T 0905/05 (Gene expression/U. OF FLORIDA) 01-02-2007

Article 123(2) EPC; added subject-matter

1. Respondent II' s arguments as regards added subject-matter was that the two expressions "bacterial host cell" (claim 1) and "encoding a plurality of polypeptides" (claim 4) could not be found in the application as filed.

2. The purpose of Article 123(2) EPC is explained in the Enlarged Board's decision G 1/93 (OJ EPO 1994, 541, point 9 of the decision):

"With regard to Article 123(2) EPC, the underlying idea is clearly that an applicant shall not be allowed to improve his position by adding subject-matter not disclosed in the application as filed which would give him an unwarranted advantage and could be damaging to the legal security of third parties relying on the content of the original application."

3. Applying Article 123(2) EPC is, thus, not a question of whether or not the same words are present in a claim and in the application as filed but whether the claimed technical teaching ("subject-matter") is clearly and unambiguously derivable from that application.

4. That the now claimed process is to be performed with recombinant bacterial host cells is, in fact, the gist of the invention as described in the application as filed, starting on page 1, lines 19 to 24 going to eg, the passage bridging page 8, line 14 to page 9, line 13, page 16, lines 2 to 8, page 18, lines 6 to 26, etc.... Furthermore, the examples describe the isolation of recombinant E.coli bacterial host cells overproducing Z.mobilis gene products involved in the production of ethanol.

5. In the same manner, the fact that bacterial host cells may be transformed by a heterologous polynucleotide segment encoding a plurality of polypeptides is straightforwardly derivable from the technical teaching in the application as filed that the segment may comprise a plurality of genes, eg. on page 6, lines 30 to 32:

"The present invention pertains to recombinant host cells that express chromosomally-integrated heterologous genes encoding useful polypeptides at high levels."

6. For these reasons, the requirements of Article 123(2) EPC are fulfilled.

Article 84 EPC; clarity

7. Respondent I argued that, in claim 1, the wording "and said heterologous polynucleotide segment is under the transcriptional control of the endogenous promoter by virtue of being integrated into a host cell chromosome on the downstream side of the promoter" left some doubts as to which promoter was being referred to in the second half of the sentence. For the board, it is unambiguous that the "promoter" must be the endogenous promoter referred to in the first half of the sentence, it being in any case the only promoter mentioned in the claim.

8. Respondent II argued that in that same passage the wording "on the downstream side of the promoter" could mean "outside of the promoter, downstream from it" or "within the promoter, in the downstream part of it" and that, therefore, there was uncertainty as to which integration event the claimed process would involve. In support of this argument, he observed that the polynucleotide segment which was made use of in the examples comprised in the upstream flanking sequence 140 base pairs containing a promoter sequence where homologous recombination could take place as well as in the rest of said sequence.

9. In accordance with the case law (T 860/93, OJ EPO 1995, 47), the description may be used to interpret the claims. Here the description in its generic part makes a clear and consistent distinction between the promoter and the gene which "follows" it. It teaches that the heterologous DNA segment should be integrated within the gene (eg. page 8, lines 17 to 24, page 14, lines 20 to 25). As for the above mentioned examples, they are wholly silent as to the presence of a promoter in the 140 base pairs fragment. Thus, irrespective of whether or not an active promoter is indeed found in this fragment, the skilled person is not made aware of it by reading the description. Accordingly, the board concludes that on the basis of the technical disclosure provided, he/she would understand the expression "on the downstream side of" as meaning "outside of the promoter, downstream from it".

10. The requirements of Article 84 EPC are fulfilled.

Article 54 EPC; novelty

11. Lack of novelty was the reason for refusal of the then pending main request (granted claims, see I above) by the opposition division. Claim 1, which was directed to a process for producing a recombinant host cell that produced high levels of a desired polypeptide, was found to lack novelty over the teachings of document (17) which related to S.cerevisiae. Present claim 1 (and dependent claims thereof) is limited to a process to be carried out in bacteria (see section VII supra). Document (17) is no longer relevant to novelty and as there are no other prior art documents on file disclosing a process such as that now claimed, the requirements of Article 54 EPC are fulfilled.

Article 56 EPC; inventive step

12. Documents (3) or (17) were referred to as the closest prior art. The former is concerned with large scale production of a polypeptide in E.coli, the latter deals with the isolation and characterisation of mutants which show an oversecretion phenotype in S.cerevisiae.

13. In accordance with the case law (T 606/89 of 18 September 1990), the closest prior art for assessing inventive step is normally a prior art document disclosing subject-matter conceived for the same purpose or aiming at the same objective as the claimed invention and having the most relevant technical features in common.

14. The purpose of the present invention is to produce high levels of a desired polypeptide in bacteria. Therefore, the closest prior art is document (3). This document teaches that overproduction of a desired peptide is most often achieved by expressing the corresponding gene in host cells from multicopy plasmids. The poor reproducibility of the system is pointed out (page 501, left-hand column) and the authors suggest a different method involving the integration of the gene of interest into the chromosome downstream from a strong inducible promoter. The general procedure for integration, which is mentioned in the paragraph bridging pages 503 and 504, requires that the gene of interest be flanked by sequences homologous to the host gene which is naturally expressed from a strong promoter. After transformation of the host cells, the integration at the gene locus on the chromosome is said to occur by homologous recombination downstream from the ribosome-binding sequence, namely downstream from the strong promoter. The results obtained with the recA gene as a model case for large scale production of a polypeptide (recA protein) show that, while somewhat lower than that obtained by expression from multicopy plasmids, the yield of recA protein obtained by expression from the chromosome is much more reproducible (passage bridging pages 501 and 502). There is no hint in document (3) that the method therein described is in need of any improvement.

15. Starting from the closest prior art, the problem to be solved may be defined as providing another method for producing a polypeptide at high levels in bacterial cells.

16. The solution provided is a method in which the step of integration into the chromosome by means of homologous recombination on the downstream side of an endogenous promoter is followed by a mutagenesis step coupled to the selection of highly producing clones.

17. In the absence of any suggestion in document (3) that the method proposed therein is not fully satisfactory, the skilled person wanting to solve the above mentioned problem would most probably follow the provided teaching that strong promoters were the answer. Thus, the solution immediately coming to mind would be to try and isolate constructs which would be expected to integrate downstream from any other E.coli promoters known to be strong. Unless associated with unexpected effects, this course of action would be obvious.

18. However, that was not the solution chosen by the appellant. Instead, a step of mutagenesis was added and a high level of expression was achieved. At the priority date, mutagenesis was only one amongst the many methods which had already been tried for altering the expression level of a bacterial gene. As already mentioned, one of the "preferred ones" was using multicopy plasmids, but one could also try to achieve gene duplication in the chromosome (document (1)), to adapt codon usage etc... Thus, in the board's judgment, the present method which, in fact, consists of the combination of, firstly, putting oneself in the unfavourable position of expressing only one copy of the gene - albeit possibly strongly - and, secondly, "correcting" the level of expression obtained by altering the genetic background of the host cells rather than manipulating the gene of interest, was unexpected.

19. In this context, reference was made to document (23) as evidence that mutagenesis was a favoured approach for improving gene expression (page 349, left-hand column). However, this document is a review of the expression of foreign genes in the methylotrophic yeast Pichia pastoris. For the board, such a document could only be found and its teachings combined with those of document (3) with hindsight knowledge of the invention. And, besides, as already mentioned, inventive step does not reside in any one of the steps of the claimed method taken separately but in their combination.

20. The claimed subject-matter was also argued to be obvious in the light of the teachings of document (17) combined with those of document (1). Document (17) is a research article on the possible genetic determinants of the oversecretion phenotype in S.cerevisiae. Two kinds of mutants are isolated which oversecrete a protein encoded by a foreign gene integrated by homologous recombination into the yeast chromosome downstream from a strong promoter. The purpose of the study is to investigate their genetic identity: one of them is found to be altered in the secretory pathway whereas the other exhibits an increased transcription level.

21. In the board's judgment, the skilled person would have no reason to consider document (17) when wanting to achieve overproduction of a protein in a bacterial host. And the information in document (1) - which was relied upon by respondent I in combination with document (17) - that bacteria are industrial microorganisms does not change anything in this respect.

22. In this context, reference was also made to the earlier decision T 455/91 (supra). In this case, vectors suitable for expressing any exogenous gene in yeast cells were claimed, which comprised genetic elements corresponding to those of vectors as were known in the bacterial art for the same purpose. The then competent board concluded that a skilled person working in one area of genetic engineering would regard a means found possible in a neighbouring area of genetic engineering as being useable in his own area, if such a transfer of technical knowledge appeared to be easy and to involve no obvious risks and, on that basis, inventive step was denied. That situation, however, is quite different from the present insofar as the then relevant bacterial art did address the same problem as the then patent in suit and, thus, "came to mind" when trying to solve the technical problem. As this is not presently the case - see paragraph 20 supra-, the findings of T 455/91 (supra) do not apply.

23. Finally, the argument was presented that the patent in suit did not provide any evidence that the problem mentioned in point 15, supra had been solved because the examples given were not suitable to illustrate the claimed solution. There existed the possibility that in the heterologous polynucleotide segment which was exemplified, homologous recombination took place within the 140bp part of the flanking sequence which contained a promoter. In other words, the gene of interest would not necessarily be under the transcriptional control of the endogenous promoter by virtue of being integrated on the downstream side from the promoter. However, it was not denied that the relevant flanking sequence contained much more DNA than the 140bp fragment nor that the rest of this DNA was homologous to the gene sequence downstream from the promoter on the chromosome and, thus, may equally participate in homologous recombination. Accordingly, and in the absence of any evidence that homologous recombination would only take place in the 140 base pairs fragment, it must be concluded that the argument is not adequately substantiated.

24. For these reasons, inventive step is acknowledged.

Article 83 EPC; sufficiency of disclosure

25. At oral proceedings, the fact that the techniques needed to put the claimed method into practice, namely homologous recombination and mutagenesis, were routine techniques in the bacterial field was not challenged.

26. The first argument which was raised against sufficiency of disclosure was that the scope of claim 1 was much too wide and, thus, unwarranted, taking into account the scant technical teaching provided. In this context, the findings in the earlier decision T 19/90 (supra, point 3.3 of the decision) are particularly relevant:

"However, the mere fact that a claim is broad is not in itself a ground for considering the application as not complying with the requirement for sufficient disclosure under Article 83 EPC. Only if there are serious doubts, substantiated by verifiable facts, may an application be objected to for lack of sufficient disclosure."

27. Although it was suggested that it would be inevitable that the invention could not be reproduced in at least some embodiments, no serious doubts were raised and no verifiable facts were produced, and , accordingly, the argument is not found convincing.

28. Decision T 694/92 (supra) was also referred to, wherein sufficiency of disclosure was denied to a method for genetically modifying a plant cell by transferring into it a T-DNA comprising a plant promoter and a plant structural gene so that the protein encoded by the plant gene would be expressed. In this earlier case, it was known from the art that no previous attempts to transfer genes into plant cells had resulted in the expression of these genes. The method itself involved a new approach and the expression of only one plant gene - encoding phaseolin - was observed but at barely detectable levels. The then competent board reasoned that the technical teaching provided was not sufficient for the skilled person to reliably achieve without undue burden the technical effect of expression in any plant cell of any plant gene under the control of any promoter. The facts of that case are, however, clearly not comparable to those of the present case and, thus, the conclusion also has no bearing on the present case.

29. Yet another argument was that the skilled person was given no technical means to find whether or not he/she was reproducing the invention, a situation which, in accordance with the case law (T 256/87, supra) amounted to the requirement of Article 83 EPC not being fulfilled. This argument was illustrated at oral proceedings by a diagram depicting what would happen if integration occurred into the chromosome by means of one cross-over with the flanking sequence situated downstream from the gene of interest in the heterologous polynucleotide segment - ie. the sequence carrying the transcription terminator. From this diagram, it was deduced that once integrated into the chromosome, the gene of interest would not be expressed from the endogenous promoter yet would nonetheless be expressed. In the respondent's opinion, this implied that the depicted event could not be distinguished from that which the claimed process involved.

30. In the board's judgment, sketching a possible recombination event on a flipchart, without providing any experimental evidence that if it occurred in vivo and was followed by a step of mutagenesis, it would result in bacterial cells such as obtained by the claimed process - producing high levels of a desired polypeptide in a stable manner- illustrates no more than an assumption that it would. Mere assumptions are not adequate evidence for a conclusion of lack of sufficient disclosure. Accordingly, the findings in decision T 256/87 (supra) are not relevant to the present case.

31. For these reasons, and in the absence of any evidence to the contrary, sufficiency of disclosure is acknowledged.

Further matter:

32. In addition to the method claim 1 and its dependent claims 2 to 23, the claim request comprises claims 24 to 29 which relate to specific, deposited recombinant E.coli host cells as defined by ATCC deposit numbers. These six claims are those which were accepted by the opposition division. The respondents did not appeal against the decision of the opposition division. Therefore, these claims are not for consideration by the board (Enlarged Board of Appeal decision G 4/93 OJ EPO 1994, 875).