阅读说明：本技术 针对细胞内抗原的单结构域抗体 (Single domain antibodies to intracellular antigens ) 是由 S·辛格 于 2016-11-02 设计创作，主要内容包括：本发明涉及针对细胞内抗原的单结构域抗体。具体而言,涉及一种抗埃博拉VP24单结构域抗体(sdAb)。本发明包括所述sdAb用于预防性、治疗性或诊断性目的的应用。(The present invention relates to single domain antibodies directed against intracellular antigens. In particular, it relates to an anti-ebola VP24 single domain antibody (sdAb). The invention comprises the use of said sdAb for prophylactic, therapeutic or diagnostic purposes.)

1. An anti-ebola VP24 single domain antibody (sdAb).

2. An anti-Ebola VP24 sdAb, wherein the anti-Ebola VP24 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 55.

3. Use of an anti-ebola VP24 sdAb according to claim 1 or 2 in the manufacture of a medicament for treating, preventing progression of, or preventing relapse of a disease in a subject.

4. The use of claim 3, wherein the anti-Ebola VP24 sdAb is administered in combination with one or more compounds.

5. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 55.

6. An antibody directed against the polypeptide of claim 5.

7. A method of determining the level of an anti-ebola VP24 sdAb in a sample from a subject, the method comprising the steps of:

a) generating a mouse monoclonal antibody directed against one or more domains of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 55;

b) obtaining a sample from the subject;

c) performing a quantitative immunoassay with the mouse monoclonal antibody and the sample to determine the amount of sdAb in the subject; and

d) quantifying the amount of sdAb in the subject.

8. An anti-arachidonic acid 12-lipoxygenase (ALOX12) single domain antibody (sdAb).

9. An anti-ALOX 12 sdAb, wherein the anti-ALOX 12 sdAb comprises the amino acid sequence set forth in SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, or SEQ ID NO 52.

10. Use of an anti-ALOX 12 sdAb according to claim 8 or 9 in the manufacture of a medicament for treating a disease, preventing disease progression, or preventing disease recurrence in a subject.

11. The use of claim 10, wherein the anti-ALOX 12 sdAb is administered in combination with one or more compounds.

12. An isolated polypeptide comprising the amino acid sequence shown as SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, or SEQ ID NO 52.

13. An antibody directed against the polypeptide of claim 12.

14. A method of determining the level of an anti-ALOX 12 sdAb in a sample from a subject, the method comprising the steps of:

a) generating a mouse monoclonal antibody directed against one or more domains of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 or SEQ ID NO 52;

b) obtaining a sample from the subject;

c) performing a quantitative immunoassay with the mouse monoclonal antibody and the sample to determine the amount of sdAb in the subject; and

d) quantifying the amount of sdAb in the subject.

Technical Field

The present invention relates to single domain antibodies directed against intracellular antigens.

Background

The use of single domain antibodies (sdabs) as single antigen-binding proteins or as antigen-binding domains in larger proteins or polypeptides offers a number of significant advantages over the use of conventional antibodies or antibody fragments. Advantages of sdabs include: only a single domain is required to bind antigen with high affinity and high selectivity; sdabs can be expressed from a single gene and do not require post-translational modification; sdabs are highly stable to denaturing agents or conditions including heat, pH, and proteases; sdAb is inexpensive to prepare; and sdabs can reach targets and epitopes inaccessible to conventional antibodies.

There are many diseases or conditions caused by abnormal intracellular or transmembrane components (such as nucleotides and proteins), such as viral infections or cancer. Elimination of the abnormal component may be utilized to prevent or treat the disease or condition. There are many pharmaceutical compounds available to treat such diseases, but these compounds may be inefficient, undeliverable, or toxic to unaffected cells.

Other therapies include the use of therapeutic proteins or agents that contain exogenous targeting sequences to enable the agent to be recognized by receptors of the cell membrane, thereby enabling the agent to cross the cell membrane and enter the cell. Once inside the cell, the therapeutic agent is able to interact with the target component to treat the disease. However, the use of exogenous targeting sequences may limit the cell types targeted by the therapeutic agent and increase the cost of preparing the therapeutic agent.

For the reasons described above, there is a need for compositions and methods for treating or preventing diseases as follows: it does not rely on exogenous targeting sequences or chemical compositions to enter cells, and effectively targets only affected cells in vivo.

The present invention relates to single domain antibodies (sdabs), proteins and polypeptides comprising sdabs. The sdabs are directed against targets that cause a condition or disease. The invention also includes nucleic acids encoding the sdabs, proteins and polypeptides comprising the sdabs, and compositions. The invention includes the use of the composition, sdAb, protein or polypeptide for prophylactic, therapeutic or diagnostic purposes. The invention also includes the use of monoclonal antibodies directed against the sdabs of the invention.

Disclosure of Invention

The present invention relates to sdabs for use in treating a condition or disease. One embodiment relates to an anti-human immunodeficiency virus type 1 (HIV-1) reverse transcriptase single domain antibody (sdAb). In one aspect, the anti-HIV-1 reverse transcriptase sdAb comprises the amino acid sequence set forth in SEQ ID NO 27. The invention also includes methods of treating, preventing progression of, or preventing relapse of a disease in a subject using an anti-HIV-1 reverse transcriptase sdAb by administering to a subject in need thereof an effective amount of an anti-HIV-1 reverse transcriptase sdAb. The subject may be a mammal, e.g., a human. The anti-HIV-1 reverse transcriptase sdAb can be administered in combination with one or more compounds (e.g., protease inhibitors). Administration of an effective amount of an anti-HIV-1 reverse transcriptase sdAb to a subject in need thereof can be performed by intravenous administration, intramuscular administration, oral administration, rectal administration, enteral administration, parenteral administration, intraocular administration, subcutaneous administration, transdermal administration, administration as eye drops, administration as nasal spray, administration by inhalation or aerosolization, topical administration, and administration as an implantable drug.

In another embodiment, the invention relates to an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO. 27. In another embodiment, the invention includes an antibody directed against the polypeptide of SEQ ID NO. 27.

It is further envisaged that the present invention comprises a method of determining the level of an anti-HIV-1 reverse transcriptase sdAb in a sample from a subject, the method comprising the steps of: a) generating a mouse monoclonal antibody directed against one or more domains of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 27; b) obtaining a sample from a subject; c) performing a quantitative immunoassay with the mouse monoclonal antibody and the sample to determine the amount of sdAb in the subject; thereby determining the amount of sdAb in the subject. In one aspect, the quantitative immunoassay comprises an enzyme-linked immunosorbent assay (ELISA), Specific Analyte Labeling and Recapture Assay (SALRA), liquid chromatography, mass spectrometry, fluorescence activated cell sorting, or a combination thereof.

Another embodiment of the invention relates to an anti-ebola VP24 sdAb. In one aspect, the anti-Ebola VP24 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 55. The invention also includes methods of treating, preventing progression of, or preventing relapse of a disease in a subject using an anti-ebola VP24 sdAb by administering to a subject in need thereof an effective amount of an anti-ebola VP24 sdAb. The subject may be a mammal, e.g., a human. The anti-ebola VP24 sdAb can be administered in combination with one or more compounds (e.g., protease inhibitors). Administration of an effective amount of an anti-ebola VP24 sdAb to a subject in need thereof can be performed by intravenous administration, intramuscular administration, oral administration, rectal administration, enteral administration, parenteral administration, intraocular administration, subcutaneous administration, transdermal administration, administration as eye drops, administration as nasal spray, administration by inhalation or aerosolization, topical administration, and administration as an implantable drug.

In another embodiment, the invention relates to an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 55. In another embodiment, the invention includes an antibody directed against the polypeptide of SEQ ID NO: 55.

It is further envisaged that the invention includes a method of determining the level of an anti-ebola VP24 sdAb in a sample from a subject, the method comprising the steps of: a) generating a mouse monoclonal antibody directed against one or more domains of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 55; b) obtaining a sample from a subject; c) performing a quantitative immunoassay with the mouse monoclonal antibody and the sample to determine the amount of sdAb in the subject; thereby determining the amount of sdAb in the subject. In one aspect, the quantitative immunoassay comprises ELISA, SALRA, liquid chromatography, mass spectrometry, fluorescence activated cell sorting, or a combination thereof.

Yet another embodiment of the invention relates to an anti-arachidonic acid 12-lipoxygenase (ALOX12) sdAb. In one aspect, the anti-ALOX 12 sdAb comprises the amino acid sequence set forth in SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, or SEQ ID NO 52. The invention also includes methods of treating, preventing progression of, or preventing relapse of a disease in a subject using an anti-ALOX 12 sdAb by administering to a subject in need thereof an effective amount of an anti-ALOX 12 sdAb. The subject may be a mammal, e.g., a human. The anti-ALOX 12 sdAb can be administered in combination with one or more compounds. Administration of an effective amount of an anti-ALOX 12 sdAb to a subject in need thereof can be performed by intravenous administration, intramuscular administration, oral administration, rectal administration, enteral administration, parenteral administration, intraocular administration, subcutaneous administration, transdermal administration, administration as eye drops, administration as nasal spray, administration by inhalation or aerosolization, topical administration, and administration as an implantable drug.

In another embodiment, the invention relates to an isolated polypeptide having the amino acid sequence shown as SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 or SEQ ID NO 52. In another embodiment, the invention includes antibodies to the polypeptide of SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 or SEQ ID NO 52.

It is further envisaged that the present invention comprises a method of determining the level of an anti-HIV-1 reverse transcriptase sdAb in a sample from a subject, the method comprising the steps of: a) generating a mouse monoclonal antibody directed against one or more domains of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 27; b) obtaining a sample from a subject; c) performing a quantitative immunoassay with the mouse monoclonal antibody and the sample to determine the amount of sdAb in the subject; thereby determining the amount of sdAb in the subject. In one aspect, the quantitative immunoassay comprises ELISA, SALRA, liquid chromatography, mass spectrometry, fluorescence activated cell sorting, or a combination thereof.

Drawings

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:

FIGS. 1 and 2 depict the results of an ELISA using HIV1-9 anti-HIV-1 RT sdAb (SEQ ID NO: 27);

FIGS. 3 and 4 depict the results of an ELISA using serial dilutions of HIV1-9 anti-HIV-1 RT sdAb (SEQ ID NO: 27);

FIGS. 5 to 8 depict the results of an ELISA using VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO: 55); and is

FIGS. 9 and 10 depict the results of an ELISA using serial dilutions of VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO: 55).

Detailed Description

As used herein, the following terms and variations thereof have the meanings given below, unless a different meaning is explicitly intended in the context in which such terms are used.

As used herein, the terms "a" and "an" and "the" and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated by the context in which they are used.

The term "epitope" refers to an epitope on an antigen that is recognized by an antigen binding molecule (e.g., an sdAb or polypeptide of the invention), more specifically, by an antigen binding site of the antigen binding molecule. The terms "epitope" and "epitope" may be used interchangeably. An amino acid sequence that binds to, has affinity for, and/or is specific for a particular epitope, antigen or protein is referred to as "anti" or "directed against" the epitope, antigen or protein.

As used herein, the term "comprising" and variations of the term (such as "comprises" and "comprising") are not intended to exclude other additives, components, integers or steps.

It is contemplated that the sdabs, polypeptides, and proteins described herein may contain so-called "conservative" amino acid substitutions, which are generally described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue that has a similar chemical structure and little or no effect on the function, activity, or other biological property of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions are those in which one amino acid in the following groups (a) to (e) is replaced by another amino acid of the same group: (a) small aliphatic nonpolar or low polar residues: ala, Ser, Thr, Pro, and Gly; (b) polar negatively charged residues and their (uncharged) amides: asp, Asn, Glu and Gln; (c) polar positively charged residues: his, Arg and Lys; (d) large aliphatic apolar residues: met, Leu, Ile, Val, and Cys; and (e) aromatic residues: phe, Tyr, and Trp. Other conservative substitutions include: ala with Gly or Ser; arg to Lys; asn for Gln or His; asp for Glu; cys for Ser; gln for Asn; glu is substituted by Asp; gly by Ala or Pro; his for Asn or Gln; ile to Leu or Val; leu by Ile or Val; lys for Arg, Gln or Glu; met for Leu, Tyr for Ile; phe for Met, Leu or Tyr; ser for Thr; thr to Ser; trp to Tyr; tyr is replaced by Trp; and/or Phe for Val, Ile or Leu.

As used herein, "domain" generally refers to, or consists essentially of, a globular region of an antibody chain, particularly a globular region of a heavy chain antibody.

The amino acid sequence and structure of sdabs typically consists of four framework regions or "FRs", which are referred to as "framework region 1" or "FR 1", respectively; "framework region 2" or "FR 2"; "framework region 3" or "FR 3"; and "framework region 4" or "FR 4". The framework regions are interrupted by three complementarity determining regions, or "CDRs," referred to as "complementarity determining region 1" or "CDR 1," respectively; "complementarity determining region 2" or "CDR 2"; and "complementarity determining region 3" or "CDR 3".

As used herein, the term "humanized sdAb" refers to an sdAb in which one or more amino acid residues in the amino acid sequence of a naturally occurring VHH sequence are replaced by one or more amino acid residues present at corresponding positions in a VH domain from a human conventional 4-chain antibody. This can be done by methods well known in the art. For example, the FR of an sdAb can be replaced by a human variable FR.

As used herein, an "isolated" nucleic acid or amino acid is separated from at least one other component with which it is ordinarily associated (e.g., its source or culture medium, another nucleic acid, another protein/polypeptide, another biological component or macromolecule or contaminant, impurity, or minor component).

The term "mammal" is defined as an individual belonging to the class mammalia and includes, but is not limited to, humans, domestic and farm animals, and zoo, sports and pet animals, such as cattle, horses, sheep, dogs and cats.

As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and delayed absorption agents and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of the standard reference book Remington's Pharmaceutical Sciences in the art. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, PBS (phosphate buffered saline), and 5% human serum albumin. Liposomes, cationic lipids and non-aqueous vehicles such as fixed oils (fixed oils) may also be used. The use of such media and agents in pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the therapeutic agents defined above, its use in the compositions of the invention is contemplated.

"quantitative immunoassay" refers to any means of determining the amount of antigen present in a sample by using an antibody. Methods for performing quantitative immunoassays include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), Specific Analyte Labeling and Recapture Assays (SALRAs), liquid chromatography, mass spectrometry, and fluorescence-activated cell sorting, among others.

The term "solution" refers to a composition comprising a solvent and a solute, and includes true solutions and suspensions. Examples of solutions include solids dissolved in a liquid, liquids or gases, and particles or micelles suspended in a liquid.

The term "specificity" refers to the number of different types of antigens or antigenic determinants that a particular antigen binding molecule or antigen binding protein molecule is capable of binding. The specificity of an antigen binding protein can be determined based on affinity and/or avidity (avidity). The affinity, expressed by the dissociation equilibrium constant (KD) of an antigen to an antigen binding protein, is a measure of the strength of binding between an epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen-binding molecule (alternatively, affinity may also be expressed as an affinity constant (KA), which is 1/KD). It will be clear to one skilled in the art that affinity may be determined based on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen-binding molecule and an antigen. Avidity relates both to the affinity between an epitope and its antigen binding site on the antigen binding molecule and to the number of associated binding sites present on the antigen binding molecule. Specific binding of an antigen binding protein to an antigen or epitope can be determined by any known means, e.g., Scatchard assays and/or competitive binding assays such as Radioimmunoassay (RIA), Enzyme Immunoassay (EIA), and sandwich competition assays.

As used herein, the term "recombinant" refers to the use of genetic engineering methods (e.g., cloning and amplification) for producing the sdabs of the invention.

A "single domain antibody", "sdAb" or "VHH" can be generally defined as a polypeptide or protein comprising an amino acid sequence consisting of four framework regions interrupted by three complementarity determining regions. It is represented by FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. The sdabs of the invention also include polypeptides or proteins comprising the amino acid sequence of the sdabs. Typically, sdabs are produced in camelids such as llamas, but may also be produced synthetically using techniques well known in the art. As used herein, the variable domains present in naturally occurring heavy chain antibodies are also referred to as "VHH domains" in order to distinguish them from heavy chain variable domains referred to as "VH domains" present in conventional 4 chain antibodies and light chain variable domains referred to as "VL domains" present in conventional 4 chain antibodies. "VHH" and "sdAb" are used interchangeably herein. Numbering of amino acid residues of sdabs or polypeptides is performed according to the general numbering of VH domains given by Kabat et al ("Sequence of proteins of immunological interest," US Public Health Services, NIH Bethesda, MD, publication No. 91). According to this numbering, FR1 of the sdAb includes amino acid residues from position 1 to 30, CDR1 of the sdAb includes amino acid residues from position 31 to position 36, FR2 of the sdAb includes amino acid residues from position 36 to position 49, CDR2 of the sdAb includes amino acid residues from position 50 to position 65, FR3 of the sdAb includes amino acid residues from position 66 to position 94, CDR3 of the sdAb includes amino acid residues from position 95 to position 102, and FR4 of the sdAb includes amino acid residues from position 103 to position 113.

The term "synthesis" refers to production by in vitro chemical synthesis or enzymatic synthesis.

The term "target" as used herein refers to any component, antigen or moiety recognized by an sdAb. The term "intracellular target" refers to any component, antigen or moiety that is present within a cell. A "transmembrane target" is a component, antigen or moiety located within the cell membrane. An "extracellular target" refers to a component, antigen, or moiety that is located outside of a cell.

As used herein, "therapeutic composition" refers to a substance intended to have a therapeutic effect, such as pharmaceutical compositions, genetic material, biological agents, and other substances. Genetic material includes substances intended to have a direct or indirect genetic therapeutic effect, such as genetic vectors, genetic regulator elements, genetic structural elements, DNA and RNA, and the like. Biological agents include substances that are or are derived from living matter intended to have a therapeutic effect.

As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in treating, preventing, or managing a disease or overt symptoms of a disease. A therapeutically effective amount can treat a disease or condition, a symptom of a disease, or a predisposition to a disease, and the specific amount that is targeted to cure, alleviate, alter, remedy, improve, ameliorate, or affect the disease, the symptom of a disease, or the predisposition to a disease can be readily determined by the ordinary medical practitioner and may vary according to factors known in the art, such as the type of disease, the patient's history and age, the stage of the disease, and the administration of other therapeutic agents.

The present invention relates to single domain antibodies (sdabs) against viral and intracellular components as well as proteins and polypeptides comprising the sdabs and nucleotides encoding the proteins and polypeptides. The invention can also relate to sdabs directed against intracellular, transcellular and extracellular targets or antigens. The invention also includes nucleic acids encoding the sdabs, proteins and polypeptides comprising the sdabs, and compositions. The invention comprises the use of said composition, sdAb, protein or polypeptide for prophylactic, therapeutic or diagnostic purposes.

sdabs have many unique structural and functional properties, which make them highly advantageous for use as functional antigen-binding domains or proteins. sdabs bind to antigen in the absence of a light chain variable domain and can act as single, relatively small, functional antigen binding building blocks, domains or proteins. This distinguishes sdabs from the domains of conventional antibodies, which do not themselves act as antigen binding proteins or domains, but rather need to be combined with conventional antibody fragments, such as antigen binding fragments (Fab) or single chain variable fragments (ScFv) to facilitate antigen binding.

sdabs can be obtained by methods well known in the art. For example, one method of obtaining an sdAb comprises: (a) immunizing a camelid with one or more antigens, (b) isolating peripheral lymphocytes from the immunized camelid, obtaining total RNA and synthesizing the corresponding complementary dna (cDNA), (c) constructing a library of cDNA fragments encoding the VHH domain, (d) transcribing the cDNA encoding the VHH domain obtained in step (c) into messenger RNA using PCR, converting the mRNA into ribosome display form and selecting the VHH domain by ribosome display, and (e) expressing the VHH domain in a suitable vector and optionally purifying the expressed VHH domain.

Another method for obtaining the sdabs of the invention is performed by: nucleic acids encoding sdabs are prepared using nucleic acid synthesis techniques and subsequently expressed in vivo or in vitro. In addition, the sdabs, polypeptides, and proteins of the invention can be prepared using synthetic or semi-synthetic techniques for preparing proteins, polypeptides, or other amino acid sequences.

The sdabs of the invention typically bind to analogs, variants, mutants, alleles, portions, and fragments of all naturally occurring or synthetic targets, or at least those analogs, variants, mutants, alleles, portions, and fragments that bind to targets containing one or more epitopes or epitopes that are substantially identical to the epitope or epitope bound by the sdAb of the invention in the wild-type target. The sdabs of the invention can bind to the analogs, variants, mutants, alleles, moieties, and fragments with the same, higher, or lower affinity and/or specificity as the sdabs of the invention bind to the wild-type target. Furthermore, the binding of the sdabs of the invention to some but not others of the analogs, variants, mutants, alleles, portions and fragments of the target is also contemplated within the scope of the invention. In addition, the sdabs of the invention can be humanized, and can be monovalent or multivalent, and/or multispecific. In addition, the sdabs of the invention can bind to phosphorylated forms of the target protein as well as to non-phosphorylated forms of the target protein. The sdAb can be linked to other molecules such as albumin or other macromolecules.

In addition, it is also within the scope of the invention that the sdAb is multivalent, i.e., the sdAb may have more than two proteins or polypeptides directed against more than two different epitopes of the target. In such multivalent sdabs, the proteins or polypeptides can be directed against, for example, the same epitope, a substantially equivalent epitope, or different epitopes. The different epitopes may be located on the same target, or may be located on two or more different targets.

Furthermore, it is also contemplated that the sequence of one or more sdabs of the invention can be linked or joined to one or more linker sequences. The linker may be, for example, a protein sequence containing a combination of serine, glycine, and alanine.

Furthermore, the use of parts, fragments, analogues, mutants, variants, alleles and/or derivatives of the sdabs of the invention also falls within the scope of the invention, as long as they are suitable for the described use.

As the sdabs of the invention are primarily intended for therapeutic and/or diagnostic use, they are directed against mammalian, preferably human targets. However, the sdabs described herein can be cross-reactive with targets from other species, e.g., targets of one or more other species (e.g., mouse, rat, rabbit, pig, or dog) from primates or other animals, particularly in animal models of diseases and conditions associated with target-related diseases.

In another aspect, the invention relates to a nucleic acid encoding an sdAb of the invention. Such nucleic acids may be in the form of, for example, genetic constructs.

In another aspect, the invention relates to a host or host cell expressing or capable of expressing an sdAb of the invention and/or comprising a nucleic acid encoding an sdAb of the invention. The sequence of the sdAb can be used to insert into the genome of any organism to create a Genetically Modified Organism (GMO). Examples include, but are not limited to, plants, bacteria, viruses, and animals.

The invention also relates to methods of making or producing the sdabs, nucleic acids encoding the sdabs, host cells expressing or capable of expressing the sdabs, products and compositions comprising the sdabs of the invention.

The invention also relates to the use and uses of the sdabs, nucleic acids encoding the sdabs, host cells, products and compositions described herein. Such products or compositions may for example be pharmaceutical compositions for the treatment or prevention of diseases, or products or compositions for diagnostic use. sdabs can be used in a variety of assays, such as ELISA assays and mass spectrometry assays for determining serum and tissue levels of sdabs.

In another aspect, a nucleic acid encoding one or more sdabs of the invention can be inserted into the genome of an organism to treat or prevent a disease.

The present invention relates generally to sdabs and proteins or polypeptides comprising or consisting essentially of one or more such sdabs, which can be used for prophylactic, therapeutic and/or diagnostic purposes.

The methods and compositions detailed herein can be used to treat the diseases described herein and can be used in any dosage and/or formulation described or known herein and any route of administration described herein or known to those of skill in the art.

The sdabs of the invention can be used to treat and prevent diseases caused by viruses or by abnormal cellular proteins. The sdabs of the invention can also be used for the treatment and prevention of diseases. The sdabs of the invention can be used to target diseases where there is overexpression of an intracellular molecule. It can also be used to treat viral infections by targeting intracellular viral proteins in infected cells. Blocking the production of viral proteins (e.g., HIV-1 reverse transcriptase) can block the viral life cycle.

The sdabs of the invention can also target intracellular viral proteins such as ebola VP24 and thereby block the ability of ebola to stop the host's antiviral immune response.

The sdabs of the invention can be used with one or more compounds. For example, an sdAb of the invention can be used with a JAK/STAT inhibitor, e.g., curcumin, resveratrol, cucurbitacin A, B, E, I, Q, Flavopiridol (Flavopiridol), doxycycline, cyclopentenone derivatives, N-acyl homoserine lactones, indirubin derivatives, methylisonidi (meiisonicondigo), tyrphostin inhibitors (Tyrphostins), platinum-containing compounds (e.g., IS3-295), peptidomimetics, antisense oligonucleotides, S3I-201, phosphotyrosine tripeptide derivatives, HIV protease inhibitors (e.g., nelfinavir, indinavir, saquinavir, and ritonavir), JSI-124, XpYL, Ac-pllpqtv-NH 2, ISs 610, CJ-1383, pyrimethamine, metformin, avimod, S3I-M2001, STX 0119; n- [2- (1,3, 4-oxadiazolyl) ] -4-quinolinecarboxamide derivatives, S3I-1757, LY 5; 5, 8-dioxo-6 (pyridin-3-ylamino) -5, 8-dihydro-naphthalene-1-sulfonamide, withacistin, static, STA-21, LLL-3, LLL12, XZH-5, SF-1066, SF-1087, 17o, cryptotanshinone, FLL32, FLL62, C188-9, BP-1108, and BP-1075, Sarcodon (Galiellactone), JQ1, 5, 15DPP, WP1066, niclosamide, SD1008, Nifuroxazide (Nifuroxazide), cryptotanshinone, BBI quinone, and ruxolitinib phosphate. The one or more compounds can increase the therapeutic response and improve the effectiveness of the sdabs of the invention. Additionally, the effectiveness of sdabs can be increased by combining them with peptides, peptidomimetics, and other drugs (such as, but not limited to, cimetidine, atorvastatin, celecoxib, metformin, and cimetidine).

It is also contemplated that one or more sdabs of the invention can be combined, or that an sdAb of the invention can be combined with other sdabs.

It is contemplated that certain sdabs of the invention are capable of passing through a cell membrane and into a cell without the aid of a targeting protein sequence on the additional sdAb, and without the aid of an exogenous compound that directs the sdAb to bind to a cell surface receptor and pass through the cell membrane.

After crossing the cell membrane, these sdabs are able to target transmembrane or intracellular molecules or antigens. These targets can be, for example, proteins, carbohydrates, lipids, nucleic acids, muteins, viral proteins, and prions. sdAb targets can function as enzymes, structural proteins of a cell, intracellular portions of cell membrane molecules, molecules within membranes of organelles, RNA molecules of any type, any region of DNA or chromosome, methylated or unmethylated nucleic acids, partially assembled molecules within the synthetic machinery of a cell, second messenger molecules, and molecules within the signaling machinery of a cell. Targets may include all molecules in the cytoplasm, nucleus, organelles, and cell membrane. Molecules targeted for secretion or retention in the cell membrane can be targeted within the cytoplasm before leaving the cell.

The sdAb target can be in humans, animals, plants, fungi, parasites, protists, bacteria, viruses, prions, prokaryotic cells, and eukaryotic cells. Some examples of intracellular and intercellular signaling molecules and proteomes that can be targeted by the sdabs of the invention are: oncogenic products, hormones, cytokines, growth factors, neurotransmitters, kinases (including tyrosine, serine and threonine kinases), phosphatases, ubiquitin, cyclic nucleotides, cyclases (adenylyl and guanylyl), proteins G, phosphodiesterases, the GTPase superfamily, immunoglobulins (antibodies, Fab fragments, conjugates, sdabs), the immunoglobulin superfamily, phosphoinositide lipids, steroid receptors, calmodulin, CD groups (e.g., CD4, CD8, CD28, etc.), transcription factors, TGF- β, TNF- α and β, TNF ligand superfamily, notch receptor signaling molecules, hedgehog receptor signaling molecules, Wnt receptor signaling molecules, toll-like receptor signaling molecules, caspase, actin, myosin, myostatin, 12-lipoxygenase, 15-lipase, Lipoxygenase superfamily, reverse transcriptase, viruses and their proteins, amyloid, collagen, G protein coupled receptors, mutated normal proteins, prion, Ras, Raf, Myc, Src, BCR/ABL, MEK, Erk, Mos, Tpl2, MLK3, TAK, DLK, MKK, p38, MAPK, MEKK, ASK, SAPK, JNK, BMK, MAP, JAK, PI3K, cyclooxygenase, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6, Myc, p53, BRAF, NRAS, KRAS, HRAS and chemokines.

HIV is a retrovirus responsible for the Acquired Immune Deficiency Syndrome (AIDS) in humans. AIDS causes progressive failure of the immune system of the infected individual, which leads to the development of life-threatening opportunistic infections and cancer. The mean survival time after infection with HIV is expected to be 9 to 11 years without treatment.

HIV is delivered as a single-stranded, positive-sense, enveloped RNA virus. Upon entry into the target cell, the viral RNA genome is reverse transcribed into double-stranded DNA by the virally encoded Reverse Transcriptase (RT) that is transported with the viral genome in the viral particle. RT is an RNA-dependent DNA polymerase and has RNaseH activity. The resulting viral DNA is then introduced into the host cell nucleus and integrated into the cellular DNA by the viral-encoded integrase and host co-factors. Once integrated, the virus may remain latent for months or even years. Alternatively, the virus may be transcribed, producing a new RNA genome and viral proteins, which are encapsulated and released from the cell as new viral particles.

Two classes of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is more virulent, more infectious and is the cause of most HIV infections worldwide. HIV-2 is primarily restricted to Western Africa.

anti-HIV RT sdabs have been developed to target HIV-1 reverse transcriptase. anti-HIV-1 RT sdabs can successfully treat individuals infected with HIV alone or with HIV and other retroviral agents. Recombinant HIV-1 reverse transcriptase protein (Creative Biomart, Shirley, NY) (SEQ ID NO:1) was used to generate sdAbs against HIV-1RT or an epitope that binds thereto using methods well known in the art.

The protein sequence of the recombinant HIV-1 reverse transcriptase protein (SEQ ID NO:1) for camelid immunization is

PISPIETVPVKLKPGMDGPKVKQWPLT

EEKIKALVEICAELEEEGKISRIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSIPLDEDFRKYTAFTIPSTNNETPGTRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYVDDLYVGSDLEIGQHRTKVEELRQHLWRWGFYTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQK。

As a result of immunization, several sdabs were obtained and screened. The DNA sequence of the anti-HIV-1 RT sdAb is listed below:

HIV1-1(SEQ ID NO:2):5’-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctgtttacagctacaacacaaactgcatgggttggttccgccaggctccagggaaggagcgcgagggggtcgcagttatttatgctgctggtggattaacatactatgccgactccgtgaagggccgattcaccatctcccaggagaatggcaagaatacggtgtacctgacgatgaaccgcctgaaacctgaggacactgccatgtactactgtgcggcaaagcgatggtgtagtagctggaatcgcggtgaggagtataactactggggccaggggacccaggtcaccgtctcctca-3’

HIV1-2(SEQ ID NO:3):5’-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagactctctgagactctcctgtgcagcctctggaaacactgccagtaggttctccatgggctggttccgccaggctccagggaaggagcgcgagggggtcgcggctatttctgctggtggtaggcttacatactatgccgactccgtgaagggccgattcaccatctcccgagacaacgccaagaacacgctgtatctggacatgaacaacctgaaacctgaggacactgccatgtactactgtgccgcaattagtgaccggatgactggtattcaggctcttgcggctctacccagacttcgcccagaagactacggtaactggggccaggggaccctggtcaccgtctcctca-3’

HIV1-7(SEQ ID NO:4):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggaccctggtcaccgtctcctca-3’

HIV1-8(SEQ ID NO:5):5’-caggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggccaggggacccaggtcaccgtctcctca-3’

HIV1-6(SEQ ID NO:6):5’-caggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccaatatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggccaggggaccctggtcaccgtctcctca-3’

HIV1_28(SEQ ID NO:7):5’-aggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggccaggggaccctggtcaccgtctcctca-3’

HIV1-21(SEQ ID NO:8):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

HIV1-37(SEQ ID NO:9):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

HIV1-3(SEQ ID NO:10):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcactgtctcctca-3’

HIV1-5(SEQ ID NO:11):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaggcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctaccattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgctaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

HIV1-10(SEQ ID NO:12):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcagcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

HIV1_29(SEQ ID NO:13):5’-gaggtgcagctggtggagtctgggggagactcagtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

HIV1_32(SEQ ID NO:14):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

HIV1-9(SEQ ID NO:15):5’-gaggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctgtttacagctacaacacaaactgcatgggttggttccgccaggctccagggaaggagcgcgagggggtcgcagttatttatgctgctggtggattaacatactatgccgactccgtgaagggccgattcaccatctcccaggagaatggcaagaacacggtgtacctgacgatgaaccgcctgaaacctgaggacactgccatgtactactgtgcggcaaagcgatggtgtagtagctggaatcgcggtgaggagtataactactggggccaggggacccaggtcactgtctcctca-3’

HIV1-16(SEQ ID NO:16):5’-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggaaacacctacagtagtagctactgcatgggctggttccgccaggctccagggaaggaccgcgagggggtcgcgcgtattttcactcgaagtggtaccacatactatgccgactccgtgaagggccgattcaccatttcccgtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaagacgctgccatgtactactgtgcggcagcccaggggggtgcctgcatttcgtttacttcgttcgcgaagaatttcgtgtaccggggccaggggaccctggtcactgtctcctca-3’

HIV1-13(SEQ ID NO:17):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggtcctctgactataactactggggtgaggggaccctggtcaccgtctcctca-3’

HIV1_35(SEQ ID NO:18):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggtcctctgactataactactggggtgaggggaccctggtcaccgtctcctca-3’

HIV1-11(SEQ ID NO:19):5’-caggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcactgtctcctca-3’

HIV1_22(SEQ ID NO:20):5’-caggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

HIV1-4(SEQ ID NO:21):5’-catgtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggaccctggtcaccgtctcctca-3’

HIV1_38(SEQ ID NO:22):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccaactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactatgactactggggtgaggggaccctggtcaccgtctcctca-3’

HIV1_23(SEQ ID NO:23):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatggacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

HIV1_25(SEQ ID NO:24):5’-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaggcctctggatacacctacaatagtagagtcgatatcagatctgtgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtctcctca-3’

the amino acid sequence of the anti-HIV-1 RT sdAb is shown below:

HIV1-1(SEQ ID NO:25):DVQLVESGGGSVQAGGSLRLSCAASVYSYNTNCMGWFRQAPGKEREGVAVIYAAGGLTYYADSVKGRFTISQENGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS

HIV1-2(SEQ ID NO:26):QVQLVESGGGSVQAGDSLRLSCAASGNTASRFSMGWFRQAPGKEREGVAAISAGGRLTYYADSVKGRFTISRDNAKNTLYLDMNNLKPEDTAMYYCAAISDRMTGIQALAALPRLRPEDYGNWGQGTLVTVSS

HIV1-9(SEQ ID NO:27):EVQLVESGGGSVQAGGSLRLSCAASVYSYNTNCMGWFRQAPGKEREGVAVIYAAGGLTYYADSVKGRFTISQENGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS

HIV1-16(SEQ ID NO:28):QVQLVESGGGSVQAGGSLRLSCAASGNTYSSSYCMGWFRQAPGKDREGVARIFTRSGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDAAMYYCAAAQGGACISFTSFAKNFVYRGQGTLVTVSS

HIV1-27(SEQ ID NO:29):EVQLGESGGGSVQAGGSLRLSCAASVYSYTTNCMGWFRQAPGKEREGVAVIYSAGGLTYYADSVKGRFTISQDNGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS

HIV1-30(SEQ ID NO:30):QVQLVESGGGSVQAGGSLRLSCAASVYSYNTNCMGWFRQAPGKEREGAAVIYAAGGLTYYADSVKGRFTISQENGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS

HIV1-21(SEQ ID NO:31):EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSS

HIV1-4(SEQ ID NO:32):HVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTLVTVSS

HIV1-6(SEQ ID NO:33):QVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGQGTLVTVSS

HIV1-7(SEQ ID NO:34):EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTLVTVSS

HIV1-8(SEQ ID NO:35):QVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGQGTQVTVSS

HIV1-11(SEQ ID NO:36):QVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSS

HIV1-13(SEQ ID NO:37):EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRSSDYNYWGEGTLVTVSS

HIV1-23(SEQ ID NO:38):EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMDALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSS

HIV1-24(SEQ ID NO:39):HVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPGDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGQGTLVTVSS

HIV1-25(SEQ ID NO:40):EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSVGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSS

HIV1-31(SEQ ID NO:41):DVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSS

HIV1-38(SEQ ID NO:42):EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYANSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYDYWGEGTLVTVSS

HIV1-39(SEQ ID NO:43):EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPTRYGIRPSDYNYWGQGTQVTVSS

one or more mouse monoclonal antibodies can be raised against one or more domains of the anti-HIV-1 RT sdAb of the invention. Mouse monoclonal antibodies can be produced by methods known to those of skill in the art, e.g., mouse monoclonal antibodies can be produced by mouse hybridomas. Mouse monoclonal antibodies can be used for diagnostic assays, e.g., the antibodies can be used in immunoassays such as ELISA or mass spectrometric assays to determine the amount of anti-HIV-1 RT sdAb present in a sample from a patient.

Sdabs can also be produced against recombinant arachidonic acid 12-lipoxygenase (ALOX 12). ALOX12 is also known as the platelet-type 12-lipoxygenase, arachidonic acid oxygen 12-oxidoreductase, delta 12-lipoxygenase, 12 delta-lipoxygenase, C-12 lipoxygenase, leukotriene A4 synthase, and LTA4 synthase. ALOX12 is a lipoxygenase-type enzyme involved in the metabolism of arachidonic acid. ALOX12 has been associated with the development and complications of diet-induced and/or genetically-induced diabetes, adipocyte/tissue dysfunction, and obesity. ALOX12 is also believed to regulate vasoconstriction, dilation, pressure, remodeling and angiogenesis. Inhibition of ALOX12 prevented the development of angiogenesis, and therefore ALOX12 is a target for reducing neovascularization that contributes to arteriosclerosis, steatohepatitis, and other arthritic and cancer diseases. An elevated amount of ALOX12 may contribute to the development of alzheimer's disease.

The invention provides sdabs, proteins and polypeptides directed against the ALOX12 protein.

It is contemplated that the anti-ALOX 12 sdabs and polypeptides of the invention can be used to prevent and/or treat diseases and disorders associated with and/or mediated by ALOX12, such as diabetes, adipocyte dysfunction, obesity, atherosclerosis, steatohepatitis, arthritis, and cancer.

Recombinant human ALOX12 protein was used to generate sdabs against ALOX12 or epitopes that bind thereto. To generate the anti-ALOX 12 sdAb, recombinant human ALOX12 was expressed in e.coli and used as the target antigen.

The recombinant ALOX12 protein sequence (SEQ ID NO:44) used for camel immunization was:

MGRYRIRVATGAWLFSGSYNRVQLWLVGTRGEAELELQLRPARGEEEEFDHDVAEDLGLLQFVRLRKHHWLVDDAWFCDRITVQGPGACAEVAFPCYRWVQGEDILSLPEGTARLPGDNALDMFQKHREKELKDRQQIYCWATWKEGLPLTIAADRKDDLPPNMRFHEEKRLDFEWTLKAGALEMALKRVYTLLSSWNCLEDFDQIFWGQKSALAEKVRQCWQDDELFSYQFLNGANPMLLRRSTSLPSRLVLPSGMEELQAQLEKELQNGSLFEADFILLDGIPANVIRGEKQYLAAPLVMLKMEPNGKLQPMVIQIQPPNPSSPTPTLFLPSDPPLAWLLAKSWVRNSDFQLHEIQYHLLNTHLVAEVIAVATMRCLPGLHPIFKFLIPHIRYTMEINTRARTQLISDGGIFDKAVSTGGGGHVQLLRRAAAQLTYCSLCPPDDLADRGLLGLPGALYAHDALRLWEIIARYVEGIVHLFYQRDDIVKGDPELQAWCREITEVGLCQAQDRGFPVSFQSQSQLCHFLTMCVFTCTAQHAAINQGQLDWYAWVPNAPCTMRMPPPTTKEDVTMATVMGSLPDVRQACLQMAISWHLSRRQPDMVPLGHHKEKYFSGPKPKAVLNQFRTDLEKLEKEITARNEQLDWPYEYLKPSCIENSVTI

as a result of immunization, several sdabs were obtained and screened. The DNA sequence of sdabs is listed below:

ALOX_21(SEQ ID NO:45):5’-gaggtgcagctggtggagtctgggggaggttcggtgcaggctggagggtctctgaggatctcctgtacagcctctggattcacttttgatgacactgacatgggctggtaccgccagactctaggaaatgggtgcgagttggtttctcagattagtaatgatggtagtacattctatagagattccgtgaagggccgattcaccatctcctgggaccgcgtcaacaacacggtgtatctgcaaatgagcgccctgagacctgaggacacggccatgtattactgcaatatcaacgggtgtaggagaccctcgtacaatcttcacttgaacgcatggggccaggggacacaggtcaccgtctcctca-3’

ALOX_41(SEQ ID NO:46):5’-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgacactgtcctgtgtagcctctggatacggctacagtgccacgtgcatgggctggttccgccaggctccagggaaggagcgcgagggggtcgcgtctatttcaccttatggtgttagaaccttctatgccgactccgcgaaaggccgattcaccgtctcccgagacaacgccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacacgtccgtgtactactgtgcggccggttcgggcgttggtgtttgttcactttcgtatccatacacctactggggccaggggacccaggtcaccgtctcctca-3’

ALOX_43(SEQ ID NO:47):5’-caggtgcagctggtggagtctgggggaggctcggtgcgggctggagagtctctgagactctcctgtgtagcctctagatccatctatgtttggtactgcatgggctggttccgccaggctgcagggaaggagcgcgagggggtcggaagtatgttcgttggtggcggtaggacatattatgacgactccgtcaagggccgattcaccatctcccaagacaaggccaagaacacgctgtatctgcaaatggacaacctggcacctgaagacactgccatgtattactgtgcggctgggcgctgcggtggcaactggctgagaagcaatgctttcgacaaatggggccaggggacactggtcaccgtctcctca-3’

ALOX_46(SEQ ID NO:48):5’-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagccactggaaacacctacattagccgctgcatgggctggttccgccagcctccagggaaggagcgcgaggtggtcgcacgtatttataccgactctggtaatacatactatcccgacgccgtggagggccgattcaccatctcccaagacaacgccaagaacacgatatatctgcaaatgaacagcctgaaacctgacgacaccgccgtgtactactgtgtgctctcagaggccgtctgtacaaaagaacctggggactttcgttactggggccaggggacccaggtcactgtctcctca-3’

the protein sequence of the anti-ALOX sdAb is as follows:

ALOX_21(SEQ ID NO:49):EVQLVESGGGSVQAGGSLRISCTASGFTFDDTDMGWYRQTLGNGCELVSQISNDGSTFYRDSVKGRFTISWDRVNNTVYLQMSALRPEDTAMYYCNINGCRRPSYNLHLNAWGQGTQVTVSS

ALOX_41(SEQ ID NO:50):QVQLVESGGGSVQAGGSLTLSCVASGYGYSATCMGWFRQAPGKEREGVASISPYGVRTFYADSAKGRFTVSRDNAKNTLYLQMNSLKPEDTSVYYCAAGSGVGVCSLSYPYTYWGQGTQVTVSS

ALOX_43(SEQ ID NO:51):QVQLVESGGGSVRAGESLRLSCVASRSIYVWYCMGWFRQAAGKEREGVGSMFVGGGRTYYDDSVKGRFTISQDKAKNTLYLQMDNLAPEDTAMYYCAAGRCGGNWLRSNAFDKWGQGTLVTVSS

ALOX_46(SEQ ID NO:52):DVQLVESGGGSVQAGGSLRLSCAATGNTYISRCMGWFRQPPGKEREVVARIYTDSGNTYYPDAVEGRFTISQDNAKNTIYLQMNSLKPDDTAVYYCVLSEAVCTKEPGDFRYWGQGTQVTVSS

one or more monoclonal antibodies can be raised against one or more domains of the anti-ALOX 12 sdAb of the invention. Mouse monoclonal antibodies can be generated by methods known to those of skill in the art, e.g., mouse monoclonal antibodies can be produced by mouse hybridomas. Mouse monoclonal antibodies can be used in diagnostic assays, e.g., the antibodies can be used in immunoassays such as ELISA or mass spectrometry assays to measure the amount of anti-ALOX 12 sdAb present in a sample from a patient.

Ebola, also known as Ebola Virus Disease (EVD) and Ebola Hemorrhagic Fever (EHF), is a viral hemorrhagic fever of humans and other primates caused by ebola virus. The risk of mortality for the disease is high, typically killing 25% to 90% of infected persons 6 to 16 days after symptoms appear.

Ebola interferes with the proper functionality of the natural immune system of the infected individual. Ebola proteins attenuate the immune system's response to viral infection by interfering with the ability of cells to produce and respond to interferon proteins such as interferon-alpha, interferon-beta, and interferon gamma. The structural proteins VP24 and VP35 of ebola play a key role in this infection. The V24 protein blocks the production of antiviral proteins by the host cell. By suppressing the host's immune response, ebola rapidly spreads throughout the body.

As described herein, an anti-VP 24 sdAb was developed to target ebola VP24 protein. The anti-VP 24 sdAb can successfully treat individuals infected with ebola alone or in combination with other retroviral agents. Recombinant VP24 protein (SEQ ID NO:53) was used to produce sdabs against VP24 or capable of binding to its epitope, using methods well known in the art.

The protein sequence of the recombinant VP24 protein (SEQ ID NO:53) used for camel immunization is:

AKATGRYNLISPKKDLEKGVVLSDLCNFLVSQTIQGWKVYWAGIEFDVTHKGMALLHRLKTNDFAPAWSMTRNLFPHLFQNPNSTIESPLWALRVILAAGIQDQLIDQSLIEPLAGALGLISDWLLTTNTNHFNMRTQRVKEQLSLKMLSLIRSNILKFINKLDALHVVNYNGLLSSIEI ILEFNSSLAI

as a result of the immunization, an anti-VP 24 sdAb, VP24_5, that binds to VP24 was obtained and screened. The DNA sequence of VP24_5(SEQ ID. NO:54) is:

5’-ATGGGTGAT GTGCAGCTGGTGGAGTCT GGGGGAGAC TCGGTGCGG GCTGGAGGG TCTCTTCAAATGGGTGAT GTGCAGCTG GTGGAGTCT GGGGGAGAC TCGGTGCGGGCTGGAGGGTCTCTTCAA CTCTCCTGT AAAGCCTCT GGATACACC TACAATAGTAGAGTCGATATCAGATCT ATGGGCTGG TTCCGCCAG TATCCAGGA AAGGAGCGCGAGGGGGTCGCTACTATT AATATTCGT AATAGTGTC ACATACTAT GCCGACTCCGTGAAGGGCCGATTCACC ATCTCCCAA GACAACGCC AAGAACACG GTGTATCTGCAAATGAACGCCCTGAAA CCTGAGGAC ACTGCCATG TACTACTGT GCGTTGTCAGACAGATTCGCGGCGCAG GTACCTGCC AGGTACGGAATACGGCCC TCTGACTAT AACTACTGG GGTGAGGGG ACCCTGGTC ACCGTCTCC TCAAGCTCT GGTCTCGAG-3’

the amino acid sequence of VP24_5sdAb (SEQ ID NO:55) is shown below, with the CDRs underlined:

MGDVQLVESGGDSVRAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADS VKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTLVTVSSSSGLE

one or more monoclonal antibodies can be generated against one or more domains of the anti-VP 24 sdAb of the invention. Mouse monoclonal antibodies can be generated by methods known to those of skill in the art, e.g., mouse monoclonal antibodies can be produced by mouse hybridomas. The mouse monoclonal antibody may be used in a diagnostic assay, for example, the antibody may be used in an immunoassay such as an ELISA or mass spectrometric assay to measure the amount of anti-VP 24 sdAb present in a sample from a patient.

Examples

Example 1 Generation of sdabs

Sdabs are produced by camels immunized with several proteins including ALOX12(SEQ ID NO:44), VP24(SEQ ID NO:53), and HIV-1 reverse transcriptase (SEQ ID NO: 1).

Phage display libraries were constructed using pCdisplay-3M vector (Creative Biogene, Shirley, NY) and M13K07 helper phage (New England Biolabs, Ipswich, Mass.) using standard techniques. Single clones of sdabs were confirmed by ELISA and DNA and protein sequences were determined using standard methods.

Example 2 binding of HIV1-9(SEQ ID NO:27) sdAb to HIV-1 reverse transcriptase and Ebola VP-24

Protein binding experiments were performed at 25 ℃ on a Biacore 3000(General Electric Company, Fairfield, CT). The assay buffer contained 10mM HEPES buffer (pH 7.4), 150mM NaCl, 3mM EDTA, 0.05% P20. The regeneration buffer contained 10mM glycine HCl, pH 1.75, and the fixation buffer contained 10mM sodium acetate, pH 5.0. The flow rate for capturing the ligand was 5. mu.L/min. The flow rate for kinetic analysis was 30. mu.L/min.

Ligands used for protein binding experiments were HIV1-9(SEQ ID NO:27) and STAT3-VHH 14(SEQ ID NO: 56). Ligands were immobilized on flow cells 2 and 4 of CM5 sensor chips in 1200 and 550 Response Units (RU) respectively by amine coupling (EDC/NHS). The flow cell 1 is left empty andused as background subtraction. Unoccupied sites on the CM5 chip were blocked with 1M ethanolamine. For the binding assay, the analyte rHIV-1(SEQ ID NO:1) was flowed over the sensor chip. The binding of the analyte to the ligand is monitored in real time. The affinity constant (K) was calculated from the observed association rate (ka) to the dissociation rate (kd)_D＝kd/ka) as shown in table 1.

Negative controls for protein binding experiments were anti-STAT 3 sdAb, VHH14(SEQ ID NO: 56):

QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAALSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAAREGWECGETWLDRTAGGHTYWGQGTLVTVSS

chi-squared (χ) between the actual sensorgram and the sensorgram generated by BIAnalysis software²) Analyzed to determine the accuracy of the analysis. X within 1-2²Values are considered to be accurate and values below 1 are considered to be extremely accurate.

TABLE 1

Ligands

Analyte

ka(1/Ms)

kd(1/s)

Rmax

KD(M)

Concentration (nM)

χ2

HIV1-9 VHH

rHIV-1

8.91x104

3.79x10-4

71.3

4.25x10-9

100

0.0321

STAT3 VHH14

rHIV-1

N/A

N/A

N/A

N/A

100

N/A

Full kinetic analysis was performed as shown in table 2 and with 2-fold serial dilutions of the highest analyte concentration. HIV1-9 anti-RT sdabs bind to HIV-1 and Ebola VP24 analytes.

TABLE 2

Example 3 binding of HIV1-9(SEQ ID NO:27) sdAb to HIV-1 reverse transcriptase in ELISA

Two different samples of HIV1-9 anti-HIV-1 RT sdAb (SEQ ID NO:27) were evaluated at1 μ g/mL in an ELISA against a checkerboard of coating antigen, secondary antibody (2 ° antibody) and HRP concentration. The coating antigen was recombinant HIV-1RT (creative BioMart) (SEQ ID NO:1) at 0.5. mu.g/mL, 0.025. mu.g/mL and 0.125. mu.g/mL per well. The secondary antibodies were rabbit anti-llama biotinylated antibodies diluted 1:5,000 and 1:10,000, and HRP diluted 1:25,000 and 1:50,000. Signal to noise ratio >20 was seen with several concentrations. The results of the ELISA are shown in FIGS. 1 and 2.

Three combinations were selected to evaluate serial dilutions (1. mu.g/mL to 0.0001. mu.g/mL) of HIV1-9 anti-HIV-1 RT sdAb (SEQ ID NO: 27).

Coating antigens	Second antibody	HRP
			0.5μg/mL	1:10,000	1:25,000
0.5μg/mL	1:5,000	1:50,000
			0.5μg/mL	1:10,000	1:50,000

The results are shown in fig. 3 and 4. The two preparations of HIV1-9 anti-HIV-1 RT sdAb (SEQ ID NO:27) used had very similar results. The binding of HIV1-9 anti-HIV-1 RT sdAb (SEQ ID NO:27) to HIV 1RT (SEQ ID NO:1) was shown with 0.5 μ g/mL coating, 1:5,000 diluted secondary antibody and 1:50,000 diluted HRP, with the highest signal-to-noise ratio and slightly lower blank value.

Example 4 VP24-5(SEQ ID NO:55) sdAb binding to VP24

Protein binding experiments were performed as described in example 2. The ligands used for protein binding were VP24-5(SEQ ID NO:55) and STAT3-VHH 14(SEQ ID NO: 56). By passingAmine coupling (EDC/NHS) ligands were immobilized at 427 and 550 Response Units (RU) on flow cells 2 and 4, respectively, of a CM5 sensor chip. Flow cell 1 was left blank and used as background subtraction. Unoccupied sites on the CM5 chip were blocked with 1M ethanolamine. For the binding assay, analyte VP24(SEQ ID NO:53) was flowed over the sensor chip and monitored in real time. The affinity constant (K) was calculated from the observed association rate (ka) to the dissociation rate (kd)_D＝kd/ka) as shown in Table 3.

TABLE 3

Ligands

Analyte

ka(1/Ms)

kd(1/s)

Rmax

KD(M)

Concentration (nM)

χ2

VP24-5-VHH

VP-24

1.39x105

8.77x10-4

6.84

6.31x10-9

100

0.0481

STAT3 VHH14

VP-24

NA

NA

NA

NA

100

NA

As shown in table 4, full kinetic analysis was performed at different analyte concentrations serially diluted 2-fold of the highest analyte concentration.

TABLE 4

Example 5 VP24-5(SEQ ID NO:55) sdAb binds to Ebola VP24 target in an ELISA

Two different samples of VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO:55) were evaluated at1 μ g/mL in an ELISA against a checkerboard of coating antigen, secondary antibody and HRP concentration. The coating antigen was recombinant Ebola VP24(Creative BioMart) (SEQ ID NO:53) at 0.5. mu.g/mL, 0.025. mu.g/mL, and 0.125. mu.g/mL per well. The secondary antibodies were biotinylated antibodies against llama in rabbit diluted 1:5,000 and 1:10,000. HRP was used at 1:10,000 and 1:25,000 dilutions. The ELISA results are shown in FIGS. 5 and 6. The signal to noise ratio was low and the analysis was repeated at higher concentrations.

The ELISA was repeated with 1. mu.g/mL and 0.5. mu.g/mL VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO: 55). Recombinant VP24(SEQ ID NO:53) was used at 0.5. mu.g/mL or 1. mu.g/mL per well. The secondary antibodies were rabbit anti-llama biotinylated antibodies diluted at 1:4,000, 1:10,000, and 1:10,000. HRP was used at 1:25,000 and 1:50,000 dilutions. The ELISA results are shown in FIGS. 7 and 8.

Three combinations were selected to evaluate serial dilutions (1. mu.g/mL to 0.0001. mu.g/mL) of VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO: 55).

Coating antigens	Second antibody	HRP
			0.5μg/mL	1:1,000	1:1,000
0.5μg/mL	1:10,000	1:25,000
			1μg/mL	1:4,000	1:25,000

The results are shown in fig. 9 and 10. The two VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO:55) preparations used had very similar results and showed binding of VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO:55) to recombinant VP24(SEQ ID NO: 53).

Although the present invention has been described in considerable detail with reference to certain preferred versions thereof, other versions are possible. For example, the steps disclosed for the method of the present invention are not intended to be limiting, and it is not intended to specify that each step is absolutely necessary for the method, but is merely an exemplary step. Therefore, the scope of the appended claims should not be limited to the description of the preferred embodiments contained in this disclosure. All documents applied herein are incorporated by reference in their entirety.