WO2008137165A1 - Anti-glycated cd59 antibodies and uses thereof - Google Patents

Abstract

This invention includes, in part, methods of preparing glycated polypeptide antigens and antibodies that specifically recognize glycated epitopes on polypeptides. The invention also relates, in part, to the preparation and use of antibodies that specifically recognize and bind with high affinity to glycated epitopes on glycated CD59 polypeptides, including, but not limited to a K41-glycated epitope on CD59. In some aspects, the invention also includes, hybridoma cell lines that produce antibodies that specifically bind glycated CD59; antibodies and antigen-binding fragments thereof produced with the methods of the invention; and methods of using the antibodies and antigen-binding fragments thereof for diagnosis and treatment of diseases and conditions such as abnormal glycemic control, pre-diabetes, diabetes, and diabetes-associated conditions.

Description

ANTI-GLYCATED CD59 ANTIBODIES AND USES THEREOF

Related Application

This application claims the benefit under 35 U.S.C §119 (e) of US Provisional Patent Application serial number 60/928,122 filed May 7, 2007, the entire contents of which is incorporated herein by reference.

Government Support

This invention was made with government support under Grant No. DK052855 and Grant No.: DK062994 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

Field of the Invention

This invention relates generally to preparation of glycated polypeptide antigens and antibodies that specifically recognize glycated epitopes on polypeptides and proteins. The invention also relates, in part, to the preparation and use of antibodies that specifically recognize glycated epitopes on glycated CD59 polypeptides. Aspects of the invention also relate, in part, to antibodies or antigen-binding fragments thereof that bind specifically to glycated CD59.

Background of the Invention

Diabetes Mellitus (diabetes) is a leading cause of morbidity and mortality in the adult population. This is primarily because diabetic patients tend to develop vascular complications that involve the kidneys (diabetic nephropathy), the retina (diabetic retinopathy), as well as large and small blood vessels in other organs (macro- and microvascular disease) including nerves (diabetic neuropathy). It is well established that the vascular complications of diabetes are caused by elevated blood glucose levels over long periods of time. Elevated blood glucose levels affect proteins by a process known as glycation. Different "glycated" proteins have been identified in diabetic subjects, including albumin, hemoglobin and others. Measurement of the extent of protein "glycation" of certain proteins is considered a valuable clinical tool to assess long-term glycemic control and thereby the efficacy of diabetes treatment. Glycation, the non-enzymatic attachment of glucose to proteins, is considered a major pathophysiological mechanism causing tissue damage in diabetic subjects. Glycation involves the reaction of glucose and/or other reducing sugars with amino groups in proteins resulting in the formation of a Schiff base or aldimine. This labile adduct can tautomerize via the Amadori rearrangement to the more stable ketoamine. The function of the glycated protein may be impaired, depending on the location of the amino group(s) affected. For example, amino-terminal glycation of the β-chains of hemoglobin gives rise to the glycated hemoglobins (HbAl) in which responsiveness to 2,3-diphosphoglycerate is decreased and oxygen affinity increased. Glycation of the major thrombin inhibitor of the coagulation system, antithrombin III, decreases its affinity for heparin, and has been postulated to contribute to the hypercoagulable state associated with diabetes.

Hemoglobin glycation and thrombin inhibitor glycation do not account for the vascular complications of diabetes. The mechanism which results in such complications remains unknown. Currently, protein glycation in diabetic subjects is measured in blood by estimating the amount of glycated hemoglobin (hemoglobin AIc) through a complicated clinical test that requires extraction of a blood sample. Accordingly, there is a need for a simplified and less invasive method for rapid monitoring of protein glycation levels.

Summary of the Invention

The present invention relates, in part, to methods and compositions for making high- affinity antibodies that specifically bind to glycated epitopes on polypeptides. The invention, in some aspects, relates to methods making synthetic glycated polypeptide antigens. Glycated-polypeptide synthesis methods of the invention may include preparation of glycated building-blocks to be used in the stepwise assembly of polypeptide and synthetic methods to prepare a glycated polypeptide antigens. In some embodiments of the invention, glycated polypeptides may also be produced using post-synthetic polypeptide modification methods. A glycated polypeptide antigen of the invention may be synthesized on a solid support or may be synthesized in solution. In some aspects of the invention, a building block molecule used in synthesis of a glycated polypeptide may be a protected glucitol lysine. In some embodiments, a synthetic glycated polypeptide of the invention may be prepared by reductively alkylating on the e-amino of a lysine residue of the polypeptide. In certain embodiments, the reductive alkylation is on the e-amino of a CD59 lysine and in some embodiments, the reductive alkylation is on the e -amino of K41 of CD59.

The invention includes, in part, methods for preparing a synthetic polypeptide that includes a protected glucitol lysine residue. In some aspects, the invention includes compounds for preparing a synthetic glycated polypeptide. Preparative compounds of the invention, include, but are not limited to compound (1):

Compound (2):

and Compound (3):

One or more of these compounds may be used in the synthesis of a glycated polypeptide using methods provided herein or alternative synthetic methods and conditions known in the art. The compound drawings provided herein are representative drawings and are not intended to follow the stereochemical Fisher convention. A glycated polypeptide that may be produced using the methods and compounds of the invention maybe a glycated CD59 polypeptide, which may be a full-length CD59 polypeptide or a fragment of a full-length polypeptide. In some embodiments, glycated CD59 compounds or fragments thereof are glycated at the residue that corresponds to the K41 residue of full-length CD59 polypeptide. - A -

K41-glycated CD59 polypeptides prepared using the methods of the invention may be used as antigens to prepare antibodies that specifically bind K41-glycated CD59.

The invention disclosed herein describes novel methods of producing antibodies that specifically bind glycated polypeptides, including antibodies that specifically bind to glycated CD59 protein. In some embodiments, glycated CD59 of the invention is K41-glycated CD59 and the invention relates in part to the production and use of antibodies that specifically bind to K41-glycated CD59 polypeptides. The invention also includes in some aspects compositions for detecting and measuring glycated CD59 levels, particularly as they relate to glycemic levels. The invention also relates, in part, to methods of preparing (e.g. synthesizing) glycated CD59 polypeptides and the use of such synthetic glycated CD59 polypeptides for preparing high-affinity antibodies that specifically recognize glycated CD59. The invention also includes in some aspects generating hybridoma cell lines that produce antibodies that specifically bind glycated polypeptides (e.g. glycated CD59 polypeptides); and methods of making antibodies and antigen-binding fragments thereof that specifically bind glycated polypeptides (e.g. glycated CD59 polypeptides) for diagnosis and selection of and/or assessment of treatments of pre-diabetes, diabetes, and related conditions.

The present invention relates, in part, to high-affinity antibodies and antigen-binding fragments thereof that specifically bind glycated CD59 polypeptides. The invention includes, in some aspects, the use of antibodies or antigen-binding fragments thereof either singly or in combination. Antibodies that are useful in methods of the invention include, but are not limited to antibodies or antigen-binding fragments thereof that specifically bind glycated CD59 polypeptides with high affinity. The invention includes, in some aspects, methods of using antibodies and antigen-binding fragments thereof that specifically bind glycated polypeptides for diagnosis and selection of and/or assessment of treatments of pre-diabetes, diabetes, and related conditions. In some embodiments, glycated CD59 antibodies of the invention are antibodies that specifically recognize and bind to CD59 polypeptides or fragments thereof that are glycated at the residue that corresponds to the K41 residue of a full-length CD59 polypeptide. The discovery of high affinity antibodies that specifically bind to a glycated CD59 polypeptide facilitates analysis of diseases in which the amount of CD59 glycation differs from normal levels. For example, it has been discovered that the level of glycation of CD59 is elevated in diabetes (see US Patent Nos. 6,835,545 and 7,049,082, each of which is incorporated by reference in its entirety herein). Thus, onset, progression, and/or regression of diabetes or other diseases can be monitored by monitoring levels of glycated CD59 in a subject. It also has been determined, that CD59 is present in urine, saliva, tissue, etc. (see US Patent Nos. 6,835,545 and 7,049,082, each of which is incorporated by reference in its entirety herein). Antibodies and antigen-binding fragments thereof of the invention may be used to measure levels of glycated CD59 (e.g., K41-glycated CD59) in urine or other samples without requiring a blood sample.

According to one aspect of the invention, a composition of matter is provided. The composition of matter includes purified compound (1) set forth as:

According to another aspect, methods of making compound (1) set forth as:

are provided. The methods include combining Nα-Fmoc-L- Lysine, and D-glucose, under conditions to make compound (1). In some embodiments, the method also includes purifying compound (1), which is set forth as:

In certain embodiments, wherein the conditions include the presence of sodium cyanoborohydride (NaCNBH₃) in THF/H₂O. According to yet another aspect of the invention, a composition of matter is provided. The composition of matter includes purified compound (2), which is set forth as:

According to another aspect of the invention, methods of making compound (2), which is set forth as:

are provided. The methods include combining compound (1), which is set forth as:

with Boc anhydride (BoC₂O) under conditions to make compound (2). In some embodiments, the methods also include purifying compound (2), which is set forth as:

According to one aspect of the invention, a composition of matter is provided. The composition of matter includes purified compound (3), which is set forth as:

According to another aspect of the invention, methods of making compound (3), which is set forth as:

are provided. The methods include combining Nα-Fmoc-Lys-OH with D-glucose under conditions to make compound (3). In some embodiments, the methods also include purifying compound (3), which is set forth as:

According to yet another aspect of the invention, methods of incorporating compound (2), which is set forth as:

onto a resin-bound polypeptide are provided. The methods include: adding compound (2) to a resin-bound polypeptide, and incubating under suitable conditions to incorporate the compound (2), onto the resin-bound polypeptide. In some embodiments, incorporating includes forming an amide bond between the carboxylic function of compound (2) and a free Nα-terminal amino group of the resin-bound polypeptide. In certain embodiments, the methods also include washing the resin-bound polypeptide incorporating compound (2). In some embodiments, the suitable conditions include the presence of pyBOP and DEEA in the suspended resin-bound polypeptide solution. In some embodiments, the resin-bound polypeptide is a CD59 polypeptide or fragment thereof. In some embodiments, the resin- bound polypeptide includes the amino acid sequence set forth as: FEHANFNDC (SEQ ID NO:44). In certain embodiments, the resin-bound polypeptide incorporating compound (2), set forth as:

includes an amino acid sequence set forth as: KFEHANFNDC (SEQ ED NO:45), and compound (2), is incorporated onto the lysine residue of the amino acid sequence. In some embodiments, the resin-bound polypeptide is a CD59 polypeptide. In some embodiments, the compound (2), set forth as:

is incorporated onto a residue of the resin-bound polypeptide that corresponds to residue K41 of a full-length CD59 polypeptide. In certain embodiments, the CD59 polypeptide is the polypeptide set forth as SEQ ED NO:43.

According to yet another aspect of the invention, methos of incorporating isolated compound (3), set forth as:

onto a resin-bound polypeptide, are provided. The methods include adding compound (2) to a resin-bound polypeptide, and incubating under suitable conditions to incorporate the compound (3), onto the resin-bound polypeptide. In some embodiments, incorporating includes forming an amide bond between the carboxylic function of compound (3) and a free N-terminal amino group of the resin-bound polypeptide. In some embodiments, the resin- bound polypeptide is in solution, hi certain embodiments, the methods also include washing the resin-bound polypeptide incorporating compound (3). hi some embodiments, the suitable conditions include the presence of pyBOP and DIEA in the suspended resin-bound polypeptide solution, hi some embodiments, the resin-bound polypeptide is a CD59 polypeptide or fragment thereof. In some embodiments, the resin-bound polypeptide includes the amino acid sequence set forth as: FEHANFNDC (SEQ ID NO:44). hi certain embodiments, the resin-bound polypeptide incorporated with compound (3), set forth as:

includes an amino acid sequence set forth as: KFEHANFNDC (SEQ DD NO:45), and wherein compound (3) is incorporated onto the lysine residue in the amino acid sequence, hi some embodiments, the resin-bound polypeptide is a CD59 polypeptide. In some embodiments, the compound (3), set forth as:

is incoφorated onto a residue of the resin-bound polypeptide that corresponds to residue K41 of a full-length CD59 polypeptide. In some embodiments, the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:43.

According to another aspect of the invention, methods of extending a polypeptide incorporating compound (2) set forth as:

or compound (3), set forth as:

and bound to a resin are provided. The methods include deprotecting the polypeptide incorporating compound (2) or compound (3), adding Fmoc-amino acid-OH to the deprotected polypeptide, and incubating the Fmoc-amino acid-OH and the polypeptide under suitable conditions to extend the sequence of the polypeptide. In certain embodiments, the suitable conditions include the presence of pyBOP and DIEA. In some embodiments, the Fmoc-amino acid-OH is Fmoc-Lysine-OH. hi some embodiments, the polypeptide incorporating compound (2) or compound (3) includes a CD59 polypeptide or fragment thereof. In some embodiments, the polypeptide is a polypeptide incorporating compound (3), set forth as:

In certain embodiments, the polypeptide incorporating compound (2) includes the amino acid sequence set forth as: KFEHANFNDC (SEQ ID NO:45), and wherein compound (2), set forth as:

is attached to the lysine residue of the amino acid sequence. In some embodiments, the polypeptide is a polypeptide incorporating compound (3), set forth as:

In some embodiments, the polypeptide incorporating compound (3) has the amino acid sequence set forth as: KFEHANFNDC (SEQ ID NO:45), and compound (3), set forth as:

is attached to the lysine residue of the amino acid sequence. In certain embodiments, the polypeptide is a CD59 polypeptide. In some embodiments, compound (2) or compound (3) is incorporated at a residue of the polypeptide that corresponds to residue K41 of a full-length CD59 polypeptide. In some embodiments, the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:43.

According to yet another aspect of the invention, methods of preparing a glycated polypeptide are provided. The methods include steps of, (a) incorporating compound (2) set forth as:

or compound (3), set forth as:

onto a resin-bound polypeptide, (b) deprotecting the incorporated polypeptide, (c) extending the sequence of the deprotected polypeptide, (d) N-terminally capping the extended polypeptide, and (e) cleaving the capped, extended polypeptide from the resin.

In certain embodiments, incorporating includes forming an amide bond between the carboxylic function of compound (2) or compound (3) and a free N-terminal amino group of the resin-bound polypeptide. In some embodiments, the methods also include purifying the cleaved polypeptide. In some embodiments, the methods also include repeating steps (b) and (c) one or more times to lengthen the polypeptide. In certain embodiments, the deprotecting includes treatment with piperidine. In some embodiments, compound (2) set forth as:

is incorporated onto the polypeptide resin using the method of any embodiment of any of the aforementioned aspects of the invention. In some embodiments, compound (3) set forth as:

is incorporated onto the polypeptide resin using the method of any embodiment of any of the aforementioned aspects of the invention. In some embodiments, the polypeptide incorporating compound (2) or compound (3) is extended using the method of any embodiment of any aforementioned aspect of the invention. In certain embodiments, the resin-bound polypeptide is a CD59 polypeptide. In some embodiments, compound (2) or compound (3) is incorporated onto the resin-bound polypeptide at a residue that corresponds to residue K41 of a full-length CD59 polypeptide, hi some embodiments, the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:43. In some embodiments, the compound (2) or compound (3) is incorporated into the polypeptide set forth as SEQ ID NO:43 at residue K5 of the amino acid sequence of SEQ ID NO:43.

According to yet another aspect of the invention, methods of preparing a glycated polypeptide are provided. The methods include the steps of: (a) N-terminally capping a resin-bound polypeptide, (b) glycating the Nα-capped resin-bound partially protected polypeptide,

(c) deprotecting the capped resin and (d) cleaving the capped polypeptide from the resin. In certain embodiments, the methods also include purifying the cleaved polypeptide, hi some embodiments, the resin-bound polypeptide is a CD59 polypeptide. In some embodiments, the capped polypeptide is glycated at a residue that corresponds to residue K41 of a full- length CD59 polypeptide, hi some embodiments, the deprotecting includes treatment with hydrazine. In certain embodiments, the CD59 polypeptide is the polypeptide set forth as SEQ E) NO:43. In some embodiments, the resin-bound polypeptide is glycated at the residue that corresponds to K41 of mature CD59 polypeptide.

According to another aspect of the invention, methods of preparing a glycated polypeptide in solution are provided. The methods include the steps of: (a) N-terminally capping a polypeptide in solution, (b) glycating the Nα-capped, partially protected polypeptide, and (c) deprotecting the capped polypeptide. In some embodiments, the methods also include purifying the deprotected polypeptide. In some embodiments, the polypeptide in solution is a CD59 polypeptide, hi certain embodiments, the capped polypeptide is glycated at a residue that corresponds to residue K41 of a mature CD59 polypeptide. In some embodiments, the deprotecting includes treatment with hydrazine, hi some embodiments, the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:8. According to yet another aspect of the invention, isolated antibodies or antigen- binding fragments thereof are provided. The isolated antibodies or antigen-binding fragments thereof bind specifically to a glycated epitope of glycated CD59 with an affinity that is from between about 1 X 10^"5M to about 1 X 10^"12M, wherein the epitope includes a glycated lysine. In certain embodiments, the glycated lysine corresponds to K41 of CD59. hi some embodiments, the antibody is a recombinant antibody, hi some embodiments, the antibody or antigen-binding fragment thereof is tagged with a detectable label, hi certain embodiments, the detectable label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label, hi some embodiments, the antibody or antigen-binding fragment thereof is lyophilized. hi some embodiments, the antibody or antigen-binding fragment thereof is in an aqueous medium. According to certain aspects of the invention, a nucleic acid sequence that encodes the antibody of any embodiment of the aforementioned antibody or antigen-binding fragment thereof is provided, hi some aspects of the invention expression vectors that include any of the aforementioned isolated nucleic acid molecules encoding the antibody or antigen-binding fragment thereof are provided, hi some aspects a host cell transformed by or transfected with any of the aforementioned expression vectors are provided, hi certain aspects of the invention, a plasmid which produces any of the antibodies or antigen-binding fragment thereof of any of the aforementioned aspect of the invention are provided. According to yet another aspect of the invention, kits for detecting the presence of glycated CD59 are provided. The kits include a package including a container containing the isolated antibody or antigen-binding fragment thereof any embodiment of an aforementioned aspect of the invention, and instructions for use of the antibody or antigen-binding fragment thereof to detect the presence of glycated CD59. hi some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody or antigen-binding fragment thereof is attached to a detectable label. In some embodiments, the detectable label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label. In certain embodiments, the antibody or antigen-binding fragment thereof is lyophilized. hi some embodiments, the antibody or antigen-binding fragment thereof is packaged in an aqueous medium. In some embodiments, the kit also includes a container containing a second antibody or antigen-binding fragment thereof that specifically binds non-glycated CD59 or non-K41 -glycated CD59, and instructions for using the second antibody as a control antibody.

According to yet another aspect of the invention, kits are provided. The kits include a package including a container containing a hybridoma that includes a nucleic acid sequence that encodes an antibody of any aforementioned aspect of the invention, and instructions for producing the antibody. According to yet another aspect of the invention, immunogenic polypeptide are provided. The polypeptide includes 1) the amino acid sequence set forth as WKFEH (SEQ ID NO:6), and is prepared using the method of any embodiment of any aforementioned aspect of the invention or 2) the amino acid sequence of the polypeptide is a modified amino acid sequence of SEQ ID NO: 5 or fragment thereof wherein the modification of the amino acid sequence set forth as SEQ ID NO:5 is the presence of one or more N^glycated-lysine residues, or the replacement of one or more cysteine residues with alanine residues, or the addition of a cysteine residue to the C-terminus, or combinations thereof. In some embodiments, the amino acid sequence of the immunogenic polypeptide is set forth as SEQ ID N0:8. According to yet another aspect of the invention, methods of making an antibody that specifically binds to glycated CD59 but not to nonglycated CD59 are provided. The methods include preparing an immunogenic polypeptide of any aforementioned aspect of the invention, and immunizing an animal with the immunogenic polypeptide, hi certain embodiments, the animal is a mouse, hi some embodiments, the immunogenic polypeptide has the amino acid sequence set forth as SEQ ID NO: 8.

According to yet another aspect of the invention, methods of making an antibody that specifically binds K41 -glycated CD59 are provided. The methods include inoculating an animal with an antigenic glycated polypeptide made using any of the aforementioned synthetic methods and obtaining an antibody from the animal, hi some embodiments, the antibody binds K41-glycated CD59 with at with sub-nanomolar affinity and in some embodiments, has an affinity from between 1 X 10^"5M to about 1 X 10^"12M, wherein the epitope includes a glycated lysine, hi certain embodiments, the glycated lysine corresponds to K41 of CD59. hi some embodiments, the glycated lysine is a lysine that has been reductively alkylated on the e-amino of a lysine residue of the CD59 polypeptide, hi some embodiments of any of the aforementioned aspects of the invention, the glycated polypeptide additionally may be conjugated to a molecule to increase antigenicity, hi some embodiments the molecule to increase antigenicity is KLH or BSA. hi some embodiments of any of the aforementioned aspects of the invention, a glycated lysine includes a N*-glucytol.

These and other aspects of the invention will be described in further detail in connection with the detailed description of the invention.

Brief Description of the Drawings

Fig. 1 depicts a schematic of a kit according to the invention.

Fig. 2 shows traces of a typical FfPLC at 214 nm, gradient 5-95% in 10 min (Fig. 2 A: crude product Fig. 2B: purified compound (I)) and ESI - mass spec (Fig. 2C: corresponding to component eluted at 3.80 min with [M+H] ⁺ corresponding to diglycated compound (3)- Fig. 2D: corresponding to component eluted at 3.97 min with [M+H] ⁺ corresponding to monoglycated compound (I)- and Fig. 2E: at 4.13 min with [M+H] ⁺ corresponding to the starting material Fmoc-Lys-OH) .

Fig. 3 shows traces of a typical HPLC at 214 nm, gradient 5-95% in 10 min (Fig. 3 A: crude product. Fig. 3B: purified compound (2)) and ESI - mass spec (Fig. 3C: corresponding to component eluted at 5.71 min with [M+H] ⁺ corresponding to Compound 2). Fig. 4 shows traces of a typical HPLC at 214 nm, gradient 5-95% in 10 min (Fig. 4 A: crude product. Fig. 4B: purified compound (3)) and ESI - mass spec (Fig. 4C: corresponding to component eluting at 3.69 min with [M+H] ⁺) corresponding to Compound (3)).

Fig. 5 shows traces of a typical HPLC at 214 nm, gradient 5-35% in 20 min (Fig. 5A: crude product. Fig. 5B: purified mono-glycated polypeptide 7 (compound (7)) and ESI - mass spec (Fig. 5C: corresponding to component eluting at 9.73 min with [M+2H]²⁺ corresponding to mono-glycated polypeptide 7).

Fig. 6 shows traces of a typical HPLC at 214 nm, gradient 5-35% in 20 min (Fig. 6A: crude product. Fig. 6B: purified non-glycated polypeptide 8 (compound (8)) and ESI - mass spec corresponding to component eluting at 9.75 min with [M+2H]²⁺ (Fig. 6C) and [M+H] ⁺ (Fig.. 6D), which result form the mono- and di-protonated non-glycated polypeptide 8.

Fig. 7 shows traces of a typical HPLC at 214 nm, gradient 10-70% in 25 min (Fig. 7 A: purified ivDde-protected polypeptide 9 (compound (9)) and ESI - mass spec (Fig. 7B: corresponding to component eluting at 8.76 min with [M+2H]²⁺) corresponding to polypeptide 9).

Fig. 8 shows traces of a typical HPLC at 214 nm, gradient 0-50% in 25 min (Fig. 8 A: crude product. Fig. 8B: purified ivDde-protected and glycated polypeptide 10 (compound (10)) and ESI - mass spec (Fig. 8C: corresponding to component eluting at 13.22 min with [M+2H]²⁺ corresponding to the ivDde-protected diglycated polypeptide; Fig. 8D: corresponding to component eluting at 13.44 min with [M+2H]²⁺ corresponding to compound (10)); and Fig. 8E: corresponding to component eluting at 13.70 min with [M+2H]²⁺ corresponding to compound 9 the starting material.

Fig. 9 shows traces of a typical HPLC at 214 nm, gradient 0-35% in 10 min (Fig. 9A) and ESI - mass spec (9B: corresponding to component eluting at 5.43 min with [M+2H]²⁺ corresponding to the glycated polypeptide 11 and Fig. 9C: corresponding to component eluting at 8.61 min with [M+2H]²⁺ corresponding to compound (10)). Fig. 10 shows traces of a typical HPLC at 214 nm, gradient 10-70% in 25 min (Fig. 10A) and ESI - mass spec (Fig. 10B) corresponding to the component eluting at 10.05 min with [M+2H]²⁺ corresponding to Compound 12 the ivDde "Lys⁴¹ "-protected polypeptide.

Fig. 11 shows traces of a typical HPLC at 214 nm, gradient 0-35% in 25 min (Fig. 1 IA), , and ESI - mass spec (Fig. 1 IB: corresponding to component eluting at 10.90 min with [M+2H]²⁺) corresponding to polypeptide 13).

Fig. 12 shows traces of a typical HPLC at 214 nm, gradient 0-35% in 25 min (Fig. 12A), , and ESI - mass spec (Fig. 12B: corresponding to component eluting at 10.92 min with [M+2H]²⁺) corresponding to the "Lys⁴ '"-glycated polypeptide 14.

Fig. 13 shows a Coomasee stained SDS-PAGE gel providing results of analysis of maleimido-BSA-"Lys⁴¹ "-glycated polypeptide conjugate (11) (center lane), maleimido- activated BSA (right lane) and molecular weight markers, (left lane)

Fig. 14 shows a dot-blot membrane providing results of dot-blot analysis of maleimido-KLH and maleimido-BSA and their conjugation products to the "Lys⁴¹ "-glycated polypeptide. The membrane was blotted with 1789 rabbit anti-glucitolysine polyclonal antibody and probed with IR-800 tagged goat anti-rabbit poly-clonal antibodies.

Fig. 15 shows a dot-blot membrane providing dot blot analysis of anti-sera from three mice (A, B and C), immunized with "Lys⁴¹ "-glycated polypeptide 14 conjugated to maleimido- KLH, with (from left to right) BSA, "Lys⁴¹ "-glycated polypeptide 14-maleimido BSA conjugate, KLH and "Lys⁴¹ "-glycated polypeptide 14-maleimido KLH conjugate. The dot blot was probed with IR-800 tagged goat anti-rabbit poly-clonal antibodies.

Fig. 16 shows a dot-blot membrane providing dot blot analysis of anti-sera (1/500 dilution) raised in two rabbits (numbered as 4879 and 4880) using "Lys⁴¹ "-glycated polypeptide 7

(compound 7) conjugated to KLH as immunogen (10 weeks after immunization). For control, the pre-immune serum collected from the same animals was used to probe against (left to right) glycated-polypeptide conjugated with BSA, non glycated-polypeptide 8 (compound 8) conjugated with BSA, in-vitro glycated ghost prepared from Chinese hamster ovary (CHO) cells that were expressing human CD59, same ghosts but not glycated that serve as control, glycated CHO ghost expressing Lys41Gln mutant human CD59, same ghost but not glycated that serve as control and glycated BSA (Sigma Inc, St Louis, USA). Goat anti-rabbit antibody conjugated with Alexa-fluor 680 was used as detection antibody.

Fig. 17 shows digitized images of immunoblots prepared using a rabbit polyclonal antibody prepared with the Lys⁴¹-glycated CD59 polypeptide prepared using methods described in the Examples section. Blot A was probed with a pre-immune sera from rabbit #4880 and blot B was probed with sera from rabbit #4880 10 weeks after immunization with glycated-CD59- derived peptide-KLH conjugate. 1 and 1 ' = MW markers; 2 and 2' = Glycated CD59 IP with BRIC and MEM 43/5; 3 and 3' = Non-Glycated CD59 IP with BRIC and MEM 43/5; Fig. 17A was probed with preimmune sera from rabbit # 4880 at a dilution of 1 :50; Fig. 17B was probed with sera from rabbit # 4880 10 weeks after immunization with glycated-CD59- derived peptide-KLH conjugate at a dilution of 1:100

Detailed Description of the Invention

The present invention provides antibodies or antigen-binding fragments thereof which bind specifically to glycated CD59-derived polypeptides, compositions containing one or a combination of such antibodies or antigen-binding fragments thereof, hybridoma cell lines that produce the antibodies, and methods of making and using the antibodies or antigen- binding fragments thereof for diagnosis and selection and/or assessment of treatment of diabetic conditions and diabetes-related conditions. The invention, in part also includes, synthetic polypeptide antigens that can be used to produce antibodies, methods of preparing synthetic polypeptide antigens, intermediate compounds that may be used to prepare synthetic polypeptide antigens and the methods for preparing the intermediate compounds, hi some embodiments, the synthetic polypeptides are K41 glycated polypeptides.

In contrast to markers of glycation such as hemoglobin, glycation of CD59 polypeptide is believed to be involved in the pathogenesis of the vascular complications of diabetes. Accordingly, clinical evaluation of glycated CD59 polypeptide is a more direct measure for vascular complications of diabetes induced by glycation. As used herein, CD59 (also known as membrane inhibitor of reactive lysis [MIRL], protectin, HRF20 and H 19) is a polypeptide with a amino acid sequence set forth as Accession No. AAA60957, (Davies, A., et al., Journal J. Exp. Med. 170 (3), 637-654 (1989)). The nucleic acid sequence that encodes human CD59 set forth as Genbank Accession No. AAA60957 also is provided by Davis, A, et al. and has Genbank Accession No. M95708. The sequence of the 128 amino acid CD59 polypeptide of Accession No. AAA60957 is provided herein as SEQ ED NO:1. The nucleic acid sequence that encodes SEQ ID NO:1 and is set forth as Genbank Accession No. M95708 is set provided herein as SEQ ID NO:2. The immature, full-length CD59 polypeptide sequence includes a 25 amino acid signal polypeptide that is cleaved when CD59 polypeptide is produced. The CD59 amino acid sequence in which the 25 amino acid signal sequence has been cleaved is set forth herein as SEQ ID NO:3. The sequence of a CD59 polypeptide that has the 25 amino acid signal sequence cleaved and is glycated at K41 is set forth as SEQ ID NO:4. In addition to cleavage of the signal peptide, immature CD59 may be further processed by cleavage of a 26 amino acid sequence from its C-terminus (residues 102-128 of SEQ ID NO:3). In a living cell, cleavage of the C-terminal amino acid sequence may be followed by the addition of a glycosylphosphatidylinositol (GPI) modification on to the peptide's C-terminus. The GPI may serve to anchor the protein on the extracellular side of a plasma membrane. Thus, the amino acid sequence of an immature CD59 polypeptide set forth as SEQ ID NO:1 may be reduced in length by the 25 amino acid signal sequence and be further reduced by an additional 26 amino acids cleaved from its C-terminus. The resulting mature sequence of CD59 polypeptide following such cleavage is the 77 amino acid polypeptide set forth herein as SEQ ID NO:5. A synthetic CD59 useful in the methods and products of the invention may be a 77 amino acid long CD59 polypeptide having the sequence set forth as SEQ ID NO:5. The CD59 polypeptide set forth as SEQ ID NO:5 includes six lysine residues that are K14, K30, K38, K41, K65, and K66. SEQ ID NOs: 46-51 are CD59 polypeptides with the same amino acid sequence as SEQ ID NO:5 but with a glycated lysine residue that corresponds to K14, K30, K38, K41, K65, or K66. Those of ordinary skill in the art will recognize that in some CD59 polypeptides (synthetic and natural) more than one lysine may be glycated in the CD59 polypeptide, hi some embodiments, each or any combination of the lysines that correspond to K14, K30, K38, K41, K65, or K66 of mature CD59 may be glycated. A synthetic CD59 polypeptide of the invention may be referred to herein as "soluble recombinant CD59".

One of ordinary skill in the art will recognize that the drawings of compounds provided herein are representative drawings and are not intended to follow, or be limited by, the stereochemical Fisher convention. As provided herein, compounds (7), (11), and (14) are the same mono-glycated polypeptide but each has been prepared using different methods. They have been provided different compound numbers to simplify reference to figures, data, and experimental results herein. As used herein the term "function" when used in reference to a chemical compound means a chemical group that can participate in a chemical reaction". A nonlimiting example of use of the term may be found in the phrase "carboxylic function of compound (2)", in which the carboxylic function is a chemical group of compound (2) that is a carboxylic functional group.

As used herein, the term "synthetic" means artificially prepared. A synthetic polypeptide is a polypeptide that is synthesized and is not a naturally produced polypeptide molecule (e.g., not produced in an animal or organism). It will be understood that the sequence of a natural polypeptide (e.g., an endogenous polypeptide) may be identical to the sequence of a synthetic polypeptide, but the latter will have been prepared using at least one synthetic step.

As used herein, a synthetic glycated polypeptide is a polypeptide glycated with a synthetic method, which may be, but is not limited to a method of the invention. A glycated polypeptide of the invention may include a synthetic glycated epitope. Although a synthetic glycated polypeptide may differ from a natural glycated polypeptide in subtle ways, an antibody raised against a synthetic polypeptide of the invention will specifically bind with high affinity the synthetic polypeptide epitope against which it was raised, and will also specifically bind with high affinity to the natural epitope in a polypeptide. For example, an antibody of the invention raised against a synthetic glycated polypeptide prepared using methods of the invention, and including the amino acid sequence WKFEH (SEQ ED NO:6), is able to specifically bind with high affinity to a synthetic glycated polypeptide that comprises the amino acid sequence WKFEH (SEQ ID NO: 6) and also is able to specifically bind to a natural glycated-polypeptide or protein that comprises the amino acid sequence WKFEH (SEQ ID NO:6). Thus, even though the glycated epitope of a synthetic polypeptide may differ slightly from the same epitope in a natural glycated polypeptide or protein, an antibody raised against a synthetic glycated epitope of the invention prepared using the methods of the invention specifically binds with high affinity the natural glycated epitope. Antibodies of the invention generated using a synthetic glycated polypeptide prepared using the methods of the invention, specifically bind with high affinity to natural and synthetic glycated polypeptides and are able to distinguished between natural glycated and natural non- glycated polypeptides. An anti-glycated CD59 antibody made using a synthetic glycated CD59 antigen of the invention is useful in methods to distinguish between glycated and non- glycated CD59. hi some aspects, the invention may include the synthesis of glycated polypeptides. Synthesis methods of the invention may include building-block synthesis. Building-block synthetic methods may include the preparation of building-block molecules for addition to a polypeptide. In some embodiments, a building-block molecule used in synthesis of a glycated polypeptide of the invention may be a glycated amino acid monomer. One or more building-block molecules may be added in sequence to a polypeptide under conditions to result in a synthetic glycated polypeptide, hi some embodiments, building-block synthesis includes preparation of a glycated polypeptide from a polypeptide that is a resin-bound polypeptide and in certain embodiments, building-block synthesis may include preparation of a glycated polypeptide from a polypeptide in solution.

Steps in a building-block synthetic method to make an extended synthetic polypeptide may include, but are not limited to, incorporation of an intermediate compound onto a resin- bound polypeptide, or a polypeptide in solution, deprotecting the incorporated polypeptide, extending the amino acid sequence of the deprotected polypeptide, and N-terminally capping the extended polypeptide, hi some embodiments, incorporating of a compound onto a polypeptide may include attaching the compound onto the polypeptide by forming an amide bond between the carboxylic terminus of the compound/building block and a free terminal amine of the polypeptide, hi some embodiments, if an originating polypeptide is a resin- bound polypeptide, an additional step in the polypeptide synthesis may include cleaving the capped, extended polypeptide from the resin, hi some embodiments the synthetic glycated polypeptide is purified following its preparation. In some embodiments, synthetic steps described herein may be repeated one or more times, to further elongate a polypeptide.

Synthetic building-block methods of the invention may include the use of one or more intermediate compounds for synthesis of glycated polypeptides. Intermediate compounds that may be incorporated onto the polypeptide may include, but are not limited to: compound 2, set forth herein as:

or compound 3, set forth herein as:

. Alternative intermediate molecules may also be used in a synthetic method for building blocks of the invention. Alternative intermediate molecules may include variants of the intermediate molecules presented herein and useful variants will be variants that retain a function of the intermediate molecules disclosed herein, namely, retain functionality in synthetic methods of the invention that are useful for preparation of a synthetic glycated polypeptide. Variant building blocks useful in methods of the invention may present different chirality at the Ca and or different protecting groups or combination of protecting groups. For example, the chirality at the Ca may be either L, which is the natural form, or S; the Na protection could be Boc and in such a case the preferable side chain Nε protection would be 2-chlorobenzyloxycarbonyl. Examples of synthetic methods that utilize protecting groups such as tert-Butyloxycarbonyl (BOC) groups and 9-Fluorenylmethyloxycarbonyl (FMOC) groups are provided herein. Methods for their use in peptide synthesis are well known in the art. It will be understood that alternative protection groups may be used. Other protection schemes can be used and are known to those of ordinary skill in the art of polypeptide synthesis.

Deprotecting, neutralization, and extension steps of building-block syntheses of the invention may be done using art-known chemical synthesis procedures. For example, deprotecting methods useful in the methods of the invention may be methods set forth herein in the Examples section and may also include alternative deprotection methods that result in the effect of deprotection of a polypeptide. In some embodiments, an agent for deprotection that may be useful in the methods of the invention may be piperidine or hydrazine. Those of ordinary skill in the art will recognize alternative deprotection agents and conditions that can be used in the methods of the invention for the synthesis of a glycated polypeptide of the invention. Thus, alternative deprotection methods and agents are envisioned for use within the scope of the invention. For example, Fmoc deprotection can be carried out by morpholine, DCHA or DIPA etc. dissolved in DCM or DMF. Use of Nα-Boc protection will require deprotection employing for example solutions of TFA in DCM or other solvents. Deprotection of Nε-2-chlorobenzyloxycarbonyl may be carried out in the presence of liquid HF.

Methods for extending, capping, and/or cleaving a polypeptide sequence in a synthetic method of the invention may include a method set forth herein in the Examples section, or may be an alternative method for extending, capping, and/or cleaving a polypeptide sequence. Those of skill in the art will recognize that various methods and conditions may be used to reach the effect of extension, capping and/or cleavage of a polypeptide in synthetic procedure. Thus, alternative extension, capping, and cleavage methods are envisioned for use within the scope of the invention. For example, extension may be carried out by employing a variety of N-protected amino acid active esters, or using mixed anhydride methods etc.; capping can be carried out by biotin or long alkyl chain containing aliphatic carboxylic acids etc.

Thus, a number of different specific conditions may be used in methods of the invention for making a synthetic glycated polypeptide. Variants of molecules and compounds may be used in methods of the invention as long as the molecules and compounds retain their function in the synthetic methods of the invention and their use in methods of synthesis set forth herein result in the production of a synthetic glycated polypeptide. The invention, in some aspects, includes the use of post-synthetic modifications of polypeptides for preparation of glycated polypeptides. It will be clear to those of ordinary skill in the art that a polypeptide prepared using a post-synthetic modification method of the invention, will be a synthetic polypeptide. The post-synthetic modification methods may include building an extended, resin-bound polypeptide or polypeptide in solution using non- glycated monomers, followed by the glycation of specific monomers after completing the assembly of the polypeptide. Examples, of methods of post-synthetic modification of a polypeptide to make a glycated polypeptide are provided herein. One of ordinary skill in the art will appreciate that alternative conditions may also be used for post-synthetic modification steps within the scope of the invention.

The synthetic methods of the invention may be used to generate synthetic glycated polypeptides, including, but not limited to, synthetic glycated CD59 polypeptides. In some aspects of the invention, a synthetic glycated polypeptide antigen is a polypeptide that is reductively alkylated on the €-amino of a lysine residue of the polypeptide. Reductive alkylation is an approximation of the Schiff base formation, Amadori rearrangement and/or both in the context of Millard reaction. One of ordinary skill in the art will recognize that the invention also encompasses alternative methods of preparing antigenic glycated polypeptides that be used to generate high-affinity antibodies that specifically bind to glycated polypeptides.

The invention, in some aspects, includes synthetic glycated polypeptides. In some embodiments, a synthetic glycated polypeptide is a synthetic glycated CD59 polypeptide. As used herein, "glycated polypeptide" means a polypeptide that has been glycated at one or more residues. The term, "glycated CD59" means CD59 that has been glycated at one or more residues. In some embodiments of the invention, a polypeptide may be glycated by reductive alkylation of the e-amino of a lysine residue of the polypeptide.

In some embodiments, a synthetic glycated CD59 is a CD59 polypeptide that has been glycated at the amino acid residue that corresponds to the amino acid residue number 41 of mature CD59, which is the 77 amino acid polypeptide set forth herein as SEQ ID NO:5. The amino acid residue in position 41 of the mature CD59 is a lysine, and this lysine in the mature CD59 and the residue that corresponds to this position in CD59 polypeptide fragments is referred to herein as "K41". CD59 in which the K41 residue is glycated is referred to herein as K41 -glycated CD59. It has been determined that natural glycation of CD59, including, but not limited to

K41 glycation of CD59, is correlated to abnormal blood sugar levels in animals, including humans, and that glycation of CD59, in particular on K41, interferes with the normal activity of CD59. CD59 functions normally by binding to the terminal components of the membrane attack complex of complement (MAC), thereby interfering with membrane insertion and polymerization of the C9 component of complement in a process that leads to a pore formation, which may result in cell lysis. Natural glycation at the K41 of CD59 interferes with CD59's ability to prevent the assembly of the MAC. While not wishing to be bound by any theory, it is believed that, as a result of glycation of CD59, the MAC is permitted to be activated and leads to the development of proliferative chronic diabetic complications. Indeed, the present inventor has shown that the membrane attack complex stimulates proliferation of fibroblasts, smooth muscle, mesangial and other cells, in part by releasing growth factors such as FGF and PDGF from MAC-targeted endothelium. The MAC also induces increased synthesis of extracellular matrix proteins by mesangial cells. Thus, increased MAC deposition in diabetic tissues is believed to induce growth factor release from endothelium, which stimulates cell proliferation in the vascular wall and contributes to the expansion of the extracellular matrix and to the glomerulosclerosis that characterizes diabetic nephropathy.

The invention includes in one aspect, methods and compositions for preparation of antibodies that specifically bind synthetic and natural glycated CD59. The invention includes, in part, methods for preparing synthetic glycated CD59 polypeptides, including, but, not limited to synthetic K41 -glycated CD59 polypeptides. Synthetic glycated CD59 polypeptides, including, but not limited to those prepared with the methods of the invention, can be used as antigens to make antibodies that specifically bind synthetic and natural glycated CD59. Compositions useful for making an antibody of the invention include a synthetic glycated CD59 polypeptide molecule. As used herein, a synthetic glycated CD59 polypeptide or fragment thereof means a synthetic glycated full-length CD59 polypeptide, or a fragment of a full-length CD59 that is a glycated fragment. One such synthetic glycated CD59 polypeptide that is useful in methods of the invention is the polypeptide set forth as NH₂-NKAWKFEHANFNDC (SEQ ID NO:8) In SEQ ID NO:8, the lysine (K) that is residue 5 of SEQ ID NO:8 corresponds to the lysine that is residue 41 (K41) of the mature CD59 polypeptide sequence set forth as SEQ ID NO:5. In SEQ ID NO:8, the K5 residue is glycated.

The invention also involves fragments of the foregoing proteins. A fragment of K41- glycated CD59 comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more contiguous amino acids of CD59 having a consecutive sequence found in CD59 or a modified CD59 sequence as described herein. In some embodiments, a fragment includes K41, which may or may not be glycated K41. Fragments of glycated CD59 can be prepared using the synthetic methods of the invention and are useful for a variety of purposes, including in the preparation of molecules that bind specifically to synthetic and natural glycated CD59 and in immunoassays well known to those of ordinary skill in the art, including competitive binding immunoassays.

The methods of the invention include methods to make an antibody that specifically binds to a glycated CD59 polypeptide. As used herein, the term "glycated CD59" polypeptide includes a natural or synthetic mature CD59 polypeptide with one or more glycated lysine (K) residues, hi some embodiments, the glycated lysine residue of CD59 is residue K41 of mature CD59. One of ordinary skill in the art will understand that a fragment of CD59 can be compared to mature CD59, and the presence of a residue in that fragment is said to "correspond" to the residue of mature CD59 (e.g., the 77 amino acid sequence set forth herein as SEQ ID NO:5). As used herein therefore, residue positions for lysines are identified as they occur in mature CD59 (SEQ BD NO:5), whether that residue is part of mature CD59 or part of a fragment or modified fragment. Thus, K41 maintains that designation in mature CD59 or fragments thereof. In some embodiments, the glycated lysine- residue in a fragment of CD59 is K41. In certain embodiments of the invention, the glycated residue of CD59 or a fragment thereof is or corresponds to K14, K30, K38, K41, K65, and/or K66 of mature CD59 polypeptide (e.g., SEQ ID NO:5). hi some embodiments, more than one lysine (K) residue is glycated.

The methods of the invention include use of a synthetic immunogenic polypeptide for the production of an anti-glycated CD59 antibody, hi some embodiments, a synthetic antigenic polypeptide can be as small as 5 amino acids in length. For example, WKFEH (SEQ ED NO:6) is a synthetic glycated antigenic fragment that may be used to generate antibodies that specifically recognize natural glycated CD59. hi some embodiments, when the size of the polypeptide is less than about 8 amino acids in length, a second carrier molecule, e.g. keyhole limpet hemocyanin (KLH), may be attached to the polypeptide to increase antigenicity of the polypeptide. Thus, small fragments of CD59 that include the desired epitope for antibody production can be used in the production of an antibody that specifically binds to the epitope. As set forth herein, SEQ ID NO:7 is WKFEH, wherein the K residue is not glycated. hi one embodiment, antibodies that bind specifically WKFEH (SEQ ID NO:6), are provided. In the preparation of antibodies that specifically bind to glycated CD59, WKFEH (SEQ ID NO:6) can be used. SEQ ED NO:6 can be used in conjunction with a second molecule, e.g. KLH as described above, as an antigenic polypeptide with which to prepare antibodies that specifically bind to the WKFEH (SEQ ID NO:6) epitope.

In addition, one or more amino acids that correspond to amino acids of CD59 or a modified CD59 as described herein can be added to either or both ends of the WKFEH (SEQ ID NO:6) sequence to make additional synthetic immunogenic polypeptides for use in making an antibody of the invention. For example, one or more amino acids may be added to the N-terminal end and/or one or more amino acids may be added to the C-terminal end of SEQ ID:6 for the production of an immunogenic fragment useful in the methods of the invention. It will be understood that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids that correspond to an amino acid of CD59 or a modified CD59 as described herein can be added to one or both ends of the amino acid sequence of WKFEH (SEQ ID NO:6). Therefore, an immunogenic fragment of the invention may include WKFEH (SEQ ID NO:6) with from 1 to 39 amino acids that correspond to amino acids of CD59 or a modified CD59 as described herein added to the N-terminal end and/or from 1 to 59 amino acids that correspond to amino acids of CD59 or a modified CD59 as described herein added to the C-terminal end.

Non-limiting examples of synthetic fragments that include K41, may be WKFEHCNFNDVTTRLREN (SEQ ID NO:9); CWKFEHCNFNDVTTRLRENELTY (SEQ ID NO:10); AGLQVYNKCWKFEHCNFNDVTTRLRENELT (SEQ ID NO:11); QVYNKCWKFEHCNFND (SEQ ID NO: 12); AGLQVYNKCWKFEHCNF (SEQ ID NO: 13); DFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYC (SEQ ID NO: 14); KCWKFEHCNFND VTTRLR (SEQ ID NO: 15);

KCWKFEHCNFNDVTTRLRENELTYYC (SEQ ID NO: 16); VYNKCWKFEHCNF (SEQ ID NO: 17); GLQVYNKCWKFEHCNFND (SEQ ID NO: 18); YNKCWKFEHCNFNE (SEQ ID NO: 19); AGLQVYNKCWKFEHCNFN (SEQ ID NO:20); and NKCWKFEHC (SEQ ID NO:21). hi some embodiments, the synthetic fragment is a synthetic K41 -glycated fragment.

The invention also includes synthetic fragments of CD59 that include a lysine that is K14, K30, K38, K65, or K66. In some embodiments of the invention a particular a lysine in a fragment is glycated and in some embodiments of the invention the lysine is not glycated. A fragment of CD59 that may be useful in the invention is at least five amino acids in length and includes Kl 4, with between 1 and 13 amino acids added to the N-terminal side of Kl 4 and/or from 1 to 63 amino acids added to the C-terminal side of Kl 4, wherein the amino acids that are added to the N- terminal and/or C-terminal side of Kl 4 correspond to amino acids in the same positions in CD59 or a modified CD59 as described herein. Examples of synthetic fragments that include Kl 4, although not intended to be limiting may be: PNPTADCKTAVNC (SEQ ID NO:22); DCKTAVNC (SEQ ID NO:23); PNPTADCKTAVNC (SEQ ID NO:24); and LQCYNCPNPTADCK (SEQ ID NO:25). In some embodiments, a synthetic fragment is a synthetic K14-glycated fragment.

Another synthetic fragment of CD59 that may be useful in the methods of the invention is at least five amino acids in length and includes K30, with between 1 and 29 amino acids added to the N-terminal side of K30 and/or from 1 to 47 amino acids added to the C-terminal side of K30, wherein the amino acids that are added to the N-terminal and/or C-terminal side of K30 correspond to amino acids in the same positions in CD59 or a modified CD59 as described herein. Examples of synthetic fragments that include K30, although not intended to be limiting may be: DFDACLITKAGLQ (SEQ ID NO:26); FDACLITKAGLQVY (SEQ ID NO:27); CLITKAGLQVYN (SEQ ID NO:28); and DFDACLITKAG (SEQ ID NO:29). In some embodiments, a synthetic fragment is a synthetic K30-glycated fragment.

Another synthetic fragment of CD59 that may be useful in the invention is at least five amino acids in length and includes K38, with between 1 and 37 amino acids added to the N-terminal side of K38 and/or from 1 to 39 amino acids added to the C-terminal side of K38, wherein the amino acids that are added to the N-terminal and/or C-terminal side of the K38 lysine correspond to amino acids in the same positions in CD59 or a modified CD59 as described herein. Examples of synthetic fragments that include K38, although not intended to be limiting may be: QVYNKCW (SEQ ID NO:30); VYNKCW (SEQ ID NO:31); AGLQVYNKCW (SEQ ID NO:32); and AGLQVYNKCWKFEHC (SEQ ID NO:33). In some embodiments, a synthetic fragment is a synthetic K38-glycated fragment.

Another synthetic fragment of CD59 that may be useful in the invention is at least five amino acids in length and includes K65, with between 1 and 64 amino acids added to the N-terminal side of K65 and/or from 1 to 12 amino acids added to the C-terminal side of K65, wherein the amino acids that are added to the N-terminal and/or C-terminal side of K65 correspond to amino acids in the same positions in CD59 or a modified CD59 as described herein. A synthetic fragment of CD59 that may be useful in the invention is at least five amino acids in length and includes K66, with between 1 and 65 amino acids added to the N- terminal side of K66 and/or from 1 to 11 amino acids added to the C-terminal side of K66, wherein the amino acids that are added to the N-terminal and/or C-terminal side of K66 correspond to amino acids in the same positions in CD59 or a modified CD59 as described herein. Examples of synthetic fragments that include K65 and K66, although not intended to be limiting may be: LTYYCCKKDLCNFNEQ (SEQ ED NO:34); NELTYYCCKKDLCNF (SEQ ID NO:35); LRENELTYYCCKKDLC (SEQ ID NO:36); CNFNDVTTRLRENELTYYCCKKDLC (SEQ ID NO:37); YCCKKDLC (SEQ ID NO:38); TTRLRENELTYYCCKKDLC (SEQ ID NO:39); VTTRLRENELTYYCCKKDLCN (SEQ ID NO:40); and FNDVTTRLRENELTYYCCKKD (SEQ ID NO:41). In some embodiments, a synthetic fragment is a synthetic K65- and/or a K66-glycated fragment.

It will be understood by those of ordinary skill in the art that it is preferable that a synthetic fragment of CD59 for use as an immunogenic fragment in the methods of the invention be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length. If a synthetic fragment of CD59 includes more than one lysine residue, it is desirable that in some embodiments, only one of the lysine residues is a glycated lysine residue. One of ordinary skill in the art will be able to use the guidance provided herein to make additional fragments of CD59 that can be used in the methods of the invention. An amino acid sequence of a synthetic polypeptide for use in methods of the invention to produce an antibody that specifically binds to glycated CD59 can be modified in one or more ways. Modifications of the amino acids sequence may be the substitution of one or more lysine residues in a polypeptide with a glycated lysine residue, substituting of one or more cysteine residues with alanine or serine residues, and/or adding a cysteine residue to the C-terminus of a polypeptide. An example of a synthetic fragment of CD59 that can be modified is NKCWKFEHCNFND (SEQ ED NO:42). SEQ ID NO:42 may be modified to include a glycated lysine residue in place of the lysine residue position 5 of the sequence, hi addition, the cysteine residues in positions 3 and 9 of SEQ ED NO:42 can be replaced with alanine residues, to avoid of spontaneous formation of intramolecular and intermolecular disulfide bridges. Further, a cysteine residue may be added to the C-terminus of SEQ ED

NO:42 to create a specific conjugation site for either a maleimido modified carrier protein or a appropriately modified matrix that will serve as an affinity media. The resulting modified synthetic polypeptide fragment of CD59 is set forth as NKAWKFEHANFNDC (SEQ ID NO:43), and is useful in the antibody-production methods of the invention. In some embodiments the sequence set forth as SEQ ID NO:43 may include a glycated residue that corresponds to the K41 residue of SEQ ID NO:3. One of ordinary skill in the art will recognize that there are additional synthetic polypeptide fragments of CD59 that can be used and/or modified and used in the methods of the invention. Thus, the invention includes synthetic polypeptides with an epitope of interest, e.g. WKFEH (SEQ ID NO:7) that may be flanked on either or both sides with one or more additional amino acids that correspond to an amino acid sequence of CD59 and may include modifications from the amino acid sequence of CD59 as described herein.

Aspects of the invention, in part, include methods for making glycated polypeptides. In some embodiments, glycated polypeptides may be made using a synthetic method for building blocks to synthesize CD59 glycated polypeptides. Glycated polypeptides may be synthesized using a synthetic method for building blocks of the invention on a solid support (e.g. on a polypeptide resin) or in solution. The synthesis of a glycated polypeptide may include preparation of intermediary compounds such as compounds (1), (2), and (3), which are provided herein. Examples of synthetic methods of preparing glycated polypeptides and methods of preparing the intermediate compounds (e.g. (compounds (1), (2), and (3)) are provided in the Examples section. As used herein with respect to polypeptides, proteins or fragments thereof, "isolated" means separated from its native environment or synthetic environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may be, but need not be, substantially pure. The term "substantially pure" means that the proteins or polypeptides are essentially free of other substances with which they may be found in synthetic mixtures, nature, or in vivo systems to an extent practical and appropriate for their intended use. Substantially pure polypeptides may be obtained naturally or produced using methods described herein and may be purified with techniques well known in the art. Because an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight of the preparation. The protein is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, i.e. isolated from other proteins.

Fragments of a protein (e.g. a CD59 protein) preferably are those fragments that retain a distinct functional capability of the protein. Functional capabilities which can be retained in a fragment include interaction with antibodies, and interaction with other polypeptides or fragments thereof. Other protein fragments, e.g., can be synthesized using methods of the invention, and tested for function using the methods exemplified herein.

Modifications to a polypeptide (e.g., a CD59 polypeptide) may be made by modification of the nucleic acid that encodes the polypeptide. These modifications may include deletions, point mutations, truncations, amino acid substitutions and/or additions of amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the polypeptide's amino acid sequence. In general, modified polypeptides (e.g. modified CD59 polypeptides) include polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Synthetic polypeptides of the invention may be modified using post-synthetic modifications (including, but not limited to those described herein). An example, though not intended to be limiting, of a modified CD59 polypeptide that includes modifications is the CD59 polypeptide set forth as NKAWKFEHANFNDC (SEQ ED NO:43), in which cysteine residues in two positions corresponding to C39 and C45 of mature CD59 (SEQ ID NO:5) have been replaced by alanine residues.

Polypeptide modifications can be made by selecting an amino acid substitution, deletion, addition. Modified polypeptides then can be tested for one or more activities (e.g., antibody binding, antigenicity, etc.) to determine which modification provides a modified polypeptide with the desired properties.

The skilled artisan will also realize that conservative amino acid substitutions may be made in a polypeptide to provide a functionally equivalent polypeptide, i.e., modified CD59 polypeptides that retain a functional capability of a CD59 polypeptide. As used herein, a

"conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Modified polypeptides (e.g., modified synthetic CD59 polypeptides) can be prepared according to methods for altering polypeptide sequence and known to one of ordinary skill in the art such. Exemplary functionally equivalent CD59 polypeptides include conservative amino acid substitutions of synthetic SEQ ID NO:5, or fragments thereof, such as a modified synthetic CD59 polypeptide. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Conservative amino-acid substitutions in a polypeptide (e.g., CD59 polypeptide) typically are made by alteration of a nucleic acid encoding the polypeptide. Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis, or by chemical synthesis of a gene encoding the polypeptide (e.g., the CD59 polypeptide). Where amino acid substitutions are made to a small fragment of a polypeptide, the substitutions can be made by directly synthesizing the polypeptide. The activity of functionally equivalent fragments of polypeptides (e.g., CD59 polypeptides) can be tested by cloning the gene encoding the altered polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered polypeptide, and testing for a functional capability of the polypeptide as disclosed herein.

The methods of the invention may include use of synthetic polypeptide fragments of CD59 for the production of antibodies that specifically bind to glycated CD59. In some embodiments, the glycated residue on CD59 that is part of the epitope specifically recognized by the antibody is the lysine residue that corresponds to K41 of CD59. The invention also includes nucleic acid sequences that encode the polypeptide sequences of the invention. For example, the invention includes nucleic acid sequences that encode a CD59 polypeptide or fragment thereof, and includes the use of the nucleic acid sequences for the production of polypeptide sequences, which in some embodiments may be further modified, etc. The full- length nucleic acid sequence of CD59 is set forth herein as SEQ ID NO:2. Fragments of SEQ ID NO:2 that encode polypeptides of CD59 that can be used to produce polypeptides that are useful to generate synthetic polypeptide antigens that can be used to raise antibodies that recognize glycated CD59 polypeptides are useful in methods of the invention.

Additional nucleic acids of the invention include nucleic acids that encode an antibody or antigen-binding fragment thereof of the invention. In certain embodiments, a nucleic acid of the invention is a nucleic acid molecule that is highly homologous to a nucleic acid that encodes an antibody or antigen-binding fragment thereof of the invention. Preferably the homologous nucleic acid molecule comprises a nucleotide sequence that is at least about 90% identical to the nucleotide sequence that encodes the antibody or antigen- binding fragment thereof. More preferably, the nucleotide sequence is at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleotide sequence that encodes an antibody or antigen-binding fragment thereof of the invention. The homology can be calculated using various, publicly available software tools well known to one of ordinary skill in the art. Exemplary tools include the BLAST system available from the website of the National Center for Biotechnology Information (NCBI) at the National Institutes of Health.

One method of identifying highly homologous nucleotide sequences is via nucleic acid hybridization. Thus the invention also includes antibodies having glycated CD59- binding properties and other functional properties described herein, which are encoded by nucleic acid molecules that hybridize under high stringency conditions to a nucleic acid that encodes an antibody or antigen-binding fragment thereof of the invention. Identification of related sequences can also be achieved using polymerase chain reaction (PCR) and other amplification techniques suitable for cloning related nucleic acid sequences. Preferably, PCR primers are selected to amplify portions of a nucleic acid sequence of interest, such as a CDR. The term "high stringency conditions" as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references that compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. One example of high-stringency conditions is hybridization at 65°C in hybridization buffer (3.5X SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH₂PO₄(pH7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid. After hybridization, a membrane upon which the nucleic acid is transferred is washed, for example, in 2X SSC at room temperature and then at 0.1 - 0.5X SSC/0.1X SDS at temperatures up to 68°C. As used herein, the term "antibody" refers to a glycoprotein that may include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C_HI , C_H2 and C_H3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or V_L) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The V_H and V_L 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 V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, 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 (CIq) of the classical complement system.

The term "antigen-binding fragment" of an antibody as used herein, refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., glycated CD59 and in some embodiments, the glycated CD59 is K41 -glycated CD59). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term

"antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and C_HI domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and CHl domains; (iv) a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et ah, (1989) Nature 341:544-546) which consists of a V_H domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V_L and V_H, 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 V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et a (1988) Science 242:423-426; and Huston et a (1988) Proc. Natl. Acad. ScL USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional procedures, such as proteolytic fragmentation procedures, as described in J. Goding, Monoclonal Antibodies: Principles and Practice, pp 98-118 (N. Y. Academic Press 1983), which is hereby incorporated by reference as well as by other techniques known to those with skill in the art. The fragments are screened for utility in the same manner as are intact antibodies.

Isolated antibodies of the invention encompass various antibody isotypes, such as IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, IgE. As used herein, "isotype" refers to the antibody class (e.g. IgM or IgGl) that is encoded by heavy chain constant region genes. Antibodies of the invention can be full length or can include only an antigen-binding fragment such as the antibody constant and/or variable domain of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD or IgE or could consist of a Fab fragment, a F(ab')₂ fragment, and a Fv fragment.

Antibodies of the present invention can be polyclonal, monoclonal, or a mixture of polyclonal and monoclonal antibodies. Antibodies of the invention can be produced by methods disclosed herein or by a variety of techniques known in the art. An example of a method to produce a monoclonal antibody that specifically binds K41 -glycated CD59 is provided in the Examples section and is discussed further herein. In some embodiments, the epitope recognized by a monoclonal antibody of the invention includes glycated lysine that corresponds to the K41 in mature CD59. In some embodiments, the epitope recognized by a monoclonal antibody of the invention includes synthetic WKFEH (SEQ ID NO:6).

Monoclonal antibody production may be effected by techniques described in the Examples section and by using alternative methods that are known in the art. The Examples section provides methods of producing a monoclonal antibody that specifically binds to K41- glycated CD59. The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. The term "polyclonal antibody" refers to a preparation of antibody molecules that comprises a mixture of antibodies active that specifically bind a specific antigen. A process of monoclonal antibody production may include obtaining immune somatic cells with the potential for producing antibody, in particular B lymphocytes, which have been previously immunized with the antigen of interest either in vivo or in vitro and that are suitable for fusion with a B-cell myeloma line. Mammalian lymphocytes typically are immunized by in vivo immunization of the animal (e.g., a mouse) with the desired protein or polypeptide, e.g., with glycated CD59 or a fragment thereof, or K41 -glycated CD59 or a fragment thereof in the present invention, hi some embodiments, the polypeptide is a modified polypeptide as described herein, hi some embodiments the polypeptide comprises the sequence set forth as SEQ ED NO:6. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Once immunized, animals can be used as a source of antibody-producing lymphocytes. Following the last antigen boost, the animals are sacrificed and spleen cells removed. Mouse lymphocytes give a higher percentage of stable fusions with the mouse myeloma lines described herein. Of these, the BALB/c mouse is preferred. However, other mouse strains, rat, rabbit, hamster, sheep, goats, camels, llamas, frogs, etc. may also be used as hosts for preparing antibody- producing cells. See; Goding (in Monoclonal Antibodies: Principles and Practice, 2d ed., pp. 60-61, Orlando, FIa., Academic Press, 1986). Mouse strains that have human immunoglobulin genes inserted in the genome (and which cannot produce mouse immunoglobulins) can also be used. Examples include the HuMAb mouse strains produced by Medarex/GenPharm International, and the XenoMouse strains produced by Abgenix. Such mice produce fully human immunoglobulin molecules in response to immunization. Those antibody-producing cells that are in the dividing plasmablast stage fuse preferentially. Somatic cells may be obtained from the lymph nodes, spleens and peripheral blood of antigen-primed animals, and the lymphatic cells of choice depend to a large extent on their empirical usefulness in the particular fusion system. The antibody-secreting lymphocytes are then fused with (mouse) B cell myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature 256:495 (1975), which is hereby incorporated by reference.

Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of the desired hybridomas. Examples of such myeloma cell lines that may be used for the production of fused cell lines include, but are not limited to Ag8, P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4.1, Sp2/0-Agl4, FO, NSO/U, MPC-I l, MPC11-X45-GTG 1.7, S194/5XX0 BuI, all derived from mice; R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210 derived from rats and U-266, GM1500-GRG2, LICR-L0N-HMy2, UC729-6, all derived from humans (Goding, in Monoclonal Antibodies: Principles and Practice, 2d ed., pp. 65-66, Orlando, FIa., Academic Press, 1986; Campbell, in Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology Vol. 13, Burden and Von Knippenberg, eds. pp. 75-83, Amsterdam, Elsevier, 1984). Those of ordinary skill in the art will be aware of numerous routine methods to produce monoclonal antibodies.

Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol ("PEG") or other fusing agents (See Milstein and Kohler, Eur. J. Immunol. 6:511 (1976), which is hereby incorporated by reference).

Procedures for raising polyclonal antibodies are well known to those of ordinary skill in the art. As a non-limiting example, anti-glycated CD59 polyclonal antibodies may be raised by administering glycated CD59 protein subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The glycated CD59 can be injected at a total volume of 100 μl per site at six different sites, typically with one or more adjuvants. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is collected 10 days after each boost. Polyclonal antibodies are recovered from the serum, preferably by affinity chromatography using glycated CD59 to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et al., editors, Antibodies: A

Laboratory Manual (1988), which is hereby incorporated by reference. Those of ordinary skill in the art will be aware of numerous routine methods to produce polyclonal antibodies. In some embodiments, the epitope recognized by the polyclonal antibody includes glycated lysine that corresponds to the K41 in mature CD59. In some embodiments, the epitope recognized by the polyclonal antibody includes WKFEH (SEQ ID NO:6).

In other embodiments, the antibodies may be recombinant antibodies. The term "recombinant antibody", as used herein, is intended to include antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for another species' immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.

The present invention further provides nucleic acid molecules encoding anti-glycated CD59 antibodies (e.g. anti-K41 -glycated CD59 antibodies) and vectors comprising the nucleic acid molecules as described herein. The vectors provided can be used to transform or transfect host cells for producing anti-glycated CD59 antibodies with the specificity of antibodies described herein. In some embodiments, the vectors can include an isolated nucleic acid molecule encoding a heavy chain and/or a light chain of an antibody of the invention encoded by a nucleic acid molecule. In a further embodiment, plasmids are given which produce the antibodies or antigen-binding fragments described herein. Antibodies or antigen-binding fragments of the invention are, preferably, isolated.

"Isolated", as used herein with respect to antibodies and antigen-binding fragments thereof, is intended to refer to an antibody (or antigen-binding fragment thereof) that is substantially free of other antibodies (or antigen-binding fragments) having different antigenic specificities (e.g., an isolated antibody that specifically binds to glycated CD59 is substantially free of antibodies that specifically bind antigens other than glycated CD59). An isolated antibody that specifically binds to an epitope, isoform or variant of a glycated polypeptide (e.g., glycated CD59) may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., CD59 species homologs). Moreover, an isolated antibody (or antigen- binding fragment thereof) may be substantially free of other cellular material and/or chemicals.

Antibodies of the invention include, but are not limited to antibodies that specifically bind to a glycated polypeptide. In some embodiments, an antibody of the invention specifically binds glycated CD59. In certain embodiments, an antibody of the invention specifically binds CD59 that is glycated at the K41 residue. As used herein, "specific binding" refers to antibody binding to a predetermined antigen with a preference that enables the antibody to be used to distinguish the antigen from others to an extent that permits the diagnostic assays described herein. Specific binding to glycated CD59 means that the antibody not only preferentially binds CD59 versus other proteins, but also that it preferentially binds a glycated CD59 versus a CD59 that is not glycated. Typically, the antibody binds with an affinity that is at least two-fold greater than its affinity for binding to antigens other than the predetermined antigen. In some embodiments, an antibody or antigen-binding fragment thereof of the invention specifically binds to K41-glycated CD59 and in other embodiments an antibody of the invention or antigen-binding fragment thereof specifically binds to a CD59 that is glycated at lysine residue that does not correspond to K41 ofCD59.

Anti-K41 -glycated CD59 antibodies or antigen-binding fragments thereof, of the invention, can specifically bind endogenous K41-glycated CD59 with sub-nanomolar affinity. The binding affinities may be about lxlO^"5M, Ix 10^'6M, 1x10^'7M, 1x10^"8M, IxIO ⁹M, IxIO^"10, or less. A binding affinity for glycated CD59 of an antibody of the invention , may be about IxIO ¹¹M or less, or may be about IxIO ¹²M or less. In a particular embodiment the binding affinity is less than about 5xl0^"nM. In some aspects of the invention, the antibody or antigen-binding fragment thereof binds to a conformational epitope within the glycated CD59 molecule. To determine if the selected anti-glycated CD59 antibodies bind to conformational epitopes, each antibody can be tested in assays using native protein (e.g., non-denaturing immunoprecipitation, flow cytometric analysis of cell surface binding) and denatured protein (e.g., Western blot, immunoprecipitation of denatured proteins). A comparison of the results will indicate whether the antibodies bind conformational epitopes. Antibodies that bind to native protein but not denatured protein are those antibodies that bind conformational epitopes, and are preferred antibodies.

In some embodiments, antibodies of the invention competitively inhibit the specific binding of a second antibody to its target glycated epitope on glycated CD59. In some embodiments, the target epitope includes the sequence set forth as synthetic WKFEH (SEQ ID NO:6), which is a glycated sequence. To determine competitive inhibition, a variety of assays known to one of ordinary skill in the art can be employed. For example, competition assays can be used to determine if an antibody competitively inhibits binding to glycated CD59 (or K41 -glycated CD59) by another antibody. These methods may include cell-based methods employing flow cytometry or solid phase binding analysis. Other assays that evaluate the ability of antibodies to cross-compete for glycated CD59 (or K41 -glycated CD59) molecules in solid phase or in solution phase, also can be used.

Certain antibodies competitively inhibit the specific binding of a second antibody to its target epitope on glycated CD59 (or K41 -glycated CD59) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. Inhibition can be assessed at various molar ratios or mass ratios; for example competitive binding experiments can be conducted with a 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold or more molar excess of the first antibody over the second antibody.

Other antibodies include antibodies that specifically bind to an epitope on glycated CD59 defined by a second antibody. To determine the epitope, one can use standard epitope mapping methods known in the art. For example, fragments (polypeptides) of K41 -glycated CD59 antigen (preferably synthetic polypeptides) that bind the second antibody can be used to determine whether a candidate antibody binds the same epitope. In some embodiments, the epitope is WKPEH (SEQ ID NO:6), which includes the glycated lysine that corresponds to K41 of mature CD59. For linear epitopes, overlapping polypeptides of a defined length (e.g., 8 or more amino acids) may be synthesized. The polypeptides preferably are offset by 1 amino acid, such that a series of polypeptides covering every 8 amino acid fragment of the glycated CD59 protein sequence are prepared. Fewer polypeptides can be prepared by using larger offsets, e.g., 2 or 3 amino acids. In addition, longer polypeptides (e.g., 9-, 10- or 11- mers) can be synthesized. Binding of polypeptides to antibodies can be determined using standard methodologies including surface plasmon resonance (BIACORE) and ELISA assays. For examination of conformational epitopes, larger glycated CD59 fragments, including in some embodiments K41-glycated CD59, can be used. Other methods that use mass spectrometry to define conformational epitopes have been described and can be used (see, e.g., Baerga-Ortiz et al., Protein Science 11:1300-1308, 2002 and references cited therein). Still other methods for epitope determination are provided in standard laboratory reference works, such as Unit 6.8 ("Phage Display Selection and Analysis of B-cell Epitopes") and Unit 9.8 ("Identification of Antigenic Determinants Using Synthetic Polypeptide Combinatorial Libraries") of Current Protocols in Immunology, Coligan et al., eds., John Wiley & Sons. Epitopes can be confirmed by introducing point mutations or deletions into a known epitope, and then testing binding with one or more antibodies to determine which mutations reduce binding of the antibodies. Antibodies that bind to glycated CD59 may be tested in in vitro or in vivo models (e.g., in mice) to determine their efficacy in binding to glycated CD59. These antibodies can be selected, for example, based on the following criteria, which are not intended to be exclusive: 1) binding to live cells or fixed cells that express glycated CD59; 2) high affinity of binding to glycated CD59; 3) binding to a unique epitope on CD59 (e.g., a K41 glycated epitope); 4) binding to a conformational epitope on glycated CD59; and 5) minimal cross- reactivity with cells or tissues that do not express glycated CD59; Preferred antibodies of the invention meet one or more, and preferably all, of these criteria.

An antibody or antigen-binding fragment thereof of the invention can be linked to a detectable label. Detectable labels useful in the invention include, but are not limited to: a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label. The detectible labels of the invention can be attached to the antibodies or antigen-binding fragments thereof by standard protocols known in the art. In some embodiments, the detectible labels may be covalently attached to an anti-CD59 antibody or antigen-binding fragment thereof of the invention. The covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging moieties. Many bivalent or polyvalent agents are useful in coupling protein molecules to other proteins, polypeptides or amine functions, etc. For example, the literature is replete with coupling agents such as carbodiimides, diisocyanates, glutaraldehyde, and diazobenzenes. This list is not intended to be exhaustive of the various coupling agents known in the art but, rather, is exemplary of the more common coupling agents.

A wide variety of detectable labels are available for use in methods of the invention and may include labels that provide direct detection (e.g., fluorescence, colorimetric, or optical, etc.) or indirect detection (e.g., enzyme-generated luminescence, epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, labeled antibody, etc.). A variety of methods may be used to detect a detectable label depending on the nature of the label and other assay components. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for using and detecting labels are well known to those of ordinary skill in the art. Methods of the invention may be used for in vivo, in vitro, and/or ex vivo imaging, including but not limited to real- time imaging. The presence of a labeled antibody in a subject can be detected by in vivo, ex vivo, or in vitro imaging using standard methods. Examples of detection methods include, but are not limited to, MRI, functional MRI, X-Ray detection, PET, CT imaging, immunohistochemistry, Western blot of tissues or cells, or by any other suitable detection method.

The term "detectable label" as used here means a molecule preferably selected from, but not limited to, the group consisting of fluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent, and bioluminescent molecules. As used herein, a detectable label may be a colorimetric label, e.g., a chromophore molecule. In some aspects of the invention, an antibody may be detectably labeled with a single or with two or more of the foregoing detectable labels.

Radioactive or isotopic labels may be, for example, ¹⁴C, ³H, ³⁵S, ¹²⁵I, and ³²P. Fluorescent labels may be any compound that emits an electromagnetic radiation, preferably visible light, resulting from the absorption of incident radiation and persisting as long as the stimulating radiation is continued.

Examples of fluorescent labels that may be used on antibodies of the invention and in methods of the invention include but are not limited to 2,4-dinitrophenyl, acridine, cascade blue, rhodamine, 4-benzoylphenyl, 7-nitrobenz-2-oxa-l,3-diazole, 4,4-difluoro-4-bora-3a,4a- diaza-3-indacene and fluorescamine. Absorbance-based labels may be molecules that are detectable by the level of absorption of various electromagnetic radiation. Such molecules may be, for example, the fluorescent labels indicated above.

Chemiluminescent labels in this invention refer to compounds that emit light as a result of a non-enzymatic chemical reaction. Methods of the invention may also include the use of a luminescent detectable diagnostic molecule such as enhanced green fluorescent protein (EGFP), luciferase (Luc), or another detectable expression product.

Enzymatic methods for detection may be used including the use of alkaline phosphatase and peroxidase. Additional enzymes may also be used for detection in methods and kits of the invention.

As used herein, fluorophores include, but are not limited to amine-reactive fluorophores that cover the entire visible and near-infrared spectrum. Examples of such fluorophores include, but are not limited to, 4-methylumbelliferyl phosphate, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), BODIPY dyes; Oregon Green, rhodamine green dyes; the red-fluorescent Rhodamine Red-X, Texas Red dyes; and the UV light-excitable Cascade Blue, Cascade Yellow, Marina Blue, Pacific Blue and AMCA-X fluorophores. Fluorophores may also include non-fluorescent dyes used in fluorescence resonance energy transfer (FRET). A labeled antibody of the invention can be prepared from standard moieties known in the art. As is recognized by one of ordinary skill in the art, the labeling process for preparing a detectable labeled antibody or fragment thereof may vary according to the molecular structure of the antibody and the detectable label. Methods of labeling antibodies with one or more types of detectable labels are routinely used and are well understood by those of ordinary skill in the art.

In some embodiments, it is contemplated that one may wish to first derivatize the antibody, and then attach the detectible label to the derivatized product. Suitable cross- linking agents for use in this manner include, for example, SPDP (N-succinimidyl-3-(2- pyridyldithio)propionate), and SMPT, 4-succinimidyl-oxycarbonyl-methyl-(2- pyridyldithio)toluene. In some embodiments, a radionuclide may be coupled to an antibody or antigen-binding fragment thereof by chelation.

Compositions (antibodies to glycated CD59 and derivatives/conjugates thereof) of the present invention have diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or to a sample obtained from a subject to diagnose a variety of disorders. As detailed herein, the antibodies or antigen- binding fragments thereof of the invention may be used for example to isolate and identify CD59 protein and/or glycated and/or nonglycated CD59 protein. The antibodies may be coupled to specific diagnostic labeling agents for imaging of the protein or fragment thereof. The antibodies or antigen-binding fragments thereof of the invention may also be used for immunoprecipitation, immunoblotting CD59 and/or glycated CD59 using standard methods known to those of ordinary skill in the art.

In some embodiments, an antibody or antigen-binding fragment thereof of the invention that specifically binds to a glycated polypeptide may be attached to a substrate (e.g. a dipstick, microtiter plate, multiwell plate, plastic, slide, card, etc.). A sample from a subject may then be applied to the substrate and the substrate is then processed to assess whether specific binding occurs between the antibody and a polypeptide or other component of the sample. As used herein a substrate may be made of a material including any synthetic or natural material. Examples of substrates of the invention may include, but are not limited to: glass, plastic, nylon, metal, paper, cardboard, filter paper, filter membranes, etc., and can be in numerous forms including, but not limited to, tubes, centrifuge tubes, cuvettes, cards, slides, dipsticks, beads, coverslips, multiwell plates, Petri plates, etc. One of ordinary skill in the art will recognize that numerous additional types of surfaces can be used in the methods of the invention.

As will be understood by one of skill in the art, a binding assay using an antibody of the invention may also be performed in solution by contacting a sample from a subject with an antibody or antigen-binding fragment thereof of the invention when the antibody or antigen-binding fragment thereof, for example in a 96-well plate, a tube, a drop on a slide, etc.

As used herein the term "attached to a surface" means physically linked (e.g. by chemical means) to the surface and not freely removable from a surface. Examples of attachment, though not intended to be limiting are covalent binding between the substrate and an antibody, attachment via specific binding or the like. For example, "attached" in this context includes chemical linkages, etc. As used herein the term "covalently attached" means attached via one or more covalent bonds. As used herein the term "specifically attached" means an antibody or fragment thereof is chemically or biochemically linked to a surface as described above with respect to the definition of "attached," but excluding all non-specific binding. In the methods of the invention, an antibody that is attached to a substrate is attached such that the antibody is not removable from the substrate without specific stripping methods or solutions. Such stripping methods may include, but are not limited to, physical methods such as scraping or heating, enzymatic methods, and chemical methods, which may include but are not limited to contacting the attached antibody and substrate with a solution such that the link between the substrate and the surface is broken and the substrate is released.

In some embodiments of the invention, an antibody or antigen-binding fragment thereof is attached to a substrate, for example a dipstick, and is dipped into a body fluid sample (e.g., a blood or urine sample) from a subject. The surface of the substrate may then be processed using procedures well known to those of skill in the art, to assess whether specific binding occurred between the antibody and a polypeptide (e.g. a glycated CD59 polypeptide) in the subject's sample. For example, procedures may include, but are not limited to, contact with a secondary antibody, or other method that indicates the presence of specific binding.

The invention, in some aspects, includes various assays to determine levels of glycated CD59. The methods of the invention that are useful to determine levels of glycated CD59 in cells, tissues, and samples from subjects, include, but are not limited to: binding assays, such as described in the examples below; specific binding assays, such as using antibodies or antigen-binding fragments thereof of the invention that bind specifically to glycated CD59; gel electrophoresis; mass spectrometry; NMR; and the like. Immunoassays may be used according to the invention including, but not limited to, sandwich-type assays, competitive binding assays, one-step direct tests and two-step tests such as described in the examples.

Methods and assays of the invention (e.g. binding assays, gel electrophoresis; mass spectrometry; NMR; and the like) may be used to monitor changes in blood sugar levels in a subject over time. Thus, the methods of the invention may be used to examine changes in glycated CD59 levels in a subject over time. This allows monitoring of glycated CD59 levels in a subject who is believed to be at risk of developing a diabetic condition and also enables monitoring in a subject who is known to have a diabetic condition. Thus, the methods of the invention may be used to diagnose or assess a diabetic condition in a subject and may also be used to assess the efficacy of a therapeutic treatment of a diabetic condition by the assessment of the level of glycated CD59 in a subject at various time points. For example, a level of a subject's glycated CD59 can be obtained prior to the start of a therapeutic regimen (either prophylactic or as a treatment of an existing diabetic condition), during the treatment regimen and/or after a treatment regimen, thus providing information on the effectiveness of the regimen in the patient. It will be understood that a therapeutic regimen may be either prophylactic or a treatment of an existing diabetic condition in a subject. Thus, methods of the invention may be used to monitor a subject's response to prophylactic therapy and/or treatment for a diabetic condition provided to a subject. Methods of the invention (e.g. binding assays, gel electrophoresis; mass spectrometry; NMR; and the like) may also be useful to monitor the onset, progression, or regression of a diabetic condition in a subject. The level of glycated CD59 may be determined in two, three, four, or more samples obtained from a subject at separate times. The level of glycated CD59 in the samples may be compared and changes in the levels over time may be used to assess glycemic control (e.g., the status of diabetes) in the subject.

One aspect of the present invention relates to the use of the antibodies and/or antigen- binding fragments thereof of the invention for detecting glycated CD59 proteins or portions thereof in a biological sample (e.g., histological or cytological specimens, body fluid samples, biopsies and the like), and, in particular, to distinguish the level of glycated CD59 from the level of non-glycated CD59 in a sample or a subject. This method involves providing an antibody or an antigen-binding fragment thereof, which specifically binds to glycated CD59, e.g., an anti-glycated CD59 antibody. The anti-CD59 antibody may be bound to a label that permits the detection of the glycated CD59. In some embodiments, a biological sample may be contacted with a labeled anti-glycated CD59 antibody under conditions effective to permit binding of the anti-glycated CD59 antibody to glycated CD59 in the sample. The presence of glycated CD59 in the biological sample is detected by detection of the label. In some embodiments, the contact between the anti-glycated CD59 antibody and the biological sample is carried out in samples from a subject. Samples to which the methods of the invention can be applied include tissue and body fluid samples.

Thus, the anti-glycated CD59 antibodies of the present invention can be used in immunohistochemical techniques to examine human tissue, cell and bodily fluid specimens. In some embodiments, the samples are fresh samples. In some embodiments, slides containing cryostat sections of frozen, unfixed tissue biopsy samples or cytological smears are air dried, formalin or acetone fixed, and incubated with the monoclonal antibody preparation in a humidified chamber at room temperature. The slides are then washed and further incubated with a preparation of a secondary antibody directed against the monoclonal antibody, which may be some type of anti-mouse immunoglobulin if the monoclonal antibodies used are derived from the fusion of a mouse spleen lymphocyte and a mouse myeloma cell line. This secondary antibody is tagged with a detectable compound, for instance a fluorescent compound such as rhodamine or fluorescein isothiocyanate, that fluoresces at a particular wavelength. The staining pattern and intensities within the sample are then determined by standard imaging methods such as microscopy and optionally photographically recorded.

As yet another alternative, computer enhanced fluorescence image analysis or flow cytometry can be used to examine tissue specimens or cells using the anti-glycated CD59 antibodies of the invention. The anti-glycated CD59 antibodies of the invention are particularly useful in assessing samples obtained from subjects which can be evaluated using a fluorescence image analyzer or with a flow cytometer.

The antibodies and/or antigen-binding fragments thereof of the present invention can be used to screen patients for diseases associated with the presence of elevated levels of glycated CD59. As used herein, the term "elevated" means higher, for example elevated versus a control level. In addition, the antibodies of the invention can be used to identify the recurrence of a disease associated with an elevated level of glycated CD59. The antibodies of the invention are particularly useful in assays to differentiate whether or not a subject has a diabetic condition, because the glycated CD59 protein to which the anti-glycated CD59 antibodies bind is present in increased amounts in tissues and body fluids of subjects who have a diabetic condition. The percent of glycated CD59 in a sample can be used to determine the presence and/or status of a diabetic condition. The antibodies of the invention can be used to obtain useful prognostic information by providing an early indicator of disease onset and /or progression.

In some embodiments of the invention, antibodies of the present invention can be used in combination with other known antibodies to provide additional information regarding the level of glycated CD59 as a percentage of the level of total CD59 in a sample. For example, an antibody that binds CD59 (glycated and non-glycated) can be used to determine the total amount or level of CD59 in a sample, can be used in conjunction with an antibody of the invention that specifically binds a glycated CD59 to determine what percentage of the total CD59 in a sample is glycated CD59.

The step of contacting an antibody or antigen-binding fragment thereof of the invention with a sample to be tested can be carried out in a sample of saliva, urine, serum or other body fluids, to detect the presence of glycated CD59 in the body fluid. When the contacting is carried out in a saliva, urine, or serum sample, it is preferred that the antibody or antigen-binding fragment thereof of the invention recognize substantially no antigens in the sample other than glycated CD59. In some embodiments, it is preferred that the antibody or antigen-binding fragment thereof of the invention recognize substantially no antigens in the sample other than K41 -glycated CD59.

Antibodies and antigen-binding fragments thereof suitable for detecting glycated CD59 include anti-glycated CD59 antibodies, such as monoclonal or polyclonal antibodies. In addition, antibody fragments, half-antibodies, hybrid derivatives, probes, and other molecular constructs may be utilized. In some embodiments, the antibodies are anti-K41- glycated CD59 antibodies.

The antibodies or antigen-binding fragments thereof of the invention may also be used in a variety of assays based upon detecting levels of glycated CD59 in subjects. The assays include (1) characterizing the impact of blood sugar levels on glycation levels in a subject; (2) evaluating a treatment for regulating blood sugar levels in a subject; (3) selecting a treatment for regulating blood sugar levels in a subject; and (4) determining onset, progression, and/or regression of a condition characterized by abnormal levels of glycated protein in a subject. Thus, subjects can be characterized, treatment regimens can be monitored, treatments can be selected and diseases can be better understood using the assays of the present invention. For example, the antibodies or antigen-binding fragments thereof of the invention are useful in one aspect in methods for measuring the level of glycated CD59 in a subject, which is a direct indicator of the level of the subject's glycemic control. The impact of blood sugar levels or glycation levels thus can be measured due to the positive correlation between the level of circulating blood glucose and the amount of glycation of endogenous CD59. The level of glycated CD59 thus correlates with the level of glycemic control in the subject. Relatively low levels of glycated CD59 reflect well-controlled circulating blood sugar levels and selectively high levels of glycated CD59 reflect poorly controlled glycemic levels.

The antibodies or antigen-binding fragments thereof of the invention may be used in assays described herein, which are carried out on samples obtained from subjects. As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In all embodiments, human subjects are preferred. The samples used herein are any body tissue or body fluid sample obtained from a subject. Body fluids that may be used as samples in the methods and with the antibodies or antigen-binding fragments thereof of the invention include, but are not limited to, lymph, saliva, blood, urine, and the like.

Particularly, important subjects to which the present invention can be applied are diabetic subjects. The term "diabetic" as used herein, means an individual who, at the time the sample is taken, has a primary deficiency of insulin and/or an abnormal (e.g. reduced) ability to metabolize glucose, e.g. impaired glucose tolerance versus a normal subject. The term diabetic includes, but is not limited to, individuals with juvenile diabetes (Type 1 diabetes), adult-onset diabetes (Type 2 diabetes; also known as noninsulin-dependent diabetes), gestational diabetes, and any other conditions of insulin deficiency or reduction in the ability to metabolize glucose. The term "diabetic" is a term of art, known and understood by those practicing in the medical profession, a formal definition of which can be found in Harrison's Principles of Medicine (Harrisons, VoI 14, Principles of Internal Medicine, Eds. Fauci, A.S., E. Braunwald, KJ. Isselbacher, J.D. Wilson, J.B. Martin, D. L. Kasper, S.L.Hauser, D.L. Longo, McGraw-Hill, New York, 1999).

Methods and antibodies of the invention may also be used to detect elevated blood glucose levels subjects with blood glucose levels that are higher than normal but not yet in the range associated with a diagnosis of diabetes. Such subjects may be considered to have "pre-diabetes." and methods of the invention can be used for diagnosis of a pre-diabetes in a subject. The onset, progression, and/or regression of pre-diabetes may also be monitored using methods and antibodies of the invention. Pre-diabetes is also referred to by those of skill in the art as "impaired fasting glucose" (IFG) or "impaired glucose tolerance" (IGT). Subjects with pre-diabetes have a higher risk of developing type 2 diabetes, often within 10 years without appropriate intervention - such as diet and/or activity level changes. Health effects associated with diabetes may include vascular complications that involve the kidneys (diabetic nephropathy), the retina (diabetic retinopathy), as well as large and small blood vessels in other organs (macro- and micro-vascular disease) including nerves (diabetic neuropathy); heart attack; stroke; blindness; deafness; amputations; kidney failure; burning foot syndrome; venous insufficiency with ulceration and stasis dermatitis. Subjects with prediabetes also have a higher risk of heart disease.

Assays described herein may include the use of antibodies or antigen-binding fragments thereof of the invention and involve measuring levels of glycated CD59. Levels of glycated CD59 can be determined in a number of ways when carrying out the various methods of the invention. In one particularly important measurement, the level of glycated CD59 is measured in relation to nonglycated CD59. Thus, the measurement may be a relative measure, which can be expressed, for example, as a percentage of total CD59. Another measurement of the level of glycated CD59 is a measurement of absolute levels of glycated CD59. This could be expressed, for example, in terms of grams per liter of body fluid. Another measurement of the level of glycated CD59 is a measurement of the change in the level of glycated CD59 over time. This may be expressed in an absolute amount or may be expressed in terms of a percentage increase or decrease over time. Antibodies or antigen- binding fragments of the invention may be used in diagnostic methods alone or in conjunction with certain antibodies already known in the art. Known antibodies may include anti-CD59 antibodies as well as anti-glycation-moiety antibodies, for example, Anti-CD-59 YTH53.1, and the anti-hexitol-lysine antibody, which binds to glycated CD59. Various examples of the use of known antibodies in the methods of the invention are provided in the Examples section.

Importantly, levels of glycated CD59 can be determined using the antibodies or antigen-binding fragments thereof of the invention and are advantageously compared to controls according to the invention. The control may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as in groups having normal amounts of circulating insulin and groups having abnormal amounts of circulating insulin. Another example of comparative groups may be groups having a particular disease, condition or symptoms and groups without the disease, condition or symptoms. Another comparative group may be a group with a family history of a condition and a group without such a family history. The predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest risk or amounts of glycated protein and the highest quadrant or quintile being individuals with the highest risk or amounts of glycated protein.

The predetermined value, of course, will depend upon the particular population selected. For example, an apparently healthy population will have a different 'normal' range than will a population that is known to have a condition related to abnormal protein glycation. Accordingly, the predetermined value selected may take into account the category in which an individual falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. As used herein, "abnormal" means not normal as compared to a control. By abnormally high it is meant high relative to a selected control. Typically the control will be based on apparently healthy normal individuals in an appropriate age bracket.

In measuring the relative amount of glycated CD59 to nonglycated CD59, those of ordinary skill in the art will appreciate that the relative amount may be determined by measuring either the relative amount of glycated CD59 or the relative amount of nonglycated CD59. In other words, if 90% of an individual's CD59 is nonglycated CD59, then 10% of the individual's CD59 will be glycated CD59. Thus, measuring the level of glycated CD59 may be carried out using an antibody or antigen-binding fragment thereof of the invention in methods to measure the relative amount of nonglycated CD59.

It will also be understood that controls according to the invention may be, in addition to predetermined values, samples of materials tested in parallel with the experimental materials. Examples include samples from control populations or control samples generated through manufacture to be tested in parallel with the experimental samples. As mentioned above, it is also possible to use the antibodies or antigen-binding fragments thereof of the invention to characterize blood sugar levels by monitoring changes in the absolute or relative amounts of glycated CD59 over time. For example, it is expected that an increase in glycated CD59 correlates with increasing dysregulation of glycemic levels. Accordingly one can monitor glycated CD59 levels over time to determine if glycemic levels of a subject are changing. Changes in relative or absolute glycated CD59 of greater than 0.1% may indicate an abnormality. Preferably, the change in glycated CD59 levels, which indicates an abnormality, is greater than 0.2%, greater than 0.5%, greater than 1.0%, 2.0%, 3.0% , 4.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more. Reductions in amounts of glycated CD59 over time may indicate improved glycemic control. The antibodies or antigen-binding fragments thereof of the invention may also be used in diagnostic methods to determine the effectiveness of treatments for abnormal glycemic levels. The "evaluation of treatment" as used herein, means the comparison of a subject's levels of glycated CD59 measured in samples collected from the subject at different sample times, preferably at least one day apart. In some embodiments, the time to obtain the second sample from the subject is at least 5, 10, 20, 30, 40, 50, minutes after obtaining the first sample from the subject. In certain embodiments, the time to obtain the second sample from the subject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, 96, 120 or more hours after obtaining the first sample from the subject. The antibodies or antigen-binding fragments thereof of the invention may be used to allow the comparison of levels of glycated CD59 in two or more samples, taken at different times, which is a measure of level of the subject's glycemic control and allows evaluation of the treatment to regulate blood sugar levels. The comparison of a subject's levels of glycated CD59 measured in samples obtained at different times and/or on different days provides a measure of glycemic control to determine the effectiveness of any treatment to regulate blood sugar levels.

As will be appreciated by those of ordinary skill in the art, the evaluation of the treatment also may be based upon an evaluation of the symptoms or clinical end-points of the associated disease, such as the vascular complications of diabetes. Thus, the antibodies or antigen-binding fragments thereof of the invention are useful for determining the onset, progression or regression of a condition that is characterized by abnormal levels of glycated protein, including those characterized by abnormal levels of glycated CD59. In some instances, antibodies or antigen-binding fragments thereof of the invention can be used to test glycemic control in subjects already diagnosed as having a particular condition or disease. In other instances, antibodies or antigen-binding fragments thereof of the invention can be used to obtain measurements that represent the diagnosis of the condition or disease. In some instances, the subjects will already be undergoing drug therapy for regulating blood sugar levels, while in other instances the subjects will be without present drug therapy for regulating blood sugar levels.

According to still another aspect of the invention, antibodies or antigen-binding fragments thereof of the invention can be used in methods for treating a subject to reduce the risk of a disorder associated with abnormally-high levels of glycated CD59. The method involves selecting and administering to a subject who is known to have an abnormally-high level of glycated CD59, an agent for treating the disorder. Preferably, the agent is an agent for reducing glycated CD59 levels and is administered in an amount effective to reduce glycated CD59 levels.

In this aspect of the invention, the treatments are based upon selecting subjects who have unwanted, elevated levels of glycated CD59 and the selection can be done using the antibodies or antigen-binding fragments thereof of the invention. Such subjects may already be receiving a drug for regulating blood sugar levels, but, according to the invention, are now candidates for an elevated level of the drug based upon the presence of the elevated levels of glycated CD59. It may be appropriate according to the invention to alter a therapeutic regimen for a subject, based upon the measurement of the level of glycated CD59. This can be understood in connection with treatment of diabetics. Diabetics may be treated in at least three different ways, or with a combination thereof. Some diabetics are treated only with non-drug therapy, such as exercise and diet. Other diabetics are treated with oral drug therapy, but not with insulin that is injected. Finally, some diabetics are treated with insulin or analogs of insulin by injection. According to the present invention, as a result of determining an elevated level of glycated CD59, an individual undergoing only non-drug therapy may be a candidate for drug therapy as a result of the glycated CD59 test. Likewise, a subject receiving only oral drug therapy, may be a candidate for an insulin-based injectable therapy, due to testing with antibodies or antigen-binding fragments thereof of the invention to determine levels of glycated CD59. Finally, a subject may be free of any present treatment but may be a candidate for blood sugar level regulating treatment as a result of the use of the antibodies or antigen-binding fragments thereof of the invention in a test for glycated CD59. Thus, subjects may be selected and treated with elevated levels of the same drugs or with different therapies as a result of assays that utilize the antibodies or antigen-binding fragments thereof of the invention.

According to the present invention, some subjects may be free of symptoms otherwise calling for treatment with a particular therapy. This means that absent the use of the antibodies or antigen-binding fragments thereof of the invention to assess glycated CD59, the subject would not according to convention as of the date of the filing of the present application have symptoms calling for treatment with a particular therapy. It is only as a result of the measuring the level of glycated CD59 that the subject becomes a candidate for treatment with the therapy.

Drug therapies for regulating blood sugar levels include oral therapies with hypoglycemic agents an/or oral anti-diabetic agents, injectable therapies, and the like. Non- drug therapies for regulating blood sugar level include, but are not limited to, dietetic and/or exercise control measures. Diet and exercise alterations include, but are not limited to, reducing caloric intake, and/or increasing fiber intake, and/or decreasing fat intake, and/or increasing exercise level.

Oral drug therapies for regulating blood sugar levels include hypoglycemic agents that may include, but are not limited to:

Acarbose; Acetohexamide; Chlorpropamide; Darglitazone Sodium: Glimepiride; Glipizide; Glyburide, Repaglinide; Troglitazone; Tolazamide; Tolbutamide.

Oral drug therapies for regulating blood sugar levels include antidiabetic agents that may include but are not limited to: Acarbose, Acetohexamide; Buformin; Butoxamine Hydrochloride ; Camiglibose; Chloφropamide; Ciglitazone; Englitazone Sodium; Etoformin Hydrochloride; Gliamilide; Glibomuride; Glicetanile Gliclazide Sodium; Gliflumide; Glipizide; Glucagon; Glyburide; Glyhexamide; Glymidine Sodium; Glyoctamide; Glyparamide; Insulin; Insulin, Dalanated; Insulin Human; Insulin Human, Isophane; Insulin Human Zinc; Insulin Human Zinc, Extended; Insulin, Isophane; Insulin Lispro; Insulin,

Neutral; Insulin Zinc; Insulin Zinc, Extended; Insulin Zinc, Prompt; Linogliride; Linogliride Fumarate; Metformin; Methyl Palmoxirate; Palmoxirate Sodium; Pioglitazone Hydrochloride; Pirogliride Tartrate; Proinsulin Human; Repaglinide; Seglitide Acetate; Tolazamide; Tolbutamide; Tolpyrramide; Troglitazone; Zopolrestat. Injectable therapies for regulating blood sugar levels include, but are not limited to:

Fast-Acting Insulin:

Insulin Injection: regular insulin; Prompt Insulin Zinc Suspension; Semilente® insulin. These categories include preparations such as: Humalog® Injection; Humulin® R;. Iletin II; Novolin R, Purified Pork Regular Insulin; Velosulin BR Human Insulin Intermediate-acting Insulin:

Isophane Insulin Suspension: NPH insulin, isophane insulin; Insulin Zinc Suspension Lente® Insulin. These categories include preparations such as: Humulin® L; Humulin® R; Humulin® N NPH; Iletin® II, Lente®; Iletin® II, NPH; Novolin® L, Novolin® N, Purified Pork Lente® insulin, Purified Pork NPH isophane insulin. Intermediate and Rapid -acting Insulin Combinations:

Human Insulin Isophane Suspension/Human Insulin Injection;. This category includes preparations such as: Humulin® 50/50; Humulin ®70/30; Novolin ®70/30 Long-acting Insulin:

Protamine Zinc Insulin Suspension; Extended Insulin Zinc Suspension. These categories include preparations such as: Ultralente® Insulin, Humulin® U.

Reducing the risk of a disorder associated with abnormally high levels of glycated CD59 means using treatments and/or medications to reduce glycated CD59 levels, therein reducing, for example, the subject's risk of vascular complications including but not limited to: diabetic nephropathy, diabetic retinopathy, macro- vascular disease, micro-vascular disease, and diabetic neuropathy.

In a subject determined to have an abnormally high level of glycated CD59, an effective amount is that amount effective to reduce glycated CD59 levels in the subject. A response can, for example, also be measured by determining the physiological effects of the hypoglycemic, antidiabetic, or insulin composition, such as the decrease of disease symptoms following administration of the hypoglycemic, antidiabetic, or insulin. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response to a treatment. The amount of a treatment may be varied for example by increasing or decreasing the amount of a therapeutic composition, by changing the therapeutic composition administered, by changing the route of administration, by changing the dosage timing and so on. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration, and the like factors within the knowledge and expertise of the health practitioner. For example, an effective amount may depend upon the degree to which an individual has abnormally elevated levels of glycated CD59.

An "effective amount" of a drug therapy is that amount of a hypoglycemic, antidiabetic, or insulin or insulin analog that alone, or together with further doses, produces the desired response, e.g. reduction of glycemic level or glycated CD59 levels. Typically an effective amount of a drug or other therapy will be determined in clinical trials, establishing an effective dose for a test population versus a control population in a blind study. In some embodiments, an effective amount will be that amount that diminishes or eliminates a negative effect (e.g., symptom and/or physiological effect and/or clinical effect) of pre- diabetic or diabetic condition in a subject. In the case of treating a particular disease or condition the desired response is inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the hypoglycemic, antidiabetic, or insulin composition (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of hypoglycemic, antidiabetic, or insulin for producing the desired response in a unit of weight or volume suitable for administration to a patient. The doses of hypoglycemic, antidiabetic, or insulin administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

Various modes of administration will be known to one of ordinary skill in the art which effectively deliver the hypoglycemic, antidiabetic, or insulin to a desired tissue, cell or bodily fluid. Preferred methods for administering the hypoglycemic and antidiabetic are oral. The preferred method of administering insulin is by injection. Administration includes: topical, intravenous, oral, intracavity, intrathecal, intrasynovial, buccal, sublingual, intranasal, transdermal, intravitreal, subcutaneous, intramuscular and intradermal administration. The invention is not limited by the particular modes of administration disclosed herein. Standard references in the art (e.g., Remington 's Pharmaceutical Sciences, 18th edition, 1990) provide modes of administration and formulations for delivery of various pharmaceutical preparations and formulations in pharmaceutical carriers. Other protocols which are useful for the administration of hypoglycemic, antidiabetic, or insulin will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration (e.g., intra-organ) and the like vary from those presented herein. Administration of hypoglycemic, antidiabetic, or insulin to mammals other than humans, e.g. for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above. It will be understood by one of ordinary skill in the art that this invention is applicable to both human and animal diseases which can be treated by hypoglycemic, antidiabetic or insulin. Thus this invention is intended to be used in husbandry and veterinary medicine as well as in human therapeutics. When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. Preferred components of the composition are described above in conjunction with the description of the hypoglycemic, antidiabetic, or insulin compositions of the invention.

A hypoglycemic, antidiabetic, or insulin composition may be combined, if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the hypoglycemic, antidiabetic, or insulin, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents, as described above, including: acetate, phosphate, citrate, glycine, borate, carbonate, bicarbonate, hydroxide (and other bases) and pharmaceutically acceptable salts of the foregoing compounds.

The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal. The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.

Compositions suitable for parenteral administration conveniently comprise hypoglycemic, antidiabetic, or insulin. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non- toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

The application of the invention to a diabetic subject under treatment with an oral blood sugar regulating agent and otherwise free of symptoms calling for any oral blood sugar regulating agent, as used herein means a subject treated with oral blood sugar regulators whose glycemic-control levels appear normal based on standard diagnostic criteria, including but not limited to measurement of glycated hemoglobin levels.

The application of the invention to a diabetic subject under treatment with insulin (including analogs thereof) and otherwise free of symptoms calling for any insulin, as used herein means a subject treated with insulin whose glycemic-control levels appear to be normal based on standard diagnostic criteria, including but not limited to measurement of glycated hemoglobin levels. Dosages of blood sugar regulating agents are well-known to those of ordinary skill in the art and documented in the literature.

Also within the scope of the invention are kits comprising the compositions of the invention and instructions for use. The kits can further contain at least one additional reagent, such as one or more additional antibodies of the invention (e.g., an antibody having a complementary activity which binds to an epitope in glycated CD59 epitope distinct from the first antibody).

Kits containing the antibodies or antigen-binding fragments thereof of the invention can be prepared for in vitro diagnosis, prognosis and/or monitoring a diabetic condition or complication by the immunohistological, immunocytological and immunosero logical methods described above. The components of the kits can be packaged either in aqueous medium or in lyophilized form. When the antibodies or antigen-binding fragments thereof are used in the kits in the form of conjugates in which a label moiety is attached, such as an- enzyme or a radioactive metal ion, the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user or the kit. In some embodiments of a kit of the invention, an antibody or antigen- binding fragment thereof may be attached to a substrate, for example a dipstick, card, slide, plate, dish, tube, vial, etc.

A kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like. A first of said container means or series of container means may contain one or more anti-glycated CD59 antibodies or antigen-binding fragments thereof or glycated CD59. A second container means or series of container means may contain a label or linker-label intermediate capable of binding to the primary anti-glycated CD59 antibodies (or fragment thereof).

Referring to Fig. 1, a kit according to the invention is shown. The kit 11 includes a package 15 housing a container 17 which contains an agent for determining the level of - glycated CD59 in a sample. The kit also includes a control 19. The kit also may further comprise instructions 21, as described above. The instructions typically will be in written form and will provide guidance for carrying-out the assay embodied by the kit and for making a determination based upon that assay. Antibodies and antigen-binding fragments of the invention may also be useful in methods of screening for candidate agents that modulate levels of glycated CD59. The methods can include mixing the candidate agent with cells or tissues or in a subject and using the antibodies of the invention to determine the level of glycated CD59 before and after contact with the candidate agent. A decrease in the amount of glycated CD59 formed in comparison to a control is indicative of an agent capable of reducing the production of glycated CD59. An increase in the amount of product formed in comparison to a control is indicative of an agent capable of enhancing the production of glycated CD59.

The assay mixture comprises a candidate agent. The candidate agent is preferably an antibody, a small organic compound, or a polypeptide, and accordingly can be selected from combinatorial antibody libraries, combinatorial protein libraries, or small organic molecule libraries. Typically, a plurality of reaction mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one.of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.

Candidate agents encompass numerous chemical classes, although typically they are organic compounds, proteins or antibodies (and fragments thereof that bind antigen). In some preferred embodiments, the candidate agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500. Candidate agents comprise functional chemical groups necessary for structural interactions with polypeptides and/or nucleic acids, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate agents also can be biomolecules such as polypeptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random or non-random polypeptides, combinatorial libraries of proteins or antibodies, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents.

A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc., which may be used to facilitate optimal protein-protein and/or protein-agent binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.

The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40°C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours. After incubation, the presence or absence of and/or the level of glycated CD59 is detected by any convenient method available to the user. For example, the level of glycated CD59 can be determined through the measure of a detectible label using standard methods and as described herein.

Examples Example 1

Introduction

A polypeptide was prepared that can be used for the production of an antibody that specifically binds to K41 -glycated CD59. The polypeptide was made using a building block that included glycated and appropriately protected amino acid derivatives and stepwise synthesis leading to the preparation of antibodies that specifically recognized and bound to K41- glycated CD59. Note that as described above, compounds (7), (11), and (14) provided below are all the same mono-glycated polypeptide but have been produced using different synthetic procedures. Each has been set forth with a different compound number to clarify the data presented including the figures and HPLC results, etc.

Methods

Compound (1) [Na-FmOC-LyS(N⁶- glucito I)] :

compound (2) [Na-Fmoc-Lys(N⁶-Boc,N^ε-glucitol)]:

and compound (3) [Nα-Fmoc-Lys^^-diglucitol)]:

were prepared as described herein.

Synthesis of Building Blocks

Production of compound (1):

D-glucose Nα-Fmoc-Lys

2-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-(2,3,4,5,6-pentahydroxy-hexylam ino)- hexanoic acid [Fmoc-Lys(glucose)-OH] [N^Fmoc-N⁶- (l-Deoxy-D-glucitol-l-yl)-L-Lysine or Nα-Fmoc-Lys(N^ε-glucitol)] (compound 1) (see Donald J. Walton, et al., Carbohydrate Research, 1984, 128 (1), 37-49)

A solution of Nα-Fmoc-L-lysine (1.84 g, 5 mmol) and sodium cyanoborohydride (378 mg, 6 mmol), in THF/H₂O (50/50, 3OmL) was adjusted to pH 7.0 with 0.1N hydrochloric acid, then mixed with an aqueous solution (2.5 mL) of D-glucose (2.7 g, 15 mmol). The solution was stirred for 72 h at 37⁰C. Then the solution was acidified with a 10% acetic acid aqueous solution until pH=5, then concentrated. Pure compound (1) was obtained as a powder (2.29 g, 55%) after purification by C-18 silica gel chromatography (linear gradient: 100% H₂O 0.1%AcOH, 0% ACN 0.1%AcOH, to 70% H₂O 0.1%AcOH, 30% ACN 0.1 %AcOH).

[See Fig. 2 for HPLC and mass spec results for Compound (1)].

Conversion of compound (1) into compound (2):

6-[tert-Butoxycarbonyl-(2,3,4,5,6-pentahydroxy-hexyl)-amino]-2-(9H-fluoren-9- ylmethoxycarbonylamino)-hexanoic acid [N^α-Fmoc-N^ε-Boc-N^ε-( 1 -Deoxy-D-glucitol- 1 -yl)-L- Lysine or Nα-Fmoc-Lys(N^ε-Boc,N^ε-glucitol)] (compound 2) To a solution of compound (1) (1.34 g, 2.5 mmol) in MeOH/TEA 10% was added Boc anhydride (1.15 mL, 5 mmol). Some drops OfH₂O were added as necessary to completely dissolve the mixture. After 16h under agitation at room temperature the solution was concentrated under vacuum. The product was then purified on C- 18 silica gel (linear gradient: 100% H₂O, 0% ACN, to 60% H₂O, 40% ACN). Pure product (compound 2) was obtained after lyophilization as a white powder (1.49g, 95%). [See Fig. 3 for HPLC and mass spec results for Compound (2)].

Production of compound (3):

6-[Bis-(2,3,4,5,6-pentahydroxy-hexyl)-amino]-2-(9H-fluoren-9-ylmethoxycarbonyl-amino)- hexanoic acid [N^Fmoc-N⁶- (l,l-Dideoxy-D-glucitol-l-yl)-L-Lysine or Nα-Fmoc- LystN^ISf-diglucitol) ] (compound 3)

To a solution of Fmoc-Lys-OH (5 mmol, 1.85g) in MeOH/ 10% AcOH was added D- Glucose (9g, lOeq), then NaBH₃CN (1.57g, 5eq). After 7 days under agitation at 40°C (non complete reaction), the solution was concentrated under vacuum. The product was then purified on C-18 silica gel (linear gradient: 100% H₂O, 0% ACN, to 70% H₂O, 30% ACN). The pure product was obtained after lyophilization as a white powder (1.4g, 40%). The compound was then relyophilized 3 times in a solution H₂O/ACN/TFA 50/50/0.1 to give the corresponding TFA salt. [See Fig. 4 for HPLC and mass spec results for Compound (3)].

Example 2

Compounds 2 and 3 as described in Example 1, were used to prepare a polypeptide that included glycated lysine. Building Block incorporation on polypeptide-resin

2-Chlorotrityl chloride resin (Trt resin) was utilized as a carrier for the solid synthesis of the polypeptide. Glu(tBu) stands for Glutamic acid γ-tert-butyl ester residue; His(Trt) stands for Histidyl(N^im-trityl); Asn(Trt) stands for Asparginyl(N^β-trityl); Cys(Trt) stands for Cysteinyl(S-trityl). Asp(tBu) stands for Aspartic acid γ-tert-butyl ester residue. Each of these would be familiar to one of ordinary skill in the art of polypeptide synthesis.

Phe-Glu(tBu)-His(Trt)-Ala-Asn(Trt)-Phe-Asn(Trt)-Asp(tBu)-Cys(Trt)-

Fmo sp(tBu)-Cys(Trt)-

(6) X= Boc Y= H

Compounds (4), (5), (6) have the X and Y identities shown above, the top sequence as SEQ ID NO:46 and the lower sequence SEQ ID NO:47.

The resin Trityl-PS bound polypeptide (Ig, 0.21mmol/g) was suspended in DMF (2OmL). Then, compound Nα-Fmoc-Lys(N^ε-Boc)-OH, or compound (2) or compound (3) (3 eq.), pyBOP (330mg, 3 eq.) and DIEA (366 μL) were added and the mixture was stirred at room temperature for 35 min. The reaction was monitored by Kaiser Test [E.Kaiser, et al.,

Anal. Biochem., 34, 595 (1970)]. The resin was classically washed with 2xDMF, lxiPrOH and 2xDCM.

Polypeptide Extension on solid support - General procedure

1- Elongation

After deprotection with piperidine/DMF 20/80 (v/v) for 20 minutes and washes

(2xDMF, lxiPrOH and 2xDCM), the resin bound polypeptide (Ig, 0.21mmol/g) was suspended in DMF (2OmL). Then, Fmoc-amino acid (3 eq.), pyBOP (330mg, 3 eq.) and

DIEA (366 μL) were added and the mixture was stirred at room temperature for 35 min. The reaction was monitored by Kaiser Test. The resin was classically washed (2xDMF, lxiPrOH and 2xDCM). In some embodiments, the Fmoc-amino acid was Nα-Fmoc-LysineCN^Boc)- OH.

The N- terminus acetylation was accomplished in presence of Ac₂O/DCM (20/80, v/v) during 10 min. In the synthesis of the glycated polypeptide (7) , the resin was then treated with hydrazine 2% in DMF during 6h.

2- Cleavage and purification conditions:

The resin was stirred in a solution TFA: TIS (triisopropylsilane): H₂O: DODT (3,6- dioxa-l,8-octanedi thiol) (95:2.5:2.5:1, v/v) for 4h. Then, the resin was filtered using a sintered funnel and the filtrate concentrated under reduced pressure. The resulting oil was precipitated in ether to yield the desired product as a solid. The corresponding product was identified by LC/MS with a XTerra MS C₁₈ column, 5μm, 3* 100mm, using a linear gradient of eluting solvents - A: 0.1% AcOH in acetonitrile and B : 0.1% AcOH in water - 0 to 35% B in 30 min, flowrate 1 mL - and monitored at λmax=214 nm (crude: 50-70% purity).

The product was purified by HPLC-MS, with a X-Terra Prep MSC₁₈ column, 5μm, 30* 100mm, using a linear gradient eluting solvents - A: 0.1% AcOH in acetonitrile and B: 0.1% AcOH in water; 5 to 25% of B in 30 min) flow rate 20 mL- and monitored at λmax=214 nm to give the desired product in high purity (>95%) (Yield —20 %).

Example 3

Glycated polypeptide preparation (Compound (7), which is also referred to herein as polypeptide 7)

A monoglycated polypeptide (compound 7) was prepared using the components prepared as described above. [See Fig. 5 for HPLC and mass spec results for the monoglycated polypeptide].

SEQ ID NO:8

>o 5 referred to herein as polypeptide (8).

A non-glycated polypeptide (compound 8) was prepared using the resin-bound polypeptide described herein. [See Fig. 6 for HPLC and mass spec results for the non-glycated polypeptide].

I o Ac-Asn-Lys- Ala-Trp-Lys-Phe-Glu-His- Ala- Asn-Phe- Asn-Asp-Cys-OH

Exact mass: 1764.77

[M+2H]⁺²/2=883.4

SEQ ID NO:48

' 5 Example 4

Post-synthetic modification of polypeptides.

Methods to post-synthetic modify polypeptides in solution and on solid support are described below.

20 Preparation in solution

Compounds (9), (10), and (11) [top to bottom, also referred to herein as polypeptides (9, SEQ ID NO:49) (10, SEQ ID NO:50) and (11, SEQ ID N0:51), respectively.]

Compound (9) See Fig. 7 for HPLC and mass spec results for Compound (9).

Exact mass: 1970.90

[M+2H]⁺²/2=986.45

[M+3H]⁺³/3=657.97

SEQ ID NO:52 Compound (10) See Fig. 8 for HPLC and mass spec results for Compound (10).

Exact mass: 2134.97

[M+2H]⁺²/2=1068.49

[M+3H]⁺³/3=712.66

SEQ ID NO:53 1-Reductive amination ofLys⁵ corresponding to Lys⁴¹in hCD59

To a solution of polypeptide in DMF/10%AcOH (5 mg in 500 μL, 0.5 mM), is added.

10 mg of D-glucose (final concentration^.1 M) and 2.5 mg OfNaBH₃CN (final concentration=75 mM).and the reaction is maintained under agitation at RT for 4 days.

Then the solution is lyophilized and the product was purified by HPLC-MS, with a X-Terra Prep MSC₁₈ column, 5 μm, 30*100 mm, using linear gradient composed of eluting solvents:

A: 0.1% AcOH in acetonitrile and B: 0.1% AcOH in water (5 to 50% of A in 30 min) to give the desired product in high purity (>95%) (Yield =70 %).

Compound (11) See Fig. 9 for HPLC and mass spec results for Compound (11)

Ac-Asn-Lys-Ala-Trp- is-Ala-Asn-Phe-Asn-Asp-Cys-OH

Exact mass: 1928.84

[M+2H]⁺²/2=965.42

SEQ ID NO:8

2-Deprotection ofLys² corresponding to Lys³⁸ in hCD59

To a solution of polypeptide in MeOH (0.5 mg in 500 uL, 0.5 mM), is added 10 μL of hydrazine hydrate (2%, 0.4 M) and the reaction is maintained under agitation at RT for Ih. Then the solution is neutralized with acetic acid 10% aqueous solution (crude purity at 214 nm = 45%).

Preparation of the resin-bound partially protected Λc[Ala 39,45 ,C r-y,,s.50 ]-,hCD59(37-5O)

Phe-Glu(tBu)-His(Trt)-Ala-Asn(Trt)-Phe-Asn( _{Tr t})-Asp(tBu)-Cys(Trt)-

)-Asp(tBu)-Cys(Trt)

Compounds (12), (13), (14) after cleavage of a resin aliquot in TFA/TIS/H₂O/DODT (95/2/2/1 )[top to bottom beginning at the second compound from the top, compounds (12), (13), and (14) are also referred to herein as polypeptides (12, SEQ ID NO:54) , (13, SEQ ID NO:55), and (14, SEQ ID NO:56), respectively. The top compound is SEQ ID NO:46]

\-Elongation The resin bound polypeptide (Ig, 0.21 mmol/g) was suspended in DMF (20 mL).

Then, Fmoc-Lys(ivDde)-OH (362 mg, 3 eq.), pyBOP (330 mg, 3 eq.) and DIEA (366 μL) were added and the mixture was stirred at room temperature for 35 min. The reaction was monitored by Kaiser Test. The resin was washed consecutively with DMF, iPrOH, and DCM. The resin was then treated with morpholine/DMF (1 :1, 2x15 min), and washed in the standard manner.

The resin was suspended in DCM containing Fmoc-Trp(Boc)-OH (332 mg, 2eq), DIEA (293 μL, 8eq) and the agitated mixture was cooled to -40⁰C prior the addition of PyBop (benzotriazole-lyl-oxytripyrrolidinophosphonium hexafluorophosphate) (219 mg, 2eq). The mixture was maintained in the cold bath for 30 min at -40°C, then for 2h at RT.

The resin was washed with DCM and DMF, and the subsequent elongation/deprotection steps were continued in the standard manner. The N-terminus acetylation was accomplished in presence of Ac₂O/DCM (20/80) during 10 min.

2- Selective removal ofivDde [l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3-methylbutyl]

The peptidyl-resin was treated with 2% hydrazine monohydrate in DMF (25 mL) 3x20 min at RT. Then the resin is washed with DMF and DCM (purity at 214 nm of the crude after cleavage of an aliquot of resin = 61%).

3- Reductive aminatiόn

The peptidyl-resin was suspended in DMF 5% AcOH. Then, D-Glucose (190 mg, 5eq) and NaBH₃CN (67 mg, 5eq) were added and the mixture was stirred at RT for 2 days (purity at 214 nm of the crude after cleavage of an aliquot of resin = 58%).

Compound (12) See Fig. 10 for HPLC and mass spec results for Compound (12).

Exact mass: 1970.90 [M+2H]⁺²/2=986.45 [M+3H]⁺³/3=657.97

SEQ ID NO:57

Compound (13) See Fig. 1 1 for HPLC and mass spec results for Compound (13). Ac-Asn-L ..yyss--AAllaa--Tlφφ--LLyysε-Phe-Glu-His-Ala-Asn-Phe-Asn-Asp-Cys-OH (CH₂)₄ (CH₂)₄

NH₂ NH₂

Exact mass: 1764.77

[M+2H]⁺²/2=883.4

SEQ ID NO:58

Compound (14) See Fig. 12 for HPLC and mass spec results for Compound (14).

Ac-Asn-Lys-Ala-Tφ-Lys-Phe-Glu-His-Ala-Asn-Phe-Asn-Asp-Cys-OH

NH

OH

Exact mass: 1928.84

Example 5

Conjugation of Polypeptides to BSA and KLH

Conjugation of both the non-glycated Asn-Lys-Ala-Tφ-Lys-Phe-Glu-His-Ala-Asn-

Phe-Asn-Asp-Cys-OH (SEQ ID NO: 43; compound (8)) and glycated Ac-Asn-Lys-Ala-Tφ- LysOSf-GlucytoO-Phe-Glu-His-Ala-Asn-Phe-Asn-Asp-Cys-OH (SEQ ID NO: 8); compound

(7)) polypeptides to BSA were carried out according to protocols provided by the Pierce

Biotechnology Inc, the supplier of Imject® maleimide activated BSA kit (cat. No. 77116) with the following modifications.

Conjugation of the glycated polypeptide (compound (7)) to KLH was carried out according to protocols provided by the same manufacturer the supplier of Imject® maleimide activated mcKLH kit (Pierce Biotechnology, Inc, Rockford, MD, cat. No. 77611) with the following modifications.

Glycated polypeptide 7 (compound (7)) was completely soluble in water. The commercially obtained non-glycated polypeptide 8 (compound (8)) was solubilized by adding few microliters of IM acetic acid and adjusting the pH to 7.0 with 1 N NaOH. Both polypeptides had a C-terminal cysteine residue. The presence of cysteine was validated by performing a quantitative Ellman test. Preparations where the cysteine content was less than

30% were incubated in the presence of small amounts of immobilized-TECP (from Pierce Biotechnology Inc., Cat No. 77712) to reduce the disulfides prior to conjugation with malemide-activated BSA and KLH (keyhole limpet protein). Conjugation and purification was performed essentially as described by the manufacturer. An excess of polypeptide versus malemide-activated carrier protein was used to ensure complete consumption of all malemide groups. The quality of BSA-conjugates was verified by running samples over 10% SDS- PAGE. (Fig. 12).

Fig. 13 shows a Coomasee stained SDS-PAGE gel providing results of analysis of maleimido-BSA-"Lys⁴¹"-glycated polypeptide conjugate (11) (center lane), maleimido- activated BSA (right lane) and molecular weight markers, (left lane) Conjugation of glycated-polypeptide with KLH was verified on dot-blot by anti- glucitollysine antibody (C6C9). Antibody C6C9 is a murine monoclonal antibody that specifically binds to plasma proteins that were glucosylated in the presence of glucose and the reducing agent sodium cyanoborohydride. (see Curtiss LK, Witztumm JL, J. Clin. Invest. 72: 1427-1438, 1983). Due to the high water solubility of compound (7) its excess was removed away during the washing step.

Fig. 14 shows a dot-blot membrane providing results of dot-blot analysis of maleimido-KLH and maleimido-BSA and their conjugation products to the "Lys⁴¹ "-glycated polypeptide. The membrane was blotted with 1789 rabbit anti-glucitolysine polyclonal antibody and probed with IR-800 tagged goat anti-rabbit poly-clonal antibodies.

Example 6 Antibody Testing

The rabbit polyclonal anti-glycated CD59Ab produced using methods described herein, a 1/500 sera dilution was tested using a Dot-Blot on nitrocellulose membrane. Samples (4 μL) were spotted and allowed to dry. The membrane was blocked for Ih with non-fat dry milk (2% in TBS buffer). The blocked membrane was then exposed for 3h to anti-glycated CD59 Ab (1:500 dilution), washed 3-times with TBS-Twin 20 0.05% and then exposed to secondary anti-rabbit Ab (1:2000 dilution) in non-fat dry milk (2% in TBS-Twin 20 0.05%) followed by 3 washes with TBS-Twin 20 0.05%. The membrane was read in an Odyssey Imaging System. (LI CORE Biosciences, Lincoln, NE) .

Fig. 15 shows a dot-blot membrane providing dot blot analysis of anti-sera from three mice (A, B and C), immunized with "Lys⁴¹ "-glycated polypeptide 14 conjugated to maleimido-KLH, with (from left to right) BSA, "Lys⁴¹ "-glycated polypeptide 14-maleimido BSA conjugate, KLH and "Lys⁴ '"-glycated polypeptide 14-maleimido KLH conjugate. The dot blot was probed with IR-800 tagged goat anti-rabbit poly-clonal antibodies.

Further experiments were performed to assess polyclonal antibodies prepared using methods described herein. Fig. 16 shows a dot-blot membrane providing dot blot analysis of anti-sera (1/500 dilution) raised in two rabbits (numbered as 4879 and 4880) using "Lys⁴¹"- glycated polypeptide 7 (compound 7) conjugated to KLH as immunogen (10 weeks after immunization). For control, the pre-immune serum collected from the same animals was used to probe against (left to right) glycated-polypeptide conjugated with BSA, non glycated- polypeptide 8 (compound 8) conjugated with BSA, in-vitro glycated ghost prepared from Chinese hamster ovary (CHO) cells that were expressing human CD59, same ghosts but not glycated that serve as control, glycated CHO ghost expressing Lys41Gln mutant human CD59, same ghost but not glycated that serve as control and glycated BSA (Sigma Inc, St Louis, USA). Goat anti-rabbit antibody conjugated with Alexa-fluor 680 was used as detection antibody. Fig. 17 shows results of blots probed with preimmune serum from rabbit #4880 (Fig. 17A) or sera from rabbit #4880 10 weeks after immunization with glycated- CD59-derived peptide-KLH.

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose and variations can be made by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety.

We Claim:

Claims

CIaims

1. A composition of matter comprising purified compound (1) set forth as:

2. A method of making compound (1) set forth as:

the method comprising combining Nα-Fmoc-L-Lysine, and D-glucose, under conditions to make compound (1).

3. The method of claim 2, further comprising purifying compound (1), which is set forth as:

4. The method of claim 2, wherein the conditions include the presence of sodium cyanoborohydride (NaCNBH₃) in THF/H₂O.

5. A composition of matter comprising purified compound (2), which is set forth as:

6. A method of making compound (2), which is set forth as:

the method comprising combining compound (1), which is set forth as:

with Boc anhydride (BoC₂O) under conditions to make compound (2).

7. The method of claim 6, further comprising purifying compound (2), which is set forth as:

8. A composition of matter comprising purified compound (3), which is set forth as:

9. A method of making compound (3), which is set forth as:

the method comprising combining Nα-Fmoc-Lys-OH with D-glucose under conditions to make compound (3).

10. The method of claim 9, further comprising purifying compound (3), which is set forth as:

11. A method of incorporating compound (2), which is set forth as:

onto a resin-bound polypeptide, the method comprising: adding compound (2) to a resin-bound polypeptide, and incubating under suitable conditions to incorporate the compound (2), onto the resin- bound polypeptide.

12. The method of claim 11 , wherein incorporating comprises forming an amide bond between the carboxylic function of compound (2) and a free Nα-terminal amino group of the resin-bound polypeptide.

13. The method of claim 11, wherein the resin-bound polypeptide is in solution.

14. The method of claim 11 , further comprising washing the resin-bound polypeptide incorporating compound (2).

15. The method of claim 13, wherein the suitable conditions include the presence of pyBOP and DIEA in the suspended resin-bound polypeptide solution.

16. The method of claim 11, wherein the resin-bound polypeptide is a CD59 polypeptide or fragment thereof.

17. The method of claim 11, wherein the resin-bound polypeptide comprises the amino acid sequence set forth as: FEHANFNDC (SEQ ID NO:44).

18. The method of claim 11, wherein the resin-bound polypeptide incorporating compound (2), set forth as:

comprises an amino acid sequence set forth as: KFEHANFNDC (SEQ ID NO:45), and compound (2), is incorporated onto the lysine residue of the amino acid sequence.

19. The method of claim 11, wherein the resin-bound polypeptide is a CD59 polypeptide.

20. The method of claim 19, wherein the compound (2), set forth as:

is incorporated onto a residue of the resin-bound polypeptide that corresponds to residue K41 of a full-length CD59 polypeptide.

21. The method of claim 19, wherein the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:43.

22. A method of incorporating isolated compound (3), set forth as:

onto a resin-bound polypeptide, the method comprising: adding compound (2) to a resin-bound polypeptide, and incubating under suitable conditions to incorporate the compound (3), onto the resin- bound polypeptide.

23. The method of claim 22, wherein incorporating comprises forming an amide bond between the carboxylic function of compound (3) and a free N-terminal amino group of the resin-bound polypeptide.

24. The method of claim 22, wherein the resin-bound polypeptide is in solution.

25. The method of claim 22, further comprising washing the resin-bound polypeptide incorporating compound (3).

26. The method of claim 24, wherein the suitable conditions include the presence of pyBOP and DEEA in the suspended resin-bound polypeptide solution.

27. The method of claim 22, wherein the resin-bound polypeptide is a CD59 polypeptide or fragment thereof.

28. The method of claim 22, wherein the resin-bound polypeptide comprises the amino acid sequence set forth as: FEHANFNDC (SEQ ID NO:44).

29. The method of claim 22, wherein the resin-bound polypeptide incorporated with compound (3), set forth as:

comprises an amino acid sequence set forth as: KFEHANFNDC (SEQ ID NO:45), and wherein compound (3) is incorporated onto the lysine residue in the amino acid sequence.

30. The method of claim 22, wherein the resin-bound polypeptide is a CD59 polypeptide.

31. The method of claim 30, wherein the compound (3), set forth as:

is incorporated onto a residue of the resin-bound polypeptide that corresponds to residue K41 of a full-length CD59 polypeptide.

32. The method of claim 30, wherein the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:43.

33. A method of extending a polypeptide incorporating compound (2) set forth as:

or compound (3), set forth as:

and bound to a resin, the method comprising: deprotecting the polypeptide incorporating compound (2) or compound (3), adding Fmoc-amino acid-OH to the deprotected polypeptide, and incubating the Fmoc-amino acid-OH and the polypeptide under suitable conditions to extend the sequence of the polypeptide.

34. The method of claim 33, wherein the suitable conditions comprise the presence of pyBOP and DIEA.

35. The method of claim 33, wherein the Fmoc-amino acid-OH is Fmoc-Lysine-OH.

36. The method of claim 33, wherein the polypeptide incorporating compound (2) or compound (3) comprises a CD59 polypeptide or fragment thereof.

37. The method of claim 33, wherein the polypeptide is a polypeptide incorporating compound (3), set forth as:

38. The method of claim 37, wherein the polypeptide incorporating compound (2) comprises the amino acid sequence set forth as: KFEHANFNDC (SEQ ID NO:45), and wherein compound (2), set forth as:

is attached to the lysine residue of the amino acid sequence.

39. The method of claim 33, wherein the polypeptide is a polypeptide incorporating compound (3), set forth as:

40. The method of claim 39, wherein the polypeptide incorporating compound (3) has the amino acid sequence set forth as: KFEHANFNDC (SEQ ID NO:45), and compound (3), set forth as:

is attached to the lysine residue of the amino acid sequence.

41. The method of claim 33, wherein the polypeptide is a CD59 polypeptide.

42. The method of claim 33, wherein compound (2) or compound (3) is incorporated at a residue of the polypeptide that corresponds to residue K41 of a full-length CD59 polypeptide.

43. The method of claim 41, wherein the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:43.

44. A method of preparing a glycated polypeptide, the method comprising the steps of, (a) incorporating compound (2) set forth as:

or compound (3), set forth as:

onto a resin-bound polypeptide,

(b) deprotecting the incorporated polypeptide,

(c) extending the sequence of the deprotected polypeptide,

(d) N-terminally capping the extended polypeptide, and

(e) cleaving the capped, extended polypeptide from the resin.

45. The method of claim 44, wherein incorporating comprises forming an amide bond between the carboxylic function of compound (2) or compound (3) and a free N-terminal amino group of the resin-bound polypeptide.

46. The method of claim 44, further comprising purifying the cleaved polypeptide.

47. The method of claim 44, further comprising repeating steps (b) and (c) one or more times to lengthen the polypeptide.

48. The method of claim 44, wherein the deprotecting comprises treatment with piperidine.

49. The method of claim 44, wherein compound (2) set forth as:

is incorporated onto the polypeptide resin using the method of claim Dl.

50. The method of claim 44, wherein compound (3) set forth as:

is incorporated onto the polypeptide resin using the method of claim El.

51. The method of claim 44, wherein the polypeptide incorporating compound (2) or compound (3) is extended using the method of claim Fl.

52. The method of claim 44, wherein the resin-bound polypeptide is a CD59 polypeptide.

53. The method of claim 52, wherein compound (2) or compound (3) is incorporated onto the resin-bound polypeptide at a residue that corresponds to residue K41 of a full-length

CD59 polypeptide.

54. The method of claim 52, wherein the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:43.

55. The method of claim 54, wherein the compound (2) or compound (3) is incorporated into the polypeptide set forth as SEQ ID NO:43 at residue K5 of the amino acid sequence of SEQ ID NO:43.

56. A method of preparing a glycated polypeptide, the method comprising the steps of:

(a) N-terminally capping a resin-bound polypeptide,

(b) glycating the Nα-capped resin-bound partially protected polypeptide,

(c) deprotecting the capped resin and

(d) cleaving the capped polypeptide from the resin.

57. The method of claim 56, further comprising purifying the cleaved polypeptide.

58. The method of claim 56, wherein the resin-bound polypeptide is a CD59 polypeptide.

59. The method of claim 56, wherein the capped polypeptide is glycated at a residue that corresponds to residue K41 of a full-length CD59 polypeptide.

60. The method of claim 56, wherein the deprotecting comprises treatment with hydrazine.

61. The method of claim 58, wherein the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:43.

62. The method of claim 61, wherein the resin-bound polypeptide is glycated at the residue that corresponds to K41 of mature CD59 polypeptide.

63. A method of preparing a glycated polypeptide in solution, the method comprising the steps of:

(a) N-terminally capping a polypeptide in solution,

(b) glycating the Nα-capped, partially protected polypeptide, and

(c) deprotecting the capped polypeptide.

64. The method of claim 63, further comprising purifying the deprotected polypeptide.

65. The method of claim 63, wherein the polypeptide in solution is a CD59 polypeptide.

66. The method of claim 63, wherein the capped polypeptide is glycated at a residue that corresponds to residue K41 of a mature CD59 polypeptide.

67 The method of claim 63, wherein the deprotecting comprises treatment with hydrazine.

68. The method of claim 65, wherein the CD59 polypeptide is the polypeptide set forth as SEQ ID NO:8.

69. An isolated antibody or antigen-binding fragment thereof, that binds specifically to a glycated epitope of glycated CD59 with an affinity that is from between about 1 X 10^"5M to about 1 X 10^"12M, wherein the epitope includes a glycated lysine.

70. The isolated antibody or antigen-binding fragment thereof of claim 69, wherein the glycated lysine corresponds to K41 of CD59.

71. The isolated antibody or antigen-binding fragment thereof of claim 69, wherein the antibody is a recombinant antibody.

72. The isolated antibody or antigen-binding fragment thereof of claim 69, tagged with a detectable label.

73. The isolated antibody or antigen-binding fragment thereof of claim 72, wherein the detectable label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

74. The isolated antibody or antigen-binding fragment thereof of claim 69, wherein the antibody or antigen-binding fragment thereof is lyophilized.

75. The isolated antibody or antigen-binding fragment thereof of claim 69, wherein the antibody or antigen-binding fragment thereof is in an aqueous medium.

76. A nucleic acid sequence that encodes the antibody of claim 69.

77. An expression vector comprising an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of claim 69.

78. A host cell transformed by or transfected with the expression vector of claim 77.

79. A plasmid which produces the antibody or antigen-binding fragment thereof of any one of claims 69-71.

80. A kit for detecting the presence of glycated CD59 comprising a package including a container containing the isolated antibody or antigen-binding fragment thereof of claim 69, and instructions for use of the antibody or antigen-binding fragment thereof to detect the presence of glycated CD59.

81. The kit of claim 80, wherein the antibody is a recombinant antibody.

82. The kit of claim 80, wherein the antibody or antigen-binding fragment thereof is attached to a detectable label.

83. The kit of claim 82, wherein the detectable label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

84. The kit of claim 80, wherein the antibody or antigen-binding fragment thereof is lyophilized.

85. The kit of claim 80, wherein the antibody or antigen-binding fragment thereof is packaged in an aqueous medium.

86. The kit of claim 80, further comprising a container containing a second antibody or antigen-binding fragment thereof that specifically binds non-glycated CD59 or non-K41- glycated CD59, and instructions for using the second antibody as a control antibody.

87. A kit comprising a package including a container containing a hybridoma that comprises a nucleic acid sequence that encodes the antibody of any one of claims 69-71, and instructions for producing the antibody.

88. An immunogenic polypeptide, wherein

1) the polypeptide comprises the amino acid sequence set forth as WKFEH (SEQ ED NO:6), and is prepared using the method of any one of claims 44-55 or 56-62, or 2) the amino acid sequence of the polypeptide is a modified amino acid sequence of

SEQ ID NO:5 or fragment thereof wherein the modification of the amino acid sequence set forth as SEQ ID NO:5 is the presence of one or more N^glycated-lysine residues, or the replacement of one or more cysteine residues with alanine residues, or the addition of a cysteine residue to the C-terminus, or combinations thereof.

89. The immunogenic polypeptide of claim 88, wherein the amino acid sequence of the immunogenic polypeptide is set forth as SEQ ID NO:8.

90. A method of making an antibody that specifically binds to glycated CD59 but not to nonglycated CD59, comprising: preparing an immunogenic polypeptide of claim 88, and immunizing an animal with the immunogenic polypeptide.

91. The method of claim 90, wherein the animal is a mouse.

92. The method of claim 90, wherein the immunogenic polypeptide has the amino acid sequence set forth as SEQ ID NO:8.

93. The isolated antibody or antigen-binding fragment thereof of claim 69, wherein the glycated epitope comprises the amino acid sequence set forth as SEQ ID NO:6.

94. A method of making an antibody comprising, using a method set forth in any one of claims 44-55 or 56-62 to prepare an antigenic polypeptide comprising the amino acid sequence set forth as SEQ ID NO:6, inoculating an animal with the antigenic polypeptide, obtaining the antibody from the inoculated animal.

95. The method of claim 94, wherein the antigenic polypeptide comprises the sequence set forth as SEQ ID 8.