METHODS FOR USING ANTIBODIES AND ANALOGS THEREOF

Abstract

Camelid and shark single-domain heavy chain antibodies lacking the light chains, and their analogs are disclosed. Methods for using the antibodies and their analogs to detect antigens are disclosed. Methods to derivatize such antibodies and analogs to develop diagnostic assays are described. Also provided are kits, and methods of using such diagnostics assays.

Claims

1. A method for detecting the presence or absence of an antigen in a sample comprising: a) obtaining a sample suspected of having said antigen, b) detecting the presence or absence of said antigen in said sample utilizing a polypeptide, wherein said polypeptide comprises all or a portion of at least one variable antigen-binding (Vab) domain of camelid and/or shark single-domain heavy chain antibodies lacking light-chains, at least ten contiguous amino acids derived from a source other than camelid and/or shark single-domain heavy chain antibodies lacking light-chains, wherein said polypeptide comprises at least one binding site for an antigen, wherein said polypeptide binds specifically to said antigen, wherein said binding is indicative of the presence of said antigen.

2. The method of claim 1, wherein said polypeptide comprises at least two variable antigen-binding (Vab) domains of camelid and/or shark single-domain heavy-chain antibody lacking the light chains.

3. The method of claim 2, wherein said two variable antigen-binding (Vab) domains bind to two different antigens.

4. The method of claim 1, wherein said polypeptide has three or more variable antigen-binding (Vab) domains of camelid and/or shark single-domain heavy-chain antibody lacking the light chains.

5. The method of claim 1, wherein said polypeptide has improved cellular uptake, blood brain barrier permeability, biodistribution and retention.

6. The method of claim 1, wherein said polypeptide is immobilized on a solid support prior to binding to said antigen.

7. The method of claim 1, wherein said polypeptide binds to said antigen to form a complex, and wherein said complex is immobilized on a solid support.

8. The method of claim 1, wherein said polypeptide is linked to at least one entity other than an antibody.

9. The method of claim 8, wherein said entity is selected from a group consisting of solid support, radioisotope, enzyme, detectable label, ligand, fluorophore, biotin, digoxegenin, avidin, streptavidin, Fc region of IgGs, a therapeutic agent, toxin, hormone, peptide, protein, vector, siRNA, micro-RNA and nucleic acid.

10. The method of claim 8, wherein said solid support is selected from the group consisting of beads, biosensors, nanoparticles, microchannels, microarrays, and microfluidic devices, glass slides, glass chambers, and gold particles.

11. The method of claim 8, wherein said enzyme is selected from the group consisting of alkaline phosphatase (AP), horse-raddish-peroxidase (HRP), Luciferase, and beta-galactosidase.

12. The method of claim 1, wherein said polypeptide is selected from the group consisting of structures 5, 5 a, 7, 7 a, 14, 14 a, 15, 15 a, 19, 20, 31, 32, 33.

13. The method of claim 1, wherein said antigen is selected from the group consisting of A42, Tau, ABAD (Abeta-binding alcohol dehydrogenase), a mitochondria regulating protein (MRP), Cyclophilin-D (Cyp-D: MRP), TOM (Translocase of Outer Mitochondria Membrane: MRP), hPReP (Human Presequence Protease: MRP), NMDAR (MRP), PtDS (MRP), mSOD1 (MRP), mHTT (MRP), ApoE4 (Demyelination Regulating Protein: dMRP), integrin-41 (dMRP), integrin-47 (dMRP), PPAR-gamma (dMRP), MAdCAM-1 (dMRP), -synuclein, TDP-43 (TAR-DNA binding protein-43), ubiquitin, APP, ALZAS, gamma secretase, BACE (-secretase), Apo-A1, Apo-H, PV-1, PEDF, BDNF, Cystatin C, VGF nerve growth factor inducible, APO-E, GSK-3 binding protein, TEM1, PGD2, EGFR, EGFRT790M, Notch-4, ALDH-1, ESR-1, HER-2/neu, P53, RAS, KLKB1, SMAD4, Smad7, TNF-alfa, HPV, tPA, Mucin, Cadherin-2, FcRn alpha chain, TNF-alfa, Thrombin, cytokerratin 1-20, Celuloplasmin, Apo AII, VGF, Vif, LEDGF/p75, TS101, gp120, CCR5, CXCR4, HIV protease, HIV integrase, OST-577, H1N1, CD3, CD11a, CD20, CD33, CD25, CD52, Protein C5, VEGF, alfa-4-integrin, EPCA2, PSMA, PSA, TMPRSS2-ERG, PCA3, HAAH, AMACR, Glycoprotein IIb/IIIA, AP-1, VEGF-A, IgG-E, Bacillus anthracis protein, NadD (Nicotinate Mononucleotide Adenyltransferase, a enzyme implicated in drug-resistant bacteria), Plasmodium falciparum, STDs, TB, cGMP directed phosphodiestrase, chain B of Clostridium botulinum neurotroxin type E protein, Borrelia VlsE protein, ACE2 receptor, TTHY, AIAT, AFMN, APOE, SFRS4, SAMP, CD 71, GPA, epsilon- and gamma-glycophorins, TIMP-1, RGIA, EXTL3, biomarkers for: lung cancer, bladder cancer, gastric cancer, brain cancer, breast cancer, prostate cancer, cervical cancer, colorectal cancer, oral cancer, leukemia, childhood neuroblastoma, Non-Hodgkin lymphoma, Alzheimer's disease, Parkinson's disease, and AIDS.

14. A method for diagnosing an individual with a disease, said method comprising: a) obtaining a sample from said individual b) detecting the presence or absence of one or more biomarkers associated with said disease, wherein said detection comprises utilizing a polypeptide, wherein said polypeptide comprises all or a portion of at least one variable antigen-binding (Vab) domain of camelid and/or shark single-domain heavy chain antibodies lacking light-chains, at least ten contiguous amino acids derived from a source other than camelid and/or shark single-domain heavy chain antibodies lacking light-chains, wherein said polypeptide binds specifically to at least one of said biomarkers; and said binding of said polypeptide to one or more of said biomarkers is indicative of the presence of said one or more biomarkers in said sample, c) identifying said individual as having said disease when said one or more biomarkers are present in said individual's sample.

15. The method of claim 14 further comprising determining the amount of said biomarker in said sample and comparing said amount to a reference value, wherein an amount higher than said reference value is indicative of a disease.

16. The method of claim 14, wherein, the said polypeptide is capable of binding specifically to a biomarker selected from the group consisting of: biomarkers associated with Alzheimer's Disease, wherein said biomarkers for Alzheimer's disease is selected from the group consisting of Amyloid-beta (A), ALZAS, Tau, Cyclophilinp-D, ABAD, TOM, hPReP, PtDS, PLSCR1, mSOD1, mHTT, integrin-41, integrin-47, PPAR-gamma, MAdCAM-1, NMDAR, integrin-DJ-1, Bax-1, PEDF, HPX, Cystatin-C, Beta-2-Microglobulin, BDNF, Tau-Kinase, gamma-Secretase, beta-Secretase, Apo-E4, and VGF-Peptide; biomarkers associated with Parkinson's Disease, wherein said biomarkers for Parkinson's disease is selected from the group consisting of Apo-H, Cerulopasmin, Chromogranin-B, VDBP, Apo-E, Apo-AII, and alfa-Synuclein; biomarkers associated with Brain Cancer, wherein said biomarkers for Brain cancer is selected from the group consisting of TEM1, Plasmalemmal Vesicle (PV-1), Prostaglandin D Synthetase, and (PGD-S); biomarkers associated with HIV/AIDS, wherein said biomarkers for HIV/AIDS is selected from the group consisting of gp120, Vif, LEDGF/p75, TS101, HIV-Integrase, HIV-Reverse Transcriptase, HIV-Protease, CCR5, and CXCR4; biomarkers associated with Lung Cancer, wherein said biomarkers for lung cancer is selected from the group consisting of KRAS, Ki67, EGFR, KLKB1, EpCAM, CYFRA21-1, tPA, ProGRP, Neuron-specific Enolase (NSE), and hnRNP; biomarkers associated with Prostate Cancer, wherein said biomarkers for prostate cancer is selected from the group consisting of AMACR, PCA3, TMPRSS2-ERG, HEPSIN, B7-H3, SSeCKs, EPCA-2, PSMA, BAG-1, PSA, MUC6, hK2, PCA-1, PCNA, RKIP, and c-HGK; biomarkers associated with Breast Cancer, wherein said biomarkers for breast cancer is selected from the group consisting of EGFR, EGFRT790M, HER-2, Notch-4, ALDH-1, ESR1, SBEM, HSP70, hK-10, MSA, p53, MMP-2, PTEN, Pepsinigen-C, Sigma-S, Topo-11-alfauKPA, BRCA-1, BRCA-2, SCGB2A1, and SCGB1D2; biomarkers associated with Colorectal Cancer, wherein said biomarkers for colorectal cancer is selected from the group consisting of SMAD4, EGFR, KRAS, p53, TS, MSI-H, REGIA, EXTL3, p1K3CA, VEGF, HAAH, EpCAM, TEM8, TK1, STAT-3, SMAD-7, beta-Catenin, CK20, MMP-1, MMP-2, MMP-7,9,11, and VEGF-D; biomarkers associated with Ovarian Cancer, wherein said biomarkers for ovarian cancer is selected from the group consisting of CD24, CD34, EpCAM, hK8, 10, 13, CKB, Cathesin B, M-CAM, c-ETS1, and EMMPRIN; biomarkers associated with Cervical Cancer, wherein said biomarkers for cervical cancer is selected from the group consisting of HPV, CD34, ERCC1, Beta-CF, Id-1, UGF, SCC, p16, p21WAF1, PP-4, and TPS; biomarkers associated with Bladder Cancer is selected from the group consisting of CK18, CK20, BLCa-1, BLCA-4, CYFRA21-1, TFT, BTA, Survivin, UCA1, UPII, FAS, and DD23; biomarkers associated with a disease causing bacteria, wherein said bacteria or biomarker associated with disease causing bacteria is selected from the group consisting of Clostridium Botulinum, Bacillus Anthracis, Salmonella Typhi, Treponema Pallidum, Plasmodinum, Chlamadyia, Borrelia B, Staphyloccus Aureus, Tetanus, Meningococcal Meningitis, and Mycobacterium tuberculosis, and Nicitinate Mononuceleotide adenyltransferase (NadD); biomarkers associated with a disease causing virus, wherein said virus or biomarker associated with a disease causing virus is selected from the group consisting of Pandemic Flu Virus H1N1 strain, Influenza virus H5N1 strain, Hepatitis B virus (HBV) antigen OSt-577, HBV core antigen HBcAg (HBV), HBV antigen Wnt-1, Hepatitis C Virus (HCV) antigen Wnt-1, and HCV RNA.

17. A method for detecting the presence or absence of circulating cells in a sample comprising a) obtaining a sample suspected of having circulating cells, b) detecting the level of one or more antigen associated with said circulating cell in said sample utilizing a polypeptide, wherein said polypeptide comprises all or a portion of at least one variable antigen-binding (Vab) domain of camelid and/or shark single-domain heavy chain antibodies lacking light-chains, at least ten contiguous amino acids derived from a source other than camelid and/or shark single-domain heavy chain antibodies lacking light-chains, wherein said polypeptide binds specifically to said one or more antigens, and wherein said binding of said polypeptide to said antigens is indicative of the presence of circulating cells in said sample.

18. The method of claim 17, wherein said circulating cells are circulating tumor cells.

19. The method of claim 18, wherein one or more antigens associated with tumor cell is selected from the group consisting of MUC-1, VCAM-1, EpCAm-1, CD44, CD133, E-Cadherin, VEGF, bFGF, sFASL, CD95, p53, Bc1-2 CyclinD1, Cyclin E, TNF-alfa, TGF-beta1, Her-2, EGFR, IGF-1 and IGF-1R, 1L-2R, Ras, and cMyc.

20. The method of claim 17, wherein said circulating cells are circulating fetal cells.

21. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0085] FIG. 1 shows structural differences between camel, shark, and mouse immunoglobulins (IgGs). The notations CH1, CH2, CH3, CH4, CH5 represent constant domain 2, 3, 4 of single domain heavy chain antibody of the respective species. The notations Vab and VNAR represent variable domain of camelid and shark single domain heavy chain antibodies respectively.

[0086] FIG. 2 shows the structure of exemplary analogs of camelid single-domain antibodies without the light-chains: mini-antibody 1 and its analogs 1a; micro-antibody 4 and its analogs 4a; sub-nano-antibody 5 and its analogs 5a; nano-antibody 6 and its analogs 6a; dimeric nano-antibody 7 and its analogs 7a; trimeri-nanoantibody 8 and its analogs; and tetrameric nano-antibody 9 and its analogs 9a. The notation Rn represents all or portion of the hinge region of camelid or shark single domain antibodies. CHX represents segment of human IgG CH1 domain or CH2 domain of camelid antibody. S stands for a spacer or linker. L is a ligand.

[0087] FIG. 3 shows the structure of exemplary analogs of shark single-domain antibodies without the light-chains: Hark IgNAR 2 and its analogs 2a; shark mini-antibody 11 and its analogs 11a; shark micro-antibody 12 and its analogs 12a; shark sub-nano-antibody 13 and its analogs 13a; shark dimeric nano-antibody 14 and its analogs 14a; and shark tetrameric nano-antibody 15 and 15a. The notation Rn represents all or portion of the hinge region of shark single domain antibodies. CHX represents segment of human IgG or CH1 domain of shark antibody. S stands for a spacer or linker. L is a ligand.

[0088] FIG. 4 shows the steps involved in the chemical synthesis of exemplary analogs represented by structures 1a and 4a, respectively, of camelid mini-antibody 1 and micro-antibody 4. The notation Rn represents all or portion of the hinge region of camelid or shark single domain antibodies.

[0089] FIG. 5 shows the steps involved in the chemical transformation of exemplary sub-nano-antibody 5 into its analogs represented by structure 5a, and the synthesis of dimeric camelid nano-antibody 7 and its analogs represented by generic structure 7a.

[0090] FIG. 6 shows the steps involved in the transformation of exemplary camelid dimeric nano-antibody 7 into trimeric and tetrameric nano-antibodies. The notation Rn represents all or portion of the hinge region of camelid or shark single domain antibodies.

[0091] FIG. 7 shows the steps involved in the cloning and expression of exemplary shark IgNAR 2, exemplary shark micro-antibody 12, exemplary shark sub-nano-antibody 13 and shark-nano-antibody 30. The notation Rn represents all or portion of the hinge region of camelid or shark single domain antibodies.

[0092] FIG. 8 shows the steps involved in the chemical synthesis of exemplary analogs of shark antibodies without the light chains: Shark IgNAR analogs represented by structure 2a; shark mini-antibody analogs represented by structure 11a; shark micro-antibody analogs represented by structure 12a; shark sub-nano-antibody analogs represented by 13a; shark dimeric nano-antibody analogs represented by 14a; and shark tetrameric-nano-antibody analogs represented by 15a. The notation Rn represents all or portion of the hinge region of camelid or shark single domain antibodies.

[0093] FIG. 9 shows the steps involved in the chemical synthesis of exemplary shark dimeric nano-antibody 14 and its conversion into exemplary shark trimeric and tetrameric nano-antibodies 32 and 31.

[0094] FIG. 10 shows the steps involved in the immobilization of exemplary single-domain camelid and shark antibodies deprived of light chains having the structures 1, 2, 4, 5, 6, 7, 8, 9, 11, 12,13, 14, 15, 19, 20, 31, and 32.

[0095] FIG. 11 shows an exemplary scheme of capturing and detecting antigens/biomarkers associated with a disease using camelid and shark heavy chain only antibodies and their analogs.

[0096] FIG. 12 shows an exemplary scheme of capturing and detecting antigens/biomarkers associated with a disease using immobilized shark single-domain IgNAR and their analogs.

[0097] FIG. 13 shows an exemplary scheme of capturing and detecting <200 copies of antigens/biomarkers associated with a disease using camelid and shark heavy chain only antibodies and their analogs using immuno-PCR.

[0098] FIG. 14 shows an exemplary scheme of capturing and detecting circulating tumor cells from bodily fluid using camelid and shark antibodies.

[0099] FIG. 15 shows an exemplary scheme of detecting prenatal genetic disorder using captured circulating fetal cells using camelid and shark heavy chain only antibodies and their analogs.

[0100] FIG. 16 shows an exemplary scheme of detecting chromosomal translocation using captured circulating tumor cells using camelid and shark heavy chain only antibodies and their analogs.

[0101] FIG. 17 shows an exemplary nucleic acid sequence encoding human Cyclophilin D.

[0102] FIG. 18 shows an exemplary nucleic acid sequence encoding alpha beta binding Mitochondrial Alcohol Dehydrogenase (ABAD).

[0103] FIG. 19 shows an exemplary nucleic acid sequence encoding Translocase of the Outer Membrane (TOM).

[0104] FIG. 20 shows an exemplary nucleic acid sequence encoding Prosequence Protease (hPreP).

[0105] FIG. 21 shows an exemplary nucleic acid sequence encoding Homo sapiens integrin beta 1.

[0106] FIG. 22 shows an exemplary nucleic acid sequence encoding Homo sapiens mucosal vascular addressin cell adhesion molecule 1 (MADCAM1).

[0107] FIG. 23 shows an exemplary nucleic acid sequence encoding Cu/Zn-superoxide dismutase (mSOD1).

[0108] FIG. 24 shows an exemplary nucleic acid sequence encoding Mus musculus mRNA for MPTPdelta.

[0109] FIG. 25 shows an exemplary nucleic acid sequence encoding Homo sapiens huntingtin (HTT).

[0110] FIG. 26 shows an exemplary nucleic acid sequence encoding N-Methyl-D-Aspartate Receptor (NMDAR).

[0111] FIG. 27 shows an exemplary nucleic acid sequence encoding Phosphatidylserine Synthase (PTDS).

DETAILED DESCRIPTION OF THE INVENTION

[0112] The present invention discloses the use of camelid and/or shark single-domain heavy-chain only antibodies and their analogs for ultrasensitive detection of antigens. The method is useful for diagnosing human diseases at an early stage of their manifestation, when the concentration of antigens associated with such diseases is very low for example, 200 or fewer molecules in 0.1 ml of the bodily fluid. The invention also teaches methods for the development of nano-biomedical technology platforms utilizing camelid and/or shark heavy-chain only antibodies and their analogs for in-vitro diagnosis of human and animal diseases with such antibodies.

Camelid and Shark Antibodies

[0113] The hetero-tetrameric structure of antibodies exists in humans and most animals but the single-domain heavy-chain only dimeric structure, without the light-chains, is considered characteristic of camelids and sharks [Nature Biotechnology, 23, 1126 (2005)]. These antibodies are relatively simple molecules but with unique characteristics. Their size is about rd the size of traditional antibodies, hence a lower molecular weight (about 90 KDa), with similar antigen binding affinity, but with water solubility 100 to 1000 folds higher than the conventional antibodies. Because of the lower molecular weight, the authors of this application call these antibodies as Single-domain Mini-antibodies (sdMnAbs) or simply Mini-Antibodies (MnAbs).

[0114] Another characteristic of the single-domain antibodies derived from sharks and camelids is that they have very high thermal stability compared to the conventional mAbs. For example, camel antibodies can maintain their antigen binding ability even at 90 C. [Biochim. Biophys. Acta., 141, 7 (1999)]. Furthermore, complementary determining region 3 (CDR3) of camel Vab region is longer, comprising of 16-21 amino acids, than the CDR3 of mouse VH region comprising only of 9 amino acids [Protein Engineering, 7, 1129 (1994)]. The larger length of CDR3 of camel Vab region is responsible for higher diversity of antibody repertoire of camel antibodies.

[0115] In addition to being devoid of light chains, the camel heavy-chain antibodies also lack the first domain of the constant region called CH1, though the shark antibodies do have CH1 domain and two additional constant domains CH4 and CH5 [Nature Biotech. 23, 1126 (2005)]. Furthermore, the hinge regions of camel and shark antibodies have an amino acid sequence different from that of normal heterotetrameric conventional antibodies [(S. Muyldermans, Reviews in Mol. Biotech., 74, 277 (2001)]. Without the light chain, these antibodies bind to their antigens by the variable antigen-binding domain of the heavy-chain immunoglobulin, which is referred to as Vab by the authors of this application (VHH in the literature), to distinguish it from the variable domain VH of the conventional antibodies. The single-domain Vab is amazingly stable by itself without having to be attached to the parent antibody. This smallest intact and independently functional antigen-binding fragment Vab, with a molecular weight of 12-15 KDa, is referred to as nano-antibody by the authors of this application. In the literature, it is known as nanobody [(S. Muyldermans, Reviews in Mol. Biotech., 74, 277 (2001)].

[0116] The genes encoding these full length single-domain heavy-chain antibodies and antibody-antigen binding fragment Vab (camel and shark) can be cloned in phage display vectors, and selection of antigen binders by panning and expression of selected VHH in bacteria offer a very good alternative procedure to produce these antibodies on a large scale. Also, only one domain has to be cloned and expressed to produce in vivo an intact, matured antigen-binding fragment.

[0117] There are structural differences between the variable regions of single domain antibodies and conventional antibodies. Conventional antibodies have three constant domains while camel has two and shark has five constant domains. The largest structural difference is, however, found between a VH (conventional antibodies) and Vab (heavy-chain only antibodies of camel and shark) (see below) at the hypervariable regions. Camelid Vab and shark V-NAR domains each display surface loops which are larger than for conventional murine and human IgGs, and are able to penetrate cavities in target antigens, such as enzyme active sites and canyons in viral and infectious disease biomarkers [PNAS USA., 101, 12444 (2004); Proteins, 55, 187 (2005)]. In human and mouse, the VH loops are folded in a limited number of canonical structures. In contrast, the antigen binding loop of Vab possess many deviations of these canonical structures that specifically bind into such active sites, therefore, represent powerful tool to modulate biological activities [(K. Decanniere et al., Structure, 7, 361 (2000)]. The high incidence of amino acid insertions or deletions, in or adjacent to first and second antigen-binding loops of Vab will undoubtedly diversify, even further, the possible antigen-binding loop conformations.

[0118] Though there are structural differences between camel and shark parent heavy-chain antibodies (FIG. 1), the antigen-antibody binding domains, Vab and V-NAR, respectively, have similar binding characteristics. The chemical and/or protease digestion of camel and shark antibodies results in Vab and V-NAR domains, with similar binding affinities to the target antigens [Nature Biotechnology, 23, 1126 (2005)].

[0119] Other structural differences are due to the hydrophilic amino acid residues which are scattered throughout the primary structure of Vab domain. These amino acid substitutions are, for example, Leu45 to R (arginine) or Leu45 to C (cysteine); Va137 to Y (Tyr); G44 to E(Glu), and W47(Trp) to G (Gly). Therefore, the solubility of Vab is much higher than the Fab fragment of conventional mouse and human antibodies.

[0120] Another characteristic feature of the structure of camelid Vab and shark V-NAR is that it often contains a cysteine residue in the CDR3 in addition to cysteines that normally exist at positions 22 and 92 of the variable region. The cysteine residues in CDR3 form SS bonds with other cysteines in the vicinity of CDR1 or CDR2 [Protein Engineering, 7 1129 (1994)]. CDR1 and CDR2 are determined by the germline V gene. They play important roles together with CDR3 in antigenic binding [Nature Structural Biol., 9, 803 (1996); J. Mol. Biol., 311, 123 (2001)]. Like camel CDR3, shark also has elongated CDR3 regions comprising of 16-27 amino acids residues [Eur. J. Immunol., 35, 936 (2005)].

[0121] The germlines of dromedaries and llamas are classified according to the length of CDR2 and cysteine positions in the V region [Nguyen et al., EMBO J., 19, 921 (2000); Harmsen et al., Mol. Immun., 37, 579 (2000)].

[0122] Immunization of camels with enzymes generates heavy-chain antibodies (HCAb) significant proportions of which are known to act as competitive enzyme inhibitors that interact with the cavity of the active site [(M. Lauwereys et al., EMBO, J. 17, 3512 (1998)]. In contrast, the conventional antibodies that are competitive enzyme inhibitors cannot bind into large cavities on the antigen surface. Camel antibodies, therefore, recognize unique epitopes that are out of reach for conventional antibodies.

[0123] Production of inhibitory recombinant Vab that bind specifically into cavities on the surface of variety of enzymes, namely, lysozyme, carbonic anhydrase, alfa-amylase, and beta-lactamase has been achieved [M. Lauwereys, et al., EMBO, J. 17, 3512 (1998)]. Hepatitis C protease inhibitor from the camelised human VH has been isolated against an 11 amino acid sequence of the viral protease [F. Martin et al., Prot. Eng., 10, 607 (1997)].

Novel Analogs of Single-Domain Heavy-Chain Camelid and Shark Antibodies:

[0124] FIGS. 2 and 3 outlines the analogs of new generation of camelid and shark antibodies and their analogs, respectively, which will assist us to develop ultrasensitive and ultraspecific diagnostic assays for the detection/identification of the pathological proteins and antigens.

Production of Parent Single-Domain Heavy-Chain Mini-Antibodies (sdmnAbs) of Structure 1

[0125] Host animals such as camel, llama, or alpaca will be immunized with the desired antigen(s), for example HER-2 protein, a biomarker for breast cancer or A42 antigenic peptide for detecting amyloid plaque, following the procedures described by Murphy et al, in 1989 [Am. J. Vet. Res., 50, 1279 (1989)], but with slight modification. Immunization of camels will be done with 250 ug antigenic peptide per injection will be used, followed by 4 booster shots every two weeks 4 weeks after the initial injection. For baby sharks, 10 ug antigen/injection will be used. One antigen per animal for immunization will be used, though it may be feasible to immunize an animal simultaneously with multiple antigens to raise an immune response to each antigen separately, which can make the production cost effective [EMBO, J., 17, 3512 (1998); J. Immunol. Methods, 240,185 (2000)].

[0126] After immunization, 100 ml camel blood (or 5 ml from shark) will be withdrawn from the animal and the total IgGs will be precipitated out using ammonium sulfate precipitation procedure. Using size exclusion chromatography over Sephadex G-25, the conventional IgGs, MW 150 KDa, will be removed from the single-domain mini-IgG, 1, with MW of 90 to 100 K Da. Affinity purification to obtain high affinity sdmnAb, I, will be done by magnetic beads coated with the antigenic peptide.

Recombinant Production of Camelid and Shark Single-Domain Antibodies:

[0127] Recombinant production of single-domain heavy-chain parent camelid antibodies 1, 4, 5, 6, 7, 8, 9 (FIG. 2) and shark antibodies 2, 11, 12, 13, 14, and 15 will be done according to protocols and procedures described in pending U.S. patent application Ser. No. 12/563,330.

Chemical Synthesis of Analogs of Single-Domain Camel Antibodies

Derivatization and Immobilization of Camelid Mini-Antibody 1:

[0128] Schematics for derivatization and immobilization of mini-antibodies 1 are shown in FIG. 4. Mini-antibody 1 (1 mg, 11 nmols) will be treated with commercial NHS-(PEG)-Mal (11 ul of 10 mM stock=110 nanomoles) wherein n=1-50, in 50 mM MOPS/150 mM NaCl, pH 6.8, at RT for 1 hour to obtain the pegylated conjugate of FIG. 4 (structure not shown) which will be desalted by dialysis on C-3 Amicon filters to remove excess NHS-PEG-Mal reagent.

[0129] While the pegylation is underway, the ligand will be treated with 10 folds of Traut's Reagent in MOPS buffer, pH 6.8, containing 5% EDTA, at RT for 1-2 hours. The thiolated ligand will then be purified either by dialysis (if ligands is chemical or biochemical entity) or by washing with MOPS buffer if ligand is a solid matrix.

[0130] The pegylated intermediate will be immediately conjugated with 10-20 folds excess of thiolated ligand: SH-L in MOPS buffer, pH 6.8 buffer containing 5 mM EDTA for 2-3 hours at room temperature (RT); where L may be enzyme (HRP, AP, Luciferase, galactosidase), protein, peptide, biotin, fluorophore, DNA, RNA, and solid matrix such as, magnetic beads, glass slides, gold nanoparticles, microchannels, microfluidic device.

[0131] When the ligand is a chemical or biochemical entity, for example, fluorophore, biotin, enzyme, protein, etc, the purification of the conjugate will be done by reverse-phase C8 HPLC.

[0132] Nucleic acid conjugates of mini- and nano-antibodies 1-15a will also be prepared using their pegylated conjugates followed by treatment with the thiolated-DNA/RNA molecules of interest (FIG. 4).

[0133] When the ligand is a solid matrix, such as, magnetic beads, glass slide, microchannels, etc., which we will use to immobilize the camelid antibodies, all we need to do is to wash the excess reagent with the appropriate buffer.

[0134] The activity and the amount of camelid antibody loaded onto the solid matrix will be determined by ELISA and commercially available protein assay kits.

Single-Domain Heavy-Chain Camelid Micro-Antibody 4 and its Analogs 4a:

[0135] Micro-antibody, 4, will be prepared by treating mini-antibody 1 (2 mg) with 1.0 ml of 10 mM TCEP (tris-carboxyethyl-phosphine) in 20 mM Phosphate/150 mM NaCl, pH7.4 at room temperature (RT) for one hour. The resulting micro-antibody 4 will be desalted on centricon-3 to remove the excess reagent and the buffer and stored at 4 C. in 1PBS.

[0136] Derivatization of 4 into 4a will be accomplished by the method described above for conversion of 1 into 1a.

Single-Domain Heavy-Chain Camelid Sub-Nano-Antibody 5 and its Analogs of Structure 5a:

[0137] Micro-antibody 4 will be treated with trypsin or pepsin under controlled conditions at RT to cleave the CH2-CH3 domains from the antibody. After deactivation of the proteolytic enzyme with fetal calf serum, the subnano-antibody 5 will be isolated using size exclusion chromatography.

[0138] Derivatization of 5 into 5a will be accomplished by the method described above for conversion of 1 into 1a.

Single-Domain Heavy-Chain Camelid Nano-Antibody 6 and its Analogs of Structure 6a:

[0139] Sub-nano-antibody 5 will be treated with pepsin at a low pH of 4.5 in 2M sodium acetate buffer under mild conditions for 1-8 hours to cleave the CH2-CH3 domains from the antibody. After deactivation of the proteolytic enzyme with fetal calf serum, the nano-antibody 6 will be isolated using size exclusion chromatography.

[0140] Derivatization of 6 into 6a will be accomplished by the method described above for conversion of 1 into 1a.

Single-Domain Heavy-Chains Bivalent Nano-antibody 7 and its Analogs of Structure 7a:

[0141] Schematics for the chemical synthesis of dimeric nano-antibodies and heir analogs are displayed in FIG. 5. Nano-antibody 5 will first be oxidized with 1% iodine in 20% tetrahydrofuran/70% water/10% pyridine for 5-10 minutes to transform into the dimeric nano-antibody 7. Treatment of 7 will be done with commercial NHS-(PEG).sub.n-Mal, wherein n=1-50, in 50 mM MOPS/150 mM NaCl, pH 6.8, at RT for 1 hour to obtain the pegylated intermediate with maleimido group (structure not shown in FIG. 5) which, after purification by dialysis on C-3 Amicon filters, will be immediately conjugated with thiolated-ligand in MOP buffer containing 5% EDTA at pH6.8 for 2-3 hours at RT to obtain, after purification, 7a. The dimeric conjugate 7a will then be characterized by ELISA and Western blot assays.

Trivalent and Tetravalent Camelid Nano-Antibodies and Analogs:

[0142] Schematics for the chemical synthesis of trivalent and tetravalent camelid nano-antibodies without the light-chains, and their analogs are shown in FIG. 6.

Protocol for Developing Trivalent 8 and Tetravalent 9 Camelid Nano-Antibodies:

[0143] Bivalent nano-antibody, 7, prepared by oxidative dimerization or chemical ligation, will be conjugated with NHS-(PEG)3-Mal (10 folds excess) in MOPS buffer at pH 7.0 for 1 hour at RT. Chemical ligation of the resulting monomeric and dimeric pegylated products 16 and 17 with the thiolated nano-antibody 18 (FIG. 6) will be carried out by combining the two at pH 6.8 buffer containing 5 mM EDTA and allowing the reaction to occur at RT for at least 2 hours. The so formed trivalent, 19, and tetravalent nano-antibody 20 will be purified by size exclusion chromatography and stored at 4 C. in PBS containing 0.02% NaN3.

[0144] The attachment of a ligand to 19 and 20 can be readily done by making use of the lysine(s) of the hinge region to conjugate with the NHS-L.

[0145] Pentavalent and higher analogs of nano-antibodies (Vab domains of camel antibodies) can be similarly prepared.

Production of Single-Domain Heavy-Chain Shark IgNAR (Structure 2):

[0146] Immunization of Sharks and Isolation of Shark IgNAR: Baby sharks will be immunized with the desired antigen(s), for example ALZAS, Tau, A42 peptide which are the potential biomarkers for Alzheimer's disease, following the protocol described by Suran et al [J. Immunology, 99, 679 (1967)]. Briefly, the antigen (20 ug per kg animal weight), dissolved in 20 mg/ml keyhole limpet hemocyanin (KLH) supplemented with 4 mg/ml complete Freund's adjuvant, will be injected intramuscularly. Four booster shots every two weeks four weeks after the initial injection will be administered.

[0147] After immunization, 3-5 ml shark blood will be withdrawn from the animal and the total IgGs will be precipitated out using 50% ammonium sulfate, followed by centrifugation at 2000 RPM for 10 minutes. After discarding supernatant, the precipitate will be dissolved in 20 mM PBS/150 mM NaCl containing 0.02% sodium azide and size fractionated on Sephadex G200. The conventional IgGs, MW 230 KDa, will be separated out from the shark IgNAR with MW of 180 K Da. Alternatively, the conventional IgG fraction will first be depleted with protein G bound to magnetic beads, followed by isolation of V-NAR protein with magnetic beads coated with protein-A. Affinity purification to obtain high affinity shark Ig-NAR, 2, will be done by magnetic beads coated with antigenic peptide.

[0148] After determining the amino acid sequence of IgNAR, 2, nucleic acid sequence will be derived based from the amino acid sequence and recombinant DNA protocols will be established to produce the shark single-domain antibody 2 on a large scale. Schematics for cloning and expression of IgNAR are shown in FIG. 7.

Isolation of RNA from Immunized Shark's Lymphocytes and Cloning:

[0149] Isolation of total RNA, 21, from immunized sharks will be done from 3-5 ml of shark blood using commercially available RNA extraction kits such as Bio-Rad's AquaPure RNA Isolation kit. Reverse transcription using oligo-dT primer will be achieved by PCR using high fidelity DNA polymerase to obtain the IgNAR cDNA, 22, shown in FIG. 7.

Recombinant Production of Shark Heavy Chain Only Antibodies and Their Analogs

[0150] An exemplary cloning strategy is shown in FIG. 7. Amplicons for IgNAR cDNA, 22 and its analogs will be performed using the following protocol:

IgNAR cDNA=1.0 ug
Primers Mix=10 pmol (forward and reverse primers)
1 mM dTNPs=10 ul

10 mM MgCl2=5 ul

10PCR Buffer=5 ul

Taq DNA Polymerase=0.6 ul

Water to =50 ul

[0151] After first denaturation round of 94 C. for 10 minutes, 35 to 36 cycles of amplification will be performed under conditions as described below:

Denaturation: 20 seconds at 94 C.

Annealing: 30 Seconds at 56 C.

[0152] Extension: 50 seconds at 72 C.
Final Extension: 10 min, 72 C.

[0153] All or portions of IgNAR cDNA using different combinations of the following forward and reverse primers.

TABLE-US-00002 Forwardprimers (SEQIDNO:1) 5-gcatgggtagaccaaacaccaag-3 (SEQIDNO:2) 5-gcgtcctcagagagagtcccta-3 (SEQIDNO:3) 5-gagacggacgaatcactgaccatc-3 (SEQIDNO:4) 5-gggtagaccaaacaccaagaacagc-3 Reverseprimers (SEQIDNO:5) 5-gttctagccaataggaacgtatag-3 (SEQIDNO:6) 5-gtttgcacaagagagtagtctttac-3 (SEQIDNO:7) 5-cctaaattgtcacagcgaatcatg-3 (SEQIDNO:8) 5-gtgcagttccctagaagtcttg-3

[0154] After amplification, the amplicon will be purified on 1.5% agarose. The amplicon will be extracted from the gel and its 5-end kinased with gamma-ATP for blunt-end ligation with the phage-display vector using T4 DNA-ligase following standard ligation protocols.

[0155] Library or Plasmid Construction: Prior to cloning, the PCR amplicon encoding IgNAR gene will be digested with Sfi1 and Not1 (Roche) following the cocktail:

V-NAR-CH1-CH2-CH3-CH4-CH5DNA=5ug

10 Restriction Buffer 5 ul

Sfi1 (10 U/ul) 8 ul

Water to 50 ul

[0156] Incubate 50 C. for 8 hour

Not1 35 U

Reaction Buffer 4.5

Water to 60 ul

[0157] Incubate at 37 C. for 4-5 hours.

Ethanol Precipitate at 70 C. Pellet

Water to 50 ul

[0158] Agarose gel (1.5%) purification

Pure DNA Encoding Shark IgNAR Antibody

Vector Ligation:

IgNAR DNA=200 ng

Vector DNA=1000 ng

10 Ligase Buffer=5 ul

T4 DNA Ligase=10 U

Water to 50 ul

[0159] Incubate 15 hours at 4 C.

Ethanol Precipitate at 70 C.

[0160] Suspend pellet in 10 ul.

Electroporation:

[0161] 250 ul of E. Coli TG1 cells will be made electrocompetent with BRL Cell-Porator following vendor protocol.

[0162] Panning of Phage-Displayed IgNAR-Antibody 2 Library: Electroporated TG1 cells will be transfected with the phagemid-IgNAR DNA insert. Approximately, 1010 cells will be grown to mid-logarithmic phase before injection with M13K07 helper phages. Virions will be prepared as described in the literature [Andris-Widhopf J., et al, J. Immunology Methods, 242, 159 (2002)] and used for panning at a titer of 1013/ml. Specific IgNAR antibody against the antigenic peptide will be enriched by five consecutive rounds of panning using magnetic beads conjugated with antigenic peptide. Bound phage particles will be eluted with 100 mM TEA (pH 10.00), and immediately neutralized with 1M Tris.HCl (pH 7.2) and will be used to reinfect exponentially growing E. Coli TG1 cells.

[0163] The enrichment of phage particles carrying antigen-specific IgNAR antibody will be assessed by ELISA before and after five rounds of panning. After the fifth panning, individual colonies will be picked up to analyze the presence of the virion binding by anti-M13-HRP conjugate.

Expression and Purification of the Single-Domain IgNAR 2:

[0164] The selected positive clones will be used to infect a new bacterial strain, HB 2151, a non-suppressor strain that recognizes the amber codon as a stop codon for soluble protein production. The HB2151 cell harboring the recombinant phagemids will be grown at 28 C. in 250 ml 2YT-ampicillin, 1% glucose in culture flasks until OD600 0.7. The cells will be washed and resuspended in 250 ml 2YT-ampicillin, supplemented with 1 mM isopropyl beta D-thiogalactopyranoside (IPTG), and incubated over night at 22 C. to induce protein expression.

[0165] Before adding IPTG to the cultures, a portion will be spotted on an LB/ampicillin plate for future analysis of the clones. The culture will be then be centrifuged at 4000 RPM for 15 minutes to pellet the bacterial cells. The culture supernatant will then be screened by ELISA for antigen-specific IgNAR protein 2.

Chemical Synthesis of Single-Domain Heavy-Chain Shark Only Antibodies and Their Analogs

[0166] 2a, 11, 11a, 12, 12a, 13, 13a, 14, 14a, 15, 15a:

Derivatization of Shark IgNAR 2 to Obtain Analogs of Structure 2a:

[0167] Schematics for derivatization of shark IgNAR 2 are shown in FIG. 8. Shark antibody 2 (1 mg, 6 nmols) will be treated with commercial NHS-(PEG).sub.n-Mal (6 ul of 10 mM stock=60 nanomoles) wherein n=1-50, in 50 mM MOPS/150 mM NaCl, pH 6.8, at RT for 1 hour to obtain the pegylated conjugate (structure not shown) which will be desalted by dialysis on C-3 Amicon filters to remove excess NHS-PEG-Mal reagent.

[0168] While the pegylation is underway, the ligand will be treated with 10 folds of Traut's Reagent in MOPS buffer, pH 6.8, containing 5% EDTA, at RT for 1-2 hours. The thiolated ligand will then be purified either by dialysis (if ligands is a chemical or biochemical entity) or by washing with MOPS buffer if ligand is a solid matrix.

[0169] The pegylated intermediate will be immediately conjugated with 4-5 folds excess of thiolated ligand: SH-L in MOPS buffer, pH 6.8 buffer containing 5 mM EDTA for 2-3 hours at room temperature (RT); where L may be enzyme (HRP, AP, Luciferase, galactosidase), protein, peptide, biotin, fluorophore, DNA, RNA, and solid matrix such as, magnetic beads, glass slides, gold nanoparticles, microchannels, microfluidic device.

[0170] When the ligand is a chemical or biochemical entity, for example, fluorophore, biotin, enzyme, protein, etc, the purification of the conjugate 2a will be done by reverse-phase C8 HPLC.

[0171] Nucleic acid conjugates of shark IgNAR 2 and analogs 11,12,13,14, and 15 will also be prepared using their pegylated conjugates followed by treatment with the thiolated-DNA/RNA molecules of interest.

[0172] When the ligand is a solid matrix, such as, magnetic beads, glass slide, microchannels, etc., which we will use to immobilize the camelid antibodies, all we need to do is to wash the excess reagent with the appropriate buffer.

[0173] The activity and the amount of single-domain shark antibody loaded onto the solid matrix will be determined by ELISA and commercially available protein assay kits.

Single-domain Heavy-Chain Shark Mini-Antibody 11 and its Analogs 11a:

[0174] Mini-antibody, 11, will be prepared by treating the IgNAR 2 (2 mg) with 1.0 ml of 10 mM TCEP (tris-carboxyethyl-phosphine) in 20 mM Phosphate/150 mM NaCl, pH7.4 at room temperature (RT) for one hour. The resulting micro-antibody 11 will be desalted on centricon-3 to remove the excess reagent and the buffer and stored at 4 C. in 1PBS.

[0175] Derivatization of 11 into 11a will be accomplished by the method described above for conversion of 2 into 2a.

[0176] Single-Domain Heavy-Chain Shark Micro-antibody 12 and its Analogs of Structure 12a:

[0177] Mini-antibody 11 will be treated with trypsin or pepsin under controlled conditions at RT to cleave the CH3-CH4-CH5 domains from the antibody. After deactivation of the proteolytic enzyme with fetal calf serum, the shark micro-antibody 12 will be isolated using size exclusion chromatography.

[0178] Derivatization of 12 into 12a will be accomplished by the method described above for conversion of 2 into 2a.

Single-Domain Heavy-Chain Shark Sub-nano-antibody 13 and its Analogs of Structure 13a:

[0179] Micro-antibody 12 will be treated with trypsin or pepsin under controlled conditions at RT to cleave the CH2 domain from the antibody. After deactivation of the proteolytic enzyme with fetal calf serum, the shark sub-nano-antibody 13 will be isolated using size exclusion chromatography.

[0180] Derivatization of 13 into 13a will be accomplished by the method described above for conversion of 2 into 2a.

[0181] Single-Domain Heavy-Chain Shark Dimeric-Nano-antibody 14 and its Analogs of Structure 14a:

[0182] Dimeric V-NAR will be prepared by the oxidation of monomeric V-NAR, 13 to obtain 14 as described in FIG. 9. These protocols are general and do not need detailed explanation.

[0183] Likewise, the transformation of 14 into its analogs of structure 14a will be accomplished as described above for the preparation of 2a from 2.

[0184] Tetrameric and Trimeric V-NAR Nano-antibodies 31 and 32:

[0185] Dimeric V-NAR nano-antibody 14 will be treated with 4-5 molar equivalent of NHS-PEG-Mal to obtain a mixture of tri- and tetra-pegylated derivatives of dimeric V-NAR nano-antibody (FIG. 9).

[0186] After purification, the tri- and tetrameric pegylated products will be treated with thiolated V-NAR to obtain, after purification by HPLC or just by dialysis, tetrameric and trimeric V-NAR nano-antibodies 31 and 32.

Immobilization of Single-Domain Camelid Mini-Antibody and Shark IgNAR Antibody and Analogs onto Solid Matrixes:

[0187] Immobilization of single-domain heavy-chain only shark and camelid native antibodies and their analogs onto solid matrixes, such as gold particles, magnetic particles, microchannels, glass particles and other solid surfaces will be accomplished using the steps outlined in FIG. 10. Aminated solid matrix 33 will first be derivatized with NHS-(PEG)n-Mal, 10, where n=20 (20 fold molar excess) at pH 7.0 for 1 hour at RT. The solid matrix will then be washed thoroughly with the same buffer (50 mM MOPS/150 mM NaCl, pH 7.0). Any unconjugated amine groups will be masked with sulfoNHS-Acetate (Pierce) by incubated the solid matrix with 40 fold excess of the reagent at pH 7.0 for 60 minutes. After washing off the excess masking reagent, the pegylated matrix 34 will then be conjugated with thiolated single-domain heavy-chain antibody, 36, (10 excess) over the starting amine concentration. The conjugation will be performed at pH 6.5 for 2 hours at RT with gentle shaking of the matrix. The unused antibody will be recovered, and the matrix very well washed with 1PBS/0.5% Tween-20 to obtain complex 37 in which nano-antibody is covalently bound to a solid matrix. The activity of the bound heavy-chain antibody will be measured using ELISA.

Biomarkers for Various Diseases

[0188] Single-domain heavy-chain only shark and camelid native antibodies and their analogs can be used to detect the presence or absence of one or more antigens or can be used for diagnosis of one or more diseases. Single-domain heavy-chain only shark and camelid native antibodies and their analogs can bind specifically to the antigens or one or more biomarkers for various diseases. Exemplary sequences of various biomarkers or antigens are disclosed in U.S. application Ser. No. 12/563,330 filed Sep. 21, 2009. Those sequences are incorporated by reference to its entirety. Exemplary sequences of nucleic acids encoding additional biomarkers for Alzheimer's Disease are disclosed in FIG. 17-27.

Example 1

[0189] Capture and Detection of Pathogenic Antigens/Proteins Using Shark and Camel Single-Domain Antibodies (sdAbs)

[0190] Serum from patient blood (10 ml), collected in EDTA tubes will be treated with shark and camelid heavy chain only antibodies and their analogs coated magnetic beads for 1-2 hours on a rotator with gentle rotation to bind the antigen. The beads will be separated using a magnetic rack and subsequently washed very well with PBS/1% BSA. The antigen-microantibody complex so formed will be treated with complex, detection antibody bound to an enzyme (AP, HRP, Luciferase, beta-galactosidase, gold particles) or DNA to sandwich the antigen between the shark and camelid heavy chain only antibodies and their analogs and the detection antibody forming the complex which will be detected either using an enzyme substrate or AgNO3 if the detection antibody is conjugated to gold particles. Exemplary schematics of the process is shown in FIG. 25. Alternatively, the detection antibody could be conjugated to DNA molecules which can then be amplified by PCR to obtain detection sensitivity equivalent to the detection of DNA by PCR as shown in FIG. 26.

Example 2

[0191] Non-Invasive Detection of Prenatal Genetic Disorders from Captured Circulating Fetal Cells (CFCs) Using Heavy-Chain Antibodies

[0192] Blood (10 ml) from a pregnant woman will be treated at RT for 1 hour with sdAb conjugated to magnetic beads, with gentle shaking. The beads will be allowed to settle down in a magnetic rack and then subsequently washed with a wash buffer containing 20 mM PO4-2/150 mM NaCl/0.1% Triton X-100 (32 ml) to ensure complete removal of blood and serum. The beads will then be washed with 1PBS to remove triton. The bound DNA will then be eluted by hot 10 mM Tris.HCl, pH7.0 or by protease digestion.

[0193] This fetal DNA will then be analyzed by real-time PCR using Y-chromosome primers to test the gender and by chromosome 21 primers to test for Down syndrome.

Example 3

[0194] In-Vitro Capture of Pathological Proteins with Single-Domain Camelid and/or Shark Antibodies and Detection by Enzymatic Signal Amplification:

[0195] The high specificity of camelid and shark antibodies can be exploited to detect proteins at a much lower concentrations than what is currently possible. These antibodies are stable and functional at higher temperatures (80 to 90 C.). Also, they are stable in the presence detergents and denaturing agents. This allows us to capture the pathological antigens under stringent conditions such as performing the capture reaction at elevated temperatures and using detergents (say for an example the use of up to 10% TritonX-100-), followed by high temperature stringent washings containing detergents to minimize non-specific capture. Such use of stringent conditions is only possible in immunoassays utilizing camelid and shark antibodies. When combined with enzymatic signal as shown in FIG. 11, the camelid and shark antibodies should be able to detect 0.1 to 1.0 attomoles of target molecules in 25 ul reaction volume, which itself is a much improvement over the existing proteomic detection technologies.

[0196] In the representative example shown in FIG. 11, the patient serum was incubated with magnetic beads coated with camel micro-antibody 39 (0.5 ml beads containing at least 1.0 ug camelid micro-antibody) with gentle shaking of the reaction contents at RT for 45 minutes. After 45 minutes, the beads were allowed to settle down and washed with 2SSC buffer containing 1.0% Tween-20. Detection of beads bound pathological antigen from 40 was accomplished by incubating the beads with a AP conjugate 41 of detection antibody for 1 hour at RT on a rocker. The beads were then thoroughly washed (52 ml) with preheated 2SSC buffer (60 C.) containing 0.5% NP-40 to remove any non-specifically bound complex 41 (other commercially available detergents such as Triton X-100, Tween-20, SDS, LiDS, IGEPAL, Luviquat, DTPO, Antifoam 204, etc. can also be used.). The washings of the beads can also be done at temperature above 60 C. all the way up to 85 C. to remove any contaminants from the complex 42. The detection of complex 42 was then accomplished with Attophase (100 ul of 1.0 micromolar solution, 37 C. for 30 minutes), a fluorescence substrate for AP. The liberated green fluorescence was measured using a 96 microwell plate fluorimeter. 0.1 attomole of serum PSA antigen could be readily detected with 3:1 signal to background ratio.

Example 4

In-Vitro Capture of Pathogens by Single-Domain Shark IgNAR in Solution Phase and Detection by Enzymatic Signal Amplification:

[0197] In this technology format, the biotinylated shark IgNAR, 2a (FIG. 12), can be added to the patient serum and allowed to react with the pathogen at 37 C. for about one hour while the reactants are gently stirred or rotated on a orbital shaker. Anti-biotin camelid antibody (or shark antibody) bound to magnetic beads 45 can be added to reaction mixture to capture the so formed shark-IgNAR-Antigen complex 44 forming a complex of structure 46. The magnetic beads will be allowed to settle down in a magnetic rack and washed very well with a preheated (60 C.) wash buffer containing at least 1% NP-40. The complex 46 can then be detected by incubating with AP-IgG (sec) camelid complex using Attophos as a substrate as described above.

[0198] Other camelid and/or shark antibodies and their analogs can also be used the same way.

Example 5

Ultra-Sensitive Signal Amplification Using Single-Domain Heavy-Chain Only Camelid and Shark Antibodies for In-Vitro Detection of Pathogens by Immuno-PCR:

[0199] The two vital components of the method of this invention are: #1) Ultra-specific capture of pathological proteins by the new generation of single-domain camelid and shark antibodies lacking the light-chains (heavy-chain only antibodies), and #2) an ultra-sensitive signal amplification technology to detect fewer than 200 molecules of protein biomarkers to diagnose diseases at a very early stage of the their manifestation. Therefore, in its preferred embodiment, this invention incorporates inherently highly specific heavy-chain antibodies for capturing the pathological proteins/antigens with high specificity with low to zero cross-reactivity, followed by detection of the captured antigen by enzymatic signal amplification, preferably immuno-PCR, to develop an ultra-sensitive, highly specific and reliable diagnostic assay to detect fewer than 200 copies of the pathological proteins from biological samples.

[0200] FIG. 13 outlines the steps of the process involved. The protocol involves capturing the antigen from bodily fluid utilizing camelid and/or shark antibody coated magnetic beads by bringing in contact the said sample containing antigen with the beads. In the example shown in FIG. 13, camelid mini-antibody coated magnetic beads 48 are mixed with the serum for 1-2 hours with reactants constantly but slowly mixing all the time. The beads are then allowed to settle down in a magnetic rack and very well washed to ensure complete removal of the serum.

[0201] The detection of the captured antigen will be done by adding conjugate, 49, of secondary antibody that is conjugated to 100-120 bases long DNA via a hydrophilic linker that is at least 5 nanometer long to diminish and/or remove any stearic affects in the subsequent enzymatic amplification. The reaction between the captured antigen and the conjugate 49 will be allowed to take place for 2-3 hours after which the beads will be thoroughly washed to remove any unreacted conjugate 49.

[0202] The subsequent amplification of the attached DNA molecule by PCR using PCR kit from Applied Biosystems will allow for the indirect detection of the antigen with sensitivity almost equal to the sensitivity of detection of DNA by PCR.

[0203] There are many possible permutations and combinations of this technology. For instance, the antigen can be detected in solution phase by biotinylated or digoxigenin labeled camelid or shark antibody as described above following the steps of figure outlined in FIG. 12. The antigen-antibody complex so formed can be immobilized onto some solid matrix using camelid or shark anti-biotin or anti-digoxigenin antibody. Detection can be done by Immuno-PCR by forming a complex of the immobilized antigen-camelid-antibody with the secondary antibody bound to DNA which can be amplified by PCR.

[0204] Alternatively, the detection antibody in FIG. 13 can be conjugated with camelid or shark anti-biotin Mini- or nano-antibody. Biotinylated DNA can be used as a detection agent which will be amplified by PCR as outlined in FIG. 13.

Example 6

In-Vitro Capture and Detection of Rare Cells Using Shark and Camelid Heavy Chain Only

Antibodies and Their Analogs

[0205] Fresh 5 ml patient blood will be diluted with 20 ml 1PBS/1% BSA to 25 ml. To capture circulating tumor cells (CTCs), this sample will then be passed through a micro-fluidic device coated with an appropriate shark and camelid heavy chain only antibodies and their analogs, such as, anti-EpCAM-micro-antibody (camelid) 51 following flow rate recommended by the manufacturer of microfluidic device. To ensure that antibodies or its analogs do not lose any activity upon conjugation, all solid matrixes will first be coated with a hydrophilic polymer, such as, NHS-PEG-Mal (MW 5000). The conjugation of the thiolated shark and camelid heavy chain only antibodies and their analogs with maleimido-group of the polymer can be achieved at pH 6.8 in a buffer containing 5% EDTA. Exemplary schematics of the process are shown in FIG. 14.

[0206] Alternatively, magnetic beads coated with EpCAM can be used. EpCAM (epithelial cell adhesion molecules) is frequently over expressed by carcinomas of lung, colorectal, breast, prostate, head and neck, liver, and is absent from hematological cells. The captured cells can be washed with 1% PBS (no BSA). The cell can be fixed with methanol, and then DAPI stained following CK8 or CK18 and CD45. Identification and enumeration will be done by fluorescence microscopy based upon the morphological characteristics, cell size, shape, and nuclear size. DAPI+, CK+, and CD45-cells will be classified as CTCs.

Alternative Strategies to Capture Circulating Tumor Cells (CTCs):

[0207] Patient's blood (2-3 ml) (or urine 15-20 ml after centrifugation to pellet down the cells and suspending them in 1-2 ml HBSS media) will be incubated with an appropriate biotinylated mini-sdAb (1.5 ug/ml blood sample) at RT for one hour. For example, to capture epithelial cancer cells, such as from breast, prostate, and ovarian cancers, biotinylated-anti-EpCAM-mini-antibody (camel antibody against EpCAM antigens) will be used to label the circulating cancer cells in the blood. After diluting with HBSS or RPMI-1640 media or 1PBS/2.5% BSA to lower the sample viscosity, the diluted blood is then passed through a microfluidic device coated with antibiotin-mini-camelid or shark antibody at a flow rate allowing maximum cell capture. The captured CTCs can then be fixed by fixing with methanol, followed by fixing with 1% PFA using any standard cell fixing procedures. Enumeration will then be done by DAPI staining followed by immunohistochemical staining with commonly used mouse mHCAb such as CK-7 but more preferably mini-CK-7 for higher specificity. CTCs have to be CD45 negative.

[0208] Alternatively, most of the RBCs from the blood sample can be first lysed using ammonium chloride solution (155 mM NH4Cl/10 mM NaHCO3). After pelleting, the washed cells will be suspended in HBSS media (1-2 ml) and passed through the microfluidic device coated with heavy-chain antibody specific for the cell type one needs to capture and analyze.

[0209] Alternatively, the diluted blood sample after incubation with the biotinylated-antiEpCAM-mini-antibody or micro-antibody will be treated with the anti-biotin-mini-camelid antibody coated magnetic particles (Miltenyl) for 30 minutes while the sample is being gently rotated on a rotating wheel. After pulling down the magnetic particles with a magnet, the CTCs bound to the particles will be washed with PBS/1% BSA. The CTCs can then be enumerated by spreading them in a unilayer on a glass slide, drying them for one to two hours, followed by fixing with methanol, 1% PFA and staining the CTCs with CK-7.

[0210] Furthermore, these captured CTCs can be analyzed for the gene expression. For example, in case of prostate cancer patient, one can look for TMPRSS2-ERG translocation using PCR primers. TMPRSS2-ERG transcript is present in about 50% of the prostate cancer patients. Similarly, one can look for HER-2 expression in case of breast cancer.

Example 7

Capture and Analysis of Fetal Cells:

[0211] To capture fetal cells from the blood of pregnant mothers, 5 ml blood from pregnant mothers can be diluted to 15 ml with HAM-F 12 media containing 1% BSA and passed through the microfluidic device coated with the camelid and/or shark antibodies against the fetal cell surface antigens, CD71, glycophorin-A (GPA), CD133, and CD34. The captured fetal cells be analyzed for fetal gender, and genetic abnormalities using either PCR but preferably FISH probes for chromosomes X, Y, 13, 18 and 21 as shown in FIG. 15. For FISH analysis, the captured cells will be fixed with methanol followed by fixation with 1% PFA. After staining with epsilon-hemoglobulin, the cells be hybridized with Vysis FISH Fetal male gender can be readily detected by the appearance of XY fluorescence signal under the fluorescence microscope. Cells stained with epsilon-hemoglobulin showing XX signal will be identified as female fetal cells. Trisomies can be readily identified also based upon whether two or three chromosomes are giving the fluorescence signals.

[0212] Alternatively, most of the RBCs can either be carefully lysed using a mild treatment with ammonium chloride lysis reagent (155 mM NH4C1/10 mM NaHCO3) to enrich for fetal nucleated red blood cells (fnRBCs) before incubating the sample with a mixture of biotinylated antibodies.

[0213] Still another option will be the use of a density gradient such as Ficol 1.073 or Percol 1.073. The buffy coat can then be processed as above to yield fetal nRBCs.

Example 8

[0214] Detection of Chromosomal Translocations from Captured Circulating Tumor Cells (CTCs) Using Shark and Camelid Heavy Chain Only Antibodies and Their Analogs

[0215] Cells will be captured as described above and also shown in FIG. 16. Enumeration of can be done using an appropriate FISH probes. For example, to test for the presence of TMPRESS2/ERG translocation in case of prostate cancer, FISH probes designed to hybridize with the junction region will be used. Similarly, in case of CML, bcr-Abl FISH probe will be used. An exemplary schematics of the process is shown in FIG. 16.

[0216] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All nucleotide sequences provided herein are presented in the 5 to 3 direction.

[0217] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms comprising, including, containing, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0218] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0219] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0220] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0221] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0222] Other embodiments are set forth within the following claims.