Broadly neutralizing human monoclonal antibodies to dengue virus

Abstract

The present invention provides monoclonal antibodies against Dengue virus serotypes, which are capable of effectively neutralizing Dengue virus serotypes, 1 through 4. Also provided in the present invention are expression systems, plasmids and polynucleotide / polypeptide fragments useful in generation of said monoclonal antibodies.

Description

[0001] BROADLY NEUTRALIZING HUMAN MONOCLONAL ANTIBODIES TO DENGUE VIRUS FIELD OF INVENTION

[0002] The present invention generally relates to the field of immunology and monoclonal antibody technology. In particular, the present invention provides monoclonal antibodies and reagents useful in inhibition of Dengue virus.

[0003] BACKGROUND OF THE INVENTION

[0004] India has a disproportionate burden of dengue virus infections. While three live-attenuated dengue vaccines have been licensed for human use in the last few years, however, each of the vaccines have shown variable efficacy, and in some cases, no efficacy at all against one or more of the four dengue virus serotypes. Furthermore, none of these vaccines are available in India.

[0005] Despite increasing burden of dengue cases every year, there are no available antivirals. Antibody therapy is a promising field of research for combating various diseases, especially where vaccines may be found to be ineffective or sufficiently effective in combating the disease. Such therapy avenues are also useful and promising where vaccine development is difficult and / or does not provide favorable results for various reasons.

[0006] Therefore, with regard to dengue virus, there is a need to develop effective broad activity antibodies, which can effectively neutralize dengue virus serotypes.

[0007] BRIEF DESCRIPTION OF SEQUENCES SEQ ID NO: 1 depicts amino acid sequence of heavy chain variable region and CDR3 of a first monoclonal antibody, in accordance with an embodiment of the present invention.

[0008] MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFNFNTYAMTWVRQ APGKGLEWVSTIGAAAGDTYYADSVRGRFNISRDNSKNTLHLQMNSLRAEDTAVYYCA NLYNSDWLVGS SEQ ID NO: 2 depicts amino acid sequence of heavy chain variable region and CDR3 of a second monoclonal antibody, in accordance with an embodiment of the present invention.

[0009] MEFGLSWVFLVALLRGVQCQVHLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQ APGKGLEWVATISYDVTDIYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGWNY SEQ ID NO: 3 depicts amino acid sequence of heavy chain variable region and CDR3 of a third monoclonal antibody, in accordance with an embodiment of the present invention.MDWTWRILFLVAAATGVHSQVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMYWVR QAPGQGLEWMGRINPYSGGTNYAQKFQGRVTMTRDTSISTIYMDLTRLRSDDTAVYYCV RNKWSGYWDRDTFNI SEQ ID NO: 4 depicts amino acid sequence of light chain variable region and CDR3 of a first monoclonal antibody, in accordance with an embodiment of the present invention.

[0010] MAWTPLFLFLLTCCPGSTSQAVVTQESSMTVSPGGTVTLTCGSSTGPVTSGHYPYWFQQ KPGQAPRTLIYDTSKKDSWTPARFSGSLLGGKAALTLSDAQPADEADYYCFLSFGAAGP VFGGGTKLTVL SEQ ID NO: 5 depicts amino acid sequence of light chain variable region and CDR3 of a second monoclonal antibody, in accordance with an embodiment of the present invention.

[0011] MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDKVTITCRASQSVSIWLAWYQ QKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTVSSLQPDDFATYYCQQYLSYSTFG QGTKLEIKR SEQ ID NO: 6 depicts amino acid sequence of light chain variable region and CDR3 of a third monoclonal antibody, in accordance with an embodiment of the present invention.

[0012] MDMRVPAQLLGLLLLWLSGARCDIQMTQSPSSLSASVGDRVTITCQASQDIGNSLNWYQ QKPGKAPKLLINDVSNLETGVSSRFSGSGSGTDFTFTINSLQPEDFATYYCQQYDNLPAIT FGQGTRLDIKR SEQ ID NO: 7 depicts amino acid sequence of heavy chain variable region, CDR3, and heavy chain constant region of a first monoclonal antibody, in accordance with an embodiment of the present invention.

[0013] MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFNFNTYAMTWVRQ APGKGLEWVSTIGAAAGDTYYADSVRGRFNISRDNSKNTLHLQMNSLRAEDTAVYYCA NLYNSDWLVGSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 8 depicts amino acid sequence of heavy chain variable region, CDR3, and heavy chain constant region of a second monoclonal antibody, in accordance with an embodiment of the present invention.

[0014] MEFGLSWVFLVALLRGVQCQVHLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQ APGKGLEWVATISYDVTDIYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGWNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TISI< AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 9 depicts amino acid sequence of heavy chain variable region, CDR3, and heavy chain constant region of a third monoclonal antibody, in accordance with an embodiment of the present invention.

[0015] MDWTWRILFLVAAATGVHSQVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMYWVR QAPGQGLEWMGRINPYSGGTNYAQKFQGRVTMTRDTSISTIYMDLTRLRSDDTAVYYCV RNKWSGYWDRDTFNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 10 depicts amino acid sequence of light chain variable region, CDR3, and light chain constant region of a first monoclonal antibody, in accordance with an embodiment of the present invention.

[0016] MAWTPLFLFLLTCCPGSTSQAVVTQESSMTVSPGGTVTLTCGSSTGPVTSGHYPYWFQQ KPGQAPRTLIYDTSKKDSWTPARFSGSLLGGKAALTLSDAQPADEADYYCFLSFGAAGP VFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSEQ ID NO: 11 depicts amino acid sequence of light chain variable region, CDR3, and light chain constant region of a second monoclonal antibody, in accordance with an embodiment of the present invention.

[0017] MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDKVTITCRASQSVSIWLAWYQ QKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTVSSLQPDDFATYYCQQYLSYSTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 depicts amino acid sequence of light chain variable region, CDR3, and light chain constant region of a third monoclonal antibody, in accordance with an embodiment of the present invention.

[0018] MDMRVPAQLLGLLLLWLSGARCDIQMTQSPSSLSASVGDRVTITCQASQDIGNSLNWYQ QKPGKAPKLLINDVSNLETGVSSRFSGSGSGTDFTFTINSLQPEDFATYYCQQYDNLPAIT FGQGTRLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

[0019] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0020] Figure 1 depicts the FACS sorting of plasmablasts coming from dengue infected patient and single cell sorting, in accordance with an embodiment of the present invention.

[0021] Figure 2a-b depicts the binding and neutralization of Dengue virus by a first monoclonal antibody (clone 162-18), in accordance with an embodiment of the present invention.

[0022] Figure 3a-b depicts the binding and neutralization of Dengue virus by a second monoclonal antibody (clone 162-19), in accordance with an embodiment of the present invention.

[0023] Figure 4a-b depicts the binding and neutralization of Dengue virus by a third monoclonal antibody (clone 140-3), in accordance with an embodiment of the present invention.

[0024] SUMMARY OF THE PRESENT INVENTION

[0025] This summary is not intended to identify essential characteristics of the claimed subject matter, nor is it intended to determine or limit the scope of the claimed subject matter.In an aspect of the present invention, there is provided an expression system comprising: (a) a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0026] In an aspect of the present invention, there is provided a host cell comprising an expression system, said expression system comprising: (a) a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0027] In an aspect of the present invention, there is provided a plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. In another aspect of the present invention, there is provided a monoclonal antibody comprising a heavy chain variable region and CDR3, comprising amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a light chain variable region and CDR3, comprising sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0028] In another aspect of the present invention, there is provided a polynucleotide fragment encoding a polypeptide comprising amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0029] In yet another aspect of the present invention, there is provided a method of inhibiting at least a serotype of Dengue virus, said method comprising: (a) obtaining a therapeutically effective amount of at least a monoclonal antibody comprising a heavy chain variable region and CDR3, comprising amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a light chain variable region and CDR3, comprising sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and (b) contacting said antibody with at least a serotype of Dengue virus.In still another aspect of the present invention, there is provided a medicament comprising an effective amount of a monoclonal antibody comprising a heavy chain variable region and CDR3, comprising amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a light chain variable region and CDR3, comprising sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0030] In an aspect of the present invention, there is provided a monoclonal antibody comprising a heavy chain variable region and CDR3, comprising amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a light chain variable region and CDR3, comprising sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 for use in treatment of preparing a medicament for at least a serotype of Dengue virus.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] For convenience, before the detailed description of the present invention, certain terms used in the specification and examples are collected here. These definitions should be read in light of the remainder of the disclosure and understood by a person skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would normally be understood by a person of ordinary skill in the art. Terms used throughout this specification are defined as follows, unless otherwise limited in specific cases.

[0033] As used in the specification and claims, the singular forms "a", "an", and "the" include the plural referents unless the context clearly specifies otherwise.

[0034] The present disclosure should not have its scope limited by the specific modalities described here, which are intended exclusively for exemplification purposes.

[0035] Functionally equivalent processes and methods are clearly within the scope of the disclosure as described herein.

[0036] The present invention provides an expression system comprising: (a) a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0037] In a preferred embodiment, the expression system comprises a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ IDNO: 1; and a second plasmic comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 4.

[0038] In another preferred embodiment, the expression system comprises a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 2; and a second plasmic comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 5.

[0039] In yet another preferred embodiment, the expression system comprises a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 3; and a second plasmic comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 6.

[0040] In an embodiment, the expression system comprises a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0041] In a preferred embodiment, the expression system comprises a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 7; and a second plasmic comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 10.

[0042] In another preferred embodiment, the expression system comprises a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 8; and a second plasmic comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 11.

[0043] In yet another preferred embodiment, the expression system comprises a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 9; and a second plasmic comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 12.

[0044] In an embodiment, SEQ ID NO: 1 is comprised in SEQ ID NO: 7, where SEQ ID NO: 1 contains heavy chain variable region sequence and CDR3 sequence of a first monoclonal antibody, and SEQ ID NO: 7 comprises heavy chain constant region sequence of the first monoclonal antibody. In an embodiment, SEQ ID NO: 2 is comprised in SEQ ID NO: 8, where SEQ ID NO: 2 contains heavy chain variable region sequence and CDR3 sequence of a second monoclonal antibody, andSEQ ID NO: 8 comprises heavy chain constant region sequence of the second monoclonal antibody.

[0045] In an embodiment, SEQ ID NO: 3 is comprised in SEQ ID NO: 9, where SEQ ID NO: 3 contains heavy chain variable region sequence and CDR3 sequence of a third monoclonal antibody, and SEQ ID NO: 9 comprises heavy chain constant region sequence of the third monoclonal antibody. In an embodiment, SEQ ID NO: 4 is comprised in SEQ ID NO: 10, where SEQ ID NO: 4 contains light chain variable region sequence and CDR3 sequence of a first monoclonal antibody, and SEQ ID NO: 10 comprises light chain constant region sequence of the first monoclonal antibody. In an embodiment, SEQ ID NO: 5 is comprised in SEQ ID NO: 11, where SEQ ID NO: 5 contains light chain variable region sequence and CDR3 sequence of a second monoclonal antibody, and SEQ ID NO: 11 comprises light chain constant region sequence of the second monoclonal antibody.

[0046] In an embodiment, SEQ ID NO: 6 is comprised in SEQ ID NO: 12, where SEQ ID NO: 6 contains light chain variable region sequence and CDR3 sequence of a third monoclonal antibody, and SEQ ID NO: 12 comprises light chain constant region sequence of the third monoclonal antibody. The present invention provides a host cell comprising the expression system as substantially described herein. In an embodiment, the host cell is selected from the group consisting of mammalian host cell, fungal host cell, yeast host cell, avian host cell, insect host cell, bacterial host cell, and plant host cell. In a preferred embodiment, the host cell is mammalian host cell. In an embodiment, a first plasmid and a second plasmid as substantially described herein are comprised within the host cell.

[0047] The present invention provides a plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0048] In an embodiment, there is provided a plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0049] The present invention provides a monoclonal antibody comprising a heavy chain variable region and CDR3 comprising amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a light chain variable region and CDR3 comprising amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.In an embodiment, the monoclonal antibody comprises a heavy chain variable region and CDR3, comprising amino acid sequence as set forth in SEQ ID NO: 1; and a light chain variable region and CDR3, comprising amino acid sequence as set forth in SEQ ID NO: 4.

[0050] In an embodiment, the monoclonal antibody comprises a heavy chain variable region and CDR3, comprising amino acid sequence as set forth in SEQ ID NO: 2; and a light chain variable region and CDR3, comprising amino acid sequence as set forth in SEQ ID NO: 5.

[0051] In an embodiment, the monoclonal antibody comprises a heavy chain variable region and CDR3, comprising amino acid sequence as set forth in SEQ ID NO: 3; and a light chain variable region and CDR3, comprising amino acid sequence as set forth in SEQ ID NO: 6.

[0052] In an embodiment, the monoclonal antibody comprises a heavy chain variable region, CDR3, and heavy chain constant region comprising amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; and a light chain variable region, CDR3, and light chain constant region comprising amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0053] In an embodiment, the monoclonal antibody comprises a heavy chain variable region, CDR3, and heavy chain constant region comprising amino acid sequence as set forth in SEQ ID NO: 7; and a light chain variable region, CDR3, and light chain constant region comprising amino acid sequence as set forth in SEQ ID NO: 10.

[0054] In an embodiment, the monoclonal antibody comprises a heavy chain variable region, CDR3, and heavy chain constant region comprising amino acid sequence as set forth in SEQ ID NO: 8; and a light chain variable region, CDR3, and light chain constant region comprising amino acid sequence as set forth in SEQ ID NO: 11.

[0055] In an embodiment, the monoclonal antibody comprises a heavy chain variable region, CDR3, and heavy chain constant region comprising amino acid sequence as set forth in SEQ ID NO: 9; and a light chain variable region, CDR3, and light chain constant region comprising amino acid sequence as set forth in SEQ ID NO: 12.

[0056] In an embodiment, the monoclonal antibody inhibits at least one serotype of Dengue virus. In an embodiment, the monoclonal antibody inhibits a two or more serotypes of Dengue virus. In an embodiment, the Dengue serotype is Dengue Virus 1. In an embodiment, the Dengue serotype is Dengue Virus 2. In an embodiment, the Dengue serotype is Dengue Virus 3. In an embodiment, the Dengue serotype is Dengue Virus 4.The present invention provides a method of inhibiting at least a serotype of Dengue virus, said method comprising obtaining a therapeutically effective amount of at least a monoclonal antibody as substantially described herein; and contacting said antibody with at least a serotype of Dengue virus. In an embodiment, said contacting of at least a monoclonal antibody as substantially described herein with at least a serotype of Dengue virus is in vivo in a patient. In an embodiment, said contacting of at least a monoclonal antibody as substantially described herein with at least a serotype of Dengue virus is by systemic treatment in a patient

[0057] The present invention provides a medicament comprising at least a monoclonal antibody as substantially described herein. In an embodiment, said medicament is a liquid.

[0058] The present invention also provides use of at least a monoclonal antibody as substantially describe herein for use in treatment of a person diagnosed with at least one serotype of Dengue virus. The present invention also provides use of at least a monoclonal antibody as substantially describe herein for use in preparation of a medicament for use in treatment of Dengue infection in a patient.

[0059] EXAMPLES

[0060] The invention will now be illustrated with working examples, which is intended to illustrate the working of invention and not intended to take restrictively to imply any limitations on the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0061] Dengue virus (DENV) infected patient blood was collected from the Dept, of Paediatrics, AIIMS (New Delhi, India). Peripheral blood mononuclear cells (PBMC’s) and plasma were isolated and were cryo-preserved. Plasma was tested for the presence of antibodies that can neutralize Dengue virus, and the PBMC’s were tested for the presence of plasmablasts, that are transient effector B cells which produce antibodies against an ongoing infection, in this case, Dengue.

[0062] Plasmablasts staining and single cell sorting

[0063] Five million PBMC’s from each dengue sample were washed with FACS buffer and then incubated with efluor780 Fixable Viability (Live Dead) dye (Life Technologies, #65-0865-14) and antihuman CD3, CD 19, CD20, CD27, CD38 and IgD antibodies (BD Biosciences) for 30 minutes. Cells were washed twice with FACS buffer and acquired on BD FACS ARIA Fusion (BD Biosciences) and the cells exhibiting CD3" CD19+CD20" IgD" CD27++CD38++phenotype were single-cell sorted into a 96-well plate containing 10 pl of lysis buffer. The lysis buffer consists 20U RNase inhibitor (Promega), and 10 mM Tris pH 8.0 buffer. The plates with the sorted single cellswere centrifuged gently at 2000 rpm at 4°C and stored immediately at -80°C for at least 1 h before performing the cDNA synthesis. Flow cytometry data was analysed using FlowJo software. 376 single plasmablast cells were sorted from four dengue patient samples and their respective heavy and light chains were amplified. 143 of the cells yielded both heavy and light chain pairs, which were cloned into their respective antibody vector and taken forward for antibody expression. Out of 143, 110 of the antibodies expressed and were tested against DENV antigen using ELISA. 60 out of 110 bound the DENV antigen, and only these antibodies were used for further analysis. As seen in Fig. 1, from the left, Plot 1 shows the live cells population; Plot 2 shows lymphocytes; Plot 3 shows total B cells; Plot 4 shows CD19+CD20'B cells; Plot 5 shows Plasmablast cells and Plot 6 shows single cells sorting of plasmablasts.

[0064] Antibody genes amplification and cloning

[0065] First cDNA synthesis was performed using Maxima H minus First Strand cDNA synthesis kit (Cat#K1652, Thermo Fisher Scientific, USA) as per manufacturer’s protocol. Briefly, 4 pl of 5X RT buffer, 1 pl RT enzyme mix, 1 pl of 10 mM dNTPs mix and 1 pl oligo-dT in a total volume of 20 pl was added to the plate containing sorted cells and lysis buffer. The RT program was set as followed: 20 min at 42°C, 20 min 50°C, 20 min at 65°C, 10 cycles of 2 min at 50°C & 2 min at 42°C, 5 min at 85°C, and infinity 4°C. The cDNA was stored at -20°C until further analysis. The V(D)J variable regions of the antibody genes are amplified from sorted plasmablast single cells, using primers described previously (Tiller et. al., 2009). 3 pl of cDNA in a total volume of 25 pl for 3 min 95°C, 35 cycles of 20 s at 98°C, 15 s at 65°C, 15 s at 72°C, followed by infinite min at 4°C. The nested PCR was performed with and Kapa HiFi polymerase master mix (Roche #KK2602 07958935001) and 1 pl of PCR 1 product in a total volume of 25 pl for 3 min at 95°C, 35 cycles of 20 s at 98°C, 15 s at 65°C followed by infinite min at 4°C. The amplified genes were analyzed on 1.5% agarose gel. Antibody V(D)J of HC and LC genes obtained from a single cell were PCR purified and cloned into respective AbVec6W vector / plasmid (Davis et al 2019, Cell), containing the constant regions of the human IgGl for the heavy chain (AbVec6WhIgGl) or light chain (AbVec6W kappa), using Gibson Assembly and transformed into XL- 10 Gold ultracompetent cells (Agilent). The Gibson Assembly was carried out with an in-house Gibson mix consisting of 2x Gibson mix (0.2 U T5 exonuclease (NEB), 12.5 U Phusion polymerase (NEB), Gibson reaction buffer (0.5 g PEG-8000 (Sigma Life Sciences), 1 M Tris / HCl pH 7.5, 1 M MgCl2, 1 M DTT, 100 mM dNTPs, 50 mM NAD (NEB) and performed for 60 min at 50°C. Next day, 4 colonies from each transformed plate were randomly picked and insert was checked by performing colony PCR using nested PCR primers (Tiller et. al., 2009). The sequence integrity of the plasmids was verified by Sanger sequencing (Macrogen sequencing, South Korea).Expression of monoclonal antibodies

[0066] For small scale transfection, Human expi293 cells (Thermo Fisher Scientific #A14635) were maintained in Expi293 expression medium and transfected at a density of 2 million cells / ml in a volume of 4ml culture per well of a 6 well cell culture plate (Coming). The transfection mix consisted of a 1:1.5 HC / LC ratio using a 1:3 ratio with Img / ml PEImax (Polyscienes) in 200 pL Opti-MEM. After 15-minute incubation at RT, the transfection mix was added onto the cells. Supernatants were harvested 72-96 h post-transfection and clarified supernatants were tested by enzyme-linked immunosorbent assay (ELISA). Supernatant with positive DENV binding signals was next purified using Protein G beads (Thermo Fisher Scientific).

[0067] Dengue virus direct ELISA

[0068] For assessment of DENV specific immunoglobulins, commercial fixed dengue vims from Microbix (Cat number) was coated on MaxiSorp plates (Thermo Fisher Scientific, #439454) at a concentration of 2 pg / mL in 100 pL PBS at 4°C overnight. The plates were washed extensively with PBS containing 0.05% Tween-20. Three-fold serially diluted purified monoclonal antibodies were added to the plates and incubated at room temperature for Ihr. After incubation, the plates were washed and DENV specific IgG was detected by incubating with horseradish peroxidase (HRP) conjugated - anti-human IgG (Jackson ImmunoResearch Labs, #109-036-098). Plates were then washed thoroughly and developed with o-phenylenediamine (OPD) substrate (Sigma, #P8787) in 0.05M phosphate-citrate buffer (Sigma, #P4809) pH 5.0, containing with 0.012% hydrogen peroxide (Thermo Fisher Scientific, #18755) just before use. Absorbance was measured at 490 nm.

[0069] Dengue virus neutralization assay

[0070] Neutralization assay for dengue vims was carried out pre-attachment, i.e., the antibodies were allowed to bind to the vims before putting the vims onto the Vero cells. The antibodies that inhibited vims entry into the Vero cells by blocking it receptor binding domain or viral fusion loop are capable of neutralizing the vims.

[0071] Dengue antibodies were tested against all four dengue serotypes i.e. DENV1-4. Starting with 20pg / ml, antibodies designated as 162-18, 162-19, and 140-3 were two-fold diluted in Opti-MEM and incubated with 1 MOI of dengue vims diluted in Opti-MEM for 1 hour at 37°C in 96-well round bottom plate. The antibody -vims mixture was transferred onto Vero cells, which were seeded a day before in 96-well flat bottom plate. Incubate 2 hrs at 37°C with 5% CO2. Add lOOul of 2% DMEM to make up the final volume as 150pl. The plates were incubated for 24 hours at 37°C with5% C02. Cells were harvested by trypsinization, then washed twice with 200 pl FACS buffer and fixed with 50 pl fixation buffer and kept on ice for 5 min. Cells were washed twice with perm buffer before addition of 50 pl of dengue specific 2H2 antibody conjugated with Alexa flour 488. Incubate for 45 min at 37°C with 90 RPM. The plates were then washed twice with 200 pl FACS buffer and resuspended in 50ul of FACS buffer. Cells were acquired using flow cytometer. Data was analysed using flow jo version 10. The concentration at which 50% of the dengue virus is inhibited is called IC50 of the antibody. IC50 of antibodies is calculated GraphPad prism.

[0072] As seen in Fig 2a-b, this monoclonal antibody comprises heavy chain variable sequence and CDR3 as set forth in SEQ ID NO: 1 and light chain variable sequence and CDR3 as set forth in SEQ ID NO: 4. The said antibody exhibits IC50 value of 0.21pg / ml against Dengue virus 1 (DENV1); 0.32 pg / ml against Dengue virus 2 (DENV2); 0.12 pg / ml against Dengue virus 3 (DENV3); and 0.71 pg / ml against Dengue virus 4 (DENV4).

[0073] As seen in Fig 3a-b, this monoclonal antibody comprises heavy chain variable sequence and CDR3 as set forth in SEQ ID NO: 2 and light chain variable sequence and CDR3 as set forth in SEQ ID NO: 5. The said antibody exhibits IC50 value of 0.45 pg / ml against Dengue virus 1 (DENV1); 0.04 pg / ml against Dengue virus 2 (DENV2); 0.28 pg / ml against Dengue virus 3 (DENV3); and 1.3 pg / ml against Dengue virus 4 (DENV4).

[0074] As seen in Fig 4a-b, this monoclonal antibody comprises heavy chain variable sequence and CDR3 as set forth in SEQ ID NO: 3 and light chain variable sequence and CDR3 as set forth in SEQ ID NO: 6. The said antibody exhibits IC50 value of 0.35 pg / ml against Dengue virus 1 (DENV1); 0.37 pg / ml against Dengue virus 2 (DENV2); 0.24 pg / ml against Dengue virus 3 (DENV3); and 1.2 pg / ml against Dengue virus 4 (DENV4).

[0075] Overall, these figures and experimental data exhibit that the monoclonal antibodies of the present invention show wide-spectrum inhibition of the various serotypes of the Dengue virus, in view of which, the said monoclonal antibodies can be effective for therapeutic applications.

Claims

I / We claim:

1. An expression system comprising:a. a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; andb. a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

2. The expression system as claimed in claim 1, comprising:a. a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 1; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 4; orb. a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 2; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 5; orc. a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 3; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 6.

3. The expression system as claimed in claim 1, comprising:a. a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; andb. a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

4. The expression system as claimed in claim 3, comprising:a. a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 7; and a second plasmidcomprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 10; orb. a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 8; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 11; orc. a first plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 9; and a second plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 12.

5. A host cell comprising an expression system as claimed in claim 1, wherein the host cell is selected from the group consisting of mammalian cell, yeast cell, fungal cell, avian cell, bacterial cell, plant cell, and insect cell.

6. A plasmid comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

7. The plasmid as claimed in claim 6, comprising a polynucleotide fragment encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

8. A monoclonal antibody comprising:a. a heavy chain variable region and CDR3, comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and b. a light chain variable region and CDR3 comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

9. The monoclonal antibody as claimed in claim 8, comprising:a. a heavy chain variable region and CDR3, comprising a sequence as set forth in SEQ ID NO: 1; and a light chain variable region and CDR3 comprising a sequence as set forth in SEQ ID NO: 4; orb. a heavy chain variable region and CDR3, comprising a sequence as set forth in SEQ ID NO: 2; and a light chain variable region and CDR3 comprising a sequence as set forth in SEQ ID NO: 5; orc. a heavy chain variable region and CDR3, comprising a sequence as set forth in SEQ ID NO: 3; and a light chain variable region and CDR3 comprising a sequence as set forth in SEQ ID NO: 6.

10. The monoclonal antibody as claimed in claim 8, comprising:a. a heavy chain variable region, CDR3, and constant region, comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; andb. a light chain variable region, CDR3, and constant region, and constant region, comprising a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

11. The monoclonal antibody as claimed in claim 10, comprising:a. a heavy chain variable region, CDR3, and constant region, comprising a sequence as set forth in SEQ ID NO: 7; and a light chain variable region, CDR3, and constant region comprising a sequence as set forth in SEQ ID NO: 10; or b. a heavy chain variable region, CDR3, and constant region, comprising a sequence as set forth in SEQ ID NO: 8; and a light chain variable region, CDR3, and constant region comprising a sequence as set forth in SEQ ID NO: 11; or c. a heavy chain variable region, CDR3, and constant region, comprising a sequence as set forth in SEQ ID NO: 9; and a light chain variable region, CDR3, and constant region, comprising a sequence as set forth in SEQ ID NO: 12.

12. A polynucleotide fragment encoding a polypeptide comprising amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

13. A method of inhibiting at least a serotype of Dengue virus, said method comprising: a. obtaining a therapeutically effective amount of at least a monoclonal antibody as claimed in claim 8; andb. contacting said antibody with at least a serotype of Dengue virus.

14. A medicament comprising at least a monoclonal antibody as claimed in claim 8.

15. A monoclonal antibody as claimed in claim 8 for use in treatment or preparing a 5 medicament for at least a serotype of Dengue virus.

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