# US 2004/0006204 A1 · Synthetic active peptide fragments

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- Number: US 2004/0006204 A1
- Document ID: US20040006204A1
- Status: Published application
- Kind: A1
- Application number: 10/113,946
- Filed: 2002-04-02
- Published: 2004-01-08
- Earliest claimed: 2002-04-02
- Inventors: Miriam Tendler; Richard Charles Garrat; Naftale Katz; Andrew John George Simpson; Frank Jefferson Alarcon de Barrientos; Monica Magno Vilar; Marilia Sirianni dos Santo Almeida
- Web page: https://patentgrep.com/patent/US20040006204A1

## CPC

- A61K 39/0003: {Invertebrate antigens}
- C07K 14/4354: {from nematodes}
- C07K 14/43559: {from trematodes}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- C07K 2319/00: Fusion polypeptide
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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)
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to peptide fragments which have one or more shared and/or similar amino acid sequences to amino acid sequences of specific portions of the 14 kDa protein of S. mansoni (Sm14) or related FABPs (Fatty Acid Binding Proteins), the said peptide fragments functioning as continuous or discontinuous epitopic regions of the molecule or mimicking its biological activity. More particularly, the present invention relates to a method for constructing active peptide fragments, peptide fragments, immunogenic composition and diagnostic kit using said peptide fragments.

## Claims

### Claim 1

1) Method for constructing peptides on the basis of the three-dimensional structures of homologous molecules wherein said peptides are able to mimic or prevent the interaction between helminth pathogens and receptors, said method consisting of:
(i) selecting regions of the parent protein which contain a high spatial density of residues which form continuous or discontinuous epitopes;
(ii) give priority to maintaining the previously predicted epitopic residues, responsible for stimulating the desired immuno/antigenic response, within the selected peptide sequence;
(iii) elaborate sequences which, whilst maintaining a high percentage of previously identified epitopic residues, are of limited size varying from 8 to 28 residues;
(iv) two peptides may be chosen, which may correspond to regions which are distant in the primary structure (amino acid sequence) but spatially close in the tertiary (three-dimensional) structure, the said regions are chosen on the basis that the three-dimensional structure indicates that they can be readily united assuming that they retain their original structures.
(v) modifications may be made to the peptide, be it derived from a single continuous stretch of amino acids or be it derived in the manner described in (iv), so as to favour the three-dimensional conformation as seen in the original protein, such modifications may include substitutions of amino acids as well as insertions or deletions of amino acids;
(vi) residues may be used in order to restrict the conformational freedom of the final peptide.

### Claim 2 (depends on 1)

2) Method according to claim 1 wherein in step (vi) ½-cystines residues are used to restrict the conformational freedom of the final peptide.

### Claim 3 (depends on 1)

3) Method according to claim 1 wherein the three-dimensional structure selected is that corresponding to a molecule of a Fatty Acid Binding Protein—FABP.

### Claim 4 (depends on 1)

4) Method according to claim 1 wherein the three-dimensional structure selected is that of a molecule/protein from helminths, more preferentially, Sm14 and homologous molecules.

### Claim 5

5) Peptide fragments comprising a sequence of amino acids derived from a 14 kDa protein of Schistosoma mansoni (Sm14) or its recombinant (rSm14) or related FABPs wherein the said peptide fragments are preferentially but not exclusively selected from the cross-reactive epitopic regions in the C-terminal third of the Sm14 or rSm14 molecule.

### Claim 6 (depends on 5)

6) Peptide fragments according to claim 5 wherein said fragments are composed of parts of the β-strands 9 and 10 of the Sm14 molecule and the connection between these β-strands.

### Claim 7 (depends on 6)

7) Peptide fragments according to claim 6 wherein the peptide fragment is contained within the amino acid sequence VTVGDVTA and its functional equivalents.

### Claim 8 (depends on 5)

8) Peptide fragments according to claim 5 wherein said fragments are composed of an α-helix located in the large connection between β-strands 1 and 2 of the Sm14 molecule.

### Claim 9 (depends on 8)

9) Peptide fragments according to claim 8 wherein the peptide fragment is contained within the amino acid sequence NFDAVMSKLG and its functional equivalents.

### Claim 10 (depends on 5)

10) Peptide fragments according to claim 5 wherein the fragments are a direct fusion of the β-hairpin between and including strands 9 and 10 together with the first (α-helix located in the large connection between β-strands 1 and 2 of the Sm14 molecule.

### Claim 11 (depends on 10)

11) Peptide fragments according to claim 10 wherein the peptide fragment is contained within the amino acid sequence VTVGDVTANFDAVMSKLG and its functional equivalents.

### Claim 12 (depends on 5)

12) Peptide fragments according to claim 5 wherein the fragments are a direct fusion of the β-hairpin between and including strands 9 and 10 together with the first a-helix located in the large connection between β-strands 1 and 2 of the Sm14 molecule, said fragments derived by the substitution of selected amino acid residues.

### Claim 13 (depends on 12)

13) Peptide fragments according to claim 12 wherein the peptide fragment is contained within the amino acid sequence VTVGDVTGGSDAVMSKLG and its functional equivalents.

### Claim 14 (depends on 12)

14) Peptide fragments according to claim 12 wherein the fragment is modified to introduce glycine residues at its centre.

### Claim 15 (depends on 5)

15) Peptide fragments according to claim 5 wherein said fragments are derived from two β-hairpins wherein the first β-hairpin is composed of β-strands 6 and 7 and the second β-hairpin is composed of β-strands 8 and 9, in both cases together with their connecting loops.

### Claim 16 (depends on 15)

16) Peptide fragments according to claim 15 wherein said fragments are derived from the first β-hairpin, between the strands 6 and 7.

### Claim 17 (depends on 16)

17) Peptide fragments according to claim 16 wherein the peptide fragment is contained within the amino acid sequence EKNSESKLTQ and its functional equivalents.

### Claim 18 (depends on 15)

18) Peptide fragments according to claim 15 wherein the said fragments are derived from the second β-hairpin, between the strands 8 and 9.

### Claim 19 (depends on 18)

19) Peptide fragments according to claim 18 wherein the peptide fragment is contained within the amino acid sequence IVREVDGDTMKTT and its functional equivalents.

### Claim 20 (depends on 15)

20) Peptide fragments according to claim 15 wherein the said fragments are a direct fusion of the first β-hairpin between strands 6 and 7 and the second β-hairpin between strands 8 and 9.

### Claim 21 (depends on 20)

21) Peptide fragments according to claim 20 wherein the peptide fragment is contained within the amino acid sequence EKNSESKLTQIVREVDGDTMKTT and its functional equivalents.

### Claim 22 (depends on 15)

22) Peptide fragments according to claim 15 wherein the fragments are a direct fusion of the first β-hairpin between and including strands 6 and 7 and the second β-hairpin between and including strands 8 and 9, wherein said fragments undergo a substitution of at least one amino acid at a position previously chosen and/or an insertion of at least one residue in its main chain.

### Claim 23 (depends on 22)

23) Peptide fragments according to claim 22 wherein the peptide fragment is contained within the amino acid sequence EKFSESKLTSDPTGIVREVDGATMKTT and its functional equivalents.

### Claim 24 (depends on 22)

24) Peptide fragments according to claim 22 wherein the peptide fragment is contained within the amino acid sequence EKFSESKLTFDGIVREVDGATMKTT and its functional equivalents.

### Claim 25 (depends on 1)

25) Immunogenic compositions consisting of the peptide fragments obtained according to the method described in claim 1 and a pharmaceutical carrier, said immunogenic compositions being able to confer at least partial protection against infection with pathogenic helminths, and thus serve as vaccines against same.

### Claim 26 (depends on 25)

26) Immunogenic composition according to claim 25 where the pathogenic helminths are Schiostosoma mansoni and/or Fasciola hepatica.

### Claim 27

27) Immunogenic composition consisting of peptide fragments selected from the cross-reactive epitopic regions in the C-terminal third of the Sm14 or rSm14 molecule, said immunogenic compositions being able to confer at least partial protection against infection with pathogenic helminths, and thus serve as vaccines against same.

### Claim 28 (depends on 27)

28) Immunogenic composition according to claim 27 where the pathogenic helminths are Schiostosoma mansoni and/or Fasciola hepatica.

### Claim 29 (depends on 1)

29) Direct or indirect diagnostic test for helminth infection diagnoses using peptides fragments obtained as described in claim 1 as the active ingredient wherein the said peptide fragments have similar or conservatively substituted amino acid sequences to those found in epitopic portions of the Sm14 antigen or related FABPs.

### Claim 30 (depends on 29)

30) Direct or indirect diagnostic test according to claim 29 where the helminth is Schiostosoma mansoni and/or Fasciola hepatica.

### Claim 31 (depends on 1)

31) Direct or indirect diagnostic test for helminth infection diagnoses using mono- or policlonal antibodies of any origin raised against fragments obtained as described in claim 1 wherein the said peptide fragments have similar or conservatively substituted amino acid sequences to those found in epitopic portions of the Sm14 antigen or related FABPs.

### Claim 32 (depends on 31)

32) Direct or indirect diagnostic test according to claim 31, where the helminth is Schiostosoma mansoni and/or Fasciola hepatica.

### Claim 33 (depends on 29, 30, 31, 32)

33) Direct or indirect diagnostic test for helminth infection diagnostics according to one of claims 29 through 32 wherein the said diagnostic test can be a parasitic or immunological test using any kind of substrate including excrement, urine, serum, saliva.

### Claim 34 (depends on 33)

34) Direct or indirect diagnostic test according to claim 33, where the helminth is Schiostosoma mansoni and/or Fasciola hepatica.

### Claim 35

35) Diagnostic kit comprising at least a peptide fragments obtained as described in precedent claims.

### Claim 36 (depends on 35)

36) Diagnostic kit according to claim 35 wherein it is further used medical and veterinary vehicles.