RU2810002C2 - Single-domain antibodies against intracellular antigens - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: single-domain antibody to STAT3. Also disclosed is an isolated polypeptide, a pharmaceutical composition containing said antibody. A method for diagnosing a STAT3-mediated disorder; a method for preventing or treating a STAT3-mediated disease using said antibody; use of pharmaceutical composition for the treatment of a STAT3-mediated disorder.

EFFECT: ability to effectively treat diseases associated with STAT3.

9 cl, 14 dwg, 41 tbl, 16 ex

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This international patent application claims priority to U.S. Provisional Patent Application No. 62/067908, filed Oct. 23, 2014, U.S. Provisional Patent Application No. 62/148656, filed April 16, 2015, U.S. Provisional Patent Application No. 62/188353, filed July 2, 2015, and US Provisional Patent Application No. 62/210795, filed August 27, 2015, the contents of which are incorporated herein by reference in their entirety.

LIST OF SEQUENCES

[0002] This application is filed together with a Sequence Listing in electronic format. The sequence listing is provided as a file called "Sequence_Listing_STP25.txt", created on September 30, 2015, last modified on October 22, 2015, which is 83,000 bytes in size. Information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE ART

[0003] The use of single domain antibodies (SDAbs) as individual antigen binding proteins or as an antigen binding domain in a larger protein or polypeptide provides a number of significant advantages over the use of conventional antibodies or antibody fragments. Advantages of OdAbs include: only one domain is required to bind antigen with high affinity and selectivity; sdAbs can be expressed from a single gene and do not require post-translational modification; OdAbs are highly resistant to heat, pH, proteases, and other denaturing agents or conditions; obtaining ODAT is inexpensive; and OdAbs can reach targets and epitopes that are inaccessible to conventional antibodies.

[0004] There are a number of diseases or pathological conditions, such as cancer, that are caused by aberrant intracellular or transmembrane components such as nucleotides and proteins. Elimination of aberrant components can be used to prevent or treat these diseases or pathological conditions. There are a number of pharmacological compounds available for treatment, but these compounds may be ineffective, unable to be delivered, or toxic to unaffected cells.

[0005] Other methods of treatment include the use of therapeutic proteins or agents that contain an exogenous target sequence so that the therapeutic agent can be recognized by receptors in the cell membrane, allowing the therapeutic agent to pass through the cell membrane and enter the cell. Once the therapeutic agent enters the cell, the therapeutic agent can interact with the target component to treat the disease. However, the use of an exogenous target sequence may limit the type of cells that are exposed to the therapeutic and increases the cost of obtaining the therapeutic.

[0006] For the above reasons, there is a need for compositions and methods for treating or preventing disease that do not rely on exogenous target sequences or chemical compositions for cell entry, and that are effective in targeting only affected cells in the body.

[0007] The present invention relates to single domain antibodies (SdAbs), proteins and polypeptides containing these SdAbs. These OdAbs are directed against intracellular components that cause a pathological condition or disease. This invention also includes nucleic acids encoding OdAb, proteins and polypeptides, as well as compositions containing OdAb. This invention includes the use of compositions, OdAbs, proteins or polypeptides for prophylactic, therapeutic or diagnostic purposes. The present invention also includes the use of monoclonal antibodies directed to the OdAb of the present invention.

SUMMARY OF THE INVENTION

[0008] One embodiment of the present invention is a single domain antibody (SdAb) directed against an intracellular component. The intracellular component may be, for example, a protein, nucleic acid, lipid, carbohydrate, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6, TNF-alpha and KRAS.

[0009] In another embodiment, the present invention is directed to an anti-STAT3 sdAb. Optionally, the STAT3 sdAb contains the amino acid sequence set forth in SEQ ID No.: 3 or SEQ ID No.: 4.

[0010] In another embodiment, the present invention is directed to an isolated polypeptide comprising an amino acid sequence encoding an anti-STAT sAb, such as, for example, the polypeptide set forth in SEQ ID No.: 3 or SEQ ID No.: 4.

[0011] In yet another embodiment, the present invention is directed to a host cell, and the host cell expresses an OdAb amino acid sequence, such as, for example, the amino acid sequence set forth in SEQ ID No.: 3 or SEQ ID No.: 4.

[0012] One embodiment of the present invention is a pharmaceutical composition comprising an OdAb or polypeptide, as well as a pharmaceutically acceptable carrier. Optionally, the sAb includes an anti-STAT3 sdAb containing the amino acid sequence set forth in SEQ ID No.: 3 or SEQ ID No.: 4, and the polypeptide includes an isolated polypeptide containing the amino acid sequence set forth in SEQ ID No.: 3 or SEQ ID No.: 4.

[0013] Another embodiment of the present invention is a method of diagnosing a STAT3-mediated disease in a subject, the method comprising the steps of: a) contacting a biological sample with a sdAb or polypeptide; b) determining the amount of STAT3 in the biological sample; and c) compare the amount determined in step (b) with the reference amount, with the difference in amount indicating the presence of a disorder.

[0014] Another embodiment of the present invention is a method for preventing or treating a disease or disorder, or preventing recurrence of a disease, mediated by STAT3, or for use in treating cancer or diseases caused by abnormal cell proliferation, comprising administering an anti-STAT3 sdAb or polypeptide to a subject in need of this. Optionally, the sAb includes an anti-STAT3 sdAb containing the amino acid sequence set forth in SEQ ID No.: 3 or SEQ ID No.: 4, and the polypeptide includes an isolated polypeptide containing the amino acid sequence set forth in SEQ ID No.: 3 or SEQ ID No.: 4.

[0015] One embodiment of the present invention is an anti-TNF-alpha sdb. Optionally, the TNF-alpha sdAb contains the amino acid sequence set forth in SEQ ID No.: 5, SEQ ID No.: 6, or SEQ ID No.: 7. The invention also provides an isolated polypeptide containing the amino acid sequence set forth in SEQ ID No.: 5 , SEQ ID No.: 6 or SEQ ID No.: 7.

[0016] Another embodiment of the present invention is a host cell expressing the amino acid sequence set forth in SEQ ID No: 5, SEQ ID No: 6 or SEQ ID No: 7.

[0017] In another embodiment, the present invention also provides a pharmaceutical composition comprising an OdAb or polypeptide, as well as a pharmaceutically acceptable carrier. Optionally, the sAb includes an anti-TNF-alpha sdAb containing the amino acid sequence set forth in SEQ ID No.: 5, SEQ ID No.: 6, or SEQ ID No.: 7, and the polypeptide includes an isolated polypeptide containing the amino acid sequence set forth in SEQ ID No.: 5, SEQ ID No.: 6 or SEQ ID No.: 7.

[0018] Another embodiment of the present invention is a method of diagnosing a TNF-alpha mediated disorder in a subject, the method comprising the steps of: a) contacting a biological sample with a sdAb or polypeptide; b) determine the amount of TNF-alpha in the biological sample; and c) compare the amount determined in step (b) with the reference one, with the difference in amount indicating the presence of the disease.

[0019] In one embodiment, the present invention describes a method for preventing or treating a disease or disorder, or relapse of a disease or disorder, mediated by TNF-alpha, or for use in the treatment of cancer or diseases caused by abnormal cell proliferation, comprising administering an anti-TNF-alpha sdAb or polypeptide to a mammal in need thereof. Optionally, the anti-TNF-alpha sdAb comprises the amino acid sequence set forth in SEQ ID No.: 5, SEQ ID No.: 6 or SEQ ID No.: 7, and the polypeptide includes an isolated polypeptide containing the amino acid sequence set forth in SEQ ID No.: 5, SEQ ID No.: 6 or SEQ ID No.: 7.

[0020] One embodiment of the present invention is a KRAS CDAb. Optionally, the KRAS sdAb contains the amino acid sequence set forth in SEQ ID No.: 2. In one aspect, the present invention provides an isolated polypeptide, wherein the isolated polypeptide comprises the amino acid sequence set forth in SEQ ID No.: 2. In another aspect, the invention provides to a host cell expressing the amino acid sequence shown in SEQ ID No: 2.

[0021] Another embodiment of the present invention is a pharmaceutical composition comprising an OdAb or polypeptide, as well as a pharmaceutically acceptable carrier. Optionally, the sAb includes an anti-KRAS sdAb containing the amino acid sequence set forth in SEQ ID No.: 2, and the polypeptide includes an isolated polypeptide containing the amino acid sequence set forth in SEQ ID No.: 2.

[0022] An additional embodiment of the present invention is a method of diagnosing a KRAS-mediated disorder in a subject, the method comprising the steps of: a) contacting a biological sample with a sdAb or polypeptide; b) determine the amount of KRAS in the specified biological sample; and c) compare the amount determined in step (b) with the reference amount, with the difference in amount indicating the presence of a disorder. Optionally, the sAb includes an anti-KRAS sdAb containing the amino acid sequence set forth in SEQ ID No.: 2, and the polypeptide includes an isolated polypeptide containing the amino acid sequence set forth in SEQ ID No.: 2.

[0023] This invention also provides a method of treating a disease using an anti-KRAS sdAb, the method comprising administering an effective amount of an anti-KRAS sdAb to a subject in need thereof.

[0024] In one embodiment, the present invention describes a method for preventing or treating a disease or disorder, or relapse of a disease or disorder, mediated by KRAS, or for use in treating cancer or diseases caused by abnormal cell proliferation, comprising administering an anti-KRAS sdAb or polypeptide to a mammal, who needs it. Optionally, the KRAS sAb contains the amino acid sequence set forth in SEQ ID No.: 2, and the polypeptide includes an isolated polypeptide containing the amino acid sequence set forth in SEQ ID No.: 2.

[0025] In one embodiment, the present invention describes a method of administering the OdAb of the present invention, which method includes intravenous administration, intramuscular administration, oral administration, rectal administration, intraocular administration, enteral administration, parenteral administration, subcutaneous administration, transdermal administration, administration as eye drops, administration as a nasal spray, administration by inhalation or nebulizer, topical administration and administration as an implantable drug.

[0026] In another embodiment, the present invention describes a method of treating a disease, preventing a disease, or preventing the relapse of a disease using the OdAb of the present invention in combination with one or more compounds. Optionally, one or more compounds are a transcription inhibitor.

[0027] In another embodiment, the present invention describes a method for measuring levels of OdAb, the method comprising the steps of: a) generating a mouse monoclonal antibody directed against one or more domains of OdAb; b) perform an immunological analysis to determine the number of OdAbs in the subject; and c) perform a quantitative analysis of the DAT in the subject.

GRAPHIC MATERIALS

[0028] These and other features, aspects and advantages of the present invention will become better understood in view of the following description, the accompanying claims and the accompanying drawings, in which:

In FIG. 1 illustrates a schematic map of the expression vector pTT21-stt VHH13 VHH13-ODAb to STAT3;

In FIG. Figure 2 illustrates a schematic map of the expression vector pTT21-stt VHH14 VHH14-ODAb to STAT3;

In FIG. 3 illustrates the results of an immunoprecipitation assay using bacteria-derived VHH13 (SEQ ID No: 3) to STAT3 and bacteria-derived VHH14 (SEQ ID No: 4) to STAT3;

In FIG. 4 illustrates the results of an immunoprecipitation assay using bacterially derived VHH13 (SEQ ID No: 3) to STAT3;

In FIG. 5 illustrates growth inhibition of bacterially derived VHH13-OdAb (SEQ ID No: 3) to STAT3 in the MDA-MB-231 xenograft model at a dose of 0.5 mg/kg/day;

In FIG. 6 illustrates growth inhibition of bacterially derived VHH13-OdAb (SEQ ID No. 3) to STAT3 in the MDA-MB-231 xenograft model at doses ranging from 1 mg/kg twice daily to 2 mg/kg twice daily or 2 mg/kg/day;

In FIG. 7 illustrates growth inhibition of bacterially derived VHH13-OdAb (SEQ ID No: 3) to STAT3 in the MDA-MB-231 xenograft model at a dose of 5 mg/kg/twice daily;

In FIG. 8 illustrates the growth inhibition of bacterially derived VHH13-OdAb (SEQ ID No: 3) to STAT3 in the DU145 xenograft model at a dose of 5 mg/kg/twice daily;

In FIG. 9 illustrates growth inhibition of bacterially derived VHH13-OdAb (SEQ ID No: 3) to STAT3 in a PANC-1 xenograft model at a dose of 5 mg/kg/twice daily;

In FIG. 10 illustrates growth inhibition of bacterially derived VHH13-OdAb (SEQ ID No: 3) to STAT3 in the MCF-7 xenograft model at a dose of 1 mg/kg/trid;

In FIG. 11 illustrates growth inhibition of bacterially derived VHH13-OdAb (SEQ ID No: 3) to STAT3 in the BT-474 xenograft model at a dose of 1 mg/kg/trid;

In FIG. 12 illustrates the cytotoxicity of TNF-alpha in U937 cells;

In FIG. 13 illustrates the cytotoxicity of staurosporine in U937 cells; And

In FIG. 14 illustrates the inhibition of TNF-alpha cytotoxicity by TNF-alpha OdAb.

DESCRIPTION

[0029] When used throughout this document, the following terms and variations thereof have the meanings set forth below unless a different meaning is clearly implied by the context in which the term is used.

[0030] Terms in the singular and similar referents used herein are to be construed to include both the singular and plural unless their use in the context indicates otherwise.

[0031] The term “antigenic determinant” means an epitope on an antigen recognized by an antigen-binding molecule (such as an OdAb or a polypeptide of the present invention), and more particularly by the antigen-binding region of an antigen-binding molecule. The terms "antigenic determinant" and "epitope" can also be used interchangeably. An amino acid sequence that can bind to, has affinity and/or specificity for, against, or directed against a particular antigenic determinant, epitope, antigen, or protein is believed to have affinity and/or specificity for the antigenic determinant, epitope, antigen, or protein.

[0032] When used throughout this document, the term “include” and variations of this term such as “including” and “includes” are not intended to exclude other additives, components, integers, or steps.

[0033] It is contemplated that the OdAbs, polypeptides and proteins described herein may contain so-called "conservative" amino acid substitutions, which can be generally described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and which have little or no effect on the function, activity or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions are substitutions in which one amino acid in the following groups (a)-(e) is replaced by another amino acid within the same group: (a) small aliphatic, non-polar or slightly 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, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Other conservative substitutions include: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly on Ala or on Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu on Ile or on Val; Lys to Arg, to Gln or to Glu; Met on Leu, on Tyr or on Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

[0034] The term “domain,” as used herein, generally means a globular region of an antibody chain, and in particular a globular region of an antibody heavy chain, or a polypeptide that is substantially composed of such a globular region.

[0035] The amino acid sequence and structure of an OdAb typically consists of four framework regions or “FRs,” which are referred to as “framework region 1” or “FR1”; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively. The framework regions are interrupted by three complementarity determining regions or "CDRs", which are called "complementarity determining region 1" or "CDR1"; “complementarity determining region 2” or “CDR2”; and "complementarity determining region 3" or "CDR3", respectively.

[0036] As used herein, the term “humanized sdAb” means a sdAb in which one or more amino acid residues in the amino acid sequence of the native VHH sequence have been replaced by one or more amino acid residues that occur at the corresponding position in the VH domain of the common 4 - human chain antibody. This can be accomplished using methods that are well known in the art. For example, the OdAb FRs may be replaced by human variable FRs.

[0037] As used herein, an "isolated" nucleic acid or amino acid has been separated from at least one other component with which it is normally associated, such as its source or medium, another nucleic acid, another protein/polypeptide, another biological component or macromolecule, or contaminant, impurity or minor component.

[0038] The term “mammal” means an individual belonging to the class Mammals and includes, but is not limited to, humans, domestic and farm animals, as well as zoo, sporting and companion animals such as cows, horses, sheep, dogs and cats.

[0039] As used herein, the term “pharmaceutically acceptable carrier” is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, the standard reference in the field. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, PBS (phosphate buffered saline), and 5% human serum albumin. Liposomes, cationic lipids and non-aqueous carriers such as fixed oils can also be used. The use of such media and means for pharmaceutically active substances is well known in the art. Their use in the composition of this invention is contemplated unless the conventional medium or agent is incompatible with the therapeutic agent described above.

[0040] The term "quantitative immunoassay" means any means of measuring the amount of antigen present in a sample using an antibody. Methods for performing quantitative immunoassays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), specific analyte labeling repeat capture assay (SALRA), liquid chromatography, mass spectrometry, fluorescence activated cell sorting, and the like.

[0041] The term "solution" means a composition containing a solvent and a solute, and includes true solutions and suspensions. Examples of solutions include solid, liquid or gaseous substances dissolved in a liquid, and solid particles or micelles suspended in a liquid.

[0042] The term "specificity" includes a number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein molecule can bind. The specificity of an antigen binding protein can be determined based on affinity and/or avidity. Affinity, expressed as the equilibrium constant for the dissociation of antigen and antigen-binding protein (KD), is an indicator of the strength of binding between the antigenic determinant and the antigen-binding site on the antigen-binding protein: the lower the KD value, the stronger the strength of binding between the antigenic determinant and the antigen-binding molecule (alternatively, affinity can also be expressed as an affine constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, affinity can be determined depending on the particular antigen in question. Avidity is a measure of the strength of binding between an antigen-binding molecule and an antigen. Avidity refers to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding molecule and the number of corresponding binding sites present on the antigen-binding molecule. The specific binding of an antigen binding protein to an antigen or antigenic determinant can be determined using any known methods such as, for example, the Scatchard assay and/or competitive binding assays such as radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and competitive sandwich assays.

[0043] As used throughout this document, the term “recombinant” refers to the use of genetic engineering techniques (eg, cloning and amplification) used to produce the OdAb of this invention.

[0044] The term "single domain antibody", "sdAb" or "VHH" can generally mean a polypeptide or protein containing an amino acid sequence that consists of four framework regions interrupted by three complementarity determining regions. This is represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The OdAb of the present invention also includes a polypeptide or protein that contains the amino acid sequence of the OdAb. Typically, OdAbs are produced in camelids such as llamas, but can also be synthesized using methods that are well known in the art. When used throughout this document, the variable domains present in natural heavy chain antibodies are also referred to as “VHH domains” to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies, referred to as “VH domains.” ", and from the light chain variable domains that are present in conventional 4-chain antibodies, called "VL domains". The terms "VHH" and "OdAt" are used interchangeably in this document. The numbering of the amino acid residues of the OdAb or polypeptide follows the general numbering for VH domains set forth by Kabat et al. (“Sequence of proteins of immunological interest”, US Department of Health, NIH Bethesda, Maryland, publication No. 91). In accordance with this numbering, FR1 OdAb contains amino acid residues in positions 1-30, CDR1 OdAb contains amino acid residues in positions 31-36, FR2 OdAb contains amino acids in positions 36-49, CDR2 OdAb contains amino acid residues in positions 50-65, FR3 OdAb contains amino acid residues at positions 66-94, CDR3 OdAb contains amino acid residues at positions 95-102, and FR4 OdAb contains amino acid residues at positions 103-113.

[0045] The term "synthetic" means produced by chemical or enzymatic synthesis in vitro.

[0046] The term “target,” as used herein, means any component, antigen, or fragment that is recognized by the SdAb. The term "intracellular target" means any component, antigen or fragment present within a cell. A "transmembrane target" is a component, antigen, or fragment that is located within the cell membrane. "Extracellular target" means a component, antigen or fragment that is outside the cell.

[0047] The term “therapeutic composition,” as used herein, means a substance that is expected to have a therapeutic effect, such as pharmaceutical compositions, genetic materials, biologicals, and other substances. Genetic materials include substances intended to have a direct or indirect genetic therapeutic effect, such as genetic vectors, genetic regulatory elements, genetic building blocks, DNA, RNA and the like. Biologics include substances that are living matter, or derived from living matter, that are intended to have a therapeutic effect.

[0048] As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” mean an amount that provides a therapeutic effect in the treatment, prevention or management of a disease or significant disease symptom. A therapeutically effective amount may treat a disease or pathological condition, a symptom of a disease, or a predisposition to a disease, with the purpose of treating, healing, ameliorating, ameliorating, modifying, eliminating, improving, normalizing, or influencing the disease, symptoms of a disease, or predisposition to a disease. The specific amount that is therapeutically effective can be readily determined by a person of ordinary skill in the art, and may vary depending on factors known in the art, such as, for example, the type of disease, history and age of the patient, stage of the disease, and administration of other therapeutic agents. .

[0049] The present invention relates to single domain antibodies (SdAbs) that are directed against intracellular components, as well as proteins and polypeptides containing SdAbs and nucleotides encoding these proteins and polypeptides. This invention may also relate to OdAbs that are directed against intercellular, transcellular and extracellular targets or antigens. This invention also includes nucleic acids encoding OdAb, proteins and polypeptides, as well as compositions containing OdAb. This invention includes the use of compositions, OdAbs, proteins or polypeptides for prophylactic, therapeutic or diagnostic purposes.

[0050] OdAbs have a number of unique structural characteristics and functional properties that make OdAbs highly suitable for use as functional antigen binding domains or proteins. sdAbs functionally bind to antigen in the absence of a light chain variable domain, and can function as a separate, relatively small, functional antigen-binding structural unit, domain, or protein. This distinguishes sdAbs from conventional antibody domains, which do not themselves function as an antigen-binding protein or domain, but must combine with conventional antibody fragments, such as Fab fragments or a ScFv fragment, to bind antigen.

[0051] OdAbs can be prepared using methods that are well known in the art. For example, one method for obtaining OdAb involves (a) immunizing camelids with one or more antigens, (b) isolating peripheral lymphocytes from immunized camelids, obtaining total RNA and synthesizing the corresponding cDNAs, (c) creating a library of cDNA fragments encoding VHH domains, (d ) transcribing the VHH domain-encoding cDNAs obtained in step (c) from mRNA using PCR, converting the mRNA into ribosomal display format and selecting the VHH domain using ribosomal display, and (e) expressing the VHH domain into a suitable vector and, optionally, purification expressed VHH domain.

[0052] Another method for producing the OdAb of this invention is by preparing the nucleic acid encoding the OdAb using nucleic acid synthesis methods, followed by expression of the nucleic acid in vivo or in vitro . In addition, the OdAbs, polypeptides and proteins of this invention can be produced using synthetic or semi-synthetic methods to produce proteins, polypeptides or other amino acid sequences.

[0053] The sdAbs of this invention will generally bind to all natural or synthetic analogs, variants, mutants, alleles, portions and fragments of the target, or at least those analogs, variants, mutants, alleles, portions and fragments of the target that contain one or more antigenic determinants or epitopes that are substantially the same as the antigenic determinant or epitope to which the OdAb of the present invention binds in a wild-type target. The sdAbs of the present invention can bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and/or specificity that is the same as, or which is higher or lower than the affinity and specificity with which the sdAbs of the present invention bind to wild type target. It is also within the scope of the present invention that the OdAbs of the present invention bind to certain analogs, variants, mutants, alleles, portions and fragments of the target, but do not bind to others. In addition, the OdAb of the present invention may be humanized, and may be monovalent or polyvalent, and/or multispecific. In addition, the OdAbs of the present invention can bind to the phosphorylated form of the target protein as well as to the non-phosphorylated form of the target protein. OdAb may be associated with other molecules such as albumin or other macromolecules.

[0054] It is also within the scope of this invention that the OdAbs are multivalent, that is, the OdAbs may have two or more proteins or polypeptides that are directed against two or more different epitopes of the target. In such a multivalent OdAb, the protein or polypeptide may be directed, for example, against the same epitopes, substantially equivalent epitopes, or different epitopes. Different epitopes may be located on the same target, or they may be located on two or more different targets.

[0055] It is also contemplated that the sequence of one or more OdAbs of this invention may be linked or linked to one or more linker sequences. The linker may be, for example, a protein sequence containing a combination of serines, glycines and alanines.

[0056] It is also within the scope of this invention to use parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the OdAb of this invention, provided they are suitable for the applications described.

[0057] Since the OdAbs of this invention are primarily intended for therapeutic and/or diagnostic use, they are directed against mammalian, preferably human, targets. However, it is possible that the OdAbs described herein cross-react with targets from other species, for example, with targets from one or more other primate species or other animals (e.g., mouse, rat, rabbit, pig, or dog) and, in particular, in animal models for diseases and disorders associated with disease associated with these targets.

[0058] In another aspect, the present invention relates to a nucleic acid that encodes the OdAb of the present invention. Such a nucleic acid may be present, for example, in the form of a genetic construct.

[0059] In another aspect, the present invention relates to a host or host cell that expresses or can express the OdAb of the present invention and/or which contains a nucleic acid encoding the OdAb of the present invention. OdAb sequences can be used for insertion 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.

[0060] This invention further relates to methods for producing or creating OdAbs, nucleic acids encoding OdAbs, host cells expressing or capable of expressing such OdAbs, products and compositions containing the OdAbs of the present invention.

[0061] In addition, this invention relates to methods of application and use of OdAb, nucleic acids encoding OdAb, host cells, products and compositions described herein. Such a product or composition may, for example, be a pharmaceutical composition for the treatment or prevention of a disease, or be a product or composition intended for diagnosis. OdAb can be used in a variety of assays, such as ELISA assays and mass spectrometry assays to measure OdAb levels in serum and tissue.

[0062] In another aspect, a nucleic acid encoding one or more SdAbs of the present invention can be inserted into the genome of an organism to treat or prevent diseases.

[0063] This invention generally relates to sdAbs, as well as proteins or polypeptides containing or essentially consisting of one or more such sdAbs, which can be used for prophylactic, therapeutic and/or diagnostic purposes.

[0064] The methods and compositions described in this invention can be used to treat the disease described herein and can be used in any dosage and/or formulation described herein or otherwise known, as well as with any route of administration described herein or otherwise known to one skilled in the art.

[0065] The OdAbs of this invention, in particular, VHH to STAT3, VHH to KRAS and VHH to TNF-alpha according to this invention, can be used for the treatment and prevention of malignant diseases, including, but not limited to: multiple myeloma, leukemia ( HTLV-1-dependent, erythroleukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) and large granular lymphocyte leukemia (LBGL)), lymphomas (EBV-associated/Burkitt, mycosis fungoides, cutaneous T-cell lymphoma, non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL)), breast cancer, triple negative breast cancer, head and neck cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, sarcoma, osteosarcoma, Kaposi's sarcoma , Ewing's sarcoma, hepatocellular cancer, glioma, neuroblastoma, astrocytoma, colorectal cancer, Wilms tumors, kidney cancer, bladder cancer, endometrial cancer, cervical cancer, esophageal cancer, squamous cell skin cancer, basal cell cancer and any metastatic cancers . OdAbs may be used in cancer patients to help prevent or reduce cancer-related weight loss or cachexia.

[0066] The sdAbs, in particular the anti-STAT3 and anti-TNF-alpha sdAbs of this invention, may also be used for the treatment and prevention of diseases such as, but not limited to: autoimmune diseases (e.g., rheumatoid arthritis, ulcerative colitis, Crohn's disease, bacterial colitis, asthma, scleroderma, lupus, encephalomyelitis, arteritis, vasculitis, glomerulonephritis, uveitis, uveoretinitis, multiple sclerosis), polycystic kidney disease, dermatological diseases (eg, psoriasis, alopecia areata, atopic dermatitis, keloids/hypertrophic scars, lipoma, Paget's disease and actinic keratosis), hidradenitis suppurativa, transplantation (eg, solid organ, bone marrow, hand, face, limb, any part of the body), muscular dystrophy and muscular atrophy due to cancer and aging, endometriosis, macular degeneration, retinal degeneration, stroke , epilepsy, traumatic brain and spinal cord injuries, hypertension, cardiac hypertrophy, Alzheimer's disease, pulmonary hypertension, type 2 diabetes mellitus and ankylosing spondylitis. In addition, OdAbs may have an impact on orphan diseases. Examples of such rare orphan diseases include, but are not limited to, triple-negative breast cancers, pancreatic cancers, AML (acute myeloid leukemia), head and neck cancers, multiple myeloma and chemoresistant cancers.

[0067] Viral infections can be treated by targeting intracellular viral proteins in infected cells. Viral proteins, such as HIV reverse transcriptase, can block the viral life cycle. The OdAb of this invention can also target intracellular viral proteins, such as VP24 of the Ebola virus, and thereby block the ability of the Ebola virus to stop the host's antiviral immune response. The OdAbs of this invention can be used to target diseases in which the intracellular molecule is overexpressed. Huntington's disease can be treated with OdAb.

[0068] The ODAT of this invention can be used with one or more compounds. For example, the OdAb of this invention can be used with JAK/STAT inhibitors, such as, for example, curcumin, resveratrol, cucurbitacin A, B, E, I, Q, flavopiridol, deoxytetrangomycin, cyclopentenone derivatives, N-acyl-homoserine lactone, indirubin derivatives , meisoindigo, tyrphostin, platinum-containing compounds (eg, IS3-295), peptidomimetics, antisense oligonucleotides, S3I-201, phosphotyrosine tripeptide derivatives, HIV protease inhibitors (eg, nelfinavir, indinavir, saquinavir and ritonavir), JSI-124, XpYL, Ac-pYLPQTV-NH2, ISS 610, CJ-1383, pyrimethamine, metformin, atiprimod, S3I-M2001, STX-0119; N-[2-(1,3,4-oxadiazolyl)]-4 quinoline carboxamide derivative, S3I-1757, LY5; 5,8-dioxo-6(pyridin-3-ylamino)-5,8-dihydro-naphthalene-1-sulfonamide, vitaminstin, stattic, STA-21, LLL-3, LLL12, XZH-5, SF-1066, SF -1087, 17o, cryptotanshinone, FLL32, FLL62, C188-9, BP-1108 and BP-1075, galliellalactone, JQ1, 5, 15 DPP, WP1066, niclosamide, SD1008, nifuroxazide, cryptotanshinone, BBI quinone and ruxolitinib phosphate. One or more compounds may enhance the therapeutic effect and enhance the effectiveness of the OdAb of the present invention. In addition, the effectiveness of OdAb can be increased by combining it with peptides, peptidomimetics and other drugs, such as, for example, but not limited to, cimetidine, atorvastatin, celecoxib, metformin and cimetidine. Additionally, regarding radiotherapy, STAT3 sdAbs can convert radioresistant cancers into radiosensitive cancers.

[0069] It is also contemplated that one or more SdAbs of the present invention may be combined, or that SdAbs of the present invention can be combined with other SdAbs.

[0070] It is contemplated that some of the OdAbs of this invention can cross the cell membrane and enter the cell without additional targeting of the OdAb by protein sequences and without exogenous compounds that direct the OdAb to bind to cell surface receptors and pass through the cell membrane.

[0071] After passing through the cell membrane, these OdAbs can target transmembrane or intracellular molecules or antigens. These intracellular or transmembrane targets can be, for example, proteins, carbohydrates, lipids, nucleic acids, mutant proteins, viral proteins and prions. OdAb targets can perform the function of enzymes, structural proteins of the cell, intracellular regions of cell membrane molecules, molecules in organelle membranes, any type of RNA molecule, any regions of DNA or chromosome, methylated or unmethylated nucleic acids, partially assembled molecules within the cell's synthesis mechanism, secondary molecules intermediaries and molecules in cellular signaling mechanisms. Targets may include all molecules in the cytoplasm, nucleus, organelles, and cell membrane. Targeting molecules intended for secretion or placement in the cell membrane can be carried out in the cytoplasm before exiting the cell.

[0072] OdAb targets can be in humans, animals, plants, fungi, parasites, protozoa, bacteria, viruses, prions, prokaryotic cells and eukaryotic cells. Some examples of inter- and intracellular signaling molecules and protein groups that may be targets of the OdAbs of this invention are: oncogene products, hormones, cytokines, growth factors, neurotransmitters, kinases (including tyrosine kinase, serine kinase and threonine kinase), phosphatases, ubiquitin , cyclic nucleotides, cyclases (adenylate and guanylate), G proteins, phosphodiesterases, GTPase superfamily, immunoglobulins (antibodies, Fab fragments, binders, OdAbs), immunoglobulin superfamily, inositol phosphate lipids, steroid receptors, calmodulin, CD group (e.g. CD4 , CD8, CD28, etc.), transcription factors, TNF-beta, TNF-alpha and beta, TNF ligand superfamily, Notch receptor signaling molecules, Hedgehog receptor signaling molecules, Wnt receptor signaling molecules, Toll-like receptor signaling molecules, caspases, actin, myosin, myostatin, 12-lipoxygenase, 15-lipoxygenase, lipoxygenase superfamily, reverse transcriptase, viruses and their proteins, amyloid proteins, collagen, G-protein coupled receptor, mutant normal proteins, prions, 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.

[0073] KRAS is a homolog of the Kirsten Ras oncogene of the mammalian Ras family of genes. KRAS encodes a protein that is a member of the small GTP superfamily. This protein has been implicated in a variety of malignancies, including lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, and colorectal carcinoma. Under normal conditions, members of the Ras family influence cell growth and differentiation events through a signaling system based on compartmentalization of the intracellular membrane. However, oncogenic Ras can deregulate processes that regulate cell proliferation and apoptosis.

[0074] KRAS sdAbs have been designed to target wild-type KRAS and mutant KRAS (G12D) to block its role in malignant cells, such as, for example, cells associated with colorectal cancer, pancreatic cancer, cancer bile ducts, lung cancer, leukemia and other metastatic malignant tumors. While not bound by a specific mechanism, it is believed that the KRAS sdAb binds KRAS and blocks KRAS downstream signaling in malignant cells. In addition, KRAS sdAb can successfully treat cancers that are resistant to anti-EGFR biologics (eg, cetuximab and panitumumab).

[0075] Using methods that are well known in the art, recombinant human KRAS (G12D) mutant protein has been used to generate SdAbs that are directed against or can bind to an epitope of KRAS or mutant KRAS (G12D) or other KRAS mutants. In addition, OdAbs can be generated for other KRAS mutants. To generate the KRAS sAb, recombinant full-length human KRAS (Gene ID: 3845) was expressed in Escherichia coli .

[0076] Several OdAbs were obtained and screened. The DNA sequence of one KRAS sAb (G12D), referred to as KRAS_13 (SEQ ID No: 1), is shown below:

5'Gaggtgcagctggtggagtctgggggaggctcggtgcagactggagggtctctgagactctcctgtgcagtttctggaaatatcggc agcagctactgcatgggctggttccgccaggctccagggaagaagcgcgaggcggtcgcacgtattgtacgtgatggtgccactggcta cgcagactacgtgaagggcc gattcaccatctcccgagacagcgccaagaacactctgtatctgcaaatgaacaggctgatacctgagg acactgccatctactactgtgcggcagacctgcccccaggttgtttgactcaggcgatttggaattttggttatcggggccagggaaccctg gtcaccgtctcctca-3'

[0077] The amino acid sequence of the OdAb (SEQ ID No. 2) to KRAS (G12D), KRAS_13, is shown below, with the CDRs underlined:

EVQLVESGGGSVQTGGSLRLSCAVSGNIGS SYCMG WFRQAPGKKREAVA

RIVRDGATGYADYVKGRFT ISRDSAKNTLYLQMNRLIPEDTAIYYCAA

DLPPGCLTQAIWNFGY RGQGTLVTVSS

[0078] In addition, the present invention includes one or more murine monoclonal antibodies that are directed against one or more domains of the KRAS sdAb of the present invention. A mouse monoclonal antibody can be generated using methods known to those skilled in the art, for example, a mouse monoclonal antibody can be generated using a mouse hybridoma. The mouse monoclonal antibody can be used in diagnostic assays, for example, the antibody can be used in an immunoassay, such as an ELISA or a mass spectrometry assay, to measure the amount of KRAS sdAb in a patient's blood serum. The cytotoxicity of the KRAS sdAb (G12D) was tested on PANC-1 human pancreatic cancer cells as described below.

[0079] STAT3 is a member of the signal transporter and activator of transcription (STAT) family of proteins that mediate both signal transduction and activation of transcriptional functions. STAT3 is widely expressed and activated through tyrosine and/or serine phosphorylation as a DNA binding protein in response to various cytokines and growth factors such as EGF, IL-6, TGF, IL-2, and G-CSF. STAT3 phosphoprotein forms homodimers and heterodimers with other members of the STAT family and translocates into the nucleus to modulate the transcription of various genes, and as a result plays a key role in many cellular processes such as cell growth, apoptosis, angiogenesis, immune escape and survival.

[0080] Anti-STAT3 sdAbs can be given to patients and other organisms to treat diseases caused by phosphorylated and non-phosphorylated STAT3, as well as to prevent the development of disease or relapse of disease. For example, patients who have had organ transplants and bone marrow transplants are at higher risk for SCCAs and BCCAs of the skin due to the immunosuppressive medications they are taking. Administration of an anti-STAT3 sdAb may reduce or eliminate this risk. Patients treated for cancer who are at risk of relapse will benefit from treatment with a STAT3 sdAb. Based on family history and HLA type, some individuals will be at increased risk for certain types of autoimmune diseases and may benefit from treatment with an OdAb with a reduced risk of developing that autoimmune disease. The risk of breast cancer can be reduced by administration of anti-STAT3 drugs such as GLG-302, as shown in a recent NCI study.

[0081] In addition to inhibiting STAT3, the STAT3 sdAb can also inhibit STAT1, STAT2, STAT4, STAT5a, STAT5b and STAT6 due to the high degree of homology between these molecules.

[0082] Recombinant human STAT3 protein was used to generate anti-STAT sdAbs that were directed against or could bind to an epitope of STAT3. To create the STAT3 sdAb, recombinant full-length human STAT3 (Gene ID: 6774) was expressed by baculovirus in Sf9 insect cells. The STAT sAbs have been cloned into vectors that can be expressed in both bacterial and mammalian cells, as shown in FIG. 1 and 2.

[0083] The anti-STAT3 sdAb of the present invention can be used to target STAT3 and all other STAT molecules within a cell to inhibit cell growth, such as, for example, inhibiting the growth of cancer cells. Additionally, STAT3 sdAb can inhibit cell growth in other proliferative diseases such as psoriasis and macular degeneration via VEGF.

[0084] Without being limited to a specific mechanism of action, it is believed that a STAT3 sdAb may reverse cancer-induced suppression of the immune response by reducing STAT3 levels in antigen presenting cells, such as host dendritic cells. Inhibition of STAT3 promotes the anticancer response by the patient's innate and acquired immune system (i.e., dendritic cells, macrophages, neutrophils, T cells, NK cells, and B cells).

[0085] Using methods that are well known in the art, several STAT sAbs were prepared and screened for the ability to inhibit cancer cell growth and induce apoptosis in cancer cell lines, as described below. The cytotoxicity and antiproliferative activities of OdAb to STAT3 were tested. In addition, the tolerance of OdAbs to STAT3 was tested in vitro and in vivo. The generation of murine monoclonal antibodies directed against one or more domains of the anti-STAT sAb is described below.

[0086] The amino acid sequence of one anti-STAT3 sAb called VHH13 (SEQ ID No. 3) is shown below:

HVQLVESGGGSVQAGGSLRLSCAASGANGG RSCMG WFRQVPGKEREGVS GISTGGLI

TYYADSVKGRFT ISQDNTKNTLYLQMNSLKPEDTAMYYCAT SRFDCYRGSWFNRYM

YNS WGQGTQVTVSS

Three CDRs are underlined.

[0087] The amino acid sequence of the second STAT3 Ab, referred to as VHH14 (SEQ ID No. 4), is shown below:

QVQLVESGGGSVQAGGSLRLSCVASTY TGCMG WFRQAPGKEREGVA ALSSRGFAGH

YTDSVKGRFS ISRDYVKNAVYLQMNTVKPEDAAMYYCAA REGWECGETWLDRTAG

GHTY WGQGTLVTVSS

[0088] Again, three CDRs are underlined. The protein sequences of other STAT3 sAbs that have been obtained are as shown below:

STAT3_10 (SEQ ID #5):

(1) DVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98) GWECGETWLDRTAGGHTYWGQGTQVTVSS STAT3_34 (SEQ ID No. 6):

(1) DVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGGHTYWGQGTQVTVSS

STAT3_19 (SEQ ID No. 7):

(1) HVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGGHTYWGQGTQVTVSS

STAT3_14 (SEQ ID No. 8):

(1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGGHTYWGQGTLVTVSS

STAT3_35 (SEQ ID No. 9):

(1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGGHTYWGQGTLVTVSS

STAT3_9 (SEQ ID No. 10):

(1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGGHTYWGQGTLVTVSS

STAT3_30 (SEQ ID No. 11):

(1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGGHTYWGQGTLVTVSS

STAT3_23 (SEQ ID No. 12):

(1)QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGSHTYWGQGTLVTVSS

STAT3_24 (SEQ ID No. 13):

(1) EVQLVESGGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGGHTYWGQGTLVTVSS

STAT3_36 (SEQ ID No. 14):

(1) DVQLVESGGGSVQAGDSLRLSCVASTYTGCMGWFRQAPGKEREGVAA

(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAARE

(98)GWECGETWLDRTAGGHTYWGQGTLVTVSS

STAT3_12 (SEQ ID No. 15):

(1)QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTLVTVSS

STAT3_16 (SEQ ID No. 16):

(1)QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTNNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTLVTVSS

STAT3_11 (SEQ ID No. 17):

(1) EVQLVESGGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTLVTVSS

STAT3_20 (SEQ ID No. 18):

(1) DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTLVTVSS

STAT3_2 (SEQ ID No. 19):

(1) DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTLVTVSS

STAT3_15 (SEQ ID No. 20):

(1) DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTLVTVSS

STAT3_6 (SEQ ID No. 21):

(1) HVQLVESEGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTLVTVSS

STAT3_33 (SEQ ID No. 22):

(1)QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

STAT3_17 (SEQ ID No. 23):

(1)QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

STAT3_25 (SEQ ID No. 24):

(1) EVQLVESGGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMSSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

STAT3_32 (SEQ ID #25):

(1) DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

STAT3_13 (SEQ ID No. 26):

(1) HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

STAT3_39 (SEQ ID No. 27):

(1) HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

STAT3_4 (SEQ ID No. 28):

(1) HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

STAT3_29 (SEQ ID No. 29):

(1) HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSG

(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSR

(101)FDCYRGSWFNRYMYNSWGQGTQVTVSS

[0089] The corresponding DNA sequences of the STAT3 sAb are as follows:

Stat3_VHH-10 (SEQ ID No. 30):

5'-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctgagactctcctgtgtagcctctacatacaccggc tgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtg aagggccgattctccat ctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgc agcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggacccaggtcaccgtct cctca-3'

Stat3_VHH-14 (SEQ ID No. 31):

5'-caggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctctcctgtgtagcctctacatacaccggc tgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtg aagggccgattctccatct cccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgc agcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggaccctggtcaccgtctc ctca-3'

Stat3_VHH-12 (SEQ ID No. 32):

5'-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctctcctgtgcagcctctggagccaatgg tggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccg actccgtgaagggccgattcacca tctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgt actactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcct ca-3'

Stat3_VHH-13 (SEQ ID No. 33):

5'-catgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctctcctgtgcagcctctggagccaacgg tggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccg actccgtgaagggccgattcacca tctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgt actactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcactgtctcct ca-3'

Stat3_VHH-20 (SEQ ID No. 34):

5'-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctctcctgtgcagcctctggagccaatgg tggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgaggggtttctggtatttcaaccggtggtcttattacatactatgccg actccgtgaagggccgattca ccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgt actactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcct ca-3'

Stat3_VHH-23 (SEQ ID No. 35):

5'-caggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggc tgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagcagccgtggttttgccgggcactataccgactccgtg aagggccgattctccat ctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgc agcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggagccatacctactggggccaggggaccctggtcaccgtctc ctca-3'

Stat3_VHH-24 (SEQ ID No. 36):

5'-gaggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctctcctgtgtagcctctacatacaccgg ctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgt gaagggccgattctccatct cccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtg cagcacgggagggatgggagtgcggtgagacctggttggaccgaaccgccgggggccatacctactggggccaggggaccctggtcaccgtct cctca-3'

Stat3_VHH-25 (SEQ ID No. 37):

5'-gaggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctctcctgtgcagcctctggagccaatg gtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgaggggtttctggtatttcaaccggtggtcttattacatactatgcc gactccgtgaagggtcgatt caccatctcccaagacaacaccaagaacacgctgtatctgcaaatgagcagcctgaaacctgaggacactgccatg tactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcc tca-3'

Stat3_VHH-19 (SEQ ID No. 38):

5'-catgtgcagctggtggagtctggggggggctcggtgcaggctggaggctctctgagactctctcctgtgtagcctctacatacaccggc tgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtg aagggccgattctccatct cccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgc agcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggacccaggtcaccgtct cctca-3'

Stat3_VHH-32 (SEQ ID No. 39):

5'-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctctcctgtgcagcctctggagccaatg gtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgaggggtttctggtatttcaaccggtggtcttattacatactatgcc gactccgtgaagggccgatt caccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatg tactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcc tca-3'

Stat3_VHH-33 (SEQ ID No. 40):

5'-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctctcctgtgcagcctctggagccaatg gtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgaggggtttctggtatttcaaccggtggtcttattacatactatgcc gactccgtgaagggccgattca ccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatg tactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcc tca-3'

Stat3_VHH-36 (SEQ ID No. 41):

5'-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagactctctgagactctctcctgtgtagcctctacatacaccggc tgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtg aagggccgattctccatct cccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgc agcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggaccctggtcactgtctc ctca-3'

Stat3_VHH-11 (SEQ ID No. 42):

5'-gtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtg gtcggagctgcatgggctggttccgccaggttccagggaaggagcgtgagggggtttctggtatttcaaccggtggtcttattacatactatgccgac tccgtgaagggccgattca ccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtact actgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcactgtctcctca-3'

Stat3_VHH-6 (SEQ ID No. 43):

5'-gtgcagctggtggagtctgagggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtg gtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgaggggtttctggtatttcaaccggtggtcttattacatactatgccgac tccgtgaagggccgattca ccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtact actgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcctca-3'

Stat3_VHH-1 (SEQ ID No. 44):

5'-gtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtg gtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgaggggtttctggtatttcaaccggtggtcttattacatactatgccgac tccgtgaagggccgattca ccatctcccaagacaacaccaataacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtact actgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcactgtctcctca-3'

[0090] In addition, the present invention includes one or more murine monoclonal antibodies that are directed against one or more domains of the STAT3 sdAb of the present invention. A mouse monoclonal antibody can be generated using methods known to those skilled in the art, for example, a mouse monoclonal antibody can be generated using a mouse hybridoma. The mouse monoclonal antibody can be used in diagnostic assays, for example, the antibody can be used in an immunoassay, such as an ELISA, to measure the amount of STAT3 sdAb in a patient's blood serum. It should be understood that this method is not limited to STAT3 sdAbs, and can be used to produce murine antibodies directed to any of the sdAbs of the present invention.

[0091] The TNF-alpha gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. This cytokine is involved in the regulation of a wide range of biological processes, including growth regulation, differentiation, inflammation, viral replication, oncogenesis, and autoimmune diseases; and in viral, bacterial, fungal and parasitic infections. In addition, by inducing hemorrhagic necrosis of tumors, TNF has been found to be involved in oncogenesis, tumor metastasis, viral replication, septic shock, fever, inflammation, cachexia and autoimmune diseases, including Crohn's disease and rheumatoid arthritis, as well as graft-versus-host disease. .

[0092] The present invention relates to sdAbs, proteins and polypeptides that are directed against TNF-alpha, in particular against human TNF-alpha, within a cell or cell membrane so as to prevent the cells from secreting TNF-alpha.

[0093] It is contemplated that the anti-TNF-alpha sdAb and polypeptides of this invention may be used for the prevention and/or treatment of diseases and disorders associated with and/or mediated by TNF-alpha, such as inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmune hepatitis, autoimmune parotitis, type 1 diabetes mellitus, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, male infertility, multiple sclerosis , myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis and weight loss due to cancer and cachexia.

[0094] TNF-alpha exists in various forms; there are monomeric and multimeric forms, including a trimeric form. It is within the scope of this invention that the OdAbs, proteins and polypeptides of this invention bind to TNF-alpha in its various forms, i.e. monomeric form or multimeric forms. Thus, while the sdAbs, proteins and polypeptides of the present invention are directed against TNF-alpha, it should be understood that this also includes sdAbs, proteins and polypeptides directed against TNF-alpha in its trimeric form.

[0095] It is known that signal transduction by TNF involves the cross-linking of TNF receptors by a trimer of TNF molecules that contains three receptor binding sites (see, for example, Peppel et al., J. Exp. Med., 174 (1991), 1483- 1489).

[0096] Recombinant human TNF-alpha protein has been used to create OdAbs that are directed against or can bind to an epitope of TNF-alpha. To generate the TNF-alpha sdAb, recombinant full-length human TNF-alpha (Gene ID: 7124) was expressed in Escherichia coli and used as the target antigen.

[0097] Thirty-five sdAbs to the TNF-alpha protein were obtained. These anti-TNF-alpha antibodies were divided into three groups based on sequence homology.

[0098] The amino acid sequence of the first TNF-alpha sdAb, referred to as VHH66-TNF-alpha sdAb (SEQ ID No. 45), is shown below:

HVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMG WFRQADGKEREGVA TIDIDGLT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RG-QGTLVTVSS

Three CDRs are underlined.

[0099] The amino acid sequence of the second TNF-alpha sdAb, referred to as VHH69-TNF-alpha sdAb (SEQ ID No. 46), is shown below:

EVQLVESGGGSVLAGGSLRLSCVASGFTSR YNYMA WFRQAPGKEREGVA TIGTASGS ADYYGSVKDRFT ISQDNAKNTVYLQMNSLKPEDTAMYYCAA RTYGTISLTPSDYRY WGQGT LVTVSS

Three CDRs are underlined.

[0100] The amino acid sequence of the third anti-TNF-alpha sdAb, referred to as VHH62-sdAb (SEQ ID No. 47) against TNF-alpha, is shown below:

QVQLVESGGGPVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCS WAQG TQGTLVTVSS

[0101] Three CDRs are underlined. Other TNF-alpha sdAbs that have been discovered include the sequences below, and again the CDRs are underlined:

TNF_2 (SEQ ID No. 48):

QVQLVESGGGSVEAGRSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGQT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQ VTVSS

TNF_46 (SEQ ID No. 49):

QVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGQT THADSVKGRFT ISRDNVKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQ VTVSS

TNF_71 (SEQ ID No. 50):

QVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGLT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGTQ VTVSS

TNF_21 (SEQ ID No. 51):

QVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGQT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGTQ VTVSS

TNF_38 (SEQ ID No. 52):

EVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGQT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGTQ VTVSS

TNF_18 (SEQ ID No. 53):

EVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGLT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGTL VTVSS

TNF_37 (SEQ ID No. 54):

DVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGQT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGTL VTVSS

TNF_66 (SEQ ID No. 55):

HVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGLT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGTL VTVSS

TNF_68 (SEQ ID No. 56):

HVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADGKEREGVA TIDIDGLA THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGTL VTVSS TNF_78 (SEQ ID No. 57):

HVQLVESGGGSVEAGGSLRLSCAASGFRYA AYCMGW FRQADRKEREGVA TIDIDGQT THADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGTQ VTVSS

TNF_67 (SEQ ID No. 58):

HVQLVESGGGSVQAGGSLRLSCAASGFRYA AYCMGW FRQADGKVREGVA TIDIDGQ TTHADSVKGRFT ISRDNAKNTLSLQMNDLKPEDTAMYYCAA DRDRCGSIWTYAYKY RGQGT LVTVSS

TNF_6 (SEQ ID No. 59):

QVQLVESGGGSVQAGGSLRLSCAASGFIDS FGVMA WFRQAPGKEREGVA AVYRRAG DTYYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDSAMYYCAA RTYGSVSSWTGYKY WGQG TQVTVSS

TNF_7 (SEQ ID No. 60):

DVQLVESGGGSVQAGGSLRLSCAASGFIDS FGVMA WFRQTPGKEREGVA AVYRRAG DTYYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDSAMYYCAA RTYGSVSSWTGYKY WGQG TQVTVSS

TNF_13 (SEQ ID No. 61):

DVQLVESGGGSVQVGGSLTLSCAVSGYTDS YGVMA WFRQAPGKEREGVA SIYRNSGI TYYPDSVKGRFT ISRDNAKNTVLLQMNSLKPEDSATYYCAV RSFGSVSTWAGYVY WGQGTQ VTVSS

TNF_60 (SEQ ID No. 62):

DVQLVESGGGSVQAGGSLRLSCAASGFIDS FGVMA WFRQAPGKEREGVA AVYRRAG DTYYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDSAMYYCAA RTYGSVSSWTGYKY WGRG TQVTVSS

TNF_73 (SEQ ID No. 63):

DVQLVESGGGSVRAGGSLRLSCTASGDTSK SDCMA WFRQAPGKERERVG AIYTRNGY THYADSVNGRFT ISQDNAKNALYLQMSGLKPEDTAMYYCAA RFRIYGQCVEDDDIDY WGQG TLVTVSS

TNF_69 (SEQ ID No. 64):

EVQLVESGGGSVLAGGSLRLSCVASGFTSR YNYMA WFRQAPGKEREGVA TIGTASGS ADYYGSVKDRFT ISQDNAKNTVYLQMNSLKPEDTAMYYCAA RTYGTISLTPSDYRY WGQGT LVTVSS

TNF_76 (SEQ ID No. 65):

QVQVVEYGGGSVQAGETVRLSCTASGFTFA EADMG WYRQAPGHEWELVS NITTEGIT SEASSSYADSVRGRFT IFDNAKNMVYLQMNSLKHEDTAVYYCAP DPYAYSTYREYCT WAQG TQGTLVTVSS

TNF_62 (SEQ ID No. 66):

QVQLVESGGGPVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCS WAQG TQGTLVTVSS

TNF_43 (SEQ ID No. 67):

QVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTLVTVSS

TNF_15 (SEQ ID No. 68):

QVQPVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG AQGTLVTVSS

TNF_11 (SEQ ID No. 69):

QVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCS WAQG TQGTQVTVSS

TNF_17 (SEQ ID No. 70):

QVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTQVTVSS

TNF_63 (SEQ ID No. 71):

QVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTLVTVSS

TNF_20 (SEQ ID No. 72):

HVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTQVTVSS

TNF_58 (SEQ ID No. 73):

EVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGALVTVSS

TNF_27 (SEQ ID No. 74):

EVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTLVTVSS

TNF_28 (SEQ ID No. 75):

EVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCS WAQG TQGTQVTVSS

TNF_4 (SEQ ID No. 76):

EVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTQVTVSS

TNF_14 (SEQ ID No. 77):

DVQLVESRGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTLVTVSS

TNF_3 (SEQ ID No. 78):

DVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHVCELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCS WAQG TQGTQVTVSS

TNF_1 (SEQ ID No. 79):

DVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGLECELVS TITTEGITS EASSYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSEYCT WAQGT QGTLVTVSS

TNF_45 (SEQ ID No. 80):

DVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS EASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTLVTVSS

TNF_22 (SEQ ID No. 81):

DVQLVESGGGSVQAGETLRLSCTASGFTFA EADMG WYRQAPGHECELVS TITTEGITS VASSYYADSVRGRFT ISRDNAKNMVYLQMNSLKPEDTAVYYCAP DPYAYSTYSDYCT WAQG TQGTQVTVSS

[0102] The growth inhibition of several anti-TNF-alpha sdAbs was tested in vitro as described below. In addition, the present invention includes one or more murine monoclonal antibodies that are directed against one or more domains of the TNF-alpha sdAb of the present invention. The mouse monoclonal antibody can be generated using methods known to those skilled in the art, as described below. The mouse monoclonal antibody can be used in diagnostic assays, for example, the antibody can be used in an immunoassay, such as an ELISA, to measure the amount of TNF-alpha sdAb in a patient's blood serum.

[0103] RAF proteins are a family of serine/threonine-specific kinases that serve as a central intermediate in transducing extracellular signals to the mitogen-activated protein kinase pathway, which regulates cell growth, differentiation and survival. BRAF is a member of the RAF family that is activated by members of the Ras family upon growth factor-induced stimulation. Active Ras can induce heterodimerization of cRaf and BRAF, and this may explain the observed cooperative effect of cRaf and BRaf in cells responding to growth factor signals. Activating mutations in the BRAF gene are present in a large percentage of human malignant melanomas and proportionately in colon cancers. The vast majority of these mutations result in a valine to glutamic acid substitution at residue 599 in the BRAF activation segment.

[0104] BRAF sdAbs have been designed to target wild-type and mutant BRAF to block its role in malignant cells, such as, for example, cells associated with colon cancer and other malignancies.

[0105] Using methods that are well known in the art, recombinant human BRAF protein has been used to create OdAbs that are directed against or can bind to a BRAF epitope.

[0106] In addition, the present invention includes one or more murine monoclonal antibodies that are directed against one or more domains of the BRAF sdAb of the present invention. The mouse monoclonal antibody can be generated using methods known to those skilled in the art. The mouse monoclonal antibody can be used in diagnostic assays, for example, the antibody can be used in an immunoassay, such as an ELISA, to measure the amount of BRAF OdAb in a patient's blood serum.

EXAMPLES

EXAMPLE 1: VHH13-ODAT (SEQ ID NO. 3) BINDS STAT3 TO STAT3

[0107] In this example, the affinity of two VHH targets for STAT3 was measured using a marker-free Octet-based binding assay. In this assay, VHH13-ODAb (SEQ ID No: 3) to STAT3, to KRAS (negative control) and GST-STAT3 (16 kDa monovalent antigen, Creative BioMart No. STAT3-1476H) were used as antigen probes. GST-STAT3 protein at a concentration of 20 μg/mL was immobilized in PBS using aminopropylsilane (APS) Dip and Read biosensors specifically designed for the hydrophobic protein. Then the probes were immersed in wells containing GST-STAT3, VHH13-ODAb (SEQ ID No: 3) to STAT3 or to KRAS at the indicated concentration. The rate of association (rate of formation) of the antigen was measured. The sensors were quenched by 1% BSA in water. The probes were immersed in assay buffer (PBS) and the dissociation rate (decay rate) was measured.

[0108] The affinity represented by the equilibrium constant for the dissociation of antigen and antigen binding protein (KD) was determined from the obtained affinity constant (KA), and KD using a 1:1 global approximation method using Fortebio software, as shown below in Table 1 Affinity was determined by averaging KD values for curves with R2 values > 0.95. The result for 250 nM VHH13 to STAT3 has been omitted because it represents an outlier value. It was found that the affinity of VHH13-OdAb (SEQ ID no. 3) for STAT3 was 1.16×10 ^-7 . The affinity of VHH for KRAS has not been determined.

EXAMPLE 2: IMMUNOPRECIPITATION STUDIES

[0109] The specificity of sdAb to STAT3 in human breast cancer cells was analyzed. In this example, MDA-MB-231 human breast cancer cells were grown to 50%-70% confluency. Cells were then disrupted in freshly prepared ice-cold lysis buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 0.1% NP-40, 10% glycerol, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 mM dithiothreitol , 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin) for 45 minutes on ice. The lysates were then centrifuged, the supernatant was collected, and the protein concentration was determined using the modified Lowry method (Bio Rad, Hercules, CA). Total protein (1 mg) was incubated with 1.5 mg Dynabeads (Invitrogen) with STAT3 sdAb, positive control (STAT3, cat. no. SC-482, Santa Cruz Biotechnology, Dallas, TX) or negative control (STAT-1, Cat. No. 9172, Cell Signaling, Danvers, Massachusetts) for 1 hour at 4°C. The granules were then washed. After the last wash, 60 μl of lysis buffer was added and the resulting supernatant was subjected to Western blotting. Briefly, samples were separated on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked and then incubated with appropriate primary and secondary antibodies. The anti-STAT3 antibody used as a positive control was an antibody from Cell Signaling (cat. no. 4904, Danvers, MA). The chemiluminescence reaction was performed using an ECL system from Santa Cruz Biotechnology (Dallas, TX).

[0110] As illustrated in FIG. 3, endogenous STAT3 immunoprecipitated with all sdAbs was tested in different amounts. M represents a marker lane containing a marker, lane 1 contained VHH13 (SEQ ID No: 3) to STAT3, obtained and isolated from mammalian cells, lane 2 contained VHH14 (SEQ ID No: 4) to STAT3, obtained and isolated from mammalian cells , lane 3 contained VHH13 (SEQ ID No: 3) to STAT3, obtained and isolated from bacterial cells, lane 4 contained VHH14 (SEQ ID No: 4) to STAT3, obtained and isolated from mammalian cells, lane 5 was positive to STAT3 antibody, lane 6, using STAT-1 as a negative control, showed no band.

EXAMPLE 3: BACTERIAL VHH13 STAT3 BINDS WITH HIGH AFFINITY TO CELL LINES CONTAINING CONSTITUTIVELY ACTIVATE STAT3

[0111] The specificity of bacterially derived VHH13 (SEQ ID No: 3) for STAT3 was analyzed using constitutively activated STAT3 in human (PANC-1 and DU145) and mouse (4T1) cell lines. Commercially available HeLa cells were also treated with interferon gamma (INFG) to induce phosphorylated STAT3. The PC-3 STAT3 null cell line was used as a negative control.

[0112] Cells were grown until 50%-70% confluent and then disrupted in freshly prepared ice-cold lysis buffer as described above for 45 minutes on ice. The lysates were then centrifuged, the supernatant was collected, and the protein concentration was determined as described above. Total protein (1 mg) was incubated with 1.5 mg Dynabeads (Invitrogen) with bacteria-derived VHH13 (SEQ ID no: 3) to STAT3 or negative control (KRAS, Creative Biolabs, Shirley, NY) for 1 hour at 4°C. The granules were then washed. After the last wash, 60 μl of lysis buffer was added and the resulting supernatant was subjected to Western blotting as described in Example 2.

[0113] As illustrated in FIG. 4, endogenous STAT3 was immunoprecipitated from bacteria VHH13 (SEQ ID No: 3) to STAT3 in constitutively activated STAT3 cell lines: PANC-1 (lane 1), DU145 (lane 2) and 4T1 (lane 4). In addition, anti-STAT3 derived from the bacterium VHH13 (SEQ ID No: 3) bound to phospho-STAT3 in HeLa lysate (lane 3). No bands were observed for either KRAS PANC-1, lane 3, or PC-3 (negative control), lane 6.

EXAMPLE 4: STUDIES OF ODATE CYTOTOXICITY TO STAT3 IN MDA-MB-231 CANCER CELLS

[0114] In this example, the antiproliferative effect of the STAT3 sdAb was analyzed using the breast cancer cell line MDA-MB-231. For experiments, MDA-MB-231 cells were grown until they reached 90% confluence. At the same time, cells were washed, trypsinized, and counted using a Coulter Counter (Beckman, Brea, CA). Proliferation studies were performed using the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide (MTT) method. To do this, cells were seeded in a 96-well plate at a density of 5 x 10 ³ per well as specified by the manufacturer (Roche Diagnostics Corporation, Indianapolis, Indiana). Cells were allowed to adhere for 24 hours and then OdAb was added at appropriate concentrations (i.e., 0, 0.5, 1.0, 10.0, or 100 μg/ml). Cells were counted on day 3. For cells treated for 5 days, the culture media containing OdAb was renewed on the 3rd day. At the end of the period, 10 μL of MTT reagent (0.5 mg/mL) was added to each well as specified by the manufacturer. After a 4-hour incubation period, 100 μL of solubilization solution was added and the plate was placed in the incubator overnight. All plates were read at 570 nm using a Biotek plate reader (Winooski, Vermont).

[0115] All data were analyzed using GraphPad InStat 3 (GraphPad Software, Inc., La Jolla, California). Treatment groups were compared with vehicle controls using one-way analysis of variance. If a significant difference was observed (p < 0.05), a Tukey-Kramer multiple comparison test was performed.

[0116] Based on the MTT experience, it was found that the bacterially produced VHH13-ODAb to STAT3 (SEQ ID No. 3) was effective in inhibiting cell growth on days 3 and 5 after treatment, as shown in Tables 2- 5 below.

EXAMPLE 5: ODATE CYTOTOXICITY STUDY TO STAT3 IN HUMAN BREAST (MDA-MB-231) AND PANCREAS (PANC-1) CANCER CELL LINES

[0117] In this example, the antiproliferative effects of VHH13-OdAb (SEQ ID No. 3) and VHH14-OdAb (SEQ ID No. 4) to STAT3 were analyzed using the human breast cancer cell line MDA-MB-231 and the human pancreatic cancer cell line PANC-1. For experiments, MDA-MB-231 and PANC-1 cells were grown until they reached 90% confluence. At the same time, cells were washed, trypsinized, and counted using a Coulter Counter (Beckman, Brea, CA). Proliferation studies were performed using the MTT method described above. In cells treated for 5 days, on the 3rd day, the culture media containing sdAb to STAT3 was renewed.

[0118] All data were analyzed using GraphPad InStat 3. Treatment groups were compared with a vehicle control group using one-way analysis of variance. If a significant difference was observed (p < 0.05), a Tukey-Kramer multiple comparison test was performed.

[0119] Based on the MTT assay, both VHH13 (SEQ ID No. 3) and VHH14 (SEQ ID No. 4) were found to inhibit cell growth in both MDA-MB-231 and PANC-1 cancer cells, as shown in Tables 6-13 below.

EXAMPLE 6: ANTIPROLIFERATIVE EFFECT OF ODATE TO STAT3 IN HUMAN BREAST CANCER AND HUMAN PROSTATE CANCER CELL LINES

[0120] The antiproliferative effect of VHH13-ODAb (SEQ ID No. 3) to STAT3 was analyzed in the human breast cancer cell line MDA-MB-231 and human prostate cancer cell lines DU145. For experiments, cancer cells were grown until they reached 90% confluency. At the same time, cells were washed, trypsinized, and counted using a Coulter Counter (Beckman, Brea, CA). Proliferation studies were performed using the MTT method as described above.

[0121] The antiproliferative properties of the bacterially derived VHH13-OdAb (SEQ ID No. 3) to STAT3 in MDA-MB-231 cells were compared with its actions in DU145 cells. As shown in Table 14, MDA-MB-231 cells treated with STAT3 OdAb (SEQ ID No: 3) showed average growth inhibition of 29.6 and 91.2 at 50.0 and 100 μg/ml, respectively. In DU145 cells, similar growth inhibition was observed (31.2 and 92.1% at 50.0 and 100 μg/ml, respectively) as shown in Table 15.

EXAMPLE 7: ANTIPROLIFERATIVE EFFECT OF VHH13-ODATE (SEQ ID NO. 3) TO STAT3 ON HUMAN CANCER CELL LINES

[0122] Human cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, BT474 and DU145 were used to test the anti-proliferative effects of VHH13-ODAb (SEQ ID No. 3) against STAT3, as shown in Table 16.

[0123] All human cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cell lines were maintained and cultured in RPMI 1640 (MDA-MB-231, MDA-MB-468, MCF-7, BT474) or MEM-E (DU145) medium containing 10% fetal bovine serum, 2 mM L-glutamine and 1 % solution of antibiotic and antifungal agent (10 units/ml penicillin, 10 µg/ml streptomycin and 25 µg/ml amphotericin B). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Consumable cell cultures were obtained from Life Technologies, Inc., (Grand Island, NY). MTT reagent was purchased from Sigma Aldrich (St. Louis, MO).

[0124] For experiments, cancer cells were grown until they reached 90% confluence. At the same time, cells were washed, trypsinized, and counted using a Coulter Counter (Beckman, Brea, CA). Proliferation studies were performed using the MTT method as described above.

[0125] The antiproliferative properties of bacterially derived VHH13-OdAb (SEQ ID No. 3) to STAT3 were assessed in five breast cancer cells representing different classifications (Table 34). As shown in Table 17, all cell lines showed significant growth inhibition 72 hours after treatment. For all cell lines, the greatest growth inhibition was observed at doses of 100 and 200 μg/ml. The half-maximal inhibitory concentration (IC50) for growth in the cell lines tested were: 10.1 ± 2.4, 12.36 ± 1.5, 14.8 ± 1.6 and 25.2 ± 14.7 in the case of MDA cell lines -MB-231, MDA-MB-468, MCF-7 and BT474, respectively. These data suggest that triple negative breast cancer cell lines require the lowest concentration of VHH13-ODAb (SEQ ID No: 3) to achieve IC50 compared to estrogen/progesterone positive cell lines ( i.e. , MCF-7) or cell lines with HER2 gene amplification ( ie , BT474).

[0126] The effect of bacterially derived VHH13-ODAb (SEQ ID No: 3) against STAT3 was also evaluated in the human prostate cancer cell line DU145, as shown in Table 18. Bacterially derived VHH13-ODAb (SEQ ID No: 3) against STAT3 exhibited dose-dependent growth inhibition in all cancer cells tested.

EXAMPLE 8: MAXIMUM TOLERATED DOSE OF BACTERIALLY PRODUCED VHH13 (SEQ ID NO: 3) TO STAT3 IN BALB/C MICE

[0127] In this Example, the tolerance of bacterially derived VHH13-OdAb (SEQ ID No. 3) to STAT3 was analyzed in experimental animals using the human breast cancer cell line MDA-MB-231. For the experiment, a total of 9 female BALB/C nude mice (6-7 weeks old) were divided into three groups based on body weight. (Table 19) Mice (n = 3) received either vehicle (PBS) or bacterially derived VHH13-ODAb (SEQ ID No: 3) to STAT3 at a dose of 250 or 500 μg/kg body weight/day for five days. During the study, mortality/morbidity occurred twice daily. Body weight was recorded on days 1, 4 and 6 of the study, as well as on the day the study stopped (day 13). Toxicity was assessed by body weight measurements and mouse behavior compared to control vehicle-treated mice. After completion of the treatment phase, animals were observed for an additional week to note any abnormalities in body weight and/or general health after treatment.

[0128] As shown in Table 20, body weights were not significantly different between the groups, and the bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3 did not result in any drug-related deaths at any dosage level . In addition, no changes in behavior were observed in animals treated with the bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3 compared to control mice.

EXAMPLE 9: ACTIVITY OF BACTERIALLY-derived VHH13 (SEQ ID No: 3) TO STAT3 IN TUMOR XENOTRANSPLANTS IN BALB/C NAKED MICE AND IN HUMAN BREAST CANCER AND HUMAN PANCREAS CANCER CELLS

[0129] In this example, the anti-STAT3 activity of the bacterially derived VHH13-ODAb (SEQ ID NO: 3) was assessed in mice using the human breast cancer cell line MDA-MB-231. Briefly, the anti-STAT3 activity of the bacterially derived VHH13-ODAb (SEQ ID No: 3) was assessed using: the MDA-MB-231 human breast cancer xenograft model and the PANC-1 human pancreatic cancer xenograft model. Dosing regimens were as follows: Group 1 (n = 6; PBS, i.p.), once daily for 14 days [OD x 14]; and Group 2 (n-12; 500 mcg/kg bw, i.p.), once daily for 14 days [OD x 14]. The drug administration was followed by an observation period of 5 days.

[0130] Human breast cancer cell lines (MDA-MB-231 and PANC-1) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). MDA-MB-231 cells were grown in MEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA) and penicillin-streptomycin-glutamine (Life Technologies, Grand Island, NY NY). PANC-1 cells were grown in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS and penicillin-streptomycin-glutamine. All cells were grown in an incubator in the presence of 5% CO2 at 37°C.

[0131] Male Nude-Foxnl ^nu nude mice, 4 to 5 weeks old, were purchased from Harlan Laboratories (Indianapolis, Indiana). Animals were quarantined for one week and housed five mice per cage under a 12-hour light-dark cycle and 50% relative humidity. Drinking water and food were provided to the animals in unlimited quantities. All animals were kept under sterile conditions and experiments were carried out with the approval of the Bioethics Committee of the IIT Research Institute. To study the MDA-MB-231 xenograft, cells (4 x 10 ⁶ ) in a final volume of 100 μl MEM medium were injected subcutaneously into the right flank of mice. For the PANC-1 xenograft assay, cells (5 x 10 ⁶ ) in a final volume of 100 μl RPMI medium were injected subcutaneously into the right flank of mice. Tumor measurements for both models began as soon as the tumors became palpable. Tumors were then measured twice weekly. Animals were randomized into groups when tumors reached a size ranging from 75-175 mm ³ , control (n = 6) and treatment (n = 12) groups were randomized using a stratified random sampling algorithm. Treatment (derived in bacteria VHH13-ODAb (SEQ ID No: 3) to STAT3) or vehicle (PBS) was initiated the day after randomization. The treatment was well tolerated and did not lead to drug-related deaths. No significant weight loss was noted.

[0132] For the MDA-MB-231 xenograft study, the randomization mean (±SE) tumor size was: 103.01 ± 11.89 and 102.61 ± 9.60 for the control and treatment groups, respectively. Mean body weights (± SE) at randomization were: 32.08 ± 0.76 and 30.27 + 0.75 for Group 1 and Group 2, respectively. Table 21 presents mean body weights (± SE) for the entire study.

[0133] On day 14 of dosing, the mean tumor size (±SE) for the control group was 179.11 ± 19.39 compared to 118.86 ± 15.94 for the treatment group. Mean body weights (± SE) at the time of discontinuation were: 31.98 ± 0.71 and 30.55 ± 0.74 for Group 1 and Group 2, respectively. Table 22 summarizes tumor volumes (± SE) for the entire study. The % mean tumor growth inhibition in the treatment group was 33.64%. Tumor doubling times were as follows: Group 1: 44.27 days; and Group 2: 61.06 days. In FIG. Figure 5 illustrates growth inhibition of bacterially derived VHH13-ODAb (SEQ ID No: 3) to STAT3 in the MDA-MB-231 xenograft model. Bacteria-derived VHH13-OdAb (SEQ ID No: 3) to STAT3 demonstrated significant growth inhibition (p = 0.047). Thus, VHH13-ODAb (SEQ ID No: 3) obtained in bacteria against STAT3 has chemotherapeutic activity in the human breast cancer model system MDA-MB-231.

[0134] For the PANC-1 xenograft study, randomization mean (+SE) tumor sizes were 107.01 ± 4.54 in the control group and 110.58 ± 6.18 in the treatment group. Mean body weights (± SE) at randomization were: 29.0 ± 0.81 and

28.5 ± 0.70 for Group 1 and Group 2, respectively. Mean body weights (± SE) at the time of discontinuation were: 31.2 ± 0.99 and 30.1 ± 0.75 for Group 1 and Group 2, respectively. Table 23 summarizes the mean body weights (± SE) for the entire study. On day 14 of dosing, the mean tumor size (±SE) for the control group was 287.30 ± 33.94 compared to 318.74 ± 29.76 for the treatment group. Table 24 summarizes tumor volumes (± SE) for the entire study.

[0135] Tumor doubling times were as follows: Group 1: 22.44 days; and Group 2: 23.02 days. Bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3 did not show significant growth inhibition in the PANC-1 human pancreatic cancer model system.

EXAMPLE 10: STUDY OF MDA-MB-231 XENOTRANSPLANT

[0136] This Example further assessed the effectiveness of the bacterially derived VHH13-ODAb (SEQ ID No: 3) to STAT3 in the human breast cancer xenograft model MDA-MB-231. Dosing regimens were as follows: Group 1 (n = 4; PBS; i.p.) twice daily for 14 days [DD x 14]; Group 2 (n = 4; 1 mg/kg bw; i.p.), twice daily for 14 days [DD x 14]; Group 3 (n = 4; 2 mg/kg bw; i.p.) twice daily for 14 days [DD x 14]; and Group 4 (n = 4; 2 mg/kg bw; i.p.) once daily for 14 days [OD x 14]. Administration was followed by an observation period of 7 days.

[0137] The MDA-MB-231 human breast cancer cell lines and Nude-Foxnl ^nu female nude mice were described above.

[0138] MDA-MB-231 cells were injected at a density of 5×10 ⁶ subcutaneously into the right flank of mice in a final volume of 100 μl in MEM medium. Tumor measurements began as soon as the tumors became palpable. Tumors were then measured twice weekly. Animals are randomized into groups using a stratified random sampling algorithm when tumors reach a size in the range of 55-150 mm ³ . Treatment (bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3) or vehicle (PBS) was initiated the day after randomization.

[0139] Randomization mean (± SE) tumor size was: 92.08 ± 13.24, 82.38 ± 5.17, 77.47 ± 7.17, and 104.71 ± 14.64 for Groups 1, 2, 3 and 4, respectively. As shown in Table 25, mean body weights (± SE) at randomization were: 23.65 ± 0.72, 23.45 ± 0.66, 23.10 ± 0.20, and 22.45 ± 1.25 for Groups 1, 2, 3 and 4, respectively.

[0140] As shown in Table 26, on day 14 of dosing, the mean tumor size (±SE) for the control group was 221.51±57.32 compared to 67.12±10.66, 58.27± 22.54 and 131.44 ± 22.86 for treatment groups 2, 3 and 4, respectively. At the time of discontinuation (day 42), the mean tumor size (± S.E.) was: 255.42 ± 65.46, 55.98 ± 6.94, 41.15 ± 13.21 and 145.51 ± 52.32. for groups 1, 2, 3 and 4, respectively. Mean body weights (± SE) at the time of discontinuation were: 24.80 ± 0.49, 23.25 ± 1.20, 24.00 ± 0.32 and 23.2 ± 1.46 for Groups 1, 2, 3 and 4, respectively. The maximum mean % net weight loss (day) was: 0.7 (36), 1.5 (23), 1.8 (36) and 2.2 (29) for Groups 1, 2, 3 and 4, respectively.

[0141] In addition, as shown in Table 26, the average growth inhibition in the treatment groups was 78.3, 75.2 and 55.9 for Groups 2, 3 and 4, respectively. Tumor doubling times were: Group 1: 20.56 days; Group 2: 34.54 days; Group 3: 30.07 days; and Group 4: 27.17 days. Growth delays of 13.99, 9.52 and 6.61 days were observed for Groups 2, 3 and 4, respectively. The % treatment/control ratio values for the treatment groups were: Group 2: -33.75 (tumor growth arrest); Group 3: -54.4 (tumor regression); and Group 4: 10.28 (tumor growth inhibition). In FIG. 6 illustrates growth inhibition of bacterially derived VHH13-OdAb (SEQ ID No: 3) to STAT3 in the MDA-MB-231 xenograft model.

[0142] Administration of bacteria-derived VHH13-OdAb (SEQ ID No: 3) to STAT3 caused significant growth inhibition in Group 2 (p = 0.02) [1 mg/kg; DD x 14] and Group 3 (p = 0.02) [2 mg/kg; DD x 14]. In addition, three of the four tumors showed significant regression. Based on these data, it can be concluded that the bacteria-derived VHH13-OdAb (SEQ ID No: 3) to STAT3 has chemotherapeutic activity in the MDA-MB-231 human breast cancer model system.

EXAMPLE 11: EFFECTIVENESS OF BACTERIALLY DERIVED VHH13-ODATE (SEQ ID NO: 3) TO STAT3 IN THREE XENOTRANSPLANT MODELS OF HUMAN CANCER

[0143] This Example assessed the effectiveness of the bacterially derived VHH13-ODAb (SEQ ID No: 3) to STAT3 in xenograft models of human breast cancer MDA-MB-231, pancreatic PANC-1 and prostate cancer DU145.

[0144] Nude-Foxn1 ^nu nude mice, MDA-MB-231 breast cancer cells, PANC-1 pancreatic cancer cell lines, and DU145 prostate cancer cell lines were described above. On day 1 of the study, the body weight of mice ranged from 17 to 19 g (34 females) and from 21 to 23 g (16 males).

[0145] Cells at early passages (4-10) were used for implantation into mice and harvested during logarithmic growth phase. MDA-MB-231 (5×10 ⁶ ), DU145 (5×10 ⁶ ), and PANC-1 (1.5×10 ⁶ ) were injected subcutaneously into the right flank of mice in a final volume of 100 μl of medium. Tumor measurements began as soon as the tumors became palpable. Tumors were then measured twice weekly.

[0146] Animals were randomized into groups using a stratified random sampling algorithm when tumors reached a size ranging from: 74-120 mm ³ (MDA-MB-231), 89-146 mm ³ (DU145) or 60-160 mm ³ (PANC-1). Treatment (containing bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3 and referred to herein as SBT-100) or vehicle (PBS) was initiated the day after randomization, referred to as day 1.

[0147] The bacterially produced VHH13-OdAb (SEQ ID No: 3) to STAT3 was delivered as a premixed solution at a concentration of 0.651 mg/ml and stored at -20°C until ready for use. The stock solution was diluted in sterile PBS pH 7.6 to give 5 mg/kg in a dosage volume of 10 ml/kg. The working solution was prepared every 7 days, aliquoted into seven tubes and stored at 4°C. On each day of treatment, only the required tube was brought to room temperature. All remaining OdAb material was stored at 4°C in case the next dose was needed. On day 8, any remaining OdAt material was discarded and a fresh batch was prepared.

[0148] Two groups (control and SBT-100) of mice in the tumor model were dosed according to the protocol shown in Table 27. The dosing regimens were as follows: Group 1 (n = 4; PBS) twice daily for 14 days [0148] DD x 14]; Group 2 (n = 4; SBT-100, 5 mg/kg b.w.), twice daily for 14 days [DD x 14]. Both vehicle (PBS pH 7.6) and SBT-100 were administered intraperitoneally (i.p.) twice daily, six hours apart, for fourteen days. Dosing was carried out according to the weights of individual animals. The administration was followed by a recovery period of 7 days.

[0149] Study Log Study Director Animal Study Management software (San Francisco, California) was used to randomize animals into groups, collect data (eg, dosage, body weight, tumor measurements, clinical observations), and perform data analysis.

[0150] In the MDA-MB-231 xenograft tumor model, animals were randomized to groups on day 23 post tumor inoculation with mean (±SE) sizes of: 77.98 ± 21.58 and 84.71 ± 5.56 for Groups 1 and 2, respectively. Mean body weights (± SE) at randomization were: 20.04 ± 0.62 and 23.7 ± 1.84 for Groups 1 and 2, respectively. Table 28 summarizes the mean body weights (± SE) for the entire study. On the last day of dosing (day 14), the mean tumor size (±SE) for the control group was 168.28 ± 51.57 compared to 83.81 ± 22.65 for SBT-100-treated mice. Table 29 summarizes tumor volumes (± SE) for the entire study. At the time of discontinuation (day 28), the mean tumor size (± S.E.) was: 270.49 ± 112.35 and 91.72 ± 33.17, for Groups 1 and 2, respectively. Mean body weights (± SE) at the time of discontinuation were: 25.36 ± 1.07 and 24.25 ± 1.68 for Groups 1 and 2, respectively. At the end of the study, the mean tumor growth inhibition in the SBT-100-treated group was 85.8% (p = 0.006). In FIG. Figure 7 illustrates the average tumor volume. Tumor doubling time was 25.78 days compared to 111.6 days for Group 1 and Group 2, respectively. The % treatment/control ratio for Group 2 was 13.35 (tumor inhibition).

[0151] In the DU145 xenograft tumor model, animals were randomized to groups on day 17 post tumor inoculation with a mean (±SE) size of: 111.87 ± 20.53 and 111.23 ± 25.16 for Groups 1 and 2, respectively. Mean body weights (± SE) at randomization were: 29.10 ± 1.94 and 30.68 ± 1.56 for Groups 1 and 2, respectively. Table 30 summarizes the arithmetic mean body weights (± SE) for the entire study. On the last day of dosing (day 14), the mean tumor size (±SE) for the control group was

621.81 ± 276.25 compared to 364.14 ± 51.64 for SBT-100-treated mice. Table 31 summarizes tumor volumes (± SE) for the entire study. At the time of termination (day 28), the mean tumor size (± S.E.) was: 819.42 ± 351.88 and 601.83 ± 131.51, for Groups 1 and 2, respectively. Mean body weights (± SE) at the time of discontinuation were: 29.20 ± 2.33 and 29.60 ± 1.04 for Groups 1 and 2, respectively. At the end of the study, the mean tumor growth inhibition in the SBT-100-treated group was 26.6% (p = 0.29). In FIG. Figure 8 illustrates the average tumor volume. Tumor doubling time was 14.57 days compared to 18.19 days for Group 1 and Group 2, respectively. % treatment/control ratio for

Group 2 was 74.8.

[0152] In the PANC-1 xenograft tumor model, animals were randomized to groups on day 22 after tumor inoculation with a mean (±SE) size of: 78.74 ± 40.21 and 93.84 ± 36.31 for Groups 1 and 2, respectively. Mean body weights (± SE) at randomization were: 22.50 ± 1.47 and 24.23 ± 1.63 for Groups 1 and 2, respectively. Table 32 summarizes the mean body weights (± SE) for the entire study. On the last day of dosing (day 14), the mean tumor size (±SE) for the control group was 204.95 ± 178.90 compared to 159.03 ± 28.01 for SBT-100-treated mice. Table 33 summarizes tumor volumes (± SE) for the entire study. At the time of termination (day 28), the mean tumor size (± S.E.) was: 284.77 ± 288.88 and 203.02 ± 30.34, for Groups 1 and 2, respectively. Mean body weights (± SE) at the time of discontinuation were: 27.38 ± 1.07 and 26.23 ± 1.19 for Groups 1 and 2, respectively. At the end of the study, the mean tumor growth inhibition in the SBT-100-treated group was 41.78% (p = 0.35). In FIG. Figure 9 illustrates the average tumor volume. Tumor doubling time was 18.51 days compared to 35.70 days for Group 1 and Group 2, respectively. The % treatment/control ratio for Group 2 was 52.79.

EXAMPLE 12: EFFECTIVENESS OF BACTERIALLY PRODUCED VHH13-ODATE (SEQ ID NO: 3) TO STAT3 IN A XENOTRANSPLANT MODEL OF HUMAN BREAST CANCER RE+/RP+ (MCF-7)

[0153] This Example demonstrates the effectiveness of the bacterially derived VHH13-ODAb (SEQ ID No: 3) to STAT3 in the MCF-7 xenograft model of human breast cancer in nude mice.

[0154] Female athymic nude mice (Crl:NU(Ncr) -Foxn1nu , Charles River) were twelve weeks old and had body weights (bw) ranging from 23.0 to 30.1 g/day. th day of the study. Animals were fed and housed as described above.

[0155] Human breast carcinoma cell line MCF-7 was prepared and cultured as described above and used for the mouse xenograft model. Three days before tumor cell implantation, estrogen pellets (0.36 mg estradiol, 60-day extended release, Innovative Research of America, Sarasota, Florida) were implanted subcutaneously in the interscapular region of each experimental animal using a sterilized trocar.

[0156] Tumor cells used for implantation were collected during logarithmic growth phase and resuspended in phosphate buffered saline (PBS) at a concentration of 1×10 ⁸ cells/ml. On the day of implantation, each experimental mouse was injected with 1 x 10 ⁷ MCF-7 cells (0.1 ml cell suspension) implanted subcutaneously in the right flank and tumor growth was monitored as the average size approached the target range of 100-150 mm ³ . After a twenty-one-day period, taken as the 1st day of the study, the animals were divided into two groups, each of which consisted of four mice with individual tumor volumes ranging from 108-144 mm ³ and group-average tumor volumes of 117-123 mm ³ .

[0157] Bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3 was provided as a premixed, ready-to-administer solution at a concentration of 0.41867 mg/mL in 1 mL aliquots and stored at -20°C until the need arises. The 0.41867 mg/ml solution provided a 1 mg/kg dose in a dosage volume of 23.88 ml/kg. On each day of treatment, only the necessary tubes with the VHH13-OdAb (SEQ ID No: 3) obtained in the bacterium to STAT3 were brought to room temperature. All remaining doses of suspensions were stored at 4°C in case the next dose was needed.

[0158] Two groups of athymic nude mice were dosed according to the protocol shown in Table 34. All doses of vehicle (control) and bacteria-derived VHH13-OdAb (SEQ ID No: 3) to STAT3 were administered intraperitoneally (i.p. ) three times daily, six hours apart, for fourteen days, with two doses given on day 1 and one dose given on the morning of day 15 (TD x 14, 2 doses on day 1). Dosing volume for carrier and bacteria-derived VHH13-ODAb (SEQ ID No.:

3) to STAT3 was 0.478 ml per 20 grams of body weight (23.88 ml/kg), and was selected in proportion to the body weight of each individual animal. Group 1 received vehicle and served as a reference group for tumor engraftment and progression and as a control. Group 2 received bacterially produced VHH13-ODAb (SEQ ID No: 3) to STAT3 at a dose of 1 mg/kg.

[0159] Tumors were measured twice weekly, and each animal was sacrificed when its tumor reached a predetermined end volume (1000 mm ³ ) or at the end of the study, on day 39, whichever occurred first. When the tumor reached a predetermined final volume, the animal was recorded as sacrificed due to tumor progression (TP), with the date of sacrifice indicated. Time to clinical trial endpoint (TTE) for each mouse was calculated using the following equation:

where TTE is expressed in days, the prespecified final volume is expressed in mm ³ , b is the intercept of the regression equation, and m is the slope of the line obtained by linear regression of the log-transformed tumor growth data set. The data set consists of the first observation that exceeded the preset endpoint used in the analysis and three consecutive observations that immediately preceded reaching that preset endpoint. The estimated TTE is typically less than the TP date, the day on which the animal was sacrificed due to tumor size. Animals that did not reach the prespecified endpoint volume were assigned a TTE value equal to the last day of the study (D39). Any animal classified as dying from treatment-related (TR) causes was assigned a TTE value equal to the day of death. Any animal classified as dying from non-treatment-related (NTR) causes had to be excluded from TTE calculations.

[0160] Treatment efficacy was determined by tumor growth deceleration (TGD), which is defined as the increase in median TTE, expressed in days, for the treatment group compared to the control group:

TGD = T - C

The percentage increase in median TTE, relative to the control group, is

Where:

T = median TTE for treatment group, and

C = median TTE for the designated control group.

[0161] Treatment efficacy in each group can be indicated by the median tumor volume, MTV(n), which was defined as the median tumor volume on the last day of the study (D39) among the remaining animals (n) whose tumors did not reach the predetermined target volume.

[0162] The effectiveness of treatment can also be determined by the number of cases and intensity of regression reactions observed during the study. Treatment can cause partial regression (PR) or complete regression (CR) of the tumor in the animal. In the PR reaction, the tumor volume was 50% or less of its volume in D1 in the case of three consecutive measurements during the study, and was not less than 13.5 mm ³ in one or more of these three measurements. In the CR response, tumor volume was less than 13.5 mm ³ for three consecutive measurements during the study. Any animal with a CR response at the end of the study was further classified as a complete tumor resolution survivor (TFS).

[0163] Animals were weighed daily for the first five days and then twice weekly for the remainder of the study. Mice were frequently examined for health status and significant signs of any adverse side effects associated with TR treatment, and noteworthy clinical observations were recorded. Individual weight loss was monitored per protocol, and any animal with a weight loss greater than 30% in one measurement or greater than 25% over three measurements was euthanized for health reasons as TR death. If a group's mean body weight was restored, dosing could be resumed in that group, but at a lower dose or less frequent dosing schedule. Acceptable toxicity was defined as the average bw loss. in the group, representing less than 20% during the study, and no more than one TR death per ten treated animals, or 10%. Any regimen that results in greater toxicity is considered to be in excess of the maximum tolerated dose (MTD). Death was classified as TR if it was associated with side effects of treatment, as evidenced by clinical signs and/or autopsy, or could also be classified as TR if it occurred from unknown causes during the drug administration period or within 14 days of the last drug administration doses. Death was classified as NTR if there was evidence that the death was related to the tumor model rather than treatment. NTR deaths were further classified as NTRa (due to accident or human error), NTRm (due to autopsy-proven spread due to invasion or metastasis), and NTRu (due to unknown causes).

[0164] Prism 6.07 (GraphPad) for Windows was used for graphical analyses. Statistics were not used due to the small sample size.

[0165] A scatterplot was generated to show TTE values for individual mice, by group; this chart shows the NTR of deaths that have been excluded from all other figures. Tumor volumes of individual animals and median and group mean tumor volumes were plotted as functions of time. When an animal withdrew from the study due to tumor size or TR of death, its last recorded tumor volume was included with the data used to calculate the median volume at subsequent time points. A Kaplan-Meier plot was generated to show the percentage of animals in each group remaining in the study as a function of time. Tumor growth curves were truncated after two TR deaths occurred in the same group. Average changes in b.m. in the study group were plotted as percent change ± SEM from day 1. Tumor growth curves and changes in b.m. were truncated after more than half of the analyzed mice in the group dropped out of the study. In FIG. Figure 10 illustrates the average tumor volume in the study.

[0166] Table 35 provides the average bw losses, TR and NTR death in mice. During observation, clinical signs were recorded as shown in Tables 36-38. No TR deaths occurred during the study. Weight losses were variable, significant for one animal in each group, and occurred as a result of the estrogenic effect. Clinical observations, including weight loss, enlarged uterine horns and bladder salt, were present in both groups and were also due to the estrogenic effect. Estrogen toxicity resulted in two non-treatment-related deaths in each group. The treatments evaluated in the study were acceptable tolerability.

[0167] Since two out of four mice in the control group as well as in the treatment group died from estrogen toxicity, no statistical conclusion can be drawn. Based on the data, median tumor growth and mean tumor volume decreased in the treatment group compared to the control group. This difference was present throughout the 14 days of treatment and up to the 25th day of the study. The control group took 25 days for their tumors to reach a volume of 1000 mm ³ while the treatment group took 36 days for their tumors to reach a volume of 1000 mm ³ . This indicates that the bacterially produced VHH13-ODAb (SEQ ID No: 3) to STAT3 slows down the growth of the MCF-7 tumor in vivo . Throughout the study, both the control and treatment groups maintained similar weights. This indicates that the bacteria-derived VHH13-OdAb (SEQ ID No: 3) to STAT3 did not cause weight loss toxicity.

EXAMPLE 13: TREATMENT OF HER2+ HUMAN BREAST CANCER (BT474) WITH BACTERIALLY DEVELOPED VHH13-ODATE (SEQ ID NO: 3) TO STAT3 IN XENOTRANGLANT MICE

[0168] In this Example, the potency of a bacterially derived VHH13-ODAb (SEQ ID No: 3) to STAT3 was determined in a human breast cancer xenograft BT474 in CB.17 SCID mice.

[0169] Two groups of 8-12 week old CB.17 SCID mice containing xenografts of 1 mm ³ BT474 tumor fragments in their flank were treated according to the protocol shown in Table 39 when the tumors reached an average size of 100-150 mm ³ . All doses of vehicle (PBS control) and bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3 (shown in Table 39 as SB-01) were administered intraperitoneally (i.p.) three times daily, at six-hour intervals. for fourteen days, with two doses administered on day 1 (TD x 14, 2 doses on day 1). The dosage volume for the vehicle and bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3 was 0.478 ml per 20 grams of body weight (23.88 ml/kg), and was adjusted in proportion to the body weight of each individual animal. Group 1 received vehicle and served as a reference group for tumor engraftment and progression and as a control. Group 2 received bacterially produced VHH13-ODAb (SEQ ID No: 3) to STAT3 at a dose of 1 mg/kg.

[0170] During the first 14 days of the study, the treatment group received B VHH13 to STAT3, and the control group received vehicle alone. As shown in Table 40, during this time, the treatment group maintained and gained weight throughout the study, while the control group maintained lower body weight throughout the study. This indicates that the treatment group did not experience toxicity from the bacteria-derived VHH13-ODAb (SEQ ID NO: 3) to STAT3 in relation to weight loss. The mean tumor volume and median tumor volume were similar in both groups, and exactly the same at day 15 of the study. On day 59 of the study, both groups achieved a tumor volume of 700 mm ³ . This indicates that the bacteria-derived VHH13-OdAb (SEQ ID No: 3) to STAT3 did not reduce the growth of BT474 tumors in vivo compared to the control group. In FIG. Figure 11 illustrates the average tumor volume in the group.

EXAMPLE 14: PREPARATION OF MOUSE MONOCLONAL ANTIBODY DIRECTED AGAINST BACTERIALLY DEVELOPED VHH13-ODATE (SEQ ID NO: 3) TO STAT3

[0171] In this Example, mouse monoclonal antibodies to the OdAb of the present invention were generated. Animals used were female BALB/c mice, 8-10 weeks old. A water-soluble adjuvant (CBL) was used. The HATs and HTs used were purchased from Sigma-Aldrich.

[0172] Bacteria-derived VHH13-ODAb (SEQ ID No: 3) to STAT3 was used to immunize three mice and generate hybridoma cell lines. Mice were immunized three times, each time with a water-soluble adjuvant. In one mouse, serum titers reached 1/51200. The mouse was sacrificed and hybridoma cell lines were generated by fusing spleen cells with the Sp2/0 myeloma cell line.

[0173] Confluent cells were seeded into 96-well plates by serial dilution. The fusion cells were cultured in the presence of HAT, and 651 individual clones were tested. It was found that 27 positive clones out of 651 individual clones specifically bound to the bacterial antigen VHH13-ODAb (SEQ ID No: 3) to STAT3.

EXAMPLE 15: CYTOTOXICITY OF SINGLE-DOMAIN KRAS ANTIBODIES (G12D) IN HUMAN PANCREAS CANCER PANC-1 CELLS

[0174] This Example demonstrates the antiproliferative effects of the KRAS sdAb (G12D) (SEQ ID No: 2) using the human pancreatic cancer cell line PANC-1. For experiments, PANC-1 cells were grown until they reached 90% confluence. At the same time, proliferation studies were performed using the MTT method as described above.

[0175] Table 41 shows the antiproliferative properties of the KRAS sdAb (G12D) (SEQ ID No: 2) in PANC-1 cells three days after treatment. PANC-1 cells treated with KRAS sdAb (G12D) (SEQ ID No: 2) showed average growth inhibition of 19.9 and 37.7 at 50.0 and 100 μg/ml, respectively.

[0176] In summary, the KRAS sdAb (G12D) (SEQ ID No: 2) showed dose-dependent growth inhibition in human pancreatic cancer PANC-1 cells.

EXAMPLE 16: IN VITRO GROWTH INHIBITION WITH ODATE TO TNF-ALPHA

[0177] This Example demonstrates the development of a methodology for determining TNF-alpha concentration and assessing inhibition of TNF-alpha function. The concentration of TNF-alpha required to show measurable modulation of activity in the human lung lymphoblastic cell line U937 was assessed by quantifying the ATP present, which signals the presence of metabolically active cells, using the CellTiter-GJo® luminescent cell viability assay from Promega.

[0178] U937 cells were seeded into a 96-well clear polystyrene microculture plate (Corning® Costar® 96-well Flat Bottom Plate, Cat. No. 3997) in a total volume of 90 μl/well. After 24 hours of incubation in a humidified incubator at 37°C with 5% CO ₂ and 95% air, 5 μl was added to each well in duplicate (10-point dose-response curve, highest concentration 20 ng/ml) 20 times serially diluted in TNF-alpha growth medium. In addition, 5 μl of 20-fold diluted staurosporine in growth medium was added to each well in duplicate (1 nM concentration).

[0179] After 24 hours of culture in the presence of test substances, the concentration of compound required to show measurable modulation of TNF-alpha activity in the U937 cell line was assessed by quantifying the ATP present. Percentage cell growth was calculated relative to untreated control wells. All tests were performed in duplicate at each concentration level.

[0180] The EC50 value for the test substances was estimated using Prism 6.05 by fitting the data using the following four-parameter logistic equation:

where Top is the maximum % control absorbance, Bottom is the minimum % control absorbance at the highest concentration of the substance, Y is the % control absorbance, X is the concentration of the substance, IC50 is the concentration of the substance that inhibits cell growth by 50% compared with control cells, and n represents the slope of the curve.

[0181] In FIG. 12 and 13 illustrate that TNF-alpha is cytotoxic to U937 cells. The IC50 for TNF-alpha against U937 is 95.10 pg/ml. The TNF-alpha curve shows the cytotoxic effect of dose titration.

[0182] In FIG. 14 illustrates that the cytotoxicity of TNF-alpha to U937 is inhibited by three different VHHs to TNF-alpha. When VHH62-OdAb (SEQ ID No.: 47) to TNF-alpha, VHH66-OdAb (SEQ ID No.: 45) to TNF-alpha and VHH69-OdAb (SEQ ID No.: 46) to TNF-alpha were incubated with a constant dose of TNF -alpha, at EC50, all three VHHs to TNF-alpha inhibited the cytotoxic activity of TNF-alpha against U937. The IC50 of VHH62-ODAb (SEQ ID No: 47) to TNF-alpha was about 713.6 μg/ml. The IC50 of VHH69-ODAb (SEQ ID No: 46) to TNF-alpha was more than 208.055 μg/ml. The IC50 of VHH66-ODAb (SEQ ID No: 45) to TNF-alpha could not be determined because it completely inhibited the cytotoxicity of TNF-alpha at concentrations ranging from about 1 x 10 ² μg/ml to 1 x 10 ² μg/ml VHH66 -ODAT (SEQ ID No: 45) to TNF-alpha. In this concentration range of VHH66-ODAb (SEQ ID No: 45) to TNF-alpha, there is an increase in the growth of U937 cells and, thus, complete inhibition of TNF-alpha activity.

[0183] Although the present invention has been described with reference to certain preferred embodiments in some detail, other embodiments are possible. The steps described for the methods of this invention, for example, are not intended to be limiting, nor are they intended to indicate that each step is necessarily integral to the method, but rather represent typical steps only. Accordingly, the scope of the appended claims should not be limited to the description of the preferred embodiments contained herein. All references contained in this document are incorporated by reference in their entirety.

--->

LIST OF SEQUENCES

<110> Singh, Sunanda

<120> Single domain antibodies to intracellular antigens

<130> 7304-51293-4PCT

<150> US 62/067908

<151> 2014-10-23

<150> US 62/148656

<151> 2015-04-16

<150> US 62/188353

<151> 2015-07-02

<150> US 62/210795

<151> 2015-08-27

<160> 81

<170> PatentIn version 3.5

<210> 1

<211> 372

<212> DNA

<213> Artificial sequence

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<223> Camelids

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gaggtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60

tcctgtgcag tttctggaaa tatcggcagc agctactgca tgggctggtt ccgccaggct 120

ccagggaaga agcgcgaggc ggtcgcacgt attgtacgtg atggtgccac tggctacgca 180

gactacgtga agggccgatt caccatctcc cgagacagcg ccaagaacac tctgtatctg 240

caaatgaaca ggctgatacc tgaggacact gccatctact actgtgcggc agacctgccc 300

ccaggttgtt tgactcaggc gatttggaat tttggttatc ggggccaggg aaccctggtc 360

accgtctcct ca 372

<210> 2

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Asn Ile Gly Ser Ser Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu Ala Val

35 40 45

Ala Arg Ile Val Arg Asp Gly Ala Thr Gly Tyr Ala Asp Tyr Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Arg Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Ala Asp Leu Pro Pro Gly Cys Leu Thr Gln Ala Ile Trp Asn Phe Gly

100 105 110

Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

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His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 4

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 5

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 6

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 7

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His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 8

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 9

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 10

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 11

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 12

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Ser

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 13

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 14

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Thr Tyr Thr Gly Cys Met Gly

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu

35 40 45

Ser Ser Arg Gly Phe Ala Gly His Tyr Thr Asp Ser Val Lys Gly Arg

50 55 60

Phe Ser Ile Ser Arg Asp Tyr Val Lys Asn Ala Val Tyr Leu Gln Met

65 70 75 80

Asn Thr Val Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Ala Arg

85 90 95

Glu Gly Trp Glu Cys Gly Glu Thr Trp Leu Asp Arg Thr Ala Gly Gly

100 105 110

His Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 15

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 16

<211> 127

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Asn Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 17

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<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 18

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 19

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 20

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 21

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His Val Gln Leu Val Glu Ser Glu Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 22

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 23

<211> 127

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 24

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 25

<211> 127

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 25

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 26

<211> 127

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 26

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 27

<211> 127

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 27

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 28

<211> 127

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 28

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 29

<211> 127

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 29

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Asn Gly Gly Arg Ser

20 25 30

Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Gly Ile Ser Thr Gly Gly Leu Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Thr Ser Arg Phe Asp Cys Tyr Arg Gly Ser Trp Phe Asn Arg Tyr

100 105 110

Met Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 30

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 30

gatgtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggaggctc tctgagactc 60

tcctgtgtag cctctacata caccggctgc atgggctggt tccgccaggc tcctggaaag 120

gagcgcgagg gagtcgcagc tcttagtagc cgtggttttg ccgggcacta taccgactcc 180

gtgaagggcc gattctccat ctcccgagac tacgtcaaga atgcggtgta tctgcaaatg 240

aacactgtga aacctgagga cgctgccatg tactactgtg cagcacggga gggatgggag 300

tgcggtgaga cctggttgga ccggaccgcc gggggccata cctactgggg ccaggggacc 360

caggtcaccg tctcctca 378

<210> 31

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 31

caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggaggctc tctgagactc 60

tcctgtgtag cctctacata caccggctgc atgggctggt tccgccaggc tcctggaaag 120

gagcgcgagg gagtcgcagc tcttagtagc cgtggttttg ccgggcacta taccgactcc 180

gtgaagggcc gattctccat ctcccgagac tacgtcaaga atgcggtgta tctgcaaatg 240

aacactgtga aacctgagga cgctgccatg tactactgtg cagcacggga gggatgggag 300

tgcggtgaga cctggttgga ccggaccgcc gggggccata cctactgggg ccaggggacc 360

ctggtcaccg tctcctca 378

<210> 32

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 32

caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60

tcctgtgcag cctctggagc caatggtggt cggagctgca tgggctggtt ccgccaggtt 120

ccagggaagg agcgcgaggg ggtttctggt atttcaaccg gtggtcttat tacatactat 180

gccgactccg tgaagggccg attcaccatc tcccaagaca acaccaagaa cacgctgtat 240

ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gacgagtcgg 300

tttgactgct atagaggctc ttggttcaac cgatatatgt ataacagttg gggccagggg 360

accctggtca ccgtctcctc a 381

<210> 33

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 33

catgtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60

tcctgtgcag cctctggagc caacggtggt cggagctgca tgggctggtt ccgccaggtt 120

ccagggaagg agcgcgaggg ggtttctggt atttcaaccg gtggtcttat tacatactat 180

gccgactccg tgaagggccg attcaccatc tcccaagaca acaccaagaa cacgctgtat 240

ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gacgagtcgg 300

tttgactgct atagaggctc ttggttcaac cgatatatgt ataacagttg gggccagggg 360

acccaggtca ctgtctcctc a 381

<210> 34

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 34

gatgtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60

tcctgtgcag cctctggagc caatggtggt cggagctgca tgggctggtt ccgccaggtt 120

ccagggaagg agcgcgaggg ggtttctggt atttcaaccg gtggtcttat tacatactat 180

gccgactccg tgaagggccg attcaccatc tcccaagaca acaccaagaa cacgctgtat 240

ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gacgagtcgg 300

tttgactgct atagaggctc ttggttcaac cgatatatgt ataacagttg gggccagggg 360

accctggtca ccgtctcctc a 381

<210> 35

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 35

caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggaggctc tctgagactc 60

tcctgtgtag cctctacata caccggctgc atgggctggt tccgccaggc tcctggaaag 120

gagcgcgagg gagtcgcagc tcttagcagc cgtggttttg ccgggcacta taccgactcc 180

gtgaagggcc gattctccat ctcccgagac tacgtcaaga atgcggtgta tctgcaaatg 240

aacactgtga aacctgagga cgctgccatg tactactgtg cagcacggga gggatgggag 300

tgcggtgaga cctggttgga ccggaccgcc gggagccata cctactgggg ccaggggacc 360

ctggtcaccg tctcctca 378

<210> 36

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 36

gaggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggaggctc tctgagactc 60

tcctgtgtag cctctacata caccggctgc atgggctggt tccgccaggc tcctggaaag 120

gagcgcgagg gagtcgcagc tcttagtagc cgtggttttg ccgggcacta taccgactcc 180

gtgaagggcc gattctccat ctcccgagac tacgtcaaga atgcggtgta tctgcaaatg 240

aacactgtga aacctgagga cgctgccatg tactactgtg cagcacggga gggatgggag 300

tgcggtgaga cctggttgga ccgaaccgcc gggggccata cctactgggg ccaggggacc 360

ctggtcaccg tctcctca 378

<210> 37

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 37

gaggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60

tcctgtgcag cctctggagc caatggtggt cggagctgca tgggctggtt ccgccaggtt 120

ccagggaagg agcgcgaggg ggtttctggt atttcaaccg gtggtcttat tacatactat 180

gccgactccg tgaagggtcg attcaccatc tcccaagaca acaccaagaa cacgctgtat 240

ctgcaaatga gcagcctgaa acctgaggac actgccatgt actactgtgc gacgagtcgg 300

tttgactgct atagaggctc ttggttcaac cgatatatgt ataacagttg gggccagggg 360

acccaggtca ccgtctcctc a 381

<210> 38

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 38

catgtgcagc tggtggagtc tggggggggc tcggtgcagg ctggaggctc tctgagactc 60

tcctgtgtag cctctacata caccggctgc atgggctggt tccgccaggc tcctggaaag 120

gagcgcgagg gagtcgcagc tcttagtagc cgtggttttg ccgggcacta taccgactcc 180

gtgaagggcc gattctccat ctcccgagac tacgtcaaga atgcggtgta tctgcaaatg 240

aacactgtga aacctgagga cgctgccatg tactactgtg cagcacggga gggatgggag 300

tgcggtgaga cctggttgga ccggaccgcc gggggccata cctactgggg ccaggggacc 360

caggtcaccg tctcctca 378

<210> 39

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 39

gatgtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60

tcctgtgcag cctctggagc caatggtggt cggagctgca tgggctggtt ccgccaggtt 120

ccagggaagg agcgcgaggg ggtttctggt atttcaaccg gtggtcttat tacatactat 180

gccgactccg tgaagggccg attcaccatc tcccaagaca acaccaagaa cacgctgtat 240

ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gacgagtcgg 300

tttgactgct atagaggctc ttggttcaac cgatatatgt ataacagttg gggccagggg 360

acccaggtca ccgtctcctc a 381

<210> 40

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 40

caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60

tcctgtgcag cctctggagc caatggtggt cggagctgca tgggctggtt ccgccaggtt 120

ccagggaagg agcgcgaggg ggtttctggt atttcaaccg gtggtcttat tacatactat 180

gccgactccg tgaagggccg attcaccatc tcccaagaca acaccaagaa cacgctgtat 240

ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gacgagtcgg 300

tttgactgct atagaggctc ttggttcaac cgatatatgt ataacagttg gggccagggg 360

acccaggtca ccgtctcctc a 381

<210> 41

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 41

gatgtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagactc tctgagactc 60

tcctgtgtag cctctacata caccggctgc atgggctggt tccgccaggc tcctggaaag 120

gagcgcgagg gagtcgcagc tcttagtagc cgtggttttg ccgggcacta taccgactcc 180

gtgaagggcc gattctccat ctcccgagac tacgtcaaga atgcggtgta tctgcaaatg 240

aacactgtga aacctgagga cgctgccatg tactactgtg cagcacggga gggatgggag 300

tgcggtgaga cctggttgga ccggaccgcc gggggccata cctactgggg ccaggggacc 360

ctggtcactg tctcctca 378

<210> 42

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 42

gtgcagctgg tggagtctgg gggaggctcg gtgcaggctg gagggtctct gagactctcc 60

tgtgcagcct ctggagccaa tggtggtcgg agctgcatgg gctggttccg ccaggttcca 120

gggaagggagc gtgagggggt ttctggtatt tcaaccggtg gtcttattac atactatgcc 180

gactccgtga agggccgatt caccatctcc caagacaaca ccaagaacac gctgtatctg 240

caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcgac gagtcggttt 300

gactgctata gaggctcttg gttcaaccga tatatgtata acagttgggg ccaggggacc 360

ctggtcactg tctcctca 378

<210> 43

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 43

gtgcagctgg tggagtctga gggaggctcg gtgcaggctg gagggtctct gagactctcc 60

tgtgcagcct ctggagccaa tggtggtcgg agctgcatgg gctggttccg ccaggttcca 120

gggaagggagc gcgagggggt ttctggtatt tcaaccggtg gtcttattac atactatgcc 180

gactccgtga agggccgatt caccatctcc caagacaaca ccaagaacac gctgtatctg 240

caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcgac gagtcggttt 300

gactgctata gaggctcttg gttcaaccga tatatgtata acagttgggg ccaggggacc 360

ctggtcaccg tctcctca 378

<210> 44

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Camelids

<400> 44

gtgcagctgg tggagtctgg gggaggctcg gtgcaggctg gagggtctct gagactctcc 60

tgtgcagcct ctggagccaa tggtggtcgg agctgcatgg gctggttccg ccaggttcca 120

gggaagggagc gcgagggggt ttctggtatt tcaaccggtg gtcttattac atactatgcc 180

gactccgtga agggccgatt caccatctcc caagacaaca ccaataacac gctgtatctg 240

caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcgac gagtcggttt 300

gactgctata gaggctcttg gttcaaccga tatatgtata acagttgggg ccaggggacc 360

ctggtcactg tctcctca 378

<210> 45

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 45

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Leu Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 46

<211> 124

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Leu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Ser Arg Tyr Asn

20 25 30

Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Gly Thr Ala Ser Gly Ser Ala Asp Tyr Tyr Gly Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Arg Thr Tyr Gly Thr Ile Ser Leu Thr Pro Ser Asp Tyr Arg

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 47

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Pro Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Ser Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 48

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 48

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Gln Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 49

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 49

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Gln Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 50

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 50

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Leu Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 51

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 51

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Gln Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 52

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Gln Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 53

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Leu Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 54

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Gln Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 55

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 55

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Leu Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 56

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 56

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Leu Ala Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 57

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 57

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Arg Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Gln Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 58

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 58

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Tyr Ala Ala Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Asp Gly Lys Val Arg Glu Gly Val

35 40 45

Ala Thr Ile Asp Ile Asp Gly Gln Thr Thr His Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser Leu

65 70 75 80

Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Arg Asp Arg Cys Gly Ser Ile Trp Thr Tyr Ala Tyr Lys Tyr

100 105 110

Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 59

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 59

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Asp Ser Phe Gly

20 25 30

Val Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Val Tyr Arg Arg Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Arg Thr Tyr Gly Ser Val Ser Ser Trp Thr Gly Tyr Lys Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 60

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 60

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Asp Ser Phe Gly

20 25 30

Val Met Ala Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Val Tyr Arg Arg Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Arg Thr Tyr Gly Ser Val Ser Ser Trp Thr Gly Tyr Lys Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 61

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 61

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Tyr Thr Asp Ser Tyr Gly

20 25 30

Val Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ser Ile Tyr Arg Asn Ser Gly Ile Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Leu

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr Tyr Cys

85 90 95

Ala Val Arg Ser Phe Gly Ser Val Ser Thr Trp Ala Gly Tyr Val Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 62

<211> 123

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 62

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Asp Ser Phe Gly

20 25 30

Val Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Val Tyr Arg Arg Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Arg Thr Tyr Gly Ser Val Ser Ser Trp Thr Gly Tyr Lys Tyr

100 105 110

Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 63

<211> 125

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 63

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Arg Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asp Thr Ser Lys Ser Asp

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

Gly Ala Ile Tyr Thr Arg Asn Gly Tyr Thr His Tyr Ala Asp Ser Val

50 55 60

Asn Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Ala Leu Tyr

65 70 75 80

Leu Gln Met Ser Gly Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Arg Phe Arg Ile Tyr Gly Gln Cys Val Glu Asp Asp Asp Ile

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 64

<211> 124

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 64

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Leu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Ser Arg Tyr Asn

20 25 30

Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Gly Thr Ala Ser Gly Ser Ala Asp Tyr Tyr Gly Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Arg Thr Tyr Gly Thr Ile Ser Leu Thr Pro Ser Asp Tyr Arg

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 65

<211> 128

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 65

Gln Val Gln Val Val Glu Tyr Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Val Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Trp Glu Leu Val

35 40 45

Ser Asn Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Ser Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Phe Asp Asn Ala Lys Asn

65 70 75 80

Met Val Tyr Leu Gln Met Asn Ser Leu Lys His Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Arg Glu Tyr

100 105 110

Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 66

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 66

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Pro Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Ser Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 67

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 67

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 68

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 68

Gln Val Gln Pro Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Ala Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 69

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 69

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Ser Trp Ala Gln Gly Thr Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 70

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 70

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 71

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 71

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 72

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 72

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 73

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 73

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Ala Leu Val Thr Val Ser

115 120 125

Ser

<210> 74

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 75

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 75

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Ser Trp Ala Gln Gly Thr Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 76

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 76

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 77

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 77

Asp Val Gln Leu Val Glu Ser Arg Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 78

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 78

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Val Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Ser Trp Ala Gln Gly Thr Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 79

<211> 128

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 79

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Leu Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Ala

50 55 60

Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

65 70 75 80

Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Glu Tyr

100 105 110

Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 80

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 80

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Glu Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 81

<211> 129

<212>PRT

<213> Artificial sequence

<220>

<223> Camelids

<400> 81

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ala Glu Ala

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Glu Gly Ile Thr Ser Val Ala Ser Ser Tyr Tyr

50 55 60

Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75 80

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Pro Asp Pro Tyr Ala Tyr Ser Thr Tyr Ser Asp

100 105 110

Tyr Cys Thr Trp Ala Gln Gly Thr Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<---

Claims (12)

1. A single domain anti-STAT3 antibody comprising the amino acid sequence set forth in SEQ ID No: 3 for the treatment of diseases or pathological conditions mediated by intracellular components by eliminating said intracellular components, wherein the intracellular component is STAT3. 2. A single-domain antibody according to claim 1, characterized in that the intracellular component of STAT3 may contain a nucleic acid, protein, lipid, carbohydrate, or a combination thereof. 3. An isolated polypeptide containing the amino acid sequence set forth in SEQ ID No: 3, encoding a single domain anti-STAT3 antibody, used for the treatment of diseases or pathological conditions mediated by intracellular components by eliminating said intracellular components, wherein the intracellular component is STAT3, and said polypeptide consists of four framework regions interrupted by three complementarity determining regions. 4. A pharmaceutical composition for use as a medicament for the treatment of diseases or pathological conditions caused by intracellular components, wherein the intracellular component is STAT3, by eliminating said intracellular components, containing a single domain antibody according to any one of paragraphs. 1, 2, or a polypeptide as claimed in claim 3, and a suitable pharmaceutically acceptable carrier selected from water, saline, Ringer's solutions, dextrose solution, phosphate buffered saline, and 5% human serum albumin, liposomes, cationic lipids and non-aqueous vehicles such as fixed oils. 5. A method of diagnosing a STAT3-mediated disorder in a subject using the single domain anti-STAT3 antibody of claim 1 or the polypeptide as claimed in claim 3, comprising the steps of: (a) contacting a biological sample taken from a subject with the single domain antibody of claim 1 or the polypeptide of claim 3; (b) determining the amount of intracellular component in the biological sample; And (c) compare the amount determined in step (b) with the reference one, and the difference in the amount between the sample and the standard allows the disease to be diagnosed. 6. A method for preventing or treating a disease, disorder, preventing relapse of a disease or condition mediated by STAT3, comprising administering an effective amount of a single domain anti-STAT3 antibody as claimed in claim 1, or a polypeptide as claimed in claim 3, or a pharmaceutical composition as claimed in claim 1. 4, to a subject in need thereof by administration by one of the known routes including intravenous administration, intramuscular administration, oral administration, rectal administration, enteral administration, parenteral administration, intraocular administration, subcutaneous administration, transdermal administration, eye drop administration, administration as a nasal spray, administration by inhalation or nebulizer, topical administration and administration as an implantable drug. 7. The method according to claim 6, characterized in that the disease includes breast cancer, pancreatic cancer, prostate cancer, 8. The method according to claim 6, characterized in that the disease is a disease caused by abnormal cell proliferation. 9. Use of a pharmaceutical composition as claimed in claim 4 for the treatment or prevention of a STAT3-mediated disease or condition, or the prevention of relapse of a STAT3-mediated disease, for the treatment of breast cancer, pancreatic cancer or prostate cancer or diseases, caused by abnormal cell proliferation, comprising administering the single domain anti-STAT3 antibody of claim 1 or the polypeptide as claimed in claim 3 to a subject in need thereof.