KR20230021391A - Novel anti-B7H3 Chimeric Antigen Receptor and Immune Cell Expressing the Same - Google Patents

Abstract

The present invention relates to: a chimeric antigen receptor comprising an antigen-binding variable fragment of an antibody specifically binding to B7-H3; and immune cells expressing the chimeric antigen receptor. An objective of the present invention is to provide the novel chimeric antigen receptor capable of amplifying cytotoxic activity against cancer cells when expressed in the immune cells.

Description

Novel anti-B7H3 Chimeric Antigen Receptor and Immune Cell Expressing the Same}

The present invention relates to a chimeric antigen receptor comprising an antigen-binding variable fragment of an antibody specifically binding to B7-H3, and an immune cell expressing the chimeric antigen receptor.

Methods for treating cancer have gone through a process of steady development and change, and methods such as surgery, chemotherapy, and radiation therapy are still being used, but these existing cancer treatment methods are only used in the early stage when the cancer has not metastasized. In most cases, it is effective, and in a state where metastasis has already progressed, there is a problem that, for example, even if surgical operation is performed, the possibility of recurrence is high. Accordingly, studies on methods of using an immune response to treat cancer have recently been conducted.

Among them, there is a growing interest in cell therapy methods that use immune cells to reinforce them or genetically modify them and inject them back into the patient. For example, tumor infiltrating lymphocytes (TIL), chimeric antigen receptors (Chimeric antigen receptor, CAR) and T-cell receptor (TCR) technologies are being studied. In particular, in the case of a chimeric antigen receptor, which is an artificial receptor designed to deliver antigen specificity to T cells or natural killer cells (NK cells), the receptor binds to a cancer cell-specific antigen to activate the immune cells and achieve specific immunity. It consists of an extracellular domain that can be provided, a transmembrane domain, and an intracellular signaling domain. And T cells expressing these chimeric antigen receptors were named CAR-T cells (Kershaw et al. , Nat. Rev. Immunol. , 5 (12): 928-940 (2005); Restifo et al. , Nat. Rev. Immunol. , 12 (4): 269-281 (2012)), in the case of natural killer cells were named CAR-NK cells.

The intracellular signaling domain of the chimeric antigen receptor is mainly based on the intracellular signaling domain of CD3zeta, a signaling subunit of the T cell receptor (first-generation CAR). And it has evolved in the form of adding an intracellular signaling domain of a co-stimulatory molecule that promotes the growth and differentiation of immune cells. For example, currently marketed CAR-T cell therapeutics use the intracellular signaling domains of CD28 and 4-1BB co-stimulatory molecules, respectively (second-generation CARs), followed by CD28 and 4-1BB intracellular signaling domains. A CAR (3rd generation CAR) containing at the same time has been attempted (Stegen et al. , Nat. Rev. Drug Discov. , 14 (7): 499-509 (2015)).

Meanwhile, B7-H3 (B7-Homolog3) is a protein expressed from the human CD276 gene and is known to be overexpressed in various types of cancer and is known to promote cancer progression, metastasis, and invasion. In addition, the B7-H3 plays a role of suppressing adaptive immunity, and in particular, it can help the development of tumors by suppressing the immune response to cancer-related antigens. Under this technical background, another aspect of strategies and methods for treating cancer by developing antibodies targeting the B7-H3, which is particularly overexpressed in cancer cells, or the CAR-T and CAR-NK therapeutics using the same The need for research emerges.

An object of the present invention is to provide a novel chimeric antigen receptor capable of amplifying its cytotoxic activity against cancer cells when expressed in immune cells.

In addition, an object of the present invention is to provide a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

In addition, an object of the present invention is to provide an immune cell with excellent therapeutic effect against cancer by expressing the chimeric antigen receptor on its surface.

In addition, an object of the present invention is to provide a pharmaceutical composition for treating cancer using the immune cells.

In order to achieve the above object, one aspect of the present invention, B7-H3 (B7-Homolog3) extracellular domain containing an antigen binding site that specifically binds (extracellular binding domain); transmembrane domain; And an intracellular signaling domain; as a Chimeric Antigen Receptor (CAR) containing, the antigen binding site that specifically binds to the B7-H3 is SEQ ID NO: 1 or SEQ ID NO: 1 A heavy chain variable region comprising heavy chain CDR1 having an amino acid sequence of 9, heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11 and SEQ ID NO: 4 or sequence A light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14 - A chimeric antigen receptor that is a single chain variable fragment (scFv) of the B7-H3 antibody is provided.

Another aspect of the present invention provides a polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor and an expression vector comprising the polynucleotide.

Another aspect of the present invention provides an immune cell expressing the chimeric antigen receptor on its surface.

Another aspect of the present invention provides a pharmaceutical composition for cancer treatment comprising the immune cells.

The chimeric antigen receptor using the antigen-binding fragment of the antibody of the present invention can specifically bind to B7-H3, which is mainly expressed in cancer cells, and thus signal transduction in immune cells expressing the chimeric antigen receptor As it occurs, it significantly improves the cytotoxicity of immune cells and exhibits the effect of promoting the secretion of cytokines. In addition, this has an effect of increasing degranulation of cancer cells co-cultured with the immune cells.

Therefore, the chimeric antigen receptor using the antibody of the present invention and the immune cells expressing it can be usefully used for cancer treatment.

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a schematic diagram showing the structure of a chimeric antigen receptor of the present invention comprising an antigen-binding variable fragment (scFv) that specifically binds to B7-H3 as an extracellular domain.
Figure 2 is a natural killer cell (B7H3-40F10 and B7H3-51H04) in which two types of genes of the chimeric antigen receptor of the present invention were introduced into natural killer cell NK92 and expressed therein, and the extracellular domain of the chimeric antigen receptor was deleted and introduced Measurement of the expression of the chimeric antigen receptor of the present invention in natural killer cells (ΔECTO-CAR), natural killer cells (Puro-NK92) expressing an empty vector, and NK92 cells without vector introduction It shows the result of comparison.
Figure 3 is co-cultivation of natural killer cells according to the description of Figure 2 with A498, a cancer cell line expressing B7-H3, and Jurkat, a cancer cell line not expressing B7-H3, respectively, and then measuring the cytotoxicity of the natural killer cells. This is a comparison graph.
4 is a cytokine secreted by the natural killer cells after co-culture of the natural killer cells according to the description of FIG. It is a graph comparing the amount of caine (IFN-γ) measured.
Figure 5 is a degranulation indicator in the cancer cell line after co-culture of natural killer cells according to the description of Figure 2 with A498, a cancer cell line expressing B7-H3, and Jurkat, a cancer cell line not expressing B7-H3, respectively. It is a graph comparing the expression level of phosphorus CD107α.

Hereinafter, the present invention will be described in detail.

1. Anti-B7-H3 antibody, antigen-binding fragment thereof, and chimeric antigen receptor (CAR) containing the same, and polynucleotides and expression vectors for expressing the same

One aspect of the present invention provides anti-B7-H3 antibodies and antigen-binding fragments thereof capable of specifically binding to B7-H3.

In the present invention, the term "antibody" refers to an immunoglobulin molecule having a reactivity by specifically binding to an epitope of an antigen immunologically. The antibody may include all of monoclonal antibodies, polyclonal antibodies, antibodies having a full-length chain structure (full-length antibodies), functional fragments having at least an antigen-binding function (antigen-binding fragments), and recombinant antibodies. Specifically, the antibody of the present invention may be a monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. The full-length antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain may be connected to the heavy chain through a disulfide bond. The antibody includes a heavy chain (HC) and a light chain (LC) polypeptide, and the heavy chain and light chain may include a variable region and a constant region.

The constant region is a region that mediates binding of the antibody to various types of cells (T-cells, etc.) of the immune system, host tissues including components of the complement system, and the like. The constant region, if it is the same type of antibody derived from the same species, has the same function regardless of the type of antigen, and the amino acid sequence constituting it also has the same or high similarity for each antibody. The constant region may be divided into a heavy chain constant region (which may be abbreviated as CH) and a light chain constant region (which may be abbreviated as CL). The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and / or epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), It has gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and/or alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types. IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.

The variable region is an antibody region having specificity for an antigen, and may be divided into a heavy chain variable region (which may be abbreviated as VH) and a light chain variable region (which may be abbreviated as VL). The variable region may include three complementary-determining regions (CDRs) and four framework regions (FRs). The CDR may be a ring-shaped region involved in antigen recognition, and specificity for the antigen may be determined according to the amino acid sequence of the CDR. The CDRs may be referred to as CDR1, CDR2, and CDR3 according to their order, and depending on whether the heavy chain or light chain is a CDR of any polypeptide, in the case of a heavy chain variable region, CDR-H1, CDR-H2, CDR-H3, In the case of the light chain variable region, it may be referred to as CDR-L1, CDR-L2, or CDR-L3. Similarly, FR can be referred to as FR-H1, FR-H2, FR-H3, and FR-H4 for the heavy chain variable region, and FR-L1, FR-L2, FR-L3, and FR-L4 for the light chain variable region. there is. In addition, the CDRs and FRs may be arranged in the following order in each variable region.

As used herein, the term "antigen-binding fragment" refers to any fragment of the humanized antibody of the present invention that retains the antigen-binding function of the antibody. The antigen-binding fragment may be interchangeably referred to with terms such as "fragment" and "antibody fragment", and the antigen-binding fragment may be Fab, Fab', F(ab') ₂ , Fv, etc., but is not limited thereto. .

The Fab has a structure having light and heavy chain variable regions, a light chain constant region, and a first constant region (CH1 domain) of a heavy chain, and has one antigen binding site. The Fab' is different from the Fab in that it has a hinge region including one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab') ₂ is generated when a cysteine residue in the hinge region of Fab' forms a disulfide bond. The Fv refers to a minimum antibody fragment having only a heavy chain variable region and a light chain variable region. Double-chain Fv (two-chain Fv) has a heavy chain variable region and light chain variable region connected by a non-covalent bond, and single-chain Fv (single-chain Fv) generally has a heavy chain variable region and a light chain variable region covalently bonded through a peptide linker. or directly linked at the C-terminus, it can form a dimer-like structure like double-chain Fv. The antigen-binding fragment may be prepared using a proteolytic enzyme (for example, Fab may be obtained by restriction digestion of whole antibody with papain, and F(ab')2 fragment may be obtained by digestion with pepsin), or It can be produced through genetic recombination technology, but is not limited thereto.

The linker may be a peptide linker and may have a length of about 10 to 25 amino acids. For example, the linker may include a hydrophilic amino acid such as glycine (G) and/or serine (S). The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n and m are each 1 to 10), and for example, (GnS)m( n and m may each be 1 to 10), but are not limited thereto.

As used herein, the term "epitope" refers to a specific site on an antigen that can be specifically recognized and bound by an immunoglobulin, antibody, or antigen-binding fragment thereof. The epitope may be formed from contiguous amino acids or from discontinuous amino acids juxtaposed by tertiary folding of a protein.

The “specifically binding” may mean binding to another molecule with a binding affinity greater than background binding, for example, the extracellular domain has an affinity or Ka of about 10 ⁻⁵ M or higher for the target antigen. (equilibrium dissociation constant of a specific binding interaction having a unit of 1/M). The affinity may have an equilibrium dissociation constant (Kd) of a specific binding interaction having a unit of M of 10 ⁻⁵ M to 10 ⁻¹³ M or less than the above range.

The antibody or antigen-binding fragment thereof of the present invention is a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11 a heavy chain variable region comprising a heavy chain CDR3 having the sequence; and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 14. region; and specifically binds to B7-H3 (B7-Homolog3).

The heavy chain variable region may have an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 15.

The light chain variable region may have an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16.

The antibody or antigen-binding fragment thereof of the present invention may further include, for example, a heavy chain constant region and/or a light chain constant region of an antibody derived from a human, and the antibody or antigen-binding fragment thereof is specifically for B7-H3. The heavy chain constant region and/or light chain constant region of the human-derived antibody may be used without limitation in type or amino acid sequence, as long as the binding properties are not impaired.

The above-described amino acid sequences may include variants having different sequences due to deletion, insertion, substitution, or combination thereof of amino acid residues within a range that does not affect the structure, function, activity, etc. of the polypeptide containing the same. . In addition, the amino acid sequences may include amino acids with conventional modifications known in the art, and the amino acid modifications include, for example, phosphorylation, sulfation, acrylation, glycosylation, methylation ( methylation), farnesylation, and the like. The humanized antibody or antigen-binding fragment thereof of the present invention includes those having amino acid sequences substantially identical thereto, as well as those comprising the above-described amino acid sequences, or variants thereof. The meaning of having the substantially identical amino acid sequence is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more of the amino acid sequence described above. % or more, 99% or more, or may include an amino acid sequence having a homology of 99.5% or more, but is not limited thereto.

In the present invention, the term “Chimeric antigen receptor (CAR)” is a synthetic complex designed to induce an immune response to a target antigen and a cell expressing the antigen when recognized and bound thereto. A chimeric antigen receptor may include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The chimeric antigen receptor is expressed on the surface of immune cells and recognizes and binds to a specific antigen, such as an antigen specifically expressed on the surface of cancer cells, through an antigen binding site included in an extracellular domain, thereby signaling within the immune cell. Transduction can cause changes in the activity of immune cells, and an immune response can be induced by targeting only a specific antigen.

The chimeric antigen receptor (CAR) of the present invention includes an extracellular binding domain including an antigen binding site that specifically binds to B7-H3 (B7-Homolog3); transmembrane domain; and an intracellular signaling domain.

The antigen-binding site that specifically binds to B7-H3 is a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and SEQ ID NO: 3 or sequence The heavy chain variable region comprising the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 11 and the light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, the light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and SEQ ID NO: 6 or It may be a single chain variable fragment (scFv) of an anti-B7-H3 antibody including a light chain variable region including a light chain CDR3 having the amino acid sequence of SEQ ID NO: 14.

When the chimeric antigen receptor of the present invention is expressed on the surface of an immune cell, when the target antigen, B7-H3, binds to the receptor, signal transduction occurs in the immune cell, and the cytotoxicity of the immune cell is improved. and/or stimulation of cytokine secretion of the immune cells may be induced. The enhancement of cytotoxicity or promotion of cytokine secretion may be exhibiting cytotoxicity or secreting cytokines at a higher level than cytotoxicity or cytokine secretion exhibited by immune cells in the absence of antigen.

The extracellular domain may further include at least one selected from the group consisting of a hinge domain and a spacer domain. The antigen-binding site of the extracellular domain may be linked to the transmembrane domain through a hinge domain and/or a spacer domain.

The hinge domain is the antigen-binding site from the surface of the immune cell on which the chimeric antigen receptor is expressed, so as to enable proper cell/cell contact, binding of the appropriate antigen/antigen-binding site, and activation of the appropriate chimeric antigen receptor. As a part that allows to be physically separated, it can play an important role in determining the location of the extracellular domain. The chimeric antigen receptor may include one or more hinge domains between the extracellular domain and the transmembrane domain. Hinge domains may be derived from natural, synthetic, semi-synthetic or recombinant sources. A hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. The altered hinge region is (a) a naturally occurring hinge region with up to 30% amino acid change (e.g., amino acid substitutions or deletions of up to 25%, 20%, 15%, 10% or 5%), (b) up to 30% Natural at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) in length with % amino acid changes (e.g., amino acid substitutions or deletions of up to 25%, 20%, 15%, 10%, or 5%) part of the hinge region, or (c) the core hinge region (which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14 or 15 amino acids in length). In certain embodiments, one or more cysteine residues in a naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (eg, one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue of a wild-type immunoglobulin hinge region substituted with another amino acid residue, such as cysteine. The hinge domain may be a hinge region derived from the extracellular domain of type 1 membrane proteins such as CD8, CD4, CD28 and CD7, but if the antigen binding site, the transmembrane domain, and the intracellular signaling domain can be linked between the cell membrane Any of these may be used without limitation. Also, it can be the wild type hinge region from these molecules or it can be changed.

The spacer domain may be referred to as a linking domain, and may include, for example, a hinge domain derived from CD28 and/or a hinge domain derived from CD8, and the entire or It may contain some.

The hinge domain and/or spacer domain may be at least one selected from the group consisting of a Myc epitope, a CD8 hinge domain, and Fc, and may specifically include a Myc epitope and a CD8 hinge domain. More specifically, the Myc epitope may include the amino acid sequence of SEQ ID NO: 17, and the CD8 hinge domain may include the amino acid sequence of SEQ ID NO: 18.

The transmembrane domain refers to a partial region that connects and fuses an extracellular domain and an intracellular signaling domain with each other and serves to anchor a chimeric antigen receptor to the plasma membrane of an immune cell. The transmembrane domain may be from natural, synthetic, semisynthetic or recombinant sources. The transmembrane domain is the alpha (α), beta (β) or zeta (ζ) chain of the T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22 , CD33, CD37, CD64, CD80, CD86, CD134, CD137 and may be any one selected from the group consisting of CD154, but is not limited thereto.

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

The intracellular signaling domain functions of immune cells (e.g., activation including the release of cytotoxic factors for target cells bound to chimeric antigen receptors and antigens, cytokine production, proliferation and cytotoxic activity, or other cellular response induced by the antigen binding), it corresponds to a part that serves to transmit a signal generated according to the binding of the chimeric antigen receptor to the inside of the immune cell. The intracellular signaling domain may be a part of a protein that transmits an effector signal and directs the cell to perform a specific function.

As the intracellular signaling domain, an intracellular signaling domain previously used in the development process of a chimeric antigen receptor may be used. Specifically, it may include only CD3ζ used in the first generation CAR (chimeric antigen receptor). And, as used in the second-generation CAR, a form in which a co-stimulatory domain (CD28 or CD137/4-1BB) and CD3ζ are combined can be used to improve responsiveness to immune cells. In addition, two or more co-stimulatory domains may be used as in the third-generation CAR, and at this time, to achieve expansion and persistence of immune cells including the CAR in vivo, the co-stimulatory domain is 4-1BB, CD28 or OX40, etc. can be combined. Furthermore, as used in the 4th generation CAR, additional genes encoding cytokines such as IL-12 or IL-15 can be included to allow the expression of additional CAR-based immunoproteins of cytokines, and the 5th generation CAR As used in, an interleukin receptor chain, for example, IL-2Rβ may be further included to enhance immune cells.

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 It may include at least one selected from the group consisting of zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d.

More specifically, activation of the immune cell by the intracellular signaling domain may be mediated by two different classes of intracellular signaling domains. For example, immune cell activation can be mediated by a primary signaling domain that initiates an antigen-dependent primary activation and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal. Accordingly, the intracellular signaling domain may include a primary signaling domain and a co-stimulatory signaling domain.

The primary signaling domain refers to a signaling domain that regulates immune cell activation in a stimulating or inhibiting manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. The ITAM containing the primary signaling domain may be TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD66d, etc., but is not limited thereto. More specifically, the primary signaling domain may be CD3ζ(zeta), but is not limited thereto.

The co-stimulatory signaling domain refers to an intracellular signaling domain of a co-stimulatory molecule. The costimulatory signaling domain is 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, may include a co-stimulatory signaling domain selected from the group consisting of CD83, etc., but is limited thereto It is not. Specifically, the co-stimulatory molecule may be DAP10, but is not limited thereto.

The chimeric antigen receptor may include two or more intracellular signaling domains, and in the case of including two or more intracellular signaling domains, the intracellular signaling domains may be serially connected to each other. Alternatively, they may be connected through a polypeptide linker consisting of 2 to 10 amino acids, and the linker sequence may be, for example, a glycine-serine continuous sequence. The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n and m are each 1 to 10), and for example, (GnS)m( n and m may each be 1 to 10), but are not limited thereto.

The chimeric antigen receptor may further include an immune function stimulating factor of immune cells, and for example, the immune function stimulating factor of immune cells may be an interleukin signal sequence. The interleukin signal sequence may induce expression of IL-12, IL-8, IL-2, etc., but is not limited thereto. In addition, when the immune cells are T cells, the immune function promoting factor may be IL-7, CCL19, etc., but is not limited thereto.

In a specific embodiment of the present invention, scFvs of two types of anti-B7-H3 antibodies (40F10 and 51H04) having the same amino acid sequences as described above are included in the extracellular domain, and CD28 linked to Myc and hinge domains are included in the extracellular domain. The chimeric antigen receptor of the present invention was prepared by designing a chimeric antigen receptor comprising a transit domain and an intracellular signaling domain, and CD3-zeta and DAP10 linked to the intracellular signaling domain.

As described above, when the chimeric antigen receptor of the present invention is expressed on the surface of immune cells and recognizes and binds to B7-H3, it can be induced to enhance cytotoxicity of immune cells or promote cytokine secretion of immune cells. . Therefore, when the antigen-binding site of the chimeric antigen receptor recognizes and binds to an antigen when cancer cells expressing B7-H3 are present, cytotoxicity of immune cells is enhanced and/or cytokine secretion is promoted by signal transduction. Therefore, it can be usefully used as a chimeric antigen receptor with excellent cytotoxicity to attack cancer cells.

Another aspect of the present invention provides a polynucleotide and an expression vector for expressing the chimeric antigen receptor.

The polynucleotide includes a nucleotide sequence encoding the chimeric antigen receptor.

In the present invention, the term "polynucleotide" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units, include not only natural nucleotides, but also analogs in which sugar or base sites are modified. do.

Encoding the chimeric antigen receptor means that the polynucleotide undergoes a normal protein expression process such as transcription and translation to encode genetic information that can synthesize a protein having the amino acid sequence of the chimeric antigen receptor of the present invention. means there is At this time, as described above, as well as a protein having an amino acid sequence completely identical to that of the chimeric antigen receptor, a protein having substantially the same amino acid sequence as the protein, or a protein encoding a protein having the same and/or similar activity as the protein Even polynucleotides may be included in the scope of the present invention.

Specifically, the polynucleotide of the present invention may include a nucleotide sequence encoding an antigen-binding variable fragment of an anti-B7-H3 antibody that specifically binds to the B7-H3, and more specifically, SEQ ID NO: 19 or sequence It may include the nucleotide sequence of number 20.

In addition, the polynucleotide of the present invention includes not only a nucleotide sequence encoding the antigen-binding variable fragment, but also a nucleotide sequence encoding another extracellular domain portion linked thereto, the transmembrane domain, and/or the intracellular signaling domain. can

Descriptions of the chimeric antigen receptors, such as the antibodies, antigen-binding variable fragments, extracellular domains, transmembrane domains, and intracellular signaling domains, are the same as those described above.

The polynucleotide may include the nucleotide sequence of SEQ ID NO: 21 or SEQ ID NO: 22. In a specific embodiment of the present invention, scFvs of two types of anti-B7-H3 antibodies (40F10 and 51H04) are included in the extracellular domain, and CD28 linked to Myc and hinge domains are transmembrane domains and intracellular signal transduction A polynucleotide containing the nucleotide sequence of SEQ ID NO: 21 or 22 was prepared to design and express a chimeric antigen receptor in which CD3-zeta and DAP10 are linked to an intracellular signaling domain.

The polynucleotide of the present invention may include a nucleotide sequence substantially identical to the nucleotide sequence listed above. The substantially identical nucleotide sequence includes, for example, cases in which the same amino acid can be synthesized when transcribed and translated, and is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more than the listed nucleotide sequences. It may be a nucleotide sequence having a homology of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, but is not limited thereto.

The polynucleotide encoding the chimeric antigen receptor may include an optimized nucleotide sequence according to the type of organism to be introduced and expressed, and the expression system of the organism such as transcription and translation. This is due to the degeneracy of codons, and therefore, there may be various combinations of nucleotide sequences capable of encoding proteins to be expressed, and all of them are included in the scope of the present invention. Modification of the polynucleotide according to the codon optimization may be determined according to the type of organism to which the chimeric antigen receptor of the present invention is to be expressed and applied. For example, the polynucleotide of the present invention is optimized for codon selection of mammals and primates. It may be a modified polynucleotide, and more specifically, it may be a polynucleotide optimized and modified to be suitable for expression and function in humans.

The expression vector of the present invention contains the above polynucleotide.

Since the polynucleotide contains the nucleotide sequence encoding the chimeric antigen receptor of the present invention, the expression vector containing the nucleotide sequence can be used to express and prepare the chimeric antigen receptor, and the chimeric antigen receptor It can serve to move the polynucleotide to a specific cell or organism so that it can be expressed, or can be used to store and store the polynucleotide.

The expression vector may be constructed using a prokaryotic or eukaryotic cell as a host.

For example, when the expression vector uses a prokaryotic cell as a host, a strong promoter capable of promoting 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. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator regions of the E coli tryptophan biosynthetic pathway (Yanofsky, C, J Bacteriol, (1984) 158: 1018-1024) , and the leftward promoter of phage λ (pLλ promoter, Herskowitz, I and Hagen, D, Ann Rev Genet, (1980) 14:399-445) can be used as a control region. When Bacillus bacteria are used as host cells, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl Environ Microbiol (1998) 64: 3932-3938; Mol Gen Genet (1996) 250: 734-741) or expression in Bacillus bacteria Any possible promoter can be used as a regulatory region. The expression vector is a plasmid often used in the art (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series , pET series and pUC19, etc.), phages (eg, λgt4·λB, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, SV40, etc.).

When the expression vector is a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, β-actin promoter, human hemoglobin promoter and human muscle creatine promoter) or mammalian Promoters derived from animal viruses (e.g., adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR of HIV A promoter, a promoter of Moloney virus, a promoter of Epstein Barr virus (EBV) and a promoter of Rouss sarcoma virus (RSV)) may be used, and may generally have a polyadenylation sequence as a transcription termination sequence. The expression vector may have a CMV promoter.

In addition, the expression 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). In addition, since the protein expressed by the expression vector of the present invention is a chimeric antigen receptor, considering its characteristics, the expressed protein can be easily purified through a protein A column or the like without an additional sequence for purification.

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

2. Immune cells expressing a chimeric antigen receptor for B7-H3 on their surface

Another aspect of the present invention provides immune cells characterized by enhanced cytotoxicity against cancer cells expressing B7-H3.

The immune cell expresses the above-described chimeric antigen receptor of the present invention on its surface.

The chimeric antigen receptor is described in “1. Novel anti-B7-H3 antibodies, antigen-binding fragments thereof, and chimeric antigen receptors (CARs) containing the same, and polynucleotides and expression vectors for expressing the same” are the same as described above. Specifically, the chimeric antigen receptor may include an antigen binding site capable of specifically binding to the B7-H3 antigen expressed in cancer cells.

Any cell that can induce the desired therapeutic effect by inducing immunity can be used as the immune cell without limitation. For example, natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytotoxic cells It may be any one selected from the group consisting of cytokine induced killer cells (CIK), macrophages and dendritic cells, but is not limited thereto. Therefore, immune cells expressing the chimeric antigen receptor according to the present invention on the cell surface include CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cell), CAR-T cells (Chimeric Antigen Receptor T Cell), and CAR-NKT cells (Chimeric Antigen Receptor Cell). Receptor Natural killer T Cell), CAR-macrophage (Chimeric Antigen Receptor Macrophage), and the like.

The T cells may be cytotoxic T lymphocytes (CTL), tumor infiltrating lymphocytes (TIL), T cells isolated from peripheral blood mononuclear cells (PBMC), etc., but are limited thereto. It is not.

The CAR-NK cell refers to a cell into which a chimeric antigen receptor is introduced into a natural killer cell, and a problem due to the persistent toxicity of cancer immunotherapy when using a conventional T cell-based CAR-T therapeutic agent , the risk of autoimmune disease, the problem of graft-versus-host disease (GVHD) for xenogeneic cell transplantation, the problem of off-target toxicity, etc. can be solved through the on/off switch, as well as various cancers. It has the advantage of being able to be used as a general-purpose therapeutic agent by allowing it to target cells.

The immune cell of the present invention is a chimeric antigen receptor comprising, as an extracellular domain, an antigen-binding variable fragment (scFv) of an antibody capable of specifically recognizing and binding to B7-H3, which can be specifically expressed in cancer cells. is expressed on the cell surface. Accordingly, when cancer cells expressing the B7-H3 are present, signal transduction may occur by the chimeric antigen receptor, and through this, the cytotoxicity of immune cells is further improved and the amount of cytokine secretion is increased. Therefore, the immune cells of the present invention may have an activity to attack and treat cancer cells.

In a specific embodiment of the present invention, a chimeric antigen receptor comprising the antigen-binding variable fragments of two types of B7-H3-specific antibodies of the present invention as extracellular domains was expressed on the surface of natural killer cells. When each of the two natural killer cells was co-cultured with the B7-H3-expressing cancer cell line A498, cytotoxicity was improved and the amount of cytokine secretion was significantly increased. In addition, the degree of degranulation of the A498 cells also increased, confirming that the immune cells of the present invention have an excellent therapeutic effect on cancer cells expressing B7-H3.

3. Therapeutic use of the immune cells of the present invention for cancer

Another aspect of the present invention provides a pharmaceutical composition for treating cancer comprising the immune cells.

The description of the immune cells, chimeric antigen receptors expressed therefrom, and the like, 'a novel anti-B7-H3 antibody, an antigen-binding fragment thereof, and a chimeric antigen receptor (CAR) containing the same and expressing the same' polynucleotides and expression vectors for processing' and '2. Immune cells expressing the chimeric antigen receptor for B7-H3 on their surface' are the same as those described for them, so description is omitted to avoid repetitive explanation.

In the present invention, the term “cancer” is used synonymously with “tumor” and refers to or refers to a mammalian physiological condition typically characterized by unregulated 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. For example, the cancer is lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colon cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, blood cancer, and multiple myeloma. , acute myelogenous leukemia, malignant lymphoma, thymic cancer, osteosarcoma, fibrotic tumors, and at least one selected from the group consisting of brain cancer, but is not limited thereto, and an antigen that can be recognized by the second chimeric antigen receptor Any cancer cell containing a cancer cell can be applied without limitation in the present invention.

In the present invention, the term "treatment" means suppression of the development of cancer, relief or elimination of symptoms.

The pharmaceutical composition may include 1 to 10 times, 2 to 10 times, or 5 to 8 times the number of immune cells compared to the number of tumor cells of the subject to be treated, but is not limited thereto.

In addition to pharmaceutical compositions, the composition may be in the form of quasi-drug compositions, health food compositions, and the like.

The composition for treating cancer of the present invention may further include a pharmaceutically acceptable carrier. The meaning of 'pharmaceutically acceptable' is that it does not inhibit the activity of the active ingredient and does not have toxicity more than is adaptable to the subject of application (prescription), and the 'carrier' facilitates the addition of the compound into cells or tissues. defined as a compound that

The pharmaceutical composition of the present invention may be administered alone or in combination with any convenient carrier, and such dosage form may be a single or repeated dosage form. The pharmaceutical composition may be a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, and suppositories. Solid preparations may include carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like, but are not limited thereto. Liquid preparations include solutions, suspensions, and emulsions such as water and propylene glycol solutions, but are not limited thereto, and may be prepared by adding appropriate colorants, flavoring agents, stabilizers, viscosifiers, and the like. For example, a powder may be prepared by simply mixing a tri-hydroxy derivative of polyunsaturated fatty acid, which is an active ingredient of the present invention, with a suitable pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose. Granules are obtained by mixing the tri-hydroxy derivative of the polyunsaturated fatty acid of the present invention, a suitable pharmaceutically acceptable carrier, and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone or hydroxypropylcellulose, followed by mixing water, It may be prepared using a wet granulation method using a solvent such as ethanol or isopropanol or a dry granulation method using compression force. In addition, tablets may be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then tableting them using a tableting machine.

The pharmaceutical composition is an oral agent, an injection (eg, intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalant, nasal agent, It may be administered as a vaginal agent, a rectal agent, a sublingual agent, a transdermal agent, a topical agent, and the like, but is not limited thereto. Depending on the route of administration, it may be formulated into an appropriate dosage unit formulation containing a pharmaceutically acceptable carrier, excipients, and vehicles that are commonly used and non-toxic.

The pharmaceutical composition may be administered at about 0.0001 mg/kg to about 10 g/kg daily, and at a daily dosage of about 0.001 mg/kg to about 1 g/kg. However, the dosage may vary depending on the degree of purification of the mixture, the patient's condition (age, sex, weight, etc.), the severity of the condition being treated, and the like. If necessary, for convenience, the total daily dose may be administered in several divided doses throughout the day.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example 1]

Construction of a single-chain variable fragment of an antibody that specifically binds to B7-H3

Among the anti-B7-H3 antibody sequences that can specifically bind to the B7-H3 (B7-Homolog3) protein expressed in cancer cells, a single chain variable fragment (single chain variable fragment) using a sequence that plays an important role in specific binding Two types of variable fragment (scFv) (40F10, 51H04) were prepared.

In the case of 40F10 scFv, a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and amino acids of SEQ ID NO: 4 It was designed to include a light chain variable region including light chain CDR1 having the sequence, light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 6. And in the case of 51H04 scFv, a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 9, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 10, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 11, and SEQ ID NO: 12 It was designed to include a light chain variable region including light chain CDR1 having the amino acid sequence, light chain CDR2 having the amino acid sequence of SEQ ID NO: 13, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 14.

In addition, a chimeric antigen receptor (CAR) was designed with an extracellular domain containing the two scFvs as antigen binding sites. Specifically, an extracellular domain containing the two scFvs as antigen-binding sites and CD28 as a transmembrane domain were linked to Myc and a hinge domain, and the transmembrane domain was linked with CD3-zeta as an intracellular signaling domain. The chimeric antigen receptor of the present invention was designed to have an intracellular signaling domain of a chimeric antigen receptor classified as a so-called third-generation CAR by adding and linking CD28 DAP10 as a co-stimulatory molecule. And, the nucleotide sequence encoding it was inserted into the lentiviral vector (FIG. 1).

[Example 2]

Preparation of immune cells expressing a chimeric antigen receptor for B7-H3 on the surface

Immune cells capable of expressing the chimeric antigen receptor of the present invention designed in Example 1 on the surface were prepared. A vector encoding the chimeric antigen receptor was introduced into NK92 cells, which are natural killer cells (NK cells), using lentivirus, and specifically, a natural killer expressing a chimeric antigen receptor expressing 40F10 scFv in the extracellular domain was introduced. cells (B7H3-40F10-Myc-PE), natural killer cells (B7H3-51H04) introduced with a chimeric antigen receptor expressing 51H04 scFv in the extracellular domain, and introduced with a chimeric antigen receptor with the extracellular domain sequence removed. Natural killer cells (ΔECTO-CAR), natural killer cells (Puro-NK92) introduced with a virus expressing an empty vector, and NK92 cells without vector were prepared. And, using the expression of myc present in the extracellular domain of the chimeric antigen receptor, the expression level of the chimeric antigen receptor was confirmed in natural killer cells. Specifically, the natural killer cells were stained using a Myc antibody attached to a PE fluorescent substance, and then the degree of staining of each natural killer cell was confirmed using flow cytometry (FIG. 2).

[Example 3]

Confirmation of cytotoxicity of B7-H3-CAR NK92 cells against cancer cells expressing B7-H3

Natural killer cells expressing the chimeric antigen receptor of the present invention on their surface as prepared in Example 2 contain scFv that specifically binds to B7-H3 as an antigen-binding site. Cytotoxicity to cancer cells expressing H3 was confirmed.

To this end, cancer cell lines expressing B7-H3 were identified and selected using flow cytometry, and then cytotoxicity of each natural killer cell was confirmed using the Calcein-AM method. In the Calcein-AM method, after staining the cancer cell line selected above with Calcein, co-cultured with the natural killer cells prepared in Example 2 for 4 hours according to each ratio, secreted into the culture medium It is a method to evaluate the toxicity of cells by measuring the amount of calcein. Among cancer cells, A498 renal cancer cell line expressing B7-H3 was selected as a cancer cell expressing B7-H3, and as a control, a Jurkat leukemia cell line that does not express B7-H3 was selected and co-cultured with natural killer cells. was used for As controls, natural killer cells (Puro92) into which an empty vector was introduced as prepared in Example 2 and natural killer cells (ΔECTO) into which an extracellular domain was deleted were used.

As a result, as shown in FIG. 3, when A498 cancer cells and natural killer cells were co-cultured for 2 hours, natural killer cells expressing chimeric antigen receptors containing 40F10 and 51H04 scFv of the present invention on their surfaces, respectively. showed significantly higher cytotoxicity compared to the control group. On the other hand, in the case of the Jurkat cell line with low expression of B7-H3, there was little difference in the level of cytotoxicity among natural killer cells (FIG. 3).

In addition, after co-culture of natural killer cells and A498 or Jurkat cancer cell lines as described above, the amount of cytokines secreted from natural killer cells was measured using an ELISA method and compared. As a result of confirming the amount of cytokine secretion through ELISA using an antibody that specifically binds to IFN-γ, as can be seen in FIG. 4, when compared to the amount of secretion when only natural killer cells exist without cancer cell lines, A498 expressing B7-H3 When co-cultured with cells, it was found that the amount of cytokine (IFN-γ) was significantly increased in two types of natural killer cells expressing the chimeric antigen receptor of the present invention. In contrast, in co-culture with Jurkat cells that hardly express B7-H3, the above-described increase in cytokines was not observed (FIG. 4).

Furthermore, natural killer cells secrete granules (degranulation) and use them to exhibit cytotoxicity to target cells. Therefore, in order to further confirm the cytotoxicity of the natural killer cells, it was confirmed how much degranulation of natural killer cells occurs during co-culture with cancer cells. The degree of degranulation of natural killer cells was determined by co-cultivating natural killer cells and cancer cells at a constant ratio for 4 hours, staining the expression level of CD107a, an indicator of degranulation, using a CD107a antibody conjugated with a fluorescent dye, and performing a flow cytometer analysis. can be used for analysis.

As a result of confirming the expression level of CD107α through flow cytometry, when the natural killer cells of the present invention expressing B7-H3 were co-cultured with the A498 cancer cell line, compared with the case of co-culture with the control natural killer cells, A498 It was confirmed that the expression level of CD107α was significantly increased. However, in the case of the Jurkat cancer cell line, which hardly expresses B7-H3, CD107α was expressed at a level similar to that of the control group cultured alone despite co-culture with natural killer cells (FIG. 5).

In view of the above experimental results, the scFvs (40F10, 51H04) of the two anti-B7-H3 antibodies of the present invention not only show specific binding ability to B7-H3, but also express the B7-H3. When recognizing and binding to B7-H3 of cancer cells that contain the scFv, the scFv-containing chimeric antigen receptor induces signal transduction in immune cells (natural killer cells) expressed on the surface, and thus immune cells kill cancer cells. It was confirmed that there is an effect of inducing various immune responses that can attack.

In the above, the present invention has been described in detail only for the described embodiments, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the technical idea of the present invention, and it is natural that these changes and modifications fall within the scope of the appended claims.

<110> Korea Research Institute of Bioscience & Biotechnology <120> Novel anti-B7H3 Chimeric Antigen Receptor and Immune Cell Expressing the Same <130> 2021DPA4253 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR1 of 40F10 scFv <400> 1 Gly Tyr Thr Phe Ser Ser Tyr Trp 1 5 <210> 2 <211> 8 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR2 of 40F10 scFv <400> 2 Ile Asn Pro Gly Asn Gly His Thr 1 5 <210> 3 <211> 16 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR3 of 40F10 scFv <400> 3 Val Ala Asp Pro Arg Arg Pro Lys Val Pro Thr Ala Leu Phe Val Tyr 1 5 10 15 <210> 4 <211> 6 <212> PRT <213> artificial sequence <220> <223> light chain CDR1 of 40F10 scFv <400> 4 Gln Gly Ile Gly Thr Trp 1 5 <210> 5 <211> 3 <212> PRT <213> artificial sequence <220> <223> light chain CDR2 of 40F10 scFv <400> 5 Ala Ala Ser One <210> 6 <211> 9 <212> PRT <213> artificial sequence <220> <223> light chain CDR3 of 40F10 scFv <400> 6 Gln Gln Ala Ile Asn Phe Pro Ile Thr 1 5 <210> 7 <211> 123 <212> PRT <213> artificial sequence <220> <223> heavy chain variable region of 40F10 scFv <400> 7 Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Ile Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ala Asp Pro Arg Arg Pro Lys Val Pro Thr Ala Leu Phe Val Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 107 <212> PRT <213> artificial sequence <220> <223> light chain variable region of 40F10 scFv <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Ala 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ile Asn Phe Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 9 <211> 8 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR1 of 51H04 scFv <400> 9 Gly Phe Asn Phe His Asp Tyr Ala 1 5 <210> 10 <211> 10 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR2 of 51H04 scFv <400> 10 Ile Arg His Gln Arg Tyr Gly Gly Thr Thr 1 5 10 <210> 11 <211> 15 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR3 of 51H04 scFv <400> 11 Ala Arg Gly Ser Ser Ser Ser Ser Ser Trp Tyr Leu Pro Asn Asp Tyr 1 5 10 15 <210> 12 <211> 6 <212> PRT <213> artificial sequence <220> <223> light chain CDR1 of 51H04 scFv <400> 12 Gln Asp Ile Ser Thr Trp 1 5 <210> 13 <211> 3 <212> PRT <213> artificial sequence <220> <223> light chain CDR2 of 51H04 scFv <400> 13 Lys Ala Ser One <210> 14 <211> 8 <212> PRT <213> artificial sequence <220> <223> light chain CDR3 of 51H04 scFv <400> 14 Gln Gln Tyr Asn Arg Phe Trp Thr 1 5 <210> 15 <211> 123 <212> PRT <213> artificial sequence <220> <223> heavy chain variable region of 51H04 scFv <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Thr Ser Gly Phe Asn Phe His Asp Tyr 20 25 30 Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Arg His Gln Arg Tyr Gly Gly Thr Thr Gln Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Gly Ile 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Ser Ser Ser Ser Ser Trp Tyr Leu Pro Asn Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 <210> 16 <211> 106 <212> PRT <213> artificial sequence <220> <223> light chain variable region of 51H04 scFv <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Arg Phe Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 10 <212> PRT <213> artificial sequence <220> <223> Myc epitopes <400> 17 Ala Asn Lys Asn Ser Ser Gln Lys Arg Ile 1 5 10 <210> 18 <211> 68 <212> PRT <213> artificial sequence <220> <223> CD8 hinge domain <400> 18 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> 19 <211> 759 <212> DNA <213> artificial sequence <220> <223> nucleotide sequence of full length 40F10 scFv <400> 19 caggtgcagc tggtagagtc tggggctgaa gtgaagaagc ctggggcttc agtgaagctg 60 tcctgcaagg cttctggcta caccttcagc agctactgga tgcactgggt gcgccaggcc 120 cctggacaac gccttgagtg gatgggagag attaatcctg gcaacggtca tactaactac 180 aacgagaagt tcaagtcacg cgtgacaatc actgtagaca aatccgcgag cacagcctac 240 atggagctca gcagcctgag atctgaggac acggccgtgt attactgtgt tgcagatccg 300 agaaggccta aagtaccaac tgctttgttt gtctattggg gccagggaac cctggtcacc 360 gtctcctcag ggctcggggg cctcggagga ggaggtagtg gcggaggagg ctccggtgga 420 tccagcggtg tgggttccga catccagatg acccagtctc catcttccgt gtctgcatct 480 gtaggagaca gagtcaccat cagttgtcgg gcgagtcagg gtattggcac ctggttagcc 540 tggtatcagc agaagccagg taaagcccct agactcttga tctatgctgc atccagtttg 600 gacagtgggg tcccatcgag attcagcgcc agtggatctg ggacagattt cactctcacc 660 atcagcagcc tgcagcctga agattttgca acttactact gtcaacaggc tatcaatttc 720 ccgatcacct tcggccaagg gacacgactg gaaatcaaa 759 <210> 20 <211> 759 <212> DNA <213> artificial sequence <220> <223> nucleotide sequence of full length 51H04 scFv <400> 20 caggtgcagc tggtggagtc tgggggaggc ttggtacagc caggacggtc cctgagactc 60 tcctgtacaa cctctggatt caatttccat gattatgctc tgtcctgggt ccggcaggct 120 ccagggaagg ggctggaatg ggtgagtttc attagacacc aacgttatgg tgggacaacg 180 cagtacgccg cgtctgtgaa aggcagattc accatctcaa gagacgattc caaaggcatc 240 gcatacctgc aaatgaacag tctgagagcc gaggacacgg ccgtgtatta ctgtgcgaga 300 ggatcttcca gcagcagctg gtaccttcca aatgactact ggggccaggg aaccctggtc 360 accgtctcct cagggctcgg gggcctcgga ggaggaggta gtggcggagg aggctccggt 420 ggatccagcg gtgtgggttc cgacatccag atgacccagt ctccttccac cctgtctgca 480 tctgtaggag acagagtcac catcacttgc cgggccagtc aggacacattag tacctggttg 540 gcctggtatc agcagaagcc agggaaagcc cctaaactcc tgatctataa ggcgtctagt 600 ttacaaagtg gggtcccatc aagattcagc ggcagtggat ctgggacaga attcactctc 660 actatcagca gcctgcagcc tgatgatttt gcaacttatt actgccaaca gtataatagg 720 ttttggacat tcggccaagg gaccaaggtg gaaatcaaa 759 <210> 21 <211> 1776 <212> DNA <213> artificial sequence <220> <223> nucleotide sequence of full length 40F10 scFv-CAR <400> 21 atggccttac cagtgaccgc cttgctcctg ccgctggcct tactgctcca cgccgccagg 60 ccggcggccc agccggccca ggtgcagctg gtagagtctg gggctgaagt gaagaagcct 120 ggggcttcag tgaagctgtc ctgcaaggct tctggctaca ccttcagcag ctactggatg 180 cactgggtgc gccaggcccc tggacaacgc cttgagtgga tgggagagat taatcctggc 240 aacggtcata ctaactacaa cgagaagttc aagtcacgcg tgacaatcac tgtagacaaa 300 tccgcgagca cagcctacat ggagctcagc agcctgagat ctgaggacac ggccgtgtat 360 tactgtgttg cagatccgag aaggcctaaa gtaccaactg ctttgtttgt ctattggggc 420 cagggaaccc tggtcaccgt ctcctcaggg ctcgggggcc tcggaggagg aggtagtggc 480 ggaggaggct ccggtggatc cagcggtgtg ggttccgaca tccagatgac ccagtctcca 540 tcttccgtgt ctgcatctgt aggagacaga gtcaccatca gttgtcgggc gagtcagggt 600 attggcacct ggttagcctg gtatcagcag aagccaggta aagcccctag actcttgatc 660 tatgctgcat ccagtttgga cagtggggtc ccatcgagat tcagcgccag tggatctggg 720 acagatttca ctctcaccat cagcagcctg cagcctgaag attttgcaac ttactactgt 780 caacaggcta tcaatttccc gatcaccttc ggccaaggga cacgactgga aatcaaaggc 840 ccgggaggcc gcgaacaaaa actcatctca gaagaggatc tgaagcttgg ggtcaccgtc 900 tcttcagcgc tgagcaactc catcatgtac ttcagccact tcgtgccggt cttcctgcca 960 gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1020 cagcccctgt ccctgcgccc agaggcatgc cggccagcgg cggggggcgc agtgcacacg 1080 agggggctgg ataagcttgt gaaagggaaa cacctttgtc caagtcccct atttcccgga 1140 ccttctaagc ccttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 1200 ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1260 agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1320 tatgccccac cacgcgactt cgcagcctat gaattcctgt gcgcacgccc acgccgcagc 1380 cccgcccaag aagatggcaa agtctacatc aacatgccag gcaggggcgc ggccgctaga 1440 gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1500 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1560 gaccctgaga tgggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1620 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1680 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1740 gacgcccttc acatgcaggc cctgccccct cgctaa 1776 <210> 22 <211> 1776 <212> DNA <213> artificial sequence <220> <223> nucleotide sequence of full length 51H04 scFv-CAR <400> 22 atggccttac cagtgaccgc cttgctcctg ccgctggcct tactgctcca cgccgccagg 60 ccggcggccc agccggccca ggtgcagctg gtggagtctg ggggaggctt ggtacagcca 120 ggacggtccc tgagactctc ctgtacaacc tctggattca atttccatga ttatgctctg 180 tcctgggtcc ggcaggctcc agggaagggg ctggaatggg tgagtttcat tagacaccaa 240 cgttatggtg ggacaacgca gtacgccgcg tctgtgaaag gcagattcac catctcaaga 300 gacgattcca aaggcatcgc atacctgcaa atgaacagtc tgagagccga ggacacggcc 360 gtgtattact gtgcgagagg atcttccagc agcagctggt accttccaaa tgactactgg 420 ggccagggaa ccctggtcac cgtctcctca gggctcgggg gcctcggagg aggaggtagt 480 ggcggaggag gctccggtgg atccagcggt gtgggttccg acatccagat gacccagtct 540 ccttccaccc tgtctgcatc tgtaggagac agagtcacca tcacttgccg ggccagtcag 600 gacattagta cctggttggc ctggtatcag cagaagccag ggaaagcccc taaactcctg 660 atctataagg cgtctagttt acaaagtggg gtcccatcaa gattcagcgg cagtggatct 720 gggacagaat tcactctcac tatcagcagc ctgcagcctg atgattttgc aacttattac 780 tgccaacagt ataataggtt ttggacattc ggccaaggga ccaaggtgga aatcaaaggc 840 ccgggaggcc gcgaacaaaa actcatctca gaagaggatc tgaagcttgg ggtcaccgtc 900 tcttcagcgc tgagcaactc catcatgtac ttcagccact tcgtgccggt cttcctgcca 960 gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1020 cagcccctgt ccctgcgccc agaggcatgc cggccagcgg cggggggcgc agtgcacacg 1080 agggggctgg ataagcttgt gaaagggaaa cacctttgtc caagtcccct atttcccgga 1140 ccttctaagc ccttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 1200 ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1260 agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1320 tatgccccac cacgcgactt cgcagcctat gaattcctgt gcgcacgccc acgccgcagc 1380 cccgcccaag aagatggcaa agtctacatc aacatgccag gcaggggcgc ggccgctaga 1440 gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1500 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1560 gaccctgaga tgggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1620 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1680 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1740 gacgcccttc acatgcaggc cctgccccct cgctaa 1776

Claims (14)

an extracellular binding domain containing an antigen binding site that specifically binds to B7-H3 (B7-Homolog3);
transmembrane domain; and
Including; intracellular signaling domain (intracellular signaling domain)
As a chimeric antigen receptor (CAR),
The antigen-binding site that specifically binds to B7-H3 is a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and SEQ ID NO: 3 or sequence The heavy chain variable region comprising the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 11 and the light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, the light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and SEQ ID NO: 6 or A chimeric antigen receptor that is a single chain variable fragment (scFv) of an anti-B7-H3 antibody comprising a light chain variable region comprising a light chain CDR3 having the amino acid sequence of SEQ ID NO: 14. The method of claim 1,
The antigen-binding site that specifically binds to the B7-H3,
A chimeric antigen receptor that is a single chain variable fragment of an anti-B7-H3 antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 15. The method of claim 1,
The antigen-binding site that specifically binds to the B7-H3,
A chimeric antigen receptor that is a single chain variable fragment of an anti-B7-H3 antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16. The method of claim 1,
The extracellular domain further comprises at least one selected from the group consisting of a hinge domain and a spacer domain. The method of claim 4,
The hinge domain or spacer domain is at least one selected from the group consisting of a Myc epitope, a CD8 hinge domain, and Fc, the chimeric antigen receptor. The method of claim 1,
The transmembrane domain is the alpha (α), beta (β) or zeta (ζ) chain of the T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, Any one selected from the group consisting of CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, chimeric antigen receptor. The method of claim 1,
The intracellular signaling domain,
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 chimeric antigen receptor comprising at least one selected from the group consisting of CD22, CD79a, CD79b and CD66d. The method of claim 1,
The intracellular signaling domain,
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, At least one selected from the group consisting of CD22, CD79a, CD79b and CD66d as a primary signaling domain;
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 at least one selected from the group consisting of DAP12, PD-1, LIGHT, NKG2C, B7-H3 and CD83 as a co-stimulatory signaling domain. A polynucleotide comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1 to 8. An expression vector comprising the polynucleotide of claim 9. An immune cell expressing the chimeric antigen receptor according to any one of claims 1 to 8 on its surface. The method of claim 11,
The immune cell is any one selected from the group consisting of natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine induced killer cells (CIK), macrophages and dendritic cells. Which is, immune cells. A pharmaceutical composition for treating cancer, comprising the immune cells of claim 11. The method of claim 13,
The cancers include lung cancer, stomach cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, colon cancer, colon cancer, esophageal cancer, skin cancer, thyroid cancer, kidney cancer, liver cancer, head and neck cancer, bladder cancer, prostate cancer, blood cancer, multiple myeloma, acute myeloid leukemia , Malignant lymphoma, thymic cancer, osteosarcoma, at least one selected from the group consisting of fibrotic tumors and brain cancer, the pharmaceutical composition for the treatment of cancer.

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