Human anti-human epidermal growth factor receptor antibody and encoding gene and application thereof

Abstract

The present invention provides human anti-human epidermal growth factor receptor (EGFR) antibodies and their encoding genes and applications thereof. By gene engineering means and phage surface display technology, the present invention screens anti-human EGFR gene engineering single chain antibody from fully synthetic single chain human antibody library, and obtains the antibody variable gene sequence thereof, and based thereon, constructs intact human monoclonal antibody, to further obtain high-purity antibody protein. The binding affinity of the antibody of the present invention with human EGFR is no more than 1 nM, and the mutants affinity thereof is no more than 10 nM; and the identification of the immunity activity and bioactivity of antibodies protein IgG is completed, confirming that the antibody of the present invention has good bioactivity of inhibiting the tumor growth of EGFR expressing cell A431 tumor-bearing model mouse. The antibodies of the present invention provides specific antibody drugs for preventing and treating EGFR targeted tumor and other diseases such as inflammation and autoimmune diseases.

Claims

1. A human anti-human EGFR antibody wherein: (a) the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, (b) the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, wherein the amino acid of position 26 of SEQ ID NO: 20 is replaced with Gly and the amino acid of position 89 of SEQ ID NO: 20 is replaced with Ser, (c) the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, wherein the amino acid of position 26 of SEQ ID NO: 20 is replaced with Lys and the amino acid of position 89 of SEQ ID NO: 20 is replaced with Ser, (d) the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, wherein the amino acid of position 26 of SEQ ID NO: 20 is replaced with Gly, and the amino acid of position 89 of SEQ ID NO: 20 is replaced with Ala, (e) the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, wherein the amino acid of position 26 of SEQ ID NO: 20 is replaced with Lys and the amino acid of position 89 of SEQ ID NO: 20 is replaced with Ala, (f) the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, wherein the amino acid of position 26 of SEQ ID NO: 20 is replaced with Lys, the amino acid of position 89 of SEQ ID NO: 20 is replaced with Ser, and the amino acid of position 107 of SEQ ID NO: 21 is replaced with Ser, (g) the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, wherein the amino acid of position 89 of SEQ ID NO: 20 is replaced with Ala, or (h) the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, wherein the amino acid of position 26 of SEQ ID NO: 20 is replaced with Lys, the amino acid of position 89 of SEQ ID NO: 20 is replaced with Ala, and the amino acid of position 107 of SEQ ID NO: 21 is replaced with Ser.

2. The antibody according to claim 1, wherein the antibody is selected from the group consisting of single chain antibodies, Fabs, and intact antibody immunoglobulins: IgG1, IgG2, IgA, IgE, IgM, IgG4, and IgD.

3. A method of treating a tumor expressing EGFR comprising administering an antibody according to claim 1 to a subject in need thereof and inhibiting tumor growth.

4. A drug or detection reagent comprising the antibody according to claim 1 combined with one or more antibodies.

5. An antibody targeted drug molecule, comprising the antibody according to claim 1 linked to a cytotoxic agent.

6. The antibody targeted drug molecule according to claim 5 wherein the cytotoxic agent is linked to the antibody by antibody labeling, in-vitro cross-linking or molecular coupling.

7. The antibody targeted drug molecule according to claim 5 wherein the cytotoxic agent comprises chemical molecules, radioactive isotopes, polypeptides, toxins and other substances having the properties of killing cells or inducing apoptosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows analysis of the specificity of Ame55 phage antibody.

(2) FIG. 2 shows a SDS-PAGE electrophoretogram of Ame55 treated by one-step purification of Protein A column, wherein, 1: Pr Marker; 2: Erbitux; 3: Ame55; 4: Nimotuzumab.

(3) FIG. 3 shows the binding of Ame55 to EGFR identified by Western Blot, wherein, 1: non-denatured EGFR-Erbitux; 2: denatured EGFR-Erbitux; 3: non-denatured EGFR-Ame55; 4: denatured EGFR-Ame55; 5: non-denatured EGFR-Nimotuzumab; 6: denatured EGFR-Nimotuzumab.

(4) FIG. 4 includes images showing the indirect immunofluorescence assay for the cytomixis of Ame55 and A431, wherein, A: avastin-A431; B: Ame55-A431; C: Erbitux-A431; D: Nimotuzumab-A431.

(5) FIG. 5 is a graph showing the analysis of binding of Ame55 to EGFR on A431 cell surface by flow cytometry, in FIG. 5A, antibody concentration: 3 μg/ml; in FIG. 5B: antibody concentration: 0.3 μg/ml; in FIG. 5C: antibody concentration: 0.03 μg/ml, 1 represents antibody Nimotuzumab; 2 represents antibody Ame55; 3 represents antibody Erbitux.

(6) FIG. 6 is a graph showing affinity of ame55 to EGFR-Fc as measured by Biacore.

(7) FIG. 7 shows the competitive inhibition by Ame55 and Erbitux, wherein, FIG. 7A: 1: EGFR-Fc binding; 2: PBST washing; 3: Ame55-binding; 4: PBST washing; FIG. 7B: 1: EGFR-Fc binding; 2: PBST washing; 3: Erbitux-binding; 4: PBST washing; FIG. 7C: 1: EGFR-Fc binding; 2: PBST washing; 3: Erbitux-binding; 4: PBST washing; FIG. 7D: 1: EGFR-Fc binding; 2: PBST washing; 3: Ame55-binding; 4: PBST washing; for FIG. 7A and FIG. 7B, the stationary phase is Ame55, for FIG. 7C and FIG. 7D, the stationary phase is Erbitux.

(8) FIG. 8 is a graph showing the competitive inhibition of the EGR binding to EGFR by Ame55. 1: blank control; 2: the molar ratio of Ame55 to EGFR is 3:1; 3: the molar ratio of Ame55 to EGFR is 2:1; 4: the molar ratio of Ame55 to EGFR is 1:1; 5: Ame55; 6: EGFR; 7: the molar ratio of Erbitux 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: the molar ratio of unrelated antibody control to EGFR is 3:1; 12: the molar ratio of unrelated antibody to EGFR is 2:1; 13: the molar ratio of unrelated antibody to EGFR is 1:1; 14: unrelated antibody control.

(9) FIG. 9 is a graph showing the inhibition of Ame55, Erbitux, Nimotuzumab, Adalimumab and blank control on migration ability of A431 cells.

(10) FIG. 10 is a graph showing the inhibition of Ame55, Erbitux, Nimotuzumab and PBS control on A431 cell growth.

(11) FIG. 11 is a graph showing the inhibition of Ame55 on tumor growth in A431 tumor-bearing mic.

(12) FIG. 12 is a graph showing the inhibition of Ame55 on tumor in A431 tumor-bearing mice.

(13) FIG. 13 is a graph showing the inhibition of Ame55 on tumor in A431 tumor-bearing mice.

(14) FIG. 14 is a graph showing the comparison of inhibitory activity of Ame55 and mutant antibodies thereof on tumor growth in tumor-bearing mice, A: dynamic observation of tumor volume; B: statistical results of tumor weight measurement. 1: Ame55; 2: AmeA1C1; 3: AmeA1C3; 4: AmeA1C3-HI2; 5: AmeA2; 6: AmeA2C1; 7: AmeA2C3; 8: AmeA2C3-HI2; 9: Erbitux; 10: IgG control.

(15) FIG. 15 is graph showing affinity test of mutant Ame-9B formed by combining Ame55 light chain with another unrelated heavy chain.

(16) FIG. 16 is a schematic view of the vector for eukaryotic transient expression plasmid pABL and pABG1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(17) The following examples further describe the present invention, however, they should not be understood to limit the invention. The modifications and substitutions of the methods, procedures or conditions of the invention made without departing from the spirit and essence of the invention fall into the scope of the invention.

(18) Unless particularly indicated, the technique means used in the examples are those well known to the skilled person in the art. All the materials, reagents and the like used in the following examples could be commercially available, unless otherwise expressly stated.

Example 1

Screening of Specific Antibodies from a Large Capacity Phage Antibody Library

(19) I. Material and Method

(20) 1. Material: A large capacity fully synthetic phage single chain antibody library is constructed by the Chinese people's liberation army military academy of medical sciences (ZL200910091261.8), and the library capacity reaches 1.35×10.sup.10. The antigens for screening the antibody library are purified EGFR-Fc fusion protein (at a concentration of 1 mg/ml) expressed in constructed mammalian cells and the strain is XL1-Blue (Stratagene, USA); the phage used is M13KO7 (Invitrogene, USA). The eukaryotic expression vectors pABG1 and pABL are stored in the room, and the structure information of the vector is shown in FIG. 16. The mammalian cell HEK293T cell is purchased from Invitrogene.

(21) 2. Method

(22) 2.1 Expression and Purification of EGFR-Fc Fusion Protein

(23) Full-length extracellular region of EGFR was chosen as object, which was purchased from Sino Biological Inc. (NCBI sequence: NM_005228.3). The sense primer and reverse primer as shown in SEQ ID NO 11 and SEQ ID NO 12 were designed. PCR amplification was performed to obtain full-length genes of extracellular region, which were cloned into the eukaryotic expression vector pABG1 at the restriction enzyme sites EcoR I and Nhe I. Sequencing of the constructed recombinant plasmid was performed to identify the validity thereof and then the plasmid was extracted. HEK293T cells were transfected to perform transient expression, the supernatant after expression was purified using Protein A affinity column, and the protein was quantified for the purified product by Bradford method.

(24) 2.2 EGFR-his Expression and Purification

(25) This step was accomplished on the basis of the method in section 2.1. the primer EGFRR1 (SEQ ID NO 13) was designed. The primers EGFRF and EGFRR1 were used to amplify the full-length genes of extracellular region of EGFR, which were cloned into the eukaryotic expression vector pABG1 at the restriction enzyme sites EcoR I and BamH I. Sequencing of the constructed recombinant plasmid was performed to identify the validity thereof and then the plasmid was extracted. HEK293T cells were transfected with the plasmid to perform transient expression, the supernatant after expression was purified using Ni+ affinity column, and the protein was quantified for the purified product by Bradford method.

(26) 2.3 Presentation of phage antibody library, which was performed according to the user instruction of recombinant phage selection of Pharmacia (Cat.NO.XY-040-00-05) with a minor modification. The details for modification are as follows:

(27) Cryopreserved antibody library was taken and cultured in 200 mL 2xYTCG medium (glucose concentration of 0.5%) at 37° C. to OD.sub.600=0.5. Helper phage M13KO7 was added at a ratio of MOI=20:1 and maintained at room temperature for 20 min. At 37° C., slowly shake cultivation was performed at 150 rpm for 1 h, kanamycin was added to reach the final concentration of 50 μg/ml, and IPTG was added to reach the final concentration of 0.1 mmol/L. At 30° C., shake cultivation was performed at 220 rpm for 10-12 h. In the next day, the cultured supernatant was collected by centrifugation and ⅕ volume of PEG8000 buffer (20% PEG+2.5 mol/L NaCl) was added therein. After mixing uniformly, the mixed supernatant was placed on the ice for 45 min for precipitating the phages. At 4° C., centrifugation was performed at 10000 g for 20 min, and the supernatant was discarded. The precipitation was resuspended in 5 ml PBS buffer containing 2% BSA and 15% glycerol, and the suspension was cryo-preseved at 70° C. for use.

(28) 2.4 Determination of Phage Titre

(29) The monoclone of host bacteria (XL1-Blue) was selected and seeded in LB culture medium, followed by shake cultivation at 37° C. until logarithmic phase (OD.sub.600=0.5). 50 μl prepared phage suspension was taken and gradiently diluted 10 folds with LB culture medium. The host bacterial cells at logarithmic phase were added into the suspension which had been diluted into a fold, followed by shake cultivation at 37° C. and at 150-180 rpm for 1 h. 100 or 200 μl of cultured bacteria liquid was coated onto LB plate and cultured at 37° C. overnight. In the next day, the number of colony was counted and the titre was calculated.

(30) 2.3 Bio-panning of anti-EGFR specific antibody, which was performed according to the user instruction of recombinant phage selection of Pharmacia (Cat.NO.XY-040-00-05) with a minor modification. The details for modification are as follows:

(31) Recombinant protein antigen EGFR-Fc was diluted to 20 μg/ml with coating buffer, and the dilution solution was used to coat immune tubes, with 1 ml/tube. The immune tubes were coated at 4° C. overnight. In the next day, the immune tubes were washed with PBS twice, with 2 min for each, and then blocked with 2% BSA at 37° C. for 2 h. The prepared phage antibody library was blocked with blocking buffer (2% BSA, 0.1% Tween 20) at 37° C. for 30 min. The pre-blocked phage antibody library was added into the blocked immune tubes and maintained at 4° C. overnight. The solutions in the immune tubes were discarded and washing was performed. The first round of washing involves washing with PBST for 10 times, with 5 min/time, and washing with PBS for 5 times, with 5 min/time; the second round of washing involves washing with PBST for 15 times, with 5 min/time, and PBS for 10 times, with 5 min/time; the third round of washing involves washing with PBST for 15 times, with 5 min/time, and PBS+NaCl for 15 times, with 5 min/time. 1 ml 0.2 mol/L glycine-HCl (pH2.2) was added for elution. After the elution performed at room temperature for 10 min, the solution was immediately neutralized to pH 7.4 with 1 mol/L Tris. Direct infection with E. coli XL1-Blue at 37° C., and shake cultivation was performed at 150 rpm for 1 h. 2xYTCTG plate was coated with the residual bacterial solution and cultured overnight at 37° C. The bacterial colony was taken with liquid 2xYTCTG medium. An appropriate amount of bacterial solution was charged into 100 ml of 2xYTCTG liquid medium, and shake cultivation was performed at 37° C. until OD.sub.600=0.5. The helper phages were added at a ratio of MOI=20:1 and maintained at room temperature for 20 min. Shake cultivation was performed at 150 rpm for 1 h at 30° C. The solution was added with the same volume of 2xYTCT medium, with kanamycin to a final concentration of 50 μ/ml, and with IPTG to a final concentration of 0.15 mM. Then the solution was cultivated on the shaker at 200 rpm under 30° C. for 10 h. The supernatant was collected via centrifugation and added with ⅕ volume of PEG8000 buffer solution (20% PEG +2.5 mol/L NaCl). After mixed homogeneously, the solution was placed on ice for 45 min to precipitate the phages. The solution was centrifuged at 10000 g for 20 min, and the supernatant was removed. The precipitation was resuspended in 5 ml PBS buffer solution containing 2% BSA and cryopreserved at −70° C. for use. 3 portions of phages ware determined for the titer, combined in proportion, and put into the next selection.

(32) 2.5 Identification of Positive Clones by Phage ELISA

(33) The selected clones were placed in 96-well deep-well plates, with 250 μl 2xYTCTG medium per well. After shake cultivation at 37° C. until OD.sub.600=0.5, 1×10.sup.8 cfu helper phages was added and maintained at room temperature for 15 min Shake cultivation was performed at 37° C. and at 150 rpm for 1 h, and the same volume of 2xYTCTKI (kanamycin of 50 μg/ml, IPTG of 0.2 mmol/L) was added. Shake cultivation was performed at 30° C. overnight. In the next day, the supernatant was collected via centrifugation, added with BSA to a final concentration of 2%, added with Tween-20 to a final concentration of 0.1%, and maintained at 37° C. for 15 min for use. The antigen (EGFR) was diluted with coating solution to 1 μg/ml, added into 96-well assay plate, with 50 μl/well, for coating overnight at 4° C. In the next day, the coating solution was discarded. The assay plate was rinsed twice with PBST and once with PBS, 3 min for each rinse. The plate was blocked with 2% BSA+0.1% Tween-20 at 37° C. for 2 h, with 200 μl/well. The blocking solution was discarded, and then each well was added with blocked monoclonal phage antibody at 37° C. with 50 μl/well and maintained for 1 h. The liquid was discarded. The plate was rinsed twice with PBST and once with PBS, with 200 μl/well, 5 min for each rinse. HRP-labelled murine anti-M13 antibody was diluted with PBST at a dilution ratio of 1:5000. At the same time, BSA at a final concentration of 2% was added and the antibody was pre-blocked at 37° C. for 15 min. The pre-blocked murine anti-M13 antibody was added into the assay plate with 50 μl/well and maintained at 37° C. for 30 min. The liquid was discarded and the assay plate was rinsed twice with PBST and once with PBS, with 200 μl/well, 5 min for each rinse. The rinsing solution was discarded. OPD substrate developing solution was added, with 50 μl/well, and maintained at room temperature for development. The development was terminated by 2N H.sub.2SO.sub.4. The absorbance values were measured by microplate reader.

(34) Coating buffer (pH 9.6): Na.sub.2CO.sub.3 1.59 g, NaHCO.sub.3 2.93 g, supplemented with double distilled water to 1 L;

(35) Blocking solution: 1xPBS+2% BSA+0.1% Tween 20; rinsing solution: 1×PBS +0.1% Tween 20;

(36) OPD substrate diluting solution: 0.2M Na.sub.2HPO.sub.4 (28.4 g/L) 25.7 ml, 0.1M citric acid (19.2 g/L) 24.3 ml, distilled water 50 ml.

(37) 2.6 Conversion of Phage Single Chain Antibody into Intact Antibody:

(38) Vectors pABG1 and pABL were used to clone the variable region genes of antibody heavy chain (e.g. SEQ ID NO 10) and light chain (e.g. SEQ ID NO 9) respectively. Primers H5F (SEQ ID NO 14) and HR(SEQ ID NO 15) were used to amplify variable region gene of VH5 respectively; and L3F (SEQ ID NO 16) and LR(SEQ ID NO 17) were used to amplify variable region gene of Vλ3. The variable region gene of Vλ3 was cloned into vector pABL using restriction enzyme cutting sites Bsr GI and HindIII. The variable region gene of VH5 was cloned into vector pABL using restriction enzyme cutting sites Afl II and NheI. E. coli DH5α was transformed with the recombinant plasmid. PCR of the bacterial solution and sequencing identification were performed on the recombinant plasmid, to obtain light and heavy chain expression vectors of the correctly constructed intact antibody. After extraction of the plasmid in large quantity, the HEK293T cells were tranfected with the light and heavy chains of the antibody at a molar ratio of 1:1, to perform the transient expression of the intact antibody, the supernatant after expression was purified by Protein A affinity column, and then the purified antibodies were subjected to the electrophoretic analysis and affinity and specificity assays.

(39) II. Results

(40) 1. Selection and Identification of Ame55 Phage Antibody

(41) The monoclonal antibodies obtained from the third selection were identified to obtain specific phage antibody. The nucleotide and amino acid sequences of Ame55 single chain antibody are shown as SEQ ID NO 18 and SEQ ID NO 19. The nucleotide sequences of light and heavy chain variable regions of Ame55 single chain antibody are shown as SEQ ID NO 9 and SEQ ID NO 10, respectively. The amino acid sequences of light and heavy chain variable regions of Ame55 single chain antibody are shown as SEQ ID NO 20 and SEQ ID NO 21, respectively.

(42) The specificity of Ame55 was analysed at the level of phage using EGFR-Fc as positive antigen and CD4-D2, AHC, TNF, VEGF, TTC, PBS as control antigen. The assay plate was coated at 4° C. overnight. The phage supernatant was added and the specific binding activity of Ame55 phage supernatant was tested by ELISA using HRP-labelled anti-M13 antibody as secondary antibody. The result showed that Ame55 only specially binds to EGFR-Fc. (FIG. 1)

(43) 2. Expression and Purification of the Intact Antibody

(44) The variable region genes of light and heavy chains of phage antibody Ame55 were respectively cloned into the transient expression vectors pABL and pABG1 for intact antibody to construct the recombinant expression vectors for the intact antibody. The transient secretory expression of mammalian cells was accomplished by HEK293T cell transient expression system. The intact antibody protein samples were obtained by purification through Protein A column with a molecular weight of 150 KD. (FIG. 2)

Example 2

Assay of Binding Activity of Ame55

(45) I. Material and Method

(46) 1. Material: human epidermal squamous cancer cells were purchased from Shanghai cell bank, Chinese academy of sciences; Erbitux(CT), a product from Merck & Co. Inc, Germany; Nimotuzumab(Hr-3) was an anti-EGFR human antibody marketed by Beijing Baitai Biotech Pharmaceuticals Co., Ltd.; Avastin (Bevacizumab, rhuMAb-VEGF) was purchased from Roche, Germany; CM5 chip was a product from GE; and the anti-human immunoglobulin Fc capture sensor and streptavidin sensor were purchased from FortéBio, Inc. (Shanghai). Recombinant human epidermal growth factor (EGF) was purchased from Sinobio Bio, Inc., Shanghai. FreeStyleTM293-F cell line, expression medium and transfection reagents were purchased from Invitrogen.

(47) II. Method

(48) 2.1 Immunofluorescence Assay of Binding Activity of Antibodies to EGFR on A431 Cell Surface

(49) (1) A431 cells were seeded in 96-well plate with 10.sup.4 cells/well.

(50) (2) After culture overnight, the medium was discarded and then washing was performed with PBS twice.

(51) (3) 5% skim milk powder (PBS formulation) was blocked at 37° C. for 1 h and then placed at 4° C.

(52) (4) the primary antibody to be measured was diluted with 5% skim milk powder (PBST formulation) to 1 μg/ml, and pre-blocked at 37° C. for 30 min and then placed at 4° C. Erbitux (Ct) and Nimotuzumab (Hr-3), which are commercial drugs, were used as positive antibody control, and avastin (Avs) as negative antibody control.

(53) (5) The blocking solution was discarded and the blocked antibody was added and incubated at 4° C. for 1 h.

(54) (6) Washing was performed with PBST for 3 times at 240 rpm, with 3 min/time.

(55) (7) The supernatant was discarded, FITC-labelled goat anti-human IgG secondary antibody which was pre-blocked by 5% skim milk powder was added, and bound at 4° C. for 30 min.

(56) (8) Washing was performed with PBST for 3 times at 240 rpm, with 3 min/time.

(57) (9) Observation was conducted under fluorescence microscope.

(58) 2.2 Western Blotting

(59) (1) Reducing and non-reducing SDS-PAGE gel electrophoresis were applied to EGFR-his antigen, and the gel concentrations for running were 10% respectively;

(60) (2) the proteins were tranferred from gel into nitrocellulose membrane by semi-dry method. After the end of electrophoresis, the gel for electrophoresis was transferred to nitrocellulose membrane for 2 h via semi-dry electric transfer method at 20 mA. After the transfer to the membrane, the membrane was blocked with 5% skim milk at 37° C. for 2 h;

(61) (3) after the membrane was blocked, the membrane were cut off and added with 10 μg/ml Ct, hR-3 and Ame55 respectively for binding at 4° C. overnight;

(62) (4) washing was performed with TBST for 4 times, with 10 min/time;

(63) (5) HRP-labelled goat anti-human IgG (which was pre-blocked at 37° C. for 30 min) diluted with 5% skim milk powder at 1:5000 was added, and incubated at 37° C. for 40 min;

(64) (6) after incubation of the secondary antibody, washing was performed with TBST for 3 times, with 10 min/time.

(65) (7) development: ECL luminous fluid was added thereto and development and exposure were performed in a dark room.

(66) 2.3 Measurement of Affinity of Antibody by Biacore 3000 System

(67) 10 mM NaAC were formulated with a pH of pH4.0, pH4.5, pH5.0 and pH5.5. The antibodies to be measured were diluted at appropriate times and pre-concentrated on CM5 chip. The NaAc solution with the optimal pH was selected as coating dilution solution, and the dilution concentration of the antibody was determined, which is most suitable for coating.

(68) Coating: the antibodies to be measured were diluted with NaAc solution with the the optimal pH. The optimal dilution concentration was selected, one channel in CM5 chip was selected for coupling, wherein the target value of coupling antibody was 2000RU, and another channel was selected as control. The experimental condition included temperature of 25° C., flow rate of 20 μL/min and HBS-EP (pH7.4, Bia-Certified) as buffer solution. After the chip-coupling antibody reached the target value, the chip surface was blocked.

(69) Analysis of regeneration conditions: 100 nM EGFR-Fc was gotten to flow through the surface of the chip, such that it binds to the antibodies on the surface of the chip. After stabilization, 10 mM glycine-HCl at pH 3.5, pH 2.5, pH 2.0, pH 1.5 and borate saline buffer at pH 8.5 flowed successively through the surface of the chip until the optimal regeneration effect was achieved, such that the optimal regeneration condition can be determined.

(70) Dynamic analysis of binding of antibody to antigen: the concentration of antigen EGFR-Fc was measured by Broford method. The antigen was diluted with HBS-EP buffer solution. EGFR-Fc dilutions with different concentrations (5 different concentrations of dilutions were taken between 0-100 nM) were taken and gotten to flow the channels of antibodies to be measured and the channels of control antibody in a flow rate of 20 μL/min, with binding time of 3 min, stablizing time of 1 min and dissociation time of 15 min. Regeneration condition for each cycle included: 10 mM glycine-HCl, pH 1.5 and pH 8.5 of borate saline buffer, flow rate of 20 μL/min Each solution was regenerated for 30 s.

(71) Calculation of affinity constant: the kenetic constant of binding of the antibody to antigen was calculated by fitting Langmuir binding model of the resultant sensorgram with the ratio of 1:1 using Bia-evalutation analysis software 4.

(72) 2.4 Flow Cytometry

(73) (1) A431 cells with EDTA trypsinization in good growth state were resuspended in pre-cool PBS, counted after washing, and formulated into a concentration of 6×10.sup.6 cells/ml. the resultant solution was subpackaged into 1.5 ml centrifuge tubs, with 100 μL/tube, and the tubes were placed on ice.

(74) (2) The antibody to be measured was diluted with pre-cool PBS to 150 μg/ml, 15 μg/ml, 1.5 μg/ml and 0.15 μg/ml. 100 μL of each dilution solution was taken and mixed with pre-cool cells respectively, and incubated on ice for 40 min.

(75) (3) The resultant mixture was centrifuged in pre-cooled centrifuge at 4° C., the supernatant was drawn lightly, 1 ml pre-cooled PBS solution was added for washing for 3 times.

(76) (4) Pre-blocked FITC-rabbit anti-human IgG secondary antibody diluted at a ratio of 1:100 was added into each tube and the tubes were incubated on ice for 30 min.

(77) (5) Step (3) was repeated and washing was performed for 3 times.

(78) (6) 500μL pre-cooled PBS solution was added into each tube with cells, blowed and beated lightly and uniformly, the samples were placed on the ice and the fluorometric analysis was performed by flow cytometer. The sample without antibody to be measured was used as control.

(79) II. Result

(80) 1. Antibody Ame55 Specifically Binds to Non-Denatured EGFR.

(81) Reducing and non-reducing SDS-PAGE gel electrophoresis were applied to EGFR-his. Western blot was performed using 10 μg/ml of Ame55, Erbitux and Nimotuzumab as primary antibodies and HRP-labelled goat anti-human IgG as secondary antibody respectively. The results are shown in FIG. 3. Ame55 only specifically binds to non-denatured EGFR.

(82) 2. Ame55 Specifically Binds to EGFR on A431 Cell Surface.

(83) Cellular immune fluorescence assay was performed using A431 cell expressing EGFR as antigen, Amet55, unrelated antibody Avastin, control antibody Erbitux and Nimotuzumab as primary antibodies, and goat anti-human IgG as secondary antibody. The results are shown in FIG. 4, and indicates that Ame55 (B in FIG. 4), positive control antibody Erbitux (C in FIG. 4) and Nimotuzumab (D in FIG. 4) can specifically bind to EGFR on A431 cell surface, but negative control antibody Avastin (A in FIG. 4) did not bind. The binding of Ame55 to A431 cells was further analyzed by flow cytometry. The result indicates that this antibody, in a certain concentration range, binds to A431 cells in a dose-dependent way. In the case of a concentration of less than 1 μg/ml, the fluorescence intensity of Ame55 is between control Erbitux and Nimotuzumab (FIG. 5A, FIG. 5B and FIG. 5C).

(84) 3. Measurement of Affinity Ame55 for EGFR-Fc by Biacore 3000.

(85) Ame55 was coated on the surface of CM5 chip. Affinity of Ame55 for EGFR-Fc was measured by Biacore 3000 system using different concentrations of EGFR-Fc as mobile phase. The results are shown in FIG. 6. The affinity constant K.sub.D after fitting is 0.231 nM, wherein Ka=4.21×10.sup.5, Kd=9.73×10.sup.−5.

Example 3

In Vitro Activity and Epitope Analysis of Ame55

(86) I. Method

(87) 1 Inhibition Assay of Antibody Competitive Binding

(88) Ame 55 as stationary phase was coated on CM5 chip, wherein the conditions for coating and regeneration are shown in the method of Example 2. 50 nM EGFR-Fc as first mobile phase was gotten to flow through the sample channel with duration of 3 min Upon washing with PBS-T for 3 min, 1000 nM of Erbitux as second mobile phase was gotten to flow through the sample channel with duration of 3 min and the sample channel was then washed with PBST for 3 min. Furthermore, the above experiment was performed again using 1000 nM of Ame55 as the second mobile phase, which is used as self-competitive inhibition control. Likewise, the above experiment was repeated using Erbitux as stationary phase.

(89) 2. Competitive Inhibition Assay of Binding of EGR to EGFR

(90) EGF was coated at 1 μg/well in 96-well plate overnight at 4° C. Ame55, the positive control antibody Erbitux and unrelated control antibody (anti-IL-6R antibody selected in the present invention) were mixed with 2.5 μg/well of EGFR-Fc at a molar ratio of 3:1, 2:1 and 1:1 respectively, and pre-blocked at 37° C. for 30 min, and were added into blocked EGR-coating ELISA plate, for binding at 37° C. for 1 h. Washing was performed with PBST for 3 times and with PBS once, with 5 min/time. The pre-blocked HRP-labelled goat anti-human IgG was used as secondary antibody and reacted at 37° C. for 30 min After washing with PBST and PBS, OPD substrate developing solution was added for development. The reaction was stopped by addition of 2N H.sub.2SO.sub.4, and the absorbance value was measured at OD.sub.450 nm.

(91) 3. A431 Cell Scratch Healing Assay

(92) Straight lines were marked at the bottom of 6-well plate, for quinquesection of each well. A431 cells were seeded in the marked 6-well plate with 10.sup.5 cells/well. When the cells entered into logarithmic phase, the surface of cells was marked with line perpendicular to the straight lines using 200 μL of pipette. After marking, the surface was washed twice with PBS, and each well was added with the antibody Ame55 to be measured, positive control antibody Erbitux and negative control antibody Adalimumab (Abbott, USA) until a final concentration of 25 μg/ml was reached. A blank well was set up as blank control. At the observation time of 0 h, 12 h and 24 h, the fixed location was maked with straight lines and the photos are taken for the dynamic process.

(93) 4. A431 Cell Growth Inhibition Assay

(94) 1) seeding of the cells: the A431 cells were trypsinized, blown fully and counted, and the cell concentration was adjusted to 5000 cells/ml with 1640 culture medium containing 0.5% new-born calf serum, added into 96-well plate with 200 μL per well (finally 800-1000 cells were uniformly distributed in each well), and incubated at 37° C. and at 5% CO.sub.2 for 24 h until cells completely extended to adherence;

(95) 2) Culture upon adding the antibody to be measured: the cell supernatant in the 96-well plate was discarded, and the palate was washed with PBS once. 200 nM of Erbitux(CT), Nimotuzumab (hR3) and Ame55 were prepared, and PBS was used as control, followed by adding them into 96-well plate with 200 μl/well 3 multiple-wells for each concentration. 5 pieces of 96-well plates were added in parallel, cultured at 37° C. under 5% CO.sub.2, and then the solutions were exchanged on day 3 and 5 respectively. One culture plate was selected randomly for staining on day 0, 3, 4, 5, 6 and 7.

(96) 3) Crystal violet staining: the medium in the 96-well plate was removed and the plate was washed once with PBS; each well was added with 100 μL 4% paraformaldehyde and maintained for 15 min at room temperature for immobilization; the formaldehyde was removed and 0.5% crystal violet solution diluted with PBS (which was formed by diluting 5% crystal violet solution formulated with absolute ethyl alcohol into 10 fold) was added, with 100 μL/well, then the plated was maintained at room temperature for 15 min for staining; the crystal violet solution was removed and the plate was immerged into tap water and washed for several times; the plate was immerged into distilled water at room temperature, and washed on the horizontal shaker at 560 r/min for 15 min; the moisture was removed and the plate was dried overnight.

(97) 4) Cell density determination: the 6 96-well phates on Day 1 to 7 were collected for decoloration and determination of OD value for the same batches. 200 μL sorenson's solution was added into each well; the plates were placed in horizontal shaker at 400 r/min for 30 min for decoloration; 100 μL solution was taken from each well and added into a new 96 well-plate, and the absorbance value was measured at 590 nm with absorbance value at 630 nm as internal reference. The cell growth curve was plotted.

(98) Sorenson's formulation method: Solution A: 8.967 g natrium citricum was dissolved in 305 ml ultrapure water; Solution B: 195 ml 0.1N HCl (1.6 ml 38% HCl was added in 193.4 ml ultrapure water, which is known as “addition of acid into water”); Solution C: 500 ml 95% ethyl alcohol (475 ml absolute ethyl alcohol was added with 25 ml ultrapure water); Solution A+B+C was mixed up to a total volume of 1 litre.

(99) II. Result

(100) 1. Competitive Inhibition of Binding of Ame55 and Erbitux to EGFR

(101) The epitopes of two antibodies were analyzed preliminarily, the condition for analysis included: using Ame55 as stationary phase and EGFR as first mobile phase (1), washing with PBST for a period of time (2), using Ame55(A) and Erbitux (B) as second mobile phase (3), then washing again with PBST (4); on the contrary, using Erbitux as stationary phase and EGFR as first mobile phase (1), washing with PBST for a period of time (2), using Ame55(D) and Erbitux (C) as second mobile phase (3), then washing again with PBST (4). The results (see FIGS. 7A, 7B, 7C and 7D) indicate that EGFR bound to Ame55 can not further bind to Erbitux (FIGS. 7B and 7A show self-inhibition control of Ame55). That is, Ame55 can inhibit the binding of EGFR to Erbitux. On the contrary, upon binding to Erbitux, EGFR no longer binds to Ame55 (FIGS. 7D and 7C show self-inhibition control of Erbitux). That is, Erbitux can also inhibit the binding of EGFR to Ame55. The result reveals that there may be competitive inhibition of antigen epitopes between two antibodies. It is probably because the two antigen epitopes have highly similar or identical structure.

(102) 2. Competitive Inhibition of Binding of EGF to EGFR by Ame55

(103) The competitive inhibition of binding of EGF to EGFR by Ame55 was analyzed by competitive ELISA. The result indicates that Ame55 significantly inhibits the binding of EGF to EGFR when a ratio of EGFR to antibody is 1:1, 1:2 and 1:3, but the negative control has no inhibition effect. The inhibitary activity of Ame55 was less than that of Erbitux as control at the same concentration (see FIG. 8).

(104) 3 Inhibition of A431 Cell Migration by Ame55

(105) As described for method 3 in the example, 25 μg/ML of experimental antibody group was dynamically observed for 24 h at fixed sites for the cell scratch healing (FIG. 9). For the inhibition of A431 cell migration, Ame55 exhibits the same inhibitory activity as Erbitux, whereas another negative control antibody Adalimumab, a commercial available antibody drug against TNFα, has no inhibitory activity on the A431 cell migration. The results indicate that, in FIG. 10, Ame55 does have the function of inhibiting biological activity of EGFR, revealing that it may have good effect in the therapeutic application of clinical metastases.

(106) 4 Inhibition of A431 Cell Growth by Ame55

(107) The cells expressing EGFR, such as A431, act on the EGFR by autocrine EGF and therefore activate downstream pathway, which leads the dephosphorylation of receptor and promote cell growth and migration. Accordingly, the effect of Ame55 on the growth of cell expressing EGFR was further evaluated by A431 cell proliferation assay, and the cell growth tendency was observed continuously. The assay was performed as described for method 4 in the example. The results indicate that, in FIG. 10, Ame55 has an inhibition effect on A431 cell growth, but the inhibition degree of Ame55 is less than that of control antibody Erbitux, and is comparable to another control antibody Nimotuzumab.

Example 4

Detection of In-Vivo Tumor Inhibition Activity of Ame55

(108) 47 female nude mice (about 20 g) were subcutaneously inoculated at back with 100 μL of A431 cell at a concentration of 5×10.sup.7. When the tumor grew to 50-150 mm.sup.3, the mice were weighed, the tumor size was measured and grouped into 4 groups, i.e. normal saline group, Erbitux group, Adalimumab group and Ame55 group respectively, wherein Erbitux is a marketed anti-EGFR monoclonal antibody and was set as positive control; Adalimumab is a marketed anti-TNFα antibody drug and was set as negative control; and the normal saline was set as tumor-bearing control. The dose of antibody for administration was 1 mg/mouse, once every 3 days, and before each administration, the mice were weighed and the tumor size was measured. The tumor calculation method is: π/6*long diameter*short diameter.sup.2. After 36 days, the mice were sacrificed to collect the tumors, the tumors were weighed, and then statistical analysis was performed.

(109) A431 cell transplantation tumor was adopted as research object to evaluate the inhibition effect of Ame55 on the tumor growth of a tumor-bearing mouse. Experimental results show that Ame55 has significant inhibition effect on the growth of the A431 transplantation tumor at the dose of 1 mg/mouse (FIG. 11); and the tumor weight results, which were obtained after continuously administering for 6 times and stopping administration for observation 4 weeks, showed that: the average tumor weight of the control group was 3.65 g, the average tumor weight of the Ame55 group was 0.32 g, and its tumor inhibition rate was above 90% (FIGS. 12 and 13).

Example 5

Evaluation Method for Ame55 Light and Heavy Chain Variable Region Mutant

(110) 1. Alanine Scanning

(111) Site-directed mutagenesis primers were designed respectively to perform alanine scanning on CDRL1, CDRL3, CDRH2 and CDRH3, and if it is alanine at the site, the alanine was mutated into Gly and Ser respectively. The method used is a site-directed mutagenesis method for plasmid, with reference to the document [WANG Ronghao, CHEN Ruichuan, LIU Runzhong. A modified method of quickly site-directed mutagenesis. Journal of Xiamen University (Natural Science), 2008, Vol 47, sup 2, 282-285].

(112) 2. Detection of Antibody Affinity Via ForteBio QKe

(113) EGFR-Fc fusion protein was biotinylated using a biotin kit, and the biotinylated EGFR-Fc was diluted with PBS to a concentration of 100 nM, and coated onto the surface of a streptavidin sensor for 20 min. The sensor was cleaned using HEPES EP for 5 min, and mutant antibody and parent antibody Ame55 to be detected were placed in detection pores at a concentration of 20 nM into detection holes, and bound to EGFR-Fc on the surface of the cleaned streptavidin sensor for 10 min After the binding reached balance, the complex was dissociated in HEPES EP, wherein the dissociation time was 20 min. ForteBio software package was adopted to perform affinity analysis.

(114) 3. Tumor Inhibition Activity Comparison of Mutant and Parent Antibody

(115) The experimental method was the same as in Example 4, and the group and the dosage were set as follows: normal saline control, Erbitux control, Ame55 parent antibody control and various Ame55 mutant antibodies (Ametumumabs) such as AmeA1C1, AmeA1C3, AmeA1C3-HI2, AmeA2, AmeA2C1, AmeA2C3, AmeA2C3-HI2, total 10 groups, 8 mice for each group, dose for administration was 0.2 mg/mouse, 3 d/time), wherein the administration was performed for 6 times, and 6 days after stopping drug administration, the mice were sacrificed to collect tumors for weighing.

(116) II. Results

(117) 1. Affinity Analysis of Mutants

(118) Based on Ame55 amino acid sequence, the mutations were performed for partial amino acids of the light and heave chain variable regions and framework regions. The mutants were designed by alanine scanning and site-directed mutation. A ForteBio QK.sup.e protein molecule interaction system was used to further screen and evaluate the affinity of the mutants. Results showed (FIG. 1) that, compared to parent antibody Ame55, multiple amino acid sites of light chains CDR1 and CDR3 and heavy chains CDR2 and CDR3 regions are all essential for maintaining the binding capability of the antibody to EGFR, for which the reason may be that the amino acid at the site directly participates the interaction, or that the amino acid at the site is essential for maintaining the correct whole space structure of the antibody. Among the sites where amino acids were modified, amino acids of the light chain at site 26 and site 89, and amino acids of the heavy chain at site 54 and site 107 are most important. After single amino acid substitution firstly, the present invention found that after the amino acid of the light chain at site 89 was modified from Val into Ala or Ser, the affinity was increased by 4 times, and after further combining Gly or Lys of the light chain at site 26, the affinity was further improved by above 5 times compared to the affinity of the parent antibody; or after the amino acids of the heavy chain at site 54 an site 107 were substituted for Ser respectively, and were expressed in combination with light chain 89A/S, the affinities of the antibodies were improved at different degrees based on 89A/S. In order to further confirm the effect of the affinity improvement on the bioactivity of the antibody, further, in-vivo activity of the following 7 mutant antibodies were further evaluated: AmeA1C1 (the mutated light chain has Gly at site 26 and Ser at site 89), AmeA1C3 (the mutated light chain has Lys at site 26 and Ser at site 89), AmeA1C3-HI2 (the mutated light chain has Lys at site 26 and Ser at site 89, whilst the mutated heavy chain has Ser at site 107), AmeA2 (the mutated light chain has Aly at site 89), AmeA2C1 (the mutated light chain has Gly at site 26 and Ala at site 89), AmeA2C3 (the mutated light chain has Lys at site 26 and Ala at site 89), and AmeA2C3-HI2 (the mutated light chain has Lys at site 26 and Ala at site 89, whilst the mutated heavy chain has Ser at site 107).

(119) 2. Tumor Inhibition Activity Comparison Between Mutants and Ame55

(120) In order to further evaluate the difference between the activities of the mutants and the parent antibody, the dose for administration was decreased to a low dosage of 0.2 mg/mouse, and the tumor inhibition activities of several mutants with affinity improved at different degrees and parent antibodies were compared on mice with transplantation tumors. Results showed that the average tumor weight of the control antibody group was 1.854 g; at the administration dosage of 0.2 mg/mouse, the tumor inhibition activity of the parent antibody Ame55 group (average tumor weight is 0.71 g) is decreased to about 60%; moreover, the tumor inhibition activity of the mutant antibody is obviously better than that of the parent antibody, and the tumor inhibition rates thereof are 67%-93% respectively, and the activities of some antibodies are equivalent to that of commercial antibody Erbitux (tumor inhibition rate of 91%). The results indicate that the improvement of the mutant affinity is indeed beneficial for the improvement of the in-vivo bioactivity thereof. The tumor growth conditions and the statistic analysis on tumor weight of different groups are shown in FIG. 14.

(121) TABLE-US-00001 TABLE 1 statistical table for Ame55 mutant affinity change condition Light chain Heavy chain Mutation site Affinity change Mutation site Affinity change 13A 1 50A Not binding 26G(C1) 0.5 50R Not binding 26N(C2) 1 51A >10 26K(C3) 0.3 52A Not binding G28P >100 52D >10 29E >100 53A 5 32I 1 54A >10 32V 2 54S 0.5 33Y 2 55A >100 51G 1 56A 1 55P 2 57A Not binding 88Q 2 57Y Not binding 88A >10 58A >10 88Y 5 59A >10 88L 5 59N >10 88G 3 60A 5 88N >10 66H 1 89S(A1) 0.5 99A >10 89A(A2) 0.25 99S >10 89L 5 100A 5 90A >10 101A 4 91A >10 102A Not binding 92A 2 103A Not binding 92S 4 104A 3 92P 1 105A >10 93A >10 106A >10 94A >10 107G 1.5 95A >10 107S 0.2 95S >10 108A 5 96A >10 97A >10 26G + 89S <0.3 26K + 89S <0.2 26G + 89A <0.2 26K + 89A <0.1 Note: comparing mutant antibody with parent antibody, the affinity of the parent antibody is set to be 1, and the reduction fold of the affinity is calculated.

Example 6

Detection of Specific Binding Capability of Ame55 Light Chain to EGFR

(122) In order to verify whether the Ame55 light chain has specific binding capability to EGFR, an individual light chain of Ame55 (the amino acid sequence of SEQ ID NO.20) was combined with an antibody heavy chain from VH3 germline gene family (the sequence is shown by SEQ ID NO. 22) in the example, and expressed tansiently in an HEK293 transient expression system, so as to obtain another new antibody Ame-9B containing Ame55 light chain by purifying with Protein A column. The binding capability of Ame-9B to EGFR was detected via a Fortebio protein interaction system, with reference to Example 5 for specific method. Results showed that although the affinity of Ame-9B was decreased (by about 6 times), Ame-9B still maintained the binding capability to EGFR (refer to FIG. 15).

INDUSTRIAL APPLICATION

(123) The binding affinity of the antibody of the present invention for human EGFR is no more than 1 nM, and the affinity of the mutants thereof is no more than 10 nM; and the identification of the immunity activity and bioactivity of antibody protein IgG is completed, verifying that the antibody of the present invention has good bioactivity of inhibiting the tumor growth of EGFR expressing cell A431 tumor-bearing model mouse. The antibody of the present invention provides specific antibody drugs for preventing and treating EGFR targeted tumor and other diseases such as inflammation and autoimmune diseases.