WO2024210072A1

WO2024210072A1 - Chimeric protein, nucleic acid, vector, t cell or nk cell, pharmaceutical composition, combined product, combined pharmaceutical, and target antigen binding protein

Info

Publication number: WO2024210072A1
Application number: PCT/JP2024/013334
Authority: WO
Inventors: 友活伊川; 司重廣
Original assignee: 学校法人東京理科大学
Priority date: 2023-04-06
Filing date: 2024-03-29
Publication date: 2024-10-10

Abstract

Provided are: a chimeric protein which comprises an extracellular domain capable of recognizing and binding to a peptide tag comprising the amino acid sequence represented by SEQ ID NO:1, a transmembrane domain, and an intracellular domain containing a signal domain capable of activating the cytotoxicity of a T cell or an NK cell when the extracellular domain is bound to the peptide tag; and a nucleic acid, a vector, a T cell or an NK cell, a pharmaceutical composition, a combined product, a combined pharmaceutical, and a target antigen binding protein, each associated with the chimeric protein.

Description

Chimeric proteins, nucleic acids, vectors, T cells or NK cells, pharmaceutical compositions, combinations, combination drugs, and target antigen-binding proteins

The present disclosure relates to chimeric proteins, nucleic acids, vectors, T cells or NK cells, pharmaceutical compositions, combinations, combination drugs, and target antigen binding proteins.

In recent years, research has been conducted into CAR-T and CAR-NK, which involve introducing a chimeric antigen receptor (CAR) into T cells or NK cells, and using the cytotoxic activity of the T cells or NK cells to damage target cells. CAR-T and CAR-NK technologies have been developed primarily for cancer treatment, in which cancer-related antigens expressed on the surface of cancer cells are targeted to attack the cancer cells. However, based on the principle, if there is an antigen expressed on the surface of the target cell, it is possible to damage the target cell by using a CAR against that antigen, and therefore research is also being conducted into the treatment of viral infections by targeting virus-infected cells (see, for example, Non-Patent Documents 1 and 2).

CARs often contain modified antibodies that specifically recognize and bind to antigens expressed on the surface of target cells. However, when CARs contain modified antibodies specific to target antigens, it is necessary to prepare CARs for each target antigen and introduce them into T cells or NK cells for use. Therefore, methods using CAR-T that utilize specific binding such as avidin-biotin (Non-Patent Documents 3 and 4), leucine zipper (Non-Patent Document 5), or FITC-anti-FITC antibody (Non-Patent Document 6), rather than target antigen-target cell-specific antibodies, have also been attempted. In these modified methods, T cells introduced with modified genes cannot directly recognize and bind to target cells. However, for example, in CARs, T cells that specifically bind to biotin can be prepared by adding avidin instead of target cell-specific antibodies. If a target antigen-specific antibody labeled with biotin is administered and allowed to bind to target cells, the avidin-introduced T cells can specifically recognize and bind to biotin, bind to the target cells, and attack the target cells.

In the above modified method, there is no need to prepare T cells or NK cells for each target antigen, and it is possible to target multiple target antigens and attack cells expressing the target antigens simultaneously or at different times. In addition, when NK cells are used, there is no need to consider rejection reactions due to tumor histocompatibility antigens, so not only autologous but also allogeneic NK cells can be used.

On the other hand, there is a demand for the development of further technologies based on cytotoxic activity. In view of this situation, the present disclosure relates to the provision of a novel chimeric protein capable of exerting cytotoxic activity; a nucleic acid encoding the chimeric protein; a vector containing the nucleic acid; a T cell or NK cell expressing the chimeric protein; a pharmaceutical composition, a combination, and a combination drug using these; and a target antigen-binding protein that can be used in combination with the chimeric protein.

As a result of extensive research into new methods that can exert cytotoxic activity, the inventors discovered that high cytotoxic activity can be obtained by using a chimeric protein that has an extracellular domain that recognizes and binds to a peptide tag containing the amino acid sequence of SEQ ID NO:1, and thus completed the present invention.

The present disclosure includes the following aspects.
<1> An extracellular domain that recognizes and binds to a peptide tag comprising the amino acid sequence of SEQ ID NO: 1;
A transmembrane domain;
an intracellular domain including a signal domain that activates the cytotoxic activity of a T cell or a NK cell when the extracellular domain binds to the peptide tag;
A chimeric protein comprising:
<2> The chimeric protein according to <1>, wherein the extracellular region does not contain a receptor or a ligand that binds to a ligand or a receptor other than the peptide tag.
<3> The chimeric protein according to <1> or <2>, wherein the extracellular domain comprises a single-chain antibody comprising a light chain variable region and a heavy chain variable region of an immunoglobulin that recognizes and binds to the peptide tag.
<4> The chimeric protein according to any one of <1> to <3>, wherein the intracellular domain comprises a signal domain of CD3ζ.
<5> A nucleic acid encoding the chimeric protein according to any one of <1> to <4>.
<6> A vector comprising the nucleic acid according to <5>.
<7> A T cell or NK cell expressing the chimeric protein according to any one of <1> to <4>.
<8> A pharmaceutical composition for damaging a target cell, comprising the chimeric protein according to any one of <1> to <4>, the nucleic acid according to <5>, or the T cell or NK cell according to <7>.
<9> The pharmaceutical composition according to <8> for treating cancer.
<10> The pharmaceutical composition according to <8> or <9>, which is administered to a subject simultaneously or sequentially in combination with a target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen, and a peptide tag comprising the amino acid sequence of SEQ ID NO: 1.
<11> The pharmaceutical composition according to <10>, wherein the target antigen is a tumor-associated antigen or a viral antigen.
<12> The chimeric protein according to any one of <1> to <4>, the nucleic acid according to <5>, or the T cell or NK cell according to <7>,
a target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen and a peptide tag comprising the amino acid sequence of SEQ ID NO:1;
A combination comprising:
<13> A pharmaceutical composition comprising the chimeric protein according to any one of <1> to <4>, the nucleic acid according to <5>, or the T cell or NK cell according to <7>;
a pharmaceutical composition comprising a target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen and a peptide tag comprising the amino acid sequence of SEQ ID NO:1;
A combination medicine comprising:
<14> A target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen, and a peptide tag comprising the amino acid sequence of SEQ ID NO: 1.
<15> The target antigen-binding protein according to claim 14, further comprising a ligand or receptor that recognizes a receptor or ligand on a T cell or a NK cell.
<16> A pharmaceutical composition comprising the target antigen-binding protein according to <14> or <15>, which is used to express the target antigen-binding protein in a target cell by introducing a gene encoding the target antigen-binding protein into the target cell.

The present disclosure provides a novel chimeric protein capable of exerting cytotoxic activity; a nucleic acid encoding the chimeric protein; a vector containing the nucleic acid; a T cell or NK cell expressing the chimeric protein; a pharmaceutical composition, a combination, and a combination drug using these; and a target antigen-binding protein that can be used in combination with the chimeric protein.

FIG. 1 shows a schematic diagram of a chimeric protein and a target antigen binding protein in one embodiment of the present disclosure. 1 shows the results of evaluating the activity of chimeric protein-expressing Jurkat cells targeting CD19-expressing K562 cells in Example 1. 1 shows the results of evaluating the activity of chimeric protein-expressing Jurkat cells targeting SARS-Cov-2 spike protein-expressing 293T cells, NALM6 cells, SK-N-SH cells, or CHP134 cells in Example 1. 1 shows the cytotoxic activity of chimeric protein-expressing NK92MI cells targeting NALM6 cells in Example 2. 1 shows the cytotoxic activity of chimeric protein-expressing NK92MI cells targeting MDA-MB-453 cells in Example 3. 1 shows the cytotoxic activity of chimeric protein-expressing mouse T cells targeting hCD19-B16 cells in Example 4. FIG. 1 shows a schematic diagram of a portion of a plasmid encoding a target antigen-binding protein gene containing ULBP1 or ULBP2 and a 3×FLAG® tag in Example 5. 13 is a graph showing the results of flow cytometry demonstrating that NK92MI cells expressing a chimeric protein express NKG2D in the same manner as NK92MI cells not expressing a chimeric protein (Mock) in Example 5. 1 shows the cytotoxic activity of chimeric protein-expressing NK92MI cells targeting MDA-MB-453 cells in Example 5. FIG. 1 is a graph showing the results of flow cytometry demonstrating high expression of NKG2D in CD8+ T cells. 1 shows the cytotoxic activity of chimeric protein-expressing human T cells targeting MDA-MB-453 cells in Example 6. 1 shows the amino acid and DNA sequences of a target antigen binding protein in one embodiment. 1 shows the amino acid and DNA sequences of a target antigen binding protein in one embodiment. 1 shows the amino acid and DNA sequences of a target antigen binding protein in one embodiment. 1 shows the amino acid and DNA sequences of a target antigen binding protein in one embodiment. 1 shows the amino acid and DNA sequences of a target antigen binding protein in one embodiment. 1 shows the amino acid and DNA sequences of a target antigen binding protein in one embodiment. The amino acid sequence and DNA sequence of the chimeric protein used in the examples are shown below.

Hereinafter, embodiments of the present disclosure will be described.
In the present disclosure, a numerical range expressed using "to" means a range including the numerical values described before and after "to" as the lower and upper limits. In the numerical ranges described in stages in the present disclosure, the upper or lower limit described in a certain numerical range may be replaced with the upper or lower limit of another numerical range described in stages. In addition, in the numerical ranges described in the present disclosure, the upper or lower limit described in a certain numerical range may be replaced with a value shown in the examples.
In the present disclosure, the "identity" of an amino acid sequence or a base sequence refers to the ratio of the number of identical amino acid residues or the number of identical nucleic acid residues to the total number of amino acid residues or the total number of bases when the two sequences to be compared are aligned by inserting appropriate gaps into one or both of them as necessary so as to maximize the number of matches. The alignment can be performed using a well-known alignment tool such as BLAST, FASTA, or CLUSTAL W. For example, the alignment can be evaluated with the default parameters of BLAST.
In the present disclosure, the term "antibody" includes not only full-length antibodies, but also minibodies (including antibody fragments and modified antibodies) that recognize and bind to antigens.
In the present disclosure, even if an element is described in the singular form, it does not exclude the presence of a plurality unless there is a technical contradiction, unless otherwise expressly stated.
When an embodiment of the present disclosure is described with reference to the drawings, the configuration of the embodiment is not limited to the configuration shown in the drawings. In addition, the size of the members in each drawing is conceptual, and the relative relationship between the sizes of the members is not limited to this.

Chimeric proteins
The chimeric protein of the present disclosure comprises an extracellular domain that recognizes and binds to a peptide tag comprising the amino acid sequence of SEQ ID NO:1 (hereinafter also simply referred to as "peptide tag"), a transmembrane domain, and an intracellular domain that comprises a signal domain that activates the cytotoxic activity of a T cell or NK cell when the extracellular domain binds to the peptide tag.

If the chimeric protein of the present disclosure is expressed in T cells or NK cells, for example, it is not necessary to prepare T cells or NK cells for each target antigen, and by using an antigen-binding protein with a peptide tag in combination, it becomes possible to recognize the target antigen and damage the target cell. This makes it possible to target, for example, multiple types of antigens (e.g., two or more types, or three or more types), and to effectively damage the target cell. In addition, the chimeric protein of the present disclosure uses a peptide tag containing the amino acid sequence of SEQ ID NO: 1. The present inventors have found that the CAR application technology using the chimeric protein of the present disclosure unexpectedly expresses high cytotoxicity. Therefore, the chimeric protein of the present disclosure may be particularly useful for treatment targeting a specific antigen, such as treatment of cancer or infectious disease.
In addition, the immunogenicity of avidin poses an obstacle to clinical application in the avidin-biotin techniques described in Non-Patent Documents 3 and 4. On the other hand, the technique disclosed herein, which uses peptide tags, can reduce the problem of immunogenicity.

FIG. 1 shows a schematic diagram of a chimeric protein and a target antigen-binding protein in one embodiment. The chimeric protein 10 comprises extracellular domains 1a and 1b, a transmembrane domain 3 that penetrates the cell membrane CM, and an intracellular domain 5. The target antigen-binding protein 20 used in combination comprises a peptide tag 11 and target antigen-binding domains 13a and 13b.

In one aspect, it is preferable that the chimeric protein does not contain a receptor or ligand that binds to a ligand or receptor other than the peptide tag in the extracellular region. For example, it is preferable that the chimeric protein does not contain an immunoglobulin light chain variable region (VL region) and/or heavy chain variable region (VH region) that binds to an antigen other than the peptide tag in the extracellular region. In particular, it is preferable that the chimeric protein does not contain a target antigen-binding domain that recognizes and binds to a target antigen other than the peptide tag in the extracellular region. Even if the chimeric protein does not contain a target antigen-binding domain itself, it is possible to target multiple types of antigens (e.g., two or more types, or three or more types) and damage cells by using it in combination with a target antigen-binding protein described below.

Each region of the chimeric protein is described in detail below. Each region of the chimeric protein shown below may be obtained by introducing a mutation into the natural protein from which it is derived, to the extent that the function of each region can be maintained, and may have an amino acid sequence identity of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more with the natural protein. Alternatively, each region of the chimeric protein may be obtained by replacing 1 to 30, 1 to 15, 1 to 10, or 1 to 5 amino acids of the natural protein from which it is derived with other amino acids, to the extent that the function of each region can be maintained.

<Extracellular domain>
The extracellular domain of the chimeric protein recognizes and binds to the peptide tag. The extracellular domain preferably includes an antibody or ligand that recognizes and binds to the peptide tag, and more preferably includes an antibody that recognizes and binds to the peptide tag. Examples of the antibody include full-length antibodies and low molecular weight antibodies (including antibody fragments or modified antibodies) that recognize and bind to antigens. Examples of the low molecular weight antibodies include F(ab') ₂ , Fab', Fab, Fv, rIgG, single chain antibodies (scFv), VHH antibodies, diabodies, and the like. The extracellular domain preferably includes a low molecular weight antibody such as scFv, Fab, or diabody that includes the variable regions of the light and heavy chains of immunoglobulin that specifically recognize the peptide tag, and more preferably includes an scFv that includes the variable regions of the light and heavy chains of immunoglobulin that specifically recognize the peptide tag. ScFv is often used in conventional chimeric antigen receptors and is a preferred low molecular weight antibody.

Antibodies can be prepared by known methods. Alternatively, an already prepared antibody can be used. For example, total RNA can be extracted from a hybridoma producing the desired antibody, cDNA can be synthesized, and the variable regions of the heavy and light chains can be amplified by PCR and cloned to determine the gene sequence. The gene sequence of the constant region of the antibody can also be obtained by a similar method. Alternatively, full-length cDNA of the heavy and light chains of the antibody can be prepared by the RACE method. A desired antibody can be selected from an antibody phage library to obtain a minibody. The obtained gene sequence can also be incorporated into an appropriate vector to prepare other types of minibodies or complete antibodies.

For example, an scFv antibody can be prepared by linking the VH and VL regions with an appropriate peptide linker sequence. The sequence of the peptide linker is not particularly limited, and preferably has 2 to 20 amino acid residues, more preferably 5 to 15. For example, known peptide linkers such as the GS linker [(GGGGS) ₃ ] (SEQ ID NO: 3) and the 218 linker [STSGSGKPGSGEGSTKG] (SEQ ID NO: 4) can be used.

The peptide tag that can be recognized and bound by the extracellular domain contains the amino acid sequence of SEQ ID NO: 1 shown below. The peptide having the amino acid sequence of SEQ ID NO: 1 is also called a FLAG (registered trademark) tag.

DYKDDDDK (sequence number 1)

The peptide tag may consist of only the amino acid sequence of SEQ ID NO: 1, or may have a sequence that combines the amino acid sequence of SEQ ID NO: 1 with other amino acid sequences, so long as it is recognized and bound by the extracellular domain of the chimeric protein. For example, the peptide tag may be a peptide tag having the amino acid sequence of SEQ ID NO: 2. A peptide having the amino acid sequence of SEQ ID NO: 2 is also called a 3xFLAG (registered trademark) tag.

MDYKDHDGDYKDHDIDYKDDDDK (Sequence Number 2)

The peptide tag is preferably a peptide tag of 8 to 100 amino acids in length that contains the amino acid sequence of SEQ ID NO: 1 or 2, more preferably a peptide tag of 8 to 50 amino acids in length that contains the amino acid sequence of SEQ ID NO: 1 or 2, and even more preferably a peptide tag having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The extracellular domain may be any domain that recognizes and binds to a peptide tag. The extracellular domain preferably includes an antibody or ligand that recognizes DYKDDDDK (SEQ ID NO: 1). An extracellular domain that recognizes and binds to a peptide tag can be obtained, for example, by producing an antibody specific to the peptide tag using the method described above. A ligand that recognizes a peptide tag can be prepared using a known method. For example, a desired ligand may be selected from a phage library.

An example of an extracellular domain that recognizes and binds to a peptide tag containing the amino acid sequence of SEQ ID NO:1 is an extracellular domain containing the FLAG (registered trademark) M2 antibody having the following amino acid sequence and DNA sequence described in J Proteome Res 20 (7), 3559-3566 (2021). The FLAG (registered trademark) M2 antibody is an antibody against the FLAG (registered trademark) tag having the amino acid sequence of SEQ ID NO:1. The following sequence is shown in the order [VL region]-[218 linker]-[VH region] from the N-terminus.

Amino acid sequence (SEQ ID NO:5)
SDVLMTQIPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIRRASTSGSGKPGSGEGSTKGHVSQVQLQQSAAELARPGASVKMSCKASGYSFTTYTIHWVKQRPGQGLEWIGYINPSSGYAAYNQNFKDETTL TADPSSSTAYMELNSLTSEDSAVYYCAREKFYGYDYWGQGATLTVSSA

DNA sequence (SEQ ID NO:6)

Furthermore, the amino acid sequence of the antibody in the extracellular domain may have 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more identity to SEQ ID NO: 5. Similarly, the DNA sequence encoding the antibody in the extracellular domain may have 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more identity to SEQ ID NO: 6.

When the extracellular domain contains an antibody or ligand that recognizes and binds to the peptide tag, the extracellular domain may contain other components. In addition, the antibody or ligand of the extracellular domain and the transmembrane domain may be linked via a spacer. As the spacer, for example, a polypeptide having 2 to 300 amino acid residues, preferably 10 to 100, and more preferably 20 to 50 amino acid residues can be used.

<Transmembrane domain>
The transmembrane domain fixes the chimeric protein to the cell membrane of T cells or NK cells described below and links it to the intracellular domain. Examples of the transmembrane domain include, but are not limited to, the transmembrane domains of membrane proteins such as CD2, CD3ε, CD3, CD4, CD5, CD8α, CD8β, CD28, CD134, CD137, ICOS, NKG2D, and CD154. Among the transmembrane domains, the transmembrane domains of CD8, CD28, and NKG2D, which have been used in chimeric antigen receptors, can be particularly preferably used.

The amino acid sequences and DNA sequences of the transmembrane domains of CD8, CD28, and NKG2D are publicly known. Below are examples of the amino acid sequences and DNA sequences of CD8, CD28, and NKG2D. Note that some of the DNA sequences shown below have been codon-optimized. In addition, among the DNA sequences shown below, the hinge region is an extracellular region that is connected to the transmembrane domain, and is also described in this section.

CD8A hinge region (NM_001768, AA132-182)
Amino acid sequence (SEQ ID NO:7)
GTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

DNA sequence (SEQ ID NO:8)
ggtaccaccacaacgcccgctcctcggccaccgacgccagcgccaactattgcgagtcagcctctcagtctgcgacctgaggcttgtcgaccagcagccggaggcgcagtgcacacgagggggctggacttcgcctgtgat

CD8A transmembrane domain (NM_001768, AA183-203)
Amino acid sequence (SEQ ID NO:9)
IYIWAPLAGTCGVLLLSLVIT

DNA sequence (SEQ ID NO: 10)
atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcacc

CD28 hinge region (NM_006139, AA114-150)
Amino acid sequence (SEQ ID NO:11)
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

DNA sequence (SEQ ID NO:12)
attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc

CD28 transmembrane domain (NM_006139, AA153-179)
Amino acid sequence (SEQ ID NO:13)
FWVLVVVGGVLACYSLLVTVAFIIFWV

DNA sequence (SEQ ID NO:14)
ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg

NKG2D transmembrane domain (NM_007360, AA52-72)
(Although it is a type II transmembrane protein, the left side represents the N-terminus.)
Amino acid sequence (SEQ ID NO: 15)
PFFFCCFIAVAMGIRFIIMVT

DNA sequence (SEQ ID NO: 16)
acggttatgataattttccgaattggaatggcagtcgcgatcttctgctgtttcttctttcca

<Intracellular domain>
The intracellular domain contains a signaling domain that has the function of activating the cytotoxic activity of T cells or NK cells when the extracellular domain of the chimeric protein binds to the peptide tag.

Whether or not a signal domain activates the cytotoxic activity of T cells or NK cells can be confirmed by the following methods. After culturing T cells or NK cells on cells presenting a target antigen or on a plate to which the target antigen is bound, the expression levels of CD69, CD107, IFNγ, and other cytotoxic activity factors of T cells or NK cells can be analyzed using flow cytometry, ELISA, ELISPOT, Western blotting, and the like. In addition, after co-culturing T cells or NK cells with cells presenting a target antigen, the viability of the target cells can be measured by a chromium release assay, a fluorescent dye release assay, a dead cell staining method, a fluorescent imaging method, and the like.

As a signal domain having the function of activating the cytotoxic activity of T cells or NK cells, a known signal domain that is known to activate the cytotoxic activity of T cells or NK cells may be used. The signal domain can be designed and produced by the following method. A gene encoding the signal domain can be obtained by producing the intracellular region of an activating receptor of T cells or NK cells by PCR or chemical synthesis. The intracellular domain can be introduced into the 3' end of the transmembrane domain of the chimeric protein by PCR, restriction enzyme method, assembly method, etc. Furthermore, when producing a chimeric protein having two or more signal domains, the order is not limited, and they can be linked using PCR, restriction enzyme method, gene assembly method, etc.

In the present disclosure, the "signal domain" included in the intracellular domain means an intracellular signaling region. For example, the signal domain of CD3ζ means the signaling region of CD3ζ.
Examples of the signal domain include the signal domains of CD3ζ, CD2, CD4, CD5, CD8α, CD8β, CD27, CD28, CD134, CD137, ICOS, OX40, CD244, CD226, and CD154. The intracellular domain may contain one signal domain or may contain two or more signal domains. When the intracellular domain contains two or more signal domains, the positional relationship between them is not particularly limited. A fusion of the signal domain of CD28 and the signal domain of CD3ζ, which are often used in chimeric antigen receptors, and a fusion of the signal domain of CD137 (4-1BB) and the signal domain of CD3ζ can be particularly preferably used.

The amino acid and nucleic acid sequences of the intracellular domains exemplified above are publicly known. Below are shown, as examples, the amino acid and DNA sequences of the signal domains of CD3ζ, CD28, CD137, CD244, and CD226.

CD3ζ (NM_198053, AA51-164)
Amino acid sequence (SEQ ID NO:17)
LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

DNA sequence (SEQ ID NO:18)
ctgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggc ggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

CD28 (NM_006139, AA180-220)
Amino acid sequence (SEQ ID NO: 19)
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

DNA sequence (SEQ ID NO:20)
aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccggggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc

CD137 (NM_001561, AA214-255)
Amino acid sequence (SEQ ID NO:21)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

DNA sequence (SEQ ID NO:22)
aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg

CD244 (NM_016382, AA246-365)
Amino acid sequence (SEQ ID NO:23)
WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS

DNA sequence (SEQ ID NO:24)
tggaggagaaagaggaaggagaagcagtcagagaccagtcccaaggaatttttgacaatttacgaagatgtcaaggatctgaaaaccaggagaaatcacgagcaggagcagacttttcctggaggggggagcaccatctactctatgatccagtcccagtcttctgctcccacgtcacaagaacctgcatatacatattatattcattaattcagcct tccaggaagtctggatccaggaagaggaaccacagcccttccttcaatagcactatctatgaagtgattggaaagagtcaacctaaagcccagaaccctgctcgattgagccgcaaagagctggagaactttgatgtttattcc

CD226 (NM_006566, AA276-336)
Amino acid sequence (SEQ ID NO:25)
NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV

DNA sequence (SEQ ID NO:26)
aacagaaggagaaggagagagagaagagatctatttacagagtcctgggatacacagaaggcacccaataactatagaagtcccatctctaccagtcaacctaccaatcaatccatggatgatacaagagaggatatttatgtcaactatccaaccttctctcgcagaccaaagactagagtt

The intracellular domain may contain other domains. The signal domain and the transmembrane domain, or multiple signal domains may be linked via a spacer. As a spacer, for example, a polypeptide having 2 to 300 amino acid residues, preferably 10 to 100, and more preferably 20 to 50 amino acid residues can be used.

<Nucleic acid encoding chimeric protein and vector>
In one embodiment, a nucleic acid encoding the chimeric protein and a vector containing the nucleic acid are also useful. The details of the chimeric protein are as described above. The details of the nucleic acid and the vector are described in the section on T cells or NK cells.

<Target antigen-binding protein>
In one embodiment, the chimeric protein is used in combination with a target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen and a peptide tag comprising the amino acid sequence of SEQ ID NO: 1. The chimeric protein of the present disclosure is versatile because it can be used in combination with various (e.g., two or more, or three or more) target antigen-binding domains. The target antigen-binding protein is described in detail below.

The target antigen-binding protein comprises a target antigen-binding domain that recognizes and binds to the target antigen, and a peptide tag that comprises the amino acid sequence of SEQ ID NO: 1. There are no particular limitations on the positional relationship between the target antigen-binding domain and the peptide tag, and the N-terminal side may be either the target antigen-binding domain or the peptide tag.

Each region of the target antigen-binding protein shown below may be a region obtained by introducing a mutation into the natural protein from which it is derived, and may have an amino acid sequence identity of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more with the natural protein, to the extent that the function of each region can be maintained. Alternatively, each region of the target antigen-binding protein may be a region obtained by replacing 1 to 30, 1 to 15, 1 to 10, or 1 to 5 amino acids of the natural protein from which it is derived with other amino acids, to the extent that the function of each region can be maintained.

<Target antigen binding domain>
The target antigen-binding domain may be any domain that binds to a target antigen. Examples of the target antigen-binding domain include antibodies (including full-length antibodies and minibodies) that specifically bind to a target antigen, and ligands that bind to a target antigen. The target antigen-binding domain may be any domain that specifically binds to a target antigen, and does not need to have any other function, although additional functions are not excluded.

The antibody that specifically binds to the target antigen may be an antibody such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, or may be a low molecular weight antibody (minimolecular weight antibody). Examples of the low molecular weight antibody include F(ab') ₂ , Fab', Fab, Fv, rIgG, scFv, and VHH antibodies having the variable region of a heavy chain antibody (HCAb).
The method for preparing the antibody is not particularly limited. For example, the antibody production method described above in the section on the extracellular domain of the chimeric protein can be applied.

Ligands that bind to target antigens include the antigen-binding domains of ACE2, M-CSF, IL-34, and CD4.

Target antigens include antigenic biological substances such as proteins and sugar chains expressed on the surface of target cells. Examples include tumor-associated antigens (including tumor-specific antigens) and viral antigens. Viral antigens can be viral antigens expressed on the surface of virus-infected cells.

Tumor-associated antigens include CD19, CD20, CD22, CD123, B-cell maturation antigen (BCMA), HER2, Claudin-6, SLAMF7, EPHB4 receptor, cadherin 17, ROR1, mesothelin, PSMA, EGFR, CSF1R, GD2, GPC2, CD171, etc.

Viral antigens include membrane proteins derived from viruses. Examples of membrane proteins derived from viruses include gp120, SARS-Cov-1 spike protein, and SARS-Cov-2 spike protein.

<Peptide tag>
The target antigen-binding protein comprises a peptide tag comprising the amino acid sequence of SEQ ID NO: 1. Details of the peptide tag are the same as those described in the section on the extracellular domain of the chimeric protein.

<Other configurations>
The target antigen-binding protein may have other configurations in addition to the target-binding domain and peptide tag. For example, the target antigen-binding protein may have a target-binding domain and a peptide tag, or a peptide linker (e.g., a peptide linker having 2 to 20 amino acid residues) that links these to other regions. In addition, the target antigen-binding protein may have a tag peptide (His tag, GST tag, HA tag, etc.) for purification of the target antigen-binding protein. When designing the target antigen-binding protein, a signal peptide for transport, secretion, etc. may be added.

For example, polyethylene glycol (PEG) or the like may be added to the target antigen-binding protein to extend the half-life after administration. Alternatively, the target antigen-binding protein may be modified with a sugar chain, liposome, fluorescent substance, enzyme, or the like depending on the intended use.

The target antigen-binding protein may further comprise a ligand or receptor that recognizes a receptor or ligand on a T cell or NK cell. In particular, when a T cell or NK cell expressing a chimeric protein of the present disclosure is used in combination with a target antigen-binding protein, it is believed that the ligand or receptor of the target antigen-binding protein that recognizes a receptor or ligand on the T cell or NK cell and the peptide tag can exert a cooperative action.
Examples of receptors or ligands on T cells or NK cells include NKG2D, CD16, NKp30, NKp44, NKp46, 2B4, DNAM-1, 4-1BB, OX40, ICOS, CD28, CD30, and CD40.
Examples of the ligand or receptor that recognizes a receptor or ligand on a T cell or NK cell include ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, MICA, or MICB, which are ligands for NKG2D; an antibody Fc region, which is a ligand for CD16; B7-H6, which is a ligand for NKp30; an NKp44 ligand, which is a ligand for NKp44; an NKp46 ligand, which is a ligand for NKp46; CD48, which is a ligand for 2B4; CD155 or CD112, which is a ligand for DNAM-1; a 4-1BB ligand, which is a ligand for 4-1BB; an OX40 ligand, which is a ligand for OX40; an ICOS ligand, which is a ligand for ICOS; CD80 or CD86, which are ligands for CD28; a CD30 ligand, which is a ligand for CD30; and a CD40 ligand, which is a ligand for CD40.

In one example, the target antigen-binding protein may have, from the N-terminus, a target antigen-binding domain, a linker, a tag peptide for purification, and a peptide tag, in this order, and when designing the target antigen-binding protein, a signal peptide may be added to the N-terminus of the above sequence.
In a further example, the target antigen-binding protein may have, in this order from the N-terminus, a target antigen-binding domain, a linker, a ligand or receptor that recognizes a receptor or ligand on T cells or NK cells, a tag peptide for purification, and a peptide tag, and when designing the target antigen-binding protein, a signal peptide may be added to the N-terminus of the above sequence.

Examples of target antigen-binding proteins are shown in Figures 11 to 16 and below. The following example is an example of a target antigen-binding protein having the sequence [signal peptide]-[target antigen-binding domain]-[linker]-[6xHis]-[3xFLAG (registered trademark)]. For reference, Figures 11 to 16 show the position of each region in the sequence shown below, with the boxed letters indicating each region in the brackets above and the shaded area indicating [6xHis]. Note that although the sequence below includes the signal peptide sequence, the signal peptide sequence has been removed from the target antigen-binding protein produced. Furthermore, the stop codon has been omitted from the gene sequence below.

CD19scFv (clone FMC63)-3×FLAG
Amino acid sequence (SEQ ID NO:27)
MDMRVPAQLLGLLLLWLRGARCGSSLEDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSSWIRQPPRKGLEWLGVIWG SETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:28)

GD2scFv (clone 14g2a, PDB 4TUJ)-3×FLAG
Amino acid sequence (SEQ ID NO:29)
MDMRVPAQLLGLLLLWLRGARCGSSLEDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRSTSGSGKPGSGEGSTKGEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWI GAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSAKAAAGGGGSGGGGSGGGGSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:30)

GD2VHH (clone T25)-3×FLAG (see Chinese Patent No. 110551218)
Amino acid sequence (SEQ ID NO:31)
MDMRVPAQLLGLLLLWLRGARCGSSLEDVQLQESGGGLVQPGGSLRLSCAASGFTFRNYIMSWVRQAPGKGLERVSVINSGGGSTYYADSVKGRFTISRDNAKNTLYLRLNSLKTEDTAMYYCALGDARSGGRAIFRGQGTQVTVSSAAAGGGGSGGGGSGGGGSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:32)

GPC2VHH (clone LH7)-3xFLAG (see Canadian Patent Application Publication No. 3031559)
Amino acid sequence (SEQ ID NO:33)
MDMRVPAQLLGLLLLWLRGARCGSSLEQVQLVQSGGGLVQPGGSLRLSCAASDFYFYDYEMSWVRQAPGKGLEWIGTVSYSGSTYYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAMYYCARGYSYDDSRYFDYWGQGTLVTVSSAAAGGGGSGGGGSGGGGSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:34)

CovSscFv (clone CR3022 PDB 7JN5)-3×FLAG
Amino acid sequence (SEQ ID NO:35)
MDMRVPAQLLGLLLLWLRGARCGSSLEDIQLTQSPDSLAVSLGERATINCKSSQSVLYSSINKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKVEIKSTSGSGKPGSGEGSTKQMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGL EWMGIIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAGGSGISTPMDVWGQGTTVTVAAAGGGGSGGGGSGGGGSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:36)

sACE2(WT)-3×FLAG (Reference: Chan KK et al, Science 2020 369(6508):1261-1265)
Amino acid sequence (SEQ ID NO:37)

DNA sequence (SEQ ID NO:38)

sACE2v2.4-3×FLAG (Reference: Chan KK et al, Science 2020 369(6508):1261-1265)
Amino acid sequence (SEQ ID NO:39)

DNA sequence (SEQ ID NO:40)

EGFRscFv (Cetuximab, PDB 1YY9_C)-3×FLAG
Amino acid sequence (SEQ ID NO:41)
MDMRVPAQLLGLLLLWLRGARCGSSLEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTSTSGSGKPGSGEGSTKGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDY NTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASAAAGGGGSGGGGSGGGGSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:42)

HER2scFv (clone Trastuzumab PDB 6OGE)-3×FLAG
Amino acid sequence (SEQ ID NO:45)
MDMRVPAQLLGLLLLWLRGARCGSSLEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI YPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASAAAGGGGSGGGGSGGGGSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:46)

The following is an example of a target antigen binding protein with the sequence: [signal peptide]-[target antigen binding domain]-[linker]-[ULBP1]-[6xHis]-[3xFLAG (registered trademark)].

CD19scFv-ULBP1-3×FLAG
Amino acid sequence (SEQ ID NO:47)
MDMRVPAQLLGLLLLWLRGARCGSSLEDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSSWIRQPPRKGLEWLGVIWGSET TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGG SYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGGSGWVDTHCLCYDFIITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAFASLGKKVNVTKTWEEQTETLRDVVDFLKGQLLDIQVENLIPIEPLTLQARMSCEHEAHGHGRGSWQFLFNGQKFLLFDSNNRKWTALHPGAKKMTEKWEKNRDVTMFFQKISLGDCKMWLEEFLMY WEQMLDPTKPPSLAPHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:48)

GD2scFv-ULBP1-3×FLAG
Amino acid sequence (SEQ ID NO:49)
MDMRVPAQLLGLLLLWLRGARCGSSLEDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRSTSGSGKPGSGEGSTKGEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLE WIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCV SGMEYWGQGTSVTVSSAKAAAGGGGSGGGGSGGGGSGGWVDTHCLCYDFIITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAFASLGKKVNVTKTWEEQTETLRDVVDFLKGQLLDIQVENLIPIEPLTLQARMSCEHEAHGHGRGSWQFLFNGQKFLLFDSNNRKWTALHPGAKKMTEKWEKNRDVTMFFQKISLGDCKMWLEEFLMY WEQMLDPTKPPSLAPHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:50)

HER2scFv-ULBP1-3×FLAG
Amino acid sequence (SEQ ID NO:51)
MDMRVPAQLLGLLLLWLRGARCGSSLEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI YPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDG FYAMDYWGQGTLVTVSSASAAAGGGGSGGGGSGGGGGSGWVDTHCLCYDFIITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAFASLGKKVNVTKTWEEQTETLRDVVDFLKGQLLDIQVENLIPIEPLTLQARMSCEHEAHGHGRGSWQFLFNGQKFLLFDSNNRKWTALHPGAKKMTEKWEKNRDVTMFFQKISLGDCKMWLEEFLMY WEQMLDPTKPPSLAPHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:52)

The following is an example of a target antigen binding protein with the sequence: [signal peptide]-[target antigen binding domain]-[linker]-[ULBP2]-[6xHis]-[3xFLAG (registered trademark)].

CD19scFv-ULBP2-3×FLAG
Amino acid sequence (SEQ ID NO:53)
MDMRVPAQLLGLLLLWLRGARCGSSLEDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSSWIRQPPRKGLEWLGVIWG SETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS YAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSGRADPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTTAWKAQNPVLREVVDILTEQLRDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSFDGQIFLLFDSEKRMWTTVHPGARKMKEKWENDKVVAMSFHYFSMGDCIGWLEDFLMGMDSTLEPSA GAPLAMSSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:54)

GD2scFv-ULBP2-3×FLAG
Amino acid sequence (SEQ ID NO:55)
MDMRVPAQLLGLLLLWLRGARCGSSLEDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRSTSGSGKPGSGEGSTKGEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWI GAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVS GMEYWGQGTSVTVSSAKAAAGGGGSGGGGSGGGGSGRADPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTTAWKAQNPVLREVVDILTEQLRDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSFDGQIFLLFDSEKRMWTTVHPGARKMKEKWENDKVVAMSFHYFSMGDCIGWLEDFLMGMDSTLEPSA GAPLAMSSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:56)

HER2scFv-ULBP2-3×FLAG
Amino acid sequence (SEQ ID NO:57)
MDMRVPAQLLGLLLLWLRGARCGSSLEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI YPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGF YAMDYWGQGTLVTVSSASAAAGGGGSGGGGSGGGGSGRADPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTTAWKAQNPVLREVVDILTEQLRDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSFDGQIFLLFDSEKRMWTTVHPGARKMKEKWENDKVVAMSFHYFSMGDCIGWLEDFLMGMDSTLE PSAGAPLAMSHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

DNA sequence (SEQ ID NO:58)

The amino acid sequences and DNA sequences of HER2scFv, ULBP1, and ULBP2 contained in the above target antigen-binding proteins are as follows:

HER2scFv (clone Trastuzumab PDB 6OGE)
Amino acid sequence (SEQ ID NO:59)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAS

DNA sequence (SEQ ID NO:60)

ULBP1 (NM_025218)
Amino acid sequence (SEQ ID NO:61)
MAAAASPAFLLCLPLLHLLSGWSRAGWVDTHCLCYDFIITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAFASLGKKVNVTKTWEEQTETLRDVVDFLKGQLLDIQVENLIPIEPLTLQARMSCEHEAHGHGHGRGSWQFLFNGQKFLLFDSNNNRKWTALHPGAKKMTEKWEKNRDVTMFFQKISLGDCKMWLEEFLMYWEQMLDPTKPPSLA PGTTQPKAMATTLSPWSLLIIFLCFILAGR

DNA sequence (SEQ ID NO:62)

ULBP2 (NM_025217)
Amino acid sequence (SEQ ID NO:63)
MAAAAATKILLCLPLLLLLSGWSRAGRADPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTTAWKAQNPVLREVVDILTEQLRDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSFDGQIFLLFDSEKRMWTTVHPGARKMKEKWENDKVVAMSFHYFSMGDCIGWLEDFLMGMDSTLEPSAGAPLAMSSGTTQLR ATATTLILCCLLIILPCFILPGI

DNA sequence (SEQ ID NO:64)

The method for preparing the target antigen-binding protein is not particularly limited. For example, the target antigen-binding protein can be prepared by inserting a gene encoding the target antigen-binding protein containing each of the above domains into a vector and expressing it in an appropriate cell. The gene encoding the target antigen-binding protein may contain a leader sequence that is not ultimately contained in the target antigen-binding protein. It may also contain a sequence encoding a signal peptide on the N-terminus of the target antigen-binding protein.

In one embodiment, the target antigen binding protein may be expressed in a cell. For example, a gene encoding the target antigen protein is introduced into a target cell and expressed in the target cell. This allows the target cell itself to produce the target antigen binding protein and exert cytotoxic activity when used in combination with the chimeric protein of the present disclosure, a nucleic acid encoding the chimeric protein, a chimeric protein-expressing T/NK cell, or a pharmaceutical composition containing any of these.
To introduce a gene encoding a target antigen protein into a target cell, a suitable carrier can be used, such as a virus vector (adenovirus vector, adeno-associated virus vector, etc.) or an artificial nanoparticle (liposome, micelle, etc.).
When a gene encoding a target antigen protein is introduced into a target cell in a living body, the gene incorporated in the appropriate carrier can be administered by an appropriate administration route such as intratumoral administration, intravenous administration, subcutaneous administration, or peritoneal administration.

<T cells or NK cells expressing chimeric proteins>
In one aspect of the present disclosure, T cells or NK cells express the chimeric protein of the present disclosure described above. Hereinafter, T cells or NK cells expressing the chimeric protein of the present disclosure are also referred to as "chimeric protein-expressing T/NK cells."

The origin of the T cells or NK cells is not particularly limited. For example, T cells or NK cells may be collected from peripheral blood by apheresis or the like and used. Alternatively, T cells or NK cells prepared by inducing differentiation from stem cells collected from biological tissues or iPS cells may be used.

When administering T cells expressing a chimeric protein to an individual animal, it is preferable to use autologous T cells. Autologous T cells can be, for example, T cells taken from the individual receiving the T cells expressing the chimeric protein, or T cells prepared from cells derived from the individual.

When NK cells expressing a chimeric protein are administered to an animal individual, the NK cells may be autologous cells or allogeneic cells. As autologous NK cells, for example, NK cells collected from the individual receiving the NK cells expressing the chimeric protein, or NK cells prepared from cells derived from the individual, can be used. As allogeneic NK cells, allogeneic NK cells collected or prepared from an individual other than the individual to which the cells are to be administered can be used.

The method for preparing chimeric protein-expressing T/NK cells is not particularly limited. For example, a nucleic acid encoding a chimeric protein may be introduced into T cells or NK cells using a suitable vector such as a viral vector, a plasmid vector, an episomal vector, or an artificial chromosome vector. Those skilled in the art can select a suitable vector depending on the host selected.
Examples of viral vectors include Sendai virus vectors, retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, etc. Examples of plasmid vectors include pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, etc.

When introducing a nucleic acid encoding a chimeric protein into a vector, nucleic acids encoding control sequences such as a promoter, enhancer, or polyA addition signal, an origin of replication, a protein that binds to the origin of replication to control replication, a drug resistance gene, or a marker such as a fluorescent protein may be added.

T cells and NK cells are known to exert cytotoxic activity through a similar mechanism (Albinger et al., Gene Therapy (2021) 28: 513-527). Therefore, the chimeric protein disclosed herein can be used in common for T cells and NK cells.

Pharmaceutical compositions, combinations, and combination drugs
In one embodiment, the chimeric protein of the present disclosure, the nucleic acid encoding the chimeric protein, or the chimeric protein-expressing T/NK cell may be used as a pharmaceutical composition. The pharmaceutical composition may be used, for example, to damage a target cell. The pharmaceutical composition may also be used for cancer treatment.

In one embodiment, the pharmaceutical composition may be administered to a subject in combination with a target antigen-binding protein, either simultaneously or sequentially. Details of the target antigen-binding protein are as described above.

In one embodiment, the chimeric protein-expressing T/NK cells may be used as a combination comprising such a chimeric protein, a nucleic acid encoding the chimeric protein, or a chimeric protein-expressing T/NK cell and a target antigen binding protein.
In one aspect, the chimeric protein-expressing T/NK cells of the present disclosure may be used as a combination medicine comprising a pharmaceutical composition comprising such chimeric protein, a nucleic acid encoding the chimeric protein, or a chimeric protein-expressing T/NK cell, and a pharmaceutical composition comprising a target antigen binding protein.

The chimeric protein-expressing T/NK cells and the target antigen-binding protein may be used, for example, as a research reagent or as a pharmaceutical. For example, it is not necessary to prepare CAR-T cells or CAR-NK cells for each target antigen in order to evaluate the target antigen, and the target antigen can be more easily evaluated by preparing a target antigen-binding protein corresponding to the target antigen.

In one embodiment, a pharmaceutical composition containing a target antigen-binding protein is also provided, which is used to express the target antigen-binding protein in a target cell by introducing a gene encoding the target antigen-binding protein into the target cell. Details of the introduction and expression of the gene are as described above.

As an example of a method for using a pharmaceutical composition or a combination drug, an example will be described in which an antigen expressed on the surface of cancer cells or pathogen-infected cells in a subject is targeted. For example, chimeric protein-expressing T/NK cells are administered to a subject in combination with a target antigen-binding protein. In the subject's body, the target antigen-binding protein binds to an antigen expressed on the surface of the target cells. In addition, the chimeric protein-expressing T/NK cells that specifically recognize and bind to the peptide tag of the target antigen-binding protein bind to the target antigen-binding protein. This activates the cytotoxic activity of the T cells or NK cells, allowing them to inflict damage on the target cells.

The subject of use of the pharmaceutical composition or combination medicine is not particularly limited, and examples include humans and non-human animals (e.g., mammals such as monkeys, cows, pigs, horses, donkeys, sheep, goats, deer, dogs, cats, rabbits, mice, rats, guinea pigs, hamsters, and squirrels, and birds such as chickens, ducks, and wild ducks). Among these, mammals are preferred, and humans are more preferred.

The target disease of the pharmaceutical composition or the combined medicine is not particularly limited, so long as it is a disease for which an effect can be expected by damaging target cells.
Target diseases include cancer, more specifically, blood cancers such as acute lymphocytic leukemia, large cell lymphoma, some follicular lymphomas, and multiple myeloma; and solid cancers such as lung cancer, breast cancer, colon cancer, gastric cancer, pancreatic cancer, liver cancer, gallbladder cancer, bile duct cancer, and neuroblastoma.
Other examples include viral infections such as the new coronavirus, which target antigens expressed in cells infected with pathogens.

The route of administration of the pharmaceutical composition or combination drug is not particularly limited, and examples include intravenous administration, subcutaneous administration, intratumoral administration, and intraperitoneal administration. Among these, intravenous administration is preferred.

The order and frequency of administration of the pharmaceutical compositions or combination drugs are not particularly limited. When a pharmaceutical composition comprising a chimeric protein, a nucleic acid encoding a chimeric protein, or a chimeric protein-expressing T/NK cell, and a pharmaceutical composition comprising a target antigen-binding protein are administered in combination, they may be administered simultaneously or sequentially.
While T cells or NK cells may persist in the body of a subject for an extended period of time, the target antigen binding protein may have a shorter half-life, and thus, for each administration of a pharmaceutical composition comprising chimeric protein-expressing T/NK cells, multiple administrations of a pharmaceutical composition comprising a target antigen binding protein may be administered.

The target antigen is not limited to one type, and target antigen-binding proteins for multiple target antigens and chimeric proteins, nucleic acids encoding chimeric proteins, or chimeric protein-expressing T/NK cells may be used. Chimeric proteins, nucleic acids encoding chimeric proteins, or chimeric protein-expressing T/NK cells can be used universally regardless of the type of target antigen. Pharmaceutical compositions containing multiple target antigen-binding proteins may be administered simultaneously or sequentially.

The dosage of the pharmaceutical composition containing the chimeric protein, the nucleic acid encoding the chimeric protein, or the chimeric protein-expressing T/NK cells, the pharmaceutical composition containing the target antigen-binding protein, or a combination thereof may be appropriately determined taking into consideration the therapeutic effect, etc.

The administration of a pharmaceutical composition containing chimeric protein-expressing T/NK cells may follow the administration methods of tisagenlecleucel, axicabtagene siloleucel, etc., and the administration of a pharmaceutical composition containing a target antigen-binding protein may follow the administration methods of existing antibody pharmaceuticals.
Furthermore, as described above, when NK cells are used, there is no need to prepare chimeric protein-expressing NK cells from individual subjects, and they may be used as an off-the-shelf product.

In one aspect, a method for treating a disease is provided, comprising administering to a subject at least one selected from the group consisting of a chimeric protein, a nucleic acid, a T cell or NK cell, a pharmaceutical composition, a combination, a combination drug, and a target antigen-binding protein described in the present disclosure. Examples of diseases and subjects include those described above.

In one aspect, at least one selected from the group consisting of a chimeric protein, a nucleic acid, a T cell or NK cell, a pharmaceutical composition, a combination, a combination drug, and a target antigen binding protein described in the present disclosure is provided for use in treating a disease. Examples of diseases and subjects include those described above.

In one aspect, there is provided the use of at least one selected from the group consisting of a chimeric protein, a nucleic acid, a T cell or NK cell, a pharmaceutical composition, a combination, a combination drug, and a target antigen binding protein described in the present disclosure in the treatment of a disease. Examples of diseases and subjects include those described above.

In one aspect, there is provided the use of the chimeric proteins, nucleic acids, T cells or NK cells, pharmaceutical compositions, combinations, combination pharmaceuticals, and target antigen binding proteins described in the disclosure in the manufacture of a medicament for treating a disease. Examples of diseases and subjects include those described above.

　Embodiments of the present disclosure are described below using examples, but the embodiments of the present disclosure are not limited to these examples.

Example 1
1. Preparation of target antigen-binding proteins Plasmids encoding scFv or VHH antibody genes containing 3xFLAG (registered trademark) tags were prepared as follows.
A vector plasmid (pMYs-His-FLAG-IG) was prepared by inserting genes encoding human IgκVIII signal peptide, 3×(GGGGS) linker region, 6×His tag, and 3×FLAG (registered trademark) tag into the pMYs-IG plasmid.
The target antigen-binding protein gene was inserted into pMYs-His-FLAG-IG by restriction enzyme cloning using XhoI and NotI or by infusion cloning to prepare a plasmid encoding the target antigen-binding protein gene containing a 3×FLAG (registered trademark) tag.

The target antigen-binding protein genes used are as follows:
CD19scFv (clone FCM63) (SEQ ID NO: 28)
GD2scFv (clone 14g2a, PDB 4TUJ) (SEQ ID NO: 30)
GD2VHH (clone T25) (SEQ ID NO: 32)
GPC2VHH (clone LH7) (SEQ ID NO: 34)
CovSscFv (clone CR3022 PDB 7JN5) (SEQ ID NO: 36) sACE2 (WT) (SEQ ID NO: 38)
sACE2v2.4 (SEQ ID NO: 40)
EGFRscFv (Cetuximab) (SEQ ID NO: 42)

A plasmid containing the target antigen-binding protein gene was introduced into 293T cells using a retrovirus. 293T cells highly expressing the selection marker GFP were isolated using a cell sorter (FACSAria® III). Culture supernatants from 293T cells stably expressing scFv containing a 3xFLAG® tag were collected and sterilized using a 0.22 μm filter.

The scFv containing the 3xFLAG (registered trademark) tag was purified using an HA tagged Protein Purification kit (Medical and Biological Laboratories) as follows.
The culture supernatant of 293T cells collected as described above was mixed with anti-HA-tag beads, and mixed by inversion at 4 ° C. for 1 hour, and the target antigen-binding protein and the beads were bound. The mixture was centrifuged at 400 × g for 5 minutes, and the supernatant was removed. The anti-HA-tag beads were transferred to a spin column and centrifuged at 15,000 × g for 10 seconds. After washing the column three times with a washing solution, the eluted peptide solution was added to the anti-HA-tag beads and allowed to stand at 4 ° C. for 5 minutes. The spin column was centrifuged at 15,000 × g for 10 seconds, and the target antigen-binding protein purified solution was collected. The anti-HA-tag beads were mixed with the eluted peptide solution again and allowed to stand at 4 ° C. for 1 minute. The spin column was centrifuged at 15,000 × g for 10 seconds, and the target antigen-binding protein purified solution was collected. The purification of the target antigen-binding protein was confirmed by SDS-PAGE / Western blotting method.

2. Preparation of Jurkat Cells Expressing Chimeric Proteins T cells expressing a chimeric protein comprising an scFv against a 3×FLAG (registered trademark) tag, a CD8 hinge region, a CD28 transmembrane domain, a CD3ζ signal domain, and a 4-1BB intracellular signal domain were prepared as follows.
A gene containing scFv against the 3xFLAG (registered trademark) tag, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular signal domain, and a CD3ζ signal domain was introduced into Jurkat cells using lentivirus. Jurkat cells expressing the selection marker Venus (chimeric protein-expressing Jurkat cells) were isolated using a cell sorter.

The chimeric protein is structured as follows. The following sequence has the sequence [signal peptide]-[FLAG M2 VL]-[218S linker]-[FLAG M2 VH]-[CD8 hinge region]-[CD28 transmembrane domain]-[4-1BBL signal domain]-[CD3ζ signal domain]. For reference, FIG. 17 shows the position of each region in the sequence shown below, with the boxed letters indicating the regions in the brackets above, and for convenience, [FLAG M2 VL], [FLAG M2 VH], and [4-1BBL signal domain] are shown in bold and shaded. Note that the following sequence includes the signal peptide sequence, but the signal peptide sequence is removed from the chimeric protein to be produced. Also, the stop codon is omitted in the following gene sequence.

Amino acid sequence (SEQ ID NO:43)
MALPVTALLLPLALLLHAARPSDVLMTQIPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIRRASTSGSGKPGSGEGSTKGHVSQVQLQQSAAELARPGASVKMSCKASGYSFTTYTIHWVKQRPGQGLEWIGYINPSSGY AAYNQNFKDETTLTADPSSSSTAYMELNSLTSEDSAVYYCAREKFY GYDYWGQGATLTVSSAAAAGTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRRRPPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELASLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Gene sequence (SEQ ID NO:44)

3. Preparation of target cell model (1) CD19-K562 cells Using K562 cells, a chronic myeloid leukemia cell line, K562 cells expressing CD19 (hereinafter referred to as "CD19-K562 cells") and K562 cells expressing CD19 and 3xFLAG (registered trademark) tag as a positive control (hereinafter referred to as "FLAG-CD19-K562 cells") were prepared as follows.
The CD19 gene or the CD19 gene tagged with 3xFLAG (registered trademark) at the N-terminus was introduced into K562 cells by retrovirus, and K562 cells highly expressing CD19 were isolated using a cell sorter.

(2) SARS-Cov-2-Spike 293T cells 293T cells expressing SARS-Cov-2 spike protein (hereinafter referred to as "SARS-Cov-2-Spike 293T cells") were prepared using 293T cells, which are human fetal kidney epithelial cells, as follows.
The SARS-Cov-2 spike gene was retrovirally introduced into 293T cells, and cells positive for the marker protein CFP were isolated using a cell sorter.

(3) NALM6 Cells NALM6 cells, which are B cell leukemia cells, were prepared. NALM6 cells express CD19.
(4) SK-N-SH Cells SK-N-SH cells, which are cells derived from human neuroblastoma, were prepared. SK-N-SH cells express EGFR.
(5) CHP134 Cells CHP134 cells, which are cells derived from human neuroblastoma, were prepared. CHP134 cells express GD2.

4. Activity evaluation of chimeric protein-expressing cells (1) Activity of chimeric protein-expressing Jurkat cells was evaluated based on the expression level of CD69 as follows. CD69 is a T cell activation marker, and when Jurkat cells expressing the chimeric protein recognize a target antigen, an activation signal is transmitted and CD69 is highly expressed.
Jurkat cells expressing the chimeric protein were mixed with target cells at different ratios, and activation of Jurkat cells by the chimeric protein was evaluated. Specifically, 1 x ¹⁰⁴ Jurkat cells and different numbers of target cells were seeded on a U-bottom 96-well plate and cultured for 18 hours in a medium containing anti-CD19scFv-3xFLAG antibody. After co-culture, Jurkat cells were stained with anti-CD69 antibody, and the CD69 expression level of Jurkat cells was evaluated by flow cytometry.

As a result of evaluating the activity of Jurkat cells expressing the chimeric protein, when anti-CD19scFv-3xFLAG antibody was added as the target antigen-binding protein to CD19-K562 cells, the expression level of CD69 in Jurkat cells expressing the chimeric protein increased. In the FLAG-CD19-K562 cells used as a positive control, an increase in CD69 expression was observed even without the addition of the target antigen-binding protein. Therefore, it was confirmed that Jurkat cells expressing the chimeric protein were activated specifically in response to the FLAG antigen (Figure 2).

As shown in Figure 2, CD69 expression was significantly increased in "CD19-K562+αCD19(scFv)-FLAG," a co-culture of CD19-K562 cells and chimeric protein-expressing Jurkat cells with anti-CD19scFv-3×FLAG antibody added, compared to "FLAG-CD19-K562," a co-culture of FLAG-CD19-K562 cells and chimeric protein-expressing Jurkat cells.

The meanings of the notations in FIG. 2 are as follows:
Unstimul.: No co-culture of target cells CD19-K562: Co-culture with CD19-K562 cells CD19-K562+αCD19(scFv)-FLAG: Co-culture with CD19-K562 cells, with anti-CD19scFv-3×FLAG antibody added as a target antigen-binding protein FLAG-CD19-K562: Co-culture with FLAG-CD19-K562 cells E:T ratio: Number ratio of Jurkat cells (E) to target cells (T)

(2) NALM6 cells, SK-N-SH cells, CHP134 cells, and SARS-Cov-2-Spike 293T cells were also co-cultured with Jurkat cells expressing chimeric proteins in the same manner as above, and the activity of Jurkat cells was evaluated. The target antigen-binding proteins used for evaluation were CD19scFv-3xFLAG for NALM6 cells, EGFRscFv for SK-N-SH cells, GD2scFv for CHP134 cells, and sACE2(WT) and sACE2v2.4 for SARS-Cov-2-Spike 293T cells.

As a result of activity evaluation, it was confirmed that the expression of CD69 was increased when each target antigen-binding protein was added ( FIG. 3 ). Therefore, it is considered that the activation of T cells was induced by the peptide tag for the target antigen.
Figure 3 shows the results of evaluating the activity of Jurkat cells expressing chimeric proteins using each target cell. The meanings of each notation in Figure 3 are as follows.
"Flag-CAR-Jurkat cell" represents Jurkat cells expressing the chimeric protein.
"Control" indicates the result of the negative control in which no target antigen-binding protein was added.

Example 2
1. Preparation of NK92MI cells expressing chimeric protein NK92MI cells were cultured for 2 days in a medium containing 100 IU/mL IL-2, 10 ng/mL IL-15, and 10 ng/mL IL-21. The same chimeric protein gene as in Example 1 was introduced into the NK92MI cells by retrovirus.

2. Evaluation of Cytotoxic Activity of NK92MI Cells Expressing Chimeric Proteins The cytotoxic activity of NK92MI cells expressing chimeric proteins was evaluated as follows.
NALM6 cells were labeled with Cell Trace Far-Red. Specifically, 1 x ¹⁰⁴ target cells and different numbers of NK92MI cells were seeded in a U-bottom 96-well plate and cultured for 18 hours in a medium containing anti-CD19scFv-3xFLAG antibody. After co-culture, the cells were stained with Live/Dead Near-IR, and the proportion of dead cells was evaluated by flow cytometry.

As a result of evaluating the cytotoxic activity of NK92MI cells, when anti-CD19scFv-3xFLAG antibody was added as a target antigen-binding protein to NALM6 cells (Fig. 4, black circle), the cell lysis rate was significantly increased compared to when the anti-CD19scFv-3xFLAG antibody was not added (Fig. 4, white circle). In other words, it is considered that the chimeric protein-expressing NK92MI cells showed high cytotoxic activity by recognizing and activating the peptide tag.
In FIG. 4, "Flag-CAR-NK92 cell" represents chimeric protein-expressing NK92MI cell.

Example 3
1. Preparation of target antigen-binding protein In a similar manner to Example 1, a target antigen protein (SEQ ID NO: 45) containing HER2scFv (anti-HER2scFv-3×FLAG antibody) was prepared.
2. Evaluation of Cytotoxic Activity of NK92MI Cells Expressing Chimeric Proteins The cytotoxic activity of NK92MI cells expressing chimeric proteins was evaluated as follows.
MDA-MB-453 cells, a human-derived breast cancer cell line expressing HER2, were labeled with Cell Trace Far-Red. Specifically, 1 x ¹⁰⁴ target cells and different numbers of NK92MI cells were seeded in a U-bottom 96-well plate and cultured for 18 hours in a medium containing anti-HER2scFv-3xFLAG antibody. After co-culture, the cells were stained with Live/Dead Near-IR, and the proportion of dead cells was evaluated by flow cytometry.

As a result of evaluating the cytotoxic activity of NK92MI cells, when anti-HER2scFv-3xFLAG antibody was added to MDA-MB-453 cells as the target antigen-binding protein (Figure 5, open squares), the cell lysis rate was significantly increased compared to when the anti-HER2scFv-3xFLAG antibody was not added (Figure 5, open circles). In other words, it is believed that the chimeric protein-expressing NK92MI cells showed high cytotoxic activity by recognizing and activating the peptide tag.

The meaning of each notation in FIG.
"Flag-CAR-NK92 cell" represents chimeric protein-expressing NK92MI cells.
"Mock" indicates the results when target cells were co-cultured with NK92MI cells that do not express the chimeric protein.
"HER2CAR" represents the results when target cells were co-cultured with NK92MI cells expressing a HER2-specific chimeric protein composed of [anti-HER2 scFv]-[CD8 hinge region]-[CD28 transmembrane domain]-[4-1BBL signal domain]-[CD3ζ signal domain].
"Flag-CAR" represents the results when chimeric protein-expressing NK92MI cells and target cells were co-cultured without the addition of anti-HER2scFv-3×FLAG antibody.
"Flag-CAR+HER2scFv" represents the results when chimeric protein-expressing NK92MI cells and target cells were co-cultured with the addition of anti-HER2scFv-3×FLAG antibody.

Example 4
1. Preparation of hCD19-B16 Cells B16 cells expressing human CD19 (hereinafter referred to as "hCD19-B16 cells") were prepared using B16 cells, a mouse melanoma cell line, as follows.
The human CD19 gene was introduced into B16 cells by retrovirus, and B16 cells highly expressing CD19 were isolated using a cell sorter.

2. Preparation of CD19scFv-Expressing hCD19-B16 Cells CD19scFv was introduced into hCD19-B16 cells using a retrovirus to prepare hCD19-B16 cells that forcibly express CD19scFv (hereinafter referred to as "CD19scFv-hCD19-B16 cells").

3. Preparation of chimeric protein-expressing mouse T cells To prepare an anti-CD3/CD28 antibody-coated plate, 10 μg/mL anti-CD3 antibody and 10 μg/mL anti-CD28 antibody were added to the plate and left to stand overnight at 4° C. C57BL/6-derived spleen cells were seeded on the anti-CD3/CD28 antibody-coated plate and cultured for 2 days in a medium containing 50 IU/mL rhIL2, 5 ng/mL rmIL-7, and 5 ng/mL rmIL-15 to expand the T cells. A chimeric protein gene was introduced into the T cells using a retrovirus. Mouse T cells expressing GFP, a selection marker (chimeric protein-expressing T cells) were isolated using a cell sorter.

The chimeric protein is structured as follows: The sequence below is [signal peptide]-[FLAG M2 VL]-[218S linker]-[FLAG M2 VH]-[CD28 hinge and transmembrane domain]-[4-1BBL signal domain]-[CD3ζ signal domain]. The signal domain used was derived from mouse. Note that although the sequence below includes the signal peptide sequence, the signal peptide sequence is removed from the chimeric protein that is produced. Furthermore, the stop codon has been omitted from the gene sequence below.

Amino acid sequence (SEQ ID NO:65)
MGVPTQLLGLLLLWITDAICSDVLMTQIPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIRRASTSGSGKPGSGEGSTKGHVSQVQLQQSAAELARPGASVKMSCKASGYSFTTYTIHWVKQRPGQGLEWIGYIN PSSGYAAYNQNFKDETTLTADPSSSTAYMELNSLTSEDSAVYY CAREKFYGYDYWGQGATLTVSSAAAAIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTSVLKWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYELRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEA YSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR

Gene sequence (SEQ ID NO:66)

4. Evaluation of cytotoxic activity of mouse T cells expressing chimeric proteins The cytotoxic activity of mouse T cells expressing chimeric proteins was evaluated as follows.
hCD19-B16 cells and CD19scFv-hCD19-B16 cells were labeled with Cell Trace Far-Red. Specifically, 1×10 ⁴ target cells and 5×10 ⁴ chimeric protein-expressing mouse T cells were seeded in a U-bottom 96-well plate and cultured for 18 hours in a medium containing anti-CD19scFv-3×FLAG antibody. After co-culture, the cells were stained with Live/Dead Near-IR, and the proportion of dead cells was evaluated by flow cytometry.

As a result of evaluating the cytotoxic activity of mouse T cells (Figure 6), when anti-CD19scFv-3xFLAG antibody was added to hCD19-B16 cells as a target antigen-binding protein, the cell lysis rate was significantly increased compared to when the anti-CD19scFv-3xFLAG antibody was not added. In other words, it is believed that the chimeric protein-expressing mouse T cells showed high cytotoxic activity by recognizing and activating the peptide tag.

Furthermore, when the anti-CD19scFv-3xFLAG gene was introduced into hCD19-B16 cells, and the B16 cells were made to produce anti-CD19scFv-3xFLAG antibodies, the cell lysis rate increased significantly when the cells were co-cultured with chimeric protein-expressing mouse T cells without the addition of anti-CD19scFv-3xFLAG antibodies. This suggests that even without the addition of anti-scFv-3xFLAG antibodies, by forcing the expression of the chimeric protein in the cancer cells themselves, chimeric protein-expressing T cells can recognize the peptide tag and exert cytotoxic activity.

The meaning of each notation in FIG.
"FlagCAR-T cell" represents chimeric protein-expressing mouse T cells.
"Mock" represents the results when target cells were co-cultured with mouse T cells that do not express the chimeric protein.
"CD19CAR" represents the results when target cells were co-cultured with mouse T cells expressing a chimeric protein composed of [anti-CD19 scFv]-[CD28 hinge and transmembrane domain]-[4-1BBL signal domain]-[CD3ζ signal domain].
"FlagCAR" represents the results when chimeric protein-expressing mouse T cells and target cells were co-cultured without the addition of anti-CD19scFv-3×FLAG antibody.
"FlagCAR+scFv" represents the results when chimeric protein-expressing mouse T cells and target cells were co-cultured with the addition of anti-CD19scFv-3×FLAG antibody.
"CD19scFv-hCD19-B16" represents the results when B16 cells producing anti-CD19scFv-3xFLAG antibody were used.

Example 5
1. Preparation of NKG2D Ligand-Fused scFv-FLAG Antibody Preparation of Plasmid The ULBP1 gene or ULBP2 gene was inserted upstream of the 6xHis tag gene region of the pMYs-His-FLAG-IG plasmid by the infusion cloning method to prepare pMYs-ULBP1-His-FLAG-IG or pMYs-ULBP2-His-FLAG-IG plasmid. Both ULBP1 and ULBP2 are NKG2D ligands expressed in NK cells, etc. A target antigen-binding protein gene was inserted by restriction enzyme cloning or infusion cloning method to prepare a plasmid encoding a target antigen-binding protein gene containing ULBP1 or ULBP2 and a 3xFLAG (registered trademark) tag (FIG. 7).
By the same method as in Example 1, anti-HER2scFv-ULBP1-3xFLAG antibody (SEQ ID NO: 51) and anti-HER2scFv-ULBP2-3xFLAG antibody (SEQ ID NO: 57) were prepared.

2. Evaluation of Cytotoxic Activity of NK92MI Cells Expressing Chimeric Proteins The cytotoxic activity of NK92MI cells expressing chimeric proteins was evaluated as follows.
First, the expression level of NKG2D in NK92MI cells expressing the chimeric protein was evaluated by flow cytometry.
Next, MDA-MB-453 cells, a human-derived breast cancer cell line expressing HER2, were labeled with Cell Trace Far-Red. Specifically, 1×10 ⁴ target cells and different numbers of NK92MI cells were seeded in a U-bottom 96-well plate and cultured for 4 hours in a medium containing anti-HER2scFv-3×FLAG antibody or anti-HER2scFv-ULBP1-3×FLAG antibody. After co-culture, the cells were stained with Live/Dead Near-IR, and the proportion of dead cells was evaluated by flow cytometry.

NK92MI cells expressing the chimeric protein (FlagCAR) were found to express NKG2D, similar to NK92MI cells not expressing the chimeric protein (Mock) (Figure 8A).

As a result of evaluating the cytotoxic activity of NK92MI cells, the addition of anti-HER2scFv-ULBP1-3xFLAG antibody as the target antigen-binding protein to MDA-MB-453 cells (Figure 8B, open squares) significantly increased the cell lysis rate compared to the addition of the anti-HER2scFv-3xFLAG antibody (Figure 8B, closed squares). In other words, it is believed that the anti-HER2scFv-ULBP1-3xFLAG antibody cooperatively activates signals mediated by the peptide tag and the NKG2D ligand, resulting in the chimeric protein-expressing NK92MI cells exhibiting superior cytotoxic activity.

The meaning of each notation in FIG. 8B is as follows.
"Flag-CAR-NK92 cell" represents chimeric protein-expressing NK92MI cells.
"Mock" indicates the results when target cells were co-cultured with NK92MI cells that do not express the chimeric protein.
"W/o scFv" represents the results when chimeric protein-expressing NK92MI cells and target cells were co-cultured without addition of either anti-HER2 scFv-3xFLAG antibody or anti-HER2 scFv-ULBP1-3xFLAG antibody.
"scFv" represents the results when chimeric protein-expressing NK92MI cells and target cells were co-cultured with the addition of anti-HER2scFv-3xFLAG antibody.
"scFv-ULBP1" represents the results when chimeric protein-expressing NK92MI cells and target cells were co-cultured with the addition of anti-HER2scFv-ULBP1-3xFLAG antibody.

Example 6
1. Preparation of chimeric protein-expressing human T cells Human umbilical cord blood cells were cultured for 2 days in a medium containing 50 ng/mL anti-CD3 antibody (eBioscience, Clone OKT3), 300 IU/mL rhIL-2, 10 ng/mL rhIL-15, and 10 ng/mL rhIL-7 to expand the T cells. Using lentivirus, genes containing scFv against the 3xFLAG (registered trademark) tag, CD8 hinge region, CD28 transmembrane domain, 4-1BB intracellular signal domain, and CD3ζ signal domain were introduced into the T cells. T cells expressing GFP, a selection marker (chimeric protein-expressing T cells) were isolated using a cell sorter.
The expression level of NKG2D in the chimeric protein-expressing T cells was evaluated by flow cytometry, and it was found that NKG2D was highly expressed in CD8 ⁺ T cells ( FIG. 9 ).

The meaning of each notation in FIG.
"Untransduced" represents the results in human T cells that were not transduced with genes.
"Mock" represents the results in human T cells that do not express the chimeric protein.
"HER2CAR" represents results in HER2-specific chimeric protein-expressing human T cells.
"FlagCAR" represents the results in chimeric protein-expressing human T cells.

2. Evaluation of cytotoxic activity of chimeric protein-expressing human T cells The cytotoxic activity of human T cells expressing the chimeric protein was evaluated as follows.
MDA-MB-453 cells, a human-derived breast cancer cell line expressing HER2, were labeled with Cell Trace Far-Red. Specifically, 1×10 ⁴ target cells and 1×10 ⁴ human T cells were seeded in a U-bottom 96-well plate and cultured for 48 hours in a medium containing anti-HER2scFv-3×FLAG antibody, HER2scFv-ULBP1-3×FLAG antibody, or HER2scFv-ULBP2-3×FLAG antibody. After co-culture, the cells were stained with Live/Dead Near-IR, and the proportion of dead cells was evaluated by flow cytometry.

As a result of the evaluation of the cytotoxic activity of human T cells (Figure 10), when anti-HER2scFv-3xFLAG antibody was added, chimeric protein-expressing human T cells showed the same level of cytotoxic activity as HER2CAR-T cells. Furthermore, when anti-HER2scFv-ULBP1-3xFLAG antibody or anti-HER2scFv-ULBP2-3xFLAG antibody was used, a higher cell lysis rate was shown than chimeric protein-expressing human T cells or HER2CAR-T cells to which anti-HER2scFv-3xFLAG antibody was added. This suggests that anti-HER2scFv-ULBP1-3xFLAG antibody or anti-HER2scFv-ULBP2-3xFLAG antibody activated the NKG2D signal of chimeric protein-expressing human T cells and improved the cytotoxic activity.

The meaning of each notation in FIG. 10 is as follows.
"FlagCAR-T cell" represents chimeric protein-expressing human T cells.
"HER2scFv-ULBP1/2" represents anti-HER2scFv-ULBP1-3xFLAG antibody or anti-HER2scFv-ULBP2-3xFLAG antibody.
"Mock" represents the results when target cells were co-cultured with human T cells that do not express the chimeric protein.
"HER2CAR" represents the results when HER2-specific chimeric protein-expressing human T cells were co-cultured with target cells.
"W/o scFv" represents the results when chimeric protein-expressing human T cells and target cells were co-cultured without addition of any of anti-HER2scFv-3xFLAG antibody, anti-HER2scFv-ULBP1-3xFLAG antibody, and anti-HER2scFv-ULBP2-3xFLAG antibody.
"scFv" represents the results when chimeric protein-expressing human T cells and target cells were co-cultured with the addition of anti-HER2scFv-3xFLAG antibody.
"scFv-ULBP1" represents the results when chimeric protein-expressing human T cells and target cells were co-cultured with the addition of anti-HER2scFv-ULBP1-3xFLAG antibody.
"scFv-ULBP2" represents the results when chimeric protein-expressing human T cells and target cells were co-cultured with the addition of anti-HER2scFv-ULBP2-3xFLAG antibody.

The disclosure of Japanese Patent Application No. 2023-062411, filed on April 6, 2023, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

1a, 1b Extracellular domain 3 Transmembrane domain 5 Intracellular domain 10 Chimeric protein 11 Peptide tag 13a, 13b Target antigen binding domain 20 Target antigen binding protein CM Cell membrane

Claims

An extracellular domain that recognizes and binds to a peptide tag comprising the amino acid sequence of SEQ ID NO:1;
A transmembrane domain;
an intracellular domain including a signal domain that activates the cytotoxic activity of a T cell or a NK cell when the extracellular domain binds to the peptide tag;
A chimeric protein comprising:
The chimeric protein of claim 1, wherein the extracellular domain does not contain a receptor or ligand that binds to a ligand or receptor other than the peptide tag.
The chimeric protein of claim 1, wherein the extracellular domain comprises a single-chain antibody comprising a light chain variable region and a heavy chain variable region of an immunoglobulin that recognizes and binds to the peptide tag.
The chimeric protein of claim 1, wherein the intracellular domain comprises a signal domain of CD3ζ.
A nucleic acid encoding the chimeric protein of claim 1.
A vector comprising the nucleic acid according to claim 5.
T cells or NK cells expressing the chimeric protein of claim 1.
A pharmaceutical composition for damaging a target cell, comprising the chimeric protein according to any one of claims 1 to 4, the nucleic acid according to claim 5, or the T cell or NK cell according to claim 7.
The pharmaceutical composition according to claim 8 for the treatment of cancer.
The pharmaceutical composition according to claim 8, which is administered to a subject simultaneously or sequentially in combination with a target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen and a peptide tag comprising the amino acid sequence of SEQ ID NO:1.
The pharmaceutical composition according to claim 10, wherein the target antigen is a tumor-associated antigen or a viral antigen.
A chimeric protein according to any one of claims 1 to 4, a nucleic acid according to claim 5, or a T cell or NK cell according to claim 7;
a target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen and a peptide tag comprising the amino acid sequence of SEQ ID NO:1;
A combination comprising:
A pharmaceutical composition comprising the chimeric protein according to any one of claims 1 to 4, the nucleic acid according to claim 5, or the T cell or NK cell according to claim 7;
a pharmaceutical composition comprising a target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen and a peptide tag comprising the amino acid sequence of SEQ ID NO:1;
A combination medicine comprising:
A target antigen-binding protein comprising a target antigen-binding domain that recognizes and binds to a target antigen, and a peptide tag comprising the amino acid sequence of SEQ ID NO:1.
The target antigen binding protein of claim 14, further comprising a ligand or receptor that recognizes a receptor or ligand on a T cell or NK cell.
A pharmaceutical composition comprising the target antigen-binding protein according to claim 14 or 15, which is used to express the target antigen-binding protein in a target cell by introducing a gene encoding the target antigen-binding protein into the target cell.