Patent Description:
The present invention relates to biotechnology, particularly the producing method of an anti-EGFR monoclonal antibody.

Tumor, particularly malignant tumor, is a disease which cause serious harm to human health in today's world, and is the <NUM>nd deadly among all diseases. But in recent years, the incidence rate was significantly increased. The malignant cancer has poor treatment, accompanied with high metastasis rate at late stage and poor prognosis. Current conventional clinical treatment methods including radiotherapy, chemotherapy and surgery, which although largely alleviate the pain and prolong the survival time, have significant limitations, and are difficult to improve their efficacy further.

Proliferation of normal cells is strictly controlled by respective ligands activating their growth factor receptors, such as growth factor receptor tyrosine kinases. Cancer cell proliferation is also through its factor receptor activation, but it loses the strict control of normal proliferation. This loss of control may be caused by many reasons, such as growth factor over-expression, overexpression of growth factor receptors, or spontaneous activation of biochemical pathways regulated by growth factors. Oncogenic receptors include epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), insulin-like growth factor receptor ((IGFR), nerve growth factor receptor (NGFR), and fibroblast growth factor receptor ((FGF) etc..

Epidermal growth factor receptor (EGFR) is also known as c-erbB1/HER1, whose family members are growth factor receptor tyrosine kinases, their cell surface with specific growth factors or natural ligand interactions, such as with EGF or TGF ci interactions, thereby activating the receptor tyrosine kinases. The first member of the family has been found to be a glycoprotein with apparent molecular weight of 165KD.

EGFR plays an important role in the regulation of tumor cell growth, repair and survival, angiogenesis, invasion and metastasis, and is expressed in a considerable number of human tumors. In many malignant tumors, the expression of EGFR is often associated with a poor prognosis and a low survival rate. Based on this, if there is a drug which can block EGFR activity, it will inhibit the phosphorylation and signal transduction, thus play an anti-tumor functions in multiple aspects, and increase the anti-tumor chemotherapy and radiotherapy treatment. In some studies, EGFR inhibitors show addictive and synergistic effects when used in combined treatment with various chemotherapy drugs and radiation therapy drugs for certain cancers.

EGFR inhibitors include monoclonal antibodies, tyrosine kinase inhibitors, quinazoline pyrrolo-/ pyrrolo-/pyridopyridines, ligand-toxin and immunotoxin complexes, as well as antisense oligonucleotides and EGFR/ligand mediated vaccines.

It was demonstrated in some in vivo and in vitro experiments that the anti-EGFR antibody can successfully inhibit the growth of EGFR-expressing tumor cell lines. In treatment of solid tumors, the results from some anti-EGFR monoclonal antibodies alone or their combination with traditional treatment methods are encouraging.

Glycosylation is a protein important post-translational modifications. Protein molecular surface sugar chains can have a profound impact on the structure and function of the protein molecules, glycosylation as an important post-translation process, has a great impact on proper proteins folding, localization, immunogenicity and biological activity. The glycosylation and glycan structure of mAb antibody have strong correlation with its function, by affecting the binding of IgG molecules to FcRs, Clq and FeRn to regulate the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and half-life of IgG molecules. Glycosylation also affects the safety features of mAb, particularly non-human glycans, and has potential immunogenicity. The glycans located in Fab functional region can affect both the safety and efficacy features of these drugs.

Glycosylation is highly dependent on cell expression system and subclone selection, and many factors during cell culture, for example medium components, culture conditions will affect glycosylation, thereby affecting the biological activity, efficacy, immunogenicity and pharmacokinetics of therapeutic proteins.

Among the therapeutic monoclonal antibodies currently marketed, the vast majority is produced by recombinant DNA technology, and the vast majority use in vitro cell culture technology. Because of the complexity of mammalian cell structure, function and gene expression regulation, there is a big difference between the expression of exogenous genes in mammalian cells and that in prokaryotes, consequently, the machinery for efficient expression of exogenous genes is also different from that for prokaryotes cells. Expression of exogenous gene in mammalian cells includes gene transcription, mRNA translation and post-translational modifications etc. Post-translational modifications include glycosylation, phosphorylation, oligomerization, as well as the formation of intra- or intermolecular disulfide bonds between protein molecules. Post-translational modification is crucial to the function of the protein, so it may be necessary to express certain proteins with biological functions in mammalian cells, such as membrane proteins, antibodies and enzymes having specific catalytic function. CHO cells and mouse myeloma cells (NS0, SP2/<NUM>) expression system has currently become the golden standard as cell engineering system for therapeutic antibody and Fc-fusion proteins. According to statistics, <NUM>% of currently approved therapeutic monoclonal antibodies are expressed in CHO cells, while <NUM>% are expressed in murine cells (<NUM>% NS0 cells, <NUM>% SP2/<NUM> cells, <NUM>% hybridoma cells). Although the integrity of polypeptide chains in different expression systems and culture conditions seems unchanged, the changes of glycosylation types cannot be ignored.

Cetuximab (Erbitux®, C225 mab), is a recombinant chimeric monoclonal antibody specifically targeting epidermal growth factor receptor (EGFR), and was approved in many countries for the treatment of metastatic colorectal cancer and head and neck squamous cell carcinoma. However, a number of studies have reported that the drug hypersensitivity reactions occur at very high incidence in clinical applications. Drug specific IgE antibodies were found in the serum of most patients with hypersensitivity reactions, and it specifically reacts against α-Gal. Further research found that, Erbitux® is expressed and prepared in mammalian cells (mouse myeloma cells SP2/<NUM>), and this murine cell line containing an additional α1, <NUM>-galactosidase transferase, which primarily mediates the transfer of galactose residue is from UDP-Gal of α conformation to the terminal galactose residues, thereby generating α-Gal. α-Gal is a harmful non-human disaccharide, found in certain glycans on mAb, especially mAb expressed in the murine cell lines. High levels of anti-α-Gal IgE antibodies were found in some patients. If using mAb with glycan containing α-Gal units for treatment, there will be serious hypersensitivity reactions. Further, the difference of murine cell IgG glycosylation from human is that, murine cells not only have the biosynthetic machinery to produce α-Gal epitope, but also produce N-hydroxyethyl neuraminidase (NGNA), rather than N-acetyl phenol neuraminidase (NANA). The distinction of NGNA and NANA is there is an additional oxygen atom in NGNA, and glycoproteins are considered to be closely associated with the immunogenicity in humans if they contain NGNA residues. Some marketed therapeutic glycoproteins have cause serious adverse reactions in the patients because they contains NGNA residues. <CIT> relates to a method for producing a protein molecule composition having a defined glycosylation pattern. <CIT> discloses production of cetuximab with a human glycosylation profile in a human myeloid leukemia cell line. Said antibody displayed low rates of adverse reactions in clinical studies.

In order to overcome the disadvantage of using SP2/<NUM> cells as the host cell to produce anti-EGFR monoclonal antibody, it's necessary to use a suitable host cell and optimize the culture conditions to reduce the differences between proteins expressed in cell culture and natural human proteins, so as to improve the drug safety for human.

The present inventors use CHO cells as host cells, culture cells in serum-free condition, successfully produce genetically engineered anti-EGFR antibody (CMAB009 mAb) with different glycan structures. Because this antibody does not contain the α-Gal glycan structure, it would not cause drug-specific IgE antibody-mediated hypersensitivity; there are no endogenous retrovirus particles in the engineered cells, there is no contamination in the antibody obtained from the cell culture of the engineered cells. The anti-EGFR monoclonal antibody prepared by this method has better clinical safety than Erbitux® mAb.

The present invention is defined by the appended claims; specifically, the present invention provides:.

Disclosed herein, but not forming part of the invention, is a method of producing an anti-EGFR monoclonal antibody, said method comprising:.

Wherein preferably:
The coding sequences for the light chain and heavy chain of the novel anti-EGFR monoclonal antibody are designed and synthesized according to the codons mostly preferred by Chinese hamster.

The host cell is Eukaryotic mammalian CHO cell.

The cell culture temperature is <NUM> ~ <NUM>, preferably <NUM>.

The pH of the cell culture growth media is <NUM>~<NUM>, preferably pH6.

The osmotic pressure of the cell culture growth media is <NUM> mOsm/kg~ <NUM> mOsm/kg, preferably <NUM> mOsm/kg.

The culture media is serum-free culture media, and the host cell is cultured in serum-free condition.

Also disclosed is a composition comprising the antibody of CMAB009, and a pharmaceutically acceptable carrier.

Also disclosed is a method of producing drug comprising the novel anti-EGFR monoclonal antibody of CMAB009 to treat tumors expressing epidermal growth factor receptor (EGFR).

Treating tumors expressing epidermal growth factor receptor (EGFR) with drug comprising the composition of CMAB009 and a pharmaceutically acceptable carrier.

Further comprising administering in combination with other drugs treating tumors expressing epidermal growth factor receptor (EGFR).

Also disclosed is a liquid pharmaceutical composition comprising water and an anti-EGFR antibody, wherein the anti-EGFR antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM> and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>, wherein the antibody has a z-average (z-avg) of about <NUM>-<NUM> as determined by dynamic light scattering (DLS) analysis, and wherein the anti-EGFR antibody does not comprise an N-glycolylneuraminic acid (NGNA), does not comprise a Gal-α(<NUM>,<NUM>)-Gal glycan, and/or does comprise a Gal-α(<NUM>, <NUM>/<NUM>)-Gal glycan.

Also disclosed is a composition comprising water and an anti-EGFR antibody as described above, wherein the z-avg of the antibody is <NUM>-<NUM>.

It is further disclosed that cancer can be treated, or the progression of cancer can be inhibited, in a human subject, by administering the composition described above.

In particular wherein:
The cancer is squamous cell carcinoma of the head and neck (SCCHN) or colorectal cancer. The colorectal cancer is K-Ras Wild-type, EGFR-expressing colorectal cancer.

The antibody as described above is administered in combination with FOLFIRI (irinotecan, <NUM>-fluorouracil, leucovorin).

The antibody as described above is administered in combination with irinotecan.

The subject has recurrent or metastatic squamous cell carcinoma of the head and neck and has failed prior platinum-based therapy.

The subject has locally or regionally advanced squamous cell carcinoma of the head and neck.

The antibody as described above is administered in combination with radiation therapy for the initial treatment of the cancer.

The subject has recurrent locoregional disease or metastatic squamous cell carcinoma of the head and neck.

The antibody as described above is administered in combination with platinum-based therapy with <NUM>-FU.

The antibody as described above is administered in combination with an additional therapeutic agent.

The additional therapeutic agent is a chemotherapeutic agent.

The subject has failed oxaliplatin and fluoropyrimidine-based chemotherapy.

Also disclosed is the treatment or inhibition of the progression of colorectal cancer in a subject having colorectal cancer, by administering an anti-EGFR antibody and irinotecan, such that colorectal cancer is treated, wherein the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>, comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>, and contains a Gal-α(<NUM>, <NUM>/<NUM>)-Gal glycan.

Also disclosed is the treatment or inhibition of the progression of colorectal cancer in a subject having colorectal cancer, by administering an anti-EGFR antibody and irinotecan, such that colorectal cancer is treated, wherein the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>, comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>, and does not contain either an N-glycolylneuraminic acid (NGNA) glycan or a Gal-α(<NUM>,<NUM>)-Gal glycan.

In particular wherein:
The colorectal cancer is advanced colorectal cancer.

The antibody as described above is administered via infusion to the subject at an initial dose of <NUM>/m<NUM> followed by a weekly dose of <NUM>/m<NUM>.

The antibody as described above is produced in a Chinese Hamster Ovary (CHO) cell.

Also disclosed is a liquid pharmaceutical composition comprising water and an anti-EGFR antibody, wherein the anti-EGFR antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM> and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>, wherein the anti-EGFR antibody is produced in a Chinese Hamster Ovary (CHO) cell, and wherein the composition does not comprise a polysorbate and/or a saccharobiose.

Also disclosed is a liquid pharmaceutical composition consisting essentially of water, an anti-EGFR antibody, sodium chloride, sodium dihydrogen phosphate dihydrate, and disodium phosphate dihydrate, wherein the anti-EGFR antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM> and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>, and wherein the anti-EGFR antibody does not comprise an N-glycolylneuraminic acid (NGNA) glycan, does not comprise a Gal-α(<NUM>,<NUM>)-Gal glycan, and/or does comprise a Gal-α(<NUM>, <NUM>/<NUM>)-Gal glycan.

The invention is based, at least in part, on the therapeutic advantages of producing an anti-EGFR antibody in Chinese Hamster Ovary (CHO) cells. CMAB009 is an anti-EGFR antibody that is produced in CHO cells and has the amino acid sequences of cetuximab. In comparison to Erbitux® (cetuximab), administration of CMAB009 to patients having cancer showed reduced immunogenicity reactions and improved efficacy, including an increase in the time in which the disease progressed.

As used herein, the term "cetuximab" refers to an anti-EGFR antibody having a light chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>, and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>. The sequences of the cetuximab light and heavy chains are described below:
<IMG>
<IMG>
<IMG>.

As used herein, the term "CMAB009" refers to a cetuximab antibody which is produced in a CHO cell. Thus, the CMAB009 antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM> and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: <NUM>. Further, the CMAB009 antibody does not contain either an N-glycolylneuraminic acid (NGNA) glycan or a Gal-α(<NUM>,<NUM>)-Gal glycan. The CMAB009 antibody does contain glycans associated with CHO cell expression, including, for example, a Gal-α(<NUM>, <NUM>/<NUM>)-Gal glycan.

As used herein, the term "in combination" when used in reference to administration of therapie,s refers to the use of two or more therapeutic agents, e.g., CMAB009 and irinotecan, to treat a disorder, e.g., metastatic colorectal cancer. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject with cancer. For example, a first therapy can be administered before (e.g., <NUM> minute, <NUM> minutes, <NUM> minutes, <NUM> minutes, <NUM> hour, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> week, <NUM> weeks, <NUM> weeks, <NUM> weeks, <NUM> weeks, <NUM> weeks, <NUM> weeks, or <NUM> weeks), concurrently, or after (e.g., <NUM> minute, <NUM> minutes, <NUM> minutes, <NUM> minutes, <NUM> hour, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> week, <NUM> weeks, <NUM> weeks, <NUM> weeks, <NUM> weeks, <NUM> weeks, <NUM> weeks, or <NUM> weeks) the administration of a second therapy to a subject who has had or has cancer. Any additional therapy can be administered in any order with the other additional therapies.

The invention is based on the use of CHO cells to produce an improved anti-EGFR antibody that is more effective and safer than anti-EGFR antibodies produced in, for example, myeloma cells. The glycosylation mechanism in CHO cells is very similar to the IgG glycosylation mechanism in human, earlier studies suggest that the CHO cells lack biosynthetic mechanism of α-Gal epitope-containing glycoprotein, recent studies have reported the presence of α1,<NUM> half galactosidase transferase gene in CHO cells, but it is at no or low expression state during the clone selection process, and it is unclear how this glycoside is α1,<NUM>-galactosidase transferase gene is activated in CHO cell line, presumably it associated with the transfection process, similar to other glycoside transferases. Based on this, we designed and selected CHO expression system and successfully prepared genetically engineered anti-EGFR antibody (CMAB009 mAb) with different glycan structures. By structure analysis, it was confirmed the Erbitux glycan contains a lot of α-Gal, and mostly NGNA as the terminal sialic acid, which has very high immunogenicity. CMAB009 mAb glycan does not contain α-Gal, and terminal sialic acid is mainly in the form of NANA. Subsequent clinical studies have confirmed that the antibody has a good tolerance, with no drug-related hypersensitivity observed, no IgE specific ADA detected. At the same time of greatly reduced immunogenicity, the characteristics of CMAB009 monoclonal antibody in vivo clearance is in line with the in vivo metabolic of chimeric antibodies, and the pharmacokinetic parameters are consistent with those of Erbitux®. CMAB009 monoclonal antibody has initially achieved significant clinical efficacy, and is expected to bring the greatest benefits to potential patients of with hypersensitivity.

Compared with Erbitux® monoclonal antibody, CMABOO9 monoclonal antibody has the same amino acid primary structure, while does not contain α-Gal, and the terminal sialic acid is mainly the common human sialic acid form of N-acetylneuraminic acid (NANA). This is consistent with the better tolerance we observed in clinical studies, while no drug-related hypersensitivity observed. At the same time of greatly reduced immunogenicity, the characteristics of CMAB009 monoclonal antibody in vivo clearance is in line with the in vivo metabolic of chimeric antibodies, and the pharmacokinetic parameters are consistent with those of Erbitux®.

This study demonstrates that it is effective to reducing the immunogenicity of monoclonal antibodies to prevent the occurrence of hypersensitivity, by modifying mAb glycosylation structure, while not affecting the biological activity and clearance characteristics of monoclonal antibody. This can reduce the incidence of clinical adverse reactions, and is expected to bring the greatest benefits to potential patients of hypersensitivity, and provide potential safe, tolerable and effectively targeting drugs.

The following examples of the present invention are described in further details.

Preferred codons of Chinese hamster were chosen for making most efficient eukaryotic expression vector, so as to obtain more efficient expression in Chinese hamster ovary expression system. Hamsters preferred codons are shown in Table <NUM>.

Signal peptide is selected from Chinese hamster B cell antigen receptor complex associated protein β chain. MATMVPSSVPCHWLLFLLLLFSGSS (SEQ ID NO: <NUM>), ATG GCC ACC ATG GTG CCC TCT TCT GTG CCC TGC CAC TGG CTG CTG TTC CTG CTG CTG CTG TTC TCT GGC TCT TCT (SEQ ID NO: <NUM>)∘.

Designed and synthesized according to the most preferred codons of Chinese hamster, the CMAB009 light chain comprises the nucleotide sequence of SEQ ID NO: <NUM> and the amino acid sequences of SEQ ID NO: <NUM>, the CMAB009 heavy chain comprises the nucleotide sequence SEQ ID NO: <NUM> and the amino acid sequence SEQ ID NO <NUM>. The said light chain and heavy chain above were ligated into the highly efficient Eukaryotic cell expression vector to obtain the light chain and heavy chain Eukaryotic expression vector.

In the biopharmaceutical field selection of host cells needs to focus on several important aspects: glycosylation and other post-translational modifications types to avoid causing immunogenicity; host cells suitable for large-scale cultivation in bioreactors, and can grow to high density in chemically defined and animal component free (ACDF) medium; virus safety; suitable for cloning and pressure screening in the ACDF.

CHO cell can grow at high density in bioreactors, is easy for genetic manipulation, has N-glycosylation similar to humans, lower the risk of virus transmission, and is widely used in the biopharmaceutical field. The most commonly used clone for industrial production is the CHO-K1, CHO-DXB11 and CHO-DG44. CHO-K1 is similar to the primary CHO cell, while DG44 and DXB11 were manipulated through random mutagenesis to remove DHFR gene, so they can be used for gene amplification via metabolic defects. CHO-K1 uses CS selection system, but has a lower screening efficiency because of the endogenous CS expression in CHO-K1.

The more widely used CHO cells were chosen as host cells which are more suitable for industrial production of therapeutic antibodies, and performed proper engineering of CHO-K1. We used CRTSPR/Cas techniques to knockout the CS gene of CHO-K1, and obtained cell line designated as CHO-CR-GS-/-, eliminating the expression of the endogenous CS, which is therefore more beneficial for screening of high expression cell clones.

Liposome based cotransfection of CHO-CR-GS-/-, screening under the pressure of CS selection system were performed to obtain stable cell clones with highly efficient expression of anti-EGFR monoclonal antibody. After several rounds of transfection and screening, cell clones were obtained with expressing amount greater than 20pg/cell.

We have developed universal basal medium for CHO-CR-G5-/-, which is chemically defined type of medium (Chemical Defined, CD), i.e. the medium is made by combining amino acids, vitamins, inorganic salt, glucose and trace elements according to cell growth needs and certain percentages. This basal medium can meet the initial growth needs of the engineered cells obtained from screening. In order to further improve the desired antibody yield from the engineered cells, optimizations were performed for the basal medium, including adding hormones, genetically engineered recombinant growth factors, adjusting amino acids amounts.

The culture PH is: <NUM> ~ <NUM>, preferably pH6. <NUM>; culture temperature is: <NUM> ~ <NUM>, preferably <NUM>; osmolality is: <NUM> mOsm/kg ~ <NUM> mOsm/kg, preferably <NUM> mOsm/kg.

After multiple comparisons and optimization, the culture (CHOM-B09) and supplemented medium (CHOM-S09) were ultimately determined suitable for the large scale serum-free culture of the engineered cells expressing anti-EGFR monoclonal antibody, with culture conditions: pH6. <NUM>, temperature <NUM>, and osmotic pressure of <NUM> mOsm/kg.

The expression yield of the engineered cells is greater than 30pg/cell. day in the optimized medium, using Fed-batch culture mode. The yield of the desired antibody may be greater than <NUM>/L in the culture supernatant harvested after <NUM> weeks of culture period.

The high expression clone obtained from the screening was cultured in expanded scale with serum-free culture medium, supernatant was collected, centrifuged at 9000rpm * <NUM>, <NUM>, pellet and the cell debris was discarded. concentrated by ultrafiltration using ultrafiltration packets of 50KB membrane from Millipore Corporation, then centrifuged at 9000rpm * <NUM>, <NUM> to remove cell debris, filtered with <NUM> membrane, used rProtein A (recombinant protein a) by affinity chromatography to do preliminary purification, in-situ wash buffer is <NUM> GuCl, the binding buffer for the column is <NUM> PB + <NUM> NaC1 pH7. <NUM>, after balancing with three to five column volumes, using three to five column volumes of elution buffer <NUM> Citric Acid (citrate buffer) pH3. <NUM> to elute. Column is stored in <NUM> % EtOH after equilibration and washing. The eluted desired protein from rProteinA was desalted and buffer exchanged using Hitrap G25 (GE Healthcare), the column elution buffer is PBS (<NUM> PB + <NUM> NaC1 pH7. <NUM>), in-situ washing solution is <NUM> NaOH. All of the above purification steps were performed on ice, the antibodies obtained from purification were concentrated with 50KD ultrafiltration centrifuge tubes (Merck Millipore) and to give CMAB009 monoclonal antibody.

Following purification, CMAB009 was characterized according to standard dynamic light scattering (DLS) analysis. It was determined that CMAB009 has a more homogenous size distribution in comparison to Erbitux. The z-average (z-avg) for Erbitux was determined to be <NUM>, while the z-avg. for CMAB009 was <NUM>. Furthermore, the polydispersity index ( PDI)of Erbitux was determined to be <NUM>, versus <NUM> for CMAB009. The characterization of CMAB009 versus Erbitux using DLS methods to determine size distribution was shown in <FIG>.

LC/MS, MS/MS techniques were used for the comparative analysis of the sugar chains of CMAB009 monoclonal antibody and Cetuximab (Erbitux®, C225 monoclonal antibody).

Sample preparation: Fc fragment and oligosaccharide from Fab were prepared after glucosidase digestion; oligosaccharides exonuclease treatment of oligosaccharides on Fab; <NUM>-AB fluorescence labeling of oligosaccharides; After HILIC solid phase extraction to remove excess <NUM>-AB, oligosaccharides were obtained with fluorescence labeled sugar chains, then analyzed via LC/MS and MS/MS chromatography.

The free glycans from glycosidase treatment of MAb, after fluorescent labeling, will be analyzed respectively by LC/MS, MS/MS and oligosaccharide exonuclease treatment. The results show that, CMAB009 antibody and the original antibody Cetuximab (Erbitux®) each have two glycosylation sites, with exactly the same glycan chain structure on their Fc segments, results in <FIG>. But Fab segments have different glycan chain structures, with mostly the sialic acid NANA glycan chain structure on CMAB009 Fab fragment, and mostly the sialic acid NGNA glycan chain structure on original Cetuximab Fab fragment; The glycans of CMAB009 Fab do not contain α-galactose, while the glycans of original Cetuximab Fab contain a large amount of α-galactose. LC/MS analysis of the glycan structure of the heavy chain Fab fragment is shown in <FIG>.

An initial study enrolled a total of <NUM> subjects, with <NUM>, <NUM>, <NUM> subjects each assigned to dose groups of <NUM>/m<NUM> dose, <NUM>/m<NUM> dose and <NUM>/m<NUM> dose, respectively, in the study of single intravenous administration. Among the subjects enrolled in single dose study, <NUM> subjects withdrew due to disease progression, according to the study design the remaining <NUM> subjects multiple administration inclusion criteria were enrolled in the multiple dose group meeting, with <NUM> extra subjects were enrolled to multiple dose (Table <NUM>)
<IMG>.

Subjects enrolled in this study were refractory to effective conventional treatment methods, experienced failure from conventional treatment or patients with relapse of advanced cancers, including <NUM> cases of colorectal cancer, <NUM> cases of lung cancer, <NUM> case of gastric cancer, the demographic statistical characteristics and prior treatment of the subjects are shown in Table <NUM>.

Comparison and analysis were performed on subjects' baselines, and the age, height, weight, body surface, ECOG score of subjects from the three groups of single dose and the two groups of multiple doses. The results are shown in Table <NUM> with no statistically significant difference.

The results showed that the CMAB009 monoclonal antibody was well tolerated. Among the <NUM> subjects, there was no grade III-IV drug-related toxicity as showed in Table <NUM>, with all occurring drug-related toxicity at grade I-II, and the incidence of toxicity was independent of the doses or the dosing frequency. No dose-limiting toxicity was observed, and no drug-related hypersensitivity was observed.

There was no CMAB009 antibody related hypersensitivity observed in this study, while the findings by Paula M. Fracasso and others indicated that the incidence of hypersensitivity reactions associated with Erbitux® reached <NUM>%, of which class III-IV hypersensitivity incidence is <NUM>%. Christine H. Chung and others conducted research on the hypersensitivity occurring in administering of original Erbitux® (<NPL>). Among <NUM> subjects who received Erbitux® treatment, <NUM> subjects had hypersensitivity, with hypersensitivity incidence reached <NUM>%, which is consistent with the results by Paula M. Christine H. Chung's study confirmed Erbitux® related hypersensitivity is α-Gal-specific IgE-mediated.

CMAB009 monoclonal antibody clinical safety: most adverse events were drug-related rash, there were no clinically significant new toxicity observed, and there no was severe hypersensitivity observed among the <NUM> subjects.

Immunogenicity is an important aspect in biopharmaceutical safety assessment. Traditional ELISA can be used for immunogenicity analysis, but the problem is, theoretically, the coated Fab segments for capturing antibody should be oriented to the optimal confirmation to facilitate the antigen-antibody interaction, the Fab fragments for capturing sometimes ae partially or entirely bound to microtiter plates, which results in the reduction of antibody capturing activity.

In this study, the biosensors made with biofilm interference technology and optical fibers were employed, in which the bottom was covered with SA ligands conjugated with biomolecule compatible layers. Once the captured biotinylated antibody is bound to the ligands, the biofilm thickness increases, reflected light interference spectral curve drift a measurable distance, thereby enabling real-time measurement of intermolecular interactions. This method is equivalent to the self-assembly process of the captured antibodies, which formed a range of optimal conformations at a certain density for capturing antibody on the surface of the biosensor, which not only improves the analytical sensitivity but also increases the linear range, which helps reduce the false-positive reactions from non-specific binding.

Fortebio Octet immunogenicity analysis: examination of ADA in clinical serum samples, the results shown in <FIG>, <FIG>: cut point value analysis showed that there were <NUM> subjects of potentially positive in <NUM> subjects (HPC highly positive, MIPC is positive, LPC is low positive, NC negative).

As to the immunogenicity analysis of CMAB009 monoclonal antibody in this study, the results showed that there was ADA detected in <NUM>% (<NUM>/<NUM>) of the subjects, with IgG type confirmed by subtype analysis, which are not the IgE type ADA mediated by hypersensitivity. The results of this study is consistent with the results of clinical safety evaluation, since there was no severe hypersensitivity reactions observed among subjects in clinical studies.

CMAB009 was administered to patients having metastatic colorectal cancer in a Phase <NUM>/<NUM> study to determine the efficacy and immunogenicity of CMAB009. As described below, the results from the study were then compared to similar studies performed using Erbitux® (cetuximab). Surprisingly, it was determined that CMAB009 has additional efficacy beyond that known for Erbitux®. For example, CMAB009 was able to increase the overall survival and length of time to disease progression in patients. The below study is comparable to the Erbitux® (cetuximab) study described in <NPL>.

The CMAB009 study was initiated by screening patients to identify those with <NUM>) histological confirmed metastatic colorectal adenocarcinoma, <NUM>) KRAS wild-type tumors, EGFR-expressing or EGFR-noexpressiong by immunohistochemistry, <NUM>)has measurable lesion, at least <NUM> in diametre by CT or MRI, at least <NUM> diameter by physical examination or other iconography, <NUM>) ECOG performance status <NUM> to <NUM>, <NUM>) failure (disease progression/discontinuation due to toxicity ) of fluoropyrimidine and oxaliplatin treatment, stop at least one month thereafter, irinotecan-naive. <NUM> patients were identified and randomized in a <NUM>:<NUM> manner to group <NUM> or group <NUM>. Group <NUM> included <NUM> patients who were administered a combination of CMAB009 and irinotecan. Specifically, the patients in group <NUM> were administered an initial dose of <NUM>/m<NUM> of CMAB009 followed by weekly infusions of <NUM>/m<NUM> thereafter. Irinotecan doses were maintained <NUM>/m2 every <NUM> weeks. Group <NUM> included <NUM> patients who were administered irinotecan monotherapy at a dose consistent with the patient's therapy prior to the study. Patients in both groups were treated until the disease progressed or the patient reached an unacceptable level of toxicity. Patient baseline characteristics are provided in Table <NUM>.

Patients were evaluated for radiologic response in both group <NUM> and group <NUM>. The results are described in Table <NUM>. Note the overall response rate (ORR) in Table <NUM> was determined according to the sum of the rate of CR and PR, and the disease control rate (DCR) was determined according to the sum of the rates of CR, PR, and SD.

When compared to data reported for Erbitux® (cetuximab) from a similar study (see <NPL>), patients receiving CMAB009 showed better overall survival (<NUM> months for patients receiving Erbitux® (cetuximab) + irinotecan vs. <NUM> months for patients receiving CMAB009 + irinotecan) and an incrased time to disease progression (<NUM> months for patients receiving Erbitux® (cetuximab) + irinotecan vs, <NUM> months for patients receiving CMAB009 + irinotecan).

When compared to reported data for Erbitux®, surprisingly the overall survival of the patients was greater in the patients receiving CMAB009, i.e., <NUM> months for Erbitux (cetuximab) + irinotecan vs. <NUM> months for CMAB009 + irinotecan. Data showing an increase in disease progression from this CMAB009 study is also provided in <FIG> (compare to published Erbitux (cetuximab) data; see <FIG> of <NPL>. Data showing an increase in overall survival from this CMAB009 study is also provided in <FIG> (compare to published Erbitux (cetuximab) data; see <FIG> of <NPL>).

A safety evaluation of the study is provided below in Table <NUM>.

Notably, adverse events from the CMAB009 study were lower than those reported for Erbitux® (cetuximab). The grade <NUM>-<NUM> adverse events are described below in Table <NUM> (compare to Table <NUM> of <NPL>).

Claim 1:
A method of producing an anti-EGFR monoclonal antibody, wherein the anti-EGFR monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: <NUM> and a heavy chain comprising the amino acid sequence of SEQ ID NO: <NUM>, said method comprising:
a) providing a nucleic acid encoding the light chain and the heavy chain of the anti-EGFR monoclonal antibody;
b) constructing recombinant plasmid using the nucleic acid of a), transfecting host cell, screening for a high-expressing clone; wherein said host cell is eukaryotic mammalian CHO cell; and
culturing the high-expressing clone in large scale to produce an anti-EGFR monoclonal antibody, isolating and purifying; wherein the cell culture growth medium is serum-free cell culture growth media and wherein said host cell is cultured in serum-free condition; the cell culture temperature is <NUM>, the pH of the cell culture growth media is <NUM> and the osmotic pressure of the cell culture growth media is <NUM>-<NUM> mOsm/kg; wherein the antibody obtained does not comprise an N-glycolylneuraminic acid (NGNA), does not comprise a Gal-α(<NUM>,<NUM>)-Gal glycan, and includes a Gal-α(<NUM>, <NUM>/<NUM>)-Gal glycan.