KR102119783B1 - Antibody and Fragment Thereof Binding Human Epidermal Growth Factor Receptor Variant Ⅲ - Google Patents

Abstract

The present invention relates to antibodies that bind to human epithelial growth factor receptor variant III and antibody fragments thereof. The antibody according to the present invention and its antibody fragments have the characteristics of minimizing binding to EGFR and specifically recognizing EGFRvIII. Since EGFRvIII is a highly cancer-specific antigen, the antibodies and antibody fragments developed in the present invention can be applied to early diagnosis, prevention and treatment of cancer. In addition, it is possible to maximize the therapeutic efficacy by applying Antibody-Drug Conjugates (ADC) or Chimeric Antigen Receptor-T (CAR-T) technology that combines drugs or T cells with antibodies or antibody fragments based on its high cancer specificity. It is possible to minimize the toxicity that can occur to cells. Early diagnosis of EGFRvIII mutations is possible through a method of directly binding radioisotopes or fluorescent substances to an antibody or antibody fragment according to the present invention and imaging, or through ELISA or IHC.

Description

Antibody and Fragment Thereof Binding Human Epidermal Growth Factor Receptor Variant III

The present invention relates to antibodies that bind to human epithelial growth factor receptor variant III and antibody fragments thereof.

Existing anti-cancer therapies have side effects such as limited and non-specific anti-cancer effects or severe systemic toxicity. This has created a need for new cancer treatments. Recently, as understanding of molecular biological mechanisms and signaling mechanisms for the development of cancer has increased, interest in molecular targeted therapy targeting specific molecules has increased, and signaling substances that affect cancer development Much research has been done on treatments targeting.

In the above targeted therapeutic areas, the use of monoclonal antibodies for the treatment of cancer has been successful. Antibody drug conjugates have become a powerful new treatment option for the treatment of lymphomas and solid cancers, and immunomodulatory antibodies have also had significant clinical success in recent years. The development of therapeutic antibodies is based on a deep understanding of cancer serology, protein engineering techniques and their action, mechanisms of resistance, and interactions between the immune system and cancer cells.

Among them, the Epidermal Growth Factor Receptor (EGFR) is one of the most studied molecular targets in various carcinomas. EGFR is a 170 kilodalton membrane lipoprotein product of the proto-oncogene c-erb B. The sequence of the EGFR gene is known. The EGFR gene is a homolog of the erbB oncogene originally identified in the avian erythroblastosis virus. Activation of the tumor gene by gene amplification has been observed in various human tumors.

It has been reported that EGFR is overexpressed, amplified, and mutated in many cancer species. It has been reported that such overexpression, mutated EGFR promotes cancer cell growth and survival, and makes cancer cells resistant to chemotherapy. Overexpression of EGFR has been observed in some lung, breast, colon, stomach, brain, bladder, head and neck, ovary, kidney and prostate carcinomas. It has been found that epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) bind to EGFR to induce cell proliferation and tumor growth. In addition, size variant EGFR genes and amplification have been reported in some human cancers.

EGFR mutants are caused by gene rearrangement accompanied by EGFR gene amplification. There are eight major EGFR variants known as: (i) EGFRvI lacks most of the extracellular domain of EGFR, and (ii) EGFRvII is 83 aa in-frame in the extracellular domain of EGFR. ) Consists of deletions, (iii) EGFRvIII consists of 267 aa in-frame deletions in the extracellular domain of EGFR, (iv) EGFRvIV contains a deletion in the cytoplasmic domain of EGFR, (v) EGFRvV is EGFR Contains a deletion in the cytoplasmic domain of, (vi) EGFR.TDM/2-7 contains replication of exon 2-7 to the extracellular domain of EGFR, and (vii) EGFR.TDM/18-25 is a cell of EGFR. Exon 18-26 replication to the foreign domain, and (viii) EGFR.TDM/18-26 includes replication of exon 18-26 to the tyrosine kinase of EGFR. There is also a rare second EGFRvIII mutant (EGFRvIII/Δ12-13) with a second deletion that introduces a new histidine residue at the junction of exons 11 and 14.

In the case of Epidermal Growth Factor Receptor Variant III (EGFRvIII), which is one of the mutations of EGFR, exons 2 to 7 of the wild type EGFR are lost. EGFRvIII is a variant of epithelial growth factor (EGF) that most commonly occurs in human cancer. It is expressed in about 30% of patients with glioblastoma multiforme (GBM) and not in normal tissues. More specifically, when expressed on the cell surface, amino acids 6 to 273 of wild type EGFR disappear, and amino acids 5 and 274 are conjugated, and glycine amino acid is inserted between them. In addition to glioblastoma, a mutation frequently found in patients with lung cancer, EGFRvIII is a part of the ligand-binding site present in the extracellular region and is unable to bind to the ligand, but shows more enhanced tumorigenic potential. That is, it contributes to tumor progression through structural signaling in a ligand-independent manner.

Meanwhile, the correlation between mutant proteins and cancer stem cells is not clear. Glioblastoma tumors are known to frequently express EGFRvIII, an EGFR variant expressed through gene rearrangement and amplification. Since tyrosine phosphorylation site is always present in an activated form, it shows strong tumorigenicity. However, despite these modifications, expression of EGFRvIII is limited. In cancer stem cells, EGFRvIII is highly expressed with CD133, and EGFRvIII+/CD133+ cells are defined as having high regeneration and tumor initiation ability. EGFRvIII+ cells were associated with stem/progenitor markers, while differentiation markers were found in EGFRvIII- cells. EGFRvIII expression was lost in normal cell culture but maintained in tumor protoplast culture, and cell cultured cells simultaneously express and regenerate EGFRvIII+/CD133+ and have tumor initiation ability.

Approaches to inhibit the growth of cancer cells by inhibiting the EGFR signaling system include a monoclonal antibody or a tyrosine kinase inhibitor. EGFRvIII, a mutant form of EGFR, is an attractive diagnostic and therapeutic target due to its high cancer specificity, which is not expressed in normal cells but only in cancer cells. In general, in the group of cancer patients with the EGFRvⅢ mutation, EGFR is expressed together. In summary, since EGFRvIII is a protein that is not expressed in normal cells as a mutation frequently occurring in cancer patients where EGFR is amplified/overexpressed, it is considered that EGFRvIII is a cancer-specific antigen optimized for cancer diagnosis and target treatment in a group of cancer patients expressing EGFRvIII.

However, it is known that the previously developed EGFR targeted therapeutics do not show efficacy against cancer cells with the EGFRvIII mutant type. In particular, EGFR-specific tyrosine kinase inhibitors developed to date have been found to have little efficacy in carcinomas with the EGFRvIII mutation.

Gefitinib (trademark) is Iressa, an EGFR inhibitor sold by AstraZeneca and Teva. Indications are drugs such as breast cancer and lung cancer. When Gefitinib was treated with EGFRvIII mutant cancer cells, it was reported that it could not inhibit the phosphorylation of EGFRvIII and its sub-signal transporter, Akt, and therefore it could not inhibit the growth of cancer cells.

Erlotinib (trade name) is commercially available from Genentech, Roche under the trade name Tarceva, and the indication is tyrosine kinase inhibitor drug, non-small cell lung cancer (NSCLC). In the case of erlotinib, as in the above gefitinib, EGFRvIII mutant carcinoma does not show cancer cell growth inhibitory ability.

Cetuximab (Cetuximab) is the abbreviation Erbitux, an EGFR inhibitor sold by Bristol-Myers Squibb and Merck. The indications are metastatic colorectal cancer, metastatic arsenic cell lung cancer, head and neck cancer, etc. Cetuximab is a chimeric (mouse-human) monoclonal antibody. However, cetuximab has a problem in that antigen specificity to EGFRvIII mutant cancer cells is very poor and cancer cell growth inhibitory ability is not exhibited.

In addition, since the therapeutic agents developed to date target EGFR and EGFRvIII at the same time, there is a need to increase specificity, and many studies have been conducted to develop diagnostics and therapeutic agents specifically targeting EGFRvIII.

Under this technical background, the inventors of the present application have tried to develop an antibody for cancer treatment that specifically binds to EGFRvIII. As a result, the present inventors developed an anti-EGFRvIII antibody that binds to EGFRvIII with high affinity using phage display technology, confirming that the anti-EGFRvIII antibody can significantly inhibit cancer cell migration, and completed the present invention. Did.

One aspect provides a novel antibody or antigen binding fragment thereof that binds EGFRvIII.

Another aspect provides a nucleic acid encoding the antibody or antigen-binding fragment thereof, an expression vector containing the nucleic acid, and a cell transformed with the expression vector.

Another aspect provides a composition for preventing or treating cancer comprising the antibody or antigen-binding fragment thereof.

Another aspect provides a composition for diagnosing cancer comprising the antibody or an antigen-binding fragment thereof, and a kit for diagnosing cancer comprising the same.

One aspect provides a novel antibody or antigen binding fragment thereof that binds EGFRvIII. In one embodiment, the sequence of the new antibody has a heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 1 or 7, CDRH2 comprising SEQ ID NO: 2 or 8, or CDRH3 comprising SEQ ID NO: 3 or 9, SEQ ID NO: It may have a light chain variable region comprising CDRL1 comprising 4 or 10, CDRL2 comprising SEQ ID NO: 5 or 11, or CDRL3 comprising SEQ ID NO: 6, 12 or 21. In this case, the CDRL3 light chain variable region represented by SEQ ID NO: 21 may be substituted with some amino acids of the CDRL3 light chain variable region represented by SEQ ID NO: 6. Specifically, some aspartic acids in the CDRL3 light chain variable region represented by SEQ ID NO: 6 may be substituted with glutamic acid. The antibody having the CDRL3 light chain variable region represented by SEQ ID NO: 21 is characterized by high binding affinity with the EGFRvIII antigen. That is, since EGFRvIII is a highly cancer-specific antigen, the antibody has a high binding affinity to the EGFRvIII antigen, and thus can be applied to early diagnosis, prevention and treatment of cancer.

EGFR may be a 170 kilodalton membrane lipoprotein product of the proto-oncogene c-erb B. As used herein, the term "EGFRvIII" refers to mutant type III of the epidermal growth factor receptor recognized by MR1 scFv and characterized by an 801 base pair inframe deletion of 2-7 exons near the amino terminus. EGFRvIII is highly expressed in about 50-60% of glioblastomas and appears to be present in about 70-80% of breast and ovarian carcinomas and about 16% of non-small cell lung carcinomas. EGFRvIII is expressed on the cell surface and produces a tumor specific cell surface epitope at the deletion junction.

"Epitope" means a protein determinant to which an antibody can specifically bind. Epitopes usually consist of a group of chemically active surface molecules, for example amino acid or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge properties. The steric epitopes and non-stereoscopic epitopes are distinguished in that the binding to the former is lost in the presence of a denaturing solvent, but not to the latter.

As used herein, the term "antibody (antibody)" refers to a glycoprotein that inactivates antigens such as viruses and bacteria and induces extracellular stimulation against microorganisms that have invaded the body, and in particular, an immune antibody. Since the present invention relates to an anti-EGFRvIII antibody, the term'antibody' used without modification may refer to an anti-EGFRvIII antibody specifically binding to the epitope of EGFRvIII, unless otherwise specified. The scope of the present invention includes not only complete antibody forms that specifically bind to EGFRvIII, but also antigen-binding fragments of the antibody molecules. A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain connected by a heavy chain and a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

The antigen-binding fragment or antibody fragment of an antibody means a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced by cysteine residues in the hinge region of Fab' forming disulfide bonds. Fv is a minimal antibody fragment that has only a heavy chain variable region and a light chain variable region. Recombination technology for generating Fv fragments is described in PCT International Patent Applications WO88/10649, WO88/106630, WO88/07085, WO88/07086 and WO88/09344 It is disclosed. The double-chain Fv (two-chain Fv) is a non-covalently linked heavy chain variable region and a light chain variable region, and the single-chain Fv (single-chain Fv, scFv) is a heavy chain variable region and a light chain variable region through a peptide linker. These covalent bonds or C-terminals can be linked directly to form dimer-like structures such as double-chain Fvs. These antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the whole antibody with papain yields Fab and cleavage with pepsin yields F(ab')2 fragment), gene It can also be produced through recombinant technology.

In one embodiment, the antibody according to the invention is in Fv form (eg, scFv), or in complete antibody form (IgG). In addition, the heavy chain constant region can be selected from any of the isotypes of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be of kappa or lambda type.

As used herein, the term “heavy chain (High Chain: HC or CH)” refers to the variable region domain VH and three amino acid sequences comprising an amino acid sequence having a variable region (VR) sequence sufficient to confer specificity to the antigen. It means both the full-length heavy chain and fragments thereof including the constant region domains CH1, CH2 and CH3. In addition, as used herein, the term “light chain (Low Chain: LC or CL)” includes a variable region domain VL and a constant region domain CL comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. Refers to both the full-length light chain and fragments thereof.

Antibodies of the invention are monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-binding Fvs (sdFv) And anti-idiotype (anti-Id) antibodies, or epitope-binding fragments of the antibodies, but are not limited thereto. In particular, the present invention may be a monoclonal antibody and a complete human antibody.

The monoclonal antibody refers to the same, except for antibodies obtained from a population of substantially homogeneous antibodies, ie possible naturally occurring mutations that may be present in trace amounts in the individual antibodies occupying the population. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

The non-human (eg murine) antibody of the “humanized” form is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin. In most cases, the humanized antibody is a non-human species (donor antibody) that retains the desired specificity, affinity, and ability for residues from the hypervariable region of the recipient, such as a mouse, rat, rabbit, or non-human. It is a human immunoglobulin (receptor antibody) replaced by a residue from the hypervariable region of a primate.

The "human antibody" is a molecule derived from human immunoglobulin, and means that all of the amino acid sequences constituting the antibody including the complementary determining region and the structural region are composed of human immunoglobulin. In one embodiment, the present invention relates to a fully human antibody capable of minimizing an unintended immune response.

Said "chimeric antibody" is a portion of the heavy and/or light chain derived from a particular species or identical or homologous to the corresponding sequence in an antibody belonging to a particular antibody class or subclass, while the remaining chain(s) are from another species Included are "chimeric" antibodies (immunoglobulins) that are identical or homologous to the corresponding sequence in an antibody belonging to or belong to another antibody class or subclass, as well as fragments of the antibody that exhibit the desired biological activity.

“Antibody variable domain” as used herein refers to the light and heavy chains of an antibody molecule comprising the Complementary Determining Region (CDR. ie CDR1, CDR2, and CDR3), and the amino acid sequence of the framework region (FR). Refers to the part. VH refers to the variable domain of the heavy chain (Variable Region of High Chain). VL refers to the variable domain of the light chain (Variable Region of Low Chain).

“Complementary Determining Regions (CDRs. ie CDR1, CDR2, and CDR3)” may refer to amino acid residues in antibody variable domains, beings required for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.

As used herein, the term “frame region” (FR) refers to variable domain residues other than CDR residues. Each variable domain typically has 4 FRs identified as FR1, FR2, FR3 and FR4.

In one embodiment, the antibody according to the present invention has a heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 1, CDRH2 comprising SEQ ID NO: 2, and CDRH3 comprising SEQ ID NO: 3, comprising SEQ ID NO: 4 It may be an antibody binding to EGFRvIII having a light chain variable region comprising CDRL1, CDRL2 comprising SEQ ID NO: 5, and CDRL3 comprising SEQ ID NO:6, or an antigen-binding fragment thereof.

In another embodiment, the antibody according to the invention has a heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 7, CDRH2 comprising SEQ ID NO: 8, and CDRH3 comprising SEQ ID NO: 9, comprising SEQ ID NO: 10 It may be an antibody binding to EGFRvIII having a light chain variable region comprising CDRL1, CDRL2 comprising SEQ ID NO: 11, and CDRL3 comprising SEQ ID NO: 12, or an antigen-binding fragment thereof.

In another embodiment, the antibody according to the present invention has a heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 1, CDRH2 comprising SEQ ID NO: 2, and CDRH3 comprising SEQ ID NO: 3, comprising SEQ ID NO: 4 It may be an antibody binding to EGFRvIII having a light chain variable region comprising CDRL1, CDRL2 comprising SEQ ID NO: 5, and CDRL3 comprising SEQ ID NO: 21, or an antigen-binding fragment thereof.

In another embodiment, the antibody according to the present invention may include a framework region (FR) having one or more sequences selected from the group consisting of SEQ ID NOs: 13-20. More specifically, HC F1 comprising SEQ ID NO: 13, HC F2 comprising SEQ ID NO: 14, HC F3 comprising SEQ ID NO: 15, HC F4 comprising SEQ ID NO: 16, LC F1 comprising SEQ ID NO: 17, LC F2 comprising SEQ ID NO: 18, LC F3 comprising SEQ ID NO: 19, LC F4 including SEQ ID NO: 20.

In the present invention, "antibody-binding fragment" means a fragment of an antibody having an antigen-binding ability. An “Fv” fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly virtually covalently associated with, for example, scFv. The “Fab” fragment contains the variable and constant domains of a light chain and the variable and first constant domains (CH1) of a heavy chain. F(ab')2 antibody fragments generally include a pair of Fab fragments that are covalently linked near their carboxy terminus by hinge cysteines between them. "Single chain Fv" or "scFv" antibody fragments include the VH and VL domains of an antibody, which domains are in a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain and the VL domain that allows scFv to form the desired structure for antigen binding.

As used herein, the term “phage display” refers to a technique for displaying a variant polypeptide as a fusion protein with at least a portion of an envelope protein on the surface of a phage, eg, fibrous phage particles. The usefulness of phage display lies in the fact that it can target large libraries of randomized protein variants, and quickly and efficiently classify sequences that bind the target antigen with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to identify polypeptides with specific binding properties.

Phage display technology has provided a powerful tool for generating and screening new proteins that bind specific ligands (eg, antigens). Using phage display technology, large libraries of protein variants can be generated and sequences that bind with high affinity to the target antigen can be quickly sorted. The nucleic acid encoding the variant polypeptide is fused with a viral envelope protein, such as a nucleic acid sequence encoding a gene III protein or gene VIII protein. A monovalent phage display system has been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein. In monovalent phage display systems, gene fusions are expressed at low levels and wild type gene III protein is also expressed to maintain particle infectivity.

Demonstrating the expression of peptides on the surface of fibrous phage and the expression of functional antibody fragments in the periplasm of E. coli is important in developing antibody phage display libraries. Libraries of antibodies or antigen-binding polypeptides have been prepared in a number of ways, for example by altering a single gene by inserting a random DNA sequence or cloning a related gene sequence. The library can be screened for expression of antibodies or antigen-binding proteins with desired characteristics.

Phage display technology has several advantages over conventional hybridoma and recombinant methods for producing antibodies with desired characteristics. This technique makes it possible to generate large antibody libraries with various sequences in a short time without using animals. Production of hybridomas or humanized antibodies may require several months of production. In addition, since no immunity is required, the phage antibody library can generate antibodies against antigens that are toxic or have low antigenicity. Phage antibody libraries can also be used to generate and identify new therapeutic antibodies.

Techniques can be used to generate human antibodies from immunized, non-immunized human, germline sequences, or unsensitized B cell Ig repertory using phage display libraries. Various lymphoid tissues can be used to prepare unsensitized or non-immune antigen binding libraries.

Techniques for identifying and isolating high affinity antibodies from phage display libraries are important for the isolation of new therapeutic antibodies. Separation of the high affinity antibody from the library can depend on the size of the library, production efficiency among bacterial cells and the diversity of the library. The size of the library is reduced by inadequate folding of the antibody or antigen-binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be suppressed if the antibody or antigen binding domain is not properly folded. Expression can be improved by alternating mutations at the surface of the variable/constant interface or at selected CDR residues. The sequence of the framework region is one element to provide for proper folding when generating antibody phage libraries in bacterial cells.

In high affinity antibody isolation, it is important to generate various libraries of antibodies or antigen-binding proteins. CDR3 regions have been found to often participate in antigen binding. CDR3 regions on the heavy chain vary considerably in size, sequence, and structural conformation, and thus can be used to prepare various libraries.

In addition, diversity can be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position. The use of all 20 amino acids can generate variant antibody sequences with great diversity and increase the chances of identifying new antibodies.

Antibodies or antibody fragments of the present invention may include not only the sequence of the anti-EGFRvIII antibody of the present invention described herein, but also a biological equivalent thereof within a range capable of specifically recognizing EGFRvIII. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletion, insertion and/or substitution of amino acid sequence residues of the antibody. These amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically equivalent.

In introducing the variation, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index according to hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5). The hydrophobic amino acid index is very important in conferring the protein's interactive biological function. It is well known that amino acids with similar hydrophobicity indexes must be replaced to retain similar biological activity. When introducing a variation with reference to the hydrophobic index, substitution between amino acids showing a hydrophobic index difference of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5 is performed.

On the other hand, it is also known that substitution between amino acids having a similar hydrophilicity value results in proteins having equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0±1); Glutamate (+3.0±1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5±1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). Amino acid exchange in proteins that do not entirely alter the activity of the molecule is known in the art (H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.

Considering the above-described biological equivalent activity variation, the antibody of the present invention or a nucleic acid molecule encoding the same can be interpreted as including a sequence showing substantial identity with the sequence set forth in SEQ ID NO. The substantial identity above, when aligned with the above-described sequence of the present invention and any other sequence to the maximum correspondence, and analyzed the aligned sequence using an algorithm commonly used in the art, a minimum of 61% Refers to a sequence that exhibits homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. BLAST can be accessed at https://blast.ncbi.nlm.nih.gov/Blast.cgi. The sequence homology comparison method using this program can be found at https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome.

Another aspect provides a nucleic acid encoding the antibody or antigen-binding fragment thereof, an expression vector containing the nucleic acid, and a cell transformed with the expression vector.

The nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention can be isolated to recombinantly produce the antibody or antigen-binding fragment thereof. The nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression. Based on this, the present invention relates to a vector containing the nucleic acid in another aspect.

The term "nucleic acid" used in the present invention has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and a nucleotide that is a basic structural unit in a nucleic acid is not only a natural nucleotide, but also a sugar or base site modified analogue. (analogue). The sequence of nucleic acids encoding the heavy and light chain variable regions of the invention can be modified. Such modifications include addition, deletion, or non-conservative substitutions or conservative substitutions of nucleotides.

The nucleic acid of the present invention is also interpreted to include a nucleotide sequence showing substantial identity to the nucleotide sequence. Substantial identity aligns the nucleotide sequence of the present invention with any other sequence to the maximal correspondence, and when the aligned sequence is analyzed using an algorithm commonly used in the art, at least 80% homology, more Preferably, it means a nucleotide sequence exhibiting at least 90% homology, most preferably at least 95% homology.

The DNA encoding the antibody is readily separated or synthesized using conventional procedures (eg, by using an oligonucleotide probe capable of specifically binding DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

As used herein, the term "vector" means a plasmid vector as a means for expressing a target gene in a host cell; Cosmid vectors; Viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated virus vectors. The nucleic acid encoding the antibody in the vector is operably linked to a promoter.

"Operatively linked" refers to functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence is the other nucleic acid. It controls the transcription and/or translation of sequences.

프로모터, pR

프로모터, rac5 프로모터, amp 프로모터, recA 프로모터, SP6 프로모터, trp 프로모터 및 T7 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 또한, 예를 들어, 진핵 세포를 숙주로 하는 경우에는, 포유동물 세포의 지놈으로부터 유래된 프로모터(예: 메탈로티오닌 프로모터, β-액틴 프로모터, 사람 헤로글로빈 프로모터 및 사람 근육 크레아틴 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터(예: 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40프로모터, 사이토메갈로 바이러스(CMV) 프로모터, HSV의 tk 프로모터, 마우스 유방종양 바이러스(MMTV) 프로모터, HIV의 LTR 프로모터, 몰로니 바이러스의 프로모터엡스타인바 바이러스(EBV)의 프로모터 및 로우스 사코마 바이러스(RSV)의 프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 갖는다. 경우에 따라서, 벡터는 그로부터 발현되는 항체의 정제를 용이하게 하기 위하여 다른 서열과 융합될 수도 있다. 융합되는 서열은, 예컨대 글루타티온 S-트랜스퍼라제(Pharmacia, USA), 말토스 결합 단백질(NEB, USA), FLAG(IBI, USA) 및 6xHis(hexahistidine; Quiagen, USA) 등이 있다. 상기 벡터는 선택표지로서 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함하며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성유전자가 있다.When a prokaryotic cell is used as a host, a strong promoter (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL) that can progress transcription

Promoter, pR

It is common to include a promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding sites for initiation of translation, and transcription/translation termination sequences. In addition, for example, when a eukaryotic cell is a host, a promoter derived from a genome of a mammalian cell (for example, a metallothionine promoter, a β-actin promoter, a human heteroglobin promoter, and a human muscle creatine promoter) or a mammal Promoters derived from animal viruses (eg adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse breast tumor virus (MMTV) promoter, HIV's LTR promoter , Promoter of Moloney virus Epstein Barr virus (EBV) promoter and promoter of Loews sacoma virus (RSV) can be used, and generally has a polyadenylation sequence as a transcription termination sequence. In some cases, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Fused sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6xHis (hexahistidine; Quiagen, USA). The vector includes an antibiotic resistance gene commonly used in the art as a selection marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There are resistant genes.

In another aspect, the invention relates to cells transformed with the above-mentioned vector. The cells used to generate the antibodies of the invention can be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells. Bacillus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (e.g., Staphylococcus carnosus) can be used. However, the interest in animal cells is the most, and examples of useful host cell lines are COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0 , U20S, or HT1080, but is not limited thereto.

The present invention (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells. The cells can be cultured in various media. It can be used as a culture medium without limitation among commercially available mediums. All other essential supplements known to those skilled in the art may also be included in suitable concentrations. Culture conditions, such as temperature, pH, etc., have already been used with host cells selected for expression, which will be apparent to those skilled in the art. The recovery of the antibody or antigen-binding fragment thereof can remove impurities by, for example, centrifugation or ultrafiltration, and the resultant product can be purified, for example, using affinity chromatography. Additional other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and the like can be used.

Another aspect provides a composition for preventing or treating cancer comprising the antibody or antigen-binding fragment thereof.

The present invention includes, for example, (a) a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof against EGFRvIII according to the present invention; And (b) it may be a pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier. The present invention also relates to a method for preventing or treating cancer comprising administering an antibody or antigen-binding fragment thereof against EGFRvIII according to the present invention in an effective amount required for a patient.

Since the composition uses the above-described anti-EGFRvIII antibody of the present invention or an antigen-binding fragment thereof as an active ingredient, descriptions in common between the two are omitted.

As demonstrated in the Examples below, the antibody or antigen-binding fragment thereof of the present invention can bind to EGFRvIII with high affinity, thereby inhibiting the migration of cancer cells overexpressing EGFRvIII, and thus can be used for the prevention and treatment of cancer. "Prevention" means any action that inhibits or delays the progression of cancer by administration of a composition according to the present invention, and "treatment" means suppression of cancer development, alleviation of cancer, or elimination of cancer.

“Cancer overexpressing EGFRvIII” refers to cancer having EGFRvIII on the surface of cancer cells at significantly higher levels compared to non-cancerous cells of the same tissue type. Cancer that is a disease applied to the composition is a cancer that overexpresses EGFRvIII, for example, glioblastoma, astrocytoma, glioma, neuroblastoma, testicular cancer, colon cancer, melanoma, pancreatic cancer, lung cancer, breast cancer, esophageal cancer, lung cancer, Ovarian cancer, prostate cancer and squamous cell carcinoma.

In another aspect, the present invention may be a composition for inhibiting metastasis or invasion of cancer cells comprising an antibody or antigen-binding fragment thereof against EGFRvIII according to the present invention. The present invention also relates to a method for inhibiting metastasis or invasion of cancer cells by treating an antibody against EGFRvIII or an antigen-binding fragment thereof according to the present invention.

The pharmaceutically acceptable carrier included in the composition of the present invention is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto. The composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally. Preferably, it may be parenteral administration, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered. . In one embodiment, it may be administered in the form of an intravenous injection. When administered orally, proteins or peptides are digested, 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 can be administered by any device capable of transporting the active substance to target cells.

Suitable dosages of the compositions according to the invention vary by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity, usually As a result, a skilled doctor can easily determine and prescribe a dose effective for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg. The term "pharmaceutically effective amount" as used herein means an amount sufficient to prevent or treat cancer.

The pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of ex, powder, granule, tablet or capsule, or additionally include a dispersant or stabilizer.

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.

Another aspect provides a composition for diagnosing cancer comprising the antibody or an antigen-binding fragment thereof, and a kit for diagnosing cancer comprising the same. The present invention also relates to a method for diagnosing cancer by treating an antibody against EGFRvIII according to the present invention or an antigen-binding fragment thereof.

Cancer can be diagnosed by measuring the level of EGFRvIII expression in a sample through an antibody against EGFRvIII according to the present invention. The expression level can be measured according to a conventional immunoassay method, radioimmunoassay using an antibody against EGFRvIII, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA , Can be measured through inhibition or hardwood analysis, sandwich analysis, flow cytometry, immunofluorescence staining and immunoaffinity purification, but is not limited thereto. By analyzing the intensity of the final signal by the immunoassay process, cancer can be diagnosed. That is, when the protein of the marker of the present invention is highly expressed in a biological sample and the signal is stronger than a normal biological sample (eg, normal gastric tissue, blood, plasma or serum), it is diagnosed as cancer.

In another aspect, the present invention relates to a cancer diagnostic kit comprising the composition for diagnosing cancer. The kit according to the present invention includes an antibody against EGFRvIII according to the present invention, and a sample and an antibody react to analyze a signal indicated to diagnose cancer. In this case, the signal may include, but is not limited to, an enzyme bound to the antibody, for example, alkaline phosphatase, β-galactosidase, horse radish peroxidase, luciferase or cytochrome P450. In this case, the substrate for the enzyme is bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol), when alkaline phosphatase is used as the enzyme. When chromogenic reaction substrates such as -AS-B1-phosphate) and ECF (enhanced chemifluorescence) are used, and when hose radish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, and lucigenin (Bis-N-methylacridinium nitrate), Resorphine benzyl ether, Luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazin), HYR (p-phenylenediamine-HCl and pyrocatechol) , Tetramethylbenzidine (TMB), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m Substrates such as -PMS (phenzaine methosulfate) may be used, but are not limited thereto.

In addition, the kit according to the present invention may include a label that generates a detectable signal, wherein the label is a chemical (eg, biotin), an enzyme (alkaline phosphatase, β-galactosidase, hose radish Peroxidase and cytochrome P450), radioactive materials (e.g. C14, I125, P32 and S35), fluorescent materials (e.g. fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET). It may include, but is not limited to. Measurement of the activity or signal of the enzyme used for diagnosing cancer may be performed according to various methods known in the art. Through this, EGFRvIII expression can be qualitatively or quantitatively analyzed.

The antibody according to the present invention and its antibody fragments have the characteristics of minimizing binding to EGFR and specifically recognizing EGFRvIII. Since EGFRvIII is a highly cancer-specific antigen, the antibodies and antibody fragments developed in the present invention can be applied to early diagnosis, prevention and treatment of cancer. In addition, it is possible to maximize the therapeutic efficacy by applying Antibody-Drug Conjugates (ADC) or Chimeric Antigen Receptor-T (CAR-T) technology that combines drugs or T cells with antibodies or antibody fragments based on high cancer specificity. It is possible to minimize the toxicity that can occur to cells. Early diagnosis of EGFRvIII mutations is possible through a method of directly binding radioisotopes or fluorescent substances to an antibody or antibody fragment according to the present invention and imaging, or through ELISA or IHC.

1 is a result confirming the expression pattern of EGFR/EGFRvIII in glioblastoma patient-derived cells by Western blotting.
Figure 2 is the result of selecting 55 antibody fragments that show more than three times the binding capacity to EGFRvⅢ compared to EGFR.
Figure 3 shows the results of analysis after production by converting the final two clones into the form of human IgG1 antibody.
4 is a graph showing the results of confirming the binding specificity for EGFRvIII by ELISA method.
Figure 5 is a micrograph of the binding pattern taken in GBM4 EGFRvIII mutant glioblastoma tissue (EGFRvIII +/EGFR +) using immunohistochemical staining.
6 is a graph showing the results of confirming the response unit (response unit) of H1L2 antibody (a) and H1L2mut antibody (b).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Cell panning using EGFRvIII specific antibody

Antibody screening was performed by a phage display-based cell panning method. For antibody screening, the antibody library presented in patent WO2016114567 was used. This human antibody library consists of two sub-libraries (OPAL-L, OPAL-K).

The cancer patient-derived cells used for antibody screening were used by pre-sale from Samsung Medical Center, Institute for Refractory Cancer Research. All of the patient-derived cells derived from the glioblastoma multiforme are derived from GBM1, GBM2, GBM3, GBM4, GBM5, GBM6, GBM7, and GBM8. The expression level of EGFRvIII in these cells was confirmed by Western blotting experiment using 20 μg lysate of each cell. GBM7 (EGFR -/EGFRvIII -) and GBM8 (EGFR -/EGFRvIII +) were used as cancer-derived cells used for cell panning. GBM7 cells were used for negative selection of antibody fragments that showed non-specific binding to the cell surface, and GBM8 cells were used to secure antibody fragments that bound to EGFRvIII.

In detail, to recover a phagemid vector into which various human antibody variable region genes introduced into E. coli host TG1 are inserted, in the form of a phage expressed on the surface of an antibody fragment, two sub-libraries (OPAL -L, OPAL-K) samples are added into each 400 mL culture medium (SB/ampicillin/2% glucose) and incubated at 37°C and 220 rpm for about 2 hours. Host cells cultured with absorbance of 0.5 in OD600 were centrifuged at 5,000 g for 20 minutes to remove the supernatant, and then suspended in 400 mL of secondary culture medium (SB/ampicillin), followed by 10 ¹² pfu (plaque forming unit). The helper phage (VCSM13) was added and incubated for 1 hour at 37°C and 120 rpm. Then, after adding kanamycin antibiotic (an antibiotic gene introduced in the helper phage) to a final concentration of 70 µg/mL, phages expressing human antibody fragments are made in host cells through overnight culture at 30°C and 220 rpm. To lose. The next day, the culture centrifuged using PEG (polyethylene glycol) 8000 solution to precipitate only the phage particles to recover the phage library. To count the phage recovered from each sub-library, each sample is diluted and then infected with host cells (TG1) to count in LB/ampicillin solid culture medium.

Phage display screening was performed through repeated panning. The counted sub-library was collected to a level of about 1.0 x 10 ¹³ pfu, and then treated with GBM7 cells not expressing EGFR and EGFRvIII antigen to bind for 1 hour at 4°C, and the upper layer containing phages not binding to GBM7 cells. Only liquid is recovered. This is a step of preventing nonspecific binding other than EGFRvIII by removing phages that bind to cell membrane proteins other than EGFRvIII in the antibody phage library. The recovered supernatant was treated with GBM8 (EGFR -/EGFRvIII +) cells to bind at 4°C for 1 hour. After washing cells 5 with cold complete medium to remove non-specific binding, the cells are transferred to 37° C. and incubated for 30 minutes to induce receptor-mediated endodytosis of the antigen-antibody complex. The cells are washed with cold PBS, and finally washed with 0.1 M Glycine buffer (pH 2.0) to remove non-internalized phage particles bound to the cell surface. Neutralization was performed using 2 M Tris buffer (pH 8.0), 0.5 ml of 100 mM TEA (Triethylamine) was added, and the cells were allowed to stand for 10 minutes to lyse the cells, and neutralization was performed with 1 ml of 2 M Tris buffer (pH 8.0). Order. Prepare TG1 E. coli at OD600 = 0.5 to 0.8 in advance, mix 1.5 ml of the neutralized recovered phage solution with 8.5 ml of TG1 E. coli culture, and incubate at 120 rpm at 37°C for 1 hour to infect the phage recovered in TG1 E. coli And counted in LB/ampicillin solid medium. The remaining recovery solution was smeared on a 15 cm LB/ampicillin solid medium and cultured overnight, and the next day 5 mL of SB culture medium was added to collect and store the colonies (-80°C).

For continued panning, 50 μL of the previous phage solution stored was taken to perform phage particle amplification. After incubation in the host cell, the phage particles recovered by adding helper phage (VCSM13) were prepared through precipitation using PEG8000, and the next panning was performed in the same manner as the previous panning. Cell panning was performed up to 4 times to enrich the phage expressing human antibody fragments specifically binding to GBM8 cells.

Example 2. Screening of EGFRvIII specific antibody fragments

After 4 times of cell panning using GBM8 cells with EGFRvIII mutation, phage particles recovered at the final round were identified as colonies in LB/ampicillin solid culture medium through infection with host cells (TG1). These colonies were taken and cultured after each inoculation in a 96 well plate containing 200 μL SB/ampicillin culture medium (37° C., ~3 hours). Thereafter, in order to induce the expression of scFv-pIII protein, IPTG having a final concentration of 1 mM was treated in each well and incubated overnight at 30°C. Thereafter, the culture plate was centrifuged and the supernatant was removed, and then TES solution (20% w/v sucrose, 50 mM Tris, 1 mM EDTA, pH maintained at 40 µL 4°C to recover the periplasm region of the cultured cells in each well) 8.0) and lysed by standing at 4° C. for 30 minutes. Subsequently, 60 μL of the 0.2X TES solution was left for 30 minutes again, and finally the scFv-pIII protein was produced on a small scale by centrifuging the plate and recovering the supernatant. On the other hand, the recombinant EGFRvⅢ ECD-Fc protein and EGFR ECD-Fc protein were coated on a 96 well plate, and subsequently, a blocked 96 well plate was simultaneously prepared using 3% skim milk, and then 25 μL of the recovered periplasm sites were taken and each After adding to the well, it was bound for 1 hour. Using TBST, after 3 to 4 washings, HRP-coupled anti-HA antibody was bound for 1 hour, and then washed again and induced a color reaction using a TMB substrate, and then measured at OD450. Finally, 55 clones (EGFRvIII/EGFR binding capacity multiple> 3) were selected.

Thereafter, DNA sequence analysis was performed, and two EGFRvIII specific antibody fragment clone sequences were obtained.

Example 3. Conversion, production and purification of EGFRvIII specific antibody fragments into IgG

3-1. Production and purification of H1L2 and H2L1 antibodies

The polynucleotide encoding the heavy chain of the two antibodies selected in Example 2 above is composed of'EcoRI-signal sequence-VH-NheI-CH1-Hinge-CH2-CH3-stop codon-XhoI', and the light chain is'EcoRI -signal sequence-VL-HindⅢ-CL-stop codon-XhoI' was designed to construct genes in IDT.

Subsequently, the DNA fragment having the nucleotide sequence corresponding to the heavy chain was added to the pOptiVEC ^TM -TOPO TA Cloning Kit included in the OptiCHO ^TM Antibody Express Kit (Invitrogen) from Invitrogen, and the PCDNA ^TM 3.3-TOPO TA Cloning Kit corresponds to the light chain. A DNA fragment having a nucleotide sequence to be cut was cut using EcoRI (NEB) and XhoI (NEB) restriction enzymes, respectively, and cloned using an Infusion-HD cloning kit (Takara) to construct a vector for expression of fully human IgG1 antibody. Did.

Each of the constructed vectors was secured using Qiagen Maxiprep kit (Cat no. 12662), and temporary expression was performed using Expi293 ^TM Expression System (Invitrogen). The cell line used was Expi293 cell, and was cultured in a suspension culture method at 37°C, 80% humidity, 8% CO ₂ , 130 rpm using Expi293 ^TM Expression Medium as a medium. Antibody production was carried out on a 50 mL scale, and for transient expression, cells were prepared at a concentration of 2.9x10 ⁶ cells/mL, and then transfection was performed by the liposomal reagent method using Expi293 transfection reagent (Invitrogen) heavy chain DNA:light chain DNA = 2 (33.3 ㎍): 1 (16.7 ㎍) was performed using the protocol provided by the manufacturer, and cultured for 5 days. The cultured cells were centrifuged, 50 mL of each supernatant was taken, filtered using a 0.2 μm filter system, and then purified using AKTA Prime plus (GE healthcare). After installing the HiTrap MabSelect SuRe column (GE healthcare) in AKTA Prime plus (GE healthcare) and flowing the culture solution at a flow rate of 5 mL/min, the PBS was washed at a flow rate of 5 mL/min for 6 minutes. It was eluted using IgG elution buffer (Thermo Scientific). This was exchanged with PBS buffer using Amicon Ultra-15 Centrifugal Filter Unit (Millipore) to complete the purification of the final two antibodies, named as H1L2 and H2L1, respectively, and the sequence is as follows (Table 1).

The suggested sequences were classified based on KABAT numbering. Antibodies were analyzed under SDS-PAGE experiment under reducing and non-reducing conditions, and it was confirmed that stable pairing under non-reducing conditions.

3-2. Production and purification of H1L2mut antibody

When preparing a protein containing an antibody in a mammalian cell, a change in the physicochemical or biochemical activity of the protein may occur by a post-translational modification process. N-glycosylation, deamidation, isomerization, oxidation, and non-enzymatic cleavage are the major post-transcriptional modifications that greatly affect protein activity. For example, when an N-glycosylation motif (Asn-Xaa-Ser/Thr) is present in the CDR region of an antibody, the binding capacity of the antibody decreases due to the generation of large sugar chains in Asparagine (Asn). In addition, if an Asp-Asp motif is present in the therapeutic protein, isomerization and racemization, non-enzymatic post-translational modifications, occur at a specific composition or storage condition (pH 4.5 to 7.2, temperature 2 to 37°C), degrading protein activity. (Jennifer Zhang et al., Analytical Biochemistry, 2010). The CDR region of the antibody is an important site for recognition of the target antigen, and in the case of H1L2 antibody, an Asp-Asp motif (DD sequence) is present. Therefore, in order to prevent the degradation of activity due to post-translational modification, H1L2 mutant antibodies partially substituted with the amino acid sequence of the CDR-L3 region of the light chain purified in Example 2 above were produced and purified. The H1L2 mutant antibody was named H1L2mut, and the sequence is as follows (Table 1).

Antibody amino acid sequence H1L2 H1L2mut H2L1 HC F1 EVQLLESGGGLVQPGGSLRLSCAASGFTFS EVQLLESGGGLVQPGGSLRLSCAASGFTFS EVQLLESGGGLVQPGGSLRLSCAASGFTFS CDR-H1 SYGMH SYGMH DYAMH F2 WVRQAPGKGLEWVS WVRQAPGKGLEWVS WVRQAPGKGLEWVS CDR-H2 AISSSGDSTYYADSVKG AISSSGDSTYYADSVKG AISGSGGSTYYADSVKG F3 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR CDR-H3 DWSLMDV DWSLMDV GGSSRPFDY F4 WGQGTLVTVSS WGQGTLVTVSS WGQGTLVTVSS LC F1 QSVLTQPPSASGTPGQRVTISC QSVLTQPPSASGTPGQRVTISC QSVLTQPPSASGTPGQRVTISC CDR-L1 SGNNIGSKSVH SGNNIGSKSVH SGSSSNIGNNIVN F2 WYQQLPGTAPKLLIY WYQQLPGTAPKLLIY WYQQLPGTAPKLLIY CDR-L2 TTNNKHS TTNNKHS QDSKRPS F3 GVPDRFSGSKSGTSASLAISGLRSEDEADYYC GVPDRFSGSKSGTSASLAISGLRSEDEADYYC GVPDRFSGSKSGTSASLAISGLRSEDEADYYC CDR-L3 AAWDDSLSGYV AAWD E SLSGYV AAWDDSLKGWV F4 FGGGTKLTVL FGGGTKLTVL FGGGTKLTVL

Example 4. Enzyme immunoassay (ELISA) EGFRv Ⅲ Specific antibody identification

To confirm the specificity for EGFRvIII using the two produced human antibodies, the binding patterns for EGFRvIII ECD-Fc recombinant protein and EGFR ECD-Fc (R&D systems) recombinant protein were confirmed by ELISA. After coating each protein with 100 μL at a concentration of 1 μg/mL in a 96-well ELISA plate, blocking with 3% skim milk was performed, and each produced H1L2, H2L1 human antibody was 1/2 at 25 nM concentration. The antibody was treated with 3 repetitions by dilution and bound at room temperature for 1 hour. After washing with 0.1% PBST solution three times, the secondary antibody, anti-human (Fab')2-HRP antibody (Thermo), was treated for 1 hour to detect H1L2 and H2L1 human antibodies bound to each antigen. Did. After 3 washes, 100 μL of TMB (Thermo) as a substrate was treated, and after 5 minutes, the reaction was stopped using a cell-stopping solution (Cell signaling technology), and absorbance was measured at OD450.

As a result, as shown in Figure 4, H1L2 and H2L1 showed a specific binding pattern only to the EGFRvIII recombinant protein, whereas Cetuximab used as a control antibody was confirmed to bind to both EGFRvIII-ECD and EGFR-ECD.

Example 5. Immunohistochemical staining method ( Immunohistochemistry )through EGFRv Ⅲ Confirmation of specificity

Paraffin-embedded tissue was prepared for immunohistochemical staining. BALB/c-nude mice (5 weeks old) were purchased from Orient Bio Co., Ltd. to prepare paraffin embedded tissues. After the mouse was quarantined at the Experimental Animal Research Center of Samsung Seoul Hospital for one week after arrival, a xenograft model using a glioblastoma patient-derived cell was produced at an age of 7 weeks after an additional purification period of 1 week. In BALB/c-nude mice (7 weeks of age), 2 x 10 ⁶ GBM4 cells were diluted with 200 μL of a 1:1 solution of Matrigel ^® Basement Membrane Matrix (Corning ^® ) and Hank's Balanced Salt Solution (Gibco). Then, 100 μL was implanted into the subcutaneous layer on the right side of the mouse using a 1 cc syringe. GBM4 cells are glioblastoma cells expressing EGFRvIII mutation and EGFR together. After 30 days, the mice were sacrificed by cervical dislocation, and the tumor tissue produced by subcutaneous transplantation was placed in a paraffin cassette and fixed in a 500 mL storage container for 24 hours. After washing the paraffin cassette with the secondary DW flowing after 24 hours, the paraffin cassette was fabricated after placing the paraffin cassette in a 500 mL storage container filled with the secondary DW. The produced subcutaneous animal model tumor paraffin block of GBM4 cells was sliced to a thickness of 4 μm using a Leica slide section device. The section generated by cutting was picked up with a brush and tweezers and placed in a floating hot water bath at 45°C to extend the section. The stretched section was placed in a silane coated micro glass slide (MUTO PURE CHEMICAL Co.) and dried for 24 hours. After placing the section slide prepared for the immunohistochemical staining in a new electric machine maintained at 45° C., waiting for 1 hour, confirming that the paraffin section was completely extended, and having a rest period at room temperature for 10 minutes. The section slides were completely immersed in a container containing Xylene (Daejung Gold), waited for 10 minutes, and then transferred to a new soil containing clean xylene to remove paraffin for 10 minutes. Subsequently, the section slide was transferred to a container containing 100% alcohol, completely immersed, and after 10 minutes, transferred to a container containing clean 100% alcohol, waited for 10 minutes and then proceeded to the next step. In the same way, a section slide was sequentially immersed in a container containing 95%, 90%, and 80% alcohol to hydrate, and finally transferred to a container containing DW to wait for 5 minutes to complete the rehydration operation. The re-hydrated fragment slide undergoes antigenic retrieval to solve the problem of antigenic loss that occurs during paraffin block production. For revival of antigenicity, 1x DAKO REAL ^TM target retrieval solution (DAKO) was used. 1x DAKO REAL ^TM target retrieval solution (DAKO) was placed in a 500 mL container, the section slides were completely immersed, heated in a microwave oven at 700 W for 1 minute, and then repeated 3 times with a rest for 10 minutes at room temperature. In the last round, after heating, suspend for 30 minutes at room temperature and wait for the solution to reach room temperature. Section slides that had undergone antigenic revival were soaked in TBST solution (Bio world) containing 0.2% Tween20 and washed twice for 10 minutes. After this, after placing the slide container in a wet condition to suppress endogenous peroxidase activity, a 30% H ₂ O ₂ solution (JUNSEI) was prepared as a 3% H ₂ O ₂ solution using methanol and sectioned using a 1 mL pipette. It was applied to the slide, waited for 12 minutes at room temperature, and then proceeded to the next step. To remove the applied 3% H ₂ O ₂ solution, it was completely immersed in TBST solution and washed twice for 10 minutes. Next, the prepared section slides were placed on slide slides under wet conditions, and then 1% Bovine Serum Albumin solution (Santa Cruz Biotechnology) was applied and waited for 1 hour. For antibody application, it was prepared by diluting the antibody in TBST solution. Antibody dilutions were prepared such that H1L2 and H2L1 were 1 μg/mL, respectively, and Cetuximab was prepared at 1 μg/mL and 100 μg/mL. For comparison with EGFR expression, commercially available EGFR target antibodies for immunohistochemistry were purchased from cell signaling and prepared at 1:200 dilution. The 1% Bovine Serum Albumin solution applied to the section slide was removed by inhalation, and the prepared antibody was applied to each slide. Each antibody-coated slide is placed in a slide container in wet conditions and reacted in a refrigerator at 4° C. for 24 hours. After the reaction, immerse in Tris-buffered saline solution containing 0.2% Tween20 and wash twice for 10 minutes. Horseradish Peroxidase (HRP)-coupled anti-human IgG antibody secondary antibody is prepared for each antibody and applied, and reacted at room temperature for 1 hour. After the reaction time, it was completely immersed in TBST solution and washed twice for 10 minutes. After diluting the A and B solutions of the VECTASTAIN ABC HRP Kit (VECTOR) to 1:50 each with a TBST solution, mix the diluted solution 1:1 to prepare an A/B mixture, and pipette 1 mL on each slide. A / B mixture was applied using. After application, the slides were reacted for 1 hour in a slide container under wet conditions. At the end of the reaction, wash twice with TBST solution for 10 minutes. Each slide was coated with 1 mL of DAB solution (Sigma) using a pipette to develop color for 10 minutes, and soaked in DW for 10 minutes to complete the reaction. For cell nuclei staining, it was immersed in DAKO ^® Mayer's hematoxylin (DAKO) solution for 1 second, and then washed in DW for 10 minutes. After this, for dehydration of the fragment slides, dehydration was performed by sequentially immersing in 80% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol solutions for 5 minutes each. After the dehydration was finished, soaked in the Xylene solution for 10 minutes and waited, then the slide was moved to a clean Xylene solution and waited for 10 minutes. Afterwards, the slide contained in the xylene solution is taken out and the slide is placed on two sheets of KIMTECH SCIENCE ^® Wipers (Yuhan-Kimberly) so that the remaining xylene solution is absorbed. At this time, the slide surface with the sliced tissue section is placed facing the sky. To enclose the section slide, drop a drop of Permount mounting solution (Fisher Scientific) onto the tissue section using a dropper. Thereafter, the slide cover glass was covered with a tissue piece using tweezers. At this time, the Permount mounting solution was uniformly spread between the slide and the cover glass, and when bubbles were formed, the cover glass was removed by pressing the cover glass with tweezers. The enclosed section slides were completely fixed for 24 hours. When fixing of the enclosed section slide was completed, the section slide was analyzed by scanning the entire section slide using a ScanScope ^® AT (Aperio) instrument.

5, Cetuximab was confirmed to bind to both EGFR and EGFRvIII as known. That is, it was confirmed that the staining was not performed when staining with GBM4 tissues (EGFR +/EGFRvIII +) at 1 µg/mL, but the dyeing was overall performed at a high concentration of 100 µg/mL. On the other hand, it was confirmed that the H1L2 antibody according to the present invention is strongly stained at a specific position when staining proceeds at a concentration of 1 μg/mL. Since GBM4 tissue is co-expressed with EGFRvIII and EGFR, the H1L2 clone appears to specifically bind to the population expressing EGFRvIII, and at the same concentration, it was confirmed that H2L1 does not undergo immunohistochemical staining. Accordingly, the antibodies according to the present invention, which minimize binding to EGFR and specifically bind to EGFRvIII, are applied to CAR-T cell technology to specifically target EGFRvIII + tumor cells to maximize efficacy and minimize side effects I confirmed that I could do it.

Example 6. Confirmation of binding ability of H1L2 mutant to EGFRvIII

The binding ability of the H1L2 and H1L2mut antibodies prepared in Example 3 to the EGFRvIII protein was measured. Specifically, the binding constants of the H1L2 and H1L2mut antibodies were measured using BIACORE T200 (GE), an SPR analysis equipment. First, after the CM5 sensor chip (GE) was mounted on the BIACORE T200, antigen fixation was performed using an Amine coupling kit (GE). The EGFRvIII recombinant protein used in the analysis was expressed in 293F (Gibco) based on the published sequence, and purified using affinity chromatography. Thereafter, 300 RU of EGFRvIII recombinant protein was immobilized on a CM5 sensor chip. As for the analysis, HBS-EP+ (GE) was performed in the mobile phase, and each antibody used in the analysis was at a flow rate of 30 uL/min, association time was 180 seconds, and dissociation time was 180 seconds. The binding constant was calculated using BIAEVALUATION software (GE).

Ag : rhEGFRvIII K _a (1/Ms) K _d (1/s) K _D (nM) H1L2 2.132E+05 7.615E-03 35.71 H1L2mut 9.694E+04 4.777E-05 4.93

Surface plasmon resonance sensorgrams for the interaction of antigen (EGFRvIII) and antibody (H1L2mut, H1L2) are shown in FIGS. 6A and 6B. As shown in Figure 6a, H1L2 antibody interacts with the antigen in the association section of 0 to 180 seconds, it can be confirmed that the pattern deviates from the dissociation section of 180 to 360 seconds. In addition, as shown in Figure 6b, it was confirmed that the H1L2mut antibody showed a stable binding pattern not only in the association section of 0 to 180 seconds, but also in the dissociation section of 180 to 360 seconds. Table 2 shows K _a , K _d , and K _D calculated based on these results. As shown in Table 2, it can be seen that the H1L2 antibody and H1L2mut antibody have excellent binding ability to the EGFRvIII recombinant protein.

<110> Samsung Life Public Welfare Foundation <120> Antibody and Fragment Thereof Binding Human Epidermal Growth Factor Receptor Variant III <130> PN122945KR <150> KR 10-2017-0103217 <151> 2017-08-17 <160> 21 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR H1 <400> 1 Ser Tyr Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR H2 <400> 2 Ala Ile Ser Ser Ser Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR H3 <400> 3 Asp Trp Ser Leu Met Asp Val 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR L1 <400> 4 Ser Gly Asn Asn Ile Gly Ser Lys Ser Val His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR L2 <400> 5 Thr Thr Asn Asn Lys His Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR L3 <400> 6 Ala Ala Trp Asp Asp Ser Leu Ser Gly Tyr Val 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR H1 <400> 7 Asp Tyr Ala Met His 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR H2 <400> 8 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR H3 <400> 9 Gly Gly Ser Ser Arg Pro Phe Asp Tyr 1 5 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR L1 <400> 10 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ile Val Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR L2 <400> 11 Gln Asp Ser Lys Arg Pro Ser 1 5 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR L3 <400> 12 Ala Ala Trp Asp Asp Ser Leu Lys Gly Trp Val 1 5 10 <210> 13 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> HC F1 <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> HC F2 <400> 14 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 15 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> HC F3 <400> 15 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HC F4 <400> 16 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 17 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> LC F1 <400> 17 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys 20 <210> 18 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LC F2 <400> 18 Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 19 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> LC F3 <400> 19 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 1 5 10 15 Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LC F4 <400> 20 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 mutation <400> 21 Ala Ala Trp Asp Glu Ser Leu Ser Gly Tyr Val 1 5 10

Claims (10)

A heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 1, CDRH2 comprising SEQ ID NO: 2 and CDRH3 comprising SEQ ID NO: 3; And a light chain variable region comprising CDRL1 comprising SEQ ID NO: 4, CDRL2 comprising SEQ ID NO: 5 and CDRL3 comprising SEQ ID NO: 6;
A heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 7, CDRH2 comprising SEQ ID NO: 8 and CDRH3 comprising SEQ ID NO: 9; And a light chain variable region comprising CDRL1 comprising SEQ ID NO: 10, CDRL2 comprising SEQ ID NO: 11 and CDRL3 comprising SEQ ID NO: 12; or
A heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 1, CDRH2 comprising SEQ ID NO: 2 and CDRH3 comprising SEQ ID NO: 3; And EGFRvIII (Epidermal Growth Factor Receptor Variant III) having a light chain variable region comprising CDRL1 comprising SEQ ID NO: 4, CDRL2 comprising SEQ ID NO: 5 and CDRL3 comprising SEQ ID NO: 21, or an antigen-binding fragment thereof . The antibody or antigen-binding fragment thereof according to claim 1, comprising a framework region (FR) having a sequence selected from the group consisting of SEQ ID NOs: 13 to 20. The antibody according to claim 1, wherein the antibody is a monoclonal antibody and a fully human antibody, or an antigen-binding fragment thereof. A nucleic acid encoding the antibody of any one of claims 1 to 3 or an antigen-binding fragment thereof. An expression vector comprising the nucleic acid of claim 4. Cells transformed with the expression vector of claim 5. A composition for preventing or treating cancer comprising the antibody of any one of claims 1 to 3 or an antigen-binding fragment thereof. The composition for preventing or treating cancer according to claim 7, wherein the cancer is selected from the group consisting of lung cancer, colon cancer, stomach cancer, kidney cancer, prostate cancer, breast cancer, glioblastoma, and ovarian cancer. A composition for diagnosing cancer comprising the antibody of claim 1 or an antigen-binding fragment thereof. A cancer diagnostic kit comprising the composition for diagnosing cancer of claim 9.

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