T 0960/02 (Transfection/TRANSKARYOTIC THERAPIES) 16-11-2004

I. European patent No. 0 649 464 with the title "Transfection of vertebrate cells e.g. by homologous recombination" was granted with 22 claims for all designated Contracting States on the basis of the European patent application No. 92 924 367.3 published as WO 93/09222. The latter contained ninety-nine claims of which claims 65 and 78 (relevant to the present decision) read as follows:

"65. A method of introducing exogenous DNA into a preselected site of the genome of a primary or a secondary cell of vertebrate (e.g., mammalian) origin, comprising the steps of:

a) transfecting the primary or the secondary cell with a DNA construct comprising exogenous DNA which includes DNA sequences homologous to genomic DNA sequences of the primary or secondary cell, thereby producing transfected primary or secondary cells and

b) maintaining transfected primary or secondary cells under conditions appropriate for homologous recombination between DNA sequences in the DNA construct and genomic DNA to occur;

thereby producing homologously recombinant primary or secondary cells."

"78. A method of targeting exogenous DNA into a preselected site in genomic DNA of a primary or secondary cell of vertebrate origin, comprising the steps of:

a) providing a DNA construct comprising: 1) exogenous DNA selected from the group consisting of:

a) DNA sequences which repair, alter, delete or replace a resident gene in the primary or secondary cell;

b) DNA sequences encoding a product not normally expressed in the primary or secondary cells or not expressed in significant levels in the primary or secondary cells as obtained;

c) DNA sequences which repair, alter, delete or replace a regulatory sequence present in the primary or secondary cells;

d) DNA sequences which encode a regulatory sequence not normally functionally linked to a gene to be expressed in the primary or secondary cells as obtained; and

e) DNA sequences which inactivate or remove a gene or gene portion in the primary or secondary cells;

2) DNA sequences homologous with genomic DNA sequences in the primary or secondary cells; and

3) DNA sequences encoding at least one selectable marker;

b) transfecting primary or secondary cells with the DNA construct provided in (a), thereby producing transfected primary or secondary cells containing the DNA construct provided in (a); and

c) maintaining primary or secondary cells produced in (b) under conditions appropriate for homologous recombination to occur between DNA sequences homologous with genomic DNA sequences and genomic DNA sequences,

thereby producing primary or secondary cells of vertebrate origin having the DNA construct of (a) integrated into genomic DNA of the primary or secondary cells."

Granted claim 14 which is relevant for the present decision read as follows:

"14. A barrier device containing a cell strain obtainable by the process of any one of the preceding claims, wherein the barrier device is made of a material which permits passage of the therapeutic agent (e.g. erythropoietin, insulinotropin or human growth hormone) produced by the cell strain contained therein into the circulation or tissues of a mammal and prevents contact between the immune system of the mammal and the cell strain contained within the barrier device to a sufficient extent to prevent a deleterious immune response by the mammal."

II. Five oppositions were filed under Article 100(a) to (c) EPC. The opposition division revoked the patent for lack of novelty and lack of inventive step of respectively, the main and the auxiliary requests then on file.

III. The appellant (patentee) filed an appeal and submitted a statement of grounds of appeal together with a main request (granted claims) and three auxiliary requests.

IV. Respondents II, III and V (opponents 2, 3 and 5) answered to the statement of grounds of appeal.

V. On 29 June 2004, the board sent a communication pursuant to Article 11(1) of the Rules of Procedure of the Boards of Appeal, indicating its preliminary, non- binding opinion.

VI. With letters dated 14 and 15 October respectively, respondents II and III sent further submissions in answer to this communication. With letters dated 19. October 2004 and 8 September 2004 respectively, respondents I and IV informed the board that they would not take part in the oral proceedings.

VII. With letter dated 15 October 2004, the appellant submitted a new main request and 11 auxiliary requests in replacement of all requests then on file and informed the board that the appellant would not take part nor be represented at oral proceedings, which took place on 16 November 2004.

Claim 15 of the main request and auxiliary requests 1 to 3 read as follows:

"15. A method of introducing exogenous DNA into a preselected site of the genome of a primary or secondary cell of vertebrate (e.g., mammalian) origin, comprising the steps of:

(a) transfecting the cell with a DNA construct comprising exogenous DNA which e.g. encodes a therapeutic product and which includes DNA sequences homologous to genomic DNA sequences of the cell (and which e.g. also encodes a selectable marker), thereby producing transfected primary or secondary cells;

(b) maintaining the transfected cells of step (a) under conditions appropriate for homologous recombination between DNA sequences in the DNA construct and genomic DNA to occur; thereby producing homologously recombinant primary or secondary cells which express an endogenous gene at a higher level than occurs in the corresponding non- transfected cells; and

(c) propagating in vitro the homologously recombinant cells of step (b) to produce a clonal strain of homologously recombinant secondary cells; or

(d) exposing the homologously recombinant cells of step (b) to a selective agent which selects for a selectable marker present in the DNA construct whereby cells that have not properly integrated the selectable marker are killed and cells that have stably integrated the marker can survive and form colonies, followed by screening the colonies to identify homologously recombinant primary or secondary cells, wherein for example the selective marker and selective agent is neo and G418 respectively."

Claim 15 of auxiliary requests 4 to 7 read as follows:

"15. A method of introducing exogenous DNA into a preselected site of the genome of a primary or secondary cell of vertebrate (e.g., mammalian) origin, comprising the steps of:

(a) transfecting the cell with a DNA construct comprising exogenous DNA which is selected from:

(i) sequences which repair, alter, delete or replace a resident gene or regulatory sequence in the primary or secondary cell;

(ii) sequences which encode a regulatory sequence not normally functionally linked to a gene to be expressed in the cell as obtained; and

(iii) sequences which inactivate or remove a gene or gene portion in the cells;

and which also includes DNA sequences homologous to genomic DNA sequences of the cell (and which e.g. also encodes a selectable marker), thereby producing transfected primary or secondary cells;

(b) to (d): [as in claim 15 of the main request]".

Claim 1 of auxiliary request 8 read as follows:

"1. A process for producing a cell strain comprising human transfected primary or secondary somatic cells for delivering a therapeutic product in vivo comprising the steps of:

(a) providing primary or secondary human somatic cells;

(b) transfecting the primary or secondary cells with DNA, which DNA encodes a therapeutic product;

(c) isolating a non-immortalized cell strain from the transfected cells of step (b); and

(d) expanding the cell strain of step (c) in vitro."

Claims 2 to 13, 15 and 16 of this request related to further features of the process of claim 1. Claim 14 related to a barrier device containing a cell strain obtainable by the process of any one of the preceding claims and claim 17 related to a process for producing a therapeutic product in vitro.

Claim 1 of auxiliary request 9 was identical to claim 1 of the eighth auxiliary request but for step (d) which read:

"1. (d) expanding the cell strain of step (c) in vitro, comprising maintaining the cell strain under appropriate conditions for a sufficient time for at least 20 cell doublings to occur."

Claim 1 of auxiliary request 10 read as follows:

"1. Use, in a manufacture of a vehicle or delivery system for delivering a therapeutic product in vivo of a cell strain comprising human transfected primary or secondary somatic cells, said use comprising the steps of:

(a) to (d): [as in claim 1 of the eighth auxiliary request]".

Claim 1 of auxiliary request 11 read as follows:

"1. Use, in a manufacture of a vehicle or delivery system for delivering a therapeutic product in vivo of a cell strain comprising human transfected primary or secondary somatic cells, said use comprising the steps of:

(a) to (d): [as in claim 1 of the ninth auxiliary request]".

VIII. The documents mentioned in the present decision are the following:

(2): Fountain, J.W. et al., Gene, Vol. 68, pages 167 to 172, 1988;

(12): WO-A-91/06667;

(76): First declaration of Dr D. Barnes received on 29 January 2001;

(77): Dusty-Miller, A., Blood, Vol. 76, No. 2, pages 271 to 278, 1990 attached to the declaration of Prof. K. Kurachi dated 11. January 2001;

(98): Declaration of Prof. D. Kipling dated 1. October 2002;

DB2: Cristofalo, V.J. and Stanulis-Praeger, B.M., Advances in Cell Culture, Vol. 2, pages 1 to 83, 1982, Academic Press Inc;

DB4: Goldstein, S., The Journal of Investigative Dermatology, Vol. 73, pages 19 to 23, 1979;

DK3: Holliday, R. et al., Science, Vol. 213, pages 1503 to 1505, 1981.

IX. The appellant's arguments in writing insofar as they are relevant to the present decision may be summarized as follows:

Article 84 EPC

Claim 15; main request, auxiliary requests 1 to 3 From the wording of the claim it would be immediately apparent to the skilled person that the sequence encoding a therapeutic product (part (a) of the claim) was not related to those parts of the constructs that brought about the technical effect now introduced in part (b) of the claim. Therefore, the claim was clear.

Claim 15; auxiliary requests 4 to 7 The claim was limited to aspects of the DNA constructs that might bring about the technical effect (part (b)). The claimed sequences (part (a) of the claim) could clearly result in up regulation of an endogenous gene. Therefore, the claim was clear.

Article 123(2) EPC; Article 84 EPC

Claim 1; auxiliary request 8 It was abundantly clear from page 16, line 33 and also from the many examples given in the application as filed that the claimed process was to be carried out in particular with human cells. For this reason, the requirements of Article 123(2) EPC were fulfilled.

Claim 1; auxiliary request 9 This claim corresponded to claim 1 limited to incorporate the features of claim 13. This limitation rendered clear and unambiguous the requirement of an extended expansion period (step (c)) when producing cells suitable for the delivery of a therapeutic product in vivo.

Claim 1; auxiliary requests 10 and 11 These claims had been reformulated as use claims to explicitly emphasize the need to develop a cell strain beyond an initial production point to a point where there is sufficient cells for delivery of a therapeutic product in vivo. Basis for the amendment could be found in the application as filed for example at page 8, line 16 to page 9, line 5 as well as at page 36, line 17 to page 38 line 19. Production of a delivery system was described in further detail from page 33 onwards.

Article 54 EPC, novelty

Claim 1; auxiliary requests 8 and 9 The limitation of claim 1 of auxiliary requests 8 or 9 to human cell lines imparted novelty to said claims.

Article 56 EPC, inventive step

Claim 1; auxiliary requests 8 and 9 Despite being part of the preamble and, of course, not limiting the claims to the use of those cell strains for delivering a therapeutic product in vivo, the feature "for delivering a therapeutic product in vivo" provided the absolute requirement that cell strains produced according to the processes in claim 1 of auxiliary requests 8 or 9 are suitable for delivering a therapeutic product in vivo. Accordingly, the nature of the product of the process must be relevant to the assessment of inventive step.

Because of the nature of the starting material (primary or secondary human cells), the skilled person would have been of the opinion that the experimental step of expanding or maintaining the cell strain to a point at which it can be ascertained reliably that the appropriate strain has been generated, and in sufficient quantity to deliver a therapeutic product in vivo would be jeopardized in view of the then unpredictable nature of cells having undergone the onset of senescence. Otherwise stated, the onset of senescence would have been seen as a serious barrier to the provision of a cell strain suitable for the delivery of a therapeutic product in vivo. Therefore, the skilled person would not have had a reasonable expectation of success while trying to implement a process as claimed.

In claim 1 of auxiliary request 9, the amount of time for cell expansion was further specified to be that necessary for at least 20 doublings. When added to the time necessary to accomplish steps (a) to (c), this explicit time requirement took the cell strain well beyond the point at which the onset of senescence will have occurred.

X. The respondents' arguments in writing and during oral proceedings insofar as they are relevant to the present decision may be summarized as follows:

Articles 123(2)(3) and 84 EPC

Claim 1; auxiliary request 8

- The application as filed provided isolated examples of transfecting human fibroblasts with a gene encoding a therapeutic product and expressing said product in said cells but a generic disclosure relating to human cells in general was missing.

Furthermore, step (c) of the claimed method was not described in the application as filed and could not be implicitly derived from the fact that the cells were human cells i.e. from the fact that they would not immortalize while cultured in vitro. Indeed, the eventuality that the biopsy providing the primary cells also contained tumorigenic cells i.e. that some cells would be immortalized from the start could not be discarded. Such cells would be transfected just as non- immortalized cells were. Step (c) was thus compulsory to discard immortalized (tumorigenic) transfected human cells, which step was not disclosed in the application as filed.

For both these reasons, the requirements of Article 123(2) EPC were not fulfilled.

- The claimed process found no support in the description as required under Article 84 EPC since the examples concerned with the delivery of the therapeutic product were carried out in mice and not in humans and, thus, there was no evidence that the cell strain would deliver a therapeutic product in vivo.

Claim 1; auxiliary request 9

The appellant had failed to point out a basis in the application as filed for the feature that the cells should be maintained in culture for at least twenty cell doublings. The closest process to that claimed was found in originally filed claim 43 referring to claim 41. Yet, even then, the originally claimed process was one of producing a clonal cell strain (rather than a mixture of transfected cells) starting from secondary cells (rather than from primary or secondary cells). The other alleged basis on page 104 of the application as filed concerned mammalian epithelial cells and did not provide for the claimed generalisation.

Claim 1; auxiliary requests 10 or 11

In accordance with the case law (T 279/93 of 12. December 1996), a use claim had to be construed as a process claim. Claim 1 of both requests thus related to a process for making a vehicle or delivery system comprising using a cell strain comprising transfected cells. This claim was not one of the granted claims and, thus the requirements of Article 123(3) EPC were not fulfilled.

Article 54 EPC; novelty

Claim 1 of auxiliary requests 8 and 9

Document (12) was detrimental to the novelty of claim 1 as it disclosed on pages 12 and 13 the transfection of normal human diploid skin fibroblasts with a plasmid vector comprising the gene encoding dihydrofolate reductase (DHFR) as well as the expansion of the transfected cells. The fact that the DHFR gene was a gene encoding a therapeutic product was known from document (77), page 273 which taught that "protection of normal marrow from methotrexate toxicity might allow higher doses of methotrexate in chemotherapy for cancer".

Article 56 EPC; inventive step

Claim 1 of auxiliary requests 8 and 9

- The closest prior art was document (2) as it disclosed transfection of primary human skin fibroblasts with a plasmid comprising a selective marker (CAT) by electroporation. The same steps were disclosed as steps (a) to (c) in claim 1 of auxiliary requests 8 or 9 and the cell strain was expanded for three weeks after transfection i.e. for about 35 doublings.

The difference with the claimed process was that the latter was directed to transfection with a DNA encoding a therapeutic product.

Starting from document (2), the problem to be solved could be defined as devising an alternative implementation of the transfection method.

The proposed solution was to transfect the human cells with a gene encoding a "therapeutic product". As the nature of the transfected gene would not be expected to affect the transfection process, the above mentioned difference did not impart inventive step.

As for the appellant's argument that inventive step resulted from the fact that for the delivery of a therapeutic product in vivo, an important number of cells had to be available, i.e. from the fact that the necessary number of in vitro doublings would be so high that it would be perceived by the skilled person as incompatible with obtaining cells which would still be normally metabolically active (i.e. not senescent), it was based on the false premise that the feature "for delivering a therapeutic product in vivo" necessarily brought with it a limitation to a high number of available cells. This was not correct as it could readily be understood from the patent itself (page 12, lines 5 to 8).

Even if step (c) of the claimed process was carried out for at least 20 doublings (claim 1 of auxiliary request 9), this did not mean that in the overall time necessary for carrying out (a) to (c), the cells would necessarily have become senescent. It would depend on the type of human cells which were used as well as on the origin of the primary cells. Thus, inventive step could not be justified on the basis that the skilled person would have refrained from using a population of senescent cells for delivery of a therapeutic product in vivo.

XI. The appellant requested in writing that a decision be taken on the written material in the case, that the decision under appeal be set aside and the patent be maintained on the basis, in order of preference, of the main request or auxiliary requests 1 to 11, all filed on 15 October 2004.

The respondents requested that the appeal be dismissed.