Monomer pseudo-antibody and NK cell

Abstract

Provided is a novel substance that stably binds to a surface protein expressed in NK cells. The present invention provides a monomeric pseudo-antibody which has an amino acid sequence of any one of (1) to (3) and which binds to a surface protein of an NK cell. (1) An amino acid sequence represented by any one of SEQ ID NO: A1 to A12; (2) an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence other than the arbitrary amino acid in the amino acid sequence represented by any one of SEQ ID NO: A1 to A12; and (3) an amino acid sequence having 80% or more identity to an amino acid sequence other than the arbitrary amino acid sequence among the amino acid sequences represented by any one of SEQ ID NO: A1 to A12 (in SEQ ID NO: A1 to A12, X1, X2, X3, X4, X5, and X6 are each independently an arbitrary amino acid, and n1, n2, n3, n4, n5, and n6 are each independently an arbitrary natural number. ) [Chemical Formula 1] # imgabs0 #

Description

Technical Field

[0001] The present invention relates to monomeric pseudoantibodies (monobodies), in particular to monomeric pseudoantibodies capable of binding to NK cells. Background Art

[0002] As a treatment method for cancer, immunotherapy that uses one's own immunity to destroy cancer has attracted attention. As one of the immunotherapies, NK cell therapy using natural killer (NK) cells is known. NK cell therapy is a treatment method that selectively proliferates and activates NK cells from the patient's peripheral blood mononuclear cells (PBMCs), increases their cytotoxic activity, and then returns them to the patient's body.

[0003] NK cells bind to the Fc region of antibodies through Fcγ receptors to exert ADCC (Antibody-Dependent Cellular Cytotoxicity) activity to destroy tumor cells. Therefore, it is expected that NK cell therapy will exert a high synergistic effect in combination with antibody drugs that target antigens on tumor cells. In combination with antibody drugs, when antibody drugs bind to antigens, activated NK cells and other immune cells accumulate, causing ADCC activity and ADCP (Antibody-Dependent Cellular Phagocytosis), thereby killing tumor cells.

[0004] However, there are also cells with target antigens expressed on tumor cells among normal cells. When antibody drugs bind to these cells, sometimes other immune cells other than NK cells attack normal cells (ontarget / off tumor effect). As a method to suppress this side effect, it is proposed to make a cross-linked structure connecting NK cells and antibody drugs, so that cells and antibodies are bound in advance (Patent Document 1).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2022 / 004805 Summary of the invention

[0008] Problems to be solved by the invention

[0009] However, in Patent Document 1, scFv is used as the NK binding domain of the cross-linked structure, so there is room for improvement in the affinity between the NK binding domain and the target molecule on the surface of NK cells.

[0010] Therefore, an object of the present invention is to provide a novel substance that stably binds to a surface protein expressed on NK cells.

[0011] Means for solving problems

[0012] The inventors of the present application have conducted intensive studies to solve the above-mentioned problems and have found that a monomeric mimetics having a predetermined amino acid sequence can stably bind to a surface protein expressed on NK cells, thereby completing the present invention.

[0013] That is, the present invention is as follows. [1]

[0015] A monomeric mimetibody having an amino acid sequence of any one of (1) to (3) will bind to a surface protein of a NK cell.

[0016] (1) The amino acid sequence represented by any one of SEQ ID NOs: A1 to A12

[0017] (2) An amino acid sequence represented by any one of SEQ ID NOs: A1 to A12, wherein one or more amino acids are deleted, substituted and / or added in the amino acid sequence other than any amino acid

[0018] (3) An amino acid sequence having 80% or more identity with any amino acid sequence other than any amino acid in the amino acid sequence represented by any of SEQ ID NOs: A1 to A12

[0019] (In serial numbers A1 to A12, X 1 , X 2 , X 3 , X 4 , X 5 and X 6 Each independently represents any amino acid, and n1, n2, n3, n4, n5 and n6 each independently represent any natural number.)

[0020] [Chemical formula 1]

[0021] [2]

[0023] The monomeric mimetibody according to [1], wherein the surface protein is at least one selected from the group consisting of NKp46, NKp30 and 2B4.

[0024] [3] A monomeric mimetic antibody as described in [1] or [2], wherein, among the amino acid sequences represented by sequence numbers A1 to A12, the amino acid sequences represented by sequence numbers 1 to 12 are present in the BC loop or the CD loop, and the amino acid sequences represented by sequence numbers 13 to 24 are present in the FG loop.

[0025] [Chemical formula 2]

[0026] [4]

[0028] A NK cell, wherein the monomeric mimetibody according to any one of [1] to [3] is bound to a surface protein.

[0029] Effects of the Invention

[0030] According to the present invention, a novel substance capable of stably binding to a surface protein expressed on NK cells can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] [ Figure 1 ] shows the expression vector constructed in the examples.

[0032] [ Figure 2 ] shows each monomeric pseudoantibody library.

[0033] [ Figure 3 ] shows the recovery rate of each round of monomeric mimetic antibody screening in the target mix (BC-FG: left CD-FG: right).

[0034] [ Figure 4 ] shows the recovery rate of each round of monomeric mimetic antibody screening for each target (BC-FG: upper row CD-FG: lower row).

[0035] [ Figure 5 ] shows the determination of the dissociation constants of 5 clones showing binding to NKp46.

[0036] [ Figure 6 ] shows the determination of the dissociation constants for the four clones that showed binding to 2B4.

[0037] [ Figure 7 ] shows the determination of the dissociation constants of the three clones showing binding to NKp30. DETAILED DESCRIPTION

[0038] Hereinafter, a specific embodiment of the present invention (hereinafter referred to as “the present embodiment”) will be described in detail, but the present invention is not limited thereto and various modifications can be made without departing from the gist of the invention.

[0039] The monomeric mimetic antibody of the present invention has an amino acid sequence of any one of (1) to (3) and binds to the surface protein of NK cells. 1 , X 2 , X 3 , X 4 , X5 and X 6 Each independently represents any amino acid, and n1, n2, n3, n4, n5 and n6 each independently represent any natural number.

[0040] (1) The amino acid sequence represented by any one of SEQ ID NOs: A1 to A12

[0041] (2) An amino acid sequence represented by any one of SEQ ID NOs: A1 to A12, wherein one or more amino acids are deleted, substituted and / or added in the amino acid sequence other than any amino acid

[0042] (3) An amino acid sequence having 80% or more identity with any amino acid sequence other than any amino acid in the amino acid sequence represented by any of SEQ ID NOs: A1 to A12

[0043] [Chemical formula 3]

[0044]

[0045] In this specification, the so-called "monomeric mimetic antibody" refers to an artificial protein with a molecular weight of about 10 kDa (more specifically, 7 kDa to 15 kDa) obtained by artificially adding mutations to a partial sequence using a protein as a scaffold. The molecular weight of the monomeric mimetic antibody of the present invention is, for example, 7 kDa to 15 kDa, preferably 13.6 kDa to 14.0 kDa.

[0046] The number of amino acids constituting the monomeric mimetibody of the present invention is, for example, 50 to 230, preferably 70 to 180, and more preferably 90 to 150. Within the above range, the number of amino acids may be in the range obtained by arbitrarily combining the upper and lower limits described above.

[0047] The protein used as the scaffold of the monomeric mimetic antibody of the present invention is not particularly limited, for example, it can be a part of fibronectin, it can be a part of human fibronectin, it can be a human fibronectin type III domain. That is, the monomeric mimetic antibody of the present invention is a substance obtained by changing the partial amino acid sequence in a part of fibronectin, a part of human fibronectin, or a human fibronectin type III domain. The monomeric mimetic antibody of the present invention can be a substance obtained by changing the sequence of the BC loop or CD loop of the human fibronectin type III domain, it can be a substance obtained by changing the sequence of the FG loop, or it can be a substance obtained by changing the sequence of the BC loop or CD loop and the FG loop. The monomeric mimetic antibody of the present invention can be a polypeptide that does not contain cysteine. The same skeleton as the human fibronectin type III domain (FN3) can be found in many animal proteins involved in ligand binding. The folding mode of the human fibronectin type III domain is similar to the VH domain of immunoglobulin, and its molecular weight is about 10kDa, so it has high tissue infiltration. In addition, it is heat-resistant and does not contain cysteine, so it does not form a disulfide bond.

[0048] The portion derived from the human fibronectin type III domain in the monomeric mimetic antibody of the present invention may have an amino acid sequence represented by SEQ ID NO: 82, an amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 82, or an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 82. The BC loop of the human fibronectin type III domain may be a sequence portion corresponding to PAVT (SEQ ID NO: 83) in the amino acid sequence represented by SEQ ID NO: 82, the CD loop may be a sequence portion corresponding to GNSP (SEQ ID NO: 84) in the amino acid sequence represented by SEQ ID NO: 82, and the FG loop may be a sequence portion corresponding to GRGDSPASSK (SEQ ID NO: 85) in the amino acid sequence represented by SEQ ID NO: 82. The monomeric mimetibody of the present invention may be a monomeric mimetibody having an amino acid sequence of any one of formulas (1) to (3) by altering a portion of the amino acid sequence represented by SEQ ID NO: 82, an amino acid sequence having one or more amino acid deletions, substitutions and / or additions in the amino acid sequence represented by SEQ ID NO: 82, or an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 82, and inserting any one of the amino acid sequences represented by SEQ ID NOs: 1 to 24, an amino acid sequence having one or more amino acid deletions, substitutions and / or additions in the amino acid sequence represented by SEQ ID NOs: 1 to 24, or an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NOs: 1 to 24. The altered position of the amino acid sequence derived from the type III domain of human fibronectin may be a portion of the amino acid sequence corresponding to the BC loop or CD loop of the type III domain of human fibronectin, may be a portion of the amino acid sequence corresponding to the FG loop, or may be a portion of the amino acid sequence corresponding to both the BC loop or CD loop and the FG loop.

[0049] [Chemical formula 4]

[0050]

[0051] A monomeric mimetibody may have multiple β chains and loops connecting the chains. The position where the sequence in the monomeric mimetibody is altered is not particularly limited, but preferably the sequence of the loop portion is altered, and more preferably the sequence of two or more loop portions is altered.

[0052] The monomeric mimetic antibody may have 7 or more β chains, and the sequence of at least two of the BC loop connecting the 2nd and 3rd chains, the CD loop connecting the 3rd and 4th chains, and the FG loop connecting the 6th and 7th chains may be changed. The sequences of both the BC loop and the FG loop may be changed, and the sequences of both the CD loop and the FG loop may be changed.

[0053] When the sequence of the loop portion is changed, a part of the amino acids constituting the loop may be changed, or all of the amino acids constituting the loop may be changed. Here, the so-called "change" of the amino acid sequence refers to the deletion, substitution and / or addition of one or more amino acids relative to the amino acid sequence before the change (i.e., the natural one).

[0054] The monomeric mimetibody of the present invention has the amino acid sequence of any one of (1) to (3) above.

[0055] In serial numbers A1 to A12, X 1 , X 2 , X 3 , X 4 , X 5 and X 6 Each independently represents any amino acid. Therefore, in SEQ ID NOs. A1 to A12, when n1, n2, n3, n4, n5, and n6 are natural numbers greater than 2, X 1 , X 2 , X 3 , X 4 , X 5 and X 6 Each forms an amino acid sequence consisting of any amino acids. In the case where the monomeric mimetic antibody of the present invention has the amino acid sequence represented by SEQ ID NO: 82, an amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 82, or an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 82, X 1 , X 2 , X 3 , X 4 , X 5 and X 6 Each of them becomes an amino acid corresponding to the amino acid sequence represented by sequence number 82, an amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence represented by sequence number 82, or an amino acid sequence having 80% or more identity with the amino acid sequence represented by sequence number 82.

[0056] In this specification, the term "amino acid" includes not only natural amino acids, but also artificial amino acid mutants and derivatives. As amino acids, for example, proteinaceous L-amino acids, non-proteinaceous amino acids, and chemically synthesized compounds having properties known in the art as amino acid characteristics can be cited.

[0057] Proteinogenic amino acids are represented by three-letter letters known in the art, such as Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val. Proteinogenic amino acids are represented by single-letter letters known in the art, such as R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, and V.

[0058] Non-proteinogenic amino acids include natural or unnatural amino acids other than proteinogenic amino acids.

[0059] Examples of non-natural amino acids include amino acids whose main chain structures are different from those of natural ones (α,α-disubstituted amino acids (α-methylalanine, etc.), N-alkyl amino acids, D-amino acids, β-amino acids, γ-amino acids, δ-amino acids, and α-hydroxy acids, etc.); amino acids whose side chain structures are different from those of natural ones (norleucine, homohistidine, etc.); amino acids having extra methylene groups in the side chains (“homo” amino acids, homophenylalanine, and homohistidine, etc.); and amino acids whose carboxylic acid functional groups in the side chains are substituted with sulfonic acid groups (cysteic acid, etc.).

[0060] X 1 , X 2 , X 3 , X 4 , X 5 and X 6 Each independently may be a proteinogenic amino acid, or may be any of the 19 proteinogenic amino acids excluding cysteine. 6 Can also be used with X 3 same.

[0061] In the sequence numbers A1 to A12, n1, n2, n3, n4, n5 and n6 each independently represent an arbitrary natural number. Each of n1, n2, n3, n4, n5 and n6 can be, for example, 1 to 150, preferably 3 to 100.

[0062] n1 is preferably 5 to 50, more preferably 10 to 30, and even more preferably 13 to 25.

[0063] n2 is preferably 10 or more and 100 or less, more preferably 30 or more and 70 or less, further preferably 40 or more and 60 or less.

[0064] n3 is preferably 3 or more and 60 or less, more preferably 5 or more and 55 or less, and further preferably 10 or more and 50 or less.

[0065] n4 is preferably 10 to 80, more preferably 20 to 70, and even more preferably 25 to 50.

[0066] n5 is preferably 10 to 80, more preferably 20 to 70, and even more preferably 25 to 50.

[0067] n6 is preferably 3 to 60, more preferably 5 to 55, and still more preferably 10 to 50. n6 may be equal to n3.

[0068] In the amino acid sequence of (2), an amino acid sequence other than any amino acid refers to a sequence consisting of X 1 n1 , X 2 n2 , X 3 n3 , X 4 n4 , X 5 n5 and X 6 n6 Therefore, the amino acid sequence of (2) is: in the amino acid sequence represented by sequence numbers A1 to A12, at position X 1 n1 With X 2 n2 Sequences between and / or located at X 2 n2 With X 3 n3 A sequence between or between X 4 n4 With X 5 n5 Sequences between and / or located at X 5 n5 With X 6 n6 An amino acid sequence having one or more amino acids deleted, substituted and / or added in the sequence between them.

[0069] In the present specification, the number of amino acids deleted, substituted and / or added in the "amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence" is not particularly limited as long as it is one or more. The so-called "plurality" in the multiple amino acids is a natural number greater than 2, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0070] As 1 or more, it can be 1 or more and 1 or less, preferably 1 or more and 8 or less, and more preferably 1 or more and 5 or less. The upper limit of the number of amino acids deleted, substituted and / or added may be, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. In addition, the lower limit of the number of amino acids deleted, substituted and / or added may be, for example, 2, 3, or 4. Within the above range, the number of amino acids deleted, substituted and / or added may also be set within the range obtained by arbitrarily combining the above upper limit and lower limit.

[0071] In the amino acid sequence of (3), the amino acid sequence other than any amino acid is the amino acid sequence consisting of X 1 n1 , X 2 n2 , X 3 n3 , X 4 n4 , X 5 n5 and X 6 n6 Therefore, the amino acid sequence of (3) is: in the amino acid sequence represented by sequence numbers A1 to A12, the amino acid sequence corresponding to the position X 1 n1 With X 2 n2 Sequences between and / or located at X 2 n2 With X 3 n3 A sequence between or between X 4 n4 With X 5 n5 Sequences between and / or located at X 5 n5 With X 6 n6 The sequences have more than 80% identity with each other in terms of amino acid sequence.

[0072] In the present specification, the term "an amino acid sequence having 80% or more identity with an amino acid sequence" when described as "having Y% or more identity with an amino acid sequence X" means that when the amino acid sequence having the identity is aligned (compared) in a manner that maximizes the rate of consistency between the amino acid sequence having the identity and the amino acid sequence X, the ratio of the number of common amino acid residues to the total number of amino acids comprising the amino acid sequence having the identity is Y% or more.

[0073] The identity may be 80% or more, and may be 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more. The upper limit of the identity is not particularly limited, and may be 99%, 98%, 97% or 95%. The identity may also be 100%.

[0074] Search and analysis involving the identity of amino acid sequences can be performed using algorithms or programs known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, Clustal W). With regard to the parameters when using the programs, those skilled in the art can appropriately set them, or can also use the default parameters of each program.

[0075] Regarding the amino acid sequence of any one of (1) to (3), it is preferred that, among the amino acid sequences represented by SEQ ID NOs: A1 to A12, the amino acid sequences represented by SEQ ID NOs: 1 to 12 are present in the BC loop or the CD loop, and the amino acid sequences represented by SEQ ID NOs: 13 to 24 are present in the FG loop. In particular, the amino acid sequences represented by SEQ ID NOs: 1 to 3, 6 to 7, and 10 to 11 are preferably present in the BC loop, and the amino acid sequences represented by SEQ ID NOs: 4 to 5, 8 to 9, and 12 are preferably present in the CD loop.

[0076] [Chemical formula 5]

[0077]

[0078] In addition, in the present invention, the monomeric mimetics may also be (1) a monomeric mimetics in which the amino acid sequence represented by SEQ ID NOs: 1 to 12 is present in the BC loop or the CD loop, and the amino acid sequence represented by SEQ ID NOs: 13 to 24 is present in the FG loop. In this case, the monomeric mimetics have any one of the following combinations:

[0079] Serial number 1 and serial number 13;

[0080] Serial number 2 and serial number 14;

[0081] Serial number 3 and serial number 15;

[0082] Serial number 4 and serial number 16;

[0083] Serial number 5 and serial number 17;

[0084] Serial number 6 and serial number 18;

[0085] Serial number 7 and serial number 19;

[0086] Serial number 8 and serial number 20;

[0087] Serial number 9 and serial number 21;

[0088] Serial number 10 and serial number 22;

[0089] Serial number 11 and serial number 23;

[0090] Serial number 12 and serial number 24.

[0091] The monomeric mimetibody may also be (2) a monomeric mimetibody having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence represented by any one of the combinations of SEQ ID NOs: 1 to 12 and SEQ ID NOs: 13 to 24.

[0092] The monomeric mimetibody may also be (3) a monomeric mimetibody having an amino acid sequence that is 80% or more identical to the amino acid sequence represented by any one of the combinations of SEQ ID NOs: 1 to 12 and SEQ ID NOs: 13 to 24.

[0093] In the present specification, in the amino acid sequence represented by any one of SEQ ID NOs. A1 to A12, the amino acid sequence other than any amino acid is an amino acid sequence consisting of X 1 , X 2 , X 3 , X 4 , X 5 and X 6 Specifically, in SEQ ID NOs. A1 to A12, each group of amino acid sequences correspondingly selected from the amino acid sequences represented by SEQ ID NOs. 1 to 12 and the amino acid sequences represented by SEQ ID NOs. 13 to 24 are amino acid sequences other than any amino acid.

[0094] For example, when SEQ ID NO: A1 is used as an example, the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 13 are amino acid sequences other than any amino acid in SEQ ID NO: A1.

[0095] The so-called (2) amino acid sequence having one or more amino acid deletions, substitutions and / or additions in the amino acid sequence other than any amino acid in the amino acid sequence represented by any one of SEQ ID NOs A1 to A12, when SEQ ID NO A1 is used as an example, it is an amino acid sequence having one or more amino acid deletions, substitutions and / or additions in the amino acid sequences of SEQ ID NOs 1 and 13. It should be noted that the amino acid sequence may be an amino acid sequence having one or more amino acid deletions, substitutions and / or additions in the amino acid sequence of SEQ ID NOs 1 (there is no mutation in SEQ ID NO 13), an amino acid sequence having one or more amino acid deletions, substitutions and / or additions in the amino acid sequence of SEQ ID NOs 13 (there is no mutation in SEQ ID NO 1), or an amino acid sequence having one or more amino acid deletions, substitutions and / or additions in the amino acid sequences of SEQ ID NOs 1 and 13 (there are mutations in both SEQ ID NOs 1 and 13).

[0096] The so-called (3) amino acid sequence having 80% or more identity with an amino acid sequence other than any amino acid in the amino acid sequence represented by any one of SEQ ID NOs. A1 to A12 is, when SEQ ID NO. A1 is used as an example, an amino acid sequence having 80% or more identity with the amino acid sequences of SEQ ID NOs. 1 and 13. It should be noted that the amino acid sequence may be an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO. 1 in the amino acid sequence (there is no mutation in SEQ ID NO. 13), an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO. 13 (there is no mutation in SEQ ID NO. 1), or an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO. 1 and SEQ ID NO. 13 (there are mutations in both SEQ ID NOs. 1 and 13).

[0097] Regarding the combinations of predetermined amino acid sequences possessed by SEQ ID NOs. A2 to A12, the description in SEQ ID NO. A1 also applies.

[0098] In the serial numbers A1 to A12, X 1 n1 The amino acid sequence represented is not particularly limited, but is preferably an amino acid sequence represented by sequence number 25, an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence represented by sequence number 25, or an amino acid sequence that has 80% or more identity with the amino acid sequence represented by sequence number 25.

[0099] In the serial numbers A1 to A12, X 1 n1 The amino acid sequence represented is more preferably the amino acid sequence represented by SEQ ID NO: A25, or an amino acid sequence having one or more amino acid substitutions in the amino acid sequence represented by SEQ ID NO: A25 and having 80% or more identity with the amino acid sequence represented by SEQ ID NO: A25. 7 Each independently represents any amino acid, and n7 is a natural number of 1 to 10, preferably a natural number of 1 to 5. 7 n7 The amino acid corresponding to the start codon (e.g., methionine) and the insertion sequence may be included. The amino acid substituted in the amino acid sequence represented by SEQ ID NO: A25 may not include X 7 n7 The amino acids other than E at the 5th position, S at the 7th position, I at the 11th position, and Q at the 12th position from the N-terminus are included.

[0100] In the serial numbers A1 to A12, X 1 n1The amino acid sequence represented is further preferably the amino acid sequence represented by SEQ ID NO: 26, or an amino acid sequence having one or more amino acid substitutions in the amino acid sequence represented by SEQ ID NO: 26 and having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 26. The amino acid substituted in the amino acid sequence represented by SEQ ID NO: 26 may be an amino acid other than E at position 9, S at position 11, I at position 15, and Q at position 16 from the N-terminus.

[0101] In the serial numbers A1 to A12, X 2 n2 The amino acid sequence represented is not particularly limited, but is preferably an amino acid sequence represented by sequence number 27, an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence represented by sequence number 27, or an amino acid sequence that has 80% or more identity with the amino acid sequence represented by sequence number 27.

[0102] In the serial numbers A1 to A12, X 3 n3 The amino acid sequence represented is not particularly limited, but preferably includes the amino acid sequence represented by sequence number 28, an amino acid sequence having a deletion, substitution and / or addition of one or more amino acids in the amino acid sequence represented by sequence number 28, or an amino acid sequence having more than 80% identity with the amino acid sequence represented by sequence number 28.

[0103] In the serial numbers A1 to A12, X 3 n3 The amino acid sequence represented is more preferably the amino acid sequence represented by SEQ ID NO: A28, or an amino acid sequence having one or more amino acid substitutions in the amino acid sequence represented by SEQ ID NO: A28 and having 80% or more identity with the amino acid sequence represented by SEQ ID NO: A28. 8 Each independently represents any amino acid, and n8 is an integer of 0 to 40, preferably an integer of 0 to 30. 8 n8 A linker sequence may be included.

[0104] In the serial numbers A1 to A12, X 4 n4 The amino acid sequence represented is not particularly limited, but is preferably an amino acid sequence represented by sequence number 29, an amino acid sequence having a deletion, substitution and / or addition of one or more amino acids in the amino acid sequence represented by sequence number 29, or an amino acid sequence having more than 80% identity with the amino acid sequence represented by sequence number 29.

[0105] In the serial numbers A1 to A12, X4 n4 The amino acid sequence represented is more preferably the amino acid sequence represented by SEQ ID NO: A29, or an amino acid sequence having one or more amino acid substitutions in the amino acid sequence represented by SEQ ID NO: A29 and having 80% or more identity with the amino acid sequence represented by SEQ ID NO: A29. 7 Each independently represents any amino acid, and n7 is a natural number of 1 to 10, preferably a natural number of 1 to 5. 7 n7 The amino acid corresponding to the start codon (e.g., methionine) and an insertion sequence may be included. The amino acid sequence represented by SEQ ID NO: A29 does not include X 7 n7 The ZIZ at positions 26 to 28 from the N-terminus may be DIY in sequence number A4, VIS in sequence number A5, GIY in sequence number A8, EIL in sequence number A9, or VIT in sequence number A12. The amino acid to be substituted in the amino acid sequence represented by sequence number A29 may be any amino acid not including X. 7 n7 The amino acids other than E at position 5, S at position 7, I at position 11, Q at position 12, and ZIZ at positions 26 to 28 from the N-terminus are included.

[0106] In the serial numbers A1 to A12, X 4 n4 The amino acid sequence represented is further preferably the amino acid sequence represented by SEQ ID NO: 30, or an amino acid sequence having one or more amino acid substitutions in the amino acid sequence represented by SEQ ID NO: 30 and having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 30. Here, in SEQ ID NO: 30, each Z is independently any amino acid. ZIZ at positions 30 to 32 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 30 may be DIY in SEQ ID NO: A4, VIS in SEQ ID NO: A5, GIY in SEQ ID NO: A8, EIL in SEQ ID NO: A9, or VIT in SEQ ID NO: A12. The amino acid substituted in the amino acid sequence represented by SEQ ID NO: 30 may be an amino acid other than E at position 9, S at position 11, I at position 15, Q at position 16, and ZIZ at positions 30 to 32 from the N-terminus.

[0107] In the serial numbers A1 to A12, X 5 n5The amino acid sequence represented is not particularly limited, but is preferably an amino acid sequence represented by SEQ ID NO: 31, an amino acid sequence having one or more amino acids deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 31, or an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 31. Here, in SEQ ID NO: 31, Z is each independently any amino acid.

[0108] In the serial numbers A1 to A12, X 5 n5 The amino acid sequence represented is more preferably the amino acid sequence represented by SEQ ID NO: 31, or an amino acid sequence having one or more amino acid substitutions in the amino acid sequence represented by SEQ ID NO: 31 and having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 31. The Z at the third position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 31 may be A in SEQ ID NO: A4, G in SEQ ID NO: A5, E in SEQ ID NO: A8, Y in SEQ ID NO: A9, or Q in SEQ ID NO: A12. The amino acid substituted in the amino acid sequence represented by SEQ ID NO: 31 may be an amino acid other than the Z at the third position from the N-terminus.

[0109] By X 6 n6 The amino acid sequence represented by X is not particularly limited and can be 3 n3 The amino acid sequences represented are identical.

[0110] The number of amino acids deleted, substituted and / or added, or the number of substituted amino acids in the amino acid sequences represented by SEQ ID NOs: 25 to 31, A25, A28, and A29 is 1 to 10, preferably 1 to 8. The upper limit of the number of amino acids deleted, substituted and / or added, or the number of substituted amino acids may be, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In addition, the lower limit of the number of amino acids deleted, substituted and / or added, or the number of substituted amino acids may be, for example, 2, 3, or 4. Within the above range, the number of amino acids deleted, substituted and / or added, or the number of substituted amino acids may be set to any combination of the above upper and lower limits.

[0111] [Chemical formula 6]

[0112]

[0113] The amino acid sequences represented by sequence numbers A4 to A5, A8 to A9, and A12 are preferably sequence numbers A32 to A35, and more preferably sequence numbers A36 to A41. In addition, the amino acid sequences represented by sequence numbers A1 to A3, A6 to A7, and A10 to A11 are preferably sequence numbers A42 to A48. In sequence numbers A32 to A48, the loop portion modified from the human fibronectin type III domain is underlined. In sequence numbers A32 to A48, X 7 n7 It may contain an amino acid corresponding to the start codon (e.g., methionine) and an insertion sequence, X 8 n8 A linker sequence may be included. 7 and X 8 Each independently represents an arbitrary amino acid, n7 represents a natural number of 1 to 10, and n8 represents an integer of 0 to 40. n7 is preferably an integer of 1 to 5, and n8 is preferably an integer of 0 to 30.

[0114] [Chemical formula 7]

[0115]

[0116] [Chemical formula 8]

[0117]

[0118] Any one of the following combined amino acid sequences included in sequence numbers A32 to A48 can be used as a monomeric mimetic antibody of the present invention. It can also be a monomeric mimetic antibody having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in an amino acid sequence other than the amino acid sequence of the following combination. It can also be a monomeric mimetic antibody in which the amino acid sequence other than the amino acid sequence of the following combination is an amino acid sequence that has more than 80% identity with the above-mentioned corresponding amino acid sequence.

[0119] Serial number 1 and serial number 13;

[0120] Serial number 2 and serial number 14;

[0121] Serial number 3 and serial number 15;

[0122] Serial number 4 and serial number 16;

[0123] Serial number 5 and serial number 17;

[0124] Serial number 6 and serial number 18;

[0125] Serial number 7 and serial number 19;

[0126] Serial number 8 and serial number 20;

[0127] Serial number 9 and serial number 21;

[0128] Serial number 10 and serial number 22;

[0129] Serial number 11 and serial number 23;

[0130] Serial number 12 and serial number 24.

[0131] In addition, as a monomeric mimetic antibody of the present invention, the following monomeric mimetic antibody may have the amino acid sequence of the above-mentioned combination, or may have an amino acid sequence having a mutation or the like in the amino acid sequence of the above-mentioned combination. With respect to the monomeric mimetic antibody, in the amino acid sequences of sequence numbers A32 to A48, the amino acid sequences other than the amino acid sequence of the above-mentioned combination are the same amino acid sequences; or the amino acid sequences other than the amino acid sequence of the above-mentioned combination have one or more amino acids deleted, substituted and / or added; or the amino acid sequences other than the amino acid sequence of the above-mentioned combination are amino acid sequences that have more than 80% identity with the above-mentioned corresponding amino acid sequences.

[0132] As an amino acid sequence having a mutation etc. in the amino acid sequence of the above combination, when the above combination is set to sequence number X1 and sequence number X2, it can be an amino acid sequence having a mutation etc. in sequence number X1, it can be an amino acid sequence having a mutation etc. in sequence number X2, or it can be an amino acid sequence having a mutation etc. in both sequence numbers X1 and X2.

[0133] The amino acid sequence having a mutation or the like in SEQ ID NO: X1 may be an amino acid sequence having a deletion, substitution and / or addition of one or more amino acids in SEQ ID NO: X1, or an amino acid sequence having 80% or more identity with SEQ ID NO: X1.

[0134] The amino acid sequence having a mutation or the like in sequence number X2 may be an amino acid sequence having a deletion, substitution and / or addition of one or more amino acids in sequence number X2, or an amino acid sequence having 80% or more identity with sequence number X2.

[0135] The serial number X1 and the serial number X2 may be any combination thereof.

[0136] The amino acid sequence having a mutation or the like in SEQ ID NO: X1 and SEQ ID NO: X2 is preferably an amino acid sequence in which one or more amino acids in SEQ ID NO: X1 and SEQ ID NO: X2 are substituted, or an amino acid sequence having 80% or more identity with SEQ ID NO: X1 and SEQ ID NO: X2.

[0137] The monomeric mimetic antibody of the present invention may also be further bound to a tag sequence and / or a fusion protein. The binding site is not particularly limited, for example, it may be the C-terminus of the monomeric mimetic antibody, or it may be X in sequence numbers A32 to A48. 8 n8 .

[0138] The monomeric mimetic antibodies of the present invention can be manufactured by previously known methods such as translational synthesis based on cell lines and cell-free systems, and chemical synthesis, preferably by translational synthesis based on cell lines. In the translational synthesis based on cell lines, for example, the insert DNA appropriately designed according to the desired amino acid sequence can be introduced into a suitable vector, cloned by a suitable method, and then expressed and purified using suitable cells such as competent cells, thereby manufacturing a monomeric mimetic antibody having a desired amino acid sequence. More specifically, it can be manufactured using the method described in the examples.

[0139] The monomeric mimetibodies of the present invention will bind to the surface proteins of NK cells.

[0140] So-called NK cells are large granular lymphocytes that do not express T cell receptor (TCR), CD3 as a general marker of T cells, and B cell receptor as membrane immunoglobulin, which are usually CD16 positive in humans and CD56 positive. Regarding whether it is a NK cell, those skilled in the art can easily judge based on the expression pattern of cell surface markers, etc. NK cells have cytotoxic activity, and the presence or absence of the cytotoxic activity and the degree can be measured by known various methods.

[0141] Unless otherwise specified, the NK cells in the present invention include the general NK cells described above. In addition, NK cells include peripheral blood NK cells, cord blood NK cells, primary NK cells, cultured NK cells, highly active NK cells, and the NK cells and NK-like cells described in [1], [2], [3], and [4] below.

[0142] [1] NK cells having the following characteristics (i) and (ii):

[0143] (i) CD16 positive, high expression of CD56, and CD57 negative.

[0144] (ii) NKG2C-positive, NKG2A-negative to low-expressing, and CD94-positive.

[0145] The highly active NK cells of [1] may also have high expression of CD16. In addition, regardless of whether or not they have high expression of CD16, the highly active NK cells of [1] may also have the following characteristics.

[0146] (iii) The cytotoxic activity was 50% or more when the NK cells were used as effector cells (E) and K562 cells were used as target cells (T) at a mixing ratio (E:T) of 1:1.

[0147] [1] Highly active NK cells can also be expressed as follows:

[0148] NK cells having the above-mentioned characteristics (i) and (iii) are obtained by culturing a cell population obtained by removing CD3-positive cells from peripheral blood mononuclear cells from healthy subjects using CD3 beads (e.g., CliniMACS CD3, Miltenyi Biotec, catalog number 130-017-601), LD columns (e.g., Miltenyi Biotec, catalog number 130-042-901) and separation buffer (e.g., PBS containing 0.5% human AB type serum (inactivated) and 2 mM EDTA) in an appropriate culture medium (e.g., COSMEDIUM008 supplemented with 5% human AB type serum (inactivated)) for 14 days.

[0149] For details on the characteristics of highly active NK cells [1] and a more specific production method, please refer to Japanese Patent Application Publication No. 2018-193303.

[0150] [2] The following cells:

[0151] These are CCR5-positive, CCR6-positive, and CXCR3-positive cells, and are CD3-negative.

[0152] [2] The cells may further have high expression of CD11c.

[0153] [2] The cell can also be represented as follows:

[0154] The cells are CCR5-positive, CCR6-positive, CXCR3-positive, integrin α1-positive, integrin α3-positive, integrin β3-negative, and CD3-negative. Alternatively, the cells are CCR5-positive, CCR6-positive, CXCR3-positive, highly express CD11a and highly express CD11c, and CD3-negative (high expression is determined by comparing with the expression in the NK cell population obtained from peripheral blood without substantial culture).

[0155] The cells of [2] exhibit extremely high cytotoxic activity against solid cancers that have formed tumor masses. For details on the characteristics of the cells of [2] and a more specific production method, please refer to Japanese Patent Application Publication No. 2019-170176.

[0156] [3] Highly active NK cells, which can be obtained by the following method:

[0157] CD3 beads (e.g., CliniMACS CD3, Miltenyi Biotec, 130-017-601 (1x10 7 When frozen apheresis blood is used, CD34 beads (e.g., CliniMACS CD34, Miltenyi Biotec, 130-017-501 (1×10 cells per 5 μL)) are further added. 7 The cells were counted as 2.5 μL) and suspended, incubated at 4°C for 15 minutes, and then fully suspended in a separation buffer (e.g., PBS containing 0.5% human AB serum (inactivated at 56°C for 30 minutes) and 2 mM EDTA) and centrifuged. The supernatant was removed to make a maximum of 1×10 cells per LD column (e.g., Miltenyi Biotec, 130-042-901). 8 Suspend in 0.5 mL of separation buffer in such a way that the number of cells is 5×10 cells. Add 2 mL of separation buffer in advance, then add the cell suspension to the LD column and recover the eluate from the LD column. Further add 1 mL of separation buffer to the LD column and recover the eluate. Centrifuge the recovered liquid, remove the supernatant, and, if using peripheral blood, centrifuge the cells to a concentration of 5×10 5 When using frozen apheresis blood, cells are divided into 1×10 6 The cells are suspended in a suitable culture medium (e.g., KBM501 medium containing 5% human AB serum (inactivated at 56°C for 30 minutes) or 5% UltraGRO (AventaCell, HPCPLCRL10) supplemented with 2U / mL heparin sodium) at a concentration of 1 cell / mL, the culture medium is replaced appropriately, and the cells are cultured until the 14th day.

[0158] For the specific method for producing highly active NK cells [3], reference can be made to Japanese Patent Application Publication No. 2021-136883.

[0159] [4] Cells obtained by the method for obtaining cells of [1] to [3], wherein any one selected from the group consisting of IL-12, IL-15, and IL-18 is added simultaneously with IL-2 or instead of IL-2 and cultured. For a specific method for producing such cells, see Leong JW et al. Biol Blood Marrow Transplant 20 (2014) 463-473.

[0160] The so-called surface protein of NK cells bound by the monomeric mimetic antibody of the present invention refers to a protein expressed on the surface of NK cells, which may be a receptor or a membrane protein. Such proteins include NKp46, NKp30, NKG2D, IL-15R, IL-2R, KIR3DS1, NKG2C, NKp80, NKp65, NKp44, LALRA1, LILRA2, DNAM-1, and 2B4, preferably at least one selected from the group consisting of NKp46, NKp30, and 2B4.

[0161] NKp46, NKp30, and 2B4 are receptors that regulate cytotoxicity. NKp46 (CD335) and NKp30 (CD337) are both NK cytotoxicity receptors (Natural Cytotoxicity Receptor: NCR). When ligands bind to these receptors, NK cells are activated through signal transduction, cytokine production increases, and apoptosis of target cells is induced through the release of perforin and granzymes. NKp46 uses hemagglutinin present on the surface of viruses as a ligand, and NKp30 uses B7-H6 expressed on tumor cells as a ligand. 2B4 (CD244) transmits both activation and inhibition signals by binding to CD48 expressed on other immune cells.

[0162] When the monomeric mimetibody of the present invention has the amino acid sequence of any one of (1a) to (3a), the monomeric mimetibody can also bind to NKp46.

[0163] (1a) The amino acid sequence represented by any one of SEQ ID NOs: A1 to A5

[0164] (2a) An amino acid sequence represented by any one of SEQ ID NOs: A1 to A5, wherein one or more amino acids are deleted, substituted and / or added in the amino acid sequence other than any amino acid

[0165] (3a) An amino acid sequence having 80% or more identity with an amino acid sequence other than any amino acid in the amino acid sequence represented by any of SEQ ID NOs: A1 to A5.

[0166] When the monomeric mimetibody of the present invention has the amino acid sequence of any one of (1b) to (3b), the monomeric mimetibody can also bind to 2B4.

[0167] (1b) The amino acid sequence represented by any one of SEQ ID NOs: A6 to A9

[0168] (2b) An amino acid sequence represented by any one of SEQ ID NOs: A6 to A9, wherein one or more amino acids are deleted, substituted and / or added in the amino acid sequence other than any amino acid.

[0169] (3b) an amino acid sequence having 80% or more identity with an amino acid sequence other than any amino acid in the amino acid sequence represented by any of SEQ ID NOs: A6 to A9

[0170] When the monomeric mimetibody of the present invention has the amino acid sequence of any one of (1c) to (3c), the monomeric mimetibody can also bind to NKp30.

[0171] (1c) The amino acid sequence represented by any one of SEQ ID NOs: A10 to A12

[0172] (2c) An amino acid sequence represented by any one of SEQ ID NOs: A10 to A12, wherein one or more amino acids are deleted, substituted and / or added in the amino acid sequence other than any amino acid.

[0173] (3c) an amino acid sequence having 80% or more identity with any amino acid sequence other than any amino acid in the amino acid sequence represented by any of SEQ ID NOs: A10 to A12

[0174] In the amino acid sequences of (1a) to (3a), (1b) to (3b) and (1c) to (3c), the definitions and preferred embodiments of each term and symbol are the same as those in the amino acid sequences of (1) to (3).

[0175] The monomeric mimetic antibody of the present invention binds to the surface protein of NK cells. Therefore, NK cells can be stably bound to any protein via a cross-linked body comprising the monomeric mimetic antibody of the present invention. The protein that binds to NK cells via a cross-linked body comprising the monomeric mimetic antibody of the present invention is not particularly limited, for example, an antibody, preferably an antibody drug. The antibody drug can be an antibody drug for treating cancer or an infectious disease.

[0176] Therefore, the present invention also provides NK cells to which the monomeric mimetic antibodies of the present invention are bound to surface proteins. In the NK cells, the monomeric mimetic antibodies of the present invention recognize the surface proteins of NK cells and bind to NK cells. The monomeric mimetic antibodies of the present invention can be directly or via a linker to any compound at, for example, the C-terminus. As a compound linked to a monomeric mimetic antibody, it can be a compound that recognizes a desired protein bound to NK cells, or a marker for detecting NK cells. In the case of binding an antibody to a NK cell, the monomeric mimetic antibody of the present invention can also be bound to an antibody or an antigen-binding fragment of an antibody that will bind to the Fc region of the antibody at the C-terminus. As an antigen-binding fragment of an antibody, single-chain Fv fragments (scFv), dimeric scFv (di-scFv), double-chain antibodies (diabody), three-chain antibodies (triabody), four-chain antibodies (tetrabody), Fab, F(ab')2, Fv, and monomeric mimetic antibodies can be cited.

[0177] The present invention also provides a pharmaceutical composition comprising NK cells to which antibodies are bound via a cross-linked body comprising the monomeric mimetibody of the present invention.

[0178] The pharmaceutical composition may be in the form of NK cells combined with antibodies via a cross-linked body comprising a monomeric mimetic antibody of the present invention suspended in a solution. The solution used to suspend NK cells may be, for example, a freezing protection solution comprising DMSO, saline, phosphate buffered saline (PBS), culture medium, serum, etc. The solution may also include a pharmaceutically acceptable carrier as a pharmaceutical or quasi-drug.

[0179] The pharmaceutical composition provided by the present invention can be applied to the treatment of various diseases with high sensitivity of NK cells, etc. Examples of such diseases are cancer and infectious diseases, which are not particularly limited, and specifically, skin cancer, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, kidney cancer, ovarian cancer, bladder cancer, prostate cancer, neuroblastoma, and leukemia, as well as infectious diseases caused by viruses, bacteria, etc. can be cited.

[0180] The immunotherapy based on the pharmaceutical composition of the present invention may be carried out alone or in combination with surgical therapy, chemotherapy, radiotherapy, and / or antibody drugs.

[0181] One embodiment of the present invention includes:

[0182] A method of treating, comprising administering to a patient having the above-mentioned disease an effective amount of NK cells bound to an antibody via a cross-linked body comprising a monomeric mimetibody of the present invention;

[0183] NK cells bound to antibodies via a cross-linked body comprising a monomeric mimetibody of the present invention, which are used for the treatment or prevention of the above-mentioned diseases;

[0184] Use of NK cells bound to antibodies via a cross-linked product comprising a monomeric mimetibody of the present invention for the manufacture of a pharmaceutical composition for use in the treatment or prevention of the above-mentioned diseases;

[0185] The therapeutic or preventive agent for the above-mentioned disease comprises NK cells to which antibodies are bound via a cross-linked body comprising the monomeric mimetibody of the present invention.

[0186] The above-mentioned aspects described in this specification may be aspects obtained by arbitrarily combining all aspects including aspects described in this specification and aspects described as preferred aspects in this specification.

[0187] Example

[0188] Hereinafter, the present invention will be described in more detail using Examples and Comparative Examples. The present invention is not limited to the following Examples at all.

[0189] [Device]

[0190] The following apparatus were used in the examples.

[0191] PCR device: BIO-RAD T100 Thermal Cycler

[0192] RT-PCR device: Roche Light Cycler 96

[0193] Absorbance meter: Thermo Nano Drop 200c

[0194] Gel imaging device: BIO-RAD EZ Imager

[0195] Gel fluorescence imaging device: BIO RAD Pharos FXTM Molecular Imager (excitation light 532nm, detection wavelength 605nm: HEX)

[0196] Next-generation sequencer: Ion PGM TM system

[0197] Fluorescence meter: JASCO Spectrofluorometer FP-8500

[0198] Protein purification device: NGC TM Chromatography system

[0199] Dissociation constant determination: Octet (registered trademark) RED 96

[0200] [Reagents]

[0201] Synthetic oligonucleotides (Table 4) and DNA sequence analysis were purchased from FASMAC, Dynabeads TM M-280 Streptavidin, Dynabeads TM M-270 Streptavidin, and Dynabeads TM Protein G was purchased from Thermo Scientific.

[0202] Biotinylated Human NKp46 / NCR / CD335 Protein as target protein, Fc, Avitag TM (NC1-H82F9), biotinylated human 2B4 / CD244 / SLAMF4 protein, Fc, Avitag TM (2B4-H82F0), and human NKp30 / NCR3 / CD337 protein, Fc Tag (NC3-H5259) were purchased from AcroBIOSYSTEMS.

[0203] As the library mRNA having the sequence described below, mRNA prepared according to a previous report was used.

[0204] The details of the reagents used in the Examples are as follows.

[0205] · 5× PfSH buffer

[0206] 50mM Tris-HCl pH8.4, 500mM KCl, 0.5% (v / v) Triton (registered trademark) X-100, 10mM MgSO4

[0207] · HsolA

[0208] 18.4 mM GTP, 18.4 mM ATP, 9.18 mM CTP, 9.18 mM UTP, 184 mM creatine phosphate, 459 mM HEPES-KOH pH 7.6, 919 mM potassium acetate, 121 mM magnesium acetate tetrahydrate, 18.4 mM spermidine, 9.18 mM DTT, 13.8 mg / mL tRNA mix, 14 mM Mg(OAc)2

[0209] · 3×RT mixture

[0210] 3×RT buffer, 16.5 mM DTT, 15 mM dNTPs, 7.5 μM sAbS.R28, 250 mM MgCl2, 12% (v / v) HMLV

[0211] · PCRSyber

[0212] 1×PCR dNTPs,1 / 200000Cyber ​​green I solvent solvent,1 / 6000PfSH、0.375µMT7SD8M2.F44,0.375µM concentration (RealTime) concentration,2%(v / v)DMSO

[0213] · 2×DEP PCR Mix

[0214] 1×PCR dNTPs,1 / 3000PfSH,in25µM T7SD8M2P.F47,Mos-G5R-T-an21-3.R45.4%(v / v)DMSO

[0215] · HsolB v12.5(-T7)

[0216] 0.73μM AlaRS,0.03μM ArgRS,0.38μM AsnRS,0.13μM AspRS,0.02μM CysRS,0.06μM GlnRS,0.23μM GluRS,0.09μM GlyRS,0.02μM HisRS,0.40μM IleRS,0.04μM LeuRS,0.11μM LysRS,0.03µMMetRS,0.68µM PheRS,0.16µM ProRS,0.04µM SerRS,0.09µM ThrRS,0.03µM TrpRS,0.02µM TyrRS,0.02µM ValRS,0.6µM MTF,0.26µMEF-G,0.25µM RF2,0.17µMRF3,0.5µM RRF,4µg / mL CK,0.1µM Adk,0.1µM CiPPase,0.1µM NDK,2.7µM IF1,0.4µMIF2,1.5µM IF3,20µMEF-Tu / Ts,0.5µM DnaJ,1.2µM DnaK,0.5µM GrpE,1.2μM solvent,10mMMg(OAc)2

[0217] · HsolB v12.5(+T7)

[0218] 0.69μM AlaRS, 0.03μM ArgRS, 0.36μM AsnRS, 0.12μM AspRS, 0.019μM CysRS, 0.06μM GlnRS, 0.22μM GluRS, 0.09μM GlyRS, 0.02μM HisRS, 0.38μM IleRS, 0.04μM LeuRS, 0.10μM LysRS, 0.03μM MetRS, 0.64μM PheRS, 0.15μM ProRS, 0.04μM SerRS, 0.09μM ThrRS, 0.03μM TrpRS, 0.02μM TyrRS, 0.02μM ValRS, 0.57μM MTF, 0.25μMEF-G, 0.24μM RF2, 0.16μM RF3,0.47μM RRF, 3.87 μg / mL CK, 0.09 μM Adk, 0.09 μM CiPPase, 0.09 μM NDK, 2.55 μM IF1, 0.38 μM IF2, 1.4 μM IF3, 18.9 μM EF-Tu / Ts, 0.47 μM DnaJ, 1.13 μM DnaK, 0.47 μM GrpE, 1.13 μM ribosome, 1 μM T7 RNA polymerase, 10 mM Mg(OAc)2

[0219] · HBST

[0220] 50 mM Hepes-K pH 7.5, 300 mM NaCl, 0.05% (v / v) Tween 20

[0221] · 2× DNA loading buffer

[0222] 50×TAE 0.6mL, 80% glycerol 3.75mL, ultrapure water 25.65mL

[0223] · 10×RT buffer

[0224] 500mM Tris-HCl (pH8.4), 30mM MgCl2, 750mM KCl

[0225] · 10× Annealing Buffer

[0226] 250μM Hepes-K, 2M AcOK

[0227] · 3×RT mix (-enzyme, -primer)

[0228] 10×RT buffer (Mg:fc 3mM), 5.5mM DTT, 0.5μM dNTPs 18mM MgCl2 (sel)

[0229] · 3×RT mix (+enzyme, +primers)

[0230] 80.55 μL of 3×RT mixture (-enzyme-primer) was mixed with 7.5 μL of 100 μM RT primer and 12 μL of HMLV reverse transcriptase (RNaseH-)1710.

[0231] · 2×SDS loading buffer

[0232] Add 60 μL of SDS loading buffer, 40 μL of 10% SDS, 2 μL of 1 M DTT and mix.

[0233] · RT mix for round 1 st )

[0234] To 250 μL of 2M Tris-HCl pH 8.4 (0.1M rt) (fc 46.6 mM) were added 250 μL of 3M KCl (fc 70 mM), 85.5 μL of 2.5M MgCl2 (fc 19.9 mM), and 50 μL of 1M DTT (fc 4.67 mM) and mixed.

[0235] · 1×IMAC B1(-glycerol)

[0236] 20mM Hepes-K pH7.8, 300mM KOAc, 5mM imidazole, 0.2mM DTT

[0237] · 1×IMAC B1

[0238] 20mM Hepes-K pH7.8, 300mM KOAc, 5mM imidazole, 10% glycerol, 0.2mM DTT

[0239] · 1×IMAC B1K

[0240] 20mM Hepes-K pH7.8, 300mM KOAc, 1M KCl, 5mM imidazole, 10% glycerol, 0.2mM DTT

[0241] · 1×IMAC B1K(+ATP)

[0242] 20mM Hepes-K pH7.8, 300mM KOAc, 1M KCl, 5mM imidazole, 10% glycerol, 2mM ATP, 10mM MgSO4, 0.2mM DTT

[0243] · 1×IMAC B3

[0244] 500mM imidazole pH 7.8, 300mM KOAc, 10% glycerol, 0.2mM DTT

[0245] [Method for screening monomeric mimetic antibodies that specifically bind to target molecules based on TRAP display method]

[0246] Round 1

[0247] (Annealing, translation, reverse transcription) Annealing solution was prepared according to the mRNA concentration of each library of BC-FG and CD-FG. Annealing was performed using a PCR device under the conditions of [95°C (3 minutes) -25°C (5 minutes, 0.5°C / second)]. Using this solution, 500 μL of translation solution [11.3% (v / v) HsolA, 0.5mM 20aa, 15.4% (v / v) HsolB (-T7), 1μM mRNA / PuLin-an21 (OMe)] was prepared on ice water. After reacting in a 37°C water bath for 10 minutes, 41.7 μL of 200mM EDTA (pH 8.0) was added and mixed thoroughly, and allowed to stand at room temperature for 3 minutes. To the remaining solution, add 41.1 μL of the first round RT mixture, followed by 66.7 μL of 5 mM dNTPs, 9 μL of 111 μM Mos-G5R-T.R28, and 27.5 μL (4% v / v) of H-MMLV (RNaseH-, 28.7 μM), mix thoroughly, and perform reverse transcription reaction at 42°C on a heating block for 15 minutes.

[0248] (Gel filtration) was applied to a 2 mL column (Zeba TM After adding 690 μL of the reverse transcription solution to a spin desalting column (Spin Desalting Columns 7K, MWCO), the column was centrifuged at 2300 rpm for 2 minutes.

[0249] (Negative selection) Dynabeads washed with HBST TM M-280 streptavidin) 200 μL and magnetic beads (Dynabeads TM To the mixture of 100 μL of protein G was added 690 μL of the gel-filtered solution, and the mixture was mixed by rotation using a rotor in a thermostatic bath at 25° C. for 10 minutes.

[0250] (Positive selection) Use a strong magnet to collect the magnetic beads, recover the supernatant, add each target to a final concentration of 10 nM, mix, and incubate at room temperature for 10 minutes. At this time, prepare the solution so that the final concentration of each target is 10 nM. Add the solution to the magnetic beads (Dynabeads TM M-280 streptavidin) 200 μL and mixed. Only the supernatant was recovered and added to the magnetic beads (Dynabeads TM Protein G) 30 μL. Wash the support with 500 μL HBST. Repeat the bead washing operation once more, add 690 μL 1×PCR dNTPs to the beads from which the supernatant has been removed and suspend, and heat at 95°C for 5 minutes. After returning to room temperature, recover the supernatant and use it as the positive solution.

[0251] (Real-time PCR) 2 μL of each of the gel-filtered solution prepared with 0.5×PfSH at 1 / 10000 and the forward solution was added, and 18 μL of PCR Syber (primers: T7SD8M2.F44, MoS-RealTime.R20) was added. The cDNA contained in the sample was quantified by real-time PCR at 94°C (15 seconds) - [94°C (15 seconds) - 60°C (15 seconds) - 72°C (30 seconds)] × 40 cycles.

[0252] (PCR) Add an equal amount of 2×DEP PCR mixture to 675 μL of the forward solution. On ice, dispense 70 μL into 20 tubes and perform PCR under the conditions of 94°C (1 minute)-[94°C (20 seconds)-65°C (20 seconds)-72°C (45 seconds)]×12 cycles. Perform ΦOH / CHCl3 treatment and iPrOH precipitation (0.81 vol). Dissolve the precipitate in 135 μL of 10 mM Tris-AcOH pH 7.8 and use it as 10×1 st DNA.

[0253] Transcription for Round 2

[0254] (Transcription) Mix 10 μL of 10× T7 buffer, 1 μL of 1 M DTT, 1 μL of 2.5 M MgCl2, 20 μL of 25 mM NTPs, 1.5 μL of 2N KOH, and 39.8 μL of ultrapure water, and then add 20 μL of 10×1 stDNA, 2.5 μL of 1 μM T7 RNA polymerase. React in a 37°C thermostat for 4.5 h. Add 6 μL of 0.5 M EDTA pH 8.0 to 95.8 μL of the transcribed sample, and then add 100 μL of 0.6 M NaCl. Perform ΦOH / CHCl3 treatment and iPrOH precipitation (0.75 vol). Dissolve the precipitate in 10 μL of ultrapure water and use it as 10×1 st After determining the concentration by UV measurement, prepare 9.6 μM 1 st The second round of reaction was reduced to 7 μL and carried out in the same manner as the first round. However, RNA transcription was not performed.

[0255] Round 3

[0256] (Transcription, annealing, translation, reverse transcription) Prepare 35 μL of translation solution [11.3% (v / v) HsolA, 0.5 mM 20aa, 20 μM PuLin-an21-3 (OMe), 16.7% (v / v) HsolB (+T7), 25 nM 2 nd DNA]. Incubate in a 37°C water bath for 30 minutes and perform transcription, annealing, and translation reactions. Add 6.8 μL of 100 mM EDTA (pH 8.0) to terminate the translation reaction. Add 1 / 2 (v / v) of 3×RT mixture (+ enzyme, + primer: Mos G5R-T.R28) and incubate at 42°C for 15 minutes.

[0257] (Gel filtration) was applied to a 500 μL column (Zeba TM After adding 61.2 μL of the reverse transcription reaction solution to a centrifugal desalting column (7K, MWCO), 15 μL of HBST was added from above and centrifuged at 1500 g for 2 minutes.

[0258] (Negative screening) using magnetic beads (50% Dynabeads TM M-270+Biotin labeled human IgG1 Fc protein, Avitag TM / Dynabeads TM M-280) 55 μL was used for three negative screenings.

[0259] (Forward screening - Nkp46, 2B4) Add 112nM Fc to 92.4μL of the solution. Take 9μL of each and add 1μL of 100nM of each target, and incubate in a 25°C thermostat for 5 minutes (target concentration is 10nM). Add the solution to magnetic beads (Dynabeads TMM-270 streptavidin) 0.5 μL, the magnet was washed twice with HBST. 40 μL of 1×PCR dNTPs was added to the beads and suspended, and heated at 95°C for 5 minutes. After returning to room temperature, the supernatant was recovered and used as the forward solution.

[0260] (Positive screening - Nkp30) Add 2.11 μL of HBST to 16.8 μL of the solution. Then, take 9 μL of each and add 1 μL of 100 nM of each target, and incubate in a thermostat at 25°C for 5 minutes. Add the solution to the magnetic beads (Dynabeads TM Protein G) 0.5 μL, the magnet was washed twice with HBST. 40 μL of 1×PCR dNTPs was added to the beads from which the supernatant was removed and suspended, and heated at 95°C for 5 minutes. After returning to room temperature, the supernatant was recovered and used as the forward solution. Real-time PCR and PCR were performed in the same manner as the second round.

[0261] After the 4th round, the reaction scale was set to 5 μL, and the experiment was performed in the same manner. In addition, after the 6th round, the target concentration was set to 1 nM. When a further bound monomeric mimic antibody was obtained, the bead washing conditions were made more stringent and the screening operation was performed. The obtained DNA was analyzed using a next-generation sequencer.

[0262] clone

[0263] The MuPaC method (Taniguchi, N.; Nakayama, S.; Kawakami, T.; Murakami, H., Patchcloning method for multiple site-directed and saturation mutagenesis. BMC Biotechnol. 2013, 13: 91.) was used to construct Figure 1 The expression vector shown in .

[0264] Expression and purification of monomeric mimetic antibodies

[0265] The obtained plasmid was used to transform BL21(DE3)LysS and expressed with 0.5mM IPTG at 25°C. 30mL of developing solution was added to the cells, and ultrasonic disruption was performed on ice water. 1.8mL of 80% glycerol was added to the disrupted liquid, and centrifuged at 4°C and 13000rpm for 10 minutes. Filtered, and NGC TM The purification was carried out by chromatography system (BIO-LAD).

[0266] Evaluation of Monomeric Antibodies Based on Dissociation Constant Determination

[0267] The binding ability and specificity were confirmed using Octet (registered trademark) RED96 (ForteBio) using biomembrane interferometry. The measurement was performed with a program of immersion (Dip) for 50 seconds, loading (Load) for x seconds (until the signal reached 1.0nm (NKp46, 2B4) or 0.8nm (Nkp30)), baseline (Baseline) for 60 seconds, association (Association) for 600 seconds, and dissociation (Dissociation) for 600 seconds. At this time, the concentration of monomeric pseudo antibodies was all unified to 40nM. The obtained data was analyzed using ForteBio DataAnalysis 10.0. The dissociation constant was determined under various conditions only for monomeric pseudo antibodies for which binding ability and specificity were observed. The measurement conditions are shown in the table below. Immersion, baseline, binding, and dissociation were performed at the same time as when the binding ability and specificity were confirmed. As for the concentration of monomeric pseudo antibodies, 5 concentrations were prepared for measurement by serial dilution in a 2-fold series starting from the most concentrated concentration.

[0268] [Table 1]

[0269] Table 1 Conditions for determination of dissociation constants

[0270]

[0271] [Example 1: Sorting using a monomeric mimetic antibody library]

[0272] The human fibronectin type III domain (FN3) (PDB: 1FNA) was used as a scaffold (skeleton) to randomize a portion of the sequence to prepare a monomeric pseudo-antibody library, and the TRAP display method (Transcription-translation coupled with Association of Puromycin-linker display) was used to screen out monomeric pseudo-antibodies that specifically bind to three proteins on NK cells (NKp46, 2B4, and NKp30). It should be noted that the human fibronectin type III domain has 7 β chains, and there are 6 loops connecting each β chain. In this embodiment, a BC library with changed BC and FG loops and a CD library with changed CD and FG loops were prepared. Figure 2 The libraries are shown in .

[0273] The following table shows the mRNA sequence and translated amino acid sequence of the library used. The ATG in bold is the start codon, the underlined part is the insertion sequence, the bold and underlined part is the flexible linker sequence, the shaded part is the random part, and the subscript number represents the number of randomized amino acids. The random part is designed by inserting 20% ​​Tyr, 10% Ser, 10% Gly, 15% Trp, and 3% other amino acids other than Cys.

[0274] [Table 2]

[0275] mRNA sequences of BC-FG library

[0276]

[0277] In addition, the primers used are shown below.

[0278] [Table 3]

[0279] Table 3 List of synthetic oligonucleotides (5'->3')

[0280]

[0281] In terms of protein diversity, the BC-FG library was 2.6 × 10 13 , CD-FG is 4.4×10 13 In addition, the recovery rate of cDNA in each round obtained by TRAP display method for each target is shown in Figure 3 and 4 The recovery rate was calculated by dividing the amount of cDNA recovered by the amount of puromycin linker contained in the system. The first and second rounds were screened with mixed targets, and then each target was screened separately. The target concentration was initially set to 10 nM, and the recovery rate increased significantly, which was then reduced to 1 nM. Then, in order to obtain k off The blue graph on the right side of the results for each target shows the recovery rate in the negative screening when the target is not bound to the beads, and the orange graph shows the recovery rate in the positive screening when the target is bound to the beads. Figure 4 Among all targets, the recovery rate of the 7th round of positive screening was higher than that of the negative screening, indicating that a monomeric pseudo-antibody binding to the target was successfully obtained.

[0282] [Example 2: Sequence analysis based on next generation sequencer]

[0283] The cDNAs recovered from the seventh round of positive screening and negative screening were analyzed using a next generation sequencer, and then several monomeric mimetics were screened out. The screened monomeric mimetics are shown in Tables 4 to 6.

[0284] It should be noted that, when NKp46 was used as the target, 3 monomeric mimetics with a P / N ratio of 3.0 or more were screened for the BC-FG library. The total number of reads was 370854. For the CD-FG library, 2 monomeric mimetics with a P / N ratio of 5.0 or more were screened. The total number of reads was 251088.

[0285] When 2B4 was used as the target, two monomeric mimetics with a P / N ratio of 4.0 or higher were screened for the BC-FG library. The total number of reads was 502168. Two monomeric mimetics with a P / N ratio of 2.5 or higher were screened for the CD-FG library. The total number of reads was 237279.

[0286] When targeting NKp30, two monomeric mimetics with a P / N ratio of 5.0 or higher were screened for the BC-FG library. The total number of reads was 424,980. One monomeric mimetics with a P / N ratio of 4.0 or higher was screened for the CD-FG library. The total number of reads was 256,719.

[0287] [Table 4]

[0288] Table 4 Results of sequence analysis of monomeric mimetic antibody libraries targeting NKp46 based on next generation sequencers (BC-FG: upper row CD-FG: lower row)

[0289]

[0290]

[0291] [Table 5]

[0292] Table 5 Results of sequence analysis of monomeric mimetic antibody libraries targeting 2B4 based on next generation sequencers (BC-FG: upper row CD-FG: lower row)

[0293]

[0294]

[0295] [Table 6]

[0296] Table 6 Results of sequence analysis of monomeric mimetic antibody libraries targeting NKp30 based on next generation sequencers (BC-FG: upper row CD-FG: lower row)

[0297]

[0298]

[0299] In the above table, the P / N ratio is calculated by dividing the appearance rate of a certain sequence in the positive screening solution by the appearance rate in the negative screening solution. When the value is low, it can be considered that the possibility of non-specific binding is increased. The P / N ratio ' is calculated by multiplying the value obtained by dividing the recovery rate of the positive screening by the recovery rate of the negative screening by the P / N ratio.

[0300] [Example 3: Expression and purification]

[0301] The monomeric mimetic antibody screened in Example 2 was fused with the Sortase A recognition site and Histag and expressed using NGC TM Purification was performed by chromatography system. Success of purification was confirmed by SDS-PAGE. Table 7 shows the primers used in cloning of monomeric mimetibodies. In addition, Table 8 shows an overview of the yield of clones.

[0302] [Table 7-1]

[0303] Table 7 List of synthetic oligonucleotides (5'->3')

[0304]

[0305] [Table 7-2]

[0306]

[0307] [Table 8]

[0308] Table 8 Summary of clone yields

[0309]

[0310] [Example 4: Determination of dissociation constant based on biomembrane interferometry]

[0311] The binding ability and specificity of all clones expressed and purified in Example 3 were confirmed, and the dissociation constant was further determined. Figures 5 to 7 The measurement results are shown in . Figures 5 to 7 In the upper row of graphs, the upper line shows the result of measuring the binding ability of the monomeric pseudo-antibody by binding the target to the front end of the sensor, and the lower line shows the result of confirming whether the monomeric pseudo-antibody is bound to the sensor without binding the target. Mb in the graph is an abbreviation for the concentration of the monomeric pseudo-antibody. All clones were confirmed to bind to the monomeric pseudo-antibody when the target was fixed on the sensor, and no monomeric pseudo-antibody binding was confirmed when the target was not fixed. In addition, Figures 5 to 7 The lower row of graphs shows the results of measuring the dissociation constants at five different concentrations.

[0312] The following table shows the evaluation results of the clones such as binding ability and the presence or absence of specificity.

[0313] [Table 9]

[0314] Table 9 Summary of binding results of clones

[0315]

[0316] As described above, two libraries, BC-FG and CD-FG, obtained by randomizing two loops, were used to screen for monomeric pseudo-antibodies that bind to proteins on NK cells using the TRAP display method. In the 7th round, the recovery rate of the positive screening in which the target was fixed to the magnetic beads was higher than the recovery rate of the negative screening in which the target was not fixed to the magnetic beads. Therefore, it can be seen that a monomeric pseudo-antibody that binds to the target as the target has been successfully obtained. Then, the cDNA obtained from the positive screening and negative screening in the 7th round was analyzed using a next-generation sequencer, and the expressed clones were screened out based on the appearance rate and P / N ratio. Based on the appearance rate, it was confirmed that the monomeric pseudo-antibody was successfully concentrated. In addition, using the expressed and purified clones and measuring the dissociation constant using the biomembrane interferometry method, a strongly binding monomeric pseudo-antibody (K D =1.0×10 -2 This is almost the same as the dissociation constant of antibodies used as antibody drugs, and therefore it is considered to be a sufficient dissociation constant value for use. off Slow to 1.0×10 -6 s -1 ~5.0×10 -4 s -1 , it can be said that a target monomeric pseudo-antibody was produced that binds to proteins on NK cells and is not easily dissociated.

Claims

1. A monomeric mimetic antibody having an amino acid sequence of any one of (1) to (3) and binding to a surface protein of a NK cell, (1) the amino acid sequence represented by any one of SEQ ID NOs: A1 to A12; (2) an amino acid sequence represented by any one of SEQ ID NOs: A1 to A12, wherein one or more amino acids are deleted, substituted and / or added in the amino acid sequence other than any amino acid; (3) an amino acid sequence having 80% or more identity with any amino acid sequence other than any amino acid in the amino acid sequence represented by any of SEQ ID NOs: A1 to A12; In serial numbers A1 to A12, X 1 , X 2 , X 3 , X 4 , X 5 and X 6 Each independently represents any amino acid, n1, n2, n3, n4, n5 and n6 each independently represent any natural number, [Chemical formula 1] X 1 n1 PIWGRYGT X 2 n2 TVRGQTYYWWML X 3 n3 (Serial No. A1) X 1 n1 WTWLGYEYYW X 2 n2 WDSKPGAIGI X 3 n3 (Serial No. A 2 ) X 1 n1 TYWDWGFR X 2 n2 WSWARRTHISWF X 3 n3 (Serial No. A3) X 4 n4 PGYYMWQRGVIN X 5 n5 GQERAYGHPEHF X 6 n6 (Serial No. A4) X 4 n4 RYKYHRRIN X 5 n5 GTQWYVFNEAGDVAGY X 6 n6 (Serial No. A5) X 1 n1 GPIVSYSWWYHW X 2 n2 VGYVNEKYDAWQSYVQ X 3 n3 (Serial No. A6) X 1 n1 QAYWFEYTWW X 2 n2 LEWVYDDQWTSG X 3 n3 (Serial No. A7) X 4 n4 TSPWVYNGY X 5 n5 DQYKSWNLEEFH X 6 n6 (Serial No. A8) X 4 n4 TPPWIHKGW X 5 n5 VTPYGRKWVGMNGW X 6 n6 (Serial No. A9) X 1 n1 LWHYYGDQYW 2 n2 WMYVPEYYFNPWX 3 n3 (Serial No. A10) X 1 n1 TDRNYDITYGMQ X 2 n2 YWWDGYSYSEEAWYY X 3 n3 (Serial No. A11) X 4 n4 WSWESYWHGTY X 5 n5 TFMKDIPSYY X 6 n6 (Serial number A12).

2. The monomeric mimetic antibody according to claim 1, wherein The surface protein is at least one selected from the group consisting of NKp46, NKp30 and 2B4.

3. The monomeric mimetic antibody according to claim 1, wherein Among the amino acid sequences represented by SEQ ID NOs: A1 to A12, the amino acid sequences represented by SEQ ID NOs: 1 to 12 are present in the BC loop or the CD loop, and the amino acid sequences represented by SEQ ID NOs: 13 to 24 are present in the FG loop, [Chemical formula 2] PIWGRYGT (Serial No. 1) WTWLGYEYYW (Serial No. 2) TYWDWGFR (Serial No. 3) PGYYMWQRGVIN (Serial No. 4) RYKYHRRIN (Serial No. 5) GPIVSYSWWYHW (Serial No. 6) QAYWFEYTWW (Serial No. 7) TSPWVYNGY (Serial No. 8) TPPWIHKGW (Serial No. 9) LWHYYGDQYW (serial number 10) TDRNYDITYGMQ (Serial No. 11) WSWESYWHGTY (Serial No. 12) TVRGQTYYWWML (Serial No. 13) WDSKPGAIGI (Serial No. 14) WSWARRTHISWF (Serial No. 15) GQERAYGHPEHF (Serial No. 16) GTQWYVFNEAGDVAGY (serial number 17) VGYVNEKYDAWQSYVQ (Serial No. 18) LEWVYDDQWTSG (Serial No. 19) DQYKSWNLEEFH (serial number 20) VTPYGRKWVGMNGW (serial number 21) WMYVPEYYFNPW (serial number 22) YWWDGYSYSEEAWYY (serial number 23) TFMKDIPSYY (serial number 24). 4.NK cells, among which The monomeric mimetibody according to any one of claims 1 to 3 is bound to a surface protein.

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