WO2008152537A2

WO2008152537A2 - Humanized monoclonal antibodies against human epidermal growth factor receptor, use and method thereof

Info

Publication number: WO2008152537A2
Application number: PCT/IB2008/052038
Authority: WO
Inventors: Mitali Samaddar; Kemburu Prasanna Kumar; Gosala Jaya Lakshmi; Chigurupati Jayaram
Original assignee: Zenotech Laboratories Limited
Priority date: 2007-06-14
Filing date: 2008-05-23
Publication date: 2008-12-18
Also published as: WO2008152537A3

Abstract

Humanized MAbs are designed, created and produced using a series of innovative processes comprising of computational analysis, modeling studies, rationale assessment and selection of humanized sequences, an efficient recombinant DNA expression system and functional studies to interrogate the therapeutic properties of the MAbs. The invention also provides an economic and scalable process of producing the monoclonal antibodies in large quantities and find use in the treatment of human diseases like cancer. Also described is a panel of novel humanized MAbs directed against the human EGFR that acts as receptor antagonists and are substantially non-immunogenic.

Description

HUMANIZED MONOCLONAL ANTIBODIES AGAINST HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR, USE

AND METHOD THEREOF

Field of the Invention

[1] The present invention is related to humanized MAbs directed against human EGFR that act as receptor antagonists and disrupts the ligand induced signal transduction pathway. The MAbs directed against human EGFR are created using a series of innovative approaches comprising of computational analysis, structure-based modeling studies, a predefined selection criteria, an efficient recombinant DNA expression system and functional interrogation of the MAbs for their therapeutic properties.

[2] Background of the Invention

[3] Therapeutic monoclonal antibodies (MAbs) have come a long way since its beginnings in 1980's and presently has evolved to become the most promising therapy for diseases like cancer and inflammation. To begin with, it was the pioneering work of Kohler and Milstein in 1975 that provided the method for creating murine hybridomas capable of secreting MAbs directed against specific antigen. Unfortunately initial attempts to use murine MAbs as therapeutic reagents failed miserably because of severe side effects caused due to immunogenicity and poor efficacy due to impaired ability to recruit immune effector mechanisms called the human anti mouse antibody (HAMA) response. The mouse monoclonal antibodies failed to elicit the effector functions in humans and more importantly substantial stretches of non-human amino acid sequences were immunogenic in humans. These drawbacks severely hampered further development of monoclonal antibodies as therapeutic molecules.

[4] This problem was overcome to some extent by generation of chimeric MAbs where genetic engineering was used to replace the constant regions of the murine heavy chain and light chain with human constant regions (Morrison, S. L. et. al. Chimeric human antibody molecules: Mouse antigen-binding domains with human constant. Proc. Naltl. Acad. Sci. USA 81, 6851-6855 (1984) and Boulianne, G. L. et. al. Production of functional chimaeric mouse/human antibody region domains. Nature 312, 643-646 (1984)). Chimeric MAbs did prove to be somewhat successful in the clinic, but the problem of immunogenicity still remained to some extent; called the human anti- chimeric antibody (HACA) response. In addition, other efforts to generate human hybridomas capable of secreting human MAbs against desired antigen remained unsuccessful. Major advances in genetic engineering and molecular biology have been utilized to produce MAbs with human framework regions combined with comple- mentarity determining regions (CDR's) from a donor mouse or rat immunoglobulin produced by hybridoma technology.

[5] It was in 1986 that Jones and his colleagues (Replacing the complementarity-determining regions in a human antibody with those from a mouse, Nature, 321, 522-525 (1986)) reported humanization of rodent antibodies through a technique known as Complementarity Determining Region (CDR) grafting. In this process, the antigen binding sites, which are formed by the three CDRs of the heavy chain and three CDRs of the light chain, are excised from the rodent MAb sequence and grafted into the DNA coding for the framework of a human antibody. This process made the MAbs more human like or humanized, thus reduced immunogenicity but more often led to loss of its binding affinity for the target antigen. This is highly undesirable for therapeutic MAbs as it means reduced efficacy of the drug.

[6] Thus this process has been further improved by molecular biologists to include changes referred to as 'reshaping' and 'veneering'. This technology was pioneered by Winter's group (Reshaping human antibodies for therapy, Nature 1988; 332: 323-337) that involved homology modeling and back mutations to retain binding affinities while reducing immunogenicity. Since then attempts have been made in the art to improve methods using altered strategies to produce humanized antibodies with increased affinity for the specific antigen and substantially low immunogenicity. These strategies does help to some extent but at the same time provides scope for improved and novel methods that enables creation and production of humanized MAbs that specifically react with the target antigens with high affinity, besides being substantially non- immunogenic in humans.

[7] The human Epidermal Growth Factor Receptor (EGFR and also called Erbl or Herl) belongs to the family of transmembrane, growth factor tyrosine kinase receptors that are important mediators of cell growth, differentiation and survival (Maria F. Structure and activation of EGF receptor. Endocrine Regulations 2002; 36, 87-93). The EGFR signaling pathway plays a crucial role through development and is found to be deregulated in many human cancers. This deregulation is predominantly attributed to EGFR being over-expressed in cancer cells and that is associated with more aggressive clinical behavior, bad prognosis and resistance to chemotherapy. Thus, interference with signaling through the EGFR pathway represents a highly attractive therapeutic approach with potentially broad clinical applications. In recent years EGFR has emerged as an important and viable target for therapeutic interventions in many types of cancer (Mendelsohn J. Targeting the epidermal growth factor receptor for cancer therapy. J Clin Oncol 2002; 20, 1-13 and Herbst RS, Shin DM. Monoclonal antibodies to target epidermal growth factor receptor-positive tumors; a new paradigm for cancer therapy. Cancer 2002; 94, 1593-1611). This growth factor receptor is being targeted clinically with both antibody and small molecule based approaches as investigations by various groups have shown strong evidence of aberrant EGFR signaling in cancer cells predominantly due to its over-expression. MAb-based approach has a unique advantage of exquisite specificity and thus ability to selectively target the cancer cells.

[8] Several murine monoclonal antibodies directed against the human EGFR has been described in the art (US Patent No. 4,943,533) that effectively prevents the endogenous ligands, Epidermal Growth Factor (EGF) and Transforming Growth factor a (TGF a ), from binding to EGFR and disrupts the signaling pathway. Also described in the art (WO 2004/085474 A2) is a chimeric MAb developed from the murine MAb directed against EGFR with therapeutic potential for solid cancers. However there is a need of efficient humanization of such MAbs to improve the overall efficacy of the drug by ensuring substantially low immunogenicity without any loss of affinity for the cognate antigen. Such humanized MAbs needs to be expressed in high levels by recombinant DNA technology in order for them to be developed and commercialized as therapeutic drugs. The present invention fulfills these needs in the art and in addition provides an efficient method of humanization and production of MAbs directed against any therapeutic target using recombinant DNA technology.

[9] Summary of the Invention

[10] The present invention discloses humanized monoclonal antibodies (MAbs) directed against the therapeutic target, human epidermal growth factor receptor (EGFR) using a series of rationale and innovative processes. The innovative processes comprises of a novel strategy to design humanized MAbs, a rationale approach based on predefined set of selection criteria to identify a subset of humanized MAbs for experimental evaluation and an efficient cloning and recombinant expression strategy to ensure high-level expression of recombinant humanized monoclonal antibodies in mammalian cells. In addition, the invention also provides methods to functionally interrogate the recombinant humanized MAbs for their therapeutic properties.

[11] More specifically, the invention provides novel humanized MAbs directed against

EGFR that are substantially non-immunogenic and binds to its cognate antigen with high affinity, prevents binding of endogenous ligands and disrupts the human EGFR signaling pathway.

[12] The strength of this invention lies in the series of innovative processes that are used for creating and producing humanized monoclonal antibodies. A new strategy and rationale has been applied to generate humanized antibody sequences for a murine MAb directed against human EGFR. Unlike the methods described in prior art where in the donor murine Complementarity Determining Regions (CDRs) are grafted onto the acceptor human framework regions that are selected using various approaches, this method uses the same parent murine backbone sequences where in the murine framework amino acid residues are substituted rationally with human amino acid residues using a profile of preferred amino acids at specific positions in the human variable region. This position specific amino acid preference profile is created from analysis of multiple sequence alignment of human variable regions available in the protein database. A similar position specific amino acid preference profile is also created for murine variable region so as to identify any highly conserved residues in the frame work regions.

[13] Each of the human preferred substitutions is carefully evaluated against their suitability to the local structural properties in the parent murine MAb structure such as location of phi psi angles of residues, packing interfaces of the secondary structural elements, interaction of residues with other residues of CDRs or with other residues in the antibody. From this analysis a panel of humanized sequences is derived and represents a spectrum of 'hard humanization' (with highest possible profile scores) to 'soft humanization' (with low profile scores). These humanized sequences are further evaluated for their structural compatibility using energy minimization followed by molecular dynamic simulations. The structures obtained at the end of the molecular dynamic simulations were compared with the parent structure in order to assess the extent of structural deviations.

[14] A subset of humanized sequences were selected based on rationale assessments of predefined criteria like contact interactions with its cognate antigen, potential new glycosylation sites, root mean square deviation (rmsd) values and percent identity to human sequences.

[15] Once designed, the humanized MAbs can be produced readily by recombinant DNA technology. This embodiment also describes recombinant expression and extensive characterization of the selected subset of humanized MAbs directed against the human EGFR. The DNA sequences encoding the selected subset of humanized MAbs are appropriately cloned into mammalian expression vectors. The coding sequences are optimized for high-level expression in mammalian cells like CHO (Chinese Hamster Ovary), BHK (Baby Hamster Kidney) or HEK (Human Embryonic Kidney). A gene amplification strategy and a fermentation process are employed to yield expression levels of about lg/litre of spent media. Extensive interrogation of the recombinant anti- EGFR humanized MAbs shows that they will be particularly useful as therapeutic antagonists for EGFR in treating cancers.

[16] Brief Description of the Figures

[17] Figure 1 describes the humanized sequences (variable regions of the light and heavy chains) selected for experimental evaluation.

[18] Figure 2 demonstrates SDS-PAGE analysis of purified MAbs under reducing and non-reducing conditions [19] Figure 3 demonstrates immunological identity of the recombinant humanized MAbs and their quantification.

[20] Figure 4a shows dose dependent binding of the MAb HZED 8 to purified EGFR

(erbl) when immobilized onto high-binding ELISA plates.

[21] Figure 4b shows dose dependent binding of HZED 8 to membrane bound EGFR

(erbl) present on A431 cells. The MAb do not bind to MDA-MB-453 cells known to be negative for erbl.

[22] Figure 4c shows specific binding of HZED8 to A431 cells and not to MDA-MB-453 cells by flow cytometric analysis.

[23] Figure 4d shows the ability of the MAb HZED 8 to inhibit binding of the endogenous ligand, human EGF to EGFRl present on A431 cells.

[24] Figure 5 shows the dose-dependent inhibition of proliferation of A431 cells and not of MDA-MB453 by the MAb HZED8.

[25] The invention will now be described with reference to the accompanying figures

[26] Detailed Description of the Invention

[27] In accordance with the present invention, novel humanized MAbs directed against human EGFR are provided that are substantially non-immunogenic, binds to its cognate antigen with high affinity, prevents binding of endogenous ligands and disrupts the human EGFR signaling pathway. The novel humanized MAbs are created using a series of rationale and innovative processes comprising of computational analysis, structure-based modeling studies, rationale assessment and selection of humanized sequences using predefined selection criteria, an efficient recombinant DNA expression system and studies to functionally interrogate their therapeutic properties. The invention also provides an economic and scalable process of producing the monoclonal antibodies in large quantities and find use in the treatment of human diseases like cancer.

[28] In order that the invention is more completely understood the following definitions are set forth. As used herein, the term 'antibody' is a protein consisting of two pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions together form the antigen binding region and the constant regions are responsible for antibody effector functions. An antibody light or heavy chain variable region consists of four 'framework' regions flanking the three 'CDR' regions. The extent of the framework regions and the CDR regions has been defined by Kabat et. al., 1983, Sequences of proteins of Immunological Interest. The amino acid sequences of the framework regions of various heavy and light chains are relatively conserved within a species and functions to structurally position the CDR regions that are primarily responsible for making specific contacts to the epitope of the antigen. [29] Chimeric antibodies are those that have been genetically engineered to have the constant region of the heavy and light chain from another species. For example, the constant regions of a murine antibody can be swapped with that of the human antibody to generate a Chimeric antibody.

[30] As used herein, the term humanized antibody refers to an antibody comprising of human framework regions flanking the CDRs from a non-human (murine) antibody. The constant regions are from human. Thus humanized antibody except for the CDRs is substantially identical to corresponding human antibody.

[31] As used herein, the term monoclonal antibody refers to an antibody that is directed against a specific epitope and is produced by a single B cell or a single hybridoma cell line, which is formed by the fusion of a lymphocyte cell with a myeloma cell.

[32] In 1986, Jones et. al., 'Replacing the complementarity-determining regions in a human antibody with those from a mouse.', Nature (1986); 321, 522-525, reported hu- manization of rodent antibodies through a technique known as Complementarity Determining Region (CDR) grafting. In this process, the DNA sequence coding for the three CDRs of the heavy and the light chain of a murine MAb are excised and grafted into the DNA coding for the framework of the human antibody. This technique had limited success primarily due to loss of binding affinity of the humanized MAb. When mouse CDRs are combined with human framework regions, the canonical structure of the CDRs may not be conserved due to interactions with the amino acid residues in the vicinity of the CDRs and thus, may fail to make effective contacts with the cognate antigen similar to those made by CDRs in the parent murine MAb. In addition, sometimes the amino acid residues in the murine framework regions may make contact with the antigen and contribute to the affinity. Such functionally important residues are lost when humanization is carried out by CDR grafting.

[33] The present invention uses a novel approach using a combination of computational analysis, structure-based modeling studies and rational analysis to generate a panel of novel humanized MAbs for the murine MAb M225 (ATCC No. HB-8508) directed against human EGFR, clone and express the same at high levels in mammalian cells using recombinant DNA technology. The first step in the method comprises of determining the structure of the murine MAb using comparative modeling approach. Any standard software like Modeller can be used to determine the structural templates and perform the modeling studies. It is also possible that in some cases that the X-ray crystal structure data is available in the public domain and in such cases the crystal structure can be used for the study. The second step in the method is to identify key residues for substitutions in the murine MAb. This is done by first generating a profile of position specific preferred amino acid residues for human variable region. This profile is aligned with the murine sequence to identify the key residues for substitution. Each of the human preferred substitutions is carefully evaluated for their structural properties in order to allow substitutions of the identified residues in the murine sequence. Finally the structural stability of the humanized sequences was tested using energy minimization studies and molecular dynamic simulations.

[34] The present embodiment also describes a method to assess and evaluate the humanized MAbs based on a set of predefined criteria. Modeling studies are carried out to ensure that the selected humanized sequences retained contact interactions with its cognate antigen. This assessment eliminates the humanized sequences that directly or indirectly abrogate the binding interactions. The humanized sequences are scanned for any new potential N-linked or O-linked glycosylation sites. The humanized sequences predicted to have any new glycosylation sites are eliminated as such sequences may have altered binding properties. In addition, humanized sequences with minimum root mean square deviation values with reference to the parent structure and also those showing maximum identity to human consensus sequences are selected. Thus a subset of humanized sequences are selected to be carried forward for experimental evaluation based on specific criteria that are important for retaining substantially high binding affinity and also being substantially non-immunogenic.

[35] The present embodiment also describes an efficient method of expressing the humanized MAbs in an appropriate mammalian cell host or any other host capable of expressing MAbs in a biologically active form. The DNA sequences encoding the heavy and light chains of the humanized MAbs fused in-frame with appropriate signal sequences are extensively interrogated and designed to achieve high levels of the recombinant MAbs. A gene amplification strategy and a fermentation process are employed to yield expression levels of about lg/litre of spent media. Extensive interrogation of the recombinant anti-EGFR humanized MAbs shows that they will be particularly useful as therapeutic antagonists for EGFR in treating cancers.

[36] The method of producing humanized antibodies as described in this invention can also be used to humanize a variety of non-human antibodies. Such humanized antibodies can be used as therapeutic molecules and also for diagnostic purposes.

[37] Example 1

[38] Designing of Humanized MAbs

[39] This example describes the procedure followed to design the humanized monoclonal antibodies using the parent murine MAb directed against human EGFR.

[40] a) Determination of three dimensional structure of the murine MAb

[41] Structure-based comparative modeling approach was used to determine the three dimensional structure of the Fab fragment of the murine MAb M225. The structural templates were identified and modeling was carried out using the software MODELLER. [42] b) Identification of key residues for substitutions in the murine MAb

[43] The key amino acid residues in the murine framework regions were identified and rationally substituted with more human like residues. The profile of the human MAb variable region was calculated from the analysis of multiple sequence alignments of the variable regions of known human MAbs from the protein database. The profile was then aligned to the variable regions of the murine MAb. This analysis identified the amino acid residues in the murine framework region that were to be rationally substituted with human preferred residues.

[44] c) Selection of residues for substitutions

[45] Even though the previous exercise suggested key residue substitutions all can not be considered for substitutions owing to the fact that they may not be conducive for the three dimensional structure of the murine MAb. For example proline may be the most preferred residue by the human at say position 10 but the murine structure can not accommodate proline because at that position conformation is not suitable for the placement of proline. Therefore, each of the human preferred substitutions were carefully weighed against their suitability to the local structural properties in the murine structure such as, location of residue within the secondary structure, spatial arrangement of residues i.e., phi psi angles of residues, packing interfaces of the secondary structural elements, interactions of residues with other CDR's or with other residues in the antibody.

[46] d) Modeling studies for rational substitutions of amino acid residues

[47] Based on the analysis described above, amino acid substitutions were made with most reasonable human preferred residue in the three dimensional structure of the murine MAb. As the combinations may be many, choices were made such that they represent a spectrum from 'hard humanization' (with highest possible profile scores) to 'soft humanization' (with low profile scores but potentially not causing any minimal disruption to the murine MAb structure). Although the hard and soft humanizations were clearly identified the choice of sequences within the range was a subjective selection.

[48] e) Testing of the modeled humanized sequences for structural compatibility

[49] The structural models of the humanized sequences were subjected to energy minimization followed by molecular dynamics simulations. This enabled to select sequences that were structurally compatible. The structures obtained at the end of the molecular dynamic simulations were compared with the parent murine MAb structure in order to assess the extent of structural divergence which might have occurred as a consequence of human preferred amino acid substitutions made at key positions.

[50] Example 2

[51] This example provides the important criteria based on which a subset of humanized MAbs were selected for experimental evaluation [52] 1. a) Contact interactions with its cognate antigen

[53] Modeling studies were carried out to select humanized sequences that retained the contact interactions with its cognate antigen.

[54] 1. b) Potential new glycosylation sites

[55] The humanized sequences were scanned for absence of any new potential N-linked or O-linked glycosylation sites.

[56] 1. c) Root mean square deviation (rmsd) values

[57] The structural superposition of the models for humanized sequences with the parent murine MAb structure enabled assessment of structural deviations from the parent structure. The humanized sequences with least rmsd values were selected for further evaluations.

[58] 1. d) Percent identity to human consensus sequence

[59] The percent identity to the human consensus sequence for the framework regions were also used as one of the criteria for selecting the subset of humanized sequences for experimental evaluation.

[60] Figure 1 describes the humanized sequences (variable regions of the light and heavy chains) selected for experimental evaluation.

[61] Example 3

[62] This example describes the method used in the present embodiment for design of the

DNA sequence, cloning and expression of the humanized MAbs directed against human EGFR.

[63] The DNA coding sequence encoding the variable region of the heavy and light chains of the humanized sequences were analyzed and adapted to codon usage in mammalian cells besides avoiding regions of very high (> 80%) or very low (< 30%) GC content. Also during the optimization process other cis-acting sequence motifs like internal TATA-boxes, chi sites, ribosomal entry sites, repeat sequences, RNA secondary structures, ARE (autosomal replicating sequences), INS (inhibitory sequences) or CRS (cis-acting repressive sequence) elements, cryptic splice donor and acceptor sites or branch points are avoided. Such gene optimized sequences are cloned in-frame with their respective human constant regions also optimized using the same procedure as described for the variable regions. More specifically the heavy chain variable region is cloned in-frame with the gamma 1 constant region and the light chain variable region is fused in-frame with the kappa constant region. The human gamma 1 isotype is chosen as it has been found to be the preferred human isotype for supporting antibody dependent cellular toxicity (ADCC) and complement dependent cytotoxicity (CDC) (Riechmann, L et al., (1988) Nature 332:323-327). A signal sequence encoding a signal peptide is fused to the N-terminus of the heavy and light chain coding sequences. The signal peptides have specific cleavage sites that are processed during secretion of the humanized MAbs from the host cell.

[64] The assembled coding sequences for the heavy and light chains of the humanized sequences are cloned into a mammalian expression vector containing antibiotic selection markers for bacterial host and the mammalian host. Alternatively the same selection marker can be used for both prokaryotic and the eukaryotic host. In addition the expression vector also contains a selection marker for gene amplification once integrated into the host genome.

[65] Appropriate mammalian host like Chinese Hamster Ovary (CHO) cells, Baby

Hamster Kidney (BHK) cells and Human Embryonic Kidney (HEK) cells are transfected with the constructs containing the humanized sequences for heavy and light chains. A sequential stepwise protocol is used to amplify the loci containing the expression cassettes for high level expression of the humanized MAbs. For each of the humanized sequences stable clones, adapted to serum free media and expressing high levels of the MAbs were carried forward for further investigations. The antibodies were purified from the spent media by protein A affinity chromatography. The eluted antibody was buffer-exchanged into phosphate buffered saline, concentrated, sterile filtered and stored at 4 ° C. The protein A affinity purified MAbs gave a single band of about 150 kDa, as expected, when analyzed by SDS Polyacrylamide Gel Electrophoresis (PAGE) under non-reducing conditions and stained with Coomassie blue dye. The MAbs when analyzed by SDS-PAGE under reducing conditions and stained using Coomassie blue dye showed a band of about 50 kDa as expected for the heavy chain and a second band on about 25 kDa as expected for the light chain.

[66] Figure2: SDS analysis of purified MAbs under reducing and non-reducing conditions

[67] Example 4

[68] This example provides methods to interrogate the functional properties of the recombinant humanized MAbs directed against the extra cellular domain of human EGFR.

[69] a) Immunological identity and quantification

[70] The mammalian cell derived humanized MAbs were shown to specifically bind to anti human IgG Fab fragment specific antibody and also to anti-human IgG Fc gamma fragment that is used for detecting the bound MAbs in a sandwich ELISA. Human IgG is used as the reference standard for quantification of the MAbs (Figure 3).

[71] b) Binding characteristics

[72] The recombinant humanized MAbs could specifically bind to purified EGFR (erbl) from human carcinoma A431 cells shown using ELISA and also directly to membrane bound EGFR (erbl) present on A431 cells shown using cell based assay and flow cytometry. The human breast cancer cell line MDA-MB-453 known to be negative for erbl is used as a negative control in the experiment. As expected the recombinant humanized MAbs does not bind to MDA-MB-453 cells as shown by cell based assay and also flow cytometry. The binding of the humanized MAbs to the EGFRl receptors present on A431 cells also inhibits binding of the endogenous ligand human EGF.

[73] The EC50 values (101 ng/ml for HZED8 and 91 ng/ml for 225) for the receptor binding experiment (Fig. 4b) and IC50 values (0.26 nM for HZED8 and 0.29 nM for 225) for the ligand inhibition assay (Fig. 4d) are comparable for HZED8 and the reference anti-EGFR MAb 225. More over the data also shows that the MAb HZED8 is highly specific and does not have crossreactivity with a cell line that is negative for the EGFRl receptor.

[74] Figure 4a shows dose dependent binding of HZED8 to purified EGFRl when immobilized onto high-binding ELISA plates.

[75] Figure 4b shows dose dependent binding of MAbs to membrane bound EGFR (erbl) present on A431 cells. The MAbs do not bind to MDA-MB-453 cells known to be negative for erbl.

[76] Figure 4c shows specific binding of the MAbs to A431 cells and not to MDA-

MB-453 cells by flow cytometry analysis.

[77] Figure 4d shows the ability of HZED8 to inhibit binding of the endogenous ligand human EGF to EGFRl present on A431 cells.

[78] c) Cell growth inhibition

[79] The ability of the recombinant humanized MAbs to inhibit cell proliferation of erbl positive cells (A431) and not for erbl negative cell line MDA-MB-453 showed that HZED 8 is capable of specifically disrupting the EGFRl signaling pathway and inhibiting cell proliferation.

[80] Figure 5 shows the dose-dependent inhibition of proliferation of A431 cells and not of MDA-MB453 by recombinant humanized MAbs. The EC50 value for HZED8 is 128 ng/ml and is comparable to that for the reference MAb 225 (116 ng/ml). The experiment also shows specificity of HZED8 in terms of its biological activity as it fails to inhibit proliferation of the MDA-MB-453 cells.

Claims

Claims[1] We claim:

1. A method of preparing substantially non-immunogenic humanized monoclonal antibodies comprising the steps of

- creating a profile of human preferred amino acids in the variable region,

- substituting rationally the non-human antibody framework amino acid residue with human amino acid residues using said profile of preferred amino acids at specific positions in the human variable region,

- evaluating said human preferred substitutions against their suitability to the local structural properties in the parent non-human antibody structure, and

- producing recombinant monoclonal antibodies containing said suitable substitutions.

2. The method of preparing substantially non-immunogenic humanized monoclonal antibodies as claimed in claim 1 wherein said profile of human preferred amino acids in the variable region is created by analysis of multiple sequence alignment of human variable regions available in protein database.

3. The method of preparing substantially non-immunogenic humanized monoclonal antibodies as claimed in claim 1 wherein said humanized sequences are evaluated for their structural compatibility to the parent non-human monoclonal antibody using energy minimization and molecular dynamics studies.

4. The method of preparing substantially non-immunogenic humanized monoclonal antibodies as claimed in claim 1 wherein said humanized sequences are evaluated for retaining key contact interactions with its cognate antigen, absence of any potential new glycosylation sites, minimum rmsd values with reference to the parent structure and maximum identity to human consensus sequence.

5. The method of preparing substantially non-immunogenic humanized monoclonal antibodies as claimed in claim 1, wherein the step of producing recombinant monoclonal antibodies is by humanized DNA sequences optimized for expression and appropriately cloned into suitable expression vectors carrying a selection and a gene amplification marker.

6. The method of preparing substantially non-immunogenic humanized monoclonal antibodies as claimed in claim 4 wherein said cloned humanized sequences are expressed in large quantities in a suitable host like mammalian cells or yeast.

7. The method of preparing substantially non-immunogenic humanized monoclonal antibodies as claimed in claims 1-6 wherein said non-human antibody framework is that of murine origin.

8. The non-immunogenic humanized monoclonal antibodies whenever prepared by the method as claimed in claims 1-7.

9. Humanized monoclonal antibodies directed against human epidermal growth factor receptor EGFR that acts as receptor antagonists and disrupts the human EGFR signaling pathway said antibody being substantially non-immunogenic and optionally capable of acting as receptor antagonists for cancer therapy.

10. Humanized monoclonal antibodies directed against human epidermal growth factor receptor EGFR that acts as receptor antagonists and disrupts the human EGFR signaling pathway as claimed in claim 9 wherein said antibody light chain sequence is selected from SEQ ID: HZED-7A, HZED-8A AND HZED- 13A.

11. Humanized monoclonal antibodies directed against human epidermal growth factor receptor EGFR that acts as receptor antagonists and disrupts the human EGFR signaling pathway as claimed in claim 9 wherein said antibody heavy chain sequence is selected from HZED-7B, HZED-8B AND HZED- 13B.

12. Humanized monoclonal antibodies directed against human epidermal growth factor receptor EGFR that acts as receptor antagonists and disrupts the human EGFR signaling pathway as claimed in claim 9 wherein said antibody structure is that of HZED7, HZED8 and HZED13.

13. A method of preparing substantially non-immunogenic humanized monoclonal antibodies directed against human epidermal growth factor receptor EGFR that acts as receptor antagonists and disrupts the human EGFR signaling pathway as claimed in claim 9 comprising the steps of

- creating a profile of human preferred amino acids in the variable region of the antibody,

- substituting rationally the non-human antibody framework amino acid residue of antibody against EGFR with human amino acid residues using said profile of preferred amino acids at specific positions in the human variable region,

14. The method of preparing substantially non-immunogenic humanized monoclonal antibodies directed against human epidermal growth factor receptor EGFR as claimed in claim 13 wherein said non-human antibody framework is that of murine origin.

15. The non-immunogenic humanized monoclonal antibodies directed against human epidermal growth factor receptor EGFR prepared by the method as claimed in claim 13 and 14.

16. The Humanized monoclonal antibodies as claimed in claim 15 wherein said recombinant humanized anti-EGFR monoclonal antibodies are capable of acting as receptor antagonists for cancer therapy.

17. Method of preparing substantially non-immunogenic humanized monoclonal antibodies substantially as herein described with respect to accompanying examples and drawings.

18. Humanized monoclonal antibodies directed against human epidermal growth factor receptor EGFR that acts as receptor antagonists and disrupts the human EGFR signaling pathway and method of preparing the same substantially as herein described with respect to accompanying examples and drawings.