# Patent

## Title
Monoclonal antibodies for Ebola and Marburg viruses

## Abstract

Described herein are a number of Ebola and Marburg monoclonal antibodies.

## Background
PRIOR APPLICATION INFORMATION
The instant application claims the benefit of U.S. Provisional Patent Application 61/025,491, filed Feb. 1, 2008.


BACKGROUND OF THE INVENTION
Ebola and Marburg viruses are highly pathogenic and virulent viruses causing rapidly fatal haemorrhagic fever in humans.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a monoclonal antibody comprising an amino acid sequence deduced from 1H3-light (SEQ ID No. 2); 2G4-light (SEQ ID No. 4); 4G7-light (SEQ ID No. 6); 5D2-light (SEQ ID No. 8); 5E6-light (SEQ ID No. 10); 7C9-light (SEQ ID No. 12); 7G4-light (SEQ ID No. 14), 10C8-light (SEQ ID No. 16), 1H3-heavy (SEQ ID No. 1); 2G4-heavy (SEQ ID No. 3); 4G7-heavy (SEQ ID No. 5); 5D2-heavy (SEQ ID No. 7), 5E6-heavy (SEQ ID No. 9), 7C9-heavy (SEQ ID No. 11), 7G4-heavy (SEQ ID No. 13) and 10C8-heavy (SEQ ID No. 15).
According to a second aspect of the invention, there is provided a method of preparing a chimeric antibody comprising:
providing an expression vector comprising a nucleic acid molecule encoding a constant region domain of a human light chain or heavy chain genetically linked to a nucleic acid encoding a light chain variable region selected from the group consisting of 1H3-light (SEQ ID No. 2); 2G4-light (SEQ ID No. 4); 4G7-light (SEQ ID No. 6); 5D2-light (SEQ ID No. 8); 5E6-light (SEQ ID No. 10); 7C9-light (SEQ ID No. 12); 7G4-light (SEQ ID No. 14) and 10C8-light (SEQ ID No. 16) or a heavy chain variable region selected from the group consisting of 1H3-heavy (SEQ ID No. 1); 2G4-heavy (SEQ ID No. 3); 4G7-heavy (SEQ ID No. 5); 5D2-heavy (SEQ ID No. 7), 5E6-heavy (SEQ ID No. 9), 7C9-heavy (SEQ ID No. 11), 7G4-heavy (SEQ ID No. 13) and 10C8-heavy (SEQ ID No. 15);
expressing the expression vector in a suitable host; and
recovering the chimeric antibody from said host.
According to a third aspect of the invention, there is provided a method of preparing a recombinant antibodies comprising:
providing a nucleotide sequence selected from the group consisting of 1H3-light (SEQ ID No. 2); 2G4-light (SEQ ID No. 4); 4G7-light (SEQ ID No. 6); 5D2-light (SEQ ID No. 8); 5E6-light (SEQ ID No. 10); 7C9-fight (SEQ ID No. 12); 7G4-light (SEQ ID No. 14), 10C8-light (SEQ ID No. 16), 1H3-heavy (SEQ ID No. 1); 2G4-heavy (SEQ ID No. 3); 4G7-heavy (SEQ ID No. 5); 5D2-heavy (SEQ ID No. 7), 5E6-heavy (SEQ ID No. 9), 7C9-heavy (SEQ ID No. 11), 7G4-heavy (SEQ ID No. 13) and 10C8-heavy (SEQ ID No. 15);
modifying said nucleic acid sequence such that at least one but fewer than about 30 of the amino acid residues encoded by said nucleic acid sequence has been changed or deleted without disrupting antigen binding of said peptide; and
expressing and recovering said modified nucleotide sequence.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Kaplan-Meier survival curve of mice infected with MA-ZEBOV and treated with MAbs 1 day after infection. Survival curve of MA-Ebola virus-infected mice treated with 100 μg of MAbs. Mice were intraperitoneally treated with 100 μg of each MAb on day 1. Control mice were given equal volumes of PBS.
FIG. 2. Weight changes of GPA-Ebola infected guinea pigs treated with MAbs. Weight changes of virus-infected guinea pigs treated with cocktail of MAbs. Guinea pigs were intraperitoneally treated with either 5D2, 5E6, 7C9, 7G4 or 10C8 (3 mg/treatment) on day 1 and 4G7+1H3+2G4 [(2 mg+1 mg+1 mg)/treatment] on day 2. Control guinea pig were given equal volume of PBS. The results are shown as the means and standard deviations of 6 guinea pigs.
FIG. 3. Weight changes of GPA-Ebola infected guinea pigs treated with MAbs. Weight changes of virus-infected guinea pigs treated with cocktail of MAbs. Guinea pigs were intraperitoneally treated with either 5D2, 5E6, 7C9, 7G4 or 10C8 (3 mg/treatment) on day 1 and 4G7+1H3+2G4 [(2 mg+1 mg+1 mg)/treatment] on day 2. Control guinea pig were given equal volume of PBS. The results are shown as the group weight of 6 guinea pigs.
FIG. 4. Immunoprecipitation. 293T cells were transfected with pCAGGS-ZEbovGP1,2 by using Fugene 6. After 48 hrs, cells were collected and washed 2× with cold PBS before being lysed with 2× RIPA buffer. After clarifying the cell lysate, 100 μg protein was added to each McAb (5 μg) coupled protein A+G beads. The IP samples were run 10% SDS-PAGE and transferred to Hybond-P membrane. The blot was probed with mouse ant-EBOV-GP1.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
DEFINITIONS
As used herein, “neutralizing antibody” refers to an antibody, for example, a monoclonal antibody, capable of disrupting a formed viral particle or inhibiting formation of a viral particle or prevention of binding to or infection of mammalian cells by a viral particle.
As used herein, “diagnostic antibody” or “detection antibody” or “detecting antibody” refers to an antibody, for example, a monoclonal antibody, capable of detecting the presence of an antigenic target within a sample. As will be appreciated by one of skill in the art, such diagnostic antibodies preferably have high specificity for their antigenic target.
As used herein, “humanized antibodies” refer to antibodies with reduced immunogenicity in humans.
As used herein, “chimeric antibodies” refer to antibodies with reduced immunogenicity in humans built by genetically linking a non-human Variable region to human constant domains.
Described herein are a number of Ebola and Marburg monoclonal antibodies. Specifically, antigens were developed using a live replicating vector vesicular stomatitis virus described in PCT Application PCT/CA03/001125.
The VSV based vaccine delivery system was used to develop monoclonal antibodies in mice.
Specifically, described herein are monoclonal antibodies 1H3, 2G4, 4G7, 5D2, 5E6, 7C9, 7G4 and 10C8. As discussed below, 1H3 comprises 1H3-heavy chain (SEQ ID No. 1) and 1H3-light chain (SEQ ID No. 2); 2G4 comprises 2G4-heavy chain (SEQ ID No. 3) and 2G4-light chain (SEQ ID No. 4); 4G7 comprises 4G7-heavy chain (SEQ ID No. 5) and 4G7-light chain (SEQ ID No. 6); 5D2 comprises 5D2-heavy chain (SEQ ID No. 7) and 5D2-light chain (SEQ ID No. 8); 5E6 comprises 5E6-heavy chain (SEQ ID No. 9) and 5E6-light chain (SEQ ID No. 10); 7C9 comprises 7C9-heavy chain (SEQ ID No. 11) and 7C9-light chain (SEQ ID No. 12); 7G4 comprises 7G4-heavy chain (SEQ ID No. 13) and 7G4-light chain (SEQ ID No. 14); and 10C8 comprises 10C8-light chain (SEQ ID No. 16) and 10C8-heavy chain (SEQ ID No. 15).
These antibodies also appear to have high affinity and avidity to Ebola glycoproteins, which means that they could be used as highly sensitive diagnostic tools.
For example, as shown in FIG. 1, mice infected with MA-ZEBOV and subsequently treated with the monoclonal antibodies described above showed increased survival compared to mice treated with PBS. Results are summarized in Tables 1 and 2.
FIGS. 2 and 3 show weight changes in guinea pigs treated with the monoclonal antibodies or mixtures thereof post infection. As can be seen, guinea pigs treated with the monoclonal antibodies showed consistent weight while those treated with PBS showed significant weight loss. Results are summarized in Table 3.
The nucleotide sequences of the heavy and light chains of 1H3, 2G4, 4G7, 5D2, 5E6, 7C9, 7G4 and 10C8 follow. As will be appreciated by one of skill in the art, the amino acid sequences of these antibodies can easily be deduced from the nucleotide sequences. Accordingly, in some embodiments, the invention is directed to amino acid sequences deduced from 1H3-light (SEQ ID No. 2); 2G4-light (SEQ ID No. 4); 4G7-light (SEQ ID No. 6); 5D2-light (SEQ ID No. 8); 5E6-light (SEQ ID No. 10); 7C9-light (SEQ ID No. 12); 7G4-light (SEQ ID No. 14), 10C8-light (SEQ ID No. 16), 1H3-heavy (SEQ ID No. 1); 2G4-heavy (SEQ ID No. 3); 4G7-heavy (SEQ ID No. 5); 5D2-heavy (SEQ ID No. 7), 5E6-heavy (SEQ ID No. 9), 7C9-heavy (SEQ ID No. 11), 7G4-heavy (SEQ ID No. 13) and 10C8-heavy (SEQ ID No. 15).







mAb 1H3 heavy chain sequence: 373 bp


(SEQ ID No. 1)


TGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACA


 


GCTTCTGGCTTCAACATTAAAGACACCTATATACATTGGGTGAAACAGG


 


GGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGG


 


TAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATCACAGCA


 


GACACATCCTCCAATACAGCCTACCTGCAGCTCAGCGGCCTGACATCTG


 


AGGACACTGCCGTCTATTACTGTGCTAGGGAGTCGAGGATATCTACTAT


 


GCTTACGACGGGGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACA


 


GTCTCCTCAGCCAAAACAACAGCCCCATCG


 


mAb 1H3 light chain sequence: 303 bp


(SEQ ID No. 2)


GCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTG


 


CCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATC


 


CTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGCTTCTGGAGTC


 


CCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAA


 


TCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTG


 


GAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA


 


CGGGCTGAT


 


mAb 2G4 heavy chain sequence: 364 bp


(SEQ ID No. 3)


TGGAGGAGGCTTGATGCAACCTGGAGGATCCATGAAACTCTCCTGTGTT


 


GCCTCAGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGT


 


CTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAA


 


TAATTATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATT


 


TCAAGAGATGATTCCAAAAGGAGTGTCTACCTGCAAATGAATACCTTAA


 


GAGCTGAAGACACTGGCATTTATTACTGTACCCGGGGGAATGGTAACTA


 


CAGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA


 


GCCAAAACAACACCCCCATCA


 


mAb 2G4 light chain sequence: 306 bp


(SEQ ID No. 4)


GCCTCCCTATCTGTATCTGTGGGAGAAACTGTCTCCATCACATGTCGAG


 


CAAGTGAGAATATTTACAGTAGTTTAGCATGGTATCAGCAGAAACAGGG


 


AAAATCTCCTCAGCTCCTGGTCTATTCTGCAACAATCTTAGCAGATGGT


 


GTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACTCAGTATTCCCTCA


 


AGATCAACAGCCTGCAGTCTGAAGATTTTGGGACTTATTACTGTCAACA


 


TTTTTGGGGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA


 


AAACGGGCTGAT


 


mAb 4G7 heavy chain sequence: 358 bp


(SEQ ID No. 5)


TGGACCTGAGCTGGAGATGCCTGGCGCTTCAGTGAAGATATCCTGCAAG


 


GCTTCTGGTTCCTCATTCACTGGCTTCAGTATGAACTGGGTGAAGCAGA


 


GCAATGGAAAGAGCCTTGAGTGGATTGGAAATATTGATACTTATTATGG


 


TGGTACTACCTACAACCAGAAATTCAAGGGCAAGGCCACATTGACTGTG


 


GACAAATCCTCCAGCACAGCCTACATGCAGCTCAAGAGCCTGACATCTG


 


AGGACTCTGCAGTCTATTACTGTGCAAGATCGGCCTACTACGGTAGTAC


 


TTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAA


 


ACAACAGCCCCATCG


 


mAb 4G7 light chain sequence: 306 bp


(SEQ ID No. 6)


GCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAG


 


CAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGG


 


AAAATCTCCTCAGCTCCTGGTCTATAATGCCAAAACCTTAATAGAGGGT


 


GTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGA


 


AGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTTCTGTCAACA


 


TCATTTTGGTACTCCATTCACATTCGGCTCGGGGACAGAGTTGGAAATA


 


AAACGGGCTGAT


 


mAb 5D2 heavy chain sequence: 340 bp


(SEQ ID No. 7)


GGGACCTGGCCTGGTGAGACCTTCTCAGTCTCTGTCCCTCACCTGCACT


 


GTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGC


 


AGTTTCCAGGAAACAAACTGGAGTGGCTGGGCTATATAACCAACACTGG


 


TAGCACTGGCTTCAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGA


 


GACACATCCAAGAACCAGTTCTTCCTGCAGTTGATTTCTGTGACTACTG


 


AGGACACAGCCACATATCACTGTGCAAGGGGCCTTGCTTACTGGGGCCA


 


AGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAGCCCCATCG


 


mAb 5D2 light chain sequence: 321 bp


(SEQ ID No. 8)


CTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGT


 


CAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATCTGAATTGGTT


 


GTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCT


 


AAACTGGACTCTGGAGTCACTGACAGGTTCACTGGCAGTGGATCAGGGA


 


CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGT


 


TTATTATTGTTGGCAAGGTACACACTCTCCATTCACGTTCGGCTCGGGG


 


ACAAAGTTGGAAATAAAACGGGCTGAT


 


mAb 5E6 heavy chain sequence: 370 bp


(SEQ ID No. 9)


TGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCA


 


GCCTCTGGATCCGCTTTCAGTAGATATGACATGTCTTGGGTTCGCCAGA


 


CTCCGGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTCGTGGTGGTGG


 


TTTCATCTACTATCCAGACACTGTGAAGGGCCGATTCACCATCTCCAGA


 


GACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTG


 


ACGACACAGCCATGTATTACTGTGCAAGACACGTTTACTACGGTAGTAG


 


CCCCCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTC


 


TCCTCAGCCAAAACAACAGCCCCATCG


 


mAb 5E6 light chain sequence: 324 bp


(SEQ ID No. 10)


TCAGCCTCTTTCTCCCTGGGAGCCTCAGCAAAACTCACGTGCACCTTGA


 


GTAGTCAGCACAGTACGTTCACCATTGAATGGTATCAGCAACAGCCACT


 


CAAGCCTCCTAAGTATGTGATGGAGCTTAAGAAAGATGGAAGCCACAGT


 


ACAGGTGATGGGATTCCTGATCGCTTCTCTGGATCCAGCTCTGGTGCTG


 


ATCGCTACCTTAGCATTTCCAACATCCAGCCTGAAGATGAAGCAATATA


 


CATCTGTGGTGTGGGTGATACAATTAATGAACAATTTGTGTATGTTTTC


 


GGCGGTGGAACCAAGGTCACTGTCCTAGGT


 


mAb 7C9 heavy chain sequence: 358 bp


(SEQ ID No. 11)


TGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACA


 


GCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGGAGA


 


GGCCTGACAAGGGCCTGGAGTGGATTGGAAGGATTGATCCAGCGAATGG


 


TAATACTAAATGTGACTCGAGGTTTCAGGGCAAGGCCACTATAACAGCA


 


GACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTG


 


AGGACACTGCCGTCTATTACTGTGCTAGAAGGATCTACTTTGGTAAGGG


 


CTTTGACTTTTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAA


 


ACAACAGCCCCATCG


 


mAb 7C9 light chain sequence: 324 bp


(SEQ ID No. 12)


TCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGT


 


CCAGTCAGAGTCTGTTTAACAGTGGAGATCAAAAGAACTACTTGGCCTG


 


GTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACGGGGCA


 


TCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTG


 


GAACCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGC


 


AGTTTATTACTGTCAGAATGATCAATTTTATCCTCCCACGTTCGGTGAT


 


GGGACCAAGCTGGACCTGAAACGGGCTGAT


 


mAb 7G4 heavy chain sequence: 367 bp


(SEQ ID No. 13)


TGGAGGGGGCTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCA


 


ACTTCTGGCTTCACCTTTACTGATCACTACATGGGCTGGGTCCGCCAGC


 


CTCCAGGAAAGGCACTTGAGTGGTTGGCTTTTGTTAGATACAAAGCTAA


 


GGGTTACACAACAGAGTACACTGCATCTGTGAAGGGTCGGTTCACCATC


 


TCCAGAGATAATTCCCAAAGCATCCTCTATCTTCAAATGAACACCCTGA


 


GAACTGAGGACAGTGCCACTTATTACTGTGCAAGAGATAGAGGGGGTTA


 


CGTGGGAGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC


 


TCAGCCAAAACGACACCCCCATCT


 


mAb 7G4 light chain sequence: 321 bp


(SEQ ID No. 14)


CTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGAT


 


CTAGTCAGAGCCTTGTACACAGGAATGGAAACACCTATTTCCATTGGTA


 


CCTGCAGAAGCCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCC


 


AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGA


 


CAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGT


 


TTATTTCTGCTCTCAAAGTACACATGTTCCGTACACTTTCGGAGGGGGG


 


ACCAAGCTGGAAATAAAACGGGCTGAT


 


mAb 10C8 heavy chain sequence: 352 bp


(SEQ ID No. 15)


TGGGGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACA


 


TCTTCTGGCTTCAACATTAAAGACTACTTTCTACACTGGGTGAAACAGA


 


GGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGG


 


TGATACTGAATATGCCCCGAAGTTCCAGGACAAGGCCACTATGACTGCA


 


GACACATCCTCCAACACAGCCTACCTGCACCTCAGCAGCCTGACATCTG


 


AGGACACTGGCGTCTATTACTGTAATGCAGATGGTAACTACGGGAAGAA


 


CTACTGGGGCCAAGGCACCACTCTCACCGTCTCCTCAGCCAAAACAACA


 


GCCCCATCG


 


mAb 10C8 light chain sequence: 324 bp


(SEQ ID No. 16)


CTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGAT


 


CTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTTTTTACATTGGTA


 


CCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAGAGTTTCC


 


AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGA


 


CAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGT


 


TTATTTCTGCTCTCAAAGTACACATGTTCCTCCGTACACGTTCGGAGGG


 


GGGACCAAGCTGGAAATAAAACGGGCTGAT






In another embodiment of the invention, one or more of the nucleic acid sequences described above encoding the antibody are subjected to humanization techniques or converted into chimeric human molecules for generating a variant antibody which has reduced immunogenicity in humans. Humanization techniques are well known in the art—see for example U.S. Pat. No. 6,309,636 and U.S. Pat. No. 6,407,213 which are incorporated herein by reference specifically for their disclosure on humanization techniques. Chimerics are also well known, see for example U.S. Pat. No. 6,461,824, U.S. Pat. No. 6,204,023, U.S. Pat. No. 6,020,153 and U.S. Pat. No. 6,120,767 which are similarly incorporated herein by reference.
In one embodiment of the invention, chimeric antibodies are prepared by preparing an expression vector which comprises a nucleic acid encoding a constant region domain of a human light or heavy chain genetically linked to a nucleic acid encoding a light chain variable region selected from the group consisting of 1H3-light (SEQ ID No. 2); 2G4-light (SEQ ID No. 4); 4G7-light (SEQ ID No. 6); 5D2-light (SEQ ID No. 8); 5E6-light (SEQ ID No. 10); 7C9-light (SEQ ID No. 12); 7G4-light (SEQ ID No. 14) and 10C8-light (SEQ ID No. 16) or a heavy chain variable region selected from the group consisting of 1H3-heavy (SEQ ID No. 1); 2G4-heavy (SEQ ID No. 3); 4G7-heavy (SEQ ID No. 5); 5D2-heavy (SEQ ID No. 7), 5E6-heavy (SEQ ID No. 9), 7C9-heavy (SEQ ID No. 11), 7G4-heavy (SEQ ID No. 13) and 10C8-heavy (SEQ ID No. 15). It is of note that all of these sequences are described above.
In another embodiment of the invention, there are provided recombinant antibodies comprising at least one modified variable region, said region selected from the group consisting of 1H3-light (SEQ ID No. 2); 2G4-light (SEQ ID No. 4); 4G7-light (SEQ ID No. 6); 5D2-light (SEQ ID No. 8); 5E6-light (SEQ ID No. 10); 7C9-light (SEQ ID No. 12); 7G4-light (SEQ ID No. 14), 10C8-light (SEQ ID No. 16), 1H3-heavy (SEQ ID No. 1); 2G4-heavy (SEQ ID No. 3); 4G7-heavy (SEQ ID No. 5); 5D2-heavy (SEQ ID No. 7), 5E6-heavy (SEQ ID No. 9), 7C9-heavy (SEQ ID No. 11), 7G4-heavy (SEQ ID No. 13) and 10C8-heavy (SEQ ID No. 15), in which at least one but fewer than about 30 of the amino acid residues of said variable region has been changed or deleted without disrupting antigen binding. It is of note that all of these sequences are described above.
In yet other embodiments, immunoreactive fragments of any of the above-described monoclonal antibodies, chimeric antibodies or humanized antibodies are prepared using means known in the art, for example, by preparing nested deletions using enzymatic degradation or convenient restriction enzymes.
It is of note that in all embodiments describing preparation of humanized antibodies, chimeric antibodies or immunoreactive fragments of monoclonal antibodies, these antibodies are screened to ensure that antigen binding has not been disrupted. This may be accomplished by any of a variety of means known in the art, but one convenient method would involve use of a phage display library. As will be appreciated by one of skill in the art, as used herein, ‘immunoreactive fragment’ refers in this context to an antibody fragment reduced in length compared to the wild-type or parent antibody which retains an acceptable degree or percentage of binding activity to the target antigen. As will be appreciated by one of skill in the art, what is an acceptable degree will depend on the intended use.
It is of note that as discussed herein, any of the above-described antibody or humanized variant thereof may be formulated into a pharmaceutical treatment for providing passive immunity for individuals suspected of or at risk of developing hemorrhagic fever comprising a therapeutically effective amount of said antibody. The pharmaceutical preparation may include a suitable excipient or carrier. See, for example, Remington: The Science and Practice of Pharmacy, 1995, Gennaro ed. As will be apparent to one knowledgeable in the art, the total dosage will vary according to the weight, health and circumstances of the individual as well as the efficacy of the antibody.
While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.







TABLE 1







Dose-dependent protective efficacy of McAbs in mice











Dose
Meantime to



Treatmenta
(μg/treatment)
deathb
No. of survivors/total













McAb 4G7
100
7.00 (n = 1)
5/6



50
7.00 (n = 1)
5/6



25
6.00 (n = 3)
3/6



12.5
6.80 (n = 5)
1/6



6.25
8.20 (n = 5)
2/6


McAb 5D2
100
N/Ac
6/6



50
N/Ac
6/6



25
N/Ac
6/6



12.5
N/Ac
6/6



6.25
7.50 (n = 2)
4/6


McAb 5E6
100
N/Ac
6/6



50
N/Ac
6/6



25
N/Ac
6/6



12.5
6.50 (n = 2)
4/6



6.25
6.67 (n = 3)
3/6


McAb 7C9
100
N/Ac
6/6



50
N/Ac
6/6



25
7.00 (n = 1)
5/6



12.5
7.00 (n = 1)
5/6



6.25
6.50 (n = 4)
2/6


McAb 7G4
100
N/Ac
6/6



50
7.50 (n = 1)
4/6



25
7.00 (n = 1)
5/6



12.5
7.60 (n = 5)
1/6



6.25
6.60 (n = 5)
1/6


McAb 10C8
100
7.00 (n = 1)
5/6



50
7.00 (n = 1)
5/6



25
7.50 (n = 4)
2/6



12.5
7.00 (n = 5)
1/6



6.25
6.40 (n = 5)
1/6


PBS

5.80 (n = 5)
0/5





aMice were intraperitoneally treated with antibodies 1 day after challenge with 1000 LD50 of the mouse-adapted Ebola virus.


bData for animals that died (numbers of animals are shown in parentheses).


cN/A: not applicable.













TABLE 2







Time dependency of the protective efficacy of MAbs in mice










MAbs
Day of treatmenta
Mean time to deathb
No. of survivors/total













1H3
−4
6.70 ± 0.61 (n = 10)
0/10


100 μg
−1
6.60 ± 0.61 (n = 10)
0/15



+1
8.10 ± 0.74 (n = 9)
6/15



+2
6.60 ± 0.80 (n = 5)
5/10



+3
6.40 ± 0.97 (n = 10)
0/10


2G4
−4
7.40 ± 0.63 (n = 10)
0/10


100 μg
−1
7.86 ± 0.74 (n = 14)
1/15



+1
8.00 (n = 6)
9/15



+2
7.30 ± 0.47 (n = 3)
7/10



+3
5.70 ± 1.13 (n = 10)
0/10


4G7
−4
7.42 ± 0.46 (n = 7)
3/10


100 μg
−1
7.08 ± 0.74 (n = 14)
1/15



+1
8.25 ± 0.43 (n = 4)
11/15 



+2
n/ac
10/10 



+3
5.67 ± 1.34 (n = 9)
1/10


5D2
−4
7.00 (n = 1)
9/10


100 μg
−1
8.00 ± 1.00 (n = 2)
13/15 



+1
n/a
15/15 



+2
7.00 (n = 4)
6/10



+3
6.30 ± 1.05 (n = 10)
0/10


5E6
−4
7.00 (n = 2)
8/10


100 μg
−1
8.25 ± 0.43 (n = 4)
11/15 



+1
7.00 (n = 1)
14/15 



+2
6.00 (n = 1)
9/10



+3
5.80 ± 1.03 (n = 10)
0/10


7C9
−4
7.00 (n = 1)
9/10


100 μg
−1
7.75 ± 0.43 (n = 4)
11/15 



+1
8.00 ± 0.82 (n = 3)
12/15 



+2
7.00 (n = 1)
9/10



+3
6.10 ± 0.67 (n = 10)
0/10


7G4
−4
8.20 ± 0.71 (n = 10)
0/10


100 μg
−1
8.07 ± 0.59 (n = 14)
1/15



+1
n/a
15/15 



+2
7.10 ± 0.57 (n = 9)
1/10



+3
6.70 ± 0.44 (n = 10)
0/10


10C8
−4
7.83 ± 0.64 (n = 6)
4/10


100 μg
−1
7.64 ± 1.17 (n = 14)
1/15



+1
8.50 ± 0.50 (n = 2)
13/15 



+2
6.83 ± 0.37 (n = 6)
4/10



+3
6.30 ± 1.13 (n = 10)
0/10


17F8d
−4
6.00 ± 1.10 (n = 9)
1/10


100 μg
−1
6.13 ± 0.88 (n = 15)
0/15



+1
7.21 ± 0.86 (n = 14)
1/15



+2
6.10 ± 0.83 (n = 10)
0/10



+3
6.00 ± 1.13 (n = 10)
0/10


PBS
−4
5.40 ± 1.43 (n = 10)
0/10



−1
6.60 ± 0.80 (n = 5)
0/5 



+3
5.00 ± 0.60 (n = 10)
0/10





aMice were intraperitoneally treated with each MAb at indicated time before or after challenge with 1000 LD50 of the mouse-adapted Ebola virus.


bData for animals that died (numbers of animals are shown in parentheses).


cN/A: not applicable.


dControl Mab: anti-MAR GP.













TABLE 3







Protective efficacy of MAbs in guinea pigs











Day of
Meantime
No. of


Treatment
treatmenta
to deathb
survival/Tatalc





Cocktail of





5D2(3 mg) +
1




4G7(2 mg) + 1H3(1 mg) +
2
N/Ad
6/6


2G4(1 mg)





Cocktail of





5E6(3 mg) +
1




4G7(2 mg) + 1H3(1 mg) +
2
N/A
6/6


2G4(1 mg)





Cocktail of





7C9(3 mg) +
1




4G7(2 mg) + 1H3(1 mg) +
2
N/A
6/6


2G4(1 mg)





Cocktail of





7G4(3 mg) +
1




4G7(2 mg) + 1H3(1 mg) +
2
N/A
6/6


2G4(1 mg)





Cocktail of





10C8(3 mg) +
1




4G7(2 mg) + 1H3(1 mg) +
2
9.00(n = 1)
5/6


2G4(1 mg)





Cocktail of





PBS +
1




PBS
2
7.00(n = 6)
0/6





aGuinea pigs were intraperiotoneally treated with the MAbs as showed dose in the table on the indicated days after challenge with 1000 LD50 of the guinea pig-adapted Ebola virus.


bData for all animals that died(numbers of animals are shown in parentheses).


cSurvival rate on day 28 after challenge.


dN/A: not applicable.













TABLE 4







Summary of ELISA Result of Anti-Ebola-GP McAbs









Antigen



















Rf-GP1
Mucin







sGP
sub-f-D
domain
GP1





eGP1,2
1-
157-
333-
1-


McAb
Isotype
eVLPs
ΔTm
295aa
369aa
458aa
501aa





1H3
IgG2a,κ
+
+
+
−
−
+


2G4
IgG2b,κ
+
+
−
−
−
−


4G7
IgG2a,κ
+
+
−
−
−
+


5D2
IgG2a,κ
+
+
−
+
+
+


5E6
IgG2a,λ
+
+
−
−
+
+


7C9
IgG2a,κ
+
+
−
+/−
+
+


7G4
IgG1,κ
+
+
−
−
+/−
+


10C8
IgG2a,κ
+
+
−
−
+/−
+





Antigens (0.3 μg/well) were coated in 96 well microtitre plate then blocking with 2% skim milk. Serial dilutions of each MAb were applied to the plate followed by HRP-conjugated goat anti-mouse IgG. After incubabing with substrate, the asorbance awas read at OD405. Cut off was 2X background.













TABLE 5







Prolonged survival seen in McAb-treated Guinea pigs











Treatmenta
Mean time to deathb
Student's t-test






MAb 1H3
11.7 ± 2.18 (n = 5)
p = 0.0181



MAb 2G4
11.5 ± 1.50 (n = 2)
N/Ac



MAb 4G7
10.5 ± 1.50 (n = 2)
N/Ac



MAb 5D2
 9.4 ± 1.02 (n = 5)
p = 0.0244



MAb 5E6
10.8 ± 1.47 (n = 5)
p = 0.0092



MAb 7C9
 9.6 ± 0.80 (n = 5)
p = 0.0056



MAb 7G4
 9.6 ± 0.80 (n = 5)
p = 0.0056



MAb 10C8
 9.4 ± 1.20 (n = 5)
p = 0.0428



PBS
7.67 ± 0.75 (n = 6)
N/Ac





aGuinea pigs were intraperiotoneally treated with 5 mg of the MAb as showed in the table on day 1 after challenge with 1000 LD50 of the guinea pig-adapted Ebola virus.


bData for all animals that died (numbers of animals are shown in parentheses).


cN/A: not applicable.













TABLE 6







Protective efficacy of MAbs in guinea pigs













No. of



Day of

survival/


Treatment
treatmenta
Meantime to deathb
Tatalc





Cocktail of 4G7(2 mg) +
−1
11.17 ± 3.09 (n = 3)
3/6


1H3(1.5 mg) +





2G4(1.5 mg)





Cocktail of 4G7(2 mg) +
+1
 7.92 ± 0.42 (n = 3)
3/6


1H3(1.5 mg) +





2G4(1.5 mg)





Cocktail of 4G7(2 mg) +
+2
N/Ad
6/6


1H3(1.5 mg) +





2G4(1.5 mg)





Cocktail of 4G7(2 mg) +
+3
11.17 ± 3.09 (n = 3)
4/6


1H3(1.5 mg) +





2G4(1.5 mg)





PBS
+2
 6.58 ± 0.59 (n = 6)
3/6





aGuinea pigs were intraperiotoneally treated with the MAbs as showed dose in the table on the indicated days before or after challenge with 1000 LD50 of the guinea pig-adapted Ebola virus.


bData for all animals that died(numbers of animals are shown in parentheses).


cSurvival rate on day 28 after challenge.


dN/A: not applicable.













TABLE 7







Epitopes bound by ZEbov GP McAbs











Ebola GPs with

epitope


mAb name
epitope
epitope sequence
position





1H3(IgG2a/κ):
sGPa
SNTTGKLIWKVNPEI
267-280aa




(SEQ ID NO: 17)



2G4(IgG2b/κ):
GP2a
REAIVNAQPKCNPNL
502-516aa




(SEQ ID NO: 18)



4G7(IgG2a/κ):
GP2a
REAIVNAQPKCNPNL
502-516aa




(SEQ ID NO: 19)



5D2 (IgG2a/κ):
GP1b,c,d
DPGTNTTTEDHKIMA
329-343aa




(SEQ ID NO: 20)



5E6 (IgG2a/λ):
GP1b,c,d
ATQVEQHHRRTDNDS
401-415aa




(SEQ ID NO: 21)



7C9(IgG2a,κ):
GP1b.c
unknown
unknown


7G4(IgG1, κ):
GP1b,c
unknown
unknown


10C8(IgG2a,κ):
GP1b.c
unknown
unknown





adetermined by using recombinant vesicular stamatitis virus(VSV) containing ZEbov GP gene to identify the amino acid changes in antigenic variants that escape antibody neutralization;


bdetermined by Western blot reactivity with Ebola Zaire 1976 or VLPs,


cdetermined by ELISA using recombinant GP1 protein;


ddetermined by ELISA using peptide library.

## Claims

1. A monoclonal antibody that binds Ebola glycoprotein comprising a light chain variable region comprising the amino acid sequence deduced from the nucleic acid molecule as set forth in SEQ ID NO: 6 and a heavy chain variable region comprising the amino acid sequence deduced from the nucleic acid molecule as set forth in SEQ ID NO:5.

2. A method of preparing a chimeric antibody that binds Ebola glycoprotein comprising:
providing an expression vector comprising a nucleic acid molecule encoding a constant region domain of a human light chain genetically linked to a nucleic acid molecule encoding a light chain variable region comprising the nucleic acid molecule as set forth in SEQ ID No: 6; and
providing an expression vector comprising a nucleic acid molecule encoding a constant region domain of a human heavy chain genetically linked to a nucleic acid molecule encoding a heavy chain variable region comprising the amino acid sequence deduced from the nucleic acid molecule as set forth in SEQ ID No. 5;
expressing the expression vectors in a suitable host; and
recovering the chimeric antibody that binds Ebola glycoprotein from said host.

3. A method of preparing a chimeric antibody that binds Ebola glycoprotein comprising:
providing an expression vector comprising a nucleic acid molecule encoding a constant region domain of a human light chain genetically linked to a nucleic acid molecule encoding a light chain variable region comprising the nucleic acid molecule as set forth in SEQ ID No: 6; and a nucleic acid molecule encoding a constant region domain of a human heavy chain genetically linked to a nucleic acid molecule encoding a heavy chain variable region comprising the amino acid sequence deduced from the nucleic acid molecule as set forth in SEQ ID No. 5;
expressing the expression vector in a suitable host; and
recovering the chimeric antibody that binds Ebola glycoprotein from said host.

4. A pharmaceutical composition comprising the monoclonal antibody of claim 1 and a pharmaceutically acceptable excipient or carrier.