WO2010047509A9 - Anticorps dirigé contre le dr5 présentant une meilleure affinité et une meilleure stabilité, et composition destinée a prévenir ou traiter le cancer et contenant cet anticorps - Google Patents

Anticorps dirigé contre le dr5 présentant une meilleure affinité et une meilleure stabilité, et composition destinée a prévenir ou traiter le cancer et contenant cet anticorps Download PDF

Info

Publication number
WO2010047509A9
WO2010047509A9 PCT/KR2009/006036 KR2009006036W WO2010047509A9 WO 2010047509 A9 WO2010047509 A9 WO 2010047509A9 KR 2009006036 W KR2009006036 W KR 2009006036W WO 2010047509 A9 WO2010047509 A9 WO 2010047509A9
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cancer
seq
amino acid
chain variable
Prior art date
Application number
PCT/KR2009/006036
Other languages
English (en)
Korean (ko)
Other versions
WO2010047509A2 (fr
WO2010047509A3 (fr
Inventor
김용성
권명희
이승현
박경진
Original Assignee
아주대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020090066518A external-priority patent/KR101117070B1/ko
Application filed by 아주대학교 산학협력단 filed Critical 아주대학교 산학협력단
Publication of WO2010047509A2 publication Critical patent/WO2010047509A2/fr
Publication of WO2010047509A9 publication Critical patent/WO2010047509A9/fr
Publication of WO2010047509A3 publication Critical patent/WO2010047509A3/fr

Links

Images

Classifications

    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present invention specifically binds to a death receptor 5 (hereinafter referred to as "DR5"), anti-DR5 antibody having improved affinity and stability for effectively killing various cancer cells, and a composition for preventing or treating cancer comprising the same. It is about.
  • DR5 death receptor 5
  • anti-DR5 antibody having improved affinity and stability for effectively killing various cancer cells
  • composition for preventing or treating cancer comprising the same. It is about.
  • TRAIL TNF-related apoptosis inducing ligand
  • TNFR protein p53-independent tumor necrosis factor receptor
  • TRAIL recombinant TRAIL
  • TRAIL-sensitive cancer cells cancer cells killed by TRAIL
  • TRAIL-resistant cancer cells cancer cells that do not die by TRAIL are called TRAIL-resistant cancer cells.
  • Antibodies having affinity specific for DR5 include TRA-8 (mouse-derived IgG), a humanized antibody developed from a mouse-derived monoclonal antibody (Walczak et al., Nat. Med., 5: 157-161) and AD5-10 (rat IgG) (Guo et al., J. Biol. Chem., 280: 41940-41952, 2005), HGS-ETR2 (human IgG1), a human-derived monoclonal antibody (Georgakis et al., Br. J. Haematol. , 130: 501-510, 2005) and KMTR2 (human IgG4) (Motoki et al. , Clin. Cancer Res. , 11: 3126-3135, 2005).
  • TRA-8 mouse-derived IgG
  • AD5-10 rat IgG
  • HGS-ETR2 human IgG1
  • KMTR2 human IgG4
  • Apoptosis of eukaryotic cells is characterized by three mechanisms, namely apoptosis, autophagy and necrosis, depending on morphological and biochemical characteristics (Kromer G et al. (2005), Cell Death Differ. 12 Suppl 2: 1463-1467). Among these, apoptosis is classified as type II programmed cell death and is increased by nutrient starvation, environmental stress, or various compounds, and the process is called endoplasmic.
  • reticulum or a newly synthesized double-membrane structure is created, which forms autophagosomes while isolating intracellular organelles (such as mitochondria) or cytoplasmic proteins, ultimately lysosomes and Fusion and destruction (Kondo Y et al. (2005). Nat. Rev. Cancer. 5: 726-734). If the child action is above a certain level, it causes apoptosis, and the development of cancer treatments using such apoptosis is attracting attention (Martinet W et al. (2009), Clinical Science. 116: 697-712).
  • the V fragment of the variable region of the antibody is a fragment that occupies most of the antibody sequence, and there are various subtypes.
  • VH heavy chain variable region
  • VH1a, VH1b, and VH5 are intermediate. Position, and VH2, VH4 and VH6 fall in terms of stability.
  • VL light chain variable region
  • Vk3 and Vk1 are known to be the most stable forms.
  • the CDR grafting method is remarkable, as it shows the results of the studies that improved the thermodynamic stability of antibodies by grafting CDRs of less stable antibodies to subtypes having relatively higher thermodynamic stability and yield of antibody production (Jung et al. , Protein.Eng . , 10: 959-966, 1997).
  • the improvement of the affinity of the therapeutic antibody can be expected to reduce the single dose, it can lead to a comprehensive treatment efficiency, such as cost reduction and increased efficacy, reduced side effects.
  • an increase in ligand-receptor binding affinity in agonists is not necessarily associated with biological activity (Jones et al . , Trends. Biotechnol. , 26: 498-505, 2008).
  • the inventors of the present invention while studying to develop an anti-DR5 antibody with improved affinity and stability than the existing anti-DR5 antibody, and produced a variant of the existing anti-DR5 human antibody, HW1, and the affinity and The stability was improved more than the existing HW1 and confirmed that effectively induce apoptosis by the progeny action on TRAIL-sensitive cancer cells expressing DR5 or TRAIL-resistant cancer cells expressing DR5, and completed the present invention.
  • the present invention is to provide an anti-DR5 antibody with improved affinity and stability.
  • the present invention is to provide a DNA encoding the anti-DR5 antibody.
  • the present invention is to provide a cell transformed with the DNA or an expression vector comprising the same.
  • the present invention is to provide a composition for preventing or treating cancer containing the anti-DR5 antibody as an active ingredient.
  • FIG. 1 is a diagram illustrating primers of VH-CDR2, VH-CDR3, and VL-CDR3 for improving affinity of HW1, an anti-DR5 human antibody.
  • Figure 2 is a diagram showing the manufacturing process of a variant library for improving the affinity of the anti-DR5 human antibody HW1.
  • 3 is a diagram analyzing the process of selecting a high affinity variant using the FACS from the variant library of HW1.
  • FIG. 4 is a diagram analyzing the affinity of the finally selected variants from the variants library of HW1 and HW1 again using FACS.
  • 5 is a diagram showing the entire nucleotide and amino acid sequence of the antibody AU11 (where the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2, VL-CDR3).
  • Figure 6 is a view showing a result of comparing the amino acid sequence of the AU11 finally improved the affinity and stability of the existing antibody HW1 (* indicates amino acid residues are changed according to affinity improvement in CDR).
  • FIG. 7 is a diagram showing the entire nucleotide and amino acid sequence of the antibody AU12 (where the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2, VL-CDR3).
  • FIG. 8 is a diagram showing the results of comparative analysis of the amino acid sequences of AU11 and AU12 which finally improved the affinity and stability of the existing antibody HW1.
  • FIG. 9 is a diagram showing the results of analyzing the size and purity of purified antibodies (AU11 and AU12) by reducing SDS-PAGE and non-reducing SDS-PAGE.
  • FIG. 10 is a diagram showing the results of analyzing purified antibodies (AU11 and AU12) by size exclusion chromatography.
  • FIG. 11 is a diagram showing the result of quantifying the affinity of AU11 and AU12 for DR5 using SPR and comparing the affinity with HW1.
  • Figure 13 is a diagram showing the result of measuring the same antigen binding site between HW1 and AU11 or AU12 using a competitive ELISA.
  • FIG. 14 is a diagram showing the results of measuring the cross-reactivity of AU11 and AU12 against the target antigen DR5 antigen and other analogues (DR4, DcR1, DcR2) by ELISA.
  • FIG. 15 shows the binding affinity between total DR5 and DR5 fragments CRD2 (residues 97-137) and CRD3 (residues 139-180) and HW1, AU11, AU12, TRAIL having binding capacity to them using ELISA.
  • Figure 1 shows the results.
  • FIG. 16 is a diagram showing the results of comparing and evaluating the degree of apoptosis of HW1, AU11, and AU12 in HCT116, a human colorectal cancer cell line, and U87MG, a human neurotumor cell line, by MTT-assay.
  • FIG. 17 shows MTT-assay of apoptosis ability of HW1, AU11 and AU12 in the presence of various inhibitors (Z-VAD, SP600125, SB203580, 3-MA, Chloroguine) in human colon cancer cell line HCT116 and human neurotumor cell line U87MG. It is a figure which shows the result of analysis.
  • Fig. 18 shows the results of fluorescence microscopy observation of LC3-GFP aggregates by treatment of AU11 and AU12 to U87MG cancer cell lines expressing LC3-GFP.
  • the present invention provides an anti-DR5 antibody that specifically binds to DR5, which has the amino acid sequence of SEQ ID NO: 7 or 39.
  • the present invention also provides a DNA encoding the anti-DR5 antibody.
  • the present invention also provides a cell transformed with the DNA or an expression vector comprising the same.
  • the present invention also provides a composition for preventing or treating cancer containing the anti-DR5 antibody as an active ingredient.
  • Anti-DR5 antibody according to the present invention, the amino acid residues and light chain variable VH-CDR2, VH-CDR3 in the heavy chain variable region (heavy chain variable region) of the anti-DR5 human antibody HW1 (Korean Patent No. 10-0847010) Mutating amino acid residues of VL-CDR3 in the light chain variable region to enhance affinity for DR5, as well as replacing the framework in the heavy chain variable region from the VH6 subtype to the VH3 subtype, and light chain variable It is characterized by improving the stability by substituting the structure in the region with the structural part of Vk1 or Vk3.
  • apoptotic receptor-5 (DR5) protein means a member of the tumor necrosis factor (TNF) receptor family and binds TRAIL and has an intracellular death domain at the C-terminus ( Pan et al., Science, 277: 815-818, 1997).
  • TNF tumor necrosis factor
  • DR5 binds to TRAIL, it is known to induce apoptosis in TRAIL-sensitive cancer cells and to increase apoptosis when DR5 is overexpressed, but not to induce apoptosis in normal cells.
  • DR5 includes any protein having the above characteristics, but may be, for example, having an amino acid sequence described in US Pat. No. 6,872,568, but is not limited thereto.
  • an "antibody” may be an entire form of an antibody ("antibody”) or a functional fragment thereof.
  • the whole antibody may be in the form of a monomer or a multimer in which two or more whole antibodies are bound.
  • the functional fragment of the antibody is an antibody having the heavy and light chain variable regions of the whole antibody, which means to recognize substantially the same epitope that the whole antibody recognizes.
  • Functional fragments of the antibody include, but are not limited to, single chain variable region fragments (scFv), (scFv) 2 , Fab, Fab 'and F (ab') 2 and the like, and scFv is preferred in the present invention.
  • the single chain variable region (scFv) refers to an antibody fragment in which a heavy chain variable region and a light chain variable region are linked through a linker peptide to take the form of a single chain polypeptide.
  • the antibody can be generated using methods known in the art, such as phage display methods or yeast cell surface expression systems.
  • Antibodies of the invention may be derived from any animal, including mammals, birds, and the like, including humans.
  • the antibody may be a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken antibody.
  • the human antibody is an antibody having an amino acid sequence of human immunoglobulin, which includes an antibody isolated from a human immunoglobulin library or an antibody isolated from an animal transfected against one or more human immunoglobulins and not expressing an endogenous immunoglobulin ( US Pat. No. 5,939,598).
  • Antibodies of the present invention may be conjugated to enzymes, fluorescent materials, radioactive materials and proteins, but are not limited thereto. In addition, methods of conjugating such materials to antibodies are well known in the art.
  • Anti-DR5 antibody according to the present invention, the heavy chain variable region having complementary determining region CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 1, 2 and 3 and the complementarity determining region CDR1, CDR2 and CDR3 are SEQ ID NO: 4, 5, respectively And a light chain variable region having the amino acid sequence of 6, specifically binding to DR5 only.
  • “specifically binds” means that the antibody of the present invention binds only to the DR5 antigen and does not substantially bind to DR5 (death receptor 4), death decoy receptor 1 (DcR1) and DcR2, which are DR5-like antigens. it means.
  • the light chain variable region has an amino acid sequence of SEQ ID NO: 27 or 37
  • the heavy chain variable region has an amino acid sequence of SEQ ID NO: 28 or 38.
  • the anti-DR5 antibodies of the present invention are AU11 or AU12, which are scFv antibodies having an amino acid sequence of SEQ ID NO: 7 or 39, which in turn comprise CDR1 to CDR3 of the heavy chain variable region, linker oligopeptide and CDR1 to CDR3 of the light chain variable region, respectively.
  • the antibody is preferably a monoclonal antibody.
  • Anti-Dr5 antibodies of the present invention bind to DR5 with higher affinity than HW1, and the equilibrium dissociation constant ( K D ) shows 1.17 ⁇ 10 ⁇ 8 M and 9.17 ⁇ 10 ⁇ 9 M, respectively.
  • the anti-Dr5 antibody AU11 or AU12 is relatively higher in stability than HW1, and similarly to HW1, a single molecule scFv form induces apoptosis by progeny.
  • the present invention provides DNA encoding AU11 or AU12, which is an anti-DR5 antibody.
  • the DNA may be DNA encoding an scFv having an amino acid sequence of SEQ ID NO: 7 or 39, preferably DNA having a nucleotide sequence of SEQ ID NO: 8 or 40.
  • DNA sequences encoding the antibodies of the invention can be obtained by methods well known in the art. For example, based on DNA sequences or corresponding amino acid sequences encoding portions or all of the heavy and light chains of the antibody, oligonucleotide synthesis techniques well known in the art, for example site-directed mutagenesis And the polymerase chain reaction (PCR) method.
  • PCR polymerase chain reaction
  • the present invention also provides a cell transformed with the DNA or an expression vector comprising the same.
  • the DNA or expression vector comprising the same can be delivered to an appropriate host cell using methods known in the art, such as viral transfection or non-viral based techniques.
  • the introduction of the DNA or expression vector includes, but is not limited to, adenovirus transformation, gene gun, liposome-mediated transformation and retrovirus or lentiviral-mediated transformation, plasmid, adeno-associated virus, and the like. It may be performed by any known technique.
  • the cells can be transplanted with a suitable carrier material capable of releasing or delivering the gene to the cells for a long time.
  • the cells transformed with the DNA or the expression vector including the same may be cultured under appropriate conditions to express and isolate the antibody to produce antibody molecules.
  • the antibody molecule may accumulate in the cytoplasm of the cell, be secreted from the cell, or may be targeted to periplasm or extracellular medium by an appropriate signal sequence, among which is targeted to periplasm or extracellular medium. desirable. It is also desirable to refold the produced antibody molecules and to have functional conformation using methods well known to those skilled in the art.
  • a single vector comprising a sequence encoding the heavy or light chain polypeptide is transduced into the host cell, and in order to produce an antibody including both the heavy and light chain polypeptides.
  • a first vector encoding the light chain polypeptide and a second vector encoding the heavy chain polypeptide or a single vector comprising both the light chain and heavy chain polypeptide sequences. It may be.
  • the present invention also provides a composition for preventing or treating cancer containing the anti-DR5 antibody as an active ingredient.
  • the anti-DR5 antibodies (AU11 and AU12) according to the present invention bind TRDR-sensitive cancer cells or DR5 that specifically bind to the DR5 antigen with a higher affinity than the existing anti-DR5 antibodies (HW1), and have relatively high stability and express DR5.
  • HW1 existing anti-DR5 antibodies
  • Cancer caused by the DR5 expression may include both TRAIL-sensitive cancer cells and TRAIL-resistant cancer cells, specifically, hematologic cancer, lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, Uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, Urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumors of childhood, differentiated lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal contraction tumor, brain stem glioma or pituitary gland Adenomas and the like, but is not limited thereto.
  • composition of the present invention may contain one or more known active ingredients having an anticancer effect together with the anti-DR5 antibody.
  • composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as an antioxidant, buffer And other conventional additives such as bacteriostatic agents can be added.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.
  • composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease.
  • the daily dose of the anti-DR5 antibody is about 0.01 to 50 mg / kg, preferably about 0.1 to 20 mg / kg, and more preferably administered once to several times a day.
  • composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention and treatment of cancer.
  • Example 1 Construction of variant library for improving affinity of HW1
  • CDR Complementarity Determining Region
  • Mutant library was designed by giving priority to CDR2, and the vector for expressing the variant on the yeast surface was used pCTCON, a yeast surface expression vector, and random nucleotide sequences were assigned to CDR2 of the heavy chain variable region as shown in FIG. .
  • the ratio of wild-type bases is 70% instead of the base mixtures of 25% each of the four bases generally used.
  • a base mixture was used (for example, adenine 70% adenine, guanine, cytosine, thymine each 10% instead of the base mixture containing 25% adenine, guanine, cytosine and thymine at the site where the wild type base is adenine (A)). Variants were constructed using the base mixture contained thick). In the same manner as described above, a primer (fragment 1-SEQ ID NO: 9, SEQ ID NO: 12; fragment 2-SEQ ID NO: 11, SEQ ID NO: 10) (Gennotek, Korea) was finally prepared so that the retention rate of each amino acid was higher than a predetermined level.
  • variants with improved affinity were selected from the mutant library of heavy chain variable CDR2, and the primers (heavy chain variable CDR3: fragment) were selected in the heavy chain variable CDR3 and light chain variable CDR3 sites, respectively, using the selected antibodies as templates.
  • Two libraries have been built to further enhance affinity. Primer sequence numbers and sequences used to construct variants for improving affinity are shown in Table 1 below.
  • the first fragment is 5'-GGT GGT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 '(SEQ ID NO: 9) and 5'-CCT TCC CAG CCA CTC AAG GCC-3 as reverse primer.
  • SEQ ID NO: 12 was used to perform a polymerase chain reaction (PCR).
  • the second fragment is 5'-GGC CTT GAG TGG CTG GGA AGG 135 415 415 325 435 115 425 415 115 215 TAT GCA GTA TCT GTG AAA AGT-3 '(SEQ ID NO: 11) with forward primer and 5'-GAT CTC with reverse primer.
  • GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3 '(SEQ ID NO: 10) was prepared by PCR method. PCR was performed using pfu polymerase (Intron, Korea), and the reaction of 40 seconds at 94 ° C, 30 seconds at 55 ° C, and 40 seconds at 72 ° C was repeated 30 times.
  • fragments for the variants of the heavy chain variable CDR3 and light chain variable CDR3 sites, respectively, were prepared in the same manner as above.
  • the first fragment of heavy chain variable CDR3 was 5'-GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 '(SEQ ID NO: 9) as the forward primer, and 5'-ACA GTA ATA GAC GGC as the reverse primer.
  • CGT GTC-3 '(SEQ ID NO: 14) was used, and the second fragment was a 5'-GAC ACG GCC GTC TAT TAC TGT 235 125 215 335 215 235 225 125 225 235 445 215 145 TGG GGC CAA GGG ACC ACG GTC -3 '(SEQ ID NO: 13), 5'-GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3' (SEQ ID NO: 10) as a reverse primer was prepared by the PCR method.
  • the first fragment of the light chain variable CDR3 was 5'-GGT GGT GGT GGT TCT GGT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 '(SEQ ID NO: 9) as the forward primer, 5'-ACA GTA ATA AAC TGC as the reverse primer AAA ATC-3 '(SEQ ID NO: 16) was used, and the second fragment was a 5'-GAT TTT GCA GTT TAT TAC TGT 315 315 325 125 115 425 335 335 325 235 245 TTC GGC CAA GGG ACA CGA CTG-3 '(SEQ ID NO: 15), 5'-GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3' (SEQ ID NO: 10) as a reverse primer was prepared by the PCR method.
  • Each of the two fragments prepared was electrophoresed on a 1% agarose gel and purified using an agarose gel purification kit (Intron, Korea).
  • the purified two fragments were mixed in the same amount by 10 ⁇ M each, and then, as shown in FIG. 2, overlap extension PCR was performed to prepare a mutated whole scFv gene product.
  • the PCR was performed using pfu polymerase (Intron, Korea), and the reaction of 40 seconds at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C was repeated 30 times.
  • Mutant-inserted scFv antibody gene library (10 ⁇ g / ⁇ l) and scFv yeast surface expression vector pCTCON (1 ⁇ g / ⁇ l) were mixed, and then, electroporation was performed to the yeast EBY100 strain (Invitrogen, USA). Transformed.
  • the transformed yeast EBY100 strain was selected as SD-CAA (-ura, -trp; 20 g / l glucose, YNB without 6.7 g / l amino acid (BD, USA), 5.4 g / l Na 2 Library inducing cell surface expression of scFv in SG-CAA medium substituted with glucose to galactose after inoculation with HPO 4 , 8.56 g / L NaH 2 PO 4 H 2 O and 5 g / L casamino acid) Were screened. Cells containing the library were diluted 10-fold in SD-CAA medium stepwise to determine library size.
  • the scFv antibody library size was each constructed at about 2 ⁇ 10 7 .
  • Example 2 Screening for Improved Affinity Variants from Constructed HW1 Variant Library
  • Anti-c-myc mAb (Ig therapy, Korea) and biotin label diluted 1: 100 to select scFv variants showing high affinity to DR5 from the variant library of HW1 constructed in Example 1 above Biotin-labeled DR5 was added to PBS (PBSB, pH 7.4) with 0.2 mg of 1 mg / mL BSA using the kit (EZ-LINK TM Sulfo-NHS-LC-Biotinylation kit, Pierce, USA). Then, incubated for 30 minutes at 25 °C.
  • nucleotide sequences of the selected variants were forward primer 5'-GTT CCA GAC TAC GCT CTG CAG G-3 '(SEQ ID NO: 17) and reverse primer 5'-GAT TTT GTT ACA TCT ACA CTG TTG-3' (SEQ ID NO: 18) After analysis using the amino acid sequence was determined and compared with the amino acid sequence of the wild type.
  • the affinity of the HW1 variant was increased for each selection step.
  • the amino acid sequence of the variants selected from each library was determined and the changed CDRs of the selected wildtypes were respectively combined to finally produce new variants with the most affinity.
  • a variant derived from heavy chain variable CDR3 was used as a template, and 5'-GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 '(SEQ ID NO: 9) and 5'- as reverse primer. Fragments were prepared by PCR using ACA GTA ATA AAC TGC AAA ATC-3 ′ (SEQ ID NO: 16).
  • 5'-GAT TTT GCA GTT TAT TAC TGT-3 '(SEQ ID NO: 19) as the forward primer 5'-GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA as the reverse primer A fragment was prepared by PCR using AAT AAG CTT TTG TTC GGA TCC-3 ′ (SEQ ID NO: 10), and the reaction was repeated 30 times at 40 ° C. at 94 ° C., 30 seconds at 55 ° C., and 50 seconds at 72 ° C.
  • Each of the two fragments prepared was electrophoresed on a 1% agarose gel and purified using an agarose gel purification kit (Intron, Korea), and the two purified fragments were mixed in equal amounts by 10 ⁇ M each, and then superimposed. Extended PCR was performed to finally produce a variant whole scFv gene product with improved affinity. The PCR was repeated 30 times 40 minutes at 94 °C, 30 seconds at 55 °C, 1 minute at 72 °C.
  • the mutant scFv product thus prepared was introduced into the yeast surface expression vector pCTCON (Colby et al., Methods Enzymol., 388: 348-358, 2004) using the restriction enzyme Nhe I / BamH I (NEB, USA). The plasmid was transformed into yeast EBY100 strain (Invitrogen).
  • the final variant to which each variant was bound showed the highest affinity than the other variants.
  • HW1 an anti-DR5 human antibody
  • VH6 subtype for the heavy chain variable region and Vk3 for the light chain variable region
  • VH6 subtype is compared to other subtypes, particularly VH3. It is known that the stability is low.
  • the VH6 subtype was substituted with the VH3 subtype.
  • the whole gene was divided into three fragments and each fragment was prepared.
  • the first fragment is a forward primer 5'-TTC GCT AGC GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC CTG GTA CAG CCT GGA GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA GAC AGT GTC TCT AGC ACC ACT GTT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTC-3 '(SEQ ID NO: 20), 5'-ACA GTA ATA CAC AGC CGT GTC CTC GGC TCT CAG GCT GTT CAT TTG CAG ATA CAG GGT GTT CTT GGA ATT GTC TCT GGA GAT GGT GAA CCG GCC CTT CAC GGA GTC CGC ATA GTA ATT ATA CCA CTT CGA CCT ATA ATA GAT CGC TGA GAC CCA CTC CAG CCC CTT CCC-3 '(SEQ ID NO: 21).
  • the second fragment is 5'-GAG GAC ACG GCT GTG TAT TAC TGT-3 '(SEQ ID NO: 22) with forward primer, 5'-TTC CCC TGG AGA CAA AGA CAG GGT GGC TGG AGA CTG GGT CAA TAC AAT ATC CGA with reverse primer Produced using CCC GCC ACC GCC GCT GCC ACC-3 '(SEQ ID NO: 23).
  • the third fragment is 5'-ACC CTG TTG TCT TTG TCT CCA GGG GAA-3 '(SEQ ID NO: 24) with forward primer, 5'-CGT GGA TCC ACG TTT GAT TTC CAC CTT TGT CCC TTG GCC GAA GAC CGC CCG- It was produced using 3 '(SEQ ID NO: 25).
  • 4 glycine and 1 serine is repeated three times, using a (G 4 S) 3 linker consisting of a total of 15 amino acids (SEQ ID NO: 26).
  • Each fragment thus prepared was subjected to overlap extension PCR to finally prepare a variant whole scFv gene product having improved stability.
  • PCR was repeated 30 times 40 minutes at 94 °C, 30 seconds at 55 °C, 1 minute at 72 °C. Subsequently, valine # 101 and # 102 serine in the CDR3 region of the heavy chain variable region were replaced with aspartic acid and isoleucine to finally prepare AU11, an antibody having improved affinity and stability. Primer sequence numbers and sequences used to improve stability with antibody AU11 are shown in Table 2 below.
  • FIG. 5 Schematic representation of the entire nucleotide and amino acid sequence of antibody AU11 is shown in FIG. 5 (wherein the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2,
  • FIG. 6 A comparison of the amino acid sequence of AU11 with improved affinity and stability with the existing antibody HW1 is shown in FIG. 6 (* is an amino acid residue which is changed according to affinity improvement in CDR). ).
  • Vk3 and Vk1 are known to be the most stable, and many Vk1 subtypes exist in various mouse-derived humanized antibodies commercially approved by the US FDA (Carter et al., Proc Natl Acad). Sci USA , 10; 89: 4285-4289, 1992; Werther et al ., J Immunol , 11; 157: 4986-4995, 1996; Presta et al ., J Immunol 5; 151: 2623-2632, 1993).
  • vernier zone residues contribute to thermodynamic stability in antigen binding, and are known as sites that modulate the structural entropy changes of antibodies and consequently have a significant effect on antigen binding (Makabe et al.
  • the first fragment was 5'-TTC GCT AGC GAG GTG CAG CTG GTG GAG TCT GGG-3 '(SEQ ID NO: 29) with forward primer, 5'-TGG AGA CTG GGT CAT CTG AGA GTC with reverse primer -3 '(SEQ ID NO: 30) was used, and the second fragment was a 5'-GAC TCT CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC-3 '(SEQ ID NO: 31), 5'-CTT AGG GGC TTT CCC TGG TTT CTG CTG ATA CCA GGC TAA GTG GCT-3' (SEQ ID NO: 32) was used as the reverse primer, and the third fragment was a forward primer 5'-AAA GCC CCT AAG CTC CTG ATC TAT GGT GCA TCC AGC
  • AU11 As a linker linking the heavy chain variable region and the light chain variable region, (G 4 S) 3 linker in which 4 glycine and 1 serine was repeated three times was used. Each fragment thus prepared was subjected to overlap extension PCR to finally prepare a variant whole scFv gene product having improved stability. In the PCR, AU12 was finally produced by repeating a reaction of 40 seconds at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C for 30 times. Primer sequence numbers and sequences used in the preparation of AU12 are shown in Table 3 below.
  • FIG. 7 A schematic of the entire nucleotide and amino acid sequence of antibody AU12 is shown in FIG. 7 (wherein the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2, VL-CDR3), and comparing the amino acid sequence of AU11 and AU12 finally improved affinity and stability with the existing antibody HW1 is shown in FIG. 7 (wherein the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2, VL-CDR3), and comparing the amino acid sequence of AU11 and AU12 finally improved affinity and stability with the existing antibody HW1 is shown in FIG.
  • Antibodies with improved affinity and stability were introduced into the E. coli expression vector pKJ1 in-frame using Nhe I / BamH I. At this time, the E. coli expression vector was designed to have a T7 promoter-pelB targeting sequence-AU11 or AU12 scFv-flag tag-6X his tag.
  • antibodies AU11 and AU12 have a molecular weight of about 29 kDa. It was confirmed that the purity was 99% or more.
  • the elution buffer is PBS (50 mM phosphate, pH 7.4, 150 mM NaCl), flow rate 0.5 ml / min, column 280 nm using SuperdexTM 200 10/300 GC (GE, USA) and Agilent 1100 HPLC instrument Absorbance was measured at. Reducing SDS-PAGE was performed using 15% acrylamide gel with 1 mM DTT added to the sample buffer and non-reducing SDS-PAGE was performed with 15% acrylamide gel without 1 mM DTT added to the sample buffer, 150 volts. Analyze by loading.
  • the purified AU11 and AU12 scFv antibodies were free of multimolecular forms with non-natural disulfide bonds in reducing SDS-PAGE and non-reducing SDS-PAGE.
  • AU11 and AU12 were measured using a Biacore 2000 surface Plasmon resonance (SPR) biosensor (GE, USA). About 0.5 mg / ml of antigen and negative control group BSA (bovine serum albumin) were immobilized on a carboxymethylated dextran surface chip (CM5) at about 2,000 response units, and then the antibodies AU11 and AU12 diluted at various concentrations were added. Interaction with antigen was quantified by injection into the chip for 2 minutes at a rate of 30 ⁇ l / min.
  • SPR surface Plasmon resonance
  • HW1 was prepared at a concentration of 50nM, AU11 and AU12 at 25nM, respectively, and injected at the same time.
  • the surface of the chip was regenerated with 1M NaCl / 20mM NaOH, BIA evaluation ver. Using the 3.2 software to obtain the affinity to the movement rate constants (k on and k off) and the equilibrium dissociation constant (K D).
  • AU11 and AU12 showed affinity about 17-22 times higher than HW1 (K D : about 200nM).
  • simultaneous analysis of HW1, AU11, and AU12 at the same concentration showed that AU11 and AU12 bind more strongly in the dissociation interval than HW1.
  • HW1 The stability of AU11 and AU12 against HW1 was measured using a fluorescence spectrometer (JASCO, Japan).
  • HW1, AU11 scFv, and AU12 scFv were prepared by expression and purification in E. coli, and the prepared proteins were dissociated into guanidine-HCl (guanidine-HCl) in 0.3M units from 0M to 4.2M, respectively, to give a final concentration of 10 ⁇ g / ml.
  • an excitation wave length of 280 nm, an emission wave length of 290 to 400 nm, and a measurement rate of 250 nm / min were repeated three times.
  • the relative maximum wavelength (normalized l max ) was plotted by normalizing the wavelength at the highest value for each guanidine-HCl concentration.
  • HW1 has a median value at a concentration of about 1.64M guanidine-HCl
  • AU1 and AU12 have a median value at a concentration of about 2.53M and 2.45M, respectively.
  • Ewert S et al., Biochemistry , 42: 1517-1528, 2003 the relative maximum wavelength increases as the protein is denatured by a protein denaturation sample such as guanidine-HCl.
  • the biotin-labeled HW1 was immobilized at 30 ⁇ g / ml, the concentration of AU11 or AU12 was 5 to 300 ⁇ g / ml, mixed and added to each well, followed by incubation at 37 ° C for 1 hour, and the plate was After washing, an anti-biotin antibody conjugated with alkaline phosphatase (anti-biotin mAb AP conjugated, Sigma) was added and reacted at 37 ° C. for 1 hour. After washing three times, 50 ⁇ l of pNPP (Sigma.) was added to the substrate, and the absorbance was measured at 405 nm.
  • ELISA was performed to confirm the cross-reactivity of AU11 and AU12 against DR5, the target antigen, and DR4, DcR1, DcR2, the other antigens.
  • antigens namely DR5, DR4, DcR1 and DcR2
  • DR5 antigens
  • DcR1 antigens
  • DcR2 antigens
  • the anti-DR5 antibody of the present invention is an antibody that specifically binds to the DR5 antigen.
  • CRD2 cysteine-rich domains
  • CRD3 cysteine-rich domains
  • TRAIL showed a stronger binding force to CRD2 than CRD3, but HW1, AU11 and AU12 bound to each antigen with similar binding force for both CRD2 and CRD3. Therefore, it can be seen that the degree of effect of CRD2 and CRD3 on TRAIL or antibodies differs in the binding between TRAIL-DR5 and the binding between HW1, AU11, and AU12-DR5.
  • HCT116 As cancer cells, HCT116, a human colon cancer cell line, and U87MG, a human glioma cancer cell line, were used.
  • the cell lines stored in liquid nitrogen were taken out, dissolved rapidly at 37 ° C., and centrifuged to remove the cryopreservation medium.
  • the cells obtained from the culture medium were cultured with DMEM medium containing 10% FBS, 100unit / ml penicillin and 100 ⁇ g / ml streptomycin. (Welgene) was used to cultivate the culture flask.
  • TE buffer was treated for 3 to 5 minutes, and then the reaction of TE buffer was stopped with 5 ml of DMEM medium containing 10% FBS, followed by centrifugation to recover the cells.
  • the recovered cells were each resuspended in the same culture medium, and the cells per well were dispensed in 96-well plates by 1 ⁇ 10 4 (100 ⁇ l), incubated for 24 hours, and used for MTT analysis.
  • Human colon cancer cell line HCT116 and human neural tumor cell line U87MG to a 96-well plate at 1 ⁇ 10 4 gae added and for 10% FBS DMEM medium is put 100 ⁇ l is added 5% CO 2, at 37 °C 2 il Culture was stabilized.
  • 10 ⁇ M of Z-VAD Pan-caspase inhibitor, Santacruz
  • 10 ⁇ M of SP600125 JNK inhibitor, calbiochem
  • 10 ⁇ M of SB203580 p38 inhibitor, calbiochem
  • 10 ⁇ M of 3-MA 100 Autophagic cell death inhibitor, sigma
  • Chloroguine Autophagic cell death inhibitor (sigma) 10 ⁇ M was added and pretreated for 1 hour.
  • AU11 and AU12 were added to 30 ⁇ g / ml, respectively, and then cultured at 5% CO 2 and 37 ° C for 40 hours to measure the degree of cell death through MTT-assay.
  • AU11 and AU12 significantly reduced apoptosis in the presence of SP600125 (JNK inhibitor), 3-MA and Chloroquine in human colon cancer cell line HCT116 and human neuronal tumor cell line U87MG, Z-VAD
  • SP600125 JNK inhibitor
  • 3-MA Chloroquine
  • HCT116 human colon cancer cell line
  • human neuronal tumor cell line U87MG Z-VAD
  • SB203580 p38 inhibitor
  • a typical feature of apoptosis by apoptosis is the accumulation of intracellular LC3 proteins in or on the vacuole membrane.
  • U87MG cancer cell lines TRAIL-resistant, human glioma cell lines
  • LC3-GFP expressing LC3-GFP
  • AU11 and AU12 were added to 30 ⁇ g / ml, respectively, and incubated at 5% CO 2 , 37 ° C for 30 hours, and observed with a fluorescence microscope (Axiovert 200M, Carl Zeiss, Germany).
  • the above ingredients were mixed and filled in an airtight cloth to prepare a powder.
  • a tablet was prepared by a direct tableting method.
  • the powder was prepared by mixing the above components, the powder was filled in a hard capsule according to a conventional method for preparing a capsule to prepare a capsule.
  • the amount of the above-mentioned ingredient was prepared per ampoule (2 ml).
  • Each component was added to and dissolved in purified water according to the conventional method for preparing a liquid, and lemon flavor was added appropriately, followed by mixing the above components. Then, purified water was added thereto to adjust the total volume to 100 ml, and filled into a brown bottle and sterilized to prepare a liquid.
  • the anti-DR5 antibody according to the present invention has a higher affinity than a conventional anti-DR5 antibody, specifically binds to a DR5 protein, has a relatively high stability, and is a TRAIL-sensitive cancer cell expressing DR5 or a TRAIL-resistant cancer cell expressing DR5.
  • a conventional anti-DR5 antibody specifically binds to a DR5 protein
  • has a relatively high stability and is a TRAIL-sensitive cancer cell expressing DR5 or a TRAIL-resistant cancer cell expressing DR5.
  • the anti-DR5 antibody according to the present invention can be expected to reduce costs and improve efficiency by reducing the single dose.
  • acagtaatac acagccgtgt cctcggctct caggctgttc atttgcagat acagggtgtt 60

Abstract

L'invention concerne un anticorps dirigé contre le DR5 présentant une meilleure affinité et une meilleure stabilité, qui se lie spécifiquement au récepteur de mort 5 (DR5) pour détruire efficacement des cellules cancéreuses. Cette invention se rapporte également à une composition destinée à prévenir ou traiter le cancer, qui contient ledit anticorps. Cet anticorps dirigé contre le DR5 provoque des mutagenèses dans les résidus amino-acides de VH-CDR2 and VH-CDR3 au sein de la région variable de la chaîne lourde et dans les résidus amino-acides de VL-CDR3 dans la région variable de la chaîne légère de HW1 qui est ledit anticorps dirigé contre le DR5, et remplace la charpente au sein de la région variable de la chaîne lourde de manière à passer d'un sous-type VH6 à un sous-type VH3 et remplace la charpente au sein de la région variable de la chaîne légère par la partie de charpente Vk1 ou Vk3 de manière à obtenir une meilleure affinité et une meilleure stabilité pour ledit anticorps dirigé contre le DR5, HW1. En outre, cet anticorps dirigé contre le DR5 induit efficacement l'apoptose par rapport à des cellules cancéreuses sensibles au TRAIL qui expriment le DR5 et des cellules cancéreuses résistantes au TRAIL qui expriment le DR5 par autophagie, de sorte que cet anticorps puisse être employé utilement pour empêcher ou traiter des cancers provoqués par l'expression du DR5.
PCT/KR2009/006036 2008-10-24 2009-10-20 Anticorps dirigé contre le dr5 présentant une meilleure affinité et une meilleure stabilité, et composition destinée a prévenir ou traiter le cancer et contenant cet anticorps WO2010047509A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20080104737 2008-10-24
KR10-2008-0104737 2008-10-24
KR10-2009-0066518 2009-07-21
KR1020090066518A KR101117070B1 (ko) 2008-10-24 2009-07-21 친화도와 안정성이 향상된 항 ｄｒ5 항체, 및 이를 포함하는 암 예방 또는 치료용 조성물

Publications (3)

Publication Number Publication Date
WO2010047509A2 WO2010047509A2 (fr) 2010-04-29
WO2010047509A9 true WO2010047509A9 (fr) 2010-09-02
WO2010047509A3 WO2010047509A3 (fr) 2010-10-21

Family

ID=42119820

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2009/006036 WO2010047509A2 (fr) 2008-10-24 2009-10-20 Anticorps dirigé contre le dr5 présentant une meilleure affinité et une meilleure stabilité, et composition destinée a prévenir ou traiter le cancer et contenant cet anticorps

Country Status (1)

Country Link
WO (1) WO2010047509A2 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140118682A (ko) 2013-03-29 2014-10-08 (주)셀트리온 2 이상의 인플루엔자 ａ 바이러스 중화 결합 분자를 포함하는 조성물
KR101796277B1 (ko) 2016-04-12 2017-11-13 앱클론(주) 안정성이 개선된 ｈｅｒ２에 특이적으로 결합하는 항체
WO2018048234A1 (fr) * 2016-09-08 2018-03-15 (재)스크립스코리아항체연구원 Anticorps déglycosylé se liant spécifiquement à clec14a et ses utilisations
KR101926834B1 (ko) * 2017-03-21 2018-12-07 동아에스티 주식회사 항-dr5 항체 및 그의 용도
JP7403733B2 (ja) 2017-09-04 2023-12-25 国立感染症研究所長 インフルエンザｈａスプリットワクチンの製造方法
CN109837243A (zh) * 2017-11-25 2019-06-04 深圳宾德生物技术有限公司 一种敲除pd1的靶向dr5的嵌合抗原受体t细胞及其制备方法和应用
CN113501876B (zh) * 2021-07-16 2022-10-18 四川大学华西医院 一种特异性结合蛋白激酶p38δ的纳米抗体、核酸、表达载体、宿主细胞及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6872568B1 (en) * 1997-03-17 2005-03-29 Human Genome Sciences, Inc. Death domain containing receptor 5 antibodies
TWI318983B (en) * 2000-05-02 2010-01-01 Uab Research Foundation An antibody selective for a tumor necrosis factor-related apoptosis-inducing ligand receptor and uses thereof
US8029783B2 (en) * 2005-02-02 2011-10-04 Genentech, Inc. DR5 antibodies and articles of manufacture containing same
KR100847010B1 (ko) * 2006-07-05 2008-07-17 아주대학교산학협력단 세포사멸 수용체 5 (ｄｒ5)에 특이적으로 결합하는 항체 및이를 포함하는 암 예방 또는 치료용 조성물

Also Published As

Publication number Publication date
WO2010047509A2 (fr) 2010-04-29
WO2010047509A3 (fr) 2010-10-21

Similar Documents

Publication Publication Date Title
WO2010047509A2 (fr) Anticorps dirigé contre le dr5 présentant une meilleure affinité et une meilleure stabilité, et composition destinée a prévenir ou traiter le cancer et contenant cet anticorps
WO2018194381A1 (fr) Molécule de liaison spécifique à protéine lrig-1 et son utilisation
WO2019225787A1 (fr) Anticorps anti-b7-h3 et son utilisation
WO2019107812A1 (fr) Anticorps inhibant ras activé dans une cellule par internalisation dans de cytosol de la cellule, et son utilisation
WO2022039490A1 (fr) Anticorps bispécifiques anti-b7-h4/anti-4-1bb et leurs utilisations
WO2016137108A1 (fr) Nouvel anticorps se liant à la tfpi et composition le comprenant
WO2018026248A1 (fr) Nouvel anticorps dirigé contre la protéine programmée de mort cellulaire (pd-1) et son utilisation
WO2019050362A2 (fr) Anticorps dirigé contre dlk1 humain et utilisation associée
WO2021020846A1 (fr) Anticorps bispécifique anti-her2/anti-4-1bb et son utilisation
WO2020080854A1 (fr) Molécule de liaison spécifique de la protéine lrig-1 et son utilisation
WO2022177394A1 (fr) Anticorps bispécifique à domaine unique dirigé contre pd-l1 et cd47 et son utilisation
WO2020101365A1 (fr) Anticorps anti-c-met présentant une stabilité améliorée ou des fragments de liaison à l'antigène de celui-ci
WO2021101346A1 (fr) Anticorps bispécifiques anti-ror1/anti-4-1bb et leurs utilisations
WO2019190206A1 (fr) Anticorps se liant de manière spécifique à l'ecl-2 de claudine 3, fragment de celui-ci et utilisation associée
WO2021020845A1 (fr) Anticorps bispécifique anti-egfr/anti-4-1bb et son utilisation
WO2019125070A1 (fr) Anticorps ou fragment de liaison à l'antigène de celui-ci reconnaissant spécifiquement des tumeurs malignes de lymphocytes b, récepteur antigénique chimérique le comprenant, et utilisations associées
WO2020022782A1 (fr) Composition visant à prévenir et traiter des maladies liées au système immunitaire
WO2018026249A1 (fr) Anticorps dirigé contre le ligand 1 de mort programmée (pd-l1) et son utilisation
WO2019132579A2 (fr) Immunotoxine comprenant une ribonucléase fusionnée à un cytotransmab
WO2021101349A1 (fr) Anticorps se liant à ror1 et à b7-h3, conjugué anticorps-médicament le contenant et utilisation associée
WO2022035200A1 (fr) Protéine de fusion comprenant il-12 et un anticorps anti-cd20 et utilisation associée
WO2022124866A1 (fr) Anticorps anti-pd-1 et ses utilisations
WO2021071319A1 (fr) Protéine de fusion multispécifique et utilisation associée
WO2021006714A1 (fr) Composition pour la prévention, l'atténuation ou le traitement du cancer résistant aux inhibiteurs de point de contrôle immunitaire
WO2023239228A1 (fr) Protéine de fusion comprenant un anticorps anti-tigit, l'interleukine-15 et le domaine alpha sushi du récepteur de l'interleukine-15 et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09822185

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09822185

Country of ref document: EP

Kind code of ref document: A2