# US 10,309,973 B2 · Surrogates of post-translationally modified proteins and uses thereof

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- Number: US 10,309,973 B2
- Document ID: US10309973B2
- Status: Granted
- Kind: B2
- Application number: 15/204,749
- Filed: 2016-07-07
- Published: 2017-01-26
- Granted: 2019-06-04
- Earliest claimed: 2011-02-10
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Michael Chorev; Jose A. Halperin
- Web page: https://patentgrep.com/patent/US10309973B2

## CPC

- G01N 33/6893: {related to diseases not provided for elsewhere}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids}
- G01N 33/564: for pre-existing immune complex or autoimmune disease {, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9}
- G01N 33/6872: {Intracellular protein regulatory factors and their receptors, e.g. including ion channels}
- G01N 2333/70596: Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
- G01N 2440/00: Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N 2440/38: addition of carbohydrates, e.g. glycosylation, glycation
- G01N 2800/042: Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention provides compounds that are surrogates of post-translationally modified proteins and uses thereof. Numerous diseases are associated with post-translationally modified proteins that are difficult to obtain in homogenous form and in quantities needed for immunization and use as convenient standards, calibrators, and/or reference compounds that facilitate the detection and analysis of endogenous post-translationally modified proteins. The surrogate compounds of the invention typically comprise antigenic epitopes (one of which carries a post-translational modification) that are tethered by a flexible and hydrophilic linker. The resulting compound behaves like a surrogate of the post-translationally modified protein because it preserves the character of the included antigens and allows recognition by specific antibodies targeting the individual antigens. The surrogate compounds may be prepared by covalently joining two or more polypeptide epitopes using one or more linkers, wherein at least one of the epitopes comprises a post-translational modification. In one aspect, the surrogate compounds of the invention comprise a C-terminal epitope and a glycated epitope of human CD59. The inventive methods allow quantification of the levels of glycated CD59 in the serum in human subjects, particularly those with diabetes or pre-diabetes. This technological platform of post-translationally modified protein surrogates can be applied to other diseases associated with post-translationally modified proteins (e.g., autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus). In another aspect, the invention provides antibodies that bind specifically to the compounds of the invention and methods for producing such antibodies.

## Claims

### Claim 1

A kit for detecting the presence of a post-translationally modified protein in a biological sample, said kit comprising:
a first container with a reference compound, said reference compound comprising:
a first peptide comprising a non-glycated epitope of human CD59, the first peptide comprising the amino acid sequence ACNFNDVTTRLRENELTYYCAAK (SEQ ID NO: 11),
a second peptide comprising a glycated epitope of human CD59, wherein the glycated epitope comprises amino acid residue K41 of mature human CD59, and
a linker that joins the first peptide to the second peptide, wherein the linker comprises the formula:
wherein t is an integer from 1 to 12, inclusive, and k is 0 or an integer from 1 to 100, inclusive; and
instructional material for use of said kit.

### Claim 2 (depends on 1)

The kit of claim 1, wherein said first peptide comprises a disulfide bond between cysteine residues.

### Claim 3 (depends on 2)

The kit of claim 2, wherein the linker comprises the formula:

### Claim 4 (depends on 3)

The kit of claim 3, wherein said second peptide comprises the sequence R1—K41—R2, wherein:
R1 is absent or a peptide sequence derived from residues 30-40 of mature human CD59; and
R2 is absent or a peptide sequence derived from residues 42-60 of mature human CD59.

### Claim 5 (depends on 4)

The kit of claim 4, wherein said second peptide comprises the sequence of SEQ ID NO: 10.

### Claim 6 (depends on 5)

The kit of claim 5, wherein K41 of SEQ ID NO: 10 is glycated on the ε-amino to form a glycation motif, said glycation motif comprising a moiety selected from the group consisting of:

### Claim 7 (depends on 6)

The kit of claim 6, wherein the side chain of the C-terminal residue of SEQ ID NO: 10 of said second peptide is joined to the succinimidyl moiety of said linker.

### Claim 8 (depends on 7)

The kit of claim 7, wherein the side chain of the C-terminal residue of SEQ ID NO: 11 of said first peptide is linked with said linker.