T 0919/99 (Neonatal hematopoietic stem cells/PHARMASTEM THERAPEUTICS) 07-04-2003

Admissibility of the appeals

1. The appeal by the patentee meets the requirements of Articles 106 to 108 and Rule 64 EPC and is thus admissible. By virtue of Article 107 EPC, opponents O1 to O5 are thus parties as of right in the appeal proceedings relating to this appeal by the patentee.

2. Opponent O1 has filed an appeal asking that the reasons for revoking the patent be based on "additional and/or different grounds" (see paragraph IV supra). For a party to be adversely affected within the meaning of Article 107 EPC, the opposition division must have refused some request of the party appealing. Here the decision under appeal revoked the patent, thus allowing the opponent's request. Accordingly as no request of the opponent appellant has been refused, its appeal is inadmissible (see for example T 224/96 of 19 December 2001). As respondent to the patentee's appeal it is, of course, open to opponent 01 to argue that those "additional and/or different grounds" are further reasons for dismissing the patentee's appeal, but opponent 01 has no independent right to appeal.

3. Further, opponent O4 has also filed an appeal. According to the file the Notice of Appeal was received on 15 October 1999 and the appeal fee was paid that same day. The time limit, however, expired on 21. September 1999. Thus, the two month time limit from the deemed date of notification of the written decision under appeal laid down by Article 108 EPC was not observed. This entails the consequence that this appeal is deemed not to have been filed, and the appeal fee is to be reimbursed.

Article 123(2) EPC

4. The respondents and the other parties argue that the restriction of claim 1 at issue to human neonatal hematopoietic stem cells, thus excluding from the claim the use of human fetal hematopoietic stem cells infringes Article 123(2) EPC, as the application as filed made the use of human fetal hematopoietic stem cells an essential feature of the invention.

In the board's view, however, fetal stem cells were never described as indispensable for the claimed medical use, but merely as an alternative to blood collected from the umbilical cord and/or placenta (see page 25, lines 5 to 21 of the WO 89/04168 application). Therefore, it is concluded that no objection under Article 123(2) EPC has been made out.

Article 54 EPC (Novelty)

5. As in the patent in suit (see Section 6.7), the author of document (D21) performs in vitro experiments comparing cryopreserved cells from cord blood and BM. Tests are carried out on cord blood and BM for the presence of CFU-GM, BFU-E, CFU-E and CFU-Mix cells both before freezing and after thawing the cells. According to page 276 of document (D21) (under the heading "Preparation of the cell suspensions"), the marrow and cord blood mononuclear cells are isolated by the conventional Ficoll-Hypaque. It has been agreed by appellant I (see submission of 23 March 1998, page 9) that the separation method disclosed in the patent in suit (see page 37, lines 24 to 25) is the same as in document (D21). Cryopreservation is carried out according to document (D21) by adding 10% of the cryopreservative dimethylsulfoxide (DMSO). By thawing and culturing the cryopreserved cells after 1 to 5 months of storage, the colony forming capacity after cryopreservation (ie the % survival after thaw) turns out to be comparable to that found in the patent in suit (compare eg the % recovery for the CFU-GM's etc in Table 1 of document (D21) with that reported in Table V of the patent in suit).

6. In the light of the experiments performed, the author of document (D21) arrives at the conclusion (see last paragraph on page 281) that, since the number of stem cells contained in cryopreserved cord blood is comparable to that found in cryopreserved BM, cryopreserved cord blood can be useful as a source of hematopoietic progenitor cells for marrow transplantation, ie for hematopoietic reconstitution.

7. The medical use stated in claim 1 or envisaged in document (D21) being thus the same, together with the underlying experimental evidence, the board now turns to the question of whether or not the disclosure of the patent in suit provides a new element vis-à-vis the teaching of document (D21), which element is susceptible conferring novelty on the claimed subject-matter. This new element may be in the form of eg further critical experimental evidence, fundamental technical information or means for overcoming a blockage, the absence of which would have dissuaded/prevented the skilled person from practising the medical use of claim 1.

8. Appellant I maintains that a first difference between the disclosure of the patent in suit and the teaching of document (D21) lies with the investigated cells, namely multipotent progenitor cells in the latter and hematopoietic stem cells required for hematopoietic reconstitution in the former.

9. The board agrees that a hematopoietic stem cell is something different from a hematopoietic progenitor cell. Hematopoietic stem cells indeed exhibit replating efficiency indicative of self-renewal capacity and are moreover pluripotent in that they have the greatest potential, by differentiation, to produce the various cells of the different blood cell lineages, whereas progenitor cells have more limited multipotentiality and a lesser degree of proliferative capacity.

10. At the filing date of the application underlying the patent in suit, however, while in vivo hematopoietic reconstitution performed on eg lethally-irradiated mice was indicative of the presence of hematopoietic stem cells in a sample, there was no direct in vitro assay to determine the presence of the elusive stem cells. Document (D143), taken as expert opinion (see page 46 l-h column at bottom: "Unfortunately, there is no assay yet available that detects and can quantify the human long-term marrow repopulating stem cells") demonstrates that this situation persisted even twelve years later. There was, though, a surrogate assay, namely the assay for progenitor cells, ie, CFU-GM, BFU-E-1 BFU-E-2, CFU-GEMM (myeloid lineage colony-forming cells) or CFU-Mix, which was an accepted indirect proof for the presence of stem cells (see ibidem: "However, surrogate assays such as for CFU-GM, BFU-E and CFU-GEMM...are available"). That the scientific community felt confident that these colony assays (in particular the assay for CFU-GM) were meaningful "markers" that could be used to estimate the engrafting capacity of cord blood and bone marrow is shown by document (D145) (see last paragraph on page 364).

11. The board notes that both document (D21) and the patent in suit (see paragraph 6.6.3) rely on this surrogate assay. Therefore, if the test used in the patent in suit is considered appropriate to establish the presence of viable stem cells, it has also to be considered appropriate to prove the existence of viable stem cells in document (D21). In conclusion, appellant I's allegation that the patent in suit detects/quantifies stem cells required for hematopoietic reconstitution while document (D21) merely investigates progenitor cells, is not convincing.

12. A further difference, according to appellant I arises from the flawed experimental design disclosed in document (D21) because of (a) the variability of progenitor cell assays, in particular, before freezing and after thawing; (b) the too high number of mononuclear cells plated (2 x 10 5) giving rise to a number of colonies too high for it to be scored and (c) no experiment was performed on the full volume of cord blood from a single collection from an individual, and thus there was no suggestion in document (D21) that this collection would contain sufficient amounts of stem cells for achieving a complete hematopoietic reconstitution.

13. As for the variability of the assays (see (a) above), the board notes that the pre- and post-freezing values were determined using exactly the same culture conditions both in the patent in suit and in document (D21) (compare page 277, r-h column of document (D21): "in the same fashion as described above" with page 46, line 41 of the patent in suit: "in the same assay").

14. Furthermore, the board accepts that any assay for eg CFU-GM is not a true reading of the number of CFU-GM actually present but depends on the conditions under which the assay is performed (presence or absence of growth promoters such as granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3 (IL-3) (see bottom of page 41 of the patent in suit), incubation time, etc). Therefore, interlaboratory comparisons of the colony-forming cell numbers found in hematopoietic tissues have to be interpreted cautiously due to wide variation in methodology (see as expert opinion document (D34), page 190, l-h column, third paragraph and document (D148), page 1680, l-h column: "Because of the well known variability of the stem/progenitor cell assays, however, these results should be interpreted with appropriate caution").

15. But for the purpose of estimating by colony assays the (potential) engrafting capacity of cord blood, it is not the absolute number of progenitor cells (in particular the CFU-GM) found in a biological sample which is important (as seen above, this absolute number depends on the culturing conditions). Rather, it is the comparison of the number of progenitor cells found in cord blood with that of the progenitor cells present in BM. The author of document (D21) concludes that these numbers are "comparable" (see page 281, r-h column, line 1). The patent in suit comes to the same conclusion (see page 13, line 7 and page 49, lines 34 to 35). The board, therefore, is unable to accept the appellant I's contention that the results in document (D21) are flawed due to unacceptable variability of the assays, while those described in the patent in suit are not. If anything, the board agrees to the appellant II's view (see paragraph 5.b.ii of the submission of 31. March 2003) that the comparison done in the patent in suit (see Sections 5.1.1.1 and 6.8) between the number CFU-GM present in cord blood and the 0.24 million CFU-GM found in BM (necessary for successful engraftment) reported in the prior art, is also questionable, since the 0.24 million have clearly not been measured with the same assay as in the patent.

16. As regards argument (b) above, ie the too high number (2 x 10 5) of mononuclear cells (MNC) plated giving rise, in the appellant I's view, to a number of colonies too high for it to be scored, the board observes that the author of document (D145) (see page 359, r-h column) also adopted the technique of Pike and Robinson (supra) as the author of document (D21) and likewise plated without apparent difficulties 2 x 10 5 MNC for assaying CFU-GM. This appellant I's argument is thus not convincing.

17. Appellant I argues that the experiments done in document (D21) are not performed on the full volume and that there is thus no suggestion in this document that this collection would contain a sufficient amount of stem cells for achieving a complete hematopoietic reconstitution (objection (c) above).

Yet, as regards the in vitro tests performed in the patent in suit, the board notes that the CFU-GM assay described on page 41 is also not performed on the full volume (but with 1 ml containing 1 x 10 5 cells/ml). In the board's view, it is the ratio number of progenitor cells/number of plated cells which is important. The patent in suit expresses the result as number of progenitor cells (CFU-GM, etc) per 1 x 10 5 plated mononuclear cells (see page 14, line 13), whereas document (D21) expresses the result as eg CFU-GM per 2 x 10 5 plated mononuclear cells (see Table 1). As regards the results of both documents, these are clearly within the range "11-327 CFU-GM/2 x 10 5 MNC" referred to on page 360, top of r-h column of document (D145), corresponding to about 1,000 (997) CFU-GM/ml BM (see ibidem). The skilled person reading document (D21) in the light of the prior art is taught by document (D21) that cord blood contains CFU-GM in an amount comparable to BM (therefore: about 1,000 CFU-GM/ml). He/she is also aware that the dose of bone marrow to be infused to a patient (see document (D145), Table 1) is from 0.51. x 10³ to 51 x 10³/kg of CFU-GM, ie that 0.51 ml to 51. ml BM (or cord blood) provide a sufficient number of CFU-GM to the patient for successful engraftment. These blood volumes correspond to those normally available from the cord/placenta of a newborn. Document (D21) thus implicitly discloses the feature that "the collection of cord blood from a single newborn" would contain sufficient amounts stem cells for achieving a complete hematopoietic reconstitution".

As for in vivo tests performed according to the patent in suit on the "full volume", the experiment described in Section 6.12 (page 54) not only involves 150 ml neonatal blood (ie something greater than a "collection of cord blood from a single newborn") but does not provide any result either (see Section 22 infra).

18. It is argued by appellant I that the recoveries in document (D21) after thawing are too low to convince the skilled person to use them in blood cell repopulation. However, this allegation is contradicted by the fact that the % survival after thaw in document (D21) turns out to be comparable to that found in the patent in suit (see point 5 supra).

19. According to appellant I, an experimental proof performed in vivo and showing the successful engraftment is absent in document (D21). In contrast, the patent in suit provides results obtained in experiments on animal models, demonstrating that hematopoietic reconstitution was achieved in vivo.

20. The patent in suit in fact (see Sections 6.11 to 6.12) discloses a series of experiments showing hematopoietic reconstitution of lethally-irradiated mice with blood of newborn mice. However, the board agrees to the criticism raised by the then opponents against these experiments before the first instance.

21. The most serious objection is that these experiments do not involve blood which underwent cryopreservation (as stated in claim 1 at issue) but rather "fresh" blood. It has indeed strongly been emphasized by the patentee at the opposition stage that hematopoietic cells have a different sensitivity to cryopreservation (see Section VII.E of the submission dated 23 March 1998; see also document (D31), paragraphs 18 to 21) and that a sufficient amount of stem cell could not survive cryopreservation. Thus, in the board's judgement, experiments with fresh neonatal blood are not predictive of whether successful engraftment will also occur with cryopreserved neonatal blood. A further objection is that the (unknown) ratio stem cells/progenitor cells (see document (D149), paragraph 15. and document (D30), paragraph 23) might be different in mice and humans.

22. The only experiment overcoming the above two objections could have been that of Section 6.12 (page 54) of the patent since it relates to an in vivo experiment practised on a human (treatment of Franconi's anaemia) involving cryopreserved blood. However, this experiment does not provide any result.

23. In conclusion, the in vivo experiments disclosed in the patent in suit cannot be considered as a new element in the sense of point 7 supra, susceptible to confer novelty on the claimed subject-matter.

24. In view of the foregoing, the board concludes that the claimed subject-matter is not novel in view of the disclosure of document (D21).