KR102290335B1 - Chimeric antigen receptor targeting CD30 and use thereof - Google Patents

Abstract

The present invention relates to a chimeric antigen receptor targeting CD30 and uses thereof, and more particularly, to an antibody targeting the CD30, a chimeric antigen receptor comprising the antibody, a CAR-T cell expressing the chimeric antigen receptor and uses for treatment of a disease related to CD30 expression using the same.

Description

Chimeric antigen receptor targeting CD30 and use thereof

The present invention relates to a chimeric antigen receptor targeting CD30 and uses thereof, and more particularly to an antibody targeting CD30, a chimeric antigen receptor comprising the antibody, a CAR-T cell expressing the chimeric antigen receptor and It relates to the therapeutic use of CD30 expression-related diseases using the same.

Chimeric antigen receptor (CAR) T-cells are molecules that combine antibody-based specificity for a desired antigen with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits specific anti-cancer immune activity. In general, a chimeric antigen receptor (CAR) comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain. The extracellular antigen binding domain may include a single-chain variable fragment (scFv) targeting the identified tumor antigen.

CAR can be expressed on the surface of T cells, which are immune effector cells, using a gene transfection technique. Upon binding of a CAR expressed on the surface of a T cell to a target tumor antigen, the CAR can activate the T cell to initiate a specific antitumor response in an antigen-dependent manner.

On the other hand, CD30, also known as TNFRSF8, is a member of the tumor necrosis factor receptor family and is a tumor marker. CD30 is expressed in T cells and B cells, and binds to TRAF5 and TRAF2. It is reported that CD30 can lead to abnormal proliferation of B cells or T cells in abnormal signaling, leading to tumorigenesis such as Hodgkin's lymphoma.

Hodgkin's lymphoma is one of the malignant tumors occurring in lymphoid tissue, and is a malignant lymphoma characterized by Reed-Stenberg cells or abnormally proliferated lymphocytes and histocytes. It is named after the British doctor Thomas Hodgkin, who first discovered the disease. Hodgkin's lymphoma is a classical Hodgkin's lymphoma, characterized by large cells called Reed-Sternberg cells, and abnormally proliferated lymphocytes and histiocytic cells (popcorn cells). It can be classified as nodular lymphocyte-dominant Hodgkin's lymphoma.

Some treatments for Hodgkin's lymphoma are similar to treatments for other cancers, such as chemotherapy or radiation therapy, stem cell transplantation or bone marrow transplantation, targeted therapy or biological therapy. Antibody-based cell immunotherapy has been confirmed to have practical clinical benefit for patients with B-cell non-Hodgkin's lymphoma, but most patients have problems with recurrence or secondary resistance, so There is a need for effective immunotherapeutic agents for treatment.

Under this background, in the present invention, as a result of earnest efforts to develop a therapeutic agent for a disease related to CD30 expression, such as Hodgkin's lymphoma, an antibody targeting CD30 was selected, and finally, including the anti-CD30 antibody, CD30 was targeted chimeric antigen receptor and CAR-T cells were prepared, and it was confirmed that the prepared CD30 target CAR-T cells bind to CD30 and ultimately effectively kill CD30-expressing tumor cells, thereby completing the present invention.

One object of the present invention is to provide a monoclonal antibody or fragment thereof that specifically binds to CD30.

Another object of the present invention is to provide a chimeric antigen receptor comprising the monoclonal antibody or fragment thereof.

Another object of the present invention is to provide a method for treating a disease related to CD30 expression using the immune effector cell.

Another object of the present invention is to provide a composition and a diagnostic method for diagnosing a disease related to CD30 expression comprising the monoclonal antibody or fragment thereof.

Each description and embodiment disclosed herein is also applicable to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

In addition, in the entire specification of the present application, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components unless otherwise stated. do.

Hereinafter, the present invention will be described in more detail.

As an aspect for achieving the above object, the present invention provides a monoclonal antibody or fragment thereof that specifically binds to CD30.

As used herein, the term "antibody" refers to a protein molecule that serves as a receptor that specifically recognizes an antigen, including immunoglobulin molecules that are immunologically reactive with a specific antigen, polyclonal antibody, monoclonal antibody , both whole antibodies and antibody fragments. The term also includes chimeric antibodies (eg, humanized murine antibodies) and bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies and tetrabodies. The whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4. The antibody fragment refers to a fragment having an antigen-binding function, and includes Fab, Fab', F(ab')2 and Fv. The Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (CH1 domain), and has one antigen-binding site.

Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'.

Fv (variable fragment) refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In double chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in single chain Fv (scFv), the variable region of the heavy chain and the variable region of the light chain are generally covalently bonded through a peptide linker (SEQ ID NO: 9). is connected with Such antibody fragments can be obtained using proteolytic enzymes (for example, the entire antibody can be restricted with papain to obtain Fab, and pepsin can be used to obtain F(ab')2 fragments. Specifically, the papain enzyme can divide the antibody monomer into two Fab (fragment antigen binding) fragments and one Fc (fragment crystallizable) fragment, and pepsin can fly into one f(ab')2 fragment and one Fc fragment. have.

As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.

Typically, immunoglobulins have heavy and light chains, each heavy and light chain comprising a constant region and a variable region (these regions are also known as domains). The variable regions of the light and heavy chains comprise three variable regions and four framework regions called complementarity-determining regions (hereinafter referred to as "CDRs"). The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are typically called CDR1, CDR2, CDR3 sequentially, starting from the N-terminus, and are also identified by the chain in which the specific CDR is located.

As used herein, the term "human antibody" refers to a molecule derived from human immunoglobulin, wherein the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of the amino acid sequence of human immunoglobulin. Human antibodies are commonly used in the treatment of human diseases, which may have three or more potential advantages. First, it interacts better with the human immune system, for example in complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, it is possible to more efficiently destroy the target cells. Second, it has the advantage that the human immune system does not recognize the antibody as foreign. Third, it has the advantage that the half-life in the human circulation is similar to that of naturally occurring antibodies even when the drug is administered in a lower amount and less frequently. Therefore, the human monoclonal antibody according to the present invention shows a strong affinity for CD30, and shows low immunogenicity because both the heavy and light chain domains are human-derived, so it is useful in the treatment of diseases related to CD30 expression such as Hodgkin's lymphoma. can be used

The monoclonal antibody that specifically binds to CD30 may be a human monoclonal antibody that specifically binds to CD30, but is not limited thereto.

The human monoclonal antibody specifically binding to CD30 includes a heavy chain CDR1 set forth in SEQ ID NO: 1; heavy chain CDR2 set forth in SEQ ID NO:2; and a heavy chain variable region comprising a heavy chain CDR3 set forth in SEQ ID NO: 3, and a light chain CDR1 set forth in SEQ ID NO: 4; light chain CDR2 set forth in SEQ ID NO: 5; And it may be a human monoclonal antibody or a fragment thereof comprising a light chain variable region comprising the light chain CDR3 set forth in SEQ ID NO: 6, more specifically, the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region set forth in SEQ ID NO: 8 It may be a human monoclonal antibody comprising a region amino acid sequence, but is not limited thereto. In an embodiment of the present invention, a human monoclonal antibody comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 8 was designated as 1B6F11.

In addition, specifically, the 1B6F11 antibody may be encoded by the nucleotide sequence of SEQ ID NO: 10.

When the human monoclonal antibody of the present invention includes a constant region, it may include a constant region derived from IgG, IgA, IgD, IgE, IgM, a combination thereof, or a hybrid thereof.

As used herein, the term "combination" means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin constant region of the same origin forms a bond with a single-chain polypeptide of a different origin. For example, dimers or multimers may be formed from two or more constant regions selected from the group consisting of constant regions of IgG, IgA, IgD, IgE and IgM.

As used herein, the term "hybrid" means that sequences corresponding to the constant regions of immunoglobulin heavy chains of two or more different origins exist in the constant regions of single-chain immunoglobulin heavy chains, for example, IgG, IgA, IgD, Hybridization of domains consisting of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgE and IgM is possible.

On the other hand, it is also possible to combine or hybridize the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, which are subtypes of IgG.

In addition, when the CD30-specific human monoclonal antibody of the present invention includes a light chain constant region, the light chain constant region is lambda (λ or kappa (Kappa may be derived from a light chain, but is not limited thereto).

As used herein, the term "" corresponds to a tumor marker as a member of the tumor necrosis factor receptor family, also known as TNFRSF8. CD30 is expressed in T cells and B cells, and is known to bind to TRAF5 and TRAF2. The CD30 may be included without limitation as long as it is mammalian CD30, but may preferably refer to human CD30. For the purpose of the present invention, the CD30 binds to CD30 expressed in cells of a malignant tumor, specifically hematological cancer, more specifically lymphoma, and more specifically Hodgkin lymphoma, and growth, onset or progression of Hodgkin's lymphoma. It may mean a protein capable of inhibiting, but is not limited thereto. In addition, the CD30 protein includes, but is not limited to, natural or mutant CD30 protein. The native CD30 protein generally refers to a polypeptide comprising the amino acid sequence of the native CD30 protein, and the amino acid sequence of the native CD30 protein generally refers to an amino acid sequence found in naturally occurring CD30. Information on the CD30 can be obtained from a known database such as GenBank of the National Institutes of Health.

It should be understood that the protein, polypeptide and/or amino acid sequence encompassed by the present invention includes functional variants or homologues that have at least the same or similar function as the protein or polypeptide.

In the present invention, functional variants may be proteins or polypeptides obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of the protein and/or polypeptide. For example, functional variants may have an amino acid sequence that differs due to substitutions, deletions and/or insertions of one or more amino acids, such as 1 to 30, 1 to 20 or 1 to 10 or 1, 2, 3, 4 or 5. It may include a protein or polypeptide having Functional variants are capable of substantially retaining the biological properties of an unmodified protein or polypeptide (substitution, deletion or addition). For example, functional variants are at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% of the biological activity (such as antigen binding capacity) of the original protein or polypeptide. , 95%, 96%, 97%, 98%, 99% or 100%.

In the present invention, homologues have at least about 85% amino acid sequence homology with the protein and/or polypeptide (eg, about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or more) of a protein or polypeptide (eg, an antibody capable of specifically binding CD30 or a fragment thereof).

In the present invention, homology generally refers to similarity or correlation between two or more sequences.

antibody division order SEQ ID NO: 1B6F11 heavy chain variable region
(chain variable-region:VH) QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCASGSGNYYYYMDVWGKGTTVTVSSGSASAPTLTS 7 VH-CDR1 SNWWS One VH-CDR2 EIHSGSTN 2 VH-CDR3 GSGNYYYYMDV 3 light chain variable region
(chain variable-region: VL) ELVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVAWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAHWVFGGGTKLTVL 8 VL-CDR1 SGSSSNIGNNYVA 4 VL-CDR2 DNNK 5 VL-CDR3 GTWDSSLSAH 6 Linker GGGSSRSSSSGGGGSGGGG 9

DNA sequence for encoding 1B6F11 SEQ ID NO: GAGCTCGTGCTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTGGCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGCAGCCTGAGTGCGCATTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTCCTAGGTGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGTCTGGTTCGGGGAACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAACCCTTACTAGT 10

In a specific example of the present invention, the CD30-specific human monoclonal antibody 1B6F11 was prepared, and the binding ability of CD30 was examined. As a result, a mean value of 157 was shown. It was confirmed that the 1B6F11 antibody of the present invention had excellent binding ability to human CD30 ( FIGS. 1A and 1B ).

In another aspect, the present invention provides a method for preparing the human monoclonal antibody.

As used herein, the term “human monoclonal antibody” is the same as described above.

The monoclonal antibody of the present invention can be easily prepared by a known monoclonal antibody production technique. For example, a method for producing a monoclonal antibody can be performed by preparing a hybridoma using B lymphocytes obtained from an immunized animal (Koeher and Milstein, 1976, Nature, 256:495), or by phage display (phage display). display) technology, but is not limited thereto.

The antibody library using the phage display technology is a method of expressing the antibody on the surface of a phage by obtaining an antibody gene directly from B lymphocytes without producing a hybridoma. Using phage display technology, many existing difficulties related to the generation of monoclonal antibodies by B-cell immortalization can be overcome. In general, the phage display technology comprises the steps of 1) inserting an oligonucleotide of a random sequence into a gene region corresponding to the N-terminus of the coat protein pIII (or pIV) of the phage; 2) expressing a fusion protein of a portion of a native envelope protein and a polypeptide encoded by the random sequence oligonucleotide; 3) treating a receptor material capable of binding to the polypeptide encoded by the oligonucleotide; 4) eluting the peptide-phage particles bound to the receptor using a low pH or binding competitive molecule; 5) amplifying the eluted phage in a host cell by panning; 6) repeating the method to obtain the desired amount; and 7) determining the sequence of the active peptide from the DNA sequence of the phage clones selected by panning.

Specifically, the method for producing a monoclonal antibody of the present invention can be performed using phage display technology. One of ordinary skill in the art is skilled in the art of known phage display techniques, for example, Barbas et al. (METHODS: A Companion to Methods in Enzymology 2:119, 1991 and J. Virol. 2001 Jul; 75(14):6692-9) and Winter et al. (Ann. Rev. Immunol. 12:433, 1994), each step of the manufacturing method of the present invention can be easily performed with reference to the known method. Phages that can be used to construct antibody libraries include, but are not limited to, for example, filamentous phages, fd, M13, f1, If1, Ike, Zj/Z, Ff, Xf, Pf1 or Pf3 phages. it's not going to be In addition, a vector that can be used for expression of a heterologous gene on the surface of the filamentous phage includes, for example, a phage vector such as fUSE5, fAFF1, fd-CAT1 or fdtetDOG, or a phagemid such as pHEN1, pComb3, pComb8 or pSEX. vectors, but are not limited thereto. In addition, the helper phage that can be used to provide the wild-type envelope protein required for successful re-infection of the recombinant phage for amplification includes, for example, M13K07 or VSCM13, but is not limited thereto.

A polynucleotide encoding a hybridoma-derived monoclonal antibody or phage display clone of the present invention can be readily isolated and sequenced using conventional procedures. For example, oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions from hybridoma or phage template DNA may be used. Once the polynucleotide is isolated, it can be placed into an expression vector, which can then be introduced into an appropriate host cell to produce the desired monoclonal antibody from a transformed host cell (ie, a transformant). Therefore, the method for producing a human monoclonal antibody of the present invention may be a method for producing a human monoclonal antibody, comprising amplifying an expression vector containing a polynucleotide encoding a human monoclonal antibody, but is not limited thereto. does not

In another aspect, the present invention provides a chimeric antigen receptor comprising the antibody that specifically binds to CD30.

In the present invention, the term “antibody that specifically binds to CD30” is as described above.

In the present invention, a chimeric antigen receptor (CAR) targeting CD30 was prepared using the anti-CD30 antibody 1B6F11.

Specifically, the chimeric antigen receptor is a CD30-binding domain; transmembrane domain; costimulatory domain; And it may be a chimeric antigen receptor (CAR) comprising an intracellular signal transduction domain, wherein the CD30-binding domain comprises an antibody or fragment thereof capable of specifically binding to CD30. can do.

More specifically, the antibody comprises a heavy chain variable region comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 1, CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and CDR3 represented by the amino acid sequence of SEQ ID NO: 3, and SEQ ID NO: A chimeric antigen receptor comprising 1B6F11 having a light chain variable region comprising CDR1 represented by the amino acid sequence of 4, CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and CDR3 represented by the amino acid sequence of SEQ ID NO: 6 can

As used herein, the term “chimeric antigen receptor (CAR)” generally refers to a fusion protein containing an antigen and an extracellular domain having the ability to bind one or more intracellular domains, wherein the CAR is a chimeric antigen receptor T cell ( CAR-T). A CAR may comprise an antigen (eg, tumor associated antigen (TAA)) binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain. In addition, the CAR can be combined with a T cell receptor-activating intracellular domain based on the antigen (eg CD30) specificity of the antibody, wherein the genetically modified CAR-expressing T cell is specific for the target antigen-expressing malignant cell. can be identified and eliminated.

In the present invention, the term "CD3" is a member of the tumor necrosis receptor (TNFR) family and is predominantly expressed on terminally differentiated B cells, such as memory B cells and plasma cells. CD30 is expressed on tumor cells (eg, Hodgkin's lymphoma cells) or localized on the tumor cell surface. A CD30 of the invention may comprise a protein comprising mutations of the full-length wild-type CD30, eg, point mutations, fragments, insertions, deletions, and splice variants.

In the present invention, the term “CD30-binding domain” generally refers to a domain capable of specifically binding to a CD30 protein. For example, the CD30-binding domain may contain an anti-CD30 antibody or fragment thereof capable of specifically binding to a human CD30 polypeptide or fragment thereof expressed in B cells.

In the present invention, the term "binding domain" refers to "extracellular domain", "extracellular binding domain", "antigen-specific binding domain" and May be used interchangeably with "extracellular antigen-specific bidding domain", and refers to a CAR domain or fragment having the ability to specifically bind to a target antigen, CD30.

In the present invention, the anti-CD30 antibody or fragment thereof is the above-described anti-CD30 antibody, a monoclonal antibody, specifically scFv (single chain variable fragment), and 1B6F11 was used in the present invention.

In the present invention, the term "transmembrane domain" generally refers to a domain of a CAR that passes through a cell membrane and is connected to an intracellular signaling domain to play a signaling role. The transmembrane domain may be derived from a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

In the present invention, the term "costimulatory domain" generally refers to an intracellular domain capable of providing immune-stimulatory molecules, which are cell surface molecules necessary for an effective response of lymphocytes to antigens. The costimulatory domain described above may comprise a costimulatory domain of CD28, and may comprise a costimulatory domain of the TNF receptor family, such as the costimulatory domains of OX40 and 4-1BB.

In the present invention, the term "intracellular signal transduction domain" generally refers to a domain located inside a cell and capable of transmitting a signal. In the present invention, the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor. For example, the intracellular signaling domain may be selected from a CD3ζ intracellular domain, a CD28 intracellular domain, a CD28 intracellular domain, a 4-1BB intracellular domain and an OX40 intracellular domain.

In the present invention, a hinge region located between the C terminus of the CD30-binding domain and the N terminus of the transmembrane domain may be further included, and the hinge region may be derived from CD8α. The "hinge region" generally refers to a linking region between an antigen-binding region and an immune cell Fc receptor (FcR)-binding region.

In the present invention, a signal peptide may be further included at the N-terminus of the CD30-binding domain, and the "signal peptide" generally refers to a peptide chain for guiding protein transduction. . The signal peptide may be a short peptide with a length of 5 to 30 amino acids.

The polynucleotide encoding the CD30-binding domain may specifically be a polynucleotide encoding the 1B6F11 antibody, and the specific nucleotide sequence is as described above.

As used herein, the term "polynucleotide" generally refers to a nucleic acid molecule, deoxyribonucleotide or ribonucleotide, or an analog thereof, separated by any length. In one embodiment, the polynucleotide of the present invention is (1) in-vitro amplification, such as polymerase chain reaction (PCR) amplification; (2) cloning and recombination; (3) purification such as digestion and gel electrophoretic separation; (4) It can be prepared through synthesis such as chemical synthesis, and specifically, the isolated polynucleotide is prepared by recombinant DNA technology. In the present invention, the nucleic acid for encoding the antibody or antigen-binding fragment thereof is prepared by various methods known in the art, including, but not limited to, restriction fragment operation of synthetic oligonucleotides or application of SOE PCR. can be manufactured.

In one aspect, the present invention relates to a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR).

In the present invention, the term "vector (expression vector)" refers to a gene preparation including essential regulatory elements such as a promoter so that a target gene can be expressed in an appropriate host cell. The vector may be selected from one or more of a plasmid, a retroviral vector and a lentiviral vector. Upon transformation into an appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself.

In addition, the vector may contain expression control elements that allow the coding region to be accurately expressed in a suitable host. Such regulatory elements are well known to those skilled in the art and include, for example, promoters, ribosome-binding sites, enhancers and other regulatory elements for regulating gene transcription or mRNA translation. can do. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but in general, 5' ratios participating in transcription initiation and translation initiation, such as TATA box, capped sequence, CAAT sequence, etc., respectively - contains a transcribed sequence, and a 5' or 3' non-translated sequence. For example, the 5' non-transcriptional expression control sequence may include a promoter region that may include a promoter sequence for transcription and control of a functionally linked nucleic acid.

In addition, the promoter is operably linked to induce expression of an anti-hCD30 antibody (scFv), which is a CD30-binding domain, wherein “operably linked” refers to a nucleic acid expression control sequence to perform a general function. It means that the nucleic acid sequence encoding the target protein is functionally linked. The operative linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation using enzymes generally known in the art.

Methods for introducing and expressing genes into cells are known in the art. With respect to the expression vector, the vector can be easily introduced into a host cell by any method in the art. For example, the expression vector may be transferred into a host cell by physical, chemical, or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, eg, Sambrook et al, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY. A preferred method for introduction of polynucleotides into host cells is calcium phosphate transfection.

Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian, eg, human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses and adeno-associated viruses, and the like.

Chemical means for introducing polynucleotides into host cells include colloidal component acid solutions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. include An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle). Other methods are available for state-of-the-art targeted delivery of nucleic acids, such as delivery of polynucleotides using targeted nanoparticles or other suitable sub-micron sized delivery systems.

When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid preparations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. Nucleic acids associated with lipids may be encapsulated within the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, captured within the liposome, or complexed with the liposome; It may be dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with micelles, or otherwise associated with a lipid. The lipid, lipid/DNA or lipid/expression vector association composition is not limited to any particular structure in solution.

In the present invention, the term “immune effector cell” is a cell expressing the chimeric antigen receptor (CAR) of the present invention, and may be a mammal-derived cell, specifically, a T cell or a natural killer (NK) cell. . The immune effector cell expressing the chimeric antigen receptor (CAR) may be prepared by introducing the CAR vector of the present invention into an immune effector cell, for example, a T cell or NK cell.

Specifically, the CAR vector may be introduced into T cells or natural killer cells by methods known in the art such as electroporation, lipofectamine 2000, Invitrogen, and the like. For example, immune effector cells can be transfected with a lentiviral vector to integrate the viral genome carrying the CAR molecule into the host genome to ensure long-term and stable expression of the target gene. As another example, a transposon may be used to introduce a CAR transport plasmid and a transferase transport plasmid into a target cell. As another example, a CAR molecule can be added to the genome by a gene editing method (eg CRISPR/Cas9).

In a specific embodiment of the present invention, a lentiviral vector into which a polynucleotide encoding hCD30-CAR is inserted was prepared, and the prepared vector was transformed into T cells to prepare hCD30-CAR-T cells. The prepared hCD30-CAR-T cells express the chimeric antigen receptor targeting CD30 of the present invention.

Immune effector cells for preparing immune effector cells expressing the chimeric antigen receptor (CAR) can be obtained from a subject. In the present invention, the term "subject" may include a living organism from which an immune response can be elicited, such as a mammal, as other examples, humans, dogs, cats, mice, rats, and transgenic species thereof. may include.

The T cells can be obtained from a number of sources including, without limitation, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells can be obtained from blood units collected from a subject using any of a number of techniques known to those of ordinary skill in the art, for example, Ficoll™ isolation. Cells can be obtained by apheresis from blood, and apheresis products can typically contain T cells, monocytes, granulocytes, lymphocytes including B cells, other nucleated leukocytes, red blood cells, and platelets.

In addition, the cells collected by the apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by countercurrent centrifugation.

In a specific embodiment of the present invention, activated T cells are isolated from peripheral blood mononuclear cells (PBMCs), and then hCD30-CAR-T cells are transduced into T cells with hCD30-CAR lentivirus. prepared. The hCD30 peptide binding ability of the prepared hCD30-CAR-T cells was confirmed. Specifically, as shown in FIG. 2 , it was confirmed that hCD30-CAR-T cells expressing CD4 or CD8 binding to hCD30 increased as the hCD30 peptide increased ( FIG. 2 , Q2 ). This means that the hCD30-CAR-T cells prepared in the present invention effectively bind to CD30.

In addition, in another specific embodiment of the present invention, in order to confirm the activation of hCD30-CAR-T cells, the expression level of IFNγ by 1B6F11-CAR-T cells in the presence of target cells was confirmed. As a result, as shown in FIG. 3 , it was confirmed that IFNγ expression was insignificant because T cells were not activated in H929 cells that do not express CD30, and IFNγ expression also did not increase in SU-DHL1 cells that express CD30. On the other hand, as a result of confirming the apoptosis effect of the target cells by the hCD30-CAR-T cells, as shown in FIG. 4 , the hCD30-CAR-T cells showed the effect of specifically killing the CD30-expressing SU-DHL1 cells. confirmed that.

That is, it can be seen that the chimeric antigen receptor and CAR-T cells targeting CD30 of the present invention can be usefully used as a composition for preventing or treating diseases related to CD30 expression.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating a disease related to CD30 expression, comprising immune effector cells expressing the chimeric antigen receptor targeting CD30.

In the present invention, the terms “chimeric antigen receptor targeting CD30” and “immune effector cell” are as described above.

In the present invention, the term “disease associated with CD30 expression” refers to a disease associated with expression of wild-type CD30 or mutant CD30, and may be specifically cancer or malignant tumor, and more specifically, hematologic cancer, lymphoma and hosphobia. It may be selected from the group consisting of Hodgkin's lymphoma.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol. And one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

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

The pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It may have any one of the formulations.

The composition of the present invention is administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is defined as the subject type and severity, age, sex, and infected virus. Type, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

In addition, the present invention provides a method for preventing or treating a CD30 expression-related disease, specifically a malignant tumor, more specifically, Hodgkin's lymphoma, comprising administering a pharmaceutical composition according to the present invention to a subject.

The method of treating a disease or malignancy related to CD30 expression includes administering a pharmaceutical composition further comprising an immune effector cell of the present invention and a pharmaceutically acceptable carrier to an individual having a malignant tumor or suspected of having an invention. It may be a method of treating a malignant tumor comprising a, and the pharmaceutically acceptable carrier is the same as described above. Specifically, the method of treating a malignant tumor may be a method of treating a malignant tumor, comprising administering a composition including the immune effector cells to a subject suffering from the malignant tumor.

The subject refers to a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administering the pharmaceutical composition, and specifically refers to a human. In addition, it includes mammals, birds, etc. including cattle, pigs, sheep, chickens, dogs, and the like, and subjects whose malignancy is treated by administration of the composition of the present invention are included without limitation.

In this case, the composition may be administered single or multiple in a pharmaceutically effective amount. As used herein, the term 'administration' means providing the pharmaceutical composition of the present invention to an individual by any suitable method. The composition may be administered in the form of a liquid, powder, aerosol, capsule, enteric-coated tablet or capsule or suppository. Routes of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like. However, when administered orally, the peptides are digestible, so oral compositions must be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device capable of transporting the active agent to a target cell.

The pharmaceutical composition comprising the hCD30-CAR-T cell or anti-hCD30 antibody is used for preventing or treating a disease related to B cells, including the disease type, the patient's age, weight, characteristics and severity of symptoms, the type of current treatment, It may vary depending on various factors such as the number of treatments, dosage form and route, and may be easily determined by experts in the field.

The pharmaceutical composition may be administered together or sequentially with the aforementioned pharmacological or physiological component, and may be administered in combination with an additional conventional therapeutic agent, or may be administered sequentially or simultaneously with the conventional therapeutic agent. Such administration may be single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

In another aspect, the present invention provides a diagnosis of a CD30 expression-related disease, comprising the step of detecting the CD30 protein in an isolated biological sample of an individual suspected of malignancy using the human monoclonal antibody through an antigen-antibody reaction It provides a method of providing information for

The human monoclonal antibody, CD30 expression-related disease, individual, and CD30 protein are the same as described above.

The method of providing information for the diagnosis of CD30 expression-related diseases comprises reacting the CD30-specific human monoclonal antibody of the present invention with an isolated biological sample of an individual suspected of CD30 expression-related disease and detecting the formation of an antigen-antibody complex. , CD30 protein can be detected, and through this, information for the diagnosis of CD30 expression-related diseases can be provided. Since CD30 is overexpressed in cells of a malignant tumor, specifically, lymphoma such as Hodgkin's lymphoma, cancer may be diagnosed by comparing the expression level with a control group such as normal cells or tissues, but is not limited thereto.

As used herein, the term "biological sample" includes tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells and bacterial expression systems. However, it is not limited thereto. The presence or absence of CD30 protein or CD30 expression-related diseases can be confirmed by reacting these biological samples with the antibody of the present invention in an manipulated or unmanipulated state.

As used herein, the term "antigen-antibody complex" refers to a combination of the CD30 protein antigen in a sample and the monoclonal antibody according to the present invention that recognizes it, and the formation of the antigen-antibody complex is performed by a colormetric method, electrochemical method Any selected from the group consisting of an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment and a scintillation counting method method can be detected. However, it is not necessarily limited to these, and various applications and applications are possible.

Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, β-latamase, and the like. Contains fluorescein, Eu3+, Eu 3+ chelate or cryptate, etc., ligands include biotin derivatives, etc., and luminescent materials include acridinium esters and isoruminol derivatives, and microparticles colloidal gold, colored latex, and the like, and radioactive isotopes include 57 Co, 3 H, 125 I, 125I-Bonton Hunter reagent, and the like.

Specifically, antigen-antibody complexes can be detected using enzyme immunosorbent assay (ELISA). The enzyme immunosorbent assay (ELISA) includes a direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, and an indirect ELISA using a labeled secondary antibody recognizing a capture antibody in a complex of an antibody recognizing an antigen attached to a solid support. , a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody attached to a solid support and another antibody that recognizes an antigen in a complex of an antibody attached to a solid support and another antibody that recognizes an antigen in a complex Various ELISA methods are included, such as indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody.

The monoclonal antibody may have a detection label, and when it does not have a detection label, these monoclonal antibodies can be captured and confirmed by treatment with another antibody having a detection label.

In another aspect, the present invention provides a composition for diagnosis of CD30 expression-related diseases comprising the human monoclonal antibody.

The human monoclonal antibody and CD30 expression-related diseases are the same as described above. By using the diagnostic composition comprising the CD30-specific monoclonal antibody of the present invention, it is possible to diagnose a disease related to the expression or level of CD30 expression or a disease mediated by CD30, such as a malignant tumor, specifically Hodgkin's lymphoma. have.

The chimeric antigen receptor and CAR-T cells targeting CD30 prepared in the present invention not only effectively bind to CD30, but also activate CD30-bound CAR-T cells, and the prepared CAR-T cells express CD30 Since it has been confirmed that the cells of the present invention are effectively killed, the chimeric antigen receptor and CAR-T cells targeting CD30 of the present invention can be usefully used as a composition for preventing or treating diseases related to B cells or CD30 expression.

1A and 1B are graphs showing the binding ability of the 1B6F11 antibody to CD30.
2a and 2 are graphs confirming the binding ability between hCD30-CAR-T cells (1B6F11-CAR-T cells) and CD30 through reaction with anti-CD4 and anti-CD8 antibodies, respectively.
3 is a diagram confirming the expression level of IFNγ according to T cell activation in H929 cells that do not express CD30 and SU-DHL1 cells that express CD30.
4 is a diagram confirming the apoptosis effect of hCD30-CAR-T cells with respect to CD30-expressing SU-DHL1 cells.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Example 1: Preparation of anti-CD30 antibody

1-1. library phage preparation

In order to prepare a single-chain antibody that specifically binds to CD30, a library phage was prepared for phage display.

Specifically, a recombinant human CD30 (Sino, LSbio) solution was added to an immunotube to adsorb the protein to the test tube surface overnight at 4°C, and then a 1% solution of BSA (Bovine serum albumin) was added to the test tube to release CD30. The non-adsorbed surface was protected. ^{After emptying the test tube, 10 15} CFU of antibody phage library dispersed in 1% BSA solution was added to bind the antigen. After washing the non-specifically bound phage with PBS-T (Phosphate buffered saline-0.05% Tween 20) solution 5 times, the remaining antigen-specific phage antibody was recovered using 100 mM triethylamine solution.

After neutralizing the recovered phage with 1M Tris buffer (pH 7.4), E. coli was infected at 37° C. for 1 hour, and the infected E. coli was smeared on LB (Luria-Bertani) agar medium containing ampicillin and cultured at 37° C. overnight. The next day, the cultured E. coli was suspended in 4 ml of SB (superbroth)-ampicillin culture solution, 15% glycerol was added, and some was stored at -80 ° C. glucose) was added and incubated at 37 °C. When the absorbance of the culture medium reached 0.6 at 600 nm, the culture medium was removed by centrifugation, which was again suspended in 20 ml of SB-ampicillin culture medium, VCSM13 helper phage was added, and cultured at 37° C. with slow stirring. The next day, the culture solution was centrifuged, only the culture solution was taken, 4% of polyethylene glycol 8000 (PEG8000) and 3% of sodium chloride (NaCl) were added, followed by precipitation at 4° C. for 30 minutes, followed by centrifugation. The supernatant was removed and the precipitated phages were suspended in 1 ml of PBS and used as a library to amplify/concentrate antigen-specific clones by repeating the above panning process.

1-2. Panning via phage display

During panning to select an antibody that binds to human CD30 protein, panning was performed by crossing human CD30 protein and cancer cells, and the panning was performed up to three rounds. Then, plated and cultured on LB-ampicillin agar medium containing the antibody gene to obtain single colonies, inoculated and cultured in 100 μl SB-ampicillin culture medium, and induced with IPTG to induce scFv protein in the periplasm of Escherichia coli. ) was expressed. E. coli was suspended in TES solution (Tris, EDTA, sucrose), left at 4°C for 1 hour, centrifuged to extract periplasm, and this was used to confirm the binding of recombinant human CD30 antigen to scFv using ELISA technique. (Steinberger. Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1sted. ColdSpringHarborLaboratoryPress. NY. USA. pp. 11.9-11.12). The bound scFv was detected using Horseradish peroxidase (HRP)-anti-HA antibody and tetramethylbenzidine (TMB) substrate. The antigen-specific antibody clones identified therefrom were analyzed by sequencing.

Example 2: Confirmation of CD30 binding ability of anti-CD30 antibody

Each of the isolated and purified antibodies in Example 1 was named 1B6F11, and it was confirmed that they each contained a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8, respectively.

In addition, in the present invention, it was confirmed whether the anti-CD30 antibody 1B6F11 antibody prepared in Example 1 can specifically target CD30.

First, the 1B6F11 antibody was produced and purified, and the ability to bind to CD30 was verified by flow cytometer.

For this experiment, cells stably expressing human CD30 were prepared, and the degree of binding of the anti-CD30 antibody to CD30 was measured using the prepared cells and a FACS Calibur (BD Biosciences) instrument. Cells stably expressing CD30 were dissociated and washed in PBS, the number of cells was counted ^{, adjusted to 1x10 5} cells/200 μl PBS, and 10 μg of each CD30 monoclonal antibody was treated, followed by reaction at room temperature for 30 minutes.

^{After the reaction, the cells were washed in PBS, and 5 μl/1x10 5 of a} FITC-labeled constant region (Fab)-specific antibody (Alexa fluor 488 goat anti human IgG(ab)2 fragment specific jackson, concentration: 2.0mg/ml) was added. The cells/200 μl were suspended in PBS and reacted at 4° C. for 1 hour. After reaction, cells were washed in PBS and read on a FACS Calibur instrument.

As a control group, only the FITC-labeled constant region (Fab)-specific antibody was treated. In the experimental group treated with each CD30 monoclonal antibody, the reading result of the binding degree of human CD30-monoclonal antibody-FITC was compared with that of the control group.

As a result, the degree of binding of each monoclonal antibody to the human CD30 antigen is shown in FIG. 1 . As for the binding degree, the average value of the 1B6F11 antibody was 157 compared to that of the control group, which showed a mean value of 64.6 ( FIG. 1b ).

These results indicate that the 1B6F11 antibody of the present invention has excellent binding ability to human CD30.

Example 3: hCD30-CAR-T cell preparation

In the present invention, the anti-CD30-CAR expression vector prepared in Example 3 was transformed into T cells to prepare hCD30-CAR-T cells (1B6F11-CAR-T cells).

Specifically, peripheral blood mononuclear cells (PBMC) were isolated from blood, and then T cells were activated using T cell activation beads (manufacturer information).

The activated T cells were transduced with the hCD30-CAR lentivirus prepared in Example 4 into the T cells to prepare hCD30-CAR-T cells.

The hCD30 peptide binding ability of the hCD30-CAR-T cells was confirmed through flow cytometry. Specifically, the hCD30-CAR-T cells prepared above were reacted with FITC-hCD30 protein and anti-CD4 and anti-CD8 antibodies, and then the fluorescence intensity was measured using a FACS machine.

As a result, as shown in FIG. 2 , it was confirmed that hCD30-CAR-T cells expressing CD4 or CD8 binding to hCD30 increased as the hCD30 peptide increased. This means that the hCD30-CAR-T cells prepared in the present invention effectively bind to CD30.

Example 4: Confirmation of hCD30-CAR-T cell activation by hCD30 peptide

In the present invention, in order to confirm whether the hCD30-CAR-T cells prepared in Example 4 are activated by the CD30 peptide, the expression levels of IFNγ, CD4 and CD8 by 1B6F11-CAR-T cells in the presence of target cells were confirmed. .

As target cells, H929 cells that do not express CD30 (ATCC, CCL-243™) and SU-DHL1 cells that express hCD30 (ATCC, CRL-2955™) were used, and 1B6F11-CAR-T cells and target cells were used. :1, 1:1. After reacting for a certain period of time at a ratio of 0.5:1, 0:1, surface & intra antibody staining was performed for FACS measurement (CD30 protein, INF-r, CD4, CD8 staining). IFNγ, CD4 and CD8 expression levels of 1B6F11-CAR-T reacted with target cells were confirmed based on 0:1 (CAR-T only).

As a result, as shown in FIG. 3 , it was confirmed that IFNγ expression was insignificant because T cells were not activated in H929 cells that do not express CD30, and IFNγ expression also did not increase in SU-DHL1 cells expressing CD30. On the other hand, as a result of confirming the killing effect of the target cells by the hCD30-CAR-T cells, as shown in FIG. 4 , the hCD30-CAR-T cells showed the effect of specifically killing the SU-DHL1 cells expressing CD30. was confirmed (FIG. 4).

Example 5: Confirmation of the killing effect of hCD30-CAR-T cells on CD30-expressing cells

In the present invention, the killing effect of target cells by hCD30-CAR-T cells was confirmed.

Specifically, as target cells, H929 cells that do not express CD30 and U2932, K562, MOLT4, and SU-DHL1 cells that express CD30 were used, and were 1:4, 1:2, 1:1 with hCD30-CAR-T cells. , 1:0.5 and 1:0.25 were mixed, and then luminescence was measured. The degree of apoptosis was calculated using Equation 1 below as the measured value.

As a result, as shown in FIG. 4 , it was confirmed that the hCD30-CAR-T cells had a specific killing effect on CD30-expressing U2932, K562, MOLT4, and SU-DHL1 cells.

Cytotoxicity (%) H929
(CD30 -) U2932
(CD30+) K562
(CD30+) MOLT4
(CD30+) SU-DHL1
(CD30+) 1:4 26.09 31.34 41.07 42.08 56.28 1:2 19.25 20.21 36.27 35.31 46.69 1:1 11.64 13.11 26.95 27.10 38.49 1:0.5 7.730 8.840 23.79 27.21 36.41 1:0.25 9.350 7.770 17.08 15.87 29.34

That is, this suggests that the CD30-targeting chimeric antigen receptor and CAR-T cell of the present invention can be usefully used as a composition for preventing or treating diseases related to CD30 expression.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

<110> YntoAb Co., Ltd. <120> Chimeric antigen receptor targeting CD30 and use thereof <130> GP200089KRPRI <150> KR 2020-0186257 <151> 2020-12-29 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VH <400> 1 Ser Asn Trp Trp Ser 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VH <400> 2 Glu Ile Tyr His Ser Gly Ser Thr Asn 1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VH <400> 3 Gly Ser Gly Asn Tyr Tyr Tyr Tyr Met Asp Val 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VL <400> 4 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ala 1 5 10 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VL <400> 5 Asp Asn Asn Lys One <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VL <400> 6 Gly Thr Trp Asp Ser Ser Leu Ser Ala His 1 5 10 <210> 7 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VH <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Asn Trp Trp Ser Trp Val Arg Gln Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Gly Ser Gly Asn Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu 115 120 125 Thr Ser 130 <210> 8 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VL <400> 8 Glu Leu Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ala Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY LINKER <400> 9 Gly Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly <210> 10 <211> 780 <212> DNA <213> Artificial Sequence <220> <223> ANTIBODY DNA <400> 10 gagctcgtgc tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgctctg gaagcagctc caacattggg aataattatg tggcctggta ccagcagctc 120 ccaggaacag cccccaaact cctcatttat gacaataata agcgaccctc agggattcct 180 gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240 actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tgcgcattgg 300 gtgttcggcg gaggcaccaa gctgaccgtc ctaggtggtg gttcctctag atcttcctcc 360 tctggtggcg gtggctcggg cggtggtggg caggtgcagc tgcaggagtc gggcccagga 420 ctggtgaagc cttcggggac cctgtccctc acctgcgctg tctctggtgg ctccatcagc 480 agtagtaact ggtggagttg ggtccgccag cccccaggga aggggctgga gtggattggg 540 gaaatctatc atagtgggag caccaactac aacccgtccc tcaagagtcg agtcaccata 600 tcagtagaca agtccaagaa ccagttctcc ctgaagctga gctctgtgac cgccgcggac 660 acggccgtgt attactgtgc gtctggttcg gggaactact actactacat ggacgtctgg 720 ggcaaaggga ccacggtcac cgtctcctca gggagtgcat ccgccccaac ccttactagt 780 780

Claims (11)

a heavy chain variable region comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 1, CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and
An antibody that specifically binds to CD30, comprising a light chain variable region comprising a CDR1 represented by the amino acid sequence of SEQ ID NO: 4, a CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and a CDR3 represented by the amino acid sequence of SEQ ID NO: 6. or a fragment thereof.
delete According to claim 1,
The antibody is an antibody comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 7 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 8, an antibody or fragment thereof that specifically binds to CD30.
a heavy chain variable region comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 1, CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and
A light chain variable region comprising a CDR1 represented by the amino acid sequence of SEQ ID NO: 4, a CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and a CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A chimeric antigen receptor comprising an antibody or fragment thereof.
delete 5. The method of claim 4,
A chimeric antigen receptor, wherein a hinge region is further included between the C terminus of the CD30-binding domain and the N terminus of the transmembrane domain.
A polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 4 or 6.
An isolated immune effector cell comprising the polynucleotide of claim 7.
9. The method of claim 8,
The immune effector cell is a T cell or a natural killer (Natural Killer) cell, the immune effector cell.
A pharmaceutical composition for preventing or treating hematologic cancer or Hodgkin's lymphoma associated with CD30 expression, comprising the immune effector cell of claim 8 .
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