KR102666554B1 - Receptor Tyrosine-protein Kinase ErbB3-Specific Chimeric Antigen Receptor and Immune Cell Expressing the Same - Google Patents

Abstract

The present invention relates to ErbB3 (Receptor Tyrosine-protein Kinase ErbB3) specific chimeric antigen receptor, immune cells containing the same, and uses thereof. The chimeric antigen receptor according to the present invention exhibits excellent affinity and binding ability to ErbB3, and immune cells expressing the chimeric antigen receptor have excellent cytotoxicity against cancer cells expressing ErbB3, making them useful for immune cell therapy for cancer treatment. It can be useful.

Description

ErbB3-specific chimeric antigen receptor and immune cell therapeutic agent expressing the same {Receptor Tyrosine-protein Kinase ErbB3-Specific Chimeric Antigen Receptor and Immune Cell Expressing the Same}

The present invention relates to ErbB3 (Receptor Tyrosine-protein Kinase ErbB3) specific chimeric antigen receptor, immune cells containing the same, and uses thereof. The chimeric antigen receptor according to the present invention exhibits excellent affinity and binding ability to ErbB3, and immune cells expressing the chimeric antigen receptor have excellent cytotoxicity against cancer cells expressing ErbB3, making them useful for immune cell therapy for cancer treatment. It can be useful.

T cells are cells that play an important role in mediating adaptive immunity. T cells are activated through stimulation with receptors that can recognize antigens (T cell receptor, TCR), co-stimulatory molecules, and cytokines. In addition, the TCR of T cells causes an immune response through MHC molecules (major histocompatibility complex, MHC molecules) bound to antigens, and cancer cells suppress the expression of MHC molecules as a mechanism to avoid the immune response. Adoptive immune therapy (adoptive cellular therapy) is being developed using these characteristics of T cells.

The development of anticancer treatments using immune cells has been developed with a focus on T cells, and as in vitro culture and proliferation of tumor antigen-specific T cells became possible, anticancer T cell treatments have shown visible results (Gattinoni L, et al. , Nat Rev Immunol. 2006;6(5):383-93). However, since the number of tumor antigen-specific T cells present in the patient's body is very small, it has the disadvantage of requiring a long period of more than a month to secure sufficient T cells by proliferating these T cells in vitro.

Accordingly, based on recombinant antibody production technology accumulated in the field of therapeutic antibodies, a chimeric antigen receptor (chimeric antigen receptor) connects a recombinant antibody that recognizes tumor antigens expressed on the surface of cancer cells and a signaling domain that induces T cell activation. A technology was developed to secure large amounts of tumor-specific T cells in a short period of time by introducing the CAR) gene into T cells, and these T cells were named CAR-T cells (Kershaw MH, et al., Nat Rev Immunol. 2005 ;5(12):928-40; Restifo NP, et al., Nat Rev Immunol. 2012;12(4):269-81).

The CAR protein is designed in such a way that the variable region (single chain variable fragment; scFv) of an antibody that recognizes a cancer antigen is connected to an intracellular signaling domain through a spacer region (extracellular hinge + transmembrane domain). (Dotti G, et al., Immunol Rev. 2014;257(1):107-26). The intracellular signaling domain is mainly based on the intracellular signaling domain of CD3 zeta chain, a signaling subunit of the T cell receptor (first generation CAR), plus co-stimulatory molecules that promote T cell growth and differentiation. It has evolved to add an intracellular signaling domain. For example, the two CAR-T cell therapies currently on the market use the intracellular signaling domains of CD28 and 4-1BB costimulatory molecules, respectively (second-generation CARs), followed by the intracellular signaling of CD28 and 4-1BB. CARs (3rd generation CARs) containing both signaling domains are being attempted (van der Stegen SJ, etal., Nat Rev Drug Discov. 2015;14(7):499-509).

Meanwhile, ErbB3 is a member of the ErbB/HER family of receptor tyrosine kinases (RTKs). Other members of this group include EGFR (also known as ErbB1 or HER1), ErbB2 (also known as HER2 or HER2/Neu), and ErbB4 (also known as HER4). ErbB receptors regulate cell proliferation, survival and differentiation by activating intracellular signaling cascades that induce changes in gene expression. ErbB3 has been found to be overexpressed in various cancer types, including breast, gastrointestinal, and pancreatic cancer.

Under this technical background, the present inventors developed a chimeric antigen receptor containing an scFv that specifically targets cancer cells-ErbB3, confirmed that immune cells expressing it exhibit excellent anti-cancer effects, and completed the present invention.

The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that constitutes prior art already known to those skilled in the art to which the present invention pertains. It may not be possible.

The purpose of the present invention is to provide a new chimeric antigen receptor and immune cells containing the same that exhibit high anticancer effects against cancer expressing ErbB3.

Another object of the present invention is to provide a nucleic acid encoding the chimeric antigen receptor, an expression vector containing the nucleic acid, and a virus containing the expression vector.

Another object of the present invention is to provide a composition for treating cancer containing the immune cells, a method for treating cancer using the immune cells, the use of the immune cells for treating cancer, and the use of the immune cells for manufacturing a drug for treating cancer. It is to provide.

In order to achieve the above object, the present invention provides an extracellular binding domain comprising an antigen binding site that specifically binds to ErbB3 (Receptor Tyrosine-protein Kinase ErbB3); transmembrane domain; and a chimeric antigen receptor (CAR) comprising an intracellular signaling domain.

The present invention also provides a nucleic acid encoding the chimeric antigen receptor, an expression vector containing the nucleic acid, a virus containing the expression vector, and an immune cell expressing the chimeric antigen receptor.

The present invention also provides a composition for treating cancer containing the immune cells, a method for treating cancer using the immune cells, the use of the immune cells for treating cancer, and the use of the immune cells for manufacturing a drug for treating cancer. .

The chimeric antigen receptor according to the present invention exhibits excellent affinity and binding ability to ErbB3, and immune cells expressing the chimeric antigen receptor have excellent cytotoxicity against cancer cells expressing ErbB3, making them useful for immune cell therapy for cancer treatment. It can be useful.

Figure 1 shows the results of constructing a CAR construct using five ErbB3 ScFv candidates (442S1, 451M3, 472S2, 446, and 464) that specifically bind to ErbB3 (HER3) as the ectodomain of CAR, respectively. It represents.
Figure 2 shows the results of establishing an NK92 cell line expressing ErbB3-CAR.
Figure 3a shows the results of selecting cancer cell lines expressing ErbB3.
Figure 3b shows the results of confirming the cytotoxicity of each ErbB3-CAR NK92 against ErbB3-expressing cancer cells.
Figure 4 shows the results of confirming the cytotoxicity of ErbB3-CAR NK92 according to the ratio of CAR NK92 and cancer cells (E:T ratio).
Figure 5a shows the results of confirming the amount of IFN-r secretion in CAR NK92 cells co-cultured with cancer cells expressing ErbB3.
Figure 5b shows the results of confirming degranulation in CAR NK92 cells co-cultured with cancer cells expressing ErbB3.
Figure 6 shows the results confirming the efficacy of CAR NK92 at the single cell level.
Figure 7a shows the results of confirming CAR expression in each of ErbB3-CAR NK92.
Figure 7b shows the results of selecting cancer cell lines that satisfy all targets of each CAR NK92.
Figure 7c shows the results of comparing the cytotoxicity of each CAR NK92 cell.
Figure 8a shows the results confirming the binding of each ErbB3 ScFv to cancer cells.
Figure 8b shows the results of confirming the NK92 cell line expressing CD16.
Figure 8c shows the results of measuring ADCC using ErbB3 ScFv.
Figure 9a shows the results of manufacturing the ErbB3-CAR construct.
Figure 9B shows NK92 cell lines expressing ErbB3-CAR (442S1 vs. LJM716).
Figure 9c shows the results of comparing the cytotoxicity of ErbB3-CAR NK92 (442S1 vs. LJM716).

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

According to one embodiment of the present invention, five types of ErbB3 ScFv (442S1, 451M3, 472S2, 446, 464) that specifically bind to ErbB3 expressed in cancer cells are each synthesized into the ectodomain of the chimeric antigen receptor (CAR). A CAR structure was produced using . In addition, the external domain was engineered to express myc and hinge to confirm that CAR is expressed on the cell surface through myc, and the internal domain is a signaling domain with CD3-zeta and a costimulatory molecule. A third generation CAR was produced by adding CD28 and DAP10.

Accordingly, in one aspect, the present invention provides an extracellular domain comprising an antigen binding site that specifically binds to ErbB3; transmembrane domain; and a chimeric antigen receptor (CAR) containing an intracellular signaling domain.

'Chimeric antigen receptor (CAR)' is a synthetic construct designed to induce an immune response against a target antigen and cells expressing the antigen. CARs include an extracellular domain, a transmembrane domain, and an intracellular signaling domain. Cancer cells can be killed by introducing a gene encoding a receptor that recognizes a cancer cell surface antigen specifically expressed on the surface of cancer cells into immune cells. Through immune cells that contain receptors that bind to antigens specifically expressed in cancer cells, an immune response can be generated by targeting only cancer cells.

The first generation CAR contains an extracellular domain including an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and only CD3ζ was used as the signaling domain, but the therapeutic effect on cancer was minimal. There was a problem that the duration was short.

To improve responsiveness to immune cells, a second-generation CAR was manufactured combining a costimulatory domain (CD28 or CD137/4-1BB) and CD3ζ. Compared to the first-generation CAR, the number of CAR-containing immune cells remaining in the body was significantly increased. . While the second-generation CAR used one co-stimulatory domain, the third-generation CAR used two or more co-stimulatory domains. To achieve expansion and persistence of CAR-containing immune cells in vivo, the costimulatory domain can be combined with 4-1BB, CD28, or OX40.

The 4th generation CAR includes additional genes encoding cytokines such as IL-12 or IL-15, allowing additional expression of CAR-based immune proteins of the cytokine, while the 5th generation CAR contains interleukin to strengthen immune cells. It further comprises a receptor chain, such as IL-2Rβ.

The “extracellular binding domain” refers to a portion of CAR containing an antigen binding domain that has the ability to specifically bind to a target antigen of interest. An extracellular binding domain is any protein that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or component thereof), It may include polypeptides, oligopeptides or peptides. Binding domains include any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner for the biological molecule of interest.

The term "specifically binding" as used herein refers to the binding of a molecule to another molecule with a binding affinity greater than background binding. For example, if an extracellular binding domain binds or associates with a target molecule with an affinity or Ka (i.e., the equilibrium dissociation constant for a particular binding interaction with units of 1/M) of about 10 ^-5 M or greater, then the target Binds specifically to molecules. Alternatively, affinity may be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (eg, 10 ^-5 M to 10 ^-13 M or less).

The affinity of the extracellular binding domains and CARs according to the invention can be determined using conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay) or labeled ligands, or using a Biacore T100 (Peace, NJ). surface-plasmon resonance devices, such as those available from Cataway Biacore, Inc.) or combinations using optical biosensor technology, such as the EPIC system or EnSpire, available from Corning and Perkin Elmer, respectively. It can be easily measured by association or substitution analysis.

In the present invention, the binding domain that specifically binds to ErbB3 may be an anti-ErbB3 antibody or an antigen binding fragment thereof.

Preferably, the binding domain that specifically binds to ErbB3 is a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25 and 33,

Heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34,

Heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27, and 35; and

A light chain CDR1 selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28 and 36,

A light chain CDR2 selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, and 37, and

It may be a single chain variable fragment (scFv) of an anti-ErbB3 antibody comprising a light chain CDR3 selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, and 38.

In the present invention, the antibody or antigen-binding fragment thereof that binds to ErbB3 includes a heavy chain CDR1 containing the sequence of SEQ ID NO: 1, a heavy chain CDR2 containing the sequence of SEQ ID NO: 2, a heavy chain CDR3 containing the sequence of SEQ ID NO: 3, Light chain CDR1 comprising the sequence of SEQ ID NO: 4, light chain CDR2 comprising the sequence of SEQ ID NO: 5, light chain CDR3 comprising the sequence of SEQ ID NO: 6;

Heavy chain CDR1 containing the sequence of SEQ ID NO: 9, heavy chain CDR2 containing the sequence of SEQ ID NO: 10, heavy chain CDR3 containing the sequence of SEQ ID NO: 11, light chain CDR1 containing the sequence of SEQ ID NO: 12, sequence of SEQ ID NO: 13 Light chain CDR2 comprising, light chain CDR3 comprising the sequence of SEQ ID NO: 14;

Heavy chain CDR1 containing the sequence of SEQ ID NO: 17, heavy chain CDR2 containing the sequence of SEQ ID NO: 18, heavy chain CDR3 containing the sequence of SEQ ID NO: 19, light chain CDR1 containing the sequence of SEQ ID NO: 20, sequence of SEQ ID NO: 21 Light chain CDR2 comprising, light chain CDR3 comprising the sequence of SEQ ID NO: 22;

Heavy chain CDR1 containing the sequence of SEQ ID NO: 25, heavy chain CDR2 containing the sequence of SEQ ID NO: 26, heavy chain CDR3 containing the sequence of SEQ ID NO: 27, light chain CDR1 containing the sequence of SEQ ID NO: 28, sequence of SEQ ID NO: 29 Light chain CDR2 comprising, light chain CDR3 comprising the sequence of SEQ ID NO: 30; or

Heavy chain CDR1 containing the sequence of SEQ ID NO: 33, heavy chain CDR2 containing the sequence of SEQ ID NO: 34, heavy chain CDR3 containing the sequence of SEQ ID NO: 35, light chain CDR1 containing the sequence of SEQ ID NO: 36, sequence of SEQ ID NO: 37 A light chain CDR2 comprising the sequence, and a light chain CDR3 comprising the sequence of SEQ ID NO: 38.

The “antibody” refers to an anti-ErbB3 antibody that specifically binds to ErbB3. The scope of the present invention includes not only complete antibody forms that specifically bind to ErbB3, but also antigen-binding fragments of this antibody molecule.

As used herein, “antibody variable domain” refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3), and the framework region (FR) . VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

“Complementarity determining regions” (CDRs; i.e., CDR1, CDR2, and CDR3) refer to amino acid residues of an antibody variable domain that are required for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2 and CDR3.

A complete antibody has two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and is subclassed as gamma1 (γ1), gamma2 (γ2), and gamma3 (γ3). ), gamma4 (γ4), alpha1 (α1) and alpha2 (α2). The constant region of the light chain is of kappa (κ) and lambda (λ) types.

In the present invention, the “fragment” of an antibody refers to a fragment that possesses an antigen-binding function and is used to include scFv, Fab, F(ab') ₂ , and Fv fragments. Among antibody fragments, Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibody is produced when cysteine residues in the hinge region of Fab' form a disulfide bond. Fv is the smallest antibody fragment containing only the heavy chain variable region and light chain variable region. A double-chain Fv (two-chain Fv) connects the heavy chain variable region and the light chain variable region by a non-covalent bond, while a single-chain Fv (single-chain Fv, scFv) generally connects the heavy chain variable region and the light chain variable region through a peptide linker. It can be connected by a covalent bond or directly at the C-terminus to form a dimer-like structure, such as double-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.

“Single chain Fv” or “scFv” antibody fragments contain the VH and VL domains of an antibody, which are contained within a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.

In the present invention, in an scFv containing the heavy chain variable region (VH) and light chain variable region (VL) of an antibody, VH and VL may be connected through a linker. In one embodiment of the present invention, the binding domain that specifically binds to ErbB3 is an anti-ErbB3 antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, and 39. It may be a single chain variable fragment (scFv). The binding domain that specifically binds to ErbB3 is a single chain variable fragment of an anti-ErbB3 antibody comprising a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, and 40. fragment; scFv).

The antibody or antigen-binding fragment thereof that binds to ErbB3 includes a heavy chain variable region containing the sequence of SEQ ID NO: 7, a light chain variable region containing the sequence of SEQ ID NO: 8, a heavy chain variable region containing the sequence of SEQ ID NO: 15, SEQ ID NO: A light chain variable region containing the sequence of SEQ ID NO: 16, a heavy chain variable region containing the sequence of SEQ ID NO: 23, a light chain variable region containing the sequence of SEQ ID NO: 24, a heavy chain variable region containing the sequence of SEQ ID NO: 31, SEQ ID NO: 32 It may include a light chain variable region including the sequence, a heavy chain variable region including the sequence of SEQ ID NO: 39, or a light chain variable region including the sequence of SEQ ID NO: 40.

The antibody or antibody fragment of the present invention may include not only the sequence of the anti-ErbB3 antibody of the present invention described herein, but also its biological equivalent, to the extent that it can specifically recognize ErbB3. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutions shows that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.

Considering the mutations having the above-mentioned biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence shown in SEQ ID NO. The above-mentioned substantial identity is at least 90% when the sequence of the present invention and any other sequence are aligned to the maximum extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence showing homology, most preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, and at least 99% homology. Alignment methods for sequence comparison are known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLSAT can be accessed at www.ncbi.nlm.nih.gov/BLAST/. The sequence homology comparison method using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

On this basis, the antibody or antigen-binding fragment thereof of the present invention has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence or the entire sequence described in the specification. , may have 99% or more homology. Such homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence homology of a nucleic acid or protein of the invention.

The linker may be a peptide linker and may be about 10-25 aa long. For example, hydrophilic amino acids such as glycine and/or serine may be included. The linker may comprise, for example, (GS) _n , (GGS) _n , (GSGGS) _n or (G _n S) _m (n, m are each 1 to 10), for example (G _n S) _m (n and m may be 1 to 10, respectively), but are not limited thereto.

The chimeric antigen receptor may be characterized as comprising a transmembrane domain.

The “transmembrane domain” is the part of the CAR that fuses the extracellular binding portion and the intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain may be derived from natural, synthetic, semisynthetic, or recombinant sources. In the present invention, the transmembrane domain is the alpha (α), beta (β) or zeta (ζ) chain of T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, It may be characterized as being selected from the group consisting of CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, but is not limited thereto.

The transmembrane domain may be attached to the extracellular binding domain of CAR through a linker. For example, the linker may be a short oligo- or polypeptide linker of 2 to 10 amino acids in length, preferably a glycine (G)-serine (S) doublet, but is not limited thereto. .

The binding domain of a CAR is generally followed by one or more “hinge domains”. Therefore, in the present invention, the extracellular domain may be characterized as including a binding domain that specifically binds to ErbB3 and additionally a signal peptide (SP) and/or a hinge. The extracellular domain is the site where the main signal is transmitted, exists outside the cell membrane, and is a domain for specifically recognizing the target, that is, ErbB3.

As used herein, the term "hinge domain" refers to an important role in positioning the extracellular binding domain containing the antigen binding site away from the actuating cell surface to enable appropriate cell/cell contact, antigen binding and activation. It is the part of CAR in charge. CARs generally contain one or more hinge domains between the extracellular binding domain and the transmembrane domain. The hinge domain may be derived from natural, synthetic, semi-synthetic or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. “Altered hinge region” refers to (a) a naturally occurring hinge region with up to 30% amino acid change (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), (b) up to 30% amino acid change; of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) in length with a 30% amino acid change (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions) a portion of a naturally occurring hinge region, or (c) a core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6, 7, may be 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long). In certain embodiments, one or more cysteine residues in a naturally occurring immunoglobulin hinge region may be replaced with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have the proline residue of the wild-type immunoglobulin hinge region replaced with another amino acid residue (e.g., a serine residue). The hinge domain includes a hinge region derived from the extracellular region of type 1 membrane proteins such as CD8, CD4, CD28, and CD7, which may be the wild-type hinge region from these molecules or may be varied.

In the present invention, any transmembrane domain including the hinge domain can be used as long as it can connect the extracellular domain and the intracellular signaling domain between the cell membrane. Preferably, it may consist of a hinge domain and a transmembrane domain derived from CD8, but is not limited thereto.

In some cases, the binding domain and the transmembrane domain may be connected by a spacer domain.

Preferably, the spacer domain may be a hinge domain. In the present invention, the spacer domain may include a hinge domain derived from CD28 and/or a hinge domain derived from CD8, and may include all or part of the hinge domain derived from CD28 and/or a hinge domain derived from CD8. You can.

The hinge region or spacer region of the present invention may be one or more selected from the Myc epitope, CD8 hinge region, and Fc, and may preferably include the Myc epitope and the CD8 hinge region. The Myc epitope and CD8 hinge region of the present invention function as a linking domain (spacer).

The CD8 hinge region of the present invention may include the sequence GVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD (SEQ ID NO: 41). The Myc epitope may include the sequence of ANKNSSQKRI (SEQ ID NO: 42).

In the present invention, the chimeric antigen receptor may be characterized as comprising an intracellular signaling domain.

In the present invention, the intracellular signaling domain is a part located inside the cell membrane of an immune cell, that is, in the cytoplasm, and when the binding domain included in the extracellular domain binds to the target antigen, it transmits a signal within the cell to activate the immune cell. This refers to the area that activates the immune response.

In the present invention, the intracellular signaling domain may be characterized as comprising an intracellular signaling domain and/or a co-stimulatory signaling domain.

The signaling domain can induce activation of the normal effector functions of the immune cell in which the CAR is placed. For example, cytolytic activation or helper activation can be induced through the secretion of cytokines. The signaling domain may comprise a truncated fragment of the intracellular signaling domain sufficient to transduce an effector function signal.

The intracellular signaling domains include T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), and CD3 A primary signaling domain selected from the group consisting of zeta (ζ), CD5, CD22, CD79a, CD79b and CD66d; and/or CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a/CD18), GITR, MyD88 , DAP10, DAP12, PD-1, LIGHT, NKG2C, B7-H3 and CD83, and characterized in that it contains a co-stimulatory signaling domain selected from the group consisting of a ligand that specifically binds to You can.

As used herein, the term "intracellular signaling domain" refers to effector cell functions, such as activation, cytokine production, proliferation, and release of cytotoxic factors against CAR-bound target cells. refers to the portion of a CAR that is involved in the transmission of the message of effective CAR binding to a target antigen into the interior of an immune effector cell to induce cytotoxic activity, or other cellular response triggered by antigen binding to the extracellular binding domain of the CAR It is done. Effector function refers to a specific function of a cell, for example, the effector function of an immune cell may be cytolytic, may support activities including secretion of cytokines, or may be active. Accordingly, an intracellular signaling domain refers to a portion of a protein that transmits effector signals and directs the cell to perform a specific function.

Immune cell activation is mediated by two different classes of intracellular signaling domains. For example, immune cell activation is mediated by primary signaling domains that initiate antigen-dependent primary activation and costimulatory signaling domains that act in an antigen-independent manner to provide secondary signals. Accordingly, the intracellular signaling domain may be characterized as comprising a “primary signaling domain” and a “co-stimulatory signaling domain.”

As used herein, the term “primary signaling domain” refers to a signaling domain that regulates immune cell activation in a stimulatory or inhibitory manner. The primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as the immunoreceptor tyrosine-based activation motif, or ITAM. The ITAM containing the primary signaling domain may be selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b and CD66d, but is not limited thereto.

In the present invention, the primary signaling domain may preferably be CD3ζ(zeta) containing the amino acid sequence represented by SEQ ID NO: 9, but is not limited thereto. CD3ζ(zeta) may include the sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 43).

As used herein, the term “co-stimulatory signaling domain” refers to the intracellular signaling domain of a co-stimulatory molecule. Costimulatory signaling domain refers to the portion of CAR that contains the intracellular signaling domain of the costimulatory molecule. CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a/CD18), GITR, MyD88, DAP10, It may include, but is not limited to, a co-stimulatory signaling domain selected from the group consisting of a ligand that specifically binds to DAP12, PD-1, LIGHT, NKG2C, B7-H3, and CD83. no.

In the present invention, the co-stimulatory molecule may be DAP10, but is not limited thereto. DAP10 may comprise the sequence LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 44).

The chimeric antigen receptor of the present invention can exhibit a killing effect on cancer cells through high activity of NK cells by using DAP10 and CD3 zeta as intracellular signaling domains.

The chimeric antigen receptor according to the present invention may be characterized as comprising two or more intracellular signaling domains, and when it contains two or more intracellular signaling domains, the intracellular signaling domains may be connected to each other in series. there is. Alternatively, they may be connected through an oligopeptide linker or polypeptide linker consisting of 2 to 10 amino acids, and examples of such linker sequences include glycine-serine continuous sequences. The linker may comprise, for example, (GS) _n , (GGS) _n , (GSGGS) _n or (G _n S) _m (n, m are each 1 to 10), for example (G _n S) _m (n and m may be 1 to 10, respectively), but are not limited thereto.

In the present invention, the intracellular signaling domain may be characterized as comprising at least one selected from the group consisting of the costimulatory signaling domain of CD28 or DAP10 and the intracellular signaling domain of CD3 zeta. It is not limited.

In the present invention, the chimeric antigen receptor may further include an immune function promoting factor for immune cells, and the immune function promoting factor for immune cells may be an interleukin signal sequence. The interleukin signal sequence may be characterized as inducing the expression of interleukin (IL)-12, IL-8, or IL-2, but is not limited thereto. In addition, examples of the immune function promoting factors of T cells among the immune cells include IL-7 or CCL19.

When designing CAR, a signal peptide may be additionally included in front of the scFv. The nucleic acid encoding the chimeric antigen receptor may further include a nucleic acid encoding a signal peptide.

The signal peptide may be, for example, GM-CSF, CD8α signal peptide, etc. It may be CD8 alpha or mouse light kappa signal peptid, and in the case of CD8 alpha, the signal peptide of the present invention may include the sequence of MALPVTALLLPLALLLHAARP (SEQ ID NO: 45).

In the present invention, the chimeric antigen receptor may further include an interleukin receptor chain including a JAK binding motif and a STAT 3/5 association motif, and examples include IL-2Rβ. It is not limited.

In another aspect, the present invention relates to a nucleic acid encoding the chimeric antigen receptor.

“Nucleic acid” is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic structural unit of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base sites. . The sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. The modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

The nucleic acid (polynucleotide) encoding the chimeric antigen receptor according to the present invention can be modified by codon optimization, which results from the degeneracy of codons, resulting in many nucleotide sequences encoding polypeptides or variant fragments thereof. That it exists will be well understood by those skilled in the art. Some of these polynucleotides (nucleic acids) possess minimal homology to the nucleotide sequence of any naturally occurring gene.

In particular, polynucleotides (nucleic acids) that are variable due to differences in codon usage, for example, polynucleotides (nucleic acids) optimized for codon selection in humans, primates and/or mammals, are preferred.

From another aspect, the present invention relates to an expression vector containing the above nucleic acid and a virus containing the expression vector.

The term “vector” in the present invention refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, for example, inserted into, a vector nucleic acid molecule. Vectors may contain sequences that direct autonomous replication in the cell, or may contain sequences sufficient to allow integration into host cell DNA. The vector may be selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, and retroviral vector, but is not limited thereto.

Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

In the vector, the nucleic acid encoding the antibody is operably linked to a promoter.

“Operably linked” means a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence is linked to the other nucleic acid. It regulates the transcription and/or translation of the sequence.

When using a prokaryotic cell as a host, a strong promoter capable of advancing transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. Additionally, for example, in the case of eukaryotic cells as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human heroglobin promoter, and human muscle creatine promoter) or mammalian cell genomes Promoters derived from animal viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV , the promoter of Moloney virus, the promoter of Epstein-Barr virus (EBV), and the promoter of Roux Sarcoma virus (RSV) can be used, and generally have a polyadenylation sequence as the transcription termination sequence.

In some cases, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

The vector contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. There is a resistance gene.

In the present invention, “virus” means something that has been genetically modified to express the chimeric antigen receptor of the present invention for use in the treatment of cancer, etc. To be genetically modified is to add foreign genetic material in the form of DNA or RNA to the total genetic material in the cell.

In the present invention, the nucleic acid or the vector is transfected or transfected into a virus. There are a variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells to “transfect” or “transfect,” including electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection can be used.

From another aspect, the present invention relates to immune cells expressing the chimeric antigen receptor on their surface.

In the present invention, the immune cells are capable of inducing immunity and causing the desired cancer treatment effect, for example, T cells, NK cells, NKT cells, and cytokine induced killer cells (CIK). , may be selected from the group consisting of macrophages and dendritic cells, but are not limited thereto. Preferably, it may be characterized as a T cell.

Therefore, the immune cells expressing the chimeric antigen receptor according to the present invention include CAR-T cells (Chimeric Antigen Receptor T Cell), CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cell), and CAR-NKT cells (Chimeric Antigen Receptor Natural killer). T Cell) or CAR-macrophage (Chimeric Antigen Receptor Macrophage). In the present invention, the T cells include cytotoxic T lymphocytes (CTL); It may be characterized as being selected from the group consisting of T cells isolated from tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC).

In the present invention, CAR-NK cells in particular refer to cells into which a chimeric antigen receptor has been introduced into NK (Natural killer) cells. Response initiation (problems caused by persistent toxicity of cancer immunotherapy using existing CAR-T treatments, risk of autoimmune disease, graft-versus-host disease (GVHD) problem with xenogeneic cell transplantation, and off-target toxicity problem) Not only can it be solved through an on/off switch, but it also has the advantage of being able to target various cancer cells and be used as a general-purpose treatment.

From another aspect, the present invention relates to a composition for treating cancer containing immune cells (eg, T cells or NK cells) expressing the chimeric antigen receptor. The T cells include CD4 positive T cells; CD8 positive cytotoxic T lymphocyte (CTL); gamma-delta T cells; It may be characterized as being selected from the group consisting of T cells isolated from tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC), but is not limited thereto.

In the present invention, “cancer” and “tumor” are used interchangeably and typically refer to or mean a physiological condition in mammals characterized by uncontrolled cell growth/proliferation.

Cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited and includes both solid cancer and hematological cancer. Examples of such cancer include, but are not limited to, breast cancer.

The therapeutic composition of the present invention is a composition for the prevention or treatment of cancer, and the term "prevention" of the present invention refers to any action that suppresses or delays the progression of cancer by administering the composition of the present invention, and "treatment" "means inhibiting the development of cancer, alleviating or eliminating symptoms.

The composition preferably contains 1 to 10 times the number of immune cells expressing the chimeric antigen receptor according to the present invention as the number of tumor cells in the treatment target, but is not limited thereto.

A pharmaceutical composition containing immune cells expressing a chimeric antigen receptor according to the present invention may additionally contain pharmaceutically acceptable excipients. Examples of such excipients include surfactants, preferably nonionic surfactants of the polysorbate series; Buffering agents such as neutral buffered saline and human salt buffered saline; Sugars or sugar alcohols such as glucose, mannose, sucrose, dextran, and mannitol; Amino acids, proteins, or polypeptides such as glycine and histidine; antioxidant; Chelating agents such as EDTA or glutathione; penetrant; supplements; and preservatives, but are not limited thereto.

According to one embodiment of the present invention, the pharmaceutical composition of the present invention contains a number of immune cells (e.g., T cells, or NK cells) within one dose that is 1 to 10 times the number of tumor cells in the treatment subject. It could be.

Compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal other than a human. Dosage forms may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, or sterile powders.

The pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

From another aspect, the present invention relates to a cancer treatment method comprising administering immune cells expressing the chimeric antigen receptor to a subject.

In another aspect, the present invention relates to the use of said immune cells for cancer treatment.

In another aspect, the present invention relates to the use of said immune cells for the manufacture of drugs for treating cancer.

The subject may be a mammal with a tumor, and may specifically be a human, but is not limited thereto.

Immune cells expressing chimeric antigen receptors or compositions containing them according to the present invention can be administered orally, infusion, intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneally. It may be administered by intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.

The dosage of the active ingredient can be appropriately selected depending on various factors such as the route of administration, the patient's age, gender, weight, and patient severity, and the therapeutic composition according to the present invention has the effect of preventing, improving, or treating cancer symptoms. It can be administered in parallel with known compounds having .

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Production of NK cells expressing CAR with ErbB3 (HER3) ScFv as the ectodomain

A CAR structure was constructed using five ErbB3 ScFv candidates (442S1, 451M3, 472S2, 446, 464) that specifically bind to ErbB3 (HER3) expressed in cancer cells as the ectodomain of CAR.

[Table 1]

To confirm that CAR is expressed on the cell surface, myc and hinge were expressed in the ectodomain, and CD28, DAP10, and CD3zeta, which are involved in killing ability, were used as the endodomain.

Specific sequence information corresponding to each domain is shown in Table 2 below.

[Table 2]

Each of the five CAR constructs was inserted into a lentiviral vector, a CAR-expressing lentivirus was created, and a CAR-expressing NK92 cell line was established using this (Figure 2).

Example 2. Confirmation of the efficacy of ErbB3-CAR NK92 on cancer cells expressing ErbB3

To confirm the cytotoxicity of ErbB3-CAR NK92 on cancer cells, cancer cell lines expressing ErbB3 were identified and selected using flow cytometry (Figure 3a), and each cancer cell line was identified using the Calcein-AM method. The cytotoxicity of ErbB3-CAR NK92 was compared. Compared to the control group (NK92 or PURO-NK92), the cytotoxicity of each CAR NK92 against cancer cells expressing ErbB3 was confirmed to be increased, and cancer cells not expressing ErbB3 were confirmed to show cytotoxicity similar to the control group (Figure 3b) ).

In addition, among the five types of ErbB3-CAR NK92 candidates, three types (442S1, 451M3, 472S2) with high cytotoxicity were selected to determine cytotoxicity according to the ratio of CAR NK92 to cancer cells (Effector cell: Target cell; E:T ratio). was reconfirmed. In cancer cells expressing ErbB3, all three types of CAR NK92 were confirmed to exhibit cytotoxicity according to the E:T ratio, and for cancer cells not expressing ErbB3, cytotoxicity was confirmed to be similar to that of the control group (Figure 4).

Furthermore, after co-culturing each ErbB3-CAR NK92 cell with cancer cells, the amount of secreted cytokines and the degree of degranulation were confirmed. As a result of confirming the amount of cytokine (IFN-r) using ELISA, it was confirmed that CAR NK92 cells co-cultured with cancer cells expressing ErbB3 secreted a greater amount of IFN-r compared to the control group (Figure 5a), and degranulation Additionally, as a result of checking the expression level of CD107a, it was confirmed that it was expressed more in the group co-cultured with cancer cells expressing ErbB3 (Figure 5b).

As a result of confirming the cytotoxicity of ErbB3-CAR NK92, the contact frequency with cancer cells, and the killing time of ErbB3-CAR NK92 at the single cell level, the control group (PURO-NK92, Ecto; Ectodomain deleted structure) While the frequency of contact with NK92) and cancer cells was similar, cytotoxicity was higher than that of the control group, and the time taken to kill cancer cells was also confirmed to be shorter compared to the control group (Figure 6).

Example 3. Comparison of efficacy of ErbB3-CAR NK92

To compare the efficacy of ErbB3-CAR NK92, cytotoxicity was compared with CAR NK92 cell lines with different ectodomains. Among the two types of CAR NK92 targeting ErbB2 (HER2) (Cot-CAR NK92; CAR of universal concept; uses a mediator targeting HER2, HER2-CAR NK92) and ErbB3-CAR NK92, 442S1 cells are the most effective. Toxicity was compared. The expression of CAR was confirmed in each CAR NK92 cell (Figure 7a), and cancer cells expressing all targets for each CAR NK92 were selected (Figure 7b). As a result of comparing the cytotoxicity of the corresponding cancer cells, it was confirmed that the cytotoxicity of ErbB3-CAR NK92 (442S1) was significantly higher than that of other CAR NK92s (Figure 7c).

In addition, ADCC (Antibody-dependent cellular cytotoxicity) was confirmed using a third-party ErbB3 ScFv (LJM716), which has the same target as the ErbB3 ScFv (442S1) used in the ectodomain of ErbB3-CAR NK92. Whether each ScFv binds to the corresponding cancer cells was confirmed using flow cytometry (Figure 8a), and ADCC was performed using NK92 (CD16+NK92) cells expressing CD16 (Figure 8b). As a result, it was confirmed that in cancer cells expressing ErbB3, ErbB3 ScFv (442S1) used in ErbB3-CAR NK92 showed higher ADCC compared to ErbB3 ScFv (LJM716) of another company (FIG. 8c).

Furthermore, a CAR was constructed using another company's ErbB3 ScFv (LJM716), an NK92 cell line expressing CAR was established, and its efficacy was compared with ErbB3-CAR NK92 (442S1). Except for the external domain (Ectodomain) of CAR, the remaining structures (Transmembrane and endodomain) were produced in the same structure (Figure 9a), and a NK92 cell line expressing CAR was established using lentivirus (Figure 9b). As a result of comparing the cytotoxicity of each ErbB3-CAR NK92, it was confirmed that 442S1-CAR NK92 cells showed higher cytotoxicity against cancer cells expressing the target (ErbB3) than LJM716-CAR NK92 cells. Regarding cancer cells, it was confirmed that the cytotoxicity of the two CAR NK92 cells was similar (Figure 9c).

ErbB3-CAR NK92 (442S1) shows specific anticancer efficacy against cancer cells expressing ErbB3, and was confirmed to have higher efficacy compared to the existing ErbB3 ScFv (LJM716).

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Research Institute Of Bioscience And Biotechnology Isu Abxis Co., Ltd <120> Receptor Tyrosine-protein Kinase ErbB3-Specific Chimeric Antigen Receptor and Immune Cell Expressing the Same <130> P21-B093 <160> 46 <170> PatentIn version 3.2 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 1 Asp Tyr Asp Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 2 Thr Ile Asp Leu Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val Gln 1 5 10 15 Gly <210> 3 <211> 12 <212 > PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 3 Asp Leu His Met Gly Pro Glu Gly Pro Phe Asp Tyr 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 4 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Ser Val Ser 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220 > <223> L-CDR2 <400> 5 Ser Asp Asn His Arg Pro Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 6 Gln Gly Trp Asp Thr Ser Leu Ser Gly His Val 1 5 10 <210> 7 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asp Leu Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Leu His Met Gly Pro Glu Gly Pro Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser 115 120 <210> 8 <211> 111 <212> PRT < 213> Artificial Sequence <220> <223> VL <400> 8 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Ser Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asp Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Gly Trp Asp Thr Ser Leu 85 90 95 Ser Gly His Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 < 210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 9 His Tyr Asp Met Ser 1 5 <210> 10 <211> 17 <212> PRT <213 > Artificial Sequence <220> <223> H-CDR2 <400> 10 Ala Ile Tyr Tyr Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Ala Lys 1 5 10 15 Gly <210> 11 <211> 12 <212> PRT < 213> Artificial Sequence <220> <223> H-CDR3 <400> 11 Asp Arg Leu Phe Ala Ser Asp Ser Thr Phe Asp Tyr 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence < 220> <223> L-CDR1 <400> 12 Ser Gly Ser Pro Ser Asn Ile Gly Asn Asn Ser Val Thr 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223 > L-CDR2 <400> 13 Tyr Asp Ser His Arg Pro Ser 1 5 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 14 Gly Ser Trp Asp Ala Ser Leu Asn Gly Tyr Val 1 5 10 <210> 15 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 15 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Tyr Tyr Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Phe Ala Ser Asp Ser Thr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser 115 120 <210> 16 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 16 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Pro Ser Asn Ile Gly Asn Asn 20 25 30 Ser Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ser Ala Ser Leu Ala Ile Gly Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ala Ser Leu 85 90 95 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 17 Trp Tyr Asp Leu Ala 1 5 <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 18 Gly Ile Ser Tyr Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 19 Asp Pro Ser Trp Cys Leu Gln Asp Leu Cys Tyr Tyr Ala Asp Gly Met 1 5 10 15 Asp Val <210> 20 <211> 13 <212> PRT <213 > Artificial Sequence <220> <223> L-CDR1 <400> 20 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Ser Val Ser 1 5 10 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence < 220> <223> L-CDR2 <400> 21 Ala Asp Ser Asn Arg Pro Ser 1 5 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 22 Gly Ser Trp Asp Tyr Ser Leu Ser Gly Tyr Val 1 5 10 <210> 23 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 23 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 20 25 30 Asp Leu Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Tyr Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Ser Trp Cys Leu Gln Asp Leu Cys Tyr Tyr Ala Asp 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 <210> 24 < 211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 24 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Ser Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 25 Asp Tyr Asp Met Ser 1 5 <210> 26 <211 > 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 26 Leu Ile Tyr Tyr Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Ala Lys 1 5 10 15 Gly <210> 27 < 211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 27 Asp Gly Asn Gln Met Asp Ser Glu Met Phe Asp Tyr 1 5 10 <210> 28 <211> 13 <212 > PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 28 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Ser 1 5 10 <210> 29 <211> 7 <212> PRT <213 > Artificial Sequence <220> <223> L-CDR2 <400> 29 Ala Asp Asn Asn Arg Pro Ser 1 5 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L- CDR3 <400> 30 Ala Ser Trp Asp Ala Ser Leu Ser Gly Tyr Val 1 5 10 <210> 31 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 31 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Tyr Tyr Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asn Gln Met Asp Ser Glu Met Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser 115 120 <210> 32 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 32 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Ala Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 33 Asp Tyr Tyr Met Ser 1 5 <210> 34 <211> 17 < 212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 34 Ala Ile Ser His Gly Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Ala Lys 1 5 10 15 Gly <210> 35 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 35 Asp Ala Leu Ser Cys Pro Leu Thr Glu Cys Ser Tyr Tyr Asp Gly Met 1 5 10 15 Asp Val <210> 36 <211 > 13 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 36 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Ser 1 5 10 <210> 37 <211> 7 <212 > PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 37 Tyr Asn Ser His Arg Pro Ser 1 5 <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <220> < 223> L-CDR3 <400> 38 Gly Thr Trp Asp Asp Ser Leu Asn Gly Tyr Val 1 5 10 <210> 39 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 39 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser His Gly Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ala Leu Ser Cys Pro Leu Thr Glu Cys Ser Tyr Tyr Asp 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 <210> 40 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 40 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Val Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asn Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Arg 65 70 75 80 Ser Glu Asp Gly Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu 85 90 95 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 41 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> hinge <400> 41 Gly Val Thr Val Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser 1 5 10 15 His Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala 20 25 30 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 35 40 45 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 50 55 60 Arg Gly Leu Asp 65 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Myc epitope <400> 42 Ala Asn Lys Asn Ser Ser Gln Lys Arg Ile 1 5 10 <210> 43 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3 zeta <400> 43 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 44 <211> 24 <212> PRT <213> Artificial Sequence <220 > <223> DAP10 <400> 44 Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val 1 5 10 15 Tyr Ile Asn Met Pro Gly Arg Gly 20 <210> 45 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 45 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 46 <211> 84 <212> PRT <213> Artificial Sequence <220> <223> CD28 <400> 46 Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser 1 5 10 15 Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 20 25 30 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 35 40 45 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 50 55 60 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 65 70 75 80Phe Ala Ala Tyr

Claims (16)

An extracellular binding domain containing an antigen binding site that specifically binds to ErbB3 (Erb-B2 Receptor Tyrosine Kinase 3);
transmembrane domain; and
It is a chimeric antigen receptor (CAR) containing an intracellular signaling domain,
The binding domain that specifically binds to ErbB3 includes a heavy chain CDR1 containing the sequence of SEQ ID NO: 1, a heavy chain CDR2 containing the sequence of SEQ ID NO: 2, a heavy chain CDR3 containing the sequence of SEQ ID NO: 3, and the sequence of SEQ ID NO: 4. A light chain CDR1 comprising the sequence of SEQ ID NO: 5, a light chain CDR2 comprising the sequence of SEQ ID NO: 6, and a light chain CDR3 comprising the sequence of SEQ ID NO: 6;
Heavy chain CDR1 containing the sequence of SEQ ID NO: 9, heavy chain CDR2 containing the sequence of SEQ ID NO: 10, heavy chain CDR3 containing the sequence of SEQ ID NO: 11, light chain CDR1 containing the sequence of SEQ ID NO: 12, sequence of SEQ ID NO: 13 Light chain CDR2 comprising, light chain CDR3 comprising the sequence of SEQ ID NO: 14;
Heavy chain CDR1 containing the sequence of SEQ ID NO: 17, heavy chain CDR2 containing the sequence of SEQ ID NO: 18, heavy chain CDR3 containing the sequence of SEQ ID NO: 19, light chain CDR1 containing the sequence of SEQ ID NO: 20, sequence of SEQ ID NO: 21 Light chain CDR2 comprising, light chain CDR3 comprising the sequence of SEQ ID NO: 22;
Heavy chain CDR1 containing the sequence of SEQ ID NO: 25, heavy chain CDR2 containing the sequence of SEQ ID NO: 26, heavy chain CDR3 containing the sequence of SEQ ID NO: 27, light chain CDR1 containing the sequence of SEQ ID NO: 28, sequence of SEQ ID NO: 29 Light chain CDR2 comprising, light chain CDR3 comprising the sequence of SEQ ID NO: 30; or
Heavy chain CDR1 containing the sequence of SEQ ID NO: 33, heavy chain CDR2 containing the sequence of SEQ ID NO: 34, heavy chain CDR3 containing the sequence of SEQ ID NO: 35, light chain CDR1 containing the sequence of SEQ ID NO: 36, sequence of SEQ ID NO: 37 A chimeric antigen receptor characterized in that it is a single chain variable fragment (scFv) of an anti-ErbB3 antibody comprising a light chain CDR2 comprising the sequence and a light chain CDR3 comprising the sequence of SEQ ID NO: 38.
delete The method of claim 1, wherein the binding domain that specifically binds to ErbB3 is a single chain anti-ErbB3 antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, and 39. A chimeric antigen receptor characterized in that it is a variable fragment (single chain variable fragment; scFv).
The method of claim 1, wherein the binding domain that specifically binds to ErbB3 is a single chain of an anti-ErbB3 antibody comprising a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, and 40. A chimeric antigen receptor characterized in that it is a variable fragment (single chain variable fragment; scFv).
The chimeric antigen receptor according to claim 1, wherein the extracellular domain further comprises a signal peptide and/or a hinge.
The chimeric antigen receptor according to claim 5, wherein the signal peptide is the signal peptide of CD8.
The chimeric antigen receptor according to claim 5, wherein the hinge includes a hinge of CD28 or CD8.
The method of claim 1, wherein the transmembrane domain is an alpha (α), beta (β) or zeta (ζ) chain of a T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8. , a chimeric antigen receptor selected from the group consisting of CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
The method of claim 1, wherein the intracellular signaling domain is
T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, A primary signaling domain selected from the group consisting of CD22, CD79a, CD79b and CD66d; and/or
CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a/CD18), GITR, MyD88, DAP10, A chimeric antigen receptor comprising a co-stimulatory signaling domain selected from the group consisting of a ligand that specifically binds to DAP12, PD-1, LIGHT, NKG2C, B7-H3, and CD83. .
A nucleic acid encoding a chimeric antigen receptor according to any one of claims 1, 3 to 9.
An expression vector containing the nucleic acid of claim 10.
A virus comprising the expression vector of claim 11.
An immune cell expressing the chimeric antigen receptor of any one of claims 1, 3 to 9 on its surface.
The immune cell of claim 13, wherein the immune cell is selected from the group consisting of T cells, NK cells, NKT cells, Cytokine Induced Killer cells (CIK), macrophages, and dendritic cells.
A composition for treating cancer comprising the immune cells of claim 13.
The composition for treating cancer according to claim 15, wherein the cancer is breast cancer.