T 0455/91 (Expression in yeast) 20-06-1994

Reasons for the Decision

1. The appeal is admissible.

2. Formal admissibility [Article 123 (2) (3) EPC]

As a result of the amendments the subject-matter of the claims of all requests is more narrowly defined than it was in the claims as granted. Consequently, the extent of protection conferred by the claims is reduced in comparison with that conferred by the claims as granted. Thus, the requirements of Article 123 (3) EPC are met.

Also the requirements of Article 123(2) EPC are met because the said amendments find support in the original application documents. In particular:

i) the application as originally filed explicitly referred in general terms to production of a "biocompetent polypeptide" (see page 4, third paragraph). The reference to biocompetent polypeptides "other than those required for growth of the transformant" in the original application documents is to be regarded as a particular embodiment. Thus, in the Board's view, the omission of the latter expression from the independent claims does not constitute a violation of Article 123(2) EPC.

ii) As for the introduction in the claims of the expression "which flanking sequence also contains a deletion into the 5' untranslated leader sequence of said gene", the Board observes that, although the reference to the leader sequence and to deletions in the said region is found in the original application documents only in connection with the specific example of the alcohol dehydrogenase (ADH) gene, nevertheless it is unambiguously derivable from the whole context that the exemplified teaching is meant to be generally applicable (see, for example, page 24, fourth paragraph and page 25, fourth paragraph). Thus, the amendment cannot be considered to result in subject-matter which extends beyond the content of the application as filed.

iii) As for the use of the expression "provided by" in the auxiliary request II and III, the Board is of the opinion that it finds full support in the application as originally filed since this made clear that transcription terminations were supplied in the form of 3' flanking sequences of yeast genes (see page 18, sixth paragraph and page 25, second paragraph). In original claim 2 the word "provision" is found.

3. Clarity (Article 84 EPC)

In the Board's opinion, the clarity requirement of Article 84 EPC is met by all newly filed claims of all requests. The expressions objected to by Respondent III do not leave doubts as to the category and the scope of the claims. In the whole context of the claims, the said expressions are used to specify the position or the origin of a structural feature. Nor do said features render the claims obscure from a technical point of view.

4. Novelty (Article 54 EPC)

4.1. The closest prior art

The oral disclosure by Dr Guarente at the 10th International Conference of Yeast Genetics and Molecular Biology held in Louvain-La-Neuve on September 8 to 12, 1980 (24') represents the closest prior art.

The evidence available indicates that at the poster presentation and during two workshops at the said conference Dr Guarente illustrated his studies on the characterisation of the Cyc1 (iso-1-cytochrome c) promoter and on the use therefor of the lacZ' gene. The aim of these studies was the elucidation of the function of the said promoter. Dr Guarente disclosed the construction of plasmids which contained fusions of the E.coli lacZ' gene to a region flanking Cyc1 to the 5' side and which carried selectable markers and origins of replication for both E.coli and S.cerevisiae, in particular the construction of the plasmid corresponding to pLG669-Z shown in Figure 1 of the later publication (10). He reported that both E.coli and S.cerevisiae cells transformed with the said plasmids were capable of expressing ß-galactosidase under the Cyc1 promoter. The lacZ' gene represented in the said constructs the exogenous DNA sequence.

Plasmid pLG669-Z (plasmid with the inserted exogenous DNA sequence = "loaded vector") - as he disclosed - was constructed from plasmid pLG669 (plasmid before the insertion of the exogenous DNA sequence = "empty vector") by inserting into the Bam HI site the lacZ' coding sequence. This precursor plasmid pLG669 comprised the following structural elements: i) a yeast replicon; ii) a 5' flanking sequence of a yeast structural gene including its promoter (and the first four nucleotides ATGA of the Cyc1 gene, i.e. the start codon and one additional nucleotide); iii) a site downstream thereof for inserting a gene (Bam HI site); iv) a yeast marker; v) a bacterial marker and vi) a bacterial origin of replication.

Dr Guarente described also certain deletions in the said 5' flanking sequence, in particular the deletion of the XhoI(-700)-XhoI(-250) fragment. The latter deletion resulted in the reduction of expression in yeast, but not in E.coli. Thereby the probable location of the Cyc1 promoter region within the 1100 nucleotide region which preceded the CYC1 structural gene in the yeast chromosome was identified. Moreover, the location of the Hogness box (ca. 120 nucleotides upstream of the start of the Cyc1 coding sequence) was also identified.

In the course of the present proceedings there was a general consensus on the overall contents of the oral disclosure by Dr Guarente as depicted above.

4.2. None of the Respondents contested novelty in respect of any of the claims of the new requests vis-à-vis the oral disclosure (24').

It is observed that the gene encoding the lacZ' "reporter" molecule falls under the definition of "a structural gene coding for a biocompetent polypeptide ordinarily exogenous to yeast" in the same meaning as used in the present case.

The vectors according to Claims 1 and 9 as well as the method according to Claim 28 of the main request are novel vis-à-vis the vectors and methods known from (24') because:

- the "empty" vector according to Claim 1 does not contain the ATG codon of the yeast gene which affords the promoter;

- the "loaded" vector of Claims 9 and 28 bears a deletion into the 5' untranslated leader sequence of the yeast gene which affords the promoter.

Thus, no novelty objection applies to the main request.

The independent claims of auxiliary requests I to III all incorporate inter alia the feature of the deletion into the 5' untranslated leader sequence. Thus, there can be no novelty objection also in respect of these requests for the reasons already given.

5. Inventive step (Article 56 EPC)

5.1. Main request

5.1.1. The technical problem

In the light of (24') the problem to be solved by the patent-in-suit can be seen in the construction of alternative yeast expression vectors suitable for expressing in yeast any exogenous gene of choice.

5.1.2. The solution proposed

As a solution thereto, Claim 1 proposes the construct of an "empty" vector which bears downstream of the 5' flanking sequence of a yeast gene which affords the promoter a site for insertion of the exogenous DNA sequence of choice with its own start signal. In this "ready-for-use" vector, the start signal of the said yeast gene is missing. Claim 9, on the other hand, proposes a "loaded" vector bearing a deletion into the 5' untranslated leader sequence of the yeast gene which affords the promoter.

The patent specification gives examples of vector constructs in which a DNA sequence encoding leucocyte interferon D is inserted together with its start signal downstream of the 5' flanking sequence of the yeast ADH gene including the promoter and having deletions extending through the native start signal into the 5' untranslated leader region. Yeast cells transformed with such vector constructs are shown to express a biologically active product (see Table 1).

5.1.3. Assessment of inventive step

5.1.3.1. Dr Guarente provided with his oral disclosure (24') what was perceived at the conference as the first demonstration of expression in yeast of a sequence exogenous thereto under the control of a yeast gene promoter [see in this respect in particular the comments in document (124)].

5.1.3.2. As it is the normal task of the skilled person to be constantly occupied with the elimination of deficiencies, with the overcoming of drawbacks and with the achievement of improvements of known devices and/or products (in this respect see, for example, decisions T 15/81 OJ EPO 1982, 2, point 3 of the Reasons and T 195/84, OJ EPO 1986, 121, point 8.1 of the Reasons), it is realistic to assume that a skilled person aware of the presentation by Dr Guarente (24') would have readily considered the problem of providing alternatives and/or improvements to the disclosed vectors and methods.

5.1.3.3. A number of decisions in the field of biotechnology have already provided a definition of the person skilled in the art for the purpose of Article 56 EPC (see, for example, T 60/89, OJ EPO 1992, 268, see point 2.2.4 of the Reasons; T 500/91 dated 21 October 1992, see point 2.2 of the Reasons and T 223/92 dated 20. July 1993, see point 5.5 of the Reasons, both not published in the OJ EPO).

The Board considers it useful for the purpose of the present decision to make some considerations on what is believed to be the attitude of the said person skilled in the art vis-à-vis possible changes, modifications and/or adjustments in known products (e.g. a plasmid) or procedures (e.g. an experimental protocol). This with a view to provide a possibly objective answer to the question whether or not the introduction of a given change in a structure or in a procedure can be seen as obvious for the skilled person, avoiding any ex post facto analysis.

In the Board's view, the skilled person in this field is well aware of the fact that even a small structural change in a product (e.g. a vector, a protein, a DNA sequence) or in a procedure (e.g. a purification process) can produce dramatic functional changes. Therefore, the said expert would constantly be conditioned by the prior art and, before taking action, would carefully ponder any possible modification, change or adjustment against the background of the existing knowledge. Under these circumstances, in the Board's view, the skilled person would adopt a conservative attitude. However, this must not be seen in the sense of being reluctant or opposed to modify or adjust a known product or process, but rather in the sense of being cautious. For example, the skilled person in question would neither go against an established prejudice nor try to enter into "sacrosanct" or unpredictable areas nor take incalculable risks. However, within the normal design procedures, the said expert would readily seek appropriate, manifest changes, modifications or adjustments which involve little trouble or work and no risks or only calculable risks, especially for the sake of obtaining a more handy or convenient product or of simplifying a procedure. In particular, the skilled person working in one field (e.g. expression in yeast) would regard a means conveniently adopted in a neighbouring field (e.g. the bacterial art) as being readily usable also in that field, if this transfer of technical knowledge involves nothing out of the ordinary.

5.1.3.4. Taking the above considerations into account, the proper question to ask is not whether the skilled person could have tried to modify the technical teaching disclosed in document (24'), but whether he or she would have done so. In seeking an answer to this question it should be borne in mind that the skilled person in the field of expression of polypeptides in yeast had good reasons to move in the direction of the technical teaching of the patent-in-suit, because the skilled person knew how to adjust the technical teaching in (24') from an adjacent neighbouring field, namely the bacterial art. This was a sufficient incentive for an expert at least to try to transform knowledge from the bacterial art to yeast. It is observed that in this respect the expert in the bacterial art and for yeast is the same.

5.1.3.5. Turning now to the solution proposed in Claim 1 of the patent-in-suit, the Board observes that the "empty" vector of this claim differs from the "empty" plasmid disclosed in (24') only in that it does not contain the start signal (ATG) of the yeast gene which affords the promoter. The idea beyond this proposal by the present patent is to provide a "ready-for-use" vector in which a DNA sequence consisting of start signal-exogenous DNA sequence-(optionally) termination signal can be inserted. This approach, however, was already known from the bacterial art [see, for example, document (120), in particular figure 1 (A)]. Nothing in the prior art indicated to the skilled person concerned with the construction of yeast expression vectors that the said approach would not have been usable in yeast. Moreover, for the skilled person it would have made no difference whether the start signal was already in place within the vector or it was introduced together with the exogenous DNA sequence, as long as it was properly positioned within the vector.

In the Board's view, the structural change which makes the difference between the vector of Claim 1 and the "empty" plasmid of (24') is one that a skilled person occupied with the construction of alternative yeast expression vectors would have readily considered. The introduction of such a change into the known plasmid vector required for a skilled person nothing out of the ordinary and thus involved no inventive skill, all being a matter of technical convenience.

5.1.3.6. The "loaded" vector of Claims 9 and 28 differs from the "loaded" plasmid disclosed in (24') in that it contains deletions into the 5' untranslated leader sequence of the yeast gene which affords the promoter. A key question with respect to inventive step of these claims is whether a skilled person, starting from the teaching of (24'), would have readily considered making such deletions.

In this respect the Appellant substantially maintains that the skilled person would not have taken the incalculable risk of carrying out any engineering in the said region because little was known about its possible influence on expression in yeast. Moreover, in its submissions, too little was known about yeast promoters, in particular about their 3' boundaries, to render obvious for the skilled person making deletions in the 5' untranslated leader region. Thus, in the Appellant's opinion, the skilled person regarded this region as "sacrosanct".

The Board observes that in early 1981 a number of yeast genes had been cloned and sequenced and the upstream and downstream sequences therein had been identified [see, for example, document (13A), in particular Figure 25, and document (28)]. Promoter sequences were known to be located in the upper region of the 5' flanking sequences [see documents (13A) and (24')]. The model in which translation started in yeast at the first AUG downstream from the 5' terminus of the mRNA, with no other sequence requirements found wide support and acceptance [see documents (28), (30) and (120)]. In particular, the studies disclosed in document (30) had established that there was no absolute requirement for a particular sequence 5' to the initiation codon and that translation started at the AUG codon closest to the 5' end of the mRNA. The latter document is particularly relevant to the present discussion and deserves further analysis.

5.1.3.7. The studies of document (30) were carried out by performing mutations in the Cyc1 locus of a mutant yeast strain that lacked the normal ATG codon so as to obtain intragenic revertants in which the initiation codon was relocated within the region occupied by codons -3 to 9. It was observed that normal amounts of iso-1-cytochrome c occurred when translation initiated at the sites corresponding to codon positions -3, -2, 3 and 5 as well as the normal position -1 and it was therefore concluded that the initiation translation codon could be located anywhere within a region spanning 37 nucleotides and presumably at any site preceding and following the site of the normal initiation codon.

The Appellant insists that the studies reported in document (30):

- dealt with mutations, not deletions;

- were concerned with the initiation of translation, not with transcription;

- did not report any explicit DNA sequence data.

Thus, in its submissions, the said studies did not allow the conclusion that alterations could readily be introduced into the leader sequence of a yeast gene.

The Board is rather of the view that the skilled person would have readily concluded from document (30) that alterations, at least by way of mutation, in the region just upstream of the start signal (leader region) in the DNA were feasible. This is because:

- the changes in the position of the translation initiation codon reported in document (30) with reference to the mRNA sequences reflected the alterations produced by the mutations at the corresponding site into the DNA. No explicit DNA data were necessary to recognize this;

- the fact that translation had taken place with normal or near-normal efficiencies in the mutated intragenic revertants, must have implied that transcription, which precedes translation in DNA expression, had also occurred rather undisturbed.

Consequently, the Board believes that the skilled person on the basis of the teaching of document (30) would not have regarded the region immediately preceding the start signal of a yeast gene (the 5' untranslated leader region) as being "sacrosanct" or "untouchable" in spite of alleged uncertainties about a possible function of the leader sequence. The fact that the introduction of point mutations in this region had not sensibly changed translation efficiency rather indicated that alterations therein were feasible.

5.1.3.8. It still remains to be established whether a skilled person faced with the problem of providing alternative yeast expression vectors would have considered modifying those known from (24') by carrying out deletions into the 5' untranslated leader sequence of the yeast gene which afforded the promoter.

In respect of this question, it is observed that document (30) expressly indicated that the initiation codon could be located inter alia at a site preceding its normal position (e.g. at the site corresponding to codons -3, -2). This amounted for the skilled person to the teaching that by producing a change of the codons through mutation the start signal could be moved towards the promoter, upstream of its normal position, i.e. into the 5' untranslated leader region. The skilled person knew from the bacterial art [see, for example, document (120), in particular on page 1429, left hand column, the passage referring to Figure 1 (B)] that the distance between the promoter and the ATG start signal could be varied by way of resection with nucleases. From the said prior art the skilled person knew that the distance between the promoter and the start signal could be optimized. Thus, one of the possible modifications into the "loaded" plasmid of (24') which would have readily occurred to the skilled person was the changing of the distance between the start signal of the exogenous gene and the promoter that was known to be located in the upper region of the 5' flanking sequence of the yeast gene (see point 4.1, fourth paragraph, above). In order to perform such modification the skilled person had two possibilities:

- either produce by mutation a change in the codons preceding the natural start signal as taught by document (30); or

- resect or produce deletions into non-essential regions upstream of the said start signal as known from the bacterial art.

Both possibilities required for the skilled person nothing out of the ordinary in the field and involved only routine trials.

Thus, also in view of the considerations made above in point 5.1.3.3, the Board concludes that it would have been obvious for a skilled person to try with a reasonable expectation of success to modify the "loaded" plasmid of (24') by producing one or more deletions into the 5' untranslated leader sequence of the yeast gene which afforded the promoter. In view of the prior art [especially documents (24') and (30)], the skilled person would have regarded this modification as feasible and as involving only fully calculable risks, if any. Thereby the skilled person would have readily arrived at a yeast vector according to Claim 9 and at its use for expressing an exogenous polypeptide in yeast according to Claim 28. This was a matter of normal design procedures for which neither "creative thinking" nor "inventive talent" were necessary.

5.1.3.9. For the above reasons, Claims 1, 9 and 28 lack an inventive step and the main request is consequently not allowable.

5.2. Auxiliary request I

Claims 1 to 30 of this request are essentially the same as Claims 1 to 30 of the main request with the only difference that all claims are formulated as method claims. Claim 1, however, contains the feature "which flanking sequence also contains a deletion into the 5' untranslated leader sequence of said gene" which is absent in Claim 1 of the main request.

In the Appellant's submission this request represents what was done.

In the Board's view, the mere change of product claims into method claims is not enough to confer an inventive step to their subject-matter if the characterising features of the claimed methods are the same which characterise the product. This is precisely the case here. Since it has been concluded above in respect of the main request that the skilled person needed no inventive skill in order to arrive at the claimed "empty" and "loaded" vectors, it must necessarily be concluded that also the methods for preparing them did not require inventive skill. Therefore, auxiliary request I is not allowable for the same reasons given above in respect of the main request.

5.3. Auxiliary requests II and III

5.3.1. The technical problem and its solution

For both of these requests the closest prior art is represented by the oral disclosure of Dr Guarente (24').

The technical problem derivable therefrom is the same as for the main request, namely the construction of alternative yeast expression vectors suitable for expressing in yeast any exogenous gene of choice (see point 5.1.1, above).

As a solution thereto, the two requests provide in Claim 11 the construct of a "loaded" vector and in Claim 1 the construct of an "empty" vector.

As already stated (see section X, above), Claim 1 is identical in both auxiliary requests II and III. Thus, in respect of the "empty" vector the same solution is offered.

Claim 11 differs in the two requests in that the feature related to the presence of "a transcription termination sequence for said DNA downstream of said insertion site" which characterises Claim 11 of auxiliary request II is further specified in Claim 11 of auxiliary request III by the expression "provided by a flanking sequence of a yeast gene".

5.3.2. Assessment of inventive step

Inventive step is discussed here in relation to the solution as represented by the claimed "loaded" vectors of both requests (see Claim 11).

The "loaded" vectors of auxiliary requests II and III differ from the "loaded" vector of the main request (see Claim 9 therein) in that they contain a transcription termination sequence downstream of the inserted exogenous DNA, this sequence being provided by a flanking sequence of a yeast gene in the case of auxiliary request III.

In points 5.1.3.6 to 5.1.3.9 above it was concluded that the "loaded" vector according to claim 9 of the main request did not involve an inventive step. Thus, the relevant question now is whether or not the additional feature of the presence of a termination sequence confers an inventive step to the claimed vectors.

In the Board's view, a "cautious" skilled person occupied - in the light of documents (24') and (30) - with the construction of alternative yeast expression vectors would have given careful consideration to all structural elements believed to be suitable and/or necessary for expression in yeast.

The skilled person knew that sequences at the 3' end of yeast genes beyond the translation stop codon were associated with the termination of RNA transcription [see, for example, document (13A), in particular page 7, Figure 25, page 134, line 10 to 136, line 3, Table V and page 141, last paragraph to page 145, line 13; document (28), in particular Figure 2 and page 759, left hand column, paragraphs 3 and 4; document (52), in particular page 544, right hand column to page 545, left end column, first paragraph and figures 3 and 4; document (61), in particular figure 2b; document (64), in particular page 164, right hand column, last paragraph].

In the Board's opinion, although not much information on the precise structure and mechanism of action of such yeast termination sequences was available in early 1981, the skilled person occupied with the construction of alternative yeast expression vectors would have readily considered the inclusion therein of a reasonably large, discrete fragment comprising a termination sequence (cf. Claim 11 of auxiliary request II), in particular the inclusion of a fragment consisting of the 3' flanking sequence of a yeast gene (cf. Claim 11 of auxiliary request III), downstream of the exogenous DNA sequence. In view of the information available, the skilled person would have expected this structural measure to ensure proper transcription termination, i.e. the functional role normally exerted in yeast. Nothing in the art indicated that such a measure could have prejudiced expression. On the contrary, based on the cautious experimental approach "when in yeast, do like yeast does", as the Respondents put it, the skilled person would have considered such a structural measure as being appropriate for the purpose of achieving effective expression. As DNA sequences comprising termination sequences were known (see above), the construction of yeast expression vectors containing them would have involved for the skilled person routine experimental work comprising only routine trials.

5.3.3. For the above reasons, Claim 11 of both auxiliary requests II and III lacks an inventive step and the said requests are consequently not allowable.

Under these circumstances, it is not necessary to discuss inventive step in relation to the "empty" vector.