KR102058381B1 - Humanized antibody against human L1CAM and method for preparing the antibody - Google Patents

Abstract

The present invention relates to a humanized antibody against human L1CAM, a method for producing the same, and a composition for treating cancer comprising the humanized antibody.
Humanized antibodies according to the present invention have low immunogenicity and high affinity and specificity for L1CAM, which can effectively inhibit cancer cell proliferation and minimize side effects. There is this.

Description

Humanized antibody against human L1CAM and method for preparing the antibody}

The present invention relates to a humanized antibody against human L1CAM, a method for producing the same, and a composition for treating cancer comprising the humanized antibody.

The humanized antibody according to the present invention is a humanized antibody derived from a mouse known to L1CAM, and has a low immunogenicity and high affinity and specificity for L1CAM, thereby efficiently proliferating cancer cells. It can be suppressed, and side effects can be minimized.

L1 cell adhesion molecules (L1CAMs) are endogenous membrane glycoproteins belonging to immunoglobulin superfamily cell adhesion molecules (CAMs) that mediate cell-to-cell adhesion at the cell surface. One of the integral membrane glycoproteins has a molecular weight of 220 kDa. L1CAM is originally found in neurons, hematopoietic and renal cells and the like (Bateman, et al, EMBO J. 15: 6050-6059; 1996), whose functions are neuronal migration, neurogenesis growth, and cellular migration. Human L1CAM genes were isolated from human fetal brain cDNA libraries using degenerate oligonucleotides as probes in mouse and rat L1CAM homologs (Hlavin, ML et al., Genomics 11: 416). -423, 1991; US 5,872,225B). It was originally known to be expressed mainly in the brain, but it has also been found in some normal tissues and recently in many cancer cells.

In relation to the association of L1CAM with cancer, L1CAM has been reported to be expressed in several cancers including melanoma, neuroblastoma, ovarian cancer and colorectal cancer (Takeda, et al., J. Neurochem. 66 : 2338-2349, 1996; Thies et al, Eur. J. Cancer, 38: 1708-1716, 2002; Arlt et al., Cancer Res. 66: 936-943, 2006; Gavert et al., J. Cell Biol 168: 633-642, 2005) In addition to membrane bound forms, cleavage products were found to be secreted extracellularly (Gutwein et al., FASEP J. 17 (2): 292-). 4, 2003).

And recently, as one of the molecules that play an important role in cancer cell growth, has emerged as a new target of cancer therapy (Primiano, et al., Cancer Cell. 4 (1): 41-53 2003). Recently L1CAM is expressed in the invasive front of colorectal cancer (Gavert, et al., J Cell Biol. 14; 168 (4): 633-42. 2005), and antibodies against this molecule in ovarian cancer Has been shown to inhibit cancer cell growth and metastasis (Arlt, et al., Cancer Res. 66: 936-943. 2006).

The inventor of the present invention, while trying to develop an antibody that can be used for the diagnosis or treatment of biliary tract cancer, monoclonal antibody named A10-A3 that specifically binds to the surface of biliary tract cancer by vaccinating a biliary cancer cell line to mice. The protein bound by the A10-A3 antibody is L1CAM protein, and the A10-A3 antibody binds specifically to cancer cells such as breast cancer, ovarian cancer, lung cancer, colorectal cancer and skin cancer as well as biliary tract cancer. It has been confirmed that it does not bind to normal cells such as lymphocytes, hepatocytes, and blood endothelial cells (see Korean Patent No. 756,051). In particular, it has been found that L1CAM is expressed in breast cancer, ovarian cancer, colon cancer, skin cancer, etc., but it is not known that L1CAM is expressed in biliary tract cancer or lung cancer. The epitope of the A10-A3 antibody was analyzed and compared with epitopes of 5G3 and UJ127, which are monoclonal antibodies against known L1CAM, A10-A3 bound to the Ig1 domain of L1CAM, but 5G3 and UJ127 Confirmed binding to the Ig2 domain and the fibronectin type III domain, respectively, confirming that A10-A3 is a novel monoclonal antibody against L1CAM.

As described above, the A10-A3 antibody may be effectively used for the treatment of breast cancer, ovarian cancer, colon cancer, skin cancer, and biliary tract cancer and lung cancer in which L1CAM is expressed. However, the A10-A3 antibody has a mouse-derived antibody sequence. When administered to HAMA (Human Anti-Mouse Antibody) is likely to induce a problem that the therapeutic efficacy can be significantly reduced.

Therefore, there is an urgent need for humanized antibodies that can prevent immunogenicity, i.e., decrease the therapeutic efficacy due to HAMA, while maintaining or improving the affinity and specificity for A1-A3 antibodies to L1CAM.

KR 10-0756051 B1, August 30, 2007

 Arlt et al., Cancer Res. 66: 936-943, 2006; Gavert et al., J. Cell Biol. 168: 633-642, 2005  Primiano, et al., Cancer Cell. 4 (1): 41-53 2003

In order to solve the above problems, an object of the present invention is to provide a humanized antibody HzA10A3 of the A10-A3 antibody with a minimum of a mouse-derived antibody sequence.

Another object of the present invention is to provide a nucleotide encoding the humanized antibody HzA10A3, a recombinant vector comprising the same, and a host cell transformed with the recombinant vector, wherein the host cell transformed with the recombinant vector is cultured to be humanized. It is to provide a method for producing the antibody HzA10A3.

Another object of the present invention to provide a pharmaceutical composition for treating cancer comprising the humanized antibody HzA10A3 and a method for treating cancer using the same.

As one embodiment for achieving the above object, the present invention provides the CDR1, CDR2 and CDR3 regions, which are complementarity determining regions (CDRs) of the heavy and light chain variable regions of the A10-A3 antibody, and the heavy and light chain variable of the human antibody. Region, particularly humanized antibody HzA10A3-1 having heavy and light chain variable regions obtained by implantation into human antibodies IGHV1-2 * 02 and IGKV7-3 * 01, respectively, wherein the affinity for L1CAM of the humanized antibody HzA10A3-1 Humanized antibody HzA10A3 obtained by additional transplantation of five amino acid residues from the A10-A3 heavy chain variable region for substitution and further substitution of four amino acid residues of HCDR2 and one amino acid residue of LCDR2 with that of a human antibody Provides -2.2.

For humanization of the A10-A3 antibody in the present invention, as described above, CDR1 to CDR3 included in the heavy and light chain variable regions of the A10-A3 antibody are transplanted to the CDR1 to CDR3 positions of the heavy and light chain variable regions of the human antibody. CDR-grafting) can be used. Human antibodies that can be used for the transplantation of CDR1 to CDR3 included in the heavy and light chain variable regions of the A10-A3 antibody have sequences derived from humans, and the variable region sequence or three-dimensional structure is similar to that of the A10-A3 antibody. Preferred examples are not limited thereto. In particular, it is preferable to use IGHV1-2 * 02, which is a heavy chain variable region, and IGKV7-3 * 01, which is a light chain variable region. CDR1 to CDR3 included in the heavy and light chain variable regions of the A10-A3 antibody according to the present invention are IGHV1-2 * 02 and JH-4, the heavy chain variable region germline sequences of human antibodies, and IGKV7, the light chain variable region germline sequences of human antibodies. The heavy and light chain variable regions of humanized antibody HzA10A3-1 grafted to -3 * 01 and JK1, respectively, have the amino acid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 20, respectively.

In addition, the present invention provides a L1CAM-specific humanized antibody having improved affinity for L1CAM and reduced immunogenicity by substituting some of the amino acid residues in the light and heavy chain variable regions of HzA10A3-1 with other amino acid residues. At this time, a portion of the amino acid residues of the HzA10A3-1 heavy chain variable region is replaced with amino acid residues derived from A10-A3 at the corresponding position, and a portion of the amino acid residues of the HzA10A3-1 heavy chain HCDR2 and the light chain LCDR2 is replaced with amino acid residues derived from the human antibody at the position. It is preferable to substitute. In a particularly preferred embodiment, the humanized antibodies which have improved affinity for L1CAM provided in the present invention and are less likely to induce human immune responses have the amino acid sequences set forth in SEQ ID NO: 24 and SEQ ID NO: 30, respectively.

In addition, the humanized antibodies having improved affinity for L1CAM provided in the present invention and less likely to induce a human immune response have amino acid sequences set forth in SEQ ID NO: 26 and SEQ ID NO: 28, respectively.

Also,

In the light and heavy chain variable region of the L1CAM specific humanized antibody having the amino acid sequence of SEQ ID NO: 24 and SEQ ID NO: 30 or in the light and heavy chain variable region of the L1CAM specific humanized antibody having the amino acid sequence of SEQ ID NO: 26 and SEQ ID NO: 28 As long as properties consistent with the object of the present invention, such as affinity and specificity for L1CAM, are maintained, even if some amino acids are substituted, inserted and / or deleted, they fall within the scope of the present invention. It is obvious to those with ordinary knowledge (hereinafter referred to as ordinary technicians). An example is an antibody in which a conservative substitution of amino acids in a variable region occurs. Conservative substitutions mean substitutions with other amino acid residues that have properties similar to those of the original amino acid sequence. For example, lysine, arginine, and histidine have similar properties because they have a base side chain, and aspartic acid and glutamic acid are acidic. It has similar characteristics in that it has a side chain. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar characteristics in that they have a non-charged polar side chain, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine are nonpolar. Tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have side chains.

Therefore, it will be apparent to those skilled in the art that amino acid substitutions within a group having similar characteristics as described above will not show any change in characteristics, so as long as the characteristics of the antibody according to the present invention are maintained, Antibodies that have undergone mutations due to conservative substitutions in are also included in the scope of the invention.

A fragment of L1CAM specific humanized antibody having amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 30 as light and heavy chain variable regions, respectively, or L1CAM specific having amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 28 as light and heavy chain variable regions, respectively Fragments of humanized antibodies are also included in the scope of the present invention.

Fragments of the antibodies in the present invention are single-chain antibodies, diabodies, tribodies, tetrabodies, Fab fragments, F (ab ') ₂ fragments, Fd, scFv, domain antibodies, minibodies, scaffolds having a binding function to L1CAM ( single chain antibody, scAb), IgD antibody, IgE antibody, IgM antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody, derivatives of antibody constant regions, phosphorus based on protein scaffolds, and the like. It will be apparent to those skilled in the art that, as long as the binding function to L1CAM is maintained, fragments of antibodies of any form according to the invention will exhibit the same properties as antibodies according to the invention.

On the other hand, another embodiment of the present invention provides an antibody-drug conjugate (ADC: Antibody-Drug Conjugate) is bound to the L1CAM-specific humanized antibody or fragments thereof, the drug for the treatment of cancer that has the effect of inhibiting the proliferation of tumor cells.

Drugs that can be used in the antibody-drug conjugates according to the present invention include any compound, moiety or group that has a cytotoxic or cytostatic effect, and (i) a microtubulin inhibitor, mitosis inhibitor, topoisomerase inhibitor Or a chemotherapeutic agent that can function as a DNA intercalator; (ii) protein toxins that can function enzymatically; And (iii) radioisotopes (radionuclides) and the like, one or more of which may be used in the compound.

Non-limiting examples of such drugs include maytansinoids, auristatins, dolastatins, tricortesenes, CC1065, calicheamicins and other enedin antibiotics, taxanes, anthracyclines, methotrexate, adriamycin, binddesin, vinca Alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, carminomycin, aminopterin , Dactinomycin, mitomycin, bleomycin, esperamycin, 5-fluorouracil, melphalan, other nitrogen mustards and stereoisomers, isomers, homologues or derivatives thereof, cis-platinum and cis-platinum homologues , Enzymes and other fragments thereof, such as nucleases, antibiotics, toxins (enzymatically active toxins or small molecule toxins of bacterial, fungal, plant or animal origin), cisplas , But containing the various antitumor or anticancer agents such as CPT-11, doxorubicin, paclitaxel and docetaxel, but it is not limited to these. In addition, radioisotopes (radionuclides) include 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, and the like.

The antibody and the drug are preferably conjugated using a functional group such as a thiol group of an amino acid residue such as lysine or cysteine in the antibody, and when necessary, linker-mediated using a linker commonly used. It is also possible to join in the form of). Preferably, maleimide or iodine acetamide series linkers are used. When the drug is conjugated to the antibody or fragment thereof, it is preferable to conjugate the drug to the C-terminal site opposite to the antigen binding site so as not to affect the binding capacity and specificity of the antibody or fragment thereof to L1CAM. When using an antibody, it is also preferable to conjugate to an Fc region.

In another aspect of the present invention, there is provided a bispecific antibody comprising the L1CAM specific humanized antibody or fragment thereof. Bispecific antibodies are antibodies that have the ability to bind two antigens at the same time, with the most common forms of linking different heavy and light chain pairs with the ability to bind different antigens, VL and VH Bispecific single chain antibody in which single-chain antibody fragments (scFv) linked by short linker peptides are in the form of scFv1-scFv2 (-Fc), or BiTE technology of Micromet (Germany). It is also available in the form of bispecific antibodies using http://www.micromet.de).

The bispecific antibody according to the present invention is preferably in the form in which the HzA10A3-2.2 antibody or fragment thereof is bound to an antibody or fragment thereof having the ability to bind to an immunopotent cell-specific target molecule. Immunopotent cell specific target molecules are preferably selected from, but not limited to, TCR / CD3, CD16 (FcγRIIIa) CD44, Cd56, CD69, CD64 (FcγRI), CD89 and CD11b / CD18 (CR3).

The present invention also provides a gene encoding a variable region of the L1CAM-specific humanized antibody or fragment thereof according to the present invention, and a recombinant vector comprising the same. The polynucleotides encoding the light and heavy chain variable regions of the antibody or fragment thereof according to the present invention, i.e., a gene, may be viewed from the amino acid sequence of SEQ ID NO: 26 and SEQ ID NO: 28 or the amino acid sequence of SEQ ID NO: 24 and SEQ ID NO: 30 Those skilled in the art can easily derive, and in particular, it is preferable to have a sequence described in SEQ ID NO: 27 and SEQ ID NO: 29 or a sequence described in SEQ ID NO: 25 and SEQ ID NO: 31, respectively.

As used herein, the term "recombinant vector" is an expression vector capable of expressing a protein of interest in a suitable host cell, and refers to a gene construct comprising essential regulatory elements operably linked to express the gene insert. The gene encoding the L1CAM specific humanized antibody or fragment thereof may be inserted into a separate vector or may be used in a form inserted into one vector.

Specifically, polynucleotides encoding the amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 30 may be used in the form of being inserted into separate or one vectors, respectively, particularly preferably the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 31 Polynucleotides having a can be used in the form of each inserted in a separate or one vector.

In addition, polynucleotides encoding the amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 28 may be used in the form of being inserted into separate or one vectors, respectively, and particularly preferably the sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 29 The branches may be used in a form in which the polynucleotides are inserted in separate or one vectors, respectively.

In the present invention, "operably linked" means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest is functionally linked to perform a general function. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be facilitated using enzymes commonly known in the art. have

Suitable expression vectors of the invention may include signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers. Initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject and be in frame with the coding sequence when the gene construct is administered. Generic promoters can be either constitutive or inducible. Prokaryotic cells include, but are not limited to, lac, tac, T3 and T7 promoters. Eukaryotic cells include monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), for example the long terminal repeat (LTR) promoter of HIV, moronivirus, cytomegalovirus (CMV) ), Epstein Barr virus (EBV), Loose sacoma virus (RSV) promoters, as well as promoters derived from β-actin promoter, human heroglobin, human muscle creatine, human metallothionein, but are not limited thereto. .

The expression vector may comprise a selectable marker for selecting a host cell containing the vector. The selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected. In addition, when the vector is a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

As a recombinant expression vector for inserting a foreign gene, various forms of vectors such as plasmids, viruses, and cosmids can be used. The type of recombinant vector is not particularly limited as long as it functions to express a desired gene and to produce a desired protein in various host cells of prokaryotic and eukaryotic cells, but has a promoter with strong activity and strong expression, similar to natural state. Vectors that can produce large amounts of foreign protein in form are preferred.

Various expression host / vector combinations can be used to express the humanized antibodies according to the invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. . Expression vectors that can be used in bacterial hosts include Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and derivatives thereof. coli ), a plasmid with a broader host range, such as RP4, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as λgt10 and λgt11, NM989, and M13 and filamentary single strands. Other DNA phages, such as DNA phages, are included. Useful expression vectors for yeast cells are 2 ° C. plasmids and derivatives thereof. A useful vector for insect cells is pVL941.

In another aspect, the present invention provides a host cell transformed with the recombinant vector. The recombinant vector is inserted into a host cell to form a transformant. Suitable host cells of the vector are Escherichia coli, Bacillus subtilis ( Bacillus subtilis ), Streptomyces sp.), Pseudomonas sp.), Proteus Billy's Mum (Proteus mirabilis ) or the genus Staphylococcus prokaryotic cells such as sp. Also, the genus Aspergillus fungi such as sp.), blood Chiapas pastoris (Pichia pastoris ), Saccharomyces cerevisiae , Schizosaccharomyces sp.) and yeast, such as Neurospora crassa , other eukaryotic cells, and eukaryotic cells such as cells of higher eukaryotes such as cells from insects. It can also be derived from plants, mammals. Preferably, 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 HEK293 cells and the like are available, but are not limited to these. Particularly preferably CHO cells.

In the present invention, "transformation" into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue, or organ, and may be performed by selecting a suitable standard technique according to the host cell as known in the art. . These methods include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, Lipofectamine and dry / inhibited mediated transformation methods and the like.

As another aspect, the present invention provides a method for producing a humanized antibody according to the present invention by culturing the host cell transformed with the recombinant vector described above.

The method for preparing a humanized antibody comprises the steps of preparing a recombinant vector by inserting a nucleotide sequence encoding a humanized antibody of the present invention into a vector; Culturing the recombinant vector in a host cell; It may comprise the step of separating and purifying the humanized antibody from the cultured transformant.

Specifically, the humanized antibody according to the present invention can be produced in large quantities by culturing the transformant expressing the recombinant vector in a nutrient medium, and the medium and culture conditions can be appropriately selected and used according to the host cell. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for the growth of cells and the mass production of proteins during the culture.

Recombinantly produced humanized antibodies as described above may be recovered from the medium or cell lysate. If membrane bound, it may be liberated from the membrane using a suitable surfactant solution (eg Triton-X 100) or by enzymatic cleavage. Cells used to express humanized antibodies can be disrupted by a variety of physical or chemical means, such as freeze-thaw purification, sonication, mechanical disruption or cytolysis, and can be isolated and purified by conventional biochemical separation techniques. Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990). Electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent chromatography, size exclusion chromatography, etc.), isoelectric focusing and various variations and combinations thereof It is possible, but not limited to.

As another aspect, the present invention provides a composition for treating cancer comprising the humanized antibody. As used herein, the term “anticancer” includes “prevention” and “treatment”, where “prevention” means any action in which cancer is inhibited or delayed by administration of a composition comprising an antibody of the invention, and “treatment” Is any action that improves or advantageously changes the symptoms of cancer by administration of the antibody of the present invention.

Cancers or carcinomas that can be treated with the compositions of the present invention are not particularly limited and include solid and hematological cancers. Preferably gastric cancer, breast cancer, lung cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, It is selected from the group consisting of kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureter cancer, osteosarcoma, neuroblastoma, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma. More preferably, all cancers in which L1CAM is expressed are included. The composition of the present invention can treat all cancers with the expression of the gene, but more preferably, may include breast cancer, ovarian cancer, colon cancer, skin cancer, biliary tract cancer or lung cancer.

The anticancer composition of the present invention may further comprise a pharmaceutically acceptable carrier. In the case of oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances and the like can be used.In the case of injections, buffers, preservatives, analgesics, solubilizers, isotonic agents , Stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elesir, suspensions, syrups, wafers, etc., and in the case of injections, may be prepared in unit dosage ampoules or multiple dosage forms. In addition, the anticancer composition may typically include a surfactant that facilitates movement across the membrane. Such surfactants are steroid derived or cationic lipids such as N- [1- (2,3-dioleoyl) propyl-N, N, N-trimethylammonium chloride (DOTMA), or cholesterol hemisuccinate And various compounds such as phosphatidyl glycerol.

In another aspect, the present invention provides a method for treating cancer and inhibiting cancer growth by administering the humanized antibody or composition comprising the humanized antibody to a subject. The composition comprising a humanized antibody according to the present invention may be administered in a pharmaceutically effective amount to treat cancer cells or their metastases, or to inhibit the growth of cancer. It may vary depending on various factors such as the type of cancer, the age, weight of the patient, the nature and extent of symptoms, the type of current treatment, the number of treatments, the dosage form and the route, and can be easily determined by those skilled in the art. The compositions of the present invention may be administered together or sequentially with the pharmacological or physiological components described above, and may also be administered in combination with additional conventional therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. Such administration may be single or multiple administration. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

In the present invention, "individual" means a mammal suffering from or at risk of a condition or disease that can be alleviated, inhibited or treated by administering a humanized antibody according to the present invention, preferably human.

In the present invention, “administration” means introducing a predetermined substance into an individual by any suitable method, so that the route of administration of the composition comprising the humanized antibody of the present invention is administered through any general route as long as it can reach the target tissue. Can be. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. However, when orally administered, the protein is digested, so the oral composition is preferably formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.

Humanized antibodies according to the present invention have low immunogenicity and high affinity and specificity for L1CAM, which can effectively inhibit cancer cell proliferation and minimize side effects. There is this.

1 is a comparison of the amino acid sequence and the polynucleotide sequence of the heavy chain variable region of the mouse antibody A10-A3 and humanized antibody. The bold letters indicate that the amino acid residues derived from the mouse were replaced with the amino acid residues derived from the human antibody in the sequence of the humanized antibody, and the arrows indicate the mutagenic primers used to prepare HzA10A3-2.1h.
Figure 2 is a comparison of the amino acid sequence and the polynucleotide sequence of the light chain variable region of the mouse antibody A10-A3 and humanized antibody. The bold letters indicate that the amino acid residues derived from the mouse were replaced with the amino acid residues derived from the human antibody in the sequence of the humanized antibody, and the arrows indicate the mutagenic primers used to prepare HzA10A3-2k.
3 shows the expression vector of cA10-A3.
Fig. 4 shows the results of measuring the affinity of A10A3 and cA10-A3 antibodies by the competitive Elisa method.
5 is based on the human germline VH and VL sequences showing the highest similarity compared to the heavy chain (A) and light chain (B) databases of human antibodies based on the DNA sequences of the mouse antibody A10-A3. Designed sequences of humanized antibodies show comparison of amino acid sequences of A10-A3, human antibodies, and humanized antibodies.
Fig. 6 shows the results of measuring the affinity of HzA10A3-1, HzA10A3-2, HzA10A3-2.1, HzA10A3-2.2 antibodies by a competitive Elisa method.
7 is T aminotope analysis of the amino acid sequence of HzA10A3-1 and the amino acid sequence of HzA10A3-2.2 shows the comparison of MHCII class molecules detected.
FIG. 8 shows FACS assay results using cell lines expressing L1CAM and non-expressing cell lines to confirm binding specificities of HzA10A3-2.1 and HzA10A3-2.2 to L1CAM. FIG.
9 is a fluorescence image showing the movement of HzA10A3-2.2 into cells using a cell line expressing L1CAM.

Hereinafter, the present invention will be described in detail with reference to embodiments and the accompanying drawings. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited by the following examples.

Example 1 cDNA Gene Synthesis Encoding Heavy and Light Chain Variable Regions of A10-A3

Total RNA was cloned from the A10-A3 hybridoma (Registration No. 10-756051, Aug. 30, 30) for cloning the cDNA encoding the heavy chain variable region (VH) and the light chain variable region (VL) of A10-A3. The cDNA encoding the variable region and the variable region were encoded by the 5'-primer (DGH3; 5'-ATATGTCGACGAGGTGAAGCTGCAGGAGTCAGGACCTAGCCTGGTG-3 ', DGH3B; 5'-ATATGTCGACAGGTSMAACTGCAGS) of the VH of the mouse antibody J Immunol Methods 233: 167, 2000. 3 ', DGH3D; 5'-ATATGTCGACAGGTSCAGCTGCAGSAGTCWGG-3') and 3'-primer specific to Cγ1 (Cr1; 5'-CGGTC GACAGGGATCCAGAGTTCCAGGTCAC-3 ') and 5'-primer specific to Vk (MK1-1; 5' -CAGCATGTGGCCCAGGCGGCCGAYATTGTGMTSACMCARWCTMCA-3 ') and Ck specific 3'-primers (MuCk; 5'-GCAGTCGACTGAGGCACCTCCAGATGTTAAC-3') were synthesized using polymerase chain reaction (PCR), respectively.

After cDNA synthesis, PCR products were subcloned into pBluescript SK (+), respectively, and then sequenced for three clones of VH or VL cDNA. To identify the heavy and light chain N-terminal sequences of A10-A3, each chain was separated using SDS-PAGE, followed by N-terminal sequence analysis. As a result, the heavy chain was 1. glutamine (Q) 2. valine (V) 3. glutamine (Q) 4. leucine (L) 5. glutamine (Q) and the light chain was 1. asphatic acid (D) 2 Isoleucine (I) 3. valine (V) 4. leucine (L) 5. threonine (T).

The VH and VL sequences of A10-A3 obtained by combining the cloned sequences of VH and VL and the amino acid sequences obtained from the N-terminal amino acid sequence analysis are shown in FIGS. 1 and 2, respectively.

Example 2. Preparation of Chimeric Antibody A10-A3 (cA10-A3)

To synthesize the signal sequence of the light chain gene, the LHS42 primer described in SEQ ID NO: 1 and the KCleaderBack primer described in SEQ ID NO: 2 using pdCMV-dhfr-AKA / HzK (see Korean Patent Publication No. 2003-0013633) as a template. PCR was performed in pairs. In order to amplify the A10-A3 light chain variable region, PCR was performed using the VK cDNA of Example 1 as a template, the VkA10-A3 primer described in SEQ ID NO: 3, and the KCBSiWIback primer described in SEQ ID NO: 4.

Again, in order to connect the signal sequence and the A10-A3 light chain variable region, the DNA prepared by the above PCR was mixed to form a template, and recombinant PCR was performed using the LHS42 primer described in SEQ ID NO: 1 and the KCBSiWIback primer described in SEQ ID NO: 4. Was performed. At this time, PCR reaction conditions were carried out 30 times using pfu DNA polymerase (30 seconds at 94 ℃, 30 seconds, 52 seconds 30 seconds, 72 ℃ 40 seconds, after preliminary denaturation at 95 ℃ for 5 minutes. Both ends of the DNA fragment were digested with restriction enzymes Hind III and Bsi WI, and then inserted at the Hind III- Bsi WI position of pdCMV-dhfrC-AKA / HzK to prepare pdCMV-dhfrC-cA10A3-vk.

To synthesize the signal sequence of the heavy chain gene, pdCMV-dhfr-AKA / HzK (see Korean Patent Publication No. 2003-0013633), the LHS39 primer of SEQ ID NO: 5 and the HCleaderback primer of SEQ ID NO: 6 PCR was performed in pairs. In order to amplify the A10-A3 heavy chain variable region, PCR was performed using the VH cDNA of Example 1 as a template and the VHA10A3 primer shown in SEQ ID NO: 7 and the LHS11 primer shown in SEQ ID NO: 8.

In order to connect the signal sequence and the A10-A3 heavy chain variable region, the DNA of the heavy chain leader region portion prepared by PCR and the DNA of the heavy chain variable region portion are mixed to form a template, and the LHS39 primer and SEQ ID NO. Recombinant PCR was performed using the LHS11 primer described as 8. At this time, PCR reaction conditions were performed 30 times using puf DNA polymerase (30 seconds at 94 ℃, 30 seconds at 52 ℃, 40 seconds at 72 ℃ after premodification at 95 ℃ for 5 minutes). Both ends of the DNA fragment were cut with restriction enzymes Eco RI and Apa I and inserted into the Eco RI- Apa I position of pdCMV-dhfrC-cA10A3-vk, which was previously prepared, to prepare the expression plasmid of the present invention, which was' pdCMV. -dhfrC-cA10A3 'was named (see Figure 3).

In order to confirm the binding capacity of the prepared cA10-A3, HEK293T cells were transformed with 'pdCMV-dhfrC-cA10A3' expression plasmid using Lipofectamine 2000 (Invitrogen). After 6 hours, the cells were incubated for 12 hours in DMEM medium containing 10% FBS, and then changed to CD293 (Invitrogen) medium containing no serum, and then cultured in an incubator for 4 days to obtain a supernatant producing cA10-A3. The antibody was purified using a Protein A agarose column (Millipore).

Competitive ELISA (ELISA) was performed to confirm that the antigen binding capacity of the purified cA10-A3 to L1CAM is the same as A10-A3. Purified L1CAM proteins at various concentrations between 5 × 10 ^-7 M and 5 × 10 ^-13 M were prepared and reacted with 3.165 ng of cA10-A3 or A10-A3 at 37 ° C. for 3 hours, and then the mixture was treated with L1CAM. It was added to a 96 well plate coated with 100 ng / well (MaxiSorp, Nunc) and reacted at 37 ° C. for 1 hour, and the secondary antibody, anti-human IgG Fc-HRP (1: 2000) or anti-mouse IgG Fc- After reacting HRP (1: 2000) for 1 hour, color development was performed and absorbance was measured at 450 nm.

As a result, A10-A3 showed 1.8 nM and cA10-A3 showed 1.9 nM affinity (see FIG. 4).

The results indicate that the affinity between A10-A3 and cA10-A3 is the same, and thus, the VH and VL genes encoding the variable regions of the A10-A3 antibody were cloned correctly.

Example 3. Preparation and Analysis of Humanized Antibodies

Humanized antibodies have been prepared by CDR-grafting (graft) method in which complementarity determining regions (CDRs) corresponding to antigen binding regions of mouse antibody variable regions are implanted into human antibodies. The CDR-transplantation method is a method of transplanting mouse CDRs into a human antibody having homology with the amino acid sequence of a mouse monoclonal antibody variable region. However, since the antigen-binding ability of humanized antibodies prepared by only transplanting mouse CDRs is often much lower than that of the original mouse antibody, some amino acid residues of the framework region (FR) affecting the conformation of CDRs may be reduced. Humanized antibodies are prepared by substituting those from the original mouse antibodies.

In the present invention, humanized antibody HzA10A3-1 was prepared by implanting CDR1, CDR2 and CDR3 regions, which are complementarity determining regions of the heavy and light chain variable regions, of the A10-A3 antibody into the heavy and light chain variable regions of a human antibody, respectively (see FIG. 5). As a result of measuring the antigen binding affinity, it was confirmed that the affinity of cA10-A3 having the variable region of A10-A3 was 35 times lower than that of cA10-A3.

In order to further enhance the affinity of the humanized antibody HzA10A3-1 for L1CAM, five amino acid residues from the A10-A3 heavy chain variable region were additionally implanted and the four amino acid residues of HCDR2 and LCDR2 were further reduced to lower immunogenicity in the human body. Humanized antibody HzA10A3-2.2 was prepared by substituting one amino acid residue for that of a human antibody (see FIG. 5).

As a result of measuring the affinity of HzA10A3-2.2, it was confirmed that it increased about 3 times than cA10-A3 (see FIG. 6), and T epitope analysis showed that it was possible to show lower immunogenicity than HzA10A3-1 ( Referring to FIG. 7), it was confirmed that there was specificity as a result of the division of antigen-binding specificity into a cell catapult (see FIG. 8), thereby completing the present invention.

If the above results are described in more detail, the present invention prepared a humanized antibody HzA10A3-1 to which only the CDRs of A10-A3 were grafted to compare the antigen-binding affinity with the antigen-binding affinity of cA10-A3. It confirmed (refer FIG. 6). In order to improve the affinity of HzA10A3-1, the 5 FR residues of VH (Kabat No, 48, 71, 73, 93, 94) were replaced with those of the mouse antibody, and the mouse of the prepared humanized antibody In order to reduce the immune response that CDRs can induce, the human body has four residues (No. 60, 61, 64 and 65) of CDR2 (HCDR2) of VH and one residue (No. 56) of CDR2 (LCDR2) of VL. The humanized antibody HzA10A3-2.2 of the present invention was prepared by replacing the antibody with that of the antibody.

As a result of measuring the affinity of HzA10A3-2.2, it was confirmed that it was increased about 3 times than cA10-A3 (see FIG. 6). In addition, T epitope analysis (ProPred; http://www.imtech.res.in/raghava) was used to determine whether HzA10A3-2.2 is less likely to induce a human immune response than HzA10A3-1, which retains the CDR sequences of A10-A3. /propred/index.html) was confirmed to reduce the number of MHC class II molecules that can bind to the peptide sequence of the antibody.

The above results indicate that the humanized antibody HzA10A3-2.2 of the present invention has excellent antigen binding ability and low possibility of inducing a human immune response, and thus can be usefully used for cancer treatment targeting L1CAM as a therapeutic antibody.

(One) HzA10A3 -1 Preparation of Humanized Antibodies and Analysis of Antigen Binding Affinity

In order to prepare a humanized antibody, the heavy chain variable region and light chain variable region sequences of cA10-A3 were compared with the human antibody database (IMGT database; http://www.imgt.org). IGKV7-3 * 1 showed the most similar nucleotide sequence.

For the preparation of HzA10A3-1 humanized antibody, the HzA10A3-1h humanized heavy chain variable region gene (see FIG. 1) and the HzA10A3-1Vk humanized light chain variable region gene (see FIG. 2) were synthesized to synthesize pdCMV-dhfrC-cA10A3 (see FIG. 3). PdCMV -dhfrC-HzA10A3-1 was prepared by inserting into the Eco RI- Apa I position and the Hind III- Bsi WI position, respectively. This plasmid was introduced into HEK293 cells as in Example 2, cultured in CD293 medium to produce humanized antibodies, and purified using a Protein A column.

The affinity of the purified HzA10A3-1 antibody was determined to be 6.73 × 10 ⁻⁸ M by the competitive ELISA method (see FIG. 6).

The result is It means that the affinity of the HzA10A3-1 antibody is about 35 times lower than the affinity of the cA10-A3 antibody.

(2) HzA10A3 -2 Preparation and Characterization of Humanized Antibodies

In order to improve the affinity of the HzA10A3-1 antibody, four FR residues (Kabat No, 48, 71, 93, 94) of HzA10A3-1h were replaced with those of a mouse antibody and further, a mouse of the prepared humanized antibody In order to reduce the immune response that CDRs can induce, a sequence of genes in which four residues of HCDR2 (Nos. 60, 61, 64 and 65) were substituted with those of human antibodies was synthesized and named 'HzA10A3-2h'. PdCMV-dhfrC-HzA10A3-2 was prepared by subcloning the Eco RI- Apa I position sub-cloning of pdCMV-dhfrC-HzA10A3-1.

pdCMV-dhfrC-HzA10A3-2 was introduced into HEK293 cells as in Example 2 and humanized antibodies were produced in CD293 medium and purified using a Protein A column. The affinity of the purified HzA10A3-2 antibody was measured by the competitive ELISA method and showed 2.14 × 10 ⁻⁹ M (see FIG. 6). Thus, the affinity of the HzA10A3-2 antibody was about 34 times higher than the affinity of the HzA10A3-1 antibody.

In addition, T epitope analysis was performed to confirm whether HzA10A3-2 is less likely to induce a human immune response than HzA10A3-1, which contains the HCDR2 sequence of A10-A3. As a result, it was possible to bind to HCDR2 peptide of HzA10A3-2. It was found that the number of MHC class II molecules present was reduced.

The results indicate that HzA10A3-2 may be less likely to induce a human immune response than HzA10A3-1.

(3) HzA10A3 -2.1 Preparation of Humanized Antibodies and Analysis of Antigen Binding Affinity

Residue 73 of the HzA10A3-2 antibody VH FR was replaced with a threonine residue with lysine, the residue of the original mouse antibody, to test for increased affinity. To mutate residue 73, a HT73K-F primer as shown in SEQ ID NO: 9 and a HT73K-R primer as shown in SEQ ID NO: 10 were synthesized as mutagenic primers, and an LHS39 primer as shown in SEQ ID NO: 5 and a SEQ ID NO: Recombinant PCR was performed using LHS11 primers. The sequence of the synthesized gene was named 'HzA10A3-2.1h'. PdCMV-dhfrC-HzA10A3-2.1 was prepared by subcloning the Eco RI- Apa I position sub-clones of the synthesized humanized heavy chain variable region genes of pdCMV-dhfrC-HzA10A3-1.

pdCMV-dhfrC-HzA10A3-2.1 was introduced into HEK293 cells as in Example 2 and humanized antibodies were produced in CD293 medium and purified using a Protein A column. The affinity of the purified HzA10A3-2.1 antibody was measured by the competitive ELISA method, and the result was 6.22 × 10 ⁻¹⁰ M (see FIG. 6).

Thus, the affinity of the HzA10A3-2.1 antibody was about 3 times higher than that of the HzA10A3-2 antibody.

The results indicate that residue 73 of VH is an important residue that affects the structure of HCDR2.

(4) HzA10A3 -2.2 Preparation and Characterization of Humanized Antibodies

In order to further humanize the light chain LCDR2 of the prepared HzA10A3-2.1 humanized antibody, the serine residue 56 was transformed into threonine which is a human antibody to prepare HzA10A3-2.2, and antigen binding affinity and T epitope analysis were performed.

In order to mutate the residue 56, the LS56T-F primer of SEQ ID NO: 11 and the LS56T-R primer of SEQ ID NO: 12 were synthesized as mutagenic primers, and the LHS42 primer of SEQ ID NO: 1 and SEQ ID NO: 13 were described. Recombinant PCR was performed using HzA10BsiWI-R primers. The sequence of the synthesized gene was named 'HzA10A3-2k'. This synthesized humanized light chain variable region gene was subcloned at the HindIII-BsiWI position of pdCMV-dhfrC-HzA10A3-2.1 to prepare pdCMV-dhfrC-HzA10A3-2.2.

pdCMV-dhfrC-HzA10A3-2.2 was introduced into HEK293 cells as in Example 2 and humanized antibodies were produced in CD293 medium and purified using a Protein A column. The affinity of the purified HzA10A3-2.2 antibody was determined by the competitive ELISA method, which showed 5.32 × 10 ⁻¹⁰ M (see FIG. 6). This result is Affinity of the HzA10A3-2.2 antibody means similar to that of the HzA10A3-2.1 antibody.

In addition, T epitope analysis was performed to confirm whether HzA10A3-2.2 is less likely to induce a human immune response than HzA10A3-2.1, which retains the LCDR2 sequence of A10-A3. As a result, it was possible to bind to the LCDR2 peptide of HzA10A3-2.2. A decrease in the number of MHC class II molecules present (see FIG. 7).

The results indicate that HzA10A3-2.2 may be less likely to induce a human immune response than HzA10A3-2.1.

Example  4. Validation of specificity of the prepared humanized antibody

In order to confirm the specificity of the prepared humanized antibody, flow cytometry was performed using a cell line expressing L1CAM and a cell line not expressing. 293T-L1 cells overexpressing L1CAM on HEK293T cells and gallbladder cancer cell lines JCRB1033 expressing L1CAM and A375, a melanoma cell line, were used as positive cells, and HEK293T and NIH3T3 cell lines without L1CAM expression were used as negative cells. Each cell was bound to 0.5 μg of HzA10A3-2.1 or HzA10A3-2.2 antibody, and then stained using a secondary antibody, anti-human IgG (Fc specific) -FITC, and then stained with FACSCalibur (BD-Bioscience). Analyzed.

As a result, it was confirmed that HzA10A3-2.1 and HzA10A3-2.2 do not have a nonspecific binding and specifically bind only to L1CAM (see FIG. 8).

Example  5. Preparation of Humanized Antibodies cell internalization  analysis

In order to confirm whether the prepared humanized antibody binds to cells expressing L1CAM and enters into cells, 20 μg of a HzA10A3-2.2 antibody is expressed in SCK-L1 (Clin Cancer Res 16, 3571-3580, 2010) cells expressing L1CAM. After incubation at 37 ° C. for 6 hours, cells were washed twice with PBS, fixed with 2% paraformaldehyde, and punctured with PBS buffer containing 0.2% Triton X-100. . PBS added with 2% BSA and 1% horse serum was added to the cells and allowed to stand at room temperature for 1 hour. Then, goat-anti-human IgG (1: 500) bound with Alexa-488 was added and the mixture was kept at room temperature for 1 hour. I shook it. After washing the cells six times with PBS, PBS added with 0.25 μg / mL DAPI, 0.1% Triton X-100 was added and shaken at room temperature for 20 minutes. Fluorescence images were analyzed by Confocal imaging system (Carl Zeiss, Germany).

As a result, it was confirmed that HzA10A3-2.2 antibody enters SCK-L1 cells (see FIG. 9). In FIG. 9, the control refers to the cells treated with the same conditions after binding the humanized antibody to SCK-L1 cells and then placed on ice without incubating at 37 ° C.

The results indicate that the ADC (antibody drug conjugate) by combining the drug with the HzA10A3-2.2 antibody can be used in cancer treatment.

In conclusion, the humanized antibody HzA10A3-2.2 of the present invention is excellent antibody that has excellent antigen binding ability, low possibility of inducing human immune response, and maintains the specificity of the original mouse antibody A10-A3 antibody. Can be.

<110> KNU-Industry Cooperation Foundation          Korea Research Institute of Bioscience and Biotechnology <120> Humanized antibody against human L1CAM and method for preparing          the antibody <160> 31 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LHS42 primer <400> 1 tgcaaagctt cggcacgagc a 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KCleaderback primer <400> 2 tccttcaaca ccagacaac 19 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> VkA10A3 primer <400> 3 ttgtctggtg ttgaaggaga tattgtgctg acc 33 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> KCBSiWIback primer <400> 4 agccaccgta cgtaagattt ccagcat 27 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LHS39 primer <400> 5 gacgaattca ctctaaccat ggaa 24 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> HCleaderback primer <400> 6 ggagtggaca cctgtag 17 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> VHA10A3 primer <400> 7 acaggtgtcc tctcccaggt ccagctggtg 30 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> LHS11 primer <400> 8 caccggttcg gggaagt 17 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HT73K-F primer <400> 9 gtagacaagt ccattagcac agcc 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HT73K-R primer <400> 10 aatggacttg tctacagtca ttgt 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LS56T-F primer <400> 11 ctagaaactg gggtccctgc cagg 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LS56T-R primer <400> 12 gaccccagtt tctaggttgg atgc 24 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> Hz223 BsiWI-R primer <400> 13 agccaccgta cgtttgattt ccacctt 27 <210> 14 <211> 111 <212> PRT <213> Unknown <220> <223> A10-A3 VL amino acid seq <400> 14 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Leu Gly Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Gly Tyr              20 25 30 Gly Ile Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro          35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala      50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn  65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys His Gln Ser Asn                  85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Met Leu Glu Ile Leu             100 105 110 <210> 15 <211> 118 <212> PRT <213> Unknown <220> <223> A10-A3 VH amino acid seq <400> 15 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr              20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile          35 40 45 Gly Glu Ile Asn Pro Arg Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe      50 55 60 Arg Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr  65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                  85 90 95 Thr Val Tyr Tyr Gly Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Val         115 <210> 16 <211> 333 <212> DNA <213> Unknown <220> <223> A10-A3 VL gene <400> 16 gatattgtgc tgacccagtc tccagcttct ttggctgtgt ctctagggct gggggccacc 60 atatcctgca gagccagtga aagtgttgat ggttatggca ttacctttat gcactggtac 120 cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180 gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240 cctgtggagg ctgatgatgt tgcaacctat tactgtcatc aaagtaatga ggatccgtgg 300 acgttcggtg gaggcaccat gctggaaatc tta 333 <210> 17 <211> 354 <212> DNA <213> Unknown <220> <223> A10-A3 VH gene <400> 17 caggtccagc tgcagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagctg 60 tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt gaaacagagg 120 cctggacaag gccttgagtg gattggagag attaatccca ggaacggtcg tactaactat 180 aatgagaagt tcaggagcaa ggccacactg actgtagaca aatcctccag cacagcctac 240 atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtac agtctactat 300 ggttacgacg ggtttgctta ctggggccaa gggactctgg tcactgtctc tgta 354 <210> 18 <211> 111 <212> PRT <213> Unknown <220> Hz223A V-1 amino acid seq <400> 18 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly   1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Gly Tyr              20 25 30 Gly Ile Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro          35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala      50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn  65 70 75 80 Pro Val Glu Ala Gln Asp Thr Ala Asn Tyr Tyr Cys His Gln Ser Asn                  85 90 95 Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 19 <211> 333 <212> DNA <213> Unknown <220> <223> HzA10A3-1 VL gene <400> 19 gatattgtgc tgacccagtc tccagcttct ttggctgtgt ctccagggca gagggccacc 60 ataacctgca gagccagtga aagtgttgat ggttatggca ttacctttat gcactggtac 120 cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caccattaat 240 cctgtggagg ctcaggatac tgcaaattat tactgtcatc aaagtaatga ggatccgtgg 300 acgttcggtc agggcaccaa ggtggaaatc aaa 333 <210> 20 <211> 118 <212> PRT <213> Unknown <220> Hz223A V-1 amino acid seq <400> 20 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr              20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Glu Ile Asn Pro Arg Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe      50 55 60 Arg Ser Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Tyr Tyr Gly Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 21 <211> 354 <212> DNA <213> Unknown <220> <223> HzA10A3-1 VH gene <400> 21 caggtccagc tggtgcagtc tggggctgaa gtgaagaagc ctggggcttc agtgaaggtg 60 tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt gagacaggcg 120 cctggacaag gccttgagtg gatgggagag attaatccca ggaacggtcg tactaactat 180 aatgagaagt tcaggagcag ggtcacaatg actagagaca catccattag cacagcctac 240 atggaactca gcaggctgag gtctgatgac actgcggtct attactgtgc taggtactat 300 ggttacgacg ggtttgctta ctggggccaa gggactctgg tcactgtctc ttca 354 <210> 22 <211> 118 <212> PRT <213> Unknown <220> Hz223A V-2 amino acid seq <400> 22 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr              20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile          35 40 45 Gly Glu Ile Asn Pro Arg Asn Gly Arg Thr Asn Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Met Val Asp Thr Ser Ile Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Thr Val Tyr Tyr Gly Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 23 <211> 354 <212> DNA <213> Unknown <220> <223> HzA10A3-2 VH gene <400> 23 caggtccagc tggtgcagtc tggggctgaa gtgaagaagc ctggggcttc agtgaaggtg 60 tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt gagacaggcg 120 cctggacaag gccttgagtg gattggagag attaatccca ggaacggtcg tactaactat 180 gctcagaagt tccagggcag ggtcacaatg actgtagaca catccattag cacagcctac 240 atggaactca gcaggctgag gtctgatgac actgcggtct attactgtac agtctactat 300 ggttacgacg ggtttgctta ctggggccaa gggactctgg tcactgtctc ttca 354 <210> 24 <211> 118 <212> PRT <213> Unknown <220> Hz223 A3-2.1 VH amino acid seq <400> 24 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr              20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile          35 40 45 Gly Glu Ile Asn Pro Arg Asn Gly Arg Thr Asn Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Thr Val Tyr Tyr Gly Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 25 <211> 354 <212> DNA <213> Unknown <220> <223> HzA10A3-2.1 VH gene <400> 25 caggtccagc tggtgcagtc tggggctgaa gtgaagaagc ctggggcttc agtgaaggtg 60 tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt gagacaggcg 120 cctggacaag gccttgagtg gattggagag attaatccca ggaacggtcg tactaactat 180 gctcagaagt tccagggcag ggtcacaatg actgtagaca agtccattag cacagcctac 240 atggaactca gcaggctgag gtctgatgac actgcggtct attactgtac agtctactat 300 ggttacgacg ggtttgctta ctggggccaa gggactctgg tcactgtctc ttca 354 <210> 26 <211> 111 <212> PRT <213> Unknown <220> Hz223 A3-2.2 VL amino acid seq <400> 26 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly   1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Gly Tyr              20 25 30 Gly Ile Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro          35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Thr Gly Val Pro Ala      50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn  65 70 75 80 Pro Val Glu Ala Gln Asp Thr Ala Asn Tyr Tyr Cys His Gln Ser Asn                  85 90 95 Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 27 <211> 333 <212> DNA <213> Unknown <220> <223> A10A3-2.2 VL gene <400> 27 gatattgtgc tgacccagtc tccagcttct ttggctgtgt ctccagggca gagggccacc 60 ataacctgca gagccagtga aagtgttgat ggttatggca ttacctttat gcactggtac 120 cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaaact 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caccattaat 240 cctgtggagg ctcaggatac tgcaaattat tactgtcatc aaagtaatga ggatccgtgg 300 acgttcggtc agggcaccaa ggtggaaatc aaa 333 <210> 28 <211> 118 <212> PRT <213> Unknown <220> Hz223A3-2.2 VH amino acid seq <400> 28 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr              20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile          35 40 45 Gly Glu Ile Asn Pro Arg Asn Gly Arg Thr Asn Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Thr Val Tyr Tyr Gly Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 29 <211> 354 <212> DNA <213> Unknown <220> <223> HzA10A3-2.2 VH gene <400> 29 caggtccagc tggtgcagtc tggggctgaa gtgaagaagc ctggggcttc agtgaaggtg 60 tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt gagacaggcg 120 cctggacaag gccttgagtg gattggagag attaatccca ggaacggtcg tactaactat 180 gctcagaagt tccagggcag ggtcacaatg actgtagaca agtccattag cacagcctac 240 atggaactca gcaggctgag gtctgatgac actgcggtct attactgtac agtctactat 300 ggttacgacg ggtttgctta ctggggccaa gggactctgg tcactgtctc ttca 354 <210> 30 <211> 111 <212> PRT <213> Unknown <220> <223> HzA10A3-2.1 VL amino acid seq <400> 30 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly   1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Gly Tyr              20 25 30 Gly Ile Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro          35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala      50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn  65 70 75 80 Pro Val Glu Ala Gln Asp Thr Ala Asn Tyr Tyr Cys His Gln Ser Asn                  85 90 95 Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 31 <211> 333 <212> DNA <213> Unknown <220> <223> HzA10A3-2.1 VL gene <400> 31 gatattgtgc tgacccagtc tccagcttct ttggctgtgt ctccagggca gagggccacc 60 ataacctgca gagccagtga aagtgttgat ggttatggca ttacctttat gcactggtac 120 cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caccattaat 240 cctgtggagg ctcaggatac tgcaaattat tactgtcatc aaagtaatga ggatccgtgg 300 acgttcggtc agggcaccaa ggtggaaatc aaa 333

Claims (23)

delete delete HzA10A3-2.2 or a fragment of such an antibody, which is an L1CAM specific humanized antibody consisting of a light chain variable region having an amino acid sequence as set out in SEQ ID NO: 26 and a heavy chain variable region having an amino acid sequence as set out in SEQ ID NO: 28. The method of claim 3,
The antibody or fragment of the antibody may be a single-chain antibody, diabody, tribody, tetrabody, Fab fragment, F (ab ') ₂ fragment, Fd, scFv, domain antibody, minibody, single that has a binding function to FcRn. chain antibody, scAb), IgD antibody, IgE antibody, IgM antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody, derivatives of antibody constant region, and L1CAM characterized in that it is selected from phosphorus airports based on protein scaffolds Specific humanized antibodies, or fragments of such antibodies. An antibody or a fragment thereof and a conjugate of the drug according to claim 3. The method of claim 5,
The drug is a conjugate, characterized in that the drug for the treatment of cancer. The method of claim 6,
The drug is selected from the group consisting of microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or chemotherapeutic agents that can function as DNA intercalators, protein toxins and radioisotopes that can function enzymatically A conjugate, characterized in that at least one. The method of claim 6,
The drugs include maytansinoids, auristatins, dolastatins, tricortesenes, CC1065, calicheamicins and other enedin antibiotics, taxanes, anthracyclines, methotrexate, adriamycin, bindesin, vinca alkaloids (vincristine, Vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, carminomycin, aminopterin, dactinomycin, Mitomycins, bleomycins, esperamicins, 5-fluorouracil, melphalan and other nitrogen mustards, stereoisomers, isotopes, homologues or derivatives thereof, cis-platinum and cis-platinum homologues, other intercalating enzymes And fragments thereof, cisplatin, CPT-11, doxorubicin, paclitaxel, and docetaxel. A bispecific antibody comprising the L1CAM specific humanized antibody according to claim 3, or a fragment of such antibody. The method of claim 9,
A bispecific antibody, characterized in that the humanized antibody, or a fragment of such an antibody and an antibody having an ability to bind to an immune effector cell-specific target molecule. The method of claim 10,
The immuno-specific cell-specific target molecule is selected from TCR / CD3, CD16 , CD44, Cd56, CD69, CD64, CD89 and CD11b / CD18 (CR3). A composition for treating cancer comprising the antibody according to any one of claims 3 to 11, or a fragment thereof, a conjugate, or a bispecific antibody. The method of claim 12,
The cancer is gastric cancer, breast cancer, lung cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer , Kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureter cancer, osteosarcoma, neuroblastoma, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma Cancer treatment composition, characterized in that. The method of claim 13,
The cancer is a cancer treatment composition, characterized in that breast cancer, ovarian cancer, colon cancer, skin cancer, biliary tract cancer or lung cancer. A polynucleotide encoding the light chain variable region of HzA10A3-2.2, which is the humanized antibody according to claim 3. A polynucleotide encoding the heavy chain variable region of HzA10A3-2.2, which is the humanized antibody according to claim 3. The method of claim 15,
Polynucleotide having a sequence represented by SEQ ID NO: 27. The method of claim 16,
Polynucleotide having the sequence shown in SEQ ID NO: 29. A polynucleotide encoding an amino acid sequence set forth in SEQ ID NO: 26, and a polynucleotide encoding an amino acid sequence set forth in SEQ ID NO: 28; or
A polynucleotide having a sequence represented by SEQ ID NO: 27, and a polynucleotide having a sequence represented by SEQ ID NO: 29;
Recombinant vector comprising a. A host cell transformed with the recombinant vector according to claim 19. The method of claim 20,
Host cell, characterized in that selected from animal cells, plant cells, yeast, E. coli, insect cells. The method of claim 21,
Monkey kidney cells (COS7) cells, NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells, and HEK293 cells, E. coli, Bacillus subtilis (Bacillussubtilis), genus Streptomyces (Streptomyces sp.), Pseudomonas species (Pseudomonas sp.), Proteus Mira Billy's (Proteusmirabilis) or Staphylococcus genus ( Staphylococcus sp.), Aspergillus sp., Pichiapastoris , Saccharomyces cerevisiae , Schizosaccharomyces sp. And Neurospores Host cell, characterized in that selected from La la ( Carasporacrassa ). A method for producing a fragment or variable region of HzA10A3-2.2, an L1CAM specific humanized antibody comprising culturing a host cell according to any one of claims 20 to 22.

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