TW202246311A - Erbb3-specific chimeric antigen receptor and immune cell expressing the same - Google Patents

Abstract

Disclosed are an ErbB3 (receptor tyrosine-protein kinase ErbB3)-specific chimeric antigen receptor, immune cells including the same, and the use thereof. This chimeric antigen receptor exhibits high affinity and ability to bind to ErbB3, and the immune cells expressing the chimeric antigen receptor have superior cytotoxicity to cancer cells expressing ErbB3, so they are useful for adoptive immune therapy for cancer treatment.

Description

ErbB3 specific chimeric antigen receptor and immune cells expressing it

The present invention relates to an ErbB3 (receptor tyrosine-protein kinase (ErbB3) specific chimeric antigen receptor, an immune cell containing it and use thereof. The chimeric antigen receptor according to the present invention exhibits high affinity to ErbB3 and the ability to bind to ErbB3, and immune cells expressing chimeric antigen receptor have superior cytotoxicity to cancer cells expressing ErbB3, so it can be used for cancer therapy adoptive immunotherapy.

T cells play an important role in mediating adaptive immunity. T cells are activated by stimulating antigen recognition receptors (T-cell receptor (TCR)), co-stimulatory molecules and cytokines. Moreover, the TCR of T cells induces an immune response by binding to major histocompatibility complex (MHC) molecules of antigens, and cancer cells inhibit the expression of MHC molecules to escape immune responses. Adoptive immune therapy (adoptive cell therapy) utilizing these properties of T cells is being developed.

The development of anticancer therapy using immune cells is centered on T cells. As the ex vivo ( ex-vivo ) culture and proliferation of tumor antigen-specific T cells become possible, anticancer T cell therapy has shown substantial results (Gattinoni L. et al., Nat. Rev. Immunol. 2006;6(5):383-93). However, the number of tumor antigen-specific T cells in the patient is very small, so it takes a month or more to obtain a sufficient number of T cells by ex vivo proliferation of such T cells, which is not desirable .

Therefore, based on the recombinant antibody production technology developed in the field of therapeutic antibodies, a chimeric antigen receptor (CAR) gene that connects the recombinant antibody to the signaling domain is introduced into T cells to develop a product that can be obtained in a short time. The technology of a large number of tumor-specific T cells, the recombinant antibody recognizes tumor antigens expressed on the surface of cancer cells, and the signaling domain induces the activation of T cells, such T cells are named CAR-T cells (Kershaw M.H. et al ., Nat. Rev. Immunol. 2005); 5(12):928-40; Restifo N.P. 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 cancer antigens is linked to the intracellular space through a spacer (extracellular hinge + transmembrane domain) The messaging 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 the CD3ζ (zeta) chain, which is the signaling subunit of the T cell receptor (the first generation CAR). Stimulatory form of the intracellular signaling domain of the molecule. For example, two CAR-T cell therapies currently available on the market use the intracellular signaling domains of CD28 and 4-1BB co-stimulatory molecules respectively (second-generation CARs), followed by attempts to have a cell that contains both CD28 and 4-1BB. CAR in the intra-communication domain (third-generation CAR) (van der Stegen S.J. et al., 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 shown to be overexpressed in several types of cancer, including breast, gastrointestinal and pancreatic cancers.

The information disclosed in the prior art is only used to enhance the understanding of the background of the present invention, and such information should not be interpreted as being included in common knowledge or known technical contents in the technical field to which the present invention belongs.

Against this technical background, the present inventors developed a chimeric antigen receptor comprising scFv specifically targeting cancer cell ErbB3, and determined that the immune cells expressing it had superior anti-cancer effects, thus completing the present invention.

One object of the present invention is to provide novel chimeric antigen receptors exhibiting high anticancer effects against ErbB3-expressing cancers and immune cells comprising the same.

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

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

In order to achieve the above object, the present invention provides a chimeric antigen receptor (CAR), comprising: an extracellular binding domain comprising an antigen binding domain specifically binding to ErbB3 (receptor tyrosine protein kinase ErbB3); a transmembrane domain; and intracellular signaling domains.

In addition, the present invention provides nucleic acids encoding chimeric antigen receptors, expression vectors comprising nucleic acids, viruses comprising expression vectors, and immune cells expressing chimeric antigen receptors.

In addition, the present invention provides a composition for treating cancer comprising immune cells, a method for treating cancer using immune cells, a use of immune cells for cancer treatment, and a use of immune cells for manufacturing a drug for treating cancer.

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. In general, the nomenclature used herein is well known and typical in the art.

According to an embodiment of the present invention, five ErbB3 scFvs (442S1, 451M3, 472S2, 446 and 464) specifically binding to ErbB3 expressed in cancer cells were used as the ectodomain of the chimeric antigen receptor (CAR) to make CAR constructs. In addition, myc and hinge were expressed in the outer domain to confirm the expression of CAR on the cell surface through myc, and CD3ζ (zeta) as the signaling domain and CD28 and DAP10 as co-stimulatory molecules were added to the endodomain, And then create the third generation CAR.

Accordingly, one aspect of the present invention relates to a chimeric antigen receptor (CAR), comprising: an extracellular binding domain, including an antigen binding domain that specifically binds to ErbB3; a transmembrane domain; and an intracellular signaling domain.

A chimeric antigen receptor (CAR) is a synthetic construct designed to induce an immune response against a target antigen and the cells expressing the antigen. CAR includes an extracellular binding domain, a transmembrane domain and an intracellular signaling domain. Cancer cells can be killed by introducing into immune cells a gene encoding a receptor that recognizes a cancer cell surface antigen specifically expressed on the surface of the cancer cell. Immune cells containing receptors that bind to antigens specifically expressed on cancer cells can induce an immune response by targeting only cancer cells.

The first-generation CARs include: an extracellular binding domain, including a region that recognizes antigens specifically bound to cancer cells; a transmembrane domain; and an intracellular signaling domain; and it only uses CD3ζ as a signaling domain, but it is important for the treatment of cancer The effect is not significant and the duration of the effect is short, which is undesirable.

In order to improve the response to immune cells, a second-generation CAR containing co-stimulatory domains coupled to each other (CD28 or CD137/4-1BB) and CD3ζ was prepared, and the number of CAR-containing immune cells remaining in the body was compared with that of the first generation CAR significantly increased. Unlike second-generation CARs, which contain one co-stimulatory domain, third-generation CARs contain more than two co-stimulatory domains. The co-stimulatory domain can be coupled with 4-1BB, CD28 or OX40 to achieve the expansion and persistence of CAR-containing immune cells in vivo.

In the fourth-generation CARs, additional genes encoding interleukins such as IL-12 or IL-15 were included to allow the additional expression of CAR-based immune proteins of interleukins, and the fifth-generation CARs further included genes such as IL-2Rβ Interleukin (interleukin) receptor chain to enhance immune cell activity.

The term "extracellular binding domain" as used herein refers to a portion of a CAR comprising an antigen binding domain having the ability to specifically bind to a target antigen of interest. The extracellular binding domain can comprise any protein, polypeptide, oligopeptide, or peptide that retains the ability to specifically recognize and bind biomolecules such as cell surface receptors, tumor proteins, lipids, polysaccharides, other cell surface target molecules, or portions thereof ability. A binding domain comprises any naturally occurring, synthetic, semi-synthetic or recombinant binding partner of a biomolecule of interest.

As used herein, the term "specifically binds" means that a molecule binds to another molecule with a binding affinity greater than background binding. For example, an extracellular binding domain binds to or associates with a target molecule with Ka (ie, the equilibrium dissociation constant for a particular binding interaction in units of 1/M) or about 10 ⁻⁵ M The above affinity specifically binds to the target molecule. Alternatively, affinity can be defined as the equilibrium dissociation constant (Kd) in M for a particular binding interaction (eg, 10 ⁻⁵ M to below 10 ⁻¹³ M).

The affinity of the CAR according to the present invention to the extracellular binding domain can be easily determined by binding association or substitution analysis using the following common techniques, such as competitive enzyme-linked immunosorbent assay (ELISA), Labeled ligands, surface plasmon resonance using devices such as Biacore T100 (Biacore, Inc., Piscataway, New Jersey), EPIC systems available from Corning and PerkinElmer, optical biosensor technology such as EnSpire, etc. .

According to 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 specifically binding to ErbB3 is a single-chain variable fragment (scFv) of an anti-ErbB3 antibody, and the single-chain variable fragment of an anti-ErbB3 antibody comprises: heavy chain CDR1 selected from the group consisting of SEQ ID NO: 1, 9, 17, 25 and 33, a heavy chain CDR2 selected from the group consisting of SEQ ID NO: 2, 10, 18, 26 and 34, a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 3, 11, 19, 27 and 35, light chain CDR1 selected from the group consisting of SEQ ID NO: 4, 12, 20, 28 and 36, light chain CDR2 selected from the group consisting of SEQ ID NO: 5, 13, 21, 29 and 37, and A light chain CDR3 selected from the group consisting of SEQ ID NO: 6, 14, 22, 30 and 38.

According to the present invention, the antibody or antigen-binding fragment thereof that binds to ErbB3 comprises: Heavy chain CDR1 comprising the sequence of SEQ ID NO: 1, heavy chain CDR2 comprising the sequence of SEQ ID NO: 2, heavy chain CDR3 comprising the sequence of SEQ ID NO: 3, light chain comprising the sequence of SEQ ID NO: 4 CDR1, light chain CDR2 comprising the sequence of SEQ ID NO: 5, and light chain CDR3 comprising the sequence of SEQ ID NO: 6; Heavy chain CDR1 comprising the sequence of SEQ ID NO: 9, heavy chain CDR2 comprising the sequence of SEQ ID NO: 10, heavy chain CDR3 comprising the sequence of SEQ ID NO: 11, light chain comprising the sequence of SEQ ID NO: 12 CDR1, light chain CDR2 comprising the sequence of SEQ ID NO: 13, and light chain CDR3 comprising the sequence of SEQ ID NO: 14; Heavy chain CDR1 comprising the sequence of SEQ ID NO: 17, heavy chain CDR2 comprising the sequence of SEQ ID NO: 18, heavy chain CDR3 comprising the sequence of SEQ ID NO: 19, light chain comprising the sequence of SEQ ID NO: 20 CDR1, light chain CDR2 comprising the sequence of SEQ ID NO: 21 and light chain CDR3 comprising the sequence of SEQ ID NO: 22; Heavy chain CDR1 comprising the sequence of SEQ ID NO: 25, heavy chain CDR2 comprising the sequence of SEQ ID NO: 26, heavy chain CDR3 comprising the sequence of SEQ ID NO: 27, light chain comprising the sequence of SEQ ID NO: 28 CDR1, light chain CDR2 comprising the sequence of SEQ ID NO: 29, and light chain CDR3 comprising the sequence of SEQ ID NO: 30; or Heavy chain CDR1 comprising the sequence of SEQ ID NO: 33, heavy chain CDR2 comprising the sequence of SEQ ID NO: 34, heavy chain CDR3 comprising the sequence of SEQ ID NO: 35, light chain comprising the sequence of SEQ ID NO: 36 CDR1, light chain CDR2 comprising the sequence of SEQ ID NO: 37, and light chain CDR3 comprising the sequence of SEQ ID NO: 38.

The term "antibody" as used herein refers to an anti-ErbB3 antibody that specifically binds to ErbB3. Whole antibody forms and antigen-binding fragments of antibody molecules that specifically bind to ErbB3 are encompassed within the scope of the present invention.

The term "antibody variable domain" as used herein refers to an antibody molecule comprising the amino acid sequence of a complementarity-determining region (CDR; namely CDR1, CDR2 and CDR3) and a framework region (framework region, FR) Parts of the light chain and heavy chain. VH refers to the variable domain of the heavy chain and VL refers to the variable domain of the light chain.

"Complementarity determining regions" (CDRs; ie CDR1, CDR2 and CDR3) refer to the amino acid residues of an antibody variable domain that are required for antigen binding. Each variable domain typically has three CDRs, designated CDR1, CDR2 and CDR3.

A complete antibody has two full-length light chains and two full-length heavy chains, and the light chains are respectively connected to the heavy chains through disulfide bonds. The heavy chain constant region has γ (gamma), μ (mu), α (alpha), δ (delta) and ε (epsilon) types, and also has γ1 (gamma 1), γ2 (gamma 2), γ3 (gamma 3) , γ4 (gamma 4), α1 (alpha 1) and α2 (alpha 2) subclasses. The light chain constant region has κ (kappa) and λ (lambda) types.

According to the present invention, the term "fragment" of an antibody refers to a fragment having an antigen-binding function, and includes scFv, Fab, F(ab') ₂ and Fv. Among these antibody fragments, Fab has a structure including light chain and heavy chain variable regions, light chain constant region and the first heavy chain constant region (CH1), and has an antigen-binding site. Fab' differs from Fab in that Fab' has a hinge region comprising at least a cysteine residue at the C-terminus of the CH1 domain of the heavy chain. F(ab') ₂ antibodies are produced by disulfide bonds between cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment that has only the variable region of the heavy chain and the variable region of the light chain. Double-chain Fv is a fragment in which the variable region of the heavy chain and the variable region of the light chain are connected by non-covalent bonds, and the single-chain Fv (scFv) is a fragment of the variable region of the heavy chain and the variable region of the light chain, usually through a peptide between them Linker chains are covalently linked or directly linked to C-terminal fragments to form dimeric structures, such as double-chain Fv. Such antibody fragments can be obtained using proteases (for example, Fab can be obtained by restriction cleavage of whole antibody with papain, and F(ab') ₂ fragments can be obtained by restriction cleavage of whole antibody with pepsin). Obtained), or can be prepared by genetic recombination technology.

A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, such domains being present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker chain between the VH domain and the VL domain so that the scFv can form the desired structure for antigen binding.

According to the present invention, in a scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, VH and VL can be connected via a linker chain. In an embodiment of the present invention, the binding domain that specifically binds to ErbB3 may be a single-chain variable fragment (scFv) of an anti-ErbB3 antibody, and the single-chain variable fragment of an anti-ErbB3 antibody includes a heavy chain variable region, and the heavy chain may be The variable region comprises a sequence selected from the group consisting of SEQ ID NO: 7, 15, 23, 31 and 39. The binding domain specifically binding to ErbB3 may be a single-chain variable fragment (scFv) of an anti-ErbB3 antibody, and the single-chain variable fragment of an anti-ErbB3 antibody comprises a light chain variable region, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO : Sequence of groups of 8, 16, 24, 32 and 40.

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

The antibody or antibody fragment according to the present invention may contain not only the sequence of the anti-ErbB3 antibody of the present invention but also biological equivalents thereof insofar as it can specifically recognize ErbB3. For example, in order to further improve the binding affinity and/or other biological properties of the antibody, additional modifications can be made to the amino acid sequence of the antibody. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid residues of the antibody. Amino acid changes are based on the relative similarity of amino acid side chain substituents, eg, hydrophobicity, hydrophilicity, charge, size, and the like. Based on the analysis of the size, shape, and type of amino acid side chain substituents, arginine, lysine, and histidine are all positively charged residues, and alanine, glycine, and serine have similar residues. Size, phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine may be considered biologically functional equivalents, alanine, glycine, and serine may be considered biologically functional equivalents, phenylalanine, tryptamine Acids and tyrosine are considered biologically functional equivalents.

Taking into account the above variations with equivalent biological activity, the antibodies of the present invention or nucleic acid molecules encoding them are construed as comprising sequences exhibiting substantial identity to the sequences set forth in the Sequence Listing. When the sequences of the invention and other sequences are aligned to correspond as closely as possible to each other and the aligned sequences are analyzed using algorithms commonly used in the art, "substantial identity" means exhibiting at least 90% homology Similar sequences, preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is available through NBCI, among others, and can be used with sequencers available on the Internet, such as blastp, blastn, blastx, tblastn, and tblastx. BLAST is available at www.ncbi.nlm.nih.gov/BLAST/. Methods for using this program to compare sequence homology can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Based on the above, the antibody or antigen-binding fragment thereof of the present invention may have a specific sequence or all sequences described in the specification that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% or higher homology. This homology can be determined by sequence comparison and/or alignment using methods known in the art. For example, percent sequence identity for nucleic acids or proteins of the invention can be determined using sequence comparison algorithms (ie, BLAST or BLAST 2.0), manual alignment, or visual inspection.

The connecting strand can be a peptide connecting strand and can be about 10-25 amino acids in length. For example, it may comprise hydrophilic amino acids such as glycine and/or serine. The connecting chain may comprise, for example, (GS) _n , (GGS) _n , (GSGGS) _n or (G _n S) _m (where n and m are each 1 to 10), preferably (G _n S) _m (where n and m are each 1 to 10), but not limited thereto.

A chimeric antigen receptor can comprise a transmembrane domain.

The term "transmembrane domain" as used herein refers to the part of the CAR that fuses the extracellular binding domain and the intracellular signaling domain and anchors the CAR to the cell membrane of the immunizing cell. Transmembrane domains can be derived from natural, synthetic, semi-synthetic or recombinant sources. According to the invention, the transmembrane domain can be selected from T cell receptor (TCR), alpha (alpha), beta (beta) or zeta (zeta) chain, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, but not limited thereto.

The transmembrane domain can be attached to the extracellular binding domain of CAR through a linker. For example, the linking chain can be a short oligopeptide linking chain or polypeptide linking chain with a length of 2 to 10 amino acids, preferably a glycine (G)-serine (S) doublet , but not limited to this.

The binding domain of CAR is usually connected with at least one "hinge domain". Therefore, according to the present invention, in addition to the binding domain specifically binding to ErbB3, the extracellular binding domain may further include a signal peptide (SP) and/or a hinge. The extracellular binding domain is the site of primary signaling, located outside the cell membrane, and is the domain for specific recognition of targets (ie, ErbB3).

The term "hinge domain" as used herein refers to the part of the CAR that plays an important role in the localization of the extracellular binding domain, including the antigen binding domain away from the surface of the acting cell for proper cell-to-cell contact, antigen binding and activation . CARs usually contain at least one hinge domain located between the extracellular binding domain and the transmembrane domain. Hinge domains can be derived from natural, synthetic, semi-synthetic or recombinant sources. The hinge domain may comprise a naturally occurring immunoglobulin hinge region or an altered amino acid sequence of an immunoglobulin hinge region. "Altered hinge region" means (a) a naturally occurring hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions). Hinge region, (b) at least 10 amino acids in length ( e.g. at least 12, 13, 14 or 15 amino acids), or (c) comprises a core hinge region (having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or part of a naturally occurring hinge region of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length). In certain embodiments, at least a cysteine residue in the hinge region of a naturally occurring immunoglobulin may be substituted with at least one other amino acid residue (eg, at least a serine residue). An altered immunoglobulin hinge region may instead or additionally comprise a proline residue of a wild-type immunoglobulin hinge region substituted by another amino acid residue (eg, a serine residue). The hinge domain may comprise a hinge region derived from the extracellular region of a type 1 membrane protein, such as CD8, CD4, CD28, and CD7, which may be a wild-type hinge region from these molecules or which may be altered.

According to the present invention, any transmembrane domain including a hinge domain can be used as long as it can connect the extracellular binding domain and the intracellular signaling domain through the cell membrane. Preferably, it consists of a hinge domain and a transmembrane domain derived from CD8, but not limited thereto.

In some cases, the binding and transmembrane domains can be linked by a spacer domain.

Preferably, the spacer domain is a hinge domain. According to the present invention, the spacer domain may comprise a CD28-derived hinge domain and/or a CD8-derived hinge domain, and may comprise all or part of a CD28-derived hinge domain and/or a CD8-derived hinge domain.

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

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

According to the invention, a chimeric antigen receptor may comprise an intracellular signaling domain.

According to the present invention, the intracellular signaling domain is located in the cytoplasmic part, which is the interior of the cell membrane of the immune cell, and activates the immune cell by transmitting a signal to the interior of the cell when the binding domain contained in the extracellular binding domain binds the target antigen The location of the immune response.

According to the present invention, the intracellular signaling domain may comprise an intracellular signaling domain and/or a co-stimulatory signaling domain.

The signaling domain can induce the activation of the normal function of the immune cells where the CAR is located. For example, it can induce cytolytic activation or helper activation by secreting cytokines. A signaling domain may comprise a truncated fragment of an intracellular signaling domain sufficient to transduce a functional signal.

The intracellular signaling domain includes: primary signaling domain, selected from T cell receptor (TCR) ζ (zeta), FcRγ (gamma), FcRβ (beta), CD3γ (gamma), CD3δ (delta), CD3ε (epsilon), CD3ζ (zeta), CD5, CD22, CD79a, CD79b, and CD66d; and/or co-stimulatory signaling domains selected from 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 ligands that specifically bind to CD83 groups of bodies.

The term "intracellular signaling domain" as used herein refers to a part of the CAR that is involved in transducing the signal of the effective CAR bound to the target antigen to the interior of the immune cell to cause the cell function, such as activation, Interleukin production, proliferation and cytotoxic activity, including the release of cytotoxic factors to target cells bound to CAR or other cellular responses caused by antigen binding to the extracellular binding domain of CAR. The action function refers to the special function of the cell, for example, the action function of the immune cell may be cytolytic activity or auxiliary cell activity, including the secretion of cytokines. Thus, an intracellular signaling domain is a part of a protein that transmits functional information and directs the cell to perform a specific function.

Immune cell activation is mediated by two distinct classes of intracellular signaling domains. For example, immune cell activation is mediated by primary signaling domains that initiate antigen-dependent primary activation and co-stimulatory signaling domains that act in an antigen-independent manner to provide secondary signals. Therefore, the intracellular signaling domain can include "primary signaling domain" and "co-stimulatory signaling domain".

The term "primary signaling domain" as used herein refers to a signaling domain that regulates immune cell activation in a stimulatory or inhibitory manner. Primary signaling domains acting in a stimulatory manner may comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. The ITAM containing the primary signaling domain can 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.

According to the present invention, the primary signaling domain is preferably CD3ζ (zeta) comprising the amino acid sequence represented by SEQ ID NO: 9, but not limited thereto. CD3ζ(zeta) may comprise the sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 43).

The term "co-stimulatory signaling domain" as used herein refers to the intracellular signaling domain of a co-stimulatory molecule. A co-stimulatory signaling domain is a portion of a CAR comprising the intracellular signaling domain of a co-stimulatory molecule. It may comprise 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 a costimulatory signaling domain of the group consisting of ligands that specifically bind to CD83, but are not limited thereto.

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

The chimeric antigen receptor of the present invention can use DAP10 and CD3ζ as the intracellular signaling domain to exhibit the effect of killing cancer cells by virtue of high NK cell activity.

A chimeric antigen receptor according to the present invention may comprise more than two intracellular signaling domains. When more than two intracellular signaling domains are included, the intracellular signaling domains can be connected to each other in series. Alternatively, they may be linked by an oligopeptide linking chain or a polypeptide linking chain consisting of 2 to 10 amino acids, the linking chain sequence may comprise a linked glycine-serine sequence. The connecting chain may comprise, for example, (GS) _n , (GGS) _n , (GSGGS) _n or (G _n S) _m (where n and m are each 1 to 10), preferably (G _n S) _m (where n and m are each 1 to 10), but not limited thereto.

According to the present invention, the intracellular signaling domain may include one or more selected from the group consisting of the co-stimulatory signaling domain of CD28 or DAP10 and the intracellular signaling domain of CD3ζ, but is not limited thereto.

According to the present invention, the chimeric antigen receptor may further include immune function promoting factors of immune cells, and the immune function promoting factors of immune cells may include interleukin signaling sequences. The interleukin message sequence can induce the expression of interleukin-12 (interleukin-12, IL-12), IL-8 or IL-2, but not limited thereto. In addition, IL-7 or CCL19 can be exemplified as the immune function promoting factor of T cells among immune cells.

When designing a CAR, an additional message peptide can be included before the scFv. The chimeric antigen receptor-encoding nucleic acid may further comprise a nucleic acid encoding a messager peptide.

The message peptide can be, for example, GM-CSF, CD8α message peptide, etc. It may be CD8α or mouse light chain kappa message peptide. In the case of CD8α, the message peptide of the present invention may comprise the sequence of MALPVTALLLPLALLLLHAARP (SEQ ID NO: 45).

According to the present invention, the chimeric antigen receptor may further comprise an interleukin receptor chain comprising a JAK binding motif and a STAT 3/5 association motif, examples of which may include but not limited to IL-2Rβ.

Another aspect of the invention pertains to nucleic acids encoding chimeric antigen receptors.

The term "nucleic acid" as used herein has the meaning of comprehensively covering DNA (gDNA and cDNA) and RNA molecules as well as nucleotides, which are the basic building blocks of nucleic acids and include not only natural nucleotides but also sugars or bases Modified analogs of the region. Nucleic acids encoding heavy and light chain variable regions of the invention may be modified. Such modifications include additions, deletions, or conservative or non-conservative substitutions of nucleotides.

The chimeric antigen receptor-encoding nucleic acid (polynucleotide) according to the present invention can be modified by codon optimization due to codon degeneracy, and those skilled in the art can well understand a large number of encoded polypeptides or their The presence of the nucleotide sequence of the variant fragment. Some of these polynucleotides (nucleic acids) retain minimal homology to the nucleotide sequence of any naturally occurring gene.

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

Yet another aspect of the invention relates to expression vectors comprising nucleic acids and viruses comprising expression vectors.

The term "vector" as used herein refers to a nucleic acid capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to, eg, insertable, a carrier nucleic acid molecule. A vector may contain sequences that direct the cell to replicate autonomously, or may contain sequences sufficient to permit integration into the host cell DNA. The vector can be selected from the group consisting of DNA, RNA, plastid, lentiviral vector, adenoviral vector and retroviral vector, but is not limited thereto.

The vector composition generally includes but is not limited to at least one selected from a message sequence, an origin of replication, at least one marker gene, 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.

As used herein, the term "operably linked" means a functional linkage between a nucleic acid expression control sequence (such as a promoter, a message sequence, or an array of transcriptional regulator binding sites) and a different nucleic acid sequence such that the control sequence is used to control Transcription and/or translation of different nucleic acid sequences.

When prokaryotic cells are used as hosts, they usually contain strong promoters that promote transcription (such as tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter or T7 promoter, etc.), the ribosome binding site used to initiate translation, and the transcription/translation stop sequence. In addition, for example, when eukaryotic cells are used as hosts, promoters derived from the genome of mammalian cells (such as metallothionein promoter, β-actin promoter, human heme promoter, or human muscle Creatine promoter) or promoters derived from mammalian viruses (such as adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse Mammary tumor virus (mouse mammary tumor virus, MMTV) promoter, HIV LTR promoter, murine leukemia virus (Moloney virus) promoter, Eistein-Barr virus (Epstein-Barr virus, EBV) promoter or Lao Rous sarcoma virus (RSV) promoter, and usually has a polyadenylation sequence as a transcription termination sequence.

In some cases, the vector is fused to another sequence to facilitate purification of antibodies expressed therefrom. Examples of fused sequences include glutamine sulfur S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Qiagen, USA).

The vector contains antibiotic resistance genes commonly used in the art as selection markers, for example, endowing ampicillin, gentamycin, carbenzillin, chloramphenicol, streptomycin, kangmycin, geneticin (geneticin), neomycin Genes for resistance to clindamycin or tetracycline.

The term "virus" as used herein refers to a substance that has been genetically modified to express the chimeric antigen receptor of the present invention for cancer therapy. Herein, the term "genetic modification" means the addition of foreign genetic material in the form of DNA or RNA to the entire genetic material of a cell.

According to the invention, the nucleic acid or vector is transfected into a virus. A variety of different techniques commonly used to introduce exogenous nucleic acid (DNA or RNA) into prokaryotic or eukaryotic host cells for "transfection" such as electrophoresis, calcium phosphate precipitation, DEAE-glucose transfection, lipofection Dye (lipofection), etc.

Yet another aspect of the invention relates to immune cells expressing chimeric antigen receptors on their surface.

According to the present invention, immune cells can exhibit desired cancer therapeutic effect by causing immunity, and can be selected from, for example, T cells, NK cells, NKT cells, cytokine-induced killer cells (CIK), macrophages, etc. A group consisting of phagocytes and dendritic cells, but not limited thereto. The immune cells are preferably T cells.

Therefore, the immune cells expressing chimeric antigen receptors according to the present invention may include chimeric antigen receptor T cells (CAR-T cells), chimeric antigen receptor natural killer cells (CAR-NK cells), chimeric antigen receptor Human natural killer T cells (CAR-NKT cells) or chimeric antigen receptor macrophages (CAR-macrophages), etc. According to the present invention, T cells can be selected from cytotoxic T lymphocytes (CTL) and T cells isolated from tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC). composed of groups.

According to the invention, inter alia, the CAR-NK cell line introduces chimeric antigen receptors into cells of natural killer cells. These cells can solve the problems of immunotherapy using CAR-T therapy, such as persistent toxicity, risk of autoimmune diseases, grafts of xenogeneic cell transplantation, through the switch to initiate (on)/stop (off) the response Anti-host disease (graft-versus-host disease, GVHD) and non-target toxicity, and can also target a variety of cancer cells, so it can be used as a general therapeutic agent.

Yet another aspect of the invention pertains to compositions for treating cancer comprising immune cells (eg, T cells or NK cells) expressing chimeric antigen receptors. T cells can be selected from CD4+ (positive) T cells, CD8+ cytotoxic T lymphocytes (CTL), γ-δ T cells, and T cells isolated from tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) The group formed, but not limited to this.

According to the present invention, "cancer" and "tumor" are used interchangeably and refer to a physiological condition in mammals, usually characterized by unregulated cell growth and proliferation.

Cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited, and includes solid cancer and blood cancer. Examples of such cancers 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. The term "prevention" as used herein refers to any action that inhibits or delays the progression of cancer by administering the composition of the present invention. "Treatment" refers to the inhibition Cancer progression and slowing or elimination of its symptoms.

The composition preferably comprises immune cells expressing chimeric antigen receptors according to the present invention, the number of which is 1 to 10 times the number of tumor cells in the therapeutic target, but the present invention is not limited thereto.

The pharmaceutical composition comprising immune cells expressing chimeric antigen receptors according to the present invention may further comprise pharmaceutically acceptable excipients. Examples of such excipients include surfactants (preferably polysorbate series nonionic surfactants), buffers (such as neutral buffered saline, phosphate buffered saline, etc.), sugars or sugar alcohols (such as glucose , mannose, sucrose, polyglucose, mannitol, etc.), amino acids, proteins or polypeptides (such as glycine, histidine, etc.), antioxidants, chelating agents (such as EDTA or glutathione (glutathione )), penetrants, supplements and preservatives, but not limited thereto.

According to an embodiment of the present invention, the pharmaceutical composition of the present invention may contain immune cells (such as T cells or NK cells) in a single dose, the number of which is 1 to 10 times that of tumor cells in the treatment target.

The compositions of the present invention may be prepared using methods known in the art so as to provide quick, sustained or delayed release of the active ingredient after administration to mammals other than humans. The preparations may be in the form of powders, granules, lozenges, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions or sterile powders.

Pharmaceutical compositions may be provided in a variety of forms, either oral or parenteral formulations. In the case of formulations, it is prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., which are obtained by mixing one or more compounds with at least one excipient (such as starch, calcium carbonate, sucrose, lactose, gelatin, etc.) etc.) obtained by mixing. Besides simple excipients, lubricating agents such as magnesium stearate, talc, and the like can also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups and the like. In addition to simple diluents such as commonly used water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may also be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, Witepsol, polyethylene glycol (macrogol), Tween 61, cocoa butter, laurin fat, glycerin gelatin and the like can be used.

Yet another aspect of the invention relates to a method of treating cancer comprising administering to a subject an immune cell expressing a chimeric antigen receptor.

Another aspect of the present invention relates to the use of immune cells in the treatment of cancer.

Yet another aspect of the invention relates to the use of immune cells in the manufacture of medicaments for the treatment of cancer.

The subject can be a mammal with a tumor, especially a human, but not limited thereto.

The chimeric antigen receptor expressing immune cells according to the present invention or compositions comprising the same can be administered orally or via infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, rectal administration, topical administration, Administration such as intranasal injection, but not limited thereto.

The dose of the active ingredient can be properly selected according to the following factors, such as administration route, age, sex, patient weight, disease severity, etc. The therapeutic composition according to the present invention can effectively prevent, improve or treat cancer symptoms with known The compounds are administered in combination.

The invention can be better understood through the following examples. These examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, which is obvious to those having ordinary knowledge in the art.

Example 1. Construction of NK cells expressing CARs using ErbB3 (HER3) scFv as ectodomain

Each of the CAR constructs was made using five ErbB3 scFv candidates (442S1, 451M3, 472S2, 446 and 464) specifically binding to ErbB3 (HER3) expressed in cancer cells (442S1, 451M3, 472S2, 446 and 464) as the ectodomain of the CAR (Figure 1).

〔Table 1〕 name CDR1 CDR2 CDR3 442S1 VH DYDMS TIDLDSGSIYYADSVQG DLHMGPEGPFDY SEQ ID NO: 1 2 3 7 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYDMSWVRQAPGKGLEWVSTIDLDSGSIYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLHMGPEGPFDYWGQGTLVTVS VL SGSSSNIGSNSVS SDN HRPS QGWDTSLSGHV SEQ ID NO: 4 5 6 8 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNSVSWYQQLPGTAPKLLIYSDNHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQGWDTSLSGHVFGGGTKLTVLG 451M3 VH HYDMS AIYYDSGSIYYADSAKG DRLFASDSTFDY SEQ ID NO: 9 10 11 15 EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYDMSWVRQAPGKGLEWVSAIYYDSGSIYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRLFADSTFDYWGQGTLVTVS VL SGSPSNIGNNSVT YDSHRPS GSWDASLNGYV SEQ ID NO: 12 13 14 16 QSVLTQPPSASGTPGQRVTISCSGSPSNIGNNSVTWYQQLPGTAPKLLIYYDSHRPSGVPDRFSGSKSGTSASLAIGGLRSEDEADYYCGSWDASLNGYVFGGGTKLTVLG 472S2 VH WYDLA GISYDGGNTYYADSVKG DPSWCLQDLCYYADGMDV SEQ ID NO: 17 18 19 twenty three EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYDLAWVRQAPGKGLEWVSGISYDGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPSWCLQDLCYYADGMDVWGQGTLVTVS VL SGSSSNIGSNSVS ADSNRPS GSWDYSLSGYV SEQ ID NO: 20 twenty one twenty two twenty four QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNSVSWYQQLPGTAPKLLIYADSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLG 446 VH DYDMS LIYYDGGSTYYADSAKG DGNQMDSEMFDY SEQ ID NO: 25 26 27 31 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYDMSWVRQAPGKGLEWVSLIYYDGGSTYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGNQMDSEMFDYWGQGTLVTVS VL SGSSSNIGSNTVS ADNN RPS ASWDASLSGYV SEQ ID NO: 28 29 30 32 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVSWYQQLPGTAPKLLIYADNNRPSGVPDRFSGSKSGTSASLAIGGLRSEDEADYYCASWDASLSGYVFGGGTKLTVLG 464 VH DYYMS AISHGGSSKYYADSAKG DALSCPLTECSYYDGMDV SEQ ID NO: 33 34 35 39 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSAISHGGSSKYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDALSCPLTECSYYDGMDVWGQGTLVTVS VL SGSSSNIGSNDVS YNSHRPS GTWDDSLNGYV SEQ ID NO: 36 37 38 40 QSVLTQPPSASGTPGQRVTVSCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYNSHRPSGVPDRFSGSKSGTSASLAIGGLRSEDGADYYCGTWDDSLNGYVFGGGTKLTVLG

In order to confirm that CAR is expressed on the cell surface, myc and hinge are expressed in the outer domain, and CD28, DAP10, and CD3ζ involved in cytotoxicity are used as the inner domain.

The specific sequences corresponding to each domain are disclosed in Table 2 below.

〔Table 2〕 composition sequence SEQ ID NO: myc ANKNSSQKRI 42 Hinge GVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 41 CD28 (contains crotch membrane domain and co-stimulatory domain) VKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY 46 DAP10 LCARPRRSPAQEDGKVYINMPGRG 44 CD3-zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR- 43 CD8 SP MALPVTALLLPLALLLLHAARP 45

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

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

In order to confirm the cytotoxicity of ErbB3-CAR NK92 to cancer cells, flow cytometry was used to identify and select cancer cell lines expressing ErbB3 (Figure 3A), and the cytotoxicity of ErbB3-CAR NK92 to cancer cells was compared using the Calcein-AM method. It was confirmed that each CAR NK92 exhibited increased cytotoxicity against ErbB3-expressing cancer cells compared to the control group (NK92 or PURO-NK92), and exhibited cytotoxicity similar to the control group in ErbB3-nonexpressing cancer cells (Fig. 3B ).

In addition, among the 5 ErbB3-CAR NK92 candidates, 3 types with high cytotoxicity (442S1, 451M3, and 472S2) were selected, and their cytotoxicity was reconfirmed to depend on the ratio of CAR NK92 to cancer cells (acting cells: target cells ; E:T ratio). In cancer cells expressing ErbB3, it was confirmed that all three types of CAR NK92 exhibited cytotoxicity dependent on the E:T ratio, and in cancer cells not expressing ErbB3 exhibited cytotoxicity similar to that of the control group (Fig. 4 ).

Furthermore, after the ErbB3-CAR NK92 cells were co-cultured with cancer cells, the amount of secreted cytokines and the degree of degranulation were measured. Based on the measurement results of interleukin (IFN-γ) using ELISA, it was confirmed that a large amount of IFN-γ was secreted in CAR NK92 cells co-cultured with ErbB3-expressing cancer cells compared to the control group ( FIG. 5A ). Furthermore, regarding degranulation based on the degree of expression of CD107a, it was confirmed that the expression degree was higher in the group co-cultured with ErbB3-expressing cancer cells ( FIG. 5B ).

Based on measurements of ErbB3-CAR NK92 cytotoxicity at the single-cell level, frequency of contact with cancer cells, and time required to kill cancer cells, the frequency of contact with cancer cells was similar to that of the control group (PURO-NK92, Ecto ; NK92 with an ectodomain deletion structure), while the cytotoxicity was higher than that of the control group, and the time required to kill cancer cells was shorter than that of the control group (Figure 6).

Example 3. Comparing the effect of ErbB3-CAR NK92

To compare the effect of ErbB3-CAR NK92, cytotoxicity was compared with CAR NK92 cell lines with different ectodomains. Specifically, comparison of targeting ErbB2 (HER2) (Cot-CAR NK92, general concept chimeric antigen receptor; mediator using targeting HER2, HER2-CAR NK92) and ErbB3-CAR NK92 (especially 442S1 with the best effect ) of two types of CAR NK92 cytotoxicity. After confirming the expression of CAR in each CAR NK92 cell ( FIG. 7A ), cancer cells expressing all CAR NK92 targets were selected ( FIG. 7B ). When comparing the cytotoxicity against the corresponding cancer cells, it was confirmed that the cytotoxicity of ErbB3-CAR NK92 (442S1) was very high compared with other CAR NK92 ( FIG. 7C ).

Furthermore, antibody-dependent cellular cytotoxicity (ADCC) was confirmed using ErbB3 scFv (442S1) for the ErbB3-CAR NK92 ectodomain and a commercially available ErbB3 scFv (LJM716) with the same target. Binding of each scFv to the corresponding cancer cells was assessed using flow cytometry ( FIG. 8A ), and ADCC was performed using CD16-expressing NK92 (CD16+NK92) cells ( FIG. 8B ). As a result, it was confirmed that the ErbB3 scFv (442S1) for ErbB3-CAR NK92 exhibited higher ADCC in ErbB3-expressing cancer cells compared to the commercially available ErbB3 scFv (LJM716) ( FIG. 8C ).

Furthermore, commercially available ErbB3 scFv (LJM716) was used to construct CAR to establish a CAR-expressing NK92 cell line, and then its effect was compared with that of ErbB3-CAR NK92 (442S1). Except for the outer domain of CAR, the rest of the constructs (transmembrane and inner domain) were prepared with the same structure (Figure 9A), and the NK92 cell line expressing CAR was established using lentivirus (Figure 9B). As a result of comparing the cytotoxicity of the ErbB3-CAR NK92 cell line, it was confirmed that 442S1-CAR NK92 cells exhibited higher cytotoxicity against cancer cells expressing the target (ErbB3) than LJM716-CAR NK92 cells. For cancer cells not expressing ErbB3, it was confirmed that both types of CAR NK92 cells exhibited similar cytotoxicity (Fig. 9C).

ErbB3-CAR NK92 (442S1) exhibits specific anticancer activity against ErbB3-expressing cancer cells, and has a very high effect compared to the existing ErbB3 scFv (LJM716).

It is clear from the above description that the chimeric antigen receptor according to the present invention exhibits high affinity and the ability to bind to ErbB3, and immune cells expressing chimeric antigen receptor have superior cytotoxicity to cancer cells expressing ErbB3, so it can be used Adoptive immunotherapy for cancer treatment.

Although specific embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that these descriptions are only preferred exemplary embodiments and should not be construed as limiting the scope of the present invention. Therefore, the substantive protection scope of the present invention is defined by the following claims and their equivalent scopes.

none

The above and other objects, features and advantages of the present invention will be more clearly understood through the following detailed description and accompanying drawings, wherein:

Figure 1 reveals the results of making CAR constructs using five ErbB3 scFv candidates (442S1, 451M3, 472S2, 446 and 464) specifically binding to ErbB3 (HER3) as the ectodomain of CAR;

Figure 2 reveals the results of establishing NK92 cell lines expressing ErbB3-CAR;

Figure 3A reveals the results of selecting cancer cell lines expressing ErbB3;

FIG. 3B reveals results confirming the cytotoxicity of each ErbB3-CAR NK92 against ErbB3-expressing cancer cells;

Figure 4 reveals the results confirming the cytotoxicity of ErbB3-CAR NK92 depending on the ratio of CAR NK92 to cancer cells (E:T ratio);

Figure 5A reveals the results of the amount of IFN-γ secretion in CAR NK92 cells co-cultured with ErbB3-expressing cancer cells;

Figure 5B reveals results confirming degranulation in CAR NK92 co-cultured with ErbB3-expressing cancer cells;

Figure 6 reveals the results of confirming the effect of CAR NK92 at the single cell level;

FIG. 7A reveals the result of confirming CAR expression in each ErbB3-CAR NK92;

Figure 7B reveals the results of selection of cell lines satisfying all targets of each CAR NK92;

Figure 7C reveals the result of comparing the cytotoxicity of each CAR NK92 cells;

FIG. 8A reveals results confirming that each ErbB3 scFv is attached to cancer cells;

Figure 8B reveals the results of confirming the NK92 cell line expressing CD16;

Figure 8C reveals the measurement results of ADCC using ErbB3 scFv;

Figure 9A reveals the results of making ErbB3-CAR constructs;

Figure 9B reveals NK92 cell lines expressing ErbB3-CAR (442S1 vs. LJM716); and

Figure 9C reveals the results of comparing the cytotoxicity of ErbB3-CAR NK92 (442S1 vs. LJM716).

Claims (16)

A chimeric antigen receptor (chimeric antigen receptor, CAR), comprising: an extracellular binding domain, including an antigen binding domain that specifically binds to ErbB3 (Erb-B2 receptor tyrosine kinase 3); a transmembrane domain; and a Intracellular signaling domain. The chimeric antigen receptor as described in claim 1, wherein the extracellular binding domain specifically binding to ErbB3 is a single-chain variable fragment (scFv) of an anti-ErbB3 antibody, and the single chain of the anti-ErbB3 antibody can be The variable fragment comprises: a heavy chain CDR1 selected from the group consisting of SEQ ID NO: 1, 9, 17, 25 and 33, a heavy chain CDR2 selected from the group consisting of SEQ ID NO: 2, 10, 18, 26 and 34 The group, a heavy chain CDR3, selected from the group consisting of SEQ ID NO: 3, 11, 19, 27 and 35, a light chain CDR1, selected from the group consisting of SEQ ID NO: 4, 12, 20, 28 and 36 The group consisting of a light chain CDR2 selected from the group consisting of SEQ ID NO: 5, 13, 21, 29 and 37, and a light chain CDR3 selected from SEQ ID NOs: 6, 14, 22, 30 and 38 groups. The chimeric antigen receptor as described in claim 1, wherein the extracellular binding domain specifically binding to ErbB3 is a single-chain variable fragment (scFv) of an anti-ErbB3 antibody, and the single chain of the anti-ErbB3 antibody can be The variable segment comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 7, 15, 23, 31 and 39. The chimeric antigen receptor as described in claim 1, wherein the extracellular binding domain specifically binding to ErbB3 is a single-chain variable fragment (scFv) of an anti-ErbB3 antibody, and the single chain of the anti-ErbB3 antibody can be The variable fragment comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 8, 16, 24, 32 and 40. The chimeric antigen receptor according to claim 1, wherein the extracellular binding domain further comprises at least one of a message peptide and/or a hinge. The chimeric antigen receptor as described in claim 5, wherein the message peptide is the message peptide of CD8. The chimeric antigen receptor according to claim 5, wherein the hinge comprises a CD28 or CD8 hinge. The chimeric antigen receptor as claimed in claim 1, wherein the transmembrane domain is selected from T cell receptor (T-cell receptor, TCR) α, β or ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8 , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. The chimeric antigen receptor as claimed in claim 1, wherein the intracellular signaling domain comprises: a primary signaling domain selected from T cell receptor (TCR) ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5 , the group consisting of CD22, CD79a, CD79b, and CD66d; and/or a co-stimulatory signaling domain selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, Group consisting of ICAM-1, ICOS (CD278), LFA-1 (CD11a/CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, B7-H3 and ligands that specifically bind to CD83 Group. A nucleic acid encoding the chimeric antigen receptor as described in any one of claims 1-9. An expression carrier, comprising the nucleic acid as described in claim 10. A virus, comprising the expression carrier as described in Claim 11. An immune cell expressing the chimeric antigen receptor according to any one of claims 1 to 9 on its surface. The immune cell according to claim 13, wherein the immune cell is selected from T cells, NK cells, NKT cells, cytokine-induced killer cells (cytokine-induced killer cells, CIK), macrophages and dendritic cells composed of groups. A composition for treating cancer, comprising the immune cells as described in claim 13. The composition according to claim 15, wherein the cancer is breast cancer.

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