T 0451/99 (AIDS-related disease/BIO-RAD LABS.) 13-01-2005

I. European patent No. 0 220 273 with the title "Synthetic antigens for the detection of AIDS-related disease" was granted with 43 claims for all designated Contracting States on the basis of European patent application No. 86 902 998.3 filed on 21 April 1986 and published as WO 86/06414. Priorities were claimed from 29 April 1985 (US 728052), 19 August 1985 (US 767303) and 26 March 1986 (US 844485).

Granted claim 1 read as follows:

"1. A method of detecting the presence of LAV/HLTV-III virus or antibody to LAV/HTLV-III virus where a sample is combined with a composition having epitopic sites immunologically competitive with LAV/HTLV-III epitopic sites, whereby antibodies bind to such protein composition to form a specific binding pair complex and the amount of complex formation is determined, characterized by

employing in the assay medium as a reagent a composition containing at least one peptide which has at least six amino acids and fewer than 50 amino acids, at least six of those amino acids are contiguous and encoded for by part of the coding region of LAV/HTLV- III from bp 450 to bp 731 from the gag region or bp 900 to bp 1421 (from the gag region) or bp 7210 to bp 7815 (from the env region)."

II. Two oppositions were filed pursuant to Article 100(a) to (c) EPC for lack of novelty and inventive step, lack of sufficient disclosure, added subject-matter. The Opposition Division decided to maintain the patent in amended form pursuant to Article 102(3) EPC on the basis of the third subsidiary request then on file.

III. The Patent Proprietor (Appellant I) and Opponent 2 (Appellant II) filed appeals and submitted statements of grounds of appeal. The appeal by Appellant I was accompanied by one main request and two subsidiary requests.

Claim 1 of the main request read as follows:

"1. A method of detecting the presence of LAV/HLTV-III virus or antibody to LAV/HTLV-III virus where a sample is combined with a composition having epitopic sites immunologically competitive with LAV/HTLV-III epitopic sites, whereby antibodies bind to such protein composition to form a specific binding pair complex and the amount of complex formation is determined, characterized by:

employing in the assay medium as a reagent a composition containing at least one peptide which has at least six amino acids and fewer than 50 amino acids, at least six of those amino acids are contiguous and encoded for by part of the coding region of LAV/HTLV- III from bp 450 to bp 731 (from the gag region) or bp 900 to bp 1421 (from the gag region) or bp 7210 to bp 7815 (from the env region), except the following peptides:

a) peptides from the gag region defined starting from amino acid 1 = Met coded by the ATG in position 336-338 in the LAV DNA sequence:

(here follow the sequences of seven peptides from the above-mentioned gag regions)

b) peptides from the env region defined starting from aminoacid 1 = Lysine coded by the AAA at position 5746- 5748 in the LAV DNA sequence:

(here follow the sequences of six peptides from the above-mentioned env region)

IV. A first oral proceeding took place on 28 May 2002. It was then decided that the proceedings would be continued in writing, a two months term being given to the parties to propose question(s) to be referred to the Enlarged Board of Appeal regarding the admissibility of disclaimers of the kind found in claim 1 of the main request.

V. On 14 March 2003, the Board issued a decision referring questions to the Enlarged Board of Appeal (EBA) on this issue (cf the referral decision T 451/99, OJ EPO 2003, 334). The EBA answered these questions with decision G 2/03 (OJ EPO 2004, 448).

VI. A second oral proceeding was summoned for 12 and 13 January 2005.

VII. Appellants I and II filed further submissions, Appellant I also filed subsidiary requests II to VIII in replacement of subsidiary request II then on file.

VIII. On 10 January 2005, a main request and five subsidiary requests destined to replace all requests on file were faxed to the European Patent Office by Appellant I.

IX. At oral proceedings, subsidiary requests III to V were replaced by a new subsidiary request III.

The claims of relevance for this decision are as follows:

Main request, claims 1 and 2

"1. A method of detecting the presence of LAV/HLTV-III virus or antibody to LAV/HTLV-III virus where a sample is combined with a composition having epitopic sites immunologically competitive with LAV/HTLV-III epitopic sites, whereby antibodies bind to such protein composition to form a specific binding pair complex and the amount of complex formation is determined, characterized by:

employing in the assay medium as a reagent a composition containing at least one peptide which has at least six amino acids and fewer than 50 amino acids, at least six of those amino acids are contiguous and encoded for by part of the coding region of LAV/HTLV- III from bp 900 to bp 1421 (from the gag region) or bp 7210 to bp 7815 (from the env region), except the following peptides:

a) peptides from the gag region defined starting from amino acid 1 = Met coded by the ATG in position 336-338 in the LAV DNA sequence:

(here follow the sequences of four peptides from the above-mentioned gag region)

b) peptides from the env region defined starting from aminoacid 1 = Lysine coded by the AAA at position 5746- 5748 in the LAV DNA sequence:

(here follow the sequences of six peptides from the above-mentioned env region)"

"2. A method of detecting the presence of LAV/HLTV-III virus or antibody to LAV/HTLV-III virus where a sample is combined with a composition having epitopic sites immunologically competitive with LAV/HTLV-III epitopic sites, whereby antibodies bind to such protein composition to form a specific binding pair complex and the amount of complex formation is determined, characterized by: employing in the assay medium as a reagent a composition containing at least one peptide which is a variant, by conservative or non-conservative substitution, of a peptide as specified in claim 1, the variant being immunologically competitive with a native LAV/HTLV-III virus protein."

Subsidiary request I; claim 1

"1. A method of detecting the presence of LAV/HLTV-III virus or antibody to LAV/HTLV-III virus where a sample is combined with a composition having epitopic sites immunologically competitive with LAV/HTLV-III epitopic sites, whereby antibodies bind to such protein composition to form a specific binding pair complex and the amount of complex formation is determined, characterized by:

employing in the assay medium as a reagent a composition containing at least one peptide synthesized in solution or on a solid support, said peptide having at least six amino acids and fewer than 50 amino acids, at least six of those amino acids are contiguous and encoded for by part of the coding region of LAV/HTLV- III from bp 900 to bp 1421 (from the gag region) or bp 7210 to bp 7815 (from the env region)."

Subsidiary request II; claims 9, 19 and 20

"9. A peptide of the formula:

(X) (39)

Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys- Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys- Ser-Gly-Lys-Leu-Ile-Cys-X (from the env gp41 protein sequence)

where X is OH or NH2, N-terminal acetylated X, or X linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host."

"19. A peptide immunoreactive with antibodies to LAV/HTLV-III virus which has at least twelve and fewer than 50 amino acids, at least twelve of those amino acids are contiguous and within the sequence of one of the peptides as specified in claims 8 and 9."

"20. A peptide immunoreactive with antibodies to LAV/HTLV-III virus which has at least twelve and fewer than 50 amino acids, which peptide is a variant, by conservative or non-conservative substitution, of a peptide as specified in claim 19, the variant being immunologically competitive with a native LAV/HLTV-III virus protein."

New subsidiary request III

This request comprised 12 claims, each of them being directed to a specific peptide from the gag or env protein sequences. They read as follows:

"1. A peptide of the formula:

(I) (15)

Y-Asp-Cys-Lys-Thr-Ile-Leu-Lys-Ala-Leu- Gly-Pro-Ala-Ala-Thr-Leu-Glu-G1u-Met-Met- Thr-Ala-Cys-X (from the gag p25 protein sequence)

where X is OH or NH2, and Y, if present, is an amino acid added to facilitate coupling, N-terminal acetylated I, or I linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

2. A peptide of the formula:

(III) (92)

Y-Asp-Arg-Val-His-Pro-Val-His-Ala-Gly-Pro- Ile-Ala-Pro-Gly-Gln-X (from the gag p25 protein sequence)

where X is OH or NH2, and Y, if present, is an amino acid added to facilitate coupling, N-terminal acetylated III, or III linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host

3. A peptide of the formula:

(IV) (90)

Y-Tyr-Ser-Pro-Thr-Ser-Ile-Leu-Asp-Ile-Arg Gln-Gly-Pro-Lys-Glu-Pro-Phe-Arg-Asp-Tyr-Val- Asp-Arg-Phe-Tyr-Lys-Thr-Leu-Arg-Z-X (from the gag p25 protein sequence)

where X is OH or NH2, and Y and Z, if present, are amino acids added to facilitate coupling, N-terminal acetylated IV, or IV linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

4. A peptide of the formula:

(V) (88)

Y-Asn-Trp-Nor-Thr-Glu-Thr-Leu-Leu-Val-Gln- Asn-Ala-Asn-Pro-Asp-Cys-Lys-Thr-Ile-Leu-Lys- Ala-Leu-Gly-Pro-Ala-Ala-Thr-Leu-Glu-Glu-Nor- Nor-Thr-Ala-Cys-X (from the gag p25 protein sequence)

where X is OH or NH2, and Y, if present, is an amino acid added to facilitate coupling, N-terminal acetylated V, or V linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

5. A peptide of the formula:

(VI) (97)

Y-Arg-Glu-Leu-Glu-Arg-Phe-Ala-Val-Asn-Pro-Gly- Leu-Leu-Glu-Thr-Ser-Glu-Gly-Cys-Arg-Gln-Ile- Leu-Gly-Gln-Leu-Gln-Pro-Ser-Leu-Gln-Thr-X (from the gag p18 protein sequence)

where X is OH or NH2, and Y, if present, is an amino acid added to facilitate coupling, N-terminal acetylated VI, or VI linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

6. A peptide of the formula:

(VII) (71)

Y-Asp-Thr-Gly-His-Ser-Ser-Gln-Val-Ser-Gln- Asn-Tyr (from the gag p18 protein sequence)

where Y, if present, is an amino acid added to coupling, N-terminal acetylated VII, or VII linked to a peptide or protein of at least 5,000 weight, which peptide or protein does not normally bind to antibodies present in a human host.

7. A peptide of the formula:

(VIII) (36)

Val-Lys-Ile-Glu-Pro-Leu-Gly-Val-Ala-Pro- Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg- Glu-Lys-Arg-Ala-Z-X

(from the env gpll0 protein sequence)

where X is OH or NH2, and Z, if present, is an amino acid added to facilitate coupling, N-terminal acetylated VIII, or VIII linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

8. A peptide of the formula:

(IX) (56)

Ile-Lys-Gln-Leu-Gln-Ala-Arg-Ile-Leu- Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Z-X (from the env gp4l protein sequence)

where X is OH or NH2, and Z, if present, is an amino acid added to facilitate coupling, N-terminal acetylated IX, or IX linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

9. A peptide of the formula:

(X) (39)

Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys- Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys- Ser-Gly-Lys-Leu-Ile-Cys-X (from the env gp4l protein sequence)

where X is OH or NH2, N-terminal acetylated X, or X linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

10. A peptide of the formula:

(XI) (40)

Y-Lys-Ser-Leu-Glu-Gln-Ile-Trp-Asn-Asn- Met-Thr-Trp-Met-Glu-Trp-Asp-Arg-Glu- Ile-Asn-Z-X (from the env gp4l protein sequence)

where X is OH or NH2, and each of Y and Z, if present, is an amino acid added to facilitate coupling, N- terminal acetylated XI, and XI linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

11. A peptide of the formula:

(XII) (23)

Y-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn- Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu- Glu-Leu-Asp-Lys-Trp-Z-X (from the env gp4l protein sequence)

where X is OH or NH2, and each of Y and Z, if present, is an amino acid added to facilitate coupling, N- terminal acetylated XII, or XII linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host.

12. A peptide of the formula:

(XIII) (79)

Y-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly- Cys-Ser-Gly-Lys-Leu-Ile-Cys-X (from the env gp4l protein sequence)

where X is OH or NH2, and Y, if present, is an amino acid added to facilitate coupling, N-terminal acetylated XIII, or XIII linked to a peptide or protein of at least 5,000 molecular weight, which peptide or protein does not normally bind to antibodies present in a human host."

X. The following documents are mentioned in the present decision:

(1): WO 86/02383 filed on 18 October 1985 and published on 24 April 1986 claiming priority from 18 October 1984 (FR 84/16013), 16 November 1984 (GB 8429099) and 21 January 1985(GB 8501473);

(2): Chang, N.T. et al., Science, Vol. 228, 5 April 1985, pages 93 to 96;

(3): Crowl, R. et al., Cell, Vol.41, July 1985, pages 979 to 986;

(5): Cabradilla, C.D. et al., Bio/Technology, Vol. 4, February 1986, pages 128 to 133;

(7): Hopp, T.P., Molecular Immunology, Vol. 18, No. 9 1981, pages 869 to 872;

(8): Prince, A.M. et al., Proc.Natl.Acad.Sci.USA Vol. 79, January 1982, pages 579 to 582;

(9): Hopp, T.P., Molecular Immunology, Vol. 21, No. 1 1984, pages 13 to 16;

(11): Hopp, T.P. and K.R., Woods, Molecular Immunology, Vol. 19, No. 11, 1982, pages 1453 to 1463.

XI. Appellant I's submissions in writing and during oral proceedings insofar as they are relevant to the present decision may be summarized as follows:

Article 123(2) EPC; admissibility of disclaimers

Main request

Claims 1 and 6 contained disclaimers of specific peptides which had been introduced for the purpose of delimiting the claimed subject-matter against the teachings of document (1) which was relevant for novelty purposes pursuant to Article 54(3)(4) EPC. There was no clearer way to disclaim these peptides than to identify them by their sequences. The number of disclaimed peptides (11 in claim 1 and 5 in claim 6) was entirely compatible with the requirement of clarity. The disclaimers were, thus, allowable.

Article 54(3)(4) EPC; novelty

Main request: claim 2; subsidiary request II: claim 20

- Claim 2 of the main request related to a method to be carried out with at least one peptide which was a variant by conservative or non conservative substitution of a peptide specified in earlier claim 1 (ie which, in particular, was of the same size as the peptide disclosed in claim 1) and which was immunologically competitive with a LAV/HTLV-III protein. The patent specification (page 4, lines 14 to 17 and 43 to 45) made clear what kind of primary structure a variant peptide would be expected to have: it was produced to accommodate strain-to-strain variations and usually fewer than 20%, more usually fewer than 10% of its amino acids were exchanged. Indeed, four of the variant peptides described in the patent in suit were respectively 7.7%, 8.3%, 12.5% and 5.9% substituted. It would also be obvious for the skilled person that a variant had to come from the same genomic region as the peptide, it was derived from.

Thus, specific peptides disclosed in document (1) which happened:

(a) to be of a size of at least 6 amino acids and smaller than 50 amino acids (as in claim 1, section IX supra), and

(b) to be immunologically competitive with a LAV/HTLV-III protein and

(c) to have a sequence completely different from any peptide originating from the regions mentioned in claim 1,

did not answer the definition of a variant peptide as was derivable from the technical information disclosed in the patent specification. Thus, the method disclosed in document (1) did not destroy the novelty of the subject-matter of claim 2 of the main request.

- Claim 20 of subsidiary request II was directed to peptide variants by conservative or non conservative substitution of, in particular, the specific 19 and 26 amino acids long peptides disclosed in claims 8 and 9. For the same reasons as given in relation to claim 2 of the main request, specific, immunologically competitive, 19 or 26 amino acids long peptides disclosed in document (1) which had a completely different sequence from that of the specific peptides claimed in claims 8 or 9 were not detrimental to the novelty of the subject-matter of claim 20.

Subsidiary request I; claim 1

The claimed method required the use of peptides characterized by a combination of three specific features: their region of origin, their size and the way they were produced (synthesized in solution or on a solid support). This combination was not disclosed in document (1). Furthermore, it had to be kept in mind that the conformation of proteins was complex, involving a primary structure (the succession of specific amino acids), a secondary structure (for example, helical), a tertiary structure (folding upon itself) and, possibly, a quaternary structure (dimerisation). For this reason, it could equally be expected that synthetic peptides would have a different conformation from that of recombinantly synthesized peptides. In addition, synthetic peptides generally were of a smaller size (less than 49 amino acids instead of more than 50 amino acids). Thus, they would not necessarily mimic or expose the same epitope in the same manner. Moreover, as a consequence of their greater length, recombinant peptides could contain additional epitopes. Accordingly, as the claimed method and that described in document (1) were carried out with different reagents, they could not be the same. Thus, the method described in document (1) was not detrimental to the novelty of the subject-matter of claim 1.

Article 56 EPC; inventive step

New subsidiary request III

The closest prior art document was document (2) which was a study of which HTLV-III encoded peptides immunologically reacted with antibodies in sera from AIDS patients. It was determined that some gag or env peptides of a length of at least 75 amino acids had that property. It was also suggested that these peptides could ultimately be useful reagents in particular to diagnose AIDS.

Starting from these teachings and aware of other scientific publications (eg. documents (3) or (4)) which taught to use large recombinant proteins for their antigenic properties, the skilled person would have been deterred from using short peptides for the same purpose as disclosed in document (2). Indeed, it was known that in general these peptides did not retain the 3-dimensional conformational epitopes and, so, might loose a significant degree of immune reactivity. It would also have been felt that with a small peptide there was a great risk of missing antibodies directed to several retroviral strains i.e. a risk of not accommodating strain-to-strain variations. For these reasons, the skilled person would not have embarked on the project of obtaining small, immunologically competitive peptides such as claimed. Alternatively, he/she would not have had a reasonable expectation of success that small peptides could be used for the purpose they were intended to. The subject-matter of subsidiary request III as a whole was inventive.

XII. Appellant II's submissions in writing and during oral proceedings insofar as they are relevant to the present decision may be summarized as follows:

Article 123(2) EPC; admissibility of disclaimers

Main request

Claim 1 contained as many as 11 disclaimers. This number of disclaimers clearly went against the principle stated in the Enlarged Board decision G 2/03 (OJ EPO 2004, 448) that a disclaimer is not allowable if the necessary limitation can be expressed in simpler terms, or if the disclaimers are so numerous that it would put an unreasonable burden on the public to find out what is protected and what is not protected.

Article 54(3)(4) EPC; novelty

Main request: claim 2; subsidiary request II: claim 20

- Claim 2 of the main request was drafted in very wide terms: the peptide variants were neither qualitatively nor quantitatively defined. The argument to the avail that the skilled person would narrowly construe the "concept" of variants as defined in the claim (by conservative or non conservative substitution) on the basis of the technical information in the patent specification that the number of substituted amino acid residues in the variant would usually not exceed 20%, more usually not exceed 10% of the total amino acid residues was not convincing. Indeed, any feature following the term "usually" had to be understood not as a compulsory feature but as a preferred embodiment. Furthermore on page 4, lines 49 to 50, it was mentioned that the sequence of the polypeptides employed in the subject invention needed not be identical to that of any particular LAV/HLTV-III peptide. This last characteristic, of course, also implied that a variant needed not be recognizable as originating from the same region of the LAV protein as a putative corresponding native peptide. Thus, as the claimed variants could have any sequences, they were in fact solely characterized by their length and their ability to be immunologically competitive with a LAV/HTLV-III protein.

Document (1) (eg. page 14, lines 23 to 26) disclosed polypeptides comprising antigenic determinants included in the proteins encoded and expressed by the LAV genome, as well as the use of said polypeptides in an in vitro process of diagnosis for the detection of anti-LAV antibodies in sera of AIDS patients (page 44, lines 6 to 12). Many specific peptides were described which had a length of between 7 and 50 amino acids, for example, the peptides identified as "amino acids 780- 803" (claim 8 and page 30) or as "amino acids 239-264" (claim 9 and page 24). These immunogenic peptides respectively comprised 24 and 26 amino acids and, thus, were peptide variants in accordance with claim 2 of the main request or in accordance with claim 20 of subsidiary request II (dependent on claim 9). Thus, document (1) disclosed a method/a product which happened to fall within the scope of method claim 2 of the main request or within the scope of product claim 20 of the subsidiary request II. These requests were not allowable under Article 54(3)(4) EPC.

Subsidiary request I; claim 1

Appellant I had not provided any evidence to show that a synthetic peptide would be in any way different from the same peptide but recombinantly produced. It was impossible to conceive which step in either of the methods would bring a difference. This was all the more true since the peptides in question were of a small size, thus it would be most unlikely that they adopted a tertiary conformation. In this respect, it had to be noted that in the patent itself (page 8, lines 38 to 42 and 49 to 50), either method of peptide synthesis was proposed without any suggestion that it made any difference to the peptide structure. For these reasons, the feature "synthesized in solution or on a solid support" did not have any bearing on the claimed process which was not novel in view of the teaching in document (1) of a method using specific immunogenic peptides of the same length and originating from the same regions as now claimed.

Article 56 EPC; inventive step

New subsidiary request III

The closest prior art was document (2). The purpose of the study described therein was to identify regions of HTLV-III proteins which could ultimately be used for detecting anti-HTLV-III antibodies in the sera of AIDS patients. One peptide which had been recombinantly produced was described as particularly efficient: it was derived in part from the env region and was 81 amino acids in length (ORF clone 121 containing a portion of the env-lor region; page 96).

Starting from the closest prior art, the problem to be solved could be defined as isolating further peptides which would be better diagnostic reagents.

The solution given in claims 7 to 12 was specific peptides originating from the same env region as the DNA contained in clone 121, these peptides having a length from 17 to 26 amino acids.

Taking into account that the region was already identified in document (2), it needed no inventive step to look for further immunogenic peptides in this region. Moreover, the skilled person would have been perfectly aware that in order to obtain a good diagnostic reagent, one had to strike a balance between sensitivity and specificity. Thus, it would have been obvious to choose, in the specific env region, peptides of smaller size than 81 amino acids and test them for having retained sensitivity.

The entire sequence of the HTLV-III proteins was known and in accordance with the patent itself isolating the peptides was only a matter of routine work (page 8, lines 38 to 50). Consequently, no inventive step could be seen in producing them.

Appellant I's argument that the skilled person would have been deterred from producing small peptides as diagnostic reagents for fear that they may have lost sensitivity was not convincing because the state of the art provided numerous examples of the use of short peptides in diagnosis (eg in document (11)). In the same manner, as it was known from document (7) to (9) that a short peptide (11 amino acids) could bind significantly to antibodies of different subtypes of hepatitis causing viruses, the skilled person would not have regarded the intrinsic, strain-to-strain variability of the HLTV-III proteins as an obstacle to using a small peptide as diagnostic reagent.

For these reasons, inventive step was to be denied.

XIII. Appellant I requested that the decision under appeal be set aside and that the patent be maintained on the basis of the main request or the first or second subsidiary request (all filed with fax of 10 January 2005) or the new third subsidiary request filed at the oral proceedings (consisting of claims 1 to 12 and an adapted description thereto).

Appellant II requested that the decision under appeal be set aside and the patent be revoked.

The other party (opponent 1) requested that the decision under appeal be set aside and that the patent be revoked.