Ce diaporama a bien été signalé.
Nous utilisons votre profil LinkedIn et vos données d’activité pour vous proposer des publicités personnalisées et pertinentes. Vous pouvez changer vos préférences de publicités à tout moment.

Pfizer opp claims vrs Hadasit July 2015

IP Opposition to Hadasit's hESC EPO Patent by Pfizer

  • Identifiez-vous pour voir les commentaires

  • Soyez le premier à aimer ceci

Pfizer opp claims vrs Hadasit July 2015

  1. 1. BOP15P0008 - 1 - OPPOSITION AGAINST THE PATENT EP 2 147 094 An opposition is filed against the patent EP 2147 094, in the name of Hadasit Medical Research Services & Development Limited, entitled “Stem-cell derived retinal pigment epithelial cells'', on behalf of Pfizer Limited. 1. Request We request the revocation of the patent EP 2 147 094 in its entirety. We request oral proceedings pursuant to Article 116(1) EPC should rejection of our request be contemplated. 2. Grounds of opposition The opposition is grounded on Articles 100 a) and 54 EPC, 100 a) and 56 EPC, 100 b) EPC and 100 c) EPC. 3. The Patent EP 2 147 094 3.1. Status The patent EP 2 147 094 was filed on April 27, 2008 under application number 08738258.6 and claims the priority of the provisional US application 907818P filed on April 18, 2007. 3.2. Validity of the claimed priority Granted claim 1 defines a method for promoting directed differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) fate, the method comprising: a) culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA to generate differentiating cells, and b) culturing said differentiating cells in a culture system comprising a basic medium supplemented with one or more members of TGFB superfamily, whereby said human pluripotent stem cells are induced to differentiate into RPE fate. The priority application does not provide any basis for such a method including a step a) of culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA to generate differentiating cells. Claim 1 of the priority application defines a method for promoting directed differentiation of hSCs into RPE fate, the method comprising: a) providing a cell culture comprising hSCs;
  2. 2. BOP15P0008 - 2 - b) culturing cells in the culture in a culture system comprising a basic medium supplemented with one or more member of TGFB superfamily whereby said hSCs are induced to differentiate into RPE fate. Among hSCs, the Patentee specifically selected providing differentiating cells, rather than undifferentiated hSCs (page 20, lines 6-10 of the priority application). The Patentee further selected providing differentiating cells generated from pluripotent hSCs, rather than from other types of hSCs (page 12, lines 21-25 and page 13, lines 9-18 of the priority application). The Patentee finally selected generating differentiating cells by culture in a culture system comprising a basic medium supplemented with NA, among the conditions suitable to differentiate undifferentiated hSCs in an augmented, directed fashion into a predetermined fate (page 21, lines 11-13 of the priority application). Granted claim 1 thus results from a combination of at least three independent selections of features. Such a specific combination of features is not disclosed in the priority application and the method resulting from this combination is not directly and unambiguously derivable from the content of the priority application. Granted claim 1 thus does not validly benefit from the priority of the US provisional application 907818P. Furthermore, granted claim 2 recites that culturing the human pluripotent stem cells in a culture system comprising said basic medium supplemented with NA is effected for at least two days. As mentioned in section 3.3 below, the term "basic medium supplemented with NA” is open- ended and the basic medium supplemented with NA may also be supplemented with other factors, in particular with one or more member of TGFB superfamily. The feature recited in claim 2 derives from the preferred embodiment 5 of the priority application (page 6, lines 8-10), which recited: “5. The method of any one of embodiments 1 to 4, comprising culturing the hSCs in a culture system comprising the basic medium supplemented with NA for at least two days prior to culturing said cell culture in a basic medium supplemented with the one or more member of TGFQ superfamily. ” The feature “prior to culturing said cell culture in a basic medium supplemented with the one or more member of TGFB superfamily” is no longer recited in granted claim 2. Granted claim 2 thus does not validly benefit from the priority of the US provisional application 907818P.
  3. 3. BOP15P0008 - 3 - 3.3. Granted claim 1 Independent claim 1 defines a method for promoting directed differentiation of human pluripotent stem cells into RPE fate, the method comprising: a) Culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with nicotinamide to generate differentiating cells; and b) Culturing said differentiating cells in a culture system comprising a basic medium supplemented with one or members of TGFB superfamily, whereby said human pluripotent stem cells are induced to differentiate into RPE fate. The terms used in the claim do not have clear technical meaning such that the description can be used to interpret them, as confirmed by T50/90. — Human pluripotent stem cells are defined as precursor cells that have the ability to form any adult cell (§[0027] of the opposed Patent). They include multipotent adult progenitor cells, induced pluripotent stem cells and amniotic fluid stem cells, but also human embryonic stem cells, as confirmed by dependent claim 4. - The culture system is defined as a culture system suitable for the propagation of stem cells, which comprises a combination of elements, at minimum including a basic medium E one or more member of the TGFB superfamily (§[0030] of the opposed Patent). Accordingly, in view of the description, both steps of the claimed method comprise a step of culture in presence of one or more members of TGFB superfamily. Granted claim 1 encompasses two embodiments: 1/ culture step a) in presence of NA and a member of TGFB superfamily and culture step b) in presence of a member of TGFB superfamily but in absence of NA; 2/ culture step a) in presence of NA and a member of TGFB superfamily and culture step b) also in presence of NA and a member of TGFB superfamily. Accordingly, granted claim 1 covers a particular embodiment in which only one culture step is implemented, namely a culture step in presence of NA and a member of TGFB superfamily. - The basic medium is not clearly defined in the description. It is only specified that it is a cell culture medium usually comprising a defined base solution which includes salts, sugars and amino acids (§[0030] of the opposed Patent). In view of the description, it is therefore not possible to distinguish a basic medium supplemented with a particular ingredient from a basic medium comprising that particular ingredient in its defined base solution. — The term "nicotinamide" (NA) is generally used to refer to a compound of the following formula:
  4. 4. BOP15P0008 - 4 - O N‘ NH2 / (see column 17, lines 5-20 of the opposed Patent). However, the definition rovided in the o osed Patent is not limited to the above com ound. Indeed, it is mentioned in §[0052] of the opposed Patent that "in the context of the present disclosure, the term NA also denotes derivatives of NA". The term "derivative of nicotinamide (NA)" is defined in §[0053] of the opposed Patent, and refers to chemically modified derivatives of the natural NA. According to the definition, such modification may include: - substitution of pyridine ring, - substitution of the amide moiety, - deletion of a group, or — replacement of group. As highlighted in the third party observations (D22) filed on June 6, 2014 during the examination proceedings of the opposed Patent, as any group of NA can be substituted, deleted or replaced by undefined groups, the term NA defined in granted claim 1 has no structural limitation. The opposed Patent further mentions that nucleoside derivatives such as “nicotinamide adenine" are encompassed by the term NA derivatives, as well as PDE4 inhibitors disclosed in WO 03/068233 (D23), WO 02/060875 (D24), GB 2327675A (D25) or VEGF-receptor tyrosine-kinase inhibitors disclosed in WO 01l55114 (D26). The general formulae of these compounds are reminded below: nicotinamide adenine dinucleotide WO 03/068233 (D23) M». N fie ff’ livkblkl-L! -()1 I? (,2 UN OI ‘I »’. >Ul‘~lHg 0=D-v: .)ti Q tI——<: :H, , 0 I’ >. ,Il~l xIH wo 02/060875 (D24) GB2327675 (D25) 0 R I I1”: NIIIII I 2 / ‘"1 RI «:3 InT i ‘NI R n: I I; R“ / ’ NTRI -I n m ‘ to; R ‘ R1! R5
  5. 5. BOP15P0008 - 5 - wo 01/55114 (D26) w / ' l NR, RZ n TN tr’ 5' (cars: ,, --. l( These compounds are structurally remote from the natural NA and do not even have the same biological activity (PDE4 inhibitor and VEGF receptor tyrosine-kinase inhibitor). Accordingly, the definition of NA derivatives basically encompasses any type of structural modification of NA. Consequently, the definition of “Nicotinamide" provided in the opposed Patent is extremely broad and covers an infinite number of compounds. - Differentiating cells are not defined in the description. It is only specified that "the cells in the cell culture may be a population of undifferentiated hSCs or a population of cells in which at least part of the hSCs have initiated differentiation" (§[0061]). The description also refers to "differentiating hSCs", which are defined as undifferentiated hSCs, which under suitable conditions are capable of differentiating in an augmented, directed fashion into a predetermined fate (§[0032]). The differentiated cells mentioned in claim 1 thus encompass any pluripotent stem cells which have initiated differentiation towards any cell type, optionally towards a mixture of cell types, at any stage of this differentiation; and even any undifferentiated stem cells which are simply capable of differentiating into a given fate. — Finally, RPE fate is not defined in the description. It therefore encompasses any differentiation stage from pluripotent stem cells to functional RPE cells.
  6. 6. BOP15P0008 - 6 - 4. Cited documents The following documents are cited in support to the opposition. The numbering of the documents is based on the numbering used by James Poole Limited in its opposition notice. Document Reference wo 2006/070370 wo 2006/080952 D2 Vuler et al. 2007 Mechanisms of Dev. 124:807-829 D9 Fuhrmann et al. 2000 Develoment127:4599-4609 Chow et al. (2001) Annu. Rev. Cell Dev. Biol. 17:255-296 EP 2 123 244 D19 wo 03/060085 D20 Moshiri etal. 2004 Int. J. Dev. Biol. 4821003-1014 Third party observations D22 wo 03/068233 D23 wo 02/060875 GB 2327675 D25 wo 01/55114 D26 EP 1 783 205 Sigma GMEM datasheet D28 Ikeda et al. 2005 PNAS 102 :11331-11336 D29
  7. 7. BOP15P0008 - 7 - 5. Articles 100a) and 54 EPC 5.1. Lack of novelty over EP 2 128 244 (D19) D19, filed on January 11, 2008, claims the priority of a Japanese application filed on January 18, 2007 and was published on December 2, 2009. D19 is thus a prior art document under Article 54(3) EPC. 5.1.1. Claim 1 D19 discloses a method for producing primate retinal progenitor cells, in particular human retinal progenitor cells, from embryonic stem cells (see §[0005], lines 16-17), which are pluripotent stem cells. “Retinal progenitor cells" are defined as progenitor cells committed to differentiate into cells present in the retina, including neural retina and retinal pigment epithelium, and encompass retinal pigment epithelium progenitor cells (see §[0030]). D19 thus discloses a method for promoting differentiation of human pluripotent stem cells (i. e. embryonic stem cells) into retinal pigment epithelium fate. This method comprises culturing embryonic stem cells as suspension culture in a medium optionally containing serum and obtaining retinal progenitor cells from the culture (see §[0029] and [0032]). The medium used for the suspension culture preferably comprises the compound SB-431542 of the following formula (as a “Nodal signal inhibitor”): <3’ I 1), It is clearly apparent from the formula of SB-431542 that this compound is included in the definition of nicotinamide according to the opposed Patent. £57 0 NH: 122 (§[0042] and [0044]-[0045]). Indeed, as detailed at section 3.3 above, the term “nicotinamide" also denotes derivatives of nicotinamide, which cover basically any type of structural modification of NA. Therefore, SB-431542 meets the structural definition of a derivative of NA provided in the opposed Patent. This derivative of NA (Nodal signal inhibitor in D19) can be added to the medium at the start of cultivation of embryonic cells (see §[0051]).
  8. 8. BOP15P0008 - 8 - D19 further specifies that, for the purpose of promoting differentiation into retinal progenitor cells, activin A may be added to the medium used for the suspension culture (§[0054]). This member of TGFB superfamily may be added to the medium 3 to 7 days after the start of cultivation of embryonic cells (see §[0055]). D19 thus discloses a method for promoting differentiation of human pluripotent stem cells into retinal pigment epithelium fate, comprising: - culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA, namely a derivative of NA, to generate differentiating cells, and - culturing said cells in a culture medium supplemented with a member of TGFB superfamily, whereby said human pluripotent stem cells are induced to differentiate into RPE fate. The subiect-matter of claim 1 is thus not novel in view of D19. 5.1.2. Claim 2 D19 specifies that the suspension culture can last for about 3 days or more (see §[0064]). D19 thus discloses the embodiment wherein the culturing of human pluripotent stem cells in a culture comprising said basic medium supplemented with said NA is effected for at least 2 days. The subiect-matter of claim 2 is thus not novel in view of D19. 5.1.3. Claim 3 D19 includes a step of culturing the cells in a culture medium supplemented with Activin A. The subject-matter of claim 3 is thus not novel in view of D19. 5.1.4. Claim 4 The human pluripotent stem cells cultured in D19 are human embryonic stem cells. The subiect-matter of claim 4 is therefore not novel in view of D19.
  9. 9. BOP15P0008 - 9 - 5.2. Lack of novelty over WO 2006/080952 (D2) 5.2.1. Claim 1 D2 describes the production of RPE-like cells from human embryo-derived cells (see Example 5 of D2). More particularly, D2 discloses an optimized method for promoting directed differentiation of human pluripotent stem cells into RPE late, the method comprising culturing human pluripotent stem cells, on feeder cells or as embryoid bodies, in the presence of factors such as bFGF, insulin, TGF§, IBMX, BMP-2, BMP-4 or their combinations, including stepwise addition (see page 22, lines 24-26). The culturing of human pluripotent stem cells on feeder cells to obtain RPE-like cells is generally performed in a medium that includes knockout high glucose DMEM supplemented with 500 u/ ml penicillin, 500 ug/ ml streptomycin, 1% non-essential amino acids solutions, 2mM GlutaMAX1 I, 0.1 mM beta-mercaoptoethanol, 4-80 ng/ ml bFGF, 8,4-20% serum replacement (see page 9, line 34 — page 10, line 4). As demonstrated by the first Opponent James Poole Limited, high glucose DMEM and knockout high glucose DMEM contain niacinamide, i. e., NA (see section 6 of opposition statement from James Poole Limited). In addition, any chemical compound comprised in the high glucose DMEM and knockout high glucose DMEM would fall within the extremely broad definition of NA of the patent. Indeed, as mentioned in D22 (third party submission) and detailed at section 3.3 above, the definition of NA in the opposed Patent is so broad that it cannot be considered to define any structural or functional limitation. Therefore the presence of a chemical compound in the cell culture medium should be considered equivalent to the presence of nicotinamide. D2 thus describes a method for promoting directed differentiation of human pluripotent stem cells into RPE fate, the method comprising culturing human pluripotent stem cells in a medium comprising NA, and in the presence of bFGF, insulin, TGF§, IBMX, BMP-2, BMP-4 or their combinations, added stepwise. The opposed Patent specifies that “the second culture system may also comprise NA, i. e. may be the same as the initial culture system, into which the member of the TGFQ superfamily is added’ (§[0067], column 20, lines 5-8 of the opposed Patent). D2 thus discloses all the features of claim 1, namely a method for promoting directed differentiation of human pluripotent stem cells into RPE fate, the method comprising: a) culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA to generate differentiating cells, and b) culturing said cells in a culture system comprising a basic medium supplemented with one or more members of TFGB superfamily, whereby said human pluripotent cells are induced to differentiate into RPE fate. The culture of stem cells in presence of NA disclosed in D2 would inherently generate differentiating cells, since the conditions of culture disclosed in D2 are identical to the ones
  10. 10. BOP15P0008 - 10 - defined in claim 1 and the differentiating cells mentioned in claim 1 are not specifically characterized. It should further be noted that, even if D2 was interpreted as disclosing the simultaneous presence of NA and a member of the TGFB superfamily, it would still disclose all the features of claim 1. Indeed, as detailed at section 3.3 above, the opposed Patent specifies that the term "culture system“ denotes "a combination of elements, at minimum including a basic medium (. ..) fly’ the one or more member of the TGFQ superfamily of growth factors" (see §[0030], column 13, lines 2-8 of the opposed Patent). Accordingly, the culture system used in step a) of claim 1 also comprises the one or more member of the TGF§ family. The subject-matter of claim 1 is thus not novel in view of D2. 5.2.2. Claim 2 D2 specifies that the embryonic cells are grown on MEF in a MEF medium for about 6 weeks (see page 19, lines 11-12). D2 thus discloses the embodiment wherein the culturing of human pluripotent stem cells in a culture comprising said basic medium supplemented with said NA is effected for at least 2 days. The subiect-matter of claim 2 is thus not novel in view of D2. 5.2.3. Claim 4 The human pluripotent stem cells cultured in D2 are human embryonic stem cells (see for example page 8, lines 10-11). The subject-matter of claim 4 is therefore not novel in view of D2. 5.3. Lack of novelty over EP 1783205 (D27) 5.3.1. Claim 1 D27 describes the differentiation of embryonic stem cell, in particular human embryonic stem cells (column 5, line 27) into retinal precursor cells (see column 12, lines 9-11 and 25-28).
  11. 11. BOP15P0008 - 11 - More particularly, embryonic stem cells, which are pluripotent stem cells, are cultured in floating culture in the presence of activin (column 24, lines 23-25), which is added 3-7 days after the start of floating culture (column 24, lines 37-42). The floating culture involves the use of a medium such as GMEM medium (column 13, lines 11-16). As confirmed by the Sigma datasheet relative to GMEM (D28), GMEM comprises nicotinamide. In addition, any chemical compound comprised in GMEM would fall within the extremely broad definition of NA of the patent. Indeed, as mentioned in D22 (third party submission) and detailed at section 3.3 above, the definition of NA in the opposed Patent is so broad that it cannot be considered to define any structural or functional limitation. Therefore the presence of a chemical compound in the cell culture medium should be considered equivalent to the presence of nicotinamide. D27 thus describes a method for promoting directed differentiation of human pluripotent stem cells into RPE fate, the method comprising culturing human pluripotent stem cells in a medium comprising NA, and culturing said cells in the medium further comprising activin. D27 thus discloses all the features of claim 1, namely a method for promoting directed differentiation of human pluripotent stem cells into RPE fate, the method comprising: a) culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA to generate differentiating cells, and b) culturing said cells in a culture system comprising a basic medium supplemented with one or more members of TFGB superfamily, whereby said human pluripotent cells are induced to differentiate into RPE fate. The subiect-matter of claim 1 is thus not novel in view of D27. 5.3.2. Claim 2 D27 specifies that the floating culture (in GMEM, which contains NA) lasts 3 to 7 days (column 24, lines 40-42). D27 thus discloses the embodiment wherein the culturing of human pluripotent stem cells in a culture comprising said basic medium supplemented with said NA is effected for at least 2 days. The subject-matter of claim 2 is thus not novel in view of D27. 5.3.3. Claim 3 D27 describes the addition of activin in the floating culture medium (column 24, line 25).
  12. 12. BOP15P0008 - 12 - The subiect-matter of claim 3 is thus not novel in view of D27. 5.3.4. Claim 4 The human pluripotent stem cells cultured in D27 are human embryonic stem cells (see column 5, line 27). The subiect-matter of claim 4 is therefore not novel in view of D27. 5.4. Lack of novelty over W0 03/060085 (D20) 5.4.1. Claim 1 D20 discloses a method for inducing retinal differentiation from neural stem cells (see page 29, lines 7-10), which are pluripotent stem cells. “Retinal differentiation” is defined as encompassing differentiation into retinal pigment epithelium cells (see page 29, lines 13-15). D20 thus discloses a method for promoting differentiation of human pluripotent stem cells (i. e. neural stem cells) into retinal pigment epithelium fate. This method comprises culturing neural stem cells in a medium comprising a substituted deoxynucleotide or deoxynucleoside (see page 7, lines 24-33), such as bromodeoxyuridine, iododeoxyuridine, bromodeoxyguanosine or iododeoxycytosine (page 8, lines 6-10). These substituted deoxynucleotides and deoxynucleosides are included in the definition of nicotinamide according to the opposed Patent. Indeed, as detailed at section 3.3 above, the term “nicotinamide” also denotes derivatives of nicotinamide, which cover basically any type of structural modification of NA. Therefore, the substituted deoxynucleotides and deoxynucleosides meet the structural definition of a derivative of NA provided in the opposed Patent. D20 further specifies that, for the purpose of inducing retinal differentiation, TGFB3 is added to the culture medium (page 29, lines 7-10). D20 thus discloses a method for promoting differentiation of human pluripotent stem cells into retinal pigment epithelium fate, comprising: - culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA, namely a derivative of NA, to generate differentiating cells, and
  13. 13. BOP15P0008 - 13 - - culturing said cells in a culture medium supplemented with a member of TGFB superfamily, whereby said human pluripotent stem cells are induced to differentiate into RPE fate. The subiect-matter of claim 1 is thus not novel in view of D20. 5.4.2. Claim 2 D20 specifies that the cells are cultured in the presence of a substituted deoxynucleotide or deoxynucleoside for an effective period (page 7, lines 31-33), namely for about 2 to 3 days (page 16, lines 14-17). D20 thus discloses the embodiment wherein the culturing of human pluripotent stem cells in a culture comprising said basic medium supplemented with said NA is effected for at least 2 days. The subject-matter of claim 2 is thus not novel in view of D20. 5.4.3. Claim 3 D20 includes a step of culturing the cells in a culture medium supplemented with TGFB3. The subiect-matter of claim 3 is thus not novel in view of D20. 6. Articles 100a) and 56 EPC 6.1.Lack of inventive step over WO 2006/070370 (D1) in combination with W0 2006l080952 (D2), W0 03/060085 (D20), Chow et al. (D12), Fuhrmann et al. (D11) or Moshiri et al. (D21) 6.1.1. Claim 1 D1 discloses a method for differentiating stem cells into retinal pigmented epithelial cells (RPE) using nicotinamide treatment (NA). More specifically, D1 discloses that NA treatment of stem cells promotes differentiation towards RPE cells (see page 27, lines 17-21). D1 can thus be considered as the closest prior art document. The difference between the subject-matter of claim 1 and the teaching of D1 is that the cells are further cultured in a basic medium supplemented with one or more members of TGFB superfamily.
  14. 14. BOP15P0008 - 14 - The technical effect associated with this difference is that the differentiation of human stem cells into RPE cells is augmented/ promoted/ induced (§ [0020] of the opposed Patent). The objective technical problem can thus be defined as the provision of an improved method for differentiating stem cells towards RPE late. It should first be noted that, as mentioned in the opposed Patent, NA was known from the prior art as having an inhibitory effect on differentiation of stem cells into extraembryonic cells, and as promoting somatic differentiation towards neural fate (§[0051] of the opposed Patent further referring to D1 (as reference (8))). In view of these complementary properties, NA was clearly known to be used at early steps of differentiation of stem cells towards neural lineage. We submit that the skilled person would have been incited, in view of the prior art, to add a culture step in a medium comprising a member of the TGFB superfamily, such as TGFB3 or Activin A, in order to improve differentiation of RPE cells. First of all, D2 describes the use of a medium including TGFB as an optimized culture system for differentiation towards RPE fate (see Example 5 of D2). More particularly, D2 describes the culture of stem cells as embryoid bodies in the presence of TGFB to induce differentiation towards RPE cells (see page 22, lines 24-26). W0 03/060085 (D20) also discloses that treating neural stem cells with TGFB3 induce their adoption of a retinal differentiation path in vitro, in particular their differentiation into retinal epithelium cells (see page 29, lines 7-14). As reminded in D20, neural stem cells are multipotent cells of central nervous system, which can only differentiate into specific cell types (see page 15, lines 19-23). Accordingly, neural stem cells correspond to stem cells which carried out a first step of differentiation towards neural fate. Secondly, many prior art documents describe the role of Activin A in the differentiation of RPE cells. For example, Chow et al. (D12) emphasizes the ability of exogenously applied Activin A on explants to induce RPE development (page 274, lines 5-8). Similarly, Fuhrmann et al. (D11), which is referenced by D12 in the above mentioned section, discloses that Activin can substitute for the extraocular mesenchyme in promoting RPE development and downregulating expression of neural retina-specific genes in explants (see Abstract and page 4607, right column, second paragraph, lines 6-9). Moshiri et al. (D21) also discloses that activin promotes the pigmented epithelial fate by activating RPE-specific genes, like MiTF (page 1005, left column, §1). Accordingly, in view of either D2, D20, D12, D11 or D21, the skilled person would have been incited to add, in the medium of a culture system, a TGFB superfamily member such as
  15. 15. BOP15P0008 - 15 - TGFB3 or Activin A, in order to promote differentiation of stem cells, already initially differentiated towards the neural lineage, into RPE cells. The subject-matter of claim 1 thus does not involve an inventive step in view of the prior art. 6.1.2. Claim 2 D1 discloses culturing human pluripotent stem cells in presence of NA for 6 weeks (page 54, lines 12-13). The subject-matter of claim 2 thus does not involve an inventive step in view of the prior art. 6.1.3. Claim 3 As specified at point 6.1.1. above, D20 suggests using TGFB3 to induce RPE differentiation, and D11/D12 suggest using Activin A to induce RPE differentiation. The subject-matter of claim 3 thus does not involve an inventive step in view of the prior art. 6.1.4. Claim 4 D1 discloses the use of human embryonic stem cells as human pluripotent stem cells (page 34, line 6). The subject-matter of claim 4 thus does not involve an inventive step in view of the prior art. 6.2. Lack of inventive step over Ikeda et al (D29) 6.2.1. Claim 1 D29 describes the differentiation of mouse embryonic stem cell line EB5 cells, which are pluripotent stem cells, into retinal precursor cells (see abstract). More particularly, EB5 cells are cultured in ES differentiation medium, in which activin is added 4 to 6 days after the start of cultivation (page 11332, Figure 1G)). The ES differentiation medium is defined as a medium comprising GMEM, 5% KSR, 0.1 mM nonessential amino acids, 1 mM pyruvate and 0.1 mM 2-mercaptoethanol (page 11331, right column, § 3).
  16. 16. BOP15P0008 - 16 - As confirmed by the Sigma datasheet relative to GMEM (D28), GMEM comprises nicotinamide. In addition, any chemical compound comprised in GMEM would fall within the extremely broad definition of NA of the patent. Indeed, as mentioned in D22 (third party submission) and detailed at section 3.3 above, the definition of NA in the opposed Patent is so broad that it cannot be considered to define any structural or functional limitation. Therefore the presence of a chemical compound in the cell culture medium should be considered equivalent to the presence of nicotinamide. D29 thus describes a method for promoting directed differentiation of mouse pluripotent stem cells into RPE fate, the method comprising culturing mouse pluripotent stem cells in a medium comprising NA, and culturing said cells in the medium further comprising activin. D29 thus discloses all the features of claim 1, namely a method for promoting directed differentiation of pluripotent stem cells into RPE fate, the method comprising: a) culturing pluripotent stem cells in a culture system comprising a basic medium supplemented with NA to generate differentiating cells, and b) culturing said cells in a culture system comprising a basic medium supplemented with one or more members of TFGB superfamily, whereby said pluripotent cells are induced to differentiate into RPE fate, except the pluripotent stem cells are mouse pluripotent stem cells rather than human pluripotent stem cells. It would have been obvious for the skilled person to apply this method to human pluripotent stem cells. Accordingly, the subject-matter of claim 1 does not involve an inventive step in view of D29. 6.2.2. Claim 2 D29 specifies that the cultivation in the ES differentiation medium lasts for 8 to 17 days (see page 11332, Figure 1G). D29 thus discloses the embodiment wherein the culturing of pluripotent stem cells in a culture comprising said basic medium supplemented with said NA is effected for at least 2 days. The subject-matter of claim 2 thus does not involve an inventive step in view of D29. 6.2.3. Claim 3 D29 describes the addition of activin in the ES differentiation medium (see page 11332, Figure 1G). The subject-matter of claim 3 thus does not involve an inventive step in view of D29.
  17. 17. BOP15P0008 - 17 - 6.2.4. Claim 4 The pluripotent stem cells used in D29 are mouse embryonic stem cells (see Abstract). The subject-matter of claim 4 thus does not involve an inventive step in view of D29. 7. Article 100b) EPC 7.1. RPE fate Claim 1 defines a method for promoting directed differentiation of human pluripotent stem cells into RPE fate. The expression "RPE fate" is not defined in the application. Moreover, different definitions are provided concerning RPE cells. Indeed, the opposed Patent first defines retinal pigment epithelial cells (or RPE cells or RPEs) as referring to cells of a cell type functionally similar to that of native RPE cells (§[O035]). It further distinguishes hSC-derived RPE cells, which are RPE cells which are obtained by directed differentiation from hSC and include (among others) mature and functional RPE cells (§[0036]). Functional RPE cells are defined as cells exhibiting at least one of the following characteristics: - during differentiation, the cultured cells respond to TGFB signaling; - the RPE cells are mature, terminally differentiated cells as exhibited by the expression of markers indicative of terminal differentiation, such as bestrophin or RPE65, and/ or by their lack of potency to proliferate in vivo; - following transplantation, the RPE cells exhibit trophic effect supporting photoreceptors adjacent to RPE cells, - in situ, the RPE cells are capable of functioning with phagocytosis of shed receptor outer segments as part of the normal renewal process of these photoreceptors (§[0037]). RPE cells functionally similar to native RPE cells are finally defined as differentiated RPE cells which share one or more distinct morphological or functional features with native RPE cells, such as the expression of one or more markers among MITF, Z0-1, bestrophin, RPE65, 0tx2, Mertk and CRALBP or typical F-actin distribution within the cell, pigmentation by pigmented granules, polygonal shape, cobblestone-like appearance, trophic effect supporting photoreceptors adjacent to RPE cells, functionality with phagocytosis of shed photoreceptor outer segments that harbor rhodopsin or lack of potency to proliferate in vivo (§[0039]).
  18. 18. BOP15P0008 - 18 - In view of all these different definitions relating to RPE differentiation, the skilled person would not have been able to determine what the expression "RPE fate" means, in particular if it refers to an initial differentiation towards RPE cells or to a complete differentiation into functional RPE cells. The skilled person would not have been able to determine which features of the cells (morphological, functional, specific marker) are necessary or sufficient to conclude that the RPE fate was reached. The subject-matter of the whole set of claims is thus insufficiently disclosed. 7.2. Step a) of generating differentiating cells Step a) of claim 1 defines the culture of human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA to generate differentiating cells. Step a) of claim 1 is thus defined by a result to be achieved, namely generating differentially cells. However, differentiating cells are not clearly defined in the application. It is only specified that "the cells in the cell culture may be a population of undifferentiated hSCs or a population of cells in which at least part of the hSCs have initiated differentiation" (§[0061]). Additionally, the specific culture conditions of step a) (NA concentration, duration) are not disclosed either. Accordingly, the skilled person would not be able to implement step a) of claim 1 since no information is provided in the opposed Patent to determine (i) which features the differentiating cells to be obtained need to display and (ii) which conditions of culture of step a) enable obtaining differentiating cells, which can differentiate into RPE fate. The subject-matter of the whole set of claims is thus insufficiently disclosed. 8. Article 100(c) EPC 9.1 Claim 1 Granted claim 1 defines a method for promoting directed differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) late, the method comprising: a) culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA to generate differentiating cells, and b) culturing said differentiating cells in a culture system comprising a basic medium supplemented with one or more members of TGFB superfamily, whereby said human pluripotent stem cells are induced to differentiate into RPE fate.
  19. 19. BOP15P0008 - 19 - We submit that the application as filed does not provide any basis for such a method including a step a) of culturing human pluripotent stem cells in a culture system comprising a basic medium supplemented with NA to generate differentiating cells. Claim 1 as filed defined in a method for promoting directed differentiation of hSCs into RPE fate, the method comprising: a) providing a cell culture comprising hSCs; b) culturing cells in a culture system comprising a basic medium supplemented with one or more member of TGFB superfamily whereby said hSCs are induced to differentiate into RPE fate. Among hSCs, the Patentee specifically selected providing differentiating cells, rather than undifferentiated hSCs (page 23, lines 20-23 of the application as filed). The Patentee further selected providing differentiating cells generated from pluripotent hSCs, rather than from other types of hSCs (page 14, lines 5-9 and page 14, line 25 — page 15, line 3 of the application as filed). The Patentee finally selected generating differentiating cells by culture in a culture system comprising a basic medium supplemented with NA, among the conditions suitable to differentiate undifferentiated hSCs in an augmented, directed fashion into a predetermined fate (page 24, lines 26-28 of the application as filed). Granted claim 1 thus results from a combination of at least three independent selections of features. Such a specific combination of features is not disclosed in the application as filed and the method resulting from this combination is not directly and unambiguously derivable from the content of the application as filed. The sub'ect-matter of the granted claims thus extends beyond the content of the application as filed. 9.2 Claim 2 Claim 2 recites that culturing the human pluripotent stem cells in a culture system comprising said basic medium supplemented with NA is effected for at least two days. As mentioned in section 3.3, the term "basic medium supplemented with NA” is open-ended and the basic medium supplemented with NA may also be supplemented with other factors, in particular with one or more member of TGFB superfamily. The feature recited in claim 2 derives from claim 5 as initially filed, which recited: “5. The method of any one of Claims 1 to 4, comprising culturing the hSCs in a culture system comprising the basic medium supplemented with NA for at least two days prior to culturing said cell culture in a basic medium supplemented with the one or more member of TGFQ superfami/ y.” The feature “prior to culturing said cell culture in a basic medium supplemented with the one or more member of TGFL3 superfamily”is no longer recited in granted claim 2.
  20. 20. BOP15P0008 - 20 - Claim 2 extends beyond the content of the application as filed, as there is no teaching for culture in a basic medium supplemented with NA for at least two days, in general.

×