Title of Invention

A RECOMBINANT MOUSE-HUMAN CHIMERIC FAB AGAINST HEPATITIS B SURFACE ANTIGEN

Abstract

This invention relates to a recombinant chimeric Fab antibody comprising a recombinant Fd having seq id# 1 and a recombinant chimeric light chain having seq id# 2 which binds to Hepatitis B surface antigen (HBsAg) with high affinity {KD= 8.166 ± 0.14 nmol/L), generated by fusing heavy chain variable region (VH) of anti-HBsAg antibody 5S having seq id# 3 with first constant region (CHI) of human IgGl and light chain variable region (VL) of anti-HBsAg antibody 5S having seq id# 4 with constant region (CL) of human kappa chain, wherein mouse VL and human CI are linked by overlap PCR to generate chimeric light chain and mouse VH and CH1 are linked by overlap PCR to generate chimeric Fd using such primers as herein described. Supportive of their active, and folded nature and stabilize the same. This is through the sh groups present on the amino acid present on the amino acid entertain disulphide bond molecules. There are numerous cysteins in each of the chains, most form intra chain disulphide bond. However there is only

Full Text

Field of Invention; This invention relates to the generation of new recombinant mouse-human chimeric Fab antibody, which binds to the hepatitis B surface antigen with high affinity. Prior Art; Protective antibodies that appear after natural infection are mostly directed against the major antigenic 'a' determinant of Hepatitis B surface antigen (HBsAg). The immunodominant 'a' epitope is a part of a large antigenic area of HBsAg, called the major hydrophilic region and this epitope is present in all serotypes. Antibodies against HBsAg are thus advocated for passive immunotherapy against Hepatitis B infection in cases of accidental needle stick injuries, for liver transplant patients etc. Presently, human anti-HBs immune globulin (HB1G) collected from the blood of hyper immune donors is used for post exposure prophylaxis. This blood derived product is not manufactured in India. Being a blood derived product, anti-HBs HB1G is costly and can cause cross contamination. Few anti-HBsAg mouse monoclonal have been reported in literature. However such mouse antibodies are not suitable for therapeutic uses as they may generate immunogenic reaction. Several recombinant anti-HBsAg antibody fragments have also been reported in literature. However, most of them are of mouse origin and are not available for therapeutic uses. It is well documented that immunogenic reactions are predominantly directed towards the Fc region of murine antibodies. It has also been observed that creation of mouse-human chimeric antibodies, by swapping murnie constant regions with the human ones, can reduce the immunogenicity of an antibody. Such mouse-human chimeric molecules are safer for therapeutic uses and currently, several such chimeric molecules are in clinical uses for different diseases. Five different recombinant anti- HBsAg Fab antibodies have been reported in literature. They are all unique in nature and are different from the recombinant molecule of this invention. Objects of the Invention; The object of the invention is to generate a recombinant mouse-human chimeric antibody fragment, against Hepatitis B surface antigen. Other objective is to develop a recombinant mouse-human chimeric Fab antibody which can bind to Hepatitis B surface antigen with high affinity and specificity. Further objective is to show that the original monoclonal from which it is derived has binding characteristics that are very conducive to bio neutralization. Another objective is to produce a recombinant mouse-human chimeric Fab antibody which would have less antigenicity as compared to the mouse monoclonal. Yet another objective is to produce a recombinant mouse-human chimeric Fab antibody which would be more suitable for invivo use in humans. Other objective is to generate mouse-human chimeric antibody which can be safer and cheaper alternative. Further objective is to develop the recombinant molecule which can also be used with/without any modification/in combination with other molecules for generation of complete full length antibody bispecific antibodies, diabodies or any other modification of a protein molecule with containing the described Fab. Yet another objective is also to develop immuno conjugates, like conjugated with enzymes, ligands, receptors, drugs, radio isotopes, toxins or any other large or small molecule for invivo or invitro use. Detailed Description of the Invention: At the outset of the description that follows, it is to be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only an exemplary embodiment and is not intended to be taken restrictively to imply any limitation on the scope of the present invention. There are many well known methods to generate recombinant chimeric antibodies. However, every recombinant chimeric antibody is unique in nature, if and only if (a) its molecular structure as defined by the amino acid sequence is unique (b) has unique biological function as defined by specificity and affinity for the target antigen. The recombinant molecule of this invention is unique as no other molecule matches the structure and properties of this molecule. This novel molecules can be generated by many other well documented strategies too. Other recombinant molecule may have the same function as they bind the hepatitis B surface antigen but each molecule with different sequences has unique binding characteristics in terms of epitope specificity ie the precise sequence of antigen it binds to and the molecular interactions for doing the same and affinity. Here, the inventors generated a reqombinant chimeric Fab antibody, composed of a recombinant Fd and a recombinant chimeric light chain, which binds to Hepatitis B surface antigen with high affinity. Only the Fab and not the individual light and heavy chains can bind to Hepatitis B surface antigen and thus be useful for virus neutralization. Inventors have used the antibody genes from a mouse monoclonal (named as 5S), for generation of recombinant antibody fragments against Hepatitis B surface antigen. This mouse antibody binds to the immunodominant 'a' epitope of the hepatitis B surface antigen and found to be protective in a surrogate in vitro assay. This mouse monoclonal was generated using existing protocol for generation of hybridomas. However this mouse monoclonal cannot be used directly in human subjects, as it will trigger human anti-mouse immunogenic response. To reduce the immunogenicity of the antibody, inventors have generated a mouse-human chimeric Fab that retains the high affinity binding to HBsAg. Variable region genes (VH and VL) of the anti-HBs mouse monoclonal 5S were amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR). Similarly constant region of human kappa chain (CK) and first constant region of human IgGl heavy chain (CH1) were amplified using human peripheral blood lymphocytes (PBLs) as the source of antibody genes. Mouse VL and human CK were linked by overlap PCR to generate chimeric Fd. Both the chimeric antibody genes were then cloned into a bicistronic bacterial expression vector (pCOMB3H, Scripps Research Institute, La Zola, USA) to express the chimeric Fab in the periplasm of E.coli (XL1-blue). The chimeric Fab so expressed in bacterial periplasm showed high binding to HBsAg and competitive ELISA confirmed that it binds to the same epitope as that of the original mouse monoclonal. The apparent dissociation constant of the chimeric antibody was found to be close to that of the original mouse monoclonal (4.575nM). This chimeric anti-HBs Fab have been cloned and expressed in such a fashion that it can be further manipulated to generate a complete chimeric antibody by fusing the chimeric Fd with human Fc fragment. Therefore this chimeric Fab, which has unique mouse variable regions fused to human constant domains can be the starting material for generation of a therapeutically functional full-length recombinant antibody against Hepatitis B surface antigen. Being chimeric in nature, it is expected to be less immunogenic than a mouse antibody and as generated by recombinant means it can be safer and cheaper that the currently used human polyclonal antibody. Inventive steps; 1. Amplification of the variable region genes (VH and VL) of the anti-HBs mouse monoclonal 5S by reverse transcription (RT) followed by polymerase chain reaction (PCR). RNA isolated from 5S hybridoma was used as the source of these two genes. 2. Amplification of constant region of human kappa chain (Ck) and first constant region of human IgGl heavy chain (CH1) by reverse transcription (RT) followed by polymerase chain reaction (PCR). RNA isolated from human peripheral blood lymphocytes (PBLs) was used as the source of these two genes. 3. Mouse VL and human CK were linked by overlap PCR to generate chimeric light chain. Similarly mouse VH and CH1 were linked by overlap PCR to generate chimeric Fd. 4. Both of these chimeric antibody genes were cloned into a bicistronic bacterial expression vector (pCOMB3H, Scripps Research Institute, La Zola, USA) to express the chimeric Fab in the periplasm of E.coli (XLl-blue). 5. Recombinant clone was cultured in suitable medium and cell extract was prepared by the conventional method. 6. The recombinant chimeric antibody was purified by affinity chromatography using Protein G. This recombinant molecule was generated by fusing variable region genes (VH and VL) of an anti-HBsAg mouse antibody 5S and constant region genes of human (CHl region of human IgGl and the CL region of human kappa chain). The over all scheme of the invention is shown in Figure 1. Cloning and generation of the recombinant construct; The strategy for generation of the chimeric Fab is shown in Figure 2. The variable region genes of 5S hybridoma were amplified by reverse transcription followed by PCR. Primers used for all reverse transcriptions and PCRs are listed in Table 1. Total RNA was isolated from 5S cells using standard protocol and cDNAs for the VH and VL fragments were generated by reverse transcription. Primers used for reverse transcriptions were Fd3 and K3 for VH and VL respectively. The VH fragment was further amplified by PCR using primers 5H23M and Fd3. Similarly the VL fragment was amplified by PCR using primers 5L35 and K3. Conditions for both these PCRs were: 30 cycles at 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, followed by a final extension for 10 min at 72° C. For amplification of human constant domains, RNA was isolated from human peripheral blood lymphocytes (PBLs). cDNAs for the CH1 region of human IgGl and the CL region of human kappa chain were generated by reverse transcription. Primers used for reverse transcriptions were CG1Z and Ckld for CH1 and CL respectively. The CH1 fragment was further amplified by PCR using primers Fd5 and CG1Z. Human CL was amplified by PCR using primers K5 and CKld. Fd5 and K5 carry overhangs complementary to the 3' end of variable regions of 5S. conditions for both these PCRs were: 30 cycles at 94° C for 1 min, 60° C for 1 min, 72° C for 2 min, followed by a final extension for 10 min at 72° C. PCR amplified fragments were resolved in 1.5% agarose gel and respective bands were eluted out using standard protocol. PCR amplified CH1 (human IgGl) and mouse VH were used as templates for generation of the chimeric Fd by overlap PCR. Intitial assembly of equimolar amounts of the mouse VH and human CH1 was done by PCR for 20 cycles at 94°C for 1 min, 60° C for 1 min and 72° C for 2 min, followed by final extension of 10 min at 72°C. The product of the initial assembly reaction was diluted 10 times and used as the template for pull through PCR using primers 5H23M and CGIZ. Similarly human kappa CL and mouse VL fragment were joined to generate the chimeric light chain. Primers used for this reaction were 5L35 and CKld. The chimeric Fd was digested with Xhol and Spel and cloned into the phagemid vector pCOMBSH. The resulting construct (pCOMB3H-Fd) was transformed into chemically competent E.coli XLl-Blue cells by standard chemical method (Cacl2/heat shock). Transformed cells were grown on Ampicillin-Agar plates. Colonies were picked up after overnight incubation and screened for the presence of the insert by colony PCR and restriction digestion (Xhol/spel). The recombinant phagemid pCOMBSH-Fd was isolated by alkali-lysis method and digested with Sacl and Xbal. The chimeric light chain digested with these two enzymes was cloned into pCOMB3H-Fd to generate the phagemid construct pCOMB3H-Fd-L. This construct was transformed into chemically competent XL 1-Blue cells by standard chemical transformation method and after overnight incubation, recombinant clones were checked for the presence of the chimeric light chain by colony PCR and by restriction digestion (Sac 1/Xbal). Expression of the recombinant chimeric Fab; For soluble expression of the chimeric Fab, the phage gIII gsequence was removed from the recombinant phagemid construct pCOMB3H-Fd-L by double digestion with spe I and Nhe I. Double digested phagemid was self-ligated to generate the construct pCOMB3H-Fd-L-Sol and transformed in chemically competent XLl-Blue cells. For soluble expression of recombinant chimeric Fab, 1 L Super Broth with 20 mmol/L MgCl2 and Ampicillin (100 µg/ml) was inoculated with 10 ml of overnight culture of the recombinant clone and grown at 37° C till the A600 reached approximately 0.6, when over-expression of the chimeric Fab was induced by 1 mmol/L IPTG. After overnight growth at 30°C, cells were harvested by centrifugation at 4000 g. Cell pellet was re-suspended in 20 ml PBS/lmmolL ED/TA and kept on ice for 40 min. Clarified periplasmic extract was obtained by centrifugation of the re-suspended product at 10000 g. Purification of the recombinant chimeric Fab; The recombinant chimeric Fab was purified from the periplasmic extract by affinity chromatography using Protein G HP column. In brief, the protein G column (1ml) was washed thoroughly with double distilled water (5 column volumes) and equilibrated with 5 column volumes of equilibration buffer (pH 7.0). The periplasmic extract was allowed to pass through the column using a syringe at a sped of 2 ml/min. The column was washed thoroughly by 10 volume of equilibration buffer and bound chimeric Fab was eluted out by 5 volume of elution buffer (pH 2.7). Eluted fractions were immediately neutralized using neutralization buffer (77 µl/ml, pH 9). The eluted fractions were checked on SDA-PAGE. Assays of the recombinant chimeric Fab; The purified chimeric Fab was resolved by 12% SDS-PAGE, separately in reducing and non-reducing conditions. The resolved protein was stained by silver staining. As shown in Figure 3, the chimeric Fab is expressed as a heterodimer (~50 kD) of the chimeric Fd and light chain. In reducing conditions, both the chains were detected in monomeric form (~25 kD). Expression of the heterodimeric chimeric Fab was further confirmed by Western blot in non-reducing conditions (Figure 4, lane NR). In reducing conditions a band corresponding to monomeric Fd and/light chain was detected (Figure 4, lane R). Binding of the chimeric Fab was detected by solid phase ELISA and the result is shown in Figure 5. As shown in Figure 5, the binding of the antibody increases with increasing amount of the chimeric Fab, reaching a saturation level as expected for antigen-antibody interactions. Nucleotide sequence of the chimeric Fab; Antibody Fab fragment is composed of two polypeptide chains: Fd and L. Nucleotide sequence of the anti-HBsAg mouse-human chimeric Fd chains: 1 gtccagcttc tcgagcccgg ggctgagctg gcgacgcctg gggcctcatt gaagatgtcc 61 tgcaaggctt ctggctactc atttagcacc tacaacattc actgggtaaa gcagacacct 121 ggacggggcc tggaatggat tggaactatt tatccaggaa ttggtgatac ctcctacaat 181 cagaagttca aaggcaaggc cacattgact gcagacaaat cctccagcac agcctatttg 241 cacctcaaca gcctgacatc tgaggactct gcggtctatt actgtgcaag aagtgacatc 301 tactatggta actacaatgc tttggactac tggggtcaag gaacctcagt cactgtctct 361 tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 421 gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 481 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 541 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 601 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 661 cccaaatctt gtgacaaaac tagtacatgc nucleotide sequence of the anti-HBsAg chimeric Light chain; 1 agatgtgagc tcgtgatgac ccagactcca ctctccctgc ctgtcagtct tggagatcaa 61 gcctccatct cttgcagagc tagtcagagc attgtccaca gttatggaga cacctatttg 121 gaatggcacc tgcagaaacc aggccagtct ccaaagctcc tgatctacaa agtttccaac 181 cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga attcacactc 241 aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctttca acgttcatat 301 gttccgtgga cgttcggtgg aggcaccaag ctggaactca aacggactgt ggctgcacca 361 tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactge ctctgttgtg 421 tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc 481 ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac 541 agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc 601 tgcgaagtca cccatcaggg cctgagttcg cccgtcacaa agagcttcaa caggggagag 661 tgt Amino acid sequence of the chimeric Fab: Antibody Fab fragment is composed of two polypeptide chains: Fd and L. Amino acid sequence of the anti-HBsAg mouse-human chimeric Fd chains: VQLLEPGAELATPGASLKMSCKASGYSFSTYNIHWVKQTPGRGLEWIGTI YPGIGDTSYNQKFKGKATLTADKSSSTAYLHLNSLTSEDSAVYYCARSDI YYGNYNALDYWGQGTSVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTSTC Amino sequence of the anti-HBsAg chimeric Light chain: RCELVMTQTPLSLPVSLGDQASISCRASQSIVHSYGDTYLEWHLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTEFTLKISRVEAEDLGYYFCFQRSYV PWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC The Fab is formed by the precise covalent linkage of the two polypeptide fragments (chemeric Fd and chemeric light chain) at the carboxy terminal of both the fragments. This is via the SH group on the said chain of the amino acid cysteine present in each of the chains at the carboxy end. These two SH groups are condensed from a disulphide (S-S) bond in the proper redox environment to make a functional Fab. Table 1: Oligonucleotide primers used for generation of the chimeric Fd and light chaim.(Table Removed) The Fab or fragment antigen binding is a combination of the light and the first two domains of heavy chains in a very precise manner. Individually these light and heavy chains will fold in a manner that is very different from the manner they will fold in combination with each other and thus have no desired biological properties. The light chain and heavy chain have to be linked covalently in a manner that will be supportive of their active and folded nature and stabilize the same. This is through the SH groups present on the amino acid cysteine near the carboxy terminal end of the both the molecules. There are numerous cysteins in each of the chains, most form intra chain disulphide bond. However there is only one interchain disulphide bond. This can be formed only the precise alignment of the two relevant cysteines or else the molecule will be mis-folded and have no functions and also itself be antigenic. In order to make the Fab molecule, the inventors here have adopted a recombinant approach, where individual fragments have been cloned in a vectors specially designed for the purpose. Those fragments have been expressed in a host bacterium (E.coli). This particular strain of E.coli has a periplasmic space which provides an environment of a particularly favourable redox potential that will facilitate the proper interaction of the fragments, their functionally active alignment and the formation of an inter-chain disulplude bond to make a complete and functional Fab fragement. It is to be noted that the formulation of the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant formulations are intended to be within the scope of the present invention which is further set forth under the following claims:- WE CLAIM 1, A recombinant chimeric Fab antibody comprising a recombinant Fd having seq id# 1 and a recombinant chimeric light chain having seq id# 2 which binds to Hepatitis B surface antigen (HBsAg) with high affinity (KD= 8.166 ± 0.14 nmol/L), generated by fusing heavy chain variable region (VH) of anti-HBsAg antibody 5S having seq id# 3 with first constant region (CHI) of human IgG1 and light chain variable region (VL) of anti-HBsAg antibody 5S having seq id# 4 with constant region (CL) of human kappa chain, wherein mouse VL and human CI are linked by overlap PCR to generate chimeric light chain and mouse VH and CH1 are linked by overlap PCR to generate chimeric Fd using such primers as herein described. 2. A recombinant chimeric antibody, as claimed in claim 1, wherein the individual fragments have been cloned in bicistronic bacterial expression vector (pCOMBSH, Scripps Research Institute, La zola USA) and expressed in a host bacterium, a particular strain of E. Coli (XL 1-blue) having a periplasmic space, which provides an environment of a particularly favorable redox pot,ential, wherein that redox potential facilitate the proper interaction of fragments, their functionally active alignment and the formation of an interchain disulphide bond to make a complete and functional Fab fragment. 3. A recombinant chimeric Fab antibody as claimed in claimV 1, wherein only the Fab can bind to Hepatitis,B surface antigen and not the individual light and heavy chains.

Documents:

2704-del-2006-Abstract (19-03-2012).pdf

2704-DEL-2006-Abstract-(07-12-2010).pdf

2704-DEL-2006-Abstract-(11-11-2011).pdf

2704-del-2006-abstract.pdf

2704-del-2006-Claims (19-03-2012).pdf

2704-DEL-2006-Claims-(07-12-2010).pdf

2704-DEL-2006-Claims-(11-11-2011).pdf

2704-del-2006-claims.pdf

2704-DEL-2006-Correspondence Others-(11-11-2011).pdf

2704-del-2006-Correspondence-others (19-03-2012).pdf

2704-del-2006-correspondence-others 1.pdf

2704-del-2006-Correspondence-Others-(06-04-2011).pdf

2704-DEL-2006-Correspondence-Others-(07-12-2010).pdf

2704-del-2006-correspondence-others.pdf

2704-del-2006-correspondence-po.pdf

2704-del-2006-description (complete).pdf

2704-del-2006-description (provisional).pdf

2704-del-2006-drawings.pdf

2704-DEL-2006-Form-1.pdf

2704-del-2006-form-18.pdf

2704-DEL-2006-Form-2.pdf

2704-del-2006-form-5.pdf

2704-del-2006-gpa.pdf