T 1311/13 (LPS binding peptides/ NATIONAL UNIVERSITY OF SINGAPORE) 17-01-2018

Admissibility of the appeal

1. In the communication pursuant to Article 15(1) RPBA (cf. point VI supra), the board informed the parties that, in its opinion, the respondent did not challenge the admissibility of the appeal but only some of the appellant's grounds of appeal. The board noted that the respondent's acknowledgement that the requirements of Article 108, third sentence, EPC were fulfilled for at least one ground of appeal, namely inventive step, was also an acknowledgement that the admissibility of the appeal was not contested, since there is no partial admissibility of an appeal (cf. "Case Law of the Boards of Appeal of the EPO", 8th edition 2016, IV.E.2.6.9, 1108). In the reply to the board's communication, the respondent has not contested the board's provisional opinion as regards this issue and the board has thus no reason to deviate from said opinion. Therefore, the appeal is admissible under Article 108 EPC and Rules 99(2) and 101(1) EPC.

Admission of the main and first auxiliary requests

2. The main request and the first auxiliary request have been filed in reply to the board's communication pursuant to Article 15(1) RPBA shortly before the date of the scheduled oral proceedings.

3. The main request is identical to the main request filed with the appellant's statement of grounds of appeal except for the deletion of one amino acid sequence (SSCrFCES) in dependent claim 21. The main request filed with the statement of grounds of appeal was the request on the basis of which the opposition division decided to maintain the patent and was part of the proceedings from the beginning.

4. The first auxiliary request is identical to the main request filed in reply to the board's communication pursuant to Article 15(1) RPBA except for the deletion of claims 8 and 21 of the main request.

5. The deletion of subject-matter of dependent claim 21 in the main request and the deletion of dependent claims 8 and 21 in the first auxiliary request are amendments of the previous main request that intend to overcome an objection raised under Article 123(2) EPC by the board in its communication pursuant to Article 15(1) RPBA. These amendments are thus a direct reply to this communication and, by their nature, do not change the subject-matter of the appeal nor do they raise new issues in the appeal proceedings.

6. Thus, the board, exercising its discretion pursuant to Article 114(2) EPC, governed by the principles laid down in Article 13(1) RPBA, admits the main request and the first auxiliary request in the appeal proceedings.

Technical background

7. The patent describes the expression and secretion of functional endotoxin or lipopolysaccharide (LPS) binding domains of Factor C from the circulating amebocytes of the mangrove horseshoe crab Carcinoscorpius rotundicauda (CrFC). It discloses polypeptides of 20 to 34 amino acid residues in length derived from the CrFC short consensus repeats - also known as sushi domains; in particular amino acid sequences of the sushi-1, sushi-3, sushi-4, sushi-5, sushi-6-vg1, sushi-7-vg2, sushi-8-vg3, and sushi-9-vg4 polypeptides. The patent further discloses isolated nucleic acids encoding these polypeptides, a method for producing them recombinantly, and the use of these polypeptides for various purposes, such as for preserving a sample from contamination by gram negative bacteria, for purifying a sample by removal of endotoxin, for detecting gram negative bacteria or LPS in a sample, and for the preparation of a medicament for treating sepsis caused by a gram negative bacterial infection (cf. point X supra).

Main request

Admission of the objection raised under Article 123(2) EPC

8. In the statement of grounds of appeal, the appellant raised three objections under Article 123(2) EPC, the first against all independent claims, the second against dependent claims 8, 12, 13 and 17 to 24, and the third against claims 8 and 21 (cf. point XIII supra). The arguments on which the first objection is based, were already put forward in the Notice of opposition and the admission of this objection in appeal proceedings is not contested by the respondent.

9. In point 2.2.1.4 of the decision under appeal, the opposition division referred to an objection raised under Article 123(2) EPC by the opponent/appellant against claim 10 as granted (corresponding to claim 8 of the main request) and, in analogy thereto, against granted claims 14, 15 and 21 to 28 (corresponding to claims 12, 13 and 17 to 24 of the main request). Point 3.3 of the "Minutes of the oral proceedings before the opposition division" (hereinafter "the Minutes") refers to this objection and does not report any protest from the patent proprietor/respondent to the introduction of this objection into the opposition proceedings. None of the parties contested the Minutes. Thus, the board sees no reason for not admitting the second objection into the appeal proceedings.

10. The second and third objections raised under Article 123(2) EPC in the statement of grounds of appeal are directed against claims that had been discussed under the same ground of opposition in the opposition procedure. However, whilst the arguments on which the second objection is based are clearly found in both the decision under appeal and the Minutes, this is not the case for the arguments on which the third objection is based. Nevertheless, the subject matter at the basis of the second objection, namely the presence of "additional parts" (cf. first sentence of point 3.3 of the Minutes), is also at the basis of the third objection, namely the presence of the secretory signal sequence (21 residues) and the "additional" amino acids of the corresponding sushi domains in the polypeptides of claims 8 and 21.

11. In view thereof, the objections raised under Article 123(2) EPC do not introduce a fresh ground of opposition in the appeal proceedings, and all three objections are admitted.

Article 123(2) EPC

12. The first objection was raised against all independent claims, in particular claim 9. The appellant argued that the patent application did not disclose the specific peptide sequences listed in these claims.

13. Under the heading "Summary of the invention", the patent application refers to a functional LPS-binding domain (spanning 333 amino acids) of C. rotundicauda Factor C protein (SSCrFCES) and portions/fragments thereof (cf. page 3, lines 23 to 30), 34-mer synthetic sushi peptides, such as the sushi-1 (S1) and sushi-3 (S3) peptides, as well as fusion constructs (cf. page 4, line 23 to page 5, line 31; in particular, page 4, lines 27 to page 5, line 2). Likewise, under the heading "Brief description of the drawings", reference is made to the LPS-binding domain of SSCrFCES, synthetic sushi peptides, such as those of 34 amino acids in length shown in Figure 11B, and fusion constructs (cf. inter alia, page 6, line 30 to page 7, line 14 for Figures 3(A) and 3(E); page 9, lines 10 to 15 for Figures 10(a) and 10(b), and Figures 11(a) and 11(B)). The examples of the patent application refer to the LPS-binding domain of SSCrFCES, synthetic sushi peptides, and several SSCrFCsushi-GPF fusion proteins (cf. inter alia, page 21, Example 3; page 29, lines 28 to 30). The disclosure of the patent application as a whole, and in particular the amino acid sequences of the sushi peptides shown in Figure 11B of the patent application, provide a formal basis for the specific amino acid sequences of the isolated polypeptides of claim 9 and for all other independent claims of the main request.

14. The second objection was raised against dependent claims 8, 12, 13 and 17 to 24. The appellant argued that although the independent claims of the main request referred to isolated polypeptides of specific lengths and sequences, the polypeptides of the dependent claims comprised "additional elements". The patent application did however not provide a basis for the subject matter of the dependent claims.

15. Claim 10 of the patent application is directed to an isolated polypeptide comprising a LPS-binding domain of a FC protein that may be selected from those given in claim 12 which include inter alia the sushi-1, sushi-2 and sushi-3 domains as well as the sushi-1, sushi-2 and sushi-3 peptides. The isolated polypeptide may also comprise "additional elements", such as a specific (vitellogenin) secretory signal sequence (claim 13), a reporter protein or an affinity tag (claim 16). Likewise, claim 37 of the patent application is directed to an isolated nucleic acid encoding the polypeptide of claim 10, and dependent claims 38 to 40 contemplate the presence of "additional elements". Claims 44, 45 and 49 of the patent application are directed to a method for producing an isolated LPS-binding protein using an isolated nucleic acid. The board considers these claims to directly and unambiguously disclose the subject matter of claims 12, 13, 17 to 20, and 22 to 24 of the main request. Appellant's objection arising from an alleged lack of clarity - due to the use of the term "consisting" in the independent claims instead of "comprising" - is not an issue within the scope of the appeal since Article 84 EPC is not a ground for opposition and this wording was in the claims as granted.

16. The appellant argued furthermore, that the specific amino acid sequences shown in claims 8 and 21 were neither disclosed in the claims nor in the sequence listing of the patent application.

16.1 Claims 8 and 21 define fusion polypeptides containing a secretory signal sequence (the first 21 residues), a sushi (sushi-1, sushi-3, or sushi-1,2,3) domain from the C. rotundicauda FC protein, and a GFP protein (for which no amino acid sequence is shown).

Figures 10 and 28 of the patent application schematically show fragments (SSCrFCES) and fusion proteins (cf. page 9, lines 10 to 13; page 12, lines 13 to 20). With reference to SEQ ID NO: 4, Figure 10A indicates the start/end amino acids of several CrFC21 domains. Figure 10B refers to the FC fragments in pAc5/ssCrFCES-V5-His, pAc5.1/sssushi-1,2,3-EFGP, pAc5.1/sssushi-1-EFGP, and pAc5.1/sssushi-3-EFGP, but does not disclose any amino acid sequence. None of these constructs is exemplified in the patent application.

The first 21 residues of all the sequences of the polypeptides shown in claims 8 and 21 are identical, and the subsequences starting at position 22 of the first, second and third polypeptide of claim 8 (identical to the second, third and fourth polypeptide sequences of claim 21) correspond to: i) a sushi-1 domain consisting of residues 142 to 196 of SEQ ID NO: 4, ii) a sushi-3 domain consisting of residues 260 to 321 of SEQ ID NO: 4, and iii) a sushi-1,2,3 domain conisting of residues 142 to 321 of SEQ ID NO: 4 (cf. also Figure 10A).

The start/end residues of the sushi-1, sushi-3 and sushi-1,2,3 domains encoded by the vector constructs shown in Figure 10B are not indicated, only the start/end residues of subsequences (residues 171 to 204 of SEQ ID NO: 4 for sushi-1 peptide; residues 268 to 301 of SEQ ID NO: 4 for sushi-3 peptide, even though in Figure 10B it is wrongly annotated as residues 288 to 301 of SEQ ID NO: 4). These start/end residues are however different from those indicated in Figure 10A. Yet other start/end residues characterizing the sushi domains are indicated on page 15, lines 1 to 11, and in Figure 28 of the patent application, namely residues 29 to 201 for sushi-1, residues 264 to 330 for sushi-3, and residues 29 to 330 for sushi-1,2,3. Reference is also made to the functional relevance of residues 60 to 70 in the sushi-1 domain, residues 170 to 185 in the sushi-2 domain, and residues 270 to 280 in the sushi-3 domain (cf. page 15, lines 6 to 9).

16.2 The facts of the present case differ substantially from, and cannot be compared to, those of the case underlying decision T 2048/10 of 21 July 2015 (cf. points 4 and 5 of the Reasons). In said case, the indication of the start/end residues of a fragment or subsequence in a Figure of the patent application was sufficient for acknowledging direct and unambiguous disclosure of this fragment or subsequence as such. In the present case, however, the reference in Figure 10A of the patent application to the start/end residues of the sushi domains is not the sole disclosure of these domains and there is a considerable degree of ambiguity in the characterization of the respective start/end residues (supra). The board concludes therefore that the specific amino acid sequences from residue 22 onwards of the polypeptides of claims 8 and 21 are not directly and unambiguously derivable from the patent application.

16.3 Moreover, the patent application does not disclose the first 21 amino acid residues of these polypeptides which, as stated above, are identical in all the amino acid sequences of claims 8 and 21. There is no reference to this sequence in Figures 10(A) and 10(B) or Figure 28, nor in the description of these figures under the heading "Brief description of the drawings". Although there is a reference to "a novel secretory signal" on page 3, line 28 to page 4, line 2 of the patent application, the sequence of this secretory signal is disclosed only in US Patent Application No. 09/426,776, which is based on the provisional US patent application No. 60/106,402. This US patent application is also cited in Example 1 describing SSCrFCES expression in stable cell lines of Drosophila (cf. page 18, lines 13 to 15). Although a reference to a US patent application may fulfil - under certain circumstances - the conditions required by the case law for acknowledging a direct and unambiguous disclosure (cf. inter alia, J 16/13 of 22 May 2014, points 14 to 20 of the Reasons; T 2477/12 of 12 November 2015; and "Case Law", supra, II.E.1.1.2, 402), it is questionable whether the general reference to a US patent application, which in the present case is based on a provisional US patent application, suffices as a basis for the disclosure of the specific first 21 amino acid residues of the polypeptides shown in claims 8 and 21 and a combination of these residues directly fused with the sequence of the sushi domains as in claims 8 and 21. In this context, it is worth noting again that there is no signal sequence shown in the constructs of Figure 10B and there is no information on the actual components/elements defining the pAc5.1 constructs shown in this Figure.

16.4 In view of these considerations, the board concludes that the patent application does not directly and unambiguously disclose the specific amino acid sequences of the (fusion) polypeptides shown in claims 8 and 21 of the main request.

17. Thus, the main request contravenes Article 123(2) EPC.

First auxiliary request

Article 123(2) EPC

18. The first auxiliary request is identical to the main request except for the deletion of claims 8 and 21 (cf. point XI supra). The deletion of these claims overcomes the third objection raised under Article 123(2) EPC against the main request. The other two objections raised under this article against the main request, and maintained by the appellant against the first auxiliary request, are not successful (cf. points 13 and 15 supra) and thus, the first auxiliary request does not contravene Article 123(2) EPC.

Admission of objections raised under Articles 83 and 54 EPC

19. In the communication pursuant to Article 15(1) RPBA, the board drew the parties' attention to the case law concerning the substantiation of grounds of appeal (cf. "Case Law", supra, IV.E.3.2.1.h) and IV.E.3.2.1.i), 1122; and related IV.E.2.6.3.b), 1098; IV.E.2.6.4.a), 1102; IV.E.2.6.6, 1107, on admissibility of the appeal).

As stated in this case law, a mere reference to a party's earlier submissions and/or the verbatim repetition of the arguments presented in these submissions ("grounds by cut-and-paste"), including those submissions or arguments put forward at the oral proceedings before the opposition division, but without actually dealing with, or entering into a discussion of, the reasons given in the decision under appeal by the opposition division for arriving at its decision, is not enough to substantiate a ground of appeal.

20. The board informed the parties that, in view of the reasons given by the opposition division in relation to Articles 83 and 54 EPC (cf. pages 7 to 10, points 2.3.5 and 2.3.6 of the decision under appeal) and of the parties' submissions on these articles in appeal, the board was of the provisional opinion that the grounds of appeal on Articles 83 and 54 EPC were not sufficiently substantiated.

In its communication, the board further indicated that, if a discussion on these objections arose at the scheduled oral proceedings, the first issue to be discussed was their admissibility into the appeal. At the oral proceedings before the board, the appellant referred to its written submissions and did not put forward any further argument or reason that could have led the board to deviate from its provisional opinion as expressed in its communication. Thus, the grounds of appeal under Articles 83 and 54 EPC are considered not to be substantiated and therefore, not admitted into the appeal proceedings.

Article 56 EPC

21. The board agrees with the opposition division that document (6) represents the closest state of the art (cf. page 13, point 2.3.7.3 of the decision under appeal). Document (6) refers to the presence of proteins capable of LPS binding and neutralization in the circulating amebocytes of horseshoe crabs (cf. paragraph bridging pages 391 and 392) and describes the production of recombinant Factor C from C. rotundicauda (CrFC21/rFC21) and of a 762 amino acid fragment (CrFC21EE/rFC21EE) thereof comprising the cysteine-rich region, the EGF-like domain and one or two sushi domains with a "slightly reduced endotoxin-binding capacity" (cf. page 392, left-hand column, second paragraph, and right-hand column, first paragraph; paragraph bridging pages 394 and 395; page 396, right-column, first paragraph; and page 397, left-hand column, first paragraph). Document (6) refers also to "the potential of genetically engineered rFC for the detection and removal of endotoxin in the pharmaceutical industry, and protection against Gram-negative bacteria endotoxemia" (cf. page 392, left-hand column, last sentence of the second paragraph).

22. Starting from document (6), the opposition division defined the objective technical problem as the provision of alternative Factor C polypeptides with LPS-binding activity (cf. page 14, first paragraph of the decision under appeal). This formulation of the technical problem has not been contested by the parties and the board agrees with it. The claimed polypeptides, in particular those of claim 8, are the solution proposed by the patent.

23. Examples 2 and 3 of the patent show the polypeptides of claim 8 to have LPS binding activity (albeit lower than the full-length Factor C protein) and thus, to solve the technical problem as formulated above.

24. The appellant argued that, in view of the low LPS-binding activity, the polypeptides shown in Example 3, and Figures 3B to 3E of the patent, did not represent true alternatives to the CrFC21EE/rFC21EE fragment disclosed in document (6). The board, however, shares the respondent's view that Example 4 and Table 3 of the patent show the claimed polypeptides to retain an unexpectedly high affinity for LPS-binding allowing thereby the detection of the Gram-negative bacterial endotoxin and providing a significant endotoxin-neutralizing activity (anti-endotoxin potency). Moreover, contrary to the appellant, the board considers that the selection of these polypeptides is not an arbitrary selection because, as stated by the respondent and the opposition division in the decision under appeal (cf. page 15, lines 5 to 7 from the bottom), other advantageous properties (cheaper production costs, easier handling, low cytotoxicity) are associated with these polypeptides.

25. Whilst document (6) refers to the "reduction in intensity of binding of rFCEE to lipid A as compared to that of rFC" and, in this context, mentions the "downstream sushi domains" of the Factor C protein, this citation is found within a general reference stating that "further downstream sequences of Factor C", in general, "may co-operate directly or indirectly, via protein-protein interaction" (cf. page 395, right-hand column, first paragraph). This reference does not hint at or suggest that an isolated sushi domain or a polypeptide consisting of a small number of them, let alone fragments thereof, such as the claimed polypeptides, may have LPS binding ability. On the contrary, in the board's view, this reference teaches away from such expectation and, if at all, leads the skilled person towards longer rFC fragments. Moreover, although, as argued by the appellant, the method for producing rFC protein and/or rFCEE fragment described in document (6) is identical to the method disclosed in Example 12 of the patent, none of these methods results in the production of the claimed polypeptides. There is no evidence on file - nor has any been provided by the appellant at the oral proceedings before the board - of a slurry that contains any of the claimed polypeptides in any of the steps carried out in these methods. There is neither evidence on file showing the degradation or cleavage of the rFC protein and/or rFCEE fragment to shorter fragments in any of these methods, nor a reference to any degradation or cleavage of these products in either document (6) or in Example 12 of the patent. Indeed, none of them refers to a slurry containing whatever polypeptides other than the rFC protein and/or the rFCEE fragment. Thus, the board considers the claimed polypeptides not to be obvious from the disclosure of document (6) alone.

26. Furthermore, the claimed polypeptides are not obvious from a combination of the disclosure of document (6) with any of the prior art documents on file, in particular, documents (1), (5), (9) or (10) (cf. point XIII supra).

26.1 Document (1), originating from two of the three authors of document (6), discloses a cDNA encoding the full-length Factor C protein from C. rotundicauda (CrFC 26, Figure 6, 1083 residues; SEQ ID NO: 1 and 2; cf. column 5, lines 20 to 42) and several cDNAs encoding fragments derived from this protein (cf. column 5, first paragraph, and Figure 3), the longest fragment having 1019 residues (CrFC 21, Figure 8; SEQ ID NO: 3 and 4; cf. column 5, lines 49 to 57). Figure 11 shows an alignment of the two cDNA sequences and the encoded putative proteins (cf. column 6, lines 4 to 21; column 14, Example 6). Document (1) identifies "five short consensus repeats" (the five sushi domains: residues 206 to 259, 263 to 318, 324 to 385, 640 to 698 and 758 to 812) in the CrFC 26 sequence (cf. column 15, first paragraph) but discloses no biological function or activity associated with these domains. Although document (1) states that the disclosed proteins have "the same enzymatic activity as Factor C protein in assays for Gram negative bacterial endotoxin" (cf. column 2, second paragraph) and further refers to "biologically functional equivalents" as "nucleic acid sequences and polypeptides exhibiting the same or similar biological activity as the particular nucleic acid sequences and polypeptides described herein" (cf. column 16, second paragraph; column 2 and 3, last full paragraphs), the sole polypeptides for which a biological activity (endotoxin binding assay) is disclosed are the two forms (single and double-chain) of Factor C (pro)enzyme purified from the amebocytes of C. rotundicauda (cf. column 21, line 36 to column 26, first paragraph). There is no information in document (1) on the biological activity of the polypeptides encoded by the various cDNAs shown in Figure 3, and there is no indication or suggestion that all these polypeptides may have a biological activity. Indeed, the section of document (1) concerned with "biologically functional equivalents" does not refer to these polypeptides at all but only to muteins, derivatives, fragments and portions in general without characterizing or defining the essential residues and/or domains necessary for retaining the desired biological activity. In view thereof, the board does not consider the disclosure of document (1) to provide additional technical information that would allow a skilled person starting from document (6) to arrive at the claimed polypeptides in an obvious manner.

26.2 Document (5), the scientific publication corresponding to document (1), describes only part of the results shown in document (1). Thus, as stated above for document (1), the disclosure of document (5) in combination with that of document (6), does not lead a skilled person to the claimed polypeptides in an obvious manner.

26.3 Document (10), an excerpt from the UNIPROT databank, discloses the amino acid sequence (1019 residues) of the Limulus clotting Factor C precursor and identifies several domains within this sequence, including four short consensus repeats (SCR) or sushi domains (sushi-1, 2, 3 and 4; residues 142 to 195, 200 to 254, 260 to 321, and 576 to 634, respectively). There is however no indication of any biological function or activity associated with these domains, let alone with fragments or subsequences therefrom. As for bibliographic data, document (10) refers to a single scientific publication which is document (9) in the present proceedings.

26.4 Document (9) discloses the amino acid sequence of the Factor C protein from amebocytes of Tachypleus tridentatus (horseshoe crab) and identifies five putative sushi domains (residues 117 to 170, 174 to 229, 235 to 296, 551 to 609, and 615 to 723; cf. page 6556, Figure 2 and page 6558, Figures 7 and 8) which, based on studies of proteins with homologous domains, are predicted to be sites for interactions with proteins of the mammalian complement system (cf. page 6557, left-hand column, second paragraph). None of these domains has been isolated and, in the board's view, a skilled person would not be motivated to isolate them, let alone fragments or sub-sequences therefrom, based on this general statement. Moreover, the amino acid sequences of the putative sushi domains identified in document (9) do not correspond to the sequences of the polypeptides of claim 8 which represent fragments and/or combinations of several of these domains. The sushi-1 peptide of claim 8 corresponds to amino acids 146 to 179 of the sequence shown in Figure 2 of document (9) and includes several residues of the sushi-1 domain and a few residues of the sushi-2 domain (cf. Figure 6 of document (9)). In addition, the claimed sushi-1 peptide differs from the amino acid sequence shown in Figure 2 of document (9) at three positions (V152M, S165N, and K176M). The sushi-3 peptide of claim 8 corresponds to amino acids 243 to 276 of the sequence shown in Figure 2 of document (9). It represents thus a sub-fragment of this sushi domain but with one substitution (Q247K). None of the other sequences of claim 8 is present within the sequence shown in Figure 2 of document (9). It is thus not obvious for a skilled person to arrive at the specific amino acid sequences of the polypeptides of claim 8 based on the disclosure of document (9) alone or in combination with document (6).

26.5 In conclusion, the combination of document (6) with any of documents (1), (5), (9) or (10), or with other, less relevant prior art on file, would not have led the skilled person to the claimed polypeptides and thereby to the claimed subject-matter of the first auxiliary request in an obvious way.

27. Thus, the first auxiliary request fulfils the requirements of Article 56 EPC.