KR20200049594A - Manipulated immune cell - Google Patents

Abstract

The present invention relates to an engineered immune cell of which the T cell receptor (TCR) is inactivated. More specifically, the present invention relates to trapper molecule which interacts with a TCR subunit in order to engineer immune cells, and/or an immune cell including the trapper molecule, favorably, an immune cell of which the surface area of at least one cell among the TCR subunits.

Description

Manipulated immune cells

The present invention relates to engineered immune cells in which the T cell receptor (TCR) is inactivated. More specifically, the amount of the cell surface of at least one of the TCR subunits and / or immune cells comprising the Trapper subunits, preferably TCR subunits, that interact with a TCR subunit to manipulate immune cells is reduced. It relates to immune cells.

T cell adoptive immunotherapy is a promising approach for the treatment of cancer. This approach utilizes engineered isolated human T cells to enhance specificity for specific tumor-associated antigens. The engineered T cells may contain foreign nucleic acids or polypeptides that are not naturally present or may contain expression of a chimeric antigen receptor. The chimeric antigen receptor is derived from the variable domain of a monoclonal antibody and can specifically recognize the antigen of a cancer cell. Since the chimeric antigen receptor directly recognizes the cancer cell antigen, it induces a tumor immune response in a non-limiting way to the main tissue complex. To date, T cell adoptive immunotherapy has been used as a clinical therapy for many cancers, including B cell malignancies, glioblastoma, neuroblastoma, ovarian cancer, melanoma, and pancreatic cancer.

On the other hand, despite its potential usefulness as a cancer therapy, adoptive immunotherapy with CAR-T cells has been limited primarily to autologous cell therapy, primarily due to the expression of endogenous T cell receptors on the cell surface. Taga-derived CAR-T cells expressing endogenous T cell receptors can cause allograft versus host disease (GVHD) by recognizing major histocompatibility antigens and sub-histocompatibility antigens after administration to allograft patients. As a result, clinical trials to date have focused mainly on the use of autologous CAR-T cells that separate T cells from patients, transform the isolated T cells to express the chimeric antigen receptor, and then inject them back into the same patient. However, this approach is limited by both time and cost because the patient's T cell must be manipulated to produce CAR-T only after the patient is diagnosed with cancer.

Therefore, it is necessary to develop a non-alloreactive T cell that does not induce a disease such as GVHD even if a third donor T cell is used. Accordingly, the present invention is to provide an allogeneic CAR-T cell in which the endogenous T cell receptor is inactivated by trapping the endogenous T cell receptor in the cell and preventing exposure to the cell surface. These non-alloreactive T cells will be more useful for cancer treatment in the future because they can produce cancer-specific T cells before they are diagnosed and can be provided to cancer patients in need immediately.

If it is possible to develop a method for preventing the immune-activated receptor containing the TCR complex from reaching the cell surface, the immune-activated receptor can be inactivated to produce immune cells with greatly improved toxicity in terms of autoimmunity.

The present application relates to a binding domain derived from gp41 having the ability to bind to at least one of the subunits of the immune activation receptor; And it provides a trapper peptide comprising any one or more of the vesicle residual domain, Golgi residual domain and proteasome residual domain.

In one embodiment, the binding domain derived from gp41 provides a trapper peptide characterized by being one of FP (fusion protein region), NHR, Loop, CHR and TMD, and some sequences thereof.

In one embodiment, the binding domain derived from gp41 provides a trapper peptide comprising at least one amino acid sequence selected from SEQ ID NO: 27, SEQ ID NO: 34, and SEQ ID NO: 44.

In one embodiment, the endoplasmic reticulum and Golgi apparatus residual domain comprises any one or more of KDEL, KKX'X, KX'D / E, YQRL 'and KX', wherein X 'is any amino acid. It provides a trapper peptide. Furthermore, the vesicle and Golgi residual domains containing the KKX'X 'provide a trapper peptide characterized by KKMP or LYKYKSRRSFIEEKKMP.

In one embodiment, the trapper peptide provides a trapper peptide characterized in that it further comprises any one or more of a linking domain, a CD8α transmembrane domain, and a CD8α signal sequence. In one example, the linking domain is (GGGGS) n, where n is an arbitrary integer to provide a trapper peptide. In another example, the amino acid sequence of the CD8α transmembrane domain provides a trapper peptide characterized by having the amino acid sequence of SEQ ID NO: 118. In another example, the CD8α signal sequence provides a trapper peptide characterized by having the amino acid sequence of SEQ ID NO: 148.

In one embodiment, the trapper peptide is capable of binding to any one or more of TCR-alpha, TCR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD4, CD8-beta and CD8-alpha It provides a trapper peptide characterized by having a.

In one embodiment,

SNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL;

SNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP;

KKVIGFRILLLKVAGFNLGGGGSGGGGSGGGGSGGGGSAEKDEL;

KKVIGFRILLLKVAGFNLIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP;

LFIMIVGGLVGLRIVFAVLSIGGGGSGGGGSGGGGSGGGGSAEKDEL;

LFIMIVGGLVGLRIVFAVLSIIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP;

AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGGGGSGGGGSAEKDEL;

AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP;

ALPVTALLLPLALLLHAARPSNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL;

ALPVTALLLPLALLLHAARPSNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP provides a trapper peptide characterized by comprising the amino acid sequence of any one.

In addition, the present application provides an engineered immune cell comprising the trapper peptide and characterized in that the expression trait of the immune activating receptor is different from that of the wild state cell.

In one embodiment, the expression trait of the immune activating receptor is different from that of the wild state cell provides an engineered immune cell characterized in that the amount of the cell surface of at least one of the subunits of the immune activating receptor is reduced. In one example, the expression trait of the immune activating receptor differs from that of the wild state cell provides a engineered immune cell characterized in that the immune activating receptor is trapped within the cell.

In one embodiment, the engineered immune cells provide engineered immune cells, characterized in that at least one selected from the group consisting of T cells, NK cells and NKT cells.

The present application also provides a vector comprising a nucleic acid encoding the trapper peptide.

In one embodiment, the vector provides a vector characterized in that it is a viral vector. In one example, the viral vector provides a vector derived from any one or more selected from the group consisting of adenovirus, adeno-associated virus, vaccinia virus, pox virus, herpes virus, retrovirus and herpes simplex virus.

The present application also provides a method for producing an engineered immune cell comprising introducing the trapper polypeptide or a nucleic acid encoding the same into an immune cell.

In one embodiment, there is provided a method of producing an engineered immune cell, further comprising isolating cells from which a different trait is derived from a wild-state cell.

In one embodiment, the introducing step is characterized in that it is performed by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles and protein translocation domain (PTD) fusion protein methods. It provides a method for producing immune cells. Furthermore, the viral vector is derived from any one or more selected from the group consisting of adenovirus, adeno-associated virus, vaccinia virus, pox virus, herpes virus, retrovirus and herpes simplex virus. to provide.

The present application also provides a method of treating cancer comprising injecting a composition comprising the engineered immune cells into a subject.

In one embodiment, the step of administering the composition to a treatment subject provides a method of treating cancer, characterized in that it is systemic administration. Furthermore, the systemic administration provides a method of treating cancer characterized by intravenous administration.

For immune cells expressing the trapper molecule, it has been found that the amount of cell surface of the TCR subunit is significantly reduced, so that the toxicity problems such as autoimmune diseases can be greatly improved.

Figure 1 shows the domain structure of the trapper peptides used in the experiment.
Figure 2 shows the results of transformation of Jurkat cells using electroporation.
Figure 3 shows the results of transformation of Jurkat cells using lentivirus.
4 is a result of analyzing the amount of TCR on the cell surface according to the lentivirus dose confirmed by flow cytometry.
5 is a TCR negative result according to the lentivirus dose confirmed by flow cytometry.
6 is a schematic diagram of a vector used in experimenting the relationship between the expression of the trapper peptide in Jurkat cells and the amount of TCR subunits on the cell surface.
FIG. 7 is the result of the positive correlation of the expression of the trapper peptide in Jurkat cells with the amount of TCR subunits on the cell surface.
8 is a schematic diagram of a vector used in experimenting to correlate the amount of trapper peptide in Jurkat cells with the amount of TCR subunits on the surface of Jurkat cells.
FIG. 9 is the result of correlation of the amount of trapper peptide in Jurkat cells with the amount of TCR subunits on the surface of Jurkat cells.
10 is a schematic diagram of a vector used for transformation of T cells using lentivirus.
11 is a result of transformation of T cells using lentivirus.
12 is a schematic diagram of a vector used in experimenting to correlate the amount of the trapper peptide in T cells with the amount of TCR subunits on the surface of T cells.
FIG. 13 is the result of correlation of the amount of trapper peptide in T cells with the amount of TCR subunits on the surface of T cells.

1. Definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or identical to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods and examples mentioned in this specification are exemplary only and are not intended to be limiting.

As used herein, the term 'affinity' refers to the property that two specific substances are specifically or closely bound to each other, such as the relationship between the components constituting the enzyme-substrate, antigen-antibody, hormone-receptor, and protein complex. Says

As used herein, the term 'interaction' between a protein / peptide and a protein / peptide is a biochemical and / or chemical reaction caused by an electrostatic force and / or a hydrophobic effect between two molecules. / Or a physical phenomenon. In addition, the term 'combination' as used herein should be interpreted as equivalent to 'interaction' unless otherwise stated.

As used herein, the terms 'retention', 'retain', and 'retaining' refer to a specific location within a biological space when any biomolecule is located in a biological space, such as a cell, For example, it tends to have a high probability of being in a small organ.

As used herein, the term 'peptide' or 'peptide sequence' and equivalent expressions are terms not only for an amino acid sequence composed of amino acids but also for derivatives and variants thereof, and include glycoproteins, peptide derivatives, and peptides and other substances. Conjugates, but are not limited thereto. Furthermore, the term is not intended to point out only sequences that exactly match the designated amino acid sequence, but is a term to encompass an amino acid sequence having 50 to 100% homology to the designated amino acid sequence. In addition, the term is used as a concept encompassing the base sequence encoding it.

As used herein, the term 'immune activating receptor' refers to an endogenous receptor that activates immune cells to enable the immune cells to form and perform an immune response. Immune activating receptors include both wild type or naturally occurring mutated receptors naturally present in immune cells. That is, when a specific protein in a cell can bind to other molecules to generate signaling that activates an immune response, all such proteins are included.

In the present application, when the amino acid sequence of a peptide is described and there is no specific mention, an underlined amino acid residue means that the corresponding amino acid residue is a D-type amino acid, and a residue without a specific mark means an L-type amino acid. For example, in the amino acid sequence G A LFLGFLG, the underlined A and alanine residues correspond to D-type amino acids.

2. Structure and components of TCR complex

This part is intended to explain the structure and components of the TCR complex as a part to help understand the term 'T-Cell Receptor complex' used throughout the specification. The structure and components of the TCR complex described in this section are based on the wild-state TCR complex, and are merely exemplary descriptions based on the technically known contents so far. It is not intended to be limited to this.

The TCR complex is a receptor expressed on the surface of T cells, and is known to function to recognize MHC on the surface of other cells during an immune response. The TCR complex contains TCR, which directly recognizes MHC as a component, and CD3, which is involved in signal transduction. At this time, the TCR is present in the form of a heterodimer and may be composed of one TCR-α chain and one TCR-β chain, or one TCR-γ chain and one TCR-δ chain. At this time, the TCR may exist in the form of a dimer of a TCR-α chain / TCR-β chain or a dimer of a TCR-γ chain / TCR-δ chain, and each dimer is collectively referred to as a 'TCR heterodimer'. In addition, CD3 is composed of one CD3γ chain, one CD3δ chain, two CD3ε chains, and two zeta chains. Specifically, CD3 exists in the form of a CD3γ chain / CD3ε chain dimer, a CD3δ chain / CD3ε chain dimer, and a zeta chain dimer. At this time, each dimer is collectively referred to as a 'CD3 heterodimer'. The dimer of the zeta chain is also referred to as a 'zeta chain dimer'. In the present specification, individual components constituting the TCR complex are collectively referred to as a 'TCR subunit'. The protein sequence information of each exemplary TCR subunit is as follows, but this is only an example and the content of the present application is not limited thereto (TCR-α chain: https: //www.ncbi.nlm.nih. gov / gene / 6955; TCR-β chain: https://www.ncbi.nlm.nih.gov/gene/6957; TCR-γ chain: https://www.ncbi.nlm.nih.gov/gene/ 6965; TCR-δ chain: https://www.ncbi.nlm.nih.gov/gene/6964; CD3γ chain: https://www.ncbi.nlm.nih.gov/gene/917; CD3δ chain: https : //www.ncbi.nlm.nih.gov/gene/915; CD3ε chain: https://www.ncbi.nlm.nih.gov/gene/916; zeta chain: https: //www.ncbi.nlm .nih.gov / gene / 919). In addition, if necessary, a ligand recognition unit among TCR subunits is referred to as a 'ligand recognition unit', and a role that is involved in immune signal generation is referred to as a 'signal activation unit'. The ligand recognition unit includes the TCR and chains constituting it, and the signal activation unit comprises CD3 and chains constituting it.

The TCR chain contains a motif that interacts with CD3. In the case of the TCR-α chain, it is known to stably interact with CD3 in a motif composed of 9 amino acid residues [1]. The motif in which the interaction between the TCR chain and CD3 occurs is referred to herein as a 'TCR chain-CD3 interaction motif'. In addition, when there is a motif that interacts with any second TCR subunit on any first TCR subunit, this motif is collectively referred to as a 'first TCR subunit-second TCR subunit interaction motif'.

In addition, the TCR chain includes a 'transmembrane domain'. The term 'transmembrane domain' refers to the part through which the protein having binding to the cell membrane passes through the cell membrane (transmembrane). More broadly expressed, the part of the protein sequence that spans the cell membrane (membrane-spanning) Means TCR subunits other than the TCR chain also include both trans-membrane domains.

The CD3 chain contains motifs that play an important role in the generation of immune signals. Motif having this function is collectively referred to as an 'immune signal activation motif'. A representative example of an immune signal activation motif is an ITAM (immunoreceptor tyrosin-based activation motif).

Zeta chains are known to contain immune signal activation motifs. Specifically, the zeta chain is known to contain three ITAMs.

Each of these TCR subunits is known to contain characteristic amino acid residues that are involved in the assembly of the subunits into the TCR complex [2]. Characteristic amino acid residues and / or motifs involved in the assembly of such TCR complexes are referred to as 'TCR complex assembly engagement residues / motifs'. According to a specific study, each TCR subunit contains residues involved in TCR complex assembly. More specifically, for each TCR subunit, residues involved in TCR complex assembly are included in the transmembrane domain. More specifically, one TCR heterodimer contains three TCR complex assembly-response residues, and furthermore, the TCR-α chain and TCR-γ chain contain two TCR complex assembly-response residues, and the TCR-β chain and TCR- The δ chain contains one residue involved in TCR complex assembly. In addition, each of the CD3 heterodimer contains two TCR complex assembly residues, and further, the CD3γ chain, CD3δ chain and CD3ε chain each contain one TCR complex assembly residue. In addition, the zeta chain contains residues involved in the assembly of one TCR complex. At this time, the residues involved in the assembly of the known TCR complex have a common point in that they are polar amino acids, that is, acidic or basic amino acids. That is, the residues involved in TCR complex assembly may be polar residues included in the TCR transmembrane domain.

3. Formation process of TCR complex

This section is intended to illustrate the process by which TCR complexes are formed in the body to help understand the specification. The process described in this section is based on the process of forming a wild state TCR complex in normal cells, and is merely an exemplary explanation based on technically known contents so far. It is not intended to be limited to this.

The TCR complex formation process begins when the TCR subunit is genetically expressed through ribosomes. The expressed TCR subunit initially resides in the endoplasmic reticulum (ER). The TCR subunit is present in the form of a TCR heterodimer, CD3 heterodimer, zeta chain dimer before being assembled into a TCR complex.

The expressed TCR subunit remains bound to the surface of the vesicle. At this time, it is known that the TCR subunit, which is not bound to other TCR subunits, promotes the degradation process of the peptide, thus reducing stability, and cannot be secreted into the cell membrane and remains in the endoplasmic reticulum [3]. This may be due to the relationship between the residue involved in TCR complex assembly and the vesicle membrane. Specifically, in the case of a TCR heterodimer, the residues involved in TCR complex assembly have a polarity and do not equilibrate with the lipid bilayer structure of the vesicle membrane, so that the transmembrane domain of the TCR heterodimer is stably settled in the double phospholipid structure of the vesicle membrane Failure to do so can result in this. However, when the TCR heterodimer is assembled with the CD3 heterodimer, the residues involved in the assembly of the TCR complex are shielded, and the transmembrane domain having a lower polarity is stably settled in the double phospholipid structure, thereby increasing stability and consequently secreting from the endoplasmic reticulum. [2, 4].

In summary, the TCR subunit synthesized from the endoplasmic reticulum first undergoes a process of forming a temporary complex in the endoplasmic reticulum, and may be secreted from the vesicle after the complex is temporarily formed. Specifically, the process of forming a temporary complex in the vesicle membrane is unknown. The TCR heterodimer and CD3 heterodimer have been studied to be secreted from the endoplasmic reticulum in a complex. However, this does not mean that the zeta chain is secreted from the endoplasmic reticulum in the form of a zeta chain dimer, and the temporary complex is not limited to the complex of the TCR heterodimer and CD3 heterodimer. At this time, the temporary complex assembled in the endoplasmic reticulum membrane is referred to as an 'intermediate TCR complex'. At this time, the intermediate TCR complex may include a TCR heterodimer and a CD3 heterodimer. Illustratively, the intermediate TCR complex may be composed of one TCR heterodimer and one CD3 heterodimer. In another example, the intermediate TCR complex may be one TCR heterodimer and two CD3 heterodimers. In another example, the intermediate TCR complex may be one TCR heterodimer, two CD3 heterodimers, and one zeta chain dimer.

The TCR subunit and / or the intermediate TCR complex secreted from the endoplasmic reticulum penetrates the cell membrane and is initiated outside the cell. In this case, a small organ such as a golgi apparatus may be passed. In addition, if the intermediate TCR complex does not have all the components of the final TCR complex, it may include a process of assembling the intermediate TCR complex into the final TCR complex.

4. Structure of the gp41 protein

This section is intended to explain the structure of the gp41 protein to aid understanding of the specification. The structure of gp41 described in this section is based on gp41 of HIV-1 (Human Immunodeficiency Virus-1), and is only an exemplary explanation based on technically known contents so far, and gp41 and the object of the present invention It is not intended to limit the derived peptide sequence thereto.

gp41 is one of the subunits constituting a protein complex expressed on the surface of a virus belonging to the retroviral family. The gp41 is generally present in the form of a trimer in which three gp41s are assembled, and the trimer has a six-helix bundle structure. The 6-helix bundle structure is formed by domains N36 and C34 having an alpha helix structure included in gp41 [5].

Peptide sequence of gp41 can be largely classified into FP domain, NHR domain, Loop domain, CHR domain, transmembrane domain [6] The amino acid sequence of exemplary gp41 and its domain is as follows, but this is the strain NCBI Reference Sequence: NC_001802 It is obvious that it is only an example by .1 and does not mean that the technical scope of the present application does not reach to gp41 and its domains of other strains (gp41 full-length sequence: SEQ ID NO: 1; FP domain: SEQ ID NO: 2; NHR domain: SEQ ID NO: 3; Loop domain: SEQ ID NO: 4; CHR domain: SEQ ID NO: 5; transmembrane domain: SEQ ID NO: 6). Among them, an N-heptad repeat (NHR) domain is a domain having an alpha helical structure by including a repeat of a heptad motif, and includes an N36 domain (SEQ ID NO: 7). In addition, the CHR (C-heptad repeat) domain is located at the C-terminus than the NHR domain and includes a repeat of a heptad motif, and thus has an alpha-helix structure, and includes a C34 domain (SEQ ID NO: 8). In addition, the Loop domain is a domain that is located between the NHR domain and the CHR domain and connects two alpha helix structures. The linking portion of the loop domain and the CHR domain contains an immunosuppressive loop-associated determinant (ISLAD) motif (SEQ ID NO: 9), which is a highly homologous sequence between immunodeficiency vices.

It is known that gp41 induces fusion between the nuclear membrane of the retrovirus and the nuclear membrane of the host immune cell, and is involved in the suppression of the immune function of the host immune cell. It is known that many of the domains and / or motifs included in gp41 have a property of interacting with the TCR subunit. These domains can inhibit normal interactions between TCR subunits by interacting with TCR subunits, and further prevent TCR complexes from forming normal structures. More specifically, it is known that at least FP domain [7], Loop domain [8], CHR domain, transmembrane domain [9] and ISLAD motif [8] of the domain of gp41 have the ability to bind to the TCR subunit.

The molecule that binds to the immune-activated receptor so that the immune-activated receptor remains in the cell may be a 'trapper molecule'.

Hereinafter, the trapper molecule will be described.

5. Trapper molecules

One aspect of the content disclosed by this specification relates to a 'trapper molecule' for manipulating cells by interacting with a target molecule within the cell. '

 "Trapper molecule" refers to a molecule that interacts with an immune activating receptor or other target molecule. It is preferred that the trapper molecule can interact with the target molecule to confine the target molecule in the cell. That is, the trapper molecule may be a molecule of various types such as nucleic acids, peptides, polypeptides, proteins, or the like, as long as the target molecule interacts with the target molecule to prevent the target molecule from being exposed outside the cell surface, or different domains are combined. It may be an artificial recombinant polypeptide. When the trapper molecule is a peptide, it is referred to as a 'trapper peptide'.

In one embodiment, the trapper molecule can include a binding domain.

In some embodiments, the trapper molecule can include residual domains.

In some embodiments, the trapper molecule can include a linking domain.

In some embodiments, the trapper molecule can include a binding domain and a residual domain.

In some embodiments, the trapper molecule can include binding domains, residual domains and linking domains.

In one embodiment, the trapper molecule may be composed of a first structural domain having a specific conformational structure and a second structural domain having a conformational structure distinct from the first structural domain.

In some embodiments, the trapper molecule is a first structural domain having a specific conformational structure, a second structural domain having a conformational structure distinct from the first structural domain, and a conformational structure distinct from the first structural domain and the second structural domain. And a loop domain connecting the first structural domain and the second structural domain.

In the above embodiments, the first structural domain may include a binding domain and / or a residual domain. Also, the second structural domain may include a binding domain and / or a residual domain. Also, the loop domain may include a linking domain.

In one embodiment, the trapper molecule may have properties that interact with TCR subunits. Specifically, the trapper molecule can have affinity with the TCR subunit.

In some embodiments, the trapper molecule may have properties that interact with the genetic sequence encoding the TCR subunit.

In some embodiments, the trapper molecule may have a short half-life in the cell. Specifically, the trapper molecule can be a ligand for a receptor that is known to be involved in the proteolytic process within the cell.

In some embodiments, the trapper molecule may have properties that persist in certain organelles within the cell.

The bond between the trapper molecule and the target protein can be covalent or non-covalent.

When the trapper molecule is a nucleic acid, it may be DNA and / or RNA. At this time, the nucleic acid may have 15 to 3,000 or less nucleotides.

The trapper molecule may be a polypeptide composed of one consecutive amino acid sequence. At this time, the polypeptide may have an amino acid sequence of 5 to 1000 or less.

The trapper molecule can include one or more functional domains.

In one embodiment, the trapper molecule may be an artificial recombinant polypeptide in which two or more domains are bound.

The trapper molecules may have two or more identical domains. The same domain means a domain having the same function. For example, if both domains have a binding function, both domains may be the same domain. However, the same domains do not mean that the amino acid sequences are identical to each other, and even if the amino acid sequences are different, the same domains can be said to be functionally performing the same function.

The trapper molecule may have two or more different domains.

In one embodiment, the trapper molecule may be an artificial recombinant polypeptide in which a binding domain binding to a target molecule and a residual domain capable of trapping a protein bound in a cell are fused.

For example, the artificial recombinant polypeptide may be a binding domain and a vesicle residual domain.

In another embodiment, the trapper molecule may have a form in which other functional domains are added in addition to the binding domain and the residual domain.

For example, the artificial recombinant polypeptide may be in the form of a binding domain, a residual domain, and a transmembrane domain. As another example, the artificial recombinant polypeptide may have a form in which a binding domain, a residual domain, and a signal sequence domain are combined. Alternatively, as another example, the artificial recombinant polypeptide may have a form in which a binding domain, a residual domain, a transmembrane domain, and a signal sequence domain are combined.

The trapper molecule can be combined with a target molecule, so that the target molecule can be located in a specific organ or specific region in the cell. That is, it is possible to prevent the target molecule from moving to a place to function normally.

For example, the binding domain of the trapper molecule binds to the target molecule, and the residual domain of the trapper molecule can trap the target molecule in the cell by placing the target molecule in a specific organ or region within the cell.

The target molecule that the trapper molecule can bind to and confine in the cell may be an immune activation receptor.

Hereinafter, the functional domain will be described in detail.

5.1. Combined domain

The binding domain can be a polypeptide, a recombinant polypeptide, a synthesized polypeptide or a non-peptide sequence that can interact with a target, such as a ligand, antibody, receptor, peptide, and the like.

For example, the binding domain may be an antibody binding domain that binds to the antibody.

As another example, the binding domain may be a ligand binding domain that binds a ligand.

As another example, the binding domain may be a receptor binding domain that binds a receptor.

As another example, the binding domain may be a peptide binding domain that binds a peptide.

In one embodiment, the binding domain may be a binding domain that binds to a receptor or a receptor ligand.

At this time, the interaction between the binding domain and the target substance is an immunoprecipitation method, and a biochemical method including BiFC (Bimolecular fluorescence complementation), SPR (surface plasmon resonance), DPI (dual polarization interferometry), DLS (DLS) It can be measured by means such as biophysical methods including dynamic light scattering), FRET (Fluorescence resonance energy transfer), and the like, and is not limited thereto.

For example, the ligand binding domain may be a domain that binds a nuclear receptor ligand. Nuclear receptors include glucocorticoid receptors (GR), receptors for androgens (AR), receptors for mineralocorticosteroids (MR), receptors for progestins (PR), receptors for estrogens (ER), and thyroid hormones. Receptors for (TR), receptors for vitamin D (VDR), and receptors for retinoids (RAR). In addition, it may be a binding domain that binds receptors and receptor ligands such as steroids and thyroid receptors.

For another example, the binding domain may be a domain that binds a receptor present in immune cells.

Preferably, the binding domain may be a receptor present in immune cells, that is, a binding domain that binds to an immune-activated receptor.

Such immune-activated receptors include T cell receptors, B cell receptors, killer cell activating receptors, toll-like receptors (TLRs), such as TLR4 and TLR9, cytokines, chemokines, cytokine receptors, and chemokine receptors, , But is not limited to this.

The binding domain may be a polypeptide that binds all or part of the immune activation receptor.

The 'some regions' of the immune activation receptor include all essential peptides, polypeptides or domains corresponding to essential components of the immune activation receptor. Alternatively, an essential peptide, polypeptide, or domain corresponding to an essential component may also be included in another protein that forms a complex with an immune-activated receptor.

The binding domain may be a part of a polypeptide or a domain of a region that directly binds an antigen in an immune-activating receptor, a part of a cell membrane-penetrating domain, or a part of a polypeptide or a domain present inside a cell.

The binding domain may be covalently or non-covalently with some polypeptide or domain of the immunoactivating receptor.

The binding domain peptide may be composed of about 5 to 300 amino acid sequences.

According to certain embodiments, the peptide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 , 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 , 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 Two values selected from, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, and 300 It may consist of a number of amino acids in between.

According to another embodiment, the peptide may consist of 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, or 9 or more amino acids.

According to another embodiment, the peptide has 50 or more, 45 or less, 40 or less, 35 or less, 30 or less, 28 or less amino acids, 26 or less amino acids, 24 or less amino acids, 22 It may be the following amino acids, 20 or fewer amino acids, 18 or fewer amino acids, 16 or fewer amino acids, 14 or fewer amino acids, 12 or fewer amino acids, or up to 10 amino acids.

In some embodiments, the binding domain may include GLRLLMFIV (SEQ ID NO: 10) or GLRIVFAVL (SEQ ID NO: 11).

In some embodiments, the binding domain is AVGIGALF (SEQ ID NO: 12), GALFLGFLG (SEQ ID NO: 13), GAVFLGFL (SEQ ID NO: 14), GAMFLGFLG (SEQ ID NO: 15), GAVLLGFLG (SEQ ID NO: 16), GAFFLGFLG (SEQ ID NO: 17) , GAMIFGFLG (SEQ ID NO: 18) or GALLFGFLG (SEQ ID NO: 19). For example, the binding domain may be AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL (SEQ ID NO: 20), including AVGIGALF. Alternatively, the peptide may be G A LFLGFLG.

In some embodiments, the binding domain may include a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with the amino acid sequence of AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL. For example, the binding domain may be AEGIGALFLGFLGAAGSTMGARSMTLTVQARQL (SEQ ID NO: 21).

In some embodiments, the binding domain may include EQIWNHTTWMEW (SEQ ID NO: 22).

In some embodiments, the binding domain may include a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the EQIWNHTTWMEW amino acid sequence. For example, the binding domain may include EQLWNHTTWMEW (SEQ ID NO: 23). As another example, EEIWNHTTWMEW may include (SEQ ID NO: 24).

The binding domain may be a peptide sequence having the property of interacting with a TCR subunit. In this case, the TCR subunit that interacts with the binding domain may be one or more selected from TCR-α chain, TCR-β chain, TCR-γ chain, TCR-δ chain, CD3γ chain, CD3δ chain, CD3ε chain, and zeta chain. have. In one example, the binding domain may interact with a ligand recognition unit of the TCR subunit, preferably with a TCR chain, and more preferably with a TCR-α chain. In another example, the binding domain can interact with the signal activation unit of the TCR subunit, preferably with the CD3 chain.

Furthermore, the binding domain can interact with some domains / motifs within the amino acid sequence of the TCR subunit. Illustratively, the domain / motif may be an interaction motif between TCR subunits. In another example, the domain / motif may be a transmembrane domain of a TCR subunit. In another exemplary embodiment, the domain / motif may be a residue / motif involved in TCR complex assembly. In another exemplary embodiment, the domain / motif may be an immune signal activation motif included in the signal activation unit.

The length of the amino acid sequence of the binding domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 21 4, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, It may be a number between two values selected from 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, and 300. In one example, the length of the amino acid sequence of the binding domain is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102 , 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 , 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152 , 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 , 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202 , 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, It may be an amino acid between two values selected from 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, and 300. In other examples, the length of the amino acid sequence of a binding domain is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 , 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 , 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 , 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, It may be an amino acid between two values selected from 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, and 300.

In some embodiments, the binding domain can include an amino acid sequence that is homologous to the amino acid sequence of the gp41 protein. Specifically, the binding domain may include all or part of the full-length sequence of gp41. Preferably, the binding domain is an amino acid sequence homologous to the amino acid sequence of the gp41 protein, and the length of the sequence is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200. In one preferred example, the length of the amino acid sequence of the binding domain is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, Choose from 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 Become two figures It may be an amino acid in between. In other examples, the length of the amino acid sequence of a binding domain is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 , 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 Two selected from 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 Between the figures ah It can be Minosan. In another example, the length of the amino acid sequence of the binding domain is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, Two numbers selected from 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 Between Amino acids can be. In another example, the length of the amino acid sequence of the binding domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, Can be an amino acid between two numbers selected from 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 The. In another example, the length of the amino acid sequence of the binding domain is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200. In another example, the length of the amino acid sequence of the binding domain is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, It may be an amino acid between two values selected from 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200. In another example, the length of the amino acid sequence of a binding domain is 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, Two selected from 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 It can be an amino acid between dog numbers. In another example, the length of the amino acid sequence of a binding domain is 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, Between two numbers selected from 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 It may be an amino acid. However, the length of the binding domain is not limited to the numbers mentioned in this paragraph.

At this time, the binding domain homologous to the amino acid sequence of the gp41 protein is partially or completely selected from at least one amino acid sequence selected from FP domain, NHR domain, Loop domain, CHR domain, transmembrane domain, N36, and C34 of gp41. It can contain as.

In some examples, the binding domain that is homologous to the amino acid sequence of the gp41 protein may include part or all of the amino acid sequence of the FP domain of gp41. At this time, preferably, the length of the binding domain is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. Also preferably, the length of the binding domain may be an amino acid between two values selected from 33, 34, 35, 36, 37, 38, 39, and 40.

In some examples, the binding domain homologous to the amino acid sequence of the gp41 protein may include some or all of the amino acid sequence of the transmembrane domain of gp41. At this time, preferably, the length of the binding domain is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. Further preferably, the length of the binding domain is from 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 It may be an amino acid between the two values selected.

In some examples, the binding domain homologous to the amino acid sequence of the gp41 protein may include at least one amino acid sequence selected from a CHR domain, a Loop domain, and an ISLAD motif, in whole or in part, preferably an amino acid of the ISLAD motif Sequences can be included as part or all. At this time, preferably, the length of the binding domain is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. Also preferably, the length of the binding domain is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, and 40.

The binding domain may have a percent homology between the gp41 protein and two values selected from 50 to 100, preferably a percent homology between the two values selected from 60 to 100, and preferably It may have a percentage homology between two values selected from 70 to 100, preferably may have a percentage homology between two values selected from 80 to 100, preferably 90 to 100 It may have a percentage homology between two values selected from 100.

In some embodiments, the binding domain can include an amino acid sequence that is homologous to the amino acid sequence of a TCR subunit. Specifically, the binding domain may include all or a part of the full-length sequence of the TCR subunit. Preferably, the binding domain is an amino acid sequence that is homologous to the amino acid sequence of the TCR subunit, and the length of the sequence is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Choose from 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 It can be an amino acid between two levels. In one preferred embodiment, the length of the amino acid sequence of the binding domain is 18, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, It may be an amino acid between two values selected from 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. However, the length of the binding domain is not limited to the numbers mentioned in this paragraph.

At this time, the binding domain homologous to the amino acid sequence of the TCR subunit is selected from the TCR-α chain, TCR-β chain, TCR-γ chain, TCR-δ chain, CD3γ chain, CD3δ chain, CD3ε chain and zeta chain. At least one amino acid sequence may be included in part or in whole. Furthermore, the binding domain may partially or entirely include the amino acid sequence of the domain / motif included in the TCR subunit. Specifically, the domain / motif included in the TCR subunit may be, but is not limited to, an interaction motif between TCR subunits, a transmembrane domain, or a TCR complex assembly engagement motif.

In some instances, a binding domain homologous to the amino acid sequence of a TCR subunit may include some or all of the amino acid sequence of the TCR chain, wherein the TCR chain is preferably a TCR-α chain. At this time, preferably, the length of the binding domain is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, and 40.

Furthermore, the binding domain may include, in whole or in part, the amino acid sequence of the interaction motif between the TCR chain and other TCR subunits. Alternatively, the binding domain may include part or all of the amino acid sequence of the transmembrane domain of the TCR chain. Alternatively, the binding domain may include, in whole or in part, the amino acid sequence of the motif involved in TCR complex assembly of the TCR chain.

In some examples, the binding domain homologous to the amino acid sequence of the TCR subunit may include at least one amino acid sequence selected from the CD3γ chain, CD3δ chain and CD3ε chain, in whole or in part.

Furthermore, the binding domain may comprise the amino acid sequence of the CD3 chain-TCR-α chain interaction motif in part or in whole. Alternatively, the binding domain may include part or all of the amino acid sequence of the transmembrane domain of the CD3 chain. Alternatively, the binding domain may include, in whole or in part, the amino acid sequence of the motif involved in the assembly of the TCR complex of the CD3 chain.

The binding domain may have a percent homology between the TCR subunit and two values selected from 50 to 100, preferably a percent homology between the two values selected from 60 to 100, Preferably it can have a percentage homology between two values selected from 70 to 100, preferably it can have a percentage homology between two values selected from 80 to 100, preferably 90 It may have a percentage homology between two values selected from 100.

In some embodiments, the binding domain may have properties that interact with the genetic sequence encoding the TCR subunit. In this case, the binding domain is a part of a sequence encoding at least one amino acid sequence selected from TCR-α chain, TCR-β chain, TCR-γ chain, TCR-δ chain, CD3γ chain, CD3δ chain, CD3ε chain and zeta chain. Or you can interact with the whole. Preferably, the binding domain is capable of inhibiting the expression of the genetic sequence encoding the TCR subunit. Binding domains include, but are not limited to, means for interacting with commonly known genetic sequences, such as RNAi, siRNA, chemical inhibitors, and peptides that bind nucleic acids.

The trapper molecule may include one or more binding domains. For example, a trapper molecule may include two or more binding domains, and each of the binding domains may bind some domain of the immune activation receptor.

For example, the two or more binding domains may bind the same region of the same immune activation receptor. In this case, the binding domain may have the same amino acid sequence or different amino acid sequences.

As another example, the two or more binding domains may bind different regions of the same immune activation receptor. In this case, the binding domain may have the same amino acid sequence or different amino acid sequences.

For example, the two or more binding domains may bind some regions of different immune activation receptors.

The binding domain is a similar amino acid sequence as well as the above amino acid sequence, and includes all such amino acid sequences if it can perform the same function as that of the binding domain.

The binding domain may be an artificially synthesized polypeptide. At this time, the artificially synthesized binding domain may form a covalent or non-covalent bond with the immunoactivating receptor.

Artificially synthesized binding domains may have some or complete homology with some of the polypeptides present in nature.

The binding domain may be derived from some polypeptide of a naturally occurring protein. Depending on the species present in nature, a difference may exist in the amino acid sequence of the binding domain, and the binding domain may have some or complete homology with some polypeptides derived from nature.

For example, the binding domain may include a human-derived peptide sequence. For example, the binding domain may include some of the peptide sequences of the human immune activation receptor.

As another example, the binding domain may include some domains derived from viruses. In this case, the binding domain is referred to as a virus-derived binding domain. For example, the binding domain is derived from a herpes virus, a giant cell virus, a Lhasa virus, a retrovirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a pox virus, or a herpes simplex virus-derived binding domain. And so on.

Preferably, the binding domain may be a polypeptide derived from retrovirus.

For example, retroviruses include human HIV (human immunodeficiency virus), SIV (monkey immunodeficiency virus), and infectious anemia virus (EIAV) in horses.

Preferably, the binding domain may be a polypeptide derived from human immunodeficiency virus (HIV).

Human immunodeficiency virus (HIV) is a pathogen of AIDS, commonly referred to as its acronym AIDS. There are two main strains of the virus named HIV-1 and HIV-2.

The binding domain derived from HIV may be derived from HIV-1 or derived from HIV-2.

Envelope proteins in HIV-1 are glycoproteins synthesized with the gp160 protein, and then gp160 is cleaved to form gp120 and gp41 (HIV-1), respectively. Envelope proteins in HIV-2 are glycoproteins synthesized with the gp140 protein, and then gp140 is cleaved to form gp110 or gp36 (HIV-2), respectively.

In one example, the binding domain may be some or all of the proteins of gp41, gp120 or gp160 derived from HIV-1.

As another example, the binding domain may be some or all of the proteins of gp110, gp36 or gp140 derived from HIV-2.

Preferably, the binding domain may be some domain of the gp41 protein.

For example, some of the domains of the gp41 protein may bind some domains of the T cell receptor, and thus the binding domain derived from the gp41 protein may be a binding domain targeting the T cell receptor.

Hereinafter, the binding domain derived from gp41 will be described.

5.1.1. Gp41-derived binding domain

 'gp41' corresponds to one type of sugar protein, and is a sub-unit of the envelope protein complex of retroviruses, including human immune virus (HIV). gp41 is a membrane-permeable protein that contains several regions of the extracellular region required by the virus to infect host cells.

 The 'gp41' protein is composed of cytoplasmic, transmembrane and extracellular domains [14]. The extracellular domain has a region having four major functions, and two additional regions.

The extracellular domain of gp41 is i) a fusion peptide composed of 16 hydrophobic residues (referred to as 'FP') region, ii) N-terminal heptad repeat site (referred to as 'NHR') iii) C-terminal heptad repeat Site (referred to as 'CHR') iv) Includes a membrane proximal extracellular region rich in tryptophan v) a loop region.

Each of the FP, NHR, CHR and Loop can specifically bind to the T cell receptor and inhibit the proliferation of T cells.

The FP contains an immunosuppressive site that specifically binds to the T cell receptor, and the first immunosuppressive site includes an N-terminal hydrophobic region.

The NHR region is located at the N-terminus in the extracellular domain, and is located at amino acids 535-581. In addition, the NHR region contains the 'ISU' region, which is an immunosuppressive region, and the ISU is included in the 583-599 amino acid region [15].

The CHR region is located at the C-terminus in the extracellular domain, and is located at amino acids 628-662.

The Loop region is a part of the extracellular domain, and may form a disulfide bond so that the hairpin structure of gp41 is stabilized. The loop region is located at amino acids 584-628. The Loop region contains a highly conserved sequence region, an immunosuppressive loop-associated determinant (called 'ISLAD'), and the ISLAD is located at amino acids 605-641. For example, the ISLAD can bind to the T-cell receptor membrane, thereby interacting with the complex of T-cell receptors.

The transmembrane (called 'TMD') region, which is the transmembrane region of gp41, can bind to T cells and CD3 / TCR complexes and inhibit the proliferation of T cells. The TMD region is located at amino acid sequences 684-702. Peptides derived from some domains (GLVGLRIVF) of the transmembrane region can significantly inhibit T cell proliferation than the full length transmembrane domain.

The binding domain may include a part of the amino acid sequence of any one or more domains of FP, NHR, CHR, Loop or TMD.

For example, the binding domain may be a partial amino acid sequence of the FP domain, a partial amino acid sequence of the NHR domain, a partial amino acid sequence of the CHR domain, a partial amino acid sequence of the Loop domain, or a partial amino acid sequence of the TMD domain.

As another example, the binding domain may be some amino acid sequences included across the FP domain and the NHR domain.

For example, the binding domain may be some amino acid sequences included across the NHR domain and the Loop domain.

As another example, the binding domain may be some amino acid sequences included over the Loop domain and the CHR domain.

For example, the binding domain may be a partial amino acid sequence included over the CHR domain and the TMD domain.

Preferably, the binding domain binding to the T cell receptor may be any one of FP, Loop and TMD.

More preferably, the region of the loop that binds the T cell receptor may be ISLAD.

The binding domain may include about 3 to 50 amino acids of the FP domain.

In one example, the binding domain is 5 to 50 amino acids, 5 to 45 amino acids, 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 28 amino acids, 5 to 26 of the FP domains Amino acids, 5 to 24 amino acids, 5 to 22 amino acids, or 5 to 18 amino acids.

As another example, the binding domain may include 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, or 9 or more amino acids among FP domains.

For example, the binding domain is 50 or more, 45 or less, 40 or less, 35 or less, 30 or less, 28 or less amino acids, 26 or less amino acids, 24 or less amino acids, 22 or less of 22 FP domains It may be the following amino acids, 20 or fewer amino acids, 18 or fewer amino acids, 16 or fewer amino acids, 14 or fewer amino acids, 12 or fewer amino acids, or up to 10 amino acids.

Preferably, the binding domain may include 10 to 35 amino acids in the FP domain, and includes analogs or derivatives of 5 or more amino acids.

In one embodiment, the binding domain derived from the FP domain may include any one or more of AVGIGALF, GALFLGFLG, GAVFLGFL, GAMFLGFLG, GAVLLGFLG, GAFFLGFLG, GAMIFGFLG, and GALLFGFLG.

For example, the binding domain may be AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL.

As another example, the binding domain derived from the FP domain may be AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL or an amino acid sequence having at least 70% homology to AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL.

The binding domain derived from the FP domain may further include (X) n and / or (Y) m at both ends, that is, N'- (X) n AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL (Y) m-C ',. The (X) n corresponds to the N-terminal blocking group, and at least 50% or more of the amino acids are hydrophobic (X is an arbitrary amino acid sequence, n is an arbitrary integer). In addition, (Y) m corresponds to a C-terminal blocking group, and at least 50% or more of the amino acids are hydrophobic (Y is an arbitrary amino acid sequence, m is an arbitrary integer).

The m and n may be an integer of 5 to 30 or less, an integer of 6 to 28 or less, an integer of 7 to 26 or less, an integer of 8 to 24 or less, an integer of 10 to 22 or less, or an integer of 12 to 20 or less.

In another embodiment, the binding domain may be an amino acid sequence in which some mutations occur among FP domains. For example, the FP peptide can be a V2E variant. The V2E variant peptide may be AEGIGALFLGFLGAAGSTMGARSMTLTVQARQL.

The binding domain may include about 3 to 50 amino acids of the loop domain.

For example, the binding domain is 5 to 50 amino acids, 5 to 45 amino acids, 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 28 amino acids, 5 to 26 amino acids in the loop domain Amino acids, 5 to 24 amino acids, 5 to 22 amino acids, or 5 to 18 amino acids.

As another example, the binding domain may include 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, or 9 or more amino acids among loop domains.

For example, the binding domain is 50 or more, 45 or less, 40 or less, 35 or less, 30 or less, 28 or less amino acids, 26 or less amino acids, 24 or less amino acids, 22 or less of 22 of the loop domains It may be the following amino acids, 20 or fewer amino acids, 18 or fewer amino acids, 16 or fewer amino acids, 14 or fewer amino acids, 12 or fewer amino acids, or up to 10 amino acids.

Preferably, the binding domain may include 10 to 35 amino acids in the loop domain, and includes analogs or derivatives of 5 or more amino acids.

In one embodiment, the Loop-derived binding domain may include an amino acid sequence of TTAVPWNASWSNKSLEQI (SEQ ID NO: 25), WNASWSNKSLEQIWNHT (SEQ ID NO: 26), SNKSLEQIWNHTTWMEWD (SEQ ID NO: 27) or EQIWNHTTWMEWDREINN (SEQ ID NO: 28).

For example, the binding domain may be TTAVPWNASWSNKSLEQTWNHTTWMEWDREINNYTSL (SEQ ID NO: 29) or SNKSLEQIWNHTTWMEWD.

Preferably, the binding domain may be SNKSLEQIWNHTTWMEWD corresponding to the ISLAD domain among loop domains.

The binding domain may be an analog in which some amino acid sequences of the ISLAD domain are modified. For example, the ISLAD analogue can be SNKSLEQIGNHTTGMEGD (SEQ ID NO: 30) or SNKSLEQIWNHTTWMGWG (SEQ ID NO: 31).

The binding domain may include about 3 to 50 amino acids of the TMD domain.

In one example, the binding domain is 5 to 50 amino acids, 5 to 45 amino acids, 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 28 amino acids, 5 to 26 of the TMD domains Amino acids, 5 to 24 amino acids, 5 to 22 amino acids, or 5 to 18 amino acids.

As another example, the binding domain may include 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, or 9 or more amino acids among TMD domains.

For example, the binding domain is 50 or more, 45 or less, 40 or less, 35 or less, 30 or less, 28 or less amino acids, 26 or less amino acids, 24 or less amino acids, 22 or less of TMD domains It may be the following amino acids, 20 or fewer amino acids, 18 or fewer amino acids, 16 or fewer amino acids, 14 or fewer amino acids, 12 or fewer amino acids, or up to 10 amino acids.

Preferably, the binding domain may include 9 to 30 amino acids in the TMD domain, and includes analogs or derivatives of 5 or more amino acids.

In one embodiment, the TMD-derived binding domain comprises a GLVGLAIVF (SEQ ID NO: 32), GLLGLAIVF (SEQ ID NO: 33), GGVGLAQF (SEQ ID NO: 34), GGVGLAIVF (SEQ ID NO: 35), or GGLGLAIVF (SEQ ID NO: 36) amino acid sequence. Can be.

For example, the binding domain derived from TMD may be LFIMIVGGLVGLRIVFAVLSI (SEQ ID NO: 37).

The binding domain may be an analog in which some amino acid sequences of the TMD domain are modified. For example, the binding domain may be LFIMIVGGLRLLMFIVFAVLSI (SEQ ID NO: 38) or LFIMIVGGLRIVFAVLSI (SEQ ID NO: 39).

The binding domain includes not only the domain derived from gp41, but also some amino acid sequences of other virus-derived proteins, if it functions similarly to the gp41-derived binding domain.

The immunoactivated receptor to which the gp41-derived binding domain binds may be a T cell receptor. That is, the binding domain can bind all or part of the T cell receptor. At this time, the T cell receptor also includes a protein that forms a complex therewith.

The binding domain may perform a function of recognizing and binding at least one of the TCR subunits.

The trapper molecule may further include a residual domain for trapping the bound immune activation receptor in the cell.

Hereinafter, the residual domain for allowing the immune-activated receptor to be present in the cell will be described.

5.2. Residual domain

The trapper molecule for the immune activation receptor to be present in the cell may include a residual domain.

The residual domain refers to a peptide sequence having a high tendency to exist in a specific organelle or a specific region in a cell. At this time, the residual domain may be a signal sequence that retains a certain protein in a corresponding organ or a specific site from the beginning, or a signal sequence that recovers an external protein and remains in a specific organ or a specific region in a cell.

The specific organelle may be a organelle via a TCR subunit, an intermediate TCR complex, or a final TCR complex in the process of forming a TCR complex. In some instances, certain organelles may be vesicles. In some examples, certain organelles may be Golgi apparatus.

In some embodiments, the residual domain may be a peptide with a high tendency to be present in endoplasmic reticulum. At this time, the residual domain is referred to as a 'vesicle residual domain'. In one example, the vesicle residual domain may be a signal peptide of a vesicle surface protein / peptide. In another example, the vesicle residual domain may be a transmembrane domain of vesicle surface protein / peptide. Examples related to specific vesicle residual domains can be found in various well-known papers, all of which are included in the scope of the present invention [10].

In some embodiments, the residual domain may be a peptide with a high likelihood of being present in the Golgi apparatus. At this time, the residual domain is referred to as a 'Golgi body residual domain'. In one example, the Golgi apparatus residual domain may be a Golgi surface protein / peptide signal peptide. In another example, the Golgi apparatus residual domain may be a transmembrane domain of the Golgi surface protein / peptide. Examples related to specific Golgi residual domains can be found in various well-known papers, and all the examples are included in the scope of the present invention [11].

In some embodiments, the residual domain may be a peptide with a high likelihood of being present in the cell membrane. At this time, the residual domain may be a C-terminal signal sequence and / or a transmembrane domain having a feature that the N-terminal is arranged in a cytoplasmic direction [12]. The C-terminal signal sequence and / or transmembrane domain having these properties is referred to as a 'cytoplasm-directing expression domain'. Illustratively, the signal sequence and transmembrane domain may be homologous to the sequence of synaptobrevin or cytochrome b5.

The tendency that residual domains are likely to be present in specific organelles can be confirmed by commonly known methods such as immunofluorescent staining [13].

The residual domain may be located at the N-terminus, C-terminus, or adjacent regions of the trapper molecule.

For example, the vesicle residual domain may be located at the N-terminus, C-terminus, or adjacent regions of the trapper molecule.

As another example, the lysosomal residual domain may be located at the N-terminus, C-terminus, or adjacent regions of the trapper molecule.

For example, the residual domain, which is a nucleus, may be located at the N-terminus, the C-terminus, or adjacent regions thereof.

As another example, the Golgi apparatus residual domain may be located at the N-terminus, C-terminus, or adjacent regions of the trapper molecule.

For example, the proteasome residual domain may be located at the N-terminus, C-terminus, or adjacent regions of the trapper molecule.

As another example, the mitochondrial residual domain may be located at the N-terminus, C-terminus, or adjacent regions of the trapper molecule.

In one embodiment, the endoplasmic remnant signal sequence may be a signal sequence comprising KDEL (SEQ ID NO: 40) or KKXX (SEQ ID NO: 41) (X is any amino acid).

As an example, the vesicle residual signal sequence may include SEKDEL (SEQ ID NO: 42).

As another example, the vesicle residual signal sequence may include KKMP (SEQ ID NO: 43) or KKTN (SEQ ID NO: 44).

For example, the vesicle residual signal sequence is KYKSPRSFIEEKKMP (SEQ ID NO: 45), LKYKSRRSFIEEKKMP (SEQ ID NO: 46), LYKYKSRRSFIEEKKMP (SEQ ID NO: 47), LYCKYKSRRSFIEEKKMP (SEQ ID NO: 48), KYCNKYKSRRSFIEEKKMP (SEQ ID NO: 49) , LYCNKYKSRRSFIDEKKMP (SEQ ID NO: 51), LYEQKLISEEDLKYKSRRSFIEEKKMP (SEQ ID NO: 52), LYCYPYDVPDYAKYKSRRSFIEEKKMP (SEQ ID NO: 53), LYKKLETFKKTN (SEQ ID NO: 54), and any one of LYEQKLISEEDLKKLETFK, which may be any one of SEQ ID NO: 55

Residual sequence of the endoplasmic reticulum may be added with some sequences in addition to the signal sequence depending on the nature of the protein.

For example, in the case of a water-soluble protein, the sequence of amino acids 5 and 6 to the left of the vesicle residue sequence corresponds to an important sequence for retaining the water-soluble protein in the vesicle. Therefore, in the TCR / CD3 complex configuration, in the case of the portion corresponding to the water-soluble protein, not only the endoplasmic remnant sequence, but also the amino acid sequence located 5th and 6th to the left of the endoplasmic resid sequence may be included in the residual domain.

In other embodiments, other endoplasmic reticulum or Golgi apparatus signal sequence may be a signal sequence comprising KXKXX (SEQ ID NO: 56), KXD / E and / or YQRL (SEQ ID NO: 57) (X is any amino acid).

For example, the vesicle residual signal sequence is HDEF (SEQ ID NO: 58), HDEL (SEQ ID NO: 59), RDEF (SEQ ID NO: 60), RDEL (SEQ ID NO: 61), WDEL (SEQ ID NO: 62), YDEL (SEQ ID NO: 63), HEEF (SEQ ID NO: 64), HEEL (SEQ ID NO: 65), KEEL (SEQ ID NO: 66), REEL (SEQ ID NO: 67), KAEL (SEQ ID NO: 68), KCEL (SEQ ID NO: 69), KFEL (SEQ ID NO: 70), KGEL (SEQ ID NO: 71), KHEL (SEQ ID NO: 72), KLEL (SEQ ID NO: 73), KNEL (SEQ ID NO: 74), KQEL (SEQ ID NO: 75), KREL (SEQ ID NO: 76), KSEL (SEQ ID NO: 77), KVEL ( SEQ ID NO: 78), KWEL (SEQ ID NO: 79), KYEL (SEQ ID NO: 80), KEDL (SEQ ID NO: 81), KIEL (SEQ ID NO: 82), DKEL (SEQ ID NO: 83), FDEL (SEQ ID NO: 84), KDEF (SEQ ID NO: Number 85), KKEL (SEQ ID NO: 86), HADL (SEQ ID NO: 87), HAEL (SEQ ID NO: 88), HIEL (SEQ ID NO: 89), HNEL (SEQ ID NO: 90), HTEL (SEQ ID NO: 91), KTEL (SEQ ID NO: 92), HVEL (SEQ ID NO: 93), NDEL (SEQ ID NO: 94), QDEL (SEQ ID NO: 95), REDL (SEQ ID NO: 96), RNEL (SEQ ID NO: 97), RTDL (SEQ ID NO: 98), RTEL (SEQ ID NO: 99), SDEL (SEQ ID NO: 100), TDEL (SEQ ID NO: 101) and SKEL (SEQ ID NO: 102).

For example, the other vesicle or Golgi residual signal sequence is any of LYYQRL (SEQ ID NO: 103), LYEQKLISEEDLYQRL (SEQ ID NO: 104), LYKRKIIAFALEGKRSKVTRRPKASDYQRL (SEQ ID NO: 105), LYRNIKCD (SEQ ID NO: 106) and LYEQKLISEEDLRNIKCD (SEQ ID NO: 107) Can be.

In other embodiments, the proteasome residual signal sequence can be a proteasome target sequence comprising a “PEST” motif—SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 108).

The trapper molecule can include one or more residual domains.

For example, when two or more residual domains exist, two or more residual domains may be the same organelle residual domain. At this time, the residual signal sequence may be the same or different.

As another example, when two or more residual domains are present, the two or more residual domains may be different organelle residual domains.

The residual domain may be located at the N-terminus or C-terminus of the binding domain of the trapper molecule.

For example, the trapper molecule may be N '-binding domain (n)-residual domain (m)-C'.

As another example, the trapper molecule may be N '-residual domain (m)-binding domain (n)-C'.

At this time, n and m indicate integers.

5.3. Connection domain

The trapper molecule may further include an additional domain. The domain that is further included in the trapper molecule is not a necessary constitutive domain for performing the function of the trapper molecule, but may enhance or add new functions.

The domain added to the trapper molecule may be a linking domain.

The linking domains may link the same two or more functional domains of the trapper molecule or two or more different functional domains.

For example, a linking domain functions to link a binding domain with one or more residual domains.

The amino acid sequence of the linking domain is as follows.

In one embodiment, the linking domain can be about 1 to about 100 amino acids in length. At this time, the connection domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99, and 100.

Depending on the specific embodiment, the linking domain may have 3 to 20 amino acids in length. Depending on the particular embodiment, the linking domain may be from about 20 to about 40 amino acids long.

According to certain embodiments, the linking domain may comprise at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids or at least It may be 40 amino acids long. However, it is not limited thereto.

The linking domain may connect a binding domain and a residual domain using various linking domains known in the art.

The linking domain may be a polypeptide composed of hydrophilic residues of various lengths.

According to one embodiment, the linking domain may be (GGGGS) n. At this time, n may be an integer from 1 to 12.

For example, the amino acid sequence of the linking domain may be GGGGSGGGGS (SEQ ID NO: 109) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 110).

According to another embodiment, the linking domain may be a polypeptide comprising an amino acid sequence selected from the group consisting of (GGGXX) nGGGGS and GGGGS (XGGGS) n. X may be any one of the amino acid sequences of amino acids A, G, Q, P, S, and (GGGXX) n may be repeated n times.

In one embodiment, the linking domain can be any one of (GGGGA) nGGGGS, (GGGGQ) nGGGGS, (GGGPS) nGGGGS and GGGGS (PGGGS) n, where n is an integer greater than or equal to 1.

For example, the linking domain may be (GGGGA) 2GGGGS, (GGGGQ) 2GGGGS, (GGGPS) 2GGGGS or GGGGS, (PGGGS) 2.

In this case, n may be 1 to 20, or n may be 1 to 10. That is, n can be 2, n is 3, n is 4, n is 5, n is 6, n is 7, n is 8, n is 9 or n is 10.

5.4. Transmembrane domain

The trapper molecule may further include a transmembrane domain.

The transmembrane domain can help membrane anchoring of the residual domain, thereby improving the function of the residual domain.

For example, transmembrane domains include CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16 (e.g. CD16A or CD16B), OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32 For example, CD32A or CD32B), CD64 (eg, CD64A, CD64B or CD64C), VEGFR2, FAS or FGFR2B. However, it is not limited thereto. Various other residual signal sequences are known in the art and can be used.

Preferably, the transmembrane domain may be a CD8α transmembrane domain.

* In one embodiment, the amino acid sequence of the CD8α transmembrane domain may be IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 111).

The transmembrane domain may be located at the N-terminal, C-terminal or N-terminal, C-terminal region of the residual domain.

For example, the endoplasmic reticulum signal sequence and the transmembrane domain located in the region adjacent to the N-terminal or N-terminal thereof

N'- IYIWAPLAGTCGVLLLSLVITLYC KYKSPRSFIEEKKMP-C '(SEQ ID NO: 112);

N'- IYIWAPLAGTCGVLLLSLVITLYC LKYKSRRSFIEEKKMP-C '(SEQ ID NO: 113);

N'- IYIWAPLAGTCGVLLLSLVITLYC LYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 114);

N'- IYIWAPLAGTCGVLLLSLVITLYC LYCKYKSRRSFIEEKKMP-C '(SEQ ID NO: 115);

N'- IYIWAPLAGTCGVLLLSLVITLYC KYCNKYKSRRSFIEEKKMP-C '(SEQ ID NO: 116);

N'- IYIWAPLAGTCGVLLLSLVITLYC LYKYKSRRSFIDEKKMP-C '(SEQ ID NO: 117);

N'- IYIWAPLAGTCGVLLLSLVITLYC LYCNKYKSRRSFIDEKKMP-C '(SEQ ID NO: 118);

N'- IYIWAPLAGTCGVLLLSLVITLYC LYEQKLISEEDLKYKSRRSFIEEKKMP-C '(SEQ ID NO: 119);

N'- IYIWAPLAGTCGVLLLSLVITLYC LYCYPYDVPDYAKYKSRRSFIEEKKMP-C '(SEQ ID NO: 120);

N'- IYIWAPLAGTCGVLLLSLVITLYC LYKKLETFKKTN-C '(SEQ ID NO: 121); And

N'- IYIWAPLAGTCGVLLLSLVITLYC LYEQKLISEEDLKKLETFKKTN-C '(SEQ ID NO: 122) may be any one or more (underlined means transmembrane domain sequence).

For example, the endoplasmic reticulum signal sequence and its transmembrane domain located at the C-terminus or the region adjacent to the C-terminus

N'-KYKSPRSFIEEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 123);

N'-LKYKSRRSFIEEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 124);

N'-LYKYKSRRSFIEEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 125);

N'-LYCKYKSRRSFIEEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 126);

N'-KYCNKYKSRRSFIEEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 127);

N'-LYKYKSRRSFIDEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 128);

N'-LYCNKYKSRRSFIDEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 129);

N'-LYEQKLISEEDLKYKSRRSFIEEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 130);

N'-LYCYPYDVPDYAKYKSRRSFIEEKKMP IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 131);

N'-LYKKLETFKKTN IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 132); And

N'-LYEQKLISEEDLKKLETFKKTN IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 133) can be any one or more (underlined means transmembrane domain sequence).

For example, the proteasome signal sequence and its transmembrane domain located at the N-terminus or the region adjacent to the N-terminus may be N'- IYIWAPLAGTCGVLLLSLVITLYC SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV-C '(SEQ ID NO: 134) (underlined means transmembrane domain sequence) box).

In other embodiments, the transmembrane domain can be located at the N-terminus, C-terminus, or adjacent region of the binding domain.

For example, the binding domain and its transmembrane domain located at the N-terminal or N-terminal adjacent region are

N'- IYIWAPLAGTCGVLLLSLVITLYC LFIMIVGGLVGLRIVFAVLSI-C '(SEQ ID NO: 135);

N'- IYIWAPLAGTCGVLLLSLVITLYC AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL-C '(SEQ ID NO: 136); And

N'- IYIWAPLAGTCGVLLLSLVITLYC SNKSLEQIWNHTTWMEWD-C '(SEQ ID NO: 137), any one or more (underlined means transmembrane domain sequence).

For example, the binding domain and its transmembrane domain located at the C-terminus

N'-LFIMIVGGLVGLRIVFAVLSI IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 138) ;

N'-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 139); And

N'-SNKSLEQIWNHTTWMEWD IYIWAPLAGTCGVLLLSLVITLYC -C '(SEQ ID NO: 140) may be any one or more (underlined means transmembrane domain sequence).

5.5. Additional signal sequences

The trapper molecule may further include a signal sequence in addition to the residual domain.

For example, the additional signal sequence may be a CD8α signal sequence.

The CD8α chain is a visceral membrane glycoprotein that plays an essential role in the immune response and acts primarily as an auxiliary receptor for MHCI molecules in T cells. In NK cells, the CD8α allomeric dimer at the cell surface provides a mechanism involved in multiple targets and lysis, and the CD8α allomeric dimer molecule also functions to promote survival and differentiation of memory CD8 + T cells. T-lymphocyte differentiation antigen T8 / Leu-2 or CD_antigen: also called CD8a.

The CD8α signal sequence may be N'-ALPVTALLLPLALLLHAARP-C '(SEQ ID NO: 141).

The CD8α signal sequence may be located at the N-terminus, C-terminus, or adjacent regions of both ends of the trapper molecule.

In one embodiment, the CD8α signal sequence may be located at the N-terminus or a region adjacent to the N-terminus of the trapper molecule, and may be linked to a binding domain. In this case, for example, the amino acid sequence

N ' -ALPVTALLLPLALLLHAARP LFIMIVGGLVGLRIVFAVLSI-C' (SEQ ID NO: 142);

N ' -ALPVTALLLPLALLLHAARP LFIMIVGGLVGLRIVFAVLSI -C' (SEQ ID NO: 143); And

N ' -ALPVTALLLPLALLLHAARP LFIMIVGGLVGLRIVFAVLSI-C' (SEQ ID NO: 144) may be any one (underlined means CD8α signal sequence).

In another embodiment, the CD8α signal sequence may be located at the N-terminal or N-terminal region of the trapper molecule, and may be linked to a residual domain.

For example, when the residual domain is a vesicle signal sequence, the CD8α signal sequence may be linked to the vesicle signal sequence. In this case, for example, the amino acid sequence

N ' -ALPVTALLLPLALLLHAARP KYKSPRSFIEEKKMP- C' (SEQ ID NO: 145);

N ' -ALPVTALLLPLALLLHAARP LKYKSRRSFIEEKKMP- C' (SEQ ID NO: 146);

N ' -ALPVTALLLPLALLLHAARP LYKYKSRRSFIEEKKMP- C' (SEQ ID NO: 147);

N ' -ALPVTALLLPLALLLHAARP LYCKYKSRRSFIEEKKMP- C' (SEQ ID NO: 148);

N ' -ALPVTALLLPLALLLHAARP KYCNKYKSRRSFIEEKKMP- C' (SEQ ID NO: 149);

N ' -ALPVTALLLPLALLLHAARP LYKYKSRRSFIDEKKMP- C' (SEQ ID NO: 150);

N ' -ALPVTALLLPLALLLHAARP LYCNKYKSRRSFIDEKKMP- C' (SEQ ID NO: 151);

N ' -ALPVTALLLPLALLLHAARP LYEQKLISEEDLKYKSRRSFIEEKKMP- C' (SEQ ID NO: 152);

N ' -ALPVTALLLPLALLLHAARP LYCYPYDVPDYAKYKSRRSFIEEKKMP- C' (SEQ ID NO: 153);

N ' -ALPVTALLLPLALLLHAARP LYKKLETFKKTN- C' (SEQ ID NO: 154); And

N ' -ALPVTALLLPLALLLHAARP LYEQKLISEEDLKKLETFKKTN-C' (SEQ ID NO: 155) may be any one or more (underlined means CD8α signal sequence).

In one embodiment, the CD8α signal sequence may be located at the N-terminus or a region adjacent to the N-terminus of the trapper molecule, and may be linked to a transmembrane domain.

In this case, for example, the amino acid sequence may be N'-IYIWAPLAGTCGVLLLSLVITLYC ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 156) (underlined means CD8α signal sequence).

In another embodiment, the CD8α signal sequence may be located at the C-terminus or a region adjacent to the C-terminus of the trapper molecule, and may be linked to a binding domain.

In this case, for example, the amino acid sequence,

N'-LFIMIVGGLVGLRIVFAVLSI ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 157);

N '-LFIMIVGGLVGLRIVFAVLSI ALPVTALLLPLALLLHAARP -C' (SEQ ID NO: 158); And

N '-LFIMIVGGLVGLRIVFAVLSI ALPVTALLLPLALLLHAARP -C' (SEQ ID NO: 159) may be any one (underlined means CD8α signal sequence).

In another embodiment, the CD8α signal sequence may be located at a C-terminus or a region adjacent to the C-terminus of the trapper molecule, and may be linked to a residual domain.

In this case, for example, the amino acid sequence,

N'-KYKSPRSFIEEKKMP ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 160);

N'-LKYKSRRSFIEEKKMP ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 161);

N'-LYKYKSRRSFIEEKKMP ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 162);

N'-LYCKYKSRRSFIEEKKMP ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 163);

N'-KYCNKYKSRRSFIEEKKMP ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 164);

N'-LYKYKSRRSFIDEKKMP ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 165);

N '-LYCNKYKSRRSFIDEKKMP ALPVTALLLPLALLLHAARP -C' (SEQ ID NO: 166);

N'-LYEQKLISEEDLKYKSRRSFIEEKKMP ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 167);

N'-LYCYPYDVPDYAKYKSRRSFIEEKKMP ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 168);

N'-LYKKLETFKKTN ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 169); And

N'-LYEQKLISEEDLKKLETFKKTN ALPVTALLLPLALLLHAARP -C '(SEQ ID NO: 170) may be any one or more (underscore means CD8α signal sequence).

In another embodiment, the CD8α signal sequence may be located at the C-terminus or a region adjacent to the C-terminus of the trapper molecule, and may be linked to a transmembrane domain.

In this case, for example, the amino acid sequence may be N ' -ALPVTALLLPLALLLHAARP IYIWAPLAGTCGVLLLSLVITLYC- C' (SEQ ID NO: 171) (underlined means CD8α signal sequence).

5.6. Trapper molecule containing [binding domain]-[residual domain]

One aspect of the content disclosed herein relates to a trapper molecule comprising [binding domain]-[residual domain].

The trapper molecule

i) some domains of gp41 capable of binding to the immune activating receptor; And

ii) comprising any of the vesicle residual domains, Golgi residual domains, and proteasome residual domains,

It may be a polypeptide for trapping all or part of the immune activating receptor in a cell.

The description of the binding domain and the residual domain is as described above.

In one embodiment, the trapper molecule may be N'- [gp41 derived binding domain]-[residual domain] -C '.

For example, the amino acid sequence of the trapper molecule is

N'-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLKDEL-C '(SEQ ID NO: 172);

N'-SNKSLEQIWNHTTWMEWDKDEL-C '(SEQ ID NO: 173); And / or

N'-LFIMIVGGLVGLRIVFAVLSIKDEL-C '(SEQ ID NO: 174)

Can be

In other embodiments, the trapper molecule may be N'- [binding domain derived from gp41]-[link domain]-[residual domain]-C '.

For example, the amino acid sequence of the trapper molecule is

N'-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 175).

Alternatively, the amino acid sequence of the trapper molecule is

N'-TTAVPWNASWSNKSLEQIGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 176);

N'- WNASWSNKSLEQIWNHTGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 177);

N'-SNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 178); And / or

N'- EQIWNHTTWMEWDREINNGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 179);

Can be

As another example, the amino acid sequence of the trapper molecule comprising N'- [ISLAD]-[Linking domain]-[Residual domain]-C 'may be N'- SNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL-C' (SEQ ID NO: 180).

In one embodiment, the trapper molecule may be N'- [gp41 derived binding domain]-[transmembrane domain]-[residual domain]-C '.

For example, the amino acid sequence of the trapper molecule is

N'- AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 181);

N'-LFIMIVGGLVGLRIVFAVLSIIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 182);

N'- TTAVPWNASWSNKSLEQIIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 183);

N'- WNASWSNKSLEQIWNHTIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 184); And / or

N'- SNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 185)

N'- EQIWNHTTWMEWDREINNIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 186)

It may include any one of.

In other embodiments, the trapper molecule may be N'- [gp41 derived binding domain]-[transmembrane domain]-[link domain]-[residual domain]-C '.

For example, the amino acid sequence of the trapper molecule is

For example, the trapper molecule is N'-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 187);

N'-TTAVPWNASWSNKSLEQIIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 188);

N'- WNASWSNKSLEQIWNHTIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 189);

N'-SNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEE GGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 190);

N'- EQIWNHTTWMEWDREINNIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 191);

N'-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLIYIWAPLAGTCGVLLLSLVITLYCGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 192);

N'-TTAVPWNASWSNKSLEQIIYIWAPLAGTCGVLLLSLVITLYCGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 193);

N'- WNASWSNKSLEQIWNHTIYIWAPLAGTCGVLLLSLVITLYCGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 194);

N'-SNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYC GGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 195); And / or

N'- EQIWNHTTWMEWDREINNIYIWAPLAGTCGVLLLSLVITLYCGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 196)

Can be

At this time, the location of the transmembrane domain and the connection domain may be changed. For example, it may be N'- [gp41-derived binding domain]-[linking domain]-[transmembrane domain]-[residual domain]-C '.

In other embodiments, the trapper molecule may be N'- [transmembrane domain]-[gp41 derived binding domain]-[link domain]-[residual domain]-C '.

For example, the trapper molecule

N'-IYIWAPLAGTCGVLLLSLVITLYCAVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 197);

N'-IYIWAPLAGTCGVLLLSLVITLYCTTAVPWNASWSNKSLEQIGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 198);

N'-IYIWAPLAGTCGVLLLSLVITLYCWNASWSNKSLEQIWNHTGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 199);

N'-IYIWAPLAGTCGVLLLSLVITLYCSNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 200); And / or

N'-IYIWAPLAGTCGVLLLSLVITLYCEQIWNHTTWMEWDREINNGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 201);

Can be

In one embodiment, the trapper molecule may be N'- [binding domain derived from gp41]-[link domain]-[residual domain]-[transmembrane domain]-C '.

For example, the trapper molecule

N'-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYC-C '(SEQ ID NO: 202);

N'-TTAVPWNASWSNKSLEQILGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEE-C '(SEQ ID NO: 203);

N'-WNASWSNKSLEQIWNHTGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEE-C '(SEQ ID NO: 204);

N'-SNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEE-C '(SEQ ID NO: 205); And / or

N'-EQIWNHTTWMEWDREINNGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEE-C '(SEQ ID NO: 206);

Can be

The trapper molecule may further add a CD8α signal sequence.

The CD8α signal sequence is as described above.

The CD8α signal sequence can be added to any one or more of both ends of the trapper molecule.

In one embodiment, the trapper molecule may be N'- [CD8α signal sequence]-[gp41 derived binding domain]-[residual domain]-C '.

For example, the trapper molecule,

* N'-ALPVTALLLPLALLLHAARPAVGIGALFLGFLGAAGSTMGARSMTLTVQARQLKDEL-C '(SEQ ID NO: 207);

N'-ALPVTALLLPLALLLHAARPTTAVPWNASWSNKSLEQIKDEL- C '(SEQ ID NO: 208);

N'-ALPVTALLLPLALLLHAARPWNASWSNKSLEQIWNHTKDEL- C '(SEQ ID NO: 209);

N'-ALPVTALLLPLALLLHAARPSNKSLEQIWNHTTWMEWKDEL- C '(SEQ ID NO: 210); And / or

N'-ALPVTALLLPLALLLHAARPEQIWNHTTWMEWDREINNKDEL- C '(SEQ ID NO: 211);

Can be

In another embodiment, the trapper molecule may be N'- [gp41 derived binding domain]-[residual domain]-[CD8α signal sequence]-C '.

For example, the trapper molecule

N'-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLKDELALPVTALLLPLALLLHAARP-C '(SEQ ID NO: 212);

N'-TTAVPWNASWSNKSLEQIKDELALPVTALLLPLALLLHAAR-C '(SEQ ID NO: 213);

N'-WNASWSNKSLEQIWNHTKDELALPVTALLLPLALLLHAAR-C '(SEQ ID NO: 214);

N'-SNKSLEQIWNHTTWMEWDKDELALPVTALLLPLALLLHAAR-C '(SEQ ID NO: 215); And / or

N'-EQIWNHTTWMEWDREINNKDELALPVTALLLPLALLLHAAR-C '(SEQ ID NO: 216);

Can be

In one embodiment, the trapper molecule may be N'- [CD8α signal sequence]-[gp41 derived binding domain]-[link domain]-[residual domain]-C '.

For example, the trapper molecule

N'-ALPVTALLLPLALLLHAARPAVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 217);

N'-ALPVTALLLPLALLLHAARPTTAVPWNASWSNKSLEQIGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 218);

N'-ALPVTALLLPLALLLHAARPWNASWSNKSLEQIWNHTGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 219);

N'-ALPVTALLLPLALLLHAARPSNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 220); And / or

N'-ALPVTALLLPLALLLHAARPEQIWNHTTWMEWDREINNGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 221);

Can be

Alternatively, the trapper molecule may be N'- [gp41-derived binding domain]-[link domain]-[residual domain]-[CD8α signal sequence]-C '.

In one embodiment, the trapper molecule may be N'- [CD8α signal sequence]-[gp41 derived binding domain]-[transmembrane domain]-[residual domain]-C '.

For example, the trapper molecule

N'-ALPVTALLLPLALLLHAARPAVGIGALFLGFLGAAGSTMGARSMTLTVQARQLIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP- C '(SEQ ID NO: 222);

N'-ALPVTALLLPLALLLHAARPTTAVPWNASWSNKSLEQIIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 223);

N'-ALPVTALLLPLALLLHAARPWNASWSNKSLEQIWNHTIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 224); And / or

N'-ALPVTALLLPLALLLHAARPTTAVPWNASWSNKSLEQIIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP-C '(SEQ ID NO: 225);

Can be

Alternatively, the trapper molecule may be N '-[gp41 derived binding domain]-[transmembrane domain]-[residual domain]-[CD8α signal sequence]-C'.

In one embodiment, the trapper molecule may be N'- [CD8α signal sequence]-[gp41 derived binding domain]-[transmembrane domain]-[connection domain]-[residual domain]-C '.

For example, the trapper molecule

N'-ALPVTALLLPLALLLHAARPAVGIGALFLGFLGAAGSTMGARSMTLTVQARQLIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 226);

N'-ALPVTALLLPLALLLHAARPTTAVPWNASWSNKSLEQIIYIWAPLAGTCGVLLLSLVITLYCGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 227);

N'-ALPVTALLLPLALLLHAARPWNASWSNKSLEQIWNHTIYIWAPLAGTCGVLLLSLVITLYCGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 228); And / or

N'-ALPVTALLLPLALLLHAARPEQIWNHTTWMEWDREINNIYIWAPLAGTCGVLLLSLVITLYCGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 229);

Can be

Alternatively, the trapper molecule may be N '-[gp41 derived binding domain]-[transmembrane domain]-[link domain]-[residual domain]-[CD8α signal sequence]-C'.

The transmembrane domain and the connection domain may be interchanged with each other. For example, it may be N'- [CD8α signal sequence]-[gp41 derived binding domain]-[linking domain]-[transmembrane domain]-[residual domain]-C '.

In one embodiment, the trapper molecule may be N'- [CD8α signal sequence]-[transmembrane domain]-[gp41 derived binding domain]-[link domain]-[residual domain]-C '.

For example, the trapper molecule

N'-ALPVTALLLPLALLLHAARPIYIWAPLAGTCGVLLLSLVITLYCAVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 230);

N'-ALPVTALLLPLALLLHAARPIYIWAPLAGTCGVLLLSLVITLYCTTAVPWNASWSNKSLEQIGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 231);

N'-ALPVTALLLPLALLLHAARPIYIWAPLAGTCGVLLLSLVITLYCWNASWSNKSLEQIWNHTGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 232);

N'-ALPVTALLLPLALLLHAARPIYIWAPLAGTCGVLLLSLVITLYCSNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 233); And / or

N'-ALPVTALLLPLALLLHAARPIYIWAPLAGTCGVLLLSLVITLYCEQIWNHTTWMEWDREINNGGGGSGGGGSGGGGSGGGGSAEKDEL-C '(SEQ ID NO: 234);

Can be

Alternatively, the trapper molecule may be N '-[transmembrane domain]-[gp41 derived binding domain]-[link domain]-[residual domain]-[CD8α signal sequence]-C'.

In one embodiment, the trapper molecule may be N'- [CD8α signal sequence]-[gp41 derived binding domain]-[link domain]-[residual domain]-[transmembrane domain]-C '.

For example, the trapper molecule

N'-ALPVTALLLPLALLLHAARPAVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYC-C '(SEQ ID NO: 235);

N'-ALPVTALLLPLALLLHAARPTTAVPWNASWSNKSLEQIGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYC-C '(SEQ ID NO: 236);

N'-ALPVTALLLPLALLLHAARPWNASWSNKSLEQIWNHTGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYC-C '(SEQ ID NO: 237);

N'-ALPVTALLLPLALLLHAARPSNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYC-C '(SEQ ID NO: 238); And / or

N'-ALPVTALLLPLALLLHAARPEQIWNHTTWMEWDREINNGGGGSGGGGSGGGGSGGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYC-C '(SEQ ID NO: 239);

Can be

Alternatively, the trapper molecule may be N'- [gp41-derived binding domain]-[link domain]-[residual domain]-[transmembrane domain]-[CD8α signal sequence]-C '.

The N-terminus and the C-terminus of the embodiment of the trapper molecule may be interchanged.

6. Engineered cells

Cells containing foreign nucleic acids and / or polypeptides that do not exist naturally are referred to as 'engineered cells'. In this case, it is preferable that the foreign nucleic acid and / or polypeptide is a trapper peptide. Another object of the present invention is to provide an engineered cell comprising the trapper peptide.

In one embodiment, the engineered cell may include a trapper peptide in the form of a peptide. In some instances, the trapper peptide can be distributed in large quantities in the cytoplasm of the engineered cell. In some instances, the trapper peptide can be distributed in large quantities to specific organelles of the engineered cell. For example, the trapper peptide may be distributed in large quantities in the endoplasmic reticulum, Golgi, or cell membrane of the engineered cell, but is not limited thereto. In order to measure the distribution of the trapper peptide, it can be confirmed by commonly known methods such as immunofluorescence staining. In some examples, the trapper peptide may be in the form of a whole peptide sequence.

In some embodiments, the engineered cell may include a trapper peptide or base sequence encoding it. In this case, the base sequence may be DNA or RNA. In some examples, the base sequence may be included in the vector. In some instances, the trapper peptide can be incorporated into cells that have been engineered as such. In some instances, the trapper peptide may exist in a separated base sequence from the genome of the manipulated cell. In some instances, the trapper peptide may be present inserted into the genome of the engineered cell.

In some embodiments, the engineered cell may include a peptide to a trapper peptide and a nucleotide sequence encoding it. In some examples, the engineered cell may include a peptide sequence of a trapper peptide and a base sequence encoding it.

In some embodiments, the engineered cells may be identical in trait to wild-state cells. In some instances, the engineered cells may exhibit the same traits as the wild state cells in the expression form of the wild state cells and the target material.

In some embodiments, the engineered cells may be different in trait from wild-state cells.

At this time, the engineered cells may exhibit different traits from wild-state cells in the expression form of the target molecule.

In addition, the engineered cells may show traits different from wild-state cells non-permanently. Alternatively, the engineered cells may permanently show different traits from wild-state cells. Alternatively, the engineered cell may appear semi-permanently different from wild-type cells.

The types of cells that can be engineered cells are not limited. For example, the type of the engineered cell is not limited to somatic cells, germ cells, secretory cells, and nerve cells. However, preferred examples of the engineered cells are for immune cells.

7. Methods of making engineered cells

Another object of the present invention is to provide a method for producing cells manipulated using the trapper peptide.

In one embodiment, the method of manufacturing the engineered cell is

It may include introducing a trapper peptide or a nucleic acid encoding the same into a cell.

In another embodiment, the method of manufacturing the engineered cell is

Introducing a trapper peptide or nucleic acid encoding it into the cell; And

And inducing different traits from wild state cells.

In another embodiment, the method of manufacturing the engineered cell is

Introducing a trapper peptide or nucleic acid encoding it into the cell; And

And isolating cells from which different traits are derived from wild-state cells.

In another embodiment, the method of manufacturing the engineered cell is

Introducing a trapper peptide or nucleic acid encoding it into the cell;

Induce different traits from wild state cells;

And isolating cells from which the different traits are derived.

In the present invention, examples are elaborated in introducing a trapper peptide or a nucleic acid encoding the same into a cell. In one example, a trapper peptide can be introduced into the cell. In some instances, a nucleic acid encoding a trapper peptide can be introduced into a cell. In some instances, a trapper peptide and nucleic acids encoding it can be introduced into the cell. In some instances, the nucleic acid encoding the trapper peptide can be introduced into the cell as introduced into a vector capable of delivering the nucleic acid into the cell. In some instances, non-vectors can be used to deliver nucleic acids encoding trapper peptides into cells. In some instances, the trapper peptide or nucleic acid encoding it can be incorporated into a cell to be incorporated into a composition for making the engineered cell. Various other delivery means that can be used are disclosed in the following 'delivery and delivery method'.

Examples in eliciting different traits from wild-state cells in the present invention are expanded. In one example, different traits can be induced through culturing cells. In this case, the different trait may be a decrease in the amount of the cell surface of at least one of the TCR subunits.

In the present invention, examples are elaborated in isolating cells in which traits different from wild ailments are induced. In this case, the different trait may be a decrease in the amount of the cell surface of at least one of the TCR subunits. In one example, the cells from which the different traits are derived can be isolated from the cells from which the above-described traits are not induced. At this time, isolating the cells can be performed by a conventionally known method such as FACS, MACS, aptamer sensor.

In this case, the engineered cell that has undergone the introduction of the trapper peptide or the nucleic acid encoding the same into the cell may have the same trait as the wild state cell. In this case, optionally, it can be added to control the engineered cells so that the trait does not change. Controlling the engineered cells so that the trait does not change can be used, for example, conventional methods such as cryopreservation. Alternatively, the engineered cell that has undergone the introduction of a trapper peptide or a nucleic acid encoding the same into a cell may have a different trait from a wild-state cell.

The manipulation can be performed in-vivo. Alternatively, the manipulation can be performed in-vitro.

Preferably, the cells can be immune cells.

8. Compositions for preparing engineered cells

Another object of the present invention is to provide a composition for providing a method for producing the engineered cell.

In this case, the composition may include a trapper peptide and / or a base sequence encoding it.

Alternatively, the composition may include i) a trapper peptide and / or a base sequence encoding it, and ii) a vector for delivering the trapper peptide and / or a base sequence encoding it. At this time, the trapper peptide and / or the nucleotide sequence encoding the same may be enclosed in the vector. Alternatively, the trapper peptide and / or base sequence encoding it may be attached to the outside of the vector.

In one embodiment, the engineered cell may include a trapper peptide in the form of a peptide. In some instances, the trapper peptide can be distributed in large quantities in the cytoplasm of the engineered cell. In some instances, the trapper peptide can be distributed in large quantities to specific organelles of the engineered cell. For example, the trapper peptide may be distributed in large quantities in the endoplasmic reticulum, Golgi, or cell membrane of the engineered cell, but is not limited thereto. In order to measure the distribution of the trapper peptide, it can be confirmed by commonly known methods such as immunofluorescence staining.

9. Engineered immune cells

The disclosures herein are directed to engineered immune cells comprising trapper molecules and / or artificial receptors.

The engineered immune cells can exhibit the following characteristics:

i) There is no difference in the form of expression of immune-activated receptors from wild-state cells.

ii) Shows different traits from wild-type cells in the expression form of immune-activated receptors

iii) Contains artificial receptors.

However, the above characteristics are only exemplary characteristics that the engineered immune cells may have, and the characteristics thereof are not limited thereto.

 An "engineered" immune cell refers to an immune cell containing a foreign nucleic acid and / or polypeptide that does not exist naturally. In addition, if the engineered immune cells are geometrically different from wild-type immune cells or spontaneous mutant immune cells naturally occurring, this corresponds to the engineered immune cells.

Hereinafter, the characteristics of the engineered immune cells may be described in detail.

9.1. Shows / does not show different traits from wild-state cells in the expression form of immune-activated receptors

In some embodiments, the engineered immune cells may be identical in trait to wild state cells. In some instances, the engineered immune cells may exhibit the same traits as the wild state cells in the expression form of the wild state cells and TCR complex.

In some embodiments, the engineered immune cells may be different in trait from wild state cells.

At this time, the engineered immune cells may exhibit different traits from wild-state cells in the expression form of the TCR complex. In this case, the engineered immune cells may differ in the amount of any TCR subunit in the cell and / or the surface from the wild state cells.

The amount of the TCR subunit in the cell can be measured by lysing the cells and measuring the concentration of the TCR subunit in the lysate or by other conventional methods. Illustratively, the amount of TCR subunit on the cell surface can be measured by flow cytometry or by other conventional methods.

In one example, the engineered immune cell may have a reduced sum of the amount of TCR subunits in the cell and the amount of TCR subunits on the cell surface in any TCR subunit. At this time, if the sum of the amount of the amount of cells in the cell and the surface of the cell is 'net amount throughout cell', the engineered immune cells are TCR-α chain, TCR-β chain, TCR-γ chain, TCR-δ chain. , CD3γ chain, CD3δ chain, CD3ε chain, zeta chain, and the total amount of intracellular and intracellular reduction can be reduced for one or more selected from the intermediate TCR complex. In either case, the amount of engineered immune cells within the cells of the TCR subunit can be reduced. In either case, the engineered immune cells may have the same / similarity to the total amount of intracellular and extracellular TCR subunits as the wild-state cells, except for the TCR subunit with reduced total intracellular and subcellular units. In some cases, the engineered immune cells may have a reduced amount of genetic expression in at least one of the TCR subunits. In some cases, the engineered immune cells can be stimulated with a peptide elimination process of at least one of the TCR subunits. At this time, the peptide decomposition process may be by an immune response or hydrolysis, but is not limited thereto.

In either case, the engineered immune cells may have a reduced amount of cell surface in the TCR subunit. Illustratively, the amount of engineered immune cells in cells of at least one of the TCR subunits may be increased over wild-state cells. That is, the amount of cell surface can be reduced as the TCR subunit is retained in the cell. Or by way of example, the engineered immune cells may have a reduced ratio of the amount of TCR subunits on the cell surface to the amount of TCR subunits in the cell compared to wild state cells.

At this time, the possible positional relationship between the components inside the engineered immune cell will be described. The positional relationship between the components can be confirmed by a conventional method other than immunofluorescence staining. For convenience of explanation, it is assumed that the TCR subunit remains in a specific organelle inside the engineered immune cell.

In some instances, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also bound to the membrane of the organelle; In addition

The TCR subunit may be bound to the membrane of the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also bound to the membrane of the organelle; In addition

The TCR subunit may be in a state existing inside the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also bound to the membrane of the organelle; In addition

The TCR subunit may be in a state existing outside the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also located outside the organelle; In addition

The TCR subunit may be in a state existing outside the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also located outside the organelle; In addition

The TCR subunit may be bound to the membrane of the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also present inside the organelle; In addition

The TCR subunit may be bound to the membrane of the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also present inside the organelle; In addition

The TCR subunit may be in a state existing inside the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and also binds to the membrane of the organelle but is expressed toward the organelle-interior direction; In addition

The TCR subunit may bind to the membrane of the organelle, but may be in a state expressed in the inner direction of the organelle.

In some instances, engineered immune cells may have a reduced amount of TCR complex on the cell surface. In one example, engineered immune cells may have a reduced ratio of the amount of TCR complex on the cell surface to the amount of TCR complex in the cell.

In another additional example, the engineered immune cells may have an increased ratio of abnormal TCR complex to normal TCR complex. In some cases, the abnormal TCR complex may have a peptide sequence of at least one of the TCR subunits different from the wild state. In some cases, the abnormal TCR complex may not include at least one of the TCR subunits. In some cases, the abnormal TCR complex may contain external components that are not wild TCR subunits. At this time, the external component may be homologous to the trapper peptide.

The examples / examples of the different traits are not mutually exclusive, and the engineered immune cells may exhibit a combination of two or more of the modified traits.

In addition, the engineered immune cells may show non-permanent traits different from wild-state cells. Alternatively, the engineered immune cells may permanently show different traits from wild-state cells. Alternatively, the engineered immune cells may appear to have a different trait than wild-type cells.

The engineered immune cells include immune cells in which an immunoactivating receptor is trapped in the cell.

The immune cell in which the immunoactivation receptor is trapped in the cell may include a trapper molecule.

In this case, the trapper molecule may be an immune cell in which the immune-activated receptor is trapped in the cell by binding to the immune-activated receptor.

The engineered immune cells may be engineered T cells.

The engineered T cells can be engineered CD4 + T cells, engineered CD8 + T cells, engineered NK cells and engineered regulatory T cells. In addition, the engineered CD4 + T cells may be any one of the engineered Th1, Th2, Th3, Th17 or Th9 T cells.

Preferably, the T cells may be CD8 + T cells or CD4 + T cells.

The engineered T cell may be a cell in which the T cell receptor (TCR) and / or TCR / CD3 complex is trapped in the cell. At this time, the T cell receptor and / or TCR / CD3 complex is normally expressed, but may be artificially manipulated so as not to be located on the surface of the T cell.

In one embodiment, the engineered T cells may have at least one of the TCR subunits in the cell bound to a trapper molecule.

In the engineered T cell, the TCR / CD3 complex may be present between the T cell organelle, cytoplasm and / or cell membrane. That is, it may be a T cell trapped in the endoplasmic reticulum by binding the immune-activated receptor and the trapper molecule.

For example, the engineered T cells include AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 182); SNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 185); SNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 192); Alternatively, a trapper molecule having the sequence of LFIMIVGGLVGLRIVFAVLSIGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 240) binds to all and / or some domains of the immunoactivating receptor, leaving an immunoactivating receptor in the endoplasmic reticulum.

The engineered T cells have a reduced effect on the exposed immune activation receptors on the cell surface, compared to T cells that do not contain the expressed trapper molecule.

In one embodiment, the engineered T cell in which the trapper molecule is expressed in the cell has 5% or less, 7% or less, and 10% or less of the T cell receptor present on the cell surface as compared to the other T cell. Below, 15% below, 17% below, 20% below, 25% below, 27% below, 30% below, 35% below, 40% below, 45% below, 50% below, 60% below, 70% below, 80% or less, or 90% or less.

In some embodiments, the engineered immune cells may be identical in trait to wild state cells. In some instances, the engineered immune cells may exhibit the same traits as the wild state cells in the expression form of the wild state cells and TCR complex.

In some embodiments, the engineered immune cells may be different in trait from wild state cells.

At this time, the engineered immune cells may exhibit different traits from wild-state cells in the expression form of the TCR complex. In this case, the engineered immune cells may have different amounts of any TCR subunits in and / or on the surface from the wild state cells.

The amount of the TCR subunit in the cell can be measured by lysing the cells and measuring the concentration of the TCR subunit in the lysate or by other conventional methods. Illustratively, the amount of TCR subunit on the cell surface can be measured by flow cytometry or by other conventional methods.

In one example, the engineered immune cell may have a reduced sum of the amount of TCR subunits in the cell and the amount of TCR subunits on the cell surface in any TCR subunit. At this time, the sum of the amount in the cell and the amount of the cell surface is referred to as a 'net amount throughout cell'. In some cases, the engineered immune cells are directed against one or more selected from TCR-α chain, TCR-β chain, TCR-γ chain, TCR-δ chain, CD3γ chain, CD3δ chain, CD3ε chain, zeta chain, and intermediate TCR complex. The total amount of cells inside and outside can be reduced. In either case, the amount of engineered immune cells within the cells of the TCR subunit can be reduced. In either case, the engineered immune cells may have the same / similarity to the total amount of intracellular and extracellular TCR subunits as the wild-state cells, except for the TCR subunit with reduced total intracellular and subcellular units. In some cases, the engineered immune cells may have a reduced amount of genetic expression in at least one of the TCR subunits. In some cases, the engineered immune cells can be stimulated with a peptide degradation process of at least one of the TCR subunits. In this case, the peptide decomposition process may be by an immune response or hydrolysis, but is not limited thereto.

In either case, the engineered immune cells may have a reduced amount of cell surface in the TCR subunit. Illustratively, the amount of engineered immune cells in cells of at least one of the TCR subunits may be increased over wild-state cells. That is, the amount of cell surface can be reduced as the TCR subunit is retained in the cell. Or by way of example, the engineered immune cells may have a reduced ratio of the amount of TCR subunits on the cell surface to the amount of TCR subunits in the cell compared to wild state cells.

At this time, the possible positional relationship between the components inside the engineered immune cell will be described. The positional relationship between the components can be confirmed by a conventional method other than immunofluorescence staining. For convenience of explanation, it is assumed that the TCR subunit remains in a specific organelle inside the engineered immune cell.

In some instances, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also bound to the membrane of the organelle; In addition

The TCR subunit may be bound to the membrane of the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also bound to the membrane of the organelle; In addition

The TCR subunit may be in a state existing inside the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also bound to the membrane of the organelle; In addition

The TCR subunit may be in a state existing outside the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also located outside the organelle; In addition

The TCR subunit may be in a state existing outside the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also located outside the organelle; In addition

The TCR subunit may be bound to the membrane of the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also present inside the organelle; In addition

The TCR subunit may be bound to the membrane of the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and is also present inside the organelle; In addition

The TCR subunit may be in a state existing inside the organelle.

In another example, the engineered immune cell has a residual domain of the trapper peptide bound to the membrane of the organelle;

The binding domain of the trapper peptide interacts with the TCR subunit and also binds to the membrane of the organelle but is expressed toward the organelle-interior direction; In addition

The TCR subunit may bind to the membrane of the organelle, but may be in a state expressed in the inner direction of the organelle.

In some instances, engineered immune cells may have a reduced amount of TCR complex on the cell surface. In one example, engineered immune cells may have a reduced ratio of the amount of TCR complex on the cell surface to the amount of TCR complex in the cell.

In another additional example, the engineered immune cells may have an increased ratio of abnormal TCR complex to normal TCR complex. In some cases, the abnormal TCR complex may have a peptide sequence of at least one of the TCR subunits different from the wild state. In some cases, the abnormal TCR complex may not include at least one of the TCR subunits. In some cases, the abnormal TCR complex may contain external components that are not wild TCR subunits. At this time, the external component may be homologous to the trapper peptide.

The examples / examples of the different traits are not mutually exclusive, and the engineered immune cells may exhibit a combination of two or more of the modified traits.

In addition, the engineered immune cells may show non-permanent traits different from wild-state cells. Alternatively, the engineered immune cells may permanently show different traits from wild-state cells. Alternatively, the engineered immune cells may appear to have a different trait than wild-type cells.

9.1.1. Inactivated immune activating receptor

One aspect of the content disclosed by this specification relates to an inactivated immune activating receptor.

'Inactivation' generally refers to a phenomenon in which a main action decreases or disappears in a substance having a certain function or function, or includes a case that is manipulated to decrease or disappear.

The 'immune activating receptor' is a receptor that does not normally recognize or bind to tumor cells, virus-infected cells or antigen presenting cells. That is, it means that the original function of the immune-activated receptor is reduced or lost.

The original function of the immune-activated receptor is to distinguish its own cells from other cells, and when other cells bind to the receptor, generate immune signaling to activate an immune response or induce differentiation. However, when the function of the immune-activating receptor is reduced or lost, it is impossible to distinguish one's own cells from other cells, and accordingly, even if other cells are bound, it cannot activate an immune response or induce immune cell differentiation.

For example, the immune-activated receptors whose function is deteriorated or lost cannot be recognized by distinguishing autologous cells from other cells.

For example, the immune activation receptor having a modified part of the other cell recognition domain cannot normally differentiate other cells on the cell surface to induce immune signaling normally.

As another example, the immune-activated receptors whose function is reduced or lost may not be able to bind other cells.

For example, when the expressed immunoactivation receptor is trapped in a cell, the immunoactivation receptor cannot bind to other cells to generate immune signaling.

The inactivated immune activating receptor may be a receptor in which the immune activating receptor is trapped and present in the cell.

The immune-activated receptor may not be able to move out of the cell surface by a specific signal sequence originally present, for example, a naturally-synthesized signal sequence polypeptide, and may remain in the cell.

Alternatively, the immune-activated receptor may bind to an artificially synthesized polypeptide, fail to migrate to the cell surface, and may be retained in the cell.

The immune-activated receptor bound to the artificially synthesized polypeptide may be permanently bound.

Alternatively, the immune-activated receptor bound to the artificially synthesized polypeptide may be temporarily bound.

In this case, the binding may be an essential region, domain, or some sequence constituting the immune activation receptor.

The inactivated immune-activating receptor is intended to provide an allogeneic T cell.

'Allogeneic immune cells' refers to immune cells that do not undergo an immune rejection reaction among allogeneic species even if the donor's immune cells are provided to other beneficiaries. In general, since immune cells, for example, T cells, have different antigens for each person, they can differentiate themselves and others' immune cells and cause an immune response. It does not differentiate its own cells from other cells, and does not cause immune rejection.

Accordingly, the present specification is intended to provide an allogeneic immune cell that does not recognize the main tissue complex antigen by inactivating the receptor of an immune cell that recognizes an antigen and activates an immune response.

The contents disclosed by this specification are intended to provide substances, methods, and uses for suppressing the immune response of immune cells by inactivating immune-activated receptors for specific purposes.

In order to inactivate the immune-activated receptor, it is intended to provide a substance and a method for trapping the immune-activated receptor in a specific organ or specific region in a cell.

More specifically, in order to inactivate the immune-activated receptor, it is intended to provide a substance and method capable of binding to the immune-activated receptor.

Furthermore, it is intended to provide a new use by inserting an artificial receptor into a cell-immobilized immune-activated receptor. For example, an immune-activated receptor trapped in a cell can no longer bind to the antigen and cause an immune response, and only artificially engineered artificial receptors can recognize cells expressing a specific antigen. Accordingly, allogeneic CAR-T provided identically without distinction between the same species may be transplanted to remove cancer cells expressing a specific antigen without an immune rejection reaction.

Hereinafter, a 'trapped immune activating receptor' will be described.

9.1.2. Intracellular trapped immune activation receptors

One aspect of the content disclosed by this specification relates to a 'trapped immune activating receptor'.

Immune-activated receptors are normally 'expressed' in cells, and expressed receptors can be 'localized' on the cell surface to bind to pathogens or antigens and cause an immune response.

Expression (expression) refers to the entire process of forming a protein by DNA chains through transcription and translation. The protein thus formed can be secreted into the organelles or to be released or moved out of the cell for use elsewhere in the body.

Localization refers to the movement of a protein to a place to function properly or the movement of a protein to a specific location.

The 'trapped immune activating receptor' refers to a receptor in which the receptor expressed in the cell is not normally located on the cell surface. That is, the immune-activated receptor refers to a receptor that is trapped inside a cell due to some artificial manipulation or specific molecular interference.

The trapped immune-activated receptor in the cell can bind to a specific molecule that prevents it from being located on the cell surface. In this case, the position to which a specific molecule is bound may be one or more domains of the immune activation receptor or some polypeptide regions.

The immune-activated receptors trapped in the cells may remain in the organelles within the cells. Here, the immune-activated receptor trapped in the cell does not exist on the cell surface, and therefore cannot function normally. For example, a cell-immobilized immune-activated receptor is a receptor that is trapped in an organelle or a specific region in a cell or that the released protein is recovered and remains in the cell.

For another example, the immune-activated receptor trapped in the cell may be the case that is caught in the cell membrane, not inside the cell. At this time, even if the immune-activated receptor is trapped on the cell surface, it cannot bind antigen to foreign cells.

The cell to which the immune activation receptor is trapped may be an immune cell. For example, immune cells include T cells, B cells, natural killer cells, NKT cells, monocytes, macrophages or dendritic cells. However, it is not limited thereto.

For example, the T cells may be CD4 + T cells, CD8 + T cells, CD4 + memory cells, CD8 + memory cells, central memory cells, effect memory T cells, or regulatory T cells. CD4 + T cells may be any of TH1, TH2, TH3, TH17, or TH9 cells, but are not limited thereto.

At this time, the immune-activated receptor trapped in the immune cell is a T cell receptor, a B cell receptor, a killer cell activating receptor, a toll-like receptor (TLR), for example, TLR4 and TLR9, cytokines, chemokines, cytokine receptors , And chemokine receptors.

In one embodiment, the immune-activated receptor is a T-cell receptor (TCR), and more specifically, may be a TCR α / β receptor, a TCR γ / δ receptor, and / or a T cell co-receptor.

The term 'T cell receptor' refers to a receptor capable of activating T cells or inducing differentiation as a protein capable of directly binding T cells to antigen presenting cells (APCs). More specifically, according to the expression of the membrane protein CD4 or CD8, which acts as a co-receptor in addition to the T-cell receptor, the T-cell receptor recognizes a peptide antigen derived from inside the cell (MHC class I) or from outside the cell (MHC class II ) To determine whether to recognize the peptide antigen, and then start signaling of the TCR to activate the T cells.

The 'caged TCR / CD3 complex in the cell' may not bind MHC I or MHC II.

The specific region of the immune cell to which the immune-activated receptor is trapped may be any region capable of trapping the immune-activated receptor of the immune cell.

In one embodiment, a specific region within an immune cell may be an organelle or cytoplasm present in the immune cell.

The organelles in the immune cells are the nuclear membrane organelles (nucleus) or the mitochondria (mitochondria), single membrane cell organelles (endoplasmic reticulum), golgi (golgi apparatus), lysosome (lysosome), vacuoles, peroxisomes (peroxisome) or glyoxysome (glyoxysome), non-membrane organelles (nucleolus), centriole (ribosome), as well as ribosomes (ribosome), as well as intracellular giant complexes, such as hydrolysis complex (proteasome) And the like. However, it is not limited thereto.

In other embodiments, the specific region to which the immune activating receptor is trapped may be a 'one specific portion' of the organelle or cytoplasm present in the immune cell. The term 'one specific portion' may be, for example, a rough endoplasmic reticulum or a smooth endoplasmic reticulum among endoplasmic reticulum, or refers to a specific region or region within a cell organelle such as a region proximal to the cell membrane of an endoplasmic reticulum or an area adjacent to a nuclear membrane.

In one embodiment, a specific region within an immune cell may be between the cell membranes of an immune cell. For example, the immune activating receptor may be present in the region between the cell membranes or may remain interposed between the cell membranes. At this time, even if the receptor interposed between the cell membranes is partially exposed on the cell surface, it cannot bind to foreign antigens.

The engineered immune cells may include artificial receptors. In this case, the engineered immune cell may be an immune cell expressed on an artificial receptor and present on the cell surface.

The artificial receptor will be described below.

9.2. Artificial receptors

One aspect of the content disclosed in the present specification, the immune cells with suppressed immune activity may further include an artificial receptor that specifically recognizes a specific antigen. At this time, preferably, the specific antigen may be an antigen specifically expressed in cancer cells.

"Artificial receptors are not wild-type receptors, but artificially made, which are those that have the ability to recognize specific antigens and have specific functions. These artificial receptors have improved or enhanced immunity to specific antigens. By generating a response signal, the immune response can contribute to improvement.

The artificial receptor, for example, may have the following configuration.

(Iv) Antigen recognition part (ligand binding part)

The artificial receptor includes an antigen recognition portion (ligand binding portion).

"Antigen recognition part" means a site that recognizes an antigen as part of an artificial receptor.

The antigen recognition unit may have improved recognition ability for a specific antigen over a wild receptor. In this case, the specific antigen may be an antigen of cancer cells. In addition, the specific antigen may be an antigen of cells in the general body.

* The antigen recognition unit may have affinity for the antigen.

The antigen recognition unit may generate a signal while binding to the antigen. The signal may be an electrical signal. The signal may be a chemical signal.

The antigen recognition portion may include a signal sequence.

The signal sequence refers to a peptide sequence that allows the protein to be delivered to a specific position in the process of protein synthesis.

The signal sequence may be located near the N-terminal portion of the antigen recognition region. At this time, the distance from the N terminal may be around 100 amino acids. The signal sequence may be located close to the C-terminus of the antigen recognition region. At this time, the distance from the C-terminal may be around 100 amino acids.

The antigen recognition unit may have an organic functional relationship with the first signal generation unit.

The antigen recognition region may have homology to the Fab (fragment antigen binding) domain of the antibody.

The antigen recognition unit may be a single-chain variable fragment (scFv).

For example, the antigen recognition region can be selected from scFv, Fab, Fab ', Fv, F (ab') 2, and dAb, and any fragment or combination thereof.

The antigen recognition unit may be any of NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, NKp30 nNKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80.

The antigen recognition unit may recognize an antigen by itself or may recognize an antigen by forming an antigen recognition structure.

The antigen recognition structure can recognize an antigen only when a specific structure is formed, and a unit constituting the specific structure and a combination of the units can be easily understood by a person skilled in the art. In addition, the antigen recognition structure may be composed of one or two or more units.

The antigen recognition structure may be a structure in which units are connected in series, or may be a structure connected in parallel.

The structure connected in series means that two or more units are continuously connected in one direction, and the structure connected in parallel has two or more units at the same time, for example, in different directions. It means combined form.

For example,

The unit may be inorganic.

The monomer may be a biochemical ligand.

The unit may be homologous to the antigen recognition portion of the wild-type receptor.

The monomer may be homologous to the antibody protein.

The monomer may have a heavy chain of immunoglobulin or homology thereto.

The monomer may have a light chain of immunoglobulin or homology thereto.

For example, the antigen recognition unit CD19, CD20, ROR1, CD22, alpha fetal protein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL -11Ralpha, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2 and HER3 and may be all or part of the ScFv region of the antibody targeting any one or more of HER1 and HER2.

The unit may include a signal sequence.

*

On the other hand, the unit may be bonded through a chemical bond, or may be bound through a specific bond.

"Antigen recognition unit combining part" is a site where antigen recognition structural units are combined with each other, and may be an optional configuration that exists when there is an antigen recognition structure composed of two or more antigen recognition structural units. .

The antigen recognition structural unit binding unit may be a peptide. At this time, the binding portion may have a high ratio of serine and threonine.

The antigen recognition structural unit binding portion may be a chemical bond.

The antigen-recognition structural unit binding portion may have a specific length to help express the three-dimensional structure of the antigen-recognition structural unit.

The antigen-recognition structural unit binding unit may help a function of the antigen-recognition structural unit by having a specific positional relationship between antigen-recognition structural units.

(ii) receptor body

The artificial receptor includes the receptor body.

The "receptor body part" is a site that mediates the connection between the antigen recognition part and the signal generating part, and may be a physical connection between the antigen recognition part and the signal generating part.

The function of the receptor body part may be to transmit a signal generated by the antigen recognition part or the signal generating part.

In some cases, the structure of the receptor body may simultaneously have the function of the signal generator.

The function of the receptor body may be such that the artificial receptor gas is fixed to immune cells.

The receptor body portion may include an amino acid helix structure.

The structure of the receptor body portion may include a portion homologous to a part of a common receptor protein present in the body. The range of homology can be 50 to 100%.

The structure of the receptor body portion may include a portion homologous to a protein on the immune cell. The range of homology can be 50 to 100%.

For example,

The receptor body portion may be a CD8 transmembrane domain.

The receptor body portion may be a CD28 transmembrane region. At this time, if the second signal generating portion is CD28, CD28 can simultaneously function as the second signal generating portion and the receptor body portion.

The receptor body can be a CD3, or CD19 transmembrane domain.

(iii) Signal generator (signaling domain)

The artificial receptor may include a signal generator.

The “first signal generator” refers to a site that generates an immune response signal as part of an artificial receptor.

The "second signal generation unit" refers to a site that generates an immune response signal by interacting with the first signal generation unit or independently of the artificial receptor.

The artificial receptor may include a first signal generator and / or a second signal generator.

Two or more first and / or second signal generators may be included.

The first and / or second signal generators may include specific sequence motifs.

The sequence motif may be homologous to the motif of the CD (cluster of designition) protein.

In this case, the CD protein may be CD3, CD247, and CD79.

The sequence motif may be the amino acid sequence of YxxL / I.

Sequence motifs may be included multiplexed in the first and / or second signal generators.

At this time, the first sequence motif may be at a position 1 to 200 amino acids away from the starting position of the first signal generating unit. The second sequence motif may be at a position 1 to 200 amino acids away from the starting position of the second signal generator.

In addition, the spacing between each sequence motif may be 1 to 15 amino acids.

At this time, the spacing between each preferred sequence motif is 6 to 8 amino acids.

For example,

* The first and / or second signal generator may be CD3 ζ.

The first and / or second signal generator may be FcεRIγ.

The first and / or second signal generator may generate an immune response signal only when a specific condition is satisfied.

The specific condition may be that the antigen recognition unit recognizes the antigen.

The specific condition may be that the antigen recognition portion forms a bond with the antigen.

The specific condition may be to receive a signal generated when the antigen recognition portion and the antigen form a bond.

The specific condition may be that the antigen recognition unit is isolated after recognizing or binding the antigen.

The immune response signal may be a signal related to the growth and differentiation of immune cells.

The immune response signal may be a signal related to the death of immune cells.

The immune response signal may be a signal related to the activity of immune cells.

The immune response signal may be a signal related to the aid of the immune response.

The immune response signal may be specifically activated by a signal generated by the antigen recognition unit.

The immune response signal may be a signal that regulates the expression of the target gene.

The immune response signal may be a signal that suppresses the immune response.

In one example, the signal generator may include an additional signal generator.

The "additional signal generation unit" means a portion that generates an additional immune response signal with respect to the immune response signal generated by the first and / or second signal generation unit as part of the artificial receptor.

Hereinafter, the additional signal generator is referred to as an n-th signal generator according to the sequence.

The artificial receptor may include an additional signal generator in addition to the first signal generator.

Two or more additional signal generating units may be included in the artificial receptor.

Additional signal generators include 4-1BB, CD27, CD28, ICOS, OX-40, Dap10, CD40, 2B4, ITAM motif, inducible T-cell co-stimulator (ICOS), CD3 gamma, CD3 delta, CD3 emsilon, CD247 , Ig alpha (CD79a), Fc gamma receptor signaling region or other structure that generates an immune response signal, but is not limited thereto.

The conditions under which the additional signal generating unit generates the immune response signal and the characteristics of the generated immune response signal include contents corresponding to those of the first and / or second signal generating unit.

The immune response signal may be to promote the synthesis of cytokines. The immune response signal may be to promote or inhibit the secretion of cytokines. At this time, the cytokine may be preferably IL-2, TNFα or IFN-γ.

The immune response signal may be a signal that helps the growth or differentiation of other immune cells.

The immune response signal may be a signal that modulates the activity of other immune cells.

The immune response signal may be a signal that attracts other immune cells to a location where the signal occurs.

The present invention includes all possible bonding relationships of artificial receptors. Accordingly, aspects of the artificial receptor of the present invention are not limited to those mentioned herein.

The artificial receptor may be composed of an antigen recognition part-a receptor body part-a first signal generating part. The receptor body part can be optionally included.

The artificial receptor may be composed of an antigen recognition part-a receptor body part-a second signal generating part-a first signal generating part. The receptor body part can be optionally included. At this time, the positions of the first signal generator and the second signal generator may be changed.

The artificial receptor may be composed of an antigen recognition part-a receptor body part-a second signal generating part-a third signal generating part-a first signal generating part. The receptor body part can be optionally included. At this time, the positions of the first signal generator to the third signal generator may be changed.

The number of signal generating units in the artificial receptor is not limited to 1 to 3, and may be included in excess of 3.

In addition to the above examples, the artificial receptor may have an antigen recognition unit-signal generating unit-receptor body unit structure. This structure can be advantageous when it is necessary to generate an immune response signal that acts outside the cell with an artificial receptor.

The artificial receptor can function as a wild receptor.

The artificial receptor can function to form a specific positional relationship by forming a bond with a specific antigen.

The artificial receptor can function to recognize the specific antigen and generate an immune response signal that promotes an immune response to the specific antigen.

The artificial receptor can function to suppress the immune response to cells in the body by recognizing the antigens of the cells in the body.

(iv) Signal sequence

In one example, the artificial receptor may optionally include a signal sequence.

If the artificial receptor contains the signal sequence of a specific protein, it can help the artificial receptor to be easily located on the immune cell membrane. Preferably, if the artificial receptor in the immune cell contains the signal sequence of the transmembrane protein, the artificial receptor may pass through the immune cell membrane and help to locate the outer membrane of the immune cell.

The artificial receptor may include one or more signal sequences.

The signal sequence may contain many positively charged amino acids.

The signal sequence may include a positively charged amino acid at a position close to the N or C terminus.

The signal sequence may be a signal sequence of a transmembrane protein.

The signal sequence may be a signal sequence of a protein located on the outer membrane of an immune cell.

The signal sequence may be included in the structure of the artificial receptor, that is, an antigen recognition unit, a receptor body unit, a first signal generation unit, and an additional signal generation unit.

At this time, the signal sequence may be located near the N or C terminal of each structure.

At this time, the distance from the N or C terminal may be around 100 amino acids.

In one example, the engineered cells may be engineered to contain a specific chimeric antigen receptor (CAR). In one example, the CAR is a tumor associated antigen (eg, CD 19, CD20, carbonic anhydrase IX (CAIX), CD 171, CEA, ERBB2, GD2, alpha-folate receptor, Lewis Y antigen, prostate specific membrane antigen (PSMA) Or it may have a binding specificity for tumor associated glycoprotein 72 (TAG72)), but is not limited thereto.

In another example, the engineered cells may be engineered to bind one or more of the following tumor antigens, for example, by CAR.

Tumor antigens are, but are not limited to, AD034, AKT1, BRAP, CAGE, CDX2, CLP, CT-7, CT8 / HOM-TES-85, cTAGE-1, EGFR, EGFRvIII, Fibulin-1, HAGE , HCA587 / MAGE-C2, hCAP-G, HCE661, HER2 / neu, HLA-Cw, HOM-HD-21 / Galectin9, HOM-MEEL-40 / SSX2, HOM-RCC-3.1.3 / CAXII, HOXA7, HOXB6 , Hu, HUB 1, KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, KOC3, LAGE-1, MAGE-1, MAGE-4a, MPPl 1, MSLN, NNP-1, NY-BR -1, NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NY-ESO-1, NY-ESO-5, NY-LU-12, NY-REN-10 , NY-REN-19 / LKB / STK1 1, NY-REN-21, NY-REN-26 / BCR, NY-REN-3 / NY-CO-38, NY-REN-33 / SNC6, NY- REN- 43, NY-REN-65, NY-REN-9, NY-SAR-35, OGFr, PSMA, PSCA PLU-1, Rab38, RBPJkappa, RHAMM, SCP1, SCP-1, SSX3, SSX4, SSX5, TOP2A, TOP2B , Or Tyrosinase.

10. CAR cells

This application discloses engineered immune cells, allogeneic CAR cells.

The engineered immune cells include both trapper molecules and artificial receptors.

The allogeneic CAR cells

A) The trapper molecule binds to the immune-activated receptor inside the cell,

B) The chimeric antigen receptor is expressed and located on the surface of the taxa

It is characterized by.

The trapper molecule and chimeric antigen receptor are as described above.

The cells refer to immune cells, and the immune cells are as described above.

In one embodiment, the allogeneic CAR cells may be allogeneic CAR-T cells.

In some examples, the allogeneic CAR-T cell is a trapper molecule consisting of the amino acid sequence of SNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 192) and a T cell receptor and / or TCR / CD3 complex bound within the T cell, trapped in the endoplasmic reticulum, NKG2 The chimeric antigen receptor comprising the ligand binding domain of and CD3zeta signaling domain may be expressed in T cells and may exist on the cell surface.

In some examples, the Allogeneic CAR-T cell is a trapper molecule consisting of the amino acid sequence of SNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 185) and a T cell receptor and / or TCR / CD3 complex bound in T cells, trapped in vesicles, NKG2D The chimeric antigen receptor comprising the ligand binding domain of and CD3zeta signaling domain may be expressed in T cells and may exist on the cell surface.

In some examples,

Allogeneic CAR-T cells can be trapped in a endoplasmic reticulum and trapped in a vesicle by binding the T cell receptor and / or TCR / CD3 complex in the T cell with the trapper molecule consisting of the amino acid sequence of LFIMIVGGLVGLRIVFAVLSIGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 247), and bound to the endoplasmic reticulum, the ligand binding of NKG2D The chimeric antigen receptor comprising a domain and a CD3zeta signaling domain can be expressed in T cells and present on the cell surface.

The allogeneic CAR cells are wild-type immune cells, that is, compared to immune cells without artificial receptors, the engineered T cells expressing artificial receptors are 5% or more, 7% or more, 10% or more, 15% or more on the cell surface. , 17%, 20%, 25%, 27%, 30%, 35%, 40%, 45%, 50%, 60%, 70% or more Can be.

On the other hand, allgeneic CAR cells have 5% or less, 7% or less, 10% or less, 15% or less, 17 compared to wild-type immune cells, i.e., immune cells whose trapper molecules are not expressed in the cells. % Or less, 20% or less, 25% or less, 27% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 60% or less, 70% or less, 80% or less or 90% or less Can exist on the cell surface.

The allogeneic CAR cells can be used without distinction among allogenes.

In the allogeneic CAR T cell, since the T cell receptor cannot bind to MHC I or MHC II, the cell has a reduced ability to distinguish foreign antigens from its own cells. Therefore, even if T cells isolated from other subjects are manipulated and administered, they may not cause an immune response, and can be used without distinction among allogenes.

The allogeneic CAR-T cells can specifically bind only specific cells to induce an immune response. That is, even if the number of allogeneic CAR cells decreases or the number of immune-activated receptors does not exist, the signal pathway that delivers the immune response functions normally, and thus can cause an immune response against specific cells.

For example, in the case of cancer cell-specific allogeneic CAR-T cells, the artificial receptor can specifically recognize and bind only cancer cells while using the same signal pathyway of the T cell receptor. At this time, since the T-cell receptor trapped in the cell is inactivated, an immune response against other foreign antigens may not be induced.

The Allogeneic CAR-T cells are interferon gamma (IFN-gamma), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-7 receptor (IL-7R), dominant negative TGFbeta receptor (dnTGFbetaR) And interleukin-2 (IL-2).

11. CAR cell manufacturing method

One embodiment of the content disclosed by this specification relates to a method of producing CAR cells as engineered immune cells.

The method can be done in vivo, ex vivo or in vitro.

In the method for producing CAR cells, the contents of the above-described 'Method for producing engineered cells' are included in this section as a whole.

In one embodiment

The above method

Activates T cells;

Trapper molecule and / or chimeric antigen receptor (CAR);

It may include introducing into the immune cells.

Furthermore, in some embodiments, the method may additionally

And inducing different traits from wild state cells.

In some other embodiments, the method further comprises:

It can include isolating cells from which different traits have been induced.

In another embodiment, the method further comprises:

Induce different traits from wild state cells;

It can include isolating cells from which different traits have been induced.

In one embodiment, to produce an allogeneic CAR-T, i) T cells may be activated.

"T cell activation" refers to a process in which uncontacted T cells bind to the MHC: antigen complex, thereby activating specific transcription factors through dephosphorylation and phosphorylation processes in cells, thereby allowing T cells to divide and differentiate.

For example, T cell activation may activate T cells by stimulating the TCR / CD3 complex.

The term “stimuatlion” refers to a T cell response that occurs when triggering a primary activation signal (eg, stimulation via a CD3 / TCR complex or CD2) and / or an auxiliary stimulation signal from a T cell. T cell responses include lymphokine production (eg IL-2) and T cell proliferation.

In order to stimulate the TCR / CD3 complex, T cell activity can be induced and proliferated by treating the additives treated with the antigen mixture.

Alternatively, T cells can be activated by stimulating the TCR / CD3 complex using CD3 / CD28 Beads and / or CD2 / CD3 / CD28 Beads.

For example, when beads are used, T cells can be activated using beads having an anti-CD3 / CD28 antibody as a functional group.

To activate the T cells, cytokines may be further included. “Cytokine” refers to an immune activated cytokine that induces activation such that T cells differentiate and divide. For example, IL-2, IL-7, IL-15, anti-IL-4 and / or IL-21 can be added to the cell culture to optimize cell proliferation and survival.

In one embodiment, to produce an allogeneic CAR-T, ii) introducing a trapper molecule and / or a chimeric antigen receptor (CAR) into immune cells.

Wherein ii),

* Incorporating the trapper molecule into immune cells.

The introduction of the binding domain, residual domain, connecting domain and / or transmembrane domain and immune cells is described above.

For example,

SNKSLEQIWNHTTWMEWDLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 241);

LFIMIVGGLVGLRIVFAVLSILYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 242);

SNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 178); or

LFIMIVGGLVGLRIVFAVLSIIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 182);

Vectors containing nucleic acids encoding amino acid sequences, such as can be introduced into immune cells.

In addition, the ii) is,

And introducing the chimeric antigen receptor into immune cells. At this time, the method of introducing the chimeric antigen receptor into immune cells can be performed by using any suitable method known to those skilled in the art.

For example, transfection of CAR into T cells can be accomplished using a viral vector. Examples of non-limiting viral vectors include retro, lenti, adeno, adeno-ointment or viral vectors. Transduction includes, but is not limited to, electrophoresis and micro-injection of plasmid and transposon / transposase systems.

The method for intracellular delivery of the chimeric antigen receptor and the chimeric antigen receptor is as described above.

For example, two or more vectors each containing a nucleic acid encoding the NKG2D ligand binding domain and the CD3zeta signaling domain may be introduced into the immune cell.

As another example, one vector comprising a nucleic acid encoding the NKG2D ligand binding domain and the CD3zeta signaling domain can be introduced into immune cells.

Additionally, it may be added to confirm whether the trapper molecule and / or chimeric antigen receptor is expressed in the cell or is present on the cell surface.

The expression of the trapper molecule can be confirmed by the expression of the mRNA or protein of the trapper molecule.

For example, PCR can be used to confirm the mRNA expression of the trapper molecule.

For example, Western blot, ELISA, IP, Mass spectrometry, or the like can be used as a method of confirming the protein expression of the trapper molecule.

For example, it can be confirmed whether the trapper molecule is expressed and the immune-activated receptor is confined in the cell. For example, T cell receptors can be bound by using a fluorescent antibody method and then flow cytometry to confirm whether they have been exposed to the immune cell surface.

It can be confirmed whether the chimeric antigen receptor is expressed in immune cells.

For example, the expression of the chimeric antigen receptor can be confirmed by the expression of the mRNA or protein of the chimeric antigen receptor. For example, PCR can be used to confirm the mRNA expression of the chimeric antigen receptor.

For example, Western blot, ELISA, IP, etc. can be used as a method of confirming the protein expression of the chimeric antigen receptor.

Protein expression can be confirmed by whether or not the chimeric antigen receptor has appeared on the surface of the immune cell. After binding to the chimeric antigen receptor using a fluorescent antibody method, expression can be confirmed using a flow cytometer.

For example, it can be confirmed whether the trapper molecule is expressed and the immune-activated receptor is locked in the cell. For example, T cell receptors can be bound by using a fluorescent antibody method and then flow cytometry to confirm whether they have been exposed to the immune cell surface.

In one example in inducing different traits from wild state cells, different traits can be induced through culturing the cells.

In isolating cells from which different traits are induced, in one example, cells from which the different traits are derived can be isolated from cells from which the above-described traits are not induced. At this time, isolating the cells can be performed by a conventionally known method such as FACS, MACS, aptamer sensor.

In this case, the engineered immune cells that have undergone the introduction of the trapper peptide and / or chimeric antigen receptor (CAR) into the cells may have the same traits as the wild-state cells. In this case, optionally, it can be added to control the engineered immune cells so that the trait does not change. Controlling the engineered immune cells so that the trait does not change can be used, for example, conventional methods such as cryopreservation. Alternatively, the engineered cells that have undergone the introduction of the trapper peptide and / or chimeric antigen receptor (CAR) into the cells may have different traits from the wild state cells.

12. Delivery and delivery methods

In one embodiment of the content disclosed by the present specification, the trapper molecule and / or the artificial receptor can be delivered or introduced into the subject by a vector or non-vector containing sequences encoding it.

The trapper molecule and / or artificial receptor may be delivered or introduced into the subject in the form of DNA, RNA, peptide, polypeptide, and / or protein encoding it.

The trapper molecule and / or artificial receptor can be delivered in a variety of forms within the subject.

The trapper molecule and / or artificial receptor can be delivered as a vector.

The trapper molecule and / or artificial receptor may be delivered as a non-vector.

This application discloses vectors comprising a nucleic acid encoding a trapper molecule and / or an artificial receptor.

Description of the trapper molecule is as described above.

The description of the artificial receptor is as described below.

12.1. vector

The vector can be a viral or non-viral vector (eg, plasmid).

The term "vector" is a carrier capable of delivering a gene to a cell. “Vector constructs”, “expression vectors”, and “gene transfer vectors” typically refer to any nucleic acid construct capable of directing the expression of a gene of interest and delivering a gene sequence to a target cell. Accordingly, the term includes cloning, and integrating vectors as well as expression vehicles.

The vector can be a viral or non-viral vector (eg, plasmid).

The vector may include a nucleic acid sequence encoding a trapper molecule.

In one embodiment, the vector comprises a nucleic acid sequence encoding a binding domain constituting the trapper molecule. For example, the vector comprises a nucleic acid sequence that is 5'-CUUUUUAUUAUGAUUGUUGGUGGUCUUGUUGGUCUUCGUAUUGUUUUUGCUGUUCUUUCU-3 '(SEQ ID NO: 243). At this time, the sequence corresponding to the binding domain is translated into LFIMIVGGLVGLRIVFAVLS amino acid sequence.

In other embodiments, the vector may include nucleic acid sequences encoding residual domains constituting the trapper molecule. For example, the vector comprises a nucleic acid sequence that is 5'-AAAGAUGAAUUA-3 '(SEQ ID NO: 244) or 5'-AAAAAGAUGCCU-3' (SEQ ID NO: 245). At this time, the residual domain is a endoplasmic reticulum signal sequence encoding KDEL or KKMP.

Alternatively, the nucleic acid sequence may be a nucleic acid sequence encoding the proteasome residual signal sequence SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 108). Illustratively, the vector comprises a nucleic acid sequence that is 5'UCUCAUGGUUUUCCUCCUGAAGUUGAAGAACAAGAUGAUGGUACUUUACCUAUGUCUUGUGCUCAAGAAUCUGGUAUGGAUAGACAUCCUGCUGCUUGUUCCGCUAGAAUUAAUGUU-3 '(SEQ ID NO: 246).

In one embodiment, the vector may include a nucleic acid sequence encoding a transmembrane domain added to the trapper molecule. For example, a vector comprising a transmembrane domain having an amino acid sequence of IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 111), 5'-AUUUAUAUUUGGGCUCCUUUAGCUCCUUUAGCUGGUACUUGUGGUGUUUUUAUUAUUAAGUUUAGUUAUUACUUUAUAUUGU-3 ', a nucleic acid sequence of SEQ ID NO:

The vector may further include a nucleic acid sequence encoding a linking domain constituting the trapper molecule. For example, the nucleic acid sequence includes a nucleic acid sequence that is (GGGGS) n and / or (GGUGGUGGUGGUUCU) n.

The vector can include additional functional domains that make up the trapper molecule.

The trapper molecule and / or artificial receptor can be delivered or introduced into one or more vectors.

In one example, the vector may deliver a trapper molecule and / or an artificial receptor in immune cells through the same or different vectors. For example, the trapper molecule and the artificial receptor may be delivered by the same viral vector, or the trapper molecule may be delivered by the viral vector, and the artificial receptor may be carried by a non-viral vector.

For example, the trapper molecule and / or artificial receptor may be delivered or introduced into the same single vector.

Or, for example, in another embodiment, the trapper molecule and / or the artificial receptor may each be delivered or introduced into different vectors.

The trapper molecule can be delivered or introduced into immune cells via one or more vectors. For example, binding domains, linking domains, and residual domains included in the trapper molecule can be delivered in one and the same vector, or can be delivered or introduced in different vectors, respectively.

In one embodiment, the nucleic acid encoding the trapper molecule can be delivered or introduced into a single viral vector.

The artificial receptor can be delivered or introduced into immune cells using one or more vectors. For example, the domain of the antigen recognition region, the domain of the receptor body region, and the domain of the signaling region may be delivered to one and the same vector, or may be delivered or introduced to different vectors, respectively. In addition, two or more artificial receptors can be simultaneously delivered or introduced into immune cells.

For example, the artificial receptor can be a chimeric antigen receptor (CAR).

The vector for delivering or introducing the trapper molecule and / or the artificial receptor can include one or more regulatory / control elements.

In this case, the regulatory / control elements include promoter, enhancer, intron, polyadenylation signal, Kozak consensus sequence, internal ribosome entry site (IRS), splice acceptor and / or 2A sequence. It can contain.

In addition, the vector may additionally include a reporter gene (eg, GFP) or a selection marker for selecting a host cell containing the vector, or a replication origin in the case of a replicable vector.

The vector may include a Splicing Acceptor (SA) sequence at the left, right, or adjacent positions of the transgene.

The promoter may be an endogenous promoter or an exogenous promoter in the target region.

The promoter may be a promoter recognized by RNA polymerase II or RNA polymerase III.

The promoter may be a constitutive promoter.

The promoter may be an inducible promoter.

The promoter may be a target specific promoter.

The promoter may be a viral or non-viral promoter.

The promoter may be a suitable promoter depending on the control region (ie, guide nucleic acid, editor protein, or transgene). For example, a useful promoter for guide nucleic acid can be the H1, EF-1a, tRNA or U6 promoter.

The vector can be a viral vector or a recombinant viral vector.

The virus can be a DNA virus or an RNA virus.

In this case, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.

* At this time, the RNA virus may be a single-stranded RNA (ssRNA) virus.

The viral vector includes, but is not limited to, retro virus vector, lentivirus vector adenovirus vector, pox virus vector, herpes virus vector, herpes simplex virus, vaccinia virus and adeno-associated virus vector (AAV).

When a guide nucleic acid, an editor protein and / or a donor molecule is introduced into a target organism using a virus, it may be temporarily expressed in a subject. Or it may be expressed over a long period of time. For example, it can be expressed in 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, or permanently.

Virus packaging ability is at least 2kb to 50kb, which may vary depending on the type of virus. Depending on the packaging ability, a viral vector containing only the guide nucleic acid or the editor protein can be designed, or a viral vector containing both the guide nucleic acid and the editor protein can be designed. Alternatively, viral vectors containing guide nucleic acids, editor proteins, and additional components can be designed.

For example, adeno-associated virus vectors have very high transduction efficiency in various cells (muscle, brain, liver, lung, retina, ear, heart, and blood vessels) regardless of cell division, are pathogenic, and treat most of the viral genome It can be replaced by genes and elicits a relatively low immune response. In addition, AAV is inserted into the chromosome of the target cell, whereby the therapeutic protein is stably expressed for a long time. For example, in vitro production of nucleic acids and peptides is useful for introducing specific traits into target cells in vivo and ex vivo. However, AAV is small and has a packaging capacity of 4.5 kb or less.

For another example, the lentiviral vector is a retrovirus, which not only avoids the human immune response, but also enables gene transfer of about 8 kb to non-divided cells such as retina, nerve, muscle, and liver cells.

For example, a nucleic acid sequence encoding a trapper molecule and / or an artificial receptor can be delivered or introduced into a subject by a recombinant lentivirus.

As another example, a nucleic acid sequence encoding a trapper molecule and / or an artificial receptor can be delivered or introduced into a subject by a recombinant adenovirus.

The vector may include a nucleic acid phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP) modification.

12.2. Rain vector

The trapper molecule and / or artificial receptor may be delivered or introduced into the subject in a non-vector form.

The nucleic acid sequence encoding the trapper molecule and / or the artificial receptor can be delivered or introduced into the subject as a non-vector.

The non-vector may be naked DNA, DNA complex, mRNA or a mixture thereof.

The non-vector may be delivered or introduced into the subject by electroporation, lipofection, microinjection, gene gun method, virosome, liposome, immunoliposome, lipid-mediated transfection, or a combination thereof.

For example, in order to efficiently introduce a nucleic acid sequence into a target cell genome, target specific engineered nucleases such as ZFN, TALEN, CRISPR / Cas9, and Mega-TAL can be simultaneously delivered.

For example, it can be delivered in a subject as a non-vector by a positive liposome method. It is composed of positive amphiphile and neutral helper lipids DPOE to form stable liposomes. At this time, the liposome-DNA complex has a + charge and can be introduced into the cell by a cell uptake action by binding to a cell surface having a-charge.

As another example, DNA may be coated with gold particles and injected into cells.

Additionally, nucleic acids to be delivered to EnGeneIC delivery vehicles (EDV) can be packaged for delivery or introduction. Specifically, EDV is delivered to the target tissue using a bispecific antibody having one arm having specificity for the target tissue and the other arm having specificity for the EDV. Antibodies take EXV to the target cell surface, and then EDV can enter the cell by endocytosis.

Each of the trapper molecules and / or artificial receptors may be delivered or introduced into the subject in the form of a polypeptide.

The trapper molecule and / or artificial receptor may be in the form of DNA, RNA, polypeptide or a mixture thereof.

12.3. Delivery method

The vector comprising the nucleic acid encoding the trapper molecule and / or the artificial receptor can be delivered in the subject by an ex vivo method.

12.3.1. In vitro delivery method

The trapper molecule and / or artificial receptor can be transferred to immune cells obtained from individual subjects or to universal hematopoietic stem cells, and then transplanted into the subject.

The delivery method introduces a trapper molecule and / or an artificial receptor into the cell; And administering the cells directly to the subject. Furthermore, the introduction of trapper molecules and / or artificial receptors into cells; Isolating cells from which different traits are derived from wild state cells; And administering the cells directly to the subject. In this case, the different trait may be a decrease in the amount of the cell surface of at least one of the TCR subunits.

In one embodiment, the introduction into the cell may be introduced into the cell in the form of a vector containing a nucleic acid sequence encoding a trapper molecule and / or an artificial receptor. When introduced into a cell in the form of a vector, it may be performed by one or more methods selected from non-viral vector plasmid or viral vector.

In another embodiment, the introduction into the cell may introduce the trapper molecule and / or artificial receptor into the cell in the form of a non-vector. For example, the trapper molecule can be introduced into cells in the form of DNA, mRNA or protein, and in this case, the trapper molecule may be electroporation, lipofection, liposome, microinjection method, or the like.

Isolating cells in which the amount of the cell surface of at least one of the TCR subunits is reduced among cells in which the trapper molecule and / or artificial receptor has been introduced to efficiently produce cells having a reduced amount of the cell surface of at least one of the TCR subunits can do.

At this time, isolating the cells can be performed by a conventionally known method such as FACS, MACS, aptamer sensor.

Illustratively, unstained cells can be obtained through flow cytometry after staining with a TCR subunit specific antibody labeled with a fluorescent label on cells in which a trapper molecule and / or artificial receptor has been introduced. In another example, after staining with a TCR subunit specific antibody labeled with a magnetic label on a cell in which a trapper molecule and / or an artificial receptor has been introduced, MACS (magnetic activated cell sorting) is performed on unstained cells. It can be obtained through. In another example, the engineered T cells comprising the trapper molecule have a number of TCR subunits present on the cell surface through a cell separation method of 10% or less, 5% or less, 3% or less of wild-state cells, 1 % Or less, 0.5% or less, and 0.1% or less.

Direct administration of the cells to the subject means that the manipulated cells are administered directly into the subject, and may be delivered or introduced into the subject by systemic administration or topical application, but is not limited thereto.

The systemic administration may be injected, for example, intrathecal, intraperitoneally, intramuscularly, or subcutaneously. It may preferably be systemic administration via intravenous administration.

13. Use

One aspect of the content disclosed in this specification is for the treatment of diseases using a method of administering a composition comprising an engineered immune cell to a treatment target.

More specifically, it relates to the use of engineered immune cells, i.e., immune cells comprising a cell-immobilized immunoactivating receptor, or a composition comprising the same, to treat the disease through T cell adoptive immunotherapy.

In one example, the disease is cancer.

"Cancer" refers to a broad group of various diseases characterized by abnormal cells in the body that continue to divide cells without regulating the cell cycle. Unregulated cell division and growth results in the formation of malignancies that can spread to other tissues through the lymphatic system or bloodstream. “Cancer” or “cancer tissue” can include a tumor.

In this case, the treatment target may be a mammal including a primate such as a human or a monkey or a rodent such as a mouse or rat.

13.1. Pharmaceutical composition

One embodiment of the present specification is intended to be used in the treatment of diseases using a composition comprising an allogeneic CAR-T.

In one embodiment, the composition comprising the allogeneic CAR-T may be a composition for treating cancer using the same.

Allogeneic CAR-T cells included in the composition are allogeneic immune cells, and even T cells of other people can be used to treat cancer without causing an immune rejection.

The CAR can specifically recognize only cancer cells and induce an immune response, and a composition comprising allogeneic CAR-T cells can be used to treat cancer.

At this time, the subject to which the composition is administered may be a treatment patient. More specifically, it may be a cancer patient

The allogeneic CAR-T cell-related description is as described above.

.

In one embodiment, the composition for treating cancer may include:

A) a nucleic acid encoding a polypeptide comprising a binding domain and a residual domain ; And

B) A nucleic acid encoding a chimeric antigen receptor (CAR) comprising a ligand binding domain and a signaling domain

It may be a composition comprising allogeneic CAR-T cells generated by introducing a vector containing T cells.

For example,

A) X- [binding domain]-[connection domain]-[residual domain]-Y;

X- [binding domain]-[transmembrane domain]-[residual domain]-Y;

X- [binding domain]-[connection domain]-[residual domain]-Y; And

X- [residual domain]-[transmembrane domain]-[binding domain]-Y;

It may be a polypeptide bound in any one order,

X is the N-terminal or C-terminal of the amino acid,

The Y is characterized in that the amino acid N terminal or C terminal

Nucleic acids encoding polypeptides; And

B) Ligand binding domain and a nucleic acid encoding a chimeric antigen receptor comprising a signaling domain

It may be a composition comprising allogeneic CAR-T cells produced by introducing a composition comprising a vector containing T cells.

In the composition for the treatment of cancer comprising allogeneic CAR-T cells, the chimeric antigen receptor can be engineered to target a specific tumor antigen.

In one embodiment, the tumor antigen is CD19, CD20, ROR1, CD22, alpha fetal protein, CA-125, 5T4, MUC-1, epithelial tumor prostate, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutation Ras, HER2 / Neu, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, GD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, CSPG4, ERBB2, EGFRvIII, VEGFR2, combined HER2-HER3, combined HER1-HER2, and any combination thereof.

In certain embodiments, the tumor antigen is CD19.

For example, the composition for treating cancer may be a composition comprising an allogeneic CAR-T that binds to CD19 and further comprises a CD28 co-stimulatory domain and a CD3-zeta signaling region.

For example,

A) A nucleic acid encoding a domain comprising an amino acid sequence that is SNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP; And

B) A nucleic acid encoding a chimeric antigen receptor comprising a ligand binding domain NKG2D and a signaling domain CD3zeta

It may be a composition comprising allogeneic CAR-T cells produced by introducing a composition comprising a vector containing T cells.

The pharmaceutical composition may further include additional elements.

The composition may include a suitable carrier or pharmaceutical additive for delivering allogeneic CAR-T cells to the body. Examples include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, carboxymethylcellulose sodium , Sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethylcellulose (ethylcellulose), xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol ), Sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.

13.2. How to treat cancer

One aspect of the content disclosed by the present specification relates to a method for treating cancer using a composition comprising allogeneic CAR cells.

One embodiment disclosed by the present specification is a use for a method of treating cancer by activating an immune response specifically to a cancer cell without an immune rejection reaction among allogenes by administering allogeneic CAR-T cells to a treatment target.

The cancer treatment method treats cancer in a subject, reduces tumor size, kills tumor cells, prevents tumor cell proliferation, prevents tumor growth, removes tumors from patients, or prevents tumor recurrence. Can be used to prevent, prevent tumor metastasis, induce remission in patients, or any combination thereof.

In this case, the treatment target may be a mammal including a primate such as a human or a monkey or a rodent such as a mouse or a rat.

Cancers that can be treated include vascularized tumors that have not been vascularized, have not been substantially vascularized yet, or have been vascularized. Cancers can also include solid or non-solid tumors.

In one embodiment, the method is, for example, bone tumor, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma in the skin or eye, lung cancer, breast cancer, uterine cancer, liver cancer, prostate cancer, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer , Testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal giant B cell lymphoma (PMBC), diffuse giant B cell lymphoma (DLBCL), follicular lymphoma (FL), converted follicular lymphoma, splenic marginal lymphoma (SMZL), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic Or acute leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (including all non-T cells), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, myeloplastic dysplasia, multiple myeloma, bladder , Kidney or ureter cancer, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal contraction tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal cancer, squamous cell carcinoma, T-cell lymphoma, B cell It can be used to reduce the size of a tumor derived from a malignant tumor and a combination of these cancers. The cancer may be some that can be treated (reactive) by chemotherapy or radiation therapy, or some that cannot be treated (reactive) by chemotherapy or radiation therapy.

In other embodiments, the method can be used to treat tumors that are lymphoma or leukemia. Lymphoma and leukemia are blood cancers that specifically affect lymphocytes. All white blood cells in the blood are derived from a single type of pluripotent hematopoietic stem cell found in the bone marrow. These stem cells produce both myeloid progenitor cells and lymphocytic progenitor cells, after which they develop the various types of white blood cells found in the body. White blood cells that arise from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells arising from lymphocytic progenitor cells include macronuclear cells, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphoma and leukemia can affect one or more of these cell types within the patient.

In some embodiments, the lymphoma or leukemia is B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoblastic lymphoma (e.g., Waldenström macroglobulinemia), splenic marginal zone lymphoma, Hairy cell leukemia, plasma cell neoplasm (e.g. plasma cell myeloma (i.e., multiple myeloma), or plasmacytoma), extracellular lymph node marginal cell B lymphoma (e.g. MALT lymphoma), lymph node marginal cell B cell lymphoma, Follicular lymphoma (FL), switched follicular lymphoma (TFL), primary cutaneous follicular central lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), Epstein-Barr virus-positive DLBCL, lymphoma granulomatosis, primary mediastinum (Thymus) giant B-cell lymphoma (PMBCL), intravascular giant B-cell lymphoma, ALK + giant B-cell lymphoma, plasmablastic lymphoma, primary exudative lymphoma, HHV8-associated multicentre Giant B-cell lymphoma, Bucket lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell giant granulocyte leukemia, aggressive NK cell leukemia, adult T-cell leukemia / lymphoma, extra-node NK / T -Cellular lymphoma, enteropathy-associated T-cell lymphoma, hepatic spleen T-cell lymphoma, parental NK cell lymphoma, mycosis carcinoma / tridentate syndrome, primary skin anaplastic giant cell lymphoma, lymphocytic papules, peripheral T-cells Lymphoma, angioimmunoblastic T cell lymphoma, anaplastic giant cell lymphoma, B-lymphoblastic leukemia / lymphoma, B-lymphoblastic leukemia / lymphoma with recurrent genetic abnormality, T-lymphoblastic leukemia / lymphoma, And Hodgkin's lymphoma. The cancer may be a tumor that is refractory to one or more prior treatments, or a tumor that recurs after one or more prior treatments. For example, the cancer is refractory to one or more of chemotherapy, radiotherapy, immunotherapy (including antibody or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof, or the cancer recurs thereafter. It may be.

In one embodiment, a method of treating cancer targeting the CD19 antigen is

Introducing a trapper molecule into T cells; And

Isolating cells from which different traits are derived from wild state cells; Produced by

It may include administering a composition comprising an Allogeneic CAR-T in a subject.

Furthermore, the Allogeneic CAR-T may further include introducing a CAR comprising an antigen recognition unit that binds CD19, a CD28 co-stimulatory domain, and a CD3-zeta signaling region in the production process.

In this case, the trait different from the wild state cell may be different from the wild state cell in the expression form of the immune-activated receptor and / or may include an artificial receptor. At this time, the artificial receptor may be a chimeric antigen receptor.

The cancer may be leukemia.

Leukemia is a disease that occurs when normal blood cells in the bone marrow are converted into cancer cells and multiply for some reason. Leukemia cells multiply indefinitely and interfere with the production of normal white blood cells, red blood cells, and platelets, and leukemia cells from bone marrow can deposit in the liver, spleen, lymph nodes, central nervous system, and various organs through blood circulation. It mainly shows acute progression, and is a case where cancer cell changes occur mainly in lymphocyte-based white blood cells. The cells of patients with acute lymphocytic leukemia overexpress CD19 and CD20.

Allogeneic CAR-T for treating leukemia can be used for treatment by inducing an immune response to abnormal cancer cells using CAR-T that can specifically recognize CD19 and / or CD20 antigens.

For example, a method for treating the blood cancer,

Introducing a trapper molecule into T cells; And

Isolating cells from which different traits are derived from wild state cells; Produced by

And administering a composition comprising an Allogeneic CAR-T in a subject.

Furthermore, the Allogeneic CAR-T may further include introducing a CAR specific for leukemia in the production process.

In this case, the trait different from the wild state cell may be different from the wild state cell in the expression form of the immune-activated receptor and / or may include an artificial receptor. At this time, the artificial receptor may be a chimeric artificial receptor.

In one embodiment, the method for treating cancer comprises administering a composition comprising allogeneic CAR cells to a subject.

For example, the method of administering the composition comprising the allogeneic CAR cells can be performed in any convenient way, such as injection, transfusion, implantation or transplantation. Routes of administration are subcutaneously, intradermal, intratumorally, intramally, intramuscularly, intramuscularly, intravenous, intralymphatic, and intraperitoneally. (intraperitoneally).

Preferably intravenous systemic administration.

In another example, the administration may include digestion pain including the kidney, stomach, pancreas, duodenum, and / or colon of the subject to be treated; Heart; lungs; brain; Eyes comprising retinal tissue; Ears, including the inner ear; skin; muscle; bone; And / or liver '.

For another example, the allogeneic CAR cells can be administered in a therapeutically effective amount.

For example, a therapeutically effective amount of allogeneic CAR T cells is at least about 10 ⁴ cells, at least about 10 ⁵ cells, at least about 10 ⁶ cells, at least about 10 ⁷ cells, at least about 10 ⁸ cells, at least about 10 ⁹ , or at least about 10 ¹⁰ cells. or

As another example, it may be about 10 ⁴ cells, about 10 ⁵ cells, about 10 ⁶ cells, about 10 ⁷ cells about 10 ⁸ cells, about 10 ⁹ cells, or about 10 ¹⁰ cells.

In another example, a therapeutically effective amount of allogeneic CAR T cells is about 1 X 10 ⁵ cells / kg, 2 X 10 ⁵ cells / kg, about 3 X 10 ⁵ cells / kg, about 4 X 10 ⁵ cells / kg, about 5 X 10 ⁵ cells / kg, about 6 X 10 ⁵ cells / kg, about 7 X 10 ⁵ cells / kg, About 8 X 10 ⁵ cells / kg, about 9 X 10 ⁵ cells / kg, about 1 X 10 ⁶ cells / kg, 2 X 10 ⁶ cells / kg, about 3 X 10 ⁶ cells / kg, about 4 X 10 ⁶ cells / kg, about 5 X 10 ⁶ cells / kg, about 6 X 10 ⁶ cells / kg, about 7 X 10 ⁶ cells / kg, About 8 X 10 ⁶ cells / kg, about 9 X 10 ⁶ cells / kg, about 1 X 10 ⁷ cells / kg, 2 X 10 ⁷ cells / kg, about 3 X 10 ⁷ cells / kg, about 4 X 10 ⁷ cells / kg, about 5 X 10 ⁷ cells / kg, about 6 X 10 ⁷ cells / kg, about 7 X 10 ⁷ cells / kg, About 8 X 10 ⁷ cells / kg or about 9 X 10 ⁷ cells / kg.

In one particular embodiment, the therapeutically effective amount of the allogeneic CAR T cells can be about 2 X 10 ⁶ cells / kg.

In another example, a therapeutically effective amount of allogeneic CAR-T cells is from about 1 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, from about 2 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, about 3 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, about 4 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, about 5 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, about 6 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, about 7 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, about 8 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, about 9 X 10 ⁵ cells / kg to about 2 x 10 ⁸ cells / kg, about 1 X 10 ⁶ cells / kg to about 2 x 10 ⁹ cells / kg, about 2 X 10 ⁶ cells / kg to about 2 x 10 ⁹ cells / kg, about 3 X 10 ⁶ cells / kg to about 2 x 10 ⁹ cells / kg, about 4 X 10 ⁶ cells / kg to about 2 x 10 ⁹ cells / kg, about 5 X 10 ⁶ cells / kg to about 2 x 10 ⁹ Cells / kg, about 6 X 10 ⁶ cells / kg to about 2 x 10 ⁹ cells / kg, about 7 X 10 ⁶ cells / kg to about 2 x 10 ⁹ cells / kg, about 8 X 10 ⁶ It may be between 2 cells / kg to about 2 x 10 ⁹ cells / kg, about 9 X 106 cells / kg to about 2 x 10 ⁹ cells / kg. In one particular embodiment, the therapeutically effective amount of engineered CAR T cells is 2.0 X 105 cells / kg.

In one embodiment,

The symptoms of the disease may be improved by administering the Allogeneic CAR-T cells to a recipient in a sufficient number. As an example, the dosage may be 10 ⁷ to 10 ¹⁰ cells, for example 10 ⁹ cells, in a single environment. 10 ⁹ cells can be divided and administered at regular intervals, and the infusion amount can be generally injected until symptoms of the disease improve or generally accepted. Preferably, it is one or more injections. In this case, the injection may be intravenously injecting the cells at a rate of 50 to 250 ml / hour. As another example, other suitable administration methods include intraarterial injection, direct injection into the tumor and / or perfusion of the tumor layer after surgery, intrathecal Administration and intraocular administration. However, the present invention is not limited thereto, and may be appropriately prescribed in consideration of the age, health and weight of the administration target, the type of treatment simultaneously received, if any, the frequency of treatment, and characteristics of desired effects.

In addition, when the pharmaceutical composition is administered, it may be administered prophylactically or therapeutically, regardless of before or after the occurrence of clinical symptoms of the disease.

14. Examples

Hereinafter, the present invention will be described in more detail through examples.

These examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1. Sequence of the trapper peptide used in the experiment

List of trapper peptides used in the experiment Name of the trapper peptide Amino acid sequence One SNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 178) 2 SNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 185) 3 KKVIGFRILLLKVAGFNLGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 248) 4 KKVIGFRILLLKVAGFNLIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 249) 5 LFIMIVGGLVGLRIVFAVLSIGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 240) 6 LFIMIVGGLVGLRIVFAVLSIIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 182) 7 AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 175) 8 AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 181) ERret1 ALPVTALLLPLALLLHAARPSNKSLEQIWNHTTWMEWDGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 220) ERret2 ALPVTALLLPLALLLHAARPSNKSLEQIWNHTTWMEWDIYIWAPLAGTCGVLLLSLVITLYCLYKYKSRRSFIEEKKMP (SEQ ID NO: 250)

The amino acid sequence of the trapper peptide used in the following experiments is shown in Table 1, and the names of the trapper peptides mentioned in the graphs and tables showing the experimental results refer to the names located in column 1 of Table 1.

The positional relationship between domains constituting the trapper peptides 1 to 8 is shown in FIG. In addition, the positional relationship between the domains constituting ERret1 and ERret2 is shown in FIG. ERret1 and ERret2 were intended to show an improved effect compared to 1 to 8 by additionally including the CD8a signal sequence.

Example 2. Transformation results of Jurkat cells using electroporation

The Jurkat cell line (ATCC-TIB-152) was cultured in RPMI1640 medium (GeneAll) containing 10% FBS and antibiotics. Cells were cultured in an incubator at 37 ° C 5% CO ₂ .

Sequences encoding the trapper peptides corresponding to 1 to 8 in Table 1 were codon optimized and then subcloned into the lentivirus vector pLVX-IRES-puro vector (clonetech). PLVX vectors that were not subcloned as controls were also prepared. In order to transform the cells prepared in Examples 1 and 2, 10 uL tip of Neon Transfection System (ThermoFisher Scientific, Grand Island, NY) is used for gene transfer under 1,400 V, 20 ms, and 2 pulses conditions. Did. Cells were plated in 500 μl of antibiotic-free medium and incubated at 37 ° C in a 5% CO ₂ incubator.

Cell staining was performed in PBS at 4 ° C with 1% FBS added. For evaluation of CD3 and TRAC expression, Jurkat cells were cultured for 14 days after electroporation. For flow cytometry analysis, cells were washed with phosphate buffered saline (PBS) supplemented with 1% FBS and FITC mouse anti-human CD3 antibody (Myltenyi Biotec) and APC anti-human TCRα / β (Myltenyi Biotec) at 4 ° C. And stained for 30 minutes. Cells were then washed and resuspended in PBS. Data were obtained with an Attune NxT Acoustic Focusing Cytometer (Thermo Scientific) and then analyzed with FlowJo (FlowJo, LLC).

The analyzed results are shown in Table 2 and FIG. 2. Jurkat cells transduced with the trapper peptide were found to have significantly reduced the amount of TCR heterodimer, TCR heterodimer, and the cell surface of CD3. Among them, the trapper peptide corresponding to 2 showed excellent properties.

Results of flow cytometry of Jurkat cells transduced with trapper peptide by electroporation DNA transfection TCR negative (%) CD3 negative (%) pLVX 20.8 36.5 One 19 34.1 2 30.3 55.3 3 20.5 40.3 4 20.1 41.1 5 22 41.4 6 22.8 40.8 7 22.6 41.3 8 21.7 44.9

Example 3. Transformation results of Jurkat cells using lentivirus

The Jurkat cell line (ATCC-TIB-152) was cultured in RPMI1640 medium (GeneAll) containing 10% FBS and antibiotics. Cells were cultured in an incubator at 37 ° C 5% CO ₂ .

Sequences encoding ERret1 and ERret2 in Table 1 were codon optimized, and then subcloned into a lentivirus vector pLVX-IRES-puro vector (clonetech). PLVX vectors that were not subcloned as controls were also prepared. Virus was collected from the supernatant of 293T cells transfected with lentiviral vector and helper plasmid using Lipofectamine2000 (Thermo Scientific). After harvesting the supernatant, the lentivirus is soaked in 20% sucrose-containing buffer (100 mM NaCl, 0.5 mM ethylene diamine tetra acetic acid [EDTA], 50 mM Tris-HCl, pH 7.4) at a ratio of 4: 1 to 10000 xg. Centrifugation at 4 ° C for 4 hours. After centrifugation, the supernatant was removed and phosphate buffered saline (PBS) was added again to form a suspension. The lenti virus prepared by the above method was added to the culture medium of Jurkat cells and transduced. At this time, the volume of the lentivirus suspension to be added was compared and experimented with 10, 100, and 500 μl.

Cell staining was performed in PBS at 4 ° C with 1% FBS added. For evaluation of CD3 and TRAC expression, Jurkat cells were cultured for 14 days after electroporation. For flow cytometry analysis, cells were washed with phosphate buffered saline (PBS) supplemented with 1% FBS and FITC mouse anti-human CD3 antibody (Myltenyi Biotec) and APC anti-human TCRα / β (Myltenyi Biotec) at 4 ° C. And stained for 30 minutes. Cells were then washed and resuspended in PBS. Data were obtained with an Attune NxT Acoustic Focusing Cytometer (Thermo Scientific) and then analyzed with FlowJo (FlowJo, LLC).

The analyzed results are shown in Table 3 and FIG. 3. When comparing the results of pLVX and pLVX-ERret2 in FIG. 3, it can be seen that Jurkat cells transduced with ERret2 significantly reduced the amount of TCR and CD3 cell surfaces compared to wild-type cells. In the results of the numerical analysis, it was confirmed that the amount of TCR heterodimer and CD3 cell surface of TCR subunits was significantly decreased in Jurkat cells transduced with the trapper peptide (Table 3). Among them, the corresponding trapper peptide of ERret2 showed excellent properties.

Furthermore, as a result of administration by increasing the dose of the lentiviral suspension into which ERret2 was inserted, it was confirmed that the amount of TCR on the cell surface was significantly decreased (FIGS. 4 and 5). The value of the 'virus' axis of the graph represents the dose of the suspension administered (μl). This result is interpreted as the effect of the trapper peptide on the expression trait of TCR in immune cells.

Results of flow cytometry analysis of Jurkat cells transfected with lentivirus trapper peptide Lentiviral transduction TCR negative (%) CD3 negative (%) pLVX 13.2 14.6 ERret1 14.3 13.3 ERret2 54.6 54.1

Example 4. Correlation of the expression of the trapper peptide in Jurkat cells with the amount of TCR subunits on the cell surface

The Jurkat cell line (ATCC-TIB-152) was cultured in RPMI1640 medium (GeneAll) containing 10% FBS and antibiotics. Cells were cultured in an incubator at 37 ° C 5% CO ₂ .

Sequences encoding ERret1 and ERret2 in Table 1 were codon optimized, and then subcloned into a lentivirus vector pLVX-IRES-puro vector (clonetech). In addition, EGFP was subcloned to quantify the amount of trapper peptide expressed in the cell. In this case, since the EGFP is also expressed when the trapper peptide is expressed, the amount of the trapper peptide can be deduced by measuring the amount of EGFP through flow cytometry. The design of the vector used in the experiment is shown in FIG. 6. Virus was collected from the supernatant of 293T cells transfected with lentiviral vector and helper plasmid using Lipofectamine2000 (Thermo Scientific). After harvesting the supernatant, the lentivirus is soaked in 20% sucrose-containing buffer (100 mM NaCl, 0.5 mM ethylene diamine tetra acetic acid [EDTA], 50 mM Tris-HCl, pH 7.4) at a ratio of 4: 1 to 10000 xg. Centrifugation at 4 ° C for 4 hours. After centrifugation, the supernatant was removed and phosphate buffered saline (PBS) was added again to form a suspension. The lenti virus prepared by the above method was added to the culture medium of Jurkat cells and transduced.

Cell staining was performed in PBS at 4 ° C with 1% FBS added. For evaluation of CD3 and TRAC expression, Jurkat cells were cultured for 14 days after electroporation. For flow cytometry analysis, cells were washed with phosphate buffered saline (PBS) supplemented with 1% FBS and FITC mouse anti-human CD3 antibody (Myltenyi Biotec) and APC anti-human TCRα / β (Myltenyi Biotec) at 4 ° C. And stained for 30 minutes. Cells were then washed and resuspended in PBS. Data were obtained with an Attune NxT Acoustic Focusing Cytometer (Thermo Scientific) and then analyzed with FlowJo (FlowJo, LLC).

The analyzed results are shown in FIG. 7. When comparing the EGFP MFI values of pLVX-EGFP and pLVX-EGFP-ERret2, it can be seen that the amount of cell surface of EGFP expressed as ERret2 is reduced, and through this, the trapper peptide is not a cell membrane as intended in this application. It can be confirmed that it remains in the organelles. In addition, by comparing the TCR MFI value, it was confirmed that the amount of the cell surface of the TCR chain was reduced, and through this, it was confirmed that the expression of the trapper peptide decreased the amount of the cell surface of the TCR. When synthesized, it was expected that the trapper peptide remained in the organelle and bound the TCR chain, thereby preventing TCR from being exposed on the cell surface by being trapped in the cell.

Example 5. Correlation of the amount of trapper peptide in Jurkat cells with the amount of TCR subunits on the Jurkat cell surface

The Jurkat cell line (ATCC-TIB-152) was cultured in RPMI1640 medium (GeneAll) containing 10% FBS and antibiotics. Cells were cultured in an incubator at 37 ° C 5% CO ₂ .

Sequences encoding ERret1 and ERret2 in Table 1 were codon optimized, and then subcloned into a lentivirus vector pLVX-IRES-puro vector (clonetech). In addition, EGFP was subcloned to quantify the amount of trapper peptide expressed in the cell. In addition, in order to control the amount of expression of the trapper peptide, a vector in which the promoter was substituted with CMV2 from EF1a was constructed. The design of the vector used in the experiment is shown in FIG. 8. Virus was collected from the supernatant of 293T cells transfected with lentiviral vector and helper plasmid using Lipofectamine2000 (Thermo Scientific). After harvesting the supernatant, the lentivirus is soaked in 20% sucrose-containing buffer (100 mM NaCl, 0.5 mM ethylene diamine tetra acetic acid [EDTA], 50 mM Tris-HCl, pH 7.4) at a ratio of 4: 1 to 10000 xg. Centrifugation at 4 ° C for 4 hours. After centrifugation, the supernatant was removed and phosphate buffered saline (PBS) was added again to form a suspension. The lenti virus prepared by the above method was added to the culture medium of Jurkat cells and transduced.

Cell staining was performed in PBS at 4 ° C with 1% FBS added. For evaluation of CD3 and TRAC expression, Jurkat cells were cultured for 14 days after electroporation. For flow cytometry analysis, cells were washed with phosphate buffered saline (PBS) supplemented with 1% FBS and FITC mouse anti-human CD3 antibody (Myltenyi Biotec) and APC anti-human TCRα / β (Myltenyi Biotec) at 4 ° C. And stained for 30 minutes. Cells were then washed and resuspended in PBS. Data were obtained with an Attune NxT Acoustic Focusing Cytometer (Thermo Scientific) and then analyzed with FlowJo (FlowJo, LLC).

The analyzed results are shown in FIG. 9. Since the CMV2 promoter is a promoter having a higher activity than the EF1a promoter, it is expected that the expression level of the trapper peptide will increase. As a result of the actual experiment, it was confirmed that when the CMV2 promoter was used, the expression level of the trapper peptide increased and the amount of the cell surface of the TCR chain decreased. This means that the amount of the trapper peptide in the immune cells correlates positively with the TCR negative trait of the immune cells.

Example 6. Transformation results of T cells using lentivirus

Human peripheral blood pan-T cells (STEMCELL Technologies) were activated by adding Dynabeads Human T Activator anti-CD3 / 28 (Thermo Scientific) at a 3: 1 bead to cell ratio in RPMI with 10% FBS added.

Sequences encoding ERret1 and ERret2 in Table 1 were codon optimized and then subcloned into a lentivirus vector pLVX-IRES-puro vector (clonetech). In addition, EGFP was subcloned to quantify the amount of trapper peptide expressed in the cell. The design of the vector used in the experiment is shown in FIG. 10. Virus was collected from the supernatant of 293T cells transfected with lentiviral vector and helper plasmid using Lipofectamine2000 (Thermo Scientific). After harvesting the supernatant, the lentivirus is soaked in 20% sucrose-containing buffer (100 mM NaCl, 0.5 mM ethylene diamine tetra acetic acid [EDTA], 50 mM Tris-HCl, pH 7.4) at a ratio of 4: 1 to 10000 xg. Centrifugation at 4 ° C for 4 hours. After centrifugation, the supernatant was removed and phosphate buffered saline (PBS) was added again to form a suspension. The lentivirus prepared by the above method was added to the T cell culture medium and transformed.

After 72 hours of activation and transduction, beads were removed from the cell suspension and T cells were removed with 10% FBS, hIL-2 (Peprotech, 50 U / mL), IL-15 (Peptrotech, 5 ng / ml) and hIL-7 ( Peprotech, 5 ng / mL) was cultured in RPMI 1640 medium. Cells were cultured in an incubator at 37 ° C 5% CO ₂ .

Cell staining was performed in PBS at 4 ° C with 1% FBS added. For flow cytometry analysis, cells were washed with phosphate buffered saline (PBS) supplemented with 1% FBS and FITC mouse anti-human CD3 antibody (Myltenyi Biotec) and APC anti-human TCRα / β (Myltenyi Biotec) at 4 ° C. And stained for 30 minutes. Cells were then washed and resuspended in PBS. Data were obtained with an Attune NxT Acoustic Focusing Cytometer (Thermo Scientific) and then analyzed with FlowJo (FlowJo, LLC).

The analyzed results are shown in FIG. 11. When comparing the EGFP MFI values of pLVX-EGFP and pLVX-EGFP-ERret2, it can be seen that the amount of cell surface of EGFP expressed as ERret2 is reduced, and through this, the trapper peptide is not a cell membrane as intended in this application. It can be confirmed that it remains in the organelles. In addition, by comparing the TCR MFI value, it was confirmed that the amount of the cell surface of the TCR chain was reduced, and through this, it was confirmed that the expression of the trapper peptide decreased the amount of the cell surface of the TCR. When synthesized, it was expected that the trapper peptide remained in the organelle and bound the TCR chain, thereby preventing TCR from being exposed on the cell surface by being trapped in the cell. Through this, it was confirmed that the trapper peptide was successfully transduced into T cells.

Example 7. Correlation of the amount of trapper peptide in T cells with the amount of TCR subunits on the surface of T cells

Human peripheral blood pan-T cells (STEMCELL Technologies) were activated by adding Dynabeads Human T Activator anti-CD3 / 28 (Thermo Scientific) at a 3: 1 bead to cell ratio in RPMI with 10% FBS added.

Sequences encoding ERret1 and ERret2 in Table 1 were codon optimized, and then subcloned into a lentivirus vector pLVX-IRES-puro vector (clonetech). In addition, EGFP was subcloned to quantify the amount of trapper peptide expressed in the cell. In addition, in order to control the amount of expression of the trapper peptide, a vector in which promoters were substituted with CMV2 and CMV3, respectively, was prepared. The design of the vector used in the experiment is shown in FIG. 12. Virus was collected from the supernatant of 293T cells transfected with lentiviral vector and helper plasmid using Lipofectamine2000 (Thermo Scientific). After harvesting the supernatant, the lentivirus is soaked in 20% sucrose-containing buffer (100 mM NaCl, 0.5 mM ethylene diamine tetra acetic acid [EDTA], 50 mM Tris-HCl, pH 7.4) at a ratio of 4: 1 to 10000 xg. Centrifugation at 4 ° C for 4 hours. After centrifugation, the supernatant was removed and phosphate buffered saline (PBS) was added again to form a suspension. The lentivirus prepared by the above method was added to the T cell culture medium and transformed.

After 72 hours of activation and transduction, beads were removed from the cell suspension and T cells were removed with 10% FBS, hIL-2 (Peprotech, 50 U / mL), IL-15 (Peptrotech, 5 ng / ml) and hIL-7 ( Peprotech, 5 ng / mL) was cultured in RPMI 1640 medium. Cells were cultured in an incubator at 37 ° C 5% CO ₂ .

Cell staining was performed in PBS at 4 ° C with 1% FBS added. For flow cytometry analysis, cells were washed with phosphate buffered saline (PBS) supplemented with 1% FBS and FITC mouse anti-human CD3 antibody (Myltenyi Biotec) and APC anti-human TCRα / β (Myltenyi Biotec) at 4 ° C. And stained for 30 minutes. Cells were then washed and resuspended in PBS. Data were obtained with an Attune NxT Acoustic Focusing Cytometer (Thermo Scientific) and then analyzed with FlowJo (FlowJo, LLC).

The analyzed results are shown in FIG. 13. In the order of the CMV3 and CMV2 promoters, it is expected that the amount of expression of the trapper peptide will increase because the promoter is highly active. As a result of the actual experiment, it was confirmed that the amount of cell surface of the TCR chain was reduced in pLVX-CMV3-EGFP-ERret2 than in pLVX-CMV3-EGFP-ERret3. This means that the amount of trapper peptide in immune cells correlates positively with the TCR negative trait of immune cells.

<110> TOOLGEN <120> Allogeneic CAR-T <130> OPP18-032-PCT <150> US 62 / 749,928 <151> 2018-10-24 <160> 250 <170> KoPatentIn 3.0 <210> 1 <211> 345 <212> PRT <213> Human immunodeficiency virus type 1 <400> 1 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile          35 40 45 Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln      50 55 60 Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln  65 70 75 80 Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala                  85 90 95 Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp             100 105 110 Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr         115 120 125 Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys     130 135 140 Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn 145 150 155 160 Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys Leu Phe Ile Met                 165 170 175 Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Ala Val Leu Ser             180 185 190 Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr         195 200 205 His Leu Pro Thr Pro Arg Gly Pro Asp Arg Pro Glu Gly Ile Glu Glu     210 215 220 Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser Ile Arg Leu Val Asn Gly 225 230 235 240 Ser Leu Ala Leu Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser                 245 250 255 Tyr His Arg Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu             260 265 270 Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu         275 280 285 Leu Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu     290 295 300 Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu 305 310 315 320 Val Val Gln Gly Ala Cys Arg Ala Ile Arg His Ile Pro Arg Arg Ile                 325 330 335 Arg Gln Gly Leu Glu Arg Ile Leu Leu             340 345 <210> 2 <211> 33 <212> PRT <213> Human immunodeficiency virus type 1 <400> 2 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu     <210> 3 <211> 37 <212> PRT <213> Human immunodeficiency virus type 1 <400> 3 Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu   1 5 10 15 Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu              20 25 30 Gln Ala Arg Ile Leu          35 <210> 4 <211> 44 <212> PRT <213> Human immunodeficiency virus type 1 <400> 4 Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly   1 5 10 15 Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Ser              20 25 30 Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His          35 40 <210> 5 <211> 33 <212> PRT <213> Human immunodeficiency virus type 1 <400> 5 Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His   1 5 10 15 Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu              20 25 30 Leu     <210> 6 <211> 21 <212> PRT <213> Human immunodeficiency virus type 1 <400> 6 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile              20 <210> 7 <211> 36 <212> PRT <213> Human immunodeficiency virus type 1 <400> 7 Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala   1 5 10 15 Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln              20 25 30 Ala Arg Ile Leu          35 <210> 8 <211> 34 <212> PRT <213> Human immunodeficiency virus type 1 <400> 8 Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His   1 5 10 15 Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu              20 25 30 Leu Leu         <210> 9 <211> 18 <212> PRT <213> Human immunodeficiency virus type 1 <400> 9 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp         <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 10 Gly Leu Arg Leu Leu Met Phe Ile Val   1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 11 Gly Leu Arg Ile Val Phe Ala Val Leu   1 5 <210> 12 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 12 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala   1 5 10 15 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala              20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala          35 40 45 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala      50 55 60 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala  65 70 75 80 Ala     <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 13 Gly Ala Leu Phe Leu Gly Phe Leu Gly   1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 14 Gly Ala Val Phe Leu Gly Phe Leu   1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 15 Gly Ala Met Phe Leu Gly Phe Leu Gly   1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 16 Gly Ala Val Leu Leu Gly Phe Leu Gly   1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 17 Gly Ala Phe Phe Leu Gly Phe Leu Gly   1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 18 Gly Ala Met Ile Phe Gly Phe Leu Gly   1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 19 Gly Ala Leu Leu Phe Gly Phe Leu Gly   1 5 <210> 20 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 20 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu     <210> 21 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 21 Ala Glu Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu     <210> 22 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 22 Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp   1 5 10 <210> 23 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 23 Glu Gln Leu Trp Asn His Thr Thr Trp Met Glu Trp   1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 24 Glu Glu Ile Trp Asn His Thr Thr Trp Met Glu Trp   1 5 10 <210> 25 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 25 Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu   1 5 10 15 Gln Ile         <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 26 Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His   1 5 10 15 Thr     <210> 27 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 27 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp         <210> 28 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 28 Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile   1 5 10 15 Asn Asn         <210> 29 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 29 Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu   1 5 10 15 Gln Thr Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn              20 25 30 Asn Tyr Thr Ser Leu          35 <210> 30 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 30 Ser Asn Lys Ser Leu Glu Gln Ile Gly Asn His Thr Thr Gly Met Glu   1 5 10 15 Gly Asp         <210> 31 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 31 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Gly   1 5 10 15 Trp Gly         <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 32 Gly Leu Val Gly Leu Ala Ile Val Phe   1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 33 Gly Leu Leu Gly Leu Ala Ile Val Phe   1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 34 Gly Gly Val Gly Leu Ala Gln Phe   1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 35 Gly Gly Val Gly Leu Ala Ile Val Phe   1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 36 Gly Gly Leu Gly Leu Ala Ile Val Phe   1 5 <210> 37 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 37 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile              20 <210> 38 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 38 Leu Phe Ile Met Ile Val Gly Gly Leu Arg Leu Leu Met Phe Ile Val   1 5 10 15 Phe Ala Val Leu Ser Ile              20 <210> 39 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 39 Leu Phe Ile Met Ile Val Gly Gly Leu Arg Ile Val Phe Ala Val Leu   1 5 10 15 Ser Ile         <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 40 Lys Asp Glu Leu   One <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 41 Lys Lys Xaa Xaa Ala   1 5 <210> 42 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 42 Ser Glu Lys Asp Glu Leu   1 5 <210> 43 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 43 Lys Lys Met Pro   One <210> 44 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 44 Lys Lys Thr Asn   One <210> 45 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 45 Lys Tyr Lys Ser Pro Arg Ser Phe Ile Glu Glu Lys Lys Met Pro   1 5 10 15 <210> 46 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 46 Leu Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro   1 5 10 15 <210> 47 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 47 Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met   1 5 10 15 Pro     <210> 48 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 48 Leu Tyr Cys Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys   1 5 10 15 Met Pro         <210> 49 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 49 Lys Tyr Cys Asn Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys   1 5 10 15 Lys Met Pro             <210> 50 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 50 Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Asp Glu Lys Lys Met   1 5 10 15 Pro     <210> 51 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 51 Leu Tyr Cys Asn Lys Tyr Lys Ser Arg Arg Ser Phe Ile Asp Glu Lys   1 5 10 15 Lys Met Pro             <210> 52 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 52 Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys Tyr Lys Ser   1 5 10 15 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro              20 25 <210> 53 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 53 Leu Tyr Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Lys Tyr Lys Ser   1 5 10 15 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro              20 25 <210> 54 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 54 Leu Tyr Lys Lys Leu Glu Thr Phe Lys Lys Thr Asn   1 5 10 <210> 55 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 55 Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys Lys Leu Glu   1 5 10 15 Thr Phe Lys Lys Thr Asn              20 <210> 56 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 56 Lys Xaa Lys Xaa Xaa   1 5 <210> 57 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 57 Tyr Gln Arg Leu   One <210> 58 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 58 His Asp Glu Phe   One <210> 59 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 59 His Asp Glu Leu   One <210> 60 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 60 Arg Asp Glu Phe   One <210> 61 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 61 Arg Asp Glu Leu   One <210> 62 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 62 Trp Asp Glu Leu   One <210> 63 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 63 Tyr Asp Glu Leu   One <210> 64 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 64 His Glu Glu Phe   One <210> 65 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 65 His Glu Glu Leu   One <210> 66 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 66 Lys Glu Glu Leu   One <210> 67 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 67 Arg Glu Glu Leu   One <210> 68 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 68 Lys Ala Glu Leu   One <210> 69 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 69 Lys Cys Glu Leu   One <210> 70 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 70 Lys Phe Glu Leu   One <210> 71 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 71 Lys Gly Glu Leu   One <210> 72 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 72 Lys His Glu Leu   One <210> 73 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 73 Lys Leu Glu Leu   One <210> 74 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 74 Lys Asn Glu Leu   One <210> 75 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 75 Lys Gln Glu Leu   One <210> 76 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 76 Lys Arg Glu Leu   One <210> 77 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 77 Lys Ser Glu Leu   One <210> 78 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 78 Lys Val Glu Leu   One <210> 79 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 79 Lys Trp Glu Leu   One <210> 80 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 80 Lys Tyr Glu Leu   One <210> 81 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 81 Lys Glu Asp Leu   One <210> 82 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 82 Lys Ile Glu Leu   One <210> 83 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 83 Asp Lys Glu Leu   One <210> 84 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 84 Phe Asp Glu Leu   One <210> 85 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 85 Lys Asp Glu Phe   One <210> 86 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 86 Lys Lys Glu Leu   One <210> 87 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 87 His Ala Asp Leu   One <210> 88 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 88 His Ala Glu Leu   One <210> 89 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 89 His Ile Glu Leu   One <210> 90 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 90 His Asn Glu Leu   One <210> 91 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 91 His Thr Glu Leu   One <210> 92 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 92 Lys Thr Glu Leu   One <210> 93 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 93 His Val Glu Leu   One <210> 94 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 94 Asn Asp Glu Leu   One <210> 95 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 95 Gln Asp Glu Leu   One <210> 96 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 96 Arg Glu Asp Leu   One <210> 97 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 97 Arg Asn Glu Leu   One <210> 98 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 98 Arg Thr Asp Leu   One <210> 99 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 99 Arg Thr Glu Leu   One <210> 100 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 100 Ser Asp Glu Leu   One <210> 101 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 101 Thr Asp Glu Leu   One <210> 102 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 102 Ser Lys Glu Leu   One <210> 103 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 103 Leu Tyr Tyr Gln Arg Leu   1 5 <210> 104 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 104 Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Tyr Gln Arg Leu   1 5 10 15 <210> 105 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 105 Leu Tyr Lys Arg Lys Ile Ile Ala Phe Ala Leu Glu Gly Lys Arg Ser   1 5 10 15 Lys Val Thr Arg Arg Pro Lys Ala Ser Asp Tyr Gln Arg Leu              20 25 30 <210> 106 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 106 Leu Tyr Arg Asn Ile Lys Cys Asp   1 5 <210> 107 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 107 Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Arg Asn Ile Lys   1 5 10 15 Cys Asp         <210> 108 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 108 Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu   1 5 10 15 Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala              20 25 30 Cys Ala Ser Ala Arg Ile Asn Val          35 40 <210> 109 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 109 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 <210> 110 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly   1 5 10 15 Gly Gly Gly Ser              20 <210> 111 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 111 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys              20 <210> 112 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 112 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Lys Tyr Lys Ser Pro Arg Ser Phe              20 25 30 Ile Glu Glu Lys Lys Met Pro          35 <210> 113 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 113 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Lys Tyr Lys Ser Arg Arg Ser              20 25 30 Phe Ile Glu Glu Lys Lys Met Pro          35 40 <210> 114 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 114 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg Arg              20 25 30 Ser Phe Ile Glu Glu Lys Lys Met Pro          35 40 <210> 115 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 115 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Cys Lys Tyr Lys Ser Arg              20 25 30 Arg Ser Phe Ile Glu Glu Lys Lys Met Pro          35 40 <210> 116 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 116 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Lys Tyr Cys Asn Lys Tyr Lys Ser              20 25 30 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro          35 40 <210> 117 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 117 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg Arg              20 25 30 Ser Phe Ile Asp Glu Lys Lys Met Pro          35 40 <210> 118 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 118 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Cys Asn Lys Tyr Lys Ser              20 25 30 Arg Arg Ser Phe Ile Asp Glu Lys Lys Met Pro          35 40 <210> 119 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 119 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Glu Gln Lys Leu Ile Ser              20 25 30 Glu Glu Asp Leu Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys          35 40 45 Lys Met Pro      50 <210> 120 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 120 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Cys Tyr Pro Tyr Asp Val              20 25 30 Pro Asp Tyr Ala Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys          35 40 45 Lys Met Pro      50 <210> 121 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 121 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Lys Leu Glu Thr Phe              20 25 30 Lys Lys Thr Asn          35 <210> 122 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 122 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Glu Gln Lys Leu Ile Ser              20 25 30 Glu Glu Asp Leu Lys Lys Leu Glu Thr Phe Lys Lys Thr Asn          35 40 45 <210> 123 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 123 Lys Tyr Lys Ser Pro Arg Ser Phe Ile Glu Glu Lys Lys Met Pro Ile   1 5 10 15 Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser              20 25 30 Leu Val Ile Thr Leu Tyr Cys          35 <210> 124 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 124 Leu Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro   1 5 10 15 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu              20 25 30 Ser Leu Val Ile Thr Leu Tyr Cys          35 40 <210> 125 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 125 Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met   1 5 10 15 Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu              20 25 30 Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 <210> 126 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 126 Leu Tyr Cys Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys   1 5 10 15 Met Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 <210> 127 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 127 Lys Tyr Cys Asn Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys   1 5 10 15 Lys Met Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val              20 25 30 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 <210> 128 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 128 Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Asp Glu Lys Lys Met   1 5 10 15 Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu              20 25 30 Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 <210> 129 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 129 Leu Tyr Cys Asn Lys Tyr Lys Ser Arg Arg Ser Phe Ile Asp Glu Lys   1 5 10 15 Lys Met Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val              20 25 30 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 <210> 130 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 130 Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys Tyr Lys Ser   1 5 10 15 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro Ile Tyr Ile Trp Ala              20 25 30 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr          35 40 45 Leu Tyr Cys      50 <210> 131 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 131 Leu Tyr Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Lys Tyr Lys Ser   1 5 10 15 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro Ile Tyr Ile Trp Ala              20 25 30 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr          35 40 45 Leu Tyr Cys      50 <210> 132 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 132 Leu Tyr Lys Lys Leu Glu Thr Phe Lys Lys Thr Asn Ile Tyr Ile Trp   1 5 10 15 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile              20 25 30 Thr Leu Tyr Cys          35 <210> 133 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 133 Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys Lys Leu Glu   1 5 10 15 Thr Phe Lys Lys Thr Asn Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr              20 25 30 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 45 <210> 134 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 134 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Ser His Gly Phe Pro Pro Glu Val              20 25 30 Glu Glu Gln Asp Asp Gly Thr Leu Pro Met Ser Cys Ala Gln Glu Ser          35 40 45 Gly Met Asp Arg His Pro Ala Ala Cys Ala Ser Ala Arg Ile Asn Val      50 55 60 <210> 135 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 135 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Leu Phe Ile Met Ile Val Gly Gly              20 25 30 Leu Val Gly Leu Arg Ile Val Phe Ala Val Leu Ser Ile          35 40 45 <210> 136 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 136 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Ala Val Gly Ile Gly Ala Leu Phe              20 25 30 Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met          35 40 45 Thr Leu Thr Val Gln Ala Arg Gln Leu      50 55 <210> 137 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 137 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Ser Asn Lys Ser Leu Glu Gln Ile              20 25 30 Trp Asn His Thr Thr Trp Met Glu Trp Asp          35 40 <210> 138 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 138 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys              20 25 30 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 45 <210> 139 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 139 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu          35 40 45 Leu Ser Leu Val Ile Thr Leu Tyr Cys      50 55 <210> 140 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 140 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 <210> 141 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 141 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro              20 <210> 142 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 142 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu              20 25 30 Arg Ile Val Phe Ala Val Leu Ser Ile          35 40 <210> 143 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 143 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu              20 25 30 Arg Ile Val Phe Ala Val Leu Ser Ile          35 40 <210> 144 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 144 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu              20 25 30 Arg Ile Val Phe Ala Val Leu Ser Ile          35 40 <210> 145 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 145 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Lys Tyr Lys Ser Pro Arg Ser Phe Ile Glu Glu Lys              20 25 30 Lys Met Pro          35 <210> 146 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 146 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu              20 25 30 Lys Lys Met Pro          35 <210> 147 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 147 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu              20 25 30 Glu Lys Lys Met Pro          35 <210> 148 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 148 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Tyr Cys Lys Tyr Lys Ser Arg Arg Ser Phe Ile              20 25 30 Glu Glu Lys Lys Met Pro          35 <210> 149 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 149 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Lys Tyr Cys Asn Lys Tyr Lys Ser Arg Arg Ser Phe              20 25 30 Ile Glu Glu Lys Lys Met Pro          35 <210> 150 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 150 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Asp              20 25 30 Glu Lys Lys Met Pro          35 <210> 151 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 151 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Tyr Cys Asn Lys Tyr Lys Ser Arg Arg Ser Phe              20 25 30 Ile Asp Glu Lys Lys Met Pro          35 <210> 152 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 152 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu              20 25 30 Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro          35 40 45 <210> 153 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 153 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Tyr Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala              20 25 30 Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro          35 40 45 <210> 154 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 154 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Tyr Lys Lys Leu Glu Thr Phe Lys Lys Thr Asn              20 25 30 <210> 155 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 155 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu              20 25 30 Lys Lys Leu Glu Thr Phe Lys Lys Thr Asn          35 40 <210> 156 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 156 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Ala Leu Pro Val Thr Ala Leu Leu              20 25 30 Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro          35 40 <210> 157 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 157 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu              20 25 30 Ala Leu Leu Leu His Ala Ala Arg Pro          35 40 <210> 158 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 158 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu              20 25 30 Ala Leu Leu Leu His Ala Ala Arg Pro          35 40 <210> 159 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 159 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu              20 25 30 Ala Leu Leu Leu His Ala Ala Arg Pro          35 40 <210> 160 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 160 Lys Tyr Lys Ser Pro Arg Ser Phe Ile Glu Glu Lys Lys Met Pro Ala   1 5 10 15 Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala              20 25 30 Ala Arg Pro          35 <210> 161 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 161 Leu Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro   1 5 10 15 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His              20 25 30 Ala Ala Arg Pro          35 <210> 162 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 162 Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met   1 5 10 15 Pro Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu              20 25 30 His Ala Ala Arg Pro          35 <210> 163 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 163 Leu Tyr Cys Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys   1 5 10 15 Met Pro Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu              20 25 30 Leu His Ala Ala Arg Pro          35 <210> 164 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 164 Lys Tyr Cys Asn Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys   1 5 10 15 Lys Met Pro Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu              20 25 30 Leu Leu His Ala Ala Arg Pro          35 <210> 165 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 165 Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Asp Glu Lys Lys Met   1 5 10 15 Pro Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu              20 25 30 His Ala Ala Arg Pro          35 <210> 166 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 166 Leu Tyr Cys Asn Lys Tyr Lys Ser Arg Arg Ser Phe Ile Asp Glu Lys   1 5 10 15 Lys Met Pro Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu              20 25 30 Leu Leu His Ala Ala Arg Pro          35 <210> 167 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 167 Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys Tyr Lys Ser   1 5 10 15 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro Ala Leu Pro Val Thr              20 25 30 Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro          35 40 45 <210> 168 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 168 Leu Tyr Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Lys Tyr Lys Ser   1 5 10 15 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro Ala Leu Pro Val Thr              20 25 30 Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro          35 40 45 <210> 169 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 169 Leu Tyr Lys Lys Leu Glu Thr Phe Lys Lys Thr Asn Ala Leu Pro Val   1 5 10 15 Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro              20 25 30 <210> 170 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 170 Leu Tyr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys Lys Leu Glu   1 5 10 15 Thr Phe Lys Lys Thr Asn Ala Leu Pro Val Thr Ala Leu Leu Leu Pro              20 25 30 Leu Ala Leu Leu Leu His Ala Ala Arg Pro          35 40 <210> 171 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 171 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly              20 25 30 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys          35 40 <210> 172 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 172 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Lys Asp Glu Leu          35 <210> 173 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 173 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Lys Asp Glu Leu              20 <210> 174 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 174 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile Lys Asp Glu Leu              20 25 <210> 175 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 175 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser          35 40 45 Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu      50 55 <210> 176 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 176 Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu   1 5 10 15 Gln Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly              20 25 30 Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu          35 40 <210> 177 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 177 Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His   1 5 10 15 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser              20 25 30 Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu          35 40 <210> 178 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 178 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly              20 25 30 Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu          35 40 <210> 179 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 179 Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile   1 5 10 15 Asn Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly              20 25 30 Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu          35 40 <210> 180 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 180 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly              20 25 30 Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu          35 40 <210> 181 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 181 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu          35 40 45 Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg      50 55 60 Arg Ser Phe Ile Glu Glu Lys Lys Met Pro  65 70 <210> 182 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 182 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys              20 25 30 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys          35 40 45 Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro      50 55 60 <210> 183 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 183 Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu   1 5 10 15 Gln Ile Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser          35 40 45 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro      50 55 <210> 184 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 184 Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His   1 5 10 15 Thr Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu              20 25 30 Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg          35 40 45 Arg Ser Phe Ile Glu Glu Lys Lys Met Pro      50 55 <210> 185 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 185 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser          35 40 45 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro      50 55 <210> 186 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 186 Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile   1 5 10 15 Asn Asn Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser          35 40 45 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro      50 55 <210> 187 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 187 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu          35 40 45 Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg      50 55 60 Arg Ser Phe Ile Glu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser  65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu                  85 90 95 <210> 188 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 188 Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu   1 5 10 15 Gln Ile Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser          35 40 45 Arg Arg Ser Phe Ile Glu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly      50 55 60 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu  65 70 75 80 Leu     <210> 189 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 189 Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His   1 5 10 15 Thr Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu              20 25 30 Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg          35 40 45 Arg Ser Phe Ile Glu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser      50 55 60 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu  65 70 75 80 <210> 190 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 190 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser          35 40 45 Arg Arg Ser Phe Ile Glu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly      50 55 60 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu  65 70 75 80 Leu     <210> 191 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 191 Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile   1 5 10 15 Asn Asn Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser          35 40 45 Arg Arg Ser Phe Ile Glu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly      50 55 60 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu  65 70 75 80 Leu     <210> 192 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 192 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu          35 40 45 Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Gly Gly Gly Ser Gly Gly      50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys  65 70 75 80 Asp Glu Leu             <210> 193 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 193 Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu   1 5 10 15 Gln Ile Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Gly Gly Gly Ser Gly          35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu      50 55 60 Lys Asp Glu Leu  65 <210> 194 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 194 Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His   1 5 10 15 Thr Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu              20 25 30 Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Gly Gly Gly Ser Gly Gly          35 40 45 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys      50 55 60 Asp Glu Leu  65 <210> 195 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 195 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Gly Gly Gly Ser Gly          35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu      50 55 60 Lys Asp Glu Leu  65 <210> 196 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 196 Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile   1 5 10 15 Asn Asn Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Gly Gly Gly Ser Gly          35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu      50 55 60 Lys Asp Glu Leu  65 <210> 197 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 197 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Ala Val Gly Ile Gly Ala Leu Phe              20 25 30 Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met          35 40 45 Thr Leu Thr Val Gln Ala Arg Gln Leu Gly Gly Gly Gly Ser Gly Gly      50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys  65 70 75 80 Asp Glu Leu             <210> 198 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 198 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Thr Thr Ala Val Pro Trp Asn Ala              20 25 30 Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Gly Gly Gly Gly Ser Gly          35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu      50 55 60 Lys Asp Glu Leu  65 <210> 199 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 199 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Trp Asn Ala Ser Trp Ser Asn Lys              20 25 30 Ser Leu Glu Gln Ile Trp Asn His Thr Gly Gly Gly Gly Ser Gly Gly          35 40 45 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys      50 55 60 Asp Glu Leu  65 <210> 200 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 200 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Ser Asn Lys Ser Leu Glu Gln Ile              20 25 30 Trp Asn His Thr Thr Trp Met Glu Trp Asp Gly Gly Gly Gly Ser Gly          35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu      50 55 60 Lys Asp Glu Leu  65 <210> 201 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 201 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu   1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Glu Gln Ile Trp Asn His Thr Thr              20 25 30 Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Gly Gly Gly Gly Ser Gly          35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu      50 55 60 Lys Asp Glu Leu  65 <210> 202 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 202 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser          35 40 45 Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu Ile Tyr Ile Trp Ala      50 55 60 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr  65 70 75 80 Leu Tyr Cys             <210> 203 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 203 Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu   1 5 10 15 Gln Ile Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly              20 25 30 Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu Ile Tyr Ile          35 40 45 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val      50 55 60 Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile  65 70 75 80 Glu Glu         <210> 204 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 204 Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His   1 5 10 15 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser              20 25 30 Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu Ile Tyr Ile Trp Ala          35 40 45 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr      50 55 60 Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu  65 70 75 80 <210> 205 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 205 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly              20 25 30 Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu Ile Tyr Ile Trp          35 40 45 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile      50 55 60 Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu  65 70 75 80 Glu     <210> 206 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 206 Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile   1 5 10 15 Asn Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly              20 25 30 Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu Ile Tyr Ile Trp          35 40 45 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile      50 55 60 Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu  65 70 75 80 Glu     <210> 207 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 207 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu              20 25 30 Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val          35 40 45 Gln Ala Arg Gln Leu Lys Asp Glu Leu      50 55 <210> 208 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 208 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn              20 25 30 Lys Ser Leu Glu Gln Ile Lys Asp Glu Leu          35 40 <210> 209 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 209 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln              20 25 30 Ile Trp Asn His Thr Lys Asp Glu Leu          35 40 <210> 210 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 210 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr              20 25 30 Thr Trp Met Glu Trp Lys Asp Glu Leu          35 40 <210> 211 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 211 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp              20 25 30 Asp Arg Glu Ile Asn Asn Lys Asp Glu Leu          35 40 <210> 212 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 212 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Lys Asp Glu Leu Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu          35 40 45 Ala Leu Leu Leu His Ala Ala Arg Pro      50 55 <210> 213 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 213 Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu   1 5 10 15 Gln Ile Lys Asp Glu Leu Ala Leu Pro Val Thr Ala Leu Leu Leu Pro              20 25 30 Leu Ala Leu Leu Leu His Ala Ala Arg          35 40 <210> 214 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 214 Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His   1 5 10 15 Thr Lys Asp Glu Leu Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu              20 25 30 Ala Leu Leu Leu His Ala Ala Arg          35 40 <210> 215 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 215 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Lys Asp Glu Leu Ala Leu Pro Val Thr Ala Leu Leu Leu Pro              20 25 30 Leu Ala Leu Leu Leu His Ala Ala Arg          35 40 <210> 216 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 216 Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile   1 5 10 15 Asn Asn Lys Asp Glu Leu Ala Leu Pro Val Thr Ala Leu Leu Leu Pro              20 25 30 Leu Ala Leu Leu Leu His Ala Ala Arg          35 40 <210> 217 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 217 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu              20 25 30 Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val          35 40 45 Gln Ala Arg Gln Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly      50 55 60 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu  65 70 75 <210> 218 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 218 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn              20 25 30 Lys Ser Leu Glu Gln Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser          35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu      50 55 60 <210> 219 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 219 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln              20 25 30 Ile Trp Asn His Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly          35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu      50 55 60 <210> 220 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 220 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr              20 25 30 Thr Trp Met Glu Trp Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser          35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu      50 55 60 <210> 221 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 221 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp              20 25 30 Asp Arg Glu Ile Asn Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser          35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu      50 55 60 <210> 222 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 222 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu              20 25 30 Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val          35 40 45 Gln Ala Arg Gln Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys      50 55 60 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys  65 70 75 80 Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro                  85 90 <210> 223 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 223 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn              20 25 30 Lys Ser Leu Glu Gln Ile Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr          35 40 45 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr      50 55 60 Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro  65 70 75 <210> 224 <211> 78 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 224 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln              20 25 30 Ile Trp Asn His Thr Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys          35 40 45 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys      50 55 60 Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro  65 70 75 <210> 225 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 225 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn              20 25 30 Lys Ser Leu Glu Gln Ile Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr          35 40 45 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr      50 55 60 Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro  65 70 75 <210> 226 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 226 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu              20 25 30 Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val          35 40 45 Gln Ala Arg Gln Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys      50 55 60 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys  65 70 75 80 Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Gly Gly Gly Gly Ser Gly                  85 90 95 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu             100 105 110 Lys Asp Glu Leu         115 <210> 227 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 227 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn              20 25 30 Lys Ser Leu Glu Gln Ile Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr          35 40 45 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Gly      50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly  65 70 75 80 Gly Ser Ala Glu Lys Asp Glu Leu                  85 <210> 228 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 228 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln              20 25 30 Ile Trp Asn His Thr Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys          35 40 45 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Gly Gly      50 55 60 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly  65 70 75 80 Ser Ala Glu Lys Asp Glu Leu                  85 <210> 229 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 229 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp              20 25 30 Asp Arg Glu Ile Asn Asn Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr          35 40 45 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Gly      50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly  65 70 75 80 Gly Ser Ala Glu Lys Asp Glu Leu                  85 <210> 230 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 230 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly              20 25 30 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Ala Val Gly Ile          35 40 45 Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly      50 55 60 Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu Gly Gly Gly  65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly                  85 90 95 Ser Ala Glu Lys Asp Glu Leu             100 <210> 231 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 231 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly              20 25 30 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Thr Thr Ala Val          35 40 45 Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Gly Gly      50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly  65 70 75 80 Gly Ser Ala Glu Lys Asp Glu Leu                  85 <210> 232 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 232 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly              20 25 30 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Trp Asn Ala Ser          35 40 45 Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Gly Gly Gly      50 55 60 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly  65 70 75 80 Ser Ala Glu Lys Asp Glu Leu                  85 <210> 233 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 233 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly              20 25 30 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Ser Asn Lys Ser          35 40 45 Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp Asp Gly Gly      50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly  65 70 75 80 Gly Ser Ala Glu Lys Asp Glu Leu                  85 <210> 234 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 234 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly              20 25 30 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Glu Gln Ile Trp          35 40 45 Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Gly Gly      50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly  65 70 75 80 Gly Ser Ala Glu Lys Asp Glu Leu                  85 <210> 235 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 235 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu              20 25 30 Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val          35 40 45 Gln Ala Arg Gln Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly      50 55 60 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu Ile  65 70 75 80 Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser                  85 90 95 Leu Val Ile Thr Leu Tyr Cys             100 <210> 236 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 236 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn              20 25 30 Lys Ser Leu Glu Gln Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser          35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu      50 55 60 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu  65 70 75 80 Ser Leu Val Ile Thr Leu Tyr Cys                  85 <210> 237 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 237 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln              20 25 30 Ile Trp Asn His Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly          35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu Ile      50 55 60 Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser  65 70 75 80 Leu Val Ile Thr Leu Tyr Cys                  85 <210> 238 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 238 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr              20 25 30 Thr Trp Met Glu Trp Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser          35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu      50 55 60 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu  65 70 75 80 Ser Leu Val Ile Thr Leu Tyr Cys                  85 <210> 239 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 239 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu Trp              20 25 30 Asp Arg Glu Ile Asn Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser          35 40 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu      50 55 60 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu  65 70 75 80 Ser Leu Val Ile Thr Leu Tyr Cys                  85 <210> 240 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 240 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly              20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu          35 40 45 <210> 241 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 241 Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr Thr Trp Met Glu   1 5 10 15 Trp Asp Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg Arg Ser Phe Ile              20 25 30 Glu Glu Lys Lys Met Pro          35 <210> 242 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 242 Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe   1 5 10 15 Ala Val Leu Ser Ile Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser Arg Arg              20 25 30 Ser Phe Ile Glu Glu Lys Lys Met Pro          35 40 <210> 243 <211> 60 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 243 cuuuuuauua ugauuguugg uggucuuguu ggucuucgua uuguuuuugc uguucuuucu 60                                                                           60 <210> 244 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 244 aaagaugaau ua 12 <210> 245 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 245 aaaaagaugc cu 12 <210> 246 <211> 117 <212> RNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 246 ucucaugguu uuccuccuga aguugaagaa caagaugaug guacuuuacc uaugucuugu 60 gcucaagaau cugguaugga uagacauccu gcugcuuguu ccgcuagaau uaauguu 117 <210> 247 <211> 81 <212> RNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 247 auuuauauuu gggcuccuuu agcuccuuua gcugguacuu gugguguuuu auuauuaagu 60 uuaguuauua cuuuauauug u 81 <210> 248 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 248 Lys Lys Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe   1 5 10 15 Asn Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly              20 25 30 Ser Gly Gly Gly Gly Ser Ala Glu Lys Asp Glu Leu          35 40 <210> 249 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 249 Lys Lys Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe   1 5 10 15 Asn Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu              20 25 30 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr Lys Tyr Lys Ser          35 40 45 Arg Arg Ser Phe Ile Glu Glu Lys Lys Met Pro      50 55 <210> 250 <211> 78 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 250 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His   1 5 10 15 Ala Ala Arg Pro Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn His Thr              20 25 30 Thr Trp Met Glu Trp Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr          35 40 45 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Leu Tyr      50 55 60 Lys Tyr Lys Ser Arg Arg Ser Phe Ile Glu Glu Lys Lys Met  65 70 75

Claims (25)

A binding domain derived from gp41 that has the ability to bind to at least one of the subunits of the immune activating receptor; And
Comprising any one or more of vesicle residual domains, Golgi apparatus residual domains, and proteasome residual domains,
Trapper peptide.
According to claim 1,
The gp41-derived binding domain is a FP, NHR, Loop, CHR and TMD, and a trapper peptide, characterized in that any one of these sequences.
According to claim 1,
The gp41-derived binding domain is a trapper peptide comprising at least one amino acid sequence selected from SEQ ID NOs: 20, 27 and 37.
According to claim 1,
The endoplasmic reticulum and Golgi apparatus residual domains include any one or more of KDEL, KKXX, KXD / E, YQRL and KX, wherein X is any amino acid.
The method of claim 4,
The endoplasmic reticulum and Golgi apparatus domain containing the KKXX has a amino acid sequence of SEQ ID NO: 43 or 47, a trapper peptide.
According to claim 1,
The trapper peptide further comprises any one or more of a connecting domain, a CD8α transmembrane domain and a CD8α signal sequence.
The method of claim 6,
The linking domain is (GGGGS) n, wherein n is an arbitrary integer, a trapper peptide.
The method of claim 6,
The amino acid sequence of the CD8α transmembrane domain has a trapper peptide having the amino acid sequence of SEQ ID NO: 111.
The method of claim 6,
The CD8α signal sequence is a trapper peptide, characterized in that it has the amino acid sequence of SEQ ID NO: 141.
According to claim 1,
The trapper peptide is characterized by having a binding ability to any one or more of TCR-alpha, TCR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD4, CD8-beta and CD8-alpha Trapper peptide.
According to claim 1,
A trapper peptide comprising the amino acid sequence of any one of SEQ ID NOs: 175, 178, 181, 182, 185, 220, 240, 248, 249, 250.
It comprises the trapper peptide according to any one of claims 1 to 11,
Characterized in that the expression trait of the immune activating receptor is different from that of the wild state cell.
Engineered immune cells.
The method of claim 12,
The engineered immune cell, characterized in that the expression trait of the immune-activated receptor differs from the wild-state cell is that the amount of the cell surface of at least one of the sub-units of the immune-activated receptor is reduced.
The method of claim 12,
The expression trait of the immune-activated receptor differs from the wild-state cell, wherein the engineered immune cell is characterized in that the immune-activated receptor is trapped within the cell.
The method of claim 12,
The engineered immune cell is an engineered immune cell, characterized in that at least one selected from the group consisting of T cells, NK cells and NKT cells.
A vector comprising a nucleic acid encoding a trapper peptide according to any one of claims 1 to 11.
The method of claim 16,
The vector is a viral vector.
The method of claim 17,
The viral vector is a vector derived from any one or more selected from the group consisting of adenovirus, adeno-associated virus, vaccinia virus, pox virus, herpes virus, retrovirus and herpes simplex virus.
A method comprising introducing the trapper polypeptide according to any one of claims 1 to 11 or a nucleic acid encoding the same into an immune cell.
Methods of making engineered immune cells.
The method of claim 19,
A method for producing an engineered immune cell, further comprising isolating a cell from which a different trait is derived from a wild-state cell.
The method of claim 19,
The introducing step is an electroporation (electroporation), liposomes, plasmids, viral vectors, nanoparticles (nanoparticles) and PTD (Protein translocation domain) fusion protein method characterized in that it is carried out by at least one method selected from the method of producing immune cells Way.
The method of claim 21,
The viral vector is an adenovirus, adeno-associated virus, vaccinia virus, pox virus, herpes virus, retrovirus and herpes simplex virus.
A method for treating cancer comprising injecting a composition comprising the engineered immune cell according to any one of claims 12 to 15 into a subject.
The method of claim 23,
The step of administering the composition to a treatment target is a method of treating cancer, characterized in that it is systemic administration.
The method of claim 24,
The systemic administration is a method of treating cancer, characterized by intravenous administration.

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