Patent Application
20240024480
The present invention relates to a PRAME-binding molecule and the like.
Preferentially expressed antigen in melanoma (PRAME) is known to be a cancer-testis antigen and is only slightly expressed in normal adult tissues in the endometrium, ovary, and adrenal glands, in addition to the testis. Known cancers with high expression of PRAME include melanoma (95% of patients), lung cancer (50%), breast cancer (27%), acute leukemia (30%), and multiple myeloma (52%) (Non-patent Literature (NPL) 1, 2, and 3), and PRAME is attracting attention as a target for cancer treatment. The peptides that are known to be used as the target include p100-108, p142-151, p300-309, p425-433, p435-443, and the like for HLA-A2, and p301-309 for HLA-A24. T cells that recognize pMHC have been cloned, T-cell receptors (TCRs) have been isolated, and their functions have been analyzed (NPL 4, 5, and 6). However, so far no TCR that can be used for cancer treatment has been isolated.
An object of the present invention is to provide a PRAME-binding molecule.
In view of the problem above, the present inventors attempted isolation of an antibody that recognizes an HLA-A24 PRAMEp301-309 pMHC complex and production of a CAR using the complex. As a result of further research, the inventors found that the problem can be solved by a PRAMS-binding molecule that comprises a heavy-chain variable region containing a heavy-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and/or a light-chain variable region containing a light-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9, a light-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10, and a light-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11. The inventors conducted further research on the basis of this finding and completed the present invention. Specifically, the present invention includes the following subject matter.
A PRAMS-binding molecule comprising
The PRAME-binding molecule according to Item 1, comprising the heavy-chain variable region and the light-chain variable region.
The PRAME-binding molecule according to Item 1 or 2, which has binding capability to an HLA-A24 PRAMEp301-309 pMHC complex.
The PRAME-binding molecule according to any one of Items 1 to 3, wherein the binding capability of the PRAME-binding molecule to a peptide consisting of the amino acid sequence represented by SEQ ID NO: 19, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 20, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 21, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 23, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 24, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 25, or a peptide consisting of the amino acid sequence represented by SEQ ID NO: 26 is equal to or less than ½ of the binding capability of the PRAME-binding molecule to a peptide consisting of the amino acid sequence represented by SEQ ID NO: 17.
The PRAME-binding molecule according to any one of Items 1 to 4, which is a chimeric antigen receptor.
The PRAME-binding molecule according to Item 5, comprising a core domain containing an scFv domain that contains the heavy-chain variable region and the light-chain variable region, a transmembrane domain, and an intracellular domain of TCR.
The PRAME-binding molecule according to Item 6, wherein the core domain further contains an intracellular domain of a co-stimulator.
The PRAMS-binding molecule according to any one of Items 1 to 4, which is an antibody.
A polynucleotide encoding the PRAMS-binding molecule according to any one of Items 1 to 8.
A cell comprising the polynucleotide according to Item 9.
A lymphocyte cell comprising a polynucleotide encoding the PRAME-binding molecule according to any one of Items 5 to 7.
A pharmaceutical composition comprising the lymphocyte cell according to Item 11 or the PRAME-binding molecule according to Item 8.
The pharmaceutical composition according to Item 12, which is for use in the diagnosis, treatment, or prevention of cancer.
The present invention provides a PRAMS-binding molecule. The PRAME-binding molecule of the present invention has specific CDR sequences. Thus, the PRAMS-binding molecule of the present invention has excellent binding capability to PRAMS and also has an excellent anticancer effect particularly when used as a chimeric antigen receptor.
In the present specification, the terms “comprising,” “containing,” and “including” include the concepts of comprising, containing, consisting essentially of, and consisting of.
The “identity” of amino acid sequences refers to the degree to which two or more contrastable amino acid sequences match each other. Thus, the higher the degree of match between two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, by using FASTA (a tool for sequence analysis) with default parameters. Alternatively, the level of amino acid sequence identity can be determined by using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. 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 SF. Applications and statistics for multiple high-scoring segments in molecular sequences, Proc Natl Acad Sci USA. 90: 5873-7(1993)). A program called “BLASTX,” based on this BLAST algorithm, has been developed. The specific techniques of these analysis methods are known and can be found on the website of the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). The “identity” of base sequences is also defined in the same manner as above.
In the present specification, “conservative substitution” means the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, the substitution between amino acid residues having a basic side chain such as lysine, arginine, and histidine is considered to be a conservative substitution. The following substitutions between other amino acid residues are also considered to be a conservative substitution: the substitution between amino acid residues having an acidic side chain, such as aspartic acid and glutamic acid; the substitution between amino acid residues having an uncharged polar side chain, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; the substitution between amino acid residues having a nonpolar side chain, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; the substitution between amino acid residues having a beta-branched side chain, such as threonine, valine, and isoleucine; and the substitution between amino acid residues having an aromatic side chain, such as tyrosine, phenylalanine, tryptophan, and histidine.
In the present specification, “CDR” is an abbreviation for complementarity determining region. CDR is a region in the variable regions of immunoglobulins and is deeply involved in the specific binding of an antibody to its antigen. The phrase “light-chain CDR” refers to a CDR present in the light-chain variable regions of immunoglobulins, and the phrase “heavy-chain CDR” refers to a CDR present in the heavy-chain variable regions of immunoglobulins.
In the present specification, the phrase “variable region” refers to a region containing CDR1 to CDR3 (simply “CDRs 1-3” below). The order in which these CDRs 1-3 are arranged is not limited; however, the variable region preferably refers to a region in which CDR1, CDR2, and CDR3 are arranged in this order in the direction from the N-terminus toward the C-terminus or in the reverse order either consecutively or via other amino acid sequences referred to as “framework regions” (FRs), which are described later. The phrase “heavy-chain variable region” refers to a region in which heavy-chain CDRs 1-3 are arranged, and the phrase “light-chain variable region” refers to a region in which light-chain CDRs 1-3 are arranged.
The regions other than CDRs 1-3 of each variable region are referred to as “framework regions” (FRs), as mentioned above. In particular, the region between the N-terminus and CDR1 of a variable region 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 a variable region as FR4.
In an embodiment, the present invention relates to a PRAME-binding molecule comprising a heavy-chain variable region containing a heavy-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; and/or a light-chain variable region containing a light-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9, a light-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10, and a light-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11 (which may be referred to as “the PRAMS-binding molecule of the present invention” in the present specification). The PRAME-binding molecule of the present invention is described below.
The PRAME-binding molecule of the present invention can be any PRAMS-binding molecule as long as the PRAME-binding molecule comprises a heavy-chain variable region containing a heavy-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and/or a light-chain variable region containing a light-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9, a light-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10, and a light-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11, and as long as the PRAME-binding molecule is capable of binding to PRAME.
The PRAME-binding molecule of the present invention may be a molecule formed of a single type of polypeptide or a molecule formed of a complex of two or more types of polypeptides. The PRAME-binding molecule of the present invention may also be a molecule formed of a polypeptide or of a complex of polypeptides, or a molecule formed of a polypeptide or complex of polypeptides to which another substance (e.g., a fluorescent substance, a radioactive substance, or an inorganic particle) is linked.
The binding capability to PRAME can be measured in accordance with a known method, such as by ELISA (specifically, for example, by the method of Test Example 2). The binding capability of the PRAME-binding molecule of the present invention to PRAME is, for example, at least 20%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding capability of #98 antibody to PRAME in the Examples described later, which is taken as 100%.
The PRAME-binding molecule of the present invention preferably contains both the heavy-chain variable region and the light-chain variable region.
The heavy-chain variable region is preferably a heavy-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence having at least 90% (preferably at least 95%, preferably at least 98%, preferably at least 99%) identity with the amino acid sequence represented by SEQ ID NO: 4. The light-chain variable region is preferably a light-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 12, or an amino acid sequence having at least 90% (preferably at least 95%, preferably at least 98%, preferably at least 99%) identity with the amino acid sequence represented by SEQ ID NO: 12. If the amino acid sequence of SEQ ID NO: 4 or 12 is mutated, the mutation is preferably a substitution of an amino acid, and more preferably a conservative substitution of an amino acid.
The PRAME-binding molecule of the present invention can have binding capability to a HLA-A24 PRAMEp301-309 pMHC complex. The HLA-A24 PRAMEp301-309 pMHC complex is a complex of HLA-A24 and a partial peptide of PRAMS (p301-309: SEQ ID NO: 17). The embodiment of the complex is not particularly limited as long as HLA presents the peptide as an antigen.
The PRAME-binding molecule of the present invention can specifically recognize PRAMEp301-309 (SEQ ID NO: 17). From this viewpoint, the binding capability of the PRAME-binding molecule of the present invention to at least one member selected from the group consisting of peptides in which part of PRAMEp301-309 is mutated (a peptide consisting of the amino acid sequence represented by SEQ ID NO: 19, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 20, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 21, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 23, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 24, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 25, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 26) (preferably, two members or more, three members or more, four members or more, five members or more, six members or more, or seven members (all)), is preferably ½ or less (preferably, ⅕ 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 capability of the PRAME-binding molecule of the present invention to PRAMEp301-309 (SEQ ID NO: 17).
The PRAMS-binding molecule of the present invention may be chemically modified. The polypeptide that constitutes the PRAME-binding molecule of the present invention may have a carboxyl group (—COOH), carboxylate (—COO—), amide (—CONH2), or ester (—COOR) at the C-terminus. “R” in the ester is, for example, a C1-6 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, or n-butyl; a C3-8 cycloalkyl group, such as cyclopentyl or cyclohexyl; a C6-12 aryl group, such as phenyl or α-naphthyl; a phenyl-C1-2 alkyl group, such as benzyl or phenethyl; a C7-14 aralkyl group, such as an α-naphthyl-C1-2 alkyl group, such as α-naphthyl methyl; or a pivaloyloxymethyl group. The polypeptide that constitutes the PRAMS-binding molecule of the present invention may have an amidated or esterified carboxyl group (or carboxylate), which is not the carboxyl group at the C-terminus. The ester in this case may be, for example, the esters of the C-terminus described above. The polypeptide that constitutes the PRAME-binding molecule of the present invention includes polypeptides having the amino group of the N-terminal amino acid residue protected by a protective group (e.g., a C1-6 acyl group, including a C1-6 alkanoyl, such as a formyl group and an acetyl group), polypeptides having the N-terminal glutamine residue pyroglutamated that can be formed due to cleavage in vivo; and polypeptides having a substituent (e.g., —OH, —SH, an amino group, an imidazole group, an indole group, and a guanidino group) on a side chain of an amino acid in the molecule protected by an appropriate protective group (e.g., a C1-6 acyl group, including a C1-6 alkanoyl group, such as a formyl group and an acetyl group).
The PRAMS-binding molecule of the present invention may have a protein or peptide (e.g., a known protein tag or signal sequence) added. Examples of protein tags include biotin, a His tag, a FLAG tag, a Halo tag, a MBP tag, a HA tag, a Myc tag, a V5 tag, a PA tag, and a fluorescent protein tag.
The PRAME-binding molecule of the present invention may be a pharmaceutically acceptable salt formed with an acid or base. The salt can be any pharmaceutically acceptable salt, and can be either an acid salt or a basic salt. Examples of acid 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 para-toluenesulfonate; and amino acid salts, such as aspartate and glutamate. Examples of basic salts include alkali metal salts, such as sodium salts and potassium salts; and alkaline-earth metal salts, such as calcium salts and magnesium salts.
The PRAME-binding molecule of the present invention may be in the form of a solvate. The solvent can be any pharmaceutically acceptable solvent, and may be, for example, water, ethanol, glycerol, or acetic acid.
In a preferable embodiment, the PRAMS-binding molecule of the present invention is an antibody (in the present specification, the PRAMS-binding molecule of the present invention being an antibody may be referred to as “the antibody of the present invention”).
The antibody of the present invention is a monoclonal antibody.
The antibody of the present invention can be of any molecular weight. The lower limit is, for example, 20,000, preferably 50,000, preferably 100,000, and more preferably 120,000. The upper limit is, for example, 1,000,000, preferably 500,000, and more preferably 200,000.
The antibody of the present invention may be of any structure. The antibody of the present invention may contain constant regions or no constant region. If the antibody of the present invention contains constant regions, the antibody of the present invention may contain all of the constant regions of the heavy chain (CH1, CH2, and CH3) and the constant regions of the light chain (CL), or any one or a combination of two or more constant regions of these constant regions.
Specific examples of the structure of the antibody of the present invention include immunoglobulins, Fab, F(ab′)2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, and tetrabody. Of these, an immunoglobulin is preferable from the standpoint of the effect of the present invention.
An immunoglobulin has a structure formed of a combination of two structures each of which is composed of a single heavy chain that contains a heavy-chain variable region and a heavy-chain constant region and a single light chain that contains a light-chain variable region and a light-chain constant region.
“Fab” contains a fragment of a heavy chain containing the heavy-chain variable region and CH1 in the heavy-chain constant region and a light chain containing the light-chain variable region and the light-chain constant region (CL), with the heavy-chain variable region and the light-chain variable region being aggregated by non-covalent intermolecular interaction described above, or bound to each other through a disulfide bond. In Fab, CH1 and CL may be linked through a disulfide bond between the thiol groups of the cysteine residues present in CH1 and CL.
“F(ab′)2” contains two pairs of Fabs, with CH1 of one Fab linked with CH1 of the other Fab through a disulfide bond between the thiol groups of their cysteine residues.
“Minibody” refers to the structure in which two fragments each containing CH3 bound to a heavy-chain variable region constituting scFV, described below, are aggregated between CH3 and CH3 by non-covalent intermolecular interaction.
“scFv-Fc” refers to the structure in which two antibody fragments each containing scFv, CH2, and CH3 are aggregated between CH3 and CH3 by non-covalent intermolecular interaction, as with the minibody, and the fragments are linked through a disulfide bond between thiol groups of the cysteine residues contained in each CH3.
“Fv” is considered to be the smallest structural unit of an antibody with the heavy-chain variable region and the light-chain variable region being aggregated by non-covalent intermolecular interaction. In Fv, the thiol group of the cysteine residue present in the heavy-chain variable region may be linked to the thiol group of the cysteine residue present in the light-chain variable region through a disulfide bond.
“scFv” has the structure in which the C-terminus of the heavy-chain variable region and the N-terminus of the light-chain variable region are bound through a linker, or the N-terminus of the heavy-chain variable region and the C-terminus of the light-chain variable region are bound through a linker, and is also referred to as a “single-chain antibody.”
The “diabody,” “triabody,” and “tetrabody” respectively refer to a dimer, a trimer, and a tetramer formed by scFv described above and are each aggregated and structurally stabilized, for example, by non-covalent intermolecular interaction of the variable regions, as with Fv.
If the antibody of the present invention is an immunoglobulin, its class is not particularly limited. The classes include, for example, IgA, IgD, IgE, IgG, and IgM, as well as subclasses of these classes. The class of the antibody of the present invention is, for example, IgG or IgM, preferably IgG, and more preferably IgG1.
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 be a chimeric antibody (e.g., an antibody formed by replacing the amino acid sequence of the constant region of an antibody derived from a non-human organism (e.g., a mouse) with the amino acid sequence of the constant region of a human-derived antibody), a humanized antibody, or a fully humanized antibody.
The antibody of the present invention can be produced, for example, by a method including culturing a host transformed with a polynucleotide encoding the antibody of the present invention, and collecting the fraction containing the antibody of the present invention.
The polynucleotide encoding the antibody of the present invention can be any polynucleotide that expressibly contains the sequence of the antibody of the present invention, and may contain other sequences in addition to the coding sequence of the antibody of the present invention. 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-resistant-gene-coding sequence that are located adjacent to the coding sequence of the antibody of the present invention. The polynucleotide encoding the antibody of the present invention may also be a linear polynucleotide or a cyclic polynucleotide (e.g., a vector).
Specific examples of polynucleotides include (I) polynucleotides containing a base sequence encoding at least one member selected from the group consisting of the heavy chain, the heavy-chain variable region, the heavy-chain CDR1, the heavy-chain CDR2, and the heavy-chain CDR3 of the antibody of the present invention, (II) polynucleotides containing a base sequence encoding at least one member selected from the group consisting of the light chain, the light-chain variable region, the light-chain CDR1, the light-chain CDR2, and the light-chain CDR3 of the antibody of the present invention, and (III) polynucleotides containing a base sequence encoding at least one member selected from the group consisting of the heavy chain, the heavy-chain variable region, the heavy-chain CDR1, the heavy-chain CDR2, and the heavy-chain CDR3 of the antibody of the present invention, and polynucleotides containing a base sequence encoding at least one member selected from the group consisting of the light chain, the light-chain variable region, the light-chain CDR1, the light-chain CDR2, and the light-chain CDR3 of the antibody of the present invention.
The host can be any organism, and is, for example, insect cells, eukaryotic cells, or mammal cells. Of these, mammal cells, such as HEK cells, CHO cells, NS0 cells, SP2/O cells, or P3U1 cells, are preferable from the standpoint of more efficiently expressing the antibody. The methods for transformation, culture, and collection are not particularly limited, and any method known in the field of antibody production can be used. After being collected, the antibody of the present invention may optionally be purified. Purification can be performed by a method known in the field of antibody production, such as chromatography or dialysis.
In a preferable embodiment, the PRAM-binding molecule of the present invention is a chimeric antigen receptor. (In the present specification, the PRAME-binding molecule of the present invention being a chimeric antigen receptor may be referred to as “the chimeric antigen receptor of the present invention.”)
The chimeric antigen receptor (CAR) is typically a chimeric protein that has its single-chain antibody (scFv) composed of a light chain (VL) bound in tandem to a heavy chain (VH) of the variable region of a monoclonal antibody at a position closer to the N-terminus as a domain responsible for its binding capability to an antigen and its T-cell receptor (TCR) chain at a position closer to the C-terminus. T cells expressing CAR are referred to as “CAR-T cells.”
The domain responsible for the binding capability to an antigen (PRAME) (PRAM-binding domain) in the chimeric antigen receptor of the present invention is not particularly limited as long as the domain contains a heavy-chain variable region containing a heavy-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and/or a light-chain variable region containing a light-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9, a light-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10, and a light-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11.
The PRAM-binding domain preferably has the structure of scFv. The linker that links the heavy-chain variable region with the light-chain variable region can be any linker that maintains functionality of the chimeric antigen receptor. The linker is preferably, for example, a linker composed of glycine alone or glycine and serine. The number of amino acid residues of the linker is, for example, 5 to 30, preferably 10 to 20, and more preferably 15.
The chimeric antigen receptor of the present invention typically contains a core domain containing an scFv domain having a heavy-chain variable region and a light-chain variable region, a transmembrane domain, and the intracellular domain of TCR. In general, in the core domain, the scFv domain, the transmembrane domain, and the intracellular domain of TCR are arranged in this order from the N-terminus directly or via other domains.
The transmembrane domain can be of any type that does not interfere with the functionality of the chimeric antigen receptor. For example, CD28, CD3zeta, CD4, or CD8alpha, which are expressed in cells such as T cells, can be used. These transmembrane domains may be mutated as long as the functionality of the chimeric antigen receptor is not interfered with.
The intracellular domain of TCR can be, for example, an intracellular domain derived from CD3, which is also called a “TCRζ chain.” CD3 may be mutated as long as the functionality of the chimeric antigen receptor is not interfered with. Mutation of CD3 is preferably made such that CD3 contains ITAM (immunoreceptor tyrosine-based activation motif).
The chimeric antigen receptor of the present invention preferably has a spacer sequence between the scFv domain and the transmembrane domain. The spacer sequence can be of any length and can be formed of any amino acid residues as long as the functionality of the chimeric antigen receptor is not interfered with. For example, the spacer sequence can be designed so as to have about 10 to 200 amino acid residues. The spacer sequence for use is preferably the sequence of the constant region of the light chain.
The core domain in the chimeric antigen receptor of the present invention preferably further contains the intracellular domain of a co-stimulator. The intracellular domain of a co-stimulator can be of any intracellular domain derived from a co-stimulator of cells such as T cells. For example, at least one member selected from the group consisting of OX40, 4-1BB, GITR, CD27, CD278, CD28, and the like can be suitably selected and used. The intracellular domain of these co-stimulators may be mutated as long as the functionality of the chimeric antigen receptor is not interfered with. The position of the intracellular domain of a co-stimulator is not particularly limited as long as the intracellular domain is at a position closer to the C-terminus of the transmembrane domain; the intracellular domain may be on the cell membrane side of the intracellular domain of TCR or on the side opposite to the cell membrane. In a preferable embodiment of the present invention, the intracellular domain of a co-stimulator is preferably placed on the side opposite to the cell membrane of the intracellular domain of TCR.
The chimeric antigen receptor of the present invention preferably contains a ligand domain, such as a GITRL domain, a 4-1BBL domain, or an ICOSL domain, at a position closer to the C-terminus of the core domain via a self-cleaving peptide domain. This can increase the expression efficiency of the chimeric antigen receptor or the cytotoxic activity of CAR-T cells containing the chimeric antigen receptor.
In the present specification, the phrase “self-cleaving peptide” refers to a peptide sequence with cleavage activity occurring between two amino acid residues in the peptide sequence. Examples of self-cleaving peptides include 2A peptides and 2A-like peptides. For example, in 2A peptides or 2A-like peptides, cleavage occurs between the glycine residue and the proline residue of these peptides. This occurs because of the “ribosomal skipping mechanism,” in which a normal peptide linkage between the glycine residue and the proline residue does not form during translation, and this does not affect the translation downstream. The ribosomal skipping mechanism is known in the art and is used in the expression of multiple proteins encoded by a single molecular messenger RNA (mRNA). The self-cleaving peptide for use in the present invention can be obtained from 2A peptides of viruses or 2A-like peptides that have equivalent functionality. For example, the self-cleaving peptide can be selected from the group consisting of 2A peptides derived from foot-and-mouth disease virus (FMDV) (F2A), 2A peptides derived from equine rhinitis A virus (ERAV) (E2A), 2A peptides derived from porcine teschovirus (PTV-1) (P2A), and 2A peptides derived from Thosea asigna virus (TaV) (T2A). The self-cleaving peptide domain may be mutated as long as the activity of the self-cleaving peptide domain is not greatly impaired.
The techniques for producing a chimeric antigen receptor and a CAR-T cell that expresses the chimeric antigen receptor are known. Chimeric antigen receptors and CAR-T cells can be produced in accordance with a known method or an equivalent method.
In an embodiment, the present invention relates to a polynucleotide encoding the PRAME-binding molecule of the present invention (which may be referred to as “the polynucleotide of the present invention” in the present specification). The polynucleotide of the present invention is described below.
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 expressibly contains the sequence of the PRAMS-binding molecule of the present invention. Other sequences include promoter sequences, enhancer sequences, repressor sequences, insulator sequences, origins of replication, reporter protein (e.g., fluorescent proteins) coding sequences, and drug-resistant-gene-coding sequences. The polynucleotide of the present invention may be a linear polynucleotide or a cyclic polynucleotide (e.g., a vector). The vector can be a plasmid vector or a virus vector (e.g., an adenovirus or retrovirus). The vector can also be, for example, a vector for cloning or for expression. The vector for expression includes vectors for prokaryotic cells, such as Escherichia coli or actinomycetes, and vectors for eukaryotic cells, such as yeast cells, insect cells, or mammal cells.
The polynucleotide of the present invention includes not only DNA and RNA but also known chemically modified DNA or RNA as described below. To prevent the degradation by hydrolases, such as nucleases, the phosphate residue (phosphate) of each nucleotide can be substituted with, for example, a chemically modified phosphate residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. The hydroxyl group at position 2 of the ribose of each ribonucleotide may also be substituted with —OR (R represents, for example, CH3(2′-O-Me), CH2CH2OCH3 (2′-O-MOE), CH2CH2NHC (NH) NH2, CH2CONHCH3, or CH2CH2CN). Additionally, the base moiety (pyrimidine, purine) may be chemically modified, by, for example, introduction of a methyl group or a cationic functional group into positon 5 of the pyrimidine base, or substitution of the carbonyl group at position 2 with thiocarbonyl. Additionally, the polynucleotide of the present invention also includes, but is not limited to, those formed by modifying the phosphate moiety or the hydroxyl portion, for example, by biotin, an amino group, a lower alkyl amine group, or an acetyl group. The term “polynucleotide” includes not only natural nucleic acids but also BNA (bridged nucleic acid), LNA (locked nucleic acid), and PNA (peptide nucleic acid).
In an embodiment, the present invention relates to a cell comprising the polynucleotide of the present invention (which may be referred to as “the cell of the present invention” in the present specification). The cell of the present invention is described below.
The cells from which the cell of the present invention is derived are not particularly limited. For the purpose of using the cell of the present invention in the production of the PRAME-binding molecule of the present invention, for example, cells that can be used for protein expression (e.g., insect cells, eukaryotic cells, mammal cells) can be used as the origin cells.
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 cell (e.g., T cells (e.g., CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, αβ-T cells, and γδ-T cells), NK cells, and NKT cells). The lymphocyte cell is preferably a cell expressing the chimeric antigen receptor of the present invention. In a more specific embodiment of the lymphocyte cell of the present invention, the chimeric antigen receptor of the present invention is expressed on the cell membrane, and preferably expressed in such a state that the PRAME-binding domain is exposed outside the cell membrane.
A lymphocyte cell or the like expressing the chimeric antigen receptor recognizes PRAMS in the PRAME-binding domain, and then intracellularly transfers a recognition signal to activate a signal that induces cytotoxic activity. In conjunction with this, the cell can mount attacks against other cells or tissues expressing PRAME, or exert cytotoxic activity.
When a cell exhibiting such a function is a CTL, this cell is called a “chimeric antigen receptor T-cell” (“CAR-T cell”). Cells that have potential to exhibit cytotoxic activity, such as NK cells, can also exert cytotoxic activity when the PRAME-binding domain binds to PRAME, as with the chimeric antigen receptor T-cell. Thus, a host cell comprising the polynucleotide encoding the chimeric antigen receptor (in particular, a host cell having cytotoxic activity) is useful as an active ingredient of pharmaceutical compositions.
Such lymphocyte cells or the like are useful for treatment or prevention of cancer or the like because they specifically recognize cancer tissue (tumor tissue). 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 lymphocytic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma), myeloproliferative diseases, myelodysplastic/myeloproliferative tumors (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndrome, acute myeloid leukemia, multiple myeloma, and the like. Examples of solid cancer include lung cancer, colorectal 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, bladder cancer, and the like.
The cell of the present invention can be obtained by introducing the polynucleotide of the present invention into cells. If necessary, the cell containing the polynucleotide of the present invention may be concentrated, or may be concentrated using a specific marker (CD antigen, such as CD8) as an indicator.
In an embodiment, the present invention relates to a pharmaceutical composition comprising the lymphocyte cell containing the polynucleotide encoding the chimeric antigen receptor of the present invention or comprising the antibody of the present invention (which may be referred to as “the pharmaceutical composition of the present invention” in the present specification). The pharmaceutical composition of the present invention is described below.
The content of the cell or antibody in the pharmaceutical composition can be appropriately set in consideration of the type of target disease (e.g., solid cancer), desired therapeutic effects, administration method, treatment period, patient's age, patient's body weight, etc. For example, the content of the antibody in the pharmaceutical composition may be about 0.001 parts by weight to 10 parts by weight, based on 100 parts by weight of the entire pharmaceutical composition. The content of the cell in the pharmaceutical composition may be, for example, about 1 cell/mL to 104 cells/mL.
The administration form of the pharmaceutical composition is not particularly limited as long as the desired effects are obtained. The pharmaceutical composition can be administered to mammals, including humans, by any of the following administration routes: oral administration and parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, dermal administration, and local administration). Since the active ingredient is a cell, the administration form is preferably parenteral administration, and more preferably intravenous injection. The dosage forms for oral administration and parenteral administration, and their production methods, are well known to a person skilled in the art. The pharmaceutical composition can be produced according to a usual method by, for example, mixing the antibody or cell of the present invention with a pharmaceutically acceptable carrier etc.
Examples of dosage forms for parenteral administration include injection preparations (e.g., intravenous drip infusion, intravenous injection, intramuscular injection, subcutaneous injection, and endodermic injection), external preparations (e.g., ointments, cataplasms, and lotions), suppositories, inhalants, eye drops, ophthalmic ointments, nasal drops, ear drops, liposome agents, and the like. For example, an injection preparation can be prepared by dissolving or suspending an antibody or cells in distilled water for injection, and optionally adding a solubilizer, a buffer, a pH adjuster, an isotonizing agent, a soothing agent, a preservative, a stabilizer, etc. The pharmaceutical composition can also be used as a freeze-dried preparation prepared before use.
The pharmaceutical composition may further comprise other drugs effective for the diagnosis, treatment, or prevention of diseases. The pharmaceutical composition can also contain components such as sterilants, antiphlogistics, cell activators, vitamins, and amino acids, if necessary.
As the carrier used for formulating the pharmaceutical composition, excipients, binders, disintegrators, lubricants, coloring agents, and flavoring agents that are generally used in this technical field can be used; and stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, antiseptics, antioxidants, extenders, moisturizers, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, and the like can also optionally be used.
The type of disease diagnosed, treated, or prevented using the pharmaceutical composition is not particularly limited as long as the diagnosis, treatment, or prevention can be achieved. Examples of specific target diseases include 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 lymphocytic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma), myeloproliferative diseases, myelodysplastic/myeloproliferative tumors (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndrome, acute myeloid leukemia, multiple myeloma, and the like. Examples of solid cancer include lung cancer, colorectal 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, bladder cancer, and the like.
The administration target (test subject) of the pharmaceutical composition is, for example, an animal having a disease described above or an animal with a potential to develop such a disease. A “potential to develop such a disease” can be determined by a known diagnostic method. The animal is, for example, a mammal, and preferably a human.
The dose of the pharmaceutical composition can be determined by a clinical physician, taking into consideration various factors, such as administration route, the type of disease, the degree of symptoms, patient's age, sex, and body weight, severity of disease, pharmacological findings, such as pharmacokinetics and toxicological characteristics, use or non-use of drug delivery system, and whether the composition is administered as part of a combinational drug with other medicinal agents. For example, when the active ingredient is the antibody, the dose of the pharmaceutical composition can be about 1 microgram/kg (body weight) to 10 g/kg (body weight) per day. When the active ingredient is the cell, the dose can be about 104 cells/kg (body weight) to 109 cells/kg (body weight). The administration schedule of the pharmaceutical composition can also be determined taking into consideration the same factors as those for the dose. For example, the composition can be administered once a day to once a month in the daily dose described above.
The present invention is described below in detail with reference to Examples; however, the present invention is not limited to these Examples.
Using a human antibody library made in-house, an antibody library screen targeting an artificially prepared HLA-A24 PRAMEp301-309 (SEQ ID NO: 17) pMHC complex (hereafter referred to as “A24-PRAMS”) was performed, and multiple antibodies that recognize A24-PRAMS were isolated. The method was performed according to the method described in NPL 7. Specifically, the antibodies were isolated using a phage display method. In this method, scFvCL is presented as part of phage coat protein cp3, and clones with high binding capability to the antigen can be selected by ELISA. After the magnet bead screen shown in
40 clones that reacted with A24-PRAMS, which is a positive target, and did not react with A24-CMV, which is a negative target, were selected, and their recognition for two concentrations of A24-PRAMS and one concentration of A24-CMV was examined by ELISA (
To examine the binding specificity of these antibodies, ELISA was performed using multiple peptide-HLA-A24 complexes (pMHCs). Each of the HLA-A24 pMHCs, i.e., A24-PRAME p301, PRAMS 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 using the selected antibodies.
Next, the recognition for PRAMEp301-309 peptide-pulsed A24-LCL cells was examined in order to investigate whether a pMHC complex formed between cell-expressed HLA-A24 and PRAMEp301-309 was recognized.
The amino acid sequence of antibody #98 and the base sequence encoding the antibody were analyzed. The analysis results are shown below. The sequences of the CDRs were deduced by IMGIT.
LCL cells have high expression of MHC-Class I. Thus, when LCL cells are pulsed with a peptide, the peptide is trapped on HLA-A24 on the LCL cells and presented as a pMHC. Thus, #98 was allowed to react with LCL cells pulsed with each of PRAME and CMV peptides and with DMSO, and sufficient recognition was confirmed by FACS.
Next, to investigate the amino acids involved in antibody recognition, peptides in which the amino acids of PRAME p301-309 (LYVDSLFFL, Table 1, SEQ ID NO: 17) were replaced one by one with other amino acids (mainly alanine) were prepared (Table 1, SEQ ID NOs: 2 to 10). Since 2Y and 9L were predicted to be anchor amino acids, 2F, 2W, 9F, and 9I (Table 1, SEQ ID NOs: 11 to 14), which stabilize peptide binding, were also examined, and antibody recognition changes due to peptide changes were examined. Specifically, wild type, DMSO, and peptides containing 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 2F, 2W, 9F, and 9I were prepared, A24-LCL was peptide-pulsed with each of them at a concentration of 10 micromolar, reacted with #98 scFvCL-cp3, then reacted with mouse-anti-cp3 antibody, and then reacted with Alexa488-labeled anti-mouse antibody, and measurement was performed using FACSCanto.
As a result, as shown in
Next, the KD value of #98 scFvCL-cp3 pp, which was generated by converting #98 scFvCL-cp3 to pp form, was measured using Biacore X100. The KD value was determined with Biacore by measuring dynamic changes in detection sensitivity (resonance, reflecting the change in mass on the chip) over time. The dissociation rate constant (Kd) and association rate constant (Ka) were determined from the dynamic change curve, and the association constant was determined from the ratio of the two constants. Specifically, each scFv-cp3 expression plasmid was cleaved with restriction enzyme SalI, subjected to self-ligation, and used to transform E. coli DH5a, thereby obtaining a clone that produces scFv-pp. The clone was cultured in medium containing IPTG, the supernatant was collected, concentrated with ammonium sulfate, purified with IgG Sepharose 6 Fast Flow, and dialyzed with PBS, and the concentration was estimated by SDS-PAGE. SPR measurement using Biacore X100 was performed using Biotin CAPture Kit (GE) according to the manufacturer's instructions. Specifically, first, A24 PRAMEp301-309 was immobilized on the sensor chip as a ligand. Next, five concentrations, i.e., 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, of scFv-pp were sequentially reacted, and association and dissociation were measured. Global fit was performed on this, and Kon, Koff, and KD were calculated. The measurement results revealed that the association constant of #98 scFvCL-pp was 5.3 nM (
Retroviral gene transfer was performed for constitutive CAR expression. CAR-T cells were produced by constructing a retroviral vector for CAR introduction (
T2A24 cells pulsed with each of the alanine scan peptides described in Test Example 2 were co-cultured with #98 zG CAR-T cells, and the amounts of IFNg secreted into the culture supernatants were measured by ELISA (
Further, Table 2 shows genes encoding amino acid sequences that differ from the amino acid sequence of PRAMEp301-309 by three amino acids in the BLAST searches. T2A24 was pulsed with these peptides, CMV, and DMSO, and co-cultured with #98 CAR-T cells. The amounts of IFNg secreted in the culture supernatants were measured by ELISA. Note that “e alone” indicates only effector cells (
T2A24 cells were pulsed with different concentrations of PRAME peptide and co-cultured with #98 zG CAR-T cells, intracellular staining of IFNg was performed, and the recognition by #98 zG CAR-T cells was measured. This is avidity. Specifically, T2A24 cells were peptide-pulsed with PRAME p301-309 at concentrations of 10, 1, 0.1, 0.01, and 0.001 micromolar, CMV at a concentration of 10 micromolar, and DMSD, reacted and co-cultured for 6 hours with #98 CAR-T cells, PEcy7-labeled CD8+ RPE-labeled tetramer+ CAR-T cells were stained with APC-labeled anti-IFNg antibody, and the reacted CAR-T cells were measured by FACSCanto. The IC50 was about 10 nM, and it was found that there was sufficient recognition activity (
PRAME expression in the target cells used in this test was analyzed. Specifically, total RNA was extracted from cultured cells using an RNA extraction kit (Promega), followed by reverse transcription reaction. PRAME (Hs01022301_m1) and GAPDH CONTROL MIX (REF 4325792), which are TaciMan gene expression assay reagents sold by Applied Biosystems, were used for RT-PCR. The expression level of PRAME was normalized by the expression level of GAPDH.
Further, the HLA-A24 expression in the target cells used in this test was analyzed. Specifically, T2A24, SK-MEL-124, and NW-MEL-38 were stained with Bulk Monoclonal Antibody A23, 24 IgG2b (One Lambda, Inc.) as a primary antibody and Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen) as a secondary antibody. Measurement was performed by flow cytometry.
Retrovirally transduced CAR-T cells were co-cultured with peptide-pulsed target cells for 24 hours, and IFN-γ in the culture supernatants was measured. IFN-γ production was increased specifically in PRAME-pulsed LCL cells. In co-culture with CMV-pulsed T2A24 cells, a negative control, no IFN-γ production was observed (
IFNg production was observed in co-culture of #98 CAR-T cells with PRAMEp301-309 peptide-pulsed T2A24 cells, indicating that the #98 CAR-T cells recognized the positive target. In contrast, IFNg production was not observed in co-culture with CMV peptide-pulsed T2A24 cells and T2A24 cells without peptide pulsing, indicating that the #98 CAR-T cells did not recognize these negative targets (
Next, recognition for cultured cancer cells was examined. PRAME expression in the cultured cells used is shown in
NW-MEL-38-HLA-A24 (NW-MEL-38 cells were retrovirally transduced with the HLA-A24 gene to forcibly express HLA-A24; PRAME-positive HLA-A24-positive) and SK-MEL-124 (PRAMS-positive HLA-A24-positive) as positive target cells, and NW-MEL-38 (PRAME-positive HLA-A24-negative) as negative target cells were seeded on E-plates. After 24.5 hours, #98 CAR-T cells were allowed to act as effector cells, and index changes were observed using xCelligence. The details are as follows.
After 7000 negative target cells NW-MEL-38 (A24-PRAME+) were cultured on an E-plate for 24.5 hours, each kind of effector cells (100000 cells) was added and cultured, and 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 is a cell index normalized on the assumption that the number of NW-MEL-38 cells immediately before co-culture with the effector cells was 1.
After 7000 HLA-A24-transduced NW-MEL-38 cells (A24+PRAME+) were cultured on an E-plate for 24.5 hours, each kind of effector cells (100000 cells) was added and cultured, and 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 is a cell index normalized on the assumption that the number of A24 NW-MEL-38 cells immediately before co-culture with the effector cells was 1.
After 7000 SK-MEL-124 cells (A24+PRAME+) were cultured on an E-plate for 24.5 hours, each kind of effector cells (100000 cells) was 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. The normalized cell index is a cell index normalized on the assumption that the number of A24 NW-MEL-38 cells immediately before co-culture with the effector cells was 1.
As shown in
Since NOG mice lack a common γ receptor, there are no NK cells in the mice. Thus, NOG mice show notably better engraftment of human cells and human tissues than NOD-SCID mice, and human cancer and human normal tissues can be engrafted in NOG mice at high rates. In addition, human T-cell differentiation is observed after transplantation of human hematopoietic stem cells; thus, demand for NOG mice as human immune system model mice is increasing. The characteristics of NOG mice include deletion of T cells and B cells, deletion of natural killer (NK) cells, reduced dendritic cell function, reduced macrophage function, loss of complement activity, and no leakiness of T cells and B cells associated with aging. NW-MEL-124 (A24-positive PRAME-positive) cells were transplanted into the right flank of NOG mice at 4×106 cells/mouse. On day 7, PRAME #98 CAR-T cells sorted with anti-lambda antibody were infused into the mice via a tail vein at 8×106 cells/mouse. The details are as follows (the outline is shown in
Gene transfer to CAR-T cells was performed according to the method of Test Example 8. CD8 and CD4 were present at 95.2% and 2.1%, and the CAR positive percentage was 97% for CD8 and 98.8% for CD4.
An experiment was performed in the CAR-T cell infusion group (n=3) and untreated group (n=3), and the tumor diameter was measured every 3 to 4 days. Measurement was performed until day after tumor transplantation.
The results show that in the CAR-T cell infusion group, suppression of the growth of the SK-MEL-124 cells (A24-positive PRAME-positive) was observed in the 3 mice (
Since NOG mice, which were used in the test for cancer suppression by #98 zG CAR-T cells of Test Example 10, lack a common γ receptor, there are no NK cells in the mice. Thus, NOG mice show notably better engraftment of human cells and human tissues than NOD-SCID mice, and human cancer and human normal tissues can be engrafted in NOG mice at high rates. In addition, human T-cell differentiation is observed after transplantation of human hematopoietic stem cells; thus, demand for NOG mice as human immune system model mice is increasing. The characteristics of NOG mice include deletion of T cells and B cells, deletion of natural killer (NK) cells, reduced dendritic cell function, reduced macrophage function, loss of complement activity, and no leakiness of T cells and B cells associated with aging. SK-MEL-1 24 (A24-positive PRAMS-positive) cells were transplanted into the right flank of NOG mice at 4×106 cells/mouse. On day 10 after transplantation, NGM and PRAME #98 CAR-T cells were infused into the mice via a tail vein at 4×106 cells/mouse. The details are as follows (the outline is shown in
Gene transfer to CAR-T cells was performed according to the method of Test Example 8. CD8 and CD4 were present at 91.0% and 6.5%, and the CAR positive percentage was 78.5% for CD8 and 56.9% for CD4.
An experiment was performed in the CAR-T cell infusion group (n=3) and NGM group (n=3), and the tumor diameter was measured every 3 to 4 days. Measurement was performed until day 46 after tumor transplantation.
The results show that in the CAR-T cell infusion group, suppression of the growth of the SK-MEL-124 cells (A24-positive PRAME-positive) was observed in the 3 mice (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-205384 | Dec 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/044807 | 12/7/2021 | WO |
