Link:  Pharm/Biotech Resources

Title:  Monoclonal antibodies and complementarity-determining regions binding to Ebola glycoprotein

United States Patent:  6,875,433

Issued:  April 5, 2005

Inventors:  Hart; Mary Kate (Frederick, MD); Wilson; Julie (Birmingham, AL)

Assignee:  The United States of America as represented by the Secretary of the Army (Washington, DC)

Appl. No.:  226795

Filed:  August 23, 2002

Abstract

In this application are described Ebola GP monoclonal antibodies, epitopes recognized by these monoclonal antibodies, and the sequences of the variable regions of some of these antibodies. Also provided are mixtures of antibodies of the present invention, as well as methods of using individual antibodies or mixtures thereof for the detection, prevention, and/or therapeutical treatment of Ebola virus infections in vitro and in vivo.

Description of the Invention

BACKGROUND OF THE INVENTION

Ebola viruses cause acute, lethal hemorrhagic fevers for which no human-use vaccines or treatments currently exist. Knowledge about the immune mechanisms mediating protection is limited. The membrane-anchored glycoprotein (GP) is the only viral protein known to be on the surfaces of virions and infected cells, and is presumed to be responsible for receptor binding and fusion of the virus with host cells. As a result, Ebola GP may be an important target of protective antibodies. However, the contribution of antibodies to Ebola GP in disease resistance has been controversial. Negligible serum titers of neutralizing antibodies in convalescent patients, together with inconsistent results in achieving protection through experimental transfers of immune sera to animals (C. J. Peters and J. W. LeDuc, J. Infect. Dis. 179 (Suppl. 1), ix, 1999; V. V. Mikhailov et al., Vopr. Virusol. 39, 82, 1994) have led to suggestions that antibodies to Ebola GP cannot confer protection to Ebola virus (L. Xu et al., Nature Med. 4, 37, 1998).

The role of anti-GP antibodies in protection is further confounded by the observation that Ebola GP occurs in several forms. The transmembrane glycoprotein of Ebola viruses is unusual in that it is encoded in two open reading frames. Expression of GP occurs when the 2 reading frames are connected by transcriptional or translational editing (Sanchez et al., Proc. Natl. Acad. Sci. USA 93, 3602-3607, 1996; Volchkov et al., Virology 214, 421-430, 1995). The unedited GP mRNA produces a non-structural secreted glycoprotein (sGP) that is synthesized in large amounts early during the course of infection (Volchkov et al., 1995, supra; Sanchez et al., 1996, supra; Sanchez et al., J. Infect. Dis. 179 (suppl. 1, S164, 1999). Following editing, the virion-associated transmembrane glycoprotein is proteolytically processed into 2 disulfide-linked products (Sanchez et al., J. Virol. 72, 6442-6447, 1998). The amino-terminal product is referred to as GP1 (140 kDa) and the carboxy-terminal cleavage product is referred to as GP2 (26 kDa). GP1 and membrane-bound GP, covalently associate to form a monomer of the GP spike found on the surfaces of virions (V. E. Volchkov et al., Proc. Natl. Acad. Sci. U.S.A. 95, 5762, 1998; A. Sanchez et al., J. Virol. 72, 6442, 1998). GP1 is also released from infected cells in a soluble form (V. E. Volchkov. et al., Virology 245, 110, 1998). sGP and GP1 are identical in their first 295 N-terminal amino acids, whereas the remaining 69 C-terminal amino acids of sGP and 206 amino acids of GP 1 are encoded by different reading frames. It has been suggested that secreted GP 1 or sGP may effectively bind antibodies that might otherwise be protective (Sanchez et al., 1996, supra; Volchkov et al. 1998, supra).

Ebola virus GP is a type I transmembrane glycoprotein. Comparisons of the predicted amino acid sequences for the GPs of the different Ebola virus strains show conservation of amino acids in the amino-terminal and carboxy-terminal regions with a highly variable region in the middle of the protein (Feldmann et al., Virus Res. 24: 1-19, 1992). The GP of Ebola viruses are highly glycosylated and contain both N-linked and O-linked carbohydrates that contribute up to 50% of the molecular weight of the protein. Most of the glycosylation sites are found in the central variable region of GP.

Other studies have also demonstrated limited efficacy of passively transferred polyclonal antibodies in protection against Ebola challenge (Mikhailov et al, 1994, Voprosi Virusologii, 39, 82-84; Jahrling et al., 1996, Arch Virol, 11S, 135-140; Jahrling et al., 1999, J Infect Dis, 179 (Suppl 1), S224-234; Kudoyarova-Zubavichene et al., 1999, J Infect Dis, 179(Suppl 1), S218-223). However, it is difficult to determine the effective therapeutic dose of antibodies in different preparations of polyclonal antibodies. Efforts to identify the role of antibodies in protection led to the isolation of monoclonal antibodies from mice vaccinated with Ebola GP (for instance, co-pending patent application Ser. No. 09/650,086; and Wilson et al. Science 287, 1664,2000), and from convalescent people (Maruyama et al. J. Infect. Dis. 179 (suppl 1), S235, 1999; Maruyama et al. J. Virol. 73, 6024, 1999; Parren et al. J. Virol 76, 6408, 2002). These were tested in rodents and protected against lethal infection (Wilson et al. Science 287, 1664,2000; Parren et al. J. Virol 76, 6408, 2002).

SUMMARY OF THE INVENTION

This application describes protective GP-specific MAbs. The antibodies are classified into five groups based on competitive binding assays. Individual MAbs in these five groups were protective against Ebola challenge when administered prophylactically or therapeutically. (By "prophylactic", it is meant administered before challenge, and by "therapeutic", it is meant administered after challenge.) Three of the epitopes bound by protective MAbs are linear sequences on GP1 whereas the other two are conformational epitopes shared between GP1 and sGP. Ten out of 14 MAbs identified in these five competition groups protected BALB/c mice from a lethal challenge with mouse-adapted Ebola Zaire virus when 100 ug of purified MAb was administered 24 hours before challenge. Similar results were observed in a second mouse strain (C57BL/6). Protection from Ebola challenge decreased when the MAb dose was lowered to 50 or 25 ug (Please see Table 3 and Table 5 in Examples below). For the most effective MAbs, the amount required for protection was within an achievable human therapeutic dose of 3-5 mg/kg. Some of the MAbs were effective even when administered up to 2 days after challenge (please see Table 3 in Examples below), after significant viral replication had occurred (M. Bray et al., J. Infect. Dis. 178, 651, 1998). None of the MAbs were protective when 100 ug was administered 3 days after challenge, when there are high viral titers (Bray et al., 1998, supra) and possibly irreversible damage of cells and organs. The ability of the MAbs to inhibit plaque formation by Ebola virus, a standard assay of virus neutralization, did not always predict their protective efficacy. None of the protective MAbs inhibited plaque formation in the absence of complement (please see Table 6 in the Examples below).

One embodiment of this invention relates to monoclonal antibodies that protect against Ebola virus and bind to epitopes on the Ebola virus GP. Such antibodies are, for instance, produced by any one of the cell lines deposited under the Budapest Treaty at American Type Culture Collection, Manassas, Va. on Jul. 20, 1999, EGP 13F6-1-2, assigned accession no. PTA-373, EGP 6D3-1-1 assigned accession no. PTA-374, EGP 13-C6-1-1 assigned accession no. PTA-375, EGP 6D8-1-2 assigned accession no. PTA-376 and EGP 12B5-1-1 deposited on Jul. 29, 1999 and assigned accession no. PTA-436 (Table 1).

Another embodiment relates to the sequences of these monoclonal antibodies, in particular, the sequences to MAb EGP 6D8-1-2, MAb EGP 13F6-1-2, and Mab EGP 13-C6-1-1.

A further embodiment relates to the complementarity-determining regions of these three monoclonal antibodies (MAb EGP 6D8-1-2, MAb EGP 13F6-1-2, and Mab EGP 13-C6-1-1) which are involved with the binding of the monoclonal antibodies to Ebola virus.

Another embodiment of the invention relates to antibodies that are functionally equivalent to the antibodies listed above. These functionally equivalent antibodies substantially share at least one major functional property with an antibody listed above and herein described comprising: binding specificity to Ebola GP, protection against Ebola challenge when administered prophylactically or therapeutically, competition for same binding site on Ebola GP, and/or use of the same combination of complementarity determining regions. The antibodies can be of any class such as IgG, IgM, or IgA or any subclass such as IgG1, IgG2a, and other subclasses known in the art. Further, the antibodies can be produced by any method, such as phage display, or produced in any organism or cell line, including bacteria, insect, mammal or other type of cell or cell line which produces antibodies with desired characteristics, such as humanized antibodies. The antibodies can also be formed by combining a Fab portion and a Fc region from different species, or by keeping the complementarity-determining regions and modifying the framework regions to that of another species (such a human, which is described in more detail below).

The monoclonal antibodies of the present invention described below recognize epitopes on Ebola GP (SEQ ID NO: 1 and 2 describe the DNA and amino acid sequence, respectively, of Ebola GP used as an immunogen). Three epitopes are within the sequence extending from 389 to 493 and defined as:

HNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDF (SEQ ID
NO:3)

LDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLI.

More specifically, the cell line EGP 13F6-1-2 produces a monoclonal antibody 13F6 which recognizes and binds to an amino acid sequence of GP corresponding to a region extending from 401 to 417 (SEQ ID NO:4), recognizing an epitope within this region corresponding to Glu-Gln-His-His-Arg-Arg-Thr-Asp-Asn (SEQ ID NO:5). The cell line EGP 6D8-1-2 produces a monoclonal antibody 6D8 which recognizes and binds to an amino acid sequence of GP corresponding to a region extending from 389 to 405 (SEQ ID NO:6), recognizing an epitope within this region corresponding to Val-Tyr-Lys-Leu-Asp-Ile-Ser-Glu-Ala (SEQ ID NO:7). The cell line EGP 12B5-1-1 produces a monoclonal antibody 12B5 which recognizes and binds to an amino acid sequence of GP corresponding to a region extending from 477 to 493 (SEQ ID NO:8), recognizing an epitope within this region corresponding to Leu-Ile-Thr-Asn-Thr-Ile-Ala-Gly-Val (SEQ ID NO:9). The antibodies produced by cell lines EGP 13C6-1-1, 13C6, and EGP 6D3-1-1, 6D3, recognize conformational epitopes in GP sequence that may comprise discontinuous Ebola virus amino acids that are conserved between Zaire and Ivory Coast viruses and found in the 295 amino terminus of the protein (SEQ ID NO:10).

A further embodiment of the present invention provides for mixtures of the above-described antibodies, as well as to methods of using individual antibodies, or mixtures thereof for the prevention and/or therapeutic treatment of Ebola infections in vitro and in vivo, and/or for improved detection of Ebola infections.

Another embodiment relates to the treatment or prevention of Ebola virus infection by administering a therapeutically or prophylactically effective amount of one antibody of the present invention or a mixture of antibodies of the present invention to a subject in need of such treatment.

A further embodiment provides passive vaccines for treating or preventing Ebola virus infections comprising a therapeutically or prophylactically effective amount of the antibodies of the present invention which protect against Ebola virus, in combination with a pharmaceutically acceptable carrier or excipient.

Yet another embodiment provides methods for diagnosis of Ebola virus infection by assaying for the presence of Ebola in a sample using the antibodies of the present invention.

Still another embodiment provides novel immunoprobes and test kits for detection of Ebola virus infection comprising antibodies according to the present invention. For immunoprobes, the antibodies are directly or indirectly attached to a suitable reporter molecule, e.g., and enzyme or a radionuclide. The test kit includes a container holding one or more antibodies according to the present invention and instructions for using the antibodies for the purpose of binding to Ebola virus to form an immunological complex and detecting the formation of the immunological complex such that presence or absence of the immunological complex correlates with presence or absence of Ebola virus.

In another embodiment, there are provided anti-idiotypic antibodies raised against one of the present monoclonal antibodies for use as a vaccine to elicit an active anti-GP response.

In a further embodiment, there are provided antigenic epitopes as a component of a Ebola virus vaccine. The epitopes described above comprising SEQ ID NO:3-10, or conservative changes thereof which are still recognized by the antibodies, are useful for actively immunizing a host to elicit production of protective antibodies against Ebola.

DETAILED DESCRIPTION OF THE INVENTION

In the description that follows, a number of terms used in recombinant DNA, virology and immunology are extensively utilized. In order to provide a clearer and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. "Ebola viruses", members of the family Filoviridae, are associated with outbreaks of highly lethal hemorrhagic fever in humans and nonhuman primates. Human pathogens include Ebola Zaire, Ebola Sudan, and Ebola Ivory Coast. Ebola Reston is a monkey pathogen and is not considered a human pathogen. The natural reservoir of the virus is unknown and there are currently no available vaccines or effective therapeutic treatments for filovirus infections. The genome of Ebola virus consists of a single strand of negative sense RNA that is approximately 19 kb in length. This RNA contains seven sequentially arranged genes that produce 8 mRNAs upon infection. Ebola virions, like virions of other filoviruses, contain seven proteins: a surface glycoprotein (GP), a nucleoprotein (NP), four virion structural proteins (VP40, VP35, VP30, and VP24), and an RNA-dependent RNA polymerase (L) (Feldmann et al.(1992) Virus Res. 24, 1-19; Sanchez et al.,(1993) Virus Res. 29, 215-240; reviewed in Peters et al. (1996) In Fields Viroloqy, Third ed. pp. 1161-1176. Fields, B. N., Knipe, D. M., Howley, P. M., et al. eds. Lippincott-Raven Publishers, Philadelphia). The glycoprotein of Ebola virus is unusual in that it is encoded in two open reading frames. Transcriptional editing is needed to express the transmembrane form that is incorporated into the virion (Sanchez et al. (1996) Proc. Natl. Acad. Sci. USA 93, 3602-3607; Volchkov et al, (1995) Virology 214, 421-430). The unedited form produces a nonstructural secreted glycoprotein (sGP) that is synthesized in large amounts early during the course of infection. Little is known about the biological functions of these proteins and it is not known which antigens significantly contribute to protection and should therefore be used to induce an immune response.

The term "antibody" is art-recognized terminology and is intended to include molecules or active fragments of molecules that bind to known antigens. Examples of active fragments of molecules that bind to known antigens include Fab and F(ab')2 fragments. These active fragments can be derived from an antibody of the present invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23:1011-1019 (1982). The term "antibody" also includes bispecific and chimeric antibodies.

The language "monoclonal antibody" is art-recognized terminology. It is generally understood by those of skill in the art to refer to the antibody produced by one clone of B lymphocytes. The monoclonal antibodies of the present invention can be prepared using classical cloning and cell fusion techniques. The immunogen (antigen) of interest, Ebola GP protein, is typically administered (e.g. intraperitoneal injection) to wild type or inbred mice (e.g. BALB/c) or transgenic mice which produce desired antibodies, rats, rabbits or other animal species which can produce native or human antibodies. The immunogen can be administered alone, or mixed with adjuvant, or expressed from a vector (VEE replicon vector, vaccinia), or as DNA, or as a fusion protein to induce an immune response. Fusion proteins comprise the peptide against which an immune response is desired coupled to carrier proteins, such as b-galactosidase, glutathione S-transferase, keyhole limpet hemocyanin (KLH), and bovine serum albumin, to name a few. In these cases, the peptides serve as haptens with the carrier proteins. After the animal is boosted, for example, two or more times, the spleen can be removed and splenocytes can be extracted and fused with myeloma cells using the well-known processes of Kohler and Milstein (Nature 256: 495-497 (1975)) and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988)). The resulting hybrid cells can then be cloned in the conventional manner, e.g. using limiting dilution, and the resulting clones, which produce the desired monoclonal antibodies, cultured.

Monoclonal antibodies raised against Ebola GP as described in the Examples are listed in Table 1 above.

The monoclonal antibodies of this invention contain at least one "complementarity-determining region" (CDR). By "complementarity-determining region", it is meant the hypervariable regions in the heavy and light chains of an antibody molecule that form the 3-dimensional cavity by which the antibody binds to an epitope on the antigen.

The term "epitope" is art-recognized. It is generally understood by those of skill in the art to refer to the region of an antigen, such as Ebola virus GP, that interacts with an antibody. An epitope of a peptide or protein antigen can be formed by contiguous or noncontinguous amino acid sequences of the antigen. Ebola GP, like many large proteins, contains many epitopes. Examples of Ebola GP epitopes recognized by antibodies of the present invention include the region extending from 389 to 493 and defined as:

HNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDF (SEQ ID
NO:3)

LDPATTTSPQNIISETAGNNNTHHQDTGEESASSGKLGLINTIAGVAGLI.

Continuous epitopes were found within 1) the amino acid sequence of GP corresponding to a region extending from 401 to 417 (SEQ ID NO:4), for example corresponding to Glu-Gln-His-His-Arg-Arg-Thr-Asp-Asn (SEQ ID NO:5), 2) the amino acid sequence of GP corresponding to a region extending from 389 to 405 (SEQ ID NO:6), for example corresponding to Val-Tyr-Lys-Leu-Asp-Ile-Ser-Glu-Ala (SEQ ID NO:7), and 3) the amino acid sequence of GP corresponding to a region extending from 477 to 493 (SEQ ID NO:8), for example Leu-Ile-Thr-Asn-Thr-Ile-Ala-Gly-Val (SEQ ID NO:9). The epitopes or peptides recognized by the antibodies of the present invention and conservative substitutions of these peptides which are still recognized by the antibody are an embodiment of the present invention. These peptides offer a convenient method for eluting GP bound to MAb 6D8, 13F6, or 12B5 on immunoaffinity columns. For example, when an antibody which recognizes the epitope for MAb 6D8, 13F6 or 12B5 is used in an immunoaffinity column to purify Ebola GP, the peptide recognized by the antibody can be added to the immunoaffinity column to elute the Ebola GP. Further truncation of these epitopes may be possible, as would be understood by someone having ordinary skill in this art having this disclosure in hand.

Epitope mapping studies described in this application defined five competition groups of MAbs. Antibodies which compete with the monoclonal antibodies of the present invention for binding to GP are considered to recognize the epitopes of the antibodies and are considered equivalent to the antibodies of the present invention. The MAbs 13C6 and 6D3 recognize conformational epitopes comprising discontinuous Ebola virus amino acids. Antibodies which compete with MAbs 13C6 and 6D3 for binding to Ebola GP are considered to recognize discontinuous epitopes and are considered equivalent to the antibodies of the present invention. Assays for determining whether or not an antibody competes with an antibody of the present invention are known to a person with ordinary skill in the art and are described below. Table 2 below defines functional criteria of each of the monoclonal antibodies identified in the Examples below.

By further mapping of the binding site of the monoclonal antibodies described in this disclosure other peptides useful as a vaccine or a therapeutic can be determined using known methodologies. Therefore, in another aspect, this invention relates to a method for identifying protective antigenic epitopes, which method comprises the steps of (i) reacting a monoclonal antibody described herein to different overlapping fragments encompassing the complete antigen, (ii) identifying a fragment to which the protective antibody binds, (iii) narrowing the region containing sites further by reacting the monoclonal with smaller overlapping fragments encompassing the region identified in (ii), and (iv) choosing peptides to which the antibody binds as possible antigenic epitopes. The peptides can then be assayed for their ability to protect an animal from disease, or to reduce the severity of disease. Peptides defining antigenic protective epitopes can be used in a vaccine as described below and in the Examples.

The epitopes or peptides on Ebola GP to which the monoclonal antibodies bind can constitute all or part of an active vaccine. An active vaccine or therapeutic candidate might comprise these peptide sequences and others. These may be delivered as synthetic peptides, or as fusion proteins, alone or co-administered with cytokines and/or adjuvants or carriers safe for human use, e.g. aluminum hydroxide, to increase immunogenicity. In addition, sequences such as ubiquitin can be added to increase antigen processing for more effective immune responses.

Antibody molecules produced in vivo comprise two identical heavy chains that are covalently bound and two identical light chains, each of which is covalently bound to a heavy chain. Heavy and light chains each have one variable region and three constant regions. Within the variable regions of light and heavy chains are hypervariable sequences called complementarity-determining regions flanked by framework regions. The binding specificity of an antibody is conferred by its combination of complementarity-determining regions. There are three complementarity-determining regions on the light chain and three on the heavy chain of an antibody molecule. Together, these form the 3-dimensional cavity that will bind (hold) an epitope on an antigen. Although these regions are hypervariable, a particular complementarity-determining region on one antibody may also be found on antibodies with different specificities, as it is the total combination of complementarity-determining regions that is important. Generally, binding specificity is determined by the complementarity-determining regions on both chains, although it has been suggested that the complementarity-determining regions on the heavy chain do not contribute to specificity when the light chain is produced by a gene called lambda x. Identification of the complementarity-determining regions is useful for changing the "speciation" of an antibody, for example changing a mouse antibody to a humanized form suitable for human use, because one would want to preserve the complementarity-determining regions so as not to eliminate the binding specificity. Using the numbering system of Kabat et al, (NIH Publication No. 91-3242, 1991) in which the signal sequences of the heavy and light chains are indicated with negative numbers, the complementarity-determining regions of the light chain are between amino acids 24-34 (CDR1), 50-56 (CDR2) and 89-97 (95 a-f, CDR3). The complementarity-determining regions of the heavy chain are between amino acids 31-35 (35 a-b, CDR1), 50-65 (52 a-c, CDR2), and 95-102 (100 a-k, CDR3). Insertions of extra amino acids into the complementarity-determining regions can be observed and their locations are represented above in parentheses, e.g 95 a-f. Deletions are also observed, for example in CDR3 of some types of heavy chains.

Throughout this description we refer to the CDRs in terms of both the amino acid sequence and the location within the light or heavy chain. As someone having ordinary skill in this art would understand, the "location" of the CDRs is conserved between species, but through the use the well known Kabat system--an arbitrary numbering system that aligns sequences. Therefore, according to the Kabat system, the first invariant amino acid of a given type of light chain might be used to define the CDR beginning at, for example, "position 24" even if there are not 23 preceding amino acids. Therefore, for the purposes of the description of this invention we are defining CDRs as according to the Kabat system which is accepted in the art. The Kabat system aligns the Mab sequences of different species, for example mouse and human, such that all species have CDRs aligned at the same numbered "positions". Alignment of the sequences occurs through the identification of invariant residues in either the CDR or the framework regions adjacent to the CDR. There are different forms of light and heavy chain variable regions that differ in the use and location of the invariant residues, but Kabat et al. identify these. Using the nomenclature in the 1991 edition of Kabat et al, Mab EGP 6D8-1-2 uses a heavy chain variable region of the IIID type, and a kappa light chain type II. Mab EGP 13C6-1-1 uses a heavy chain variable region of the miscellaneous type, and a kappa light chain type V. Mab EGP 13F6-1-2 uses a heavy chain variable region of the IIID type, and a lambda x light chain.

The DNA sequence of the variable regions of the heavy chain of Mab EGP13C6-1-1 is represented in SEQ ID NO:11, and the amino acid sequence is represented in SEQ ID NO:12. For the heavy chain, the CDRs were identified as located at the following amino acid positions:

31-35b (where, as noted above, "b" signifies the insertion of an extra amino acid) having the amino acid sequence TSGVGVG with the last two amino acids representing insertions at 35a and 35b (SEQ ID NO:13),

50-65: having the amino acid sequence LIWWDDDKYYNPSLKS (SEQ ID NO:14), and

95-102 (includes residues at 100c,h,j,k, where, as noted above, "c,h,j,k" signifies the insertion of extra amino acids): having the amino acid sequence RDPFGYDNAMGY where DNAM are 100 c,h,j,k, respectively (SEQ ID NO:15).

It is believed that all three of these CDRs are necessary for effective binding of the Mab EGP 13C6-1-1 to the epitopes of Ebola GP.

The DNA sequence of the variable regions of the light chain of Mab EGP 13C6-1-1 is represented in SEQ ID NO:16, and the amino acid sequence is represented in SEQ ID NO:17. For the light chain, the CDRs were identified as located at the following amino acid positions:

24-34: having the amino acid sequence--KASQNVGTAVA (SEQ ID NO:18)

50-56: having the amino acid sequence--SASNRYT (SEQ ID NO:19) and

89-97: having the amino acid sequence--QQYSSYPLT (SEQ ID NO:20).

It is believed that all three CDRs are necessary for effective binding of the Mab EGP13C6-1-1 to the epitopes of Ebola GP. The invention also contemplates monoclonal antibodies having sequences that are at least 90%, and preferably 95%, homologous to the heavy and/or light chain regions described here as SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16 and SEQ ID NO:17, and which compete for binding Ebola GP. There can be a 5% variation normally in even the more conserved framework regions, and someone having ordinary skill in this art using known techniques would be able to determine without undue experimentation such homologous, competing monoclonal antibodies. The invention also contemplates monoclonal antibodies that compete with EGP13C6-1-1 for binding to Ebola GP, and which have the above-described CDRs in the appropriate positions as determined by the Kabat system in the light and/or heavy chains.

Specificity is conferred with both heavy and light chains, and not usually with just the heavy or light chain alone; therefore, it is preferred that when this monoclonal antibody is used to detect Ebola in a sample (as described below), or to prevent or treat Ebola infection (as described below), both heavy and light chains are present.

The DNA sequence of the variable regions of the heavy chain of Mab EGP6D8-1-2 is represented in SEQ ID NO:21, and the amino acid sequence is represented in SEQ ID NO:22. For the heavy chain, the CDRs were identified as located at the following amino acid positions:

31-35: having the amino acid sequence--RYWMS (SEQ ID NO:23)

50-65 (includes 52a): having the amino acid sequence--EINPDSSTINYTPSLKD (SEQ ID NO:24)

95-102 (has one deletion): having the amino acid sequence--QGYGYNY (SEQ ID NO:25)

It is believed that all three CDRs are necessary for effective binding of the Mab EGP6D8-1-2 to the epitopes of Ebola GP.

The DNA sequence of the variable regions of the light chain of Mab EGP 6D8-1-2 is represented in SEQ ID NO:26, and the amino acid sequence is represented in SEQ ID NO:27. For the light chain, the CDRs were identified as located at the following amino acid positions:

24-34, includes 27 a-e: having the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO:28)

50-56: having the amino acid sequence KASNRFS (SEQ ID NO:29) and

89-97: having the amino acid sequence LQGSHVPST (SEQ ID NO:30).

It is believed that all three CDRs are necessary for effective binding of the Mab EGP 6D8-1-2 to the epitopes of Ebola GP. The invention also contemplates monoclonal antibodies having sequences that are at least 90%, and preferably 95%, homologous to the heavy and/or light chain regions described here as SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:27, and which compete for binding Ebola GP. As noted above, there can be a 5% variation normally in even the more conserved framework regions, and someone having ordinary skill in this art using known techniques would be able to determine without undue experimentation such homologous, competing monoclonal antibodies. The invention also contemplates monoclonal antibodies that compete with EGP6D8-1-2 for binding to Ebola GP, and which have the above-described CDRs in the appropriate positions as determined by the Kabat system in the light and/or heavy chains. Specificity generally is conferred with both heavy and light chains, and not usually with just the heavy or light chain alone; therefore, it is preferred that when this monoclonal antibody is used to detect Ebola in a sample (as described below), or to prevent or treat Ebola infection (as described below), both heavy and light chains are present.

The DNA sequence of the variable regions of the heavy chain of Mab EGP 13F6-1-2 is represented in SEQ ID NO:31, and the amino acid sequence is represented in SEQ ID NO:32. For the heavy chain, the CDRs were identified as located at the following amino acid positions:

31-35: having the amino acid sequence SYDMS (SEQ ID NO:33)

50-65: having the amino acid sequence YISRGGGYTYYPDTVKG (SEQ ID NO:34)

95-102, includes 100 a-c,h-k: having the amino acid sequence HIYYGSSHYYAMDY (SEQ ID NO:35) and

It is thought that some or all of these three CDRs may not be necessary for effective binding of the Mab EGP13F6-1-2 to the epitopes of Ebola GP because the light chain of this antibody is a lambda x, which has been described as sufficient for binding. Lambda x light chains have an insertion at 27a and an insertion of four amino acids at position 54.

The DNA sequence of the variable regions of the light chain of Mab EGP 13F6-1-2 is represented in SEQ ID NO:36, and the amino acid sequence is represented in SEQ ID NO:37. For the light chain, the CDRs were identified as located at the following amino acid positions:

24-34, includes 27a: having the amino acid sequence TLSRQHSTYTIE (SEQ ID NO:38)

50-56, includes insertion at 54: having the amino acid sequence LKKDGSHSTGD (SEQ ID NO:39) and

89-97, INCLUDES 95a-d: having the amino acid sequence GVGDTIKEQFVYV (SEQ ID NO:40).

It is believed that all three CDRs are necessary, and may be sufficient for effective binding of the Mab EGP 13F6-1-2 to the epitopes of Ebola GP. Consequently, when this monoclonal antibody is used to detect Ebola in a sample (as described below), or to prevent or treat Ebola infection (as described below), the light chain may be used alone, or both heavy and light chains together may be present. The invention also contemplates monoclonal antibodies having sequences that are at least 90%, and preferably 95%, homologous to the heavy and/or light chain regions described here as SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:36 and SEQ ID NO:37, and which compete for binding Ebola GP. As noted above, there can be a 5% variation normally in even the more conserved framework regions, and someone having ordinary skill in this art using known techniques would be able to determine without undue experimentation such homologous, competing monoclonal antibodies. The invention also contemplates monoclonal antibodies that compete with EGP 13F6-1-2 for binding to Ebola GP, and which have the above-described CDRs in the appropriate positions as determined by the Kabat system in the light and/or heavy chains.

The above-described heavy and light chains for Mab EGP 13C6-1-1, Mab EGP 6D8-1-2, and EGP 13F6-1-2 are particularly useful for detecting Ebola GP in a sample suspected of containing Ebola GP, as well as use as therapeutic and prophylactic agents for treating or preventing Ebola infection in susceptible Ebola-infected subjects.

The present invention also pertains to hybridomas producing antibodies which bind to an epitope of Ebola GP. The term "hybridoma" is art recognized and is understood by those of ordinary skill in the art to refer to a cell produced by the fusion of an antibody-producing cell and an immortal cell, e.g. a multiple myeloma cell. This hybrid cell is capable of producing a continuous supply of antibody. See the definition of "monoclonal antibody" above and the Examples below for a more detailed description of the method of fusion.

The present invention still further pertains to a method for detecting Ebola GP in a sample suspected of containing Ebola GP. The method includes contacting the sample with an antibody which binds an epitope of Ebola GP, allowing the antibody to bind to Ebola GP to form an immunological complex, detecting the formation of the immunological complex and correlating the presence or absence of the immunological complex with the presence or absence of Ebola GP in the sample. The sample can be biological, environmental or a food sample.

The language "detecting the formation of the immunological complex" is intended to include discovery of the presence or absence of Ebola GP in a sample. The presence or absence of Ebola GP can be detected using an immunoassay. A number of immunoassays used to detect and/or quantitate antigens are well known to those of ordinary skill in the art. See Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612). Such immunoassays include antibody capture assays, antigen capture assays, and two-antibody sandwich assays. These assays are commonly used by those of ordinary skill in the art. In an antibody capture assay, the antigen is attached to solid support, and labeled antibody is allowed to bind. After washing, the assay is quantitated by measuring the amount of antibody retained on the solid support. A variation of this assay is a competitive ELISA wherein the antigen is bound to the solid support and two solutions containing antibodies which bind the antigen, for example, serum from an Ebola virus vaccinee and a monoclonal antibody of the present invention, are allowed to compete for binding of the antigen. The amount of monoclonal bound is then measured, and a determination is made as to whether the serum contains anti Ebola GP antibodies. This competitive ELISA can be used to indicate immunity to known protective epitopes in a vaccinee following vaccination.

In an antigen capture assay, the antibody is attached to a solid support, and labeled antigen is allowed to bind. The unbound proteins are removed by washing, and the assay is quantitated by measuring the amount of antigen that is bound. In a two-antibody sandwich assay, one antibody is bound to a solid support, and the antigen is allowed to bind to this first antibody. The assay is quantitated by measuring the amount of a labeled second antibody that can bind to the antigen.

These immunoassays typically rely on labeled antigens, antibodies, or secondary reagents for detection. These proteins can be labeled with radioactive compounds, enzymes, biotin, or fluorochromes. Of these, radioactive labeling can be used for almost all types of assays and with most variations. Enzyme-conjugated labels are particularly useful when radioactivity must be avoided or when quick results are needed. Biotin-coupled reagents usually are detected with labeled streptavidin. Streptavidin binds tightly and quickly to biotin and can be labeled with radioisotopes or enzymes. Fluorochromes, although requiring expensive equipment for their use, provide a very sensitive method of detection. Antibodies useful in these assays include monoclonal antibodies, polyclonal antibodies, and affinity purified polyclonal antibodies. Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H., et al., 1976 (Clin. Chim. Acta 70:1-31), and Schurs, A. H. W. M., et al. 1977 (Clin. Chim Acta 81:1-40). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, and others, all of which are incorporated by reference herein.

The language "biological sample" is intended to include biological material, e.g. cells, tissues, or biological fluid. By "environmental sample" is meant a sample such as soil and water. Food samples include canned goods, meats, and others.

Yet another aspect of the present invention is a kit for detecting Ebola virus in a biological sample. The kit includes a container holding one or more antibodies which binds an epitope of Ebola GP and instructions for using the antibody for the purpose of binding to Ebola GP to form an immunological complex and detecting the formation of the immunological complex such that the presence or absence of the immunological complex correlates with presence or absence of Ebola virus in the sample. Examples of containers include multiwell plates which allow simultaneous detection of Ebola virus in multiple samples.

As described in greater detail below, the present inventors have isolated five monoclonal antibodies which bind to five epitopes on Ebola GP and display in vitro and/or in vivo Ebola virus protective properties. Significantly, the reactivity of the MAbs is applicable against a broad variety of different wild type and laboratory Ebola strains of different types. Wild type strains include for example Ebola Ivory Coast, Ebola Zaire 1976 (Mayinga isolate), Ebola Zaire 1975, and Ebola Sudan (Boniface). Laboratory strains can be derived from wild type strains and include those which have been passaged or animal adapted strains such as mouse-adapted Ebola.

Given these results, monoclonal antibodies according to the present invention are suitable both as therapeutic and prophylactic agents for treating or preventing Ebola infection in susceptible Ebola-infected subjects. Subjects include rodents such as mice or guinea pigs, monkeys, and other mammals, including humans.

In general, this will comprise administering a therapeutically or prophylactically effective amount of one or more monoclonal antibodies of the present invention to a susceptible subject or one exhibiting Ebola infection. Any active form of the antibody can be administered, including Fab and F(ab')2 fragments. Antibodies of the present invention can be produced in any system, including insect cells, baculovirus expression systems, chickens, rabbits, goats, cows, or plants such as tomato, potato, corn, banana or strawberry.

Methods for the production of antibodies in these systems are known to a person with ordinary skill in the art. Preferably, the antibodies used are compatible with the recipient species such that the immune response to the MAbs does not result in clearance of the MAbs before virus can be controlled, and the induced immune response to the MAbs in the subject does not induce "serum sickness" in the subject. Preferably, the MAbs administered exhibit some secondary functions such as binding to Fc receptors of the subject.

Treatment of individuals having Ebola infection may comprise the administration of a therapeutically effective amount of Ebola antibodies of the present invention. The antibodies can be provided in a kit as described below. The antibodies can be used or administered as a mixture, for example in equal amounts, or individually, provided in sequence, or administered all at once. In providing a patient with antibodies, or fragments thereof, capable of binding to Ebola GP, or an antibody capable of protecting against Ebola virus in a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc.

In general, it is desirable to provide the recipient with a dosage of antibody which is in the range of from about 1 pg/kg-100 pg/kg, 100 pg/kg-500 pg/kg, 500 pg/kg-1 ng/kg, 1 ng/kg-100 ng/kg, 100 ng/kg-500 ng/kg, 500 ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500 ug/kg, 500 ug/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100 mg/kg-500 mg/kg, 500 mg/kg-1 g/kg, 1 g/kg-5 g/kg, 5 g/kg-10 g/kg (body weight of recipient), although a lower or higher dosage may be administered. Dosages as low as about 1.0 mg/kg may be expected to show some efficacy. Preferably, about 5 mg/kg is an acceptable dosage, although dosage levels up to about 50 mg/kg are also preferred especially for therapeutic use.

In a similar approach, another therapeutic use of the monoclonal antibodies of the present invention is the active immunization of a patient using an anti-idiotypic antibody raised against one of the present monoclonal antibodies. Immunization with an anti-idiotype which mimics the structure of the epitope could elicit an active anti-GP response (Linthicum, D. S. and Farid, N. R., Anti-Idiotypes, Receptors, and Molecular Mimicry (1988), pp 1-5 and 285-300).

Likewise, active immunization can be induced by administering one or more antigenic and/or immunogenic epitopes as a component of a subunit vaccine. Vaccination could be performed orally or parenterally in amounts sufficient to enable the recipient to generate protective antibodies against this biologically functional region, prophylactically or therapeutically. The host can be actively immunized with the antigenic/immunogenic peptide in pure form, a fragment of the peptide, or a modified form of the peptide. One or more amino acids, not corresponding to the original protein sequence can be added to the amino or carboxyl terminus of the original peptide, or truncated form of peptide. Such extra amino acids are useful for coupling the peptide to another peptide, to a large carrier protein, or to a support. Amino acids that are useful for these purposes include: tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof. Alternative protein modification techniques may be used e.g., NH2-acetylation or COOH-terminal amidation, to provide additional means for coupling or fusing the peptide to another protein or peptide molecule or to a support.

The antibodies capable of protecting against Ebola virus are intended to be provided to recipient subjects in an amount sufficient to effect a reduction in the Ebola virus infection symptoms. An amount is said to be sufficient to "effect" the reduction of infection symptoms if the dosage, route of administration, etc. of the agent are sufficient to influence such a response. Responses to antibody administration can be measured by analysis of subject's vital signs.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.

The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a phamaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A. ed., Mack Easton Pa. (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle. Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the compounds. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lacticacid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate)-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

Administration of the antibodies disclosed herein may be carried out by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), in ovo injection of birds, orally, or by topical application of the antibodies (typically carried in a pharmaceutical formulation) to an airway surface. Topical application of the antibodies to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. Oral administration may be in the form of an ingestable liquid or solid formulation. The treatment may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of treatment may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. Examples of suitable treatment schedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired responses expected to reduce disease symptoms, or reduce severity of disease.

The present invention also provides kits which are useful for carrying out the present invention. The present kits comprise a first container containing or packaged in association with the above-described antibodies. The kit may also comprise another container containing or packaged in association solutions necessary or convenient for carrying out the invention. The containers can be made of glass, plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. The kit may also contain written information, such as procedures for carrying out the present invention or analytical information, such as the amount of reagent contained in the first container means. The container may be in another container apparatus, e.g. a box or a bag, along with the written information.

The contents of all cited references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

Claim 1 of 6 Claims

What is claimed is:

1. An isolated monoclonal antibody which binds Ebola virus GP, which monoclonal antibody consists of a heavy chain variable region encoded by the DNA sequence SEQ ID NO:21.

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