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Title:
RECOMBINANT ANTIBODY COMPRISING HEAVY CHAIN GENETICALLY FUSED TO SIGNATURE PEPTIDE AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2019/118318
Kind Code:
A1
Abstract:
Provided herein are engineered recombinant antibodies and antigen binding fragments thereof ("SiGERMabs") comprising one or more heavy chains which are genetically fused to a signature peptide, as well as the use of such SiGERMabs and pairs or complexes thereof as research and diagnostic agents.

Inventors:
TAYLOR CLIVE (US)
MAHRAT REEM (US)
VILLANUEVA JACINTO (US)
Application Number:
PCT/US2018/064672
Publication Date:
June 20, 2019
Filing Date:
December 10, 2018
Export Citation:
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Assignee:
CALICO BIOLABS INC (US)
International Classes:
C07K16/30; C07K16/00; C07K19/00; G01N33/574; G01N33/58
Domestic Patent References:
WO2016127149A22016-08-11
Foreign References:
US20160258956A12016-09-08
US8785150B22014-07-22
US5892019A1999-04-06
US7846440B22010-12-07
US4816567A1989-03-28
Other References:
MICHAEL T. FURLONG ET AL: "A universal surrogate peptide to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies : Universal surrogate peptide for LC-MS/MS bioanalysis of protein drugs", BIOMEDICAL CHROMATOGRAPHY., 1 May 2012 (2012-05-01), GB, pages n/a - n/a, XP055569494, ISSN: 0269-3879, DOI: 10.1002/bmc.2759
HAIBIAO GONG ET AL: "Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells", BIOCONJUGATE CHEMISTRY, vol. 27, no. 1, 20 January 2016 (2016-01-20), US, pages 217 - 225, XP055408616, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.5b00613
HAGEN JODI ET AL: "Hapten-Anti-Hapten Technique for Two-Color IHC Detection of Phosphorylated EGFR and H2AX Using Primary Antibodies Raised in the Same Host Species", SIGNAL TRANSDUCTION IMMUNOHISTOCHEMISTRY : METHODS AND PROTOCOLS; IN: METHODS IN MOLECULAR BIOLOGY; ISSN 1064-3745 ; VOL. 1554, HUMANA PRESS, US, vol. 1554, 1 January 2017 (2017-01-01), pages 155 - 160, XP009511798, ISBN: 978-1-4939-6757-5, [retrieved on 20170210]
COONS AH ET AL., PROC. SOC. EXP. BIOL. MED., vol. 47, 1941, pages 200 - 202
COONS AH ET AL., J. IMMUNOL., vol. 45, 1942, pages 159 - 170
TAYLOR CR; BURNS J., J. CLIN. PATHOL., vol. 27, 1974, pages 14 - 20
"Immunomicroscopy: A diagnostic tool for the surgical pathologist", 2005, ELSEVIER
TAYLOR, CR; SHI S-R: "Comprehensive Diagnost. Immunohistochemistry", 2013, ELSEVIER, article "Techniques of Immunohistochemistry: Principles, Pitfalls and Standardization"
TAYLOR CR., APPLIED IMMUNOHISTOCHEM. MOL. MORPH., vol. 22, 2014, pages 555 - 561
MAITY B. ET AL., METHODS CELL BIOL, vol. 113, 2013, pages 81 - 105
PAUL NAKANE, THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, vol. 16, no. 9, 1968
STACK EC ET AL., SCIENCE DIRECT. METHODS, vol. 70, 2014, pages 46 - 58
ADAMS A ET AL., BLOOD, vol. 122, 2013, pages 2994
"Epitope Mapping Protocols in Methods in Molecular Biology", vol. 66, 1996
KABAT ET AL., J. BIOL. CHEM., vol. 252, 1977, pages 6609 - 6616
KABAT ET AL., SEQUENCES OF PROTEIN OF IMMUNOLOGICAL INTEREST, 1991
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPT. HEALTH AND HUMAN SERVICES
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
MARKS ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
ELLMAN ET AL., METH. ENZYM., vol. 202, 1991, pages 301 336
NOREN ET AL., SCIENCE, vol. 244, 1989, pages 182
HOLLIGER, P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
POLJAK, R. J. ET AL., STRUCTURE, vol. 2, 1994, pages 1121 - 1123
CRASTO CJ; FENG JA: "LINKER: a program to generate linker sequences for fusion proteins", PROTEIN ENG, vol. 13, no. 5, May 2000 (2000-05-01), pages 309 - 12, XP055092127, DOI: doi:10.1093/protein/13.5.309
GEORGE RA; HERINGA J.: "An analysis of protein domain linkers: their classification and role in protein folding", PROTEIN ENG, vol. 15, no. 11, November 2002 (2002-11-01), pages 871 - 9, XP002374925, DOI: doi:10.1093/protein/15.11.871
BARNES, L. M. ET AL., CYTOTECHNOLOGY, vol. 32, 2000, pages 109 - 123
BARNES, L.M. ET AL., BIOTECH. BIOENG., vol. 73, 2001, pages 261 - 270
DUROCHER, Y. ET AL., NUCL. ACIDS. RES., vol. 30, 2002, pages E9
ORLANDI, R. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 3833 - 3837
CARTER, P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4285 - 4289
NORDERHAUG, L. ET AL., J. IMMUNOL. METHODS, vol. 204, 1997, pages 77 - 87
SCHLAEGER, E. -J.; CHRISTENSEN, K., CYTOTECHNOLOGY, vol. 30, 1999, pages 71 - 83
SCHLAEGER, E.-J., J. IMMUNOL. METHODS, vol. 194, 1996, pages 191 - 199
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
MAKRIDES, S.C., PROTEIN EXPR. PURIF, vol. 17, 1999, pages 183 - 202
GEISSE, S. ET AL., PROTEIN EXPR. PURIF., vol. 8, 1996, pages 271 - 282
KAUFMAN, R.J., MOL. BIOTECHNOL., vol. 16, 2000, pages 151 - 161
WERNER, R.G., DRUG RES., vol. 48, 1998, pages 870 - 880
"Current Protocols in Molecular Biology", 1987, GREENE PUBLISHING AND WILEY INTERSCIENCE
VIJAYALAKSHMI, M.A., APPL. BIOCHEM. BIOTECH., vol. 75, 1998, pages 93 - 102
MCCARTY ET AL., CANCER RES, vol. 46, 1986, pages 4244 - 8
BOSMAN ET AL., J CLIN PATHOL, vol. 45, 1992, pages 120 - 4
DIESET ET AL., ANALYT QUANT CYTOL HISTOL, vol. 18, 1996, pages 351 - 4
ALLRED ET AL., MOD PATHOL, vol. 11, 1998, pages 155 - 68
HARVEY ET AL., J CLIN ONCOL, vol. 17, 1999, pages 1474 - 91
CHOUDHURY ET AL., J HISTOCHEM CYTOCHEM, vol. 58, 2010, pages 95 - 107
RIZZARDI ET AL., DIAGNOSTIC PATHOLOGY, vol. 7, 2012, pages 42 - 52
CAMP ET AL., CLIN CANCER RES, vol. 10, 2004, pages 7252 - 9
CAMP ET AL., NAT MED, vol. 8, 2002, pages 1323 - 7
CAMP ET AL., CANCER RES, vol. 63, 2003, pages 1445 - 8
GHOSH ET AL., HUM PATHOL, vol. 39, 2008, pages 1835 - 43
BOSE ET AL., BMC CANCER, vol. 12, 2012, pages 332
MASCAUX ET AL., CLIN CANCER RES, vol. 17, 2011, pages 7796 - 807
TAYLOR CR.; RUDBACK, L.: "Immunohistochemical Staining Methods", 2013
Attorney, Agent or Firm:
REMILLARD, Jane, E. et al. (US)
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Claims:
CLAIMS

We claim:

1. A recombinant antibody, or antigen binding fragment thereof, comprising a heavy chain constant domain genetically fused to a signature peptide.

2. The recombinant antibody, or antigen binding fragment thereof, of claim 1, wherein all or a portion of the heavy chain Fc region is genetically fused to the signature peptide.

3. The recombinant antibody, or antigen binding fragment thereof, of claim 2, wherein the heavy chain Fc region and the signature peptide are fused by one or more linkers.

4. The recombinant antibody, or antigen binding fragment thereof, of claim 3, wherein the linker is ten to fifty amino acids in length.

5. The recombinant antibody, or antigen binding fragment thereof, of claim 3, wherein the linker is forty amino acids in length.

6. The recombinant antibody, or antigen binding fragment thereof, of any one of the

preceding claims, wherein the signature peptide comprises synthetic or non-natural amino acids.

7. The recombinant antibody, or antigen binding fragment thereof, of any one of the

preceding claims, wherein the signature peptide comprises a tandem repeat of amino acids.

8. The recombinant antibody, or antigen binding fragment thereof, of claim 7, wherein the signature peptide comprises two to four tandem repeats of amino acids.

9. The recombinant antibody, or antigen binding fragment thereof, of claim 7 or 8, wherein the tandem repeats of the signature peptide are separated by a linker.

10. The recombinant antibody, or antigen binding fragment thereof, of claim 9, wherein the linker separating the tandem repeats is three to ten amino acids in length. 11. The recombinant antibody, or antigen binding fragment thereof, of claim 9 or 10, wherein the linker separating the tandem repeats is five amino acids in length.

12. The recombinant antibody, or antigen binding fragment thereof, of any one of claims 9- 11, wherein the linker separating the tandem repeats comprises the amino acids sequence set forth in SEQ ID NO:6.

13. The recombinant antibody, or antigen binding fragment thereof, of any one of the

preceding claims which binds to a protein of interest. 14. The recombinant antibody, or antigen binding fragment thereof, of claim 13, wherein the protein of interest is a biomarker associated with a particular disease.

15. The recombinant antibody, or antigen binding fragment thereof, of claim 13 or 14,

wherein the protein of interest is a cancer biomarker.

16. The recombinant antibody, or antigen binding fragment thereof, of claim 13, wherein the protein of interest is keratin, vimentin, CD3, CD20, Ki67, or PD-L1.

17. A pair of proteins, wherein the pair comprises:

(a) a primary recombinant protein comprising an antibody, or antigen binding fragment thereof, comprising a heavy chain genetically fused to a signature peptide; and

(b) a secondary binding protein that binds to the signature peptide of the primary recombinant antibody, or antigen binding fragment thereof.

18. The pair of proteins of claim 17, wherein the primary recombinant protein comprises an antibody, or antigen binding fragment thereof, wherein the heavy chain Fc portion is genetically fused to the signature peptide.

19. The pair of proteins of claim 18, wherein the secondary binding protein is an antibody or antigen binding fragment thereof.

20. The pair of proteins of claim 19, wherein the secondary antibody is a monoclonal

antibody.

21. The pair of proteins of claim 19, wherein the secondary antibody is a polyclonal antibody.

22. The pair of proteins of claim 19, wherein the secondary antibody is a modified antibody.

23. The pair of proteins of claim 20, wherein the secondary modified antibody is a

multivalent antibody, such as IgM.

24. The pair of proteins of claim 18, wherein the secondary binding protein is a recombinant antibody, or antigen binding fragment thereof, comprising a heavy chain, wherein the heavy chain Fc portion is genetically fused to a signature peptide.

25. The pair of proteins of any one of claims 18-24, wherein the secondary binding protein comprises a detectable moiety.

26. The pair of proteins of claim 25, wherein the detectable moiety is a luminescent label, fluorescent label, radiolabel (e.g., 99Tc, 45Ti, 112In, mIn, 3H, 121I, 125I, 131I, 14C, 18F, 36Cl, 55Co, 58Co, 51Cr, 67Cu, ^Cu, 66Ga, 68Ga, 76Br, 89Zr, 35S, 32P, 90Y, 13N, 150, 211 At, 186Re, 188Re, 75Se), enzymatic label (e.g., hydrogen peroxidase, horseradish peroxidase, alkaline phosphatase, glucose oxidase, urease, acetylcholinetransferase, luciferase, beta- galactosidase), epitope tag, chromophore label, phosphorescent label, photoaffinity molecule, ECL label, dye, biotin, or hapten.

27. The pair of proteins of any one of claims 18-25, wherein the secondary binding protein includes an active site of an enzyme, so as to directly convert a substrate added subsequently.

28. The pair of proteins of claim 27, wherein the active site of the enzyme is the active site of peroxidase, alkaline phosphatase, or glucose oxidase.

29. A multiplex of proteins comprising two or more pairs of proteins of any one of claims 17- 28.

30. The multiplex of claim 29, comprising three, four, five, six, seven, eight, nine, or ten pairs of recombinant proteins.

31. The multiplex of claim 29 or 30, wherein the primary recombinant proteins each bind to a different epitope.

32. The multiplex of claim 29 or 30, wherein the signature peptides of the primary

recombinant proteins are different.

33. A diagnostic kit comprising the recombinant antibody, or antigen binding fragment

thereof, of any one of claims 1-16 and instructions for use.

34. A diagnostic kit comprising the pair of proteins of any one of claims 17-28 and

instructions for use.

35. A diagnostic kit comprising two or more of pairs of the proteins of any one of claims 17- 28 and instructions for use.

36. A method for detecting a protein of interest in a biological sample from a patient,

comprising: (a) contacting the sample with the pair of proteins of any one of claims 17-28 and

(b) detecting binding of the primary recombinant protein to the protein of interest within the sample via the secondary protein.

37. A method for detecting one or more proteins of interest in a biological sample from a patient, comprising

(a) contacting the sample with the multiplex of any one of claims 29-32 and

(b) detecting binding of the primary recombinant proteins to the one or more proteins of interest within the sample via the secondary proteins.

38. A method for quantifying levels of one or more proteins of interest in a biological sample from a patient, comprising

(a) contacting the sample with the multiplex of any one of claims 29-32;

(b) detecting binding of the primary recombinant proteins to the one or more proteins of interest within the sample via the secondary proteins; and

(c) quantifying levels of the one more proteins of interest using Quantifiable Internal Reference Standard technology.

39. A method of monitoring expression levels of one or more proteins of interest in a

biological sample from a patient comprising:

(a) detecting expression levels of the one or more proteins of interest in a biological sample obtained from the patient at a first time point by contacting the sample with the multiplex of any one of claims 29-32,

(b) detecting expression levels of the one or more proteins of interest in a biological sample of the same type from the patient obtained at a second time point using the same multiplex used in step (a), and

(c) comparing expression levels of the one or more proteins of interest determined at the first and second time points, to assess whether the expression levels are higher or lower at the first time point relative to the second time point.

40. A method of diagnosing a patient as having a disease characterized by a protein of interest, comprising:

(a) contacting a biological sample obtained from the patient with the pair of

recombinant proteins of any one of claims 17-28, wherein the primary recombinant protein binds to the protein of interest within the sample, and

(b) diagnosing the patient as having the disease based on detection of binding of the primary recombinant protein to the protein of interest within the sample via the secondary protein.

41. A method of diagnosing a patient as having a disease characterized by one or more

proteins of interest, comprising:

(a) contacting a biological sample obtained from the patient with the multiplex of any one of claims 29-32, wherein the primary recombinant

protein binds to the protein of interest within the sample, and

(b) diagnosing the patient as having the disease based on detection of detecting binding of the primary recombinant proteins to the one or more proteins of interest within the sample via the secondary proteins.

42. The method of any one of claims 37-39 and 41, wherein the one or more proteins of interest are detected sequentially.

43. The method of any one of claims 37-39 and 41, wherein the one more proteins of interest are detected simultaneously.

44. The method of any one of claims 36-43, wherein the detection step is performed by

enzyme-linked immunosorbent assay (ELISA), immunofluorescence, flow cytometry, Western blot, lateral flow assay, and/or immunohistochemistry (IHC).

45. The method of any one of claims 36-43, wherein the biological sample is a human tissue sample.

46. The method of claim 45, wherein the human tissue sample is a tumor tissue.

47. The method of any one of claims 36-46, wherein the sample is a formalin-fixed paraffin- embedded sample or frozen tissue.

48. The method of any one of claims 36-43, wherein the biological sample is selected from the group consisting of blood, urine, tissue ( e.g ., tumor), serum, stool, urine, sputum, cerebrospinal fluid, or supernatant from cell lysate.
Description:
RECOMBINANT ANTIBODY COMPRISING HEAVY CHAIN GENETICALLY FUSED TO SIGNATURE PEPTIDE AND USES THEREOF

SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 7, 2018, is named CBJ-004PC_SL.txt and is 57,959 bytes in size.

RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No. 62/735,559, filed on September 24, 2018, and claims the benefit of U.S. Provisional Application No. 62/597,713, filed on December 12, 2017. The contents of the aforementioned applications are incorporated herein by reference in its entirety.

BACKGROUND

Immunologic detection of target molecules, or parts thereof, by microscopy in tissue sections and other cell preparations dates back to Albert Coons, who in the l930s employed a fluorescent tag on an antibody to search for Streptococcus in rheumatic fever (see e.g., Coons AH, et al., Proc. Soc. Exp. Biol. Med. 1941;47:200-202 and Coons AH, et al., J. Immunol. 1942;45:159-170). A number of targets (antigens and epitopes) were detected using this fluorescent labeled antibody method. However, for many decades, the application of this technique was restricted by the requirement for dark field fluorescence microscopy and the use of various fresh cell preparations or smears and frozen sections. Morphologic detail upon which histopathologic diagnosis depends was therefore lost, and the method was limited to research applications and some immunologic diseases of kidneys and skin.

Widespread use of labeled antibody methods in surgical pathology began forty years later, in 1974, when Clive Taylor used horseradish peroxidase with a chromogen in place of a fluorescent label to transfer the method to formalin fixed paraffin embedded material, thereby enabling examination by orthodox bright field microscopy (see Taylor CR and Burns J., J. Clin. Pathol. 27:14-20, 1974). This method is referred to as immunofluorescence (IF) and/ or immunohistochemistry (IHC). Many derivative methods and their relative modes of application were subsequently developed (see, e.g., Taylor CR., W.B. Saunders, Philadelphia, 1986; Taylor CR. Cote RJ. Editors. Immunomicroscopy: A diagnostic tool for the surgical pathologist, Third Edition. Elsevier. Saunders, Philadelphia, 2005; Taylor, CR, Shi S-R. Techniques of

Immunohistochemistry: Principles, Pitfalls and Standardization, In: Comprehensive Diagnost. Immunohistochemistry, Elsevier, David J. Dabbs (ed), Fourth Edition. 2013; and Taylor CR., Applied Immunohistochem. Mol. Morph. 2014; 22:555-561).

Immunohistochemical (IHC) methods (both IF and bright filed methods) traditionally have been used to detect a single target (e.g., protein) within the section or preparation (see, e.g., Maity B. et al., Methods Cell Biol. 2013; 113:81-105). This approach is colloquially referred to as“immuno staining”. Almost any target for which there is a specific antibody can be stained in this manner.

When it is desirable to detect more than one protein in a tissue specimen, the usual approach (both in research and diagnosis) is to cut serial parallel tissue sections and stain each for a different biomarker, followed by review and comparison of the separate slides (e.g., typically 2-10 slides or more). The sequential approach is the most commonly used method in diagnostic surgical pathology and often requires several staining runs and several days for a result.

In addition, there are often research and diagnostic circumstances where it is desirable to detect more than one biomarker in the exact same section (e.g., to closely examine the localization or co-localization at a cellular level). This feat applied to routine formalin paraffin (FFPE) sections for diagnostic purposes was first described in 1974 using a sequential double staining (multiplex) method (see Taylor CR and Bums J., J. Clin. Pathol. 27:14-20, 1974). Paul Nakane had previously used a sequential approach in a research of hormones in rat pituitary specially fixed in Bouin’s fixative (see Paul Nakane, The Journal of Histochemistry and

Cytochemistry, Vol. 16, No. 9, 1968). However, this sequential method was cumbersome and limited in terms of specificity and sensitivity.

Over the succeeding four decades many different approaches were devised for multiplex IHC. For two, three, four, or more multiplex“immunostains”, the most common approach until quite recently was to perform individual stains sequentially, seeking to avoid cross reactivity with preceding stages by careful selection of reagents, labels, etc., and, in each case preserving the colored reaction product (or image thereof) before“stripping” reagents and applying second and subsequent staining protocols (see, e.g., Taylor CR., W.B. Saunders, Philadelphia, 1986; Taylor CR. Cote RJ. Editors. Immunomicroscopy: A diagnostic tool for the surgical pathologist, Third Edition. Elsevier. Saunders, Philadelphia, 2005). Commercially available variants of this sequential method have been adapted to demonstrate four, six, or more biomarkers by immunofluorescence (see, e.g., Stack EC et al, Science Direct. Methods 20l4;70: 46-58 and Adams A et al., Blood. 122; 2994; 2013). These methods involve“stripping” or“bleaching” of preceding reactions or repeated whole slide imaging (WSI) before proceeding to the next staining step. Detection has primarily involved fluorescent labels and digital imaging with computer analysis essential for the display and interpretation of these complex color images. Disadvantages of the sequential approach include time taken to perform multiple sequential steps (e.g., days) and concerns about the denaturation or damage to later target antigens by repeated heat or chemical treatment of the slides.

Another approach for two, three, or four multiplex“immunostains” has been to use primary antibodies from different species, followed by secondary detection reagents directed to the different primary species (see, e.g., Taylor CR., W.B. Saunders, Philadelphia, 1986; Taylor CR. Cote RJ. Editors. Immunomicroscopy: A diagnostic tool for the surgical pathologist, Third Edition. Elsevier. Saunders, Philadelphia, 2005). This approach is usually limited to three biomarkers because of the difficulty in obtaining good primary antibodies from more than three different species and pairing them with appropriate specific secondary detection reagents.

Problems of low affinity, poor sensitivity, poor specificity and cross reactivity, resulting in background interference, are all common with this method.

In yet another approach, tags or labels have been attached to the primary antibody to facilitate detection by secondary reagents. The use of haptens chemically conjugated to primary antibodies, was described 30 years ago (see, e.g., Taylor CR., W.B. Saunders, Philadelphia, 1986; Taylor CR. Cote RJ. Editors. Immunomicroscopy: A diagnostic tool for the surgical pathologist, Third Edition. Elsevier. Saunders, Philadelphia, 2005; Taylor, CR, Shi S-R.

Techniques of Immunohistochemistry: Principles, Pitfalls and Standardization, In:

Comprehensive Diagnost. Immunohistochemistry, Elsevier, David J. Dabbs (ed), Fourth Edition. 2013; and Taylor CR., Applied Immunohistochem. Mol. Morph. 2014; 22:555-561)).

A labeled hapten approach designed for multiplex staining has been described as the“bridge antigen method” (see, Schwartz DA, US Patent Publication No: 20160258956). In this method, the species of origin of the primary antibody is immaterial as it is chemically linked to a peptide, and it is this peptide (hapten) that may then be detected by a secondary reagent to said peptide. By use of different peptides on different primary antibodies, with different peptide specific secondary antibodies, multiplex staining can be achieved. This method has the advantage of allowing simultaneous application of primary and secondary antibodies and thus, requires a short time to perform ( e.g ., less than three hours). Major disadvantages of this method include the fact that each primary and secondary antibody must be separately prepared, and each new batch of primary antibody must be chemically labeled. The primary antibody also must be highly purified (e.g., contaminant protein(s) also will be peptide linked), and may be damaged by chemical linking. In addition, some of the primary antibody molecules may be unlabeled and the label also may dissociate over time. In addition, the cost of this method is five to six times greater than the sequential approach.

In view of the disadvantages associated with known IHC methods, there is a need in the art for additional and improved techniques.

SUMMARY

Provided herein are engineered recombinant antibodies and antigen binding fragments thereof (also referred to herein as a“SiGERMabs”) comprising one or more heavy chains which are genetically fused to a signature peptide, as well as the use of such SiGERMabs as research and diagnostic agents. In one embodiment, the SiGERMab further comprises one or more light chains.

The signature peptide of the SiGERMab can be of any length or sequence, but must be recognizable by a secondary binding protein (e.g., a secondary antibody). In one embodiment, the signature peptide comprises naturally occurring amino acids. In another embodiment, the signature peptide comprises synthetic or non-natural amino acids. In another embodiment, the signature peptide comprises a tandem repeat of amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of amino acids). In a particular embodiment, the signature peptide comprises four tandem repeats. In another embodiment, the tandem repeats are separated by a linker (e.g., which is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids in length). In a particular embodiment, the linker is five amino acids in length. Any suitable linker can be used. In one embodiment, the linker comprises the amino acids sequence“GGGGS” (SEQ ID NO:6). Exemplary signature peptides are set forth in SEQ ID NO: 1 and SEQ ID NO: 7.

The signature peptide is genetically fused to the one or more heavy chains of the

SiGERMab (e.g., via all or a portion of the Fc region). In one embodiment, the heavy chain includes a complete Fc region. In another embodiment, the heavy chain includes a portion of an Fc region (e.g., CHI domain, the hinge, CH2 domain, CH3 domain, and/or CH4 domain). In another embodiment, the signature peptide is fused to the CH2 domain of the Fc region. In another embodiment, the signature peptide is fused to the CH3 domain of the Fc region. In another embodiment, the signature peptide is fused to the CH4 domain of the Fc region (e.g., the C-terminus of the Fc region). In further embodiments, the signature peptide is genetically fused to the heavy chain Fc region by one or more linkers. Typically, the linker is between 10 to 60 amino acids in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,

55, 56, 57, 58, 59, or 60 amino acids). In one embodiment, the linker is ten to sixty amino acids in length. In another embodiment, the linker is thirty to fifty amino acids in length. In another embodiment, the linker is twenty to fifty amino acids in length. In another embodiment, the linker is forty amino acids in length.

SiGERMabs of the invention specifically bind to a protein of interest, preferably with high affinity. In one embodiment, the protein of interest is a biomarker associated with a disease ( e.g ., a cancer, autoimmune disease, or infectious disease). In a particular embodiment, the protein of interest is a cancer biomarker. Exemplary proteins of interest include, but are not limited to, ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2;

ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCF2; BCF6; BDNF; BFNK; BFR1 (MDR15); BlyS; BMP1;

BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; Cl9orfl0 (IF27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCF1 (1-309); CCF11 (eotaxin); CCF13 (MCP-4); CCF15 (MIP-ld); CCF16 (HCC-4); CCF17 (TARC); CCF18 (PARC); CCF19 (MIP-3b); CCF2 (MCP-l); MCAF; CCF20 (MIP-3a); CCF21 (MIP-2); SFC; exodus-2; CCF22 (MDC/STC-l); CCF23 (MPIF-l); CCF24 (MPIF -2/eotaxin-2) ; CCF25 (TECK); CCF26 (eotaxin-3); CCF27 (CTACK/IFC); CCL28; CCL3 (MIP-la); CCL4 (MIP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-lRB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR 13); CCR6 (CMKBR6/CKR- L3/STRL 22/DRY6); CCR7 (CKR7/EBI1); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9- 6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDHl(E- cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p2l Wapl/Cipl); CDKN1B (p27Kipl); CDKN1C; CDKN2A (pl6INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); C-MET;

CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSFl(M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GR01); CXCL10 (IP-10);

CXCL1 l(I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3

(GPR9/CKR-L2) ; CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB21P; DES; DKFZp45U0l l8; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EGFR, EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENOl; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2;

FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF1; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-l); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GAT A3; GDF5; GFI1; GGT1; GITR; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4;

HDAC5; HDAC7A; HDAC9; HER; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-l; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1;

IL1RAPL2; IL1RL1; IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2;

IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; Ki67; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; keratin, KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair- specific type II keratin); LAMA5; LEP (leptin); Lingo-p75; Lingo- Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR- p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PC A3; PCNA; PDGF; PDGFA; PDGFB; PD-L1; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p2lRac2); RARB; RGS1;

RGS13; RGS3; RNF110 (ZNF144); ROB02; S100A2; SCGB1D2 (lipophilin B);

SCGB2Al(mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-l); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Sprl); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFB1; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin- 1); THBS2; THBS4; THPO; TIE (Tie-l); TIGIT; TIMP3; tissue factor;

TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (0X40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A

(topoisomerase Iia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; vimentin, VLA-4; XCL1 (lymphotactin); XCL2 (SCM-lb); XCR1 (GPR5/CCXCR 1 ) ; YY1; and ZFPM2.

In a particular embodiment, the protein of interest is keratin, vimentin, CD3, CD20, Ki67, or PD-L1.

Also provided herein are pairs or complexes (also referred to as“SiGERMab pairs” or “SiGERMab complexes”) which include at least one SiGERMab and a respective secondary protein that binds to the signature peptide of the SiGERMab, as well as the use of such pairs and complexes as research, diagnostic and therapeutic agents.

In one embodiment, the SiGERMab and secondary binding protein are from the same species. In another embodiment, the SiGERMab and secondary binding protein are from different species.

The secondary binding protein can be any suitable protein that binds to the signature peptide of the SiGERMab. In one embodiment, the secondary protein is an antibody, or antigen binding fragment thereof ( e.g ., a monoclonal or polyclonal antibody). In one emobidiment, the secondary protein is a monoclonal antibody. In another embodiment, the secondary protein is a polyclonal antibody. In a particular embodiment, the secondary protein is a modified antibody (e.g., a multivalent antibody, such as IgM). In another embodiment, the secondary protein is another SiGERMab.

The secondary binding protein generally includes a detectable moiety (e.g., a tag or label). Exemplary detectable moieties include, but are not limited to, a luminescent label, fluorescent label, radiolabel (e.g., 99 Tc, 45 Ti, 112 In, m In, ¾ 121 I, 125 I, 131 I, 14 C, 18 F, 36 Cl, 55 Co, 58 Co, 51 Cr, 67 Cu, ^Cu, 66 Ga, 68 Ga, 76 Br, 89 Zr, 35 S, 32 P, 90 Y, 13 N, 15 0, 211 At, 186 Re, 188 Re, 75 Se), enzymatic label (e.g., hydrogen peroxidase, horseradish peroxidase, alkaline phosphatase, glucose oxidase, urease, acetylcholinetransferase, luciferase, beta-galactosidase), epitope tag, chromophore label, phosphorescent label, photoaffinity molecule, ECL label, dye, biotin, or hapten. In one embodiment, the secondary binding protein includes an active site of an enzyme (e.g., peroxidase, alkaline phosphatase, or glucose oxidase), so as to directly convert a substrate added

subsequently.

Also provided herein are multiplexes which include two or more SiGERMab pairs or SiGERMab complexes. In one embodiment, the multiplex comprises three, four, five, six, seven, eight, nine, or ten SiGERMab pairs or complexes. In another embodiment, each SiGERMab pair or complex binds to a different epitope. In another embodiment, each SiGERMab pair or complex includes a different signature peptide. Detection and interpretation of multiplexes can be achieved using known techniques, including digital scanning and computer based analysis (e.g., via commercially available software).

In another aspect, diagnostic kits are provided. In one embodiment, the diagnostic kit includes one or more SiGERMabs or SiGERMab pairs, optionally with instructions for use. In one embodiment, the kit further includes a means for detecting the secondary binding protein (e.g., a substrate).

In another aspect, methods are provided for using the SiGERMabs, SiGERMab pairs and/or multiplexes and/or multiplexes described herein as research and diagnostic agents. In order for the methods to be accurate and function as intended, it is critical that the epitopes present in the SiGERMab signature peptide are not present in the assay that is used.

In one embodiment, methods of detecting a protein of interest in a biological sample from a patient are provided, comprising contacting the sample with a SiGERMab, SiGERMab pair and/or multiplex described herein, and detecting binding of the SiGERMab, SiGERMab pair and/or multiplex to the protein of interest within the sample.

Also provided are methods for quantifying levels of one or more proteins of interest in a biological sample from a patient are provided, comprising contacting the sample with a

SiGERMab, SiGERMab pair and/or multiplex described herein, detecting binding of the

SiGERMab, SiGERMab pair and/or multiplex to the one or more proteins of interest within the sample; and quantifying levels of the one more proteins of interest using known techniques (e.g., Quantifiable Internal Reference Standard technology, e.g., as described in US 8,785,150, the entire contents of which are expressly incorporated herein by reference). Also provided are methods of monitoring expression levels of one or more proteins of interest in a patient are provided comprising: (a) detecting expression levels of the one or more proteins of interest in a biological sample obtained from the patient at a first time point by contacting the sample with a SiGERMab, SiGERMab pair and/or multiplex described herein, (b) detecting expression levels of the one or more proteins of interest in a biological sample of the same type from the patient obtained at a second time point using the SiGERMab, SiGERMab pair and/or multiplex used in step (a), and (c) comparing expression levels of the one or more proteins of interest determined at the first and second time points, to assess whether the expression levels are higher or lower at the first time point relative to the second time point.

Also provided are methods of diagnosing a patient as having a disease characterized by a protein of interest, comprising (A) contacting a sample obtained from the patient with any of the pairs of recombinant proteins described herein, wherein the primary recombinant protein binds to the protein of interest within the sample, and (B) diagnosing the patient as having the disease based on detection of binding of the primary recombinant protein to the protein of interest within the sample via the secondary recombinant protein.

Further provided are methods of diagnosing a patient as having a disease characterized by two or more proteins of interest, comprising (A) contacting a sample obtained from the patient with a SiGERMab, SiGERMab pair and/or multiplex described herein, and (B) diagnosing the patient as having the disease based on detection of binding of the SiGERMab, SiGERMab pair and/or multiplex to their respective target proteins within the sample.

In one embodiment, the disease is a cancer, infectious disease, or autoimmune disorder, such as the exemplary diseases described herein. The proteins of interest can include, for example any of the exemplary proteins of interest described herein. In one embodiment, the protein of interest is a biomarker associated with a disease ( e.g ., a cancer, autoimmune disease, or infectious disease). In a particular embodiment, the protein of interest is a cancer biomarker.

In the methods described herein, wherein multiple proteins of interest are detected, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more proteins can be detected. In one embodiment, the one or more proteins of interest are detected sequentially. In another embodiment, the one more proteins of interest are detected simultaneously. Expressions levels of the one or more proteins of interest in the methods described herein can be detected via enzyme-linked immunosorbent assay (ELISA), immunofluorescence

(including all labeled antibody methods, such as particle labels and metal ions), and/or immunohistochemistry (IHC). ELISA, immunofluorescence, and/or IHC can also be used to detect binding of the primary recombinant proteins to the one or more proteins of interest within a sample via the secondary recombinant proteins.

The methods described herein can be applied to any suitable biological sample. In one embodiment, the biological sample is a human tissue sample ( e.g ., a tumor tissue sample). In another embodiment, the sample is a formalin-fixed paraffin-embedded sample or frozen tissue. In another embodiment, the biological sample is selected from the group consisting of blood, urine, tissue (e.g., tumor), serum, stool, urine, sputum, cerebrospinal fluid, or supernatant from cell lysate.

In another aspect, nucleic acids encoding SiGERMabs of the invention are provided, as well as expression vectors comprising such nucleic acids and host cells.

In another aspect, methods of producing SiGERMabs of the invention are provided by culturing a host cell as described above ( i.e ., transfected with a nucleic acid encoding the SiGERMab) in culture medium under conditions wherein the nucleic acid sequence is expressed, thereby producing the SiGERMab; and recovering the SiGERMab from the host cell or culture medium. In another embodiment, the SiGERMab is coexpressed with a light chain.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 depicts the use of recombinant antibodies, or antigen binding fragments thereof, comprising a heavy chain genetically fused to a signature peptide (SiGERMabs) for use in multiplex assessment of epitopes.

Figures 2A-2C depict Multiplex Immunohistochemistry (IHC) analysis of (A)

SiGERMAb2b; (B) SiGERMab3C; and (C) SiGERMAb2b + SiGERMab3C.

DETAILED DESCRIPTION

The SiGERMabs, SiGERMab pairs and/or multiplexes described herein have significant advantages over known IHC techniques, including increased speed and efficiency (e.g., the ability to detect 4 or more target epitopes in a single section, limited only by the variety of available distinguishable labels). Further, SiGERMabs, SiGERMab pairs and/or multiplexes avoid the major disadvantages of existing approaches in that they can be produced by plasmid transfer to producer cell cultures once the recombinant sequence is prepared. Accordingly, large amounts can be readily produced. Moreover, because the unique signature peptide is already incorporated into each SiGERMab, further purification and chemical conjugation (which risks denaturation) is entirely avoided. For these reasons performance and reproducibility are increased compared to traditional methods, while simultaneously minimizing costs.

In addition, the methods described herein can be applied to the use of immune reagents of the same species origin as the epitopes (antigens) and tissues or cells being tested, since the specific binding of the secondary binding protein is directed to the unique signature peptide of the SiGERMab and not to any intrinsic sequence of the reagents or tissues, thereby avoiding background interference. For example, SiGERMabs of human origin can be used to detect human proteins ( e.g therapeutic antibodies) in human cells and tissues.

One of ordinary skill in the art will appreciate that starting materials, biological and chemical materials, biological and chemical reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue

experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this disclosure.

The terms and expressions which have been employed herein are 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. Thus, it should be understood that although aspects of the present invention have been specifically disclosed by various embodiments which may include preferred embodiments, exemplary embodiments and optional features, modifications and variations of the concepts herein disclosed may be resorted to by those skilled in the art. Such modifications and variations are considered to be within the scope of embodiments of the invention as described and as may be defined by the appended claims. Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below.

As used herein, "comprising" is synonymous with "including," "containing," or

"characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be optionally replaced with either of the other two terms, thus describing alternative aspects of the scope of the subject matter. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

The term "polypeptide" as used herein, refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

The term“antibody” as used to herein includes whole antibodies and any antigen binding fragments ( i.e .,“antigen-binding portions”) or single chains thereof. An“antibody” refers, in one embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed

complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (K D ) of 10 5 to 10 11 M or less. Any K D greater than about 10 4 M is generally considered to indicate nonspecific binding. As used herein, an antibody that "binds specifically" to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means having a K D of 10 7 M or less, preferably 10 8 M or less, even more preferably 5 x 10 9 M or less, and most preferably between 10 8 M and 10 10 M or less, but does not bind with high affinity to unrelated antigens.

An immunoglobulin may be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype is divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. In certain embodiments, the antibodies described herein are of the IgGl or IgG2 subtype. Immunoglobulins, e.g., IgGl, exist in several allotypes, which differ from each other in at most a few amino acids. "Antibody" includes, by way of example, both naturally occurring and non- naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies.

“Antigen binding site” refers to a binding site that comprises the VH and/or VL domain of an antibody, or at least one CDR thereof, provided that the antigen binding site binds specifically to its target antigen. For example, an antigen binding site may comprise, consist essentially of, or consist of a VHCDR3 alone or together with a VHCDR2 and optionally a VHCDR1. In certain embodiments, an antigen binding site comprises a VH domain and a VL domain, which may be present on the same polypeptide or on two different polypeptides, e.g., the VH domain is present on a heavy chain and a VL domain is present on a light chain.

“Antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be retained by fragments of a full-length antibody.

Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab’) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment which consists of a VH domain; and (vi) an isolated

Complementarity Determining Region (“CDR”). Furthermore, although VL and VH are two domains of an Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent proteins, known as single chain Fvs (scFvs) (see, e.g., U.S. Pat. No. 5,892,019). Such single chain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.

The term“epitope” or“antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody ( i.e ., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides 4 are tested for reactivity with the given antibody. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray

crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). The term

"epitope mapping" refers to the process of identification of the molecular determinants for antibody- antigen recognition.

"Binding affinity” refers to the strength of a binding interaction and includes both the actual binding affinity as well as the apparent binding affinity. The actual binding affinity is a ratio of the association rate over the disassociation rate. The apparent affinity can include, for example, the avidity resulting from a polyvalent interaction. Dissociation constant (Kd), is typically the reciprocal of the binding affinity, and may be conveniently measured using a surface plasmon resonance assay (e.g., as determined using a ForteBio Octet platform (Pall ForteBio Corp.), a BIACORE 3000 instrument (GE Healthcare) or a cell binding assay, examples of which assays are described in Example 3 of US Patent No. 7,846,440.

“Specific binding,”“specifically binds,”“selective binding,” and“selectively binds,” as well as“binds specifically”“binds selectively,” when referring to the binding of a binding site to its target epitope or a combination of binding sites to their target epitopes, means that the binding site(s) exhibit(s) immunospecific binding to the target epitope(s). A binding site that binds specifically to an epitope exhibits appreciable affinity for a target epitope and, generally, does not exhibit cross -reactivity with other epitopes in that it does not exhibit appreciable affinity to any unrelated epitope and preferably does not exhibit affinity for any unrelated epitope that is equal to, greater than, or within two orders of magnitude lower than the affinity for the target epitope. “Appreciable” or preferred binding includes binding with a dissociation constant (Kd) of 10 8 , 10 9 M, 10 10 , 10 11 , 10 12 M, 10 13 M or an even lower Kd value. The Kd values can also be indicated as l0e-8, lOe-9 M, etc. Note that lower values for Kd (dissociation constant) indicate higher binding affinity, thus a Kd of 10 7 is a higher Kd value than a Kd of 10 8 , but indicates a lower binding affinity than a Kd of 10 8 . Dissociation constants with values of about 10 7 M, and even as low as about 10 8 M, are at the high end of dissociation constants suitable for therapeutic antibodies. Binding affinities may be indicated by a range of dissociation constants, for example, l0 6 to 10 12 M, l0 7 to 10 12 M, l0 8 to lO 12 M or better (i.e., or lower value dissociation constant). Dissociation constants in the nanomolar (l0 9 M) to picomolar (l0 12 M) range or lower are typically most useful for therapeutic antibodies. Suitable dissociation constants are Kds of 50 nM or less (i.e., a binding affinity of 50 nM or higher - e.g., a Kd of 45 nM) or Kds of 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 100 pM, 10 pM or 1 pM or less. Specific or selective binding can be determined according to any art-recognized means for determining such binding, including, for example, according to Scatchard analysis, competitive binding assays, ELISA, flow cytometry, fluorescence microscopy, Western blotting). Methods for analyzing binding affinity, cross-reactivity, and binding kinetics include standard assays known in the art, for example, Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

"CDR" or "complementarity determining region" refers to the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et ah, J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et ah, Sequences of protein of immunological interest. (1991), and by Chothia et ah, J. Mol. Biol. 196:901-917 (1987) and by MacCallum et ah, J. Mol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other.

“CHI domain” refers to the heavy chain immunoglobulin constant domain located between the VH domain and the hinge. It spans EU positions 118-215. A CH1 domain may be a naturally occurring CH1 domain, or a naturally occurring CH1 domain in which one or more amino acids have been substituted, added or deleted, provided that the CH1 domain has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to the naturally occurring sequence.

“CH2 domain” refers to the heavy chain immunoglobulin constant domain that is located between the hinge and the CH3 domain. As defined here, it spans EU positions 237-340. A CH2 domain may be a naturally occurring CH2 domain, or a naturally occurring CH2 domain in which one or more amino acids have been substituted, added or deleted, provided that the CH2 domain has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.

“CH3 domain” refers to the heavy chain immunoglobulin constant domain that is located C-terminally of the CH2 domain and spans approximately 110 residues from the N-terminus of the CH2 domain, e.g., about positions 341 -446b (EU numbering system). A CH3 domain may be a naturally occurring CH3 domain, or a naturally occurring CH3 domain in which one or more amino acids have been substituted, added or deleted, provided that the CH3 domain has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain. A CH3 domain may or may not comprise a C-terminal lysine.

“CH4 domain” refers to the heavy chain immunoglobulin constant domain that is located C-terminally of the CH3 domain in IgM and IgE antibodies. A CH4 domain may be a naturally occurring CH4 domain, or a naturally occurring CH4 domain in which one or more amino acids have been substituted, added or deleted, provided that the CH4 domain has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.

“CL domain” refers to the light chain immunoglobulin constant domain that is located C- terminally to the VL domain. It spans about Kabat positions 107A-216. A CL domain may be a naturally occurring CL domain, or a naturally occurring CL domain in which one or more amino acids have been substituted, added or deleted, provided that the CL domain has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain. A CL domain may or may not comprise a C-terminal lysine.

A“constant region” or domain of a light chain of an immunoglobulin is referred to interchangeably as a“CL,”“light chain constant region domain,”“CL region” or“CL domain.” A“constant region” or domain on a heavy chain (e.g., hinge, CH1, CH2 or CH3 domains) of an immunoglobulin is referred to interchangeably as a“CH,”“heavy chain constant domain,”“CH” region or“CH domain.”

A variable domain on an immunoglobulin light chain is referred to interchangeably as a “VL,”“light chain variable domain,”“VL region” or“VL domain.” A variable domain on an immunoglobulin heavy chain is referred to interchangeably as a “VH,”“heavy chain variable domain,”“VH region” or“VH domain.”

“Domain” refers generally to a region, e.g., an independently folding, globular region or a non-globular region (e.g., a linker domain), of a heavy or light chain polypeptide which may comprise peptide loops (e.g., 1 to 4 peptide loops) that may be stabilized, for example, by a b- pleated sheet and/or an intrachain disulfide bond. The constant and variable regions of immunoglobulin heavy and light chains are typically folded into domains. In particular, each one of the CH1, CH2, CH3, CH4, CL, VH and VL domains typically form a loop structure.

“EU” indicates that amino acid positions in a heavy chain constant region, including amino acid positions in the CH1, hinge, CH2, and CH3 domains, are numbered herein according to the EU index numbering system (see Kabat et al., in“Sequences of Proteins of Immunological Interest”, U.S. Dept. Health and Human Services, 5 th edition, 1991).

“Fab” refers to the antigen binding portion of an antibody, comprising two chains: a first chain that comprises a VH domain and a CH1 domain and a second chain that comprises a VL domain and a CL domain. Although a Fab is typically described as the N-terminal fragment of an antibody that was treated with papain and comprises a portion of the hinge region, it is also used herein as referring to a binding domain wherein the heavy chain does not comprise a portion of the hinge.

“Fc region” refers to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e. residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the antibody. Accordingly, a complete Fc region comprises at least a hinge, a CH2 domain, and a CH3 domain. Two Fc regions that are dimerized are referred to as“Fc” or“Fc dimer.” An Fc region may be a naturally occurring Fc region, or a naturally occurring Fc region in which one or more amino acids have been substituted, added or deleted, provided that the Fc region has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.

“Framework region” or“FR” or“FR region” includes the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g., using the Kabat definition of CDRs). Therefore, a variable region framework is between about 100-120 amino acids in length but includes only those amino acids outside of the CDRs. For the specific example of a heavy chain variable region and for the CDRs as defined by Rabat et ah, 1991, ibid., framework region 1 corresponds to the domain of the variable region encompassing amino acids 1-30; framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49;

framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acids 103 to the end of the variable region. The framework regions for the light chain are similarly separated by each of the light chain variable region CDRs. Similarly, using the definition of CDRs by Chothia et al. or McCallum et al. the framework region boundaries are separated by the respective CDR termini as described above. In preferred embodiments, the CDRs are as defined by Rabat.

“Full length antibody” or“full length Ab” is an antibody (“Ab”) that comprises one or more heavy chains and one or more light chains, which optionally may be connected. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains CH1, CH2, and CH3, and optionally a fourth domain, CH4. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino- terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

Immunoglobulin proteins can be of any type or class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).

“Hinge” or“hinge region” or“hinge domain” refers to the flexible portion of a heavy chain located between the CH1 domain and the CH2 domain. It is approximately 25 amino acids long, and is divided into an“upper hinge,” a“middle hinge” or“core hinge,” and a“lower hinge.” A hinge may be a naturally occurring hinge, or a naturally occurring hinge in which one or more amino acids have been substituted, added or deleted, provided that the hinge has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to the naturally occurring sequence.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cells that are uncontaminated by other immunoglobulin producing cells. Alternative production methods are known to those trained in the art, for example, a monoclonal antibody may be produced by cells stably or transiently transfected with the heavy and light chain genes encoding the monoclonal antibody.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring engineering of the antibody by any particular method. The term "monoclonal" is used herein to refer to an antibody that is derived from a clonal population of cells, including any eukaryotic, prokaryotic, or phage clone, and not the method by which the antibody was engineered. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et ah, Nature, 256:495 (1975), or may be made by any recombinant DNA method (see, e.g., U.S. Pat. No. 4,816,567), including isolation from phage antibody libraries using the techniques described in Clackson et ah, Nature, 352:624-628 (1991) and Marks et ah, J. Mol. Biol., 222:581-597 (1991), for example. These methods can be used to produce monoclonal mammalian, chimeric, humanized, human, domain antibodies, single chain diabodies, vaccibodies, and linear antibodies.

The term "chimeric" antibodies includes antibodies in which at least one portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and at least one other portion of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

"Humanized" forms of nonhuman (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, FW region residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These

modifications are made to further refine antibody performance. In general, a humanized antibody heavy or light chain will comprise substantially all of at least one or more variable domains, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FWs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

A "human antibody" can be an antibody derived from a human or an antibody obtained from a transgenic organism that has been "engineered" to produce specific human antibodies in response to antigenic challenge and can be produced by any method known in the art. In certain techniques, elements of the human heavy and light chain loci are introduced into strains of the organism derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic organism can synthesize human antibodies specific for human antigens, and the organism can be used to produce human antibody- secreting hybridomas. A human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA. A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, or in vitro activated B cells, all of which are known in the art. identical” refers to two or more nucleic acid or polypeptide sequences or subsequences that are the same (100% identical) or have a specified percentage of nucleotide or amino acid residues that are the same, when the two sequences are aligned for maximum correspondence and compared. To align for maximum correspondence, gaps may be introduced into one of the sequences being compared. The amino acid residues or nucleotides at corresponding positions are then compared and quantified. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the sequences are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % identity= # of identical positions/total # of positions (e.g., overlapping positions) x 100). In certain

embodiments, the two sequences are the same length. The determination that one sequence is a measured % identical with another sequence can be determined using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for such comparison of two sequences is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program e.g., for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Additional algorithms for sequence analysis are well known in the art and many are available online.

The term "polynucleotide(s)" refers to nucleic acids such as DNA molecules and RNA molecules and analogs thereof (e.g., DNA or RNA generated using nucleotide analogs or using nucleic acid chemistry). As desired, the polynucleotides may be made synthetically, e.g., using art-recognized nucleic acid chemistry or enzymatically using, e.g., a polymerase, and, if desired, be modified. Typical modifications include methylation, biotinylation, and other art-known modifications. In addition, the nucleic acid molecule can be single-stranded or double- stranded and, where desired, linked to a detectable moiety.

The term "variant" with respect to a reference polypeptide refers to a polypeptide that possesses at least one amino acid mutation or modification (i.e., alteration) as compared to a native polypeptide. Variants generated by "amino acid modifications" can be produced, for example, by substituting, deleting, inserting and/or chemically modifying at least one amino acid in the native amino acid sequence. An "amino acid modification" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include an amino acid substitution, insertion and/or deletion.

An "amino acid modification at" a specified position, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. By insertion "adjacent" a specified residue is meant insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue.

An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different "replacement" amino acid residue. The replacement residue or residues may be "naturally occurring amino acid residues" (i.e. encoded by the genetic code) and selected from the group consisting of: alanine (Ala);

arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine (Lys);

methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein.

A "non-naturally occurring amino acid residue" refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in El 1m an et al. Meth. Enzym. 202:301 336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al.

Science 244:182 (1989) and El 1m an et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA.

An "amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present application contemplates larger "peptide insertions", e.g. insertion of about three to about five or even up to about ten amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

The term "mutagenesis" refers to, unless otherwise specified, any art recognized technique for altering a polynucleotide or polypeptide sequence. Preferred types of mutagenesis include error prone PCR mutagenesis, saturation mutagenesis, or other site directed mutagenesis.

"Site-directed mutagenesis" is a technique standard in the art, and is conducted using a synthetic oligonucleotide primer complementary to a single-stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the single-stranded phage DNA, and the resulting double-stranded DNA is transformed into a phage supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells that harbor the phage. Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. Plaques of interest are selected by hybridizing with kinased synthetic primer at a temperature that permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques that hybridize with the probe are then selected, sequenced and cultured, and the DNA is recovered.

1. Signature Peptides

Signature peptides which can be genetically fused to the heavy chain of the SiGERMabs described herein (e.g., via all or a portion for the Fc region), can be of any length or sequence, but must be recognizable by a secondary binding protein. In one embodiment, the signature peptide comprises naturally occurring amino acids. In another embodiment, the signature peptide comprises synthetic or non-natural amino acids.

The signature peptide can comprise a single set of amino acids. Alternatively, the signature peptide can comprise a tandem repeat of amino acids. In one embodiment, the signature peptide comprises two tandem repeats. In one embodiment, the signature peptide comprises three tandem repeats. In one embodiment, the signature peptide comprises four tandem repeats. In one embodiment, the signature peptide comprises five tandem repeats. In another embodiment, the signature peptide comprises six tandem repeats. In another

embodiment, the signature peptide comprises seven tandem repeats. In another embodiment, the signature peptide comprises eight tandem repeats. In another embodiment, the signature peptide comprises nine tandem repeats. In another embodiment, the signature peptide comprises ten tandem repeats. In another embodiment, the signature peptide comprises eleven tandem repeats. In another embodiment, the signature peptide comprises twelve tandem repeats. In another embodiment, the signature peptide comprises no more than six tandem repeats. In another embodiment, the signature peptide comprises no more than five tandem repeats. In another embodiment, the signature peptide comprises no more than four tandem repeats. In another embodiment, the signature peptide comprises no more than three tandem repeats. In another embodiment, the signature peptide comprises no more than two tandem repeats.

In embodiments where the signature peptide includes tandem repeats, the tandem repeats can be separated by a linker. A variety of linkers can be used. “Linked to” refers to direct or indirect linkage or connection of, in context, amino acids or nucleotides. “Linker” refers to one or more amino acids connecting two domains or regions together. Such linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 6448; Poljak, R. L, et al. (1994) Structure 2:1121-1123). Additional linkers suitable for use can be found in the Registry of Standard Biological Parts at http://partsregistry.org/Protein_ domains/Linker (see also, e.g., Crasto CJ and Feng JA. LINKER: a program to generate linker sequences for fusion proteins. Protein Eng 2000 May; 13(5) 309-12 and George RA and Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng 2002 Nov; 15(11) 871-9).

In one embodiment, the linker between the tandem repeats is three amino acids in length. In another embodiment, the linker between the tandem repeats is four amino acids in length. In another embodiment, the linker between the tandem repeats is five amino acids in length. In another embodiment, the linker between the tandem repeats is six amino acids in length. In another embodiment, the linker between the tandem repeats is seven amino acids in length. In another embodiment, the linker between the tandem repeats is eight amino acids in length. In another embodiment, the linker between the tandem repeats is nine amino acids in length. In another embodiment, the linker between the tandem repeats is ten amino acids in length. In another embodiment, the linker between the tandem repeats is eleven amino acids in length. In another embodiment, the linker between the tandem repeats is twelve amino acids in length. Any suitable linker can be used. In one embodiment, the linker comprises the amino acids sequence“GGGGS” (set forth in SEQ ID NO:6). Exemplary signature peptides are set forth in SEQ ID NO: 1 and SEQ ID NO: 7.

2. SiGERMabs

The antibody portion of the SiGERMabs can be generated using art-recognized

techniques. In one embodiment, for example, the DNA encoding heavy and light chains are isolated and cloned from a hybridoma producing an antibody of interest. In another embodiment, DNA encoding heavy and light chains are obtained directly from B-cells present in the lymphoid tissues (such as spleen or lymph node) or blood.

The heavy chain of the antibody is genetically (recombinantly) fused ( e.g .,“linked”) to a signature peptide (thereby generating a SiGERMab), e.g., using standard restriction enzyme based technology (e.g., as discussed in Example 2). In one embodiment, the signature peptide is fused to the Fc portion of the heavy chain. This Fc portion can be fused in-frame with a linker sequence. The linker sequence can in turn be fused to the signature peptide.

Accordingly, in one embodiment, the Fc portion of the heavy chain is genetically fused to a signature peptide, e.g., by one or more linkers. As discussed above,“linked to” refers to direct or indirect linkage or connection of, in context, amino acids or nucleotides. “Linker” refers to one or more amino acids connecting two domains or regions together. A linker can be 1-10, 10- 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or at least 90-100 amino acids long.

Typically, the linker is between 10 to 60 amino acids in length (e.g., 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids). In one embodiment, the linker is thirty to fifty amino acids in length. In one embodiment, the linker is twenty to sixty amino acids in length. In another embodiment, the linker is thirty to fifty amino acids in length. In another embodiment, the linker is forty amino acids in length.

In one embodiment, the heavy chain variable domain is fused to a complete Fc region. In another embodiment, the heavy chain is fused to a portion of an Fc region (e.g., CHI1 domain, the hinge, CH2 domain, CH3 domain, and/or CH4 domain).

The DNA encoding the heavy and light chains of the antibody of interest can be transfected into mammalian cells (such as HEK293 or CHO cells). Supernatants from transfected cells can be filtered and concentrated before affinity chromatography with protein A- sepharose. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined.

The SiGERMabs described herein are capable of binding one or more proteins (“target molecules”) of interest. Exemplary proteins of interest include, but are not limited to: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; Cl9orfl0 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-ld); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-l); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-l); CCL23 (MPIF-l); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-la); CCL4 (MIP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-lRB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR 13); CCR6 (CMKBR6/CKR-L3/STRL 22/DRY6); CCR7 (CKR7/EBI1); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1);

CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDHl(E-cadherin); CDH10;

CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p2l Wapl/Cipl); CDKN1B (p27Kipl);

CDKN1C; CDKN2A (pl6INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6;

CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); C-MET;

CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSFl(M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB

(cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GROl); CXCL10 (IP-10); CXCL1 l(I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3

(GPR9/CKR-L2) ; CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB21P; DES; DKFZp45U0l l8; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EGFR, EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENOl; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2;

FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF1; FGF12; FGF12B; FGF13; FGF14; FGF16;

FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-l); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GAT A3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HER; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-l; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2;

IL1RL1; IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB;

IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair- specific type II keratin); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III) ; MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4;

NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PC A3; PCNA; PDGF; PDGFA; PDGFB;

PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG;

PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p2lRac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROB02; S100A2; SCGB1D2 (lipophilin B); SCGB2Al(mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2;

SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-l); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Sprl); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFB1; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin- 1); THBS2; THBS4; THPO; TIE (Tie-l); TIGIT; TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A;

TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (0X40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase Iia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3 ; TRAF4; TRAF5; TRAF6; TREM1;

TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-lb); XCR1 (GPR5/CCXCR1); YY1; and ZFPM2.

3. SiGERMab Pairs

Also provided herein are pairs or complexes (also referred to as“SiGERMab pairs” or “SiGERMab complexes”) which include at least one SiGERMab (also referred to as the primary SiGERMab), and a respective secondary protein that binds to the signature peptide of the SiGERMab, as well as the use of such pairs and complexes as research, diagnostic and therapeutic agents. In one embodiment, the SiGERMab and secondary binding protein are from the same species. In another embodiment, the SiGERMab and secondary binding protein are from different species.

The secondary protein can be any suitable protein that binds the signature peptide of the primary recombinant protein ( e.g ., SiGERMab). In one embodiment, the secondary recombinant protein is an antibody, or antigen binding fragment thereof. In another embodiment, the secondary protein is a monoclonal antibody. In another embodiment, the secondary recombinant protein is a polyclonal antibody. In another embodiment, the secondary recombinant protein is a modified antibody (e.g., a multivalent antibody, such as IgM). In another embodiment, the secondary recombinant protein is a second recombinant antibody, or antigen binding fragment thereof, comprising a heavy chain genetically fused to a signature peptide (e.g., via the heavy chain Fc portion), i.e., a second SiGERMab that binds to the signature peptide of the primary recombinant antibody, or antigen binding fragment thereof.

The secondary protein can include a detectable moiety (e.g., a tag or label). Exemplary detectable moieties include, but are not limited to, a luminescent label, fluorescent label, radiolabel (e.g., 99 Tc, 45 Ti, 112 In, m In, ¾ 121 I, 125 I, 131 I, 14 C, 18 F, 36 Cl, 55 Co, 58 Co, 51 Cr, 67 Cu, 64 Cu, 66 Ga, 68 Ga, 76 Br, 89 Zr, 35 S, 32 P, 90 Y, 13 N, 15 0, 211 At, 186 Re, 188 Re, 75 Se), enzymatic label (e.g., hydrogen peroxidase, horseradish peroxidase, alkaline phosphatase, glucose oxidase, urease, acetylchoiinetransferase, luciferase, beta-galactosidase), epitope tag, chromophore label, phosphorescent label, photoaffinity molecule, ECL label, dye, biotin, or hapten. In one embodiment, the secondary protein includes an active site of an enzyme (e.g., peroxidase, alkaline phosphatase, or glucose oxidase), so as to directly convert a substrate added

subsequently.

4. SiGERMab Multiplexes

Also provided herein are multiplexes which include two or more SiGERMab pairs or SiGERMab complexes. In one embodiment, the multiplex comprises three SiGERMab pairs or complexes. In another embodiment, the multiplex comprises four SiGERMab pairs or

complexes. In one embodiment, the multiplex comprises five SiGERMab pairs or complexes. In one embodiment, the multiplex comprises six SiGERMab pairs or complexes. In one

embodiment, the multiplex comprises seven SiGERMab pairs or complexes. In one embodiment, the multiplex comprises eight SiGERMab pairs or complexes. In one embodiment, the multiplex comprises nine SiGERMab pairs or complexes. In one embodiment, the multiplex comprises ten SiGERMab pairs or complexes. In one embodiment, the multiplex comprises eleven SiGERMab pairs or complexes. In one embodiment, the multiplex comprises twelve SiGERMab pairs or complexes. In one embodiment, the multiplex comprises thirteen

SiGERMab pairs or complexes. In one embodiment, the multiplex comprises fourteen

SiGERMab pairs or complexes. In one embodiment, the multiplex comprises fifteen SiGERMab pairs or complexes. In another embodiment, each SiGERMab pair or complex binds to a different epitope. In another embodiment, each SiGERMab pair or complex includes a different signature peptide. Detection and interpretation of multiplexes can be achieved using known techniques, including digital scanning and computer based analysis ( e.g ., via commercially available software).

5. Nucleic Acids, Expression Vectors, Host Cells, and Methods of Production

Further provided herein are nucleic acids, e.g., DNA and RNA, encoding the

SiGERMabs as described herein, as well as expression vectors comprising such nucleic acids and host cells. Exemplary nucleotide sequences provided herein in Examples 1 and 2.

Nucleic acids, e.g., DNA, that comprise a nucleotide sequence that is at least about 70%, 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a nucleotide sequence encoding a polypeptide described herein or a nucleotide sequence set forth herein are also encompassed herein. Such nucleotide sequences may encode a recombinant protein set forth herein or may encode a recombinant protein that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical or similar to a recombinant protein set forth herein or a portion thereof (e.g., a domain).

In certain embodiments, a nucleotide sequence encoding a polypeptide is linked to a sequence that enhances or promotes the expression of the nucleotide sequence in a cell to produce a protein. Such nucleic acids may be encompassed within a vector, e.g., an expression vector.

The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a

"plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

For the purposes of being secreted, the constructs described herein preferably comprises a signal sequence, which is normally cut off after secretion to provide a mature polypeptide.

"Leader sequence," "signal peptide," or a "secretory leader," are terms are used interchangeably, contains a sequence comprising amino acid residues that directs the intracellular trafficking of the polypeptide to which it is a part. Polypeptides contain secretory leaders, signal peptides or leader sequences, typically at their N-terminus. These polypeptides may also contain cleavage sites where the leader sequences may be cleaved from the rest of the polypeptides by signal endopeptidases. Such cleavage results in the generation of mature polypeptides. Cleavage typically takes place during secretion or after the intact polypeptide has been directed to the appropriate cellular compartment.

"Transformation" refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.

The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Preferably host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. Preferred eukaryotic cells include protist, fungal, plant and animal cells. In one embodiment, HEK293 cells and CHO cells are used as host cells. Expression in NSO cells is described by, e.g., Barnes, L. M., et al, Cytotechnology 32 109-123 (2000); Barnes, L.M., et ah, Biotech. Bioeng. 73 261- 270 (2001). Transient expression is described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30 E9 (2002). Cloning of variable domains is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 3833-3837 (1989); Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 4285 - 4289 (1992); and Norderhaug, L., et al., J. Immunol. Methods 204 77-87 (1997). An exemplary transient expression system (HEK 293) is described by Schlaeger, E. -J., and Christensen, K., in

Cytotechnology 30 71-83 (1999) and by Schlaeger, E.-J., in J. Immunol. Methods 194 191-199 (1996).

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity. For the expression of the binding proteins in a host cell, nucleic acids encoding the respective polypeptides are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells (such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells), and the binding protein is recovered from the cells

(supernatant or cells after lysis). General methods for recombinant production of antibodies are well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif 17 183-202 (1999); Geisse, S., et al, Protein Expr. Purif. 8 271-282 (1996); Kaufman, R.J., Mol. Biotechnol. 16 151-161 (2000); Werner, R.G., Drug Res. 48 870- 880 (1998).

The recombinant proteins can be suitably separated from the culture medium by conventional purification procedures. Purification can be performed in order to eliminate cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et ah, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity

chromatography), ion exchange chromatography (e.g. cation exchange (carboxylmethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A. Appl. Biochem. Biotech. 75 93-102 (1998)). DNA and RNA encoding the recombinant proteins are readily isolated and sequenced using conventional procedures.

In another aspect, methods of producing SiGERMabs of the invention are provided by culturing a host cell as described above ( i.e ., transfected with a nucleic acid encoding the SiGERMab) in culture medium under conditions wherein the nucleic acid sequence is expressed, thereby producing the SiGERMab; and recovering the SiGERMab from the host cell or culture medium. In another embodiment, the SiGERMab is coexpressed with a light chain.

6. Kits

Also provided are kits (e.g., diagnostic kits) comprising one or more SiGERMabs or SiGERMab pairs, optionally with instructions for use. In one embodiment, the kit further includes a means for detecting the secondary binding protein (e.g., a substrate). The kits can include a label indicating the intended use of the contents of the kit and optionally include instructions for use of the kit in diagnosing or treating a disease. These kits may optionally comprise a detectable label, e.g. indicator enzymes, radiolabels, fluorophores, or paramagnetic particles. Kits may also include informative pamphlets, for example, pamphlets informing one how to use reagents to practice a method disclosed herein. The term "pamphlet" includes any writing, marketing materials or recorded material supplied on or with the kit, or which otherwise accompanies the kit. In order for the kits to produce accurate results and function as intended, it is critical that the epitopes present in the signature peptide are not present in the assay that is used.

7. Methods of Detection, Quantification, and Monitoring

Further provided are methods for using the SiGERMab, SiGERMab pair and/or multiplexes described herein as research and diagnostic agents, e.g., to detect, quantify, and/or monitor levels of one or more proteins of interest in biological sample from a patient. The protein of interest (e.g.,“target molecule”) can be any suitable molecule of interest, including, but not limited to, the exemplary molecules disclosed above.

The term "sample", as used herein, is used in its broadest sense. A "biological sample", as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, serum, urine, stool, sputum, synovial fluid, cerebrospinal fluid, supernatant from cell lysate, cells, organs, tissues, bone marrow, lymph nodes and spleen. Biological samples, can be obtained from the patient using routine methods, such as, but not limited to, biopsy, surgical resection, or aspiration. Biological samples for use in the methods described herein can be fresh, frozen, or fixed. In one embodiment, the biological sample is a human tissue sample (e.g., a tumor tissue sample). In another embodiment, the sample is a formalin-fixed paraffin-embedded sample or frozen tissue.

In one embodiment, methods of detecting a protein of interest in a biological sample from a patient are provided, comprising contacting the sample with a SiGERMab, SiGERMab pair and/or multiplex described herein, and detecting binding of the SiGERMab, SiGERMab pair and/or multiplex to the protein of interest within the sample.

Also provided are methods of detecting one or more proteins of interest in a biological sample from a patient, comprising contacting the sample with a SiGERMab multiplex described herein, and detecting binding of the SiGERMab pairs to the protein of interest within the sample.

Also provided are methods for quantifying levels of one or more proteins of interest in a biological sample from a patient are provided, comprising contacting the sample with a

SiGERMab, SiGERMab pair and/or multiplex described herein, detecting binding of the

SiGERMab, SiGERMab pair and/or multiplex to the one or more proteins of interest within the sample; and quantifying levels of the one more proteins of interest using known techniques ( e.g ., Quantifiable Internal Reference Standard technology, e.g., as described in US 8,785,150, the entire contents of which are expressly incorporated herein by reference).

Also provided are methods of monitoring expression levels of one or more proteins of interest in a patient are provided comprising: (a) detecting expression levels of the one or more proteins of interest in a biological sample obtained from the patient at a first time point by contacting the sample with a SiGERMab, SiGERMab pair and/or multiplex described herein, (b) detecting expression levels of the one or more proteins of interest in a biological sample of the same type from the patient obtained at a second time point using the SiGERMab, SiGERMab pair and/or multiplex used in step (a), and (c) comparing expression levels of the one or more proteins of interest determined at the first and second time points, to assess whether

expression levels are higher or lower at the first time point relative to the second time point.

Such methods can be used to assess whether a subject has a disease, is at (increased) risk of developing a disease, or whether a treatment regimen is efficient (e.g., determine regression, progression, and/or course of a disease (such as cancer)). For example, expression levels of one or more proteins of interest which are decreased compared to those in a biological sample taken earlier from the patient may indicate a regression, a positive course, e.g., a successful treatment, or a reduced risk for an onset of a disease in a patient. Alternatively, expression levels of one or more proteins of interest which are increased compared to those in a biological sample taken earlier from the patient may indicate a progression, a negative course, e.g., an unsuccessful treatment, recurrence or metastatic behavior, an onset or a risk for an onset of a disease in said patient.

In another embodiment, expression levels of one or more proteins of interest are compared to one more reference levels, wherein a deviation from said reference level is indicative of the presence and/or stage of a disease in a subject. The reference level may be a level as determined in a control sample (e.g., from a healthy tissue or subject, in particular a patient without a disease) or a median level from healthy subjects. A "deviation" from said reference level designates any significant change, such as an increase by at least 10%, 20%, or 30%, preferably by at least 40% or 50%, or even more.

In another embodiment, expression levels of one or more proteins of interest which are increased compared to one or more reference levels, e.g., compared to a patient without a disease, indicates the presence of or risk for ( i.e ., a potential for a development of) a disease in the patient. "Being at risk" means that a subject, i.e., a patient, is identified as having a higher than normal chance of developing a disease compared to the general population. In other embodiments, increased protein expression in a subject who has had, or who currently has, a disease, e.g., compared to the level of protein expression at a previous time point, is indicative of a subject who has an increased risk for reoccurrence of a disease, or as a subject who has an increased risk of continued development of a disease.

Expression levels of the one or more proteins of interest in the methods described herein can be detected via enzyme-linked immunosorbent assay (ELISA), immunofluorescence

(including all labeled antibody methods, such as particle labels and metal ions), flow cytometry, Western blot, lateral flow assay, and/or immunohistochemistry (IHC). ELISA,

immunofluorescence, and/or IHC can also be used to detect binding of the primary recombinant proteins to the one or more proteins of interest within a sample via the secondary recombinant proteins.

In one embodiment, detection is achieved by directly or indirectly labeling the secondary recombinant protein(s) with a label that provides for detection, e.g. indicator enzymes, radiolabels, fluorophores, or paramagnetic particles. For example, the one or more secondary recombinant protein(s) may be directly or indirectly bound to a label that functions to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g. FRET (Fluorescence Resonance Energy Transfer); (iii) affect mobility, e.g., electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) provide a capture moiety, e.g., affinity, antibody/antigen, or ionic

complexation. Suitable as label are structures, such as fluorescent labels, luminescent labels, chromophore labels, radioisotopic labels, isotopic labels, preferably stable isotopic labels, isobaric labels, enzyme labels, particle labels, in particular metal particle labels, magnetic particle labels, polymer particle labels, small organic molecules such as biotin, ligands of receptors or binding molecules such as cell adhesion proteins or lectins, label- sequences comprising nucleic acids and/or amino acid residues which can be detected by use of binding agents, etc. Exemplary detectable moieties include, but are not limited to, a luminescent label, fluorescent label, radiolabel (e.g., 99 Tc, 45 Ti, 112 In, m In, 3 H, 121 I, 125 I, 131 I, 14 C, 18 F, 36 Cl, 55 Co, 58 Co, 51 Cr, 67 Cu, ^Cu, 66 Ga, 68 Ga, 76 Br, 89 Zr, 35 S, 32 P, 90 Y, 13 N, 15 0, 211 At, 186 Re, 188 Re, 75 Se), enzymatic label ( e.g ., hydrogen peroxidase, horseradish peroxidase, alkaline phosphatase, glucose oxidase, urease aeetylcholinetransferase, luciferase, beta-galaetosidase), epitope tag, chromophore label, phosphorescent label, photoaffinity molecule, ECL label, dye, biotin, or hapten. In one embodiment, the secondary protein includes an active site of an enzyme (e.g., peroxidase, alkaline phosphatase, or glucose oxidase), so as to directly convert a substrate added subsequently.

In the methods described herein wherein multiple proteins of interest are detected, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more proteins can be detected. In one embodiment, the one or more proteins of interest are detected sequentially. In another embodiment, the one or more proteins of interest are detected simultaneously.

Protein expression detected by immunohistochemistry can be scored using art-recognized scoring methods. Non-limiting examples of scoring methods are described below. For example, in certain embodiments, expression is evaluated using the H-score (histochemical score) system, which is widely used in the art and is useful given its dynamic range and use of weighted percentiles. The H-score is a semiquantitative scoring system based on the formula: 3 x percentage of strongly staining cells (3+ staining) + 2 x percentage of moderately staining (2+ staining) cells + 1 x percentage of weakly staining (1+ staining) cells + 0 x percentage of non- stained (0 staining) cells, giving a score range of 0 to 300. See, e.g., McCarty et al., Cancer Res 1986;46:4244-8; Bosman et al., J Clin Pathol 1992;45:120-4; Dieset et al., Analyt Quant Cytol Histol 1996;18:351-4. Control tissue can include, e.g., matching non-tumor tissue from the same subject.

In other embodiments, expression is evaluated using the Allred scoring system (Allred et al ., Mod Pathol 1998;11:155-68; Harvey et al., / Clin Oncol 1999;17:1474-91). This scoring system involves adding proportion and intensity scores to obtain a total score. Accordingly, in some embodiments, the proportion score is obtained based on the estimated proportion of cells that are positive for the protein of interest (0: none, 1: <1/100, 2: 1/100 to 1/10, 3: 1/10 to 1/3; 4: 1/3 to 2/3; and 5: >2/3), and the intensity score is obtained based on the average intensity of protein expression in positive tumor cells (0: none, 1: weak, 2: intermediate, 3: strong). Thus, Allred scores range from 0 to 8, with a score ranging from 3 to 8 considered positive (i.e., positive detection). In other embodiments, the scoring system is automated, e.g., computerized, and quantified by image analysis. Automated methods are well known in the art. For example, an average threshold measure (ATM) score, which obtains an average of 255 staining intensity levels, can be calculated as described in, e.g., Choudhury et al., J Histochem Cytochem

2010;58:95-107, Rizzardi et al., Diagnostic Pathology 2012; 7:42-52. Another automated scoring system is AQUA ® (automated quantitative analysis), which is performed using, e.g., tissue microarrays (TMAs), on a continuous scale. AQUA ® is a hybrid of standard

immunohistochemistry and flow cytometry that provides an objective numeric score ranging from 1-255, and involves antigen retrieval, use of primary and secondary antibodies, and multiplexed fluorescent detection. Optimal cutoff points can be determined as described in Camp et al. Clin Cancer Res 2004;10:7252-9. The AQUA ® scoring system is described in detail in Camp et al., Nat Med 2002;8:1323-7; Camp et al., Cancer Res 2003;63: 1445-8; Ghosh et al., Hum Pathol 2008;39:1835-43; Bose et al., BMC Cancer 20l2;l2:332; Mascaux et al., Clin Cancer Res 201 l;l7:7796-807. Other suitable automated immunohistochemistry platforms include commercially available platforms, such as the Leica BOND RX staining platform. The platform detects protein expression on the basis of staining intensity on the following scale: minimal, <1 cells per 20x objective field; mild, 1~<10 cells per 20x objective field; moderate, l0~<50 cells per 20x objective field, marked, 50~<200 cells per 20x objective; and intense, >200 cells per 20x objective field.

8. Diagnostic Methods

Also provided are methods of diagnosing a patient as having a disease characterized by two or more proteins of interest, comprising (A) contacting a sample obtained from the patient with a SiGERMab, SiGERMab pair and/or multiplex described herein, and (B) diagnosing the patient as having the disease based on detection of binding of the SiGERMab, SiGERMab pair and/or multiplex to their respective target proteins within the sample. The protein(s) of interest (e.g.,“target molecule(s)”) can be any suitable molecule of interest, including, but not limited to, the exemplary molecules disclosed above. The term "sample", as used herein, is used in its broadest sense and includes the exemplary samples disclosed above.

In one embodiment, the disease is cancer. Exemplary cancers include, but are not limited to melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer ( e.g ., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

In another embodiment, the disease is an autoimmune disease. An“autoimmune disease” herein is a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom. Examples of autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis,

scleroderma (including systemic scleroderma ), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, allergic conditions, allergic reaction, eczema including allergic or atopic eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid

granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis (including large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa), microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, or hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia pemiciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis,

CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome,

Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune

polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody- mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic thrombocytopenic purpura (TTP) and autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis,

Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma- associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGETS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird- fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch- Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy,

Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia- reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture,

endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta , pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, infertility due to

antispermatozoan antibodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus- associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Leishmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed

hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis,

polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult- onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital

inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

In another embodiment, the disease is an infectious disease. In one embodiment, the infectious disease relates to an agent selected from the group consisting of: a virus, a bacterium, a fungus, and a protozoan parasite. Exemplary infectious diseases include, but are not limited to human immunodeficiency viruses, hepatitis viruses class A, B and C, Eppstein Barr virus, human cytomegalovirus, human papilloma viruses, herpes viruses, leishmaniasis, toxoplasmosis, cryptosporidiosis, sleeping sickness, and malaria.

In one embodiment, the methods described herein can be used to assess“clonality” in hematopathology specimens for the purpose of distinguishing between reactive cell populations ( e.g ., polyclonal lymphocytes and/or plasma cells) and malignant populations (e.g., monoclonal cells). For example, the methods can be used to assist in early diagnosis of multiple myeloma (monoclonal) in bone marrow specimens by IHC and recognition of B cell lymphoma versus reactive lymphoid hyperplasia by IF or IHC. In another embodiment, the methods described herein can be used to distinguish rare tumor cells from reactive cells in tissue sections, including micro-metastases in sentinel lymph nodes. In another embodiment, the assessment is performed using a single section of tissue, thereby conserving tissue for other assay approaches.

In another embodiment, the methods described herein can be used to identify key diagnostic cells present in low proportion in a tissue section. For example, the methods can be used to identify Reed Sternberg cells in seeking to achieve a diagnosis of Hodgkin lymphoma.

In another embodiment, the methods described herein can be used to distinguish between non-invasive Prostatic Intraepithelial Neoplasia (PIN) and invasive cancer.

In another embodiment, the methods described herein can be used to identify triple negative breast cancer (ER, PR, HER2), including the identification of sub-types Fuminal A, B, triple negative and HER2 for prognostic and therapeutic purposes.

In another embodiment, the methods described herein can be used to identify target cell expression, e.g., PD-F1 expression.

In another embodiment, the methods described herein can be used to identify target cell co-expresslon. In another embodiment, the methods described herein can be used to measure the distance between cells and/or identify cellular subsets that require multiple markers for classification.

In another embodiment, the methods described herein can be used to identify the immune profile of a tumor in consideration of an immunotherapy approach ( e.g ., stimulation (vaccine) versus blockade (PD-l, PD-L1)).

In another embodiment, the methods described herein can be used to identify the cellular location of hormone production in lesions of endocrine glands (e.g., pituitary, islets cells and/or thyroid) with the goal of separating glandular enlargement due to hyperplasia from benign (adenoma) or malignant neoplasia.

In another embodiment, the methods described herein can be used to identify and classify anaplastic tumor cells or tumors of unknown cellular origin.

In another embodiment, the methods described herein can be used to identify metastatic and recurrent tumor populations at low levels in bone marrow. In another embodiment, the assessment is performed using a single section of tissue, thereby conserving tissue for other assay approaches.

In another embodiment, the methods described herein can be used to improve the accuracy of scoring markers, such as Ki67 in tumor cells, where the section also contains other Ki67 positive cells (e.g., a malignant lymphoma where reactive T and B cells also express Ki67 and cannot be distinguished from the lymphoma cells in the absence of a second label that aids in identification of the lymphoma cells).

In another embodiment, the methods described herein can be used to detect biomarkers present on circulating tumor cells in conjunction with flow cytometry.

In another embodiment, the methods described herein can be applied to the use of immune reagents of the same species origin as the epitopes (antigens) and tissues or cells being tested, since the specific binding of the secondary detection reagent is directed to the unique signature peptide and not any intrinsic sequence of the reagents or tissues, thereby avoiding background interference. For example, SiGERmabs of human origin can be used to detect human proteins (e.g., therapeutic antibodies) in human cells and tissues.

All references cited throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference. Any sequence listing and sequence listing information is considered part of the disclosure herewith.

The following examples should not be construed as limiting the scope of this disclosure.

EXAMPLES

Example 1: Identification of Signature Peptide Sequences.

Signature peptide sequences suitable for incorporation into the SiGERMabs described herein were designed and selected to ensure that: (1) the epitopes present within the signature peptide sequence are not present in the assay of interest; and (2) the signature peptide is specifically recognized by a secondary recombinant protein (e.g., antibody).

DNA encoding signature peptide sequences were formulated so that the signature peptides are in tandem and therefore, readily available and able to promote higher affinity binding than a single signature peptide alone. However, single signature peptides can also be used.

An exemplary signature peptide that was generated is“Signature Peptide A”. The amino acid sequence of Signature Peptide A is set forth in uppercase letters below (SEQ ID NO: 1), and the encoding DNA sequence in lowercase letters below (SEQ ID NO: 2) flanked by restriction sites. The BglII sites are underlined (SEQ ID NO: 3) and BamHI site (SEQ ID NO: 4) is in bold:

Signature Peptide A:

accaaatctaaacaaaaataaatccaacaaacaaaaaaacaacaatacaaacaaaacaaa a

P D L G G G G S S E Q K S N D A S E A G

ggcggtgggtccagcgagcagaagagcaacgatgcaagcgaagcaggtggtggaggttcc

G G G S S E Q K S N D A S E A G G G G S

agcgaacaaaaaagcaacgacgcgagcgaggcgggaggaggaggctccagcgaacagaag

S E Q K S N D A S E A G G G G S S E Q K

agcaacgatgccagcgaagcggatcctgaagatctgcg (SEQ ID NO: 2)

S N D A S E A D P E D L (SEQ ID NO: 1)

In this exemplary“Signature Peptide A”, the amino acid sequence SEQKSNDASEA (SEQ ID NO: 5) appears as four tandem repeats separated by short linkers having the amino acid sequence GGGGS (SEQ ID NO: 6).

Another exemplary signature peptide that was generated is“Signature Peptide B”. The amino acid sequence of Signature Peptide B is set forth in uppercase letters below (SEQ ID NO: 7), and the encoding DNA sequence in lowercase letters below (SEQ ID NO: 8) flanked by restriction sites. The BglII sites are underlined (SEQ ID NO: 3) and BamHI site (SEQ ID NO: 4) is in bold:

Signature Peptide B:

gccagatctaggcggaggtgggtcccaggatcagctgctggcgattagcaacgcggatct g

P D L G G G G S Q D Q L L A I S N A D L

ggctggcatattagcccgagctttaaagatggcggaggcgggtcccaggatcagctgctg

G W H I S P S F K D G G G G S Q D Q L L

gcgattagcaacgcggatctgggctggcatattagcccgagctttaaagatggcggaggt

A I S N A D L G W H I S P S F K D G G G

gggtcccaggatcagctgctggcgattagcaacgcggatctgggctggcatattagcccg

G S Q D Q L L A I S N A D L G W H I S P

agctttaaagatggcggaggcgggtcccaggatcagctgctggcgattagcaacgcggat

S F K D G G G G S Q D Q L L A I S N A D

ctgggctggcatattagcccgagctttaaagatggggatccacjcitctgcg (SEQ ID NO: 8) L G W H I S P S F K D G D P D L (SEQ ID NO: 7)

In this exemplary“Signature Peptide B”, the amino acid sequence

QDQLLAISNADLGWHISPSFKD (SEQ ID NO:9) appears as four tandem repeats separated by short linkers having the amino acid sequence GGGGS (SEQ ID NO:6).

Another exemplary signature peptide that was generated is“Signature Peptide C”. The amino acid sequence of Signature Peptide C is set forth in uppercase letters below (SEQ ID NO: 10), and the encoding DNA sequence in lowercase letters below (SEQ ID NO: 11) flanked by restriction sites. The BglII sites are underlined (SEQ ID NO: 3) and BamHI site (SEQ ID NO: 4) is in bold:

Signature Peptide C:

gccagatctaggcggaggtgggtccggcaaaaaagctctgcgtattcatagcgttgaagg t

P D L G G G G S G K K A L R I H S V E G

gaactgcgccgcaaaagcgcgggccaggaagaatggagcgggggcggcggttctggcaag

E L R R K S A G Q E E W S G G G G S G K

aaggccctacgcattcacagtgtcgagggcgaactacgtcgtaagagcgctggccaagag

K A L R I H S V E G E L R R K S A G Q E

gagtggagcggcggaggtggatcgggcaaaaaggcactccggattcacagcgtagaagga

E W S G G G G S G K K A L R I H S V E G

gaactccgacgaaagagtgccggccaggaagagtggagcggcggtggaggctcgggcaag

E L R R K S A G Q E E W S G G G G S G K

aaagcgcttcggattcatagcgtggagggggaacttcggcggaagagcgcaggccaagaa

K A L R I H S V E G E L R R K S A G Q E

gagtggagcgaggatccagatctgcg (SEQ ID NO: 11)

E W S E D P D L (SEQ ID NO: 10)

In this exemplary“Signature Peptide C”, the amino acid sequence

GKKALRIHS VEGELRRKS AGQEEW S (SEQ ID NO: 12) appears as four tandem repeats separated by short linkers having the amino acid sequence GGGGS (SEQ ID NO:6). Example 2: Genetic Engineering of Primary Recombinant Antibodies to Include Signature Peptide Sequences (SiGERMabs).

Primary antibodies were generated via standard recombinant techniques ( e.g ., as discussed above). These antibodies are specific for the epitope of interest and do not cross-react with other epitopes.

SiGERMabs were then generated using standard restriction enzyme based technology. The DNA encoding the Fc portion of the heavy chain fused to a long linker sequence (SEQ ID NO: 16) containing a Pmel restriction site at the 5’ end and a BamHI restriction site present at the 3’ end was synthesized using gene synthesis technology. This fragment was digested with Pmel and BamHI restriction enzymes and cloned into a mammalian expression vector to create an Fc/linker construct. DNA encoding Signature Peptide fragments with flanking BglII sites were digested using the BglII restriction enzyme and these fragments were cloned into the BamHI site of the vector containing the Fc/linker construct since BglII and BamHI share cohesive ends. The vectors were then subjected to DNA sequencing, and those plasmids containing the Signatures Peptides in-frame with the heavy chain Fc portion were selected to create the Fc/linker/Signature Peptide constructs. Nucleic acids encoding heavy chain variable domains were then inserted in-frame with the Fc/linker/Signature Peptide constructs using standard restriction and ligation DNA cloning to create the SiGERMab heavy chain constructs. To create a single SiGERMab, a single heavy chain construct and the corresponding unmodified light chain construct (not identified herein) are co-transfected into a mammalian cell line.

Consequently, the SiGERMab comprising of a modified heavy chain containing a Signature peptide sequence and an unmodified light chain are coexpressed and assembled to form a fully functional antibody.

“Primary Antibody 1 A” (also known as“SiGERMab 1A”) is an exemplary heavy chain constant domain of a SiGERMab that was generated which, when co-expressed with the corresponding unmodified light chain, recognizes Epitope 1 and contains Signature Peptide A genetically fused to the Fc region, as set forth below.

Primary Antibody 1A Heavy Chain Constant Domain:

accctggtcaccgtctccttagggcaacctaaggctccatcagtcttcccactggcc ccc

T L V T V S L G Q P K A P S V F P L A P

tgctgcggggacacacccagctccacggtgaccctgggctgcctggtcaaaggctac ctc

C C G D T P S S T V T L G C L V K G Y L

ccggagccagtgaccgtgacctggaactcgggcaccctcaccaatggggtacgcacc ttc P E P V T V T W N S G T L T N G V R T F

ccgtccgtccggcagtcctcaggcctctactcgctgagcagcgtggtgagcgtgacc tca

P S V R Q S S G L Y S L S S V V S V T S

agcagccagcccgtcacctgcaacgtggcccacccagccaccaacaccaaagtggac aag

S S Q P V T C N V A H P A T N T K V D K

accgttgcgccctcgacatgcagcaagcccacgtgcccaccccctgaactcctgggc ggc

T V A P S T C S K P T C P P P E L L G G

cgctctgtcttcatcttccccccaaaacccaaggacaccctcatgatctcacgcacc ccc

R S V F I F P P K P K D T L M I S R T P

gaggtcacatgcgtggtggtggacgtgagccaggatgaccccgaggtgcagttcaca tgg

E V T C V V V D V S Q D D P E V Q F T W

tacataaacaacgagcaggtgcgcaccgcccggccgccgctacgggagcagcagttc aac

Y I N N E Q V R T A R P P L R E Q Q F N

agcacgatccgcgtggtcagcaccctccccatcgcgcaccaggactggctgaggggc aag

S T I R V V S T E P I A H Q D W L R G K

gagttcaagtgcaaagtccacaacaaggcactcccggcccccatcgagaaaaccatc tcc

E F K C K V H N K A L P A P I E K T I S

aaagccagagggcagcccctggagccgaaggtctacaccatgggccctccccgggag gag

K A R G Q P L E P K V Y T M G P P R E E

ctgagcagcaggtcggtcagcctgacctgcatgatcaacggcttctacccttccgac atc

L S S R S V S L T C M I N G F Y P S D I

tcggtggagtgggagaagaacgggaaggctgaggacaactacaagaccacgccgacc gtg

S V E W E K N G K A E D N Y K T T P T V

ctggacagcgacggctcctacttcctctacagcaagctctcagtgcccacgagtgag tgg

L D S D G S Y F L Y S K L S V P T S E W

cagcggggcgacgtcttcacctgctccgtgatgcacgaggccttgcacaaccactac acg

Q R G D V F T C S V M H E A L H N H Y T

cagaagtccatctcccgctctccgggtaagcctcaaccccaaccacaaccgcaacct cag

Q K S I S R S P G K P O P O P O P O P O

ccccagccacagccgcagccgcagccacaaccccagcctcaaccacaaccccagcct caa

P O P O P O P O P O P O P O P O P O P O

cctcagtcggatctaggcggaggtgggtccagcgaacagaaaagcaacgatgcgagc gaa

P O S D L G G G G S S E Q K S N D A S E

gcgggaggcggtgggtccagcgagcagaagagcaacgatgcaagcgaagcaggtggt gga

caaaaaaacaacaataccaacaaaacaaatcccccaacctcaacctctaactaa (SEQ ID NO: 14)

Q K S N D A S E A D P P T S T S G (SEQ ID NO: 13)

The amino acid and nucleotide sequences of the heavy chain constant domain of Primary Antibody 1A are set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The amino acid sequence of the Fc portion of the heavy chain is in italics (SEQ ID NO: 15). The linker sequence between the FC portion and Signature Peptide A is underlined (SEQ ID NO: 16). The amino acid sequence of Signature Peptide A is in bold (SEQ ID NO: 17), and the amino acid sequence SEQKSNDASEA (SEQ ID NO: 5) appears as four tandem repeats in bold and italics, separated by short linkers having the amino acid sequence GGGGS (SEQ ID NO:6), in bold and underlined. The nucleotide sequence (lowercase) that is underlined and in bold denotes the BamHI restriction site (SEQ ID NO:4). In other embodiments, iterative clonings of BglII fragments containing the Signature Peptide sequence can result in very long successions of Signature Peptide sequences in tandem. In addition, if multiple and different Signature Peptides are desired, a second Signature Peptide moiety can be inserted at this site.

A second example of a heavy chain of a SiGERMab is“Primary Antibody 2B” (also known as“SiGERMab 2B”). When the Primary Antibody 2B heavy chain is co-expressed with the corresponding unmodified light chain, it recognizes Epitope 2 and contains the Signature Peptide B genetically fused to the Fc region, as set forth below.

Primary Antibody 2B Heavy Chain Constant Domain:

accctggtcaccgtctcctcagggcaacctaaggctccatcagtcttcccactggcc ccc

T L V T V S S G Q P K A P S V F P L A P

tgctgcggggacacacccagctccacggtgaccctgggctgcctggtcaaaggctac ctc

C C G D T P S S T V T L G C L V K G Y L

ccggagccagtgaccgtgacctggaactcgggcaccctcaccaatggggtacgcacc ttc

P E P V T V T W N S G T L T N G V R T F

ccgtccgtccggcagtcctcaggcctctactcgctgagcagcgtggtgagcgtgacc tca

P S V R Q S S G L Y S L S S V V S V T S

agcagccagcccgtcacctgcaacgtggcccacccagccaccaacaccaaagtggac aag

S S Q P V T C N V A H P A T N T K V D K

accgttgcgccctcgacatgcancaagcccatgtgcccaccccctgaactcctgggc ggc

T V A P S T C X K P M C P P P E L L G G

cgctctgtcttcatcttccccccaaaacccaaggacaccctcatgatctcacgcacc ccc

R S V F I F P P K P K D T L M I S R T P

gaggtcacatgcgtggtggtggacgtgagccaggatgaccccgaggtgcagttcaca tgg

E V T C V V V D V S Q D D P E V Q F T W

tacataaacaacgagcaggtgcgcaccgcccggccgccgctacgggagcagcagttc aac

Y I N N E Q V R T A R P P L R E Q Q F N

agcacgatccgcgtggtcagcaccctccccatcgcgcaccaggactggctgaggggc aag

S T I R V V S T L P I A H Q D W L R G K

gagttcaagtgcaaagtccacaacaaggcactcccggcccccatcgagaaaaccatc tcc

E F K C K V H N K A L P A P I E K T I S

aaagccagagggcagcccctggagccgaaggtctacaccatgggccctccccgggag gag

K A R G Q P L E P K V Y T M G P P R E E

ctgagcagcaggtcggtcagcctgacctgcatgatcaacggcttctacccttccgac atc

L S S R S V S L T C M I N G F Y P S D I

tcggtggagtgggagaagaacgggaaggctgaggacaactacaagaccacgccgacc gtg

S V E W E K N G K A E D N Y K T T P T V

ctggacagcgacggctcctacttcctctacagcaagctctcagtgcccacgagtgag tgg

L D S D G S Y F L Y S K L S V P T S E W

cagcggggcgacgtcttcacctgctccgtgatgcacgaggccttgcacaaccactac acg

Q R G D V F T C S V M H E A L H N H Y T

cagaagtccatctcccgctctccgggtaagcctcaaccccaaccacaaccgcaacct cag

Q K S I S R S P G K P Q P Q P Q P Q P Q

ccccagccacagccgcagccgcagccacaaccccagcctcaaccacaaccccagcct caa

P Q P Q P Q P Q P Q P Q P Q P Q P Q P Q

cctcagtcggatctaggcggaggtgggtcccaggatcagctgctggcgattagcaac gcg

P Q S D L G G G G S Q D Q L L A I S N A

gatctgggctggcatattagcccgagctttaaagatggcggaggcgggtcccaggat cag

D L G W H I S P S F K D G G G G S Q D Q

ctgctggcgattagcaacgcggatctgggctggcatattagcccgagctttaaagat ggc

L L A I S N A D L G W H I S P S F K D G

ggaggtgggtcccaggatcagctgctggcgattagcaacgcggatctgggctggcat att

G G G S Q D Q L L A I S N A D L G W H I

agcccgagctttaaagatggcggaggcgggtcccaggatcagctgctggcgattagc aac

S P S F K D G G G G S Q D Q L L A I S N

gcggatctgggctggcatattagcccgagctttaaagatggggatccagatcccccg acc

A D L G W K D G D P D P P T

tcgacctctggctaa S T S G (SEQ ID NO: 18)

The amino acid and nucleotide sequences for Primary Antibody 2B are set forth in SEQ ID NO: 18 and SEQ ID NO: 19, respectively. The amino acid sequence of the Fc portion of the heavy chain is in italics (SEQ ID NO:20). The linker sequence between the Fc portion and Signature Peptide B is underlined (SEQ ID NO: 16). The amino acid sequence of Signature Peptide B is in bold(SEQ ID NO: 21), and the amino acid sequence

QDQLLAISNADLGWHISPSFKD (SEQ ID NO: 9) appears as four tandem repeats in bold and italics, separated by short linkers having the amino acid sequence GGGGS (SEQ ID NO:6) in bold and underlined.

A third example of a heavy chain of a SiGERMab is“Primary Antibody 3C” (also known as“SiGERMab 3C”). When the Primary Antibody 3C heavy chain is co-expressed with the corresponding unmodified light chain, it recognizes Epitope 3 and contains the Signature Peptide C genetically fused to the Fc region, as set forth below.

Primary Antibody 2B Heavy Chain Constant Domain:

accctggtcaccgtctccttagggcaacctaaggctccatcagtcttcccactggcc ccc

T L V T V S L G Q P K A P S V F P L A P

tgctgcggggacacacccagctccacggtgaccctgggctgcctggtcaaaggctac ctc

C C G D T P S S T V T L G C L V K G Y L

ccggagccagtgaccgtgacctggaactcgggcaccctcaccaatggggtacgcacc ttc

P E P V T V T W N S G T L T N G V R T F

ccgtccgtccggcagtcctcaggcctctactcgctgagcagcgtggtgagcgtgacc tca

P S V R Q S S G L Y S L S S V V S V T S

agcagccagcccgtcacctgcaacgtggcccacccagccaccaacaccaaagtggac aag

S S Q P V T C N V A H P A T N T K V D K

accgttgcgccctcgacatgcagcaagcccacgtgcccaccccctgaactcctgggc ggc

T V A P S T C S K P T C P P P E L L G G

cgctctgtcttcatcttccccccaaaacccaaggacaccctcatgatctcacgcacc ccc

R S V F I F P P K P K D T L M I S R T P

gaggtcacatgcgtggtggtggacgtgagccaggatgaccccgaggtgcagttcaca tgg

E V T C V V V D V S Q D D P E V Q F T W

tacataaacaacgagcaggtgcgcaccgcccggccgccgctacgggagcagcagttc aac

Y I N N E Q V R T A R P P L R E Q Q F N

agcacgatccgcgtggtcagcaccctccccatcgcgcaccaggactggctgaggggc aag

S T I R V V S T L P I A H Q D W L R G K

gagttcaagtgcaaagtccacaacaaggcactcccggcccccatcgagaaaaccatc tcc

E F K C K V H N K A L P A P I E K T I S

aaagccagagggcagcccctggagccgaaggtctacaccatgggccctccccgggag gag

K A R G Q P L E P K V Y T M G P P R E E

ctgagcagcaggtcggtcagcctgacctgcatgatcaacggcttctacccttccgac atc

L S S R S V S L T C M I N G F Y P S D I

tcggtggagtgggagaagaacgggaaggctgaggacaactacaagaccacgccgacc gtg

S V E W E K N G K A E D N Y K T T P T V

ctggacagcgacggctcctacttcctctacagcaagctctcagtgcccacgagtgag tgg

L D S D G S Y F L Y S K L S V P T S E W

cagcggggcgacgtcttcacctgctccgtgatgcacgaggccttgcacaaccactac acg

Q R G D V F T C S V M H E A L H N H Y T

cagaagtccatctcccgctctccgggtaagcctcaaccccaaccacaaccgcaacct cag

Q K S I S R S P G K P Q P Q P Q P Q P Q

ccccagccacagccgcagccgcagccacaaccccagcctcaaccacaaccccagcct caa P Q P Q P Q P Q P Q P Q P Q P Q P Q P Q

cctcagtcggatctaccagatctaggcggaggtgggtccggcaaaaaagctctgcgt att

P Q S D L P D L G G G G S G K K A L R I

catagcgttgaaggtgaactgcgccgcaaaagcgcgggccaggaagaatggagcggg ggc

H S V E G E L R R K S A G Q E E W S G_G

ggcggttctggcaagaaggccctacgcattcacagtgtcgagggcgaactacgtcgt aag

G G S G K K A L R I H S V E G E L R R K

agcgctggccaagaggagtggagcggcggaggtggatcgggcaaaaaggcactccgg att

S A G Q E E W S G G G G S G K K A L R I

cacagcgtagaaggagaactccgacgaaagagtgccggccaggaagagtggagcggc ggt

H S V E G E L R R K S A G Q E E W S G_G

ggaggctcgggcaagaaagcgcttcggattcatagcgtggagggggaacttcggcgg aag

K K H V E K

agcgcaggccaagaagagtggagcgaggatccagatcccccgacctcgacctctggc taa

S A G Q E E W S E D P D P P T S T S G ( SEQ ID NO: 22) - taa (SEQ ID NO: 23)

The amino acid and nucleotide sequences for Primary Antibody 3Care set forth in SEQ ID NO:22 and SEQ ID NO:23, respectively. The Fc portion of the heavy chain is in italics (SEQ ID NO:24). The linker sequence between the Fc portion and Signature Peptide C is underlined (SEQ ID NO: 16). The amino acid sequence of Signature Peptide C is in bold (SEQ ID NO: 25), and the amino acid sequence GKKALRIHSVEGELRRKSAGQEEWS (SEQ ID NO: 12) appears as four tandem repeats in bold and italics, separated by short linkers having the amino acid sequence GGGGS (SEQ ID NO:6) in bold and underlined.

Expression of the modified heavy chains with their respective unmodified light chains to produce Primary Antibodies 1A, 2B, and 3C was achieved using a transient transfection method. Products obtained from these transfections were then affinity purified. Labeled Secondary Antibodies A, B, and C (see below) were used to detect Primary Antibodies 1A, 2B, and 3C, respectively.

Example 3: Development of Labeled Secondary Antibodies That Recognize Signature Peptide Sequences

Secondary antibodies were raised against the signature peptides. Suitable secondary antibodies can be either polyclonal or monoclonal in nature. In this example, recombinant monoclonal antibodies were generated that specifically recognize Signature Peptides A, B and C. These antibodies were designated Secondary Antibody A , Secondary Antibody B , and

Secondary Antibody C, respectively. Commercially available products consisting of activated forms of AP or HRP that is readily available for conjugation were used according to

manufacturer’s instructions (Biotium, Cat. No.: 92314 (AP) and Cat. No.: 92301 (HRP)), were used to label these secondary antibodies with a detectable moiety. Example 4: Binding Experiments

A number of assays can be used to determine if the Primary and Secondary Antibodies bind to their specific targets. In this example, ELISA or IHC are used to confirm that: (A)

Primary Antibodies 1A, 2B, and 3C bind to their specific targets, 1, 2, and 3, respectively, and (B) the labeled Secondary Antibodies A, B, and C recognize Primary Antibodies 1A, 2B, and 3C specifically and respectively.

A. Transfections

To confirm binding, a number of transfections are completed and the secreted antibodies are collected. The following genes are co-transfected into HEK293 cells using 293Fectin reagent:

A. Heavy and Light chains encoding Primary Antibody 1A (also known as“SiGERMab

1A”) which includes Signature Peptide A and recognizes Epitope 1.

B. Heavy and Light chains encoding Primary Antibody 2B (also known as“SiGERMab 2b”) which includes Signature Peptide B and recognizes Epitope 2.

C. Heavy and Light chains encoding Primary Antibody 3C (also known as“SiGERMab

3C”) which includes Signature Peptide C and recognizes Epitope 3.

D. Heavy and Light chains encoding“original Monoclonal Antibody 1”, which does not contain Signature Peptide A and recognizes Epitope 1.

E. Heavy and Light chains encoding“original Monoclonal Antibody 2”, which does not contain Signature Peptide B and recognizes Epitope 2.

F. Heavy and Light chains encoding“original Monoclonal Antibody 3”, which does not contain Signature Peptide C and recognizes Epitope 3.

G. Heavy and Light chains encoding“Secondary Monoclonal Antibody A”, which

recognizes“Signature Peptide A” on Primary Antibody 1A (SiGERMab 1A).

H. Heavy and Light chains encoding“Secondary Monoclonal Antibody B”, which recognizes “Signature Peptide B” on Primary Antibody 2B (SiGERMab 2B).

I. Heavy and Light chains encoding“Secondary Monoclonal Antibody C”, which recognizes “Signature Peptide C” on Primary Antibody 3C (SiGERMab 3C). Supernatants from these samples are collected and the secreted antibodies present within these supernatants are either tested by ELISA and/or IHC directly, and/or Protein A purified and then tested by ELISA and/or IHC. B. ELISA Experiments:

For the ELISA experiments, Epitope 1 and 2 are referred to as Peptide 1 and Peptide 2, respectively. ELISA testing is performed with standard procedures using the supernatants obtained from transfections listed above (A-I). Serially diluted samples from transfections A, B, C, D, A+B, and C+D are tested against Peptide 1, Peptide 2, and Peptide 1+2 to test for specificity. Secondary antibodies are labeled by two different flourophores (e.g., Dylight 488 (Green) and Dylight 649 (Far Red)), to enable multiplex analysis by ELISA. If a plate reader that detects fluorescence is not available, the Secondary antibodies are labeled with Horse Radish Peroxidase (HRP).

The layout for the ELISA experiment is set forth below in Table 1. Row B identifies the Peptide moiety used to coat the ELISA plates. Row C identifies the Primary Antibody samples used in the experiment. Rows D, E, F, and G identify the secondary antibody used in the ELISA experiments. In Row D, HRP-Donkey anti-Rabbit Ig (H+L) are used as positive control secondary. If a fluorescence plate reader is not available, then HRP labeled Secondary

Antibodies is used. Fields D3 to G20 indicate the expected ELISA results.

Table 1: Layout for ELISA Experiment

Example 5: Multiplex IHC Using SiGERMAb-Specific Labeled Secondary Antibodies

Multiplex Immunohistochemistry (IHC) was conducted on tissues wherein Epitope 2 (present on the surface of particular cancer cells) and Epitope 3 (present on the surface of T- cells) are known to be present based on previous control experiments conducted with MAb 2 and MAb 3, respectively. Secondary Antibodies B and C were labeled enzymatically with HRP or Alkaline Phosphatase (AP).

Experiments were performed to show that both SiGERMab pairs can be run concurrently in the same experiment, in the case of immunohistochemistry, on the same slide (Figure 2A-C). The Multiplex slide (Figure 2C) was compared to Mab2 (Figure 2A) and Mab3 (Figure 2B) staining.

Standard IHC methodologies were used for these experiments (see, e.g., Taylor CR. and Rudback, L. Immunohistochemical Staining Methods - Sixth Edition. 2013). Formalin-fixed paraffin embedded (FFPE) non-small cell lung cancer human tissue expressing Epitope 2 and Epitope 3 as determined from previous IHC experiments were used for these studies.

Briefly, unmasking of the antigens on tissue was performed by heat- induced antigen retrieval (HIER) using a Tris-based antigen unmasking solution (Vector Laboratories; Cat No.: H-3301) for 20 minutes at 95°C. Slides were then cooled to room temperature (22°C) and endogenous peroxidases were inactivated by incubation of the slides with Bloxall Blocking Solution (Vector Laboratories; Cat No.: SP-6000). The slides were then incubated for 30 minutes with Blocking Buffer containing 2.5% Normal Horse Serum (Vector Laboratories; as supplied with Cat No. MP-7401) and 1% BSA. Subsequently, Blocking Buffer was removed and primary antibody samples were diluted using Blocking Buffer to lpg/ml, added to the tissue sections, and then incubated for 75 minutes.

To remove the primary antibodies, the slides were washed with Tris-buffered saline with polysorbate 20 (TBST). AP labeled Anti-Rabbit Ig (Vector Laboratories, Cat No.: MP-5401) (Figure 2A), HRP labeled Anti-Rabbit Ig (Vector Laboratories, Cat No.: MP-7401) (Figure 2B), or AP and HRP labeled Secondary Antibodies B and C (which recognize SiGERMab2b and SiGERMAb3c, respectively) (Figure 2C), were applied to the slides and incubated for 1 hour. Slides were washed again (TBST) and substrates (Vector Laboratories, Cat. No.: SK-5105 (AP Substrate), Cat. No.: SK-4105 (HRP Substrate)) were added in sequence. The DAB substrate for HRP was added first for 5 minutes. The slides were then washed with TBST and then the substrate for AP was added for 30 minutes. Slides were counterstained with Hematoxylin (Vector Laboratories, Cat. No.: H-3401) according to manufacturer’s instructions. Slides were then dehydrated and cover slipped.

MAb2 recognizes particular tumor cells which express Epitope 2 (Figure 2A, white arrows). As mentioned above, the secondary antibody used was a Goat anti-Rabbit Ig

conjugated to Alkaline Phosphatase (Vector Laboratories; Cat No.: MP-5401). Primary antibody concentration was lpg/ml, and the secondary antibody was used according to manufacturer’s instructions. As depicted in Figure 2, Mab2 recognizes tumor cells with large nuclei (Figure 2A). T-cells are not stained.

MAb3 recognizes human T-cells which express Epitope 3 (Figure 2B, black arrows). As mentioned above, the secondary used was a Goat anti-Rabbit Ig conjugated to Horseradish Peroxidase (Vector Laboratories; Cat No.: MP-7401). Primary antibody concentration was lpg/ml, and the secondary antibody was used according to manufacturer’s instructions. As depicted in Figure 2, Mab3 recognizes T-cells with small nuclei (Figure 2B). Tumor cells are not stained.

As shown in Figure 2C, SiGERMab 2b and SiGERMab3c recognize tumor cells (white arrows) and T-cells (black arrows), respectively, on the same slide. Primary antibodies,

SiGERMab2b and SiGERMab3c , were used at a concentration of lpg/ml and incubated concurrently for 1 hour. SiGERMab2b stains only tumor cells with large nuclei whereas

SiGERMab3c stains only T-cells. SUMMARY OF SEQUENCE LISTING




 
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