WO2014205617A1

WO2014205617A1 - Lanthanide labeled peptide and use thereof

Info

Publication number: WO2014205617A1
Application number: PCT/CN2013/077729
Authority: WO
Inventors: Chun SONG; Jie Han; Chengcheng Zhang; Xin Zhao; Huizhen GENG
Original assignee: Shandong University; Jinan Chengcheng Biotechnology Co., Ltd.; Suzhou Baikang Biotechnology Co., Ltd.
Priority date: 2013-06-24
Filing date: 2013-06-24
Publication date: 2014-12-31

Abstract

The present invention provides lanthanide labeled peptides, such as lanthanide labeled insulin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), glucagon, glucagon-like peptide-1 (GLP-1), derivatives and/or analogs thereof, as well as the use in establishing robust biological assays.

Description

Lanthanide Labeled Peptide and Use thereof Field of Invention

The present invention relates to lanthanide labeled peptide and polypeptide, such as lanthanide labeled insulin, insulin- like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), glucagon, glucagon-like peptide-1 (GLP-1), their derivatives and/or analogs, as well as their use in establishing robust biological assays.

Background of the Invention

The ability to label and thus to detect proteins or peptides at trace levels is required and extremely important in many areas of biotechnology. In the past, tracking and detection of peptides was usually performed using radioisotopes. However, these methods employing radioisotopes are generally very laborious, time-consuming, expensive, and require the use of unstable and hazardous radioisotopes leading to problems and handling and disposal of the radioisotope labeled reagents. Therefore, interest has arisen in discovering alternative and safer methods of labeling and detection.

One such alternative has been the suggestion that enzyme catalyzed color development be employed. However, this proposed methodology has not found general acceptance because of a much lowered sensitivity than methods employing radiolabeled peptides. In addition, the enzyme catalyzed methodology was found not to have any general improved ease of performance over the radiolabeled nucleotide methods.

Therefore, as another alternative, various methods of detecting proteins or peptides based upon fluorescent emissions have been proposed or employed, e.g. by using specific fluorescent dyes. However, due to background emission from unbound dye, the detection limits cannot approach those in autoradiography. It has been proposed that elimination of the background problem due to free dye can be achieved by covalent modification of proteins with a fluorescent tag followed by separation of unreacted label. With appropriate choice of fluorophore and optimization of the optical train, sensitivities approaching or matching those of radioisotopic detection are considered to be possible. However, background fluorescence remains a problem for achieving high sensitivity.

Thus, there remains the need for labeled peptides that can be employed in various biological assays and clinical diagnosis to provide a means for sensitive and reliable detection of the peptide/protein of interest.

Summary of the Invention

Lanthanide elements are 15 elements from lanthanum (La) to lutetium (Lu) corresponding to the progressive filling of the 4f electrons in a subshell. Fluorescent lanthanide chelates can be divided into three main groups according to their luminescent characteristics that are based on the 4f energy levels. In the strong luminescence group (Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺ chelates), there is a large energy difference between the excited level and the ground level of these metal ions. Non-radioactive transition does not occur easily so the luminescence quantum yield is high. In the weakly luminescent group (Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Er³⁺, Tm³⁺, Yb³⁺ chelates), the energy difference between the excited and group levels of the metal ion is fairly small. Significant portion of the excitation energy is dissipated via non-radioactive thermal deactivation processes and the quantum yield is low. The chelates of the remaining lanthanide elements, La³⁺, Gd³⁺, Lu³⁺, are not luminescent.

The key properties of lanthanide chelates that make them especially suited for time-resolved fluorescence assays are as follows: (1) Decay time of lanthanide chelates can exceed 1,000 000 ns. Fluorescence from lanthanide chelates may last up to 200,000 times longer than from conventional fluorophores. Non-specific background fluorescence from plates, cells, and reagents in many fluorescence measurements has a decay time of only about 10 ns. It thus dies away before the sample fluorescence is measured. In a time-resolved fluorometer or multilabel reader, the sample is pulsed 1000 per second with an excitation light of 340 nm. In the period between flashes the sample fluorescence is measured for 400 after a delay time of 400 μβ. This explains the high sensitivity and gives statistically accurate results after a short and convenient measuring time. (2) Lanthanide chelates have large Stokes' shifts (the difference in wavelength between the excitation and emission light) (>200 nm). The Stokes' shift for europium is almost 300 nm. This big difference between excitation and emission peaks means that the fluorescence measurement is made at a wavelength where the influence of background is minimal. (3) The emission peak is very sharp so a detector can be set to very fine limits and the emission signals from different lanthanide chelates can be easily distinguished from each other. (4) Lanthanide chelates have high fluorescence intensity. These characteristics contribute

^— 12 o to the low background, high sensitivity, and very low detection limit (10 -10 M) of the emitted fluorescence of the lanthanide chelates.

The present invention provides various lanthanide labeled peptide and polypeptide, such as lanthanide labeled insulin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), glucagon, glucagon-like peptide- 1 (GLP-1), their derivatives and/or analogs, as well as their use in establishing robust biological assays.

Detailed Description of the Invention

Definition and terms

The following are definitions of the terms as used throughout this specification and claims. The definitions provided apply throughout the present specification unless otherwise indicated. Terms not defined herein have the meaning commonly understood in the art to which the term pertains.

"Amino acid" encompasses any molecule containing both amino and carboxyl functional groups, wherein the amino and carboxylate groups are attached to the same carbon (the alpha carbon). The alpha carbon optionally may have one or two further organic substituents. For the purposes of the present disclosure designation of an amino acid without specifying its stereochemistry is intended to encompass either the L or D form of the amino acid, or a racemic mixture. However, in the instance where an amino acid is designated by its one letter code (i.e., K), such a designation is intended to specify the native L form of the amino acid, whereas the D form will be specified by inclusion of a lower case d before the one letter code (i.e., dK).

As used herein, an amino acid "substitution" refers to the replacement of one amino acid residue by a different amino acid residue.

As used herein, the term "conservative amino acid substitution" is defined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

Asp, Asn, Glu, Gin;

III. Polar, positively charged residues:

His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

Met, Leu, He, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

Phe, Tyr, Trp, acetyl phenylalanine

"Native insulin" means mammalian insulin (e.g., human insulin, bovine insulin, porcine insulin or whale insulin) from natural, synthetic, or genetically engineered sources. Human insulin comprises a 21 amino acid A chain and a 30 amino acid B chain, which are cross-linked by disulfide bonds. A properly cross-linked human insulin includes three disulfide bridges: one between A7 and B7, a second between A20 and B 19, and a third between A6 and Al l .

"Insulin derivative" refers to a modified insulin peptide, comprising an A chain and B chain dimer, as well as single-chain insulin analogs thereof, that retains close sequence homology with native insulin. "Insulin derivative" exhibits some, all or enhanced activity relative to a corresponding native insulin or is converted in vivo or in vitro into a polypeptide exhibiting some, all or enhanced activity relative to a corresponding native insulin. "Insulin derivative" differs by substitution and/or deletion of at least one naturally occurring amino acid residue and/or addition of at least one amino acid residue and/or organic residue from the corresponding, otherwise identical naturally occurring insulin. One example of an insulin derivative is Gly(A21), Arg(B31), Arg(B32) human insulin (insulin Glargine by Sanofi-Aventis). Another example of an insulin derivative is insulin aspart, in which Pro(B28) is substituted with an aspartic acid residue. Another example of an insulin derivative is insulin lispro, in which Pro(B28) and Lys(B29) on the C-terminal end of the B chain are reversed. Insulin glulisine is a rapid-acting insulin analogue that differs from human insulin in that the amino acid asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid. Chemically, it is Lys(B3), Glu(B29) human insulin. Proinsulins, pre-proinsulins, insulin precursors, single chain insulin precursors of human and non-human animals and analogs of any of the foregoing are also referred to herein as insulin derivatives, as are non-mammalian insulins. Many insulin derivatives are known in the art. Unless the context specifically indicates otherwise (e.g., where a specific insulin is referenced, such as "human insulin" or the like), the term "insulin derivative" is used broadly to include native insulins and insulin derivatives.

"Single chain insulin derivative" encompasses a group of structurally-related proteins wherein the insulin A and B chains are covalently joined by a linker.

"IGF-1 derivative" comprises a peptide with a sequence as set forth in SEQ ID NO: 3 as well as derivatives thereof having 1-5 amino acid substitution, addition or deletion, with the proviso that the A chain and B chain do not each have the sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

"IGF-2 derivative" comprises a peptide sequence of SEQ ID NO: 4 as well as derivatives thereof having 1-5 amino acid substitution, addition or deletion, with the proviso that the A chain and B chain do not each have the sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

GLP-1 includes native GLP-1, a derivative or fragment of native GLP-1 peptide. Preferably, a GLP-1 derivative has the amino acid sequence of GLP-l(7-37)-OH or GLP-1 (7-36)-NH₂ or a fragment thereof modified so that 1,2,3,4,5 or 6 amino acids differ from the amino acid in the corresponding position of GLP-1 (7-37)-OH or GLP-l(7-36)-NH₂.

"GLP-1 derivative" encompasses polypeptides having from about twenty-five to about thirty-nine naturally occurring or non-naturally occurring amino acids that have sufficient homology to native GLP-1 (7-37)-OH such that they exhibit insulinotropic activity by binding to the GLP-1 receptor on β-cells in the pancreas. A GLP-1 derivative typically comprises a polypeptide having the amino acid sequence of GLP-1 (7-37)-OH, an analog of GLP-1 (7-37)-OH, a fragment of GLP-l(7-37)-OH or a fragment of a GLP-1 (7-37)-OH analog. GLP-1 (7-37)-OH has the amino acid sequence of SEQ ID NO: 6

(SEQ ID NO:6)

7 8 9 10 11 12 13 14 15 16 17

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-

18 19 20 21 22 23 24 25 26 27 28

Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- 29 30 31 32 33 34 35 36 37

Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

By custom in the art, the amino terminus of GLP-l(7-37)-OH has been assigned number 7 and the carboxy-terminus number 37.

GLP-1 derivatives also encompass "GLP-1 fragments." A GLP-1 fragment is a polypeptide obtained after truncation of one or more amino acids from the N-terminus and/or C-terminus of GLP-1 (7-37)-OH or an analog or derivative thereof. The nomenclature used to describe GLP-1 (7-37)-OH is also applicable to GLP-1 fragments. For example, GLP-1 (9-36)-OH denotes a GLP-1 fragment obtained by truncating two amino acids from the N-terminus and one amino acid from the C-terminus. The amino acids in the fragment are denoted by the same number as the corresponding amino acid in GLP-l(7-37)-OH. For example, the N-terminal glutamic acid in GLP-1 (9-36)-OH is at position 9; position 12 is occupied by phenylalanine; and position 22 is occupied by glycine, as in GLP-l(7-37)-OH. For GLP-1 (7-36)-OH, the glycine at position 37 of GLP-l(7-37)-OH is deleted.

GLP-1 derivatives also include polypeptides in which one or more amino acids have been added to the N-terminus and/or C-terminus of GLP-l(7-37)-OH, or fragments or analogs thereof. It is preferred that GLP-1 derivatives of this type have up to about thirty-nine amino acids. The amino acids in the "extended" GLP-1 derivative are denoted by the same number as the corresponding amino acid in GLP-1 (7-37)-OH. For example, the N-terminus amino acid of a GLP-1 derivative obtained by adding two amino acids to the N-terminal of GLP-1 (7-37)-OH is at position 5; and the C-terminus amino acid of a GLP-1 derivative obtained by adding one amino acid to the C-terminus of GLP-1 (7-37)-OH is at position 38. Thus, position 12 is occupied by phenylalanine and position 22 is occupied by glycine in both of these "extended" GLP-1 derivatives, as in GLP-l(7-37)-OH. Amino acids 1-6 of an extended GLP-1 derivative are preferably the same as or a conservative substitution of the amino acid at the corresponding position of GLP-l(l-37)-OH. Amino acids 38-45 of an extended GLP-1 derivative are preferably the same as or a conservative substitution of the amino acid at the corresponding position of glucagon or Exendin-4.

"GLP-1 derivatives" are also defined as a molecule having the amino acid sequence of GLP-1 or of a GLP-1 analog, but additionally having chemical modification of one or more of its amino acid side groups, a-carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine ε-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino group include, without limitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily-skilled protein chemist. The α-carbon of an amino acid may be mono- or dimethylated.

The term "native glucagon" refers to native human glucagon having the sequence H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala -Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-OH (SEQ ID NO: 5). Amino acids within the sequence can be considered to be numbered consecutively from 1 to 29 in the conventional N-terminal to C-terminal direction.

A "glucagon derivative" as used herein includes any peptide comprising, either the amino acid sequence as set forth in SEQ ID NO: 5, or any derivative thereof, including amino acid substitutions, additions, or deletions, or post translational modifications (e.g. methylation, acylation, ubiquitination and the like) of the peptide, that stimulates glucagon or GLP-I receptor activity.

"Polyethylene glycol" or "PEG", refers to condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH₂CH₂)_nOH, wherein n is at least 1.

In one aspect, the present invention provides a lanthanide labeled (polypeptide, which comprises at least one lanthanide label and wherein said lanthanide is attached to the N terminal or Lysine side chain amino group of said (polypeptide.

Depending on specific need, said lanthanide label may comprise Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺ , Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Er³⁺, Tm³⁺ or Yb³⁺ chelate.

Lanthanide labeled insulin and insulin derivatives

In one aspect, the present invention relates to an insulin derivative having an A chain comprising the sequence of GIVEQCCX₈SICSLYQLENYCX₂iX₂₂ (SEQ ID NO: 7) and a B chain comprising the sequence of

Ji-Ri-X₂3_-26HLCGSX₃₂LVEALYLVCGERGFFX₄₈X49X5oX5iX52X53 (SEQ ID NO: 8) Wherein,

X₈ is selected from the group consisting of threonine and histidine; X₂₁ is asparagine, glycine or alanine; X₂₂ is absent or has the general structure

Wherein

N is 1,2,3,4,5,6,7 or 8;

R_L IS an optional spacer between J and amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent;

Ji is a lanthanide label or absent;

Ri is an optional spacer between Ji and N terminal amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

X23-2₆ is phenylalanine-valine-asparagine-glutamine(SEQ ID NO: 49),

valine-asparagine-glutamine, asparagine-glutamine, glutamine or absent;

X32 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;

X₄₈ is tyrosine or absent, or has the general structure

Wherein

N is 1,2,3,4,5,6,7 or 8;

R_L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent;

threonine or absent, or has the general structure

Wherein

N is 1 ,2,3,4,5,6,7 or 8;

RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent;

X₅o is selected from the group consisting of proline, lysine, or absent, or has the general structure

Wherein

N is 1 ,2,3,4,5,6,7 or 8;

J is a lanthanide label or absent;

selected from the group consisting of proline, lysine, or absent, or has the general structure

Wherein

N is 1,2,3,4,5,6,7 or 8;

J is a lanthanide label;

threonine, alanine or absent r has the general structure

Wherein

N is 1 ,2,3,4,5,6,7 or 8;

J is a lanthanide label or absent;

absent, or has the general structure

Wherein

N is 1 ,2,3,4,5,6,7 or 8;

R_L is an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid; J is a lanthanide label, and

There is at least one lanthanide label comprised in said insulin derivative.

In accordance with one embodiment of the invention, an insulin analog is provided, wherein the A chain of the insulin peptide comprises the sequence

GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 9) and the B chain comprises a sequence selected from the group consisting of: HLCGSHLVEALYLVCGERGFF (SEQ ID NO: 10), FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 11) and FVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 12).

In one embodiment, a lanthanide label is attached to either insulin B chain N terminal or B29 Lysine side chain amino group. Our insulin receptor binding assay and insulin receptor phosphorylation assay results showed that lanthanide labeled insulin maintained full potency as native insulin.

In accordance with one embodiment of the invention, single-chain insulin analogs are provided. In this case, the carboxy terminus of the human insulin B chain, or a functional analog thereof, is covalently linked to the N-terminus of an A chain analog of the present application. In one embodiment, the B chain is linked to the A chain via a peptide linker of 4-12 or 4-8 amino acids. Said peptide linker is selected from the group consisting of:

Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 13), Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg- Ala-Pro-Gln-Thr (SEQ ID NO: 14), Arg-Arg-Gly-Pro-Gly-Gly-Gly (SEQ ID NO: 15), Gly-Gly-Gly-Gly-Gly-Lys-Arg (SEQ ID NO: 16), Arg-Arg-Gly-Gly-Gly-Gly- Gly (SEQ ID NO: 17), Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 18), Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 19), Gly-Gly-Tyr-Pro-Gly- Asp-Val-Lys-Arg (SEQ ID NO: 20), Arg-Arg-Tyr-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 21), Gly-Gly-His-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 22) and Arg-Arg- His-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 23), AGRGSGK (SEQ ID NO: 24); AGLGSGK (SEQ ID NO: 25); AGMGSGK (SEQ ID NO: 26); ASWGSGK (SEQ ID NO: 27); TGLGSGQ (SEQ ID NO: 28), TGLGRGK (SEQ ID NO: 29); TGLGSGK (SEQ ID NO: 30); HGLYSGK ( SEQ ID NO: 31 ) ; KGLGSGQ ( SEQ ID NO: 32); VGLMSGK (SEQ ID NO: 33); VGLSSGQ (SEQ ID NO: 34); VGLYSGK (SEQ ID NO: 35), VGLSSGK (SBQ (D NO: 36); VGMSSGK (SEQ ID NO: 37); VWSSSGK (SEQ ID NO: 38); VGSSSGK (SEQ ID NO: 39), and VGMSSGK (SEQ ID NO: 40).

Lanthanide labeled IGF-1 and IGF-1 derivatives

In another aspect, the present invention relates to an IGF-1 derivative having the following sequence:

Ji -Ri -GPETLCGAELVD ALQFVCGDRGFYFNX₂7PTGYGS S SRRAPQTGIVDECC FRSCDLRRLEMYCAPLX₆₅PAX₆₈SAX7i (SEQ ID NO: 41)

Wherein,

Ji is a lanthanide label or absent;

X₂7 is lysine, arginine, homoarginin r has the general structure

Wherein

N is 1,2,3,4,5,6,7 or 8;

R_L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label;

lysine, arginine, homoarginine, absent, or has the general structure

Wherein

N is 1 ,2,3,4,5,6,7 or 8;

RL IS an optional spacer between J and amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label;

lysine, arginine, homoarginine, or absent, or has the general structure

Wherein

N is 1 ,2,3,4,5,6,7 or 8;

J is a lanthanide label;

absent, or has the general structure

Wherein

N is 1 ,2,3,4,5,6,7 or 8; R_L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label; and

There is at least one lanthanide label comprised in said IGF-1 derivative.

In one embodiment, a lanthanide label is attached to either IGF-1 B domain N terminal or Lysine (at positions 27, 65, 68) side chain amino group. Our IGF-1 receptor binding assay and IGF-1 receptor phosphorylation assay results showed that lanthanide labeled IGF-1 maintained full potency as native IGF-1.

Lanthanide labeled IGF-2 and IGF-2 derivatives

The present invention also relates to an IGF-2 derivative having the following sequence:

J₁-R₁-AYRPSETLCGGELVDTLQFVCGDRGFYFSX₃₀PASRVSRRSRGIVEECCFR

SCDLALLETYCATPAX₆₅SEX₆₈ (SEQ ID NO: 42)

Wherein

Ri is an optional spacer between Ji and N terminal amino group, and it is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

Ji is a lanthanide label or absent;

X₃₀ is lysine, arginine, homoarginine or has the general structure

Wherein

N is 1,2,3,4,5,6,7 or 8; R_L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent;

X₆₅ is lysine, arginine, homoarginine, absent, or has the general structure

O

NH

I

J

Wherein

N is 1,2,3,4,5,6,7 or 8;

J is a lanthanide label or absent;

X₆₈ is absent, or has the general structure

_|__| O

NH

RL⁷

J

Wherein

N is 1,2,3,4,5,6,7 or 8;

R_Lis an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent; and

There is at least one lanthanide label comprised in said IGF-2 derivative. In one embodiment, a lanthanide label is attached to either IGF-2 B domain N terminal or Lysine (at positions 30 and 65) side chain amino group. Our IGF-2 receptor binding assay and IGF-2 receptor phosphorylation assay results showed that lanthanide labeled IGF-2 maintained full potency as native IGF-2.

Lanthanide labeled glucagon and glucagon derivatives

The present invention relates to a glucagon derivative having the following sequence:

HSQGTFTSDYSXi₂YLDSR AQX₂iFVX24WLMNX2₉X3o (SEQ ID NO: 43) wherein

X₁₂ is lysine or arginine_;

X₂₁ is aspartic acid, lysine, cysteine, homocysteine, ornithine, or has the general structure

Or

Wherein

N is 1,2,3,4,5,6,7 or 8;

RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group. Li is an optional spacer or linker. It is selected from the group consisting of long chain fatty acid and polyethylene glycol (PEG);

J is a lanthanide label or absent;

glutamine, lysine, cysteine, homocysteine, ornithine, or has the general structure

Or

Wherein

N is 1,2,3,4,5,6,7 or 8;

Ri has a formula of Mi-Li-NH-, where Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group. Li is an optional spacer or linker. It is selected from the group consisting of long chain fatty acid and polyethylene glycol (PEG);

J is a lanthanide label or absent;

threonine, lysine, cysteine, homocysteine, ornithine, or absent, or has the general structure

Or

Wherein

N is 1,2,3,4,5,6,7 or 8;

Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limieted to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide or vinylsulfone group. Li is an optional spacer or linker. It is selected from long chain fatty acids or polyethylene glycol (PEG);

J is a lanthanide label or absent;

lysine, cysteine, homocysteine, or a short peptide sequence of 1 to 5 amino acid long, and one amino acid of which is lysine, cysteine, homocysteine, or absent, or has the general structure

Or

Wherein

N is 1,2,3,4,5,6,7 or 8;

Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group. Li is an optional spacer or linker. It is long chain fatty acids or polyethylene glycol (PEG);

J is a lanthanide label or absent; and

There is at least one lanthanide label comprised in said glucagon derivative.

One embodiment of the present invention is directed to a glucagon derivative that has been modified relative to the native glucagon to improve the peptide solubility and stability in aqueous solutions, while retaining the native peptide's biological activity.

In one embodiment, a glucagon derivative is provided wherein the native glucagon sequence has been modified to contain a naturally occurring or synthetic amino acid in at least one of positions 16, 17, 20, 21 , 24 and 29 of the native sequence that is different from the corresponding amino acid of the native sequence. In one embodiment, one or more amino acids at position 16, 17, 20, 21 , 24 and 29 of the native sequence are substituted with an amino acid selected from the group consisting of lysine, arginine, cysteine, and ornithine. In accordance with one embodiment, the lysine residue at position 12 of the native peptide is substituted with arginine and an amino acid present at one of the positions 16, 17, 20, 21 , 24 and 29 is substituted by a single lysine. In one embodiment, the amino acid present at position 16, 17, 20, 21, 24 or 29 of the native peptide is substituted with cysteine.

It has been reported that certain positions of the native glucagon can be modified while retaining at least some of the activity of the parent peptide. Accordingly, one or more of the amino acids located at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29 of the peptide as set forth in SEQ ID NO: 5 can be substituted with an amino acid different from that present in the corresponding position of the native glucagon, while still retaining the biological activity of the native glucagon. In one embodiment, the substitutions at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 27, 28 or 29 are conservative amino acid substitutions.

In one embodiment, an amino acid substitution using a natural or synthetic amino acid is made at position 16, 17, 20, 21, 24 or 29 of the glucagon, wherein the substitute amino acid allows for the covalent attachment of a lanthanide label to the amino acid side chain. In one embodiment, a lanthanide label is bound to an amino acid side chain at position 16, 21 or 24 of the glucagon. In one embodiment, the substitution is made at position 21 or 24 of the glucagon. In one embodiment, a glucagon derivative is provided that comprises a lanthanide label covalently bound to the side chain of an amino acid present at position 16, 17, 20, 21, 24 or 29, wherein the glucagon derivative further comprises one, two or three amino acid substitutions at positions selected from positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29.

In another embodiment, the methionine residue present at position 27 of the native glucagon peptide is changed to leucine or norleucine to prevent oxidative degradation of the peptide.

The present invention also encompasses glucagon fusion peptides, wherein a second peptide has been fused to the c-terminus of the glucagon peptide. More particularly, the glucagon fusion peptide may comprise an amino acid sequence of SEQ ID NO: 45(GPSSGAPPPS), SEQ ID NO: 46(KRNRNNIA) or SEQ ID NO: 47(KRNR) linked to amino acid 29 of the glucagon peptide through a peptide bond.

Lanthanide labeled GLP-1 and GLP-1 derivatives

The present invention also relates to a GLP-1 derivative having the following sequence:

HX₈EGTFTSDVSSYLEGQAAX₂₆EFIAWLVX34GRX₃7X38 (SEQ ID NO: 44) Wherein

X₈ is selected from the group consisting of alanine, 2-methylalanine (Aib), serine and glycine;

X₂6 is lysine, arginine, cysteine, homoc steine, or has the general structure

Or

Wherein

N is 1,2,3,4,5,6,7 or 8;

J is lanthanide label or absent;

rginine, lysine, cysteine, homoc steine, or has the general structure

Or

Wherein

N is 1,2,3,4,5,6,7 or 8;

J is lanthanide label or absent;

-NH₂, glycine, lysine, cysteine, homocysteine, or absent, or has the general structure

Or

Wherein

N is 1,2,3,4,5,6,7 or 8;

Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group. Li is an optional spacer or linker. It is long chain fatty acids or polyethylene glycol (PEG);

J is lanthanide label or absent;

lysine, cysteine, homocysteine, absent, or a peptide with a sequence of one to five amino acids with one amino acid of which is lysine, cysteine, or homocysteine, or has the general structure

Or

Wherein N is 1,2,3,4,5,6,7 or 8;

J is lanthanide label or absent; and

There is at least one lanthanide label comprised in the GLP-1 derivative of the invention.

Improvement of lanthanide labels is still progressing, and their design not only calls for stronger luminescence, but also requires specific properties. A lanthanide label generally consists of a chelator, a lanthanide metal ion, and a linking group connecting the label with a biomolecule.

In early works, 2-naphthoyltrifluoroacetone (β-ΝΤΑ), 2-thenoyltrifluoroacetrone (TTA), and pivaloyltrifluoroacetone (PTA), BCDOT, BCOT and 4,4 ' -bis( 1 ", 1 ", 1 ",2",2",3 ",3"-heptafluoro-4",6"-hexanedion-6"-yl)-chlorosulfo-o-terp henyl (BHHCT) were used for fluorescent complexes of Eu³⁺, Sm³⁺, Tb³⁺, and Dy³⁺.

3+

The quantum yields and molar absorption coefficients of Eu^" β-diketonate

[Eu (R₁COCH₂COR₂)] are strongly dependent on the substituents Ri and R₂. When Ri and R₂ are alkyl or aryl, the complexes are weakly luminescent. The emission can be strongly enhanced when R₂ becomes CF₃. When R₂=CF_3> the overall emission intensity decreases in the order of Ri: naphthyl > thienyl > phenyl > alkyl. When Ri is an aryl group, such as phenyl, naphthyl, thienyl, and phenanthryl, the emission intensity increases in the order of R₂: CH₃ < CF₃ < C₂F₅ < C₃F₇ < C₄F9. Another factor affecting the luminescence of a lanthanide β-diketonate is the existence of a synergic agent. A synergic agent, such as tri-n-octylphosphine oxide (TOPO) or 1,10-phenanthroline, intensifies the emission through the formation of a ternary β-diketonate-Eu -TOPO complex.

Another class of chelators consists of derivatives of pyridine, 2,2'-bipyridine, 2,2'2"-terpyridine, and 1,10-phenanthroline.

4,7-bis(chlorosulfophenyl)-l,10-phenanthroline-2,9-dicarboxylic acid (BCPDA)-Eu fluorescence labels.

The chelators used in this invention also include, but not limited to, diethylenetriaminetetraacetic acid (DTTA), diethylenetriamine pentaacetic acid (DTPA), triethylenetetraamine hexaacetic acid (TTHA),

1.4.7.10- tetraazacyclododecane-N,N' ,N",N"'-tetra(acetic acid) (DOTA),

1.4.8.11- tetraazacyclotetradecane- 1,4, 8,1 l-tetra( acetic acid) (TETA) and 4,4'-bis (l",l",2",2",3",3"-heptafiuoro-4",6"-hexanedion-6"-yl)- chlorosulfo- O- terphenyl (BHHCT). Other chelators in the literature can also be used. The lanthanide ions include Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺, Tm³⁺, Nd³⁺, Ho³⁺, Er³⁺, Yb³⁺, Pm³⁺, Pr³⁺, Ce³⁺.

In order to use the lanthanide complexes for biological assays, they must have a linking group able to bind biomolecules. Isothiocyanate, sulfonyl chloride, carboxylate of N-hydroxysuccinimide, and maleimide are usually used to couple with peptides, proteins and biological molecules. These linking groups can be bound to an amino group or a thiol group of the biomolecules. Examples include

1 1 2 3 3

N -(p-iodoacetamidobenzyl)diethylenetriamine-N ,N ,N ,N -tetraacetic acid, iodoacetamido group of which reacts with free sulfhydryl groups on the proteins and peptides, forming a stable, covalent thioether bond, and

1 1 2 3 3

N -(p-isothiocyanatobenzyl)diethylenetriamine-N ,N ,N ,N -tetraacetic acid, isothiocyanato group of which reacts with free amino groups on the proteins and peptides, forming a stable, covalent thiourea bond. The DTTA group (diethylenetriamine tetraacetic acid) forms a stable complex with a lanthanide ion. Another example is

Eu -4,7-bis(chlorosulfophenyl)-l,10-phenanthroline-2,9-dicarboxylate

(Eu -BCPDA). Bifunctional bridging reagents are also available for the conjugation of amino or thiol derivatives of lanthanide complexes with biomolecules.

Furthermore, the chelate chemistry used for lanthanides can be compatible with chelation of radiolabeled metals (e.g. ^mIn, ^99mTc, ⁶⁸Ga) in lieu of photoactive lanthanides, allowing such agents to be used for γ-ray or positron emission imaging in the field of nuclear medicine. The location and concentration of these radionuclide-labeled agents can be determined using positron emission tomography (PET) or single photon emission computed tomography (SPECT) scanning.

Brief Description of the Figures

Figure 1 schematically illustrates combination of insulin chains.

Figure 2 schematically illustrates lanthanide labeled insulin.

Figure 3 schematicall illustrates lanthanide labeled IGF-1.

Figure 4 illustrates the chemical structure of selected examples of lanthanide chelators.

Figure 5 shows that lanthanide labeled insulins remains active and their biological activity is comparable to that of the native insulin.

The following examples only serve to further illustrate specific embodiments of the present invention and shall not be used in any aspect to limit the scope of the present invention.

Examples

Example 1 Peptide Synthesis

All linear precursor peptides were synthesized with solid phase method as C -terminal acids on Wang resin or as C-terminal amide on Rind amide resin using Fmoc chemistry. All peptides were synthesized on a 0.1 mmol scale using a 10-fold molar excess of Fmoc protected amino acids that were activated by using 10-fold excess of diisopropylcarbodiimide (DIC) and l-hydroxybenzotriazole (HOBT). A chelator such as diethylenetriamine-N,N,N",N"-tetra-tert-butyl acetate-N'-acetic acid (DTPA) (3equiv, 0.3 mmol) was coupled to the N-terminus of the solid phase bound peptide. When a chelator is coupled to the side chain amino group of lysine residue on the solid phase, an orthogonal deprotection scheme was used. Lysine side chain amino group is preferably protected by allyloxycarbonyl (aloe) group. Upon completion of syntheisis of the peptide sequence, aloe group(s) is preferably removed using tetrakis(triphenylphosphine)palladium(0) along with a 37:2: 1 mixture of methylene chloride, acetic acid, and N-Methylmorpholine (NMM) for 2 hours. The resin was then carefully washed with 0.5% DIPEA in DMF, 3x10 ml of 0.5% sodium diethylthiocarbamate in DMF, and then with 5x10 ml of 1 :1 DCM: DMF. Fmoc protecting groups were removed by treating the resin attached peptide with piperidine. Ninhydrin (2,2-Dihydroxyindane-l,3-dione) test was used to monitor the progress of coupling. Each resin-bound polypeptide chain was cleaved from the solid support by treatment with TFA at room temperature for two hours with triisopropylsilane (TIS) and H₂0 as scavenger. The cleaved peptide was precipitated and centrifuged in ice-cold diethyl ether.

Example 2 Sulfitolysis

In one example of the invention, an oxidative sulfitolysis step was employed, which involves addition of -SO₃ groups to the reduced sulfur residues on cysteines of peptides, preventing the formation of potentially incorrect disulfide bonds during the solubilization and early purification steps prior to correct refolding of the proteins under optimal conditions for renaturation. Incorrect disulfide bond formation during the solubilization and renaturation processes of peptide production accounts for a significant decrease in yield.

0.5 mM (about 1-1.5 g) crude cysteine containing peptide was dissolved in 150 ml fresh buffer comprising of 86g Guanidine HC1, 1.8 g Tris, 5.2 g Na₂S0₃, 3.75g Na₂S₄0₆. The pH was adjusted to about 8.5 and stirred vigorously at room temperature for 1 hour. The sample was preferably stirred rapidly enough that air is pulled into the solution.

G10 or G25 column was used for desalting. A buffer was 0.05 M ammonium bicarbonate and B buffer was 0.05 M ammonium bicarbonate with 50% acetonitrile. The correct fractions were combined, frozen, and lyophilized. Combination of human insulin A and B chain s-sulfonates

A chain s-sulfonates and B chain s-sulfonates (2: 1, w/w) were dissolved in 0.1M glycine buffer (pH 10.5) at a peptide concentration of 5-10 mg/ml. 1.2 molar equivalent (SH:SSC>3^~) of dithiothreitol (DTT) was added. The reaction was stirred at

4°C overnight in an open round bottom flask. The oxidation reaction was analyzed by analytical RP-HPLC. The reaction mixture was purified by preparative RP-HPLC.

Example 3 Synthesis of Glucagon Cysl 7(l-29)

HSQGTFTSDYSKYLDSCRAQDFVQWLMNT(SEQ ID NO: 48) and Similar MonoCys Analogs

0.2 mmole Boc Thr(OBzl) Pam resin (SynChem Inc) was added in a 60 ml reaction vessel and the sequence as described below was entered and run on a modified Applied Biosystems 430A Peptide Synthesizer using FastBoc HBTU-activated single couplings.

The following side chain protecting groups were used: Arg(Tos), Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl-Z), Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br-Z). The completed peptidyl resin was treated with 20% piperidine/dimethylformamide to remove the Trp formyl protection and then transferred to an HF reaction vessel and dried in vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were added along with a magnetic stir bar. The vessel was attached to the HF apparatus (Pennisula Labs), cooled in a dry ice/methanol bath, evacuated, and approx. 10 ml liquid hydrogen fluoride was condensed in. The reaction unit was stirred in an ice bath for 1 hr, then the HF was removed in vacuo. The residue was suspended in ethyl ether; the solids were filtered, washed with ether, and the peptide was extracted into 50 ml aqueous acetic acid. An analytical HPLC was run [0.46x5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm, A buffer of 0.1% TFA, B buffer of 0.1% TF A/90% ACN, gradient=10% B to 80% B over 10 min] with a small sample of the cleavage extract. The remaining extract was loaded onto a 2.2x25 cm Kromasil C18 preparative reverse phase column and an acetonitrile gradient was run using a Pharmacia FPLC system. 5 min fractions were collected while monitoring the UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1% TF A/50% acetonitrile. Gradient=30% B to 100% B over 450 min. The fractions containing the purest product were combined, frozen, and lyophilized to give 30.1 mg. An HPLC analysis of the product demonstrated a purity of >90% and MALDI mass spectral analysis demonstrated the desired mass of 3429.7. Glucagon Cys21, Glucagon Cys24, and Glucagon Cys29 were similarly prepared.

Example 4 Preparation of metal chelates

The peptide ligand was dissolved in water and the pH was adjusted to 5-6. The ligand concentration was determined by UV absorptions at 280nM. An equimolar amount of metal salt was added as aqueous solution and the pH was maintained at 5-6. After 30 minutes of stirring at room temperature, the pH was raised to 8 with ammonium bicarbonate solution.

The conjugate was purified on Sephadex G25 or alternatively RP-HPLC. The labeled peptide was eluted from the column in acetonitrile gradient in 0.02-0.1 mol/L triethylammonium acetate (pH 7.5). Correct fractions were combined, frozen, and lyophilized.

Preferably, there was a column for each of lanthanide label (europium, terbium, samarium, dysprosium, etc). After purification the column was decontaminated by washing with 10 mmol/L phthalate buffer (pH 4.1) containing 0.01% diethylenetriaminepentaacetic acid (DTP A)

Example 5 Preparation of As ay Buffer

Preferably, a Tris-based buffer is used. Alternatively, HEPES and phosphate buffers may also be used.

To avoid non-specific binding, the buffer contains preferably a blocking agent such as bovine serum albumin (BSA). There are many different grades of BSA and some of them contain a considerable amount of heavy metals that will eventually show as high levels of background in the assay. Purified BSA is preferred, or alternatively high grade of casein or ovalbumin may be used to block non-specific binding. A detergent such as Tween 20 or Tween 40 is also needed in the buffer to further prevent non-specific binding to the plate.

To keep the fluorescence background low as well as for maintaining high accuracy, the assay buffer should contain low concentrations of chelator such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). It is, however, essential to note that too much chelator present in the assay buffer will eventually start competing for the lanthanide and will render the assay unsuccessful. Preferably, no more than 50 μιηοΙ/L of chelator may be used.

In one example, an assay buffer composition for an assay was 50mM Tris-HCl, pH 7.5-8, containing 0.9% NaCl, 0.2-0.5% of purified BSA, 0.01-0.1% Tween (20 or 40) and 20μΜ EDTA.

Example 6 Washing solutions for assays

To achieve high sensitivity of the assays, automated plate washers were used, with typically 4-6 wash cycles prior to addition of enhancement solution. To avoid dissociation of the lanthanide while washing, neutral buffered solutions, such as Tris-HCl (pH 7.5-8) with detergents are preferred.

Example 7 Storage of labeled compounds

Labeled peptides were stored at a high concentration and in the absence of chelators or competing metals in the buffer. Preferably, diluted reagents shall not be stored. In most cases, 50 mmol/L Tris-HCl buffered saline solution containing 0.1-0.5% purified BSA will ensure the stability of the labeled compound during storage. For storage purposes, phosphate buffers must not be used due to their chelating nature. Storage should be at the optimal temperature for the peptide. If the labeled peptide requires storage at +4°C, it is preferred to add a bacteriostatic agent such as sodium azide (NaN₃) at a concentration of 0.05-0.1%.

Example 8 Purification of Insulin Receptor (IR)

Cells of Chinese hamster ovary (CHO) cell line were transfected with human insulin proreceptor gene coupled to a gene for methotrexate resistance. Cells were cultured in Eagles' minimum essential medium (without deoxynucleosides) + 10% fetal bovine serum supplemented with G418 (400 μg/ml) and methotrexate (50 nM). Under these conditions cells expressed >10⁷ IR molecules per cell. Cells were lysed by homogenizing at 4°C in 1% Triton-X-100 in 50 mM HEPES, 0.15 M NaCl, pH 7.8, 1 mM in phenylmethylsulfonyl fluoride (PMSF), 2 μg/ml aprotinin. The lysate was then centrifuged at 15,000 x g for 30 min, and the supernatant was incubated with wheat germ agglutinin (WGA)-Sepharose beads for 3 hr. The beads were transferred to a column and washed 2 with 50 mM HEPES, 0.5 M NaCl, 0.5% Octyl-P -D-glucopyranoside, pH 7.8, 1 mM PMSF, 2 μg/ml aprotinin, and the adsorbed glycoproteins were eluted with steps of 0.1 M, 0.2 M, 0.3 M N-acetyl glucosamine in the above buffer. The peak fractions eluted with 0.3 M N-acetyl glucosamine were pooled, their protein contents were measured using Bradford or BCA assays (Bio-Rad, Richmond, CA), and their purity was assessed by SDS-PAGE. The identity of the band at 94 kDa was confirmed by probing as Western blot with anti-IR β-chain antibody. A typical preparation yielded 150-200 μg of IR.

Example 9 Competition binding assays

Truncated soluble receptors Assays were performed by incubating the receptors in a total volume of 200 μΐ_^ with

125 I-IGF-l (3-10 pM) or 125 I-insulin (3 pM) and increasing concentrations of unlabelled ligand in binding buffer A [100 mM Hepes, pH 8.0, 100 mM NaCl, 10 mM MgCl₂, 0.5 % (w/v) BSA, 0.025 % (w/v) Triton X-100] for 48 h at 4 °C. Subsequently, bound ligand was precipitated with 0.2 % gama-globulin and 500μί of 25 % (w/v) poly( ethylene glycol) 8000, and the radioactivity in the pellet was measured. The concentration of the receptors was adjusted to yield 15-20 % binding when no competing ligand was added in the competition assay.

Membrane-bound receptors

Competition binding assays on membrane-bound holoreceptors were performed on BHK cells overexpressing full-length insulin or IGF-1 receptors. Equal number of transfected BHK cells (2000-5000) was seeded in each well of a 24-well plate and cultured for 24 h in Dulbecco's modified Eagle's medium (DMEM; GIBCO) containing 10% (v/v) fetal-calf serum (HyClone) before performing the binding assay. Cells were washed once with binding buffer B (DMEM containing 0.50% BSA, 20 mM Hepes, pH 7.8) before adding a total volume of 400μί with ¹²⁵I-IGF-1 (6.5 pM)

125

or I-insulin (6.5 pM) and increasing concentrations of unlabelled ligand in binding buffer B. After 3 h at 16°C, unbound ligand was removed by aspirating the buffer and washing once with 1.2 ml of cold binding buffer B. Cells were solubilized in 500 μΐ_^ of 1% (w/v) SDS, 100 mM NaCl, 25 mM Hepes (pH 7.8) and counted. The number of cells was adjusted to yield 16-28 % binding when no competing ligand was added in the assay.

Example 10 Glucagon Receptor Binding Assay

The affinity of peptides to glucagon receptor was measured in a competition binding assay utilizing scintillation proximity assay technology. Serial 3 -fold dilutions of the peptides made in scintillation proximity assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% w/v bovine serum albumin) were mixed in 96 well white/clear bottom plate (Corning Inc., Acton, Mass.) with 0.05 nM (3- [¹²⁵I]-iodotyrosyl) TyrlO glucagon (Amersham Biosciences, Piscataway, N.J.), 1-6 micrograms per well, plasma membrane fragments prepared from cells over-expressing human glucagon receptor, and 1 mg/well polyethyleneimine-treated wheat germ agglutinin type A scintillation proximity assay beads (Amersham Biosciences, Piscataway, N.J.). Upon 5 min of shaking at 800 rpm on a rotary shaker, the plate was incubated for 12 h at room temperature and then read on MicroBetal450 liquid scintillation counter (Perkin-Elmer, Wellesley, Mass.). Non-specifically bound (NSB) radioactivity was measured in the wells with a concentration of "cold" native ligand 4 times greater than the highest concentration in test samples and the total bound radioactivity was detected in the wells with no competitor. Percent specific binding was calculated as follow: % Specific Binding=((Bound-NSB)/(Total bound-NSB)) l00. IC₅₀ values were determined by using Origin software (OriginLab, Northampton, Mass.).

Example 11 GLP-1 receptor binding assay (1)

The binding affinity of GLP-1 derivatives was determined in a setting of cellular ELISA. Briefly, INS-1 cells cultured in 96 well plates (BD Biosciences) at roughly 95% confluence were rinsed with PBS and fixed with 4% paraformaldehyde (Thermo Scientific) for 10 min at room temperature and quenched for 5 min with 2% glycine in PBS, pH 7.5. For the binding capacity experiment, cells were incubated with logarithmic dilutions of GLP-1 derivative alone (1 *10^~5 to 1 * 10^~12 M) for total binding or in combination with 10 μΜ GLP-1 (Abeam Inc, USA) for non-specific binding. For the competitive binding experiment, we used fixed GLP-1 derivative concentration (10 μΜ) and competed its binding with varying concentration of GLP-1, Exendin-4 and glucagon (Bachem Americas, Inc. USA) (1 * 10^~5 to 1 * 10^~12 M). After a 4-h incubation at 4°C in a final volume of 100 μΐ, excess GLP-1 receptor agonists were washed away and the cells were blocked with 5% BSA (BD Biosciences). Bound residual GLP-1 derivative was detected by goat anti-human IgG-Fc antibody (1:4000, Southern Biotech) and detected by HRP-conjugated donkey anti-goat IgG (1:5000, Jackson ImmunoResearch). Ortho-Phenylenediamine (OPD) substrate (Fisher Scientific) was added for enzymatic reaction and the colorimetric change was analyzed by reading absorbance at 490 nm in a Beckman microplate reader. Example 12 GLP-1 Receptor Binding Assay (2)

The ability of GLP-1 derivative to bind to the GLPl receptor was assessed by a radioligand competition-binding assay with intact receptor-expressing human GLP-1 receptor-bearing CHO cells. In brief, human GLP-1 receptor-bearing CHO cells (-200,000) were incubated for 1 hr at room temperature with a constant amount of

125

radioligand, I-GLP1 (5 pM, -20,000 cpm) in the presence of increasing concentrations (ranging from 0 to 1 μΜ) of GLP-1 derivative in Krebs-Ringers/HEPES (KRH) medium (25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KC1, 2 mM CaCl₂, 1 mM KH₂P0₄, 1.2 mM MgS0₄) containing 0.01% soybean trypsin inhibitor and 0.2% bovine serum albumin. Cells were then washed twice with ice cold KRH medium containing 0.01% soybean trypsin inhibitor and 0.2% bovine serum albumin to separate bound radioligand from free radioligand before being lysed with 0.5 M NaOH and quantified using a γ-spectrometer. Nonspecific binding was determined in the presence of 1 μΜ unlabeled GLPl and represented less than 15% of total radioligand bound. Data are reported as the mean ± S.E. of duplicate determinations from a minimum of three independent experiments.

Example 13 Europium binding assays (1)

The receptor-binding assay was carried out using HEK293T cells stably transfected with insulin or IGF-1 receptor. These were plated in 96-well Isoplate wells (white wall and clear bottom; Perkin-Elmer) coated with poly L-lysine at a density of 80,000 ΰε1ΐ8/200μΕ well. At the start of the experiment, the medium was aspirated and cells were washed with 250 \L of phosphate buffered saline. Different concentrations of ligand were made up in 1% bovine serum albumen (BSA) in binding buffer (20 mM HEPES, 1.5 mM CaCl₂, 50 mM NaCl, 0.01% NaN₃; pH 7.5), and 50μί of each concentration was added to each well in triplicate. Eu-DTTA insulin or IGF-1 was diluted, and 50 μΐ, (approx. 100,000 average fluorescent units) was added to each well. The cells were allowed to stand at room temperature for 1 h, after which the cells were washed with 250 μΐ, of phosphate-buffered saline. The plate was developed for fluorescence measurements by adding 100 μΐ_^ of enhancement solution (β-nitrilotriacetic acid [β-ΝΤΑ], trioctylphosphine oxide [TOPO], and 0.1% [w/v] Triton X-100 in 0.1M acetic acid adjusted to pH 3.2) and allowed to stand at room temperature for 45 min, during which Eu was liberated from chelation with DTTA to form a highly fluorescent chelate inside a protective micelle with β-ΝΤΑ and TOPO. The fluorescence measurement was carried out on a Victor multilabel reader (Perkin-Elmer) using the measurement settings for europium (excitation at 340 nm, emission at 614 nm, delay time of 400 μβ, and measurement time of 400 μβ).

Example 14 Europium binding assays (2)

Binding assays were performed on HEK293 cells transfected with insulin or IGF-1 receptor. Cells were plated in either white or black CoStar 96-well plates at a density of 12,000 cells per well and were allowed to grow for 3 days. On the day of the experiment, media were aspirated from all wells. Then 50 μΐ of nonlabeled ligand and 50 μΐ of Eu-labeled ligand were added to each well.

Ligands were diluted in binding media (DMEM, ImM 1 ,10-phenanthroline, 200mg/L bacitracin, 0.5mg/L leupeptin, 0.3% BSA), and samples were tested in quadruplicate unless otherwise noted. Cells were incubated in the presence of ligands for 40min at 37 °C. Following the incubation, cells were washed for 3 times with wash buffer (50mM Tris-HCl, 0.2% BSA, 30mM NaCl). Enhancement solution was added (100iL /well), and the plates were incubated for at least 30min at 37 °C prior to reading. The plates were read on a Wallac Victor instrument using the standard Eu time resolved fluorescence measurement (340nm excitation, 400 μβ delay, and emission collection for 400 μβ at 615nm). Competition curves were analyzed with GraphPad Prism software using the sigmoidal dose-response (variable slope) classical equation for nonlinear regression analysis. In the case of saturation binding assays, the one site-binding (hyperbola) classical equation for nonlinear regression analysis was used. Nonspecific binding was tested in the presence of 10 μΜ native insulin or IGF-1. Example 15 Biological activity of the lanthanide labeled peptide

Using standard methods (as illustrated e.g. in Examples 1-14 above), the biological activities of the synthesized lanthanide labeled (polypeptides were tested. As shown for example in the case of labeled insulin receptors, three different lanthanide labels were used and all of them showed activities comparable to that of the unlabeled native insulin (Fig. 5). Similar results were obtained for lanthanide labeled IGF-1, IGF-2, glucagon and GLP-1 derivatives.

Claims

We claim:

1. A lanthanide labeled peptide, wherein a lanthanide label is covalently linked to a peptide, preferably the lanthanide is selected from the group consisting of terbium, samarium, europium, dysprosium and neodymium.

2. The lanthanide labeled peptide of claim 1, wherein said peptide is selected from the group consisting of insulin, IGF-1, IGF -2, glucagon, GLP-1 and the functional derivatives or analogs thereof.

3. The lanthanide labeled peptide of claim 1 or 2, which has the formula of

Pep-N*-J or

Pep-S*-J

wherein

Pep is a peptide, N* is an amine nitrogen either intrinsic to the peptide or extrinsinc and introduced as a label prior to conjugation, S* is a sulfhydryl either intrinsic to the peptide or extrinsinc and introduced as a label prior to conjugation, J is a lanthanide label.

4. The lanthanide labeled peptide of any of claims 1-3, wherein said peptide is an insulin or an insulin derivative having an A chain as set forth in SEQ ID No: 7 and a B chain comprising the sequence as set forth in SEQ ID NO: 8 Ji-Ri-X₂₃-₂₆HLCGSX₃₂LVEALYLVCGERGFFX₄₈X₄9 ₅o ₅i _{52 53}, Wherein

X₈ is selected from the group consisting of threonine and histidine;

X₂₁ is asparagine, glycine or alanine; X22 is absent or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

R_L IS an optional spacer between J and amino group, which is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent;

Ji is a lanthanide label or absent;

Ri is an optional spacer between Ji and N terminal amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

X23-2₆ is phenylalanine-valine-asparagine-glutamine (SEQ ID NO: 49), valine-asparagine-glutamine, asparagine-glutamine, glutamine or absent;

X₄₈ is tyrosine or absent, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

R_L is an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid; and

J is a lanthanide label or absent;

threonine or absent, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

R_L is an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid, or gamma-glutamic acid;

J is a lanthanide label or absent;

roline, lysine, or absent, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

R_L is an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids or polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent; roline, lysine, or absent, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

R_L is an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent;

thr nine, alanine or absent, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

J is a lanthanide label or absent;

absent or has the general structure

Where

N is 1 ,2,3,4,5,6,7 or 8;

J is a lanthanide label or absent; and

wherein at least one lanthanide label is comprised in said lanthanide labeled peptide.

5. The lanthanide labeled peptide of any of claims 1 -4, wherein the peptide is a single chain insulin analog comprising a compound of the formula B-P-A, wherein

A comprises a sequence as set forth in SEQ ID NO:7,

B comprises a sequence as set forth in SEQ ID NO: 8

Ji-Ri-X23-26HLCGSX32LVEALYLVCGERGFFX48X49X5o 5i 52 53, and

P represents a peptide linker of about 4 to about 14 amino acids, said peptide linker is selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 23.

6. The lanthanide labeled peptide of any of claims 1 -3, wherein said peptide is an

IGF-1 or an IGF- 1 derivative having a sequence as set forth in SEQ ID NO: 41

Ji -Ri -GPETLCGAELVD ALQFVCGDRGFYFNX₂₇PTGYGS S SRRAPQTGIVDECC FRSCDLRRLEMYC APLX₆₅PAX₆₈S AX₇i , wherein

Ji is a lanthanide label or absent;

X₂-₇ is lysine, arginine, homoarginine, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

J is a lanthanide label or absent;

X₆₅ is lysine, arginine, homoarginine, or absent, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

R_Lis an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent;

X₆₈ is lysine, arginine, homoar inine, or absent, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8; R_L IS an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent;

X₇₁ is absent, or has the general tructure

Where

N is 1,2,3,4,5,6,7 or 8;

R_L is an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid; J is a lanthanide label or absent; and

7. The lanthanide labeled peptide of any of claims 1-3, wherein said peptide is an IGF -2 or an IGF-2 derivative having a sequence as set forth in SEQ ID NO: 42 J₁-R₁-AYRPSETLCGGELVDTLQFVCGDRGFYFSX₃₀PASRVSRRSRGIVEECCFR SCDLALLETYCATPAX₆₅SEX₆₈, wherein

Ji is a lanthanide label or absent;

X₃₀ is lysine, arginine, homoarginine, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8;

J is a lanthanide label or absent;

Where

N is 1,2,3,4,5,6,7 or 8;

J is a lanthanide label or absent;

X₆₈ is absent, or has the general structure

Where

N is 1,2,3,4,5,6,7 or 8; RL IS an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

J is a lanthanide label or absent; and

8. The lanthanide labeled peptide of any of claims 1-3, wherein said peptide is a glucagon or a glucagon derivative having a sequence as set forth in SEQ ID NO: 43 HSQGTFTSDYSXi₂YLDSRRAQX₂iFVX₂₄WLMNX₂₉X3o, wherein

X₁₂ is lysine or arginine_;

Or

Where

N is 1 ,2,3,4,5,6,7 or 8;

RL is an optional spacer between J and the amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

Ri has a formula of Mi-Li-NH-, where Mi is a thiol reactive functional group, including maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide or vinylsulfone group; Li is an optional spacer/linker and is selected from the group consisting of long chain fatty acids and polyethylene glycol (PEG); J is a lanthanide label or absent;

X24 is glutamine, lysine, cysteine, homocysteine, ornithine, or has the general structure

Or

Where

N is 1 ,2,3,4,5,6,7 or 8;

R_L is an optional spacer between J and the amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

X29 is threonine, lysine, cysteine, homocysteine, ornithine, or absent, or has the general structure

Or

Where

N is 1 ,2,3,4,5,6,7 or 8;

RL is an optional spacer between J and amino group and is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;

is selected from the group consisting of lysine, cysteine, homocysteine, a short peptide sequence of 1 to 5 amino acids, one amino acid of which is lysine, cysteine, homocysteine or absent, or has the general structure

Or

Where

N is 1 ,2,3,4,5,6,7 or 8;

Ri has a formula of Mi-Li-NH-, where Mi is a thiol reactive functional group, including maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide or vinylsulfone group; Li is an optional spacer/linker and is selected from the group consisting of long chain fatty acids and polyethylene glycol (PEG); J is a lanthanide label or absent; and

9. The lanthanide labeled peptide of any of claims 1 -3, wherein said peptide is a GLP- 1 or a GLP- 1 derivative having the a sequence as set forth in SEQ ID NO: 44 HX₈EGTFTSDVSSYLEGQAAX₂₆EFIAWLVX₃₄GRX₃7X38, wherein

X₈ is alanine, 2-methylalanine (Aib), serine, or glycine;

X₂₆ is lysine, arginine, cysteine homocysteine, or has the general structure

Or

Where

N is 1 ,2,3,4,5,6,7 or 8;

Ri has a formula of Mi-Li-NH-, where Mi is a thiol reactive functional group, including maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide or vinylsulfone group; Li is an optional spacer/linker and is selected from the group consisting of long chain fatty acids and polyethylene glycol (PEG); J is lanthanide label or absent;

is arginine, lysine, cysteine homocysteine, or has the general structure

Or

Where

N is 1 ,2,3,4,5,6,7 or 8;

X37 is -NH₂, glycine, lysine, cysteine, homocysteine, or absent, or has the general structure

Or

Where

N is 1,2,3,4,5,6,7 or 8;

X38 is selected from the group consisting of lysine, cysteine, homocysteine, or a peptide sequence of 1 to 5 amino acids, one amino acid of which is lysine, cysteine, or homocysteine, or absent, or has the general structure

Or

Where

N is 1 ,2,3,4,5,6,7 or 8;

Ri has a formula of Mi-Li-NH-, where Mi is a thiol reactive functional group, including maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide or vinylsulfone group; Li is an optional spacer/linker and is selected from the group consisting of long chain fatty acids and polyethylene glycol (PEG); J is lanthanide label or absent; and

10. Use of the lanthanide labeled peptide as defined in any one of claims 1-9 for detection of a peptide in a biological assay, for example, for the detection of insulin, IGF-1 , IGF-2, glucagon, GLP-1 or the functional derivatives or analogs thereof.