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WO2019103531A1 - Anti-icam4 antibody and composition for diagnosis and treatment of icam4 expression cell-associated disease - Google Patents

Anti-icam4 antibody and composition for diagnosis and treatment of icam4 expression cell-associated disease Download PDF

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WO2019103531A1
WO2019103531A1 PCT/KR2018/014540 KR2018014540W WO2019103531A1 WO 2019103531 A1 WO2019103531 A1 WO 2019103531A1 KR 2018014540 W KR2018014540 W KR 2018014540W WO 2019103531 A1 WO2019103531 A1 WO 2019103531A1
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antibody
icam4
seq
amino acid
acid sequence
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PCT/KR2018/014540
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French (fr)
Korean (ko)
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송형근
김민영
선성규
박보름
이지예
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충북대학교 산학협력단
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Priority claimed from KR1020180145969A external-priority patent/KR102090407B1/en
Publication of WO2019103531A1 publication Critical patent/WO2019103531A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to an improved anti-ICAM4 antibody and its use as a diagnostic and therapeutic agent for diseases related to ICAM4 expressing cells, and more particularly to a chimeric antibody of anti-ICMA4 monoclonal antibody 1E1 and a cell line, a humanized antibody, Anti-ICMA4 antibody-drug complex, and the like.
  • Antibody-based therapy for cancer has been one of the most successful and important ways to treat patients with blood and solid tumors from the past 20 years to the present [Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012; 12 (4): 278-87].
  • FDA US Food and Drug Administration
  • Humanized antibody refers to an antibody in which the antibody sequence of a non-human species is modified to increase similarity with an antibody naturally produced in humans (Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. 1988; 332 (6162): 323-7; and Queen C, et al., A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci US A. 1989; 86 (24): 10029-33).
  • the chimeric antibody is similar to the humanized antibody in that the constant region sequence of the mouse antibody is replaced with the human antibody constant region sequence in order to enhance similarity with the human antibody, but the sequences other than the complementarity determining region (CDR) (Brekke OH, Sandlie I.
  • CDR complementarity determining region
  • CDR transplantation is a method of isolating DNA encoding a variable region sequence of a mouse monoclonal antibody, securing the CDR sequence through gene cloning, and then grafting the CDR sequence into a suitable human antibody sequence in an in silico phase (Hou S, et (1): 115-20; and Kashmiri SV, De Pascalis R, Gonzales NR, Schlom, J. Biochem. J. SDR grafting - a new approach to antibody humanization. Methods. 2005; 36 (1): 25-34).
  • the phage display method is most widely used as a method designed to make antibodies for human therapy without using a mouse or other non-human mammal.
  • HUMIRA the treatment for rheumatoid arthritis, is the first antibody approved using this technique and is the highest antibody drug currently marketed.
  • Antibody-drug complexes are one of the most advanced approaches to targeted treatment of cancer [15]. Antibody-drug complexes can deliver drugs directly to tumor tissue using a target specific reaction of the antibody (Sochaj AM, Widerska KW, Otlewski J. Current methods for synthesis of homogeneous antibody-drug conjugates. Biotechnol. ; 33 (6 Pt 1): 775-84; and Teicher BA, Doroshow JH. The promise of antibody-drug conjugates. N Engl J Med. 2012; 367 (19): 1847-8).
  • ICAM4 intercellular adhesion molecule 4
  • ICAM4 intercellular adhesion molecule 4
  • VLA-4 the adjacent red blood cells
  • ⁇ v integrin of macrophages the maturation stage where the nucleus of human red blood cells is removed.
  • Studies on the interaction between ICAM4 antigen phosphorylation and various integrins binding to ICAM4 have been carried out in patients with sickle cell disease. However, there is no known target substance for antibody treatment.
  • the present inventors developed a therapeutic agent for recombinant antibodies against ICAM4.
  • various types of anti-ICAM4 monoclonal antibody 1E1 based chimeric antibody, humanized antibody, antibody-drug complex and bispecific antibody The present inventors have developed a therapeutic agent for an ICAM4 antibody, and have discovered that they have a diagnostic or therapeutic effect on a specific disease, thus completing the present invention.
  • the present invention provides a chimeric antibody, a humanized antibody, a bispecific antibody and an antibody-drug complex using an anti-ICAM4 monoclonal antibody.
  • the present inventors secured a mouse monoclonal antibody 1E1 against human erythrocyte surface antigen ICAM4 through previous studies, and obtained a patent as Korean Patent No. 10-1627020. The entirety of which is incorporated herein by reference.
  • the anti-ICAM4 antibody of the present invention is one of a chimeric antibody, a humanized antibody, a bispecific antibody or an antibody-drug delivery vehicle for the anti-ICAM4 monoclonal antibody 1E1.
  • the form of the antibody may vary depending on the application form, Sequence.
  • the anti-ICAM4 antibody of the present invention comprises a heavy chain region of a complementarity determining region (CDR) comprising a sequence selected from the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 5; And a light chain region of a complementary crystal region (CDR) comprising a selected sequence of the amino acid sequence of SEQ ID NO: 6 to SEQ ID NO: 9.
  • CDR complementarity determining region
  • CDR complementary crystal region
  • the CDR region comprises CDR H1 consisting of the amino acid sequence of SEQ ID NO: 1; A CDR H2 consisting of a sequence selected from the amino acid sequences of SEQ ID NOS: 2 to 4; And a CDR heavy chain region comprising CDR H3 consisting of the amino acid sequence of SEQ ID NO: 5 and a CDR L1 consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7; A CDR L2 consisting of the amino acid sequence of SEQ ID NO: 8; And a CDR light chain region comprising CDR L3 consisting of the amino acid sequence of SEQ ID NO: 9.
  • the invention provides a chimeric antibody engineered to be expressed together with a variable region of the anti-ICAM4 antibody and a human antibody constant region.
  • the chimeric antibody comprises the complementarity determining region (CDR) and the structural region (FR) of the anti-ICAM4 antibody and the light and heavy chain constant regions of the human antibody.
  • the invention provides a humanized antibody of said anti-ICAM4 antibody.
  • the humanized antibody heavy chain region of the anti-ICAM4 antibody comprises VH1A consisting of the amino acid sequence of 11; VH1B consisting of the amino acid sequence of SEQ ID NO: 12; VH1C consisting of the amino acid sequence of SEQ ID NO: 23; A VH1D consisting of the amino acid sequence of SEQ ID NO: 14; VH2A consisting of the amino acid sequence of SEQ ID NO: 15; VH2B consisting of the amino acid sequence of SEQ ID NO: 16; VH2C consisting of the amino acid sequence of SEQ ID NO: 17; A VH2D consisting of the amino acid sequence of SEQ ID NO: 18; A VH2E consisting of the amino acid sequence of SEQ ID NO: 19; VH3A consisting of the amino acid sequence of SEQ ID NO: 20; VH3B consisting of the amino acid sequence of SEQ ID NO: 21; VH3C consisting of the amino acid sequence of SEQ ID NO: 22; A VH3D
  • the light chain region of the humanized antibody of the anti-ICAM4 antibody comprises VL1A consisting of the amino acid sequence of SEQ ID NO: 24; VL1B consisting of the amino acid sequence of SEQ ID NO: 25; VL1C consisting of the amino acid sequence of SEQ ID NO: 26; VL1D consisting of the amino acid sequence of SEQ ID NO: 27; VL2A consisting of the amino acid sequence of SEQ ID NO: 28; VL2B consisting of the amino acid sequence of SEQ ID NO: 29; VL2C consisting of the amino acid sequence of SEQ ID NO: 30; And VL2D consisting of the amino acid sequence of SEQ ID NO: 31.
  • the invention provides a bispecific antibody comprising the anti-ICAM4 antibody.
  • the bispecific antibody is an anti-ICAM4 antibody; And antibodies specific to other antigens are bound by a linker.
  • Antibodies specific to the other antigens of the present invention are in the form of CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM21, CEACAM16, CEACAM18 , CEACAM19, CEACAM20, and CD33, CD11b which are MDSC markers, preferably CEACAM8 or CEACAM6.
  • a bispecific antibody comprising an anti-CEACAM6 (CD66c) antibody was produced and its effect was confirmed.
  • the anti-CD66c antibody may include, without limitation, a peptide that specifically binds to CD66c, and the linker is composed of the amino acid sequence of SEQ ID NO: 10, but is not limited thereto.
  • the invention provides an Antibody-Drug Conjugate (ADC) comprising the anti-ICAM4 antibody.
  • ADC Antibody-Drug Conjugate
  • the drug is a drug exhibiting anticancer activity, and may be a substance that causes toxicity in the body.
  • drugs include, but are not limited to, saporin, emtansine, Doxorubicin, Carboplatin, Cisplatin, Cyclophosphamide, Ifosfamide, Nidran, Nitrogen mustar (Mechlorethamine HCL), Bleomycin, Mitomycin C, Cytarabine, Fluoruracil Flurouracil, Gemcitabine, Trimetrexate, Methotrexate, Etoposide, Vinblastine, vinorelbine, Alimta, May be at least one drug selected from the group consisting of Altretamine, Procarbazine, Taxol, Taxotere, Topotecan and Irinotecan, Saporin or Mantan Emtansine. ≪ / RTI >
  • the present invention provides a heavy chain comprising a heavy chain region of a complementarity determining region (CDR) comprising a selected one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 5; And an anti-ICAM4 antibody comprising a light chain region of a complementarity determining region (CDR) comprising a selected sequence selected from the amino acid sequences of SEQ ID NOs: 6 to 9, wherein the anti-ICAM4 antibody .
  • CDR complementarity determining region
  • an anti-ICAM4 antibody comprising a light chain region of a complementarity determining region (CDR) comprising a selected sequence selected from the amino acid sequences of SEQ ID NOs: 6 to 9, wherein the anti-ICAM4 antibody .
  • the diagnosis can be diagnosed through an antigen-antibody reaction in an ICAM4 expression disease through the composition, and the diagnostic composition and the diagnostic kit containing the diagnostic composition can be used.
  • the invention provides a heavy chain region of a complementarity determining region (CDR) comprising a selected sequence of amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 5; And an anti-ICAM4 antibody comprising a light chain region of a complementary crystal region (CDR) comprising a selected sequence selected from the amino acid sequences of SEQ ID NOS: 6 to 9, and a kit for the treatment of diseases associated with cells expressing ICAM4 do.
  • the disease may be leukemia, liver cancer, cholangiocarcinoma or myelodysplastic syndrome (MDS).
  • the composition may be a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • the invention provides a chimeric antibody of said anti-ICAM4 antibody; Humanized antibodies; Bispecific antibodies; Or myeloid-derived suppressor cells (MDSC) comprising an antibody-drug complex (Antibody-Drug Conjugate, ADC) in which a drug is bound to these antibodies.
  • ADC Antibody-Drug Conjugate
  • Myeloid-derived suppressor cells are immunosuppressive cells in myeloid cells. They are cells that contain a very broad range of undifferentiated myeloid cells, which are increased in the onset of tumors or inflammation. In general, It is known to exert immunosuppressive action through cell-cell contact. That is, by discriminating MDSC, it is possible to determine whether tumor or inflammation has occurred, and anti-ICAM4 monoclonal antibody 1E1 of the present invention can act as a marker binding to MDSC.
  • the ICAM4 monoclonal antibodies of the invention and their chimeric antibodies; Humanized antibodies; Bispecific antibodies; Or Antibody-Drug Conjugate (ADC), in which a drug is bound to these antibodies, can be used as a marker for MDSC, and diagnosis of diseases in which MDSC is expressed can be made by diagnosing them.
  • ADC Antibody-Drug Conjugate
  • the invention provides a monoclonal antibody and its chimeric antibody of said anti-ICAM4; Humanized antibodies; Bispecific antibodies; Or a composition for inhibiting the activity of Myeloid-derived suppressor cells (MDSC) comprising an antibody-drug complex (Antibody-Drug Conjugate, ADC) in which a drug is bound to these antibodies.
  • MDSC Myeloid-derived suppressor cells
  • ADC Antibody-Drug Conjugate
  • the present invention relates to a chimeric antibody of said anti-ICAM4 antibody; Humanized antibodies; Bispecific antibodies; Or an antibody-drug complex (Antibody-Drug Conjugate, ADC) in which a drug is bound to these antibodies, to provide information for diagnosis of a disease associated with cells expressing ICAM4.
  • ADC Antibody-Drug Conjugate
  • the method comprises the steps of: 1) contacting the antibody to a biological sample obtained from an individual; 2) detecting ICAM4 bound to the antibody; And 3) comparing the degree of expression of ICAM4 detected in step 2) with a normal control.
  • the disease associated with the cells expressing ICAM4 may be, but is not limited to, cancer. In one embodiment of the present invention, the disease associated with the cells expressing ICAM4 is leukemia, liver cancer, cholangiocarcinoma or myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • a chimeric antibody of an anti-ICAM4 antibody according to the present invention Humanized antibodies;
  • the bispecific antibody and the antibody-drug complex recognized the same antigenic site as the 1E1 monoclonal antibody of the anti-ICAM4 antibody and showed similar antigen affinity.
  • the antibody-drug complex showed better efficacy than the existing antibody-drug conjugate ,
  • the improved antibodies of the present invention can be useful for the diagnosis and treatment of diseases related to ICAM4.
  • FIG. 1 is a diagram showing a process for producing a cell line for producing an anti-ICAM4 chimeric 1E1 antibody ( chi 1E1) according to the present invention.
  • FIG. 2 shows the binding affinity of human chimeric 1E1 antibody against human ICAM4 measured by flow cytometry in a leukemia cell line.
  • Figure 3 shows the three-dimensional structure of mouse 1E1 antibody by molecular modeling.
  • the marked part represents all CDRs parts.
  • Some of the designed humanized antibody sequences cause structural changes in CDRs (Kabat positions H48 and H78).
  • Figure 4 shows the selection results of optimal humanized 1E1 antibody constructs.
  • FIG. 5 shows the result of measuring the 1E1 antigen binding affinity of the humanized antibody.
  • binding affinity to 1E1 is higher than that of chimeric antibodies.
  • FIG. 6 shows the development process of two kinds of anti-ICAM4-drug complexes.
  • (a) Development of murine 1E1 antibody (m1E1-SAP) conjugated with saporin. 1E1-SAP is chemically linked to the saprin by a disulfide bond.
  • DM1 thiol-containing maytansinoid;
  • SMCC succinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate.
  • FIG. 7 shows the results of experiments on the efficacy of two kinds of anti-ICAM4-drug complexes according to one embodiment of the present invention.
  • the m-1E1 SAP of different concentrations in the K562 cells were treated for 3 days. Experiments were performed in 3 replicates and analyzed with Ez-cytox.
  • HEL cells were treated with different concentrations of chi 1E1-DM1 for 3 days. The number of living and dead cells was measured by flow cytometry.
  • Figure 8 shows the results of an experiment on the efficacy of the anti-ICAM4-DM1 complex in a xenotransplanted mouse model.
  • In vivo efficacy of anti-ICAM4 chi 1E1-DM1 was measured in HEL tumor xenografts. SCID mice with HEL tumors were injected intravenously with 4 mg / kg of chi 1E1-DM1.
  • Figure 9 shows the measurement of the different sialylation of ICAM4 in normal RBC and leukemia tumor cells by treatment with various concentrations of neuraminidase.
  • Neuramine hydrolase was treated with tumor cells (K562) and erythrocytes at different concentrations to compare the binding affinity of chimeric 1E1 antibody bound thereto.
  • Fig. 10 shows the results (b, c) of the method (a) for producing bispecific antibodies against ICAM4 and CD66c and the antibody-dependent cytotoxicity (ADCC) function of the antibodies prepared.
  • Figure 11 shows the expression of ICAM4 in a paraffin-embedded section of human fetal tissue.
  • anti-ICAM4 1E1 antibody (10 ⁇ g / mL) and HRP-labeled secondary antibody (1/1000 dilution) were sequentially treated in a paraffin tissue section of human fetal tissue and DAB was used as a substrate.
  • the staining showed no cross reactivity for all other tissues (a) except liver (b) where extramedullary erythropoiesis was present.
  • Figure 12 shows the expression of ICMA4 in tumor tissues of MDS and liver cancer patients.
  • the ICAM4 antigen is not expressed under normal conditions except for red blood cells.
  • Analysis of clinical patient samples revealed CD45 low / CD11b + / ICAM4 + populations in (a) MDS & HCC and (b) MDS patients.
  • MDS myelodysplastic syndrome
  • HCC hepato cellular
  • FIG. 13 shows that 1E1 antigen is elevated in a sample of some cancer patients among 3 normal adults and 11 cancer patients, and thus it is confirmed that the 1E1 antibody target is not restricted to the target leukemia.
  • FIG. 14 shows the effect of anti-ICAM4 antibody on bone marrow-derived inhibitory cell (MDSC), showing that anti-ICAM4 antibody can be used as a marker for the cell.
  • the present inventors have systematically verified the possibility of developing a monoclonal antibody 1E1 against ICAM4 expressed on normal erythrocyte surface as disclosed in Korean Patent No. 10-1627020 as an antibody for tumor therapy including leukemia.
  • a cell line stably producing a chimeric antibody was prepared (Fig. 1).
  • POPtiVEC and pcDNA3.3 which were designed to express human constant regions together, were used as expression vectors and CHO DG44 was used as the host cell line.
  • MTX and G418 as a drug to improve productivity, a cell line showing productivity of 7.5 ⁇ g / mL was developed.
  • Recombinant chimeric antibody 1E1 produced in a cell line for mass production recognizes the same antigenic site as mouse antibody 1E1 and shows similar antigen affinity (FIG. 2).
  • 104 recombinant humanized antibodies in silico were produced and screened to select optimal therapeutic antibody candidates.
  • the production of a therapeutic chimeric antibody in which the variable region of the mouse antibody and the constant region of the human antibody are recombined, has several advantages such as reducing the HAMA (human anti-mouse antibody) , The antibody variable region derived from the mouse may still induce the skin reaction.
  • anti-ICAM4 chimeric antibody 1E1 In order to obtain an antibody superior to the chimeric antibody in terms of physical properties and antigen affinity in the course of screening the antibody through humanization, anti-ICAM4 chimeric antibody 1E1 .
  • CDR transplantation is an antibody humanization technique that converts all but the CDR, which is the site required for antigen recognition and binding, to human-derived sequences [Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with a mouse. Nature. 1986; 321 (6069): 522-5].
  • the heavy chain IMGT human germline IGHV4-59 * 05 60.8%
  • IGHV3-11 * 01 51.6%)
  • IGHV5-51 * 01 45.4%
  • light chain GKV1-9 * 01 75.8%
  • IGKV3-11 * 01 67.4%
  • the 13 human anti-ICAM4 monoclonal antibody 1E1 heavy chain antibody region heavy chain variable region includes the heavy chain variable region and the human antibody constant region selected from the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 5, VH1A consisting of the amino acid sequence of SEQ ID NO: 11; VH1B consisting of the amino acid sequence of SEQ ID NO: 12; VH1C consisting of the amino acid sequence of SEQ ID NO: 23; A VH1D consisting of the amino acid sequence of SEQ ID NO: 14; VH2A consisting of the amino acid sequence of SEQ ID NO: 15; VH2B consisting of the amino acid sequence of SEQ ID NO: 16; VH2C consisting of the amino acid sequence of SEQ ID NO: 17; A VH2D consisting of the amino acid sequence of SEQ ID NO: 18; A VH2E consisting of the amino acid sequence of SEQ ID NO: 19; VH3A consisting of the amino acid sequence of SEQ ID NO:
  • the light chain region of the humanized antibody of the eight anti-ICAM4 monoclonal antibodies 1E1 comprises the light chain variable region and the human antibody constant region selected from SEQ ID NOs: 6 to 9, and more specifically, the amino acid sequence of SEQ ID NO: 24 VL1A; VL1B consisting of the amino acid sequence of SEQ ID NO: 25; VL1C consisting of the amino acid sequence of SEQ ID NO: 26; VL1D consisting of the amino acid sequence of SEQ ID NO: 27; VL2A consisting of the amino acid sequence of SEQ ID NO: 28; VL2B consisting of the amino acid sequence of SEQ ID NO: 29; VL2C consisting of the amino acid sequence of SEQ ID NO: 30; VL2D consisting of the amino acid sequence of SEQ ID NO: 31.
  • the anti-ICAM4 antibody therapeutic agent 1E1 is cellular internalization. This internalization characteristic is a condition that can be a good candidate for the development of an antibody-drug complex. Previous studies have shown that about 70% of the antigen-antibody complex is internalized into the cell within one hour [Kim Min-young. Development of new therapeutic monoclonal antibodies against human leukemia. Thesis of Master's Degree of Medicine in Chungbuk National University. 2014], which indicates that the previously known anti-ICAM4 antibody therapeutic agent 1E1 has a higher efficacy than the conventional T-DM1 internalization of an about 10% antibody-drug complex identified in the gastric cancer cell line, And is likely to be a therapeutic agent [Wang H, et al. Aberrant intracellular metabolism of T-DM1 confers T-DM1 resistance in human epidermal growth factor receptor 2-positive gastric cancer cells. Cancer Sci. 2017 Apr 7 doi: 10.1111 / cas.13253].
  • an anti-ICAM4 antibody and a saponin or manganese complex were produced and tested in vivo and in vitro, and showed relatively good efficacy with IC 50 of 10 ng / mL and 3.7 ⁇ g / mL, respectively 7 and 8).
  • Sapolin is a ribosome-inactivating protein with high enzymatic activity and is physico-chemically stable and has an advantage of being easy to bind to antibodies at the laboratory level [Polito L, Bortolotti M, Mercatelli D, Battelli MG, B perfumesi A. Saporin- S6: a useful tool in cancer therapy. Toxins (Basel). 2013; 5 (10): 1698-722].
  • Mantansin is a microtubule-forming inhibitor that inhibits cell division and is used for T-DM1 (cassia) targeting HER2-positive metastatic breast cancer that has been approved by the FDA in 2013 (Bender B, Leipold DD, Xu K, Shen BQ, Tibbitts J, Friberg LE.
  • T-DM1 trastuzumab emtansine
  • ADC antibody-drug conjugate
  • the first approach is to take advantage of the ability to recognize sialic acid-dependent antigenic determinants of anti-ICAM4 antibody therapy.
  • Tumor cells are characterized by abnormal energy expenditure due to malnutrition, during which various types of polysaccharides are rapidly lost and sialic acid is used to maintain glycosylation of the cell surface. More sialic acid is present on the tumor cell surface than normal cells.
  • the level of sialylation of tumor cells and normal red blood cell surface ICAM4 antigen was differentiated by treatment with neuraminase to remove sialic acid on the cell surface.
  • Anti-ICAM4 antibody binds to tumor cells and does not bind to normal red blood cells And the concentration of neuraminase (Fig. 9).
  • ICAM4 antigens show extremely limited expression in tissues, but conversely reduce cross-reactivity concerns in extracellular target cells.
  • expression of ICAM4 was detected in normal tissues of developmental stage using human normal organs and 8 weeks old fetal paraffin embedded tissues.
  • fetal tissues expression was observed only in immature erythrocytes differentiating into erythrocytes in hepatocytes with vigorous hematopoiesis, and no expression of ICAM4 was observed in any tissues of mature differentiated normal tissues and three embryonic developmental stages 11).
  • ICAM4 is expressed in most of the erythropoiesis stages of human erythropoiesis, so it can be expressed simultaneously in myeloid immature cells, and many leukemia and myelodysplastic syndrome (MDS) cells can be targeted. This was demonstrated by analyzing clinical samples of actual bone marrow dysplasia syndrome (MDS) patients (FIG. 12). ICAM4 was also expressed in some of the cholangiocarcinoma and liver cancer cell lines (Table 2)
  • the present invention provides antibody therapeutics (including chimeric antibodies, humanized antibodies, bispecific antibodies, and antibody-drug complexes) using anti-ICAM4 monoclonal antibody 1E1.
  • antibody as used in the present invention includes immunoglobulin molecules immunologically reactive with specific antigens, and includes both polyclonal antibodies and monoclonal antibodies.
  • the term also includes forms produced by genetic engineering such as chimeric antibodies (e. G., Humanized murine antibodies) and heterologous binding antibodies (e. G., Bispecific antibodies).
  • chimeric antibody refers to the most preferred antibody derived using recombinant deoxyribonucleic acid techniques, including human (including immunologically " related " species such as chimpanzee) - Contains both human components.
  • the constant region of the chimeric antibody is most preferably consistent with the constant region of the native human antibody;
  • the variable region of the chimeric antibody is most preferably derived from a non-human source and retains the desired antigen specificity for the target antigen (ICAM4).
  • a non-human source can be any vertebrate source that can be used to generate antibodies against the ICAM4 antigen.
  • non-human sources include, but are not limited to, rodents (such as rabbits, rats, mice, etc., such as those disclosed in U.S. Patent No. 4,816,567, incorporated herein by reference) and non-human primates such as Old World monkeys, apes, For example, U.S. Patent Nos. 5,750,105 and 5,756,096, incorporated herein by reference).
  • antibody fragment refers to a specific part of an antibody that is capable of binding to a target antigen, in this case ICAM4 or a specific part thereof.
  • antibody fragments include F (ab ') 2, Fab, Fab' and Fv fragments. These can be produced conventionally using recombinant DNA technology or using conventional methods of degrading antibody proteins with papain or pepsin (CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley and Sons Coligan et al., Eds. (1991-1992)).
  • monoclonal antibody refers to a highly specific antibody directed against a single antigenic site as is known in the art. Typically, unlike polyclonal antibodies that contain different antibodies directed against different epitopes (antigenic determinants), monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays that utilize antigen-antibody binding and also have other advantages that are not contaminated by other immunoglobulins because they are synthesized by hybridoma cultures. Typically, immunoglobulins have a heavy chain and a light chain, and each heavy and light chain comprises a constant region and a variable region (the region also known as a " domain ").
  • variable regions of the light and heavy chains include three variable regions and four "framework regions” called “complementarity determining regions” (hereinafter referred to as "CDRs").
  • CDRs mainly serve to bind to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, CDR3, starting from the N-terminus and sequentially identified by the chain in which the particular CDR is located.
  • variable as used herein is used to refer to the fact that the sequence of a particular region between antibodies is significantly different and is used to indicate the binding specificity of each particular antibody to a particular antigen.
  • the variability of the antibody is centered on the CDR rather than being uniformly distributed throughout the variable domains of the antibody.
  • the heavy and light chains of the monoclonal antibody each have three CDRs, and these regions form an antigen-antibody complex by recognizing ICAM4. These CDRs have a characteristic sequence for each monoclonal antibody and one or both of these six CDRs can interact to allow a single monoclonal antibody to recognize a particular epitope.
  • the anti-ICAM4 antibody of the present invention is an antibody that specifically binds to an antigenic determinant region of ICAM4, and may include variable regions of light and heavy chains.
  • the heavy chain variable region of the antibody comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, or at least 80% homology with this sequence in the CDR region, preferably 90 Or more, and most preferably 95% or more; And / or wherein the light chain variable region comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 6 to 9, or at least 80%, preferably 90% or more, And most preferably 95% or more.
  • Antibodies specific for ICAM4 can be generated by a fusion method well known in the art (Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519).
  • an antibody of the invention can be produced by immunizing an animal with ICAM4 as an immunogen, fusing splenocytes of an immunized animal with a myeloma cell to produce a hybridoma, and producing a monoclonal antibody that selectively recognizes ICAM4 Selecting the hybridomas, culturing the selected hybridomas, and separating the antibodies from the hybridomas.
  • the hybridoma cell line can be cultured in vitro or in vivo to separate the antibody.
  • the monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma may be purified to a high purity (for example, 95% or more) according to a method well known in the technical field of the present invention . Such a purification technique can be separated from a culture medium or a plurality of solutions by using a purification method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like. As an example of the production of a monoclonal antibody using a hybridoma, a hybridoma is inserted into the abdominal cavity of a mouse, and the hybridoma is separated and purified. The separation and purification of the monoclonal antibody can be carried out using affinity chromatography such as ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or protein A column.
  • affinity chromatography such as
  • the present invention also provides a diagnostic composition for a disease or an ICAM4 mediated disease associated with the expression or degree of expression of ICAM4 comprising said anti-ICAM4 antibody.
  • the present invention also relates to a method of detecting said disease marker by contacting said anti-ICAM4 antibody with a biological sample to detect antigen-antibody complex formation. That is, the ICAM4 protein can be detected by reacting an improved monoclonal antibody of the present invention with a biological sample and detecting antigen-antibody complex formation.
  • biological sample refers to a tissue, cell, whole blood, serum, plasma, tissue autopsy sample (brain, skin, lymph node, spinal cord, etc.), urine, cell culture supernatant, ruptured eukaryotic cell, But is not limited thereto. These biological samples may be reacted with the antibodies of the invention in a manipulated or untreated state to confirm the presence of the ICAM4 protein.
  • antigen-antibody complex refers to a combination of ICAM4 protein antigen in the sample and the anti-ICAM4 antibody according to the present invention recognizing the same.
  • the formation of such an antigen-antibody complex may be performed by a colormetric method, An arbitrary one selected from the group consisting of an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment, and a scintillation counting method. . ≪ / RTI > However, various applications and applications are possible without being limited thereto.
  • markers can be used to detect an antigen-antibody complex.
  • Specific examples include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, and radioactive isotopes.
  • the antigen-antibody complexes can be detected using enzyme immunoassay (ELISA).
  • Enzyme immunoassay includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes the capture antibody in a complex of an antibody recognizing the antigen attached to a solid support , A direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support, and the other antibody recognizing the antigen in the complex of the antibody and the antigen attached to the solid support. And indirect sandwich ELISA using labeled secondary antibodies recognizing the antibody.
  • the monoclonal antibody can have a detection label, and when it does not have a detection label, it can capture another monoclonal antibody and can be identified by treating another antibody having the detection label.
  • the present invention also provides a composition for the identification of myeloid-derived suppressor cells (MDSC) comprising an anti-ICAM4 antibody.
  • MDSC myeloid-derived suppressor cells
  • the above-mentioned MDSC discrimination can use the disease diagnosis method or the disease marker detection method as discussed above.
  • the present invention also provides diagnostic compositions or diagnostic kits comprising said anti-ICAM4 antibody (comprising a chimeric antibody, a humanized antibody, a bispecific antibody and an antibody-drug complex for a monoclonal antibody of an anti-ICAM4 antibody).
  • the anti-ICAM4 antibody used in the diagnostic composition of the present invention can also use fragments of the anti-ICAM4 antibody so long as the antibody can selectively recognize ICAM4. Such antibody fragments may include F (ab ') 2, Fab, Fab', Fv fragments, and the like.
  • the diagnostic kit may include monoclonal antibodies or fragments thereof that selectively recognize ICAM4 and tools / reagents used in immunological assays.
  • Tools / reagents used in immunological assays include suitable carriers, labeling agents capable of producing detectable signals, solubilizers, detergents, and the like.
  • the labeling substance when it is an enzyme, it may include a substrate capable of measuring the enzyme activity and a reaction terminator.
  • Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.
  • the assay system for use in the detection methods and diagnostic kits of the present invention may be applied to a variety of assay systems including, but not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips and split- through devices and the like.
  • the present invention provides a composition for treating a disease or ICAM4 mediated disease associated with the expression or expression level of ICAM4, comprising the anti-ICAM4 antibody, its chimeric antibody, humanized antibody, bispecific antibody or antibody-drug complex do.
  • the disease is preferably cancer.
  • the treatable cancer of the present invention may include, without limitation, cancer that can be selectively killed using the antibody therapeutic agent of the present invention, such as skin, digestive, urinary, reproductive, respiratory, circulatory, brain or nervous system cancer Cancer, cancer of the ovary, cancer of the rectum, cancer of the stomach, cancer of the anus, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, liver cancer, liver cancer, Cancer of the uterus, cancer of the cervix, vaginal cancer, mucin cancer, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, Lymphoma, lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, Waterbody can be Zen.
  • the antibody therapeutic agent of the present invention such as skin, digestive, urinar
  • the therapeutic composition may comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound.
  • the composition of the present invention may be administered in single or multiple doses in a pharmaceutically effective amount.
  • the therapeutic composition may be administered in the form of a solution, a powder, an aerosol, a capsule, an intravaginal tablet or a capsule or a suppository.
  • Routes of administration include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, endothelial, oral, topical, intranasal, intrarectal, and the like.
  • the peptide upon oral administration, the peptide will be digested and the oral composition should be formulated to coat the active agent or protect it from degradation from above.
  • the pharmaceutical composition may also be administered by any device capable of transferring the active agent to the target cell.
  • the anti-ICAM4 antibody When the composition is formulated, the anti-ICAM4 antibody is considered to retain the desired biological activity at a given point in time. If the desired biological activity at that time is within about 30%, and preferably within about 20%, of the desired biological activity occurring within that time, the pharmaceutical composition may be used as determined in a suitable assay for the desired biological activity . Assays for measuring the desired biological activity of an anti-ICAM4 monoclonal antibody can be performed as described in the Examples herein. Schultze et al. (1998) Proc. Natl. Acad. ScL USA 92: 8200-8204; Denton et al. (1998) Pediatr. Transplant. 2: 6-15; Evans et al.
  • the anti-ICAM4 antibodies of the present invention can be formulated into liquid pharmaceutical formulations.
  • Anti-ICAM4 antibodies can be prepared using any known method and include the methods disclosed herein.
  • the anti-ICAM4 antibody was recombinantly produced in the hybridoma cell line KCLRF-BP-00303.
  • Liquid pharmaceutical formulations may comprise a therapeutically effective amount of the monoclonal antibody. The amount of these antibodies present in the formulation takes into account the route of administration and the desired dosage volume.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount &quot is meant an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prophylaxis, and the effective dosage level will vary depending on factors such as the severity of the disease.
  • the composition of the present invention can be used in combination with the composition of the present invention, including drugs used in combination with the composition of the present invention, and other medical fields including, for example, body weight, health, sex, sensitivity of the patient to the drug, May be determined according to well known factors.
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
  • Suitable buffers include, but are not limited to, conventional acids and salts thereof, wherein the counterion may be, for example, sodium, potassium, ammonium, calcium, or magnesium.
  • conventional acids and salts thereof that can be used as buffers in pharmaceutical liquid formulations include succinic or succinic acid salts, citric acid or citric acid salts, acetic acid or acetic acid salts, tartaric acid or tartaric acid salts, phosphoric acid or phosphate salts, gluconic acid or gluconic acid salts , Glutamic acid or glutamate, aspartic acid or aspartate, maleic acid or maleate, and malic or malate buffers.
  • the buffer concentration in the formulation can be from about 1 mM to about 50 mM and is about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 45 mM, 50 mM, or about 1 mM to about 50 mM.
  • the buffer concentration in the formulation is about 5 mM to about 15 mM, and about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, mM, 15 mM, or from about 5 mM to about 15 mM.
  • a liquid pharmaceutical formulation comprising an anti-ICAM4 antibody and a buffer may additionally comprise an amount of isotonic agent sufficient to impart proximity to the formulation.
  • proximal isotonicity is meant that the aqueous formulation is about 240 mmol / kg to about 360 mmol / kg, preferably about 240 to about 340 mmol / kg, more preferably about 250 to about 330 mmol / Quot; means having an osmolarity of about 260 to about 320 mmol / kg, even more preferably about 270 to about 310 mmol / kg.
  • Methods for determining the isotonicity of a solution are known to those skilled in the art. See, for example, Setnikar et al. (1959) J. Am. Pharm. Assoc. 48: 628.
  • the isotonic agent may be any reagent capable of controlling the osmotic pressure of the liquid pharmaceutical formulation of the present invention at levels equivalent to body fluids. It is preferred to use physiologically acceptable isotonic agents.
  • a liquid pharmaceutical formulation comprising a therapeutically effective amount of an anti-ICAM4 antibody and a buffer can comprise an ingredient that can be used to provide isotonicity, for example, sodium chloride; Amino acids such as alanine, valine, and glycine; Sugars and sugar alcohols (polyols) including but not limited to glucose, dextrose, fructose, sucrose, maltose, mannitol, trehalose, glycerol, sorbitol, and xylitol; Acetic acid, other organic acids or salts thereof, and relatively small amounts of citric acid salts or phosphates.
  • additional agents suitable for providing the rigidity of liquid formulations are known in the art.
  • the liquid pharmaceutical formulation comprising the anti-ICAM4 antibody and the buffer may further comprise sodium chloride as an isotonic agent.
  • the sodium chloride concentration in the formulation will depend on the contribution of the other ingredients to the rigidity.
  • the concentration of sodium chloride may range from about 50 niM to about 300 mM, from about 50 mM to about 250 mM, from about 50 mM to about 200 mM, from about 50 mM to about 175 mM, from about 50 mM to about 150 mM, from about 100 mM to about 200 mM, from about 100 mM to about 150 mM, from about 125 mM to about 175 mM, from about 125 mM to about 175 mM, from about 75 mM to about 175 mM, from about 75 mM to about 150 mM, from about 100 mM to about 175 mM, About 150 mM, about 130 mM to about 170 m
  • the liquid pharmaceutical formulation comprises a therapeutically effective amount of an anti-ICAM4 antibody, a buffer, and additionally a surfactant.
  • the liquid pharmaceutical formulation comprises an anti-ICAM4 antibody, a buffer, an isotonic agent, and may further comprise a surfactant.
  • Typical surfactants used are nonionic surfactants, such as polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); Polyoxypropylene-polyoxyethylene esters such as Pluronic F68; Polyoxyethylene alcohols such as Brij 35; Simethicone; Polyethylene glycol such as PEG 400; Lysophosphatidylcholine; And polyoxyethylene-p-t-octylphenol, such as Triton X-100.
  • the stabilization of traditional medicaments by surfactants or emulsifiers is described, for example, by Levine et al. (1991) J Parenteral Sci. Technol. 45 (3): 160-165, which is incorporated herein by reference.
  • the antibody may be combined with toxic substances to form a fusion protein in order to enhance the therapeutic effect.
  • the toxin protein may be ricin, saporin, gelonin, momordin, diphtheria toxin, or pseudomonas toxin, but the type of the toxin is not particularly limited.
  • the present invention provides a method for treating a disease or an ICAM4-mediated disease associated with the expression or degree of expression of ICAM4 comprising the step of administering the antibody therapeutic agent to a subject.
  • animal refers to a mammal such as a human, horse, dog, cat, pig, goat, rabbit, hamster, monkey, guinea pigs, rat, mouse, lizard, And any animal (e. G., Human), including birds.
  • treatment refers to the administration of a composition comprising an anti-ICAM4 monoclonal antibody (including its modified antibody) according to the present invention and a pharmaceutically acceptable carrier, Or any change that beneficially modifies it.
  • the anti-ICAM4 monoclonal antibody (including its modified antibodies) of the present invention can be used with other antibodies, biologically active agents or substances for various purposes.
  • the anti-ICAM4 monoclonal antibody of the present invention can be used in combination with a known anti-cancer drug
  • DNA encoding the variable region of the anti-ICAM4 mouse antibody 1E1 was inserted into pOptiVEC, pcDNA3.3 vector (DiNonA, Korea) which was designed to express the human antibody constant region together, and the produced recombinant vector was transformed with dihydrofolate reductase (Invitrogen, USA) reagent to CHO DG44 (Invitrogen, # A1100001), a negative control (DHFR) negative cell line. (Sigma, USA) and G418 (Invitrogen, USA) in order to select the transfected cells, and subcultured in ⁇ MEM medium containing no deoxyribonucleosides (Welgene, Korea) (LONZA, Switzerland). MTX and G418 concentrations were gradually increased and the cells were cultured in a 96-well plate (SPL, Korea) with a limiting dilution to dilute the cell culture to a level of less than 1 cell per well Respectively.
  • SPL, Korea 96-
  • anti-ICAM4 mouse antibody 1E1 was activated with N-succinimidyl-3- (2-pyridyldithio) -propionate (SPDP) and bound to thiolated saponin at a molecular weight of 1: 5.
  • SPDP N-succinimidyl-3- (2-pyridyldithio) -propionate
  • N-ethylmaleimide was added to block unbound mercapto groups (SH-), and unconjugated saponins not bound were removed by protein G affinity chromatography.
  • the conjugated antibody-drug complex was eluted with ImmunoPure Elution Buffer (Pierce Biotechnology, USA) and dialyzed against PBS. The possibility of contamination by free saponins was confirmed by SDS-PAGE.
  • the cells were treated with 5 ⁇ 10 4 K562 cells per well in a 96-well plate, and antibody-drug complexes were treated three times per concentration.
  • the cells were cultured at 37 ° C for 72 hours.
  • Ey-cytox (DOGEN, Korea) was used to measure cell viability using tetrazolium salt.
  • Well plates (SPL, Korea) were plated at a dose of 1 ⁇ 10 5 cells per well of the leukemic cell line K562 and the antibody-drug complexes were treated at different concentrations. The cells were cultured for 2 hours. Cell viability was determined by flow cytometry after propidium iodide (PI) staining.
  • PI propidium iodide
  • HEL an acute leukemia cell line
  • SCID mouse Central laboratory animal, Korea
  • HEL cells prepared at 1 ⁇ 10 7 in 100 ⁇ L of RPMI medium containing no serum were subcutaneously inoculated into the right side of the mouse.
  • the efficacy of the antibody-drug complex was tested when the size of the transplanted tumor reached 100 mm 3 .
  • the antibody-drug conjugate was administered to the tail vein four times at a dose of 2.5 mg / kg every 3 days.
  • the length and width of the tumor were measured with caliper once every 3 days. Gutman et al. The volume was calculated.
  • the bispecific antibody was prepared in the form of scFv-Fc.
  • the variable region of the anti-ICAM4 antibody and the variable region DNA of the antibody against the tumor-related target were prepared in the form of scFv by overlap PCR.
  • the linker sequence used to link the variable regions of the heavy chain and the light chain was GGSSRSSSGGGGSGGGG- (SEQ ID NO: 10), and PCR was performed 30 times at 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 45 seconds .
  • the antibody variable region scFv was inserted into a pOptiVEC expression vector (DiNonA, Korea) designed to express a constant region of a human antibody, and the scFv-Fc-type bispecific antibody was expressed by transfection into host cell HEK-293T.
  • the paraffin embedded tissues used in the test were obtained from Chungbuk National University Hospital. The paraffin was removed by paraffin treatment using xylene and ethanol at room temperature, and the antigen was regenerated with microwave of 10 mM citrate buffer (pH 6.0) for 15 minutes. The primary antibody was used at a concentration of 10 ⁇ g / mL (200 ⁇ L) and color developed using the ChemMate Detection kit (Dako, Denmark).
  • POptiVEC and pcDNA3.3 were used as expression vectors to construct a cell line stably producing anti-ICAM4 chimeric antibody 1E1, and a CHO DG44 cell line was used as a host cell (Fig. 1A).
  • a CHO DG44 cell line was used as a host cell (Fig. 1A).
  • the cell line was stably cultured in a PowerCHO2 culture medium supplemented with 1000 nM MTX and 200 ⁇ g / mL G418, and then cultured in a cell line producing chimeric antibody 1E1 Final product concentration 7.5 mg / L).
  • a series of production cell lines production schemes are illustrated in Figure 1B.
  • Anti-ICAM4 Chimeric Antibody To identify the antigenic region of chimeric antibody 1E1 produced from the cell line for production of the anti-ICAM4 chimeric antibody 1E1, the antigen of the target cell was saturated with the original mouse antibody 1E1 and then produced from the cell line for producing anti-ICAM4 chimeric antibody 1E1 As a result of confirming the reactivity of the chimeric antibody 1E1, it was confirmed that the same antigen recognition site was recognized (Fig. 2A).
  • flow cytometry analysis was performed on ICAM4 overexpressed and unexpressed cell lines. The reactivity of ICAM4 overexpressed cell line gradually increased according to the concentration gradient of the chimeric 1E1 antibody treatment .
  • the chimeric 1E1 antibody treatment had an antigen saturation concentration of 5 ⁇ g / mL, similar to the original mouse antibody 1E1.
  • Humanized antibodies were prepared and screened to reduce the immunogenicity of anti-ICAM4 chimeric antibodies. 13 heavy chain variable region to the in silico from BIOTEM captured French dog, a light chain variable region versions a total of eight to obtain a humanized antibody wherein -ICAM4 sequence of the 104 combination (Table 1).
  • anti-mouse antibody 1E1 -ICAM4 four tarpaulins composite (m 1E1-SAP) are connected to each paste given a mercapto group (SH-) as SDPD and 2-Iminothiolane for tarpaulins Mouse 1E1 antibody and use the antibody and the drug via a disulfide bond (Fig. 6a), anti-ICAM4 chimeric antibody 1E1 manganese complex ( chi 1E1-DM1) was conjugated to chimeric antibody 1E1 using succinimidyl-4- [N-maleimidomethyl] cyclohexane- 3 to 4 of which are connected to each other and are manufactured by Creative Biolabs, USA (Fig. 6B).
  • the neuraminase is treated with the tumor cell line and the erythrocyte at different concentrations so that the anti-ICAM4 antibody treatment agent reacts with cancer cells but does not react with red blood cells (Fig. 9A).
  • Fig. 9A To measure the difference in sialic acidity between tumor cell lines and erythrocyte surface ICAM4 antigen, 1000 units of neuraminase were treated with anti-ICAM4 antibody, and the reactivity of anti-ICAM4 antibody treatment was almost constant in the tumor cell line , But the reactivity in red blood cells disappeared (FIG. 9B).
  • the double antibody was developed by ligating the variable region heavy chain targeting CEACAM6 to the CH3 domain end of the whole IgG1 antibody Fc portion targeting ICAM-4 and the single chain fragment variable (scFV) portion linking the light chain to the specific sequence.
  • the heavy and light chain combinations that target ICAM-4 are the heavy chain 1A-light chain 1A (w1s2H1L3), heavy chain 1A-light chain 2A (w1s2H1L4), heavy chain 1B-light chain 1A (w1s2H2L3) and heavy chain 1B-light chain 2A (w1s2H2L4) were used.
  • the heavy chain targeting the CEAMCAM 6 used for the scFV below and the E3 sequence for the light chain portion were used.
  • the sequence of the antibody targeting ICAM-4 was ligated to the CH3 domain end of the whole IgG1 antibody Fc portion targeting CEACAM6.
  • the combination used was a combination of the E3 sequence targeting CEACAM6 and the heavy chain 1A-light chain 1A (w2s1H1L3) and heavy chain 1B-light chain 2A (w2s1H2L4) sequences targeting ICAM-4.
  • a total of 6 double antibodies were produced using Expi CHO cells, and antigen binding was confirmed using purified antibodies (FIG. 10A). This double antibody was superior to monoclonal antibody in the antibody - dependent cytotoxicity assay.
  • the w1s2H1L3 and w1s2H2L3 antibodies combined with the light chain 1A sequence of an antibody targeting ICAM-4 showed the best effect ( Figures 10b and 10c)
  • the cross-reactivity of anti-ICAM4 antibody treatment showed strong positive for erythrocytes.
  • the cross-reactivity of all tissues examined including paraffin-embedded fetal tissue (8 weeks old) and adult liver, kidney and uterus excluding red blood cells There was no reactivity (Fig. 11).
  • no cross reactivity was observed in normal human tonsil tissues and normal organs in the frozen section.
  • chimeric antibody 1E1 was found to express 20-30% in some liver cancer cells and bile duct cancer cells (Table 2) In blood cells, all cells except red blood cells were found to have no reactivity.
  • ICAM4 is not expressed in all cells except red blood cells in healthy peripheral blood cells, but as a result of screening of various clinical samples, ICAM4 is expressed in some abnormal abnormality in myeloblast of actual bone marrow dysplasia syndrome (MDS) (Fig. 12).
  • MDS bone marrow dysplasia syndrome
  • the terminal cancer patients blood 1 mL in RBC lysis buffer into a 9mL by 10 min at 36 °C after removing the red blood cells, 1 ⁇ 10 6 cells, dog CD45-APC (BD, USA) , 1E1- FITC (DiNonA, Korea) was reacted at 4 DEG C for 30 minutes. The cells were then washed with cold PBS and fixed with 1% paraformaldehyde followed by analysis using a fluorescence activated flow cytometer FACS Calibur (BD, USA).
  • PBMC Peripheral blood mononuclear cells
  • the surface markers of the most commonly used bone marrow-derived inhibitory cells are CD33 + CD11b + HLADR-, with 82.5% of CD33 + CD11b + being the surface markers of bone marrow-derived inhibitory cells in the blood of solid cancer patients.
  • HLA DR- was 70%, indicating that bone marrow-derived inhibitory cells were present in the blood of patients with solid tumors, of which ICAM4 antigen recognized by 1E1 was 37.85%. Therefore, it was confirmed that ICAM4 can be used as a surface marker of bone marrow-derived inhibitory cells as anti-ICAM4 monoclonal antibody 1E1.
  • the anti-ICAM4 antibody of the present invention and the composition for diagnosing or treating the disease expressing ICAM4 using the same have excellent antigen affinity as compared with the conventional antibody and can be applied variously according to the related disease as an improved form, Can be usefully used for diagnosis or treatment.

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Abstract

The present invention relates to an improved anti-ICAM4 antibody and a use thereof as a diagnostic and therapeutic agent for ICAM4 expression cell-associated disease and, more particularly, to a chimeric antibody of an anti-ICMA4 mouse monoclonal antibody 1E1, a cell strain producing the same, a humanized antibody, and an anti-ICMA4 antibody-drug complex. The anti-ICAM4 chimeric antibody according to the present invention recognizes the same epitope as that of IE1 and exhibits similar affinity for the antigen. The antibody-drug complex using the antibody was found to exhibit efficacy higher than preexisting antibody-drug complexes. Hence, the improved antibodies of the present invention can be useful for the diagnosis and treatment of ICAM4-associated diseases.

Description

항-ICAM4 항체 및 ICAM4 발현 세포와 관련된 질병의 진단 및 치료용 조성물 Composition for diagnosis and treatment of diseases associated with anti-ICAM4 antibody and ICAM4 expressing cells
본 발명은 개량된 항-ICAM4 항체 및 이의 ICAM4 발현 세포와 관련된 질병의 진단 및 치료제로서의 용도에 관한 것으로, 보다 구체적으로 항-ICMA4 단일클론항체 1E1의 키메릭 항체 및 이를 생산하는 세포주, 인간화항체 및 항-ICMA4 항체-약물 복합체 등에 관한 것이다. The present invention relates to an improved anti-ICAM4 antibody and its use as a diagnostic and therapeutic agent for diseases related to ICAM4 expressing cells, and more particularly to a chimeric antibody of anti-ICMA4 monoclonal antibody 1E1 and a cell line, a humanized antibody, Anti-ICMA4 antibody-drug complex, and the like.
암에 대한 항체 기반 치료는 과거 20년 전부터 현재까지 혈액암 및 고형암 환자를 치료하는 가장 성공적이고 중요한 방법 중 하나이다[Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012;12(4):278-87]. 항체 치료제의 성공적인 개발을 위해서 전임상 단계에서 항체의 물리ㆍ화학적 특성 확인, 정상조직과 악성조직 패널을 이용한 항원 발현 분석, 항체의 신호경로 및 면역효과기 기능에 관한 연구, 항체 키메라화 및 인간화 그리고 in vivo에서 항체 단독 또는 약물 결합체의 치료활성 관찰 등에 대한 과정이 수반되어야 한다. 현재까지 다양한 혈액암 및 고형암의 치료를 위해 12개의 항체치료제가 미국 식품의약국(FDA)으로부터 승인받았으며, 현재 다수의 추가 항체 치료제가 임상 시험 중에 있다.Antibody-based therapy for cancer has been one of the most successful and important ways to treat patients with blood and solid tumors from the past 20 years to the present [Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012; 12 (4): 278-87]. For the successful development of antibody therapeutics, identification of the physicochemical characteristics of antibodies at preclinical stage, analysis of antigen expression using normal tissue and malignant tissue panels, studies on the signaling pathway and immune effector functions of antibodies, antibody chimerization and humanization, and in vivo And the observation of the therapeutic activity of the antibody alone or the drug conjugate. To date, 12 antibody treatments have been approved by the US Food and Drug Administration (FDA) for the treatment of a variety of hematologic cancers and solid tumors, and a number of additional antibody therapies are currently in clinical trials.
인간화항체란 인간이 아닌 종의 항체 서열을 인간에게서 자연적으로 생산된 항체와 유사성을 높이기 위해 변형시킨 항체를 의미한다(Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature. 1988;332(6162):323-7; 및 Queen C, et al. A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci U S A. 1989;86(24):10029-33). 키메릭 항체도 인간 항체와 유사성을 높이기 위해 마우스 항체의 불변부위 서열을 인간항체 불변부위 서열로 대체한다는 점에서 인간화항체와 유사하지만 가변부위의 상보성결정부위(CDR)을 제외한 서열들이 여전히 마우스 서열이라는 점에서 인간화화항체와 구별된다(Brekke OH, Sandlie I. Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat Rev Drug Discov. 2003;2(1):52-62). 인간화항체를 제작하는 방법으로는 CDR 이식 방법과 다양한 디스플레이를 이용한 방법이 주로 이용된다. 먼저 CDR 이식이란 마우스 단클론 항체의 가변부위 서열을 암호화 하는 DNA를 분리하고 유전자 클로닝을 통하여 CDR 서열을 확보한 다음 in silico 상으로 적합한 인간항체 서열 안으로 CDR 서열을 이식하여 만드는 방법이다(Hou S, et al. Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modelling. J Biochem. 2008;144(1):115-20; 및 Kashmiri SV, De Pascalis R, Gonzales NR, Schlom J. SDR grafting--a new approach to antibody humanization. Methods. 2005;36(1):25-34). 고속으로 적합한 유전자를 선별할 수 있는 디스플레이를 이용한 항체 서열 인간화 방법은 마우스 또는 인간이 아닌 다른 포유동물을 사용하지 않고 인간 치료용 항체를 만들기 위해 고안된 방법으로 파지 디스플레이법이 가장 많이 이용되고 있다. 류마티스 관절염 치료제 휴미라　가 이 기법을 이용하여 최초로 승인된 항체이며, 현재 시장 점유율이 가장 높은 항체 의약품이다.Humanized antibody refers to an antibody in which the antibody sequence of a non-human species is modified to increase similarity with an antibody naturally produced in humans (Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. 1988; 332 (6162): 323-7; and Queen C, et al., A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci US A. 1989; 86 (24): 10029-33). The chimeric antibody is similar to the humanized antibody in that the constant region sequence of the mouse antibody is replaced with the human antibody constant region sequence in order to enhance similarity with the human antibody, but the sequences other than the complementarity determining region (CDR) (Brekke OH, Sandlie I. Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat Rev Drug Discov. 2003; 2 (1): 52-62). Methods for producing humanized antibodies include CDR transplantation methods and methods using various displays. First, CDR grafting is a method of isolating DNA encoding a variable region sequence of a mouse monoclonal antibody, securing the CDR sequence through gene cloning, and then grafting the CDR sequence into a suitable human antibody sequence in an in silico phase (Hou S, et (1): 115-20; and Kashmiri SV, De Pascalis R, Gonzales NR, Schlom, J. Biochem. J. SDR grafting - a new approach to antibody humanization. Methods. 2005; 36 (1): 25-34). Antibody Sequence Using Display for Selecting Suitable Gene at High Speed The phage display method is most widely used as a method designed to make antibodies for human therapy without using a mouse or other non-human mammal. HUMIRA, the treatment for rheumatoid arthritis, is the first antibody approved using this technique and is the highest antibody drug currently marketed.
항체-약물 복합체(ADC)는 암의 표적 치료에서 가장 진보 된 접근법 중 하나이다[15]. 항체-약물 복합체는 항체의 표적 특이적인 반응을 이용해 종양 조직에 직접적으로 약물을 전달 할 수 있다(Sochaj AM, widerska KW, Otlewski J. Current methods for the synthesis of homogeneous antibody-drug conjugates. Biotechnol Adv. 2015;33(6 Pt 1):775-84; 및 Teicher BA, Doroshow JH. The promise of antibody-drug conjugates. N Engl J Med. 2012;367(19):1847-8). 항체가 표적 항원에 결합하면 항체-약물 복합체는 세포 내부로 내재화(internalization)되면서 약물이 방출되고 결국 세포 사멸이 일어난다[Kovtun YV, Goldmacher VS. Cell killing by antibody-drug conjugates. Cancer Lett. 2007;255(2):232-40]. 현재까지 브렌툭시맙-베도틴과 트라스투주맙-엠탄신 항체-약물 복합체가 미국식품의약국(FDA) 승인을 받았고, 지난 10년간 항체-약물 복합체를 개선하기 위한 많은 노력이 있었으며 최근 60가지가 넘는 새로운 항체-약물 복합체가 임상 시험 중에 있다.Antibody-drug complexes (ADC) are one of the most advanced approaches to targeted treatment of cancer [15]. Antibody-drug complexes can deliver drugs directly to tumor tissue using a target specific reaction of the antibody (Sochaj AM, Widerska KW, Otlewski J. Current methods for synthesis of homogeneous antibody-drug conjugates. Biotechnol. ; 33 (6 Pt 1): 775-84; and Teicher BA, Doroshow JH. The promise of antibody-drug conjugates. N Engl J Med. 2012; 367 (19): 1847-8). When the antibody binds to the target antigen, the antibody-drug complex is internalized into the cell and the drug is released, resulting in apoptosis (Kovtun YV, Goldmacher VS. Cell killing by antibody-drug conjugates. Cancer Lett. 2007; 255 (2): 232-40). To date, brenituximab-betotin and trastuzumab-emtansin antibody-drug complex have been approved by the US Food and Drug Administration (FDA) and over the last decade there has been much effort to improve the antibody-drug complex, There are over twenty new antibody-drug complexes in clinical trials.
본 발명자들은 이전 연구를 통해 인간 적혈구 표면항원 ICAM4에 대한 마우스 단클론 항체 1E1을 확보하였다(김민영. 적백혈병에 대한 새로운 치료용 단클론 항체의 개발. 충북대학교 의학석사학위논문. 2014). ICAM4 (intercellular adhesion molecule 4)는 인간 적혈구 막에 발현되어 있는 시알산(sailic acid) 잔기가 붙은 당단백질로, 적혈구 외에 적혈구 및 일부 T 세포와 B 세포와 같은 다른 혈액세포의 전구체에서 발현된다. ICAM4에 대한 기능은 잘 알려져 있지 않지만, 인간 적혈구의 핵이 제거되는 성숙단계에서 인접한 적혈구의 VLA-4와 대식세포의 αv 인테그린과 결합하여 적혈구를 안정화시키는 역할을 하는 것으로 알려져 있다. 그 밖에 겸형 적혈구증 환자들에서의 ICAM4 항원의 인산화 차이나 ICAM4에 결합하는 다양한 인테그린 간의 상호작용에 관한 연구가 진행되고 있으나, 항체치료제의 표적물질로서의 연구는 알려진바 없다.The present inventors obtained a mouse monoclonal antibody 1E1 against human erythrocyte surface antigen ICAM4 through previous studies (Kim, Min-Young, Development of a new therapeutic monoclonal antibody against leukemia. ICAM4 (intercellular adhesion molecule 4) is a glycoprotein with sialic acid residues expressed on the human erythrocyte membrane. It is expressed in erythrocytes and other precursors of other blood cells such as T cells and B cells. The function of ICAM4 is not well known, but it is known to play a role in stabilizing red blood cells by binding to VLA-4 of the adjacent red blood cells and αv integrin of macrophages at the maturation stage where the nucleus of human red blood cells is removed. Studies on the interaction between ICAM4 antigen phosphorylation and various integrins binding to ICAM4 have been carried out in patients with sickle cell disease. However, there is no known target substance for antibody treatment.
이에 본 발명자들은 ICAM4에 대한 재조합 항체치료제를 개발하고자 하였고, 이를 위해서 항-ICAM4 마우스 단일클론항체 1E1을 기본으로 하는 키메릭 항체, 인간화항체, 항체-약물 복합체 및 이중특이성 항체 등 다양한 형태의 항-ICAM4 항체치료제를 개발하고, 특히 특정 질환에 대하여 진단 또는 치료 효과를 가지는 것을 발견하여 본 발명을 완성하였다.Accordingly, the present inventors developed a therapeutic agent for recombinant antibodies against ICAM4. For this purpose, various types of anti-ICAM4 monoclonal antibody 1E1 based chimeric antibody, humanized antibody, antibody-drug complex and bispecific antibody, The present inventors have developed a therapeutic agent for an ICAM4 antibody, and have discovered that they have a diagnostic or therapeutic effect on a specific disease, thus completing the present invention.
본 발명의 목적은 항-ICAM4 단일클론 항체를 이용한 키메릭 항체, 인간화항체, 이중특이성 항체 및 항체-약물 복합체를 제공하는 것이다.It is an object of the present invention to provide a chimeric antibody, a humanized antibody, a bispecific antibody and an antibody-drug complex using an anti-ICAM4 monoclonal antibody.
본 발명의 다른 목적은 상기 항체를 이용하여 ICAM4와 관련된 질병의 진단을 위한 조성물, 키트, 진단을 위한 정보 제공방법을 제공하는 것이다.It is another object of the present invention to provide a composition, a kit, and a method for providing information for diagnosis for diagnosis of diseases related to ICAM4 using the antibody.
본 발명의 또다른 목적은 상기 항체를 이용하여 ICAM4와 관련된 질병의 치료용 조성물 및 치료방법을 제공하는 것이다.It is another object of the present invention to provide a composition and a therapeutic method for the treatment of diseases related to ICAM4 using the antibody.
상기 목적을 달성하기 위하여, 본 발명은 항-ICAM4 단일클론 항체를 이용한 키메릭 항체, 인간화항체, 이중특이성 항체 및 항체-약물 복합체를 제공한다.In order to achieve the above object, the present invention provides a chimeric antibody, a humanized antibody, a bispecific antibody and an antibody-drug complex using an anti-ICAM4 monoclonal antibody.
본 발명자들은 이전 연구를 통해 인간 적혈구 표면 항원 ICAM4에 대한 마우스 단일클론 항체 1E1을 확보하였으며, 이에 대해 대한민국 등록특허 제10-1627020호로 특허등록을 받았다. 상기 등록특허는 그 전체가 본 명세서에 참조로 삽입된다. The present inventors secured a mouse monoclonal antibody 1E1 against human erythrocyte surface antigen ICAM4 through previous studies, and obtained a patent as Korean Patent No. 10-1627020. The entirety of which is incorporated herein by reference.
본 발명의 항 ICAM4 항체는 상기 항 ICAM4 단일클론항체 1E1에 대한 키메릭 항체, 인간화 항체, 이중특이성 항체 또는 항체-약물 전달체 중 하나로서, 적용되는 형태에 따라 항체의 형태가 달라질 수 있으나, 공통적인 서열을 함께 포함한다. The anti-ICAM4 antibody of the present invention is one of a chimeric antibody, a humanized antibody, a bispecific antibody or an antibody-drug delivery vehicle for the anti-ICAM4 monoclonal antibody 1E1. The form of the antibody may vary depending on the application form, Sequence.
즉, 본 발명의 상기 항 ICAM4 항체는, 서열번호 1 내지 서열번호 5의 아미노산 서열 중에 선택된 서열을 포함하는 상보성 결정 영역(CDR)의 중쇄 영역; 및 서열번호 6 내지 서열번호 9의 아미노산 서열 중 선택된 서열을 포함하는 상보성 결정 영역(CDR)의 경쇄 영역을 포함하는 것을 특징으로 한다. 보다 구체적으로 상기 CDR 영역은, 서열번호 1의 아미노산 서열로 이루어진 CDR H1; 서열번호 2 내지 서열번호 4의 아미노산 서열 중 선택된 하나의 서열로 이루어진 CDR H2; 및 서열번호 5의 아미노산 서열로 이루어진 CDR H3를 포함하는 CDR 중쇄 영역과, 서열번호 6 또는 서열번호 7의 아미노산 서열로 이루어진 CDR L1; 서열번호 8의 아미노산 서열로 이루어진 CDR L2; 및 서열번호 9의 아미노산 서열로 이루어진 CDR L3를 포함하는 CDR 경쇄 영역을 포함하는 것이다. That is, the anti-ICAM4 antibody of the present invention comprises a heavy chain region of a complementarity determining region (CDR) comprising a sequence selected from the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 5; And a light chain region of a complementary crystal region (CDR) comprising a selected sequence of the amino acid sequence of SEQ ID NO: 6 to SEQ ID NO: 9. More specifically, the CDR region comprises CDR H1 consisting of the amino acid sequence of SEQ ID NO: 1; A CDR H2 consisting of a sequence selected from the amino acid sequences of SEQ ID NOS: 2 to 4; And a CDR heavy chain region comprising CDR H3 consisting of the amino acid sequence of SEQ ID NO: 5 and a CDR L1 consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7; A CDR L2 consisting of the amino acid sequence of SEQ ID NO: 8; And a CDR light chain region comprising CDR L3 consisting of the amino acid sequence of SEQ ID NO: 9.
일 양태로서, 본 발명은 상기 항-ICAM4 항체의 가변부위와 인간 항체 불변부위가 함께 발현되도록 제작된 키메릭(chimeric) 항체를 제공한다.In one aspect, the invention provides a chimeric antibody engineered to be expressed together with a variable region of the anti-ICAM4 antibody and a human antibody constant region.
본 발명의 일실시예에 있어서, 상기 키메릭 항체는, 항 ICAM4 항체의 상보성 결정 영역(CDR) 및 구조영역(FR)과 인간 항체의 경쇄 및 중쇄 불변부위를 포함한다. In one embodiment of the invention, the chimeric antibody comprises the complementarity determining region (CDR) and the structural region (FR) of the anti-ICAM4 antibody and the light and heavy chain constant regions of the human antibody.
또 다른 양태로서, 본 발명은 상기 항-ICAM4 항체의 인간화 항체를 제공한다. In yet another embodiment, the invention provides a humanized antibody of said anti-ICAM4 antibody.
본 발명의 일실시예에 있어서, 상기 항-ICAM4 항체의 인간화항체 중쇄 영역은 11의 아미노산 서열로 이루어진 VH1A; 서열번호 12의 아미노산 서열로 이루어진 VH1B; 서열번호 23의 아미노산 서열로 이루어진 VH1C; 서열번호 14의 아미노산 서열로 이루어진 VH1D; 서열번호 15의 아미노산 서열로 이루어진 VH2A; 서열번호 16의 아미노산 서열로 이루어진 VH2B; 서열번호 17의 아미노산 서열로 이루어진 VH2C; 서열번호 18의 아미노산 서열로 이루어진 VH2D; 서열번호 19의 아미노산 서열로 이루어진 VH2E; 서열번호 20의 아미노산 서열로 이루어진 VH3A; 서열번호 21의 아미노산 서열로 이루어진 VH3B; 서열번호 22의 아미노산 서열로 이루어진 VH3C; 서열번호 23의 아미노산 서열로 이루어진 VH3D로 구성된 군으로부터 선택될 수 있고, In one embodiment of the present invention, the humanized antibody heavy chain region of the anti-ICAM4 antibody comprises VH1A consisting of the amino acid sequence of 11; VH1B consisting of the amino acid sequence of SEQ ID NO: 12; VH1C consisting of the amino acid sequence of SEQ ID NO: 23; A VH1D consisting of the amino acid sequence of SEQ ID NO: 14; VH2A consisting of the amino acid sequence of SEQ ID NO: 15; VH2B consisting of the amino acid sequence of SEQ ID NO: 16; VH2C consisting of the amino acid sequence of SEQ ID NO: 17; A VH2D consisting of the amino acid sequence of SEQ ID NO: 18; A VH2E consisting of the amino acid sequence of SEQ ID NO: 19; VH3A consisting of the amino acid sequence of SEQ ID NO: 20; VH3B consisting of the amino acid sequence of SEQ ID NO: 21; VH3C consisting of the amino acid sequence of SEQ ID NO: 22; A VH3D consisting of the amino acid sequence of SEQ ID NO: 23,
항-ICAM4 항체의 인간화 항체의 경쇄 영역은 서열번호 24의 아미노산 서열로 이루어진 VL1A; 서열번호 25의 아미노산 서열로 이루어진 VL1B; 서열번호 26의 아미노산 서열로 이루어진 VL1C; 서열번호 27의 아미노산 서열로 이루어진 VL1D; 서열번호 28의 아미노산 서열로 이루어진 VL2A; 서열번호 29의 아미노산 서열로 이루어진 VL2B; 서열번호 30의 아미노산 서열로 이루어진 VL2C; 서열번호 31의 아미노산 서열로 이루어진 VL2D로 구성된 군으로부터 선택될 수 있다. The light chain region of the humanized antibody of the anti-ICAM4 antibody comprises VL1A consisting of the amino acid sequence of SEQ ID NO: 24; VL1B consisting of the amino acid sequence of SEQ ID NO: 25; VL1C consisting of the amino acid sequence of SEQ ID NO: 26; VL1D consisting of the amino acid sequence of SEQ ID NO: 27; VL2A consisting of the amino acid sequence of SEQ ID NO: 28; VL2B consisting of the amino acid sequence of SEQ ID NO: 29; VL2C consisting of the amino acid sequence of SEQ ID NO: 30; And VL2D consisting of the amino acid sequence of SEQ ID NO: 31.
다른 측면에서, 본 발명은 상기 항-ICAM4 항체를 포함하는 이중특이성 항체를 제공한다. In another aspect, the invention provides a bispecific antibody comprising the anti-ICAM4 antibody.
본 발명의 일 실시예에서 상기 이중특이성 항체는 항-ICAM4 항체; 및 다른 항원에 특이적인 항체가 링커에 의하여 결합된 형태이며, 본 발명의 상기 다른 항원에 특이적인 항체는 CEACAM 패밀리에 속하는 CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM21, CEACAM16, CEACAM18, CEACAM19, CEACAM20, 및 MDSC 마커인 CD33, CD11b 중에서 제한없이 선택하여 포함될 수 있고, 바람직하게는 CEACAM8 또는 CEACAM6일 수 있다. 본 발명의 일 실시예에서는 항CEACAM6(CD66c) 항체를 포함하는 이중특이성 항체를 제작하여 효과를 확인하였으며, 이러한 이중특이성 항체는 항체 의존성 세포독성(antibody-dependent cellular cytotoxicity, ADCC)이 증강된 특징을 가진다. 상기 항 CD66c 항체는 CD66c에 특이적인 결합을 이루는 펩타이드가 제한 없이 포함될 수 있으며, 상기 링커는 서열번호 10의 아미노산 서열로 이루어져 있으나, 이에 제한되는 것은 아니다. In one embodiment of the invention, the bispecific antibody is an anti-ICAM4 antibody; And antibodies specific to other antigens are bound by a linker. Antibodies specific to the other antigens of the present invention are in the form of CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM21, CEACAM16, CEACAM18 , CEACAM19, CEACAM20, and CD33, CD11b which are MDSC markers, preferably CEACAM8 or CEACAM6. In one embodiment of the present invention, a bispecific antibody comprising an anti-CEACAM6 (CD66c) antibody was produced and its effect was confirmed. This bispecific antibody has an enhanced antibody-dependent cellular cytotoxicity (ADCC) I have. The anti-CD66c antibody may include, without limitation, a peptide that specifically binds to CD66c, and the linker is composed of the amino acid sequence of SEQ ID NO: 10, but is not limited thereto.
또 다른 측면으로 본 발명은, 상기 항-ICAM4 항체를 포함하는 항체-약물 복합체(Antibody-Drug Conjugate, ADC)를 제공한다. In yet another aspect, the invention provides an Antibody-Drug Conjugate (ADC) comprising the anti-ICAM4 antibody.
본 발명의 일실시예에 있어서, 상기 약물은 항암 활성을 나타내는 약물로서, 체내에서 독성을 일으키는 물질일 수 있다. 이에 제한되는 것은 아니나, 상기 약물은 사포린(saporin), 엠탄신(emtansine), 독소루비신 (Doxorubicin), 카보플라틴(Carboplatin), 시스플라틴 (Cisplatin), 시클로포스파미드 (Cyclophosphamide), 이포스파미드(Ifosfamide), 니드란 (Nidran), 질소머스타드 (메클로에타민 염산염)[Nitrogen mustar(MechlorethamineHCL)], 블레오마이신 (Bleomycin), , 미토마이신 C (Mitomycin C), 시타라빈 (Cytarabine), 플루로우라실(Flurouracil), 젬시타빈 (Gemcitabine), 트리메트렉세이트 (Trimetrexate), 메토크렉세이트 (Methotrexate), 에토포시드 (Etoposide), 빈블라스틴 (Vinblastine), 비노렐빈 (vinorelbine), 알림타 (Alimta), 알트레타민 (Altretamine), 프로카바진 (Procarbazine), 탁솔 (Taxol), 탁소텔 (Taxotere), 토포테칸 (Topotecan) 및 이리노테칸 (Irinotecan)으로 이루어진 군에서 선택된 1이상의 약물 일 수 있으며, 바람직하게는 사포린(Saporin) 또는 엠탄신(Emtansine)일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment of the present invention, the drug is a drug exhibiting anticancer activity, and may be a substance that causes toxicity in the body. Such drugs include, but are not limited to, saporin, emtansine, Doxorubicin, Carboplatin, Cisplatin, Cyclophosphamide, Ifosfamide, Nidran, Nitrogen mustar (Mechlorethamine HCL), Bleomycin, Mitomycin C, Cytarabine, Fluoruracil Flurouracil, Gemcitabine, Trimetrexate, Methotrexate, Etoposide, Vinblastine, vinorelbine, Alimta, May be at least one drug selected from the group consisting of Altretamine, Procarbazine, Taxol, Taxotere, Topotecan and Irinotecan, Saporin or Mantan Emtansine. ≪ / RTI >
본 발명은 서열번호 1 내지 서열번호 5의 아미노산 서열 중 선택된 서열을 포함하는 상보성 결정 영역(CDR)의 중쇄 영역; 및 서열번호 6 내지 서열번호 9의 아미노산 서열 중 선택된 서열을 포함하는 상보성 결정 영역(CDR)의 경쇄 영역을 포함하는 항 ICAM4 항체를 포함하는, ICAM4를 발현하는 세포와 관련된 질병의 진단용 조성물을 제공한다. The present invention provides a heavy chain comprising a heavy chain region of a complementarity determining region (CDR) comprising a selected one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 5; And an anti-ICAM4 antibody comprising a light chain region of a complementarity determining region (CDR) comprising a selected sequence selected from the amino acid sequences of SEQ ID NOs: 6 to 9, wherein the anti-ICAM4 antibody .
상기 진단은 상기 조성물을 통하여 ICAM4 발현 질환에서의 항원-항체 반응을 통하여 진단할 수 있으며, 상기 진단용 조성물 및 이를 포함하는 진단용 키트를 이용할 수 있다. The diagnosis can be diagnosed through an antigen-antibody reaction in an ICAM4 expression disease through the composition, and the diagnostic composition and the diagnostic kit containing the diagnostic composition can be used.
다른 양태로서, 본 발명은 서열번호 1 내지 서열번호 5의 아미노산 서열 중 선택된 서열을 포함하는 상보성 결정 영역(CDR)의 중쇄 영역; 및 서열번호 6 내지 서열번호 9의 아미노산 서열 중 선택된 서열을 포함하는 상보성 결정 영역(CDR)의 경쇄 영역을 포함하는 항 ICAM4 항체를 포함하는, ICAM4를 발현하는 세포와 관련된 질병의 치료용 조성물을 제공한다. 본 발명의 일실시예에 있어서, 상기 질병은 백혈병, 간암, 담관암 또는 골수형성이상증후군(MDS)일 수 있다. In another aspect, the invention provides a heavy chain region of a complementarity determining region (CDR) comprising a selected sequence of amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 5; And an anti-ICAM4 antibody comprising a light chain region of a complementary crystal region (CDR) comprising a selected sequence selected from the amino acid sequences of SEQ ID NOS: 6 to 9, and a kit for the treatment of diseases associated with cells expressing ICAM4 do. In one embodiment of the present invention, the disease may be leukemia, liver cancer, cholangiocarcinoma or myelodysplastic syndrome (MDS).
본 발명의 일실시예에 있어서, 상기 조성물은 약학적으로 허용가능한 담체를 포함하는 약학적 조성물일 수 있다. In one embodiment of the invention, the composition may be a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
또 다른 양태로서, 본 발명은 상기 항-ICAM4 항체의 키메릭 항체; 인간화 항체; 이중특이성 항체; 또는 이들 항체에 약물이 결합된 항체-약물 복합체(Antibody-Drug Conjugate, ADC)를 포함하는 골수유래 면역억제세포 (Myeloid-derived suppressor cells, MDSC)의 감별용 조성물을 제공한다. In another embodiment, the invention provides a chimeric antibody of said anti-ICAM4 antibody; Humanized antibodies; Bispecific antibodies; Or myeloid-derived suppressor cells (MDSC) comprising an antibody-drug complex (Antibody-Drug Conjugate, ADC) in which a drug is bound to these antibodies.
골수유래 면역억제세포 (Myeloid-derived suppressor cells, MDSC)는 골수성 세포 중에서 면역억제 작용을 가진 세포들로서, 매우 광범위한 미분화 골수성 세포를 포함하는 세포군으로, 종양이나 염증의 발생 상태에서 증가하게 되며, 일반적으로 세포-세포 사이의 접촉을 통하여 면역 억제 작용을 한다고 알려져 있다. 즉, MDSC를 감별함으로써, 종양 또는 염증 발생 여부를 판단할 수 있으며, 본 발명의 항 ICAM4 단일클론 항체 1E1은, MDSC에 결합하는 마커로서 작용할 수 있다. Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells in myeloid cells. They are cells that contain a very broad range of undifferentiated myeloid cells, which are increased in the onset of tumors or inflammation. In general, It is known to exert immunosuppressive action through cell-cell contact. That is, by discriminating MDSC, it is possible to determine whether tumor or inflammation has occurred, and anti-ICAM4 monoclonal antibody 1E1 of the present invention can act as a marker binding to MDSC.
따라서, 본 발명의 ICAM4 단일클론항체 및 이의 키메릭 항체; 인간화 항체; 이중특이성 항체; 또는 이들 항체에 약물이 결합된 항체-약물 복합체(Antibody-Drug Conjugate, ADC)는 MDSC에 대한 마커로서 활용가능하며, 이를 진단함으로 인하여 MDSC가 발현되는 질병에 대한 진단이 가능하다. Thus, the ICAM4 monoclonal antibodies of the invention and their chimeric antibodies; Humanized antibodies; Bispecific antibodies; Or Antibody-Drug Conjugate (ADC), in which a drug is bound to these antibodies, can be used as a marker for MDSC, and diagnosis of diseases in which MDSC is expressed can be made by diagnosing them.
또 다른 양태로서, 본 발명은 상기 항-ICAM4의 단일클론항체 및 이의 키메릭 항체; 인간화 항체; 이중특이성 항체; 또는 이들 항체에 약물이 결합된 항체-약물 복합체(Antibody-Drug Conjugate, ADC)를 포함하는 골수유래 면역억제세포 (Myeloid-derived suppressor cells, MDSC)의 활성 억제용 조성물을 제공한다. In another aspect, the invention provides a monoclonal antibody and its chimeric antibody of said anti-ICAM4; Humanized antibodies; Bispecific antibodies; Or a composition for inhibiting the activity of Myeloid-derived suppressor cells (MDSC) comprising an antibody-drug complex (Antibody-Drug Conjugate, ADC) in which a drug is bound to these antibodies.
본 발명은 상기 항-ICAM4 항체의 키메릭 항체; 인간화항체; 이중특이성 항체; 또는 이들 항체에 약물이 결합된 항체-약물 복합체(Antibody-Drug Conjugate, ADC)를 이용하는, ICAM4를 발현하는 세포와 관련된 질병의 진단을 위한 정보제공방법을 제공한다. The present invention relates to a chimeric antibody of said anti-ICAM4 antibody; Humanized antibodies; Bispecific antibodies; Or an antibody-drug complex (Antibody-Drug Conjugate, ADC) in which a drug is bound to these antibodies, to provide information for diagnosis of a disease associated with cells expressing ICAM4.
본 발명의 일실시예에 있어서, 상기 방법은 1) 개체로부터 수득한 생물학적 시료에 상기 항체를 접촉시키는 단계; 2) 상기 항체에 결합된 ICAM4를 검출하는 단계; 및 3) 상기 단계 2)에서 검출된 ICAM4의 발현 정도를 정상 대조군과 비교하는 단계를 포함한다. In one embodiment of the invention, the method comprises the steps of: 1) contacting the antibody to a biological sample obtained from an individual; 2) detecting ICAM4 bound to the antibody; And 3) comparing the degree of expression of ICAM4 detected in step 2) with a normal control.
본 발명의 일 실시예에 있어서, 상기 ICAM4를 발현하는 세포와 관련된 질병은 암일 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 일 실시예에 있어서, 상기 ICAM4를 발현하는 세포와 관련된 질병은 백혈병, 간암, 담관암 또는 골수형성이상증후군(MDS)이다.In one embodiment of the present invention, the disease associated with the cells expressing ICAM4 may be, but is not limited to, cancer. In one embodiment of the present invention, the disease associated with the cells expressing ICAM4 is leukemia, liver cancer, cholangiocarcinoma or myelodysplastic syndrome (MDS).
본 발명에 따른 항-ICAM4 항체의 키메릭 항체; 인간화 항체; 이중특이성 항체 및 항체-약물 복합체는 항-ICAM4 항체의 단일클론항체인 1E1과 동일한 항원결정부위를 인지하며 유사한 항원 친화도를 보였고, 특히 항체-약물 복합체는 기존의 항체-약물 복합체보다 우수한 효능을 나타내는 것을 확인하였으므로, 본 발명의 개량된 항체들은 ICAM4와 관련된 질병의 진단 및 치료에 유용하게 사용될 수 있다.A chimeric antibody of an anti-ICAM4 antibody according to the present invention; Humanized antibodies; The bispecific antibody and the antibody-drug complex recognized the same antigenic site as the 1E1 monoclonal antibody of the anti-ICAM4 antibody and showed similar antigen affinity. In particular, the antibody-drug complex showed better efficacy than the existing antibody-drug conjugate , The improved antibodies of the present invention can be useful for the diagnosis and treatment of diseases related to ICAM4.
도 1은 본 발명에 따른 항-ICAM4 키메릭 1E1 항체 (chi1E1) 생산용 세포주의 제작 과정을 나타낸 그림이다. (a) 사람 IgG의 constant regions을 포함하는 키메릭 1E1 항체 발현 벡터의 제작. (b) 키메릭 1E1 항체 생산을 위한 안정한 세포주 개발의 과정.1 is a diagram showing a process for producing a cell line for producing an anti-ICAM4 chimeric 1E1 antibody ( chi 1E1) according to the present invention. (a) Production of a chimeric 1E1 antibody expression vector containing constant regions of human IgG. (b) The process of developing a stable cell line for chimeric 1E1 antibody production.
도 2는 백혈병 세포주에서 키메릭 1E1 항체의 사람 ICAM4에 대한 결합친화도를 유세포분석으로 측정한 결과이다. (a) 키메릭 1E1 항체에 의해 인식되는 에피토프 부위의 식별. 마우스 1E1 항체 block의 부존재(흑색) 및 존재(적색) 하에서 키메릭 1E1 항체로 염색한 K562 세포. (b) 항체 친화성을 측정하기 위해, ICAM4 양성 세포주(K562, HEL) 및 ICAM4 음성 세포주(786-O)에 대해 키메릭 1E1 항체의 희석액을 조사하였다. 간접 염색을 통해 측정한 키메릭 1E1 항체의 포화 농도는 마우스 항체 1E1 (약 5 μg/mL, 데이터 미기재)과 유사한 결합도를 나타내었다. 염색을 위해 FITC-표지된 항-사람 IgG 항체를 사용하였다.FIG. 2 shows the binding affinity of human chimeric 1E1 antibody against human ICAM4 measured by flow cytometry in a leukemia cell line. (a) Identification of the epitope region recognized by the chimeric 1E1 antibody. K562 cells stained with chimeric 1E1 antibody under the absence (black) and presence (red) of mouse 1E1 antibody block. (b) To determine antibody affinity, dilutions of chimeric 1E1 antibody were investigated against ICAM4-positive cell line (K562, HEL) and ICAM4 negative cell line (786-O). The saturation concentration of chimeric 1E1 antibody as measured by indirect staining showed similar binding as mouse antibody 1E1 (about 5 μg / mL, data not shown). FITC-labeled anti-human IgG antibodies were used for staining.
도 3은 마우스 1E1 항체의 3차원 구조를 분자 모델링으로 나타낸 것이다. 표시된 부분은 모든 CDRs 부분을 나타낸다. 설계된 인간화 항체 서열의 일부는 CDRs에 구조적 변화를 야기한다(Kabat positions H48 및 H78). Figure 3 shows the three-dimensional structure of mouse 1E1 antibody by molecular modeling. The marked part represents all CDRs parts. Some of the designed humanized antibody sequences cause structural changes in CDRs (Kabat positions H48 and H78).
도 4는 최적의 인간화된 1E1 항체 구조물(construct)의 선별 결과를 나타낸다. (a) 104개의 인간화 항체 조합을 293T 세포에서 일시적으로 발현시키고, K562 세포에서 결합 특성을 비교하였다. (* 표시는 선택된 인간화 항체를 나타낸다.) (b) 1E1 인간화 항체에 의해 인식되는 에피토프의 식별. 마우스 1E1 항체 block의 부존재(흑색) 및 존재(적색) 하에서 1E1 인간화 항체로 염색한 K562 세포. Figure 4 shows the selection results of optimal humanized 1E1 antibody constructs. (a) 104 humanized antibody combinations were transiently expressed in 293T cells and their binding characteristics were compared in K562 cells. (* Indicates the selected humanized antibody). (B) Identification of the epitope recognized by the 1E1 humanized antibody. K562 cells stained with 1E1 humanized antibody under absence (black) and presence (red) of mouse 1E1 antibody block.
도 5는 인간화 항체의 1E1 항원 결합 친화도를 측정한 결과이다. 일부 인간화 항체의 경우, 키메릭 항체에 비해 1E1에 결합 친화도가 높아지는 경향을 보이고 있다. 5 shows the result of measuring the 1E1 antigen binding affinity of the humanized antibody. In the case of some humanized antibodies, binding affinity to 1E1 is higher than that of chimeric antibodies.
도 6은 두 종류의 항-ICAM4-약물 복합체의 개발 과정을 나타낸 것이다. (a) 사포린(saporin)이 결합된 마우스 1E1 항체(m1E1-SAP)의 개발. 1E1-SAP는 화학적으로 이황화 결합(disulfide bond)에 의해 사포린에 연결된다. (b) 엠탄신(emtansine)이 결합된 키메릭 1E1 항체(r1E1-DM1)의 구조. r1E1-DM1은 SMCC 링커를 이용하여 설계되었다. DM1: thiol-containing maytansinoid; SMCC: succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate.6 shows the development process of two kinds of anti-ICAM4-drug complexes. (a) Development of murine 1E1 antibody (m1E1-SAP) conjugated with saporin. 1E1-SAP is chemically linked to the saprin by a disulfide bond. (b) Structure of chimeric 1E1 antibody ( r 1E1-DM1) conjugated with emtansine. r 1E1-DM1 was designed using the SMCC linker. DM1: thiol-containing maytansinoid; SMCC: succinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate.
도 7은 본 발명의 일실시예에 따른 두 종류의 항-ICAM4-약물 복합체의 효능을 실험한 결과이다. (a) 항-ICAM4 m1E1-SAP 의 in vitro상 효능. K562 세포에 다른 농도의 m1E1-SAP를 3일간 처리하였다. 실험은 3반복으로 수행하였고 Ez-cytox로 분석하였다. (b) 항-ICAM4 chi1E1-DM1의 in vitro상 효능. HEL 세포에 다른 농도의 chi1E1-DM1를 3일간 처리하였다. 살아있는 세포와 죽은 세포의 수를 유세포분석(flow cytometry)으로 측정하였다. FIG. 7 shows the results of experiments on the efficacy of two kinds of anti-ICAM4-drug complexes according to one embodiment of the present invention. (a) wherein m -ICAM4 1E1-SAP of the in vitro efficacy. The m-1E1 SAP of different concentrations in the K562 cells were treated for 3 days. Experiments were performed in 3 replicates and analyzed with Ez-cytox. (b) in vitro efficacy of anti-ICAM4 chi 1E1-DM1. HEL cells were treated with different concentrations of chi 1E1-DM1 for 3 days. The number of living and dead cells was measured by flow cytometry.
도 8은 이종이식(xenotransplanted) 마우스 모델에서 항-ICAM4-DM1 복합체의 효능을 실험한 결과이다. HEL tumor xenografts에서 항-ICAM4 chi1E1-DM1의 in vivo 효능을 측정하였다. HEL 종양을 갖고 있는 SCID 마우스에 4 mg/kg의 chi1E1-DM1을 단일, 정맥내 주사하였다.Figure 8 shows the results of an experiment on the efficacy of the anti-ICAM4-DM1 complex in a xenotransplanted mouse model. In vivo efficacy of anti-ICAM4 chi 1E1-DM1 was measured in HEL tumor xenografts. SCID mice with HEL tumors were injected intravenously with 4 mg / kg of chi 1E1-DM1.
도 9는 정상 RBC 및 백혈병 종양 세포에서 ICAM4의 다른 sialylation을 다양한 농도의 뉴라민가수분해효소(neuraminidase) 처리에 의해 측정한 것이다. (a) 뉴라민가수분해효소를 농도별로 종양세포주(K562)와 적혈구에 처리하여 이들에 결합하는 키메릭 1E1 항체의 결합력을 비교한 것이다. (b) 1000 unit의 뉴라민가수분해효소를 종양세포주와 적혈구에 처리하여 이들에 결합하는 키메릭 1E1 항체의 결합력을 비교한 것이다. (c) 종양 세포와 적혈구가 혼합되었을 때 세포 표면의 시알릭산의 차이에 따른 항체 반응성을 비교한 것이다.Figure 9 shows the measurement of the different sialylation of ICAM4 in normal RBC and leukemia tumor cells by treatment with various concentrations of neuraminidase. (a) Neuramine hydrolase was treated with tumor cells (K562) and erythrocytes at different concentrations to compare the binding affinity of chimeric 1E1 antibody bound thereto. (b) Comparing the binding capacity of chimeric 1E1 antibody bound to 1000 units of neuraminase in tumor cells and erythrocytes. (c) The antibody reactivity according to the difference in sialic acid on the cell surface when tumor cells and red blood cells are mixed.
도 10은 ICAM4와 CD66c에 대한 이중특이적 항체의 제조 방법(a) 및 제조된 항체의 항체의존적세포독성(ADCC) 기능을 확인한 결과(b, c)를 나타낸 것이다.Fig. 10 shows the results (b, c) of the method (a) for producing bispecific antibodies against ICAM4 and CD66c and the antibody-dependent cytotoxicity (ADCC) function of the antibodies prepared.
도 11은 사람 태아 조직의 파라핀 포매된 섹션에서 ICAM4의 발현을 나타낸 것이다. 면역조직화학 염색을 위해 사람 태아 조직의 파라핀 조직 섹션에 항-ICAM4 1E1 항체(10 μg/mL) 및 HRP-표지된 이차 항체(1/1000 dilution)를 연속적으로 처리하고, DAB를 기질로 하였다. 염색 결과, 골수 외 적혈구 생성(extramedullary erythropoiesis)이 나타나는 간(b)을 제외하고 다른 모든 조직들(a)에 대해 교차 반응성이 나타나지 않았다.Figure 11 shows the expression of ICAM4 in a paraffin-embedded section of human fetal tissue. For immunohistochemical staining, anti-ICAM4 1E1 antibody (10 μg / mL) and HRP-labeled secondary antibody (1/1000 dilution) were sequentially treated in a paraffin tissue section of human fetal tissue and DAB was used as a substrate. The staining showed no cross reactivity for all other tissues (a) except liver (b) where extramedullary erythropoiesis was present.
도 12는 MDS 및 간암 환자의 종양 조직에서 ICMA4의 발현을 나타낸다. 말초 혈액 샘플에서 ICAM4 항원은 적혈구 세포를 제외하고는 정상 조건 하에서 발현되지 않는다. 임상 환자 샘플을 분석한 결과, (a) MDS & HCC　및 (b) MDS 환자에서 CD45low/CD11b+/ICAM4+ 개체군을 발견하였다. MDS: myelodysplastic syndrome; HCC: hepato cellular cacinoma.Figure 12 shows the expression of ICMA4 in tumor tissues of MDS and liver cancer patients. In peripheral blood samples, the ICAM4 antigen is not expressed under normal conditions except for red blood cells. Analysis of clinical patient samples revealed CD45 low / CD11b + / ICAM4 + populations in (a) MDS & HCC and (b) MDS patients. MDS: myelodysplastic syndrome; HCC: hepato cellular cacinoma.
도 13은 일반 성인 3명과 4기 암환자 11명의 혈액 샘플 중 일부 암환자의 샘플에서 1E1의 항원이 높아져 있는 것을 보여주는 결과이며, 따라서 1E1 항체 타겟이 적백혈병에만 국한되는 것은 아니라는 것을 확인한 것이다.FIG. 13 shows that 1E1 antigen is elevated in a sample of some cancer patients among 3 normal adults and 11 cancer patients, and thus it is confirmed that the 1E1 antibody target is not restricted to the target leukemia.
도 14는 골수유래 억제세포(MDSC)에 대한 항 ICAM4 항체의 발현 효과를 확인한 것으로서, 항 ICAM4 항체가 상기 세포에 대한 마커로 사용될 수 있음을 보여주는 결과이다.FIG. 14 shows the effect of anti-ICAM4 antibody on bone marrow-derived inhibitory cell (MDSC), showing that anti-ICAM4 antibody can be used as a marker for the cell.
본 발명자들은 대한민국 등록특허 제10-1627020호에 개시된 정상적혈구 표면에서 발현하는 ICAM4에 대한 단일클론항체 1E1의 백혈병을 포함한 종양 치료용 항체로의 개발 가능성에 대하여 체계적으로 검증하였다. The present inventors have systematically verified the possibility of developing a monoclonal antibody 1E1 against ICAM4 expressed on normal erythrocyte surface as disclosed in Korean Patent No. 10-1627020 as an antibody for tumor therapy including leukemia.
우선 항-ICAM4 키메릭 항체 1E1을 대량 확보하기 위해서 키메릭 항체를 안정적으로 생산하는 세포주를 만들었다(도 1). 인간 불변부위가 함께 발현되도록 제작된 pOPtiVEC과 pcDNA3.3을 발현벡터로 사용하였고, 호스트 세포주는 CHO DG44를 사용하였다. 생산성을 높이기 위한 약제로 MTX와 G418을 사용하여 7.5 μg/mL의 생산성을 보이는 세포주를 개발하였다. 대량 생산용 세포주에서 생산된 재조합 키메릭 항체 1E1은 마우스 항체 1E1과 동일한 항원결정부위를 인지하며 유사한 항원 친화도를 보였다(도 2). First, to obtain a large amount of anti-ICAM4 chimeric antibody 1E1, a cell line stably producing a chimeric antibody was prepared (Fig. 1). POPtiVEC and pcDNA3.3, which were designed to express human constant regions together, were used as expression vectors and CHO DG44 was used as the host cell line. Using MTX and G418 as a drug to improve productivity, a cell line showing productivity of 7.5 μg / mL was developed. Recombinant chimeric antibody 1E1 produced in a cell line for mass production recognizes the same antigenic site as mouse antibody 1E1 and shows similar antigen affinity (FIG. 2).
그 다음 단계로 in silico로 추정된 104개의 재조합 인간화항체를 생산하여 선별과정을 거쳐 최적의 치료용 항체 후보군을 선정하였다. 마우스 항체의 가변부위와 인간 항체의 불변부위를 재조합한 키메릭 형태의 치료용 항체를 제작하는 것도 효능은 유지하면서 HAMA (human anti-mouse antibody) 반응을 줄이고 특허에 침해받지 않는 등 여러 장점이 있지만, 마우스에서 유래한 항체 가변부위는 여전히 면연반응을 유발할 수 있고 인간화 과정을 통해 항체를 선별하는 과정에서 물성 및 항원 친화도 측면으로 키메릭 항체보다 우수한 항체를 획득하기 위해 항-ICAM4 키메릭 항체 1E1의 인간화를 진행하였다.In the next step, 104 recombinant humanized antibodies in silico were produced and screened to select optimal therapeutic antibody candidates. The production of a therapeutic chimeric antibody, in which the variable region of the mouse antibody and the constant region of the human antibody are recombined, has several advantages such as reducing the HAMA (human anti-mouse antibody) , The antibody variable region derived from the mouse may still induce the skin reaction. In order to obtain an antibody superior to the chimeric antibody in terms of physical properties and antigen affinity in the course of screening the antibody through humanization, anti-ICAM4 chimeric antibody 1E1 .
인간화항체는 CDR 이식 방법을 통하여 진행되었다. CDR 이식이란 실제로 항원을 인지하여 결합하는데 필요한 부위인 CDR 을 제외한 다른 모든 부위를 인간에서 유래한 서열로 바꾸어 주는 항체 인간화 기술이다[Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986;321(6069):522-5]. 항-ICAM4 키메릭 항체 1E1의 CDR 이식을 위하여 본래 서열과 상동성이 높은 순서로 중쇄 IMGT human germline IGHV4-59*05 (60.8%), IGHV3-11*01 (51.6%), IGHV5-51*01 (45.4%), 경쇄 GKV1-9*01 (75.8%), IGKV3-11*01 (67.4%)이 선택되어 CDR 이식이 진행되었고, in silico로 항체 구조에 영향을 줄 수 있는 아미노산 서열을 유지하는지 또는 변경하는지에 따라서 A, B, C, D, E 버전으로 중쇄 13개, 경쇄 8개 인간화항체 서열을 얻었다(표 1). 구체적으로, 상기 인간화항체 중, 13개의 항-ICAM4 단일클론항체 1E1의 인간화항체 중쇄 영역은 서열번호 1 내지 서열번호 5의 아미노산 서열에서 선택되는 중쇄 가변부위 및 인간 항체 불변부위를 포함하며, 보다 구체적으로 서열번호 11의 아미노산 서열로 이루어진 VH1A; 서열번호 12의 아미노산 서열로 이루어진 VH1B; 서열번호 23의 아미노산 서열로 이루어진 VH1C; 서열번호 14의 아미노산 서열로 이루어진 VH1D; 서열번호 15의 아미노산 서열로 이루어진 VH2A; 서열번호 16의 아미노산 서열로 이루어진 VH2B; 서열번호 17의 아미노산 서열로 이루어진 VH2C; 서열번호 18의 아미노산 서열로 이루어진 VH2D; 서열번호 19의 아미노산 서열로 이루어진 VH2E; 서열번호 20의 아미노산 서열로 이루어진 VH3A; 서열번호 21의 아미노산 서열로 이루어진 VH3B; 서열번호 22의 아미노산 서열로 이루어진 VH3C; 서열번호 23의 아미노산 서열로 이루어진 VH3D이고, Humanized antibodies proceeded through the CDR transplantation method. CDR transplantation is an antibody humanization technique that converts all but the CDR, which is the site required for antigen recognition and binding, to human-derived sequences [Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with a mouse. Nature. 1986; 321 (6069): 522-5]. For the CDR transplantation of anti-ICAM4 chimeric antibody 1E1, the heavy chain IMGT human germline IGHV4-59 * 05 (60.8%), IGHV3-11 * 01 (51.6%), IGHV5-51 * 01 (45.4%), light chain GKV1-9 * 01 (75.8%) and IGKV3-11 * 01 (67.4%) were selected for CDR transplantation and maintained the amino acid sequence that could affect the antibody structure in silico Thirteen heavy chain and eight light chain humanized antibody sequences were obtained in the A, B, C, D, and E versions, depending on whether or not the antibodies were changed (Table 1). Specifically, among the humanized antibodies, the 13 human anti-ICAM4 monoclonal antibody 1E1 heavy chain antibody region heavy chain variable region includes the heavy chain variable region and the human antibody constant region selected from the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 5, VH1A consisting of the amino acid sequence of SEQ ID NO: 11; VH1B consisting of the amino acid sequence of SEQ ID NO: 12; VH1C consisting of the amino acid sequence of SEQ ID NO: 23; A VH1D consisting of the amino acid sequence of SEQ ID NO: 14; VH2A consisting of the amino acid sequence of SEQ ID NO: 15; VH2B consisting of the amino acid sequence of SEQ ID NO: 16; VH2C consisting of the amino acid sequence of SEQ ID NO: 17; A VH2D consisting of the amino acid sequence of SEQ ID NO: 18; A VH2E consisting of the amino acid sequence of SEQ ID NO: 19; VH3A consisting of the amino acid sequence of SEQ ID NO: 20; VH3B consisting of the amino acid sequence of SEQ ID NO: 21; VH3C consisting of the amino acid sequence of SEQ ID NO: 22; A VH3D consisting of the amino acid sequence of SEQ ID NO: 23,
8개의 항-ICAM4 단일클론항체 1E1의 인간화 항체의 경쇄 영역은 서열번호 6내지 서열번호 9에서 선택되는 경쇄 가변부위 및 인간 항체 불변부위를 포함하며, 보다 구체적으로는 서열번호 24의 아미노산 서열로 이루어진 VL1A; 서열번호 25의 아미노산 서열로 이루어진 VL1B; 서열번호 26의 아미노산 서열로 이루어진 VL1C; 서열번호 27의 아미노산 서열로 이루어진 VL1D; 서열번호 28의 아미노산 서열로 이루어진 VL2A; 서열번호 29의 아미노산 서열로 이루어진 VL2B; 서열번호 30의 아미노산 서열로 이루어진 VL2C; 서열번호 31의 아미노산 서열로 이루어진 VL2D이다. 분자 모델링을 이용한 항체 가변부위의 3D 구조를 구축을 통해서 중쇄의 48번(Leu→Val), 78번(Val→Leu) 아미노산의 변화가 인간화항체 가변 부위 구조의 변화를 가져오는 결과를 예측할 수 있었고(도 3), 실제로 3D 구조 변경이 예상되는 24개의 VH2-C, VH2-D, VH2-E 조합 클론들 대부분은 항원 친화도가 없거나 매우 낮았다(도 4). The light chain region of the humanized antibody of the eight anti-ICAM4 monoclonal antibodies 1E1 comprises the light chain variable region and the human antibody constant region selected from SEQ ID NOs: 6 to 9, and more specifically, the amino acid sequence of SEQ ID NO: 24 VL1A; VL1B consisting of the amino acid sequence of SEQ ID NO: 25; VL1C consisting of the amino acid sequence of SEQ ID NO: 26; VL1D consisting of the amino acid sequence of SEQ ID NO: 27; VL2A consisting of the amino acid sequence of SEQ ID NO: 28; VL2B consisting of the amino acid sequence of SEQ ID NO: 29; VL2C consisting of the amino acid sequence of SEQ ID NO: 30; VL2D consisting of the amino acid sequence of SEQ ID NO: 31. By constructing the 3D structure of the antibody variable region using molecular modeling, it was possible to predict the change of the amino acid variable of the humanized antibody variable 48 (Leu → Val) and 78 (Val → Leu) amino acid of the heavy chain (FIG. 3), most of the 24 VH2-C, VH2-D, and VH2-E combined clones that are expected to undergo 3D structural changes are either absent or very low in antigen affinity (FIG.
항-ICAM4 항체치료제 1E1의 가장 큰 특성 중 하나는 세포 내재화이다. 이러한 내재화 특성은 항체-약물 복합체 개발의 좋은 후보물질이 될 수 있는 조건인데 이전 연구결과를 보면 1시간 안에 약 70%의 항원-항체 복합체가 세포 안으로 내재화 되고[김민영. 적백혈병에 대한 새로운 치료용 단클론 항체의 개발. 충북대학교 의학석사학위논문. 2014], 이는 기존에 알려진 T-DM1이 위암 세포주에서 확인한 약 10% 항체-약물 복합체가 내재화 되는 것보다 많은 양의 항원-항체 복합체가 내재화되는 것으로 항-ICAM4 항체치료제 1E1이 더 높은 유효성을 갖는 치료제가 될 가능성이 높다는 이론적 근거를 제공해 주고 있다[Wang H, et al. Aberrant intracellular metabolism of T-DM1 confers T-DM1 resistance in human epidermal growth factor receptor 2-positive gastric cancer cells. Cancer Sci. 2017 Apr 7. doi: 10.1111/cas.13253]. One of the greatest characteristics of the anti-ICAM4 antibody therapeutic agent 1E1 is cellular internalization. This internalization characteristic is a condition that can be a good candidate for the development of an antibody-drug complex. Previous studies have shown that about 70% of the antigen-antibody complex is internalized into the cell within one hour [Kim Min-young. Development of new therapeutic monoclonal antibodies against human leukemia. Thesis of Master's Degree of Medicine in Chungbuk National University. 2014], which indicates that the previously known anti-ICAM4 antibody therapeutic agent 1E1 has a higher efficacy than the conventional T-DM1 internalization of an about 10% antibody-drug complex identified in the gastric cancer cell line, And is likely to be a therapeutic agent [Wang H, et al. Aberrant intracellular metabolism of T-DM1 confers T-DM1 resistance in human epidermal growth factor receptor 2-positive gastric cancer cells. Cancer Sci. 2017 Apr 7 doi: 10.1111 / cas.13253].
본 발명의 일실시예에서는, 항-ICAM4 항체와 사포린 또는 엠탄신 복합체를 제작해 생체내외에서 그 유효성을 검증하였고, 각각 IC50 10 ng/mL 및 3.7 μg/mL로 비교적 양호한 효능을 보였다(도 7, 8). 사포린은 리보솜 불활성화 단백질로 높은 효소활성을 가지며 물리 화학적으로 안정하고 실험실 수준에서 항체와의 결합이 용이하다는 이점을 가지고 있고[Polito L, Bortolotti M, Mercatelli D, Battelli MG, Bolognesi A. Saporin-S6: a useful tool in cancer therapy. Toxins (Basel). 2013;5(10):1698-722], 엠탄신은 세포 분열을 억제하는 미세소관 형성 저해제로 2013년에 FDA 승인을 받은 HER2 양성 전이성 유방암을 대상으로 하는 T-DM1 (캐싸일라)에 사용된 약물로 그 안전성과 효능이 입증되어 있다[Bender B, Leipold DD, Xu K, Shen BQ, Tibbitts J, Friberg LE. A mechanistic pharmacokinetic model elucidating the disposition of trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) for treatment of metastatic breast cancer. AAPS J. 2014;16(5):994-1008]. In one embodiment of the present invention, an anti-ICAM4 antibody and a saponin or manganese complex were produced and tested in vivo and in vitro, and showed relatively good efficacy with IC 50 of 10 ng / mL and 3.7 μg / mL, respectively 7 and 8). Sapolin is a ribosome-inactivating protein with high enzymatic activity and is physico-chemically stable and has an advantage of being easy to bind to antibodies at the laboratory level [Polito L, Bortolotti M, Mercatelli D, Battelli MG, Bolognesi A. Saporin- S6: a useful tool in cancer therapy. Toxins (Basel). 2013; 5 (10): 1698-722]. Mantansin is a microtubule-forming inhibitor that inhibits cell division and is used for T-DM1 (cassia) targeting HER2-positive metastatic breast cancer that has been approved by the FDA in 2013 (Bender B, Leipold DD, Xu K, Shen BQ, Tibbitts J, Friberg LE. A mechanistic pharmacokinetic model of elucidating the disposition of trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) for treatment of metastatic breast cancer. AAPS J. 2014; 16 (5): 994-1008].
백혈병이 진행되면 조혈기능이 저하되고 정상 적혈구의 숫자도 매우 감소하기 때문에 항암치료 초기에는 정상 적혈구에서의 ICAM4 발현이 덜 문제가 되지만, 근본적으로 항-ICAM4 항체치료제를 개발하면서 정상 적혈구와의 반응성 회피 문제가 제기된다. 그래서 먼저 고안한 방법은 항-ICAM4 항체치료제의 시알산 의존적인 항원결정부위를 인지하는 특징을 이용하는 것이다. 종양 세포는 영양 결핍에 의한 비정상적인 에너지 소모를 특징으로 하며 이 과정에서 다양한 유형의 다당류가 급속히 소실되고, 세포 표면의 당화를 유지하기 위해 시알산이 이용되게 된다. 즉 정상 세포보다 종양 세포 표면에 시알산이 더 많이 존재한다. 세포 표면의 시알산을 제거하는 뉴라민가수분해효소를 농도별로 처리하여 종양 세포와 정상 적혈구 표면 ICAM4항원의 시알산화정도의 차이를 만들었고 항-ICAM4 항체가 종양 세포에는 결합하면서 정상 적혈구에는 결합하지 않는 뉴라민가수분해효소 농도를 알아내었다(도 9). Although leukemia progresses, hematopoietic function decreases and the number of normal red blood cells decreases. Therefore, ICAM4 expression in normal erythrocytes is less problematic at the early stage of chemotherapy. However, in the development of anti-ICAM4 antibody therapy, The problem arises. So the first approach is to take advantage of the ability to recognize sialic acid-dependent antigenic determinants of anti-ICAM4 antibody therapy. Tumor cells are characterized by abnormal energy expenditure due to malnutrition, during which various types of polysaccharides are rapidly lost and sialic acid is used to maintain glycosylation of the cell surface. More sialic acid is present on the tumor cell surface than normal cells. The level of sialylation of tumor cells and normal red blood cell surface ICAM4 antigen was differentiated by treatment with neuraminase to remove sialic acid on the cell surface. Anti-ICAM4 antibody binds to tumor cells and does not bind to normal red blood cells And the concentration of neuraminase (Fig. 9).
ICAM4 항원은 조직에서의 극히 제한된 발현을 보이지만 이는 반대로 표적세포 외 세포에서의 교차반응 우려를 줄여 준다. 이를 확인하기 위하여 인간 정상 장기와 8주령 태아 파라핀 포매 조직을 이용하여 발생단계의 정상조직에서 ICAM4의 발현을 검색하였다. 그 결과 태아조직에서는 조혈작용이 왕성한 간 조직에서 적혈구로 분화중인 미성숙 적혈구에서만 발현이 관찰되었고, 성숙 분화된 정상조직과 발생단계의 3개 배엽층의 어떤 조직에서도 ICAM4의 발현이 확인되지 않았다(도 11). 적응증 확대 측면에서 이 결과를 고려해보면 ICAM4가 인간 적혈구 생성 단계의 대부분에 발현하므로 골수성미성숙세포에서 동시 발현되어 많은 백혈병과 골수형성이상증후군(MDS) 세포가 대상이 될 수 있다. 이는 실제 골수형성이상증후군(MDS) 환자의 임상 시료를 분석하여 증명하였다(도 12). 또한 담관암 및 간암 세포주 일부에서 ICAM4가 발현되었다(표 2), ICAM4 antigens show extremely limited expression in tissues, but conversely reduce cross-reactivity concerns in extracellular target cells. To confirm this, expression of ICAM4 was detected in normal tissues of developmental stage using human normal organs and 8 weeks old fetal paraffin embedded tissues. As a result, in fetal tissues, expression was observed only in immature erythrocytes differentiating into erythrocytes in hepatocytes with vigorous hematopoiesis, and no expression of ICAM4 was observed in any tissues of mature differentiated normal tissues and three embryonic developmental stages 11). Considering these results in terms of indications, ICAM4 is expressed in most of the erythropoiesis stages of human erythropoiesis, so it can be expressed simultaneously in myeloid immature cells, and many leukemia and myelodysplastic syndrome (MDS) cells can be targeted. This was demonstrated by analyzing clinical samples of actual bone marrow dysplasia syndrome (MDS) patients (FIG. 12). ICAM4 was also expressed in some of the cholangiocarcinoma and liver cancer cell lines (Table 2)
따라서 본 발명은 항-ICAM4 단일클론항체 1E1을 이용한 항체치료제(키메릭 항체, 인간화항체, 이중특이성항체 및 항체-약물 복합체를 모두 포함함)를 제공한다.Thus, the present invention provides antibody therapeutics (including chimeric antibodies, humanized antibodies, bispecific antibodies, and antibody-drug complexes) using anti-ICAM4 monoclonal antibody 1E1.
본 발명에서 사용되는 용어 "항체"는 면역학적으로 특정 항원과 반응성을 갖는 면역글로불린 분자를 포함하며, 다클론항체 및 단일클론항체를 모두 포함하는 개념이다. 또한, 상기 용어는 키메라 항체(예를 들면, 인간화 뮤린 항체) 및 이종결합항체(예를 들면, 양특이성 항체)와 같은 유전공학에 의해 생산된 형태를 포함한다.The term " antibody " as used in the present invention includes immunoglobulin molecules immunologically reactive with specific antigens, and includes both polyclonal antibodies and monoclonal antibodies. The term also includes forms produced by genetic engineering such as chimeric antibodies (e. G., Humanized murine antibodies) and heterologous binding antibodies (e. G., Bispecific antibodies).
본 발명에서 사용되는 용어 "키메릭(chimeric)" 항체는 재조합 디옥시리보핵산 기법을 사용하여 유도된 것이 가장 바람직한 항체를 의미하며, 인간(면역학적으로 "유관" 종, 예컨대, 침팬지를 포함) 및 비-인간 성분 둘 모두를 포함한다. 따라서 키메릭 항체의 불변 영역은 가장 바람직하게는 실질적으로 천연 인간 항체의 불변 영역과 일치하며; 키메라 항체의 가변 영역은 가장 바람직하게는 비-인간 공급원으로부터 유도되며, 대상 항원(ICAM4)에 대한 소망하는 항원 특이성을 보유한다. 비-인간 공급원은 ICAM4 항원에 대한 항체를 생성하기 위하여 사용할 수 있는 모든 척추동물 공급원이 될 수 있다. 이러한 비-인간 공급원은 설치류(예컨대, 토끼, 레트, 마우스, 등; 예를 들면, 미국 등록 특허 번호 4,816,567, 본원에서 참고자료로서 포함됨) 및 비-인간 영장류 (예컨대, 구세계 원숭이, 유인원, 등; 예를 들면, 미국 등록 특허 번호 5,750,105 및 5,756,096; 본원에서 참고자료로서 포함됨)를 포함하나 이에 한정되지 않는다.As used herein, the term " chimeric " antibody refers to the most preferred antibody derived using recombinant deoxyribonucleic acid techniques, including human (including immunologically " related " species such as chimpanzee) - Contains both human components. Thus, the constant region of the chimeric antibody is most preferably consistent with the constant region of the native human antibody; The variable region of the chimeric antibody is most preferably derived from a non-human source and retains the desired antigen specificity for the target antigen (ICAM4). A non-human source can be any vertebrate source that can be used to generate antibodies against the ICAM4 antigen. Such non-human sources include, but are not limited to, rodents (such as rabbits, rats, mice, etc., such as those disclosed in U.S. Patent No. 4,816,567, incorporated herein by reference) and non-human primates such as Old World monkeys, apes, For example, U.S. Patent Nos. 5,750,105 and 5,756,096, incorporated herein by reference).
본 발명에서 사용되는 용어 "항체 단편"은 표적 항원, 이 경우 ICAM4 또는 이의 특이적인 부분에 결합할 수 있는 항체의 특정 부분을 말한다. 예를 들어, 항체 단편은 F(ab')2, Fab, Fab' 및 Fv 단편을 포함한다. 이들은 통상적으로 재조합 DNA 기술로 제조하거나 파파인 또는 펩신으로 항체 단백질을 분해시키는 전통적인 방법을 사용하여 제조할 수 있다(CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley and Sons Coliganet al., eds. (1991-1992)).The term " antibody fragment " as used herein refers to a specific part of an antibody that is capable of binding to a target antigen, in this case ICAM4 or a specific part thereof. For example, antibody fragments include F (ab ') 2, Fab, Fab' and Fv fragments. These can be produced conventionally using recombinant DNA technology or using conventional methods of degrading antibody proteins with papain or pepsin (CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley and Sons Coligan et al., Eds. (1991-1992)).
본 발명에서 사용되는 용어 "단일클론항체"란 당해 분야에 공지된 용어로서 단일 항원성 부위에 대해서 지시되는 고도의 특이적인 항체를 의미한다. 통상적으로, 상이한 에피토프(항원결정기)들에 대해 지시되는 상이한 항체들을 포함하는 다클론 항체와는 다르게, 단일클론항체는 항원상의 단일 결정기에 대해서 지시된다. 단일클론항체는 항원-항체 결합을 이용하는 진단 및 분석학적 분석법의 선택성과 특이성을 개선시키는 장점이 있으며, 또한 하이브리도마 배양에 의해 합성되기 때문에 다른 면역글로불린에 의해 오염되지 않는 또 다른 장점을 갖는다. 전형적으로, 면역글로불린은 중쇄 및 경쇄를 가지며 각각의 중쇄 및 경쇄는 불변 영역 및 가변 영역 (상기 부위는 "도메인"으로 또한 알려져 있음)을 포함한다. 경쇄 및 중쇄의 가변 영역은, "상보성 결정 영역"(complementarity determining region, 이하, "CDR"이라 함)이라고 불리는 3개의 가변 영역 및 4개의 "구조 영역" (framework region)을 포함한다. 상기 CDR은 주로 항원의 에피토프(epitope)에 결합하는 역할을 한다. 각각의 사슬의 CDR은 전형적으로 N-말단으로부터 시작하여 순차적으로 CDR1, CDR2, CDR3로 불리고, 또한 특정 CDR이 위치하고 있는 사슬에 의해서 식별된다.As used herein, the term " monoclonal antibody " refers to a highly specific antibody directed against a single antigenic site as is known in the art. Typically, unlike polyclonal antibodies that contain different antibodies directed against different epitopes (antigenic determinants), monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays that utilize antigen-antibody binding and also have other advantages that are not contaminated by other immunoglobulins because they are synthesized by hybridoma cultures. Typically, immunoglobulins have a heavy chain and a light chain, and each heavy and light chain comprises a constant region and a variable region (the region also known as a " domain "). The variable regions of the light and heavy chains include three variable regions and four "framework regions" called "complementarity determining regions" (hereinafter referred to as "CDRs"). The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, CDR3, starting from the N-terminus and sequentially identified by the chain in which the particular CDR is located.
본 발명에서 사용되는 용어 "가변"이란 항체들 간에 특정 영역의 서열이 크게 상이하고 특정 항원에 대해 각각의 특정 항체의 결합 특이성을 나타내는데 사용된다는 것을 일컫기 위해 사용된다. 항체의 가변성은 항체의 가변 도메인 전체에 걸쳐 균일하게 분포하는 것이 아니라 CDR에 집중된다. 단클론 항체의 중쇄 및 경쇄는 각각 3개의 CDR을 가지며 이들 영역이 ICAM4를 인식함으로써 항원-항체 복합체를 형성한다. 이러한 CDR은 각각의 단일클론항체마다 특징적인 서열을 가지며 하나의 단일클론항체가 특정 에피토프를 인식하기 위해 이들 6개의 CDR의 일부 또는 모두가 상호작용할 수 있다.The term " variable " as used herein is used to refer to the fact that the sequence of a particular region between antibodies is significantly different and is used to indicate the binding specificity of each particular antibody to a particular antigen. The variability of the antibody is centered on the CDR rather than being uniformly distributed throughout the variable domains of the antibody. The heavy and light chains of the monoclonal antibody each have three CDRs, and these regions form an antigen-antibody complex by recognizing ICAM4. These CDRs have a characteristic sequence for each monoclonal antibody and one or both of these six CDRs can interact to allow a single monoclonal antibody to recognize a particular epitope.
본 발명의 항-ICAM4 항체는 ICAM4의 항원결정부위에 특이적으로 결합하는 항체로서, 경쇄 및 중쇄의 가변 영역을 포함할 수 있다.The anti-ICAM4 antibody of the present invention is an antibody that specifically binds to an antigenic determinant region of ICAM4, and may include variable regions of light and heavy chains.
보다 구체적으로는 상기 항체의 중쇄 가변부위가 서열번호 1 내지 서열번호 5로 이루어진 군으로부터 선택된 1종 이상의 아미노산 서열을 포함하거나 또는 CDR 영역 내에서 이러한 서열과의 동일성이 80% 이상, 바람직하게는 90% 이상, 가장 바람직하게 95% 이상인 서열을 포함하거나; 또는/ 및 그들의 경쇄 가변부위가 서열번호6 내지 서열번호 9으로 이루어진 군으로부터 선택된 1종 이상의 아미노산 서열을 포함하거나 또는 CDR 영역 내에서 이러한 서열과의 동일성이 80% 이상, 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상인 서열을 포함할 수 있다.More specifically, the heavy chain variable region of the antibody comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, or at least 80% homology with this sequence in the CDR region, preferably 90 Or more, and most preferably 95% or more; And / or wherein the light chain variable region comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 6 to 9, or at least 80%, preferably 90% or more, And most preferably 95% or more.
ICAM4에 대해 특이적인 항체는 본 발명이 속하는 기술분야에 잘 알려져 있는 융합 방법(fusion method)에 의해 만들어질 수 있다(Kohler 및 Milstein (1976) European Jounral of Immunology 6:511-519 참조). 예를 들어, 본 발명의 항체는 ICAM4를 면역원으로 하여 동물을 면역화시키고, 면역화된 동물의 비장세포를 골수종 세포와 융합하여 하이브리도마를 생성하고, ICAM4를 선택적으로 인식하는 단일클론항체를 생산하는 하이브리도마를 선별하며, 선별한 하이브리도마를 배양하고, 하이브리도마의 배양액으로부터 항체를 분리함으로써 제조할 수 있다. 상기 하이브리도마 세포주는 생체 외(in vitro)에서 배양하거나, 생체 내 주입하여 항체를 분리할 수 있다. 상기한 하이브리도마가 생산하는 단일클론항체는 정제하지 않고 사용할 수도 있으나, 최선의 결과를 얻기 위해서는 본 발명이 속하는 기술분야에 잘 알려져 있는 방법에 따라 고순도(예컨대, 95% 이상)로 정제하여 사용하는 것이 바람직하다. 이러한 정제 기술로는, 예를 들어 겔 전지영동, 투석, 염 침전, 이온교환 크로마토그래피, 친화성 크로마토그래피 등의 정제 방법을 이용하여 배양 배지 또는 복수액으로부터 분리될 수 있다. 하이브리도마를 이용한 단일클론항체의 생산의 일례로, 마우스의 복강 내에 하이브리도마를 삽입하고 복수로부터 분리, 정제한다. 단일클론항체의 분리 및 정제는 배양 상층액 또는 복수를 이온교환 크로마토그래피(DEAE 또는 DE52 등), 항 면역글로불린 칼럼 또는 프로테인 A 컬럼 등의 친화성 크로마토그래피를 이용하여 실시할 수가 있다. Antibodies specific for ICAM4 can be generated by a fusion method well known in the art (Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519). For example, an antibody of the invention can be produced by immunizing an animal with ICAM4 as an immunogen, fusing splenocytes of an immunized animal with a myeloma cell to produce a hybridoma, and producing a monoclonal antibody that selectively recognizes ICAM4 Selecting the hybridomas, culturing the selected hybridomas, and separating the antibodies from the hybridomas. The hybridoma cell line can be cultured in vitro or in vivo to separate the antibody. The monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma may be purified to a high purity (for example, 95% or more) according to a method well known in the technical field of the present invention . Such a purification technique can be separated from a culture medium or a plurality of solutions by using a purification method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like. As an example of the production of a monoclonal antibody using a hybridoma, a hybridoma is inserted into the abdominal cavity of a mouse, and the hybridoma is separated and purified. The separation and purification of the monoclonal antibody can be carried out using affinity chromatography such as ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or protein A column.
본 발명은 또한, 상기 항 ICAM4 항체를 포함하는 ICAM4의 발현 여부나 발현 정도와 관련된 질환 또는 ICAM4 매개되는 질병에 대한 진단용 조성물을 제공한다.The present invention also provides a diagnostic composition for a disease or an ICAM4 mediated disease associated with the expression or degree of expression of ICAM4 comprising said anti-ICAM4 antibody.
본 발명은 또한, 상기한 항 ICAM4 항체를 생물학적 시료와 접촉시켜 항원-항체 복합체 형성을 검출함으로써, 상기 질병 마커를 검출하는 방법에 관한 것이다. 즉, 본 발명의 개량된 단일클론항체를 생물학적 시료와 반응시키고 항원-항체 복합체 형성을 검출함으로써 ICAM4 단백질을 검출할 수 있다. 본 명세서에서 사용된 용어 "생물학적 시료"란 조직, 세포, 전혈, 혈청, 혈장, 조직 부검 시료(뇌, 피부, 림프절, 척수 등), 뇨, 세포 배양 상등액, 파열된 진핵세포 및 세균 발현계 등을 들 수 있지만 이에 제한되지는 않는다. 이들 생물학적 시료를 조작하거나 조작하지 않은 상태로 본 발명의 항체와 반응시켜 ICAM4 단백질의 존재를 확인할 수 있다. 본 명세서에서 사용된 용어 "항원-항체 복합체"란 시료 중의 ICAM4 단백질 항원과 이를 인지하는 본 발명에 따른 항 ICAM4 항체의 결합물을 의미하며, 이러한 항원-항체 복합체의 형성은 비색법(colormetric method), 전기화학법(electrochemical method), 형광법(fluorimetric method), 발광법(luminometry), 입자계수법(particle counting method), 육안측정법(visual assessment) 및 섬광계수법(scintillation counting method)으로 이루어진 그룹 중에서 선택되는 임의의 방법으로 검출할 수 있다. 그러나 반드시 이들로만 제한되지 않고 다양한 응용과 적용이 가능하다.The present invention also relates to a method of detecting said disease marker by contacting said anti-ICAM4 antibody with a biological sample to detect antigen-antibody complex formation. That is, the ICAM4 protein can be detected by reacting an improved monoclonal antibody of the present invention with a biological sample and detecting antigen-antibody complex formation. As used herein, the term "biological sample" refers to a tissue, cell, whole blood, serum, plasma, tissue autopsy sample (brain, skin, lymph node, spinal cord, etc.), urine, cell culture supernatant, ruptured eukaryotic cell, But is not limited thereto. These biological samples may be reacted with the antibodies of the invention in a manipulated or untreated state to confirm the presence of the ICAM4 protein. The term " antigen-antibody complex " as used herein refers to a combination of ICAM4 protein antigen in the sample and the anti-ICAM4 antibody according to the present invention recognizing the same. The formation of such an antigen-antibody complex may be performed by a colormetric method, An arbitrary one selected from the group consisting of an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment, and a scintillation counting method. . ≪ / RTI > However, various applications and applications are possible without being limited thereto.
본 발명에서는 항원-항체 복합체를 검출하기 위하여 여러 가지 표지체를 사용할 수 있다. 구체적인 예로는 효소, 형광물, 리간드, 발광물, 미소입자, 방사성 동위원소로 이루어진 그룹 중에서 선택될 수 있으며, 반드시 이들로만 국한되지는 않는다. In the present invention, various markers can be used to detect an antigen-antibody complex. Specific examples include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, and radioactive isotopes.
바람직하게는, 항원-항체 복합체를 효소면역흡착법(ELISA)을 이용하여 검출할 수 있다. 효소면역흡착법(ELISA)에는 고체 지지체에 부착된 항원을 인지하는 표지된 항체를 이용하는 직접적 ELISA, 고체 지지체에 부착된 항원을 인지하는 항체의 복합체에서 포획 항체를 인지하는 표지된 이차 항체를 이용하는 간접적 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 표지된 또 다른 항체를 이용하는 직접적 샌드위치 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 또 다른 항체와 반응시킨 후 이 항체를 인지하는 표지된 2차 항체를 이용하는 간접적 샌드위치 ELISA 등 다양한 ELISA 방법을 포함한다. 상기 단일클론 항체는 검출 표지를 가질 수 있으며, 검출표지를 가지지 않을 경우는 이들 단일클론 항체를 포획할 수 있고 검출 표지를 가지는 또 다른 항체를 처리하여 확인할 수 있다.Preferably, the antigen-antibody complexes can be detected using enzyme immunoassay (ELISA). Enzyme immunoassay (ELISA) includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes the capture antibody in a complex of an antibody recognizing the antigen attached to a solid support , A direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support, and the other antibody recognizing the antigen in the complex of the antibody and the antigen attached to the solid support. And indirect sandwich ELISA using labeled secondary antibodies recognizing the antibody. The monoclonal antibody can have a detection label, and when it does not have a detection label, it can capture another monoclonal antibody and can be identified by treating another antibody having the detection label.
본 발명은 또한, 항-ICAM4 항체를 포함하는 골수유래 면역억제세포 (Myeloid- derived suppressor cells, MDSC) 감별용 조성물을 제공한다. 상기 MDSC 감별은 앞서 검토한 바와 같은 질병 진단 혹은 질병 마커 검출 방법을 이용할 수 있다. The present invention also provides a composition for the identification of myeloid-derived suppressor cells (MDSC) comprising an anti-ICAM4 antibody. The above-mentioned MDSC discrimination can use the disease diagnosis method or the disease marker detection method as discussed above.
본 발명은 또한, 상기 항 ICAM4 항체(항 ICAM4 항체의 단일클론항체에 대한 키메릭항체, 인간화항체, 이중특이성 항체 및 항체-약물 복합체를 포함함)를 포함하는 진단용 조성물 또는 진단 키트를 제공한다. 본 발명의 진단용 조성물에 사용되는 항 ICAM4 항체는, 이 항체가 ICAM4를 선별적으로 인지할 수 있는 한, 항 ICAM4 항체의 단편도 사용할 수 있다. 이러한 항체 단편은 F(ab')2, Fab, Fab', Fv 단편 등을 포함할 수 있다. 상기 진단키트에는 ICAM4를 선별적으로 인지하는 단일클론항체 또는 이의 단편 및 면역학적 분석에 사용되는 도구/시약이 포함될 수 있다. 면역학적 분석에 사용되는 도구/시약으로는 적합한 담체, 검출 가능한 신호를 생성할 수 있는 표지 물질, 용해제, 세정제 등이 포함된다. 또한, 표지 물질이 효소인 경우에는 효소 활성을 측정할 수 있는 기질 및 반응 정지제를 포함할 수 있다. 적합한 담체로는, 이에 한정되지는 않으나, 가용성 담체, 예를 들어 당 분야에 공지된 생리학적으로 허용되는 완충액, 예를 들어 PBS, 불용성 담체, 예를 들어 폴리스틸렌, 폴리에틸렌, 폴리프로필렌, 폴리에스테르, 폴리아크릴로니트릴, 불소 수지, 가교 덱스트란, 폴리사카라이드, 라텍스에 금속을 도금한 자성 미립자와 같은 고분자, 기타 종이, 유리, 금속, 아가로스 및 이들의 조합일 수 있다.The present invention also provides diagnostic compositions or diagnostic kits comprising said anti-ICAM4 antibody (comprising a chimeric antibody, a humanized antibody, a bispecific antibody and an antibody-drug complex for a monoclonal antibody of an anti-ICAM4 antibody). The anti-ICAM4 antibody used in the diagnostic composition of the present invention can also use fragments of the anti-ICAM4 antibody so long as the antibody can selectively recognize ICAM4. Such antibody fragments may include F (ab ') 2, Fab, Fab', Fv fragments, and the like. The diagnostic kit may include monoclonal antibodies or fragments thereof that selectively recognize ICAM4 and tools / reagents used in immunological assays. Tools / reagents used in immunological assays include suitable carriers, labeling agents capable of producing detectable signals, solubilizers, detergents, and the like. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring the enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.
본 발명의 검출 방법 및 진단키트에 사용하기 위한 검정 시스템은, 이에 한정되는 것은 아니나, ELISA 플레이트, 딥-스틱 디바이스, 면역크로마토그래피 시험 스트립 및 방사 분할 면역검정 디바이스, 및 플로우-쓰로우(flow-through) 디바이스 등을 포함한다.The assay system for use in the detection methods and diagnostic kits of the present invention may be applied to a variety of assay systems including, but not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips and split- through devices and the like.
또한, 본 발명은 상기 항 ICAM4 항체, 이의 키메릭항체, 인간화항체, 이중특이성 항체 또는 항체-약물 복합체를 포함하는, ICAM4의 발현 여부나 발현 정도와 관련된 질환 또는 ICAM4 매개되는 질환 치료용 조성물을 제공한다. 상기 질환은 바람직하게는 암이다. 본 발명의 치료 가능한 암은 본 발명의 항체치료제를 이용하여 선택적으로 사멸시킬 수 있는 암은 제한 없이 포함될 수 있으나, 그 예로 피부, 소화기, 비뇨기, 생식기, 호흡기, 순환기, 뇌 또는 신경계의 암이 있으며, 구체적으로 간암, 담관암, 폐암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 유방암, 나팔관암, 자궁내막암, 자궁경부암, 질암, 음문암, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반암종, 중추신경계 (central nervous system, CNS) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종일 수 있다. 바람직하게는 본 발명의 목적상 ICAM4을 발현하는 암일 수 있다. 또한, 상기 질환은 골수형성이상증후군일 수 있다. In addition, the present invention provides a composition for treating a disease or ICAM4 mediated disease associated with the expression or expression level of ICAM4, comprising the anti-ICAM4 antibody, its chimeric antibody, humanized antibody, bispecific antibody or antibody-drug complex do. The disease is preferably cancer. The treatable cancer of the present invention may include, without limitation, cancer that can be selectively killed using the antibody therapeutic agent of the present invention, such as skin, digestive, urinary, reproductive, respiratory, circulatory, brain or nervous system cancer Cancer, cancer of the ovary, cancer of the rectum, cancer of the stomach, cancer of the anus, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, liver cancer, liver cancer, Cancer of the uterus, cancer of the cervix, vaginal cancer, mucin cancer, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, Lymphoma, lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, Waterbody can be Zen. Preferably for the purpose of the present invention, to express ICAM4. The disease may also be a myelodysplastic syndrome.
또한, 상기 치료용 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 상기 "약학적으로 허용가능한 담체"란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. In addition, the therapeutic composition may comprise a pharmaceutically acceptable carrier. The term " pharmaceutically acceptable carrier " refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound.
본 발명의 상기 조성물은 약학적으로 유효한 양으로 단일 또는 다중 투여될 수 있다. 치료용 조성물은 액제, 산제, 에어로졸, 캡슐제, 장용피 정제 또는 캡슐제 또는 좌제의 형태로 투여할 수 있다. 투여 경로는 복강내 투여, 정맥내 투여, 근육내 투여, 피하내 투여, 내피 투여, 경구 투여, 국소 투여, 비내 투여, 폐내 투여, 직장내 투여 등을 포함하지만, 이에 제한되지는 않는다. 그러나 경구 투여시, 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 되어야 한다. 또한 제약 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The composition of the present invention may be administered in single or multiple doses in a pharmaceutically effective amount. The therapeutic composition may be administered in the form of a solution, a powder, an aerosol, a capsule, an intravaginal tablet or a capsule or a suppository. Routes of administration include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, endothelial, oral, topical, intranasal, intrarectal, and the like. However, upon oral administration, the peptide will be digested and the oral composition should be formulated to coat the active agent or protect it from degradation from above. The pharmaceutical composition may also be administered by any device capable of transferring the active agent to the target cell.
상기 조성물이 제형화되는 경우 항-ICAM4 항체가 주어진 시점에서 소망하는 생물학적 활성을 보유하도록 고려된다. 그 시간에서의 소망하는 생물학적 활성은 그 시간 내에 나타나는 소망하는 생물학적 활성의 약 30% 이내, 바람직하게는 약 20% 이내인 경우, 약학적 조성물은 소망하는 생물학적 활성에 대한 적합한 분석에서 측정된바 대로 제조된다. 항-ICAM4 단일클론항체의 소망하는 생물학적 활성을 측정하는 측정법은 본원의 실시예에서 설명된 바와 같이 수행될 수 있다. 문헌 Schultze et al. (1998) Proc. Natl. Acad. ScL USA 92:8200-8204; Denton et al.(1998) Pediatr. Transplant. 2:6-15; Evans et al. (200O) J Immunol. 164:688-697; Noelle (1998) Agents Actions Suppl. 49:17-22; Lederman et al. (1996) Curr. Opin. Hematol. 3:77-86; Coligan et al. (1991) Current Protocols in Immunology 13:12; Kwekkeboom et al. (1993) Immunology 79:439-444; 및 미국 등록 특허 번호 5,674,492 및 5,847,082에 설명된 측정법을 참조하며; 본원에서 참고자료로서 포함된다.When the composition is formulated, the anti-ICAM4 antibody is considered to retain the desired biological activity at a given point in time. If the desired biological activity at that time is within about 30%, and preferably within about 20%, of the desired biological activity occurring within that time, the pharmaceutical composition may be used as determined in a suitable assay for the desired biological activity . Assays for measuring the desired biological activity of an anti-ICAM4 monoclonal antibody can be performed as described in the Examples herein. Schultze et al. (1998) Proc. Natl. Acad. ScL USA 92: 8200-8204; Denton et al. (1998) Pediatr. Transplant. 2: 6-15; Evans et al. (200O) < / RTI > J Immunol. 164: 688-697; Noelle (1998) Agents Actions Suppl. 49: 17-22; Lederman et al. (1996) Curr. Opin. Hematol. 3: 77-86; Coligan et al. (1991) Current Protocols in Immunology 13:12; Kwekkeboom et al. (1993) Immunology 79: 439-444; And U.S. Pat. Nos. 5,674,492 and 5,847,082; Which is incorporated herein by reference.
본 발명의 항-ICAM4 항체는 액체 약학적 제형으로 제형화 될 수 있다. 항-ICAM4 항체는 공지의 모든 방법을 사용하여 제조될 수 있으며, 본 명세서에 개시된 방법을 포함한다. 본 발명의 일 실시예에서, 항-ICAM4 항체는 하이브리도마 세포 세포주 KCLRF-BP-00303 내에서 재조합적으로 생산되었다. 액체 약학적 제형은 상기 단일클론항체의 치료적 유효량을 포함할 수 있다. 제형 내에 존재하는 이들의 항체의 양은 투여 경로 및 소망하는 투여 부피를 고려한다.The anti-ICAM4 antibodies of the present invention can be formulated into liquid pharmaceutical formulations. Anti-ICAM4 antibodies can be prepared using any known method and include the methods disclosed herein. In one embodiment of the invention, the anti-ICAM4 antibody was recombinantly produced in the hybridoma cell line KCLRF-BP-00303. Liquid pharmaceutical formulations may comprise a therapeutically effective amount of the monoclonal antibody. The amount of these antibodies present in the formulation takes into account the route of administration and the desired dosage volume.
본 발명의 조성물은 약제학적으로 유효한 양으로 투여한다. '약제학적으로 유효한 양'이란 의학적 치료 또는 예방에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료 또는 예방하기에 충분한 양을 의미하며, 유효 용량 수준은 질환의 중증도, 약물의 활성, 환자의 연령, 체중, 건강, 성별, 환자의 약물에 대한 민감도, 사용된 본 발명 조성물의 투여 시간, 투여 경로 및 배출 비율 치료 기간, 사용된 본 발명의 조성물과 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 또한 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. By " pharmaceutically effective amount " is meant an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prophylaxis, and the effective dosage level will vary depending on factors such as the severity of the disease, The composition of the present invention can be used in combination with the composition of the present invention, including drugs used in combination with the composition of the present invention, and other medical fields including, for example, body weight, health, sex, sensitivity of the patient to the drug, May be determined according to well known factors. In addition, the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
적합한 완충액은 종래의 산 및 이들의 염을 포함하나 이에 한정되지 않으며, 여기서, 짝이온은, 예를 들면, 나트륨, 칼륨, 암모늄, 칼슘, 또는 마그네슘이 될 수 있다. 약학적 액체 제형의 완충제로 사용될 수 있는 종래의 산 및 이들의 염의 예는, 숙신산 또는 숙신산염, 시트르산 또는 시트르산염, 아세트산 또는 아세트산염, 타르타르산 또는 타르타르산염, 인산 또는 인산염, 글루콘산 또는 글루콘산염, 글루탐산 또는 글루타메이트, 아스파르트산 또는 아스파르트산염, 말레산 또는 말레산염, 및 말산 또는 말산염 완충액을 포함하나 이에 한정되지 않는다. 제형 내의 완충액 농도는 약 1 mM 내지 약 50 mM가 될 수 있고, 약 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 또는 약 1 mM 내지 약 50 mM의 범위 내의 다른 값을 포함할 수 있다. 본 발명의 일 구체예에서, 제형 내의 완충액 농도는 약 5 mM 내지 약 15 mM이고, 약 5 mM, 6 mM, 7mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 또는 약 5 mM 내지 약 15 mM의 범위 내의 다른 값을 포함할 수 있다.Suitable buffers include, but are not limited to, conventional acids and salts thereof, wherein the counterion may be, for example, sodium, potassium, ammonium, calcium, or magnesium. Examples of conventional acids and salts thereof that can be used as buffers in pharmaceutical liquid formulations include succinic or succinic acid salts, citric acid or citric acid salts, acetic acid or acetic acid salts, tartaric acid or tartaric acid salts, phosphoric acid or phosphate salts, gluconic acid or gluconic acid salts , Glutamic acid or glutamate, aspartic acid or aspartate, maleic acid or maleate, and malic or malate buffers. The buffer concentration in the formulation can be from about 1 mM to about 50 mM and is about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 45 mM, 50 mM, or about 1 mM to about 50 mM. In one embodiment of the invention, the buffer concentration in the formulation is about 5 mM to about 15 mM, and about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, mM, 15 mM, or from about 5 mM to about 15 mM.
근접 등장성인 액체 약학적 제형이 바람직한 경우, 항-ICAM4 항체 및 완충액을 포함하는 액체 약학적 제형은 제형에 근접 등장성을 부여하기에 충분한 등장제의 양을 추가로 포함할 수 있다. "근접 등장성"은 수성 제형이 약 240 mmol/kg 내지 약 360 mmol/kg, 바람직하게는 약 240 내지 약 340 mmol/kg, 더 바람직하게는 약 250 내지 약 330 mmol/kg, 더 더욱 바람직하게는 약 260 내지 약 320 mmol/kg, 그보다 더 바람직하게는 약 270 내지 약 310 mmol/kg의 삼투질농도(osmolarity)를 지니는 것을 의미한다. 용액의 등장성을 결정하는 방법은 당해 기술 분야의 숙련자에게 공지되어 있다. 예를 들면, 문헌 Setnikar et al. (1959) J Am. Pharm. Assoc. 48:628를 참조할 수 있다.When a proximal isotonic liquid pharmaceutical formulation is desired, a liquid pharmaceutical formulation comprising an anti-ICAM4 antibody and a buffer may additionally comprise an amount of isotonic agent sufficient to impart proximity to the formulation. By " proximal isotonicity " is meant that the aqueous formulation is about 240 mmol / kg to about 360 mmol / kg, preferably about 240 to about 340 mmol / kg, more preferably about 250 to about 330 mmol / Quot; means having an osmolarity of about 260 to about 320 mmol / kg, even more preferably about 270 to about 310 mmol / kg. Methods for determining the isotonicity of a solution are known to those skilled in the art. See, for example, Setnikar et al. (1959) J. Am. Pharm. Assoc. 48: 628.
당해 기술 분야의 숙련자는 약학적 조성물 내에 등장성의 제공에 유용한 다양한 약학적으로 허용 가능한 용질을 잘 알고 있다. 등장제는 체액과 동등한 수준으로 본 발명의 액체 약학적 제형의 삼투압을 조절할 수 있는 모든 시약이 될 수 있다. 생리학적으로 허용가능한 등장제를 사용하는 것이 바람직하다. 따라서 치료적 유효량의 항-ICAM4 항체 및 완충액을 포함하는 액체 약학적 제형은 등장성을 제공하기 위하여 사용될 수 있는 성분, 예를 들면, 염화 나트륨; 아미노산 예컨대, 알라닌, 발린, 및 글리신; 글루코스, 덱스트로스, 푸룩토오스, 슈크로오스, 말토오스, 만니톨, 트레할로스, 글리세롤, 소르비톨, 및 자일리톨을 포함하나 이에 한정되지 않는 당 및 당 알콜(폴리올); 아세트산, 다른 유기산 또는 이들의 염, 및 상대적으로 소량의 시트르산염 또는 인산염을 추가로 포함할 수 있다. 당해 기술 분야의 숙련자는 액체 제형의 강직성을 제공하기에 적합한 추가적 약제를 인식하고 있다.Those skilled in the art are familiar with various pharmaceutically acceptable solutes useful in providing isotonicity within pharmaceutical compositions. The isotonic agent may be any reagent capable of controlling the osmotic pressure of the liquid pharmaceutical formulation of the present invention at levels equivalent to body fluids. It is preferred to use physiologically acceptable isotonic agents. Thus, a liquid pharmaceutical formulation comprising a therapeutically effective amount of an anti-ICAM4 antibody and a buffer can comprise an ingredient that can be used to provide isotonicity, for example, sodium chloride; Amino acids such as alanine, valine, and glycine; Sugars and sugar alcohols (polyols) including but not limited to glucose, dextrose, fructose, sucrose, maltose, mannitol, trehalose, glycerol, sorbitol, and xylitol; Acetic acid, other organic acids or salts thereof, and relatively small amounts of citric acid salts or phosphates. Those skilled in the art are aware of additional agents suitable for providing the rigidity of liquid formulations.
또한, 본 발명의 바람직한 일 구체예에서, 항-ICAM4 항체 및 완충액을 포함하는 액체 약학적 제형은 등장제로서 염화나트륨을 추가로 포함할 수 있다. 제형 내의 염화나트륨 농도는 강직성에 대한 다른 성분의 기여도에 따라 달라질 것이다. 예를 들어, 염화나트륨의 농도는 약 50 niM 내지 약 300 mM, 약 50 mM 내지 약 250 mM, 약 50 mM 내지 약 200 mM, 약 50 mM 내지 약 175 mM, 약 50 mM 내지 약 150 mM, 약 75 mM 내지 약 175 mM, 약 75 mM 내지 약 150 mM, 약 100 mM 내지 약 175 mM, 약 100 mM 내지 약 200 mM, 약 100 mM 내지 약 150 mM, 약 125 mM 내지 약 175 mM, 약 125 mM 내지 약 150 mM, 약 130 mM 내지 약 170 mM, 약 130 mM 내지 약 160 mM, 약 135 mM 내지 약 155 mM, 약 140 mM 내지 약 155 mM, 또는 약 145 mM 내지 약 155 mM이다.Further, in one preferred embodiment of the present invention, the liquid pharmaceutical formulation comprising the anti-ICAM4 antibody and the buffer may further comprise sodium chloride as an isotonic agent. The sodium chloride concentration in the formulation will depend on the contribution of the other ingredients to the rigidity. For example, the concentration of sodium chloride may range from about 50 niM to about 300 mM, from about 50 mM to about 250 mM, from about 50 mM to about 200 mM, from about 50 mM to about 175 mM, from about 50 mM to about 150 mM, from about 100 mM to about 200 mM, from about 100 mM to about 150 mM, from about 125 mM to about 175 mM, from about 125 mM to about 175 mM, from about 75 mM to about 175 mM, from about 75 mM to about 150 mM, from about 100 mM to about 175 mM, About 150 mM, about 130 mM to about 170 mM, about 130 mM to about 160 mM, about 135 mM to about 155 mM, about 140 mM to about 155 mM, or about 145 mM to about 155 mM.
본 발명의 액체 약학적 제형의 처리과정에서 냉동 해동 또는 기계적 전단에 기한 단백질 분해는 용액-공기 접면에서의 표면 장력을 낮추기 위하여 계면활성제를 제형에 편입시킴으로써 억제될 수 있다. 따라서 본 발명의 일 구체예에서, 액체 약학적 제형은 치료적 유효량의 항-ICAM4 항체, 완충액을 포함하며, 계면활성제를 추가적으로 포함한다. 본 발명의 다른 실시 상태에서, 액체 약학적 제형은 항-ICAM4 항체, 완충액, 등장제를 포함하며, 계면활성제를 추가로 포함할 수 있다.In the treatment of the liquid pharmaceutical formulations of the present invention, freeze thaw or mechanical shear-induced proteolysis can be suppressed by incorporating the surfactant into the formulation to lower the surface tension at the solution-air interface. Thus, in one embodiment of the invention, the liquid pharmaceutical formulation comprises a therapeutically effective amount of an anti-ICAM4 antibody, a buffer, and additionally a surfactant. In another embodiment of the invention, the liquid pharmaceutical formulation comprises an anti-ICAM4 antibody, a buffer, an isotonic agent, and may further comprise a surfactant.
사용되는 전형적인 계면활성제는 비이온성 계면활성제이며, 폴리옥시에틸렌 소르비톨 에스테르 예컨대, 폴리소르베이트 80 (Tween 80) 및 폴리소르베이트 20 (Tween 20); 폴리옥시프로필렌-폴리옥시에틸렌 에스테르 예컨대, 플루로닉 F68; 폴리옥시에틸렌 알콜 예컨대, 브리지(Brij) 35; 시메티콘; 폴리에틸렌 글리콜 예컨대, PEG400; 라이소포스파티딜콜린; 및 폴리옥시에틸렌-p-t-옥틸페놀 예컨대, 트리톤 X-100을 포함한다. 계면활성제 또는 에멀젼화제에 의한 전통적인 약제의 안정화는, 예를 들면, 문헌 Levine et al. (1991) J Parenteral Sci. Technol. 45(3):160-165에서 설명하였으며, 본원에서 참고자료로서 포함되어 있다.Typical surfactants used are nonionic surfactants, such as polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); Polyoxypropylene-polyoxyethylene esters such as Pluronic F68; Polyoxyethylene alcohols such as Brij 35; Simethicone; Polyethylene glycol such as PEG 400; Lysophosphatidylcholine; And polyoxyethylene-p-t-octylphenol, such as Triton X-100. The stabilization of traditional medicaments by surfactants or emulsifiers is described, for example, by Levine et al. (1991) J Parenteral Sci. Technol. 45 (3): 160-165, which is incorporated herein by reference.
한편, 상기 항체는 치료 효과를 증진시키기 위하여 독성 물질들과 결합되어 융합단백(fusion protein)을 형성할 수 있다. 독소 단백질로는 리신(ricin), 사포린(saporin), 젤로닌(gelonin), 모로딘(momordin), 디프테리아독소, 또는 녹농균독소(pseudomonas toxin)일 수 있으나, 특별히 그 종류를 제한하는 것은 아니다. On the other hand, the antibody may be combined with toxic substances to form a fusion protein in order to enhance the therapeutic effect. The toxin protein may be ricin, saporin, gelonin, momordin, diphtheria toxin, or pseudomonas toxin, but the type of the toxin is not particularly limited.
또한, 본 발명은 상기 항체치료제를 개체에 투여하는 단계를 포함하는 ICAM4의 발현 여부나 발현 정도와 관련된 질환 또는 ICAM4 매개되는 질병의 치료 방법을 제공한다. 본 명세서에서 사용된 용어 "개체"란 포유동물로서 인간, 말, 개, 고양이, 돼지, 염소, 토끼, 햄스터, 원숭이, 기니피그(guinea pigs), 래트, 마우스, 도마뱀, 뱀, 양, 소, 물고기 및 새를 포함하여 임의의 동물(예컨대, 인간)을 의미한다. 본 발명에서 사용되는 용어, "치료"는 본 발명에 따른 항-ICAM4 단일클론항체(이의 개량된 항체를 포함함) 및 약제학적으로 허용 가능한 담체를 포함하는 조성물의 투여로 상기 질병의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다.In addition, the present invention provides a method for treating a disease or an ICAM4-mediated disease associated with the expression or degree of expression of ICAM4 comprising the step of administering the antibody therapeutic agent to a subject. The term " animal " as used herein refers to a mammal such as a human, horse, dog, cat, pig, goat, rabbit, hamster, monkey, guinea pigs, rat, mouse, lizard, And any animal (e. G., Human), including birds. As used herein, the term " treatment " refers to the administration of a composition comprising an anti-ICAM4 monoclonal antibody (including its modified antibody) according to the present invention and a pharmaceutically acceptable carrier, Or any change that beneficially modifies it.
본 발명의 항-ICAM4 단일클론항체(이의 개량된 항체를 포함함)는 다양한 목적을 위해 다른 항체, 생물학적 활성을 가지는 제제 또는 물질과 함께 사용될 수 있다. 예를 들어, 본 발명의 항-ICAM4 단일클론항체는 공지의 항암 약물과도 병용하여 사용될 수 있다The anti-ICAM4 monoclonal antibody (including its modified antibodies) of the present invention can be used with other antibodies, biologically active agents or substances for various purposes. For example, the anti-ICAM4 monoclonal antibody of the present invention can be used in combination with a known anti-cancer drug
이하 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.
실험 재료 및 방법Materials and Methods
1. 세포주1. Cell line
실험에 사용된 세포주 K562 (ATTC, #CCL-243), HEL (ATTC, #TIB-180), TF-1 (ATTC, #CRL-2003), PLC/PRF/5 (ATTC, #CRL-8024), Hep3B (ATTC, #HB-8064), SK-HEP-1 (ATTC, #HTB-52), HepG2 (ATTC, #HB-8065), C3A (ATTC, #CRL-10741), 786-O (ATTC, #CRL-1932), Huh-7 (KCLB, #60104), SNU-308 (KCLB, #00308), SNU-601 (KCLB, #00601)은 10% FBS(CORNING, USA)가 포함된 RPMI 배지(Welgene, Korea)에서 5% CO2, 37℃ 조건으로 배양하였다.(ATTC, # CRL-8024), HEL (ATTC, # TIB-180), TF-1 (ATTC, # CRL-2003), PLC / PRF / ATCC, # HB-8065), C3A (ATTC, # CRL-10741), 786-O (ATTC, # HB-8064), SK-HEP- , 10% FBS (CORNING, USA)), SNU-308 (KCLB, # 00308) and SNU-601 (KCLB, # 00601) (Welgene, Korea) at 5% CO 2 and 37 ° C.
2. 항체치료제 생산용 세포주 개발2. Development of cell line for antibody treatment
인간 항체 불변부위가 함께 발현되도록 제작된 pOptiVEC, pcDNA3.3 벡터(DiNonA, Korea)에 항-ICAM4 마우스 항체 1E1의 가변부위를 암호화하고 있는 DNA를 삽입하고, 제작된 재조합 벡터를 디히드로엽산환원효소(DHFR) 음성 세포주인 CHO DG44 (Invitrogen, #A1100001)에 lipofectamine 2000 (Invitrogen, USA) 시약을 사용하여 형질주입 하였다. 형질주입된 세포만 선별하기 위해서 데옥시리보뉴클레오사이드를 포함하지 않는 αMEM배지(Welgene, Korea)에서 계대배양 한 후, 항체 생산 수율을 높이기 위해서 MTX (Sigma, USA)과 G418 (Invitrogen, USA)을 첨가한 PowerCHO (LONZA, Switzerland)배지로 교체 하여 배양하였다. MTX와 G418 첨가 농도를 점진적으로 높여주면서 96 well plate (SPL, Korea)에서 세포 배양액을 well 당 1개 이하의 세포가 들어갈 정도로 희석하여 배양하는 한계희석배양법(limiting dilution)을 진행하여 단일 클론을 확보하였다.DNA encoding the variable region of the anti-ICAM4 mouse antibody 1E1 was inserted into pOptiVEC, pcDNA3.3 vector (DiNonA, Korea) which was designed to express the human antibody constant region together, and the produced recombinant vector was transformed with dihydrofolate reductase (Invitrogen, USA) reagent to CHO DG44 (Invitrogen, # A1100001), a negative control (DHFR) negative cell line. (Sigma, USA) and G418 (Invitrogen, USA) in order to select the transfected cells, and subcultured in αMEM medium containing no deoxyribonucleosides (Welgene, Korea) (LONZA, Switzerland). MTX and G418 concentrations were gradually increased and the cells were cultured in a 96-well plate (SPL, Korea) with a limiting dilution to dilute the cell culture to a level of less than 1 cell per well Respectively.
3. 유세포 분석3. Flow cytometry
다양한 기원의 세포주들을 5×105개씩 준비한 후에 항-ICMA4 키메릭 항체 1E1을 10 μg/mL로 넣고, 4℃에서 30분간 반응시켰다. 그 다음 차가운 PBS로 세척하고 FITC 형광이 결합된 항-인간 면역글로불린(DiNonA, Korea)을 4℃에서 20분간 반응시켰다. 다시 차가운 PBS로 세척하고 1% 파라포름알데하이드(Sigma-Aldrich, USA)로 고정한 후에 형광 활성 유세포 측정기 FACS Calibur (BD, USA)를 이용하여 분석하였다.After 5 × 10 5 cell lines of various origins were prepared, 10 μg / mL of anti-ICMA4 chimeric antibody 1E1 was added and reacted at 4 ° C. for 30 minutes. Then, the cells were washed with cold PBS and reacted with FITC fluorescence-conjugated anti-human immunoglobulin (DiNonA, Korea) at 4 ° C for 20 minutes. After washing with cold PBS and fixed with 1% paraformaldehyde (Sigma-Aldrich, USA), the cells were analyzed with a fluorescence activated flow cytometer FACS Calibur (BD, USA).
4. 인간화항체 제작4. Humanized antibody production
항-ICAM4 마우스 항체 1E1의 가변부위 아미노선 서열의 in silico 인간화를 프랑스의 BIOTEM사에 의뢰하였다. BIOTEM사에서 받은 중쇄 13개, 경쇄 8개 아미노산 서열을 코스모진텍(Seoul, Korea)에 코돈최적화를 의뢰하여 각각의 아미노산을 암호화하는 발현 최적의 DNA 서열을 확보하였다. 확보한 DNA 서열을 인간 항체 불변부위가 함께 발현되도록 제작된 pcDNA3.4 발현벡터(DiNonA, Korea)에 삽입하고, 호스트 세포 HEK-293T(ATCC, #CRL-11268)에 형질 주입하여 총 104개 조합의 인간화항체를 발현시켰다. (서열번호 11 내지 서열번호 31)Wherein the -ICAM4 mouse antibody variable region amino acid sequence of the 1E1 line in silico Humanization was commissioned to BIOTEM of France. Optimized DNA sequences encoding 13 amino acids and 13 light chains from BIOTEM were obtained by codon optimization in CosmoginTech (Seoul, Korea). The obtained DNA sequence was inserted into a pcDNA3.4 expression vector (DiNonA, Korea) which was designed to express a constant region of a human antibody, and transfected into a host cell HEK-293T (ATCC, # CRL-11268) Of humanized antibody. (SEQ ID NO: 11 to SEQ ID NO: 31)
5. 항-ICAM4 마우스 항체 1E1-사포린(5. Anti-ICAM4 Mouse Antibody 1E1-Saurin ( mm 1E1-SAP) 복합체 개발1E1-SAP) complex
항-ICAM4 마우스 항체 1E1과 사포린(Sigma-Aldrich, USA)의 결합은 관련 논문을 참고하여 진행하였다[Jeon YK, et al. Targeting of a developmentally regulated epitope of CD43 for the treatment of acute leukemia. Cancer Immunol Immunother. 2011;60(12):1697-706]. 간략히 설명하면, 항-ICAM4 마우스 항체 1E1을 N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP)로 활성화 시킨 다음 분자량 1대 5 비율로 티올레이트화된 사포린과 결합시켰다. 결합되지 않은 메르캅토기(SH-)를 차단하기 위하여 N-ethylmaleimide를 첨가하였고, 단백질 G 친화성 크로마토그래피로 결합되지 않은 비 결합 사포닌을 제거하였다. 결합된 항체-약물 복합체를 ImmunoPure 용출 완충액 (Pierce Biotechnology, USA)로 용출시키고 PBS에 투석하였다. 유리 사포닌에 의한 오염 가능성은 SDS-PAGE으로 확인하였다.The binding of anti-ICAM4 mouse antibody 1E1 to saponin (Sigma-Aldrich, USA) was performed with reference to the related paper [Jeon YK, et al. Targeting of a developmentally regulated epitope of CD43 for acute leukemia. Cancer Immunol Immunother. 2011; 60 (12): 1697-706). Briefly, anti-ICAM4 mouse antibody 1E1 was activated with N-succinimidyl-3- (2-pyridyldithio) -propionate (SPDP) and bound to thiolated saponin at a molecular weight of 1: 5. N-ethylmaleimide was added to block unbound mercapto groups (SH-), and unconjugated saponins not bound were removed by protein G affinity chromatography. The conjugated antibody-drug complex was eluted with ImmunoPure Elution Buffer (Pierce Biotechnology, USA) and dialyzed against PBS. The possibility of contamination by free saponins was confirmed by SDS-PAGE.
6. 항체-약물 복합체의 유효성 시험법6. Efficacy test of antibody-drug complex
1) 항-ICAM4 마우스 항체 1E1-사포린(1) anti-ICAM4 mouse antibody 1E1 -sporin ( mm 1E1-SAP) 유효성 시험1E1-SAP) efficacy test
96 well plate에 well 당 적백혈병 세포주 K562 5×104개씩 분주하고 항체-약물 복합체를 농도별로 3개씩 처리하였다. 37℃에서 72시간동안 배양시키고, 세포 생존능 측정은 tetrazolium salt를 이용하여 살아있는 세포의 양을 측정하는 Ey-cytox(DOGEN, Korea)를 이용하였다.The cells were treated with 5 × 10 4 K562 cells per well in a 96-well plate, and antibody-drug complexes were treated three times per concentration. The cells were cultured at 37 ° C for 72 hours. Ey-cytox (DOGEN, Korea) was used to measure cell viability using tetrazolium salt.
2) 항-ICAM4 키메릭 항체 1E1-엠탄신(2) Anti-ICAM4 chimeric antibody 1E1-Mentansin ( chichi 1E1-DM1) 유효성 시험1E1-DM1) Effectiveness test
24 well plate (SPL, Korea)에 well 당 적백혈병 세포주 K562 1×105개씩 분주하고 항체-약물 복합체를 농도별로 처리하였다. 2시간동안 배양시키고, 세포 생존능 측정은 Propidium iodide (PI) staing 한 후에 사멸세포를 유세포 분석을 통하여 진행하였다.Well plates (SPL, Korea) were plated at a dose of 1 × 10 5 cells per well of the leukemic cell line K562 and the antibody-drug complexes were treated at different concentrations. The cells were cultured for 2 hours. Cell viability was determined by flow cytometry after propidium iodide (PI) staining.
7. 적백혈병 이종이식 동물모델7. Red Leukemia xenograft animal model
적백혈병 이종이식 동물모델은 SCID 마우스(중앙실험동물, Korea)에 적백혈병 세포주인 HEL로 확립하였다. 먼저 한 마리당 혈청이 들어있지 않은 RPMI배지 100 μL에 1×107으로 준비한 HEL 세포를 마우스의 오른쪽 옆구리에 피하 접종하였다. 이식한 종양의 크기가 100 mm3가 되었을 때 항체-약물 복합체의 유효성을 시험하였다. 항체-약물 복합체를 2.5 mg/kg의 용량으로 3일 간격으로 4번 마우스 꼬리 정맥에 투여하였고, 종양의 길이와 너비를 caliper로 3 일에 한 번씩 측정하여 Gutman 등이 제안한 방법의 공식으로 종양의 부피를 산출하였다. An animal model of leukemia xenografts was established with HEL, an acute leukemia cell line, in SCID mouse (Central laboratory animal, Korea). First, HEL cells prepared at 1 × 10 7 in 100 μL of RPMI medium containing no serum were subcutaneously inoculated into the right side of the mouse. The efficacy of the antibody-drug complex was tested when the size of the transplanted tumor reached 100 mm 3 . The antibody-drug conjugate was administered to the tail vein four times at a dose of 2.5 mg / kg every 3 days. The length and width of the tumor were measured with caliper once every 3 days. Gutman et al. The volume was calculated.
8. 뉴라민가수분해효소 처리 농도 구배에 따른 항체 반응성 시험법8. Antibody reactivity test with concentration gradient of neuraminase
한 시험당 적백혈병 세포주 K562 2×105개와 정상 적혈구 4×107개에 뉴라민가수분해효소(ELPIS, Korea)를 0, 125, 500, 1000 unit로 처리한 후 37℃에서 40분 동안 반응 시켜서 세포 표면의 시알릴화 정도의 차이를 형성하였다. 시알릴화 정도의 차이는 항-ICAM4 키메릭 항체 1E1으로 유세포 분석을 통해 확인하였다.One test was carried out with 2 × 10 5 K562 cell lines and 4 × 10 7 normal red blood cells treated with 0, 125, 500 and 1000 units of neuraminase (ELPIS, Korea) and incubated at 37 ° C. for 40 minutes To form a difference in the sialylation degree of the cell surface. The difference in sialylation degree was confirmed by flow cytometry with anti-ICAM4 chimeric antibody 1E1.
9. 이중특이성 항체 제작9. Production of bispecific antibody
이중특이성 항체는 scFv-Fc 형태로 제작되었으며, 항-ICAM4 항체의 가변부위와, 종양과 관련 있는 표적에 대한 항체의 가변부위 DNA를 overlap PCR을 통해 scFv 형태로 제작하였다. 중쇄와 경쇄의 가변부위를 하나로 연결하기 위해 사용된 링커 서열은 G-G-S-S-R-S-S-S-G-G-G-G-S-G-G-G-G-(서열번호 10)이며, PCR은 95℃에서 30초, 60℃에서 30초, 72℃에서 45초 조건에서 30회 진행하였다. 항체 가변부위 scFv를 인간 항체의 불변부위가 함께 발현되도록 제작된 pOptiVEC 발현벡터(DiNonA, Korea)에 삽입하고, 호스트 세포 HEK-293T에 형질주입하여 scFv-Fc 형태의 이중특이성 항체를 발현시켰다.The bispecific antibody was prepared in the form of scFv-Fc. The variable region of the anti-ICAM4 antibody and the variable region DNA of the antibody against the tumor-related target were prepared in the form of scFv by overlap PCR. The linker sequence used to link the variable regions of the heavy chain and the light chain was GGSSRSSSGGGGSGGGG- (SEQ ID NO: 10), and PCR was performed 30 times at 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 45 seconds . The antibody variable region scFv was inserted into a pOptiVEC expression vector (DiNonA, Korea) designed to express a constant region of a human antibody, and the scFv-Fc-type bispecific antibody was expressed by transfection into host cell HEK-293T.
10. 면역조직화학염색10. Immunohistochemical staining
시험에 사용된 파라핀 포매조직은 충북대학교 병원에서 제공받았다. 상온에서 자일렌과 에탄올을 이용한 탈파라핀 과정을 통해 파라핀을 제거하고, 마이크로웨이브를 이용해 10mM 구연산 버퍼(sodium citrate, ph 6.0)로 15분간 항원을 재생시켰다. 1차 항체는 10 μg/mL 농도로 200 μL씩 사용하였고, ChemMate Detection kit (Dako, Denmark)를 이용하여 발색하였다.The paraffin embedded tissues used in the test were obtained from Chungbuk National University Hospital. The paraffin was removed by paraffin treatment using xylene and ethanol at room temperature, and the antigen was regenerated with microwave of 10 mM citrate buffer (pH 6.0) for 15 minutes. The primary antibody was used at a concentration of 10 μg / mL (200 μL) and color developed using the ChemMate Detection kit (Dako, Denmark).
실험결과Experiment result
1. 항-ICAM4 재조합 항체치료제의 개발1. Development of Anti-ICAM4 Recombinant Antibody Therapeutics
1) 항-ICAM4 키메릭 항체 1E1 (1) Anti-ICAM4 chimeric antibody 1E1 ( chichi 1E1) 생산용 세포주 개발1E1) Development of cell line for production
항-ICAM4 키메릭 항체 1E1을 안정적으로 생산하는 세포주를 만들기 위해서 발현벡터는 pOptiVEC과 pcDNA3.3을 사용하였고, 호스트 세포로는 CHO DG44 세포주를 사용하였다(도 1a). 호스트 세포에 1E1 항체치료제의 유전자가 삽입된 발현 벡터를 형질주입(transfection) 한 후 선별배지를 통하여 형질 주입된 세포만을 선별하였고, 생산성 향상을 위하여 MTX와 G418 약제를 사용하였다. 약제의 농도를 높여주면서 한계희석배양법을 진행하였고, 20계대 이상 배양 후 최종적으로 1000 nM MTX와 200 μg/mL G418이 첨가된 PowerCHO2 배지 조건에서 안정적으로 키메릭 항체 1E1를 생산하는 세포주를 확보하였다(최종생산농도 7.5 mg/L). 일련의 생산용 세포주 제작 과정을 도 1b에 도식화 하였다.POptiVEC and pcDNA3.3 were used as expression vectors to construct a cell line stably producing anti-ICAM4 chimeric antibody 1E1, and a CHO DG44 cell line was used as a host cell (Fig. 1A). After transfection of the expression vector containing the gene for the therapeutic antibody of 1E1 into the host cell, only the transfected cells were selected through the selection medium, and MTX and G418 drugs were used to improve the productivity. The cell line was stably cultured in a PowerCHO2 culture medium supplemented with 1000 nM MTX and 200 μg / mL G418, and then cultured in a cell line producing chimeric antibody 1E1 Final product concentration 7.5 mg / L). A series of production cell lines production schemes are illustrated in Figure 1B.
2) 항-ICAM4 키메릭 항체 1E1의 세포 반응성 비교분석2) Comparison of cellular reactivity of anti-ICAM4 chimeric antibody 1E1
항-ICAM4 키메릭 항체 1E1 생산용 세포주로부터 생산 된 키메릭 항체 1E1의 항원결정부위 동정을 위해서 원래의 마우스 항체 1E1로 표적 세포의 항원을 포화시킨 후 항-ICAM4 키메릭 항체 1E1 생산용 세포주로부터 생산 된 키메릭 항체 1E1의 반응성을 확인한 결과 같은 항원결정부위를 인지하는 것을 확인하였다(도 2a). 또한 키메릭 항체 1E1의 항원 친화도 분석을 위하여 ICAM4가 과발현된 세포주와 발현되지 않은 세포주를 대상으로 유세포 분석을 시행하였고, 키메릭 1E1 항체치료제의 농도 구배에 따라서 ICAM4 과발현 세포주에서의 반응성이 점차 증가하는 것을 확인하였다. 반대로 ICAM4가 발현되지 않은 세포주에서는 항체의 농도와 상관없이 반응성이 증가하지 않았다(도 2b). K562 세포에서 키메릭 1E1 항체치료제의 항원 포화농도는 5 μg/mL로 원래의 마우스 항체 1E1과 유사하였다.Anti-ICAM4 Chimeric Antibody To identify the antigenic region of chimeric antibody 1E1 produced from the cell line for production of the anti-ICAM4 chimeric antibody 1E1, the antigen of the target cell was saturated with the original mouse antibody 1E1 and then produced from the cell line for producing anti-ICAM4 chimeric antibody 1E1 As a result of confirming the reactivity of the chimeric antibody 1E1, it was confirmed that the same antigen recognition site was recognized (Fig. 2A). In order to analyze the antigen affinity of chimeric antibody 1E1, flow cytometry analysis was performed on ICAM4 overexpressed and unexpressed cell lines. The reactivity of ICAM4 overexpressed cell line gradually increased according to the concentration gradient of the chimeric 1E1 antibody treatment . Conversely, in the cell line in which ICAM4 was not expressed, the reactivity did not increase regardless of the antibody concentration (Fig. 2B). In K562 cells, the chimeric 1E1 antibody treatment had an antigen saturation concentration of 5 μg / mL, similar to the original mouse antibody 1E1.
3) 항-ICAM4 인간화항체 제작3) Production of anti-ICAM4 humanized antibody
항-ICAM4 키메릭 항체의 면역원성을 줄이기 위해 인간화항체들을 제작하고 선별하였다. 프랑스의 BIOTEM사로부터 in silico로 중쇄 가변부위 13개, 경쇄 가변부위 8개 버전 총 104개 조합의 항-ICAM4 인간화항체 서열을 얻었다(표 1).Humanized antibodies were prepared and screened to reduce the immunogenicity of anti-ICAM4 chimeric antibodies. 13 heavy chain variable region to the in silico from BIOTEM captured French dog, a light chain variable region versions a total of eight to obtain a humanized antibody wherein -ICAM4 sequence of the 104 combination (Table 1).
수용체 서열로 사용되는 인간 생식세포 유래의 가변 부위를 갖는 마우스 유전자 또는 그들의 인간화 항체의 동일성 비교(%)(%) Of mouse genes having variable regions derived from human germline cells used as receptor sequences or their humanized antibodies.
IMGT human germlineIMGT human germline HybridomaHybridoma Humanized versionsHumanized versions
A* A * BB CC DD EE
<Heavy chain><Heavy chain> IGHV4-59*05 IGHV4-59 * 05 60.860.8 77.377.3 79.379.3 79.379.3 81.481.4
IGHV3-11*01IGHV3-11 * 01 51.651.6 74.574.5 76.576.5 77.677.6 76.576.5 78.678.6
IGHV5-51*01IGHV5-51 * 01 45.445.4 73.573.5 75.575.5 74.574.5 76.576.5
<Light chain><Light chain> IGKV1-9*01IGKV1-9 * 01 75.875.8 88.488.4 90.590.5 90.590.5 92.692.6
IGKV3-11*01IGKV3-11 * 01 67.467.4 81.181.1 84.284.2 85.385.3 87.487.4
* In framework, the parental mouse residues at Kabat positions H24, H29, H48, H67, H78, L2, L4, L38 and L44 have been retained in version A. * In framework, the parental mouse residues at Kabat positions H24, H29, H48, H67, H78, L2, L4, L38 and L44 have been retained in version A.
유전자 클로닝을 통하여 pcDNA3.4 발현벡터에 중쇄 13개, 경쇄 8개 유전자를 각각 삽입하여 104개 재조합 인간화항체를 293T 세포에 임시발현 시켜 기존 키메릭 항체와의 항원친화도를 비교분석 하였다. 항원 발현 세포주에 대한 결합 친화도를 기준으로 기존 재조합 키메릭 항체와 유사한 친화도를 보이는 인간화항체 조합을 4개 선별하였다(도 4a). 키메릭 항체와 마찬가지로 선별된 인간화항체의 항원결정부위를 확인한 결과 원래의 마우스 항체 1E1과 같은 항원결정부위를 인지하는 것을 확인하였다(도 4b). 추가로 분자 모델링에 의한 마우스 항체 1E1 가변부위의 3D 구조를 구축하여 CDR-grafting 과정에서 발생 할 수 있는 항체 가변부위 구조의 변형을 예측할 수 있었다(도 3). Gene cloning was performed to insert 13 heavy chains and 8 light chains into the pcDNA3.4 expression vector, and 104 recombinant humanized antibodies were transiently expressed in 293T cells to compare the antigen affinity with existing chimeric antibodies. Based on the binding affinity for the antigen-expressing cell line, four combinations of humanized antibodies showing affinities similar to those of the existing recombinant chimeric antibody were selected (Fig. 4A). Like the chimeric antibody, the antigenic determinant site of the selected humanized antibody was confirmed, and it was confirmed that it recognized the antigenic determinant site as the original mouse antibody 1E1 (FIG. 4B). In addition, the 3D structure of the mouse antibody 1E1 variable region was constructed by molecular modeling, and the deformation of the antibody variable region structure that could occur in the CDR-grafting process was predicted (FIG. 3).
* In framework, the parental mouse residues at Kabat positions H24, H29, H48, H67, H78, L2, L4, L38 and L44 have been retained in version A. * In framework, the parental mouse residues at Kabat positions H24, H29, H48, H67, H78, L2, L4, L38 and L44 have been retained in version A.
유전자 클로닝을 통하여 pcDNA3.4 발현벡터에 중쇄 13개, 경쇄 8개 유전자를 각각 삽입하여 104개 재조합 인간화항체를 293T 세포에 임시발현 시켜 기존 키메릭 항체와의 항원친화도를 비교분석 하였다. 항원 발현 세포주에 대한 결합 친화도를 기준으로 기존 재조합 키메릭 항체와 유사한 친화도를 보이는 인간화항체 조합을 4개 선별하였다(도 4a). 키메릭 항체와 마찬가지로 선별된 인간화항체의 항원결정부위를 확인한 결과 원래의 마우스 항체 1E1과 같은 항원결정부위를 인지하는 것을 확인하였다(도 4b). 추가로 분자 모델링에 의한 마우스 항체 1E1 가변부위의 3D 구조를 구축하여 CDR-grafting 과정에서 발생 할 수 있는 항체 가변부위 구조의 변형을 예측할 수 있었다(도 3). Gene cloning was performed to insert 13 heavy chains and 8 light chains into the pcDNA3.4 expression vector, and 104 recombinant humanized antibodies were transiently expressed in 293T cells to compare the antigen affinity with existing chimeric antibodies. Based on the binding affinity for the antigen-expressing cell line, four combinations of humanized antibodies showing affinities similar to those of the existing recombinant chimeric antibody were selected (Fig. 4A). Like the chimeric antibody, the antigenic determinant site of the selected humanized antibody was confirmed, and it was confirmed that it recognized the antigenic determinant site as the original mouse antibody 1E1 (FIG. 4B). In addition, the 3D structure of the mouse antibody 1E1 variable region was constructed by molecular modeling, and the deformation of the antibody variable region structure that could occur in the CDR-grafting process was predicted (FIG. 3).
4) 항-ICAM4 인간화항체의 1E1 결합 친화도 확인 4) Confirmation of 1E1 binding affinity of anti-ICAM4 humanized antibody
상기 3)에서의 가변부위 구조적 변형에 대한 예측을 토대로, 실제 인간화항체의 결합친화도에 변화가 있는지 여부를 조사하였다. 키메릭 항체와 인간화항체의 4가지 조합을 비교한 결과, 일부 인간화항체 조합에서 1E1 결합 친화도가 증가하는 클론이 관찰되었다. 이는 특히 VH1A(서열번호 8) 및 VL2A(서열번호 25)의 조합에서 가장 증가되는 경향을 보였고, VH2A(서열번호 12) 및 VL2C(서열번호 27)의 조합에서도 결합친화도가 증가되는 경향을 보였다.(도 5)Based on the prediction of the variable site structural modification in the above 3), it was examined whether there was a change in the binding affinity of an actual humanized antibody. A comparison of four combinations of chimeric and humanized antibodies showed that clones with increased 1E1 binding affinity in some humanized antibody combinations were observed. This showed the highest increase especially in the combination of VH1A (SEQ ID NO: 8) and VL2A (SEQ ID NO: 25), and the binding affinity also increased in the combination of VH2A (SEQ ID NO: 12) and VL2C (SEQ ID NO: 27) . (Fig. 5)
5) 항-ICAM4 항체-약물 복합체 개발5) Development of anti-ICAM4 antibody-drug complex
먼저 항-ICAM4 마우스 항체 1E1 사포린 복합체(m1E1-SAP)는 마우스 항체 1E1과 사포린에 SDPD와 2-Iminothiolane으로 각각 메르캅토기(SH-)를 붙여주어 항체와 약물을 이황화 결합을 통하여 연결하였고(도 6a), 항-ICAM4 키메릭 항체 1E1 엠탄신 복합체(chi1E1-DM1)은 키메릭 항체 1E1에 succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMMC) 링커를 이용해 엠탄신이 3개에서 4개가 연결된 구조로 이루어져 있으며 미국 Creative Biolabs사에 의뢰해서 제작하였다(도 6b). First, anti-mouse antibody 1E1 -ICAM4 four tarpaulins composite (m 1E1-SAP) are connected to each paste given a mercapto group (SH-) as SDPD and 2-Iminothiolane for tarpaulins Mouse 1E1 antibody and use the antibody and the drug via a disulfide bond (Fig. 6a), anti-ICAM4 chimeric antibody 1E1 manganese complex ( chi 1E1-DM1) was conjugated to chimeric antibody 1E1 using succinimidyl-4- [N-maleimidomethyl] cyclohexane- 3 to 4 of which are connected to each other and are manufactured by Creative Biolabs, USA (Fig. 6B).
6) 항-ICAM4 항체-약물 복합체의 유효성6) Efficacy of the anti-ICAM4 antibody-drug complex
개발한 항-ICAM4 항체-약물 복합체 m1E1-SAP와 chi1E1-DM1의 유효성을 검증 해 본 결과, in vitro에서 m1E1-SAP와 chi1E1-DM1을 K562와 HEL 세포주를 대상으로 세포 생존력을 측정하였을 때 IC50값이 각각 10 ng/mL, 3.7 μg/mL로 항체-약물 복합체 농도 의존적으로 세포 생존력이 감소하였도(도 7a, 7b), 적백혈병 세포주 이종이식 SCID 마우스 in vivo 모델에서 chi1E1-DM1이 약 73%의 종양성장 억제 효능을 보였다(도 8).A development -ICAM4 wherein the antibody-drug complex with m-1E1 SAP Results present to verify the validity of the chi 1E1-DM1, 10 ng / mL in the in vitro IC 50 value, as measured by cell viability the m 1E1-SAP and chi 1E1-DM1 targets K562 and HEL cells, respectively, 3.7 μg (Fig. 7a, 7b), the chi 1E1-DM1 showed an inhibitory effect on tumor growth of about 73% in an in vivo model of SCID mouse of leukemia cell line xenograft 8).
2. 항-ICAM4 항체치료제의 독성 시험2. Toxicity test of anti-ICAM4 antibody treatment
1) 뉴라민가수분해효소 처리 농도 구배에 따른 항-ICAM4 항체의 반응성1) Reactivity of anti-ICAM4 antibody with concentration gradient of neuraminase
항-ICAM4 항체치료제가 정상 적혈구에 결합하여 생길 수 있는 부작용을 줄이기 위하여 뉴라민 가수분해효소를 농도별로 종양세포주와 적혈구에 처리하여 항-ICAM4 항체치료제가 암세포주에는 반응하지만 적혈구에는 반응하지 않는 농도를 알아내었다(도 9a). 종양 세포주와 적혈구 표면 ICAM4 항원의 시알산화 정도의 차이를 측정하기 위하여 뉴라민 가수분해효소를 각각 1000 unit 처리한 후, 항-ICAM4 항체치료제의 반응성을 측정한 결과 종양 세포주에서는 거의 변함없이 결합이 유지되었지만, 적혈구에서의 반응성이 소실되었다(도 9b). 실제 혈액암 환자 생체환경과 유사하게 정상 적혈구와 종양 세포가 혼재된 조건에서 뉴라민 가수분해효소를 1000 unit을 처리하여 정상 적혈구에 대한 항-ICAM4 항체의 반응성은 줄어들고 종양 세포에 대한 반응성이 증가함을 확인하였다(도 9c).In order to reduce the adverse effects that the anti-ICAM4 antibody treatment can cause by binding to normal red blood cells, the neuraminase is treated with the tumor cell line and the erythrocyte at different concentrations so that the anti-ICAM4 antibody treatment agent reacts with cancer cells but does not react with red blood cells (Fig. 9A). To measure the difference in sialic acidity between tumor cell lines and erythrocyte surface ICAM4 antigen, 1000 units of neuraminase were treated with anti-ICAM4 antibody, and the reactivity of anti-ICAM4 antibody treatment was almost constant in the tumor cell line , But the reactivity in red blood cells disappeared (FIG. 9B). Similar to the biological environment of patients with actual blood cancer, treatment with 1000 units of neuraminase in the presence of normal red blood cells and tumor cells decreased the reactivity of anti-ICAM4 antibody to normal red blood cells and increased reactivity to tumor cells (Fig. 9C).
2) 항-ICAM4 이중특이성 항체 개발2) Development of anti-ICAM4 bispecific antibody
Acute Myeloid Leukemia에 특이적으로 발현되는 ICAM4 (CD242)와 CEACAM6 (CD66c) 를 표적으로 하는 인간화 항체의 서열을 조합하여 새로운 물질인 이중항체를 개발하였다. 이중항체는 ICAM-4를 표적으로 하는 whole IgG1 항체 Fc 부분의 CH3 도메인 말단에 CEACAM6를 표적으로 하는 variable region 중쇄와 경쇄를 특정 서열로 연결시킨 single chain fragment variable(scFV) 부분을 연결시켜서 개발하였다. ICAM-4를 표적으로 하는 중쇄, 경쇄 조합은 pcDNA 3.4 벡터를 이용해 발현시킨 재조합 인간화 서열 104개의 조합 중 중쇄1A-경쇄1A(w1s2H1L3), 중쇄1A-경쇄2A(w1s2H1L4), 중쇄1B-경쇄1A(w1s2H2L3), 중쇄1B-경쇄2A(w1s2H2L4) 를 사용하였고 아래 scFV로 사용된 CEAMCAM 6를 표적으로 하는 중쇄, 경쇄 부분은 E3 서열을 사용하였다. 다른 조합으로는 CEACAM6를 표적으로 하는 whole IgG1 항체 Fc 부분의 CH3 도메인 말단에 ICAM-4를 표적으로 하는 항체의 서열을 연결하였다. 이때 사용한 조합은 CEACAM6를 표적으로 하는 E3서열과 ICAM-4를 표적으로 하는 중쇄1A-경쇄1A(w2s1H1L3), 중쇄1B-경쇄2A(w2s1H2L4) 서열을 조합하여 사용하였다. 총 6개 이중항체는 Expi CHO 세포를 이용하여 생산하였고, 정제된 항체를 이용하여 항원 결합까지 모두 확인하였다(도 10a). 이 이중항체는 항체 의존 세포 독성실험에서 단일 항체보다 월등한 효과를 나타내었다. 그 중 ICAM-4 를 표적으로 하는 항체의 경쇄 1A 서열로 조합된 w1s2H1L3, w1s2H2L3 항체가 가장 좋은 효과를 보였다.(도 10b, 10c)We have developed a novel double antibody that combines sequences of humanized antibodies targeting ICAM4 (CD242) and CEACAM6 (CD66c) specifically expressed in Acute Myeloid Leukemia. The double antibody was developed by ligating the variable region heavy chain targeting CEACAM6 to the CH3 domain end of the whole IgG1 antibody Fc portion targeting ICAM-4 and the single chain fragment variable (scFV) portion linking the light chain to the specific sequence. The heavy and light chain combinations that target ICAM-4 are the heavy chain 1A-light chain 1A (w1s2H1L3), heavy chain 1A-light chain 2A (w1s2H1L4), heavy chain 1B-light chain 1A (w1s2H2L3) and heavy chain 1B-light chain 2A (w1s2H2L4) were used. The heavy chain targeting the CEAMCAM 6 used for the scFV below and the E3 sequence for the light chain portion were used. In another combination, the sequence of the antibody targeting ICAM-4 was ligated to the CH3 domain end of the whole IgG1 antibody Fc portion targeting CEACAM6. The combination used was a combination of the E3 sequence targeting CEACAM6 and the heavy chain 1A-light chain 1A (w2s1H1L3) and heavy chain 1B-light chain 2A (w2s1H2L4) sequences targeting ICAM-4. A total of 6 double antibodies were produced using Expi CHO cells, and antigen binding was confirmed using purified antibodies (FIG. 10A). This double antibody was superior to monoclonal antibody in the antibody - dependent cytotoxicity assay. Among them, the w1s2H1L3 and w1s2H2L3 antibodies combined with the light chain 1A sequence of an antibody targeting ICAM-4 showed the best effect (Figures 10b and 10c)
3) 정상 조직에서의 항-ICAM4 항체의 면역 조직 화학적 성상 분석3) Immunohistochemical characterization of anti-ICAM4 antibody in normal tissues
항-ICAM4 항체치료제의 조직교차반응을 확인한 결과 적혈구에 강한 양성 소견을 보였으며, 적혈구를 제외한 파라핀 포매된 태아조직(8주령) 및 성인 간, 신장, 자궁 등 검사를 시행한 모든 조직에 대한 교차반응성은 없었다(도 11). 추가로 동결절편 검사에서 정상의 인체 편도조직 및 정상 장기에서도 교차반응성이 관찰되지 않았다.The cross-reactivity of anti-ICAM4 antibody treatment showed strong positive for erythrocytes. The cross-reactivity of all tissues examined including paraffin-embedded fetal tissue (8 weeks old) and adult liver, kidney and uterus excluding red blood cells There was no reactivity (Fig. 11). In addition, no cross reactivity was observed in normal human tonsil tissues and normal organs in the frozen section.
3. 항-ICAM4 항체치료제의 확대적응증3. Indication of anti-ICAM4 antibody treatment
1) 다양한 인간 종양 세포주 선별검사1) Screening of various human tumor cell lines
다양한 기원의 종양 세포주에 대한 항-ICAM4 키메릭 항체 1E1의 반응성을 유세포 분석법으로 조사한 결과 키메릭 항체 1E1은 일부 간암 세포주와 담관암 세포주에서 20-30%의 발현을 확인할 수 있었고(표 2), 말초혈액 세포에서는 적혈구를 제외한 모든 세포에서 반응성이 없는 것을 확인하였다.The response of anti-ICAM4 chimeric antibody 1E1 to various tumor cell lines was analyzed by flow cytometry. As a result, chimeric antibody 1E1 was found to express 20-30% in some liver cancer cells and bile duct cancer cells (Table 2) In blood cells, all cells except red blood cells were found to have no reactivity.
각종 인간 암 세포주에 대한 키메라 항체 1E1의 면역 반응성.Immunoreactivity of chimeric antibody 1E1 to various human cancer cell lines.
Cell lineCell line OriginOrigin Reactivity of Reactivity of chichi 1E11E1
K562 K562 ErythroleukemiaErythroleukemia ++++++++
HEL  HEL ErythroleukemiaErythroleukemia ++++++++
TF-1  TF-1 ErythroleukemiaErythroleukemia ++++++++
PLC/PRF/5 PLC / PRF / 5 Hepatocellular carcinomaHepatocellular carcinoma ++
Hep3B Hep3B Hepatocellular carcinomaHepatocellular carcinoma ++
SK-HEP-1 SK-HEP-1 Hepatocellular carcinomaHepatocellular carcinoma --
HepG2 HepG2 Hepatocellular carcinomaHepatocellular carcinoma ++
C3A C3A Hepatocellular carcinomaHepatocellular carcinoma ++
Huh-7 Huh-7 Hemochromatotic cellHemochromatotic cell ++
SNU-308 SNU-308 CholangiocarcinomaCholangiocarcinoma ++++
HCCC-9810 HCCC-9810 CholangiocarcinomaCholangiocarcinoma --
GIST-T1 GIST-T1 Gastrointestinal stromal tumorsGastrointestinal stromal tumors --
SNU-601 SNU-601 Gastric carcinomaGastric carcinoma --
786-O 786-O Renal cell carcinomaRenal cell carcinoma --
(% gated = -; <10%, +; 10-25%, ++; 25-50%, +++; 50-75%, ++++; 75-100%)(% gated = -; <10%, +; 10-25%, ++; 25-50%, +++; 50-75%, ++++; 75-100%
2) 임상시료분석2) Clinical sample analysis
ICAM4는 건강한 사람의 말초혈액 세포에서는 적혈구를 제외한 모든 세포에서 발현되지 않지만 여러 임상 시료를 선별검사 한 결과, 실제 골수형성이상증후군(MDS) 환자의 골수성미성숙세포(myeloblast)에서 일부 비정상적으로 ICAM4가 발현됨을 알아내었다(도 12).ICAM4 is not expressed in all cells except red blood cells in healthy peripheral blood cells, but as a result of screening of various clinical samples, ICAM4 is expressed in some abnormal abnormality in myeloblast of actual bone marrow dysplasia syndrome (MDS) (Fig. 12).
이를 확인하기 위하여, 말기 암 환자 혈액 1 mL에 RBC lysis 버퍼 9mL을 넣고 36℃ 에서 약 10분간 반응시켜서 적혈구를 제거 한 후, 세포 1×106개에 CD45-APC (BD, USA), 1E1-FITC (DiNonA, Korea)를 4℃에서 30분간 반응시켰다. 그 다음 차가운 PBS로 반응이 끝난 세포를 세척하고 1% 파라포름알데하이드로 고정한 후에 형광 활성 유세포 측정기 FACS Calibur (BD, USA)를 이용하여 분석하였다.In order to confirm this, the terminal cancer patients blood 1 mL in RBC lysis buffer into a 9mL by 10 min at 36 ℃ after removing the red blood cells, 1 × 10 6 cells, dog CD45-APC (BD, USA) , 1E1- FITC (DiNonA, Korea) was reacted at 4 DEG C for 30 minutes. The cells were then washed with cold PBS and fixed with 1% paraformaldehyde followed by analysis using a fluorescence activated flow cytometer FACS Calibur (BD, USA).
그 결과, 일부 암환자 샘플에서 항-ICAM4 항체(1E1)에 대한 항원이 높아져 있는 것을 확인하였다. 이 단핵세포 안에 MDSC가 높아져 있는 것으로 보이며, 이러한 결과는 1E1 항체의 타겟이 적백혈병에 국한되지 않는다는 것을 보여주는 것이다. (도 13)As a result, it was confirmed that the antigen for the anti-ICAM4 antibody (1E1) was elevated in some cancer patient samples. These mononuclear cells seem to have elevated MDSCs, and these results show that the target of the 1E1 antibody is not limited to leukemia. (Fig. 13)
4. 골수유래억제세포의 항- ICAM4 항체 표지 확인 4 . Identification of Anti- ICAM4 Antibody Markers in Bone Marrow-Derived Inhibitory Cells
임상시험 심사위원회(IRPB) 신청에 앞서 파일럿 조사를 위해 시험에 사용된 말초 혈액은 충북대학교 병원에서 제공받았다. 환자의 말초 혈액에서 말초 혈액 단핵구(PBMC)를 분리한 다음 항 ICAM-4 1E1 항체를 넣고 4℃에서 20분간 반응시킨다. 반응 후, 차가운 PBS로 세척한 다음 FITC 형광이 결합된 항-인간면역글로불린(GAH-FITC, DiNonA, Korea)로 처리하여 4℃에서 20분간 반응했다. 상기 반응 후, 차가운 PBS로 세척한 다음 1% 파라포름알데히드 (Sigma-Aldrich, USA)로 고정한 후에 형광 활성 유세포 측정기 FACS Calibur(BD, USA)로 측정하였다. Prior to the application of the IRBB, the peripheral blood used for the pilot study was provided at Chungbuk National University Hospital. Peripheral blood mononuclear cells (PBMC) are isolated from the patient's peripheral blood and then incubated with anti-ICAM-4 1E1 antibody at 4 ° C for 20 minutes. After the reaction, the cells were washed with cold PBS, treated with FITC-conjugated anti-human immunoglobulin (GAH-FITC, DiNonA, Korea) and reacted at 4 ° C for 20 minutes. After the reaction, the cells were washed with cold PBS, fixed with 1% paraformaldehyde (Sigma-Aldrich, USA), and then measured with a fluorescence activated flow cytometer FACS Calibur (BD, USA).
현재 가장 많이 쓰이는 골수 유래 억제 세포의 표면 마커는 CD33+CD11b+HLADR-인데, 고형암 환자의 혈액에서 골수 유래 억제 세포의 표면 마커인 CD33+CD11b+가 82.5%가 나왔다. 이 중 HLA DR- 가 70%인 것으로 보아, 고형암 환자의 혈액에는 골수유래억제세포가 존재 하는 것으로 확인할 수 있으며, 이 중 1E1 이 인지하는 ICAM4 항원이 37.85%로 나타났다. 따라서 골수 유래 억제 세포의 표면 마커로 ICAM4를 사용할 수 있음을 항-ICAM4 단일클론 항체 1E1으로 확인하였다. (도 14) The surface markers of the most commonly used bone marrow-derived inhibitory cells are CD33 + CD11b + HLADR-, with 82.5% of CD33 + CD11b + being the surface markers of bone marrow-derived inhibitory cells in the blood of solid cancer patients. Of these, HLA DR- was 70%, indicating that bone marrow-derived inhibitory cells were present in the blood of patients with solid tumors, of which ICAM4 antigen recognized by 1E1 was 37.85%. Therefore, it was confirmed that ICAM4 can be used as a surface marker of bone marrow-derived inhibitory cells as anti-ICAM4 monoclonal antibody 1E1. (Fig. 14)
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
본 발명의 항 ICAM4 항체 및 이를 이용한 ICAM4 발현 질환에 대한 진단 또는 치료용 조성물은 기존의 항체와 비교하여 우수한 항원 친화도를 가지고 있으며, 개량된 형태로서 관련 질병에 따라 다양하게 적용가능하여, 해당 질명의 진단 또는 치료에 유용하게 사용될 수 있다. The anti-ICAM4 antibody of the present invention and the composition for diagnosing or treating the disease expressing ICAM4 using the same have excellent antigen affinity as compared with the conventional antibody and can be applied variously according to the related disease as an improved form, Can be usefully used for diagnosis or treatment.

Claims (17)

  1. 서열번호 1 내지 서열번호 5의 아미노산 서열 중 선택된 서열을 포함하는 상보성 결정 영역(CDR)의 중쇄 영역; 및 서열번호 6 내지 서열번호 9의 아미노산 서열 중 선택된 서열을 포함하는 상보성 결정 영역(CDR)의 경쇄 영역을 포함하는 항 ICAM4 항체. A heavy chain region of a complementarity determining region (CDR) comprising a selected one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 5; And a light chain region of a complementarity determining region (CDR) comprising a selected one of the amino acid sequences of SEQ ID NO: 6 to SEQ ID NO: 9.
  2. 제1항에 있어서, The method according to claim 1,
    상기 항체는 키메릭 항체; 인간화 항체; 이중특이성 항체; 및 항체 약물 복합체(Antibody-Drug Conjugate, ADC)를 이루는 항체로 이루어진 군으로부터 선택되는 것을 특징으로 하는 항체. Said antibody comprising a chimeric antibody; Humanized antibodies; Bispecific antibodies; And an antibody comprising an antibody-drug complex (Antibody-Drug Conjugate, ADC).
  3. 제2항에 있어서, 3. The method of claim 2,
    상기 키메릭 항체는 인간 항체 불변부위가 함께 발현되도록 제작된 것을 특징으로 하는 항체. Wherein the chimeric antibody is engineered to be expressed together with a human antibody constant region.
  4. 제2항에 있어서, 3. The method of claim 2,
    상기 인간화 항체는 서열번호 11의 아미노산 서열로 이루어진 VH1A; 서열번호 12의 아미노산 서열로 이루어진 VH1B; 서열번호 23의 아미노산 서열로 이루어진 VH1C; 서열번호 14의 아미노산 서열로 이루어진 VH1D; 서열번호 15의 아미노산 서열로 이루어진 VH2A; 서열번호 16의 아미노산 서열로 이루어진 VH2B; 서열번호 17의 아미노산 서열로 이루어진 VH2C; 서열번호 18의 아미노산 서열로 이루어진 VH2D; 서열번호 19의 아미노산 서열로 이루어진 VH2E; 서열번호 20의 아미노산 서열로 이루어진 VH3A; 서열번호 21의 아미노산 서열로 이루어진 VH3B; 서열번호 22의 아미노산 서열로 이루어진 VH3C; 서열번호 23의 아미노산 서열로 이루어진 VH3D 로 구성된 군으로부터 선택되는 중쇄 영역을 포함하는 것을 특징으로 하는 항체. The humanized antibody comprises VH1A consisting of the amino acid sequence of SEQ ID NO: 11; VH1B consisting of the amino acid sequence of SEQ ID NO: 12; VH1C consisting of the amino acid sequence of SEQ ID NO: 23; A VH1D consisting of the amino acid sequence of SEQ ID NO: 14; VH2A consisting of the amino acid sequence of SEQ ID NO: 15; VH2B consisting of the amino acid sequence of SEQ ID NO: 16; VH2C consisting of the amino acid sequence of SEQ ID NO: 17; A VH2D consisting of the amino acid sequence of SEQ ID NO: 18; A VH2E consisting of the amino acid sequence of SEQ ID NO: 19; VH3A consisting of the amino acid sequence of SEQ ID NO: 20; VH3B consisting of the amino acid sequence of SEQ ID NO: 21; VH3C consisting of the amino acid sequence of SEQ ID NO: 22; A heavy chain region selected from the group consisting of VH3D consisting of the amino acid sequence of SEQ ID NO: 23.
  5. 제2항에 있어서, 3. The method of claim 2,
    상기 인간화 항체는 서열번호 24의 아미노산 서열로 이루어진 VL1A; 서열번호 25의 아미노산 서열로 이루어진 VL1B; 서열번호 26의 아미노산 서열로 이루어진 VL1C; 서열번호 27의 아미노산 서열로 이루어진 VL1D; 서열번호 28의 아미노산 서열로 이루어진 VL2A; 서열번호 29의 아미노산 서열로 이루어진 VL2B; 서열번호 30의 아미노산 서열로 이루어진 VL2C; 서열번호 31의 아미노산 서열로 이루어진 VL2D로 구성된 군으로부터 선택되는 경쇄 영역을 포함하는 것을 특징으로 하는 항체. Said humanized antibody comprises VL1A consisting of the amino acid sequence of SEQ ID NO: 24; VL1B consisting of the amino acid sequence of SEQ ID NO: 25; VL1C consisting of the amino acid sequence of SEQ ID NO: 26; VL1D consisting of the amino acid sequence of SEQ ID NO: 27; VL2A consisting of the amino acid sequence of SEQ ID NO: 28; VL2B consisting of the amino acid sequence of SEQ ID NO: 29; VL2C consisting of the amino acid sequence of SEQ ID NO: 30; A light chain region selected from the group consisting of VL2D consisting of the amino acid sequence of SEQ ID NO: 31.
  6. 제2항에 있어서, 3. The method of claim 2,
    상기 이중특이성 항체는 상기 제1항의 항체; 서열번호 10의 아미노산 서열로 이루어진 링커; 및 다른 항원에 특이적인 항체를 포함하는 것을 특징으로 하는 항체. Wherein said bispecific antibody is the antibody of claim 1; A linker consisting of the amino acid sequence of SEQ ID NO: 10; And antibodies specific for other antigens.
  7. 제6항에 있어서, The method according to claim 6,
    상기 이중특이성 항체에 결합된 상기 다른 항원에 특이적인 항체는, 항CEACAM1, 항CEACAM3, 항CEACAM4, 항CEACAM5, 항CEACAM6, 항CEACAM7, 항CEACAM8, 항CEACAM21, 항CEACAM16, 항CEACAM18, 항CEACAM19, 항CEACAM20, 항CD33 및 항CD11b 항체로 이루어진 군으로부터 선택되는 항체인 것을 특징으로 하는 항체. The antibodies specific for the other antigens bound to the bispecific antibody may be selected from the group consisting of anti-CEACAM1, anti-CEACAM3, anti-CEACAM4, anti-CEACAM5, anti-CEACAM6, anti-CEACAM7, anti-CEACAM8, anti- CEACAM21, anti- CEACAM16, anti- CEACAM18, &Lt; / RTI &gt; CEACAM20, anti-CD33 and anti-CD11b antibodies.
  8. 제2항에 있어서, 상기 항체-약물 복합체(Antibody-Drug Conjugate, ADC)는 제 1항의 항체 및; 사포린(saporin), 엠탄신(emtansine), 독소루비신 (Doxorubicin), 카보플라틴(Carboplatin), 시스플라틴 (Cisplatin), 시클로포스파미드 (Cyclophosphamide), 이포스파미드(Ifosfamide), 니드란 (Nidran), 질소머스타드 (메클로에타민 염산염)[Nitrogen mustar(MechlorethamineHCL)], 블레오마이신 (Bleomycin), , 미토마이신 C (Mitomycin C), 시타라빈 (Cytarabine), 플루로우라실(Flurouracil), 젬시타빈 (Gemcitabine), 트리메트렉세이트 (Trimetrexate), 메토크렉세이트 (Methotrexate), 에토포시드 (Etoposide), 빈블라스틴 (Vinblastine), 비노렐빈 (vinorelbine), 알림타 (Alimta), 알트레타민 (Altretamine), 프로카바진 (Procarbazine), 탁솔 (Taxol), 탁소텔 (Taxotere), 토포테칸 (Topotecan) 및 이리노테칸 (Irinotecan)으로 이루어진 군에서 선택된 1이상의 약물과 복합체를 형성하는 것을 특징으로 하는 항체. The antibody of claim 2, wherein the antibody-drug conjugate (ADC) comprises the antibody of claim 1; Saporin, emtansine, Doxorubicin, Carboplatin, Cisplatin, Cyclophosphamide, Ifosfamide, Nidran, Nitrogen mustard (Mechlorethamine HCL), Bleomycin, Mitomycin C, Cytarabine, Flurouracil, Gemcitabine, But are not limited to, trimetrexate, methotrexate, etoposide, vinblastine, vinorelbine, alimta, altretamine, Wherein the antibody is complexed with at least one drug selected from the group consisting of Procarbazine, Taxol, Taxotere, Topotecan and Irinotecan.
  9. 제1항 내지 제8항 중 어느 한 항의 항체를 포함하는 ICAM4 관련 질환의 진단용 조성물. 9. A composition for the diagnosis of an ICAM4-related disease comprising the antibody of any one of claims 1 to 8.
  10. 제9항에 있어서, 10. The method of claim 9,
    상기 ICAM4 관련 질환은 백혈병, 간암, 담관암 또는 골수형성이상증후군(MDS)인 것을 특징으로 하는 진단용 조성물. Wherein said ICAM4-related disease is leukemia, liver cancer, cholangiocarcinoma or myelodysplastic syndrome (MDS).
  11. 제1항 내지 제8항 중 어느 한 항의 항체를 포함하는 ICAM4 관련 질환의 치료용 조성물. 9. A composition for the treatment of an ICAM4 related disease comprising an antibody according to any one of claims 1 to 8.
  12. 제11항에 있어서, 12. The method of claim 11,
    상기 ICAM4 관련 질환은 백혈병, 간암, 담관암 또는 골수형성이상증후군(MDS)인 것을 특징으로 하는 치료용 조성물. Wherein said ICAM4-related disease is leukemia, liver cancer, cholangiocarcinoma, or myelodysplastic syndrome (MDS).
  13. 제11항에 있어서, 12. The method of claim 11,
    상기 조성물은 약학적으로 허용 가능한 담체를 포함하는 약학적 조성물인, 치료용 조성물. Wherein said composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  14. 제1항 내지 제8항 중 어느 한 항의 항체를 포함하는 골수유래면역억제세포(Myeloid- derived suppressor cells, MDSC) 감별용 조성물. 9. A composition for the differentiation of myeloid-derived suppressor cells (MDSC) comprising the antibody of any one of claims 1 to 8.
  15. 제1항 내지 제8항 중 어느 한 항의 항체를 포함하는 골수유래면역억제세포(Myeloid- derived suppressor cells, MDSC) 활성 억제용 조성물. 9. A composition for inhibiting myeloid-derived suppressor cells (MDSC) activity comprising an antibody according to any one of claims 1 to 8.
  16. a) 개체로부터 수득한 생물학적 시료에 제1항의 항체를 접촉시키는 단계; comprising the steps of: a) contacting the antibody of claim 1 to a biological sample obtained from an individual;
    b) 제1항 내지 제8항 중 어느 한 항의 항체에 결합된 ICAM4 를 검출하는 단계; 및b) detecting ICAM4 bound to the antibody of any one of claims 1 to 8; And
    c) 상기 b)에서 검출된 ICAM4의 발현 정도를 정상 대조군과 비교하는 단계를 포함하는, ICAM4를 발현하는 백혈병, 간암, 담관암 또는 골수형성이상증후군(MDS) 진단을 위한 정보 제공방법.c) comparing the degree of expression of ICAM4 detected in step b) with a normal control, wherein the method comprises the step of comparing the expression level of ICAM4 detected in step b) with a normal control.
  17. 제1항 내지 제8항 중 어느 한 항의 항체의 발현 여부를 통하여 ICAM4를 발현하는 백혈병, 간암, 담관암 또는 골수형성이상증후군(MDS)을 진단하는 방법. A method for diagnosing leukemia, liver cancer, cholangiocarcinoma or myelodysplastic syndrome (MDS) expressing ICAM4 through expression of the antibody of any one of claims 1 to 8.
PCT/KR2018/014540 2017-11-24 2018-11-23 Anti-icam4 antibody and composition for diagnosis and treatment of icam4 expression cell-associated disease WO2019103531A1 (en)

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