - a sequence excluding serine (Ser) at position 16, threonine (Thr) at position 19, leucine (Leu) at position 20, arginine (Arg) at position 28, and alanine (Ala) at position 38 in the amino acid sequence of SEQ ID NO: 11, i.e., a sequence corresponding to amino acids at positions 1 to 15, 17, 18, 21 to 27, 29 to 37, and 39 in the amino acid sequence of SEQ ID NO: 11; The sequence may be a sequence excluding the third glutamine (Gln) and the sixth leucine (Leu) in the amino acid sequence of SEQ ID NO: 13, i.e., a sequence corresponding to the first, second, fourth, fifth, and seventh to eleventh amino acids in the amino acid sequence of SEQ ID NO: 13.When the single variable region constituting the protein of the present invention comprises the amino acid sequence of SEQ ID NO: 20, the number of amino acids modified by substitution or the like in (vii) above is, for example, 1 to 15, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0032] Examples of single variable regions constituting the proteins of the present invention include humanized single variable regions, preferably humanized VHHs (humanized VHHs). VHHs are also called nanobodies, and VHHs may be referred to as nanobodies in the present specification.

[0033] The protein of the present invention may be a protein having two or more single variable regions in tandem, and the single variable regions can be linked together, for example, by a peptide linker.

[0034] The proteins of the present invention may further comprise an Fc region derived from a human immunoglobulin in addition to the single variable region. Examples of such an Fc region include those derived from human antibodies such as IgG, IgM, IgA, IgE, and IgD, and preferably an Fc region derived from human IgG4.

[0035] When the proteins of the present invention comprise an Fc region derived from a human immunoglobulin, the amino acid sequence of the Fc region may be a polypeptide consisting of the amino acid sequence of an Fc region derived from a human immunoglobulin, or a polypeptide substantially identical to a polypeptide consisting of the amino acid sequence of an Fc region derived from a human immunoglobulin. Polypeptides substantially identical to polypeptides consisting of the amino acid sequence of an Fc region derived from a human immunoglobulin include, but are not limited to, polypeptides that have been modified by known methods to improve the expected action of the proteins of the present invention, such as polypeptides in which amino acids have been added to the hinge region to improve binding affinity, or polypeptides in which amino acids have been deleted or substituted in the hinge region, and polypeptides in which amino acids have been substituted to prevent IgG4 Fab arm exchange. When the proteins of the present invention comprise an Fc region derived from human IgG4, non-limiting examples of such Fc regions include polypeptides represented by the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16.

[0036] In addition to the single variable region, the proteins of the present invention may also contain proteins other than the Fc region derived from human immunoglobulin, such peptides including human serum albumin binding proteins.

[0037] When the protein of the present invention comprises a single variable region and a human immunoglobulin Fc region, the protein of the present invention may have a structure comprising two chains each comprising the single variable region and the human immunoglobulin Fc region, and the two chains may be linked by a disulfide bond.When the protein of the present invention comprises two chains each comprising the single variable region and the human immunoglobulin Fc region, the two chains may be the same or different, and preferably the two chains are the same.

[0038] The protein of the present invention can also be provided in the form of an antibody-drug conjugate (ADC). That is, the antibody-drug conjugate of the present invention comprises at least the protein of the present invention and a drug. In this case, the protein of the present invention may consist of a single variable region, or may further comprise, in addition to the single variable region, an Fc region derived from human immunoglobulin or a protein other than the Fc region derived from human immunoglobulin. The drug may be linked to the single variable region or to a region other than the single variable region, and may be linked directly or via a linker (e.g., a peptide cleavable in a specific tissue). Examples of the drug include chemotherapeutic agents, and non-limiting examples include anticancer agents (e.g., small-molecule anticancer agents).

[0039] The protein of the present invention specifically binds to HVEM and preferably has a low dissociation constant (KD) for human HVEM. The upper limit of KD (equal to or less than 10) is, for example, 10 －６ M, 10 －７ M or 10 －８ M, and the lower limit (greater than or equal to) is not particularly limited, but for example, 10 －９ M or 10 －１０ It can be M.

[0040] The proteins of the present invention can be produced, for example, by expressing a polynucleotide encoding them in a host and recovering the expression product. Therefore, according to yet another aspect of the present invention, there is provided a host cell into which a polynucleotide encoding the protein of the present invention and a polynucleotide of the present invention or a vector to which the polynucleotide of the present invention is operably linked have been introduced. In the present invention, "polynucleotide" includes DNA and RNA, as well as modified forms thereof and artificial nucleic acids, with DNA being preferred. Furthermore, DNA includes cDNA, genomic DNA, and chemically synthesized DNA.

[0041] The polynucleotide of the present invention is not particularly limited as long as it is expressible in the host used and is composed of codons that encode a protein having HVEM-binding activity, and codon optimization may be performed to enable expression in the host used or to increase the expression level. Codon optimization can be performed by known methods commonly used in the art.

[0042] The proteins of the present invention can be expressed in various host cells, such as bacterial cells, fungi, animal cells, plant cells, baculovirus / insect cells, or yeast cells. The proteins of the present invention can also be expressed in various host cells, such as animal cells or baculovirus / insect cells, and then secreted extracellularly. Expression vectors for expressing the proteins of the present invention are known, and vectors suitable for various host cells can be used.

[0043] When extracting a protein expressed in a host from cultured bacterial cells or cultured cells, the bacterial cells or cultured cells are collected after cultivation by a known method, suspended in an appropriate buffer, and disrupted by ultrasound, lysozyme, and / or freeze-thawing, followed by centrifugation or filtration to obtain a soluble extract. The target protein can be obtained from the resulting extract by an appropriate combination of known separation and purification methods. When extracting a protein secreted extracellularly after expression in a host, the culture medium after cultivation is collected, centrifuged or filtered to obtain a soluble extract. The target protein can be obtained from the resulting extract by an appropriate combination of known separation and purification methods. Known separation and purification methods include methods that utilize solubility, such as salting out or solvent precipitation; methods that primarily utilize molecular weight differences, such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods that utilize charge differences, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize hydrophobicity differences, such as reverse-phase high-performance liquid chromatography; and methods that utilize isoelectric point differences, such as isoelectric focusing.

[0044] The proteins of the present invention have HVEM-binding activity and can inhibit the function of HVEM. Therefore, the proteins of the present invention can be used as an active ingredient in inhibitors of signal transduction pathways involving HVEM. Non-limiting examples of inhibition of the HVEM signal transduction pathway include suppression of the physiological action of HVEM ligands on HVEM and suppression of HVEM downstream signals. Here, HVEM ligands include BTLA, APRIL, LIGHT, or SALM5.

[0045] As shown in the Examples below, it was confirmed that HVEM-binding proteins inhibit the binding of BTLA to HVEM. Here, "BTLA" is an abbreviation for "B- and T-lymphocyte attenuator" (a factor suppressing B and T lymphocyte function) and is also known as CD272 (cluster of differentiation 272). BTLA is an inhibitory factor at immune checkpoints and is known to negatively regulate T cell immune responses (Watanabe et al., Nat. Immunol., 4, 670-679 (2003); Chen and Mellman, Immunity, 39, 1-10 (2013)). It is also known that targeting the interaction between BTLA and HVEM can enhance anti-tumor immunity, and that anti-cancer therapies targeting BTLA-HVEM have produced promising results (Wojciechowicz et al., Eur. J. Med. Chem., 268, 116231 (2024); Sordo-Bahamonde et al., Mol. Cancer 22, 142 (2023)). Therefore, the proteins of the present invention (particularly HVR81) can be used as agents for inducing anti-cancer immunity, as well as anti-cancer agents (e.g., agents for treating or preventing solid cancers or malignant tumors).

[0046] As shown in the Examples below, it has been confirmed that the binding of APRIL to HVEM is inhibited by HVEM-binding proteins. Here, "APRIL" is an abbreviation for "a proliferation-inducing ligand" and is also known as TNFSF13 (tumor necrosis factor (TNF) ligand superfamily member 13). In other words, "APRIL" in the present invention is synonymous with "APRIL / TNFSF13." It has been confirmed that signaling from APRIL via HVEM promotes tumor formation (particularly highly malignant brain tumors such as gliomas) (WO 2020 / 138503). Furthermore, it is expected that anticancer therapies targeting the interaction between APRIL and HVEM will yield promising results. Therefore, the proteins of the present invention (particularly HVR81) can be used as anticancer agents (e.g., agents for treating or preventing solid cancers or malignant tumors).

[0047] The HVEM-binding proteins of the present invention can inhibit the binding of BTLA to HVEM, as well as the binding of APRIL to HVEM. Therefore, the proteins of the present invention (particularly HVR81) can inhibit both the BTLA-mediated signaling pathway and the APRIL-mediated signaling pathway among HVEM-mediated signaling pathways. Therefore, the proteins of the present invention (particularly HVR81) are useful as pharmaceuticals with the dual functions of inducing anti-cancer immunity and having an anti-tumor effect.

[0048] NF-κB signaling is involved in cell proliferation and angiogenesis, and is deeply involved in cancer progression (Nowacka and Jablonska, Cytokine Growth Factor Rev. 61, 38-44 (2021)). NF-κB signaling in brain tumor cells is also known to be involved in the transition from the less malignant proneural type to the more malignant mesenchymal type (Yamini, Cells 7, 125 (2018); Bhat et al., Cancer Cell 24, 331-346 (2013)). Furthermore, APRIL has been shown to activate NF-κB signaling via HVEM, and activation of NF-κB signaling has been shown to promote tumor formation (especially in highly malignant brain tumors such as gliomas) (WO 2020 / 138503). As shown in the Examples below, it has been confirmed that HVEM-binding proteins suppress HVEM-mediated NF-κB signaling. Therefore, the proteins of the present invention (particularly HVR81) can be used as anticancer agents, for example, as agents for treating or preventing solid cancers or malignant tumors. The proteins of the present invention (particularly HVR81) can also be used as inhibitors of the HVEM signaling pathway, preferably as inhibitors of the HVEM-mediated NF-κB signaling pathway.

[0049] The proteins of the present invention have HVEM-binding activity and can inhibit tumor growth. In particular, the use of the proteins of the present invention can effectively inhibit the formation of malignant tumors (e.g., highly malignant brain tumors such as gliomas). Since substances that inhibit HVEM expression or HVEM function can be used to treat or prevent solid cancers or malignant tumors (e.g., gliomas), the proteins of the present invention can be used as neutralizing antibodies against HVEM and, therefore, can be used as active ingredients in pharmaceutical compositions (e.g., compositions for treating or preventing solid cancers or malignant tumors, e.g., compositions for treating or preventing highly malignant brain tumors such as gliomas).

[0050] According to a preferred embodiment of the present invention, the pharmaceutical composition of the present invention can be used for the treatment or prevention of glioma. Here, "glioma" is a general term for tumors arising from neuroectodermal tissue in the brain parenchyma, and is the most common brain tumor, accounting for 30-40% of primary intracranial tumors. Gliomas include oligodendroglioma, oligoastrocytoma, astrocytoma, and glioblastoma multiforme. "Glioblastoma multiforme," also known as glioblastoma or atypical glioma, is a glioma composed mainly of undifferentiated cells derived from astrocytes. Significant nuclear pleomorphism, necrosis, and vascular endothelial proliferation are observed. Glioblastoma multiforme grows rapidly, infiltrates widely, and often occurs in the adult cerebrum. Glioblastoma multiforme has been reported to be classified into four subtypes: proneural, neural, classical, and mesenchymal (Verhaak RG et al., Cancer Cell, 17, 98-110 (2010)). Since HVEM is highly expressed in mesenchymal subtype glioblastoma multiforme, the composition of the present invention can be preferably used not only for the treatment and prevention of glioblastoma multiforme (especially mesenchymal subtype glioblastoma multiforme), but also for reducing the risk of developing glioma (especially mesenchymal subtype glioblastoma multiforme) as described below.

[0051] As shown in the Examples below, inhibiting HVEM expression and function in glioblastoma multiforme cells suppressed the proliferation and invasion of glioblastoma multiforme cells. Furthermore, the proteins of the present invention containing a human immunoglobulin Fc region exhibited an extended half-life in the blood. Therefore, the proteins of the present invention or antibody-drug conjugates of the present invention can be administered to subjects (e.g., brain tumor patients) whose HVEM expression levels exceed those of healthy subjects or normal tissue samples, particularly brain tumor patients with high HVEM expression in the brain. Furthermore, the therapeutic and preventive targets of the present invention can be HVEM expression-dependent gliomas or HVEM ligand-dependent gliomas. Whether a subject expresses HVEM at a high level, whether a glioma is HVEM-dependent, or whether a glioma is HVEM ligand-dependent can be determined, for example, by evaluating brain tissue resected during brain surgery performed on a brain tumor patient, according to known procedures.

[0052] The proteins of the present invention can be used as neutralizing antibodies to HVEM. A neutralizing antibody to HVEM refers to an antibody that inhibits the function of HVEM. The neutralizing activity against HVEM can be determined, for example, using as an indicator the suppression or inhibition of HVEM function, such as the suppression of the physiological action (e.g., binding) of an HVEM ligand (e.g., BTLA, CD160, APRIL, LIGHT, or SALM5) on HVEM, or the suppression of HVEM downstream signaling (e.g., the suppression of glioblastoma multiforme cell proliferation or cell infiltration, or the suppression of NF-κB signaling).

[0053] The protein of the present invention or the antibody-drug conjugate of the present invention can also be administered to a subject at risk of developing a solid cancer or malignant tumor, such as a glioma (particularly glioblastoma multiforme), thereby reducing the risk of developing the tumor. Here, "a subject at risk of developing a solid cancer or malignant tumor" refers to a subject who does not have any symptoms of a solid cancer or malignant tumor or has not been diagnosed with a solid cancer or malignant tumor, but who may develop a solid cancer or malignant tumor in the future. Examples include those who have not been diagnosed with a solid cancer or malignant tumor, cancer patients who have undergone resection of solid cancer tissue or malignant tumor tissue, brain tumor patients who have not been diagnosed with a glioma, or brain tumor patients who have undergone resection of brain tumor tissue. Furthermore, "reducing the risk of developing a solid cancer or malignant tumor" means reducing the probability of developing a solid cancer or malignant tumor, and reducing the probability of developing a solid cancer or malignant tumor improves the prognosis of solid cancer or malignant tumor (e.g., malignant brain tumor).

[0054] That is, according to another aspect of the present invention, there is provided a composition for reducing the risk of developing solid cancer or malignant tumor (e.g., glioma), which comprises the protein of the present invention or the antibody-drug conjugate of the present invention as an active ingredient, and there is also provided a composition for use as a prognosis-improving agent in the treatment of malignant tumor (e.g., malignant brain tumor), which comprises the protein of the present invention or the antibody-drug conjugate of the present invention as an active ingredient.

[0055] The composition of the present invention can be provided as a pharmaceutical product. The composition and pharmaceutical product of the present invention comprise the protein of the present invention or the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier.

[0056] The compositions and pharmaceuticals of the present invention may contain active ingredients other than the proteins of the present invention and the antibody-drug conjugates of the present invention, or may be used in combination with active ingredients other than the proteins of the present invention and the antibody-drug conjugates of the present invention, or pharmaceuticals or pharmaceutical compositions containing them. Active ingredients other than the proteins of the present invention and the antibody-drug conjugates of the present invention include anticancer drugs including immune checkpoint inhibitors (particularly anticancer drugs intended to treat malignant brain tumors), and non-limiting examples include temozolomide, erlotinib, and nivolumab.

[0057] When the protein of the present invention or the antibody-drug conjugate of the present invention is administered to a subject, the route of administration is not particularly limited as long as a therapeutic or preventive effect against solid cancer or malignant tumor, such as glioma (particularly glioblastoma multiforme), is obtained, but parenteral administration (e.g., intravenous administration, local administration (including local administration using a catheter or continuous local administration), subcutaneous administration, intraperitoneal administration) is preferred.

[0058] For parenteral administration, an appropriate dosage form can be selected depending on the specific administration mode, and examples include injections and suppositories. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art (for example, known methods described in the General Provisions for Preparations of the 18th Edition of the Japanese Pharmacopoeia, etc.). Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc.

[0059] The dosage of the HVEM binding protein or antibody-drug conjugate of the present invention can be determined depending on the type of active ingredient, the gender, age, and weight of the recipient, symptoms, dosage form, and route of administration. When the HVEM binding protein of the present invention is administered for the purpose of treating or preventing solid cancers or malignant tumors, such as gliomas (particularly glioblastoma multiforme), the single adult dosage can be determined, for example, in the range of 0.0001 mg to 1000 mg / kg body weight, but is not limited thereto. The daily adult dosage of the HVEM binding protein can be determined depending on the type of active ingredient, the gender, age, and weight of the recipient, symptoms, dosage form, and route of administration, but for example, the above-mentioned dose of the active ingredient can be administered once a day or in 2 to 4 divided doses.

[0060] The administration interval of the HVEM-binding protein or antibody-drug conjugate of the present invention can be determined depending on the type of active ingredient, the sex, age, and weight of the subject, symptoms, dosage form, and route of administration, but can be, for example, once or twice a day, once or twice a week, or once every 2 to 4 weeks.

[0061] The composition of the present invention can be administered not only to humans in need thereof, but also to non-human mammals (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, sheep, goats, and monkeys).

[0062] In the present invention, "treatment" includes, but is not limited to, therapeutic benefit, and is used to mean measures to achieve beneficial or desired results. Furthermore, therapeutic benefit means complete cure or amelioration of the disease being treated. In the present invention, "prevention" is used to mean reducing the probability of contracting a disease or the probability of disease recurrence. In the present invention, "treatment" and "prevention" are not limited to complete treatment and prevention, but may be treatment and prevention to the extent that a person skilled in the art would recognize as having potential therapeutic and preventive effects (e.g., delaying disease progression, inhibiting disease worsening, delaying onset). Furthermore, when the disease being treated or prevented is "cancer," it also includes suppressing cancer metastasis or recurrence.

[0063] Another aspect of the present invention provides a method for treating or preventing solid cancer or malignant tumor, such as glioma, comprising administering an effective amount of a protein of the present invention or a composition comprising it, or an effective amount of an antibody-drug conjugate of the present invention or a composition comprising it to a subject in need thereof. Another aspect of the present invention also provides a method for reducing the risk of developing solid cancer or malignant tumor, such as glioma, comprising administering an effective amount of a protein of the present invention or a composition comprising it, or an effective amount of an antibody-drug conjugate of the present invention or a composition comprising it to a subject in need thereof. Another aspect of the present invention also provides a method for improving prognosis in the treatment of solid cancer or malignant tumor, such as malignant brain tumor, comprising administering an effective amount of a protein of the present invention or a composition comprising it, or an effective amount of an antibody-drug conjugate of the present invention or a composition comprising it to a subject in need thereof. Another aspect of the present invention also provides a method for inhibiting the HVEM signaling pathway, comprising administering an effective amount of a protein of the present invention or a composition comprising it, or an effective amount of an antibody-drug conjugate of the present invention or a composition comprising it to a subject in need thereof. Another aspect of the present invention provides methods for inducing anti-cancer immunity, comprising administering an effective amount of a protein of the present invention or a composition comprising same, or an effective amount of an antibody-drug conjugate of the present invention or a composition comprising same to a subject in need thereof. These methods of the present invention can be carried out as described for the protein of the present invention and the composition of the present invention.

[0064] According to another aspect of the present invention, there is provided a protein of the present invention or an antibody-drug conjugate of the present invention as a therapeutic or prophylactic agent for solid cancers or malignant tumors, such as gliomas, for use in the treatment or prevention of solid cancers or malignant tumors, such as gliomas, or for use in the therapeutic or prophylactic methods of the present invention. According to another aspect of the present invention, there is provided a protein of the present invention or an antibody-drug conjugate of the present invention as an agent for reducing the risk of developing solid cancers or malignant tumors, such as gliomas, for use in reducing the risk of developing solid cancers or malignant tumors, such as gliomas, or for use in the risk-reducing methods of the present invention. According to another aspect of the present invention, there is provided a protein of the present invention or an antibody-drug conjugate of the present invention as an agent for improving prognosis in the treatment of solid cancers or malignant tumors, such as malignant brain tumors, for use in improving prognosis in the treatment of solid cancers or malignant tumors, such as malignant brain tumors, or for use in the prognosis-improving methods of the present invention. Another aspect of the present invention provides a protein of the present invention or an antibody-drug conjugate of the present invention for use as an inhibitor of the HVEM signaling pathway, for inhibiting the HVEM signaling pathway, or for use in a method of the present invention for inhibiting the HVEM signaling pathway. Another aspect of the present invention provides a protein of the present invention or an antibody-drug conjugate of the present invention for use as an anti-cancer immunity inducer, for inducing anti-cancer immunity, or for use in a method of the present invention for inducing anti-cancer immunity. The above-mentioned HVEM-binding protein and antibody-drug conjugate can be produced according to the description for the protein of the present invention and the composition of the present invention.

[0065] Another aspect of the present invention provides use of a protein of the present invention or an antibody-drug conjugate of the present invention for the manufacture of an agent for treating or preventing solid cancers or malignant tumors, such as gliomas, for the manufacture of an agent for reducing the risk of developing solid cancers or malignant tumors, such as gliomas, for the manufacture of an agent for improving prognosis in the treatment of solid cancers or malignant tumors, such as malignant brain tumors, for the manufacture of an inhibitor of the HVEM signaling pathway, or for the manufacture of an agent for inducing anti-cancer immunity. The above uses can be carried out according to the descriptions for the protein of the present invention and the composition of the present invention.

[0066] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0067] Example 1: Human chimerization of alpaca anti-human HVEM antibody (1) Binding of alpaca anti-human HVEM antibody (VHH-3) to antigen Using VHH-3 antibody, an immunoglobulin single variable region, the presence or absence of VHH-3 antibody was measured in HVEM on the membrane surface of HVEM-expressing HEK293T cells. Specifically, 1 x 10 HEK293T cells transfected with HVEM and HEK293T cells not expressing HVEM as a negative control were transfected with 1 x 10 HEK293T cells. ４ Cells were seeded at 1000 cells / well, fixed with 4% paraformaldehyde, and then reacted with a 6xHis-tagged VHH-3 antibody. An anti-histidine tag antibody labeled with horseradish peroxidase (HRP), anti-His-tag mAb-HRP-DirectT (MBL), was reacted, and the enzymatic reaction between HRP and the substrate was measured using an ELISA POD Substrate TMB Kit (Nacalai Tesque). The effect size data obtained versus antibody concentration was subjected to nonlinear regression to a sigmoid curve using the following formula: effect size = background + maximum effect size / (1 + 10^(Hill coefficient × (log EC50 - log (antibody concentration)))) to determine the 50% effect concentration (EC50). The EC50 for VHH-3 was found to be 1.94 nM. VHH-3 has a molecular weight of approximately 12,600 and has been confirmed to exhibit neutralizing activity against human mesenchymal glioblastoma.

[0068] Table 1 shows the sequences of the three CDR and four FR regions of the alpaca anti-human HVEM antibody determined according to the AbM definition.

[0069]

[0070] (2) Design and preparation of alpaca-human chimeric anti-human HVEM antibody (VHH-3_hIgG4Fc) Human chimerization was performed on the alpaca anti-human HVEM antibody (VHH-3) shown in Table 1. By adding the constant region sequence from the hinge region of human IgG to the C-terminus of VHH-3, an alpaca-human chimeric anti-human HVEM antibody (sometimes simply referred to as "VHH-3_hIgG4Fc") shown in Table 2 was designed and prepared by known methods.

[0071]

[0072] (3) Design and preparation of alpaca-human chimeric anti-human HVEM antibody (VHH-3_hIgG4Fc_S108P) Four amino acid residues were added to the hinge region of the alpaca-human chimeric anti-human HVEM antibody (VHH-3_hIgG4Fc) shown in Table 2, and 108Ser was replaced with Pro to prevent IgG4 Fab arm exchange (the number before each three-letter amino acid code indicates the position of that amino acid residue from the start of the general hIgG4 constant region). This resulted in the design of the alpaca-human chimeric anti-human HVEM antibody shown in Table 3 (sometimes simply referred to as "VHH-3_hIgG4Fc_S108P") and preparation by known methods.

[0073]

[0074] Example 2: Humanization of an alpaca-human chimeric anti-human HVEM antibody (1) Design and preparation of a humanized alpaca-human chimeric anti-human HVEM antibody (HV80) The alpaca-human chimeric anti-human HVEM antibody (VHH-3_hIgG4Fc) obtained in Example 1 was humanized. Six amino acids of VHH-3_hIgG4Fc were humanized to design the humanized anti-human HVEM antibody (sometimes simply referred to as "humanized VHH-3_hIgG4Fc" or "HV80") shown in Table 4. Humanization was commissioned to GenScript, and the humanized antibody clone deemed most effective in terms of binding affinity to HVEM and expression efficiency in mammalian cells was selected. The molecular weight of the humanized anti-human HVEM antibody (HV80) is approximately 76,000.

[0075]

[0076] (2) Design and preparation of a humanized alpaca anti-human HVEM antibody (HV15) The humanized anti-human HVEM antibody (HV80) obtained in (1) above was designed by substituting the amino acid sequence of the Fc region (SEQ ID NO: 14) with an amino acid sequence consisting of six histidines to create the humanized anti-human HVEM antibody (sometimes simply referred to as "HV15") shown in Table 5. The molecular weight of the humanized anti-human HVEM antibody (HV15) is approximately 13,400.

[0077]

[0078] (3) Design and preparation of a humanized antibody (HVs80) of an alpaca-human chimeric anti-human HVEM antibody For the humanized anti-human HVEM antibody (HV80) obtained in (1) above, the 40Cys in the amino acid sequence of FR2 (SEQ ID NO: 6) was replaced with Ser (the number before each three-letter amino acid indicates the position of that amino acid residue according to the definition of Kabat et al.), thereby designing a humanized anti-human HVEM antibody (sometimes simply referred to as "humanized VHH-3_hIgG4Fc Cys->Ser" or "HVs80") shown in Table 6, and preparing it by known methods.

[0079]

[0080] (4) Design and Preparation of a Humanized Alpaca Anti-Human HVEM Antibody (VHH-3) (HVR80) Among the V / D / J junction sequences contained in the human germline sequence database, YAC-5+ / D6-25 / JH4d was selected as the germline sequence encoding the amino acid sequence most homologous to that of VHH-3. Within the amino acid sequence encoded by YAC-5+, within the region corresponding to the second framework of a human heavy chain antibody, amino acid residues 37V, 44G, and 45L (the numbers preceding each single-letter amino acid designation indicate the position of that amino acid residue according to the definition of Kabat et al.) with hydrophobic side chains were substituted with amino acid residues 37Y, 44Q, and 45R, which are present in the second framework sequence of VHH-3 and are responsible for the stability of the VHH as a single chain.

[0081] CDR sequences defined by AbM et al. in VHH-3 were grafted between the first to fourth framework sequences to create a prototype of a humanized VHH-3 antibody candidate. Codons were optimized to facilitate translation of the amino acid sequence into protein in human cells from the gene encoding the sequence. This humanized VHH-3 antibody candidate gene sequence was synthesized using an artificial gene, and a construct expressing an antibody in which the constant region sequence from the hinge region of human IgG4 (108Ser was replaced with Pro to prevent IgG4 Fab arm exchange) was fused to the C-terminus of VHH-3 downstream of the Igκ signal peptide sequence (the number before the three-letter amino acid code indicates the position of that amino acid residue from the start of the general hIgG4 constant region) was incorporated into the pcDNA3.4 vector to create a plasmid. In the prototype, 16 amino acids in the FR sequence in the VHH-3 region were replaced with the same amino acids as in the human sequence. High-density cultured Expi293F ＴＭ The plasmid was then transfected into the cells using ExpiFectamine. ＴＭ Transfection with 293 reagent followed by ExpiFectamine 16-18 hours later. ＴＭ The 293 transfection enhancer was added to improve transfection, cell viability, and protein expression. Seven days after transfection, the culture medium was collected, and the antibody was adsorbed to Protein G bound to the surface of Sepharose beads. The antibody was then eluted with 0.1 M glycine buffer (pH 2.7). The antibody was then immediately neutralized with 1 / 10 the volume of 1 M Tris-HCl buffer (pH 10), and the solution was replaced with PBS to obtain the original antibody.

[0082] Backmutation from the original humanized sequence was repeated until significant antigen-binding activity was obtained for the humanized VHH-3 candidate, and finally, significant antigen-binding activity was first obtained for the humanized anti-human HVEM antibody (sometimes simply referred to as "HVR80") shown in Table 7, which has a sequence in which 11 amino acids were substituted from the amino acid sequence of the alpaca-human chimeric anti-human HVEM antibody (VHH-3_hIgG4Fc). The molecular weight of HVR80 is approximately 77,000.

[0083]

[0084] (5) Design and preparation of humanized antibody (HVR81) of alpaca anti-human HVEM antibody (VHH-3) Four amino acid residues were added to the hinge region of the humanized anti-human HVEM antibody (HVR80) shown in Table 7 to design the humanized anti-human HVEM antibody (sometimes simply referred to as "HVR81") shown in Table 8, and this was prepared by known methods.

[0085]

[0086] Example 3: Evaluation of the degree of humanization of antibodies The degree of humanization of the alpaca anti-human HVEM antibody (VHH-3), the alpaca-human chimeric anti-human HVEM antibody (VHH-3_hIgG4Fc), and the humanized anti-human HVEM antibody (HVR80) was determined using the integrated antibody sequence and structure analysis program (version 3.4.1) provided by Ebisu. Specifically, abYsis (http: / / www.abysis.org / abysis / index.html), Swindells The degree of humanization was determined by annotating the amino acid sequences of VHH-3 (SEQ ID NO: 17), the single variable region of the humanized anti-human HVEM antibody (HVs80) (SEQ ID NO: 19), and the single variable region of the humanized anti-human HVEM antibody (HVR80 and HVR81) (SEQ ID NO: 20) to the original VHH-3 (SEQ ID NO: 17), the single variable region of the humanized anti-human HVEM antibody (HVs80) (HVR80 and HVR81) (SEQ ID NO: 20).

[0087] The results are shown in Figure 1. It was shown that the single variable regions of HVs80 and HVR80 and HVR81 had a higher degree of humanization than VHH-3, and that the single variable regions of HVR80 and HVR81 had a higher degree of humanization than the single variable region of HVs80.

[0088] Example 4: Evaluation of antibody binding affinity for HVEM (1) Measurement of binding affinity of humanized anti-human HVEM antibody (HVR80) for human HVEM The binding affinity for human HVEM was measured by surface plasmon resonance using a BIAcore T200. A humanized anti-human HVEM antibody candidate solution diluted to 30 μg / ml with running buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Tween 20) was flowed over a CM5 sensor chip on which an anti-human IgG antibody had been immobilized by amine coupling at a flow rate of 10 μl / min for 2 minutes, allowing the humanized anti-human HVEM antibody candidate to be adsorbed onto the sensor chip. Furthermore, human HVEM extracellular domain recombinant protein (Shinobiological Japan 10334-H08H) diluted in running buffer was run as an analyte at a flow rate of 10 μl / min for 2 minutes to measure the Ka value. The running buffer was then switched to one containing no antibody, and the Kd value was measured over the next 2 minutes. The KD value was calculated as Kd / Ka.

[0089] (2) Measurement of the binding affinity of alpaca anti-human HVEM antibody (VHH-3), alpaca-human chimeric anti-human HVEM antibody (VHH-3_hIgG4Fc), and humanized anti-human HVEM antibody (HV80, HVR81) to human HVEM. For VHH-3, a SA sensor chip was adsorbed with VHH-3 biotinylated at its C-terminus and Avitag attached. Human HVEM extracellular domain recombinant protein (Shino Biological Co., Ltd., Japan 10334-H08H) diluted in running buffer was run as an analyte at a flow rate of 10 μl / min for 2 minutes to measure the Ka value. The running buffer was then switched to one containing no antibody, and the Kd value was measured for 2 minutes. The KD value was calculated as Kd / Ka. For VHH-3_hIgG4Fc, HV80, and HVR81, the KD values ​​were calculated in the same manner as in (1) above, except that the humanized anti-human HVEM antibody candidate solution was changed to a solution containing VHH-3_hIgG4Fc, HV80, or HVR81.

[0090] (3) Results The results are shown in Table 9.

[0091] HVR80 and HVR81 showed increased HVEM binding affinity compared to HV80.

[0092] Example 5: Effect of alpaca anti-human HVEM antibody gene transfer on proliferation of mesenchymal subtype cells (1) Lentiviral gene transfer A CMV promoter, alpaca anti-human HVEM antibody (VHH-3) gene (SEQ ID NO: 29) or anti-GFP antibody (negative control) gene (SEQ ID NO: 30), and bGH polyA sequence were inserted between the BglII and XbaI sites of the pENTR4 plasmid, and subjected to LR reaction with the CS-RfA-EG plasmid. The LR reaction product plasmid, pCAG-HIVgp, and pCMV-RSV-G-Rev were transfected into HEK293FT cells using Lipofectamine 2000 Reagent (Thermo Fisher Scientific) to produce lentivirus. The culture supernatant containing lentiviral particles was collected and concentrated using a Lenti-X Concentrator (TAKARA Bio). The concentrated lentivirus was suspended in serum-free medium and used to infect GBM cells. Since GBM cells expressing the antibody gene are labeled with EGFP, EGFP-positive cells were isolated using a cell sorter. The amino acid sequence of the expressed alpaca anti-human HVEM antibody (VHH-3) was SEQ ID NO: 34, and the amino acid sequence of the anti-GFP antibody (negative control) was SEQ ID NO: 35.

[0093] (2) Cell proliferation assay: 1 x 10 mesenchymal subtype GBM cells transfected with each antibody gene obtained in (1) above were cultured in a 500-well plate. ３ The cells were seeded at a density of 100 cells / well. After seeding, the number of viable cells was quantified by CCK-8 assay at 3, 5, or 7 days, and normalized to the value quantified at the time of seeding.

[0094] (3) Results The results are shown in Figure 2. Mesenchymal subtype GBM cells transfected with the alpaca anti-human HVEM antibody (VHH-3) gene showed significantly suppressed cell proliferation compared to the negative control.

[0095] Example 6: Effect of alpaca anti-human HVEM antibody on proliferation and cell invasion of mesenchymal subtype cells (1) Cell proliferation assay Mesenchymal subtype GBM cells were cultured at 1 x 10 ３ Cells were seeded at a density of 100 cells / well and treated with an alpaca anti-human HVEM antibody (VHH-3) or an anti-KLH antibody (negative control) at a concentration (1 μM). Three, five, or seven days after seeding, the number of viable cells was quantified by CCK-8 assay, and growth curves were normalized to the value quantified at the time of seeding. Seven days after seeding, the number of viable cells was also quantified by CCK-8 assay, and response dose curves were normalized to the value quantified in the absence of antibody treatment.

[0096] (2) Cell Invasion Assay Mesenchymal subtype GBM cells were treated with CFSE, and live cells were fluorescently labeled. CFSE-labeled cells were plated on a low-cell-attachment plate at 1 × 10 ４ Mesenchymal subtype GBM cell spheroids were generated by seeding at a density of 100 cells / well and culturing for 24 hours. Mouse brains were cultured in 300-500 μm slices, and mesenchymal subtype GBM cell spheroids were placed on the mouse brain slices and the morphology of the spheroids was observed under a fluorescent microscope. Mesenchymal subtype GBM cell spheroids on the mouse brain slices were treated with alpaca anti-human HVEM antibody (VHH-3) or anti-GFP antibody (negative control) at a concentration of 1 μM and cultured for 2 days, and the morphology of the spheroids was observed under a fluorescent microscope. The invasion area of ​​the spheroids was quantified and normalized to the size of the spheroids before antibody treatment to evaluate their invasive potential.

[0097] (3) Results The results are shown in Figure 3. Cell proliferation was inhibited in mesenchymal subtype GBM cells treated with alpaca anti-human HVEM antibody (VHH-3) compared to the negative control. Furthermore, the alpaca anti-human HVEM antibody (VHH-3) significantly inhibited the infiltration of mesenchymal subtype GBM cell spheroids (U3031MG and U3054MG) into mouse brains.

[0098] Example 7: Effect of intraperitoneal administration of alpaca anti-human HVEM antibody on the in vivo proliferation ability of mesenchymal subtype GBM cells (1) Animal experiment Mesenchymal subtype GBM cells (U3031MG or U3054MG) were inoculated at 1 × 10 into the brain of a BALB / c-nu / nu mouse. ５ Before antibody administration, tumor size was quantified by in vivo luminescence imaging, and the mice were divided into two groups to ensure equal luminescence intensity. Antibody administration began on day 105 after cell transplantation for U3031MG cell-derived tumor-bearing mice, and on day 91 after cell transplantation for U3054MG cell-derived tumor-bearing mice. Alpaca anti-human HVEM antibody (VHH-3) or anti-GFP antibody (negative control) was administered intraperitoneally daily at a dose of 10 mg / kg. Tumor size was quantified by in vivo luminescence imaging 14 and 28 days after the start of antibody administration, and the effect of the antibody on in vivo growth ability was examined. The time point at which 20% or more weight loss and / or neurological symptoms were observed was defined as the endpoint for survival analysis. Note that the engraftment rate of GBM cells under these experimental conditions was 100%, so no mice were excluded due to non-engraftment.

[0099] (2) Results The results are shown in Figure 4. In the group administered with alpaca anti-human HVEM antibody (VHH-3), tumor size was significantly reduced and survival time was significantly extended compared to the negative control.

[0100] Example 8: Effect of HVEM gene deletion on anticancer drugs (1) Genome editing using the CRISPR-Cas9 system The DNA sequence of sgRNA (SEQ ID NO: 31 and SEQ ID NO: 32) targeting the human TNFRSF14 gene or the DNA sequence of a negative control sgRNA (SEQ ID NO: 33) was designed downstream of the hU6 promoter in the lentiCRISPR v2 plasmid. In addition to the prepared plasmid, pCAG-HIVgp and pCMV-VSV-G-RSV-Rev were transfected into HEK293FT cells using Lipofectamine 2000 Reagent (Thermo Fisher Scientific) to produce lentivirus. The culture supernatant containing lentiviral particles was collected and concentrated using a Lenti-X Concentrator (TAKARA Bio). The concentrated lentivirus was suspended in serum-free medium and used to infect GBM cells (U3054MG) to obtain genome-edited GBM cells (U3054MG-hCas9). Single cells were sorted using a cell sorter, and each was cultured to confirm the DNA sequence of the human TNFRSF14 gene. GBM cells lacking the human TNFRSF14 gene were obtained by genome editing.

[0101] (2) Cell proliferation assay. The GBM cells lacking the human TNFRSF14 gene obtained in (1) above were seeded and treated with DMSO (negative control), an anticancer drug (temozolomide), or an EGFR inhibitor (erlotinib) at the concentrations shown in the figure. After culturing the cells for 7 days, the number of viable cells was quantified by CCK-8 assay and normalized to the value quantified in the untreated condition to create a response dose curve.

[0102] (3) Apoptosis Detection Assay: The human TNFRSF14 gene-deficient GBM cells obtained in (1) above were treated with DMSO (negative control), an anticancer drug (temozolomide), or an EGFR inhibitor (erlotinib) at the concentrations shown in the figure and cultured for 7 days. Cells under each condition were collected and stained with Annexin V-FITC and 7-AAD, and apoptosis of mesenchymal subtype GBM cells was detected using a flow cytometer.

[0103] (4) Results The results are shown in Figure 5. Mesenchymal subtype GBM cells lacking the HVEM gene were shown to have significantly increased sensitivity to an anticancer drug (temozolomide) and an EGFR inhibitor (erlotinib).

[0104] Example 9: Effect of alpaca anti-human HVEM antibody on the interaction between human BTLA and human HVEM (1) Sandwich ELISA. Human HVEM recombinant protein was immobilized on an immunoassay plate, and then reacted with alpaca anti-human HVEM antibody (VHH-3) or anti-GFP antibody (negative control) for 1 hour. After washing, human BTLA-Fc recombinant protein was reacted for 1 hour. After washing, HRP-labeled anti-Fc antibody was reacted for 30 minutes. After further washing, 100 μL of TMB substrate solution was added and reacted for 5 minutes. The enzyme reaction was stopped by adding 50 μL of 2N sulfuric acid. The absorbance at 450 nm was measured using a plate reader, and the amount of human BTLA-Fc recombinant protein binding to human HVEM recombinant protein was quantified.

[0105] (2) Results The results are shown in Figure 6. It was demonstrated that the alpaca anti-human HVEM antibody (VHH-3) inhibited the binding of human BTLA to human HVEM in a concentration-dependent manner. This suggests that the alpaca anti-human HVEM antibody (VHH-3) not only exhibits anti-glioma activity through HVEM inhibition, but also inhibits the suppression of immune responses through BTLA inhibition.

[0106] Example 10: Pharmacokinetics of humanized anti-human HVEM antibody in cynomolgus monkeys (1) Animal Experiments Heparinized blood samples were collected from cynomolgus monkeys before antibody administration. Humanized anti-human HVEM antibody (HV15) at the concentrations shown in the figure (0.3 mg / kg, 1 mg / kg, 3 mg / kg) was intravenously or subcutaneously injected into cynomolgus monkeys, and heparinized blood samples were collected from each individual 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after antibody administration. Humanized anti-human HVEM antibody (HV80) at the concentrations shown (3 mg / kg, 10 mg / kg, 30 mg / kg) was also intravenously injected into cynomolgus monkeys, and heparinized blood samples were collected from each individual 1 hour, 6 hours, 24 hours, 3 days, 7 days, and 14 days after antibody administration. Plasma components were extracted after blood collection. This study was conducted by Ina Research.

[0107] (2) Sandwich ELISA: Human HVEM recombinant protein was immobilized on an immunoassay plate and reacted with the cynomolgus monkey plasma obtained in (1) above for 1 hour. After washing, the plate was reacted with an HRP-labeled anti-His tag antibody or anti-Fc antibody for 30 minutes. After further washing, 100 μL of TMB substrate solution was added and reacted for 5 minutes. The enzyme reaction was stopped by adding 50 μL of 2N sulfuric acid. The absorbance at 450 nm was measured using a plate reader to quantify the amount of antibody in the plasma and measure the blood concentration of the antibody.

[0108] (3) Results The results are shown in Figure 7. It was shown that the humanized anti-human HVEM antibody (HV15) and the humanized anti-human HVEM antibody (HV80) are maintained in vivo, and that the humanized anti-human HVEM antibody (HV80) has a longer half-life in the blood compared to the humanized anti-human HVEM antibody (HV15). For HV15, subcutaneous injection showed a slightly longer half-life in the blood compared to intravenous injection.

[0109] Example 11: Single-dose toxicity test of humanized anti-human HVEM antibody in cynomolgus monkeys. The humanized anti-human HVEM antibody (HVs80) was administered intravenously in a single dose of 100 mg / kg or 300 mg / kg to two male cynomolgus monkeys in one group, and toxicity changes were examined. This test was conducted by Ina Research.

[0110] (1) Animal Welfare This study complied with the Act on Welfare and Management of Animals and related guidelines, and was conducted appropriately in accordance with a study protocol reviewed by the Institutional Animal Care and Use Review Committee (IACUC). The study facility was certified by AAALAC International.

[0111] (2) Test Method The test substance, HVs80, was administered into the tail vein (administration time: 1 hour, number of administrations: 1 time) to cynomolgus monkeys (4 males, age at administration: 3 years, body weight at administration: 2.68-3.58 kg) at the doses shown in Table 10. The doses in this test were set at 3.3 and 10 times the dose of 30 mg / kg, as good results were obtained at that dose in the previous pharmacokinetic study.

[0112]

[0113] (3) Observation The observation period was from the day of animal acquisition (transfer) to the end of the test (day 14 of administration), with the day of administration counted as day 1, and the observation items were general condition, body weight, and food intake.

[0114] (4) Blood Tests Hematological tests (clotting tests and complete blood counts) and blood biochemistry tests were performed. Blood samples were collected from the femoral vein before administration (during the acclimation period), and on days 2, 8, and 14 after administration.

[0115] Complete blood count includes red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte percentage (RET%), absolute count (RET#), platelet count (PLT), white blood cell count (WBC), and differential white blood cell count. ａ） The percentage (Diff WBC%) and absolute number (Diff WBC #) were measured using a multi-parameter automated hematology analyzer XN-2000 (Sysmex Corporation) ( ａ） Neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MONO), eosinophils (EO), basophils (BASO).

[0116] In the coagulation tests, prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured using a fully automated blood coagulation analyzer CA-510 (Sysmex Corporation).

[0117] In the blood biochemistry test, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), creatine kinase (CK), glucose (GLU), total bilirubin (BIL), urea nitrogen (UN), creatinine (CK), total cholesterol (CHO), triglycerides (TG), phospholipids (PL), inorganic phosphorus (IP), calcium (CA), sodium (NA), potassium (K), chloride (CL), total protein (TP), albumin (ALB), and albumin-globulin ratio (A / G) were measured using a 7180-type automatic analyzer (Hitachi High-Technologies Corporation).

[0118] (5) Results Humanized anti-human HVEM antibody (HVs80) was administered intravenously in a single dose of 100 mg / kg or 300 mg / kg to two male cynomolgus monkeys in one group, and toxicity was examined. As a result, no effects of the administration of the test substance were observed in the general condition, body weight, food intake, hematological tests, or blood biochemistry tests in any of the animals.

[0119] Example 12: Effect of humanized anti-human HVEM antibody on proliferation of mesenchymal subtype cells (1) Cell proliferation assay Mesenchymal subtype GBM cells (U3054MG) were cultured at 1 × 10 ３ Cells were seeded at a density of 100 cells / well and treated with a humanized anti-human HVEM antibody (HVR81) or an anti-GFP antibody (negative control) at concentrations ranging from 1 nM to 1 μM. Seven days after treatment, the number of viable cells was quantified by CCK-8 assay and normalized to the value quantified in the absence of antibody treatment to create a dose response curve.

[0120] (2) Results The results are shown in Figure 8. It was shown that cell proliferation was suppressed in mesenchymal subtype GBM cells treated with the humanized anti-human HVEM antibody (HVR81) compared to the negative control.

[0121] Example 13: Effect of humanized anti-human HVEM antibody on the interaction between human BTLA and human HVEM and the interaction between human APRIL and human HVEM (1) Method Human HVEM recombinant protein was immobilized on an immunoassay plate, and then reacted with a humanized anti-human HVEM antibody (HVR81) or an anti-GFP antibody (negative control) for 1 hour. After washing, the plate was reacted with human BTLA-His or Haemaglutinin (HA)-human APRIL recombinant protein for 1 hour. After washing, the plate was reacted with an HRP-labeled anti-His antibody or anti-HA antibody for 30 minutes. After further washing, 100 μL of TMB substrate solution was added and reacted for 5 minutes. The enzyme reaction was stopped by adding 50 μL of 2N sulfuric acid. The absorbance at 450 nm was measured using a plate reader, and the amount of binding of human BTLA-His recombinant protein or HA-human APRIL recombinant protein to human HVEM recombinant protein was quantified. The amount of HVEM binding to BTLA or APRIL in the presence of antibody was evaluated in each test group, and the IC was calculated from a sigmoid curve. ５０ was calculated.

[0122] (2) Results The results are shown in Figures 9 and 10. The IC of the humanized anti-human HVEM antibody (HVR81) against BTLA-HVEM was also ５０ The IC value of the humanized anti-human HVEM antibody (HVR81) against APRIL-HVEM was 0.722 nM. ５０ The concentration of the humanized anti-human HVEM antibody (HVR81) was 19.5 nM. These results demonstrated that the humanized anti-human HVEM antibody (HVR81) inhibited the binding between human BTLA and human HVEM, and the binding between human APRIL and human HVEM, in a concentration-dependent manner. In other words, the humanized anti-human HVEM antibody (HVR81) was shown to have an anti-glioma effect through the inhibition of HVEM, and also to have the effect of inducing anti-tumor immunity through the inhibition of BTLA.

[0123] Example 14: Suppression of HVEM-mediated NF-κB signaling by humanized anti-human HVEM antibodies (1) NF-κB signaling reporter assay pCS-NF-κB-RE-minP-Luc2-CMV-hRluc was prepared by cloning the firefly luciferase gene (Luc2, Promega) downstream of a minimal promoter having an NF-κB response element and the Renilla luciferase gene (hRluc, Promega) downstream of the MV promoter. HEK293FT cells were transfected with pCS-NF-κB-RE-minP-Luc2-CMV-hRluc, pCAG-HIVgp, and pCMV-RSV-G-Rev using Lipofectamine 2000 Reagent (Thermo Fisher Scientific) to produce lentivirus. The culture supernatant containing lentiviral particles was collected and concentrated with a Lenti-X Concentrator (TAKARA Bio). The concentrated lentivirus was suspended in serum-free medium and used to infect U3054MG cells, a mesenchymal subtype GBM cell, to introduce the NF-κB-responsive Luc2 gene and the constitutively expressed hRluc gene. Mesenchymal subtype GBM cells 1 × 10 ３ Cells were seeded at a density of 100 cells / well and treated with a humanized anti-HVEM antibody (HVR81) or an anti-GFP antibody (negative control) at concentrations of 1 pM to 1 μM. 48 hours after treatment, NF-κB activity was assessed using a Dual luciferase reporter assay kit (Promega). The luminescence intensity of Luc2 in the same well was corrected by the luminescence intensity of hRluc, and then normalized to the quantitative value in the absence of antibody treatment to create a reaction dose curve. Furthermore, relative NF-κB activity in the presence of antibody was assessed, and the IC was calculated from a sigmoidal curve. ５０ was calculated.

[0124] (2) Results The results are shown in Figure 11. The IC value of the humanized anti-human HVEM antibody (HVR81) on the relative NF-κB activity was also ５０The concentration of NF-κB in mesenchymal subtype GBM cells treated with the humanized anti-human HVEM antibody (HVR81) was 14.2 nM. These results demonstrated that NF-κB activity was suppressed in the mesenchymal subtype GBM cells treated with the humanized anti-human HVEM antibody (HVR81) compared to the negative control.

[0125] Example 15: Lysosomal uptake test of humanized anti-human HVEM antibody (HVR80) in HVEM-expressing cells (1) Test method To visualize the uptake of humanized anti-human HVEM antibody (HVR80) into HVEM-expressing cells into lysosomes, humanized anti-human HVEM antibody (HVR80) and mouse IgG (negative control) were fluorescently labeled with AcidiFluor ORANGE-NHS (Goryo Chemical Co., Ltd.), which emits strong fluorescence in the acidic range found in lysosomes. Each antibody was added to a final concentration of 10 μg / ml in the culture medium in which HVEM-expressing HEK293T cells (HEK293T-HVEM) were grown. Furthermore, the labeled humanized anti-human HVEM antibody (HVR80) alone was also added to the culture medium in which HeLa cells (negative control), which have minimal HVEM expression, were grown. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% complement-inactivated fetal bovine serum (ThermoFisher). 20 hours after addition of each antibody to the cell culture medium, the cells were observed using a confocal microscope (Zeiss) (excitation wavelength: 544 nm, emission wavelength: 565 nm).

[0126] (2) Results The results are shown in Figure 12. Fluorescence (black areas) was observed only when humanized anti-human HVEM antibody (HVR80) was administered to HVEM-expressing HEK293T cells, demonstrating that the humanized anti-human HVEM antibody (HVR80) was taken up into lysosomes.

[0127] Example 16: Effect of MMAE or PNU-added humanized anti-human HVEM antibody (HVR80) on HEK293T-HVEM cells (1) Method HEK293T cells (HEK293T-HVEM) that stably express high levels of HVEM and wild-type HEK293T cells as a negative control were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% pre-complement-inactivated fetal bovine serum (ThermoFisher) at 37°C and 5% CO ２ Each cell was cultured in a 96-well plate at 1 × 10 ４ After 24 hours of culture, cells were seeded at a density of 0.001 to 10 μg / ml with either a humanized anti-human HVEM antibody (HVR80) conjugated with monomethyl auristatin E (MMAE) using Glyclick ADC MMAE (Genovis AB), a humanized anti-human HVEM antibody (HVR80) conjugated with PNU using Glyclick ADC PNU (Genovis AB), or a negative control humanized anti-human HVEM antibody (HVR80). Three days after administration, cell viability (%) in each test group was assessed using the viable cell counting reagent SF (Nacalai Tesque), and the IC was calculated from a sigmoidal curve representing cell viability. ５０ was calculated.

[0128] (2) Results The results of cell viability are shown in Figures 13 and 14. The IC of the humanized anti-human HVEM antibody (HVR80) conjugated with MMAE was also shown. ５０ The IC value of the humanized anti-human HVEM antibody (HVR80) containing PNU was 70 ng / ml (0.9 nM). ５０ The concentration of HVEM in the antibody-drug conjugate (ADC) was 30 ng / ml (0.4 nM). These results demonstrate that the antibody-drug conjugate (ADC) of the humanized anti-human HVEM antibody (HVR80) has a high growth inhibitory effect on HEK293T cells highly expressing HVEM.

Claims

1. A human HVEM binding protein comprising at least one immunoglobulin single variable region, said immunoglobulin single variable region comprising complementarity determining regions 1-3 (CDR1, CDR2 and CDR3) having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and said immunoglobulin single variable region is a humanized VHH.

2. The HVEM binding protein of claim 1, wherein the immunoglobulin single variable region comprises the amino acid sequence of (v), (vi) or (vii) below: (v) the amino acid sequence of SEQ ID NO: 18, 19 or 20; (vi) an amino acid sequence having at least 80% identity to the sequence of the framework region (excluding amino acids corresponding to the humanized region) in the sequence of (v); (vii) an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the sequence of the framework region (excluding amino acids corresponding to the humanized region) in the sequence of (v) or (vi).

3. The HVEM binding protein of claim 1, wherein the immunoglobulin single variable region comprises framework regions (FR1, FR2, FR3 and FR4) and the amino acid sequences of FR1, FR2, FR3 and FR4 are (i), (ii) or (iii) below: (i) FR1 having the amino acid sequence represented by SEQ ID NO:5, FR2 having the amino acid sequence represented by SEQ ID NO:8, FR3 having the amino acid sequence represented by SEQ ID NO:11 and FR4 having the amino acid sequence represented by SEQ ID NO:13; (ii) FR1 having the amino acid sequence represented by SEQ ID NO:5, FR2 having the amino acid sequence represented by SEQ ID NO:7, FR3 having the amino acid sequence represented by SEQ ID NO:10 and FR4 having the amino acid sequence represented by SEQ ID NO:13; (iii) FR1 having the amino acid sequence represented by SEQ ID NO:5, FR2 having the amino acid sequence represented by SEQ ID NO:6, FR3 having the amino acid sequence represented by SEQ ID NO:10 and FR4 having the amino acid sequence represented by SEQ ID NO:

13.

4. The HVEM binding protein of claim 1 or 2, further comprising a human immunoglobulin Fc region or a human serum albumin binding protein.

5. The HVEM binding protein of claim 4, wherein the immunoglobulin Fc region is a human IgG4 Fc region.

6. The HVEM binding protein of claim 5, wherein the amino acid sequence of the Fc region of human IgG4 is the amino acid sequence of SEQ ID NO: 14, 15 or 16.

7. An antibody-drug conjugate comprising the HVEM binding protein of claim 1 or 4 and a drug.

8. The antibody-drug conjugate of claim 7, wherein the drug is an anticancer drug.

9. A pharmaceutical composition comprising an HVEM binding protein according to claim 1 or 2 or an antibody-drug conjugate according to claim 7.

10. The pharmaceutical composition according to claim 9, for use in inhibiting the binding of HVEM to BTLA and / or APRIL.

11. The pharmaceutical composition according to claim 9 for use in treating or preventing solid cancer.

12. The pharmaceutical composition according to claim 9 for use in the treatment or prevention of glioma.

13. The pharmaceutical composition according to claim 12, wherein the glioma is any one of oligodendroglioma, oligoastrocytoma, astrocytoma and glioblastoma multiforme.

14. The pharmaceutical composition according to claim 13, wherein the glioblastoma multiforme belongs to any one of the proneural subtype, the neural subtype, the classical subtype, and the mesenchymal subtype.

15. The pharmaceutical composition according to claim 9 for use in combination with other anticancer drugs.

16. An inhibitor of the HVEM signaling pathway comprising an HVEM binding protein described in claim 1 or 2.

17. The inhibitor described in claim 16, wherein the HVEM signaling pathway is the HVEM-mediated NF-κB signaling pathway.

18. An anti-cancer immune-inducing agent comprising the HVEM binding protein described in claim 1 or 2.

19. A polynucleotide encoding the HVEM binding protein of claim 1 or 2.

20. An expression vector comprising the polynucleotide of claim 19.

21. A method for treating or preventing a solid cancer, comprising administering to a subject in need thereof an HVEM binding protein described in claim 1 or 2 or a composition containing same, or an antibody-drug conjugate described in claim 7 or a composition containing same.

22. Use of the HVEM binding protein described in claim 1 or 2 or the antibody-drug conjugate described in claim 7 for the manufacture of a therapeutic or prophylactic agent for solid cancer.

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