T 1270/12 (Lawsonia intracellularis cultivation vaccine infection rate/BOEHRINGER INGELHEIM) 21-07-2016

Reasons for the Decision

Scope of the appeal proceedings

1. The decision under appeal is concerned with Articles 56 and 83 EPC only. In the decision T 692/08, this board, in a different composition decided that the Main Request fulfilled the requirements of Articles 123(2),(3) and 84 EPC (cf. T 692/08, supra, point 9 of the Reasons). Novelty was not challenged in opposition proceedings (cf. page 3, point 14 of the decision under appeal).

Admissibility of new evidence

2. Documents D1-D37 were filed at the beginning of the opposition proceedings, documents D38-D91 were filed during the first appeal proceedings (cf. T 692/08, supra). Documents D91a-D97 were filed after the board remitted the case to the opposition division and documents D98-D106 have been filed during the present second appeal proceedings.

3. There is no indication on file that documents D1-D91 have not been admitted into the proceedings. Moreover, none of the parties has objected to the admissibility of any of the documents filed during the second opposition and the present second appeal proceedings. Therefore, the board does not consider it necessary to enter into a detailed discussion on the admissibility of any evidence on file.

4. However, this does not apply to the experimental data submitted by former opponent 01 with the statement setting out its Grounds of Appeal (cited therein as document D92). The admissibility of this data, which has been disputed by the respondent, is examined by the board in the section below, dealing with the requirements of Article 83 EPC with regard to Auxiliary Request IV.

Admissibility of the Main Request and Auxiliary Requests I-IV

5. In reply to the Grounds of Appeal, the respondent filed a Main Request which it considered to be identical to the request upheld by the opposition division. However, the new Main Request actually differed from this request in claim 13 where one of the deposited strains had been deleted. In reply to the board's communication pointing at this difference, the respondent corrected the request and filed a Main Request identical to the request upheld by the opposition division, which does not have to be examined as to its admissibility.

6. The amendments introduced into Auxiliary Requests I-IV consist of a deletion of claimed subject-matter (a deposited strain in all Auxiliary Requests and the method for producing attenuated L. intracellularis in Auxiliary Requests III-IV) and of a limitation of the scope of the claims by introduction of subject-matter of a dependent claim (infected host cells limited to McCoy cells in Auxiliary Requests II and IV). They are straightforward in nature, do not raise any new issues and have been made in direct reply to the board's communication pursuant to Article 15(1) RPBA.

7. Therefore, the board, exercising its discretion under Article 13(1) RPBA, decides to admit Auxiliary Requests I-IV into the procedure.

Main Request, Auxiliary Requests I and III

Article 100(b) EPC; Article 83 EPC

Claim 1

8. Claim 1 of these requests is identical and relates to "a method for cultivating L. intracellularis bacteria comprising obtaining culture cells infected with L. intracellularis", without containing any limitation to the type and nature of the infected host cells. The claim further requires to maintain "said infected cells in suspension" and that the "harvesting is done when greater than about 70% of the cells ... are infected" (cf. point X supra). Whether the claimed method is disclosed in a way that enables a skilled person to achieve an infection rate greater than 70% has to be examined under Article 83 EPC (see decision of the Enlarged Board of Appeal G 1/03; OJ EPO 2004, page 413, point 2.5.2).

9. The general part of the description of the patent contains two references to this feature, namely in paragraphs [0029] and [0033]. A further reference to the harvesting of L. intracellularis in suspension cultures which is "preferably done when 50-100% of the cells are infected" can be found in paragraph [0028]. None of these references mentions any limitation to the type and nature of the cultured cells. This is also in line with the disclosure in paragraph [0016], where it is stated that "numerous cell lines can be used", although "the preferred culture cells are HEp-2, McCoys or IEC-cells".

10. Examples 2 and 3, which are concerned with the growth of L. intracellularis in suspension cultures of HEp-2 cells and of McCoy cells, do not give any information with regard to the specific level of infection. Such information is also not disclosed in Examples 4 and 5, both describing cultures in suspension of HEp-2 cells (cf. paragraphs [0070]-[0071] and [0094]-[0095]). Only Example 6 mentions that an infection rate of "approximately 100%" is observed in L. intracellularis grown in a suspension of McCoy cells (cf. paragraph [0114]). Thus, the Examples of the patent do not support the drawing of a definite conclusion with regard to the achievement of the claimed infection rate in culture suspensions of cells other than McCoy cells.

11. This is confirmed by the experimental evidence provided in documents D91/91a and D97, which report that an infection rate of 100% at day 7 p.i. is achieved by using McCoy cells in suspension. However, this evidence also shows that the infection rate at day 7 p.i. is only 50% when IEC-18 cells, another preferred cell type according to the patent, are used in suspension (cf. Table 1 of D91/D91a and Figure 1 of D97). Although this infection rate is high when compared with the infection achieved with IEC-18 cells grown in monolayer culture (2.9% infection at day 7 p.i.; Figure 1 of document D97), it is far away from the required "greater than about 70%".

12. Document D97 discloses that "the fact that the IEC-18 suspension culture was rising steadily to an infection rate of 50% at day 7 does not exclude that a higher rate of 70% or more would have been reached upon a slightly longer cultivation. This suggests that the actual immortalised cell type ... is, unexpectedly, not a crucial factor to this highly raised infectivity rate" (emphasis added by the board) (cf. page 3, point 8 of document D97). These speculations are a far cry from certain and convincing evidence demonstrating that the infection rate required by claim 1 is actually achieved with IEC-18 cells in suspension. Rather, the data provided in this experimental evidence casts serious doubt upon whether such an infection rate can be achieved with other preferred cells disclosed in the patent, or with any other types of non-preferred host cells.

13. In fact, as shown in Figure 2 of document D5, the percentage of infected IEC-18 cells grown in monolayer cultures also rises steadily to a maximum value of 20% at day 7 p.i. However, it remains stable at this value until day 9 p.i. and declines thereafter to a significantly lower value. There is thus no reason to expect that the infection rate of IEC-18 cells in suspension will necessarily continue to rise after day 7 p.i. It is not derivable in a convincing manner from the experimental evidence disclosed in documents D91/D91a and D97 that an infection rate greater than about 70% can actually be achieved with IEC-18 cells in suspension. Indeed, prior art on file (inter alia document D3), although dealing with monolayer cell cultures, provides evidence that, for L. intracellularis and for other obligate intracellular bacteria (such as Chlamydia), both the degree of infection and the number of heavily infected cells depend to a great extent on the specific type and nature of the infected host cell (epithelial/non-epithelial cells, fast-growing/slow-growing, etc.).

14. The board, in view of the evidence on file, reaches the conclusion that the high infection rate required by claim 1 has been clearly and convincingly shown for McCoy cells in suspension only. Serious doubts substantiated by verifiable facts, exist as to whether such a high infection rate can be achieved when other types of host cells, including the preferred IEC-18 rat intestinal epithelial cells (cf. paragraph [0016] of the patent), are used.

15. Thus, the Main Request and Auxiliary Requests I and III do not fulfil the requirements of Article 83 EPC.

Auxiliary Request II

Article 100(b) EPC; Article 83 EPC

Claim 13

16. Claim 13 of Auxiliary Request II is directed to "a method for producing an attenuated L. intracellularis strain" (emphasis added by the board) (cf. point XI supra). Claims 1 and 12 of the Main Request and Auxiliary Requests I to V underlying the decision T 1846/10 were both directed to "a method for producing a live vaccine against L. intracellularis" (emphasis added by the board) (cf. T 1846/10, supra, items IV, VI and point 47 of the Reasons).

17. In the statement setting out the Grounds of Appeal and in respondent's reply thereto, both parties consistently stated that they regarded the subject-matter of claim 13 of Auxiliary Request II to be a method for producing an attenuated L. intracellularis strain "suitable as a live vaccine", i.e. having all properties of a vaccine. In its communication pursuant to Article 15(1) RPBA, the board informed the parties that the subject-matter of the claim could also be interpreted broadly, namely to refer to a method merely achieving a reduction of the pathogenicity of L. intracellularis. The board drew the parties' attention to decision T 1846/10 and stated that, if the term "attenuated" was interpreted narrowly and thus equated to "suitable as a live vaccine", it was likely to come to the same conclusion as that reached in T 1846/10, namely that the patent does not disclose this subject-matter in a manner sufficiently clear and complete for it to be carried out by a person skilled in the art (cf. T 1846/10, supra, points 4-46 of the Reasons).

18. Whilst the appellant did not reply to the board's communication, the respondent filed further submissions in reply thereto, wherein it argued that the correct interpretation of claim 13 of Auxiliary Request II required that the product of the claimed method, an "attenuated" strain, had to be seen as being "suitable as a live vaccine", in line with the "narrow" interpretation of this term. At the oral proceedings, the respondent confirmed this interpretation of the subject-matter of claim 13. Based on this interpretation, the respondent gave a detailed analysis of the reasoning of decision T 1846/10, in order to convince the board that a wrong decision had been taken. The respondent argued that the decision taken on this subject-matter by the board in decision T 1846/10 with regard to the requirements of Article 83 EPC, cannot be subject to the doctrine of res judicata for the present case since the wording of the claims is different as are the facts and evidence on file. Hence the respondent concludes that the board in the present case is not obliged to, and should not, find that claim 13 of Auxiliary Request II does not meet the requirements of Article 83 EPC (cf. point XV supra).

19. Taking account the arguments put forward by both parties in the written procedure and at the oral proceedings, the board sees no reason not to apply the "narrow" interpretation to construe the subject-matter of claim 13, which consequently, although not having exactly the same wording, is identical to the subject-matter of claim 12 of the Main Request in decision T 1846/10.

20. The application underlying the patent in the present case is the parent of the divisional application underlying T 1846/10. The appellant and respondent in the present case were also the appellant and respondent respectively in T 1846/10.

21. In T 1846/10, the board found that claim 12 of the Main Request before it did not meet the requirements of Article 83 EPC.

22. Independent claim 12 of the Main Request in T 1846/10 read as follows:

"12. A method for producing a live vaccine against L. intracellularis, wherein said vaccine comprises an attenuated L. intracellularis strain, comprising the steps:

(1) producing an attenuated L. intracellularis strain comprising obtaining culture cells infected with L. intracellularis bacteria, incubating said infected cells at an oxygen concentration of 0 percent to about 18 percent, agitating said infected cells so as to cultivate said bacteria while maintaining said infected cells in suspension, passaging at least a portion of said cultivated bacteria, harvesting at least a portion of said cultivated bacteria, and selecting for an attenuated strain to provide an attenuated L. intracellularis bacteria; and

(2) admixing said attenuated L. intracellularis in an acceptable pharmaceutical carrier."

23. Claim 13 of Auxiliary Request II in the present case reads as follows:

"13. A method for producing an attenuated L. intracellularis strain comprising obtaining culture cells infected with L. intracellularis bacteria, incubating said infected cells at an oxygen concentration of 0 percent to about 18 precent, agitating said infected cells so as to cultivate said bacteria while maintaining said infected cells in suspension, passaging at least a portion of said cultivated bacteria, harvesting at least a portion of said cultivated bacteria, and selecting for an attenuated strain to provide an attenuated L. intracellularis bacteria." (cf. points XI-XII supra).

24. The wording of claim 12 of the Main Request in case T 1846/10 is not identical to the wording of claim 13 of Auxiliary Request II in the present case. Nevertheless the board has concluded that the subject matter of these claims is the same; see points 16 to 19 above.

25. The question therefore arises whether the application of the doctrine of res judicata is appropriate in this case. If it is, the board would find that claim 13 of Auxiliary Request II did not meet the requirements of Article 83 EPC.

26. The Boards of Appeal have an established practice of applying the res judicata doctrine; see T 51/08 of 7 May 2009, points 1 to 4 of the Reasons. The res judicata doctrine undoubtedly falls under those "principles of procedural law generally recognised in the Contracting States", referred to in Article 125 EPC.

27. For res judicata to apply, the same question has to have been decided in both T 1846/10 (supra) and the present case. The "same question" requirement is not to be applied in a mechanical, formalistic way so as to require that the claims under consideration in the cases have to be identical. Rather the boards examine whether the subject matter of the claims is the same (see T 790/10 of 18 December 2012; T 1155/13 of 7 May 2014, point 6.2 of the Reasons). The board has already decided affirmatively upon this point. The compliance of these claims with Article 83 EPC was the issue in T 1846/10 and is the issue in the present case.

28. As regards the facts and evidence on file, the board considers that the essential facts in the present case are the experimental data and the results disclosed in Examples 5 and 6 of the patent; both examples concern the efficacy of a vaccine produced by the method of claim 13 in hamster and swine, respectively. Except for the declaration of Dr. S. McOrist (document D67 in T 1846/10), all other declarations, experiments, prior art and one post-published patent application explicitly referred to in decision T 1846/10 (cf. T 1846/10, supra, point III of the Facts and Submissions; documents D1, D48, D50, D55 and D64 in that decision correspond to documents D4, D91/D91a, D89, D98 and D90 in the present appeal proceedings) have also been filed in the present appeal proceedings. The board notes that although the declaration of Dr. B. Eichenmüller, D64 in T 1846/10 and D90 in the present appeal, are not identical, they have essentially the same content. There are only three other documents cited in decision T 1846/10 (post-published scientific publications D65, D66 and D68). These documents were mentioned in the context of Example 6 of the patent and were considered not relevant by the board in decision T 1846/10 (cf. T 1846/10, supra, points 30 and 41 of the Reasons).

29. In addition to the reasoning put forward by the respondent at oral proceedings before the board (cf. point 18 supra), a further decisive factor for the board to be convinced that the facts and evidence in the present case are the same as in the case underlying decision T 1846/10, are the respondent's submissions filed in reply to the board's communication pursuant to Article 15(1) RPBA (cf. point VIII supra). These submissions are a straightforward review of the conclusion arrived at by the board in decision T 1846/10, wherein reference is also made to the preliminary, non-binding opinion of the board in that case expressed in the communication pursuant to Article 15(1) RPBA (Exhibit 1) and to the respondent's reply thereto (Exhibit 2).

30. At oral proceedings before the board the respondent referred to a further declaration of a technical expert. This declaration was neither filed in reply to the Grounds of Appeal nor in reply to the board's communication. Under the particular circumstances of the present case, the board drew the respondent's attention to the possible non-admissibility of such a declaration if filed at the latest stage of the appeal proceedings. The respondent did not file this declaration and thus, it is not part of the evidence on file.

31. Thus, the board concludes that the doctrine of res judicata is applicable to this case, and that applying the doctrine, claim 13 of Auxiliary Request II does not meet the requirements of Article 83 EPC.

Auxiliary Request IV

Article 100(b) EPC; Article 83 EPC

Claims 1-12

32. Claims 1-12 of this auxiliary request are directed to methods for cultivating L. intracellularis bacteria in suspension culture wherein the infected host cells are defined as being McCoy cells. Claims relating to a method for producing attenuated L. intracellularis strains have been deleted from this request (cf. point XII supra). Thus, none of the objections raised under Article 83 EPC against the Main Request and Auxiliary Requests I-III above applies to this request.

33. Former opponent 01, by referring to experimental data filed with its Grounds of Appeal, has objected that L. intracellularis bacteria cannot be cultivated in free suspensions of McCoy cells, i.e. in suspensions without microcarriers, beads, etc. (cf. point XIV supra). According to the patent, the use of microcarriers, beads, etc. for this type of adherent (anchorage-dependent) cells, is a preferred embodiment of the invention, but is not essential for carrying out the claimed method (cf. paragraph [0031] of the patent). Indeed, whilst in Example 3 of the patent microcarriers/beads are said to be used (cf. paragraphs [0059]-[0061]), there is no reference to such material in Example 6 (cf. paragraph [0114]).

34. The patent does not give detailed information on the specific conditions of the suspension culture used in Example 6. Such information is, however, provided in the experimental evidence disclosed in documents D91/D91a and D97, wherein McCoy cells are cultured in free suspensions without microcarriers or beads. It is important to note that different appearance, cell density and infection were obtained in these experiments for the two types of adherent cells used (IEC-18 and McCoy). It is stated that the McCoy cells are "forming clusters of cells" resulting in "significant clumping issues" (cf. page 1, last paragraph and page 2, second paragraph of documents D91/D91a). Thus, the probative value of the experimental data filed with opponent 01's Grounds of Appeal is at least questionable.

35. Indeed, during the course of the first appeal procedure in which this experimental data was late filed (cf. T 692/08, supra), the respondent questioned the validity of this data and its admissibility. No decision was taken on this issue by the board in the first appeal procedure or by the opposition division after the remittal. The status of this experimental data is not clearly established in the present, second appeal procedure. It is not contained in the consolidated list of documents annexed to the Minutes of the oral proceedings before the opposition division leading to the decision under appeal. However, in view of the questionable probative value of this data (cf. point 34 supra), it is not necessary for the board to enter into further detail on this issue.

36. Thus, Auxiliary Request IV fulfils the requirements of Article 83 EPC.

Article 100(a) EPC; Article 56 EPC

Claims 1-10 and 12

37. The closest prior art document D5 discloses a method for cultivating L. intracellularis bacteria in a monolayer culture of rat enterocyte IEC-18 cells infected with L. intracellularis. The infected cells are incubated at a reduced oxygen concentration of less than 18%, and harvested when most of the cells are infected and when many heavily infected cells (HIC) are present (between days 5 and 7 p.i.). Whilst the level of infection of the IEC-18 cells exposed directly to the bacteria is reported to be between 6.29 (at 5 days p.i.) and 2.90 (at 7 days p.i.), the percentage of infected cells arrives at about 20% when the IEC-18 cells are exposed to supernatant fluid from IEC-18 cells infected directly with L. intracellularis bacteria (cf. Table 1 and Figure 2 of document D5). Document D5 acknowledges that "[t]he cell culture system described has many deficiencies, not the least of which is the apparent failure of many bacteria to established cell infection once associated with cells" (cf. page 1142, left-hand column, third paragraph from the bottom).

38. Starting from the disclosure in this document, the objective technical problem to be solved is the provision of an improved method for cultivating L. intracelluaris bacteria. The subject-matter of claim 1, over its entire scope, provides a solution to this problem, as shown by the patent itself and by the evidence on file (cf. points 9-14 and 33-35 supra).

39. A person skilled in the art of culturing bacteria was well aware of the limitations of monolayer cell cultures and knew the advantages provided by suspension cultures, in particular by the microcarrier bead culture technology, that had already been used for growing the obligate intracellular bacterial parasite Chlamydia trachomatis in McCoy cells (cf. inter alia, document D6). Thus, it remains to be answered whether it would have been obvious for him/her to amend the teaching in the closest prior art and to apply this technology to L. intracellularis with a reasonable expectation of success.

40. According to prior art on file (cf. inter alia, document D3), obligate intracellular bacteria are a phylogenetically diverse group of bacteria with different life cycles based on their specific biochemical and molecular properties. The interaction of these bacteria with their host cells involves a complex sequence of events, such as membrane attachment and invasion of the host cell (coated pits, vesicles, receptor mediated, etc.), replication inside these cells (either within vacuoles or free in the cytoplasm), and eventually leaving the host cell (cf. inter alia, page 255, paragraph bridging left and right-hand column, page 258, right-hand column, third paragraph to page 259, left-hand column, last paragraph of document D3). The characteristics and properties of all these events depend on the specific obligate intracellular bacteria and their specific host cells. Thus, for in vitro growth of obligate intracellular bacteria, conditions have often been developed to resemble the natural in vivo habit. Indeed, most, if not all, in vitro growth studies of L. intracellularis have been carried out by using monolayers of intestinal cells, i.e. the natural environment of these bacteria, the rat enterocyte IEC-18 cell line being the most preferred host cell (cf. inter alia, documents D3 to D5).

41. Moreover, while some of these obligate intracellular bacteria, such as Chlamydia, are known to have a very broad range of permissive host cells, other obligate intracellular bacteria, such as L. intracellularis, are much more restrictive. Prior art on file shows that differences in the obtained number of cells infected and heavily infected with L. intracellularis are even present between different intestinal host cells (cf. inter alia, document D3).

42. In view of the fact that fibroblast McCoy cells as host cells in suspension are far away from the natural in vivo habitat of this bacterium, it seems to be highly doubtful that a skilled person would have applied the teaching of document D6 in a straightforward manner for cultivating L. intracellularis. Moreover, the skilled person could not have any expectation to succeed in obtaining a high number of infected cells, which in fact is much higher than the level of infection achieved under conditions resembling the natural environment of this bacterium (cf. documents D5, D91/D91a and D97). Thus, the board is convinced that the selection of McCoy cells as host cells in suspension was not obvious for a skilled person and that the achievement of such high level of infection could not have been reasonably expected.

43. Therefore, the subject-matter of claims 1-10 and 12 fulfils the requirements of Article 56 EPC.

Claim 11

44. The closest prior art document D4 discloses the culturing of L. intracellularis bacteria in monolayers of rat enterocytes IEC-18 host cells and the further passaging of the bacteria in IEC-18 cells. The bacteria are then used for pig infection, demonstrating thereby that L. intracellularis reproduces and is thus the cause of proliferative enteropathy in pigs. The number of passages of bacteria grown in cells and used to infect each pig varies from as low as only 3 passages to as high as 21 passages (cf. page 4287, Table 1). Although it is disputed among the parties whether document D4 discloses attenuated L. intracellularis (see also, in this context, T 1846/10, supra, point 7 of the Reasons), the document explicitly states that "[t]he doses used were considered to be relatively small but were similar for groups receiving each passage, indicating that differences in lesions produced may be due to a diminution in pathogenecity of an isolate passaged multiply in vitro" (cf. page 4291, left-hand column, last full paragraph from the bottom).

45. Starting from this prior art, the objective technical problem is the provision of alternative L. intracellularis strains with improved (attenuated) properties.

46. In Example 6 of the patent, the ATCC 55783 strain is described as the result of growing L. intracellularis continuously in a culture of McCoy cells in suspension for 29 weeks and passaged for additional 11 weeks, i.e. a total of 40 weeks (cf. paragraph [0114] of the patent). Based on the experimental data disclosed in documents D91/D91a which show that under these conditions 100% infection is achieved at 7 days p.i., the deposited strain has most likely been passaged 40 times, i.e. about twice as often as the highest number of passages reported in document D4 (see, in this context, T 1846/10, supra, points 39-40 of the Reasons). The respondent has referred to the advantageous properties of this strain and its use as master seed of a commercial vaccine. Indeed, the advantageous properties of this strain have already been acknowledged in decision T 1846/10, wherein the board allowed a claim directed to a method for producing a live vaccine against L. intracellularis based on the attenuated L. intracellularis bacteria deposited as ATCC 55783 (cf. T 1846/10, supra, point VII of the Facts and Summary and points 48-59 of the Reasons). Thus, the L. intracellularis strain deposited as ATCC 55783 solves the objective problem formulated above. Moreover, due to the unpredictability of the attenuation process (cf. T 1846/10, supra, point 7 of the Reasons), the achievement of the L. intracellularis strain deposited as ATCC 55783 was not obvious to a skilled person. The person skilled in the art had no particular reason to expect success that such a strain could indeed be produced (cf. T 1846/10, supra, points 57-58 of the Reasons).

47. Thus, the subject-matter of claim 11 meets the requirements of Article 56 EPC.