Anti-ULBP2 antibody

Abstract

This invention provides a novel antibody that influences the interaction between ULBP2 and NKG2D. [Solution] An anti-ULBP2 monoclonal antibody or its antigen-binding fragment is provided, comprising a heavy chain variable region containing specific heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 amino acid sequences, and a light chain variable region containing specific light chain CDR1, light chain CDR2, and light chain CDR3 amino acid sequences. An anti-ULBP2 monoclonal antibody and a method for producing the same are also provided, which can increase the binding of ULBP2 to human NKG2D immobilized on a solid phase in an antibody dose-dependent manner.

Description

[Technical Field]

[0001] This disclosure relates to the field of antibodies against immune-related molecules. [Background technology]

[0002] Immune checkpoint inhibitors (ICIs) have become a core component of cancer drug therapy. However, long-term responses to ICIs are limited, and biomarkers that can predict the effectiveness of the treatment are not yet fully established. There continues to be a need for therapeutic agents that target immune-related molecules, which can serve as adjuncts, complements, or alternatives to ICIs.

[0003] NKG2D is an activating receptor expressed on the surface of immune system cells such as T cells and NK (natural killer) cells, and ULBP2 (UL-16 Binding Protein 2) is one of the ligands for NKG2D in humans. ULBP2 is expressed on the surface of many cancer cells, and when cleaved by ADAM proteases, the extracellular portion is secreted into the body fluid. ULBP2 has long been reported to be a poor prognostic factor in many cancer types, but recent comprehensive analysis of immune-related genes based on The Cancer Genome Atlas (TCGA) database has revealed that it is one of the top poor prognostic factors in solid tumors, hematological malignancies, head and neck cancers, colorectal cancers, breast cancers, and others. Human NKG2D ligands include eight types: MICA, MICB, and ULBP1-6, and ULBP2 has been reported to have higher cancer tissue specificity than other NKG2D ligands. These clinical data suggest that ULBP2 may be a potential target for new cancer immunotherapy.

[0004] In vitro experiments have shown that ULBP2-expressing cancer cells are susceptible to cytotoxicity by NK cells. However, there are seemingly contradictory experimental results regarding the effects of NKG2D ligands, including ULBP2, on tumor immunity in vivo, and their clinical significance is not yet fully understood. NKG2D stimulation with NKG2D ligands promotes the survival and proliferation of T cells and NK cells, and enhances the responsiveness of other active receptors such as T cell receptors and CD16. On the other hand, chronic NKG2D stimulation has been shown to cause unresponsiveness of NKG2D and these active receptors, ultimately leading to inhibition of NK cell activity and tumor clearance ability (Non-Patent Documents 1-3). Non-Patent Document 4 reports that mouse tumor cell lines expressing ULBP2 were able to induce an immune response in syngeneic mice. Non-Patent Document 4 also confirms that human ULBP2 can specifically bind to mouse NKG2D. Non-patent document 5 reports that when ULBP2 was expressed in mouse tumor cells, cytotoxicity by NK cells was enhanced in vitro, but in vivo, ULBP2-expressing tumors proliferated more rapidly, and tumor growth was suppressed when NKG2D was blocked by administering an anti-NKG2D antibody. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Oppenheim et al., Nat Immunol. 2005 Sep;6(9):928-37. [Non-Patent Document 2] Jelencic et al., Immunol Lett. 2017 Sep:189:48-53. [Non-Patent Document 3] Wensveen et al., Front Immunol. 2018 Mar 8:9:44. [Non-Patent Document 4] Sutherland et al., Blood. 2006 Aug 15;108(4):1313-9. [Non-Patent Document 5] Yamaguchi et al., Cancer Res (2021) 81 (13_Supplement): 1770. [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] The present disclosure provides novel antibodies that can affect the interaction between ULBP2 and human NKG2D. [Means for Solving the Problems]

[0007] In certain embodiments, the present disclosure provides (a) a heavy chain variable region comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region comprising the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 4, 5, and 6, respectively; (b) a heavy chain variable region comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 7, 8, and 9, respectively, and a light chain variable region comprising the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 10, 11, and 12, respectively; (c) a heavy chain variable region comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 13, 14, and 15, respectively, and a light chain variable region comprising the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 16, 17, and 18, respectively; or (d) a heavy chain variable region comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 19, 20, and 21, respectively, and a light chain variable region comprising the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 22, 23, and 24, respectively, and provides an anti-ULBP2 monoclonal antibody or an antigen-binding fragment thereof.

[0008] These antibody and antigen-binding fragments can be characterized by their ability to dose-dependently increase the binding of ULBP2 to solid-phase-immobilized human NKG2D.

[0009] In some embodiments, the present disclosure provides an anti-ULBP2 monoclonal antibody and a method for producing the same that can increase the binding of ULBP2 to solid-phase-immobilized human NKG2D in an antibody dose-dependent manner, the method for producing the same comprising immunizing a non-human animal with ULBP2, preparing a population of hybridomas from the immunized non-human animal, selecting a subpopulation of hybridomas from the population of hybridomas that produces an antibody capable of binding to ULBP2, and selecting a hybridoma clone from the subpopulation of hybridomas that produces an antibody capable of increasing the binding of ULBP2 to human NKG2D in an antibody dose-dependent manner, wherein the antibody produced by the hybridoma clone provides the anti-ULBP2 monoclonal antibody.

[0010] In some embodiments, nucleic acid molecules encoding an anti-ULBP2 monoclonal antibody or its antigen-binding fragment are provided, as well as expression vectors and host cells containing the same.

[0011] In some embodiments, a pharmaceutical composition comprising an anti-ULBP2 monoclonal antibody or an antigen-binding fragment thereof is provided. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the interaction between ULBP2 and NKG2D and the anti-ULBP2 monoclonal antibody of this disclosure, as shown by ELISA. [Figure 2]Figure 2 shows flow cytometry data demonstrating that the binding of hNKG2D-human Fc to cancer cells expressing ULBP2 on their surface is inhibited by the commercially available anti-ULBP2 monoclonal antibody used as a control, while it is actually increased by the anti-ULBP2 monoclonal antibody disclosed herein. Note that the control experiments, human Fc + isotype Ab and human Fc + anti-ULBP2, which did not contain hNKG2D protein, produce substantially overlapping profiles (the leftmost peak corresponding to unbound cells). [Figure 3] Figures 3a-h show the cytotoxicity exhibited by NK cells in vitro over a short time span of 4 hours against ULBP2-expressing B16F10 cells or control cells, and the effect of the antibody disclosed herein on this cytotoxicity. Figures i and j show the results of evaluating the IFN-γ production performance of CD8+ T cells by flow cytometry after co-culturing spleen cells and ULBP2-expressing B16F10 cells for 72 hours in the presence of an anti-ULBP2 antibody. In the presence of the antibody (48) disclosed herein, IFN-γ production performance is increased. [Figure 4] Figure 4 shows the effect of in vivo administration of the anti-ULBP2 monoclonal antibody of this disclosure to ULBP2-expressing B16F10 tumors. [Figure 5] Figure 5 shows experimental results demonstrating that CT26.WT tumors become resistant to checkpoint inhibitor (anti-PD-1 antibody) treatment when they express ULBP2. [Figure 6] Figure 6 shows experimental results demonstrating that the antitumor effect of checkpoint inhibitors against CT26.WT tumors is at least partially inhibited by anti-NKG2D antibodies. [Figure 7] Figure 7 shows experimental results demonstrating that the combination of a checkpoint inhibitor and the anti-ULBP2 monoclonal antibody disclosed herein has a synergistic therapeutic effect on CT26.WT tumors that have become resistant to checkpoint inhibitor therapy due to ULBP2 expression. [Modes for carrying out the invention]

[0013] In one aspect, the present disclosure provides an anti-ULBP2 monoclonal antibody or an antigen-binding fragment thereof. This antibody is (a) Each comprises a heavy chain variable region containing the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 1, 2, and 3, and a light chain variable region containing the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 4, 5, and 6, (b) Each comprises a heavy chain variable region containing the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 7, 8, and 9, and a light chain variable region containing the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 10, 11, and 12, (c) Each comprising a heavy chain variable region containing the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs. 13, 14, and 15, and a light chain variable region containing the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs. 16, 17, and 18, respectively, or (d) Each may include a heavy chain variable region containing the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs. 19, 20, and 21, and a light chain variable region containing the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs. 22, 23, and 24, respectively.

[0014] The term "anti-ULBP2" antibody means that this antibody has the ability to specifically bind to ULBP2. As will be understood by those skilled in the field of antibodies, the possibility that an antibody that "specifically binds" to one antigen may cross-react with other antigens cannot be ruled out. The antibody or its antigen-binding fragment may be an isolated or purified antibody or its antigen-binding fragment. Various chemical modifications, labels, conjugates, etc., that can be conferred to an antibody or its antigen-binding fragment are known to those skilled in the art, and one or more of these may be conferred to the antibody or its antigen-binding fragment of this disclosure. The antibody of this disclosure may have different isotypes, and may be, for example, IgG1 or IgG2b.

[0015] The basic structure of an antibody, including the heavy chain variable region, heavy chain constant region, light chain variable region, and light chain constant region, is well known to those skilled in the art. The basic structure of an antibody can also be divided into the Fab region and the Fc region. As is known to those skilled in the art, the "complementarity-determining region" (CDR) is the region within the variable region of an antibody that forms the antigen-binding site. Within the variable region, the region other than the CDR region is called the framework region. The heavy chain variable region and light chain variable region of an antibody each contain three CDRs, which are called heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 from the N-terminus of the heavy chain, and light chain CDR1, light chain CDR2, and light chain CDR3 from the N-terminus of the light chain, respectively. Heavy chain CDR1-3 are also called CDR-H1-H3, and light chain CDR1-3 are also called CDR-L1-L3. In this disclosure, the CDR of an antibody or its antigen-binding fragment is defined by the IMGT definition scheme unless otherwise specified (Dev Comp Immunol, 2003, 27(1):55-77). However, as is well known in the art, the CDR region may also be determined by other schemes based on the heavy and light chain variable region sequences, such as Kabat (Sequences of Proteins of Immunological Interest, 1991, NIH Publication No. 91-3242) and Chothia (J Mol Biol. (1987) 196:901-17, Nature (1989) 342:877-83) (Dondelinger et al., 2018, Front. Immunol. 9:2278). For example, the present disclosure provides an anti-ULBP2 monoclonal antibody or an antigen-binding fragment thereof, comprising amino acid sequences of CDR-H1-H3 and CDR-L1-L3 identified according to the definitions of IMGT, Kabat, or Chothia from SEQ ID NOs. 25-48, particularly SEQ ID NOs. 25 and 26, SEQ ID NOs. 27 and 28, SEQ ID NOs. 29 and 30, or SEQ ID NOs. 31 and 32.

[0016] Table 1 below shows the CDR amino acid sequences of sequence numbers (SEQ ID NO) 1 to 24 mentioned above. In the antibody number column of the table below, (a) to (d) are the numbers shown in the paragraph three paragraphs prior, and the numbers 48 to 136 represent the numbers of the representative antibodies exemplified in the Examples section. [Table 1]

[0017] In this disclosure, “antigen-binding fragment” refers to a polypeptide that corresponds to a part of an antibody, particularly a polypeptide containing a variable region. Antigen-binding fragments of an antibody retain a specific binding ability to a specific antigen for that antibody. Various antigen-binding fragments are known to those skilled in the art. For example, antigen-binding fragments can be obtained by digesting a full-length antibody molecule with various peptidases, or they can be produced genetically independently of the full-length antibody molecule. Genetically produced antigen-binding fragments may have a different primary sequence than simple antibody digestion fragments, and may, for example, contain a heavy-chain variable region and a light-chain variable region in a continuous polypeptide. While full-length antibodies contain an Fc region and a Fab region, examples of antigen-binding fragments of antibodies include F(ab')2, Fab', Fab, Fv, scFv, diabody, etc.

[0018] In some embodiments, particularly in more specific embodiments of antibodies characterized by the inclusion of a particular CDR, numbered (a) to (d) above, the antibody is, (1) comprising the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 25 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 26; (2) comprising the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 27 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 28; (3) comprising the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 29 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 30; or (4) The antibody may contain the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 31 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 32. The antibodies described in (1) to (4) above correspond to the representative antibodies 48, 54, 83, and 136, respectively, as illustrated in the Examples section.

[0019] The variable region amino acid sequences for sequence numbers 25-32 are shown in Table 2 below. [Table 2]

[0020] Those skilled in the art will understand that an antibody or its antigen-binding fragment may contain additional sequences in addition to those described above. For example, the sequence of the constant region may be attached to the C-terminus of the variable region. The sequence of the signal peptide may be attached to the N-terminus. In the case of a full-length antibody, an Fc region may be included at the C-terminus.

[0021] SEQ ID NO: 33 provides the amino acid sequence of the 48 antibody heavy chain, including the signal peptide up to the CH1 domain of the constant region. SEQ ID NO: 34 provides the full-length amino acid sequence of the 48 antibody light chain, including the signal peptide up to the constant region. SEQ ID NO: 35 provides the amino acid sequence of the 54 antibody heavy chain, including the signal peptide up to the CH1 domain of the constant region. SEQ ID NO: 36 provides the full-length amino acid sequence of the 54 antibody light chain, including the signal peptide up to the constant region. SEQ ID NO: 37 provides the amino acid sequence of the 83 antibody heavy chain, including the signal peptide up to the CH1 domain of the constant region. SEQ ID NO: 38 provides the full-length amino acid sequence of the 83 antibody light chain, including the signal peptide up to the constant region. SEQ ID NO: 39 provides the amino acid sequence of the 136 antibody heavy chain, including the signal peptide up to the CH1 domain of the constant region. SEQ ID NO: 40 provides the full-length amino acid sequence of the 136 antibody light chain, including the signal peptide up to the constant region. Sequence codes 41-48 provide coding nucleic acid sequences corresponding to the variable region amino acid sequences of Sequence IDs 25-32. These correspond to the nucleic acid sequences found in the 48-136 antibody-producing hybridoma described in the examples.

[0022] (5) The antibody may be based on any of the antibodies described in (1) to (4) above, that is, relative to their amino acid sequences, and may contain substitutions, insertions, or deletions of 1 to 12 amino acids (or 1 to 6 amino acids, or 1 to 3 amino acids) in the amino acid sequence of the heavy chain variable region, and / or contain substitutions, insertions, or deletions of 1 to 10 amino acids (or 1 to 5 amino acids, or 1 to 3 amino acids) in the amino acid sequence of the light chain variable region. In some embodiments, the substitutions, insertions, or deletions of multiple residues are not consecutive. (6) Alternatively, the antibody may be an antibody based on any of the antibodies in (1) to (4) above, having a heavy chain variable region having at least 90% (or at least 95%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of the heavy chain variable region, and / or having a light chain variable region having at least 90% (or at least 95%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of the light chain variable region. Even when such sequence variations are present, the CDR amino acid sequence (for example, the CDR amino acid sequences of sequence numbers 1-24 mentioned above) may remain unchanged. Methods for determining amino acid sequence identity are known to those skilled in the art, and for example, sequence identity can be determined using the BLAST software available on the National Center for Biotechnology Information website with default parameters.

[0023] Amino acid substitutions are preferably conservative amino acid substitutions. A conservative amino acid substitution means that an amino acid residue is replaced by another amino acid residue having a side chain with similar physicochemical properties, and specifically includes substitutions between amino acids having basic side chains (e.g., lysine, arginine, histidine), substitutions between amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), substitutions between amino acids having non-charged side chains (e.g., asparagine, glutamine, serine, threonine, cysteine), substitutions between amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and substitutions between amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0024] Sequence IDs 25-32 and 33-40 contain variable region sequences (and, in the case of Sequence IDs 33-40, signal peptide sequences and constant region CH1 sequences) derived from human antibodies produced in human antibody-producing mice holding a mouse artificial chromosome vector containing the complete human immunoglobulin heavy chain locus and light chain κ locus in a background of mouse immunoglobulin gene knockout. Such human antibody-producing mice are described, for example, in Satofuka et al., Nature Communications, volume 13, Article number: 1841 (2022). The antibodies of this disclosure may be provided in the form of human antibodies or chimeric antibodies. Depending on the intended use, chimeric antibodies may have non-human constant region and / or Fc region sequences, such as those derived from mice. Depending on the specific intended use, the antibodies of this disclosure may also be provided in a non-humanized (e.g., mouseized) form.

[0025] The anti-ULBP2 monoclonal antibodies and their antigen-binding fragments according to this disclosure can be characterized by their ability to increase the binding of ULBP2 to solid-phase-immobilized human NKG2D in a dose-dependent manner. Several antibodies with different CDR sequences, as illustrated in the Examples section, all possessed this ability. Since ULBP2 is a natural ligand for NKG2D, the two inherently possess the ability to bind to each other with a certain degree of affinity. This binding can be reproduced in vitro. Generally, assaying the amount of one protein-binding partner binding to another protein-binding partner immobilized on a solid phase in vitro is a routine technique for those skilled in the art, most typically performed by ELISA (enzyme-linked immunosorbent assay), and an appropriate protein concentration range enabling quantitative assays can be appropriately determined by those skilled in the art based on common knowledge. For example, NKG2D can be immobilized on a solid phase (i.e., the inner surface of the plate wells) by adsorption by adding 100 μL of 0.5 μg / mL human NKG2D protein per well of a 96-well plate and allowing it to stand overnight. By adding 0.6 mg / mL of ULBP2 protein to the well and reacting it with different concentrations of anti-ULBP2 antibody (for example, over 120 minutes), an antibody dose-dependent increase in NKG2D-ULBP2 binding can be detected. For example, an antibody dose-dependent increase in NKG2D-ULBP2 binding can be detected within antibody concentration ranges of 1-10 ng / ml, 1-50 ng / ml, or 1-100 ng / ml. Those skilled in the art can, as necessary, perform blocking to prevent nonspecific binding, remove unbound fractions, or detect binding using secondary antibodies, etc., according to conventional methods. In this disclosure, concentrations of antibodies and other proteins refer to concentrations in aqueous solutions unless otherwise specified.

[0026] Those skilled in the art know that NKG2D has an extracellular domain at its C-terminus, and ULBP2 has an extracellular domain at its N-terminus. Since binding between NKG2D and ULBP2 occurs at their respective extracellular domains, soluble proteins with extracellular domains from which the membrane tethering or transmembrane portion has been removed can be used as NKG2D and ULBP2 proteins in these assays. For example, Onda et al., Biochem Biophys Res Commun, 2001, 285(2):235-43 describes the Ser of the ULBP2 protein (also called ALCAN; GenBank access numbers AB052906.1 and NM_025217.4; amino acid sequence shown in Sequence ID No. 49). 216 -Gly 218 The sequence is described as being important for GPI (glycosylphosphatidylinositol)-mediated membrane tethering, and the soluble ULBP2 protein is Met 215 , Ser 216 , or Ser 217 It may contain an extracellular domain including residues up to . For example, the C-terminal portion from these residues may be replaced with a tag sequence such as an HA tag, a His tag, or a combination thereof, or a soluble polypeptide such as Fc. The human NKG2D protein (UniProt access number P26718; amino acid sequence shown in SEQ ID NO: 50) is understood to have a cytoplasmic domain with residues 1-51, a transmembrane domain with residues 52-72, and an extracellular domain with residues 73-216. Therefore, the soluble human NKG2D protein is Ile 73 It may contain an extracellular domain including residues from . For example, the N-terminal portion of this residue may be replaced with a tag sequence such as a His tag, an HA tag, or a combination thereof, or other soluble polypeptide. Those skilled in the art can express, isolate, and purify these soluble proteins in mammalian cells, for example, based on ordinary knowledge. Suitable soluble ULBP2 proteins (e.g., Biolegend #785306) and soluble human NKG2D proteins (e.g., ACROBiosystems #NKD-H5245) are also commercially available.

[0027] The fact that the binding of ULBP2 to NKG2D is increased in a dose-dependent manner means that, keeping other conditions constant, increasing the amount of antibody added (which can be expressed as weight, moles, or concentration) will also increase the detectable amount of NKG2D-ULBP2 binding. Generally, antibodies against receptor or ligand proteins either block or have no effect on receptor-ligand binding, so the existence of an antibody capable of dose-dependently increasing the binding of ULBP2 to NKG2D, and the acquisition of multiple such antibodies, was unpredictable.

[0028] In one embodiment, an anti-ULBP2 monoclonal antibody and its antigen-binding fragment, provided at a relatively high concentration, may also directly bind to human NKG2D immobilized on a solid phase as described above. For example, when the antibody is reacted with human NKG2D protein immobilized on a solid phase (the inner surface of a 96-well plate) as described above (for example, over 120 minutes) in the absence of ULBP2, a dose-dependent increase in antibody-NKG2D binding may be detected within an antibody concentration range of 100-1000 ng / ml or 100-10000 ng / ml.

[0029] The antibodies according to the embodiments of this disclosure can also be provided in the form of multispecific antibodies, such as bispecific antibodies. In a typical embodiment, the antibodies of this disclosure are not multispecific antibodies. As is commonly understood by those skilled in the art, a "multispecific antibody" means a new antibody that combines the specificity of each antibody by combining the antigen-binding domain of one antibody with the antigen-binding domains of one or more other antibodies within a single molecule. Each anti-ULBP2 antibody exemplified in the examples section of this disclosure appears to be able to bind to NKG2D, albeit with a lower affinity for ULBP2, but this is not a bispecific antibody as defined in this disclosure because it is originally a single antibody and not a combination of antigen-binding domains derived from different antibodies.

[0030] In one embodiment, the present disclosure provides a nucleic acid molecule encoding an anti-ULBP2 monoclonal antibody or an antigen-binding fragment thereof according to embodiments of the present disclosure. Synthesizing or isolating nucleic acids of nucleotide sequences corresponding to the amino acid sequence of a given antibody or fragment is within the ordinary skill of those skilled in the art. Such nucleic acids can also be isolated, obtained, or amplified (e.g., by PCR amplification) from a hybridoma producing the antibody. The nucleic acid molecule may be DNA or RNA, and the DNA may contain introns or may be cDNA. The nucleic acid molecule may be provided in plasmid form.

[0031] In another embodiment, the present disclosure provides an expression vector comprising the nucleic acid molecule described above. The expression vector may be a plasmid vector. It is known to those skilled in the art that an expression vector comprising a nucleic acid molecule encoding a monoclonal antibody or its antigen-binding fragment can be introduced into various host cells to make the antibody or its antigen-binding fragment expressible in that host, and thus recombinantly produced. The present disclosure also provides host cells comprising these expression vectors. The host cells may be, but are not limited to, mammalian cells, and may be, for example, bacterial, yeast, or insect cells. Those skilled in the art can, based on ordinary knowledge, select a nucleic acid vector skeletal sequence, promoter, origin of replication, selection marker, etc., suitable for the host cell. The antibodies and fragments of the present disclosure may have various chemical modifications or conjugate portions known to those skilled in the antibody field, and may also have, as appropriate, tag sequences for purification or detection, signal sequences for extracellular secretion, etc., and such sequence manipulation can also be performed through the design of the expression vector.

[0032] In another embodiment, the disclosure provides an anti-ULBP2 monoclonal antibody or its antigen-binding fragment capable of increasing the binding of ULBP2 to solid-phase-immobilized human NKG2D in an antibody dose-dependent manner, and also provides a method for producing such an antibody. Such antibodies having different CDR sequences can be independently obtained by appropriate procedures. The method for production includes immunizing a non-human animal with ULBP2, preparing a population of hybridomas from the immunized non-human animal, selecting a subpopulation of hybridomas from the population of hybridomas that produces an antibody capable of binding to ULBP2, and selecting a hybridoma clone from the subpopulation of hybridomas that produces an antibody capable of increasing the binding of ULBP2 to human NKG2D in an antibody dose-dependent manner. The antibody produced by the hybridoma clone selected herein provides the desired anti-ULBP2 monoclonal antibody capable of increasing the binding of ULBP2 to solid-phase-immobilized human NKG2D in an antibody dose-dependent manner. The antigen-binding fragment can be produced genetically or biochemically based on the antibody gene sequence of the hybridoma clone, or by enzymatically treating the obtained anti-ULBP2 monoclonal antibody.

[0033] For immunizing non-human animals, soluble ULBP2 proteins having an extracellular domain with the membrane tethering or transmembrane portion removed, as described above, can be used as ULBP2 immunogens. Similarly, soluble ULBP2 proteins can be used, but are not limited to, as screening baits used when selecting a subpopulation of hybridomas, i.e., the ULBP2 antigen to which the target antibody should bind. Immunizing non-human animals, preparing a population of hybridomas from the immunized non-human animals, and selecting a subpopulation of hybridomas from the hybridoma population that produces antibodies capable of binding to the target antigen can be carried out using conventional techniques known to those skilled in the field of monoclonal antibodies. For example, screening for selecting a subpopulation of hybridomas can typically be performed by ELISA, flow cytometry, cytoimmunostaining, or a combination thereof. Screening for selecting hybridoma clones from a subpopulation of hybridomas that produce antibodies capable of increasing ULBP2 binding to human NKG2D in an antibody dose-dependent manner can also be carried out conveniently by ELISA, but are not limited to this method. Preferred examples of assays and conditions that can provide this screening are described above. For example, human NKG2D (which may be a soluble protein with the N-terminus removed and containing an extracellular domain) can be immobilized on the surface of the well, and antibodies from each candidate hybridoma can be added together with ULBP2 (which may be a soluble protein with the C-terminus removed and containing an extracellular domain). Compared to a control with only ULBP2 added and no antibody, antibodies and corresponding hybridoma clones showing increased human NKG2D-ULBP2 binding can be identified and selected (typically, most candidate anti-ULBP2 antibodies are expected to result in a decrease or no change in human NKG2D-ULBP2 binding, rather than an increase). Dose-dependence can then be confirmed by repeating the same assay with varying antibody doses for the small number of identified hybridoma clones.

[0034] In addition to, or as an alternative to, solid-phase assays, cell-based assays such as flow cytometry can also be used for screening. For example, by adding soluble human NKG2D with or without the addition of a candidate anti-ULBP2 antibody to cells expressing ULBP2, and detecting the increase in the amount of NKG2D bound to the cell surface by flow cytometry or fluorescence microscopy, anti-ULBP2 antibodies and corresponding hybridoma clones that can increase the binding of NKG2D to ULBP2 expressed in cells in an antibody dose-dependent manner can be identified and selected. Alternatively, by adding soluble ULBP2 with or without the addition of a candidate anti-ULBP2 antibody to cells expressing human NKG2D, and detecting the increase in the amount of ULBP2 bound to the cell surface by flow cytometry or fluorescence microscopy, anti-ULBP2 antibodies and corresponding hybridoma clones that can increase the binding of ULBP2 to human NKG2D expressed in cells in an antibody dose-dependent manner can be identified and selected.

[0035] The non-human animal immunized with ULBP2 is preferably a mammal, such as a mouse, rat, rabbit, guinea pig, or monkey. In a preferred embodiment, the non-human animal immunized is a human antibody-producing mouse that holds a mouse artificial chromosome vector having the complete human immunoglobulin heavy chain locus and light chain κ locus in a background in which the mouse immunoglobulin gene is disrupted, deleted, or underexpressed compared to the wild type, as described in Satofuka et al., Nature Communications, volume 13, Article number: 1841 (2022). The artificial chromosome is also called a minichromosome.

[0036] In another embodiment, the present disclosure provides a pharmaceutical composition comprising the antibody or its antigen-binding fragment as described above. The pharmaceutical composition may comprise an active ingredient, an anti-ULBP2 monoclonal antibody or its antigen-binding fragment, and may also comprise pharmaceutically acceptable excipients or carriers. The pharmaceutical composition may be provided, for example, in the form of an aqueous solution or other solution. The pharmaceutical composition may further comprise suitable additives known to those skilled in the art, and / or additional active ingredients (e.g., antibodies other than the anti-ULBP2 antibody or its antigen-binding fragment, or other anticancer agents). For example, the pharmaceutical composition may comprise an immune checkpoint inhibitor, as described below, in addition to the anti-ULBP2 monoclonal antibody or its antigen-binding fragment. Dosage forms and routes of administration known in the field of antibody drugs, particularly in the field of antibody drugs for cancer treatment, may be used as appropriate in the pharmaceutical composition according to this embodiment.

[0037] In some embodiments, the pharmaceutical compositions of the Disclosure are pharmaceutical compositions for the treatment of cancer. In corresponding embodiments, a method for treating cancer is provided, comprising administering a therapeutically effective amount of the anti-ULBP2 monoclonal antibody or its antigen-binding fragment or pharmaceutical composition of the Disclosure to a target subject, and an anti-ULBP2 monoclonal antibody or its antigen-binding fragment or pharmaceutical composition for use in such a method is provided. Cancer may be, for example, a solid tumor or a hematological malignancy, and may be particularly ULBP2-positive cancer, and may be, but not limited to, head and neck cancer, lung cancer, urothelial carcinoma, cervical cancer, colorectal cancer, breast cancer, melanoma, etc. In some embodiments, the cancer targeted for treatment by the pharmaceutical compositions of the Disclosure is an immune checkpoint inhibitor (ICI)-resistant cancer. An ICI-resistant cancer is a cancer that does not respond to or has lost responsiveness to treatment with ICIs known to those skilled in the art (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody). Those skilled in the art can recognize ICI-resistant cancer. For example, Lim et al. Nature Communications volume 14, Article number: 1516 (2023) reported that clinical trials showed 42-45% of melanoma patients responded to PD-1-targeted ICIs, but resistance to ICIs was still common, with approximately 55% of melanoma patients being inherently resistant to PD-1 inhibitor monotherapy, and some patients developing resistance during treatment.

[0038] In some embodiments, the pharmaceutical compositions of this disclosure are administered in combination with an ICI. "Administered in combination" means that both are administered under conditions that allow their pharmacological effects to combine within the patient, and they may be administered simultaneously or at different times, and may be administered via the same route or different routes. The ICI may be, for example, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof.

[0039] The pharmaceutical compositions according to embodiments of this disclosure were able to demonstrate antitumor effects even against ICI-resistant tumors. In another example, a pharmaceutical composition containing an anti-ULBP2 monoclonal antibody according to embodiments of this disclosure, when administered in combination with an ICI, was able to demonstrate a synergistic antitumor effect that surpassed the effects obtained by the anti-ULBP2 monoclonal antibody alone and by the ICI alone. Therefore, the pharmaceutical compositions according to embodiments of this disclosure can serve as adjuncts, complements, or alternatives to ICI therapy. [Examples]

[0040] [Discovery of a novel anti-ULBP2 antibody] TC-mAb used as an immunizing host TM The mice are human antibody-producing mice that carry a mouse artificial chromosome vector containing the complete human Ig heavy chain locus (IGH) and kappa light chain locus (IGL) in a background of mouse immunoglobulin (Ig) gene knockout (Satofuka et al., Nature Communications, volume 13, Article number: 1841 (2022)). TC-mAbs immunized with ULBP2 extracellular domain antigen. TM A library of human antibody-producing hybridomas was created by fusing cells from mouse spleen and lymph node suspensions with mouse myeloma cells. The hybridomas in this library were screened to prepare a subpopulation of anti-ULBP2 antibody-producing hybridomas. Furthermore, in competitive ELISA performed to evaluate the inhibitory effect of the antibodies on ULBP2 and NKG2D binding, four hybridoma clones were identified that produced anti-ULBP2 antigen antibodies that appeared to enhance, rather than inhibit, this binding. Based on the sequencing analysis of the hybridomas, recombinant antibodies (indicated as "anti-ULBP2 mIgG2b" in the figure) were synthesized in which the Fc region was replaced with that of mouse IgG2b (mIgG2b). Hereafter, these four monoclonal antibodies, distinguished by differences in their variable region sequences, will be referred to as "48," "54," "83," and "136," respectively, and these four antibodies together are sometimes collectively referred to as "48-136."

[0041] As predicted, these four antibodies showed binding reactions in ELISA immobilized with ULBP2. However, unexpectedly, at high concentrations, they also showed binding reactions in ELISA immobilized with human NKG2D (Figure 1a). This NKG2D binding was not observed with anti-ULBP2 antibodies from other hybridoma clones obtained from the above library (as illustrated in Figure 1d), suggesting that it is a phenomenon specific to the hybridomas of these four clones.

[0042] Next, when human NKG2D was immobilized on a solid phase and mixed with ULBP2 antigen and anti-ULBP2 antibody (48-136) before being reacted with the solid phase in a competitive ELISA, the detection amount (i.e., the amount of HRP-conjugated detection antibody bound in the ELISA, measured as A490 / A655 absorbance; see Figures 1b,c) increased in a 48-136 antibody concentration-dependent manner, regardless of whether ULBP2 (Figure 1b) or 48-136 antibody (Figure 1c) was detected. This result suggests that the 48-136 antibody does not inhibit the binding of ULBP2 and human NKG2D, but rather has the ability to crosslink them during binding (see Figure 1b in particular). This ability was not observed with anti-ULBP2 antibodies from other hybridoma clones obtained from the above library (exemplified in Figures 1e and 1f, corresponding to Figures 1b and 1c, respectively), and appeared to be a phenomenon specific to the four hybridoma clones mentioned above.

[0043] A mouse colon cancer cell line CT26.WT (CT26.WT-ULBP2) that stably expresses ULBP2 was established. Using flow cytometry, it was confirmed that the 48-136 antibody specifically binds to ULBP2 on the surface of this cell (data not shown). To evaluate whether the anti-ULBP2 antibody competes with NKG2D for binding to ULBP2 on the cell surface, a solution containing hNKG2D-human Fc (a fusion protein of the extracellular region of human NKG2D and the Fc region of human IgG) and the anti-ULBP2 antibody or an isotype control antibody was reacted with CT26.WT-ULBP2 cells. Then, the hNKG2D-human Fc bound to the cell surface was detected with an anti-human Fc antibody and analyzed by flow cytometry. As a result of this experiment, a commercially available anti-ULBP2 antibody (Clone #165903, R&D) decreased the hNKG2D-binding cell fraction (Figure 2a, bottom panel), while, in sharp contrast, the 48-136 antibody increased the hNKG2D-binding cell fraction (Figure 2a, other panels). The gates and numbers shown in the upper right of each panel in Figure 2a indicate the percentage of cells (corresponding to NKG2D-binding cells) contained within this gate for the experiment in which hNKG2D-human Fc + anti-ULBP2 antibody was reacted with the cells. Graphs plotting that percentage as a comparison with the case of using an isotype control antibody are shown in Figures 2b and 2c. These results indicate that the binding between ULBP2 and hNKG2D on the cell surface is inhibited by the control antibody, whereas the 48-136 antibody rather increases the binding between them. On the other hand, it was confirmed that the binding between the mouse endogenous NKG2D ligand and mouse NKG2D, and the binding between MICB, a human NKG2D ligand different from ULBP2, and human NKG2D are not affected by the 48 antibody (data not shown).

[0044] To evaluate how the 48-136 antibody changes the NKG2D activation signal by ULBP2, 4h (4 hours)- 51Cytotoxicity activity was measured using the Cr release method. Mouse spleen cells stimulated with mouse IL-15 were used as effector cells, and B16F10-mock (mouse melanoma cell line B16F10 transfected with a mock) and B16F10-ULBP2 (B16F10 expressing ULBP2) were used as target cells. Compared to B16F10 mock, cytotoxicity activity by effector cells was enhanced when B16F10-ULBP2 was used as the target cell (Figure 3a). This cytotoxicity activity was suppressed by an anti-mouse NKG2D antibody (Figure 3b). When the effect of 48-136 antibodies on the cytotoxicity activity of B16F10-ULBP2 was evaluated with an effector / target ratio (E / T ratio) of 100 / 1, cytotoxicity activity decreased in an antibody concentration-dependent manner (Figure 3d). In Figure 3d, "277" is an anti-ULBP2 antibody from a clone obtained from the same hybridoma library but functionally not belonging to the 48-136 antibody group; however, this antibody did not reduce cytotoxic activity against B16F10-ULBP2. Furthermore, cytotoxic activity against YAC-1 cells, a typical target cell of natural killer cells, was not suppressed by the 48 antibody (Figure 3c), suggesting that the suppression of cytotoxic activity by the 48-136 antibody is specific to B16F10-ULBP2, which expresses ULBP2. When experiments were conducted again using B16F10-ULBP2 as the target cell with an E / T ratio of 200 / 1, cytotoxic activity was again reduced in a concentration-dependent manner with the 48-136 antibody. However, in the experiments with the 48 antibody and the 136 antibody, the sigmoid curve shifted upward, indicating that cytotoxic activity was higher in the presence of low concentrations of these antibodies than in the presence of isotype controls (Figure 3e). These antibodies may possess both the ability to reduce cytotoxic activity by blocking NKG2D signaling mediated by ULBP2, and the ability to increase cytotoxic activity through ADCC activity mediated by the Fc receptor.

[0045] The following primarily describes experiments using antibody 48 as a representative antibody of the 48-136 antibody group related to this disclosure, but other antibodies in the same group may exhibit similar effects. To evaluate the extent to which antibody 48 suppresses the increase in cytotoxic activity caused by ULBP2 expression in B16F10, cytotoxic activity was evaluated at an E / T ratio of 100 / 1, where the influence of ADCC activity was considered to be minimal. At 10 μg / ml, antibody 48 suppressed the increase in cytotoxic activity caused by ULBP2 expression by 78% (Figure 3f). Furthermore, antibody 48 suppressed cytotoxic activity more potently than a commercially available anti-ULBP2 antibody (clone #165903, R&D) that inhibits the binding of ULBP2 to NKG2D (Figure 3g). These results suggest that antibody 48 suppresses the ULBP2-mediated NKG2D activation signal by influencing the binding of ULBP2 to NKG2D (e.g., by crosslinking) (Figure 3h).

[0046] Next, 3 × 10^5 B16F10 wild-type cells were subcutaneously transplanted into both flanks of syngeneic mice (C57BL / 6JJcl). Twelve days after transplantation, the mice's spleens were removed, and the spleen cells and B16F10-ULBP2 were co-cultured in the presence of 48 antibody or isotype control antibody, and CD8 was determined by flow cytometry. + The IFN-γ production performance of T cells was evaluated. In a control experiment using spleen cells from control mice that had not been transplanted with B16F10, no difference was observed between the 48 antibody and the isotype antibody after 72 hours of co-culture. However, in experiments using spleen cells from B16F10-transplanted mice, the presence of the 48 antibody was observed in CD8 + The IFN-γ production capacity of T cells was increased (Figure 3i,j). These results suggest that the 48 antibody can attenuate the cytotoxic activity of NK cells against ULBP2-expressing cells in vitro, while the 48 antibody also reduces the CD8 cytotoxicity mediated by NKG2D, which is induced by prolonged exposure to ULBP2. + This suggests that it suppresses the decline in T cell function.

[0047] [Novel anti-ULBP2 antibody is used for tumor infiltration CD8 +[Increasing and activating T cells to suppress tumor growth in a B16F10-ULBP2 syngeneic tumor transplant mouse model] To evaluate the antitumor effect of the 48-136 antibody, 3 × 10^5 B16F10-ULBP2 cells were subcutaneously transplanted into the right flank of syngeneic mice, and either the 48 antibody or an isotype control antibody was administered intraperitoneally (6 mice per group). The antibody dose was 300 ug / body on the day of transplantation, and 200 ug / body at 5 and 10 days later (Figure 4a). The 48 antibody suppressed tumor growth compared to the isotype control (Figures 4b-d show reductions in tumor volume, weight, and appearance, respectively). Flow cytometry analysis of tumor-infiltrating lymphocytes revealed that tumor-infiltrating CD8 cells were associated with 48 antibody administration. + An increase in IFN-γ production capacity of T cells was observed (Figure 4e).

[0048] Next, 3 × 10^5 B16F10-ULBP2 cells were subcutaneously transplanted into the right flank of syngeneic mice, and 48 hIgG1, purified directly from the culture supernatant of hIgG1-type 48 antibody-producing hybridomas, or PBS was subcutaneously administered near the tumor. The antibody dose was 400 ug / body on the day of transplantation, 5 days later, and 10 days later. When the tumor was excised 14 days post-transplantation and tumor-infiltrating lymphocytes were analyzed, CD8 was found to be associated with 48 hIgG1 administration. + Increased T cell ratio and CD8 + Effector memory T cells (T EM An increase in ) was observed (Figure 4f-i). Based on these results, the 48-136 antibody was found to be effective in tumor infiltration CD8 + It is thought that tumor growth is suppressed by increasing and activating T cells.

[0049] [When ULBP2 is expressed in cancer cells, they become resistant to anti-PD-1 antibodies.] To evaluate the effect of ULBP2 expressed on cancer cells on PD-1 / PD-L1 blockade therapy with immune checkpoint inhibitors, the therapeutic effect of anti-PD-1 antibodies was evaluated in a syngeneic subcutaneous transplant mouse model using the aforementioned CT26.WT-ULBP2 (Figure 5a). 1 × 10^6 CT26.WT-mock and CT26.WT-ULBP2 cells were subcutaneously transplanted into the right flank of syngeneic mice (BALB / cByJJcl). These mice were administered either an isotype control antibody or an anti-PD-1 antibody, and the tumors were excised 21 days post-transplant (5 mice per group). The antibody dose was 200 ug / body at 4, 7, 11, and 14 days post-transplant. While the anti-PD-1 antibody showed antitumor efficacy against CT26.WT-mock tumors, CT26.WT-ULBP2 tumors were resistant to the anti-PD-1 antibody (Figures 5b-f).

[0050] [ULBP2 is CD8 by anti-PD-1 antibody + [Inhibition of T cell activation] Furthermore, tumor-infiltrating lymphocytes from the excised tumor were analyzed by flow cytometry. CD8 + T EM PD-1 - NKG2D + The fraction increased when anti-PD-1 antibody was administered to CT26.WT-mock tumors, but not when anti-PD-1 antibody was administered to CT26.WT-ULBP2 tumors (Figure 5g). Furthermore, when anti-PD-1 antibody was administered to CT26.WT-mock tumors, tumor-infiltrating CD8 fraction increased. + T cell T-bet + EOMES - The fraction increased, but this did not increase when anti-PD-1 antibody was administered to CT26.WT-ULBP2 tumors (Figure 5h). These data suggest that in CT26.WT tumors, anti-PD-1 antibody administration reduces the activity of CD8 that is not exhausted. + T cells increased, but in CT26.WT-ULBP2 tumors, CD8 was increased by administration of anti-PD-1 antibody. + This indicates that the increase in T cell activity was suppressed.

[0051] [NKG2D signal blockade reduces the antitumor effect of anti-PD-1 antibodies] To evaluate the effect of NKG2D signaling on PD-1 / PD-L1 blockade therapy, 5 × 10^5 CT26.WT wild-type cells were subcutaneously transplanted into the right flank of syngeneic mice, and anti-NKG2D antibody and anti-PD-1 antibody were administered either alone or in combination (6 mice per group) (Figure 6a). The antibody dose was 200 ug / body at 4, 7, 11, and 14 days after transplantation. Anti-NKG2D antibody monotherapy did not show a significant difference in tumor growth compared to isotype control therapy, while anti-PD-1 antibody monotherapy showed a marked inhibitory effect on tumor growth. However, when anti-NKG2D antibody was combined with anti-PD-1 antibody, the inhibitory effect of anti-PD-1 antibody on tumor growth was attenuated (Figure 6b-d). These results suggest that blocking the NKG2D signal leads to CD8 denaturation through PD-1 / PD-L1 signal blockade. + This suggests that T cell activation is inhibited.

[0052] [A novel anti-ULBP2 antibody overcomes anti-PD-1 antibody resistance caused by ULBP2 expressed in cancer cells.] Previous results suggest that in ULBP2-expressing tumors, long-term stimulation of NKG2D by ULBP2 leads to NKG2D unresponsiveness and CD8 + It was considered possible that this caused a decrease in T cell function, leading to resistance to anti-PD-1 antibody treatment. Therefore, the antitumor effects were investigated by administering anti-PD-1 antibody and 48 antibody, either alone or in combination, to CT26.WT-ULBP2 syngeneic subcutaneously transplanted mice (Figure 7a). 1 × 10^6 CT26.WT-ULBP2 cells were subcutaneously transplanted into the right flank of syngeneic mice, and anti-PD-1 antibody and 48 antibody were administered either alone or in combination (6 mice per group). The antibody dose was 200 ug / body at 4, 7, 11, and 14 days after transplantation. While the antitumor effects of anti-PD-1 antibody and 48 antibody alone were limited, a synergistic and significant antitumor effect was obtained when they were used in combination (Figure 7b). Furthermore, 5 × 10^5 CT26.WT wild-type cells were subcutaneously transplanted into the right flank of syngeneic mice, and spleen cells collected from the mice 28 days later were co-cultured with CT26.WT-mock or CT26.WT-ULBP2, and CD8 was measured after 24 hours.+ T cell activity was analyzed by flow cytometry of IFN-γ-producing cells. In co-culture with CT26.WT-mock, CD8 was affected by anti-PD-1 antibody monotherapy. + T cell activation was observed, but when an anti-NKG2D antibody was added, CD8 was activated by an anti-PD-1 antibody. + T cell activation was suppressed (Figure 7c). In CT26.WT-ULBP2, CD8 was suppressed by anti-PD-1 antibody monotherapy compared to CT26.WT-mock. + T cell activation was suppressed, and this activity was restored by co-administration with the 48 antibody (Figure 7d). These results suggest that the 48 antibody overcame the anti-PD-1 antibody resistance caused by the ULBP2-NKG2D interaction.

[0053] [Consideration] Recent comprehensive genetic analysis of immune-related molecules based on the TCGA database has shown that ULBP2 is the most specifically expressed molecule in cancer tissue among the eight human NKG2D ligands, that ULBP2 expression in cancer tissue is a major poor prognostic factor, and that tumor infiltration CD8 is present in ULBP2-expressing tumors. + It has become clear that T cells decrease. These results suggest that ULBP2 is a molecule that is specifically expressed on cancer cells and acts suppressively against antitumor immunity, making it a potential therapeutic target for cancer immunotherapy. However, since NKG2D is an immunoactive receptor, there are concerns that blocking this activation signal may have a detrimental effect on cancer treatment. Clinical trials of anti-MICA / B antibodies targeting NKG2D ligands are underway as cancer immunotherapy. These antibodies suppress the cleavage of MICA / B on the surface of cancer cells and increase MICA / B expression on the surface of cancer cells, aiming to induce an antitumor effect by NK cells.

[0054] The novel human anti-ULBP2 antibody obtained in this disclosure is in vitro 4h- 51In experiments using the Cr release method, the cytotoxic activity of effector cells against ULBP2-expressing cells was suppressed (Figure 3d-g). In this experimental system, the effector cells were NK cells, meaning that the cytotoxic activity of NK cells was attenuated. On the other hand, in long-term co-culture experiments of spleen cells from B16F10 wild-type transplanted mice and B16F10-ULBP2, CD8 was suppressed by ULBP2 expressed in tumors. + The antibody suppressed the decrease in T cell activity (Figure 3i-j).

[0055] B16F10 syngeneic subcutaneous graft tumors are CD8 + This tumor exhibits low T-cell infiltration, is immune-cold, and is resistant to many immune checkpoint inhibitors, including PD-1 blockade therapy. The novel antibody disclosed herein suppresses tumor growth in a B16F10-ULBP2 transplanted mouse model and inhibits CD8 infiltration into the tumor. + It increased T cells (Figure 4). Although not bound by any specific theory, this antibody is thought to suppress the functional decline of effector cells caused by chronic NKG2D stimulation by ULBP2 in cancer cells. This antibody suppresses the CD8 function decline caused by chronic NKG2D stimulation by NKG2D ligands. + We were able to provide the first cancer immunotherapy targeting T cell dysfunction. Furthermore, this antibody appears to crosslink ULBP2 and NKG2D (Figures 1 and 2), thus connecting tumor cells and CD8 + It may have the effect of bringing T cells closer together, and tumor-infiltrating CD8 + The increase in T cells may be at least partially attributable to this effect of the antibody.

[0056] CT26.WT is used in many cancer immunology studies as an anti-PD-1 antibody-sensitive cell (Figure 5). In CT26.WT-transplanted mice, blocking the NKG2D signal with an anti-NKG2D antibody reduced the antitumor effect of the anti-PD-1 antibody, suggesting that proper NKG2D signaling is necessary for antitumor immunity (Figure 6). Furthermore, this result suggests that the NKG2D activation signal is involved in resistance to anti-PD-1 antibodies. Stable expression of ULBP2 in CT26.WT resulted in the loss of the antitumor effect of the anti-PD-1 antibody (Figure 5b-d,g,h), and even in this case, the antitumor effect was obtained when the anti-ULBP2 antibody and anti-PD-1 antibody of this disclosure were used in combination (Figure 7a,b). This suggests that chronic stimulation of NKG2D by ULBP2 triggers CD8 + This suggests that T cell unresponsiveness may be the mechanism of resistance to anti-PD-1 antibodies. Furthermore, in co-culture experiments of mouse splenocytes with CT26.WT-mock or CT26.WT-ULBP2 (Figure 7c,d), (1) blocking the NKG2D signaling pathway resulted in resistance to CD8 by anti-PD-1 antibodies. + (2) T cell activation is not achieved, and CD8 is affected by anti-PD-1 antibodies through the action of ULBP2 expression. + Although T cell activation was suppressed, it was shown that activity was restored by combining the anti-ULBP2 antibody disclosed herein with an anti-PD-1 antibody, and these results were consistent with those obtained in a syngeneic tumor-transplanted mouse model. Serum ULBP2 has been reported to be a poor prognostic factor in immune checkpoint inhibitor therapy for malignant melanoma, and the experimental results described above are consistent with this finding.

[0057] Generally, antibodies that block ligand-receptor signaling are typically obtained by screening for antibodies that inhibit the binding of receptors to ligands. The inventors of this invention discovered anti-ULBP2 antibodies that crosslink receptors and ligands, and also demonstrated that these antibodies block the ULBP2-NKG2D signaling pathway. These antibodies also showed binding to NKG2D at high concentrations. While it is unclear why such antibodies were obtained in mice immunized with the ULBP2 antigen, it is possible that ULBP2-NKG2D complexes were formed in the bodies of mice administered with the ULBP2 antigen, and that B cell clones that produced antibodies reacting to this complex existed. The discovery of the antibodies disclosed herein proposes a new concept regarding ligand-receptor signaling blocking antibodies.

[0058] In summary, this study demonstrated the following: (1) A human anti-ULBP2 antibody was created that can crosslink ULBP2 and NKG2D and block the ULBP2-NKG2D activation signal. (2) This antibody was found to be effective against CD8 infiltrating B16F10-ULBP2 tumors. + (3) T cells were increased and activated, suppressing tumor growth. (4) Stable expression of ULBP2 in CT26.WT, which is sensitive to anti-PD-1 antibodies, resulted in resistance to anti-PD-1 antibodies. (5) This resistance was overcome by combining the anti-PD-1 antibody with the antibody disclosed herein. In conclusion, the antibody disclosed herein is expected to become a new cancer immunotherapy drug. Furthermore, it is expected that suppressing excessive NKG2D signaling and optimizing NKG2D signaling will become a new target for cancer immunotherapy.

[0059] [Materials and methods of the example] A description of the typical experimental materials and methods used in the examples is provided below. Details not specifically described are carried out using standard techniques known to those skilled in the art.

[0060] Production of human ULBP2 antibodies The cDNA nucleic acid sequence encoding residues 1-217 of the amino acid sequence of SEQ ID NO: 49 was cloned into the pCMV6-AC-HA-His vector (OriGene Technologies, Inc.). By transfecting human 293F cell lines with this vector, a soluble ULBP2 protein (ULBP2-HA-His) containing an extracellular domain with a Ha-His tag at the C-terminus was recombinantly expressed. This ULBP2 protein (presumably with the signal peptide cleaved) was purified from the culture supernatant according to standard procedures and used as an antigen for immunization. Human antibody-producing mice (TC-mAb TM Mice were immunized by injection with the above-mentioned ULBP2 antigen (ULBP2-HA-His). After confirming an increase in ULBP2 antibody titers in mouse serum, the spleen and lymph nodes were excised and suspended in aqueous solution. Splene and lymph node cells were fused with mouse myeloma cells to create a population of hybridomas, which were diluted and seeded in 96-well plates for culture. The culture supernatant was used for ELISA, flow cytometry, and immunohistocellular staining to select a subpopulation of anti-ULBP2 antibody-producing hybridomas.

[0061] The inhibition of ULBP2 and NKG2D binding by hybridoma-produced antibodies was evaluated by performing ELISA using the culture supernatant of selected hybridomas. The nucleotide sequences of the H and L chains, including the immunoglobulin variable region, of the hybridomas that produced the selected antibodies were determined. Based on the determined nucleotide sequences, recombinant antibodies were created by recombining the Fc region with mouse IgG2b (mIgG2b) and used in experiments with a mouse model.

[0062] cell line Mouse melanoma cells B16F10 (RCB2630) and YAC-1 were obtained from the RIKEN BioResource Research Center (RIKEN BRC). CT26.WT was obtained from the American Type Culture Collection (ATCC). Typically, cell lines were cultured at 37°C and 5% CO2 in RPMI-1640 or DMEM medium containing 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 ug / ml streptomycin.

[0063] ULBP2 stable expression cell lines The cDNA of ULBP2 (NM_025217.4) was inserted into the multi-cloning site of the pcDNA3.1 (+) IRES GFP vector (51406, Addgene). B16F10 or CT26.WT cells were transfected with this vector using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Single-cell sorting was performed, and ULBP2 expression on the cell surface was analyzed using a PE-labeled anti-ULBP2 antibody (R&D Systems). ULBP2 shed in the culture supernatant was measured using the human ULBP2 ELISA kit (DY1298, R&D Systems). Stable cell lines that highly expressed both cell surface ULBP2 and shed ULBP2 were selected.

[0064] Tumor inoculation and in vivo experiments The mice used for in vivo experiments were 6-week-old female C57BL / 6JJcl mice. BALB / cByJJcl (CLEA Japan, Inc.) Tumor growth was assessed by measuring tumor diameter with electronic calipers and calculating tumor volume using the formula: Tumor volume = 0.5 × (major diameter × minor diameter^2). The survival endpoint was when tumor weight reached 10% of body weight excluding tumor (approximately 1800 mm^3 for C57CL / 6JJcl and 2000 mm for BALB / cByJJcl). ^3 This was defined as the point at which the condition was reached.

[0065] Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) The tumor was cut into small pieces and separated into single cells using TTDR reagent (BD Horizon® Dri Tumor & Tissue Dissociation Reagent, Dickinson and Company BD Biosciences). Lymphocytes in the single-cell population were analyzed by flow cytometry using antibodies that detect antigens specifically expressed on the surface of lymphocytes. The antibodies used in this analysis included anti-mCD45 (Clone 30-F11, Biolegend), anti-mCD3 (Clone 17A2, Biolegend), anti-mCD4 (Clone, Biolegend), anti-mCD8a (Clone 53-6.7, Biolegend), anti-mCD44 (Clone IM7, Biolegend), anti-mCD62L (Clone MEL-14, Biolegend), anti-NK1.1 (Clone PK136, Biolegend), anti-mNKG2D (Clone CX5, Biolegend), and anti-mIFN-γ (Clone XMG1.2, Biolegend). Analysis was performed using a CytoFLEX S flow cytometer (Becton, Dickinson and Company BD Biosciences) with Flowjo (v10.8.1) software.

[0066] Ex vivo IFN-γ restiration assay Single-cell suspensions of tumor-infiltrating lymphocytes (TILs) were incubated with a Cell Activation Cocktail (PMA (Phorbol 12-myristate-13-acetate) and ionomycin, biolegend) without brefeldin A at 37°C for 6 hours. After staining the cell surface with antibody, intracellular IFN-γ was stained with antibody, and flow cytometry analysis was performed using CytoFLEX S and Flowjo (v10.8.1) software.

[0067] Antibody reactivity in ELISA using solid-phase ULBP2 or NKG2D In Figure 1a, ULBP2-hFc represents the ULBP2 extracellular domain protein (Biolegend #785306) with a human Fc tag at the C-terminus, hNKG2D-His represents the human NKG2D extracellular domain protein (ACROBiosystems #NKD-H5245) with a His tag at the N-terminus, and HRP-Anti mIgG Fc represents the HRP-anti-mouse IgG2b for detection (A90-131P, Bethyl Laboratories). ULBP2-hFc or NKG2D-His were added to 100 μL per well in a 96-well plate at a concentration of 0.5 ug / mL, respectively, and allowed to stand overnight at 4°C. After blocking, isotype control mIgG2b antibody or recombinant mIgG2b test antibody were reacted at different concentrations, and after 60 minutes, detection was performed with HRP-anti-mouse IgG2b.

[0068] Effect of 48-136 antibody on the binding reaction of ULBP2 and NKG2D In a 96-well plate, 100 μL of 0.5 ug / mL hNKG2D-His was added per well and allowed to stand overnight at 4°C. After blocking, 0.6 mg / mL ULBP2-HA-His (the same antigen used in the above immunization) was added together with various concentrations of recombinant mIgG2b test antibodies (48-136 antibody or other hybridoma-derived antibodies) and reacted. Binding was detected after 60 minutes of reaction. Rabbit anti-HA (GTX115044, GeneTex) and HRP-anti-rabbit IgG (SA00001-2, proteintech) were used for ULBP2 detection (Figure 1b). Biotin-anti-mouse IgG (405303, biolegend) and HRP-streptavidin (N100, Thermo Scientific) were used for recombinant mIgG2b test antibody detection (Figure 1c).

[0069] statistics Standard statistical data analysis was performed using GraphPad Prism 10 software. The data in the graphs represent the mean ± standard deviation for in vitro experiments, the mean ± standard error for mouse tumor size and weight, and the mean ± standard error for flow cytometry analysis of tumor-infiltrating lymphocytes. A p-value of <0.05 was considered statistically significant.

[0070] This disclosure includes the following embodiments. [Embodiment 1] (a) Each comprises a heavy chain variable region including heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 containing the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, and a light chain variable region including light chain CDR1, light chain CDR2, and light chain CDR3 containing the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively; (b) Each comprises a heavy chain variable region including heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 containing the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, and a light chain variable region including light chain CDR1, light chain CDR2, and light chain CDR3 containing the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12; (c) Each comprising a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, respectively, containing the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, and a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3, respectively, containing the amino acid sequences shown in SEQ ID NOs. 16, 17, and 18; or (d) Each comprises a heavy chain variable region including heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 containing the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, and a light chain variable region including light chain CDR1, light chain CDR2, and light chain CDR3 containing the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, Anti-ULBP2 monoclonal antibody or its antigen-binding fragment. [Embodiment 2] The anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to Embodiment 1, which can increase the binding of ULBP2 to human NKG2D immobilized on a solid phase in a dose-dependent manner with the antibody. [Embodiment 3] The aforementioned antibody is (1) comprising a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 26; (2) comprising a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 28; (3) comprising a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 30; or (4) An antibody comprising a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 31 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 32; or (5) An antibody based on any of the antibodies in (1) to (4) above, comprising 1 to 12 amino acid substitutions, insertions, or deletions in the amino acid sequence of the heavy chain variable region, and / or comprising 1 to 10 amino acid substitutions, insertions, or deletions in the amino acid sequence of the light chain variable region; or (6) An antibody having a heavy chain variable region having at least 90% sequence identity with respect to the amino acid sequence of the heavy chain variable region, and / or a light chain variable region having at least 90% sequence identity with respect to the amino acid sequence of the light chain variable region, based on any of the antibodies in (1) to (4) above. An anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to Embodiment 1 or 2. [Embodiment 4] The anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to any one of embodiments 1 to 3, wherein the antibody is a human antibody or a chimeric antibody. [Embodiment 5] The antibody is an anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to any one of Embodiments 1 to 4, and is not a multispecific antibody. [Embodiment 6] A nucleic acid molecule encoding an anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to any one of Embodiments 1 to 5. [Embodiment 7] An expression vector comprising the nucleic acid molecule described in Embodiment 6. [Embodiment 8] A host cell containing the expression vector described in Embodiment 7. [Embodiment 9] An anti-ULBP2 monoclonal antibody, or its antigen-binding fragment, capable of increasing the binding of ULBP2 to immobilized human NKG2D in a dose-dependent manner. [Embodiment 10] A method for producing an antibody according to Embodiment 9, Immunizing non-human animals with ULBP2, To prepare a population of hybridomas from the aforementioned immunized non-human animals, From the aforementioned population of hybridomas, select a subpopulation of hybridomas that produce antibodies capable of binding to ULBP2. A method comprising selecting a hybridoma clone from a subpopulation of hybridomas that produces an antibody capable of increasing the binding of ULBP2 to human NKG2D in an antibody dose-dependent manner, wherein the antibody produced by the hybridoma clone provides the antibody described in Embodiment 9. [Embodiment 11] The method according to Embodiment 10, wherein the non-human animal is a human antibody-producing mouse that holds a mouse artificial chromosome vector having the complete human immunoglobulin heavy chain locus and light chain κ locus in a background in which the mouse immunoglobulin gene is disrupted, deleted, or underexpressed. [Embodiment 12] A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of Embodiments 1 to 5 and 9. [Embodiment 13] A pharmaceutical composition according to Embodiment 12, for the treatment of cancer. [Embodiment 14] The pharmaceutical composition according to Embodiment 13, which is administered in combination with an immune checkpoint inhibitor. [Embodiment 15] The pharmaceutical composition according to Embodiment 13 or 14, wherein the cancer is an immune checkpoint inhibitor-resistant cancer.

Claims

1. (a) Each comprises a heavy chain variable region containing the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NOs: 1, 2, and 3, and a light chain variable region containing the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NOs: 4, 5, and 6; (b) Each comprises a heavy chain variable region containing the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NOs: 7, 8, and 9, and a light chain variable region containing the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NOs: 10, 11, and 12; (c) Each comprising a heavy chain variable region containing the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs. 13, 14, and 15, and a light chain variable region containing the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs. 16, 17, and 18, respectively; or (d) Each comprises a heavy chain variable region containing the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs. 19, 20, and 21, and a light chain variable region containing the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs. 22, 23, and 24, respectively. Anti-ULBP2 monoclonal antibody or its antigen-binding fragment.

2. The anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to claim 1, which can increase the binding of ULBP2 to human NKG2D immobilized on a solid phase in a dose-dependent manner with the antibody.

3. The aforementioned antibody is (1) comprising the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 25 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 26; (2) comprising the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 27 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 28; (3) comprising the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 29 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 30; or (4) An antibody that contains the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 31 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 32; or (5) An antibody based on any of the antibodies in (1) to (4) above, comprising 1 to 12 amino acid substitutions, insertions, or deletions in the amino acid sequence of the heavy chain variable region, and / or comprising 1 to 10 amino acid substitutions, insertions, or deletions in the amino acid sequence of the light chain variable region; or (6) An antibody having a heavy chain variable region having at least 90% sequence identity with the amino acid sequence of the heavy chain variable region, and / or a light chain variable region having at least 90% sequence identity with the amino acid sequence of the light chain variable region, based on any of the antibodies in (1) to (4) above. The anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to claim 1.

4. The anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the antibody is a human antibody or a chimeric antibody.

5. The antibody is not a multispecific antibody, and the anti-ULBP2 monoclonal antibody or its antigen-binding fragment is as described in claim 1.

6. A nucleic acid molecule encoding an anti-ULBP2 monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid molecule described in claim 6.

8. A host cell comprising the expression vector described in claim 7.

9. An anti-ULBP2 monoclonal antibody, or its antigen-binding fragment, capable of increasing the binding of ULBP2 to immobilized human NKG2D in a dose-dependent manner.

10. A method for producing an antibody according to claim 9, Immunizing non-human animals with ULBP2, To prepare a population of hybridomas from the aforementioned immunized non-human animals, From the aforementioned population of hybridomas, select a subpopulation of hybridomas that produce antibodies capable of binding to ULBP2. A method comprising selecting a hybridoma clone from a subpopulation of hybridomas that produces an antibody capable of increasing the binding of ULBP2 to human NKG2D in an antibody dose-dependent manner, wherein the antibody produced by the hybridoma clone provides the antibody described in claim 9.

11. The method according to claim 10, wherein the non-human animal is a human antibody-producing mouse that holds a mouse artificial chromosome vector having a complete human immunoglobulin heavy chain locus and light chain κ locus in a background in which the mouse immunoglobulin gene is disrupted, deleted, or low-expression.

12. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5 and 9.

13. The pharmaceutical composition according to claim 12, for the treatment of cancer.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is administered in combination with an immune checkpoint inhibitor.

15. The pharmaceutical composition according to claim 13, wherein the cancer is an immune checkpoint inhibitor-resistant cancer.

Images