Prame binding molecules

Abstract

To provide PRAME-binding molecules having moderate affinity for HLA-A24PRAMEp301-309pMHC complexes and high recognition specificity.SOLUTION: PRAME binding molecules comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain variable region comprising the heavy chain CDR3, each comprising a specific amino acid sequence, and / or a light chain CDR1 comprising a specific amino acid sequence, a light chain CDR2 comprising the amino acid sequence GKN, and a light chain variable region comprising the light chain CDR3, each comprising a specific amino acid sequence.SELECTED DRAWING: None

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. In adult normal tissues, it is expressed only to a limited 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%), making it a promising target for cancer therapy. Target peptides known to be expressed include p100-108, p142-151, p300-309, p425-433, and p435-443 for HLA-A2, while p301-309 for HLA-A24. T cells that recognize pMHC have been cloned, and T cell receptors (TCRs) have been isolated and their functions analyzed; however, no TCRs capable of cancer therapy have yet been isolated.

[0003] Under these circumstances, Patent Document 1 reports an antibody that recognizes the HLA-A24 PRAMEp301-309 pMHC complex, and a lymphocyte that extracellularly presents the variable region of this antibody. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2022 / 124282 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have found that lymphocytes extracellularly displaying the variable regions of conventional antibodies that recognize the HLA-A24 PRAMEp301-309 pMHC complex exhibit low proliferation, which is believed to be due to the antibody's excessively high affinity (too low a binding constant) and insufficient recognition specificity.

[0006] Therefore, an objective of the present invention is to provide a PRAME-binding molecule that has moderate affinity and high recognition specificity for the HLA-A24 PRAMEp301-309 pMHC complex. [Means for solving the problem]

[0007] The present inventors have conducted extensive research in light of the above-mentioned problems and have 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 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 GKN, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10. Further research based on this finding has led to the completion of the present invention. That is, the present invention encompasses the following aspects.

[0008] 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 GKN, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10; A PRAME-binding molecule comprising:

[0009] Item 2. The PRAME-binding molecule according to Item 1, comprising the heavy chain variable region.

[0010] Item 3. The PRAME-binding molecule according to Item 1 or 2, comprising the heavy chain variable region and the light chain variable region.

[0011] Item 4. The PRAME-binding molecule according to any one of Items 1 to 3, which has binding affinity to an HLA-A24 PRAMEp301-309 pMHC complex.

[0012] Item 5. The PRAME-binding molecule according to any one of Items 1 to 4, wherein the binding affinity to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 16, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 17, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 18, 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: 22, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 23, and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 24 is half or less of the binding affinity to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 15.

[0013] Item 6. The PRAME-binding molecule according to any one of Items 1 to 5, which is a chimeric antigen receptor.

[0014] Item 7. The PRAME-binding molecule according to Item 6, 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.

[0015] Item 8. The PRAME-binding molecule according to any one of Items 1 to 5, which is a hybrid TCR comprising polypeptide A comprising the light chain variable region and a TCR first subunit constant region, and polypeptide B comprising the heavy chain variable region and a TCR second subunit constant region.

[0016] Item 9. The PRAME-binding molecule according to any one of Items 1 to 5, which is an antibody.

[0017] Item 10. A polynucleotide encoding the PRAME-binding molecule according to any one of Items 1 to 9.

[0018] Item 11. A cell containing the polynucleotide according to Item 10.

[0019] Item 12. A lymphocyte cell containing a polynucleotide encoding the PRAME-binding molecule according to any one of Items 6 to 8.

[0020] Item 13. A pharmaceutical composition comprising the cells of Item 11, the lymphocytes of Item 12, or the PRAME-binding molecule of Item 9.

[0021] Item 14. The pharmaceutical composition according to Item 13, which is used to treat or prevent cancer. [Effects of the Invention]

[0022] According to the present invention, it is possible to provide a PRAME-binding molecule that has moderate affinity and high recognition specificity for the HLA-A24 PRAMEp301-309 pMHC complex. [Brief explanation of the drawings]

[0023] [Figure 1] The results of ELISA testing of antibody X 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 the background (median) level. The vertical axis shows the relative color intensity, and the horizontal axis shows the HLA-A24-associated pMHC used. [Figure 2] 1 shows the results of affinity measurement for antibody X by the SPR method (Test Example 2). [Figure 3] 1 shows the results of an alanine substitution study (Test Example 3). 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 4]A: A schematic diagram of a retroviral plasmid vector for CAR introduction (Test Example 4). B: A schematic diagram of a retroviral plasmid vector for hybrid TCR introduction (Test Example 4). [Figure 5] 1 shows the FACS results of CAR / hybrid TCR-transduced cells (Test Example 4). The transduced gene is shown above the graph, and "Untransduced" indicates that the gene was not transduced. [Figure 6]

[0039] Figure 5 shows the results of alanine substitution studies on CAR / hybrid TCR-transduced cells (Test Example 5). The horizontal axis shows the substitution site in the peptide used and the amino acid after substitution, and the vertical axis shows the IFNγ concentration. The transduced gene is shown above the graph, and "Untransduced" indicates no gene transduced. [Figure 7] 1 shows the results of measuring IFNγ concentration by ELISA in Test Example 6. The vertical axis shows IFNγ concentration, and the horizontal axis shows target cells. Effector cells are shown below the horizontal axis. A shows the results when CAR-T cells were used, and B shows the results when hybrid T cells were used. [Figure 8] The graph shows the results of observing the change in the xCelligence index after applying each effector cell to target cells (SK-MEL-124 cells (A24+PRAME+)). The vertical axis shows the number of SK-MEL-124 cells, normalized to 1 as the number of SK-MEL-124 cells immediately before co-culture with effector cells. The horizontal axis shows the time elapsed since the start of culture. The legend indicates the effector cells. The graph shows the mean (n=3, ±SD). [Figure 9] The figure shows the results of observing the change in index due to xCelligence when each effector cell was applied to target cells (SK-MEL-128 (PRAME+A24+)). The legend to the figure is the same as for Figure 8. [Figure 10] The figure shows the results of observing the change in index due to xCelligence when each effector cell was applied to target cells (NW-MEL-38 (A24-PRAME+)). The legend to the figure is the same as for Figure 8. [Figure 11]The figure shows the results of observing the change in index due to xCelligence when each effector cell was applied to target cells (SK-MEL-37 (PRAME+A24-)). The legend to the figure is the same as for Figure 8. [Figure 12] This shows the results of measuring the cell count in Test Example 8. The horizontal axis shows the time elapsed since CAR / hybrid TCR transduction (retroviral infection). The legend shows the transduced CAR / hybrid TCR transgene, and "Untransduced" indicates that the gene was not transduced. [Figure 13] 1 shows the results of measuring cell viability in Test Example 8. The transduced CAR / hybrid TCR gene is shown above the graph, and "Untransduced" indicates no gene transduction. The percentage within the graph indicates cell viability. [Figure 14] This shows the results of the co-culture test in Test Example 9. TG indicates target cells, EF indicates effector cells, and dead indicates dead cells. The above the graph shows the introduced CAR / hybrid TCR gene and the proportion of each cell type. DETAILED DESCRIPTION OF THE INVENTION

[0024] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0025] "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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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 GKN, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10. This is described below.

[0031] 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 GKN, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10, and is a molecule that has binding ability to PRAME.

[0032] 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.).

[0033] 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 3). 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 X in the Examples described below.

[0034] The PRAME-binding molecule of the present invention preferably comprises the above-mentioned heavy chain variable region, and more preferably comprises both the above-mentioned heavy chain variable region and the above-mentioned light chain variable region.

[0035] 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: 11 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: 11. When there is an amino acid mutation from SEQ ID NO: 4 or 11, the mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution.

[0036] 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: 15). 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.

[0037] The PRAME-binding molecule of the present invention can specifically recognize PRAMEp301-309 (SEQ ID NO: 15). From this perspective, it is preferable that the PRAME-binding molecule of the present invention has binding affinity to at least one (preferably, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine (all)) types of peptides formed by partial mutation of PRAMEp301-309 (a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 16, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 18, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 19, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 20, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 21, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 22, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 23, and a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 24) that is 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 its binding affinity to PRAMEp301-309 (SEQ ID NO: 15).

[0038] 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 group (-COO), or a carboxyl group (-COOH). - ), 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-naphthyl 6-12 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 7-14An 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).

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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").

[0043] The antibody of the present invention is a monoclonal antibody.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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).

[0059] 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.

[0060] 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.

[0061] 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").

[0062] 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.

[0063] 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 GKN, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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).

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Hybrid TCR In a preferred embodiment, the PRAME-binding molecule of the present invention is a hybrid TCR comprising polypeptide A comprising the light chain variable region and the first subunit constant region of a TCR, and polypeptide B comprising the heavy chain variable region and the second subunit constant region of a TCR.

[0074] TCR is a heterodimer consisting of a first subunit and a second subunit. Examples of subunits that constitute TCR include an α chain, a β chain, a γ chain, and a δ chain. The first and second subunits in the hybrid TCR of the present invention are not particularly limited as long as they constitute a TCR as a heterodimer. Examples include a combination in which the first subunit is an α chain and the second subunit is a β chain, a combination in which the first subunit is a β chain and the second subunit is an α chain, a combination in which the first subunit is a γ chain and the second subunit is a δ chain, and a combination in which the first subunit is a γ chain and the second subunit is a δ chain. Among these, particularly preferred are a combination in which the first subunit is an α chain and the second subunit is a β chain, and a combination in which the first subunit is a β chain and the second subunit is an α chain.

[0075] The constant region of the first / second subunit is a portion other than the variable region of the first / second subunit (a region containing CDRs and whose amino acid sequence differs depending on the type of antigen), and is not particularly limited as long as it has the function of the constant region of the first / second subunit (e.g., the function of penetrating the cell membrane and positioning the variable region of the first / second subunit in the appropriate orientation on the cell surface).

[0076] The light chain variable region is usually located N-terminal to the first subunit constant region, and the heavy chain variable region is usually located N-terminal to the second subunit constant region.

[0077] The light chain variable region and the first subunit constant region may be linked directly or indirectly (for example, via another sequence such as a linker).

[0078] 3. Polynucleotides In one aspect, the present invention relates to a polynucleotide encoding a PRAME-binding molecule of the present invention (sometimes referred to herein as a "polynucleotide of the present invention"). This will be explained below.

[0079] 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, lentivirus, 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.

[0080] 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.

[0081] 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.

[0082] 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.).

[0083] When the cell of the present invention comprises a polynucleotide encoding the chimeric antigen receptor of the present invention or the hybrid TCR, 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 or the hybrid TCR, and in a more specific embodiment, the lymphocyte expresses the chimeric antigen receptor of the present invention or the hybrid TCR on its cell membrane, preferably expressing the chimeric antigen receptor of the present invention or the hybrid TCR with the PRAME-binding region exposed outside the cell membrane.

[0084] Lymphocyte cells expressing chimeric antigen receptors or hybrid TCRs recognize PRAME at the PRAME-binding region, then transmit the recognition signal to the inside of T cells, etc., 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.

[0085] 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 regions bind to PRAME, similar to chimeric antigen receptor T cells. Therefore, host cells (particularly host cells with cytotoxic activity) containing a polynucleotide encoding a chimeric antigen receptor or hybrid TCR are useful as an active ingredient in pharmaceutical compositions.

[0086] 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.

[0087] 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.

[0088] 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") comprising a cell containing a polynucleotide encoding a PRAME-binding molecule of the present invention, a lymphocyte cell containing a polynucleotide encoding a chimeric antigen receptor or the hybrid TCR of the present invention, or an antibody of the present invention. This is described below.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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 μg / 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]

[0097] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0098] Test Example 1: Isolation and evaluation of antibody X that recognizes HLA-A24-PRAME Antibody screening was performed according to the method described in Test Example 1 of Patent Document 1. The outline is as follows: Using a self-made human antibody library, antibody library screening was performed using an artificially created HLA-A24 PRAMEp301-309 (SEQ ID NO: 15) pMHC complex (hereinafter abbreviated as A24-PRAME) as the target. After several hundred clones were selected, culture supernatants were prepared and ELISA was performed on the resulting scFvCL-CP3. Several dozen antibodies that reacted with the positive target A24-PRAME but did not react 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, and finally one antibody (antibody X) was selected.

[0099] To examine the binding specificity of antibody X, ELISA was performed using multiple peptide-HLA-A24 complexes (pMHC). Each of the following pMHC monomers was immobilized: HLA-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. ELISA was performed with selected antibodies. Figure 1 shows the ELISA results. Antibody X demonstrated high specificity, with no cross-recognition observed.

[0100] The amino acid sequence of antibody X and the nucleotide sequence encoding the antibody were analyzed as follows. The CDR sequences were predicted using IMGIT.

[0101] <Heavy Chain> Heavy chain CDR1 amino acid sequence: GFTFSSYA (SEQ ID NO: 1) Heavy chain CDR2 amino acid sequence: ISGGGGST (SEQ ID NO: 2) Heavy chain CDR3 amino acid sequence: AKWRSAYGSTNYYYYGMDV (SEQ ID NO: 3) Heavy chain variable region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWRSAYGSTNYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 4) Heavy chain CDR1 base sequence: GGATTCACCTTTAGCAGCTATGCC (SEQ ID NO: 5) Heavy chain CDR2 base sequence: ATTAGTGGTGGTGGTGGTAGCACA (SEQ ID NO: 6) Heavy chain CDR3 base sequence: GCGAAATGGCGTAGTGCTTATGGTTCAACAAACTACTACTACTACGGTATGGACGTC (SEQ ID NO: 7) Heavy chain variable region nucleotide sequence: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTGGTGGTGGTAGCACATACTACGCAGACTC TGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATGGCGTAGTGCTTATGGTTCAACAAACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCA (SEQ ID NO: 8).

[0102] <Light chain> Light chain CDR1 amino acid sequence: SLRKYY (SEQ ID NO: 9) Light chain CDR2 amino acid sequence: GKN Light chain CDR3 amino acid sequence: NSRDRGSNQVV (SEQ ID NO: 10) Light chain variable region amino acid sequence: SSELTQDPAVSVALGQTVTITCQGDSLRKYYASWYQQKPGQAPVFVFYGKNNRPSGIPDRFSGSSSGDTASLTITGAQAEDEADYYCNSRDRGSNQVVFGGPTRLTVL (SEQ ID NO: 11) Light chain CDR1 base sequence: AGTCTGAGAAAATATTAT (SEQ ID NO: 12) Light chain CDR2 sequence: GGTAAAAAT Light chain CDR3 base sequence: AATTCCCCGCGACAGGGGTAGTAACCAGGTGGTA (SEQ ID NO: 13) Light chain variable region nucleotide sequence: TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCACCATCACATGCCAAGGAGACAGTCTGAGAAAATATTATGCAAGTTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTGTTTGTCTTCTATGGTAAAAATAATCGGCC CTCCGGaATCCCAGACCGgTTCTCTGGCTCCAGCTCAGGAGACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAATTCCCGCGACAGGGGTAGTAACCAGGTGGTATTCGGCGGACCGACCAGGCTGACCGTCCTA (SEQ ID NO: 14).

[0103] Test Example 2: Measurement of binding constant of antibody X scFvCL-pp Next, antibody X scFvCL-cp3 was converted to the pp form to produce antibody X scFvCL-pp, and the KD value was measured using BiacoreX100. The KD value in Biacore was determined by measuring the dynamic change in detection sensitivity (resonance, reflecting mass changes 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, the procedure is as follows.

[0104] Each scFv-cp3 expression plasmid was digested with the restriction enzyme SalI and then self-ligated. This was then used to transform E. coli DH12S to obtain a clone producing scFv-pp. The clone was then cultured in a medium containing IPTG, and the supernatant was collected and concentrated with ammonium sulfate. The supernatant was purified with IgG Sepharose 6 Fast Flow and dialyzed against PBS, after which the protein concentration was measured by SDS-PAGE.

[0105] 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 (31.25 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM) were sequentially reacted, and association and dissociation were measured. A global fit was performed to calculate Kon, Koff, and KD.

[0106] The results are shown in Table 1 and FIG.

[0107] [Table 1]

[0108] Test Example 3. Specific recognition of A24-PRAME by antibody X scFvCL-cp3 LCL cells have high expression of MHC Class I, so when pulsed with a peptide, the peptide is trapped on HLA-A24 on the LCL cells and presented as pMHC. Therefore, antibody X was reacted with LCL cells pulsed with PRAME, CMV peptides, and DMSO, and sufficient recognition was confirmed by FACS.

[0109] Next, to investigate the amino acids involved in antibody recognition, peptides were prepared by substituting each amino acid in PRAME p301-309 (LYVDSLFFL, Table 2, SEQ ID NO: 15) with a different amino acid (alanine) (Table 2, SEQ ID NOs: 16-24). Because 2Y and 9L in PRAME p301-309 were predicted to be anchor amino acids, 2F, 2W, 9F, and 9I (Table 2, SEQ ID NOs: 25-28), which stabilize peptide binding, were also examined to examine the effects of peptide changes 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 μM. The resulting mixture was then reacted with antibody X scFvCL-cp3, followed by mouse anti-cp3 antibody and then Alexa488-labeled anti-mouse antibody, and analyzed using a FACS CANT.

[0110] [Table 2]

[0111] The results are shown in Figure 3. When alanine-substituted peptides were used, the MFI values ​​decreased in all cases, indicating that all nine amino acids of PRAME p301-309 are important for antibody X's recognition of PRAME p301-309.

[0112] Test Example 4. Construction of retroviral vector, transfection into PBMC, and expression study Retroviral gene transfer was performed to express constitutive CAR or hybrid TCR. Retroviral vectors carrying the CAR gene (PRAME X zG CAR gene) and hybrid TCR genes (PRAME X h-TCR HL, PRAME X h-TCR LH) were constructed using the VH and VL of antibody X (Figure 4). Retroviruses were then produced using Plat-A packaging cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers. 1x10^6 cells were stimulated in a 12-well plate (Thermo Fisher) coated with 2μg of OKT-3 (Invitrogen) and 10μg of Retronectin (Takara Bio) and cultured for 3 days in GT-T551 medium containing donor plasma. These cells were then infected twice with retroviruses carrying the PRAME X zG CAR gene, PRAME X h-TCR HL gene, or PRAME X h-TCR LH gene, and further cultured. On day 8 of culture, gene transfer efficiency was measured using PRAMEp301-309 / HLA-A*24:02 tetramer, APC / Cy-7-labeled anti-human CD8 antibody (Biolegend), and APC-labeled anti-human CD4 antibody (Bioregend), and activity was examined on day 11 of culture. The #98 zG CAR gene (Patent Document 1) was also tested in the same manner.

[0113] The results of measuring the gene transfer efficiency are shown in FIG.

[0114] Test Example 5. Specific recognition of A24-PRAME by CAR / hybrid TCR-transfected cells 1x10 T2A24 cells pulsed with the peptide used in Test Example 3 5 cells and CAR / hybrid TCR-transduced cells (Test Example 4) 1 x 10 5 The cells were co-cultured in a 96-well plate (Thermo Fisher) for 24 hours, and 30 μL of the resulting supernatant was measured by IFNγ ELISA.

[0115] The results are shown in Figure 6. It was found that the CAR / hybrid TCR-introduced cells maintained the same recognition specificity as in Test Example 3.

[0116] Test Example 6. Specific recognition of A24+PRAME+ target cells by CAR / hybrid TCR cells SK-MEL-124 cells were PRAME+A24+ positive target cells, and NW-MEL-38 cells were PRAME+A24- negative target cells. T2A24 cells reacted with 10 μM PRAME peptide were positive target cells, and T2A24 cells reacted with CMV peptide were negative target cells. Effector cells were generated by retroviral transduction of PRAME X zG CAR into peripheral blood cells from healthy volunteers. 5 x 10^4 target cells and 5 x 10^4 effector cells were mixed and cultured in a 96-well plate (Thermo Fisher) for 24 hours. The supernatant was collected and subjected to ELISA testing using ELISA Flex Human IFN-g (HRP) (MabTECH).

[0117] The results are shown in Figure 7A. PRAME X zG CAR T cells produced IFNγ when co-cultured with positive cells, but not when co-cultured with negative cells, demonstrating specific recognition of target cells.

[0118] In addition, 2x10^4 of #98 h-TCR, X h-TCR LH, and X h-TCR HL were used as effectors and mixed with 2x10^4 of positive target cells 888mel cells (PRAME+A24+) or 2x10^4 of negative target cells HCT116 (PRAME+A24-), and co-cultured for 20 hours in a 96-well U plate (Thermo Fisher). 10 ul of the resulting culture supernatant was used in an ELISA assay using ELISA Flex human IFNg (HRP) (MabTECH).

[0119] The results are shown in Figure 7B. Recognition of positive target cells was confirmed for #98 H-TCR and X h-TCR HL.

[0120] Test Example 7. Continuous cytotoxicity by CAR / hybrid TCR-transduced cells SK-MEL-124 cells (A24+PRAME+), SK-MEL-128 (PRAME+A24+), NW-MEL-38 (A24-PRAME+), and SK-MEL-37 (PRAME+A24-), which contain less PRAM EpMHC than SK-MEL-124, were cultured on E-plates for 21–23 hours, and then effector cells were added and cultured. The cell index was tracked over time. Normalized cell index was calculated by setting the cell count immediately before co-culture with effector cells as 1.

[0121] The results are shown in Figures 8 to 11. Cytotoxicity was observed in CAR / hybrid TCR-introduced cells, but no cytotoxicity was observed in PBMCs without CAR / hybrid TCR transfection.

[0122] Test Example 8. Proliferation and viability of CAR / hybrid TCR-transduced cells 1x10^5 PBMC cells stimulated with anti-human CD3 antibody (Invitrogen) were infected with the retrovirus carrying the CAR / hybrid TCR gene, and cell numbers were measured 4 and 7 days later. The results showed that the proliferation of #98 zG CAR gene-transduced T cells was slower than that of non-transduced cells, whereas the proliferation of PRAME X CAR / hybrid TCR gene-transduced T cells was equivalent to that of non-transduced cells (Figure 12).

[0123] In addition, 7 days after infection, the cells were analyzed for FSC and SSC using a BD LSR Fortessa X-20 to analyze their viability. The #98 zG CAR gene-transfected T cells showed an increase in dead cells and a lower viability compared to non-transfected cells, whereas the viability of PRAME X CAR / hybrid TCR gene-transfected T cells was comparable to that of non-CAR gene-transfected cells (Figure 13).

[0124] Test Example 9. Co-culture test of target cells and effector cells prepared from different donors Target cells (NGMC) and effector cells (#98 zG CAR, X zG CAR, X h-TCR HL, NGMC) derived from different donors with HLA-A24 were prepared at 1x10^4 each and co-cultured for 20 hours in 10% FCS RPMI 1640 in a 96-well plate (Thermo Fisher). Target cells were stained with CytoTell Red 650 (AAT Bioquest). After co-culture, cells were stained with LiveDead Yellow (Thermo Fisher) and analyzed by flow cytometry using an LSRFortessa X-200 (BD). Cell analysis was performed by first separating target and effector cells using CytoTell Red 650, and then calculating the percentage of dead cells after co-culture using LiveDead staining.

[0125] The results are shown in Figure 14. It was found that the cell death rates of both target cells and effectors increased in #98zG CAR coculture compared to NGMC coculture, but the rate of target cell death did not increase in X-hybrid TCR.

Claims

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

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

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

4. The PRAME-binding molecule of claim 1, which has binding affinity to the HLA-A24 PRAMEp301-309 pMHC complex.

5. The PRAME-binding molecule of claim 1, whose binding affinity to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 16, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 17, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 18, 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: 22, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 23, and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 24 is less than half the binding affinity to a peptide consisting of the amino acid sequence shown in SEQ ID NO:

15.

6. The PRAME-binding molecule of claim 1 , which is a chimeric antigen receptor.

7. The PRAME-binding molecule of claim 6, 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.

8. The PRAME-binding molecule of claim 1, which is a hybrid TCR comprising polypeptide A comprising the light chain variable region and the first subunit constant region of a TCR, and polypeptide B comprising the heavy chain variable region and the second subunit constant region of a TCR.

9. The PRAME-binding molecule of claim 1 , which is an antibody.

10. A polynucleotide encoding the PRAME binding molecule of claim 1.

11. A cell containing the polynucleotide of claim 10.

12. A lymphocyte cell containing a polynucleotide encoding the PRAME-binding molecule according to any one of claims 6 to 8.

13. A pharmaceutical composition comprising the cells of claim 11, the lymphocyte cells of claim 12, or the PRAME-binding molecule of claim 9.

14. The pharmaceutical composition according to claim 13, which is for the treatment or prevention of cancer.

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