T 0166/01 (In vitro method/KARO BIO AB) 16-12-2003

1. The appeal is admissible.

Admissibility of documents (D33) to (D36)

2. As far as the admissibility of the new citations in the proceedings is concerned (which were filed with the Statement of Grounds), it is pointed out that these documents are relevant for elucidating the meaning of the term "hormone- depleted" in claim 1 of all requests, since they show the effect of treating foetal calf serum with dextran coated charcoal (1 x DCC or 2 x DCC) on the presence of residual hormones. This was/is an issue relevant for the first instance/the board to arrive at the decision under appeal/present decision.

Moreover, these documents do not form a basis for a new line of attack on the patentability of the claimed in vitro method, but rather support the argumentation presented by the opponent already in its grounds for opposition. These documents have also been relied upon by the respondent himself for supporting the presence of an inventive step of the claims of the second auxiliary request (see paragraph VII supra). Whilst the board recognises that the introduction of new documents after the expiry of the nine month opposition period might in certain cases be objectionable (depending especially upon the degree of relevance and the lateness), in the present appeal proceedings the board decided to admit documents (D33) to (D36) into the appeal proceedings having regard to what is set out above. Moreover, since the respondent also argued in its own favour on the basis of these new documents (see supra), it would not be equitable that the appellant has to bear any additional cost originating from these late submissions.

Main request

Fresh grounds for opposition

3. According to the opinion of the Enlarged Board of Appeal in case G 10/91 (OJ EPO 1993, 420), fresh grounds for opposition may be considered in appeal proceedings only with the approval of the patentee (see section 18). In the present case, the respondent did not consent, neither in writing nor during the oral proceedings, to the introduction by the appellant of objections under Article 83 EPC. Therefore, the objection under Article 83 EPC raised by the appellant is not considered in these appeal proceedings.

Novelty (Article 54 EPC)

Introduction

4. Claim 1 is addressed to an in vitro method for evaluating a tissue specific pattern of agonistic versus antagonistic effects of a test substance compared to a reference substance known to have an antagonist/agonist effect, on at least two types of human cells which contain intra-cellular hormone receptors. The assay comprises two distinct phases of cell cultivation. During the first phase the cells are grown and pre-conditioned in a first hormone-depleted medium (growth medium) (steps (a) and (b)) and during the second phase the cells are incubated in a second hormone-depleted medium in four containers (or groups of containers) d1) to d4), upon exposure to: (d1) the test substance at a known concentration eg C1, (d2) a reference substance at a known concentration eg C2, (d3) a control and (d4) the test substance + the reference substance at a concentration C1 + C2. The magnitude of the cellular response is then analysed (step (g)) and the tissue specific pattern of antagonistic versus agonistic effects are obtained from comparison of the magnitudes of the cellular responses (step (h)).

5. This method is illustrated in the patent in suit (see eg page 5, under the heading "pS2"), wherein two experiments are performed using the human breast cancer cell lines MCF7 and ZR- 75-1, respectively. The selected cellular response to be analysed is the amount of expressed protein pS2 after 48 h (see page 6, line 11), the expression of which is regulated by the intra-cellular estrogen receptor. Estradiol is used as a reference substance and the effects of tamoxifen is evaluated. In the table on page 6 of the patent in suit, the amount of pS2 secreted into the medium relative to the control (d3) set at 1 (see "no hormone added") is determined for the test substance (d1), ie tamoxifen alone (see "Tam (100 nM)"), the reference substance alone (d2), ie estradiol at 10 nM (see "E2 (10 nM)") and the test substance + the reference substance (d4) (see "E2 (10 nM") + Tam (100 nM)"). The obtained tissue specific pattern of antagonistic versus agonistic effects (see page 6, lines 35 to 37) is that pS2 can be induced by 10 nM E2 in the breast cancer cell lines MCF7 and ZR-75-1. It is further concluded that 10/-7 M tamoxifen functions as agonist in both the cell lines MCF7 and ZR-75-1 in the absence of E2 and that 10/-7 M tamoxifen functions as an antagonist in the presence of 10/-8 M (ie 10 nM) E2.

Documents (D3), (D27) and (D29)

6. These documents are argued by the appellant to be novelty- destroying for the subject matter of claim 1.

7. Document (3) describes a series of in vitro investigations on the effect of tamoxifen on two cancer cells lines (MCF-7 and UM-SCC). The only relevant experiment is that disclosed in Figure 3 on page 1154 of this document, relating to the "prevention of tamoxifen-induced growth inhibition by estradiol". In brief, the MCF-7 and UM-SCC cells are plated in D5 medium (ie complete Eagle essential medium supplemented with 15% fetal bovine serum (FBS) treated with dextran-coated charcoal (DCC) to remove unconjugated steroid hormones; see under "Materials and Methods" on page 1152) and allowed to reach logarithmic growth phase. From day 4, cells are fed daily with (i) the control medium D5 with or without 0.1% alcohol (drug solvent); (ii) 5 µmol/L tamoxifen citrate and (iii) 5 µmol/L tamoxifen citrate + 0.5 µmol/L or 0.1 µmol/L estradiol at two tamoxifen: estradiol ratios (1:10 or 1:50). The results are commented on in the paragraph bridging pages 1156 and 1157: "When both 5 µmol/L tamoxifen citrate and estradiol (at 1/10 or 1/50 the tamoxifen concentration) were added to logarithmically growing cultures, the growth-inhibitory effect of tamoxifen on MCF-7 was partially blocked so that progressive growth was observed. Under the same conditions, only a slight increase in cell number, over that in tamoxifen-treated cultures was obtained with UM-SCC-5."

8. The board, however, observes that the experiment illustrated in Figure 3 lacks the further test prescribed by claim 1 (e) at issue, wherein the estradiol reference substance (d2) is used alone at the same concentration as in test (d4), in the present case at 0.1 or 0.5 µmol/L (ie 100 or 500 nM), a test which the authors of document (D3) did not even conceive. It follows that the skilled person is not taught whether the observed "progressive growth" (supra) is to be ascribed to the 5 µmol/L tamoxifen citrate or to the estradiol 0.1 or 0.5 µmol/L (ie 100 or 500 nM). Hence, the skilled person is not able to derive from the "progressive growth" shown in Figure 3 any conclusion (cf step h of claim 1) as to the agonist/antagonist behaviour of 5 µmol/L tamoxifen citrate in the presence of estradiol 0.1 or 0.5. µmol/L (ie 100 or 500 nM).

9. It is true that in a further experiment (see Figure 5 and page 1154, r-h column, first full paragraph), the effect of estradiol on MCF-7 and UM-SCC cells at concentrations between 1. and 500 nmol/L was examined, however, this experiment was done in a different and not relevant context of the "tamoxifen- inhibited cultures", not in an attempt to elucidate the agonist/antagonist behaviour of 5 µmol/L tamoxifen citrate in the presence of estradiol 0.1 or 0.5 µmol/L (ie 100 or 500 nM). Thus, although the combination of the experiment illustrated in Figure 3 with some elements of the experiment of Figure 5 of document (D3) may theoretically yield complete step e of claim 1 at issue, these "scattered elements" are not disclosed as a specific combination, contrary to the requirements set out in eg decision T 305/87 (OJ EPO 1991, 429) that a specific combination has to be pointed out by a prior art document for it to be novelty-destroying. In conclusion, document (D3) does not destroy novelty of the method of claim 1.

10. In the experiment disclosed in Figure 2 of document (D27) (see page 201), synthetic progestins CMA, CPA, MPA, MGA, NRE and NRG were added to the medium in the presence or absence of 1. nM estradiol (E2), 500 ng/ml insulin (INS) or both hormones to investigate growth stimulation of ZR-75-1 cells. However, this experiment lacks the further test prescribed by claim 1 (e) at issue, wherein the estradiol reference substance (d2) is used alone at a concentration of 1 nM. In Figure 3 on page 202 of this document, the growth medium is both estrogen- and insulin-free. Growth stimulation of ZR-75-1 cells is also measured in this medium upon addition of the estrogen NRE (norethindrone) or NRG (norgestrel) and the antiestrogen EM-139 (see legend to Figure 3 and page 202, r-h column, under the heading "ER activity"). However, this experiment lacks the further test prescribed by claim 1 (e) at issue, wherein the test substance (d1) is used alone at a concentration, in this specific example, of 300 nM. The control (d3) is also missing. The skilled person is thus not in a position to derive from the growth responses shown in Figure 3 or the ER activities shown in Figure 3 of this document any conclusion (see step h of claim 1) as to the agonist/antagonist behaviour of these synthetic progestins in the presence of the reference substance. This conclusion also applies to the experiment of Figure 4 of document (D27), which is thus not prejudicial to the novelty of the method of present claim 1.

11. Document (D29) describes an in vitro experiment wherein the induction of the progesterone receptor (PR) is measured in the endometrial human cancer Ishikawa cell line and in the human breast cancer ZR-75-1 cell line (ie on "at least two selected types of cells" according to present claim 1) after a 6. day-treatment of the cells preincubated during 6 day with, inter alia, estradiol alone, antagonist ICI 164,384 (hereafter ICI), a combination of both, or nothing. Figure 4 (a) illustrates a diagram of the PR for the Ishikawa cell line versus the added estradiol (OE 1 nM), ICI 1µM, OE + ICI and the control. Figure 5 relates to a diagram of the PR for ZR-75- 1. cancer cell line treated in the same manner as the Ishikawa. On page 217, under the heading "Effects of hormone treatment on PR concentration", the authors of document (D29) conclude (cf step h of claim 1) that OE alone stimulates PR in both cells, ICI alone increases PR over control in Ishikawa but decreases it in ZR-75-1 and ICI antagonizes PR induction by OE in both cells.

12. However, one difference between the in vitro method of claim 1 at issue and that described in document (D29) lies in the fact that in the latter the cells are preincubated in a first medium "DMEM F12 ITS", which includes 10 nM estradiol and 6.25 µg/ml insulin (see page 216, l-h column), contrary to the requirement of step a of claim 1, according to which the first medium has to be "hormone-depleted".

13. In conclusion, no prior art document discloses all the features a to h of the in vitro method of claim 1. Therefore the subject-matter of claim 1 and dependent claims 2 to 12 satisfies the requirements of Article 54 EPC.

Inventive step (Article 56 EPC)

Closest prior art

14. In order to question the inventive step, the appellant uses a plethora of different combinations of prior art documents addressing a variety of problems to be solved in the light of documents (D3), (D5), (D27), (D29) and (D32) taken each as the closest prior art. The board disagrees to this approach, as explained in the following paragraph.

15. As stated above in point 12, one difference between the in vitro method of claim 1 at issue and that described in document (D29) lies in the preincubation medium which is not "hormone-depleted", contrary to the requirement of step a of claim 1. The analysis of documents (D3) and (D27) made under sections 7 to 10 supra in the context of the novelty issue shows that the teaching of these documents is more remote (and less relevant) than that of document (D29). The same conclusion applies to documents (D5) and (D32). Although both deal with investigating the behaviour of tamoxifen (tam), tam + estradiol (E2) and E2 (see Figure 5 and Figure 3, respectively) according to step h of claim 1 at issue, the experiments are carried out upon only one cell line (MCF-7) and the incubation medium is not hormone-depleted, as the paragraph headed "Cells and culture conditions" on page 1141, l-h column of document (D5) mentions insulin and hydrocortisone. Page 2497, r-h column, lines 3 to 4 of document (D32) states "Then the medium was changed to phenol red- and insulin-free MEM". This suggests that the previous incubation medium comprised insulin. In conclusion, document (D29) must represent the starting point for any problem- solution approach since it comes closer to the claimed subject- matter than any other prior art document.

Problem to be solved

16. Departing from document (D29) as closest prior art, the problem to be solved is the provision of an improved assay to evaluate the antagonistic versus the agonistic effects of test substances.

In the board's judgement, the skilled person was aware of the fact that cells involved in such assay had to be in an unstimulated state and that the incubation medium had to cause the minimum possible hormonal interference, a source of which was both the serum and the medium itself (see document (D32), page 2496, l-h column, second paragraph). On the one hand, document (D5) (see page 1141, l-h column, last full paragraph), prescribed that phenol-red had to be removed from the incubation medium owing to its estrogenic properties (ibidem, page 1143, r-h column, lines 7 to 8). On the other hand it was known that insulin interfered with cell growth experiments (see document (D27), page 201, r-h column, last paragraph). These two documents, thus, taught the skilled reader that hormone-depleted incubation was desirable and consequently it was obvious for the skilled person to arrive at the claimed in vitro method involving a defined hormone-depleted incubation first medium. Therefore, the respondent's main request is not allowable under the provision of Article 56 EPC.

First Auxiliary Request

Added subject-matter (Article 123 (2) EPC)

17. Claim 1 of this request differs from claim 1 of the main request by the addition of the wording "having been 2 x DCC treated" before "first medium" (steps a) and "second medium" (step b). In the respondent's view these amendments have a basis on page 10, lines 1 to 3, 10 and 22 of the published application as filed (WO 93/07290).

In the board's view, however, it cannot be derived from the passages pointed out by the respondent that it is the first medium ("A") and the second medium ("B") which are "2 x DCC treated". It is rather the 10% (in medium "A") or 1% (in medium "B") fetal calf serum (FCS) which undergoes such treatment, not the first or second medium as a whole. Therefore, the respondent's first auxiliary request is not allowable under the provision of Article 123(2) EPC.

Second auxiliary request

Added subject-matter (Article 123(2) EPC)

18. Claim 1 of the second auxiliary request differs from claim 1 of the main request by the addition of the wording "including 2 x D.C.C. treated foetal calf serum" after "first medium" (steps a) and "second medium" (step b). These amendments have a basis on page 10, lines 1 to 3, 10 and 22 of the published application as filed (WO 93/07290). Moreover, this information that the first and second medium should include 2 x DCC treated foetal calf serum (FCS) belongs to the general part of the description relating to the mediums to be used in all the successive experiments.

Inventive step (Article 56 EPC)

19. Again the board considers document (D29) as closest prior art, and departing from it the problem to be solved is the provision of an improved assay to evaluate the antagonistic versus the agonistic effects of test substances.

The question to be answered is whether the twice- treatment of foetal calf serum with DCC in the mediums used in the claimed method (cf "including 2 x D.C.C. treated foetal calf serum"), a measure which the respondent views as a key-feature in order to achieve very high sensitivity, is obvious or not.

20. According to document (D5) (see page 1143, r-h column, end of first full paragraph) a single treatment of the serum with DCC was held sufficient to render the medium "virtually free of estrogens". However, once this passage is balanced with the statement in document (D32) (see page 2496, l-h column, second paragraph) that in order to eliminate sources of estrogens from sera, "considerable efforts ... have been applied toward the development of serum-free media" (emphasis by the board), it would appear that 1 x DCC-treated foetal calf serum (be it "virtually free of estrogen" or otherwise) was still not the maximum which the skilled person could aim at in the field of hormone-free mediums. There was thus still a strong incentive to take measures suited to eliminating any residual hormone activity from the serum/medium. One such measure, in the board's view, was obviously to repeat DCC treatment, thus further depleting serum from residual unconjugated steroid hormones which remained adsorbed to DCC (see document (D3), page 1152, under the heading "Dextran-Coated Charcoal Treatment of FBS").

In view of the foregoing, it was obvious for the skilled person to arrive at the claimed in vitro method involving a first and second mediums "including 2. x D.C.C. treated foetal calf serum". Therefore, the respondent's second auxiliary request is also not allowable under the provision of Article 56 EPC.