T 0192/06 (FokI endonuclease/JOHNS-HOPKINS) 06-03-2007

I. European patent no. 0 682 699 was granted on the basis of European patent application No. 94 909 526.9 (published as international application WO 94/18313) and was opposed on the grounds of Article 100(a) EPC. The patent was revoked by the opposition division for lack of inventive step (Article 56 EPC).

II. The patentee (appellant) lodged an appeal and filed the statements setting out the grounds of appeal. Three new documents were filed, among them Kim et al., 1996 (document A, Section IX infra).

III. The opponent (respondent) replied to the appellant's statements of grounds of appeal and filed five new documents.

IV. With the summons to the oral proceedings, the board sent a communication to the parties under Article 11(1) of the Rules of Procedure of the Boards of Appeal (RPBA). The parties were informed therein of the board's preliminary opinion on the relevant issues.

V. With letter of 6 February 2007, the appellant replied to the communication of the board and filed the main request (claims as granted) and auxiliary requests 1 to 10. Three expert declarations and three further documents were filed, among them Kim et al., 1994 (document B, Section IX infra).

VI. With letter of 2 March 2007, the respondent replied to the communication of the board, thereby filing three additional post-published documents.

VII. Oral proceedings took place on 6 March 2007. At the end of these oral proceedings, the appellant withdrew all auxiliary requests on file, including two auxiliary requests filed during the oral proceedings (auxiliary requests 4a and 10a), and maintained the main request as its sole request.

VIII. The claims of the sole request before the board were those as granted and corresponded to those underlying the decision under appeal as well. Claim 1 read as follows:

"1. A DNA construct comprising:

(i) a first DNA segment encoding the catalytic domain of a Type IIS endonuclease which contains the cleavage activity of said Type IIS endonuclease;

(ii) a second DNA segment encoding a sequence-specific recognition domain other than the recognition domain of said Type IIS endonuclease;

(iii) a third DNA segment comprising one or more codons, wherein said third DNA segment is inserted between said first DNA segment and said second DNA segment; and

(iv) a vector

wherein said first DNA segment, said second DNA segment and said third DNA segment are operably linked to said vector so that a single protein is produced."

Claims 2 to 7 related to further embodiments of claim 1. Claims 8 to 10 were directed to prokaryotic cells comprising the DNA construct of claim 1. Claim 11 related to an isolated hybrid Type IIS endonuclease produced by the prokaryotic cell of claim 8.

IX. The following documents are cited in the present decision:

D1: L. Li et al., Proc. Natl. Acad. Sci. USA, May 1992, Vol. 89, pages 4275 to 4279;

D2: EP-B-0 318 554 (publication date of the application: 7 June 1989);

A: Y-G. Kim et al., Proc. Natl. Acad. Sci. USA, February 1996, Vol. 93, pages 1156 to 1160;

B: Y-G. Kim et al., J. Biol. Chem., 16 December 1994, Vol. 269(50), pages 31978 to 31982.

X. The appellant's arguments may be summarized as follows:

Article 56 EPC

Document D1, the closest prior art, disclosed two separate functional domains in the Type IIS restriction endonuclease FokI. Digestion of the FokI enzyme by trypsin resulted in a 41 kDa amino terminal fragment and a 25 kDa carboxy terminal fragment which comprised, respectively, the FokI sequence-specific recognition domain (DNA binding domain) and the FokI cleavage (catalytic) domain. The isolated FokI catalytic domain cleaved, non-specifically, both methylated and non-methylated DNA substrates in presence of MgCl2. In order for site-specific cleavage to occur, a cleavage signal had to be transmitted from the DNA binding domain to the catalytic domain, probably through allosteric interactions. The presence of these interactions was confirmed by further experimental evidence, in particular by the different patterns of trypsin digestion of the FokI enzyme obtained with and without a DNA substrate. Document D1 further speculated that it could be feasible to construct hybrid endonucleases of different sequence specificity by linking other DNA binding domains to the FokI cleavage domain. Although the level of knowledge of the authors of document D1 was clearly higher than the one of the average person skilled in the art, they were by no means sure that a functional hybrid endonuclease could be constructed. Moreover, no guidance was provided of how the "linking" of other DNA binding domains to the FokI cleavage domain could be effected, particularly in a way that the allosteric regulation of the specific cleavage activity of the wild-type FokI was retained.

The patent in suit went far beyond what was disclosed in document D1 and described the insertion of linkers between the catalytic domain and the sequence-specific recognition domain of the Type IIS endonuclease FokI. The resulting recombinant FokI endonuclease was functionally active and cleaved at positions shifted from the recognition site of the wild-type FokI enzyme. Thus, the patent in suit provided further technical support for the construction of the claimed hybrid Type IIS restriction endonucleases.

Starting from this prior art, the technical problem to be solved was the provision of a functional hybrid Type IIS endonuclease with different specificity. Whereas the term "functional" required sequence-specific DNA recognition in combination with the cleavage activity of a Type IIS endonuclease (a regulated site-specific DNA cleavage), the term "different specificity" required cleavage at a site different from the cleavage site of the wild-type enzyme.

The patent in suit showed that the insertion of four or seven codons between the sequence-specific recognition and the cleavage domains of the FokI endonuclease destabilized this enzyme, which however remained functionally active. Similar studies were disclosed in the post-published document B, which reported the construction of several insertions between the DNA binding domain and the catalytic domain of the FokI endonuclease. All tested FokI insertion mutants retained their function and it was concluded that large insertions did not disrupt the activity of the enzyme. Further post-published evidence demonstrated that hybrid Type IIS endonucleases with linkers of different length and nature (with or without secondary structure) were also capable of sequence-specific cleavage of substrate DNA. There was no evidence on file showing the contrary. Thus, the technical problem was solved for the whole range of the subject-matter covered by the claims.

This solution was not rendered obvious by document D1 alone or in combination with other prior art. Document D1 referred to the presence of allosteric interactions between the two domains of the FokI endonuclease and showed that trypsin digestion of this enzyme gave different results in presence and in absence of a DNA substrate. Thus, the binding to a DNA substrate caused major structural alterations showing that regulated site-specific cleavage of DNA substrates indeed required a functional interaction between the DNA binding domain and the cleavage domain. The skilled person would have deduced therefrom that site-specific cleavage at a predetermined position from the recognition site could only have occurred if a signal was successfully transmitted from the DNA binding domain to the catalytic domain. This deduction was further supported by the observation that the isolated FokI catalytic domain had completely lost its specificity. Thus, the DNA binding domain not only was capable of suppressing the non-specifically cleavage activity of the FokI catalytic domain in presence of a DNA substrate (with recognition sites) but it was also capable of blocking the cleavage activity of the FokI catalytic domain in presence of a non-substrate DNA (without recognition sites). The effect of inserting a peptide linker (for which no guidance was given in document D1) and a heterologous DNA binding domain on these allosteric and suppressing interactions was not obvious at all. In fact, the skilled person would have understood that massive obstacles were to be overcome since those structural alterations could negatively affect any allosteric signal transmission between the sequence-specific recognition domain and the cleavage domain as well as the structural interrelationship between these domains. In the absence of this information, the reference to hybrid endonuclease in document D1 was nothing more than a mere speculation. Although the skilled person could have followed this speculation, massive obstacles had to be faced and, without any technical guidance, there was no actual motivation to follow it.

This technical guidance or information could not have been derived from other prior art, which referred to fusion proteins substantially different from the claimed hybrid Type IIS endonucleases. In particular, document D2 disclosed fusion proteins of an antigen-binding site domain and an effector domain connected by a peptide linker which preserved the functions of the individual domains and, contrary to the patent in suit, avoided any allosteric interaction between the two domains. Thus, by using a peptide linker for avoiding allosteric interactions, document D2 even taught away from the solution proposed by the patent in suit.

In the light of this prior art and of the disclosure of document D1, there was no reason to expect that the presence of a heterologous DNA binding domain and a peptide linker would modify the unregulated and non-specific DNA cleavage activity of the isolated FokI catalytic domain. It could not be expected that a hybrid Type IIS endonuclease would be functional and exhibit a sequence-specific and a cleavage activity different from the wild-type enzyme. This was supported by the expert declarations on file that reflected the opinion of the scientific community at the priority date of the patent in suit. These declarations also showed that the skilled person would have expected to encounter real technical difficulties when preparing a functional hybrid type IIS endonuclease. A hope to succeed was not be confused with a reasonable expectation of success.

XI. The respondent's arguments may be summarized as follows:

Article 56 EPC

Document D1, the closest prior, identified two distinct functional domains of the FokI endonuclease, namely a 41 kDa DNA binding domain and a 25 kDa cleavage domain (positions 1-382 and 383-578, respectively, of the FokI sequence, as derived from their N-terminal sequence disclosed in Table 1). The construction of hybrid endonucleases by linking the catalytic domain of the FokI endonuclease with a heterologous binding domain was explicitly suggested in this document and several known binding domains were explicitly indicated therein. In the context of document D1, the term "linking" was clearly understood as providing a peptide linker (a DNA coding therefor) between the two identified domains.

Starting from this prior art, the objective problem to be solved was the provision of alternative functional hybrid Type IIS endonucleases. The solution proposed by the patent in suit, namely the introduction of a linker between a catalytic domain of a Type IIS endonuclease and a heterologous sequence-specific recognition domain, was obvious to the skilled person.

Document D1 already provided all the information required for solving this technical problem, namely the identification of the distinct domains of the FokI endonuclease and the demonstration that the isolated domains were functionally active. The construction of hybrid endonucleases by linking a heterologous sequence-specific recognition domain to the Type IIS endonuclease catalytic domain was explicitly suggested by this document and the term "linking" was clearly understood by the skilled person. Different linker peptides and criteria for selecting them were well-known in the prior art as shown inter alia in document D2. Moreover, document D1 itself provided sufficient information for selecting a suitable linker, in particular, the length (maintain possible allosteric interactions) and structure (retain the original binding to DNA substrate at one face of the DNA helix and the cleavage at another point on the next helical turn). No inventive contribution could be seen in the selection of the length and/or nature of the linker. Furthermore, since none of these features was defined in the claims, none of them could be considered as an essential feature for solving the technical problem. On the other hand, there was no evidence on file showing that all possible (long, arbitrary) linkers actually solved this problem. Therefore, no inventive skill was required to follow the indications of document D1 and achieve thereby the claimed hybrid endonucleases.

Although document D1 referred to the presence of possible allosteric interactions between the two domains of the FokI endonuclease and showed the non-specific cleavage activity of the isolated FokI catalytic domain, the skilled person was not dissuaded from following the suggestion made in this document and thereby construct the proposed hybrid endonucleases. In fact, the allosteric interactions were referred to at the beginning of document D1 only as a possible mechanism of action of the FokI endonucleases. However, a cleavage-specificity linked solely to the binding of the sequence-specific recognition domain to the DNA substrate (DNA binding specificity) was also indicated as another possible model at the end of document D1. Document D1 explicitly indicated other known sequence-specific recognition domains with no structural similarity to the FokI DNA binding domain for the construction of the hybrid endonucleases. There was no reason for the skilled person to expect the first model to be more valid or true than the second one. This was all the more so, since there was no evidence supporting the first model and the document provided a clear suggestion for the skilled person to follow based on the second one. In this case, the skilled person would have expected a heterologous DNA binding domain linked to the FokI catalytic domain to redirect the non-specific cleavage activity of the isolated FokI catalytic domain to a sequence-specific cleavage. Since all elements and the required information were known, there was no reason for the skilled person not to follow the explicit suggestion made in document D1. This was in fact what was exactly done in the post-published documents on file, which further demonstrated that no technical difficulties were actually encountered when following this suggestion.

If, starting from document D1 as the closest prior art, the technical problem to be solved was defined as the provision of alternative functional hybrid Type IIS endonuclease having a different cleavage specificity than the wild-type FokI endonuclease (cleavage at 9/13 nucleotides away from the sequence-specific recognition site), then the claimed subject-matter did not solve this technical problem. From the patent in suit it could be derived that the insertion site of the linker as well as the length and nature of the linker were essential features for solving the problem. The absence of these features in the claims implied that not all claimed subject-matter solved the technical problem.

XII. The appellant (patentee) requested that the decision under appeal be set aside and that the patent be maintained as granted, all other pending requests having been withdrawn.

XIII. The respondent (opponent) requested that the appeal be dismissed.