T 0500/12 (Aminoglycoside antibiotic stop codon modulator suppressor/MAXYGEN) 24-04-2015

I. European patent number 1 716 233 was maintained by the opposition division in amended form in accordance with an Auxiliary Request 2. The opposition division considered the Main Request and Auxiliary Request 1 not to fulfil the requirements of Article 54 EPC. All requests were filed at oral proceedings before the opposition division on 1 November 2011.

II. An appeal was lodged by the opponent (appellant). In the statement setting out its Grounds of Appeal, the sole argument put forward by the appellant concerned a lack of inventive step of the request upheld by the opposition division (Article 56 EPC). As an auxiliary measure, oral proceedings were requested.

III. In reply to appellant's Grounds of Appeal, the respondents (patentees) referred only to their arguments submitted with a letter dated 28 February 2011 in reply to opponent's Notice of opposition. A copy of this letter was enclosed to the respondents' submissions. The respondents requested oral proceedings as an auxiliary measure.

IV. In a letter dated 6 May 2014, the respondents informed the board of their intention not to take further action in relation to the appeal proceedings and not to attend the payment of the national renewal fees for the patent. The request for oral proceedings was withdrawn.

V. In a communication issued on 30 June 2014, the board, with reference to Rule 84 (1) EPC, sought to clarify the appellant's intentions as regards the continuation of the appeal proceedings. The board informed the respondents that the appeal proceedings could not be terminated by informing the EPO that the opposed patent was surrendered.

VI. The appellant requested to continue the appeal proceedings. The respondents did not reply to the board's communication and did not file any submissions regarding appellant's request to continue the appeal proceedings.

VII. The board issued a communication pursuant to Rule 100(2) EPC informing the parties that appellant's Request to continue the appeal proceedings was granted (cf. "Case Law of the Boards of Appeal of the EPO", 7th edition 2013, IV.C.6.2, page 849). The board further noted the following:

i) the sole issue discussed in appellant's statement of Grounds of Appeal concerned Article 56 EPC (cf. point II supra);

ii) in its reply to appellant's Grounds of Appeal, the respondents did not address any of the appellant's arguments but referred only to their submissions made at an early stage of the first instance procedure (cf. point III supra);

iii) in the board's preliminary, non-binding opinion, the combination of documents D1 and D3 (infra) made obvious the subject-matter of the claims of Auxiliary request 2, upheld by the opposition division (Article 56 EPC). Therefore, the board intended to set aside the decision under appeal and to revoke the patent.

VIII. None of the parties replied to the communication of the board.

IX. Auxiliary Request 2, upheld by the opposition division, contained 11 claims. Independent claims 1-3, 5 and 9 were directed to five different methods. Claim 1 read as follows:

"1. A method for screening or selecting cells expressing a desired level of a polypeptide, comprising:

a) providing a plurality of eukaryotic cells each comprising an expression cassette comprising a first polynucleotide encoding the polypeptide, at least one stop codon downstream of the first polynucleotide, and a second polynucleotide encoding a cell membrane anchoring peptide downstream of the stop codon;

b) cultivating the cells in the presence of a termination suppression agent under conditions that allow expression of the polypeptide, wherein the termination suppression agent is an aminoglycoside antibiotic; and

c) using FACS to select at least one cell expressing the polypeptide fused to a cell membrane anchoring peptide."

Claims 2 and 3 were directed to a method for evaluating recombinant polypeptide expression in a population of cells and to a method for screening or selecting at least one cell expressing a polypeptide with a desired binding affinity to a ligand from cells expressing a library of polypeptide variants. Both claims comprised steps a), b) and c) similar to steps a), b) and c) of claim 1.

Claims 5 and 9 were directed to a method for alternately expressing i) a soluble, untagged polypeptide or ii) a membrane-bound polypeptide from a single cell or cell line; and to a method for alternately expressing i) a membrane-bound, untagged polypeptide or ii) a membrane-bound, tagged polypeptide from a single cell or cell line. Both claims comprised steps a), b), c), similar to steps a), b) and c) of claim 1, and an additional step d) in which the selected cell was cultivated in the absence of a termination suppression agent to obtain expression of the desired polypeptide.

X. The following documents are referred to in this decision:

D1: WO 03/014361 (publication date: 20 February 2003);

D3: M. Manuvakhova et al., RNA, 2000, Vol.6, pages 1044 to 1055.

XI. The submissions of the appellant, insofar as they are relevant to the present decision, may be summarized as follows:

Article 100(a) EPC; Article 56 EPC

The closest prior art document D1 disclosed a method for screening/selecting cell clones expressing a high level of a desired polypeptide by using an expression cassette that was based on stop codon read-through. In this cassette, the gene coding for the polypeptide of interest and a selectable marker were translationally linked so that a stop codon read-through resulted in the production of a fusion protein. In that way, host cells comprising the expression cassette were selected by positive selection on expression of the fusion protein. Approximately 0.1% of the polypeptide was naturally expressed as fusion protein (natural rate of stop codon read-through) and the rest was expressed as a polypeptide without the fusion partner. The amount of fusion protein could be modulated by using stop codon suppressor mechanisms (modulation of read-through efficiency). The use of the stop codon setting provided a strict dependence of the expression of the selectable marker gene (which enabled a positive selection) on the expression of the gene of interest. Document D1 disclosed drug resistance genes as selectable markers but, as possible alternative markers, referred to reporter genes, such as the Green Fluorescence Protein (GFP). Cells were then selected by identifying these cells expressing the reporter gene, e.g. Fluorescence Activated Cell Sorting (FACS) when using GFP. In Example 1 of document D1, GFP was not expressed as a selectable marker but as a protein of interest. However, this disclosure showed that GFP expressing cells could be selected by FACS as commonly known in the art.

For selecting high producer cells expressing secreted proteins, the expression cassette disclosed in document D1 comprised a further element ensuring that the selectable marker remained inside the host cell. This additional element, namely a stop transfer sequence (transmembrane anchor, membrane spanning domain, "M"), was located downstream of the stop codon and upstream of the selectable marker. In the transmembrane expression vector disclosed in Example 7 of document D1 (pSEAPstopMneo), the selectable marker neo was located intracellularly when the expressed polypeptide SEAP was displayed on the cell surface. Selection was performed by adding the aminoglycoside antibiotic G418 which at high concentration functions as selection agent.

The use of the word "comprising" for defining the elements of the expression cassette in claim 1 allowed the presence of additional elements downstream of the membrane anchor. Indeed, the opposed patent explicitly disclosed the presence of a reporter gene arranged downstream of the membrane anchor gene. Thus, document D1 disclosed all steps of the method of claim 1 except the use of an aminoglycoside antibiotic to increase stop codon read-through, which at low concentrations functions as stop codon suppressor. However, document D1 taught other stop codon suppression options.

Starting from document D1, the objective technical problem was the provision of a FACS based screening/sorting method where the expression of the membrane anchored variant was conditionally increased. The solution presented by claim 1, namely the use of an aminoglycoside antibiotic as a termination suppression agent (instead of the genetic elements disclosed in document D1) to conditionally increase stop codon suppression, was obvious for a skilled person.

A person skilled in the art would not have been limited to the stop codon suppression options mentioned in document D1. It was well-known in the prior art, as shown by document D3, that the extent of stop codon read-through achieved depended on the concentration of the aminoglycoside antibiotic in the culture medium. Indeed, document D3 analyzed the efficiency of different aminoglycoside antibiotics to induce stop codon read-through by testing several antibiotics in a series of read-through constructs. Document D3 taught that the level of stop codon read-through could be varied depending on the type and concentration of the termination suppression agent used (dose-response curves). Moreover, cells did not die if a low concentration of aminoglycoside antibiotic was used. Therefore, claim 1 was obvious in the light of document D1 in combination with document D3.

XII. The respondents did not file any submissions in reply to the appellant's arguments, but referred only to their submissions filed in reply to the Notice of Opposition (cf. points III and VIII supra).

XIII. The appellant (opponent) requested that the decision under appeal be set aside and that the patent be revoked.

XIV. In their reply to appellant's Grounds of Appeal, the respondents (patentees) requested that the appeal be dismissed. The request for oral proceedings was withdrawn.