PRAME-binding molecules

Abstract

The present invention addresses the problem of providing a PRAME binding molecule. Said problem is solved by a PRAME binding molecule including: a heavy chain variable region that includes a heavy chain CDR1 having an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 having an amino acid sequence represented by SEQ ID NO: 3; and / or a light chain variable region that includes a light chain CDR1 having an amino acid sequence represented by SEQ ID NO: 9, a light chain CDR2 having an amino acid sequence represented by SEQ ID NO: 10, and a light chain CDR3 having an amino acid sequence represented by SEQ ID NO: 11.

Description

[Technical Field]

[0001] The present invention relates to PRAME-binding molecules and the like. [Background technology]

[0002] PRAME (Preferentially Expressed Antigen in Melanoma) is known to be a cancer-testis antigen, and in normal adult tissues, it is only expressed to a small extent in the endometrium, ovaries, and adrenal glands, in addition to the testis. High-level PRAME expression is observed in melanoma (95% of patients), lung cancer (50%), breast cancer (27%), acute leukemia (30%), and multiple myeloma (52%) (Non-Patent Documents 1, 2, 3), making PRAME a promising target for cancer therapy. Target peptides for HLA-A2 include p100-108, p142-151, p300-309, p425-433, and p435-443, while p301-309 is known for HLA-A24. T cells that recognize pMHC have been cloned, and T cell receptors (TCRs) have been isolated and their functions analyzed (Non-patent documents 4, 5, 6), but to date no TCRs that can be used in cancer therapy have been isolated. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] N. van Baren, H. Chambost, A. Ferrant, L. Michaux, H. Ikeda, I. Millard, D. Olive, T. Boon, PG Coulie (1998), PRAME, a gene encoding an antigen recognized on a human melanoma by cytolytic T cells, is expressed in acute leukemia cells. Br. J. Haematol. 102:1376–1379.

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[0004] An objective of the present invention is to provide a PRAME-binding molecule. [Means for solving the problem]

[0005] In view of the above-mentioned problems, the present inventors isolated an antibody that recognizes the HLA-A24 PRAMEp301-309 pMHC complex and attempted to produce a CAR using the antibody. As a result of further research, the present inventors found that the above-mentioned problems can be solved by a PRAME-binding molecule comprising a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and / or a light chain variable region comprising a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11. Further research based on this finding led to the completion of the present invention. That is, the present invention encompasses the following aspects.

[0006] Item 1. A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and / or a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11; A PRAME-binding molecule comprising:

[0007] Item 2. The PRAME-binding molecule according to Item 1, comprising the heavy chain variable region and the light chain variable region.

[0008] Item 3. The PRAME-binding molecule according to Item 1 or 2, which has binding affinity to an HLA-A24 PRAMEp301-309 pMHC complex.

[0009] Item 4. The PRAME-binding molecule according to any one of Items 1 to 3, wherein the binding affinity to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 19, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 20, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 21, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 23, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 24, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 25, and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 26 is half or less of the binding affinity to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 17.

[0010] Item 5. The PRAME-binding molecule according to any one of Items 1 to 4, which is a chimeric antigen receptor.

[0011] Item 6. The PRAME-binding molecule according to Item 5, comprising an scFv domain comprising the heavy chain variable region and the light chain variable region, a transmembrane domain, and a core region comprising the intracellular domain of a TCR.

[0012] Item 7. The PRAME-binding molecule according to Item 6, wherein the core region further comprises an intracellular domain of a costimulatory factor.

[0013] Item 8. The PRAME-binding molecule according to any one of Items 1 to 4, which is an antibody.

[0014] Item 9. A polynucleotide encoding the PRAME-binding molecule according to any one of Items 1 to 8.

[0015] Item 10. A cell containing the polynucleotide according to Item 9.

[0016] Item 11. A lymphocyte cell containing a polynucleotide encoding the PRAME-binding molecule according to any one of Items 5 to 7.

[0017] Item 12. A pharmaceutical composition comprising the lymphocyte cells according to Item 11 or the PRAME-binding molecule according to Item 8.

[0018] Item 13. The pharmaceutical composition according to Item 12, which is used for the diagnosis, treatment or prevention of cancer. [Effects of the Invention]

[0019] The present invention provides a PRAME-binding molecule that has a specific CDR sequence and therefore has excellent binding properties to PRAME, and can exert excellent anti-cancer effects, particularly when used as a chimeric antigen receptor. [Brief explanation of the drawings]

[0020] [Figure 1a] A schematic diagram of the antibody library screening method (magnetic bead screening method) is shown in Test Example 1. Artificially produced HLA-A24 PRAMEp301-309 was bound to Dynabeads MyOne Streptavidin T1 (Invitrogen), and a human antibody library was reacted with this to enrich for antibody clones. [Figure 1b]A schematic diagram of the antibody library screening method (cell screening) is shown in Figure 1 (Test Example 1). Polyclonal antibody clones obtained by the method in Figure 1a were reacted with T2A24 cells pulsed with PRAME p301-309 peptide, which were then separated into an aqueous layer and a cell pellet using an organic solvent, and antibody clones that bound to the cells were selected. [Figure 2] 1 shows the ELISA results for the antibodies of each clone obtained in Test Example 1. The vertical axis shows the relative value of color intensity, and the horizontal axis shows the clone number. [Figure 3] The results of ELISA testing of #98 scFvCL-cp3 for recognition of various HLA-A24-associated pMHC are shown below (Test Example 1). It was confirmed that no pMHC other than PRAME reacted at 1.5 times or more the background (none). The vertical axis shows the relative color intensity, and the horizontal axis shows the HLA-A24-associated pMHC used. [Figure 4] A24-LCL cells without peptide pulse and A24-LCL cells pulsed with 10 micromolar PRAMEp301-309 were prepared, and reacted with #98 scFvCL-cp3 antibody, followed by anti-cp3 antibody (MBL specialty), and then Alexa488-labeled anti-mouse IgG (Invitrogen). The results of measurement using a FACS CANT are shown (Test Example 1). [Figure 5] 1 shows the results of an alanine substitution study (Test Example 2). The vertical axis shows the substitution site in the peptide used and the amino acid after substitution, and the horizontal axis shows the average fluorescence intensity. [Figure 6] The results of affinity measurement for #98 by SPR method are shown below (Test Example 3). [Figure 7] 1 shows a schematic diagram of a retroviral vector for CAR introduction (Test Example 4). [Figure 8a] 1 shows the FACS results of non-CAR-transfected cells (Test Example 4). [Figure 8b] 1 shows the FACS results of CAR-introduced cells (Test Example 4). [Figure 9]1 shows the results of an alanine substitution study on CAR-transfected cells (Test Example 5). The vertical axis shows the substitution site in the peptide used and the amino acid after substitution, and the horizontal axis shows the IFN gamma concentration. [Figure 10] 1 shows the FACS results of IFNg-stained CAR-introduced cells (Test Example 5). The vertical axis shows the IFNg-positive rate, and the horizontal axis shows the PRAME p301-309 concentration after peptide pulsing. [Figure 11a] 1 shows the results of PRAME expression analysis of target cells in Test Examples 7 and 8. The vertical axis shows the relative amount of PREME mRNA, and the horizontal axis shows the target cells. [Figure 11b] 1 shows the results of HLA-A24 expression analysis of target cells in Test Examples 7 and 8. The vertical axis shows the mean fluorescence intensity for HLA-A24, and the horizontal axis shows the target cells. [Figure 12] 1 shows the results of measuring IFN gamma concentration by ELISA in Test Examples 7 and 8. The vertical axis indicates IFN gamma concentration, and the horizontal axis indicates target cells. [Figure 13a] The graph shows the results of observing the change in the xCelligence index after applying each effector cell to negative target cells (NW-MEL-38 (PRAME-positive, HLA-A24-negative)). The vertical axis shows the number of A24 NW-MEL-38 cells, normalized to the number of NW-MEL-38 cells immediately before co-culture with effector cells, which was set at 1. The horizontal axis shows the time elapsed since the start of culture. The legend indicates the effector cells. [Figure 13b] The graph shows the results of observing the change in the xCelligence index after applying each effector cell to positive target cells (HLA-A24-transfected NW-MEL-38 cells (A24+PRAME+)). The vertical axis shows the number of A24 NW-MEL-38 cells, normalized to the number of NW-MEL-38 cells immediately before co-culture with effector cells, which is set at 1. The horizontal axis shows the time elapsed since the start of culture. The legend indicates the effector cells. [Figure 13c]The graph shows the results of observing the change in the xCelligence index after applying each effector cell to positive target cells (SK-MEL-124 cells (A24+PRAME+)). The vertical axis shows the number of A24 NW-MEL-38 cells, normalized to the number of NW-MEL-38 cells immediately before co-culture with effector cells, which was set at 1. The horizontal axis shows the time elapsed since the start of culture. The legend indicates the effector cells. [Figure 14a] The test outline of Test Example 10 is shown below. [Figure 14b] 1 shows the measurement results of the tumor area in Test Example 10. The vertical axis shows the area of ​​the tumor area, and the horizontal axis shows the number of days elapsed since the inoculation of SK-MEL-124 cells. [Figure 14c] 1 shows the results of body weight measurement in Test Example 10. The vertical axis indicates relative body weight values, and the horizontal axis indicates the number of days elapsed since inoculation of SK-MEL-124 cells. [Figure 15a] The test outline of Test Example 11 is shown below. [Figure 15b] 1 shows the results of measuring tumor size in Test Example 11. The vertical axis shows tumor size, and the horizontal axis shows the number of days elapsed since SK-MEL-124 cell inoculation (-10). [Figure 15c] 1 shows the measurement results of the CAR-T cell proportion in Test Example 11. The vertical axis shows the proportion of CAR-T cells in peripheral blood, and the horizontal axis shows the number of days elapsed since inoculation of SK-MEL-124 cells. [Figure 16] T2A24 cells were pulsed with the peptides, CMV, and DMSO listed in Table 2, and co-cultured with #98 CAR-T cells. The amount of IFNg secreted into the culture supernatant was measured by ELISA (Test Example 5). The vertical axis indicates the amount of IFNg, and the horizontal axis indicates the peptide used. Note that "e alone" indicates effector cells only. DETAILED DESCRIPTION OF THE INVENTION

[0021] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0022] "Identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Thus, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul S F. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990); Karlin S, Altschul S F. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on the BLAST algorithm. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0023] As used herein, "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitution include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having beta-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0024] As used herein, "CDR" refers to C Complementarity D etermining R CDR is an abbreviation for complementarity-determining region, and is also called complementarity-determining region. CDR is a region present in the variable region of an immunoglobulin, and is a region that is deeply involved in the specific binding of an antibody to an antigen. Furthermore, "light chain CDR" refers to a CDR present in the light chain variable region of an immunoglobulin, and "heavy chain CDR" refers to a CDR present in the heavy chain variable region of an immunoglobulin.

[0025] As used herein, the term "variable region" refers to a region comprising CDR1 to CDR3 (hereinafter simply referred to as "CDRs1-3"). The order in which these CDRs 1-3 are arranged is not particularly limited, but preferably refers to a region in which they are arranged from the N-terminus to the C-terminus in the order of CDR1, CDR2, and CDR3, or in the reverse order, either consecutively or via other amino acid sequences referred to as framework regions (FRs) described below. The term "heavy chain variable region" refers to a region in which the above-mentioned heavy chain CDRs 1-3 are arranged, and the term "light chain variable region" refers to a region in which the above-mentioned light chain CDRs 1-3 are arranged.

[0026] The regions of each variable region other than CDR1-3 are referred to as framework regions (FRs) as described above. In particular, the region between the N-terminus of the variable region and CDR1 is defined as FR1, the region between CDR1 and CDR2 as FR2, the region between CDR2 and CDR3 as FR3, and the region between CDR3 and the C-terminus of the variable region as FR4.

[0027] 2.PRAME binding molecule In one aspect, the present invention relates to a PRAME-binding molecule (sometimes referred to herein as the "PRAME-binding molecule of the present invention") comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and / or a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11. This is described below.

[0028] The PRAME-binding molecule of the present invention is not particularly limited as long as it comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and / or a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, and has binding ability to PRAME.

[0029] The PRAME-binding molecule of the present invention may be a molecule consisting of one type of polypeptide or a molecule consisting of a complex of two or more types of polypeptides. Furthermore, the PRAME-binding molecule of the present invention may be a molecule consisting of a polypeptide or a complex thereof, or may be a polypeptide or a complex thereof linked to another substance (e.g., a fluorescent substance, a radioactive substance, an inorganic particle, etc.).

[0030] The binding ability to PRAME can be measured according to known methods, for example, by ELISA (specifically, for example, by the method of Test Example 2). The binding ability of the PRAME-binding molecule of the present invention to PRAME is, for example, 20% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more, relative to the 100% binding ability to PRAME of antibody #98 in the Examples described below.

[0031] The PRAME-binding molecule of the present invention preferably comprises both the heavy chain variable region and the light chain variable region.

[0032] The heavy chain variable region is preferably a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having 90% or more (preferably 95% or more, preferably 98% or more, preferably 99% or more) identity to the amino acid sequence set forth in SEQ ID NO: 4. The light chain variable region is preferably a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having 90% or more (preferably 95% or more, preferably 98% or more, preferably 99% or more) identity to the amino acid sequence set forth in SEQ ID NO: 12. When there is an amino acid mutation from SEQ ID NO: 4 or 12, the mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution.

[0033] The PRAME-binding molecule of the present invention can bind to the HLA-A24 PRAMEp301-309 pMHC complex. The HLA-A24 PRAMEp301-309 pMHC complex is a complex of HLA-A24 and a partial peptide of PRAME (p301-309: SEQ ID NO: 17). The form of the complex is not particularly limited, as long as it is the form in which HLA presents the peptide as an antigen.

[0034] The PRAME-binding molecules of the present invention can specifically recognize PRAMEp301-309 (SEQ ID NO: 17). From this perspective, the binding affinity of the PRAME-binding molecules of the present invention to at least one (preferably, two or more, three or more, four or more, five or more, six or more, or seven (all)) of peptides obtained by partially mutating PRAMEp301-309 (peptides consisting of the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26) is preferably half or less (preferably, 1 / 5 or less, 1 / 10 or less, 1 / 20 or less, 1 / 100 or less, 1 / 500 or less, 1 / 2000 or less, or 1 / 10000 or less) of the binding affinity to PRAMEp301-309 (SEQ ID NO: 17).

[0035] The PRAME-binding molecule of the present invention may be chemically modified. The polypeptide constituting the PRAME-binding molecule of the present invention may have a C-terminus containing a carboxyl group (-COOH), a carboxylate (-COO - ), amide (-CONH2), or ester (-COOR). Here, R in the ester may be, for example, a C alkyl group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups such as phenyl and α-naphthyl6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups; α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 An aralkyl group; a pivaloyloxymethyl group, etc., are used. In the polypeptide constituting the PRAME-binding molecule of the present invention, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the ester may be, for example, the C-terminal ester described above. Furthermore, in the polypeptide constituting the PRAME-binding molecule of the present invention, the amino group of the N-terminal amino acid residue may be protected by a protecting group (e.g., a C-protecting group such as a formyl group or an acetyl group). 1-6 C such as alkanoyl 1-6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1-6 C such as alkanoyl group 1-6 Also included are those protected with an alkyl group (such as an acyl group).

[0036] The PRAME-binding molecule of the present invention may be one to which a known protein or peptide such as a protein tag or signal sequence is attached. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, and fluorescent protein tag.

[0037] The PRAME-binding molecules of the present invention may be in the form of pharmaceutically acceptable salts with acids or bases. The salts are not particularly limited as long as they are pharmaceutically acceptable, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0038] The PRAME-binding molecules of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0039] 2-1. Antibodies In one preferred embodiment, the PRAME-binding molecule of the present invention is an antibody (herein, a PRAME-binding molecule of the present invention that is an antibody may be referred to as "the antibody of the present invention").

[0040] The antibody of the present invention is a monoclonal antibody.

[0041] The molecular weight of the antibody of the present invention is not particularly limited, but the lower limit is, for example, 20,000, preferably 50,000, preferably 100,000, more preferably 120,000, and the upper limit is, for example, 1,000,000, preferably 500,000, more preferably 200,000.

[0042] The structure of the antibody of the present invention is not particularly limited. The antibody of the present invention may or may not contain a constant region. When the antibody contains a constant region, it may contain all of the heavy chain constant regions (CH1, CH2, and CH3) and the light chain constant region (CL), or may contain any one or a combination of two or more of these.

[0043] Specific examples of the antibody structure of the present invention include immunoglobulin, Fab, F(ab')2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, etc. Among these, immunoglobulin is preferred from the viewpoint of the effects of the present invention.

[0044] Immunoglobulins have a structure that combines two structures: one heavy chain having a heavy chain variable region and a heavy chain constant region, and one light chain having a light chain variable region and a light chain constant region.

[0045] Fab comprises a heavy chain fragment containing a heavy chain variable region and CH1 in the heavy chain constant region, and a light chain containing a light chain variable region and a light chain constant region (CL), with the heavy chain variable region and light chain variable region associated by the noncovalent intermolecular interaction described above or linked by a disulfide bond. In Fab, CH1 and CL may be disulfide-bonded via the thiol groups of cysteine ​​residues present in each.

[0046] F(ab')2 has two pairs of the above-mentioned Fab, and has a structure in which the CH1s are disulfide-bonded together via the thiol groups of the cysteine ​​residues contained therein.

[0047] A minibody is a structure in which two fragments, each consisting of a heavy chain variable region constituting an scFv as described below, and a CH3 linked thereto, are associated via non-covalent intermolecular interactions between the CH3s.

[0048] scFv-Fc is a structure in which two antibody fragments containing the scFv, CH2, and CH3 described below are associated by non-covalent intermolecular interactions between the CH3s, similar to the minibody described above, and the thiol groups of the cysteine ​​residues contained in each CH3 are disulfide-bonded.

[0049] Fv, also known as the smallest structural unit of an antibody, is a structure in which the heavy chain variable region and the light chain variable region are associated through noncovalent intermolecular interactions. In Fv, the thiol groups of cysteine ​​residues present in the heavy chain variable region and the light chain variable region may be disulfide-bonded.

[0050] An scFv has a structure in which the C-terminus of a heavy chain variable region and the N-terminus of a light chain variable region are linked by a linker, or a structure in which the N-terminus of a heavy chain variable region and the C-terminus of a light chain variable region are linked by a linker, and is also called a single-chain antibody.

[0051] Diabodies, triabodies, and tetrabodies are structures in which the above-mentioned scFvs form dimers, trimers, and tetramers, respectively, and associate in a structurally stable state through non-covalent intermolecular interactions between the variable regions, similar to Fvs.

[0052] When the antibody of the present invention is an immunoglobulin, its class is not particularly limited. Examples of the class include IgA, IgD, IgE, IgG, IgM, and their subclasses. Preferred classes include IgG and IgM, preferably IgG, and more preferably IgG1.

[0053] The origin of the antibody of the present invention is not particularly limited. The antibody of the present invention may be, for example, a human-derived antibody, a mouse-derived antibody, a rat-derived antibody, a rabbit-derived antibody, a monkey-derived antibody, or a chimpanzee-derived antibody. The antibody of the present invention may also be a chimeric antibody (for example, an antibody in which the amino acid sequence of the constant region of an antibody derived from a non-human organism (such as a mouse) is replaced with the amino acid sequence of the constant region of a human-derived antibody), a humanized antibody, a fully humanized antibody, or the like.

[0054] The antibody of the present invention can be produced, for example, by a method comprising the steps of culturing a host transformed with a polynucleotide encoding the antibody of the present invention and collecting a fraction containing the antibody of the present invention.

[0055] Polynucleotides encoding antibodies of the present invention are not particularly limited as long as they contain the antibodies of the present invention in an expressible state, and may contain other sequences in addition to the coding sequence for the antibodies of the present invention. Examples of other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, an origin of replication, and a drug resistance gene coding sequence located adjacent to the coding sequence for the antibodies of the present invention. Furthermore, polynucleotides encoding antibodies of the present invention may be linear polynucleotides or circular polynucleotides (e.g., vectors).

[0056] Specific examples of polynucleotides include: (I) polynucleotides comprising a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDRs 1-3 of the antibody of the present invention; (II) polynucleotides comprising a nucleotide sequence encoding at least one selected from the group consisting of the light chain, light chain variable region, and light chain CDRs 1-3 of the antibody of the present invention; (III) polynucleotides comprising a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDRs 1-3 of the antibody of the present invention; and polynucleotides comprising a nucleotide sequence encoding at least one selected from the group consisting of the light chain, light chain variable region, and light chain CDRs 1-3 of the antibody of the present invention.

[0057] The host is not particularly limited, and examples thereof include insect cells, eukaryotic cells, and mammalian cells. Among these, mammalian cells such as HEK cells, CHO cells, NS0 cells, SP2 / O cells, and P3U1 cells are preferred from the viewpoint of more efficient antibody expression. The methods for transformation, culture, and recovery are not particularly limited, and known methods for antibody production can be used. After recovery, the antibody of the present invention may be purified as necessary. Purification can be carried out by known methods for antibody production, such as chromatography and dialysis.

[0058] 2-2. Chimeric antigen receptor In a preferred embodiment, the PRAME-binding molecule of the present invention is a chimeric antigen receptor (herein, a PRAME-binding molecule of the present invention that is a chimeric antigen receptor is sometimes referred to as a "chimeric antigen receptor of the present invention").

[0059] A chimeric antigen receptor (CAR) is a chimeric protein that typically contains a single-chain fragment (scFv) consisting of the light (VL) and heavy (VH) variable regions of a monoclonal antibody linked in tandem at the N-terminus as the region responsible for antigen binding, and a T cell receptor (TCR) zeta chain at the C-terminus. T cells expressing a CAR are called CAR-T cells.

[0060] In the chimeric antigen receptor of the present invention, the region (PRAME-binding region) responsible for binding to the antigen (PRAME) is not particularly limited, as long as it comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and / or a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11.

[0061] The PRAME-binding region preferably has an scFv structure. The linker connecting the heavy chain variable region and the light chain variable region is not particularly limited and can be any linker as long as the function of the chimeric antigen receptor is maintained. Preferred examples of the linker include a linker consisting of glycine alone or glycine and serine. The number of amino acid residues in the linker is, for example, 5 to 30, preferably 10 to 20, and more preferably 15.

[0062] The chimeric antigen receptor of the present invention typically comprises an scFv domain containing a heavy chain variable region and a light chain variable region, a transmembrane domain, and a core region containing a TCR intracellular domain. In the core region, the scFv domain, transmembrane domain, and TCR intracellular domain are typically arranged in this order from the N-terminus, either directly or via other domains.

[0063] The type of transmembrane domain is not limited as long as it does not inhibit the function of the chimeric antigen receptor. For example, CD28, CD3zeta, CD4, CD8alpha, and the like, which are expressed in T cells, can be used. These transmembrane domains may be appropriately mutated as long as they do not inhibit the function of the chimeric antigen receptor.

[0064] The intracellular domain of the TCR may be, for example, an intracellular domain derived from CD3, also known as the TCR ζ chain. A suitable mutation may be introduced into CD3, as long as it does not inhibit the function of the chimeric antigen receptor. When introducing a mutation into CD3, it is preferable to introduce the mutation so that it contains an immunoreceptor tyrosine-based activation motif (ITAM).

[0065] The chimeric antigen receptor of the present invention preferably has a spacer sequence disposed between the scFv domain and the transmembrane domain. The length of the spacer sequence and the types of amino acid residues constituting it are not limited as long as they do not inhibit the function of the chimeric antigen receptor. For example, the spacer sequence can be designed to consist of approximately 10 to 200 amino acid residues. The spacer sequence preferably employs a sequence from the light chain constant region.

[0066] In the chimeric antigen receptor of the present invention, it is preferable that the core region further comprises an intracellular domain of a costimulatory factor. The intracellular domain of the costimulatory factor is not particularly limited, as long as it is an intracellular domain derived from a costimulatory factor possessed by T cells or the like. For example, one or more species selected from the group consisting of OX40, 4-1BB, GITR, CD27, CD278, CD28, etc. can be appropriately selected and used. The intracellular domain of these costimulatory factors may be appropriately mutated as long as it does not inhibit the function of the chimeric antigen receptor. The location of the intracellular domain of the costimulatory factor is not particularly limited, as long as it is located on the C-terminal side of the transmembrane domain, and may be either on the cell membrane side of the intracellular domain of the TCR or on the opposite side of the cell membrane. In a preferred aspect of the present invention, the intracellular domain of the costimulatory factor is preferably located on the opposite side of the cell membrane of the intracellular domain of the TCR.

[0067] The chimeric antigen receptor of the present invention preferably contains various ligand domains, such as a GITRL domain, a 4-1BBL domain, or an ICOSL domain, at the C-terminus of the core region via a self-cleaving peptide domain, which can further enhance the expression efficiency of the chimeric antigen receptor and the cytotoxic activity of CAR-T cells containing the same.

[0068] As used herein, the term "self-cleaving peptide" refers to a peptide sequence with cleavage activity occurring between two amino acid residues within the peptide sequence itself. Examples of self-cleaving peptides include 2A peptides or 2A-like peptides. For example, in the case of 2A peptides or 2A-like peptides, cleavage occurs between a glycine residue and a proline residue on these peptides. This occurs via a "ribosomal skipping mechanism" in which normal peptide bond formation between glycine and proline residues does not occur during translation, and downstream translation is not affected. The ribosomal skipping mechanism is known in the art and is used for the expression of multiple proteins encoded by a single messenger RNA (mRNA). The self-cleaving peptide used in the present invention can be derived from a viral 2A peptide or a 2A-like peptide with equivalent function. For example, the self-cleaving peptide domain may be selected from the group consisting of 2A peptide (F2A) derived from foot-and-mouth disease virus (FMDV), 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), 2A peptide (P2A) derived from porcine teschovirus (PTV-1), and 2A peptide (T2A) derived from Thosea asigna virus (TaV). The self-cleaving peptide domain may be mutated as appropriate, as long as its activity is not significantly impaired.

[0069] Techniques for producing chimeric antigen receptors and CAR-T cells expressing them are known, and they can be produced according to or in accordance with known methods.

[0070] 3. Polynucleotides In one aspect, the present invention relates to a polynucleotide (sometimes referred to herein as "polynucleotide of the present invention") that encodes a PRAME-binding molecule of the present invention. This will be explained below.

[0071] The polynucleotide of the present invention may contain other sequences in addition to the coding sequence of the PRAME-binding molecule of the present invention. Preferably, the polynucleotide of the present invention contains the PRAME-binding molecule of the present invention in an expressible state. Examples of such other sequences include promoter sequences, enhancer sequences, repressor sequences, insulator sequences, origins of replication, coding sequences for reporter proteins (e.g., fluorescent proteins), and coding sequences for drug resistance genes. The polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide (e.g., a vector). The vector may be a plasmid vector or a viral vector (e.g., adenovirus or retrovirus). The vector may be, for example, a cloning vector or an expression vector. Examples of expression vectors include vectors for prokaryotic cells such as Escherichia coli or actinomycetes, and vectors for eukaryotic cells such as yeast cells, insect cells, and mammalian cells.

[0072] The polynucleotides of the present invention include not only DNA and RNA, but also those that have been chemically modified by known methods, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide can be substituted with chemically modified phosphate residues, such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can also be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) can also be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Furthermore, the term "polynucleotide" encompasses not only natural nucleic acids but also bridged nucleic acids (BNAs), locked nucleic acids (LNAs), peptide nucleic acids (PNAs), etc.

[0073] 4.Cells In one aspect, the present invention relates to a cell containing the polynucleotide of the present invention (sometimes referred to herein as the "cell of the present invention"), which will be described below.

[0074] The cells of origin of the cells of the present invention are not particularly limited. If the cells of the present invention are intended to be used in producing the PRAME-binding molecules of the present invention, the cells of origin may be cells that can be used for protein expression (e.g., insect cells, eukaryotic cells, mammalian cells, etc.).

[0075] When the cell of the present invention comprises a polynucleotide encoding the chimeric antigen receptor of the present invention, the cell is preferably a lymphocyte (for example, a T cell (e.g., CD4-positive CD8-negative T cell, CD4-negative CD8-positive T cell, T cell prepared from iPS cell, αβ-T cell, γδ-T cell, etc.), NK cell, NKT cell, etc.). The lymphocyte is preferably a cell that expresses the chimeric antigen receptor of the present invention, and in a more specific embodiment, the lymphocyte expresses the chimeric antigen receptor of the present invention on its cell membrane, preferably expressing the chimeric antigen receptor of the present invention with the PRAME-binding region exposed outside the cell membrane.

[0076] Lymphocytes and other cells expressing chimeric antigen receptors recognize PRAME at the PRAME-binding region, then transmit the recognition signal to T cells and other cells, activating a signal that induces cytotoxic activity, which in turn enables the cells to attack or exert cytotoxic activity against other cells or tissues that express PRAME.

[0077] When the cells that exhibit such a function are CTLs, they are called chimeric antigen receptor T cells (CAR-T cells). Cells that have the potential to exhibit cytotoxic activity, such as NK cells, can also exhibit cytotoxic activity when their PRAME-binding domain binds to PRAME, similar to chimeric antigen receptor T cells. Therefore, host cells containing a polynucleotide encoding a chimeric antigen receptor (particularly, host cells with cytotoxic activity) are useful as an active ingredient in pharmaceutical compositions.

[0078] Such lymphocytes and the like specifically recognize cancer tissue (tumor tissue) and are therefore useful for the treatment or prevention of cancer, etc. The type of cancer is not particularly limited, and includes blood cancer and solid cancer. Examples of blood cancer include various B-cell malignant lymphomas (B-cell acute lymphoblastic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative disorders, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndrome, acute myeloid leukemia, and multiple myeloma. Examples of solid cancer include lung cancer, colon cancer, ovarian cancer, breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, melanoma, neuroblastoma, and bladder cancer.

[0079] The cells of the present invention can be obtained by introducing the polynucleotide of the present invention into cells. If necessary, cells containing the polynucleotide of the present invention may be enriched, or may be enriched using a specific marker (CD antigen such as CD8) as an indicator.

[0080] 5. Pharmaceutical Compositions In one aspect, the present invention relates to a pharmaceutical composition (sometimes referred to herein as the "pharmaceutical composition of the present invention") containing a lymphocyte containing a polynucleotide encoding a chimeric antigen receptor of the present invention or an antibody of the present invention. This is described below.

[0081] The content of the above-mentioned cells and antibodies in the pharmaceutical composition can be appropriately determined taking into consideration the type of target disease (e.g., solid cancer), the desired therapeutic effect, the administration method, the treatment period, the patient's age, and the patient's weight, etc. For example, the content of the antibody in the pharmaceutical composition can be about 0.001 to 10 parts by weight, where 100 parts by weight of the total pharmaceutical composition. The content of cells in the pharmaceutical composition can be, for example, about 1 cell / mL to 10^4 cells / mL.

[0082] The administration form of the pharmaceutical composition is not particularly limited as long as the desired effect is obtained, and it can be administered to mammals, including humans, by either oral or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, or topical administration). Because the active ingredient is cells, the preferred administration form is parenteral administration, more preferably intravenous injection. Dosage forms for oral and parenteral administration and methods for their preparation are well known to those skilled in the art, and can be prepared according to standard methods by mixing the antibody or cells of the present invention with a pharmaceutically acceptable carrier or the like.

[0083] Dosage forms for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, and lotions), suppositories, inhalants, ophthalmic preparations, eye ointments, nasal drops, ear drops, and liposomes. For example, injectable preparations are prepared by dissolving or suspending antibodies or cells in distilled water for injection, and solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like can be added as needed. The pharmaceutical composition can also be in the form of a lyophilized preparation for preparation immediately before use.

[0084] The pharmaceutical composition may further contain other drugs that are effective in diagnosing, treating, or preventing diseases. In addition, the pharmaceutical composition may also contain ingredients such as bactericides, anti-inflammatory agents, cell activators, vitamins, and amino acids, as needed.

[0085] Carriers used in formulating pharmaceutical compositions include excipients, binders, disintegrants, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, and the like that are commonly used in the art.

[0086] The type of disease to be diagnosed, treated, or prevented using the pharmaceutical composition is not particularly limited as long as the diagnosis, treatment, or prevention can be achieved. Specific target diseases include, for example, cancer. The type of cancer is not particularly limited and includes blood cancer and solid cancer. Examples of blood cancer include various B-cell malignant lymphomas (B-cell acute lymphoblastic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative disorders, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndrome, acute myeloid leukemia, multiple myeloma, etc. Examples of solid cancers include lung cancer, colon cancer, ovarian cancer, breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, melanoma, neuroblastoma, and bladder cancer.

[0087] The subject (subject) to which the pharmaceutical composition is administered is, for example, an animal that is afflicted with or may be afflicted with the above-mentioned disease. The term "possibly afflicted" can be determined by known diagnostic methods. The animal is, for example, a mammal, preferably a human.

[0088] The dosage of a pharmaceutical composition can be determined by a clinician based on various factors, such as the route of administration, the type of disease, the severity of symptoms, the patient's age, sex, and body weight, the severity of the disease, pharmacological knowledge such as pharmacokinetic and toxicological characteristics, whether a drug delivery system is used, and whether the composition is administered as part of a combination of other drugs. For example, if the active ingredient is an antibody, the dosage of the pharmaceutical composition can be approximately 1 microgram / kg (body weight) to 10 g / kg (body weight) per day. Furthermore, if the active ingredient is cells, the dosage can be approximately 10^4 cells / kg (body weight) to 10^9 cells / kg (body weight). The administration schedule of the pharmaceutical composition can also be determined taking into account factors similar to those for the dosage. For example, the above daily dosage can be administered once a day to once a month. [Example]

[0089] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0090] Test Example 1: Isolation and evaluation of antibody #98 that recognizes HLA-A24-PRAME Using a self-made human antibody library, we performed an antibody library screen targeting an artificially constructed HLA-A24 PRAMEp301-309 (SEQ ID NO: 17) pMHC complex (hereafter referred to as A24-PRAME) and isolated several antibodies that recognized A24-PRAME. The method used was as described in Non-Patent Document 7. Specifically, antibodies were isolated using phage display. In this method, scFvCL is displayed as part of the phage coat protein cp3, and clones with high antigen binding can be selected by ELISA. After performing the magnet bead screen shown in Figure 1a twice, we performed the cell screen shown in Figure 1b (Non-Patent Document 8). Approximately 400 clones were selected, and the culture supernatant was prepared. ELISA was performed on the resulting scFvCL-CP3.

[0091] Forty clones that reacted with the positive target, A24-PRAME, but not with the negative target, A24-CMV, were selected and their recognition against two concentrations of A24-PRAME and one concentration of A24-CMV was examined by ELISA (Figure 2). Specifically, the 40 clones were tested for their recognition against A24-PRAME at 100 ng / well (red), A24-PRAME at 50 ng / well (black), and A24-CMV at 100 ng / well (white) using 20 ul of supernatant. Neutravidin (Thermo) was immobilized on a Maxisorp plate (NUNC), and the selected pMHC was then immobilized. The culture supernatant from each clone was then incubated and washed. The plate was then incubated with a mouse anti-cp3 antibody (MBL proprietary product) and an HRP-conjugated anti-mouse antibody (330, MBL), and the color was evaluated using TMB. Sequence analysis of these antibodies revealed that 13 types of antibodies were obtained.

[0092] To examine the binding specificity of these antibodies, ELISA was performed using several peptide-HLA-A24 complexes (pMHC). Each of the HLA-A24 pMHC complexes, including A24-PRAME p301, PRAME p412, EBNA3A p246, MAGE-A3 p195, MAGE-A4 p143, SAGE p715, CMV p30, HTLV-1 p301, NY-ESO-1 p158, Foxp3 p323, Foxp3 p363, IDO p144, IDO p269, hTERT p461, and WT1 p235, was immobilized and ELISA was performed with the selected antibodies. The ELISA results are shown in Figure 3. No cross-recognition was observed for #98, demonstrating its high specificity.

[0093] Next, to examine whether the pMHC complex formed between cell-expressed HLA-A24 and PRAMEp301-309 was recognized, we examined the recognition of PRAMEp301-309 peptide-pulsed A24-LCL cells. The results are shown in Figure 4. It was found that #98 was fully recognized.

[0094] The amino acid sequence of antibody #98 and the nucleotide sequence encoding this antibody were analyzed and the results are shown below. The CDR sequences were predicted using IMGIT.

[0095] <Heavy Chain> Heavy chain CDR1 amino acid sequence: GGTFSSYA (SEQ ID NO: 1) Heavy chain CDR2 amino acid sequence: IIPIFGTA (SEQ ID NO: 2) Heavy chain CDR3 amino acid sequence: ARHHSNYYYYGMDV (SEQ ID NO: 3) Heavy chain variable region amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTGTAYMELSSLRSEDTAVYYCARHHSNYYYYGMDVWGQGTTVTVSR (SEQ ID NO: 4) Heavy chain CDR1 base sequence: GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO: 5) Heavy chain CDR2 base sequence: ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 6) Heavy chain CDR3 base sequence: GCGAGACACCACAGTAACTACTACTACTACGGTATGGACGTC (SEQ ID NO: 7) Heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTAC GCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGGGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACACCACAGTAACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGA (SEQ ID NO: 8).

[0096] <Light chain> Light chain CDR1 amino acid sequence: NIGSKN (SEQ ID NO: 9) Light chain CDR2 amino acid sequence: RDS (SEQ ID NO: 10) Light chain CDR3 amino acid sequence: QVWDSSHV (SEQ ID NO: 11) Light chain variable region amino acid sequence: SYELTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSHVFGTGTKVTVL (SEQ ID NO: 12) Light chain CDR1 base sequence: AACATTGGAAGTAAAAAT (SEQ ID NO: 13) Light chain CDR2 base sequence: AGGGATAGC (SEQ ID NO: 14) Light chain CDR3 base sequence: CAGGTGTGGGACAGCAGCCATGTC (SEQ ID NO: 15) Light chain variable region nucleotide sequence: TCCTATGAGCTGACTCAGCCACTCTCAGTGTCAGTGGCCCTGGGACAGACGGCCAGGATTACCTGTGGCGGAAACAACATTGGAAGTAAAAATGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATAGGGATAGCAACC GGCCCTCTGGGATCCCTGAGCGATTCTCTGGCTCCAACTCGGGGAACACGGCCACCCTGACCATCAGCAGAGCCCAAGCCGGGGATGAGGCTGACTATTACTGTCAGGTGTGGGACAGCAGCCATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 16).

[0097] Test Example 2. Specific recognition of A24-PRAME by #98 scFvCL-cp3 LCL cells express high levels of MHC Class I, and when pulsed with peptides, the peptides are trapped on HLA-A24 on the LCL cells and presented as pMHC. Therefore, LCL cells pulsed with PRAME, CMV peptides, and DMSO were reacted with #98, and sufficient recognition was confirmed by FACS.

[0098] Next, to investigate the amino acids involved in antibody recognition, peptides were created by substituting each amino acid in PRAME p301-309 (LYVDSLFFL, Table 1, SEQ ID NO: 17) with a different amino acid (mainly alanine) (Table 1, SEQ ID NOs: 2-10). Because 2Y and 9L were predicted to be anchor amino acids, we also investigated 2F, 2W, 9F, and 9I (Table 1, SEQ ID NOs: 11-14), which stabilize peptide binding, to examine the effect of peptide modification on antibody recognition. Specifically, the following steps were performed. Wild-type, DMSO, 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 2F, 2W, 9F, and 9I peptides were prepared and pulsed onto A24-LCL at a concentration of 10 micromolar. The resulting mixture was then reacted with #98 scFvCL-cp3, followed by mouse anti-cp3 antibody and then Alexa488-labeled anti-mouse antibody, and analyzed using a FACS CANT.

[0099] [Table 1]

[0100] As shown in Figure 5, the MFI value of #98 decreased with 2A, 3A, 4A, 6A, 7A, 8A, and 9A, indicating that these seven amino acids are essential for antibody recognition. Since 9F and 9I did not decrease the MFI, it was determined that these amino acids were capable of recognition (Figure 5). Therefore, the motifs considered for risk screening were xYVDxLFFL, xYVDxLFFF, and xYVDxLFFI.

[0101] Test Example 3: Measurement of binding constant of #98 scFvCL-pp Next, the KD value of #98 scFvCL-cp3pp, prepared by converting #98 scFvCL-cp3 to the pp form, was measured using a BiacoreX100. The KD value in Biacore was determined by measuring the dynamic change in detection sensitivity (reflecting resonance, or mass change on the chip) over time. The dissociation rate constant (Kd) and association rate constant (Ka) were calculated from the dynamic change curve, and the binding constant was calculated from the ratio of these two constants. Specifically, each scFv-cp3 expression plasmid was cleaved with the restriction enzyme SalI and self-ligated. This was used to transform Escherichia coli DH5a to obtain a clone producing scFv-pp. This was cultured in a medium containing IPTG, and the supernatant was collected, concentrated with ammonium sulfate, purified with IgG Sepharose 6 Fast Flow, dialyzed against PBS, and the concentration was estimated by SDS-PAGE. SPR measurements using the BIAcoreX100 were performed using the Biotin Capture Kit (GE) according to the manufacturer's instructions. First, A24 PRAMEp301-309 was immobilized on the sensor chip as a ligand. Next, five concentrations of scFv-pp (500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM) were sequentially reacted, and association and dissociation measurements were performed. A global fit was performed to calculate the K, Koff, and KD. The binding constant of #98 scFvCL-pp was found to be 5.3 nM (Figure 6).

[0102] Test Example 4. Construction of retroviral vector, transfection into PBMC, and expression study Retroviral gene transfer was performed to induce constitutive CAR expression. A retroviral vector for CAR transduction was constructed (Figure 7), and the retrovirus was produced using Plat-A packaging cells. CAR-T cells were then infected with PBMCs to generate CAR-T cells. CAR expression was confirmed by tetramer staining. Specifically, human PBMCs were stimulated with OKT3 and retronectin and cultured. On days 3 and 4, the cells were infected with the retrovirus and cultured to generate CAR T cells (Figure 8). These cells were stained with PRAME tetramer and anti-CD4 and anti-CD8 antibodies and measured using FACS CANT. The CAR-positive rate of CAR-T cells was 52.0% for CD8 and 51.7% for CD4 (Figure 8).

[0103] Test Case 5. #98 zG CAR T Cell Alanine Scan Study and Risk Assessment T2A24 cells pulsed with the alanine scan peptide described in Test Example 2 were cocultured with #98 zG CAR T cells, and the amount of IFNg secreted into the culture supernatant was measured by ELISA (Figure 9). Specifically, the #98 CAR was cocultured with 1x10^5 A24-LCL pulsed with the amino acid substitution peptide shown in Figure 5 and the same number of CAR T cells. The amount of IFNg produced over 24 hours was detected and compared by ELISA. The pattern was similar to that of alanine scan using scFvCL-cp3, with clear recognition of 1A, 5A, 9F, and 9I. Therefore, a BLAST search using the motifs xYVDxLFFL, xYVDxLFFF, and xYVDxLFFI was performed. As a result, no peptides matching these motifs were identified.

[0104] Furthermore, genes with three different amino acid sequences from PRAMEp301-309 found in the BLAST search are shown in Table 2. T2A24 cells were pulsed with these peptides, CMV, and DMSO and co-cultured with #98 CAR-T cells. The amount of IFNg secreted into the culture supernatant was measured by ELISA. Note that "e alone" indicates effector cells only (Figure 16). No reaction of #98 CAR-T cells was observed with the peptides shown in Table 2.

[0105] [Table 2]

[0106] Test Example 6. #98 zG CAR cognitive avidity assessment T2A24 cells were pulsed with various concentrations of PRAME peptide and co-cultured with #98 zG CAR T cells. IFNg intracellular staining was performed to measure the recognition activity of #98 zG CAR T cells. This is the avidity. Specifically, T2A24 cells were pulsed with 10, 1, 0.1, 0.01, or 0.001 micromolar concentrations of PRAME p301-309, 10 micromolar CMV, and DMSD. #98 CAR T cells were co-cultured for 6 hours. PEcy7-labeled CD8+ RPE-labeled tetramer+ CAR T cells were stained with APC-labeled anti-IFNg antibody and the CAR T cells were measured using FACS CANT. The IC50 was approximately 10 nM, demonstrating sufficient recognition activity (Figure 10).

[0107] Test Example 7. Specific recognition of A24+PRAME+ target cells by #98 zG CAR T cellsThe target cells used in this study were analyzed for PRAME expression. Specifically, total RNA was extracted from cultured cells using an RNA extraction kit (Promega) and then reverse-transcribed. RT-PCR was performed using PRAME (Hs01022301_m1) and GAPDH CONTROL MIX (REF 4325792), TaqMan gene expression assay reagents sold by Applied Biosystems. PRAME expression levels were normalized with those of GAPDH.

[0108] Furthermore, the HLA-A24 expression of the target cells used in this study was analyzed as follows. b) T2A24, SK-MEL-124, and NW-MEL-38 were stained with Bulk Monoclonal Antibody A23,24 IgG2b (ONE LAMBDA.INC) as the primary antibody and Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen) as the secondary antibody. Expression was measured by flow cytometry.

[0109] The results are shown in Figure 11a and b. A24-transfected NW-MEL-38 and SK-MEL-124 express HLA-A24 and PRAME and are positive targets. On the other hand, NW-MEL-38 is a negative target because it does not express HLA-A24. T2A24 cells do not express PRAME.

[0110] Retrovirally transduced CAR-T cells were co-cultured with peptide-pulsed target cells for 24 hours, and IFN-γ levels in the culture supernatant were measured. IFN-γ production was specifically increased in PRAME-pulsed LCL cells. IFN-γ production was not observed when co-cultured with the negative control, CMV-pulsed T2A24 cells (Figure 12). Furthermore, tumor-specific IFN-γ production was observed in the target cells, A24-positive, PRAME-positive SK-MEL-124 and A24-transduced NW-MEL-38, which was clearly different from the off-target NW-MEL-38 (Figure 12). Therefore, we confirmed that retroviral vector-mediated CAR gene transduction resulted in CAR expression, tumor-specific recognition, and IFN-γ production.

[0111] Test Example 8. Specific recognition of target cancer cells by CAR-T cells transfected with PRAME#98 CAR IFNg production was observed when #98 CAR T cells were co-cultured with PRAMEp301-309 peptide-pulsed T2A24 cells, indicating recognition of positive targets. On the other hand, IFNg production was not observed when co-cultured with CMV peptide-pulsed or unpulsed T2A24 cells, indicating no recognition of these negative targets (Figure 12).

[0112] Next, we investigated the recognition of cultured cancer cells. Figure 11a shows PRAME expression in the cultured cells used, and Figure 11b shows HLA-A24 expression. NW-MEL-38-HLA-A24 (NW-MEL-38 cells were retrovirally transfected with the HLA-A24 gene, resulting in forced expression of HLA-A24; PRAME-positive, HLA-A24-positive) and SK-MEL-124 (PRAME-positive, HLA-A24-positive) were positive target cells, whereas NW-MEL-38 (PRAME-positive, HLA-A24-negative) was negative target cells. As shown in Figure 12, #98 CAR T cells produced IFNg when cocultured with positive cells, but not when cocultured with negative cells. This demonstrated specific recognition of target cells.

[0113] Test Case 9. PRAME#98 Specific recognition of target cancer cells by CAR-T cells transfected with CAR NW-MEL-38-HLA-A24 (NW-MEL-38 cells retrovirally transfected with the HLA-A24 gene, resulting in forced expression of HLA-A24; PRAME-positive, HLA-A24-positive) and SK-MEL-124 (PRAME-positive, HLA-A24-positive) were plated on E-plates as positive target cells, and NW-MEL-38 (PRAME-positive, HLA-A24-negative) was plated as negative target cells. After 24.5 hours, #98 CAR T cells were added as effector cells, and the change in the xCelligence index was observed. Specifically, the procedure is as follows.

[0114] Seven thousand negative target cells, NW-MEL-38 (A24-PRAME+), were cultured on E-plates for 24.5 hours, after which 100,000 cells of each effector cell line were added and cultured. The cell index was monitored over time. The cell index reflects the number of A24 NW-MEL-38 cells on the E-plate. The normalized cell index was calculated by setting the number of NW-MEL-38 cells immediately before co-culture with effector cells as 1. The results are shown in Figure 13a. The graph shows the average (n = 3). Cytotoxicity by #98 CAR T cells was minimal.

[0115] Seven thousand HLA-A24-transduced NW-MEL-38 cells (A24+PRAME+) were cultured on E-plates for 24.5 hours, after which 100,000 cells of each effector cell line were added and cultured. The cell index was monitored over time. The cell index reflects the number of A24 NW-MEL-38 cells on the E-plate. The normalized cell index was calculated by setting the number of A24 NW-MEL-38 cells immediately before co-culture with effector cells as 1. The results are shown in Figure 13b. The graph shows the mean (n = 3). Cytotoxicity was observed with #98 zG CAR T cells, but not with CD19 zG CAR T cells or PBMCs without CAR transfection.

[0116] After 7,000 SK-MEL-124 cells (A24+PRAME+) were cultured on E-plates for 24.5 hours, 100,000 cells of each effector cell line were added and cultured, and the cell index was monitored over time. The cell index reflects the number of SK-MEL-124 cells on the E-plate. Normalized cell index was calculated by setting the number of A24 NW-MEL-38 cells immediately before co-culture with effector cells as 1. The results are shown in Figure 13c. The graph shows the mean (n = 3). Cytotoxicity was observed in #98 zG CAR T cells, but not in PBMCs without CD19 zG or CAR transfection.

[0117] As shown in Figure 13a, b, and c, cytotoxicity was observed against NW-MEL-38-HLA-A24 and SK-MEL-124, which are A24+PRAME+, but not against NW-MEL-38, which is A24-. Therefore, the cytotoxicity of #98 CAR T cells was demonstrated to be specific to the positive target cells.

[0118] Test Case 10. #98 zG CAR T Cell Tumor Suppression 1 NOG mice lack common gamma receptors and therefore lack NK cells. This significantly increases the engraftment rate of human cells and tissues compared to NOD-SCID mice, allowing for the efficient engraftment of human cancers and normal tissues. Furthermore, the differentiation of human T cells after human hematopoietic stem cell transplantation (HSCT) is observed, making them increasingly popular as a model mouse for the human immune system. NOG mice are characterized by the absence of T and B cell deficiencies, natural killer (NK) cell deficiencies, reduced dendritic cell function, reduced macrophage function, and lack of complement activity, as well as the absence of T and B cell leakage associated with aging. NOG mice were transplanted with NW-MEL-124 (A24-positive, PRAME-positive) cells at 4×10^6 cells / mouse into the right flank. On day 7, anti-lambda antibody-sorted PRAME#98 CAR-T cells were infused via the tail vein at 8×10^6 cells / mouse. The specific procedure is as follows (outlined in Figure 14a). Seven-week-old female NOG mice were purchased and acclimated for one week, and then subcutaneously transplanted with 4x10^6 SK-MEL-124 cells. Effector cells were reacted with anti-lambda antibodies, followed by RPE-labeled anti-rabbit antibodies, and sorted using anti-PE beads. The purified effector cells were then infused intravenously 7 days after target cell inoculation. Control mice were used without any infusion.

[0119] Gene transfer into CAR-T cells was performed according to the method in Test Example 8. The CD8 and CD4 abundance ratios were 95.2% and 2.1%, respectively, and the CAR-positive rates were 97% for CD8 and 98.8% for CD4.

[0120] The experiment was conducted in a CAR-T cell infusion group (n=3) and an untreated group (n=3), and tumor diameters were measured every 3-4 days up to 50 days after tumor inoculation.

[0121] As a result, in the CAR-T cell infusion group, suppression of SK-MEL-124 cell (A24-positive, PRAME-positive) proliferation was observed in three mice (Figure 14b). In contrast, cancer growth was observed in the untreated group. Therefore, the antitumor effect of the infused cells observed in this study is likely due to the #98 CAR T cells. The mice did not experience any particular weight loss, and no significant GVHD effects were observed (Figure 14c).

[0122] Test Case 11. #98 zG CAR T Cell Tumor Suppression 2 Test Example 10. Tumor Suppression by #98 zG CAR T Cells. NOG mice lack common gamma receptors and therefore lack NK cells. This allows for significantly higher engraftment of human cells and tissues than NOD-SCID mice, enabling high rates of engraftment of human cancers and normal tissues. Furthermore, human T cell differentiation is observed after human hematopoietic stem cell transplantation, leading to increased demand for NOG mice as a model mouse for the human immune system. NOG mice are characterized by the absence of T cell and B cell deficiencies, natural killer (NK) cell deficiencies, reduced dendritic cell function, reduced macrophage function, and lack of complement activity, as well as the absence of T cell and B cell leakage associated with aging. SK-MEL-1 24 (A24-positive, PRAME-positive) cells were transplanted into the right flank of NOG mice at a rate of 4 x 10^6 cells per mouse. Ten days after transplantation, NGM and PRAME#98 CAR-T cells were infused via the tail vein at a cell count of 4x10^6 per mouse. Specifically, the procedure is as follows (outlined in Figure 15a). Seven-week-old female NOG mice were purchased and acclimated for three weeks, after which 4x10^6 SK-MEL-124 cells were subcutaneously transplanted. Ten days after target cell inoculation, the cells were infused intravenously. NGM was infused as a control.

[0123] Gene transfer into CAR-T cells was performed according to the method in Test Example 8. The presence of CD8 and CD4 was 91.0% and 6.5%, respectively, and the CAR-positive rate was 78.5% for CD8 and 56.9% for CD4.

[0124] Experiments were conducted in the CAR-T cell infusion group (n=3) and the NGM group (n=3), and tumor diameters were measured every 3-4 days up to day 46 after tumor inoculation.

[0125] As a result, in the CAR-T cell infusion group, suppression of SK-MEL-124 cell (A24 positive, PRAME positive) proliferation was observed in three mice (Figure 15b). On the other hand, cancer growth was observed in the NGM group. Therefore, the antitumor effect of the infused cells observed in this study is likely due to the #98 CAR T cells. After NGM and CAR-T cell infusion, orbital blood was collected on days 7, 14, and 23, and PBMCs were isolated. hCD45, CD8, and CD4 CAR were stained and analyzed by FACS. CAR-T cells were still present in the peripheral blood on day 23 (Figure 15c).

Claims

1. a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11; A PRAME-binding molecule comprising:

2. The PRAME-binding molecule of claim 1 , comprising the heavy chain variable region and the light chain variable region.

3. The PRAME-binding molecule according to claim 1 or 2, which has binding affinity to the HLA-A24 PRAMEp301-309 pMHC complex.

4. The PRAME-binding molecule according to any one of claims 1 to 3, wherein the binding affinity to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 19, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 20, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 21, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 23, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 24, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 25, and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 26 is half or less of the binding affinity to a peptide consisting of the amino acid sequence shown in SEQ ID NO:

17.

5. The PRAME-binding molecule of any one of claims 1 to 4, which is a chimeric antigen receptor.

6. The PRAME-binding molecule of claim 5, comprising an scFv domain comprising the heavy chain variable region and the light chain variable region, a transmembrane domain, and a core region comprising the intracellular domain of a TCR.

7. The PRAME-binding molecule of claim 6 , wherein the core region further comprises the intracellular domain of a costimulatory factor.

8. The PRAME-binding molecule according to any one of claims 1 to 4, which is an antibody.

9. A polynucleotide encoding the PRAME-binding molecule of any one of claims 1 to 8.

10. A cell containing the polynucleotide of claim 9.

11. A lymphocyte cell containing a polynucleotide encoding the PRAME-binding molecule according to any one of claims 5 to 7.

12. A pharmaceutical composition comprising the lymphocyte cells of claim 11 or the PRAME-binding molecule of claim 8.

13. The pharmaceutical composition described in claim 12, which is for treating or preventing cancer including cancer cells that express PRAME.

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