FIELD OF INVENTION

The present invention is in the field of immunology in particular production of recombinant antibody fragments. The present invention specifically relates to production of recombinant human bivalent diabody protein against rabies virus.

BACKGROUND OF THE INVENTION

Rabies is an invariably fatal viral infection of the nervous system of warm blooded animals including humans. It is transmitted by the bite of an infected animal, usually from a dog (Jackson, A.C., (2003). Rabies virus infection: an update. Journal for Neurovirology, 9; 253-258). It is estimated that every year 2.5-3 million people in India require post-exposure vaccination (Hemachudha T, Phuapradit P. (1997). Rabies. Current Opinion in Neurology. 10; 260-267). Potency determination of rabies vaccines involving the use of in vivo tests in mice (WHO, 1992) and in vitro methods based on estimation of rabies virus glyco¬protein have been described (Perrin et al. (1990). In vitro rabies vaccine potency appraisal by ELISA: advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody, Biologicals. 18:321-330), Nagarajan et al. (2006), Molecular Epidemiology of Rabies Virus Isolates in India Journal of Clinical Microbiology. 44 (9): 3218-3224). Use of monoclonal antibodies for estimation of rabies virus glycoprotein involves production of antibodies from hybridoma cell lines, a process which is expensive and requires complex bio-processes. The use of recombinant antibody fragments (such as bivalent diabodies) will overcome these limitations and provide homogenous, pure reagents necessary for rabies virus glycoprotein measurement.

OBJECTS OF THE INVENTION

The first object of the present invention is to provide recombinant human bivalent diabody protein capable of recognizing rabies virus glycoprotein.

The second object of the present invention is to provide a method of production of the recombinant human bivalent diabody specific for rabies virus glycoprotein.

The third object of the present invention is to develop an ELISA for the quantification of rabies viral glycoprotein.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus.

Another aspect of the present invention provides a polynucleotide encoding recombinant human bivalent diabody, wherein nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1 shows electrophoretic analysis of PCR amplified variable heavy chain (A), variable light chain (B) and the assembled diabody product (C). These gene sequences were amplified from total RNA isolated from a hetero-hybridoma secreting a fully human monoclonal antibody; Lane: M-Molecular weight marker, VH-Variable heavy chain, VL-Variable light chain and D-Assembled diabody product.

Figure 2 shows SDS-PAGE analysis of purified bivalent diabody. The purified protein was detected by staining with Coomassie brilliant blue and was determined to be of 27 KDa by running along with protein molecular weight marker. Figure 3 shows western blot analysis of purified bivalent diabody fractions obtained after column chromatography. The blot was reacted with His probe specific for the Histidine tag of the bivalent diabody. A protein band of 27 kDa was detected.

Lanes: Lane 1-5, fractions obtained from column chromatography purification of bivalent diabody; Lane M: Prestained molecular weight marker. Figure 4 illustrates an immuno- capture ELISA using the bivalent diabody. The test sample B containing M5B4 (anti-rabies murine monoclonal antibody), PV Ag (Pasteur virus antigen), D06 (bivalent diabody) and His-Probe. A and C are the negative control. A is without diabody and C is without the PV Ag.

A → M5B4 + PV Ag + His-Probe
B → M5B4 + PV Ag + D06 + His-Probe
C → M5B4 + D06 + His-Probe

Figure 5 illustrates competitive ELISA using the bivalent diabody and the anti-rabies murine monoclonal antibody (M5B4). A contains PV Ag, E. coli Lysate, M5B4 (anti-rabies murine monoclonal antibody), and anti mouse HRP. B is without E. coli lysate.

Figure 6 illustrates the vector map showing the cloned bivalent diabody fragment.

Figure 7 shows the SDS-PAGE analysis of human monoclonal antibody secreted by heterohybridoma clones after affinity purification on protein A sepharose column. The antibodies are run along with BSA as standard.

Figure 8 illustrates the schematic representation of the constructed vector containing diabody gene cloned between EcoR1 and Not1 sites of pET 28a bacterial expression vector.

Figure 9: (a) shows the specific binding of diabody to Rabies viral structural proteins transferred onto PVDF membrane by Immunoblot assay; (b) shows the SDS-PAGE analysis of rabies virus structural proteins. Figure 10 shows the binding specificity of diabody by immunoreactivity assay; (1) Positive control (Mouse Mab, M5B4), (2) Diabody and (3) Negative control, E. coli lysate Figure 11a and 11b:

(a) Line fit plot for regression through origin analysis for GP content estimated by MAb M5B4-D06 IC-ELISA and the actual NIH potency value (IU). (b) Line fit plot for regression through origin analysis for GP content estimated by MAb M5B4 IC-ELISA and the actual NIH potency value (IU)

DETAILED DESCRIPTION OF THE INVENTION

The term "diabody" used herein refers to an engineered antibody and/or antibody fragments that are bivalent, monospecific or bispecific molecules generated by dimerization of two variable heavy-variable light fragments.

The term "bivalent" used herein refers to an antibody and/or antibody fragment having two antigen binding sites capable of binding to two molecules of same or different antigens with great avidity.

The present invention provides a method for production of recombinant bivalent diabody against rabies virus. The present invention further provides bivalent diabody rabies, composition comprising the bivalent diabody fragment and uses thereof. The bivalent diabody fragment disclosed in the present invention recognizes rabies virus glycoprotein.

The bivalent diabody were constructed using gene sequences of a fully human monoclonal antibody secreted by a human X mouse heterohybridoma.

The present invention relates to the development of a bivalent diabody fragment constructed from a heterohybridoma (human X mouse) and having two antigen binding sites that is reactive against the glycoprotein of rabies virus and its use in the development of ELISA for the quantitation of rabies virus glycoprotein.

The present invention provides a method for production of recombinant bivalent diabody fragment construction from heterohybridoma and their use in the quantification of rabies glycoprotein. Heterohybridoma was generated by immortalizing immune human B cells mediated by a human X mouse heteromyeloma (Champion et al. (2000). The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure, Journal of Immunological Methods 235(1-2): 81-90).

In the present invention, the recombinant bivalent diabody was constructed from heterohybridoma and this recombinant diabody was used in the development of an ELISA for the quantitation of rabies virus glycoprotein in the vaccine manufacture.
Total RNA was isolated from the heterohybridoma and cDNA was synthesized by RT-PCR. DNA encoding variable domains of heavy and light chains were amplified separately by PCR and assembled into diabody with a short linker by splicing overlap extension polymerase chain reaction (SOE PCR). Sequence of the recombinant diabody was verified and cloned into pET 28a vector and transformed into E. coli strain (BL21-DE3) cells for soluble expression of diabody. The soluble diabody protein was purified using immobilised metal affinity chromatography method. The purified protein was checked for their antigen binding activity using the rabies virus glycoprotein and was found to be nearly identical to the parental antibody.

The present invention further provides use of the recombinant diabody disclosed in the present invention for detection and quantification of rabies virus glycoprotein in the vaccine manufacture.

In the present invention the recombinant human diabody which was constructed from heterohybridoma, is one of the smallest recombinant bispecific antibodies consisting of two antigen binding sites devoid of constant regions and therefore provides high binding avidity and specificity to the target antigens similar to the parent antibody.

The recombinant bivalent diabody as disclosed in the present invention can be over expressed in bacteria and produced in large quantities at low cost to guarantee the supply of a consistent and well-characterized specific reagent to quantify rabies virus glycoprotein which can than be used to evaluate vaccine potency.

In accordance with the present invention in one embodiment there is provided a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus.
Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus, wherein the rabies virus is selected from genotype (GT) 1 (GT1) (PV, Flury LEP, SAD, CVS-11), GT4 {Duvenhage virus (DUV)} and GT7 {Australian bat lyssavirus (ABLV)}.

Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus, wherein the diabody is a monoclonal antibody.

Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus, wherein the diabody is a fully human monoclonal antibody.

Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus, wherein the diabody is monospecifc monoclonal antibody.

Another embodiment of the present invention provides a polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25.

Another embodiment of the present invention provides a polynucleotide encoding recombinant human bivalent diabody, wherein nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25, wherein the polynucleotide encodes recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26.

Yet another embodiment of the present invention provides a recombinant DNA expression cassette comprising the polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25, wherein the polynucleotide is operably linked to a promoter.

Yet another embodiment if the present invention provides a recombinant vector comprising the recombinant DNA expression cassette comprising the polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25, wherein the polynucleotide is operably linked to a promoter.

Yet another embodiment of the present invention provides a recombinant host cell comprising the DNA expression cassette comprising the polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25.

Yet another embodiment of the present invention provides host cell selected from a group consisting of E. coli, yeast and CHO cells.

The present invention further provides a composition comprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus.

The present invention further provides a composition corrfprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 and pharmaceutically acceptable carrier, wherein the diabody binds to rabies virus and neutralizes the virus.

The present invention also provides a vaccine composition comprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 or the polynucleotide sequence as set forth in SEQ ID NO: 25.

The present invention also provides a vaccine composition comprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26.

Another embodiment of the present invention provides a therapeutic biological composition comprising the recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 25.

Further in another embodiment of the present invention, there is provided a kit for estimation of rabies virus glycoprotein, wherein the kit comprises a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26.

A method for treating rabies virus associated disease and/or disorders is also provided herein, wherein the method comprises administering an effective amount of the recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 to the subject in need thereof.

The present invention also provides use of the recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 to the subject in need thereof for the preparation of medicament for the treatment of rabies associated disease or disorders.

Still another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody is produced in bacteria.

Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus glycoprotein.

Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus glycoprotein G.

Still another embodiment of the present invention provides a test kit for estimation of rabies virus glycoprotein, wherein the kit comprises a recombinant human bivalent diabody encoded by the polynucleotide as set forth in SEQ ID NO: 25.

Diabody holds promise in quantification of the RV GP in vaccine preparations which correlated well with the NIH mouse potency. Various vaccine batches tested by diabody based IC-ELISA showed a good correlation with the NIH mouse potency studies as seen with MAb-M5B4 based IC-ELISA (Nagarajan et al 2006, WHO 1988). Quantification of RV GP using diabody based IC-ELISA provides exact information of the natively folded RV GP antigen in vaccine preparations and in-process control samples which enables reliable estimation of RV GP. The method described in this study by using diabody provides a simple, novel and efficient option for quantification of RV GP antigen in vaccine preparations without the loss of antigen, aids in the manufacture of good quality vaccines which can reduce the cost and makes the vaccine affordable in developing countries where rabies prevention and control is a challenge. The diabody based IC-ELISA could replace the MAb based IC-ELISA due to better reagent stability and ease of production.

EXAMPLE

It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light of the specification will be suggestive to person skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Example 1

Human X mouse heterohybridoma cells producing anti rabies virus human monoclonal antibody

Rabies Virus Strain used in the present invention is the Pasteur virus (PV) strain described in literature.

Human X mouse heterohybridoma was generated by fusing immune human B cells with human X mouse heteromyeloma (Champion et al. (2000); The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure. Journal of Immunological Methods 235(1-2): 81-90). The heterohybridomas were developed by fusing primary immune peripheral blood B-cells and a heteromyeloma cell line, K6H6/B5 (Carroll et al. (1986); Mouse x human heterohybridomas as fusion partners with human B cell tumors. Journal of Immunological Methods 89(1): 61-72)). The K6H6/B5 cell line was chosen because it has been successfully used to clone human anti-viral antibodies (Siemoneit et al. (1994). Isolation and epitope characterization of human monoclonal antibodies to hepatitis C virus core antigen. Hybridoma. 13(1): 9-13; Funaro et al. (1999). Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient. Experimental Cell Research 247(2): 441-450). The immune B cells isolated from a human subject vaccinated several times with the human rabies vaccine PVRV (Abhayrab, Human Biologicals Institute, Udhagamandalam) which incorporates PV strain of rabies virus which is known to provide broad coverage against street rabies virus (SRV) isolates (Badrane H, Bahloul C, Perrin P and Tordo N (2001). Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity, Journal of Virology 75; 3268-3276) were stimulated with pokeweed mitogen, before fusion. Using the hybridoma method, 8 new human IgG antibodies (huMabs) that bind the rabies virus glycoprotein were cloned.

Characterization: The 8 new human IgG antibodies (huMabs) were characterized to determine their isotype, specificity and cross-reactivity. Each of the huMabs was of gamma 1 (yl) heavy chain and lambda (X) light chain isotype (Table 1). Their specificity to RV was demonstrated by a Cell-ELISA using unfixed mock and RV infected cultured cells. All the huMabs showed reactivity to all the fixed RVs except the BHK-21-adapted CVS strain (CVS- RV). None of the huMabs showed any reactivity to host cell protein (Table 2). The huMabs bound specifically to native form of whole virus antigens as well as purified rabies virus glycoprotein and did not react with rabies virus nucleoprotein as evident from the results of Indirect ELISA (Table 1). The huMabs recognized antigenic site HI of rabies virus glycoprotein as determined by a competitive ELISA using a mouse Mab (Dl) against antigenic site III as described elsewhere (Nagarajan et al. (2006), A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture, Biologicals. 34: 21-27).

The huMabs have been tested for their ability to neutralize various street and fixed RVs both in vitro and in vivo. In the mouse neutralization test (MNT) with Swiss albino mice, the huMabs neutralized all the four Indian street rabies viruses (SRVs) of dog origin (108, 141, 142, & 129) (Table 3). All the 8 huMabs neutralized four fixed RV strains viz. Pasteur Virus (PV), Flury LEP, SAD and CVS-11) while none of the huMabs neutralized BHK-21 adapted CVS RV when tested by rapid fluorescent focus inhibition test (RFFIT) (Table 4). The ability of the best five out of 8 huMabs with the most potent RFFIT titers to neutralize members of the Lyssavirus genus was tested.

The remaining 3 huMabs have not been taken up for characterization since they did not have the desired rabies virus neutralization spectrum. Four huMabs chosen from five huMabs also neutralized GT7 (ABLV) and GT4 (DUV) effectively in the RFFIT, although none neutralized GT3 (MOKV) or GT5 (EBLV-1) (Table 5).

Example 2

Screening of 8 new human IgG antibodies (huMabs) clones

The clones were screened for the specific human Mab secretion by indirect ELISA employing purified inactivated rabies virus antigen (PV strain).

Antibody purification

The human monoclonal antibody (Mabs) secreted by heterohybridoma clones have been affinity purified on protein A sepharose column. The purified antibody was checked on gel (Figure 7). Lane 1 shows BSA as standard whose molecular weight is 66 KDa. Lane 2-5 shows the purified R16E5 Human monoclonal antibody of molecular weight 160 KDa. The main criterion for selecting four out of 8 clones was rabies virus neutralization spectrum as determined by RFFIT.

Test to confirm production of anti rabies virus human monoclonal antibody by the heterohybridoma cells

Several tests such as indirect ELISA, cell ELISA, RFFIT, and MNT were performed to confirm the rabies virus specific nature of the huMabs secreted by the heterohybridoma cells.

Indirect ELISA

The huMabs were screened for the rabies whole virus antigen specific activity by indirect ELISA. An ELISA plate was coated with zonal purified rabies whole virus antigen (1:100 dilution) in carbonate buffer overnight at 2-8°C. The plate was washed thrice with PBST and the unsaturated sites were blocked with 1% bovine gelatin. The antigen coated on the solid phase was probed with huMabs and the presence of immune complex detected with goat anti-human IgG-peroxidase conjugate followed by the addition of TMB chromogenic substrate The plate was read at 450 nm wavelength after stopping the colour reaction with 1.25M H2SO4. Growth medium and immune mouse serum were used as negative and positive controls respectively.

The results are furnished in Table 1. It is clear from the results that the huMabs exhibited rabies whole virus antigen specific activity.

Cell ELISA

The huMabs were screened for the rabies virus specific activity by cell ELISA using rabies virus (PV strain) infected and mock infected VERO monolayer cell culture.

The unsaturated sites were blocked using 1% bovine gelatin after fixing the cell sheet with 70% cold acetone. The plate was washed thrice with PBST and the fixed cell sheet was then was probed with huMabs. The presence of immune complex was detected with goat anti-human IgG-peroxidase conjugate followed by the addition of TMB chromogenic substrate. Growth medium and immune mouse serum were used as negative and positive controls respectively. The results are furnished in Table 2. It is clear from the results that the huMabs exhibited rabies virus specific activity essentially free from any cross-reactivity with the host cell proteins.

RFFIT

The huMabs were screened for their ability to neutralize fixed strain of rabies virus by RFFIT. The huMabs were heat inactivated before setting up a neutralization reaction with 50 FFD50 of rabies virus (CVS-11 strain) by incubating at 37°C for 90 minutes. The presence of unneutralized virus in the mixture after incubation was determined by seeding it along with an indicator cell line (MNA cell line) and incubated for 20 hours. The cell sheet was fixed with 70% cold acetone and probed with rabbit anti rabies virus nucleocapsid IgG-FITC conjugate to demonstrate the presence of rabies virus. Human serum of known titer sourced from NIBSC, UK was used as the internal reference standard. The results are furnished in Table 4. It is clear from the results that the huMabs potentially exhibited rabies virus neutralizing activity.

MNT

The huMabs were screened for their ability to neutralize fixed and street rabies viruses by MNT. The huMabs were heat inactivated before setting up a neutralization reaction with 50 LD50 of either fixed or street rabies virus by incubating at 37°C for 90 minutes. The presence of unneutralized virus in the mixture after incubation was determined by intracerebrally inoculating weaned mice and observed daily for the signs of rabies such as typical hind limb paralysis for a period of 21 days. HRIG of known titer obtained from a commercial vendor was used as the internal reference standard. The results are furnished in table 3. It is clear from the results that the huMabs potentially exhibited the ability to neutralize fixed and street rabies viruses.

Example 3

Construction of diabody fragment

RNA extraction and Amplification of Antibody Variable domain sequences
Total RNA was isolated from anti rabies heterohybridoma (R16E5) and DNA was synthesized by RT-PCR. The nucleotide sequence of the amplified c-DNA is as set forth in SEQ ID NO: 25. The RT-PCR amplified cDNA was used as template for amplification of variable domains of an antibody with universal primers having nucleotide sequence as set forth in SEQ ID NO: 1 to SEQ ID NO: 20). The amplified variable domains were assembled to form diabody by splicing by overlap extension PCR and cloned in TOPO vector for sequence verification (Figure 1A, Figure IB and Figure 1C).

Primers used for amplification of variable domains of heavy chain and light chains are as follows:

Human Variable Heavy Chain Forward Primers

HuVH1a:SEQIDNO: 1
GGCGGCGGCGGCTCCGGTGGTGGTCAGGTGCAGCTGGTGCAGTCTGG

HuVH2a: SEQ ID NO: 2
GGCGGCGGCGGCTCCGGTGGTGGTCAGGTCAACTTAAGGGAGTCTGG

HuVH3a: SEQ ID NO: 3
GGCGGCGGCGCCTCCGGTGGTGGTGAGGTGCAGCTGGTGGAGTCTGG

HuVH4a: SEQ ID NO: 4
GGCGGCGGCGGCTCCGGTGGTGGTCAGGTGCAGCTGCAGGAGTCGGG

HuVH5a: SEQ ID NO: 5
GGCGGCGGCGGCTCCGGTGGTGGTGAGGTGCAGCTGTTGCAGTCTGC

HuVH6a; SEQ ID NO: 6
GGCGGCGGCGGCTCCGGTGGTGGTCAGGTACAGCTGCAGCAGTCAGG

Human Variable Heavy Chain Reverse Primers

HuJHl-2:SEQIDNO:7
GGAATTCTGAGGAGACGGTGACCAGGGTGCC

HuJH3:SEQIDNO:8
GGAATTCTGAGGAGACGGTGACCATTGTCCC

HuJH4-5:SEQIDNO:9
GGAATTCTGAGGAGACGGTGACCAGGGTTCC

HuJH6: SEQ ID NO: 10
GGAATTCTGAGGAGACGGTGACCGTGGTTCC

Human Variable Light Chain Forward Primers

HuLAM1:SEQIDNO: 11
GCCATGGCGCAGTCTGTGTTGACGCAGCCGCC

HuLAM2: SEQ ID NO: 12
GCCATGGCGCAGTCTGCCCTGACTCAGCCTGC

HuLAM3a: SEQ ID NO: 13
GCCATGGCGTCCTATGTGCTGACTCAGCCACC

HuLAM3b: SEQ ID NO: 14
GCCATGGCGTCTTCTGAGCTGACTCAGGACCC

HuLAM4: SEQ ID NO: 15
GCCATGGCGCACGTTATACTGACTCAACCGCC

HuLAM5:SEQIDNO: 16
GCCATGGCGCAGGCTGTGCTCACTCAGCCGTC

HuLAM6: SEQ ID NO: 17
GCCATGGCGAATTTTATGCTGACTCAGCCCCA'

Human Variable Light Chain Reverse Primers

HuJLAM1:SEQIDNO: 18
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACC T AGGACGGTGACCTTGGTCCC

HuJLAM2-3: SEQ ID NO: 19
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACC TAGGACGGTCAGCTTGGTCCC

HuJLAM4-5: SEQ ID NO: 20
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACC TAAAACGGTGAGCTGGGTCCC

Primers used for amplification of complete diabody fragment

Variable Heavy Chain Primers Forward: SEQ ID NO: 21
GGTGGTGGTGGTTCTGGTGGTGGTCAGGGTCAGCTGGTGCAG Variable

Light Chain Primers

Reverse: SEQ ID NO: 22
ACCACCACCAGAACCACCACCACCTAGGACGGTCAGCTTGGT

The first strand cDNA synthesized from total RNA using thermoscript RT-PCR from Invitrogen was used as template for amplification of variable domains of antibody. The variable heavy chain (VH) and light chain (VL) were amplified from the cDNA using the universal human variable primers. Human variable heavy chain (HuVH) forward primers and human joining heavy chain (HuJH) reverse primers were used to amplify variable heavy chain. Human lambda light chain (HuLam) forward primers and human joining light chain (HuJLam) reverse primers for amplifying variable light chain.

cDNA encoding the antibody variable heavy chain (VH) and variable light chain (VL) were amplified separately by PCR under standard condition, performed for 34 cycles, using Taq DNA polymerase. The VH region was amplified with primer list above as SEQ ID NO: 1 to SEQ ID NO: 10 and primer as set forth in SEQ ID NO: 11 to SEQ ID NO: 20 were used for VL region. PCR was conducted with a mixture of 10 ug product of reverse transcription, 3U Taq DNA polymerase and 1ul VH/ VL primers mix in a total volume of 50 ul. The PCR reaction conditions are kept in the following order: 95°C x 5 minutes, 92 °C x 1 minute, 63 °C x 1 minute, 72 °C x 1 minute for 34 cycles, and 72 °C x 10 minutes. After quantification of PCR products by gel electrophoresis the amplified variable heavy and variable light chain were assembled by splicing by overlap extension PCR. PCR program was arranged in the following order: 95 °C x 5 minutes, 92 °C x 1 minute, 63 °C x 1 minute, 72 °C x 1 minute for 14 cycles, and 72 °C x 10 minutes. The PCR product obtained was used as a template and amplified with primers listed above as SEQ ID NO: 11 to SEQ ID NO: 17 as forward primer and SEQ ID NO: 7 to SEQ ID NO: 10 as reverse primer. The PCR conditions followed are: 95 °C x 5 minutes, 92 °C x 1 minute, 63 °C x 1 minute, 72 °C x 1 minute for 34 cycles, and 72 °C x 10 minutes.

The resultant PCR product was amplified with the primers listed above as SEQ ID NO: 21 and SEQ ID NO: 22 to obtain the PCR product of 714 base pairs (SEQ ID NO: 25) for construction of diabody.

The amplified variable heavy and light chains were joined together with the polypeptide linker using SOE and the resulted PCR product of 714 base pairs (SEQ ID NO: 25) was cloned into TOPO-TA vector. Positive clones which showed release of 714 base pairs (bps) product after enzymatic analysis of the plasmids were sequenced and blasted the sequence by using NCBI blast search showed the presence of 363 bps of variable heavy chain, 327 bps of variable light chain and 24 bps linker region which forms a diabody of 714 bps having the polynucleotide as set forth in SEQ ID NO: 25.

The resultant PCR product of 714 base pairs (SEQ ID NO: 25) was further amplified using the primers as set forth in SEQ ID NO: 23 and SEQ ID NO: 24 to add the restriction site for cloning into pET vector.

Primers used for cloning diabody into pET28a vector

Primer 1: SEQ ID NO: 23
ATGCATGAATTCTCAGATTGCCATGGCGTC

Primer 2: SEQ ID NO:
24 ATGCGCGGCCGCCGCATCCTGCAGACGCGT

Example 4

Vector construction and cloning of diabody fragment into pET vector

The diabody fragment having polynucleotide as set forth in SEQ ID NO: 25 was cloned between EcoR\ and Noil sites of pET 28a bacterial expression vector. The vector pET 28a and the insert referred to diabody of size 714 bps was digested with EcoRl and Noil, respectively by incubating at 37°C for 12 hours. The digested products was purified using the kit provided by QIAGEN and kept for various ratios of vector to insert (i.e., 1:3 and 1:6) blunt end ligation and incubated at 22°C for 2 hours. The ligated product was incubated for further 20 minutes at 65°C in order to inactive the enzyme. The pET 28a vectors carry an N-terminal His tag/thrombin/T7 Tag configuration plus an optimal C-terminal His tag sequence. At both the ends T7 promotor and T7 terminator are situated used to sequence the single stranded insert DNA fragment of the diabody. The insert was verified by sequencing the clone from vector backbone using T7 promotor and T7 terminator primers. The obtained 714 base pairs sequence is as set forth in SEQ ID NO: 25.

E. coli Transformation

Overnight grown XL-Blue strain culture was sub-cultured and grown at 37°C, shaking until the OD of the culture reaches to 0.6 at 600nm. The culture was harvested by centrifuging at 5000 x rpm for 10 minutes at 4°C and resuspended in ice-cold 0.1 mM CaCl2 and incubated overnight on the ice, before proceeding for transformation.

The chemically competent XL-Blue cells were incubated with plasmid DNA for 30 minutes on ice. The cells were given heat shock at 42°C for 90 seconds and immediately placed in ice for 2 minutes before the media was added to cells. The cells were incubated for one hour at 37°C for recovery and plated on semi-solid media containing 50mg/ml of kanamycin. The plates were incubated for overnight and screened for positive clones by isolating the plasmids and subjected to digestion with EcoRl and Notl. The positive clones were sequence verified before the plasmid was transformed into BL-21 DE3 cells of E .coli for soluble expression of the antibody gene.

Example 5

Expression of nucleic acid sequence of diabody protein in E. coli
The recombinant diabody fragment having polynucleotide sequence as set forth in SEQ ID NO: 25 was expressed in E. coli for production of recombinant human monoclonal diabody protein having polypeptide sequence as set forth in SEQ ID NO: 26. Further, the diabody protein was purified from the soluble fraction of the lysate using Immobilized Metal Affinity Chromatography (IMAC). The purified recombinant human diabody protein was analyzed and a band of 27 KDa size was visualized on 12% reducing SDS-PAGE after staining with Coomassie brilliant blue (Figure 2) and detected with His-Probe in western blotting (Figure 3) Yield of the affinity purified protein was quantified using Bicinchoninic Acid kit and found to be on an average of 5mg/10L.

Example 6

In-vitro testing of recombinant human bivalent diabody protein

Immuno-Capture ELISA

Immuno capture ELISA was performed to check the binding affinity of the recombinant human bivalent diabody protein. A micro titer plate was coated with mouse monoclonal antibody (Mab) M5B4 (l00ng/well) in carbonate buffer by incubating over night at 4°C. The plate was washed thrice with PBST and the unbound sites in the wells were blocked with 1% bovine gelatin. Purified rabies virus glycoprotein (Pasteur virus strain) was added in different dilutions and allowed to react with M5B4. Diabody protein tagged with Histidine was added and allowed to react with captured antigen. The affinity binding of the diabody with rabies virus glycoprotein was detected by adding His-Probe and a chromogenic substrate TMB. The plate was read at 450nm after the reaction was stopped with 1.25MH2So4 (Figure 4).

It is evident from the graph (Figure 4) that the diabody specifically binds to rabies PV antigen. The graph shows that there is decline in optical density with respect to the diabody concentration used in presence of antigen and there is no such signal shown in absence of an antigen.

Immuno-capture ELISA for quantification of rabies virus glycoprotein in human rabies vaccine

1C-ELISA was performed to quantify the Rabies Virus Glyco Protein (RV GP) content in rabies vaccine formulations, according to the method described by Nagarajan et al. {Nagarajan, T., G. S. Reddy, B. Mohana Subramanian, S. Rajalakshmi, D.Thiagarajan, N. Tordo, C. Jallet, and V. A. Srinivasan. 2006. A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture. Biologicals 34:21-27.}, with a few modifications wherein the diabody (450 ng/well) was used for detection.

ELISA plate was coated with mouse monoclonal antibody (MAb) M5B4 overnight at 4°C and the un-reacted sites blocked with 1% bovine gelatin. The test vaccines and an internal reference standard (IRS) vaccine of known RV GP were subjected to 8 serial 2 fold dilutions in PBS-T. The RV GP trapped by MAb M5B4 was detected using the diabody followed by the addition of anti-1 His Probe. The plate was developed with TMB at room temperature for 10 minutes. The reaction was stopped by addition of 1.25M H2S04 and the absorbance was measured at 450 nm wave length using a micro titre plate reader (BIO-TEK, USA). The assay was performed in triplicate. The RV GP content was also estimated by MAb-M5B4 IC-ELISA previously described by Nagarajan et al. 2006, using reference standard vaccine. The RV GP content was estimated using the formula

RV GP estimate (micro grams/dose) = (XxZxAxl0)/Y

Where, X - Optical Density of Sample; Y - Optical Density of IRS equivalent to twice the mean of optical density of negative control; Z - Reciprocal of end point dilution; A - GP estimate of Y in nano grams

NIH potency test on rabies vaccine formulations

NIH potency test was carried out on the different rabies vaccine formulations in mice using standard procedures (Wilber, L. A., and M. F. A. Aubert. 1996. The NIH test for potency, p.360-368. In F. X. Meslin, M. M. Kaplan, and H. Koprowski (ed.), Laboratory techniques in rabies, 4th ed. WHO, Geneva, Switzerland.).

Statistical analysis

The estimates of RV GP antigen and predicted potency derived from the IC-ELISA using the MAb-M5B4 (Nagarajan, T., G. S. Reddy, B. Mohana Subramanian, S. Rajalakshmi, D.Thiagarajan, N. Tordo, C. Jallet, and V. A. Srinivasan. 2006. A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture. Biologicals 34:21-27) and diabody D06 were compared with the in vivo NIH potency results using ANOVA with Regression Through the Origin (RTO) model (Snedecor, G. W., and W. G. Cochran. 1989. Statistical methods applied to experiments in agriculture and biology, 8th ed. Iowa State University Press, Ames, IA.).

Estimation of PV-GP in various vaccine preparations

PV GP was estimated in 65 batches of experimental human rabies vaccine preparations using the IC-ELISA and the estimates were compared to the NIH potency values of those respective batches. ANOVA with Regression Through the Origin (RTO) model was performed to compare the potency estimates derived by M5B4-D06 IC-ELSIA and MAb-M5B4 IC-ELISA previously described by Nagarajan et al., (2006). Regression analysis was performed using the data analysis program in Microsoft Excel 2003 to compare the potency estimates derived by M5B4D06 IC-ELSIA and MAb-M5B4 IC-1 ELISA with the NIH estimate. The adjusted R2 value obtained was 0.902 and equation for predicted potency values for M5B4-D06 based IC-ELISA and MAb-M5B4 IC-ELISA were 0.5651x and 0.8044x respectively, where x is the estimate of RV GP by the IC-ELISA in ug (Figure 11a and 11b). ANOVA results showed the estimates by the two methods compared differed highly significantly (P<0.001) while the predicted potencies by the two test did not differ significantly (P>0.05).

Competitive ELISA

Competitive ELISA was performed with mouse monoclonal antibody (M5B4) to see whether the recombinant human bivalent diabody can compete to the same epitope or to the different epitope. A microtiter plate was coated with purified rabies virus antigen (Pasteur virus stain) (100 ng/well) in carbonate buffer by incubating over night at 4°C. The plate was then washed with PBST and unbound sites in the wells were blocked with 1% bovine gelatin followed by washing with PBST and diabody was added by serial dilution along with E. coli Lysate as a negative control and incubated at 37°C for 1 hour. The constant amount of mouse Mab (M5B4) was added to each well containing diabody and E.coli lysate and the plates were incubated at 37°C for 1 hour. The plate was washed with PBST as mentioned above. Goat anti-mouse IgG HRP conjugate with the working dilution of 1:5000 (as recommended by the manufacturer) was added to each well and the plates were incubated further at 37°C for 1 hour. The plate was washed five times with PBST before chromogenic substrate TMB was added. TMB substrate was prepared by dissolving one TMB tablet and 3 u.1 of H2O2 in citrate buffer and an aliquot of l00ul added to each well. The reaction was stopped by adding l00u1 of 1.25 M H2SO4 to each well and plate was read at 450 nm (Figure 5).

Competition studies indicated that (Figure 5) the recombinant human bivalent diabody and murine monoclonal antibody are competing for the same epitope when compared to E. coli lysate.

Example 7

Reactivity of the recombinant human bivalent diabody with the rabies virus

Immunoblot assay was used to determine the nature of the rabies viral antigens recognized by the diabody. Rabies viral structural proteins were separated under non-reducing conditions, in 10% SDS-PAGE, transferred onto PVDF membrane and probed with the diabody. Diabody showed a specific binding to a protein band of 65 kDa, corresponding to the molecular weight of rabies virus glycoprotein (Figure 9).

Dot blot assay

An aliquot (50ug/10ul) of rabies virus glycoprotein antigen was applied to PVDF membrane and air dried. After blocking, the membrane was probed with the diabody. The immunoreactivity was detected by His probe and developed using 3, 3'-diamino benzidine tetrahydrochloride (DAB) (Figure 10). It was found that the diabody binds specifically to glycoprotein of rabies virus as shown in Figure 10. E.. coli lysate was included as a negative control.

Table 1: Characteristics of anti-rabies virus human Mabs

Table 2: Reactivity of anti-rabies virus human monoclonal antibodies with various fixed RV strains in Cell -ELISAa

* Unfixed rabies virus infected and mock infected cell culture was used as the solid phase antigen for doing Cell-ELISA to demonstrate the specificity of huMabs to rabies virus.

Table 4: Neutralization of rabies viruses by anti-rabies virus human monoclonal antibodies


a VNA titer was determined by RFFIT using rabies virus strain CVS-11 essentially as described by Smith et al. (1996).
b Heterohybridoma culture supernatant containing huMabs was used after heat inactivation. None of the huMabs tested could neutralize a rabies virus strain CVS-BHK.

Table 3: Survivorship of Swiss albino mice subjected to mouse neutralization testb to demonstrate the ability of human monoclonal antibodies to neutralize various fixed and street rabies viruses in vivo

* Not done
"Heat inactivated heterohybridoma culture supernatant containing huMabs was used for neutralization reaction.
b Female Swiss albino mice (3-4 weeks old) were intracranialy inoculated with 30 ul of (50 MICLD50/30 \i\) either rabies virus infected culture fluid or brain homogenates from naturally infected rabid stray dogs. The inoculated mice were observed daily for symptoms typical of rabies for 21 days. The results are expressed as per centage of mice that survived after 21 days of observation.

Table 5: Neutralization of rabies-related viruses by anti-rabies virus human monoclonal antibodies

* VNA titer was determined by RFFIT at CDC, Atlanta, essentially as described by Smith et al. (1996). A rapid fluorescent focus inhibition test (RFFIT) for determining rabies virus-neutralizing antibody. In: Meslin FX, Kaplan MM, Koprowski H, editors. Laboratory techniques in rabies. 4th ed. Geneva: WHO; 1996. p. 181 - 191.
b Partially purified huMAb preparation was used.

SEQ ID NO: 25 Nucleotide sequence of diabody fragment (714 nucleotides)
Light chain

TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCACGA
TTACCTGTGGGGGAAACAACATTGGACGTAAAAGTGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGCAACACGGCCACCCTGATCATCAGTAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGCAGTAGTGAGGATTT

linker

TTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTGGTGGTGGTTCTGGTGGT

Heavy chain

GGTCAGGTGCAGCTGGTGCAGTCTGGGGGAAACCTGGTGCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCACCTTCGGAAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCAGCTATTAGTGGCAGTGGTCTTCACACATACTACGCGGACGCTGTGAAGGGCCGGTTCAGCATCTCCAGAGACAACTCCAAGAACACACTGTATTTGCAAATGAACAGCCTGAGAGCCGGGGACACGGCCATTTATTACTGTGCGAAGGATAAGGGCATAGTAGTGGCTACCATCTTCTTCTCCTGGGGCCAGGGAACCC
TGGTCACCGTCTCCTCA

SEQ ID NO: 26 Amino acid sequence of diabody fragment (238 a. a.)
Light chain
SYVLTQPPSVSVAPGQTATITCGGNNIGRKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERF

linker

SGSNSGNTATLIISRVEAGDEADYYCQVWDSSSEDFWVFGGGTKLTVLGGGGSGGGQVQLVQSGGNLVQPGGSLRLSCEASGFTFGSYAMSWVRQAPGKGLEWVAAISGSGLHTYYADAVKGRFSISRDNSKNTLYLQMNSLRAGDTAIYYCAKDKGIVVATIFFSWGQGTLVTVSS
Heavy chain

I/We Claim:

1. A recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus.

2. The recombinant antibody as claimed in claim 1, wherein the rabies virus is selected from genotype (GT) 1 (GT1) (PV, Flury LEP, SAD, CVS-11), GT4 {Duvenhage virus (DUV)} and GT7 {Australian bat lyssavirus (ABLV)}.

3. The recombinant antibody as claimed in claim 1, wherein the diabody is a monoclonal antibody.

4. A polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25.

5. The polynucleotide as claimed in claim 4, wherein the polynucleotide encodes recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26.

6. A recombinant DNA expression cassette comprising the polynucleotide as claimed in claim 4, wherein the polynucleotide is operably linked to a promoter.

7. A recombinant vector comprising the recombinant DNA expression cassette as claimed in claim 6.

8. A recombinant host cell comprising the recombinant DNA expression cassette as claimed in claim 6.

9. The host cell as claimed in claim 8 is selected from a group consisting of E. coli, yeast and CHO cells.

10. A composition comprising the recombinant human bivalent diabody as claimed in claim 1.

11. The composition as claimed in claim 10 further comprises pharmaceutically acceptable carrier.

12. A vaccine composition comprising the recombinant human bivalent diabody as claimed in claim 1 or the polynucleotide as claimed in claim 4.

13. A kit for estimation of rabies virus glycoprotein, wherein the kit comprises the recombinant human bivalent diabody as claimed in claim 1.