KR102728754B1 - GRP94 specific antibody or antigen-binding fragment thereof and uses thereof - Google Patents

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to GRP94 and uses thereof. The GRP94 antibody or an antigen-binding fragment thereof according to the present invention has very high specificity and affinity for GRP94, and has very excellent growth inhibition effects, invasion inhibition effects, and angiogenesis inhibition effects on colorectal cancer cell lines, and thus can be usefully used as a composition for treating cancer, inhibiting cancer metastasis, and inhibiting angiogenesis.

Description

{GRP94 specific antibody or antigen-binding fragment thereof and uses thereof}

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to GRP94 and their uses.

Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth leading cause of cancer-related deaths. A number of chemotherapeutic agents, including 5-fluorouracil (5-FU), irinotecan, oxaliplatin, and their combinations such as FOLFOX (leucovorin, 5-FU, and oxaliplatin) and FOLFIRI (leucovorin, 5-FU, and irinotecan), have been used as standard treatments for CRC. However, despite their clinical efficacy, these chemotherapeutic agents inhibit DNA synthesis and/or disrupt microtubule structure. As a result, they are known to cause various side effects such as alopecia, diarrhea, thrombocytopenia, and paresthesia.

Therapeutic antibodies are the most effective targeted cancer treatment. Since the approval of the mouse anti-CD3 monoclonal antibody (OKT3) by the US FDA, remarkable advances in recombinant DNA technology have produced a variety of humanized and humanized antibodies.

Recently, Imclone Systems Inc. has developed Cetuximab, a recombinant mouse/human chimeric monoclonal antibody targeting the epidermal growth factor receptor (EGFR), for the treatment of patients with colorectal cancer that expresses EGFR and wild-type KRAS (ki-ras2 kirsten rat sarcoma viral oncogene homolog). Although widely used in clinical practice, Cetuximab is not effective as a single agent, and therefore, it is recommended to be administered in combination with FOLFIRI or FOLFOX regimens.

Heinemann et al. reported that FOLFIRI plus cetuximab regimen is preferred as first-line therapy in patients with metastatic colorectal cancer. In addition, another unmet medical need in cancer treatment using cetuximab is that it is effective in only a portion of patients with colorectal cancer. Cetuximab is effective in only about 10-20% of patients with colorectal cancer, and the remaining patients show cetuximab resistance due to mutations in EGFR downstream genes, including KRAS, PI3KCA (phosphoinositide-3-kinase catalytic subunit alpha), PTEN (phosphatase and tensin homolog), and BRAF. Therefore, it is very important to discover new therapeutic targets in colorectal cancer and develop new treatment regimens to improve the clinical outcomes of colorectal cancer treatment.

PCT Patent Publication No. WO2015/023976 (Published on February 19, 2015)

The present inventors used phage display technology to isolate, produce, and purify antibodies that strongly bind to rhGRP94 from a human synthetic antibody library, and discovered four antibodies targeting GRP94. The anti-GRP94 antibodies of the present invention specifically target the GRP94 antigen on the cell surface, and were able to reduce tumor reduction, tumor invasion, and angiogenesis in cetuximab-resistant colorectal cancer, thereby completing the present invention.

Therefore, it is an object of the present invention to provide an antibody or antigen-binding fragment that specifically binds to GRP94.

Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment.

Another object of the present invention is to provide a recombinant vector comprising the nucleic acid.

Another object of the present invention is to provide a host cell comprising the recombinant vector.

Another object of the present invention is to provide a pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds to GRP94 and a pharmaceutically acceptable carrier.

Another object of the present invention is to provide a composition or kit for detecting GRP94 comprising an antibody or antigen-binding fragment that specifically binds to GRP94.

According to one aspect of the present invention, the present invention provides an antibody or antigen-binding fragment that specifically binds to GRP94.

In one embodiment of the present invention, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence represented by the following general formula 1, an HCDR2 comprising an amino acid sequence represented by the following general formula 2, and an HCDR3; and a light chain variable region comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence represented by the following general formula 3, an LCDR2 comprising an amino acid sequence represented by the following general formula 4, and an LCDR3 comprising an amino acid sequence represented by the following general formula 5.

General formula 1

GFTFSX ₆ YX ₈ MS

General formula 2

_{X 1 IX 3 ​ ​ ​ ​}

General formula 3

X ₁ GSX ₄ SNIGX ₉ NX

₁₁ VX ₁₃

General formula 4

ADX ₃ X ₄ RPS

General formula 5

X ₁ X ₂ WDX ₅ SLNX ₉

i) X ₆ X ₈ of the general formula 1 is NY, X ₁ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ of the general formula 2 are GYPNSGST, and the HCDR3 comprises an amino acid sequence of SEQ ID NO: 3, X ₁ X ₄ X ₉ X ₁₁ X ₁₃ of the general formula 3 are TSNAS, X ₃ X ₄ of the general formula 4 is SH, and X ₁ X ₂ X ₅ X ₉ of the general formula 5 is GAAA;

ii) X ₆ X ₈ of the general formula 1 is NA, X ₁ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ of the general formula 2 are GSSSSGST, and the HCDR3 comprises an amino acid sequence of SEQ ID NO: 11, X ₁ X ₄ X ₉ X ₁₁ X ₁₃ of the general formula 3 are SPSTT, X ₃ X ₄ of the general formula 4 is SH, and X ₁ X ₂ X ₅ X ₉ of the general formula 5 is ASDG; or

iii) X ₆ X ₈ of the general formula 1 is GA, X ₁ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ of the general formula 2 are ASHGGSSK, and the HCDR3 comprises an amino acid sequence of SEQ ID NO: 19, X ₁ X ₄ X ₉ X ₁₁ X ₁₃ of the general formula 3 are SSSTS, X ₃ X ₄ of the general formula 4 is NN, and X ₁ X ₂ X ₅ X ₉ of the general formula 5 is ASDA.

As used herein, "X _n " and "X _m " are used to represent amino acids at positions n and m in the above-defined sequences i) to iii), wherein n and m are integers representing amino acid positions within the sequence calculated from the N-terminus of the sequence. For example, X ₃ and X ₇ represent amino acids at the 3rd and 7th positions, respectively, from the N-terminus of the sequence.

In an embodiment according to one aspect of the present invention, X _n in sequence i) is independently selected from a group of individually defined substitutable amino acid residues. It will be appreciated by those skilled in the art that X _n may be selected from any one of the amino acid residues listed as substitutable residues, and that this selection is made independently of the choice of amino acid for X _m , wherein n and m are different. Thus, any of the listed possible residues at a position X _n may be independently combined with any other of the listed possible residues at the other various positions.

The term "GRP94 (glucose-regulated protein 94)" in this specification refers to a chaperone protein encoded by the HSP90B1 gene, also known as GP96, ERp99, or endoplasmin. GRP94 is known to play an important role in the folding of proteins in the secretory pathway, such as Toll-like receptors and integrins, and is known as an essential immune chaperone that regulates both innate and adaptive immunity. GRP94 is also known as a therapeutic target for diseases such as glaucoma, multiple myeloma, and metastatic cancer.

As used herein, the term “antibody” refers to a specific antibody against the GRP94, including complete antibody forms as well as antigen-binding fragments of antibody molecules.

A complete antibody has two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond. The heavy-chain constant regions are of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and are divided into subclasses: gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The constant regions of the light chain are of the kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

As used herein, the term "antigen-binding fragment" means a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab') ₂ , chemically linked F(ab') ₂ , and Fv. Among antibody fragments, Fab has one antigen-binding site in a structure having variable regions of the light and heavy chains, a constant region of the light chain, and a first constant region (CH1) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab') ₂ antibody is produced when cysteine residues in the hinge region of Fab' form a disulfide bond. Fv is the minimum antibody fragment having only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are disclosed in PCT International Patent Publication Nos. WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086, and WO 88/09344. In a two-chain Fv, the heavy chain variable region and the light chain variable region are non-covalently linked, and in a single-chain Fv, the variable region of the heavy chain and the variable region of the single chain are generally covalently linked through a peptide linker or directly linked at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction digestion of whole antibodies with papain yields Fab fragments, and digestion with pepsin yields F(ab') ₂ fragments), or they can be produced using genetic recombination techniques.

In the present invention, the antibody is preferably in the form of scFv or a complete antibody form. In addition, the heavy chain constant region can be selected from any one of the isotypes of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). Preferably, the constant region is gamma 1 (IgG1), gamma 3 (IgG3) and gamma 4 (IgG4), and most preferably gamma 1 (IgG1) isotype. The light chain constant region can be kappa or lambda type, and is preferably kappa type. Therefore, the preferred antibody of the present invention is in the form of scFv or IgG1 having a kappa light chain and a gamma 1 heavy chain.

As used herein, the term "heavy chain" refers to both a full-length heavy chain and fragments thereof, comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2 and CH3. Furthermore, the term "light chain" as used herein refers to both a full-length light chain and fragments thereof, comprising a variable region domain VL (Vk) comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL (Ck).

As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs. The CDRs provide the key contact residues for binding of the antibody to the antigen or epitope.

Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFV), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of the above antibodies.

In this specification, the term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is generally composed of four FR domains FR1, FR2, FR3 and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL/Vk):

(a) FRH1 (Framework region 1 of Heavy chain)-CDRH1 (complementarity determining region 1 of Heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4; and

(b) FRL1 (Framework region 1 of Light chain)-CDRL1 (complementarity determining region 1 of Light chain)-FRL2-CDRL2-FRL3-CDRL3-FRL4.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have a similar structure, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies that bind to a particular antigen can be isolated from antibodies that bind the antigen and screened against a library of complementary VL or VH domains, respectively, using the VH or VL domains.

As used herein, the term "specifically binds" or the like means that the antibody, or antigen-binding fragment thereof, or other construct, such as a scFv, forms a complex with the antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 10 ^-6 M or less, preferably 1 x 10 ^-7 M or less, more preferably 1 x 10 ^-8 M or less (e.g., a smaller K _D indicates a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, an isolated antibody that specifically binds human GRP94 may exhibit cross-reactivity to other antigens, such as GRP94 molecules from other species.

As used herein, the term "affinity" refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule Y and its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein.

Also, as used herein, the term "human antibody" refers to an antibody produced by a human or a human cell, or an antibody derived from a non-human source utilizing human antibody repertoires or other human antibody coding sequences, which has an amino acid sequence corresponding to the amino acid sequence of that antibody. This definition of human antibody excludes humanized antibodies that contain non-human antigen-binding residues.

As used herein, the term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, and the remainder of the heavy and/or light chain is derived from a different source or species.

The term "humanized antibody" as used herein refers to a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin (e.g., mouse) antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, residues from the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences. Such modifications are made to further improve and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions have the sequence of an FR region of a human immunoglobulin. The humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc region) sequence, up to substantially a human immunoglobulin constant region (Fc region) sequence.

In one embodiment of the present invention, the antibody or antigen-binding fragment comprises any one of the following i) to iii):

i) HCDR1, HCDR2, HCDR3 comprising amino acid sequences of SEQ ID NOs: 1 to 3, and LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 4 to 6;

ii) HCDR1, HCDR2, HCDR3 comprising amino acid sequences of SEQ ID NOs: 9 to 11, and LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 12 to 14; or

iii) HCDR1, HCDR2, HCDR3 comprising amino acid sequences of SEQ ID NOs: 17 to 19, and LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 20 to 22.

In another embodiment of the present invention, the antibody or antigen-binding fragment comprises any one of the following i) to iii):

i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;

ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; or

iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24.

The anti-GRP94 antibody or antigen-binding fragment thereof of the present invention may include variants of the amino acid sequence within the range that can specifically recognize GRP94, as will be appreciated by those skilled in the art. For example, the amino acid sequence of the antibody may be changed to improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and/or substitutions of amino acid sequence residues of the antibody.

The above variants are said to have "substantial similarity", meaning that the two peptide sequences share at least about 90% sequence identity, more preferably at least about 95%, 98% or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitutions.

These amino acid mutations are based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be said to be biologically functional equivalents.

In introducing mutations, the hydrophobic index of amino acids can be considered. Each amino acid is assigned a hydrophobic index according to its 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 imparting interactive biological functions to proteins. It is a known fact that amino acids having similar hydrophobic indices must be substituted in order to have similar biological activities. When introducing mutations with reference to hydrophobic indices, substitutions are preferably made between amino acids having hydrophobic indices of within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

Meanwhile, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins having equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); asphaltene (+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).

When introducing a mutation with reference to a hydrophilicity value, substitution is preferably made between amino acids showing a difference in hydrophilicity value within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

Amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

In one embodiment of the present invention, the antibody or antigen-binding fragment has a dissociation constant K _D value of 10 ^-8 M or less.

In another embodiment of the present invention, the antibody or antigen-binding fragment has the ability to inhibit the proliferation of one or more tumor cell lines selected from the group consisting of HCT-8, HT-29, LoVo, HCT-116, and Caco-2 in vitro and in vivo.

In one embodiment of the present invention, the antibody of the present invention or an antigen-binding fragment thereof can be prepared as an antibody drug conjugate (ADC) that forms a conjugate with a drug. The drug is a cytotoxic agent or a chemotherapeutic agent.

In specific embodiments of the present invention, the cytotoxic agent or chemotherapeutic agent may be selected from, but is not limited to, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-amino camptothecin, actinomycin D, amanitin, aminopterin, anthracycline, anthramycin (AMC), oristatin (monomethyl auristatin E or monomethyl auristatin F), bleomycin, busulfan, butyric acid, calicheamicin, camptothecin, cisplatin, cyclophosphamide, cytarabine, daunorubicin, doxorubicin, irinotecan, mitoxantrone, taxol, topotecan, vinblastine, vincristine, and derivatives thereof.

When the antibody or antigen-binding fragment of the present invention is prepared as an antibody-drug conjugate, the drug (payload) can be conjugated to the GRP94 antibody or antigen-binding fragment of the present invention by covalent bonding via a chemical linker. The "linker" refers to any moiety that links, connects, or associates a binding agent (e.g., an antibody or antigen-binding fragment thereof) with a drug as described herein. In general, a binding agent linker suitable for the antibody conjugate is one that is sufficiently stable to utilize the circulating half-life of the antibody while at the same time being capable of releasing its drug following antigen-mediated internalization of the conjugate. The linker can be cleavable or non-cleavable. A cleavable linker is a linker that is cleaved by cellular metabolism, such as hydrolysis, reduction, or cleavage via an enzymatic reaction, and then internalized. A non-cleavable linker is a linker that is internalized after releasing the attached drug via lysosomal degradation of the antibody. Suitable linkers include, but are not limited to, acid-cleavable linkers, enzymatically cleavable linkers, reduction-cleavable linkers, self-immolative linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, those that are or include a peptide, a glucuronide, a succinimide-thioether, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citrulline unit, and a para-aminobenzyl (PAB) unit.

In another embodiment of the present invention, the antibody of the present invention or an antigen-binding fragment thereof may be provided as a complex in which an antibody or an antigen-binding fragment thereof that specifically binds to the above-described GRP94; and a polypeptide are linked. The polypeptide is not limited and may be, for example, another antibody or an antigen-binding fragment thereof, or a target binding polypeptide. The complex is covalently linked to each other, and according to one embodiment of the present invention, the complex may be implemented in the form of a fused protein or a conjugate, and the antibody or an antigen-binding fragment thereof and the polypeptide may be directly linked, or may be indirectly linked via a linker (e.g., an amino acid linker).

In another embodiment of the present invention, the antibody of the present invention or an antigen-binding fragment thereof can function as an extracellular domain of a chimeric antigen receptor (CAR). When the antibody of the present invention or an antigen-binding fragment thereof that specifically binds to GRP94 is used as an extracellular domain of a chimeric antigen receptor, the chimeric antigen receptor comprises (a) a GRP94 antibody or antigen-binding fragment; (b) a transmembrane domain; and an intracellular signaling domain.

In a specific embodiment of the present invention, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of T-cell receptor, CD27, CD28, CD3, epsilon, CD45, CD4, CD5, CD8 (CD8α), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

In a specific embodiment of the present invention, the intracellular signaling domain is a domain derived from a CD3ζ (CD3 zeta) chain, and the intracellular signaling domain may additionally include a costimulatory molecule selected from the group consisting of OX40 (CD134), CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), and 4-1BB (CD137).

The chimeric antigen receptor of the present invention can be expressed in effector cells selected from the group consisting of dendritic cells, killer dendritic cells, mast cells, natural killer cells, B lymphocytes, T lymphocytes, macrophages and precursor cells thereof.

According to one aspect of the present invention, the present invention provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to GRP94 of the present invention as described above.

The term "nucleic acid molecule" in this specification has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base portion is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

Considering the above-described mutations having biologically equivalent activity, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the antibody or an antigen-binding fragment thereof is interpreted to also include a sequence showing substantial identity therewith. The above-described substantial identity means a sequence showing at least 60% homology, more specifically 70% homology, even more specifically 80% homology, even more specifically 90% homology, and most specifically 95% homology, when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS 5:151-3(1989); Corpet et al., Nuc. Acids Res. 16:10881-90(1988); Huang et al., Comp. Appl. BioSci. 8:155-65(1992) and Pearson et al., Meth. Mol. Biol. 24:307-31(1994).

In a specific embodiment of the present invention, the nucleic acid molecule of the present invention comprises a base sequence of SEQ ID NO: 33 to 56, a fragment thereof, or a combination thereof.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule of the present invention described above.

The term "vector" as used herein refers to a means for expressing a target gene in a host cell, including plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

According to a preferred embodiment of the present invention, in the vector of the present invention, the nucleic acid molecule encoding the light chain variable region and the nucleic acid molecule encoding the heavy chain variable region are operatively linked to a promoter.

As used herein, the term "operatively linked" refers to a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and/or translation of the other nucleic acid sequence.

The recombinant vector system of the present invention can be constructed by various methods known in the art, and specific methods therefor are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

For example, when the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, which generally has a polyadenylation sequence as a transcription termination sequence.

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

In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.

Meanwhile, the expression vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selectable marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

According to another aspect of the present invention, the present invention provides a host cell transformed with the recombinant vector described above.

Any host cell capable of stably and continuously cloning and expressing the vector of the present invention is known in the art, and any host cell can be used. For example, suitable eukaryotic host cells for the vector include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK-293 cells.

As used herein, the terms "transformed", "transduced" or "transfected" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transformed", "transduced" or "transfected" cell is a cell that has been transformed, transduced or transfected with an exogenous nucleic acid, and such cell includes the cell and progeny cells resulting from passage thereof.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) an antibody or antigen-binding fragment specifically binding to GRP94 of the present invention as described above; and ii) a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the pharmaceutical composition is a pharmaceutical composition for treating cancer, inhibiting cancer metastasis, or inhibiting angiogenesis.

The term "angiogenesis" in this specification refers to the process by which new capillaries are formed from existing microvessels. Normally, angiogenesis occurs during embryonic development, tissue regeneration and wound healing, and the corpus luteum, a periodic change in the female reproductive system, and even in these cases, it is strictly regulated and progresses (Folkman J et al., Int. Rev. Exp. Pathol., 16, pp207-248, 1976). In adults, vascular endothelial cells grow very slowly and do not divide relatively well compared to other types of cells. The process by which angiogenesis occurs generally consists of the restructuring of blood vessels by protease-induced degradation of the vascular basement membrane, migration and proliferation of vascular endothelial cells, and formation of lumen by vascular endothelial cell differentiation in response to stimulation by angiogenic factors, thereby creating new capillaries. However, there are diseases caused by angiogenesis not being regulated autonomously and growing pathologically.

Diseases related to neovascularization that appear in pathological conditions include hemangiomas, angiofibromas, vascular malformations, and cardiovascular diseases such as arteriosclerosis, vascular adhesions, and edematous sclerosis. Ophthalmic diseases caused by neovascularization include corneal transplant neovascularization, neovascular glaucoma, diabetic retinopathy, corneal diseases caused by neovascularization, macular degeneration, pterygium, retinal degeneration, macular degeneration, posterior lenticulitis, and granular conjunctivitis.

Chronic inflammatory diseases such as arthritis, dermatological diseases such as psoriasis, telangiectasia, pyogenic granulomas, seborrheic dermatitis, acne, Alzheimer's disease, and obesity are also associated with angiogenesis, and cancer growth and metastasis are necessarily dependent on angiogenesis (D'Amato RJ et al., Ophthalmology, 102(9), pp1261-1262, 1995; Arbiser JL, J. Am . Acad . Dermatol., 34(3), pp486-497, 1996; O'Brien KD et al. Circulation , 93(4), pp672-682, 1996; Hanahan D et al., Cell , 86, pp353-364, 1996).

In particular, in the case of cancer, angiogenesis plays an important role in the growth and metastasis of cancer cells. Tumors receive nutrients and oxygen necessary for growth and proliferation through new blood vessels, and new blood vessels that have penetrated the tumor provide an opportunity for metastatic cancer cells to enter the blood circulation, thereby promoting metastasis (Folkman and Tyler, Cancer Invasion and metastasis, Biological mechanisms and Therapy (S.B. Day ed.) Raven press, New York, pp94-103, 1977; Polverini PJ, Crit. Rev. Oral. Biol. Med., 6(3), pp230-247, 1995). The main cause of death in cancer patients is metastasis, and the reason why chemotherapy and immunotherapy currently used in clinical practice do not contribute to increasing the survival rate of cancer patients is precisely because of cancer metastasis.

Arthritis, a representative inflammatory disease, is caused by autoimmune abnormalities, but as the disease progresses, chronic inflammation in the synovial space between the joints induces angiogenesis, destroying cartilage. That is, with the help of cytokines that induce inflammation, synovial cells and vascular endothelial cells proliferate in the synovial space, and as angiogenesis progresses, joint pannus, a connective tissue layer that develops in the cartilage area, is formed, destroying the cartilage that acts as a cushion (Koch AE et al., Arthritis. Rheum., 29, pp471-479, 1986; Stupack DG et al., Braz J. Med. Biol. Rcs., 32(5), pp578-581, 1999; Koch AE, Arthritis. Rheum., 41(6), pp951-962, 1998).

Many ocular diseases that cause blindness in millions of people worldwide each year are also caused by angiogenesis (Jeffrey MI et al., J. Clin. Invest., 103, pp1231-1236, 1999). Representative examples of these diseases are diseases caused by angiogenesis, such as macular degeneration in the elderly, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, and corneal diseases caused by neovascularization (Adamis AP et al., Angiogenesis, 3, pp9-14, 1999). Among these, diabetic retinopathy is a complication of diabetes in which the capillaries in the retina invade the vitreous humor, ultimately leading to blindness.

Psoriasis, characterized by red spots and scaly skin, is a chronic proliferative disease of the skin that is incurable and causes pain and deformity. In normal people, keratinocytes proliferate once a month, but in psoriasis patients, they proliferate at least once a week. This rapid proliferation requires a large blood supply, so angiogenesis must occur actively (Folkman J, J. Invest. Dermatol., 59, pp40-48, 1972).

The term "cancer metastasis" in the present invention means the movement of tumor cells from one organ or part to another distantly separated place. Cancer metastasis is accomplished by the stage in which in situ tumors invade the basement membrane (invasion), the stage in which cancer cells that have passed through the basement membrane enter the blood or lymphatic circulation system (intravasation), and the stage in which cancer cells that have been captured and survived in another organ after guffb circulation go through a period of dormant single cells or latent micrometastasis and ultimately form metastatic colonies together with blood vessel formation.

In a specific embodiment of the present invention, the cancer includes solid cancer or blood cancer.

The term "solid tumor" in this specification refers to a cancer that has characteristics distinct from blood cancer and is formed by a lump of abnormal cell growth in various solid organs such as the bladder, breast, intestine, kidney, lung, liver, brain, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin.

In one embodiment of the present invention, the solid cancer is at least one cancer selected from the group consisting of gastric cancer, rectal cancer, colon cancer, colorectal cancer, inflammation-related colon cancer, liver cancer, lung cancer (non-small cell lung cancer, lung adenocarcinoma), ovarian cancer, melanoma, pancreatic cancer, uterine cancer, testicular cancer, and breast cancer, but is not limited thereto (Wu et al., Adv Cancer Res. 2016;129:165-90.; Ansa-Addo et al., Curr Top Med Chem. 2016; 16(25): 2765-2778.).

The term "blood cancer" in this specification refers to a cancer that occurs in a component that constitutes blood, and means a malignant tumor that occurs in the blood, hematopoietic organs, lymph nodes, lymphatic organs, etc.

In one specific example of the present invention, the blood cancer is at least one blood cancer selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute monocytic leukemia, multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma, but is not limited thereto.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

According to one embodiment of the present invention, the administration is intravenous administration, intravitreal administration, intrathecal administration, parenteral administration, subcutaneous administration, transdermal administration, or administration by infusion.

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a generally skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. According to a preferred embodiment of the present invention, 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 treat or inhibit the above-mentioned cancer, cancer metastasis, or angiogenesis.

The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and thereby. At this time, 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 an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally contain a dispersant or stabilizer.

Since the pharmaceutical composition of the present invention uses an antibody or an antigen-binding fragment thereof that specifically binds to the GRP94 of the present invention as an effective ingredient, common contents between the two are omitted to avoid excessive complexity of the specification due to repetitive description.

According to another aspect of the present invention, the present invention provides a composition or kit for detecting GRP94 comprising an antibody or an antigen-binding fragment thereof that specifically binds to GRP94 of the present invention as described above.

The diagnostic kit of the present invention comprises an antibody or an antigen-binding fragment thereof that specifically binds to the GRP94 of the present invention described above, and diagnoses the same disease as the pharmaceutical composition of the present invention. Therefore, the description of common contents between the two is omitted to avoid excessive complexity of the present specification due to repeated description.

Since the above-described kit contains antibodies, it can basically be manufactured to be suitable for various immunoassays or immunostainings. The immunoassays or immunostainings include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining and immunoaffinity purification. The methods of the immunoassays or immunostainings are described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay(ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which are incorporated herein by reference.

For example, when the method of the present invention is performed according to a radioimmunoassay method, antibodies labeled with radioactive isotopes (e.g., C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) can be used to detect the GRP94 protein. When the present invention is performed by an ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating a sample to be analyzed on the surface of a solid substrate; (ii) reacting the sample with an antibody of the present invention that specifically binds to GRP94 as a primary antibody; (iii) reacting the resultant of step (ii) with a secondary antibody conjugated to an enzyme; and (iv) measuring the activity of the enzyme.

Suitable solid substrates include hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gels, most specifically microtiter plates.

Enzymes conjugated to the secondary antibody include, but are not limited to, enzymes that catalyze a chromogenic reaction, a fluorescent reaction, a luminescent reaction or an infrared reaction, and include, for example, alkaline phosphatase, beta-galactosidase, horse radish peroxidase, luciferase and cytochrome P ₄₅₀ . When alkaline phosphatase is used as an enzyme binding to the secondary antibody, color reaction substrates such as bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and enhanced chemifluorescence (ECF) are used as substrates, and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), Substrates that can be used include ABTS (2,2-Azine-di[3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD), and naphthol/pyronine, glucose oxidase, and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate).

When the present invention is implemented in a capture-ELISA manner, a specific embodiment of the present invention comprises the steps of (i) coating an antibody that specifically binds to GRP94 as a capturing antibody on the surface of a solid substrate; (ii) reacting the capturing antibody with a sample; (iii) reacting the resultant of step (ii) with a detecting antibody to which a label that generates a signal is bound; and (iv) measuring a signal generated from the label.

The above detection antibodies of the present invention have a label that generates a detectable signal. The labels include, but are not limited to, chemicals (e.g., biotin), enzymes (e.g., alkaline phosphatase, beta-galactosidase, horse radish peroxidase, and cytochrome P ₄₅₀ ), radioactive substances (e.g., C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent substances (e.g., fluorescein), luminescent substances, chemiluminescent substances, and FRET (fluorescence resonance energy transfer). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

In the above ELISA method and capture-ELISA method, the final enzyme activity measurement or signal measurement can be performed according to various methods known in the art. If biotin is used as a label, the signal can be easily detected with streptavidin, and if luciferase is used, the signal can be easily detected with luciferin.

Samples that can be applied to the kit of the present invention include, but are not limited to, cells, tissues or tissue-derived extracts, lysates or purified substances, blood, plasma, serum, lymph or ascites.

The antibody of the present invention can be used for in vivo or in vitro imaging. According to another aspect of the present invention, the present invention provides an imaging composition comprising a conjugate of the antibody of the present invention described above and a label that generates a detectable signal bound to the antibody.

Labels that generate the detectable signal include, but are not limited to, T1 contrast agents (e.g., Gd chelate compounds), T2 contrast agents (e.g., superparamagnetic agents (e.g., magnetite, Fe ₃ O ₄ , γ-Fe ₂ O ₃ , manganese ferrite, cobalt ferrite, and nickel ferrite)), radioisotopes (e.g., ¹¹ C, ¹⁵ O, ¹³ N, ³² P, ³⁵ S, ⁴⁴ Sc, ⁴⁵ Ti, ¹¹⁸ I, ¹³⁶ La, ¹⁹⁸ Tl, ²⁰⁰ Tl, ²⁰⁵ Bi, and ²⁰⁶ Bi), fluorescent agents (fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5), chemiluminescent particles, magnetic particles, mass labels, or electron-dense particles.

The GRP94 antibody or antigen-binding fragment thereof according to the present invention has very high specificity and affinity for GRP94, and excellent growth inhibition effects, invasion inhibition effects, and angiogenesis inhibition effects on colorectal cancer cell lines, and thus can be usefully used as a composition for treating cancer, inhibiting cancer metastasis, and inhibiting angiogenesis.

Figure 1 is a diagram showing the results of SDS-PAGE and Coomassie staining of a recombinant human GRP94 protein produced by the inventors using Expi293 cells.
Figure 2 is a diagram showing the results of SDS-PAGE and Coomassie staining of the GRP94 IgG antibody expressed in Expi293 cells by the present inventors.
Figure 3 is a diagram showing the production amount of GRP94-specific IgG of the present invention after expression and purification in Expi293 cells, measured using Nanodrop.
Figure 4 is a diagram showing the endotoxin level of the GRP94 antibody of the present invention.
Figure 5 is a diagram showing the protein aggregation index of the GRP94 antibody of the present invention.
Figures 6 and 7 are diagrams showing the equilibrium dissociation constant (K _D ) values of four types of GRP94 IgG (B5, E5, 2H5, 3G7) of the present invention.
Figure 8 is a diagram confirming the specificity of four types of GRP94 IgG (B5, E5, 2H5, 3G7) of the present invention for recombinant human GRP94, recombinant rat GRP94, and recombinant monkey GRP94 antigens.
Figures 9 and 10 are diagrams showing the results of an in vitro tube formation test using HUVEC cells of the four antibodies of the present invention compared with cetuximab.
Figures 11 and 12 are diagrams showing the results of a transwell invasion assay for four antibodies (E5, B5, 2H5, and 3G7) of the present invention against a colorectal cancer cell line, HCT-8.
Figures 13 and 14 are diagrams showing the results of a transwell invasion assay for four antibodies (E5, B5, 2H5, and 3G7) of the present invention against a colorectal cancer cell line, HCT-116.
Figures 15 and 16 are diagrams showing the results of a transwell invasion assay for four antibodies (E5, B5, 2H5, and 3G7) of the present invention against a colorectal cancer cell line, LoVo.
Figures 17 to 19 are diagrams showing the effects of four antibodies (E5, B5, 2H5, and 3G7) of the present invention on cell growth of colorectal cancer cell lines, HCT-8, HT-29, HCT-116, and LoVo.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example

Throughout this specification, "%", when used to indicate the concentration of a particular substance, unless otherwise noted, is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (volume/volume) % for liquid/liquid.

Materials and Methods

Cell culture

Human CRC cell lines (HCT116, HT-29, LoVo, HCT-8, and Caco-2) were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). HCT116, HT-29, LoVo, and HCT-8 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). Caco-2 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with the same supplements as RPMI 1640. Human umbilical vein endothelial cells (HUVEC; Lonza, Allendale, NJ, USA) cells were cultured in endothelial growth medium-2 (EGM-2; Lonza). All cells were cultured in a humidified 37°C, 5% _CO2 atmosphere. Expi293 cells were cultured in Expi293 expression medium (Invitrogen, Carlsbad, CA, USA) in a humidified _CO2 shaking incubator (N-BIOTEK, S. Korea) at 37°C, 8% _CO2 atmosphere.

Transfection ( Transfection )

To overproduce antibody and rhGRP94, Expi293 cells were transfected with pcDNA 3.1 and pcDNA 3.4 (Invitrogen) expression vectors encoding GRP94 antibody and rhGRP94, respectively, using the ExpiFectamine transfection kit (Invitrogen) according to the manufacturer's instructions.

Enzyme-Linked Immunosorbent Assay (ELISA)

Each well of a 96-well plate was coated with 0.1 μg of recombinant human GRP94 (rhGRP94), blocked with phosphate-buffered saline (PBS) containing 3% (w/v) bovine serum albumin (BSA) at 37 °C for 2 h, and 100 μL of 20 μg/mL GRP94 target IgG was added and incubated at 37 °C for 2 h. The plate was washed three times with 0.05% PBS-T, and 100 μL of 5000:1 diluted anti-human Fc Ab (sigma-aldrich) was added as a secondary antibody. After incubation at 37°C for 1 h, the wells were washed three times with 0.05% PBS-T, and 100 μL of 3,30,5,50 -tetramethylbenzidine (1-step ultra TMB) substrate solution was added to each well. Finally, the optical density was measured at 450 nm using a microplate reader (Synergy H1, BioTek).

Example 1: Using a phage display In rhGRP94 Combining High affinity Selection of a High-Affinity Binder to rhGRP94 Using Phage Display Technology)

Prior to producing scFv that specifically binds to rhGRP94, the inventors expressed rhGRP94 protein in Expi293 cells and purified it using affinity chromatography with Ni-NTA Sepharose. The purity of the purified protein was estimated using SDS-PAGE and Coomassie Brilliant Blue staining. As shown in Fig. 1, the purity of the rhGRP94 protein produced and purified by the inventors was confirmed to be equivalent to or superior to that of the commercially available protein.

Next, we re-amplified the human synthetic scFv library. Through four rounds of biopanning, scFvs that bind to rhGRP94 with high affinity were selected using Epoxy-270 dynabeads (Invitrogen) conjugated with rhGRP94.

To produce IgG antibodies, the DNA base sequences of the selected scFvs were sequenced, and each heavy chain variable region and light chain variable region was cloned into the bicistronic mammalian expression vector pcDNA3.1. IgG antibodies (GRP94 IgG) were expressed and purified through the expi293 expression system (Invirogen).

GRP94 IgG antibody expressed in Expi293 cells was purified using affinity chromatography with protein A Sepharose. The purity of the purified antibody was predicted through SDS-PAGE and Coomassie Brilliant Blue staining. In addition, the final production amount of the antibody was confirmed using Nanodrop. As shown in Fig. 2, it was confirmed that the GRP94-specific IgG antibody produced and purified by the present inventors had high purity, and as shown in Fig. 3, it was confirmed that all of the antibodies of the present invention (B5, E5, 2H5, and 3G7) had high production efficiencies of 100 mg/L or more.

Example 2: PTS ^TM Measurement of endotoxin levels using ( Measurement of endotoxin levels using a PTS ^TM )

The endotoxin level of the produced antibody was measured using the Endosafe PTS ^TM , which consists of a spectrophotometer, a reader, and a LAL reagent cartridge. First, the produced GRP94 antibody was placed in the LAL reagent cartridge (Charles River) and the endotoxin level was measured according to the manufacturer's instructions.

As shown in Figure 4, it was confirmed that all GRP94 antibodies of the present invention exhibited endotoxin levels below the FDA safety limit of 0.005 EU/ug.

Example 3: Protein aggregation index (Protein Aggregation index)

In order to evaluate antibody aggregation after production and purification of antibodies (B5, E5, 2H5, 3G7) of the present invention, each antibody was added to PBS, and the absorbance at 280 nm and 340 nm was measured using Nano Drop 2000 (Thermoscientific). The protein aggregation index was calculated from UV absorbance by the following equation 1. As a control, GRP94 antigen, which was induced to aggregation by treating with HCl, was used.

Equation 1

Protein aggregation index = 100 x (Abs340/[Abs280-Abs340])

Antibody aggregation occurs due to partial unfolding of some domains constituting the antibody, and can lead to nucleation and growth of aggregate nuclei, and is therefore a very important factor in antibody developability. As shown in Fig. 5, it was confirmed that the antibodies of the present invention (B5, E5, 2H5, 3G7) have low protein aggregation indices and are therefore suitable for development.

Example 4: K of antibody and antigen interactions _D Value measurement (K _D Value Measurement of Antibody and Antigen Interactions (ELISA)

To measure the equilibrium dissociation constant (K _D ) values of four types of GRP94 IgG (B5, E5, 2H5, 3G7) of the present invention, 100 ng of rhGRP94 was coated on each well of a 96-well plate, and blocked with PBS containing 3% (w/v) bovine serum albumin (BSA) at 37°C for 2 hours. Next, the GRP94 IgG of the present invention was incubated for 2 hours at 37°C with a concentration gradient of 0 nM to 500 nM for each clone. The use of secondary antibodies and the measurement of optical density were measured in the same manner as the ELISA method.

BSA, recombinant human GRP94, recombinant rat GRP94, and recombinant monkey GRP94 were coated on a 96-well plate, respectively, and the degree of binding to each antibody (B5, E5, 2H5, and 3G7) was confirmed through ELISA. As shown in Fig. 8, each antibody (B5, E5, 2H5, and 3G7) of the present invention specifically bound to the recombinant GRP94 antigens of human, rat, and monkey, and in particular, it was confirmed to have very high specificity for the recombinant GRP94 antigens of human and rat.

Example 5: In Vitro Tube Formation Assays

A 48-well plate was coated with 150 μL of Matrigel (Corning) and incubated at 37°C for 30 minutes. In order to investigate the effect of the four types of GRP94 IgG of the present invention on tube formation, 1x10 ⁵ HUVEC cells cultured in EGM-2 were seeded on the Matrigel-coated plate and cultured in the presence or absence of anti-GRP94 IgG or treatment with 20 μg/mL cetuximab. Images were obtained using an IncuCyte FLR live content imaging system (Essen Bioscience, Ann Arbor, MI, USA), and tube formation was quantified by counting the number of tube branches. The results are shown in Figs. 9 and 10 . As shown in Figs. 9 and 10 , the four types of antibodies of the present invention had excellent effects on inhibiting tube formation. Therefore, the four types of antibodies of the present invention can be usefully used as angiogenesis inhibitors.

Example 6: Transwell Invasion Assay ( Transwell invasion assays)

Two transwell invasion chambers coated with Matrigel (0.1 mg/ml) (Corning) were used for in vitro transwell invasion assays of HCT-8, HCT-116, or LoVo cells. First, 200 μl of serum-free medium containing 1 × 10 ⁵ cells per well was added to the upper chamber, and 0.8 ml of medium containing 10% FBS was added to the lower chamber. After incubation at 37 °C for 24 to 48 h, non-invading cells on the upper membrane were removed with a cotton swab. Migrated or invaded cells were fixed and stained with diff quick staining Kit (sysmex). Cell number was counted using ImageJ software, and four random fields from each well were selected and photographed using an inverted microscope. Each experiment was performed independently twice.

As shown in FIGS. 11 to 16, the four antibodies of the present invention have the effect of inhibiting the invasion of colorectal cancer cell lines such as HCT-8, HCT-116, and LoVo, and it can be seen that they have a superior invasion inhibition effect compared to cetuximab. Therefore, the four antibodies of the present invention can be usefully used as cancer metastasis inhibitors.

Example 7: In In Vitro CRC Effects on cell growth (In Vitro Measurement of CRC Cell Growth)

To determine the effect of the GRP94 IgG of the present invention on the growth of CRC cells in vitro, 5x10 ³ HCT116, HT29, LoVo, HCT-8, or Caco-2 CRC cells were seeded in wells with or without 100 μg/mL of cetuximab or GRP94 IgG. Cell growth was observed for 60 hours using an IncuCyte FLR live content imaging system (Essen Bioscience, Ann Arbor, MI, USA).

The results are shown in Figures 17 to 19.

For the HCT-8 cell line, the cell growth inhibition effect was in the order of 3G7>Cetuximab>E5>B5>2H5. For the HT-29 cell line, the cell growth inhibition effect was in the order of 3G7>E5>2H5>B5>Cetuximab. For the HCT-116 cell line, the cell growth inhibition effect was in the order of 3G7>E5>Cetuximab>2H5>B5. For the LoVo cell line, the cell growth inhibition effect was in the order of 3G7>E5>2H5>B5>Cetuximab.

<110> Kookmin University Industry Academy Cooperation Foundation <120> GRP94 specific antibody or antigen-binding fragment thereof and uses its <130> PN200136 <160> 64 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT < 213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1 of GRP94 B5 <400> 1 Gly Phe Thr Phe Ser Asn Tyr Tyr Met Ser 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR2 of GRP94 B5 <400> 2 Gly Ile Tyr Pro Asn Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3 of GRP94 B5 <400> 3 Asp Pro Leu His Pro Ala Arg Phe Pro Phe Asp Tyr 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR1 of GRP94 B5 <400> 4 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Ser 1 5 10 <210> 5 <211 > 7 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR2 of GRP94 B5 <400> 5 Ala Asp Ser His Arg Pro Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR3 of GRP94 B5 <400> 6 Gly Ala Trp Asp Ala Ser Leu Asn Ala 1 5 <210> 7 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of heavy chain variable of GRP94 B5 <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Tyr Pro Asn Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Leu His Pro Ala Arg Phe Pro Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 110 <212 > PRT <213> Artificial Sequence <220> <223> Amino acid sequence of light chain variable of GRP94 B5 <400> 8 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Ala Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Ala Ser Leu 85 90 95 Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1 of GRP94 E5 <400> 9 Gly Phe Thr Phe Ser Asn Tyr Ala Met Ser 1 5 10 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR2 of GRP94 E5 <400> 10 Gly Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3 of GRP94 E5 <400> 11 Asp Arg His Pro Phe Ser Pro Asn Trp Phe Asp Tyr 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR1 of GRP94 E5 <400> 12 Ser Gly Ser Pro Ser Asn Ile Gly Ser Asn Thr Val Thr 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR2 of GRP94 E5 <400> 13 Ala Asp Ser His Arg Pro Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR3 of GRP94 E5 <400> 14 Ala Ser Trp Asp Asp Ser Leu Asn Gly 1 5 <210> 15 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of heavy chain variable of GRP94 E5 <400> 15 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg His Pro Phe Ser Pro Asn Trp Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of light chain variable of GRP94 E5 <400> 16 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Pro Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu 85 90 95 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1 of GRP94 2H5 <400> 17 Gly Phe Thr Phe Ser Gly Tyr Ala Met Ser 1 5 10 <210> 18 <211> 17 <212> PRT <213 > Artificial Sequence <220> <223> Amino acid sequence of HCDR2 of GRP94 2H5 <400> 18 Ala Ile Ser His Gly Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3 of GRP94 2H5 <400> 19 Asp Leu Leu Ser Pro Leu Gln Ser Ile Gly Ser Tyr Asp Asp Ala Met 1 5 10 15 Asp Val <210> 20 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR1 of GRP94 2H5 <400> 20 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Ser 1 5 10 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR2 of GRP94 2H5 <400> 21 Ala Asp Asn Asn Arg Pro Ser 1 5 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR3 of GRP94 2H5 <400> 22 Ala Ser Trp Asp Asp Ser Leu Asn Ala 1 5 <210> 23 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of heavy chain variable of GRP94 2H5 <400> 23 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser His Gly Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Leu Leu Ser Pro Leu Gln Ser Ile Gly Ser Tyr Asp Asp 100 105 110 Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 24 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of light chain variable of GRP94 2H5 <400> 24 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asp Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu 85 90 95 Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1 of GRP94 3G7 <400> 25 Gly Phe Thr Phe Ser Asn Tyr Ser Met Ser 1 5 10 <210> 26 <211 > 17 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR2 of GRP94 3G7 <400> 26 Gly Ile Tyr Tyr Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 27 <211> 7 <212 > PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3 of GRP94 3G7 <400> 27 Asn Leu Ala Ser Phe Asp Tyr 1 5 <210> 28 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR1 of GRP94 3G7 <400> 28 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Ser Val Asn 1 5 10 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR2 of GRP94 3G7 <400> 29 Ser Asn Ser His Arg Pro Ser 1 5 <210> 30 < 211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of LCDR3 of GRP94 3G7 <400> 30 Gly Thr Trp Asp Ser Ser Leu Ser Gly 1 5 <210> 31 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of heavy chain variable of GRP94 3G7 <400> 31 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Tyr Tyr Gly Ser Gly Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Leu Ala Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 32 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of light chain variable of GRP94 3G7 <400> 32 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Ser Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 33 <211> 30 <212> DNA < 213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR1 of GRP94 B5 <400> 33 ggattcacct ttagcaatta ttatatgagc 30 <210> 34 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR2 of GRP94 B5 <400> 34 gggatctatc ctaatagtgg tagtacatat tacgctgatt ctgtaaaagg t 51 <210> 35 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR3 of GRP94 B5 <400> 35 gatcctcttc atccggcgcg ttttccgttc gactac 36 <210> 36 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR1 of GRP94 B5 <400> 36 actggctctt catctaatat tggcaataat gctgtctcc 39 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223 > Nucleic acid sequence of LCDR2 of GRP94 B5 <400> 37 gctgatagtc atcggccaag c 21 <210> 38 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR3 of GRP94 B5 <400> 38 ggtgcttggg atgctagcct gaatgct 27 <210> 39 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of heavy chain variable of GRP94 B5 <400> 39 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctatccta atagtggtag tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagatcct 300 cttcatccgg cgcgttttcc gttcgactac tggggccagg gtacactggt caccgtgagc 360 tca 363 <210> 40 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of light chain variable of GRP94 B5 <400> 40 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc aataatgctg tctcctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gctgatagtc atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt gcttgggatg ctagcctgaa tgcttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 41 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR1 of GRP94 E5 <400> 41 ggattcacct ttagcaatta tgctatgagc 30 <210> 42 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR2 of GRP94 E5 <400> 42 gggatctctt ctagtagtgg tagtacatat tacgctgatt ctgtaaaagg t 51 <210> 43 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR3 of GRP94 E5 <400> 43 gatcgtcatc cgttttcgcc taattggttc gactac 36 <210> 44 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR1 of GRP94 E5 <400> 44 agtggctctc catctaatat tggcagtaat actgtcacc 39 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR2 of GRP94 E5 <400> 45 gctgatagtc atcggccaag c 21 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR3 of GRP94 E5 <400> 46 gcttcttggg atgatagcct gaatggt 27 <210> 47 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of heavy chain variable of GRP94 E5 <400> 47 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcgg cctctggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctcttcta gtagtggtag tacatattac 180 gctgattctg taaaaggtcg gttcaccacc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagatcgt 300 catccgtttt cgcctaattg gttcgactac tggggccagg gtacactggt caccgtgagc 360 tca 363 <210> 48 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of light chain variable of GRP94 E5 <400> 48 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctctccatc taatattggc agtaatactg tcacctggta ccagcagctc 120 ccagggaacgg cccccaaact cctcatctat gctgatagtc atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgct tcttgggatg atagcctgaa tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 49 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR1 of GRP94 2H5 <400> 49 ggattcacct ttagcggtta tgctatgagc 30 <210> 50 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR2 of GRP94 2H5 <400> 50 gcgatctctc atggtggtag tagtaaatat tacgctgatt ctgtaaaagg t 51 <210> 51 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR3 of GRP94 2H5 <400> 51 gatcttctta gtcctctgca gagtattggg tcttatgatg atgctatgga cgtc 54 <210> 52 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR1 of GRP94 2H5 <400> 52 agtggctctt catctaatat tggcagtaat actgtctcc 39 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR2 of GRP94 2H5 <400> 53 gctgataata atcggccaag c 21 <210> 54 <211> 27 < 212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR3 of GRP94 2H5 <400> 54 gcttcttggg atgatagcct gaatgct 27 <210> 55 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of heavy chain variable of GRP94 2H5 <400> 55 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc ggttatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctctcatg gtggtagtag taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatctt 300 cttagtcctc tgcagagtat tgggtcttat gatgatgcta tggacgtctg gggccagggt 360 acactggtca ccgtgagctc a 381 <210> 56 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of light chain variable of GRP94 2H5 <400> 56 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc agtaatactg tctcctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gctgataata atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgct tcttgggatg atagcctgaa tgcttatgtc 300 ttcggcgggg gcaccaagct gacggtccta 330 <210> 57 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR1 of GRP94 3G7 <400> 57 ggattcacct ttagcaatta ttctatgagc 30 <210> 58 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR2 of GRP94 3G7 <400> 58 gggatctatt atggtagtgg taataaatat tacgctgatt ctgtaaaagg t 51 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of HCDR3 of GRP94 3G7 <400> 59 aatctggctt cgttcgacta c 21 <210> 60 <211> 39 <212> DNA <213> Artificial Sequence < 220> <223> Nucleic acid sequence of LCDR1 of GRP94 3G7 <400> 60 agtggctctt catctaatat tggcagtaat tctgtcaac 39 <210> 61 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR2 of GRP94 3G7 <400> 61 tctaatagtc atcggccaag c 21 <210> 62 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of LCDR3 of GRP94 3G7 <400> 62 ggtacttggg attctagcct gagtggt 27 <210> 63 <211> 348 <212 > DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of heavy chain variable of GRP94 3G7 <400> 63 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattattcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctattatg gtagtggtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaaaaatctg 300 gcttcgttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 64 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of light chain variable of GRP94 3G7 <400> 64 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc agtaattctg tcaactggta ccagcagctc 120 ccagggaacgg cccccaaact cctcatctat tctaatagtc atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggacg aggctgatta ttactgtggt acttgggatt ctagcctgag tggttatgtc 300ttcggcggag gcaccaagct gacggtccta 330

Claims (13)

An antibody or antigen-binding fragment that specifically binds to Glucose-Regulated Protein 94 (GRP94) comprising any one of the following i) to iii):
i) HCDR1, HCDR2, HCDR3 consisting of amino acid sequences of SEQ ID NOs: 1 to 3, and LCDR1, LCDR2, and LCDR3 consisting of amino acid sequences of SEQ ID NOs: 4 to 6;
ii) HCDR1, HCDR2, HCDR3 consisting of amino acid sequences of SEQ ID NOs: 9 to 11, and LCDR1, LCDR2, and LCDR3 consisting of amino acid sequences of SEQ ID NOs: 12 to 14; or
iii) HCDR1, HCDR2, HCDR3 consisting of amino acid sequences of SEQ ID NOs: 17 to 19, and LCDR1, LCDR2, and LCDR3 consisting of amino acid sequences of SEQ ID NOs: 20 to 22.
In claim 1, the antibody or antigen-binding fragment comprises any one of the following i) to iii):
i) a heavy chain variable region consisting of an amino acid sequence of sequence number 7 and a light chain variable region consisting of an amino acid sequence of sequence number 8;
ii) a heavy chain variable region comprising an amino acid sequence of sequence number 15 and a light chain variable region comprising an amino acid sequence of sequence number 16; or
iii) A heavy chain variable region consisting of the amino acid sequence of sequence number 23 and a light chain variable region consisting of the amino acid sequence of sequence number 24.
In claim 1, the antibody or antigen-binding fragment has a dissociation constant K _D value of 10 ^-8 M or less.
In claim 1, the antibody or antigen-binding fragment has the effect of inhibiting the proliferation of one or more tumor cell lines selected from the group consisting of HCT-8, HT-29, LoVo, HCT-116, and Caco-2 in vitro and in vivo.
A nucleic acid molecule encoding an antibody or antigen-binding fragment of any one of claims 1 to 4.
A recombinant vector comprising the nucleic acid of claim 5.
A host cell comprising the recombinant vector of claim 6.
A pharmaceutical composition for treating cancer, comprising i) an antibody or antigen-binding fragment of any one of claims 1 to 4; and ii) a pharmaceutically acceptable carrier.
A pharmaceutical composition for inhibiting cancer metastasis, comprising i) an antibody or antigen-binding fragment of any one of claims 1 to 4; and ii) a pharmaceutically acceptable carrier.
A pharmaceutical composition for inhibiting angiogenesis, comprising i) an antibody or antigen-binding fragment of any one of claims 1 to 4; and ii) a pharmaceutically acceptable carrier.
A composition for detecting GRP94 comprising an antibody or antigen-binding fragment of any one of claims 1 to 4.
A kit for detecting GRP94 comprising an antibody or antigen-binding fragment of any one of claims 1 to 4.


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