WO2017209553A2 - Procédé de criblage d'anticorps à l'aide de cellules dérivées de patients - Google Patents

Procédé de criblage d'anticorps à l'aide de cellules dérivées de patients Download PDF

Info

Publication number
WO2017209553A2
WO2017209553A2 PCT/KR2017/005766 KR2017005766W WO2017209553A2 WO 2017209553 A2 WO2017209553 A2 WO 2017209553A2 KR 2017005766 W KR2017005766 W KR 2017005766W WO 2017209553 A2 WO2017209553 A2 WO 2017209553A2
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
antibody
binding fragment
patient
nrp1
Prior art date
Application number
PCT/KR2017/005766
Other languages
English (en)
Korean (ko)
Other versions
WO2017209553A3 (fr
Inventor
남도현
Original Assignee
사회복지법인 삼성생명공익재단
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
Application filed by 사회복지법인 삼성생명공익재단 filed Critical 사회복지법인 삼성생명공익재단
Priority to JP2018562986A priority Critical patent/JP6895460B2/ja
Priority to EP17807046.2A priority patent/EP3480598A4/fr
Priority to SG11201810779PA priority patent/SG11201810779PA/en
Priority to US16/306,566 priority patent/US11199536B2/en
Priority to AU2017273169A priority patent/AU2017273169B2/en
Priority to CN201780041974.6A priority patent/CN109416364B/zh
Priority to CA3026236A priority patent/CA3026236C/fr
Priority claimed from KR1020170069140A external-priority patent/KR101993892B1/ko
Publication of WO2017209553A2 publication Critical patent/WO2017209553A2/fr
Publication of WO2017209553A3 publication Critical patent/WO2017209553A3/fr

Links

Images

Definitions

  • the present invention relates to a method for screening an antibody or antigen-binding fragment thereof using a patient-derived cell, and more specifically, an antibody or antigen-binding fragment thereof that specifically binds an antigen using a patient-derived cell containing the antigen. It relates to a method of screening.
  • Antibody pharmaceuticals are growing rapidly in the biopharmaceutical field due to their high therapeutic effect and target therapeutic properties.
  • the antibody pharmaceutical market is the fastest growing of the more than 200 biopharmaceuticals, accounting for 37% of the total biopharmaceutical market, and the global market grew from US $ 50.5 billion in 2012 to 11.8% per year in 2017. It is expected to reach $ 89.9 billion (2013 Biopharmaceutical Trend Analysis Report, Ministry of Trade, Industry and Energy, 2013).
  • the technology used for identifying and securing antibody candidates which are the main active pharmaceuticals of such antibody pharmaceuticals, is used for the development of chimeric or humanized antibodies using hybridoma cell lines, and the use of transgenic mice.
  • the method and the method using the antibody display technology can be broadly divided.
  • hybridoma technology was developed to produce monoclonal antibodies from hybrid cells made by fusing cancer cells with normal cells. Active antibody drug development began, and HAMA (Human-), which appears when mouse monoclonal antibodies are applied to humans, began to develop. In order to solve the anti-mouse antibody reaction, a technique for identifying humanized antibodies and human antibodies has been developed.
  • the technical field for producing human antibodies can largely exemplify transgenic mice and antibody displays (phage display, yeast display, ribosomal display, etc.).
  • the development of antibodies using transgenic mice which have been tried a lot recently, is a technique for producing human monoclonal antibodies by applying the existing hybridoma technology to transgenic mice transplanted with human antibody genes.
  • This technology can produce in vivo affinity maturation, which can produce antibodies with high affinity, and can make human antibodies effectively.
  • the conditions for use of transgenic mice are expensive and manufactured. Technological entry, such as know-how, is difficult.
  • phage display technology is a technique for expressing and screening antibody fragments on the surface of bacteriophage, and a display technology based on M13 PIII phage is most widely used.
  • phage display technology expresses recombinant fragments expressed on the surface of bacteriophages, it is difficult to screen for proteins present on the cell surface such as G-protein receptor or antibodies that are difficult to recombinantly express.
  • the antibody-drug conjugate is a cytotoxic drug bound to the antibody through a linker. Since the monoclonal antibody shows target specific properties, the drug in the antibody-drug conjugate has a selective target ability. Can be delivered to a tumor expressing an antigen / target recognized by a monoclonal antibody having. Ideally, the antibody-drug conjugate in the prodrug state in the blood after administration should be non-toxic, and when the antibody binds to the target tumor antigen and then internalizes into the cancer cell, the drug is released in active form to kill the tumor cell.
  • targets / antigens to which antibodies bind have been cell surface proteins that predominantly express (overexpress) in tumor cells.
  • antigen expressed on the cell surface of human cancer refers to a wide range of targets that are overexpressed or mutated and selectively expressed relative to normal tissue.
  • the key problem is identifying the appropriate antigen for antibody-based therapies. These therapeutic agents mediate changes in antigen or receptor function (ie, as a stimulant or antagonist), regulate the immune system through Fc and T cell activation, and through the delivery of specific drugs that bind to antibodies targeting specific antigens. It is effective. Molecular techniques that can alter antibody pharmacokinetics, functional function, size and immune stimulation are emerging as key elements in the development of novel antibody-based therapies.
  • Evidence from clinical trials of therapeutic antibodies in cancer patients is based on optimized antibodies, including affinity and binding of the target antigen and antibodies, selection of antibody structure, therapeutic approaches (blocking signaling or immune function). It highlights the importance of approaches for choice.
  • the present inventors have made diligent efforts to develop a method for screening antibodies having a high effect on patients.
  • screening antibody libraries using patient-derived cells containing antigens has high sensitivity and high accuracy.
  • the present invention was completed by confirming that few antibodies can be selected.
  • Another object of the present invention is to provide an antibody screening method using a patient-derived cell overexpressing an antigen and an animal model including the same.
  • Another object of the present invention is to provide an antibody or antigen fragment thereof selected by the above screening method.
  • Still another object of the present invention is to provide a composition for preventing or treating cancer comprising the antibody or antigen fragment thereof selected by the screening method as an active ingredient.
  • the present invention comprises the steps of (i) treating a patient-derived cell expressing an antigen, a library comprising an antibody or antigen-binding fragment thereof, to screen for an antibody or antigen-binding fragment thereof that binds to the antigen ; (ii) reacting the screened antibody or antigen-binding fragment thereof with a patient-derived cell that does not express an antigen; And (iii) isolating and removing the antibody or antigen-binding fragment thereof from the antibody or antigen-binding fragment thereof selected from (i), or the antibody-binding fragment thereof from (ii).
  • the present invention also provides a method for preparing an antibody or antigen-binding fragment thereof, the method comprising the steps of: (i) treating a patient-derived cell expressing an antigen with a library comprising an antibody or antigen-binding fragment thereof to screen the antibody or antigen-binding fragment thereof that binds the antigen; (ii) treating said primary screened antibody or antigen binding fragment library thereof to patient derived cells that are not expressing an antigen; (iii) separating the antibody or antigen-binding fragment thereof which binds to the patient-derived cell of (ii) from the antibody or antigen-binding fragment thereof selected in (i) above in an animal model in which the patient-derived cell expressing the antigen is transplanted.
  • the present invention also provides an antibody or antigen-binding fragment thereof selected by the above screening method.
  • the present invention further provides a composition for preventing or treating cancer comprising the antibody or antigen-binding fragment thereof.
  • FIG. 1 is a conceptual diagram illustrating the method of the present invention.
  • Figure 2 illustrates a method for screening antibodies that bind to Neurophilin 1 (NRP1) antigens, according to one embodiment of the invention.
  • NRP1 Neurophilin 1
  • FIG. 3 is a diagram illustrating a method for screening in vivo an antibody that binds to NRP1 antigen.
  • Figure 4 is a block diagram of a phagemid vector for the production of anti-NRP1 antibody fragments according to an embodiment of the present invention.
  • Figure 5 shows Coomassie staining results of each anti-NRP1 antibody fragment purified according to an embodiment of the present invention.
  • Figure 6 shows the ELISA results showing the binding capacity of the three anti-NRP1 antibody fragments selected according to an embodiment of the present invention to NRP1.
  • Figure 7 shows the ELISA results showing the binding capacity to NRP1 according to the concentration of the three anti-NRP1 antibody fragments selected in accordance with an embodiment of the present invention.
  • Figure 8 shows the results of analyzing the KD value of the three anti-NRP1 antibody fragments selected according to an embodiment of the present invention using SPR.
  • FIG. 9 is a FACS analysis showing the binding capacity of the anti-NRP1 antibody fragment selected in accordance with an embodiment of the present invention, cells derived from NRP1 overexpression.
  • 10A to 10C are confocal laser scanning micrographs showing the internalization function of three anti-NRP1 antibody fragments selected according to one embodiment of the invention.
  • FIG. 11 shows the results of identifying selected anti-NRP1 antibody fragment binding epitopes according to one embodiment of the present invention.
  • Figure 12 shows the results of RNA-seq analysis performed to select cell lines overexpressing NRP1.
  • Figure 13 shows the production purity of the three anti-NRP1 IgG antibodies.
  • Figure 14 shows the endotoxin test results for the three anti-NRP1 IgG antibodies.
  • Figure 15 shows the results of measuring the KD value of the anti-NRP1 IgG antibody using ELISA.
  • Figure 16 shows the results of measuring the specific binding capacity of the three anti-NRP1 IgG antibodies to human NRP1.
  • Figure 17 shows the results confirmed that the three anti-NRP1 IgG antibodies selected according to the method of the present invention is internalized in the patient-derived cancer cells.
  • Figure 19 shows the results confirming that the three anti-NRP1 IgG antibodies selected according to the method of the present invention shows a difference in binding ability between the normal and cancer cells superior to the known NRP1 antibody.
  • Figure 20 shows the results confirmed that the three anti-NRP1 IgG antibodies selected according to the method of the present invention shows an excellent cancer cell migration inhibitory effect.
  • Figure 21 shows the results confirmed that the anti-NRP1 IgG antibody selected according to the method of the present invention shows an excellent cancer cell migration inhibitory effect.
  • Figure 22 shows the results of confirming the change in the signaling material by the anti-NRP1 IgG antibody selected according to the method of the present invention.
  • Figure 23 shows the results confirmed that the apoptosis (apoptosis) by the anti-NRP1 IgG antibody selected according to the method of the present invention through the TUNEL assay increases.
  • Figure 24 shows the results of the efficacy evaluation for glioblastoma cancer of the anti-NRP1 IgG antibody selected according to the method of the present invention.
  • Figure 25 shows the results of the efficacy evaluation for lung cancer of anti-NRP1 IgG antibody selected according to the method of the present invention.
  • Figure 26 shows the results confirming the glioblastoma specific binding of the anti-NRP1 IgG antibody selected according to the method of the present invention.
  • Figure 27 shows the distribution evaluation results for the normal tissue of the anti-NRP1 IgG antibody selected according to the method of the present invention.
  • the present inventors endeavor to develop anti-cancer therapeutic antibodies using patient-derived cells containing antigens in order to select antibodies which are highly likely to be successful in the future, and which can be internalized to function effectively inside cells. It was. As a result, the present inventors have applied phage display technology to select antibodies that bind with antigens with high affinity, internalize into cells, and confirm that these antibodies internalize into cells.
  • phage display based on cells derived from glioblastoma patients was performed to select antibodies internalized into cells by binding to NRP1 (neurophilin 1), which are known to be expressed in various cancers.
  • NRP1 neuroophilin 1
  • the present invention provides a method for treating an antibody or antigen-binding fragment thereof, comprising: (i) treating a patient-derived cell expressing an antigen with a library comprising the antibody or antigen-binding fragment thereof to screen for an antibody or antigen-binding fragment thereof that binds to the antigen; (ii) reacting the screened antibody or antigen-binding fragment thereof with a patient-derived cell that does not express an antigen; And (iii) isolating and removing the antibody or antigen-binding fragment thereof from the antibody or antigen-binding fragment thereof selected from (i), or the antibody-binding fragment thereof from (ii). A method for screening antigen binding fragments thereof.
  • the term “expression” refers to a process in which an antigen is produced from a structural gene, and includes a process in which a gene is transcription into mRNA and a mRNA is translated into an antigen.
  • certain antigens may contribute to the production of a disease, for example cancer, and overexpression of certain antigens, for example, inhibits apoptosis of cancer cells, or overexpression of antigens, for example, increases the invasiveness or migration of cancer cells.
  • the expression of the antigen may be meant to include overexpression or abnormal activity of the antigen.
  • the term "antibody” is an immunoglobulin selected from the group consisting of IgA, IgE, IgM, IgD, IgY, and IgG, and can specifically bind to a target antigen. It consists of two light chains and a heavy chain, each of which consists of a variable domain in which the amino acid sequence is variable and a constant domain having a constant sequence.
  • the antigen binding site is located at the end of the three-dimensional structure of the variable region, which is formed by the collection of complementarity determining regions, each of which is present in the light and heavy chains. Complementarity determining regions are particularly highly variable parts of the amino acid sequence among the variable region, the antibody specific for a variety of antigens can be found by this high variability.
  • the scope of the present invention includes not only complete antibody forms but also antigen binding fragments of such antibody molecules.
  • ScFv single-chain Fv, single-chain fragment antibody or antibody fragment
  • the term "ScFv (single-chain Fv, single-chain fragment antibody or antibody fragment)” is an antibody connecting the variable region of the light chain and heavy chain. In some cases, it may include a linker consisting of a peptide chain of about 15 amino acids linked, wherein the ScFv is a light chain variable region-linked region-heavy chain variable region, or heavy chain variable region-linked It may have a structure of a site-light chain variable region and has the same or similar antigenic specificity as the original antibody.
  • a complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types and subclasses gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma 3 ( ⁇ 3). ), Gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant regions of the light chains have kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • An antigen binding fragment or antibody fragment of an antibody means a fragment having an antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.
  • Fab in the antibody fragment has a structure having a variable region of the light and heavy chains, a constant region of the light chain and the first constant region (CH1) of the heavy chain has one antigen binding site.
  • F (ab ') 2 antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds.
  • Double-chain Fv is a non-covalent bond in which a heavy chain variable region and a light chain variable region are linked, and a single chain Fv (single-chain Fv, scFv) is generally a variable region of the heavy chain and the light chain through a peptide linker.
  • This covalent linkage or the C-terminus is directly linked to form a dimer-like structure such as a double-chain Fv.
  • Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragments). It can also be produced by recombinant technology.
  • antibody (or ScFv) library is a collection of various antibody genes having different sequences. Very high diversity is required to isolate antibodies specific for any antigen from an antibody library, and libraries of different antibody clones are constructed and used.
  • the antibody gene constituting such an antibody library can be cloned into a phagemid vector, for example, and transformed into a transformant (E. coli).
  • nucleic acid may be used interchangeably with a gene or nucleotide, and may be selected from the group consisting of, for example, natural / synthetic DNA, genomic DNA, natural / synthetic RNA, cDNA, and cRNA, but is not limited thereto. It doesn't happen.
  • phagemid vector is used for phage display and is a plasmid DNA having a phage origin of replication, and typically has an antibiotic resistance gene as a selection marker.
  • the phagemid vector used for phage display includes the gIII gene of M13 phage or a part thereof, and the ScFv gene is ligated at the 5 'end of the gIII gene and expressed through a transformant.
  • helper phage is a phage that provides the genetic information necessary for the phagemid to be assembled into phage particles. Since phagemid contains only gIII or a part of phage gene, the host cell (transformer) transformed with phagemid is infected with a helper phage to supply the remaining phage gene. M13K07 or VCSM13 are available and most include antibiotic resistance genes such as kanamycin, which allows the selection of transformants infected with helper phage. In addition, since there is a defect in the packaging signal, the phagemid gene is selectively assembled into the phage particle rather than the helper phage gene.
  • signal sequence used in the present invention is located at the 5 'end of the gene, and is a base sequence or an amino acid sequence corresponding to a signal required when the protein encoded from the gene is secreted to the outside.
  • phage display is a technique for displaying variant polypeptides as fusion proteins with at least a portion of the envelope protein on the surface of a phage, for example, fibrous phage particles.
  • the utility of phage display lies in the fact that a large library of randomized protein variants can be targeted to quickly and efficiently classify sequences that bind with high affinity with a target antigen. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to identify polypeptides with specific binding properties.
  • Phage display technology provided a powerful tool for generating and selecting new proteins that bind specific ligands (eg antigens). Phage display technology can be used to generate large libraries of protein variants and to quickly sort sequences that bind with high affinity to target antigens.
  • Nucleic acids encoding variant polypeptides are fused with nucleic acid sequences encoding viral envelope proteins, eg, gene III protein or gene VIII protein.
  • Monovalent phage display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein. In monovalent phage display systems, gene fusions are expressed at low levels and wild type Gene III proteins are also expressed to maintain particle infectivity.
  • Phage display technology has several advantages over conventional hybridoma and recombinant methods for preparing antibodies with the desired characteristics. This technique allows the production of large antibody libraries with various sequences in a short time without the use of animals. The preparation of hybridomas or the production of humanized antibodies may require months of preparation. In addition, since no immunity is required at all, phage antibody libraries can produce antibodies against antigens that are toxic or low antigenic. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.
  • Techniques for generating human antibodies from immunized, non-immunized human, germline sequences, or na ⁇ ve B cell Ig repertory using immunized phage display libraries can be used.
  • Various lymphoid tissues can be used to prepare na ⁇ ve or non-immune antigen binding libraries.
  • the ability to identify and isolate high affinity antibodies from phage display libraries is important for the isolation of novel therapeutic antibodies. Separation of high affinity antibodies from the library may depend on the size of the library, the efficiency of production in bacterial cells, and the diversity of the library.
  • the size of the library is reduced by inefficient folding of the antibody or antigen binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be inhibited if the antibody or antigen binding domain is not properly folded. Expression can be improved by alternating mutations at the surface of the variable / constant interface or at selected CDR residues.
  • the sequence of the backbone region is one element to provide proper folding when generating antibody phage libraries in bacterial cells.
  • CDR3 regions have been found to often participate in antigen binding.
  • the CDR3 regions on the heavy chains vary considerably in size, sequence, and structural conformation, and thus can be used to prepare a variety of libraries.
  • diversity can be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position.
  • the use of all twenty amino acids can result in highly variable variant antibody sequences and increase the chance of identifying new antibodies.
  • neurophylline or neuropilin as used herein collectively includes neuropilin-1 (NRP1, neurophilin 1), neuropilin-2 (NRP2) and their isotypes and variants.
  • Neuropilin is a 120-130 kDa non-tyrosine kinase receptor.
  • NRP-1 and NRP-2 splice variants and soluble isoforms There are a number of NRP-1 and NRP-2 splice variants and soluble isoforms.
  • the basic structure of neurophylline comprises five domains: three extracellular domains (a1a2, b1b2 and c), transmembrane domains and cytoplasmic domains.
  • the a1a2 domain is homologous to the complement components Clr and Cls (CUB), which generally contain four cysteine residues that form two disulfide bridges.
  • the b1b2 domain is homologous to coagulation factors V and VIII.
  • the central portion of the c domain is named as MAM because of its homology with meprin, A5 and receptor tyrosine phosphatase ⁇ protein.
  • the a1a2 and b1b2 domains are responsible for ligand binding, while the c domains are crucial for homo-dimerization or hetero-dimerization.
  • Neuropilin-1 mediated biological activity means a physiological or pathological condition in which neuropilin-1 plays a substantial role. For example, it may be, but is not limited to, axon guidance, angiogenesis (including angiogenesis), tumor formation, and tumor metastasis during embryonic nervous system development or nerve cell regeneration.
  • Antibodies of the invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, and the like. Antibodies of the invention include antigen binding fragments or antibody fragments of antibodies, which fragments are single-chain Fvs (scFV), single-chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fvs (sdFV) and anti- Idiotype (anti-Id) antibodies may be included.
  • scFV single-chain Fvs
  • Fab fragments single-chain antibodies
  • F (ab ') fragments fragments
  • disulfide-binding Fvs sdFV
  • anti-Id anti- Idiotype antibodies
  • Said monoclonal antibody refers to the same except for possible naturally occurring mutations in which antibodies obtained from substantially homogeneous antibody populations, ie, individual antibodies in the population, may be present in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site.
  • Non-human (eg murine) antibodies of the “humanized” form are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins.
  • humanized antibodies are non-human species (donor antibodies) that retain the desired specificity, affinity, and capacity for residues from the hypervariable region of the recipient, for example mice, rats, rabbits, or non-humans.
  • donor antibodies non-human species
  • Human immunoglobulins (receptor antibodies) replaced with residues from the hypervariable regions of primates.
  • human antibody refers to a molecule derived from human immunoglobulin, in which all of the amino acid sequences constituting the antibody including complementarity determining regions and structural regions are composed of human immunoglobulins.
  • While the heavy and / or light chain portions are the same or homologous to the corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, the remaining chain (s) are derived from another species or another antibody class or Included are "chimeric" antibodies (immunoglobulins) that are identical or homologous to the corresponding sequences in antibodies belonging to the subclass, as well as fragments of such antibodies that exhibit the desired biological activity.
  • antibody variable domain refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie CDR1, CDR2, and CDR3), and framework regions (FR). .
  • CDRs complementarity determining regions
  • FR framework regions
  • VH refers to the variable domain of the heavy chain.
  • VL refers to the variable domain of the light chain.
  • CDRs Complementarity determining regions
  • Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
  • FRs Framework regions
  • Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.
  • the present invention includes the steps of (i) treating a patient-derived cell expressing an antigen with a library comprising an antibody or antigen-binding fragment thereof to screen for an antibody or antigen-binding fragment thereof that binds to the antigen.
  • the patient-derived cells may be, for example, cells derived from cancer patients, and cancer cells derived from cancer patients exhibit different physiological characteristics from normal cells, and these physiological characteristics may be cancer cell-specific in comparison with normal cells. It is determined by the expression patterns of genes whose expression is increased or decreased, and the expression pattern of such genes may be patient specific or may indicate tissue specific differences of patients.
  • the antigen may be an antigen involved in the development, growth and migration of cancer or a tumor.
  • the antigen may be in the form of an oligomer, peptide, polypeptide or protein, for example.
  • the method for measuring the antigen content of the patient-derived cells and normal cells may be characterized by comparing the expression level of the gene or protein encoding the antigen, preferably, FACS, ELISA, whole exome sequencing and RNA sequencing It may be characterized in that it is performed by one or more methods selected from the group consisting of, but is not limited thereto.
  • the patient-derived cells may be derived from a solid cancer patient, the solid cancer is liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, kidney cell cancer, gastric cancer, breast cancer, metastasis Cancer, prostate cancer, pancreatic cancer and lung cancer may be selected from the group consisting of, but is not limited thereto.
  • the library is a collection of antibodies and / or antigen binding fragments thereof, which can be displayed for screening and can be made of full length antibodies.
  • Antibodies and / or antigen binding fragments thereof in the library can be displayed, for example, on ribosomes, phages, or cells.
  • the method of obtaining the patient-derived cells is not limited, but for example, (a) pulverizing the isolated cancer patient-derived cancer tissue, and then obtaining a cell fraction from the pulverized product; And (b) treating the obtained cell fraction with proteolytic enzymes, followed by filtration, centrifugation and suspension to single cell.
  • the protease may refer to an enzyme capable of performing proteolysis, and is an endopeptata that decomposes proteins through protein catabolism that hydrolyzes peptide bonds connecting amino acids in the protein. And an exoopeptidase that hydrolyzes peptide bonds from the N-terminus or C-terminus of the protein and the enzyme.
  • the invention also includes the step of (ii) reacting the screened antibody or antigen-binding fragment thereof with a patient-derived cell that does not express an antigen.
  • Step (ii) is for negative selection of the antibody or antigen-binding fragment thereof selected in step (i), and (iii) of the antibody or antigen-binding fragment thereof selected from (i) (ii) By separating and removing the antibody or antigen-binding fragment thereof that binds to the patient-derived cells of), through this, it is possible to screen antibodies having high selectivity to the antigen.
  • the patient-derived cells removed so that the antigen of step (ii) is not expressed may be cells which do not naturally express the antigen, may be patient-derived cells artificially manipulated so that the antigen is not expressed,
  • the artificially manipulated method may be any method as long as the antigen is not expressed, but preferably at least one selected from the group consisting of aptamer, siRNA, single-stranded siRNA, microRNA, and shRNA that bind to the antigen. It may be characterized by being obtained by treatment.
  • the step of selecting only the antibody that binds to the antigen by using a patient-derived cell that does not express the antigen is a negative screening process, performed immediately after the positive screening step to determine the accuracy of the antigen of the selected antibody It is effective to improve.
  • the separation and removal may include electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent chromatography, size exclusion chromatography, etc.), isoelectric focusing and various changes thereof. Complex methods and the like are available, but are not limited to such.
  • the separation and removal can remove impurities by centrifugation or ultrafiltration, for example, and the resultant can be purified using, for example, affinity chromatography or the like. Further other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and the like can be used.
  • the present invention provides a method for preparing an antibody comprising: (i) treating an antibody library to a patient-derived cell expressing an antigen, thereby primary screening of the antibody or antigen-binding fragment thereof that binds the antigen; (ii) treating said primary screened antibody or antigen binding fragment library thereof to patient derived cells that are not expressing an antigen; (iii) separating the antibody or antigen-binding fragment thereof which binds to the patient-derived cell of (ii) from the antibody or antigen-binding fragment thereof selected in (i) above in an animal model in which the patient-derived cell expressing the antigen is transplanted.
  • NRP1 neuroophilin 1
  • a phage display based on cells derived from glioblastoma patients is performed. Selected antibody candidate groups were injected into immunodeficiency mice containing patient derived cells, and secondary screening was performed in vivo. As a result, it was confirmed that the selected anti-NRP1 antibody was bound to NRP1 expressed on the surface of cancer cells, and then internalized into cells (FIGS. 9A to 9C), and the binding epitopes of the antibodies were different from those of the existing antibodies (FIG. 10). ).
  • the present invention particularly relates to (iii) an antibody which binds to a patient-derived cell of (ii) among the antibodies or antigen-binding fragments thereof selected from (i) in an animal model in which a patient-derived cell overexpressing the antigen is transplanted.
  • the method further comprises the step of secondary screening of the antibody or antigen-binding fragment thereof that binds to the antigen by administering the antibody or antigen-binding fragment thereof that has separated and removed the antigen-binding fragment.
  • the characteristics of the patient can be sufficiently reflected through the animal model transplanted with the patient-derived cells overexpressing the antigen, such as the selection of treatment methods for the selection and treatment of patients
  • the selection of treatment methods for the selection and treatment of patients it is possible to select antibodies or antigen-binding fragments thereof that are particularly suitable for patient characteristics with high selectivity.
  • the animal model in which the patient-derived cells overexpressing the antigen is transplanted may be any animal including the patient-derived cells, but preferably, it may be characterized as an immunodeficiency mouse.
  • the mouse may be a nude mouse, a non-obese diabetic (NOD) mouse, a combined combined immunodeficiency (SCID) mouse, a NOD-SCID mouse, or a NOG (NOD / SCID I12rg ⁇ / ⁇ ) mouse.
  • the method according to the present invention can screen, for example, but is not limited to antibodies that bind to NRP1 overexpressed in patient derived cells.
  • the antibody may be, for example, an antibody that binds to the VEGF165 domain of NRP1, but may have a different epitope that binds to a conventional antibody known as an antibody that binds to the VEGF165 domain of NRP1 (MNRP1685A antibody from Genetech).
  • the antibody or binding fragment thereof selected by the present invention may be, for example, an IgG form, a Fab 'fragment, a F (ab') 2 fragment, a Fab fragment, a Fv fragment, or a single chain Fv fragment (scFv), but preferably It can be made in IgG form.
  • the present invention relates to an antibody or antigen-binding fragment thereof screened by the above method.
  • Antibodies or antibody fragments of the invention may include not only the anti-NRP1 antibodies of the invention described herein, but also biological equivalents thereof, as long as they specifically recognize NRP1.
  • further changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody.
  • Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody.
  • Such amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape.
  • arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.
  • each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).
  • the hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is well known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference of preferably within ⁇ 2, more preferably within ⁇ 1, even more preferably within ⁇ 0.5.
  • the antibody or nucleic acid molecule encoding the same of the present invention is interpreted to include a sequence that exhibits substantial identity with the sequence described in SEQ ID NO.
  • the above substantial identity is at least 61% when the sequence of the present invention is aligned as closely as possible with any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art.
  • a sequence that shows homology more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art.
  • BLAST The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and the like and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet.
  • BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparisons using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • the antibody or antigen-binding fragment may bind to an antigen, for example, an antigen involved in the development, growth and migration of cancer or a tumor.
  • the antigen may be in the form of an oligomer, peptide, polypeptide or protein, for example.
  • it may be characterized in that it is a method for screening an antibody or antigen-binding fragment thereof that specifically binds to NRP1.
  • the present invention relates to a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof as an active ingredient.
  • the present invention provides, for example, (a) a pharmaceutically effective amount of an antibody against NRP1 or an antigen binding fragment thereof according to the present invention; And (b) it may be a pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier.
  • the present invention also relates to a method for the prevention or treatment of cancer comprising administering to a patient an effective amount necessary for an antibody against NRP1 or an antigen-binding fragment thereof according to the present invention.
  • composition uses the above-described anti-NRP1 antibody or antigen-binding fragment thereof as an active ingredient, the common content between the two is omitted in order to avoid excessive complexity of the present specification according to the repetitive description. .
  • the anti-NRP1 antibodies according to the present invention can inhibit the migration of cancer cells expressing NRP1.
  • the antibody or antigen-binding fragment thereof of the present invention can bind to NRP1 with high affinity to inhibit the migration of cancer cells overexpressing NRP1, and thus can be used for the prevention and treatment of cancer.
  • the anti-NRP1 antibody selected by the method according to the invention may exhibit cancer cell specific internalization (Example 9), increase apoptosis effect and solid cancer such as glioblastoma cancer and It was confirmed that the lung tumor can exhibit the desired tumor growth inhibitory effect (Example 11).
  • anti-NRP1 antibodies selected by the method according to the present invention can be expected to minimize the side effects by confirming the low or almost no binding to normal tissue (Example 12).
  • Prevention means any action that inhibits or delays the progression of cancer by administration of a composition according to the invention
  • treatment means the inhibition of cancer development, the reduction of cancer or the elimination of cancer.
  • Cancer that is a disease applied to the composition is a cancer that overexpresses NRP1, for example, glioblastoma, astrocytoma, glioma, neuroblastoma, testicular cancer, colon cancer, melanoma, pancreatic cancer, lung cancer, breast cancer, esophageal cancer and prostate cancer, etc. Can be.
  • Cancer overexpressing NRP1 refers to a cancer having NRP1 on the cancer cell surface at significantly higher levels compared to non-cancerous cells of the same tissue type.
  • compositions of the present invention are those commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
  • the composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
  • composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration Or the like.
  • oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.
  • Suitable dosages of the compositions according to the invention vary depending on factors such as the method of formulation, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and reaction sensitivity, and usually The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.
  • the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.
  • pharmaceutically effective amount as used herein means an amount sufficient to prevent or treat cancer.
  • compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, suppositories, powders, granules, tablets, or capsules, and may further include a dispersant or stabilizer.
  • compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.
  • NRP1 expression levels of patient-derived cells possessed by the project group (Samsung Hospital Intractor Cancer Research Group) for screening cells needed for cell panning to identify anti-NRP1 antibody fragments were determined after RNA-Seq, followed by RPKM (Reads Per Killobase Million (A) method was analyzed (FIG. 12), and cultured cells were selected by FACS (Fluorocence Activated Cell Sorting) method, and patient-derived cells were used for cell panning.
  • scFv antibody fragments that bind to human NRP1 were identified through phage display screening.
  • Four sub-library samples were incubated for 2 hours in each 400 ml culture medium (SB / ampicillin / 2% glucose) to recover phagemid vectors introduced into E. coli host ER2537 in phage form. .
  • the supernatant is removed by centrifugation at 5000 g for 20 minutes, and then suspended in 400 ml of secondary culture medium (SB / ampicillin) and then helped with 1012 pfu (plaque forming unit). Phage (VCSM13) was added and incubated for 1 hour. Then, 70 ⁇ g / ml of kanamycin antibiotic (antibiotic gene introduced into helper phage) was added and cultured overnight at 30 ° C. to allow the phage library to be secreted out of the host cell. Then, the culture obtained by centrifugation was precipitated only phage form using polyethylene glycol (PEG) solution to obtain a phage library.
  • PEG polyethylene glycol
  • the phage library thus obtained and patient-derived cells with high NRP1 expression (4 ⁇ 10 6 ) were mixed and placed in a total of 5 ml NBA (neurobasal medium), fixed in a rotator at 4 ° C., and rotated at 360 degrees for 1-2 hours. Then, the cells were separated by centrifugation at 300 g for 5 minutes to remove phage particles that did not bind to the patient-derived cells, followed by washing with 5 ml NBA. This process was repeated four times, and in the final process, 5 mL of NBA previously placed in an incubator at 37 ° C. was used to place patient-derived cells and phages in a T flask and incubated at 37 ° C. for 30 minutes to internalize phage particles on the cell surface. internalization) to enter the cells.
  • NBA neurotrophic acid
  • the cells were transferred to a 15 ml conical tube and centrifuged at 300 g for 5 minutes to separate the cells, followed by repeated washing six times with 5 ml of cold PBS (Phosphate buffered saline), followed by cell panning. Repeatedly increased the number of times. Subsequently, 5 ml of 0.1 M glycine (pH 2.2) was added, and allowed to stand at room temperature for 5 minutes to separate phage particles on the cell surface from the cell surface. Thereafter, centrifugation at 300 g for 5 minutes to separate only the cells and put 0.5 ml 100 mM TEA, which was transferred to the e-tube and placed for 15 minutes at room temperature.
  • PBS Phosphate buffered saline
  • Phage particle amplification was then performed by taking 1 ml of the previous round of phage solution stored for the repeated cell panning round. Phage particles recovered by incorporating helper phage after incubation in host cell ER2537 were separated by PEG precipitation, and the same was used for the next round panning. Three rounds of panning were performed and this cell panning process is shown in FIG. 2. As the repetition was repeated, it was confirmed that the ratio of phage particles before and after panning increased, which means that internalized phage particles were amplified through cell panning, and the results are shown in Table 1.
  • Phage particles recovered in the third round of cell panning were identified as colonies in culture medium via host cell (ER2537) infection. These colonies were taken and inoculated in 96-well plates containing 200 ⁇ l SB / ampicillin culture medium and then incubated at 37 ° C. for 2-3 hours.
  • each well was treated with IPTG (Isopropyl ⁇ -D-1-thiogalactopyranoside) at a final concentration of 1 mM and incubated overnight at 30 ° C.
  • IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
  • the cultured plate was centrifuged at 3,000 rpm for 15 minutes to remove supernatant and 40 ⁇ l of TES solution (20% w / v sucrose, 50 mM) per well to recover phage particles in cultured periplasm.
  • Tris, 1 mM EDTA, pH 8.0 was added and the cells were lysed by standing at 4 ° C for 30 minutes.
  • 60 ⁇ l of 0.2X TES solution was treated and placed at 4 ° C. for 30 minutes to decompose the cells by osmotic pressure, and the plate was centrifuged for 15 minutes at 3,000 rpm to obtain supernatant scFv-pIII protein.
  • the total number of clones analyzed was 384, of which 41 clones (binding capacity> 2) showed high binding capacity to human NRP1.
  • a BSA solution was used, and among these 41 clones, 10 clones having high binding capacity were selected through reconfirmation ELISA. Thereafter, phagemids were recovered from 10 clones, followed by DNA sequencing, and clones having a total of six different sequences were selected. Except for 3H10, the same sequence as 1C08, clones with different sequences were selected and finally 3H10, 1A03 and 4F12 clones were selected as anti-NRP1 antibody fragment candidates.
  • the amino acid sequences of the 3H10, 1A03 and 4F12 clones are shown in Tables 2 and 3.
  • Candidate antibodies recovered in the third round of cell panning in Example 1 were injected via intra-tumoral injection into mice subcutaneously implanted with patient-derived cells used in Example 1.
  • mice were sacrificed to separate cancer tissues into single cells, and then antibody fragments internalized in the cells were obtained and analyzed by ELISA as in the method of Example 2.
  • phagemid The basic composition of the phagemid can be seen in FIG. 4, and in the case of the host cell ER2537 used in the above example, scFv expression alone is impossible because it inhibits the transcriptional codon (amber codon (UAG)) located in front of phage pIII. Therefore, phagemid was transduced into the expression strain using an expression strain (TOP10F ') which is a non-suppressor strain. Subsequently, it was confirmed that each phagemid was an expression strain introduced without mutation through DNA sequencing. The expression strain was taken as a colony, and then inoculated in 3 ml of LB / ampicillin culture medium and then cultured overnight at 37 ° C.
  • the scFv protein present in the filtered solution was combined with 1 ml of Ni-NTA bead (Qiagen) for 1 hour at room temperature for His-tag purification, and then packed in a gravity column (Bio-rad) to 200 mM. Recovered through imidazole solution. After expression and purification of each clone, SDS-PAGE and coomassie blue staining (coomassie blue staining) confirmed a scFv corresponding size of about 28 kDa (FIG. 5).
  • ELISA was performed using the purified scFv to determine whether binding ability to the target NRP1 exists.
  • 96 wells coated with 200 ng NRP1 protein and 96 wells coated with 200 ng of BSA as a control were bound to 5 ⁇ g / ml level for each clone for 1 hour at room temperature by ELISA (3 repetitions). Thereafter, washing with 0.1% TBST three times, followed by treatment with HRP-bound HA antibody for 1 hour, washing again, and then standing for 5 minutes with TMB solution. The OD value was measured after stopping the color reaction with 2M sulfuric acid solution.
  • each scFv in a 96 well coated with 200 ng NRP1 or BSA 2,000ng / ml, 1,000 ng / ml, 500 ng / ml Changes in OD values were analyzed by treatment at concentrations of 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml or 15.62 ng / ml.
  • KD (M) values are shown (FIG. 8).
  • FACS analysis was performed using patient-derived cells with high NRP1 expression. 5 ⁇ 10 5 patient-derived cells per scFv were bound for approximately 1 hour at 4 ° C. and washed three times with 1 ml of FACS solution. Thereafter, 1 ⁇ g of a red fluorescent (PE; phycoerithrin) bound HA antibody was treated and bound at 4 ° C. for 30 minutes. It was then washed three times with 1 ml of FACS solution and analyzed via FACS Calibur TM system.
  • PE red fluorescent
  • Intracellular permeability of the three anti-NRP1 antibody fragments was confirmed by cell immunofluorescence staining.
  • PD-lysine solution was added to the chamber slide and coated at room temperature for 1-2 hours. The slide was then dried after the solution was removed.
  • 200 ⁇ l of an NBA solution containing 5 ⁇ 10 4 patient-derived cells was treated on the slide, and then incubated at 37 ° C. for 4-5 hours to fix the slide.
  • the NBA solution was removed, and 4% paraformaldehyde was added thereto and fixed at 4 ° C. for 10 minutes. After washing three times with PBS, 0.1% Triton X-100 treated cell permeability enhancement was performed.
  • anti-NRP1 antibody fragments were treated simultaneously with anti-human NRP1 antibody (R & D) for staining of NRP1 protein and bound at 37 ° C. for 15/30/60 minutes. After washing three times with PBS, blocking for 1 hour at room temperature with 1% BSA solution to prevent nonspecific binding. Secondary antibodies were treated with goat fluorescence (Invitrogen) labeled with green fluorescence (Alexa-Fluor 488) to view NRP1 protein, and anti-HA antibody (Santacruz biotechnology) to view anti-NRP1 antibody fragments. Treated and bound for 1 hour at room temperature. Finally, after the DAPI staining for staining the nucleus, the glass cover was fixed on the slide after the final washing, and observed using a confocal laser scanning microscope.
  • the anti-NRP1 antibody fragments attached to the surface and the anti-NRP1 antibody fragments inserted into the cells were mixed at 15 and 30 minutes, but most of the anti-NRP1 antibody fragments were passed after 60 minutes. It was confirmed that the -NRP1 antibody fragments penetrated into the cells (Figs. 10A to 10C). In particular, the cell permeability of the 4F12 antibody fragments, rather than 1A03 and 3H10, was noticeable over time (Fig. 10A).
  • the heavy and light chain genes of the NRP1 antibody fragment were transfected using the Expi 293F expression technology (life technologies).
  • the yield was 120mg / L for IRCR-101 (3H10 converted to IgG form), 66mg / L for 1A03, 4F12 was 15 mg / L.
  • Fast liquid chromatography was introduced to confirm the purity of the purified anti-NRP1 IgG antibody. Since the size of the IgG is 150kD, it corresponds to the substance coming out at 16.388 minutes from the marker peak.
  • MNRP1685A was used as a positive control because the binding domain of MNRP1685A is the VEGF domain. After coating hNRP1 protein per well on a 96 well plate, 500 nM of IRCR-101 and MNRP1685A were bound at 25 ° C. for 1 hour, washed with PBST, and biotin-bound VEGF and Sema3A were bound at room temperature for 15 minutes.
  • IRCR-101 200ng hNRP1 protein was coated per 96 well plate on each well, and 500nM of IRCR-101 and MNRP1685A were combined at 25 ° C for 1 hour, followed by washing with PBST. After biotin binding, IRCR-101 and MNRP1685A were bound at room temperature for 15 minutes.
  • the plate was washed with PBST, and then streptavidin-HRP antibody was added to confirm TMB color reaction by ELISA. As a result, the binding epitopes of the control group and IRCR-101 were confirmed to be different from each other (right panel of FIG. 11).
  • pHrodo® Red Microscale Labeling Kit (Thermo # p35363) was used to compare the internalization of three anti-NRP1 IgG antibodies in cancer cells and normal cells.
  • the principle of the above kit is to conjugate the coloring sample to the antibody (conjugated), and if the antibody is outside the cell, the color does not develop. .
  • internalized antibodies began to be observed after 20 minutes in patient-derived cancer cells (FIG. 17). ).
  • IRCR-101 and 1A03 antibodies showed cancer cell specific internalization (FIG. 18).
  • IRCR-101 and conventional NRP1 antibodies were used to compare the difference in binding capacity between normal cells and cancer cells.
  • the existing NRP1 antibody showed greater binding capacity to normal cells compared to cancer cells, whereas IRCR-101 showed cancer cell specific binding ability (FIG. 19).
  • the glioblastoma cell line U87MG and patient-derived cells were used to determine whether three anti-NRP1 IgG antibodies inhibit cancer cell migration. After incubation at 37 ° C. for 24 hours after each antibody treatment, IRCR-101 and 1A03 inhibited cancer cell migration by more than 50% in both cells, and 4F12 inhibited cancer cell migration by about 40% in patient-derived cells. (Figure 20).
  • IRCR-101 With the final anti-NRP1 IgG antibody IRCR-101, migration inhibition was observed using MBAMB231, a breast cancer cell line, and A549, a lung cancer cell line. IRCR-101 (10ug / ml) treatment was found to inhibit 60% in breast cancer model and 30% cancer cell migration in lung cancer model (FIG. 21).
  • IRCR-101 5mg / kg was injected three times per week to determine tumor size. Tumor size inhibition was 30-40% compared to the control group, and immunofluorescence confirmed the increase in apoptosis by IRCR-101 (TUNEL assay) (FIG. 23).
  • Subcutaneous administration model using the glioblastoma cell line U87MG was prepared and compared with the evaluation of efficacy with the competition antibody MNRP1685A (MNRP1685A antibody, self-produced by sequence synthesis shown in the patent (WO2011143408 A1)).
  • MNRP1685A antibody self-produced by sequence synthesis shown in the patent (WO2011143408 A1)
  • MNRP1685A showed 60% inhibition
  • IRCR-101 showed 80% tumor growth inhibition (FIG. 24).
  • MNRP1685A showed 19% inhibition and IRCR-101 showed 57% tumor growth inhibition in the 25 mg / kg twice weekly injection (FIG. 25).
  • the fluorescent material was labeled with IRCR-101 and injected into the intravenous injection to observe the change in fluorescence intensity over time.
  • IRCR-101 IRCR-101
  • fluorescence was strongly developed at the site corresponding to the tumor location at 1day and fluorescence was developed at the same location until 3day (FIG. 26).
  • IRCR-101 male and female Monkey TMA (Tissue microArray) were run along with the existing NRP1 antibody (MNRP1685A antibody, self-produced through sequence synthesis disclosed in Patent (WO2011143408 A1)). .
  • NRP1 antibody MNRP1685A antibody, self-produced through sequence synthesis disclosed in Patent (WO2011143408 A1).
  • IRCR-101 was found to have less or less binding than NRP1 antibody in most normal organ tissues. Therefore, it is expected that the side effects of IRCR-101 in the clinical trials is low because there is little or no binding force in normal tissues (FIG. 27).
  • the screening method according to the present invention can not only screen for antibodies targeting a patient specific overexpression protein by using patient-derived cells, but also can produce patient-specific antibodies through the antibodies screened according to the present invention. It is useful for developing therapeutics. In addition, antibodies or antigen-binding fragments thereof selected through the method of the present invention are expected to have a high probability of success in the future. Moreover, the screening method according to the present invention enables the selection of antibodies to be internalized, thereby allowing selection of antibodies suitable for drug-antibody conjugate (ADC) production.
  • ADC drug-antibody conjugate

Abstract

La présente invention concerne un procédé de criblage d'un anticorps ou d'un fragment de liaison à l'antigène de celui-ci à l'aide de cellules dérivées de patients et, plus particulièrement, un procédé de criblage d'un anticorps se liant spécifiquement à un antigène ou à un fragment de liaison à l'antigène de celui-ci à l'aide de cellules dérivées d'un patient contenant l'antigène.
PCT/KR2017/005766 2016-06-03 2017-06-02 Procédé de criblage d'anticorps à l'aide de cellules dérivées de patients WO2017209553A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP2018562986A JP6895460B2 (ja) 2016-06-03 2017-06-02 患者由来腫瘍スフェロイドを用いた抗体スクリーニング方法
EP17807046.2A EP3480598A4 (fr) 2016-06-03 2017-06-02 Procédé de criblage d'anticorps à l'aide de cellules dérivées de patients
SG11201810779PA SG11201810779PA (en) 2016-06-03 2017-06-02 Method for screening antibody using patient-derived tumor spheroids
US16/306,566 US11199536B2 (en) 2016-06-03 2017-06-02 Method for screening antibody using patient-derived tumor spheroids
AU2017273169A AU2017273169B2 (en) 2016-06-03 2017-06-02 Method for screening antibody using patient-derived tumor spheroids
CN201780041974.6A CN109416364B (zh) 2016-06-03 2017-06-02 使用患者来源的肿瘤球体筛选抗体的方法
CA3026236A CA3026236C (fr) 2016-06-03 2017-06-02 Procede de criblage d'anticorps a l'aide de cellules derivees de patients

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20160069355 2016-06-03
KR10-2016-0069355 2016-06-03
KR10-2017-0069140 2017-06-02
KR1020170069140A KR101993892B1 (ko) 2016-06-03 2017-06-02 환자 유래 세포를 이용한 항체 스크리닝 방법

Publications (2)

Publication Number Publication Date
WO2017209553A2 true WO2017209553A2 (fr) 2017-12-07
WO2017209553A3 WO2017209553A3 (fr) 2018-01-18

Family

ID=60477675

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2017/005766 WO2017209553A2 (fr) 2016-06-03 2017-06-02 Procédé de criblage d'anticorps à l'aide de cellules dérivées de patients

Country Status (1)

Country Link
WO (1) WO2017209553A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11820826B2 (en) 2018-10-23 2023-11-21 Regeneron Pharmaceuticals, Inc. Anti-NPR1 antibodies and uses thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
WO1988001649A1 (fr) 1986-09-02 1988-03-10 Genex Corporation Molecules de liaison de chaines de polypeptide simples
WO1988006630A1 (fr) 1987-03-02 1988-09-07 Genex Corporation Procede de preparation de molecules de liaison
WO1988007086A1 (fr) 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Sequences d'amorce pour la production de proteines recombinantes
WO1988007085A1 (fr) 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Procede de purification de polypeptides recombinants
WO1988009344A1 (fr) 1987-05-21 1988-12-01 Creative Biomolecules, Inc. Proteines mutifonctionnelles a cible predeterminee
WO2011143408A1 (fr) 2010-05-13 2011-11-17 Genentech, Inc. Utilisation d'antagonistes de la neuropiline 1 pour le traitement du cancer

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7135609B2 (en) * 2001-05-10 2006-11-14 Chugai Seiyaku Kabushiki Kaisha Non-human animal exhibiting bone metastasis of tumor cells and method of screening for bone metastasis inhibitors
UA96139C2 (uk) * 2005-11-08 2011-10-10 Дженентек, Інк. Антитіло до нейропіліну-1 (nrp1)
JP5906184B2 (ja) * 2009-06-22 2016-04-20 バーナム インスティテュート フォー メディカル リサーチ Ｃ末端エレメントを有するペプチドおよびタンパク質を使用する方法および組成物

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
WO1988001649A1 (fr) 1986-09-02 1988-03-10 Genex Corporation Molecules de liaison de chaines de polypeptide simples
WO1988006630A1 (fr) 1987-03-02 1988-09-07 Genex Corporation Procede de preparation de molecules de liaison
WO1988007086A1 (fr) 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Sequences d'amorce pour la production de proteines recombinantes
WO1988007085A1 (fr) 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Procede de purification de polypeptides recombinants
WO1988009344A1 (fr) 1987-05-21 1988-12-01 Creative Biomolecules, Inc. Proteines mutifonctionnelles a cible predeterminee
WO2011143408A1 (fr) 2010-05-13 2011-11-17 Genentech, Inc. Utilisation d'antagonistes de la neuropiline 1 pour le traitement du cancer

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANDERSEN PS ET AL., PROC NATL ACAD SCI USA, vol. 93, 1996, pages 1820 - 1824
BARRY MA ET AL., NAT MED, vol. 2, 1995, pages 299 - 305
CAI X; GAREN A, PROC NATL ACAD SCI USA, vol. 92, 1995, pages 6537 - 6541
See also references of EP3480598A4
YANG ET AL., MOL. CELLS., vol. 27, 2009, pages 225 - 235

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11820826B2 (en) 2018-10-23 2023-11-21 Regeneron Pharmaceuticals, Inc. Anti-NPR1 antibodies and uses thereof

Also Published As

Publication number Publication date
WO2017209553A3 (fr) 2018-01-18

Similar Documents

Publication Publication Date Title
AU2019229076B2 (en) Anti-TIGIT antibodies and uses thereof
WO2015005632A1 (fr) Nouvelles protéines à double cible se liant spécifiquement à dll4 et vegf et leur utilisation
WO2014119969A1 (fr) Anticorps anti-c5 et méthode de prévention et de traitement de maladies liées au complément
WO2016153276A1 (fr) Peptide liant spécifiquement la neuropiline-1, protéine de fusion fusionnée à celui-ci et utilisation correspondante
WO2017074074A1 (fr) Nouvel anticorps anti-glypicane 3 et composition pharmaceutique le contenant
WO2019004799A1 (fr) Conjugué de protéine de vegf-grab et de médicament, et son utilisation
WO2012165925A2 (fr) Anticorps anti-c-met présentant une activité hgf et son utilisation
WO2019112347A2 (fr) Anticorps ou son fragment de liaison à l'antigène pour reconnaître spécifiquement une malignité de cellule b, récepteur d'antigène chimérique le comprenant, et son utilisation
WO2016089126A1 (fr) Anticorps dirigé contre la neuropiline 1 et son utilisation
WO2017030370A1 (fr) Récepteur d'anticorps chimérique auquel est lié un anticorps anti-cotinine et utilisation de celui-ci
WO2019132533A1 (fr) Anticorps anti-pd-l1 et son utilisation
WO2022030730A1 (fr) Récepteur antigénique chimérique anti-mésothéline se liant de manière spécifique à la mésothéline
KR101993893B1 (ko) 항-nrp1 항체 스크리닝 방법
WO2017074013A1 (fr) Anticorps destiné à être réticulé à la sema3a humaine et de souris, et son utilisation
WO2017209553A2 (fr) Procédé de criblage d'anticorps à l'aide de cellules dérivées de patients
WO2019209073A1 (fr) Procédé de bioadhérence à base magnétique par fixation de bille magnétique à une cellule
WO2022216014A1 (fr) Anticorps anti-cntn4 et son utilisation
WO2017209554A2 (fr) Méthode de criblage d'anticorps anti-nrp1
WO2016084993A1 (fr) Nouvel anticorps anti-egfrviii et composition le comprenant
WO2014021693A2 (fr) Anticorps monoclonal inédit se liant spécifiquement à la protéine tm4sf5 et son utilisation
KR101993892B1 (ko) 환자 유래 세포를 이용한 항체 스크리닝 방법
WO2017142294A1 (fr) Anticorps anti-egfrviii et utilisation correspondante
WO2019151632A1 (fr) Molécule de liaison ayant une activité neutralisante contre le coronavirus du syndrome respiratoire du moyen-orient
WO2022244908A1 (fr) Anticorps anti-bcam ou fragment de liaison à l'antigène de celui-ci
WO2024014619A1 (fr) Anticorps neutralisant le sars-cov-2

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: 17807046

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 2018562986

Country of ref document: JP

Kind code of ref document: A

Ref document number: 3026236

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017807046

Country of ref document: EP

Effective date: 20190103

ENP Entry into the national phase

Ref document number: 2017273169

Country of ref document: AU

Date of ref document: 20170602

Kind code of ref document: A