WO2014075576A1 - 人源抗人表皮生长因子受体抗体及其编码基因与应用 - Google Patents
人源抗人表皮生长因子受体抗体及其编码基因与应用 Download PDFInfo
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Definitions
- the invention relates to the preparation and application of a human genetically engineered antibody for treatment, and mainly relates to an antibody against a human epidermal growth factor receptor (EGFR) and a gene encoding the same and application thereof.
- EGFR human epidermal growth factor receptor
- Epidermal growth factor receptor is a member of the epidermal growth factor gene (erbB) family and has been found in the development of various tumors such as breast cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, and prostate cancer. Significant influence. Many studies at home and abroad have shown that antibodies against EGFR can effectively inhibit the EGFR signal transduction pathway by blocking the binding of ligands, and the human tumors expressed by various EGFR, especially the head and neck squamous. Cellular cancer (80% ⁇ 100%), colorectal cancer (25% ⁇ 77%), non-small cell lung cancer (40% ⁇ 80%), pancreatic cancer, etc. have a good effect. Epidermal growth factor receptor (CSF) is one of the most intensive and well-received cancer therapeutic targets. The use of genetic engineering to develop anti-EGFR monoclonal antibodies is one of the research hotspots in cancer therapy.
- CSF Epidermal growth factor receptor
- anti-EGFR therapeutic antibodies have been approved by the US FDA for the murine-human chimeric antibody cetuximab (Cetuximab, trade name Erbitux) and the full-body antibody Panidan for EGFR.
- Anti-Panitumumab for the treatment of colorectal cancer and head and neck cancer.
- the anti-EGFR humanized antibody nimotuzumab (Nimotuzumab, trade name Taixinsheng) was awarded a Class 1 new drug certificate approved by the Chinese Food and Drug Administration (CFDA).
- the antibodies under investigation for this target include humanized monoclonal antibody Pertuzumab, human antibody Zalutumumab and Necitumumab.
- the antigenic epitopes they bind are not identical, but they all play a role in inhibiting EGFR-mediated biological functions.
- Human antibodies are the ultimate direction for the development of therapeutic antibodies.
- the clinical application of human antibodies can minimize the immunogenicity of antibodies, prolong the half-life of antibodies in vivo, and mediate immunomodulation, ADCC and CDC by means of human immunoglobulin Fc-segment. The effect, which in turn enhances the biological effects of the antibody.
- the main development tools for human antibodies include antibody library technology and transgenic mouse technology.
- the library technology is very mature, especially the phage display antibody library technology has successfully achieved the success of multiple human antibodies.
- CAT's semi-synthetic antibody library and Marphosys AG's fully synthetic antibody library are the most representative and most successful representatives.
- the applicant invented a method for efficient DNA ligation and transformation, and applied it to the construction of a large-capacity phage antibody library, and constructed a large-capacity total synthesis with a library capacity of 1.35 ⁇ 10 1 () .
- a phage single-chain human antibody resource library (ZL.200910091261.8) was used, and a plurality of specific antibodies against different targets were obtained by screening the antibody library.
- the antibody Ame55 obtained in the present invention is obtained by screening from an antibody library. Summary of the invention
- the present invention provides antibody treatments for the treatment and prevention of diseases associated with EGFR expression, particularly tumors and autoimmune diseases that express EGFR.
- a first object of the present invention is to provide an amino acid sequence of a human anti-EGFR antibody and an active fragment thereof.
- a second object of the present invention is to provide a gene encoding the above antibody or an active fragment thereof.
- a third object of the present invention is to provide the use of the above antibody and its active fragment for the treatment of a malignant tumor and an autoimmune disease drug for the expression of EGFR.
- the present invention uses a phage display technology to obtain a single-chain genetically engineered antibody specific for human EGFR by multiple rounds of bio-panning from a large-capacity fully synthetic human single-chain antibody library (single chain) Variable fragment , scFv).
- the above-mentioned antibody light heavy chain variable region is introduced into the whole antibody mammalian cell transient expression vector pABL and In pABG1, transient secretion expression was performed by co-transfection of HEK 293T cells to obtain the whole antibody Ame55.
- the anti-body variable region amino acid sequence pattern provided by the present invention is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
- FR1 ⁇ 4 represent 4 frame areas
- CDR1 ⁇ 3 represent 3 hypervariable areas.
- FR1 ⁇ 4 may be isolated from the constant region sequence (such as the most commonly used amino acids in human immunoglobulin light heavy chain, subclass or subfamily), or may be isolated from individual antibody framework regions or from different framework regions.
- the genes come together. For example, a number of human antibody framework region sequences are included in libraries such as Kabat.
- the heavy chain variable region is a human immunoglobulin subgroup human heavy chain VH V family
- the light chain variable region is a Lambda III family.
- the CDRL1 sequence was: SGDX1LGDKYX2X3 (SEQ ID NO 1)
- XI may be Ala, Gly, Asn, Lys, preferably Lys and Gly
- X2 may be Ala, Val and He, preferably Ala
- X3 may be Ser and Tyr, preferably Ser.
- the CDR2L sequence is: EDX1KRPS
- the CDRL3 sequence may be: X1X2WDX3DWX4MP (SEQ ID NO 3), where XI is Ser, Ala, Gly, Leu, Asn, Tyr and Gin, preferably Ser; X2 may be Val, Ser, Ala and Leu, preferably Ser Or Ala; X3 may be Gly, Ala and Pro, preferably Gly; X4 may be Gly and Ser, preferably Gly.
- CDRH2 sequence is X1IIYPX2DSDTRYSPSFQ (SEQ ID NO 5), where XI may be Gly and Ser, preferably Gly; X2 may be Gly and Ser, preferably Gly.
- the CDR3H sequence is GIIYPSNVX1V (SEQ ID NO 6), where XI can be Ala, Ser and Gly, preferably Ser or Ala.
- Alanine scanning of the light and heavy chain CDR regions of the antibody revealed that the light and heavy chain CDR3 and the light chain CDR1 and heavy chain CDR2 are critical for binding of the antibody to EGFR. A change in only a single amino acid can cause a change in the affinity from several to several tens of times, as described in Example 5.
- the human anti-human EGFR antibody provided by the present invention has a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3. Further, the light chain variable region thereof contains the amino acid sequences shown as SEQ ID NO 1 and SEQ ID NO 2 .
- the light chain variable region thereof contains the amino acid sequence as shown in SEQ ID NO: 7.
- the human anti-human EGFR antibody provided by the present invention has a heavy chain variable region containing
- amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO 6.
- the heavy chain variable region thereof further contains an amino acid sequence as shown in SEQ ID NO 4. Further, the heavy chain variable region thereof contains the amino acid sequence as shown in SEQ ID NO: 8.
- the human anti-human EGFR antibody provided by the present invention is a single chain antibody, Fab, minibody, chimeric antibody or whole antibody immunoglobulin IgG1, IgG2, IgA, IgE, IgM, IgG4, IgD.
- the present invention provides a source of human anti-EGFR antibody Ame55, which heavy chain variable region from V H5, whose light chain variable region from ⁇ ⁇ 3, which antibody subtype IgGl.
- the human anti-human EGFR antibody provided by the present invention has a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3, and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6.
- the human anti-human EGFR antibody provided by the present invention, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain variable region thereof comprises the amino acid shown as SEQ ID NO 5 and SEQ ID NO 6. sequence.
- the human anti-human EGFR antibody provided by the present invention has a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, and a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 8.
- the present invention provides a source of anti-human EGFR antibody with an affinity of at least about 10_ 8 M binding to human EGFR.
- This antibody inhibits the binding of EGFR ligand to human EGFR.
- the antibody binds to a cell expressing EGFR; the cell is human skin cancer cell, human liver cancer cell, human colon cancer cell, human cervical cancer cell, ovarian cancer cell, human lung cancer cell, bladder cancer cell, breast cancer cell, kidney Cancer cells, prostate cancer cells, head and neck tumor squamous cells, pancreatic cancer cells, synovial fibroblasts or keratinocytes.
- the invention provides a gene encoding the above antibody.
- nucleotide sequence of the gene encoding the variable region of the light chain is:
- stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum/O.lFicoll, 42 °C, etc.; or (3) at least 70% identity between the two sequences Preferably, at least 80% or more, more preferably 90% or more, more preferably 95% or more, hybridization occurs.
- nucleotide sequence of the gene encoding the heavy chain variable region is:
- the gene encoding the antibody of the present invention may be, but is not limited to, SEQ ID NOs 9 and 10, for example, in the coding region thereof, without changing the amino acid sequence, encoding the above
- the gene sequence of the antibody is modified to obtain a gene encoding the same antibody.
- One skilled in the art can artificially engineer genes based on the codon bias of the expression antibody host to increase the expression efficiency of the antibody.
- the present invention also provides an expression vector comprising the above gene.
- the present invention provides a host strain, host cell or expression cassette comprising the above expression vector.
- the invention provides a method for preparing the above antibody, comprising screening an anti-EGFR specific single chain antibody by using a phage display antibody library technology, obtaining an antibody light chain, a heavy chain variable region gene, and cloning into a whole antibody expression vector, by breastfeeding It is fully antibody expressed by an animal expression system or other expression system to obtain its full antibody protein.
- the use of the above antibodies for the preparation of a medicament for the treatment of diseases targeting EGFR is provided.
- the drug is an antitumor drug, an anti-inflammatory drug or a drug for treating an autoimmune disease.
- a drug or a detection reagent containing the above antibody is also within the scope of the present invention.
- the drug or detection reagent containing the above antibody may be combined with the antibody of the present invention into a mixed preparation by another antibody or antibodies.
- Another antibody or antibodies in the antibody mixture can be other antibodies directed against another antigen or against a different epitope of EGFR. Studies have shown that the combination of multiple target-related diseases is an important research direction of therapeutic antibodies.
- the combination of two targets targeting EGFR and VEGFR and targeting EGFR and ErbB2 has synergistic anti-tumor. Effect (Pennell NA, Lynch TJ. The Oncologist. 2009, 14: 399-411; Galer CE, Corey CL, Wang ZY, et al. Head Neck. 2011,
- the present invention also provides an antibody-targeting drug comprising the above human anti-human EGFR antibody linked to a cytotoxic agent in various forms.
- the various linked forms are those in which the antibody is labeled, in vivo, or molecularly coupled.
- the cytotoxic agent includes chemical molecules, radioisotopes, polypeptides, toxins, and other substances which have a killing or induction of apoptosis characteristics to cells.
- the antibodies provided by the invention are whole antibodies or various other forms of genetically engineered antibodies.
- an anti-EGFR antibody can be a full antibody or antibody fragment.
- the antibody molecule itself can be used for treatment and diagnosis.
- the antibody can be labeled, cross-linked or coupled and fused to other proteins or polypeptide molecules to form complexes (e.g., cytotoxic substances, radiotoxins, and/or chemical molecules, etc.) for diagnosis and treatment.
- the present invention further provides: an independent gene encoding the antibody, an expression vector, a vector transfected host cell-related control technique and host cell, an antibody expression sequence, and an antibody recovered in the cell culture supernatant.
- the invention also provides an antibody-containing component and a pharmacologically acceptable delivery molecule or solution.
- the therapeutic component is sterile and can be lyophilized at low temperature.
- the invention provides an anti-EGFR antibody that inhibits one or more biological activities induced by EGFR.
- This antibody acts by blocking the binding of EGFR to its ligand, and can also act by killing cells expressing EGFR, or by binding to EGFR.
- the substance is internalized to consume EGFR on the cell surface to function. All interfering functions possessed by EGFR antagonists should be considered equally for the purposes of the present invention.
- sequences set forth herein and claimed in SEQ ID NOS. 8.1 include "conservative sequence modifications", ie, nucleotide and amino acid sequences that do not significantly affect and alter the binding characteristics of the antibody or antibody comprising the amino acid sequence. Modification. Such conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. Modifications can be introduced into SEQ ID NOS. 1-8 by standard techniques in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, etc., by performing alanine scanning and pairing of the CDR regions of the antibody as in the present invention. Amino acid mutations at some sites are examples of conservative sequence modifications.
- Conservative amino acid substitutions include the replacement of an amino acid residue by an amino acid residue having a similar side chain or other amino acid residue.
- a family of amino acid residues having similar side chains has been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), with uncharged polar side Chain of cystine, tryptophan), amino acids with non-polar side chains (eg alanine, valine, leucine, isoleucine, valine, phenylalanine, methotrexate) Acid), an amino acid having a ⁇ -branched side chain (such as threonine, valine, isoleucine) and an amino acid having an aromatic side chain (such as tyrosine, phenylalanine, tryptophan, histidine) ). Therefore, it is preferred to replace the non-essential amino acid residues in the human anti-EGFR antibody with another amino
- antibodies encoded by the nucleotide sequences disclosed herein and/and antibodies comprising the amino acid sequences disclosed herein include antibodies that are substantially encoded by similar sequences modified by conserved sequences, or that contain similar sequences modified by conserved sequences. , should be considered as the scope of the present invention.
- the present invention provides a bispecific or multispecific molecule comprising a molecule of an antigen binding site of the above-described antibody or antibody provided by the present invention.
- the present invention provides a fusion protein of an antibody with other proteins or ⁇ and polypeptides, comprising a complex of the above-described antibodies and other proteins or polypeptide molecules having a certain function provided by the present invention.
- the fusion protein is a recombinant expression vector obtained by ligating an antibody gene with another antibody or antibody fragment, an immunotoxin or a cytokine gene, and obtaining a recombinant fusion protein molecule by a mammalian cell or other expression system.
- the antibody Ame55 described in the present invention has a good therapeutic application prospect, and mainly exhibits specific binding activity to recombinant human EGFR and specifically binds to native EGFR on the surface of A431 cells.
- the antibody was confirmed to have good specificity by ELISA and immunohistochemistry; Western Blot results confirmed that it only specifically binds to non-reduced EGFR, and does not bind or weakly bind to reduced EGFR.
- Biacore systems preclude the use of antibodies to detect the binding capacity of different forms of EGFR, results showed that, of the extracellular domain of EGFR recombinant antibody -Fc fusion protein affinity K D of 0.23nM (Biacore3000).
- Flow cytometric analysis The relative affinity of this antibody to EGFR binding on the surface of A431 cells was higher than that of the upper sputum antibody Taixinsheng, which was comparable to the upper sputum antibody Erbitux.
- the present invention shows that the EGFR antibody of the present invention binds to human EGFR with an affinity of not more than InM, and its mutant affinity is not more than 10 nM.
- This antibody inhibits the binding of Erbitux to EGFR in vitro. Conversely, Erbitux can also inhibit the binding of this antibody to EGFR. This suggests that the antibody may overlap with the Erbitux epitope in spatial structure. This antibody is able to compete to inhibit the binding of EGF to EGFR.
- This antibody can significantly inhibit the migration activity of A431 cells.
- the results of cell scratch healing experiments showed that the antibody significantly inhibited the scratch healing of A431 cells at a concentration of 5-5 ( ⁇ g/ml). At the same time, the antibody significantly inhibited the growth of A431 cells.
- the antibody has a significant inhibitory effect on the growth of A431 xenograft tumors.
- the tumor inhibition rate can reach more than 90%, and the tumor inhibition rate is given at a dose of 0.2 mg/3 d per mouse. More than 60%.
- Targeted site-directed mutagenesis of the antibody resulted in multiple mutant antibodies with a 5-fold increase in affinity.
- the anti-tumor activity of the mutant antibody was further improved.
- Some antibodies such as AmeA2, AmeA2C3, AmeA2C3-HI2, AmeA2Cl, and AmeAlCl were 0.2 mg. At a dose of /3d, the antitumor activity was greater than 80%.
- the present invention screens a full-antibody antibody against EGFR and a series of mutant antibodies whose affinity remains unchanged or increased by large-capacity synthetic antibody library technology, and confirms that the antibodies of the invention all specifically bind to EGFR and inhibit it. biological functions.
- the antibody can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system by using the whole human anti-EGFR genetically engineered antibody variable region gene obtained above and the whole antibody gene under the characteristics of the antibody gene, or Based on this, any other gene containing this antibody gene after reconstitution has obtained inhibition
- An antibody product that is biologically active by EGFR, or a complex obtained by in vitro labeling or cross-linking, to produce a specific antibody drug for clinical use in the treatment of malignant tumors and/or autoimmune diseases that overexpress or mutate to express EGFR. . DRAWINGS
- Figure 1 shows the specificity analysis of Ame55 phage antibody.
- Figure 2 shows the SDS-PAGE electrophoresis pattern of Ame55 after one-step purification on a Protein A column, where 1: Pr Marker; 2: Erbitux (3: Abita); 3: Ame55; 4: Nimotuzumab (Taixinsheng).
- Figure 3 shows the binding of Ame55 to EGFR by Western Blot
- 1 non-denaturing EGFR-Erbitux
- 2 denatured EGFR-Erbitux
- 3 non-denatured EGFR-Ame55
- 4 denatured EGFR-Ame55
- 5 non-denaturing EGFR - Tai Xinsheng
- 6 Denatured EGFR-Tai Xinsheng.
- Figure 4 shows the indirect immunofluorescence assay of Ame55 and A431 cells, A: Avastin-A431; B: Ame55-A431; C: Erbitux-A431; D: Taixinsheng-A431.
- Figure 5 is a flow cytometric analysis of the binding of Ame55 to A431 cells on the surface of EGFR
- Figure 5A Antibody concentration: 3 g / ml
- Figure 5B Antibody concentration: 0.3 ⁇ g / ml
- Figure 5C Antibody concentration: 0.03 ⁇ ⁇ / ⁇ 1, 1 is the antibody Taixinsheng
- 2 is the antibody Ame55
- 3 is the antibody Erbitux.
- Figure 6 is a graph showing the results of Ame55 and EGFR-Fc affinity determination by Biacore.
- Figure 7 is: Mutual competition inhibition between Ame55 and Erbitux, wherein Figure 7A: 1 : EGFR-Fc binding; 2: PBST wash; 3: Ame55-binding; 4: PBST wash; Figure 7B: 1 : EGFR-Fc binding 2: PBST wash; 3: Erbitux-binding; 4: PBST wash; Figure 7C: 1 : EGFR-Fc binding; 2: PBST wash; 3: Erbitux-binding; 4: PBST wash; 1 : EGFR-Fc binding; 2: PBST washing; 3: Ame55-binding; 4: PBST washing; Figure 7A and Figure 7B stationary phase is Ame55, Figure 7C and Figure 7D stationary phase is Erbitux.
- Figure 8 is a graph showing the competitive inhibition effect of Ame55 on the binding of EGF to EGFR.
- 1 blank control
- 2 molar ratio of Ame55 to EGFR 3:1
- 3 molar ratio of Ame55 to EGFR 2:1
- 4 molar ratio of Ame55 to EGFR is 1:1
- 5 Ame55
- 6 EGFR
- 7 love
- the molar ratio of EGFR to EGFR is 3:1
- 8 the molar ratio of Erbitux to EGFR is 2:1
- 9 The molar ratio of Erbitux to EGFR is 1:1
- 10 Erbitux
- 11 Irrelevant antibody control to EGFR molar ratio of 3:1
- 12 unrelated antibody to EGFR molar ratio of 2:1
- 13 unrelated antibody to EGFR molar ratio of 1:1
- 14 unrelated antibody Control.
- Figure 9 is a graph showing the inhibitory effect of Ame55 and Erbitux, Taixinsheng, Adalimumab, and blank controls on the migration ability of A431 cells.
- Figure 10 is a graph showing the growth inhibition of A431 cells by Ame55 and Erbitux, Taixinsheng, and PBS controls.
- Figure 11 is a graph showing the inhibitory effect of Ame55 on tumor growth of A431 tumor-bearing mice.
- Figure 12 is a graph showing the inhibitory effect of Ame55 on tumors of A431 tumor-bearing mice.
- Figure 13 is a graph showing the inhibitory effect of Ame55 on tumors of A431 tumor-bearing mice.
- Figure 14 is a comparison of tumor growth inhibitory activity of Ame55 and its mutant antibody tumor mice.
- A Dynamic observation of tumor volume
- B Statistical results of tumor weight measurement. 1 : Ame55; 2: AmeAlCl ;
- Figure 15 is a graph showing the affinity of the mutant Ame-9B in which the Ame55 light chain is combined with another unrelated heavy chain. 1 : Ame55; 2: Ame-9B.
- Figure 16 is a schematic representation of the vector of eukaryotic transient expression plasmids pABL and pABGl. detailed description
- Example 1 Specific antibody screening of large-capacity phage antibody library
- Primer EGFRR1 (SEQ ID NO 13) was designed, and the full-length gene of EGFR extracellular domain was amplified by primers EGFRF and EGFRR1, and cloned into pABG1 eukaryotic expression vector by restriction endonuclease sites EcoR l and BamH I. After the plasmid was identified by sequencing, the plasmid was extracted and transfected into HEK293T cells for transient expression. The expression supernatant was purified by Ni+ affinity chromatography column, and the purified product was quantified by Bradford method.
- a 50 ⁇ l prepared phage suspension was taken and serially diluted 10-fold with LB medium.
- the host bacteria in the logarithmic growth phase were added to the suspension diluted to a certain multiple, and cultured at 37 ° C, 150-180 rpm shaker for 1 h. 100 or 200 ⁇ M of the culture solution was applied to an LB plate, cultured at 37 ° C overnight, and the number of colonies was counted, and the titer was counted.
- the recombinant protein antigen EGFR-Fc was diluted to 2 with a coating solution ( ⁇ g/ml coated immunotube, 1 ml/tube, coated at 4 ° C overnight. The next day, the immunotube was washed twice with PBS for 2 min/pass, then Blocked with 2% BSA for 2 h at 37 ° C. The prepared phage antibody library was blocked with blocking solution (2% BSA, 0.1% Tween 20) for 30 min at 37 ° C. The pre-blocked phage antibody library was added to the blocked immunization In the tube, 4V was used overnight. The solution in the immunotube was discarded and washed.
- a coating solution ⁇ g/ml coated immunotube, 1 ml/tube, coated at 4 ° C overnight.
- the first round of PBST was washed 10 times, 5 min/time, PBS washed 5 times, 5 min/time; the second round of PBST was washed 15 times, 5 min/time, PBS Wash 10 times, 5min / time; the third round of PBST wash 15 times, 5min / time, PBS + NaCl wash 15 times, 5min / time.
- 2xYTCTKI kanamycin 5 ( ⁇ g / ml, IPTG 0.2mmol / L)
- Coating buffer pH 9.6: Na 2 C0 3 1.59g, NaHC0 3 2.93g, double distilled water to 1L; blocking solution: l xPBS + 2% BSA + 0.1% Tween 20; Washing solution: lxPBS + 0.1 % Twain
- OPD substrate dilution 0.2 M Na 2 HP0 4 (28.4 g/L) 25.7 ml, 0.1 M citric acid (19.2 g/L) 24.3 ml, distilled water, 50 ml.
- the vectors pABG1 and pABL were used to clone the antibody heavy chain (such as SEQ ID NO 10) and the light chain (such as SEQ ID NO 9) variable region genes, respectively, using primer H5F (SEQ ID NO 14 ⁇ PHR (SEQ ID NO 15) Amplification of the VH5 variable region gene; L3F (SEQ ID NO 16) and LR (SEQ ID NO 17) amplify the ⁇ 3 variable region gene.
- the ⁇ 3 variable region gene is cloned into the vector PABL using the restriction sites Bsr GI and Hindlll.
- the VH5 variable region gene was cloned into the vector pABG1 using the restriction enzyme sites Afl II and Nhel.
- the recombinant plasmid was transformed into E. coli DH5a, and the recombinant plasmid was identified by bacterial PCR and sequencing to obtain the correct expression of the full antibody light and heavy chain. After the plasmid was extracted, the antibody light and heavy chains were transfected into HEK293T cells at a molar ratio of 1:1, and the transient expression of the whole antibody was performed. The expression supernatant was purified by Protein A affinity chromatography column, and the purified whole antibody was subjected to purification. Electrophoretic analysis, affinity and specificity assays, etc.
- the monoclonal clone obtained in the third round of screening was identified to obtain a specific phage antibody, and the nucleotide and amino acid sequences of the Ame55 single-chain antibody are shown in SEQ ID NO 18 and SEQ ID NO 19.
- the light chain variable region and heavy chain variable region nucleotide sequences of the Ame55 single-chain antibody are shown in SEQ ID NO: 9 and SEQ ID NO 10, respectively.
- the light chain variable region and heavy chain variable region amino acid sequences of the Ame55 single chain antibody are shown in SEQ ID NO 20 and SEQ ID NO 21, respectively.
- the specificity of Ame55 was analyzed at the phage level.
- EGFR-Fc was used as the positive antigen
- CD4-D2 AHC, TNF, VEGF, TTC and PBS were used as control antigens.
- the enzyme-linked plates were coated overnight at 4 °C and added to the phage.
- the specific binding activity of Ame55 phage supernatant was determined by ELISA using HRP-labeled anti-M13 antibody as a secondary antibody. The results indicate that Ame55 specifically binds only to EGFR-Fc. (figure 1 )
- the light heavy chain variable region gene of phage antibody Ame55 was cloned into the whole antibody transient expression vectors pABL and pABGl, and the whole antibody recombinant expression vector was constructed, and the transient secretion expression of mammalian cells was realized by the transient expression system of HEK293T cells.
- a full antibody protein sample was obtained after purification on a Protein A column with a molecular weight of approximately 150 kD. (figure 2 ).
- Human epidermal squamous cell carcinoma A431 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, Erbitux (CT) was the product of Merck Pharmaceuticals, Germany; Taixinsheng (Hr-3) was the captain of Beijing Baitai Biopharmaceutical Company.
- Anti-EGFR humanized antibody Avastin (Avastin, Bevacizumab, rhuMAb-VEGF) was purchased from Roche, Germany; CM5 chip was GE product, anti-human immunoglobulin Fc capture sensor and streptavidin sensor were purchased. Since iResearch Biotechnology (Shanghai) Co., Ltd. Recombinant human epidermal growth factor (EGF) was purchased from Shanghai Xinbino Biotechnology Co., Ltd. FreeStyleTM 293-F cell line, expression medium and transfection reagent were purchased from Invitrogen. 2.
- A431 cells per well were seeded in 104 well plates%.
- the EGFR-his antigen was subjected to reduction and non-reduction SDS-PAGE gel electrophoresis, and the separation gel concentration was 10%;
- Color development Add ECL luminescent liquid and send it to the dark room for development exposure.
- Coating Dilute the antibody to be tested with NaAC at the optimum pH, select the optimal dilution and select one channel on the CM5 chip for coupling.
- the target value of the coupled antibody is 2000 RU and select another channel as the control.
- the test conditions were 25 ° C, the flow rate was 20 ⁇ 7 ⁇ , and the buffer was HBS-EP (pH 7.4, Bia-Certified).
- the chip-coupled antibody closes the surface of the chip after reaching the target value.
- EGFR ⁇ EGFR-Fc flows through the surface of the chip to bind to the antibody on the surface of the chip, and stabilizes lOmM glycine-hydrochloride at pH 3.5, pH 2.5, pH 2.0, pH 1.5 and pH 8.5.
- the borate buffer flows sequentially through the surface of the chip until the best regeneration is achieved to determine the optimum regeneration conditions.
- Affinity constant calculation The resulting sensorgrams were fitted with a 1 : 1 Langmuir binding pattern using Bia-evaluation analysis software 4 to calculate the kinetic constants for antigen-antibody binding.
- test antibody was diluted with pre-cooled PBS to 150 ug/ml, 15 ug/ml, 1.5 ug/ml, 0.15 ug/ml, and 100 L was mixed with the pre-cooled cells. Incubate on ice for 40 min;
- Antibody Ame55 specifically binds to non-denatured EGFR
- EGFR-his was subjected to reduction and non-reducing SDS-PAGE gel electrophoresis, and 10 g/ml of Ame55, Erbitux and Taixin were used as primary antibodies, and HRP-labeled goat anti-human IgG was used as secondary antibody for Western. By blot detection, the results are shown in Figure 3. Amet55 only binds specifically to non-reduced EGFR.
- Ame55 specifically binds to EGFR on the surface of A431 cells
- A431 cells expressing EGFR were used as antigens, and Amet55, the irrelevant antibody Avastin, the control antibody Erbitux and Taixin were used as primary antibodies, and FITC-labeled goat anti-human IgG was used as secondary antibody for cell immunofluorescence detection.
- the result is shown in Figure 4.
- the results showed that Ame55 (Fig. 4, panel B) and the positive control antibody Erbitux (Fig. 4, panel C) and Taixinsheng (Fig. 4, panel D) specifically bind to EGFR on the surface of A431 cells, while the negative control antibody Avastin (Fig. 4, panel A) has no binding.
- the binding to A431 cells was analyzed by flow cytometry, and the results showed that the antibody binds to A431 cells in a dose-dependent manner within a certain concentration range.
- concentrations less than 1 g/ml the fluorescence intensity was between the control antibody Erbitux and Taixinsheng (Fig. 5A, Fig. 5B, Fig. 5C).
- Example 3 In vitro activity and epitope analysis of Ame55
- the surface of the CM5 chip was coated with Ame55 as the stationary phase.
- the coating and regeneration conditions were as shown in Example 2.
- the 50 nM EGFR-Fc was used as the first mobile phase to flow through the sample channel for 3 min, and washed with PBS-T for 3 min.
- the second mobile phase was passed through the sample channel with ⁇ , and the time was 3 min, followed by washing with PBST for 3 min.
- the above experiment was also carried out using Ame55 of ⁇ as the second mobile phase, as a self-competitive suppression control. Again, repeat the above experiment with Erbitux as the stationary phase.
- EGF is in 96 well plates at 1 ⁇ ⁇ / well coating, 4 ° C were coated overnight.
- Ame55 and the positive control antibody Erbitux and the irrelevant control antibody were mixed at a molar ratio of 3:1, 2:1, and 1:1 with 2.5 g/well of EGFR-Fc, respectively.
- After pre-blocking for 30 min at 37 ° C it was added to the ELISA plate coated with EGF, bound at 37 ° C for 1 h, washed 3 times with PBST, washed once with PBS for 5 min / time, pre-blocked HRP labeled Goat anti-human IgG was used as secondary antibody.
- PBST and PBS were washed, and OPD substrate coloring solution was added for color development. After 2N H 2 S0 4 was terminated, 00 45 was determined. The light absorption value at the dish.
- the 6-well plates were aliquoted in advance at the bottom, and each hole was equally divided into five equal portions.
- A431 cells were seeded in labeled 6-well plates, 10 5 /well cells. When the cells were grown to logarithmic growth phase, use a 200 L pipette tip perpendicular to the straight line and scribe on the cell surface. After scribing, PBS was washed twice, and the test antibody Ame55, positive control antibody Erbitux, negative control antibody adalimumab (product of Abbott, USA) was added to each well to a final concentration of 25 g/ml, and a blank well was set. As a blank control. Take 0h, 12h, and 24h as the observation time points respectively, mark the fixed position with a straight line and take photos in a dynamic process.
- A431 cell growth inhibition experiment 1 Inoculated cells: trypsinize A431 cells, fully bleed and count them; adjust to a cell concentration of 5000 cells/ml with 1640 medium containing 0.5% newborn bovine serum, and add 6 96-well plates at 200 ⁇ M per well (final per Holes 800-1000 cells are evenly distributed), incubated at 37 ° C 5% C0 2 for 24 h until the cells are fully stretched and attached;
- a liquid 8.967 grams of sodium citrate dissolved in 305 ml of ultrapure water; B solution: 195ml 0.1N HC1 (1.6ml 38% HC1 added to 193.4ml ultrapure water, "acid into water”) C liquid: 500 ml 95% ethanol (25 ml of ultrapure water was added to 475 ml of absolute ethanol); A+B+C was mixed to a total volume of 1 liter.
- Fig. 7C is the Erbitux self-inhibition control), that is, Erbitux can also inhibit the binding of EGFR to Ame55. This result suggests that there may be mutual competition inhibition between antigenic epitopes between the two antibodies, and it is likely that the epitopes of the two antigens are structurally close or identical.
- Ame55 inhibits the binding of EGF to EGFR
- the competitive ELISA method was used to analyze the competitive inhibition effect of Ame55 on the binding of EGF to EGFR.
- the results showed that Ame55 significantly inhibited the binding of EGFR to EGF at a molar ratio of EGFR to antibody of 1:1, 1:2 and 1:3, while the negative control antibody did not inhibit.
- the Ame55 inhibitory activity was lower than the same concentration of the control antibody Erbitux. (See Figure 8)
- the cell scratch healing of the 25 ug/ML experimental antibody group was observed dynamically for 24 h (Fig. 9), and in inhibiting the migration activity of A431 cells, Ame55 showed and loved The same inhibitory activity.
- the other negative control antibody, adalimumab has no inhibitory activity on its migration, and adalimumab is an antibody against TNFa.
- Ame55 does have a function of inhibiting the biological activity of EGFR, suggesting that it may have a better effect in the therapeutic application of clinical metastases.
- mice Female nude mice, about 20 g, 47 rats, were inoculated subcutaneously with 431 ⁇ A431 cells at a concentration of 5 ⁇ 10 7 .
- the mice When the tumor grows to 50-150 mm 3 , the mice are weighed and weighed and then grouped. A total of 4 groups were included, namely saline group, Erbitux group, adalimumab group and Ame55 group.
- Erbitux was an anti-EGFR monoclonal antibody, which was set as a positive control group
- adalimumab was an antibody against TNFo in the upper sputum, which was set as a negative control
- physiological saline was used as a tumor-bearing control, and antibody administration was performed.
- the drug was administered once every 3 days, and the body weight was measured and the tumor size was measured before each administration.
- the tumor calculation method is: ⁇ /6* long diameter* short diameter 2 . After 36 days, the rats were sacrificed, the tumors were weighed, and statistical analysis was performed.
- A431 cell transplanted tumor was used as the research object to evaluate the inhibitory effect of Ame55 on tumor growth of tumor mice.
- the results showed that Ame55 had a significant inhibitory effect on the growth of A431 xenografts at a dose of 1 mg/dose (Fig. 11).
- the drug was administered continuously for 6 times.
- the tumor weight after 4 weeks of drug withdrawal showed that the control group had an average tumor.
- the weight was 3.65g, and the average tumor weight of the Ame55 group was 0.32g, and the tumor inhibition rate was over 90% (Fig. 12, 13).
- Example 5 Ame55 Light Heavy Chain Variable Region Mutant Evaluation Method
- the site-directed mutagenesis primers were designed to perform alanine scanning on CDRL1, CDRL3, CDRH2 and CDRH3, and if they were alanine at this position, they were mutated to Gly and Ser, respectively.
- the method was carried out by the method of site-directed mutagenesis of plasmids. References [Wang Ronghao, Chen Ruichuan, Liu Runzhong. Optimization method for fast point mutations. Journal of Xiamen University (Natural Science Edition), 2008, Vol 47, sup 2, 282-285]
- the EGFR-Fc fusion protein was biotinylated using the biotin kit, and the biotinylated EGFR-Fc was diluted to a concentration of 100 nM in PBS, coated on the surface of the streptavidin sensor for 20 min, using HEPES EP pair.
- the sensor was washed for 5 min, and the mutant antibody and the parent antibody Ame55 were placed in the detection well at a concentration of 20 nM, and bound to the EGFR-Fc on the surface of the washed streptavidin sensor, and the binding time was 10 min. After the combination reached the equilibrium state, the composite was dissociated in HEPES EP, and the dissociation time was 20 min. ⁇ Affinity analysis was performed using the ForteBio software package. 3. Comparison of antitumor activity between mutant and parental antibodies
- the experimental method was the same as that in Example 4.
- the group and dose were set as: saline control, Erbitux control, Ame55 parental antibody control and Ame55 mutant antibodies (Ametumumabs) AmeAlCl, AmeAlC3, AmeAlC3-HI2, AmeA2, AmeA2Cl, AmeA2C3 AmeA2C3-HI2, a total of 10 groups, 8 in each group, the dose was 0.2mg / only, 3d / time, a total of 6 times, after 6 days of drug withdrawal, weighed and weighed.
- Mutations in the light and heavy chain variable regions and partial amino acids of the framework regions were carried out on the basis of the Ame55 amino acid sequence. Mutant design was performed using alanine scanning and directed mutagenesis. Further screening evaluation of the affinity of the mutants was performed using the ForteBio QK e protein molecular interaction system. The results indicate that (Table 1), compared with the parental antibody Ame55, multiple amino acid positions in the light chain CDR1, CDR3, heavy chain CDR2 and CDR3 regions are critical for the ability of the antibody to maintain binding to EGFR, possibly due to this site. The amino acids are directly involved in the interaction, and it may also be that the amino acid at that site is critical to maintaining the correct overall spatial structure of the antibody.
- the amino acid engineered sites the most notable are the 26th and 89th amino acids of the light chain, the 54th and 107th amino acids of the heavy chain.
- the present inventors found that after the amino acid at position 89 of the light chain was transformed from Val to Ala or Ser, the affinity was increased by more than 4 times, and after further combining Gly or Lys at position 26 of the light chain, The affinity is further improved, and the affinity of the parent antibody is increased by more than 5 times; or the 54 and 107 amino acids of the heavy chain are replaced by Ser, respectively, and the affinity of the antibody is 89A/S after being combined with the light chain 89A/S. Both have been improved to varying degrees.
- the dose was reduced to a lower dose of 0.2 ⁇ 8 /, and several mutants with increased affinity and parental antibodies were inhibited on the transplanted mice.
- Comparison of tumor activity The results show that: the control group, the average tumor weight of antibody 1.854g, at the dosage administered 0.2 ⁇ ⁇ / only the parent antibody Ame55 group (mean tumor weight of 0.71 g of) anti-tumor activity is reduced to about 60%, while the mutant antibodies The anti-tumor activity was significantly better than that of the parental antibody, and the tumor inhibition rate ranged from 67% to 93%, and some antibodies were equivalent to the commercial antibody Erbitux (91% inhibition rate). This result suggests that an increase in the affinity of the mutant does benefit from an improvement in the biological activity in the body.
- Statistical analysis of tumor growth and tumor weight in different groups is shown in Figure 14.
- the present invention combines the Ame55 alone light chain (amino acid sequence set forth in SEQ ID NO. 20) with an antibody heavy chain from the VH3 germline gene family (sequences such as SEQ ID NO. 22) was combined and transiently expressed in the HEK293 transient expression system, and another new antibody Ame-9B containing the Ame55 light chain was obtained by purification on a Protein A column.
- the binding ability of Ame-9B to EGFR was detected by the Fortebio protein interaction system. For the specific method, refer to Example 5.
- the results showed that Ame-9B retained its ability to bind to EGFR despite a decrease in affinity (approximately 6-fold reduction), as shown in Figure 15.
- the antibody of the present invention binds to human EGFR with an affinity of not more than InM, and the mutant affinity is not more than ⁇ ; and the immunological activity and biological activity identification of the antibody protein IgG are completed, and it is confirmed that the antibody of the present invention has good inhibition of EGFR expression cell A431 tumor-bearing
- the biological activity of tumor growth in model mice, the antibodies of the present invention provide specific antibody drugs for the prevention and treatment of anti-tumor and other diseases such as inflammation and autoimmune diseases targeting EGFR.
- Xaa Ala or Gly or Asn or Lys
- Xaa Ser or Asn or Ala or Gly or Leu or Tyr or Gin
- Xaa lie lie Tyr Pro Xaa Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 1 5 10 15
- Xaa Ala or Gly or Asn or Lys
- Xaa Ser or Asn or Ala or Gly or Leu or Tyr or Gin ⁇ 220>
- Xaa Ser or Val or Ala or Leu
- Thr Ala Arg lie Thr Cys Ser Gly Asp Xaa Leu Gly Asp Lys Tyr Xaa
- Trp lie Gly Trp Val Arg Gin Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45
- Xaa lie lie Tyr Pro Xaa Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60
- Gcgcccctta agggcgtgca gtgcgaagtt caactggttc aaagc 45
- Thr Ala Arg lie Thr Cys Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala
- Trp lie Gly Trp Val Arg Gin Met Pro Gly Lys Gly Leu Glu Trp Met
- Gly lie lie Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
- Ala Arg Gly lie lie Tyr Pro Ser Asn Val Ala Val Trp Gly Gin Gly
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US14/436,845 US9840561B2 (en) | 2012-11-16 | 2013-11-07 | Human anti-human epidermal growth factor receptor antibody and encoding gene and application thereof |
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CN103739713B (zh) * | 2013-12-10 | 2016-03-09 | 吴炯 | 一种新型诱导肺瘤细胞凋亡的融合蛋白及其应用 |
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CN108250297B (zh) * | 2018-01-23 | 2021-07-23 | 上海生物制品研究所有限责任公司 | 抗egfr抗体、其制法及其应用 |
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JP2015535295A (ja) | 2015-12-10 |
CN102993305A (zh) | 2013-03-27 |
KR101739489B1 (ko) | 2017-05-24 |
KR20150064204A (ko) | 2015-06-10 |
PL2921503T3 (pl) | 2019-01-31 |
DK2921503T3 (en) | 2018-11-26 |
CN102993305B (zh) | 2015-05-13 |
PT2921503T (pt) | 2018-11-19 |
EP2921503A4 (en) | 2016-06-15 |
EP2921503A1 (en) | 2015-09-23 |
EP2921503B1 (en) | 2018-08-08 |
ES2689299T3 (es) | 2018-11-13 |
US20150274828A1 (en) | 2015-10-01 |
US9840561B2 (en) | 2017-12-12 |
JP6143133B2 (ja) | 2017-06-07 |
HK1215443A1 (zh) | 2016-08-26 |
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