WO2011160429A1 - Anticorps humanisés anti-p185 (her2/erbb2) - Google Patents

Anticorps humanisés anti-p185 (her2/erbb2) Download PDF

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WO2011160429A1
WO2011160429A1 PCT/CN2011/001012 CN2011001012W WO2011160429A1 WO 2011160429 A1 WO2011160429 A1 WO 2011160429A1 CN 2011001012 W CN2011001012 W CN 2011001012W WO 2011160429 A1 WO2011160429 A1 WO 2011160429A1
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antibody
amino acid
protein
sequence
seq
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PCT/CN2011/001012
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Chinese (zh)
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肖卫华
郭雨刚
常亮
胡思怡
刘兢
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中国科学技术大学
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    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an anti-P185 (HER2/ErbB2) humanized antibody.
  • P185 is a cell surface important receptor tyrosine kinase encoded by the oncogene Her-2/erbB-2 and belongs to the epidermal growth factor receptor family.
  • the P185/HER2 receptor ie, human epidermal growth factor receptor 2
  • the P185/HER2 receptor is similar to other members of the family and consists of three domains: the extracellular domain, the transmembrane domain, and the intracellular domain, of which the extracellular domain (ECD) is further divided.
  • ECD extracellular domain
  • the I and III sub-regions are leucine-rich regions
  • the ⁇ and IV sub-regions are cysteine-rich regions (Carpenter G. (1987) Receptors of epidermal growth factor and other polypeptide mi togens Annu. Rev. Biochem.
  • Epidermal growth factor family receptors are widely distributed in a variety of tissues, especially in epithelial tissues, mesenchymal tissues, and neurogenesis tissues, and play an important role in regulating cell proliferation, differentiation, development, adhesion, and migration.
  • P185/HER2 is the most important member of the signal transduction network of the epidermal growth factor receptor family, and its abnormal expression level is closely related to cell signal transduction disorders, tumorigenesis and malignant development (Nancy EH,
  • pl85/HER2 has been widely studied as a hot spot in the field of cancer therapy.
  • anti-HER2 monoclonal antibodies inhibited the growth of HER2-transformed NIH3T3 tumor cells transplanted into nude mice, and the first in vivo experiments demonstrated that anti-HER2 monoclonal antibodies may have high expression of HER2 tumors.
  • the value of clinical treatment (Drebin JA, Link VC, We inberg RA, Greene MI. (1986) Inhibi tion of tumor growth by a monoclonal ant ibody Reactive wi th an oncogene- encoded tumor antigen. Proc Natl Acad Sc i USA 83 : 9129-9133 ).
  • variable region of the mouse antibody and the human antibody constant region are linked by genetic engineering technology, that is, the constant region gene of the mouse antibody is replaced with the human antibody constant region gene, and the variable region portion bound to the antigen is still the mouse source. antibody.
  • the engineered chimeric antibody constructed by this method retains the murine antibody variable region capable of recognizing the target antigen to the utmost extent, and at the same time possesses the immunological killing effect of all the constant regions of the human antibody, and due to the chimeric antibody
  • the immunogenicity is reduced by 2/3 compared to the original murine antibody, so it can be applied to human therapy (Davi s TA. (1999) Final report on the safety and efficacy of retreatment wi th ri tuximab for patients wi th non- Hodgkin's lymphoma. Blood 94: 88a).
  • the more fully humanized method is to re-splicing the part of the murine antibody variable region involved in antigen binding with the framework region of the human antibody variable region selected for template, so as to remove the height of the antibody variable region.
  • the variable complementarity determining regions (CDRs) are derived from murine antibodies and the remainder are replaced by adult sources.
  • CDR grafting Since the antibody variable region is generally involved in antigen binding in its six complementarity determining regions (CDRs), the process is also referred to as "CDR grafting", and then in order to overcome the possible decrease in affinity due to "CDR grafting", a The series of mutations in the framework region residues gradually restore or enhance the antigen binding ability of the antibody, thereby obtaining a more humanized reconstituted antibody (Jones PT, Dear PH, Foote J.
  • a series of mutants were constructed separately for framework residues that may affect binding ability, and a suitable combination of mutations was identified. These mutants need to be independently prepared and tested for their antigen binding ability, and then the better mutations are combined, that is, repeated mutation experiments for mutations and screening of various possible residues are required, so the steps are complicated, the efficiency is low, and the effect is not satisfactory. .
  • amino acid sequence of the protein provided by the present invention is shown in SEQ ID NO: 15.
  • SEQ ID NO: 15 is amino acid at positions 33-36 from the N-terminus, NNQK, amino acids 97-100 are YSNY, and amino acids 184-188 are HISSS, SEQ ID NO: 15 is SEQ. ID NO: 3 shows the HI-2 protein (HI-2: V L + Linker + V H + Fc ).
  • SEQ ID NO: 15 is SEQ. ID NO : 11 shows the Hl-2_mut3 protein (HI- 2- mut3: V L +Linker+V H +Fc ).
  • SEQ ID NO: 15 is amino acid at positions 33-36 from the N-terminus, ERGW, amino acids 97-100 are YNQE, and amino acids 184-188 are VITSP, SEQ ID NO: 15 S ⁇ Hl-2-mut7 protein (HI- 2- mut7: SEQ ID NO: 13: V L +Linker+V H +Fc).
  • the protein provided by the present invention is any of the following proteins:
  • n a protein having the same function of substitution and/or deletion and/or addition of one or more amino acid residues of amino acids 115-134 from its N-terminus in a defined protein;
  • the coding gene is as follows:
  • the coding gene encoding the protein indicated by a) is:
  • the nucleotide sequence is a DNA molecule represented by nucleotides 1-759 from the 5' end of the sequence 4 in the sequence listing;
  • the coding gene encoding the protein shown in b) is:
  • the nucleotide sequence is the sequence in the sequence listing
  • the coding gene encoding the protein shown in the above e) is:
  • the nucleotide sequence is the DNA molecule of nucleotide sequence 1-759 from the 5' end of the sequence 2;
  • the coding gene encoding the protein indicated by f) is: the nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2 in the sequence listing;
  • the coding gene encoding the protein represented by i) is:
  • the nucleotide sequence is a DNA molecule represented by nucleotides 1-759 from the 5' end of the sequence 6 in the sequence listing;
  • the coding gene encoding the protein represented by j) is: the nucleotide sequence is the DNA molecule shown in SEQ ID NO: 6 in the sequence listing;
  • the coding gene encoding the protein shown in m) is:
  • the nucleotide sequence is the sequence in the sequence listing 12 a DNA molecule represented by nucleotides 1-759 from the 5'end;
  • the coding gene encoding the protein represented by SEQ ID No: 11 is: The nucleotide sequence is the DNA molecule shown by the sequence 12 in the sequence listing;
  • the coding gene encoding the protein represented by p) is:
  • the nucleotide sequence is a DNA molecule represented by nucleotides 1-759 from the 5' end of the sequence 14 in the sequence listing;
  • the coding gene encoding the protein represented by SEQ ID No: 13 is:
  • the nucleotide sequence is the DNA molecule shown in SEQ ID NO: 14 in the Sequence Listing;
  • a recombinant vector, recombinant strain, transgenic cell line, recombinant virus or expression cassette containing any of the above-described coding genes is also within the scope of the present invention.
  • the recombinant vector is obtained by inserting the coding gene into the multiple cloning site of the pSe C tag2A vector.
  • Another object of the invention is to provide an antibody.
  • the antibody provided by the invention comprises a light chain variable region and a heavy chain variable region
  • the light chain variable region is at least one of the following protein fragments: amino acids 1-114 of SEQ ID No: 11 , amino acids 1-114 of SEQ ID No: 13, and SEQ ID No: 3 1-114 amino acids, amino acids 1-114 of SEQ ID No: 1, amino acids 1-114 of SEQ ID No: 5;
  • the heavy chain variable region is at least one of the following protein fragments: amino acids 135-253 in SEQ ID No: 11 , amino acids 135-253 in SEQ ID No: 13, and SEQ ID No: 3 Amino acid at positions 135-253, amino acids 135-253 in SEQ ID No: 1, amino acids 135-253 in SEQ ID No: 5;
  • the above-mentioned antibody of the present invention is any form of antibody obtained by ligating the light chain variable region and the heavy chain variable region in any manner, or by a light chain variable region, a heavy chain variable region and other fragments (such as a constant region). Any of the obtained antibodies can be ligated in any manner; specifically, a chimeric antibody, an antibody having a structure similar to that of an intact natural antibody, or the like.
  • the antibody provided by the present invention may specifically be composed of two identical proteins as described above; The two identical proteins are linked by a disulfide bond.
  • the antibody is prepared as follows: The recombinant vector of any of the above is introduced into a host cell to obtain a recombinant cell, the recombinant cell is cultured, and the supernatant is collected to obtain the antibody.
  • the host cell is 293T cells or CH0 or COS-7.
  • Another object of the present invention is to provide a process for the preparation of the above antibody.
  • the preparation method comprises the steps of: introducing any of the recombinant vectors described above into a host cell to obtain recombinant cells, culturing the recombinant cells, and collecting the supernatant to obtain the antibody.
  • the host cell is a 293T cell.
  • Another object of the invention is to provide a method of humanization of antibodies.
  • the method for humanization of the antibody provided by the present invention comprises the following steps:
  • variable region spatial structure of the antibody to be engineered from the pre-humanized antibody template The humanized antibody with the highest degree of similarity is used as a template for humanized antibody;
  • amino acid sequence similarity between the heavy chain variable region and the light chain variable region of the heavy chain variable region and the light chain variable region of the antibody to be engineered are respectively 50% or more;
  • the amino acid sequence similarity between the framework region of the heavy chain variable region and the framework region of the light chain variable region and the framework region of the heavy chain variable region of the antibody to be engineered and the framework region of the light chain variable region, respectively, are 50 More than % or more than 60%;
  • variable region spatial structure is as follows: a) and / or b): a) a spatial structure formed by a heavy chain variable region and a light chain variable region; b) a framework region in the heavy chain variable region and a spatial structure formed by the frame regions in the variable region of the light chain;
  • An antibody with an unknown spatial structure can be simulated by software, including a spatial structure composed of a heavy chain variable region and a light chain variable region, and a framework region and a light chain variable region in the heavy chain variable region.
  • the frame structure of the frame area together.
  • the antibody of the unknown CDR region can be identified by integrating the sequence definitions of Kabat et al. and Chothia et al., and the comprehensive value of the identification results of the two methods is taken as the final value, that is, the minimum value to the maximum value of the two identification results are taken as CDR region.
  • the engineered humanized antibody of interest is expressed by a genetic engineering method.
  • the following step of reverting comprises: replacing at least one amino acid residue of the framework region of the antibody obtained in the step (2)
  • the obtained antibody has a stable spatial structure, and the obtained antibody is used as a humanized antibody for modification.
  • variable region is a heavy chain variable region and/or a light chain variable region
  • the amino acid residue to be replaced is an amino acid which affects the structural stability of the CDR region of the antibody obtained in the step (2) or affects the structural stability of the antigen binding region of the antibody obtained in the step (2). Residues;
  • the amino acid residue to be replaced is located in the vernier region of the antibody obtained in the step (2) and/or the amino acid residue to be replaced is located in the antibody obtained in the step (2).
  • the amino acid residue to be replaced is located in the vernier region of the antibody obtained in the step (2) and/or the amino acid residue to be replaced is located in the antibody obtained in the step (2).
  • the framework region of the antibody into which the back is obtained is non-human and different from the antibody to be engineered
  • the amino acid residue of the amino acid at the corresponding position is mutated to a human amino acid residue, and the obtained antibody is used as a humanized antibody for modification.
  • Humanized antibodies obtained by any of the methods described above are also within the scope of the invention.
  • the antibody of the present invention is a humanized antibody variant of the chimeric antibody ChA21 which specifically recognizes the tumor surface antigen p l85 ( HER2/ErbB2 ).
  • the humanized antibody variant of ChA21 provided by the present invention includes the scFv, the hinge region and the human Fc region in which the murine single-chain antibody of the original chimeric antibody ChA21 is fully humanized.
  • the amino acid sequence of the humanized scFv is:
  • DIVLTQSPDSLAVSLGERVTINC KSSQTLLYSNNQKNYLA WYQQKPGQSPKLLIS WAFTRKS GVPDRFSGSGSGTDFTLTISSVQAEDVAVYYC QQYSNYPWT FGAGTKLEIKR GGGGSGGGGS GGGGSGGGGS QVQLVQSGAEVVKPGASVKISCKAS GYSFTGYFIN WVKQNPGQRLEWIG HISSSYATSTYNQKFKG KATLTVDTSASTAYMELSSLRSEDTAVYY CVR SGNYEEYAMDY WGQGTLVTVS.
  • the underlined part is a total of 6 complementary determinants (CDRs) of the 3 light chains and 3 heavy chains of the antibody, and the black body part is the linker between the light and heavy chain variable regions.
  • CDRs complementary determinants
  • the humanized antibody engineering method of the present invention comprises the following steps:
  • the antigen-binding segment of the antibody of interest is selected.
  • the complementarity determining regions are generally selected; (the CDR sequences of the antibodies can be identified by integrating the sequence definitions of Kabat et al. and Chothia et al.) for the crystal structure of antibodies and antigen complexes.
  • Antibodies which can combine their CDR sequences and structural data to obtain their antigen binding regions.
  • the selected segment residues are substituted for the residues at the corresponding positions of the human template antibody, and the original version of the humanized anti-P185/HER2 antibody sequence is designated as HI, and the simulation of HI is obtained by computer homology simulation. Spatial structure.
  • residues include all of the vernier residues (Vernier) and all of the framework region residues within 3 angstroms of the CDR residues, and the sites that are more consistent with the structural stability of the antibody are selected for the direction in which the mutation is desired.
  • residues 4 and 49 of the light chain variable region VL of the human human antibody HI and residues 71 and 93 of the heavy chain variable region VH were selected to be mutated to the target antibody.
  • the present invention also utilizes phage display technology to establish a phage antibody library to screen humanized antibody high-affinity mutants, and obtains a series of humanized antibodies.
  • the modified antibody of the invention has higher affinity with the antigen than the original antibody, and has achieved complete humanization, and has a good clinical application prospect.
  • Figure 1 shows the variable region amino acid sequences of the light and heavy chains of the antibody ChA21, and the sequence numbers are sorted according to Kabat rules.
  • the underlined part is the complementarity determining regions (CDRs) that are defined by the integrated Kabat and Chothia sequence definitions.
  • Figure 2 shows the results of spatial structure alignment of the variable region of the single-chain variable region of the antibody ChA21 (scFv, PDB number: 2GJJ) and the selected human template antibody huCC49 (PDB number: 1ZA6).
  • Figure 3 shows the complex structure of the single-chain variable region of the antibody ChA21 and its recognition antigen partial sub-region.
  • Figure 4 is a diagram showing the amino acid sequence of the original version of the humanized antibody HI obtained by transplantation of the antigen-binding region of ChA21, and the variable region-level sequence alignment with the original murine antibody and the human template antibody.
  • Figure 5 is a molecular model of humanized antibody H1-1 obtained by computer homology simulation.
  • Figure 6 is a diagram showing the results of selection of a series of framework region residue mutations based on the HI sequence and structure of the humanized antibody, and the sequence of the obtained new humanized antibody variants H1-1 and H1-2 Comparison. The complementarity decision area is indicated by a square.
  • Figure 7 is a diagram showing the humanization of ChA21 using the humanized antibody hu4D5 (PDB number: 1FVC) obtained based on the universal template VL KI -VHIII as a human template, and the amino acid sequence of the obtained humanized antibody H2-1 .
  • Figure 8 is a schematic diagram showing the construction of a humanized antibody H1-1 and H1-2 recombinant expression vector.
  • Figure 9 is an antigen binding curve of antibody ChA21 and various humanized mutants thereof.
  • Figure 10 is a binding curve of two mutants of Hl-2-mut3 and Hl-2_mut7 to an antigen.
  • the sequence of the chimeric antibody chA21 has been described by LS Cheng et al. (Chen LS, Liu AP, Liu. J. Construction, expression and characterization of the engineered antibody against tumor surface antigen pl85 c - erbb - 2 . Cell Res. 2003, 13 : 35-48) , as shown in Figure 1.
  • the structure of the chimeric antibody chA21 Two identical protein chains are bound by a disulfide bond formed in the Fc region.
  • Each protein chain is composed of a light chain variable region (VJ, linker, heavy chain variable region ( VH ) and an Fc region in turn, and its amino acid sequence is shown as SEQ ID NO: 9, wherein, 1-114 The position is VL, the 115th-134th position is linker, the 135th-253th position is VH , and the 254th to 490th position is the Fc-containing fragment. The Fc-containing fragment is not completely Fc, and the cloning site is also produced. Excess four amino acids. 1. Selection of human antibody template
  • Sequence similarity alignment The amino acid sequence similarity of the light chain variable region of chA21 and the light chain variable region of the humanized antibody is aligned, and the heavy chain variable region of chA21 and the heavy chain of the humanized antibody can be The variable regions are subjected to amino acid sequence similarity alignment.
  • Sequence similarity searches were performed separately in the protein database (PDB, Protein Data Bank).
  • the specific search parameters are as follows: Program: blastp; Submit sequence source: mouse; Database: pdb; Alignment sequence source: huma n .
  • the number of display comparison results can be specifically selected according to the comparison result, and other parameters can be selected as default parameters.
  • Structural similarity alignment PyMOL software was used to compare the overall spatial structural similarity of the candidate-derived antibody template and the variable region of ChA21 (scFv, PDB: 2GJJ) by molecular align (ie, heavy chain in protein) Spatial junction composed of variable region and light chain variable region And the spatial structure similarity of the framework region (ie, the spatial structure X consisting of the framework region in the heavy chain variable region and the framework region in the light chain variable region, the size of the difference between the structures is represented by RMS), each candidate template
  • Table 1 the sequence numbers in all the tables are numbered by Kabat sequence).
  • template 1FVC is a humanized antibody hu4D5 obtained by human modification of antibody 4D5 based on universal human antibody template VL KI -VHIII, and its framework region sequence is substantially identical to VL KI -VH III, based on this universal template structure pair ChA21 was simultaneously humanized to compare the humanization effects of the method of the present invention and the general method.
  • the antigen binding segment of ChA21 was selected.
  • the CDR sequences of the antibody ChA21 can be identified by binding to the sequence definitions of Kabat et al. and Chothia et al., and the CDR sequence partitioning results are shown in Table 2.
  • the final CDR regions are the underlined segments in Figure 1.
  • the antigen-binding portion thereof can be visually obtained from the complex structure as shown in FIG. It can be seen that the second half of the heavy chain CDR2 is located on the surface of the antibody molecule, does not participate in antigen binding, has no effect on the structure of other CDR regions, and is not involved in the interaction between the light and heavy chains of the antibody, so humanization from the improved sequence The degree of requirement is considered as a non-antigen binding region, and this sequence on the heavy chain CDR2 of ChA21 is not transplanted.
  • the first type of residue considered is a residue that may affect the conformation or structure of the complementarity determining region, or Residues that may directly affect antigen binding, based on previous studies of such framework residues, a list of such residues (see Table 3), including nearly 30 cursors that have a potential contribution to CDR structure Residues (Vernier); in addition, all residues within 3 ⁇ of the CDR residues are selected in the mimetic HI structure, and these residues may also directly affect the conformation of the CDR region residues from a structural point of view, and they It is included in the cursor residues listed in Table 3.
  • non-antigen-binding residues were then humanized. Since the selected human template antibody huCC49 is an antibody that has been humanized, there are also residues on the framework sequence that are returned to the parental mouse antibody during humanization, and these residues are shared by some mouse antibodies. Residues and residues specific to some of the huCC49 parental murine antibody mCC49, while the latter are not necessary for other murine antibodies, so from the perspective of increasing the degree of humanization, on the basis of not affecting structural stability, These residues are mutated to the corresponding human residues. Specific to the sequence of H1-1, through sequence alignment, find that the framework sequence is different from ChA21 and different from human source.
  • huCC49 light chain template sequence LEN and huCC49 heavy chain template sequence 2 1 /28 'CL
  • LEN and huCC49 heavy chain template sequence 2 1 /28 'CL residues 5, 21 and 106 in VL and residues in VH 61, 62, 80 and 91
  • select mutations to the residues of the human template sequence LEN and 21 /28 'CL corresponding positions see Table 4
  • the humanized sequence H1-2 was finally obtained, and the alignment results of all the sequences are shown in Fig. 6.
  • the humanized antibody H2-1 (Fig. 7) was obtained by the same method using the humanized antibody hu4D5 (PDB: 1FVC, which is generally regarded as a universal template) as a template, and the humanized antibody was used as a control, and
  • the human antibody obtained by the humanized template selection method in the invention has an effect on the affinity of the antibody compared with each other.
  • hu4D5 is a humanized template (ie, a universal template)
  • H2 is an antibody obtained after CDR replacement (the replacement method is identical to step 2 above)
  • HumanVL-Kappa I is a human corresponding to the light chain variable region of antibody hu4D5.
  • the source sequence, HumanVH-III is the human sequence corresponding to the heavy chain variable region of the antibody hu4D5
  • H2-1 is the sequence after the final humanization.
  • the pSectag2A vector was purchased from Invi trogen.
  • the Fc fragment was amplified by PCR, and primers were designed to add EcoRI and Xhol cleavage sites at the N-terminus and C-terminus of the Fc fragment, and then the Fc fragment was digested with EcoRI and Xhol to recover the double-digested fragment; and pSe C was simultaneously
  • the tag2A plasmid was digested with EcoRI and Xhol to recover the double-digested fragment; the double-cut Fc fragment and the pSe C tag2A vector were ligated with T4-DNA ligase, and the ligated product was transformed into Topl O competent cells.
  • the plasmid was subjected to double enzyme digestion and sequencing, and the gene sequence inserted between the EcoRI and Xhol cleavage sites of pSectag2A was shown as nucleotides 760-1470 of SEQ ID NO: 2 (ie, Fc fragment). This indicates that the constructed recombinant vector is correct, and this vector is referred to as pSectag2A-dFc as an expression vector for the humanized antibody.
  • the scFv-Fc/pEE14 (Chinese Patent CN1613873) was used as a template, and the following primers were used for PCR amplification to obtain an Fc fragment. Or artificially synthesize nucleotides 760-1470 of SEQ ID NO: 2. The Fc fragment shown.
  • dFc-XhoI 5' -TTTCTCGAGTCATTTACCCGGAGACAGGGAGAG-3' (underlined for EcoRI site)
  • H1-1 5'-ATATGCGGCCGCAGAAACAGTAACCAAAGTACC - 3 ' was subjected to PCR amplification, and the PCR amplification product was detected by 1% agarose gel electrophoresis and the target band was recovered and named as "H1-1 " .
  • the obtained H1-1 gene fragment was digested with restriction endonucleases Sfil and Not I, respectively, and the digested fragments were ligated with the same enzyme digested plasmid pSe C tag2A-dF C by T4 DNA ligase. The ligation product is transformed into E.
  • the gene sequence inserted between the Sfil and Not I sites of P Sectag2A-dFc is SEQ ID NO: The nucleotides at positions 1-759 of 2 are shown.
  • the positive recombinant plasmid was named pSe C tag2A-dF C -Hl-1.
  • the recombinant vector comprises the gene represented by SEQ ID NO: 2, wherein the 1-34 position is VL, the 343-402 is Linker, the 403-759 is VH , and the 760-1470 is Fc;
  • the gene encodes a protein as shown in SEQ ID NO: 1. In this protein, positions 1-114 are VL, positions 115-134 are Linker, positions 135-253 are VH , and positions 254-489 are Fc-containing. Fragment of.
  • the coding sequence of H1-2 was obtained by a point mutation method based on the obtained gene sequence of H1-1.
  • the specific method is shown in Figure 8. It is carried out in two rounds of point mutation.
  • the first round of point mutation in H1-1 is first obtained to obtain the H1-V1 intermediate mutant, and the second round point mutation of H1-V1 is performed to obtain H1-2. sequence.
  • the mutation sites for humanized antibody H1-1 are shown in Table 5:
  • HI-VI T ACT
  • ATT ATT
  • ATT ATT
  • AGT ATG
  • TAC HI-VI T
  • P3 (P112I-Rv): 5'-CTAAGTTGGAAATTAAGAGAGGTGGTGG -3' was subjected to PCR amplification, and the target band was recovered and named "H1-V1-5'".
  • PCR was carried out using pMD-19T-H 1 as a template under the guidance of primers P4 ( P 1121 - Fw ): 5, - CTAAGTTGGAAATTAA GAGAGGTGGTGG - 3' and P5 (P215M-RV) : 5' - GTCTTCCGATCTCAAAGAAGACA ATTCCATGTAAGCAG-3 ' Amplification, recovery of the desired band, named "Hl-Vl-m".
  • PCR amplification was carried out under the guidance of P6 (P229Y-Fw): 5, - GTCTTCTTTGAGATCGGAAGACACTGCTGTTTACTACTGTG-3, and P7 (Hl-Rv-Notl): 5'-ATATGCGGCCGCAGAAACAGTAACCAAAGTACC -3' Increase, recycle the desired strip, named "H1-V1-3'".
  • the H1-V1 gene fragments obtained in the step 4) were separately digested with restriction endonucleases Sfil and Not I, and the digested fragments were respectively ligated with the plasmid pSe C tag 2A-dF C digested with the same enzyme.
  • the ligation product was transformed into E. coli ToplO competent cells, positive recombinants were screened, plasmid DNA was extracted, and sequenced.
  • the gene sequence inserted between the Sfil and Not I sites of pSectag2A-dFc was as in SEQ ID NO: 6 Shown at nucleotide position 759. Name the positive recombinant plasmid For pSectag2A-dFc-H1-V1.
  • the recombinant vector comprising the SEQ ID N0: 6 shown gene, the gene is the first position 1-342 V L, the first bit is the Linker 343-402, 403-759 of the bit is V H, the first bit is the Fc 760-1470
  • the gene encodes a protein as shown in SEQ ID NO: 5, where the 1-114th position is V L , the 115-134th position is Linker, the 135-253th position is VH , and the 254-489th position is Fragment containing Fc.
  • the humanized antibody Hl-2 encoding gene having the above mutation site is amplified by site-directed mutagenesis, and the specific method comprises the following steps:
  • PCR amplification was carried out under the guidance of primers PI and P8 (Hl-2-Rv): 5, - GCCTTACCCTTGAATTTTTGGTTGTAAGTAGAAG -3', and the target band was recovered and named "H1-2 -5' " .
  • PCR amplification was carried out under the guidance of primer P9 (Hl-2-Fw): 5'-CTTCTACTTACAACCAAAAATTCAAGGGTAAGGC-3, and P. p7:, and the target band was recovered and named "H1". -2-3' " .
  • PCR amplification was carried out under the guidance of primers P1 and P7 using the H1-2-5' amplified in step 1) and the H1-2-3' gene fragment amplified in step 2), and the target band was recovered and named as "Hl-2".
  • the H1-2 gene fragment obtained in the step 3) was separately digested with restriction endonucleases Sfil and Not I, and the digested fragments were respectively ligated with the plasmid pSe C tag 2A-dF C digested with the same enzyme.
  • the ligation product was transformed into E. coli ToplO competent cells, positive recombinants were screened, plasmid DNA was extracted, and sequenced.
  • the gene sequence inserted between the Sfil and Not I sites of pSectag2A-dFc was as in SEQ ID NO: 4 Shown at nucleotide position 759. Name the positive recombinant plasmid For pSectag2A-dFc-Hl-2.
  • the recombinant vector comprises the gene represented by SEQ ID NO: 4, wherein the first to the 42-42 is V L , the first to the 343-402 is Linker, the third to the 403-759 is V H , and the first to the 760-1470 is a fragment of Fc; the gene encodes a protein as shown in SEQ ID NO: 3, wherein the 1st - 1 14th position is VL, the 115th - 134th position is Linker, and the 135th - 253th position is VH , 254th - Position 489 is an Fc-containing fragment.
  • the gene shown in nucleotides 1-759 from the 5' end of SEQ ID NO: 8 was synthesized and introduced into the vector pMD-19T, and the positive recombinant was designated as PMD-19T-H2-1 by sequencing.
  • PMD-19T-H2-1 as a template in Primer P10 (PH2-Sf i 1 ) : 5, - ATAT GGCC CAGCC GGCC GACATCCAATTGACTCAATCTCCATCTTCTTTG - 3 ' and P7 ( Hl-Rv-Notl ) : 5 ' - ATATGCGGCCGCAGAAACAGTAACCAAAGTACC - 3 PCR amplification was carried out under the guidance of ', and the target band was recovered and named "H2-1".
  • the obtained H2-1 gene fragment was digested with restriction endonuclease YI and ⁇ 3 ⁇ 4 ⁇ , respectively, and the restriction fragment was ligated with the plasmid P Sectag2A-dFc digested with the same enzyme to obtain a positive recombination. Plasmid, sequencing verified, the gene sequence inserted between the Sfil and Not I sites of pSectag2A-dFc as shown in SEQ ID NO: 8 from nucleotides 1 to 759, positive recombinant plasmid Named P Sectag2A-dFc-H2-l.
  • the recombinant vector comprises the gene of SEQ ID NO: 8, wherein the 1-34 position is V L , the 343-402 is Linker, the 403-759 is V H , and the 760-1470 is Fc.
  • the gene encodes a protein as shown in SEQ ID NO: 7. In this protein, the 1-114th position is V L , the first 15-134th position is Linker, the first 135-253th position is V H , and the second position is 254-489. It is a fragment containing Fc.
  • the construction method of the chimeric antibody ChA21 expression vector is identical to that described in Chinese Patent No. CN1613873.
  • Example 3 Transient 293T cell expression of humanized antibody and purification of expression product
  • 293T cells were purchased from ATCC under the catalog number CRL-11268.
  • DMEM medium was purchased from GIBC0, catalog number 1 1960.
  • a sufficient amount of 293T cells (DMEM/10% serum/1% double antibody) was cultured in advance (double antibiotics were penicillin and streptomycin, purchased from Shanghai Shenggong, item number BS732), and when the cells reached about 80% fullness, It is used to transfect recombinant antibody plasmid DNA during the logarithmic growth phase.
  • the transfection steps were as follows: (1) Add 20 ul of Lipof ectamine 2000 to 1 ml of DMEM, mix with fingers or mix with a pipette, and let stand for 5 minutes at room temperature. (2) Then add 10 ug of plasmid DNA ready for transfection in 1 ml of DMEM and mix.
  • the supernatant contains antibodies HI-1, HI-2, H2- 1, HI-VI or ChA21.
  • the antibody ChA21 did not differ from the antibody ChA21-CH0, except that the antibody ChA21 was expressed by 293T cells, and the antibody ChA21-CH0 was expressed by the CH0-GS engineered cell line.
  • the relative affinity activity of the antibody was determined by ELISA.
  • the relative affinity activity of the antibody by ELISA is as follows:
  • Rabbit anti-human Fc polyclonal antibody was purchased from Sigma, catalog number A9544.
  • the preparation method of the antigen GST-EPI is as follows:
  • the HER2 extracellular ⁇ - ⁇ region gene was synthesized (SEQ ID NO: 10).
  • the synthetic gene was double digested with EcoR I (purchased from Promega, Cat. No. R6011) and Xho I (purchased from Promega, Cat. No.
  • pGEX/4T-l-EPI was transformed into Origami B competent (purchased from novagen, Cat. No. 71408-3) and positive expression clones were selected. The positive clones were expanded and cultured. After the 0D600 value of the bacterial solution in the culture flask reached about 0.6, IPTG (purchased from Merch, Cat. No. 420291-5GM) was added to a final concentration of 1 mM for induction expression, 16 ° C, 220 rpm.
  • ChA21-CH0 ChA21 chimeric antibody (ChA21-CH0) expressing and purifying CH0, first diluted to lug/ml with DMEM medium, and then diluted to 100, 50, 25, 12 with blocking agent 5, 6. 25, 3. 13, 1. 56, 0. 78 ng/ml, a total of 8 concentrations, used as a standard curve; for expression supernatant: diluted directly with blocking agent 10, 40, 160, 640 times . The goat anti-human/HRP secondary antibody diluted 1:2000 was added and incubated for 1 hour at room temperature with shaking, and the 0PD substrate developed color and sulfuric acid was terminated. The amount of expression of each mutant antibody was calculated from the standard curve.
  • the antigen used here is GST-EPI
  • EPI is the ErbB2 extracellular I/II region fragment, which contains the recognition epitope of ChA21 and its humanized mutant
  • EPI expressed in the form of a GST fusion protein can be used in place of the full antigen ErbB2 for affinity determination of antibody variants.
  • GST-EPI (lmg/ml stock solution), diluted 1:4000 in NaHC0 3 aqueous solution (final concentration about 0. 25ug/ml), ELISA plate, 4 °C refrigerator overnight or 37 ° C oven for 2-3 hours; 5% skim milk powder was dissolved in TPBS and blocked at 37 ° C for 1 hour.
  • Standard antibody (ChA21-CH0) and mutant expression supernatant Dilute to 1000, 333, 111, 37, 12. 33, 4. 11, 1. 37, 0.446 ng/ml with blocking agent.
  • a 1:2000 dilution of goat anti-human/HRP secondary antibody was added and incubated for 1 hour at room temperature. 0PD substrate color development, sulfuric acid Terminated.
  • the EC50 value of each mutant was calculated from the curve, and the ratio of the original standard antibody to the EC50 value of the mutant is the relative affinity of the mutant.
  • the antigen binding curves of the original antibody and each human mutant were as shown in Fig. 9.
  • the humanized mutants HI-1, HI-VI, and the original ChA21 chimeric antibody (antibody ChA21) activity expressed as 293T were used as controls.
  • the relative affinities of HI-2 and H2-1 are 0.95, 0.92, 0.93, respectively.
  • H1-U Hl-VU H1-2 substantially maintained the antigen binding ability of ChA21, and the humanized mutant H2-1 obtained by humanization based on the universal humanized template had more than doubled the antigen binding ability.
  • This experiment also showed that the ChA21 chimeric antibody (antibody ChA21) expressed in 293T cells has a comparable affinity to the antibody (antibody ChA21-CH0) expressed in the CH0-GS engineered cell line.
  • the antigen HER2 was Recombinant Human ErbB2/Fc Chimera, available from R&D Systems, Cat. No. 1129-ER.
  • CM-5 sensor strip was used to adsorb carboxymethyl dextran on the surface. Both flow paths were activated at a flow rate of 15 ⁇ l/min for 6 min, 2 ⁇ g of antigen HER2 was dissolved in a 50 ⁇ l ⁇ 7.0 phosphoric acid solution, and injected into the chip flow cell 1 at a flow rate of 10 ⁇ l/min.
  • the surface density was about 10 KRu, and the surface of the chip was blocked with 1 M ethanolamine after antigen labeling.
  • Binding conditions The concentration of each test antibody is 10 mg/ml, which is diluted to 200 ⁇ g / ml, 100 ⁇ g / ml, 50 ⁇ g I ml, 25 ⁇ g / ml, 12 ⁇ g / ml, 0 ⁇ g / ml, The two flow cells of the chip were injected at a rate of 10 ⁇ l /min for 3 min; the dissociation conditions were: flow rate 10 ⁇ l /min, dissociation for 3 min.
  • Chip regeneration conditions 10 mM NaOH, 1 M NaCl.
  • the antibody affinity constant KD was analyzed using Biacore evaluation software.
  • Example 5 Screening of high affinity mutants of humanized antibodies using phage antibody library technology 1. Design of mutant antibodies
  • the present invention utilizes phage display technology to establish a phage antibody library to screen humanized antibody high affinity mutants.
  • the specific description is as follows:
  • the humanized single-chain antibody Hl-2 obtained by CDR grafting technology was used as a template to analyze the amino acid frequency of the light chain and heavy chain CDR regions of the antibody for bioinformatics analysis, and the crystal structure information of the ErbB2/pl85 antigen complex was used to screen out Potential amino acids in each CDR region that may contribute to increased affinity.
  • a total of five CDR regions of L1, L3, HI, H2 and H3 were designed, and the codon mutation primers were designed.
  • phage mutant libraries were constructed by SOE-PCR, and each mutation library contained four randomly mutated amino acids. Subsequently, using liquid immunomagnetic bead screening technology, three rounds of biopanning were performed by gradually decreasing the antigen concentration. The recombinant clones were selected for ELISA to determine the relative affinities and multiple high-affinity clones were selected for DNA sequencing analysis. Then, the single CDR mutation library sequences with good results were combined to construct a plasmid and transfected into 293T cells, and finally a series of human sources were obtained. The single-chain antibody mutant was tested for relative affinity activity of the antibody by ELISA, wherein the affinity of the two mutants of Hl-2-mut3 and Hl-2_mut7 was increased by 8-10 fold.
  • the gene shown in SEQ ID NO: 12 was artificially synthesized, and the gene was inserted into the Sfil and No t I restriction sites of the plasmid pSectag2A-dFc, and the resulting recombinant plasmid was transformed into E. coli ToplO competent cells, and the positive recombinants were screened. Plasmid DNA was extracted and sequenced, and the gene sequence inserted between the Sfil and ⁇ 3 ⁇ 4 I sites of pSe C tag2A-dF C was shown as nucleotides 1-759 of SEQ ID NO: 12. The positive recombinant plasmid was named pS e C tag2A-dF C -Hl-2-mut3.
  • the recombinant vector comprises the gene represented by SEQ ID NO: 12, wherein the 1-34 position is V L , the 343-402 is Linker, the 403-759 is V H , and the 760-1470 is a fragment of Fc; the gene encodes a protein as shown in SEQ ID NO: 11, wherein the 1-114th position is VL, the 115th-134th position is Linker, and the 135th-253th position is VH , 254-489.
  • the bit is a fragment containing an Fc;
  • the gene shown in SEQ ID NO: 14 was artificially synthesized, inserted into the Sfil and No t I restriction sites of the plasmid pSectag2A-dFc, and the resulting recombinant plasmid was transformed into E. coli ToplO competent cells, and the positive recombinants were screened. Plasmid DNA was extracted and sequenced, and the gene sequence inserted between the Sfil and No t I sites of pSe C tag2A-dF C was shown as nucleotides 1-759 of SEQ ID NO: 14. The positive recombinant plasmid was named pS e C tag2A-dF C -Hl-2-mut3.
  • Recombinant vector The gene represented by SEQ ID NO: 14 is included, wherein the 1-34 position is V L , the 343-402 is Linker, the 403-759 is V H , and the 760-1470 is Fc-containing fragment;
  • the gene encodes a protein as shown in SEQ ID NO: 13 in which the 1-114th position is VL, the 115th-134th position is Linker, the 135th-253th position is VH , and the 254th-489th position is Fc-containing. Fragment of.
  • Hl-2, Hl-2-mut3 and Hl-2-mut7 are shown in Fig. 10.
  • the relative affinity of the humanized mutants Hl-2, Hl-2-mut3, Hl-2-mut7 was calculated by using the original ChA21 chimeric antibody (antibody ChA21) activity expressed as 293T as a control (defining its relative affinity as 1). They are 0.93, 8.55, 7.72 respectively. Therefore, the affinities of the two mutants H1-2-mut3 and HI-2- 2-mut7 were 8- to 10-fold higher than those of H1-2.
  • the method provided by the invention has the following advantages and specificities:
  • the method selects two human template sequences similar to the light chain and the heavy chain in the selection of the humanized template antibody, and selects the light and heavy chains to have higher similarity with the target antibody.
  • a human antibody is used as a template, and the candidate template antibody selects crystal structure data as much as possible, and the template antibody is finally determined by comparing the similarity of the overall structure of the candidate template antibody and the target antibody.
  • the selected light and heavy chain template is derived from the same antibody, which is derived from the natural framework. Compared with the previous method of combining light and heavy chains, it is more conducive to the stability of the structure of the light and heavy chain framework. Moreover, when selecting mutations in the framework region residues, it is no longer necessary to consider whether it is necessary to mutate the light and heavy chain interface residues to make the light and heavy chain interactions more stable.
  • the selection of templates according to structural similarity is more structurally reasonable than the previous screening of templates by sequence similarity. Because the binding of the antibody to the antigen is directly dependent on the conformation of the complementarity determining region of the antibody, and the complementarity determining region is constructed on the platform constructed by the framework region, the higher the spatial structure similarity between the human template and the target antibody is, the more favorable it is. The maintenance of the conformation of the complementarity determining region after transplantation, that is, the ability to maintain the ability of the antibody to bind antigen. Sequence similarity though To a certain extent, it can represent structural similarity, but it is not very precise. For example, in the first embodiment of the present invention, several templates having the highest sequence similarity are not the best structural similarity.
  • the modification effect of this method is higher than the existing humanization method.
  • the humanized version of the ChA21 antibody obtained by this method has an antigen binding activity comparable to that of the original antibody, and the relative affinity is about 93% of that of the original antibody.
  • the antibodies that have been engineered by the universal antibody humanization method have been reported to have high and low affinity and unstable levels (Carter P, Presta L, Gorman CM, Ri dgway JB, Henner D, Wong WL, Rowland AM, Kotts C , Carver ME, Shepard HM. ( 1992 ) Humani zation of an anti-pl85HER2 antibody for human cancer therapy.
  • the affinity version H2-l by affinity determination, found that the affinity of H2-1 was more than doubled compared with the original antibody, indicating that the modification effect of the humanized method in the present invention is significantly better than that of the antibody ChA21 in this example. There are other ways.
  • E. coli expression system the operation is simple and rapid, the cost is low, but the soluble antibody expression is low (100ug/L), the expression product can not be correctly modified, the inclusion body is easy to form, the purification is difficult, and the expression levels of different mutant clones are very different. , so that the determination of the next antibody affinity is affected by the antibody concentration, which is difficult to judge; the yeast expression system, such as the pPIC9K vector, secretes expression, and Hi s-tag can be used. Column purification, purification is more convenient, but need to construct a stable cell line, long cycle, although the expression level is higher than the E.
  • the 293T eukaryotic transient expression system used in the invention can be secreted by using pSe C tag2A as a vector, and the product can be purified by Hi-tag column or protein A column, so the recombinant plasmid DNA of the designed humanized antibody is subjected to lipid.
  • the plastids were transiently transfected into 293T cells, and the expressed products were correctly modified.
  • the purification was simple and the expression was high (3-10 mg/L), and the difference in expression between different mutant clones was small. Although it was relatively expensive, it was used.
  • the results of systematic expression of purified antibody properties were clear and stable.
  • the antibody expression and relative affinity were determined by ELISA. The results were accurate and reproducible, and the expression supernatant was not purified, which facilitated the rapid identification and selection of human mutants.
  • the present invention also utilizes phage display technology to establish a phage antibody library to screen humanized antibody high affinity mutants.
  • the humanized single-chain antibody H1-2 obtained by CDR grafting technology was used as a template, and the amino acid frequency of the light chain and heavy chain CDR regions of the antibody was bioinformatically analyzed, and potential amino acids in each CDR region which may be beneficial for affinity improvement were screened.

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Abstract

La présente invention concerne des anticorps humanisés anti-p185 (HER2/ErbB2) et leurs procédés d'humanisation. Les anticorps sont des protéines qui ont les séquences d'acides aminés choisies parmi SEQ ID No:15, SEQ ID No:3, etc. En comparaison aux anticorps originaux, les anticorps humanisés de la présente invention ont une affinité supérieure pour les antigènes et ont déjà été complètement humanisés.
PCT/CN2011/001012 2010-06-21 2011-06-17 Anticorps humanisés anti-p185 (her2/erbb2) WO2011160429A1 (fr)

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CN101928345B (zh) * 2010-06-21 2012-08-29 中国科学技术大学 一种人源化抗体及其人源化改造方法
CA2823044C (fr) * 2010-12-31 2022-08-16 Jay M. Short Humanisation express d'anticorps
CN103145834B (zh) * 2013-01-17 2015-03-18 广州泰诺迪生物科技有限公司 一种抗体人源化改造方法
NL2011406C2 (en) 2013-09-06 2015-03-10 Bionovion Holding B V Method for obtaining april-binding peptides, process for producing the peptides, april-binding peptides obtainable with said method/process and use of the april-binding peptides.
CN104225594B (zh) * 2014-09-23 2016-03-23 合肥瀚科迈博生物技术有限公司 抗her2人源化抗体及其相关的抗肿瘤组合物
NL2014108B1 (en) * 2015-01-09 2016-09-30 Aduro Biotech Holdings Europe B V Altered april binding antibodies.
CN105131116B (zh) * 2015-09-25 2018-10-09 中国人民解放军第四军医大学 靶向HER2的人源化改造的可内化单链抗体P1h3及制备方法和应用
CN105131117B (zh) * 2015-09-25 2018-10-12 中国人民解放军第四军医大学 靶向HER2的人源化改造的可内化单链抗体P1h2及应用
CN107446047B (zh) * 2017-09-04 2020-08-11 山西省生物研究院有限公司 一种双功能性抗组织因子人源化抗体及其制备方法
TW202128748A (zh) * 2020-01-24 2021-08-01 日商西斯美股份有限公司 提升抗體對抗原之親和性的方法及其用途
CN111635457A (zh) * 2020-05-22 2020-09-08 上海药明生物医药有限公司 一种纳米抗体人源化的方法
CN112608944B (zh) * 2021-01-13 2023-03-24 杭州博岳生物技术有限公司 一种人源化抗体表达载体的构建方法及其应用
WO2023078420A1 (fr) * 2021-11-04 2023-05-11 Vibrant Pharma Limited Procédés d'optimisation d'anticorps
CN117701625A (zh) * 2023-12-12 2024-03-15 合肥润初生物科技有限公司 基于转基因植株的Her-2抗体生产方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101080496A (zh) * 2004-11-08 2007-11-28 宜康公司 抗体工程改造方法
WO2009105230A2 (fr) * 2008-02-21 2009-08-27 The Regents Of The University Of Colorado Procédés de traitement du cancer à l'aide d'une thérapie de combinaison
CN101522717A (zh) * 2006-08-04 2009-09-02 阿斯利康(瑞典)有限公司 针对ErbB2的人抗体
CN101928345A (zh) * 2010-06-21 2010-12-29 中国科学技术大学 一种人源化抗体及其人源化改造方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101080496A (zh) * 2004-11-08 2007-11-28 宜康公司 抗体工程改造方法
CN101522717A (zh) * 2006-08-04 2009-09-02 阿斯利康(瑞典)有限公司 针对ErbB2的人抗体
WO2009105230A2 (fr) * 2008-02-21 2009-08-27 The Regents Of The University Of Colorado Procédés de traitement du cancer à l'aide d'une thérapie de combinaison
CN101928345A (zh) * 2010-06-21 2010-12-29 中国科学技术大学 一种人源化抗体及其人源化改造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANG BIN: "Molecular Modeling and Docking of HAbl8G/CD147 with Its Monoclonal Antibody and Antibody Humanization Design, EQ059-63", CHINESE MASTER'S THESES FULL-TEXT DATABASE MEDICINE AND HEALTH SCIENCES, no. 4, 15 April 2008 (2008-04-15), pages 9 - 64 *

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