Ex Parte Palecek et alDownload PDFBoard of Patent Appeals and InterferencesJul 8, 201010993468 (B.P.A.I. Jul. 8, 2010) Copy Citation MOD PTOL-90A (Rev.06/08) APPLICATION NO./ FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. 10/993,468 11/19/2004 Sean P. Palecek 960296.00100 EXAMINER POPA ILEANA ART UNIT PAPER NUMBER QUARLES & BRADY LLP 33 E. MAIN ST, SUITE 900 P.O. BOX 2113 MADISON WI 53701-2113 1633 MAIL DATE DELIVERY MODE 07/08/2010 Electronic Please find below and/or attached an Office communication concerning this application or proceeding. The time period for reply, if any, is set in the attached communication. UNITED STATES DEPARTMENT OF COMMERCE U.S. Patent and Trademark Office Address : COMMISSIONER FOR PATENTS P.O. Box 1450 Alexandria, Virginia 22313-1450 www.uspto.gov UNITED STATES PATENT AND TRADEMARK OFFICE _____________________________________________________________________________________ UNITED STATES PATENT AND TRADEMARK OFFICE __________ BEFORE THE BOARD OF PATENT APPEALS AND INTERFERENCES __________ Ex parte SEAN P. PALECEK, JUAN J. DE PABLO, LIN JI, and JAMES A. THOMSON __________ Appeal 2010-001157 Application 10/993,468 Technology Center 1600 __________ Before RICHARD M. LEBOVITZ, MELANIE L. McCOLLUM, and JEFFREY N. FREDMAN, Administrative Patent Judges. FREDMAN, Administrative Patent Judge. DECISION ON APPEAL1 1 The two-month time period for filing an appeal or commencing a civil action, as recited in 37 C.F.R.§ 1.304, or for filing a request for rehearing, as recited in 37 CF.R.§ 41.52, begins to run from July 8, 2010 shown on the PTOL-90A cover letter attached to this decision. Appeal 2010-001157 Application 10/993,468 2 This is an appeal under 35 U.S.C. § 134 involving claims to methods of cryopreserving human embryonic stem cells. We have jurisdiction under 35 U.S.C. § 6(b). We affirm. Statement of the Case The Claims Claims 1, 2, 5-10, 12-22, 24-38, and 40-45 are on appeal. The claims subject to each rejection have not been separately argued and therefore stand or fall together. See 37 C.F.R. § 41.37(c)(1)(vii). We selected Claim 17 as representative and Claim 17 reads as follows: 17. A method of cryopreserving stem cells, comprising the steps of: a) growing undifferentiated human embryonic stem cells on a bottom layer of solid support matrix, such that the cells adhere to the matrix; b) adding an effective amount of a carbohydrate-based cryopreservation medium over the matrix adherent cells; c) adding an effective amount of a freezing medium to the cells of step b); and d) cooling the matrix adherent cells of step c) to a temperature sufficient to cryopreserve the cells. The prior art The Examiner relies on the following prior art references to show unpatentability: Reid et al. US 2002/0182188 A1 Dec. 5, 2002 Funk et al. US 6,667,176 B1 Dec. 23, 2003 Appeal 2010-001157 Application 10/993,468 3 Ure et al., A rapid and efficient method for freezing and recovering clones of embryonic stem cells, 8 TRENDS GENETICS 6 (1992). Reubinoff et al., Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method, 16 HUMAN REPRODUCTION 2187-2194 (2001). Wolkers et al., Human platelets loaded with trehalose survive freeze-drying, 42 CRYOBIOLOGY 79-87 (2001). Voight et al., Cultured epidermal keratinocytes on a microspherical transport system are feasible to reconstitute the epidermis in full-thickness wounds, 5 TISSUE ENGINEERING 563-572 (1999). Limaye et al., Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium, 10 J. HEMATOTHERAPY STEM CELL RES. 709-718 (2001). The issues A. The Examiner rejected claims 17-20, 22, 24-27, 32, 33, 36, 38, 42, 43, and 45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, and Wolkers (Ans. 4-7). B. The Examiner rejected claims 17-22, 24-27, 32-34, 36, 38, 42, 43, and 45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, and Voigt (Ans. 7-8). C. The Examiner rejected claims 1, 2, 5-9, 12-14, 16-22, 24-33, 35-38, and 40-45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, and Reid (Ans. 8-9). Appeal 2010-001157 Application 10/993,468 4 D. The Examiner rejected claims 1, 2, 5-10, 12-14, 16-22, 24-38, and 40- 45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, Reid, and Voigt (Ans. 9-10). E. The Examiner rejected claims 1, 2, 5-9, 12-22, 24-33, 35-38, and 40- 45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, Reid, and Limaye(Ans. 10-11). A. 35 U.S.C. § 103(a) over Ure, Reubinoff, Funk, and Wolkers The Examiner finds that “the mere fact that nobody combined these well known techniques before, does not mean that the instant invention was not obvious over the prior art. It is noted that all cited references are pertinent art, i.e., they all teach cryopreservation” (Ans. 17). Appellants argue that: Although the Examiner alleged that one of skill in the art would have been motivated to use the Ure method with human ESCs [embryonic stem cells] (office action dated October 30, 2007, page 3, lines 17-18), Reubinoff explicitly discourages such combination by explaining that while “slow-rate freezing and rapid thawing methods ... are efficient for the cryopreservation of mouse ES cells, it has been observed that the survival of undifferentiated human ES cells following slow-rate freezing is very poor, and most often the cells either differentiate or die” (Reubinoff, page 2187, right column, second paragraph (emphasis added)) (App. Br. 9). Appellants argue that “Funk teaches away from freezing ESCs on a Matrigel matrix by separating the cells from the matrix before cryopreservation” (App. Br. 10). Appellants argue that “Wolkers teaches . . . that human cell lines require genetic engineering to express a bacterial pore protein to permit Appeal 2010-001157 Application 10/993,468 5 trehalose penetration into the cells . . . Therefore, a skilled artisan would . . . have no reasonable expectation of success using trehalose on cells not provided with such structures” (App. Br. 10). Appellants argue that “there is no absolute Matrigel requirement in human ESC cultures. Rather, the cells can be cultured on feeder layers or other matrices, which is explicitly stated by Funk . . .Therefore, the Examiner failed to show a motivation to combine Ure with Reubinoff and Funk” (App. Br. 11). Appellants argue that even “if the prior art could be combined as suggested by the Examiner, and even if such combination of four or more documents would teach the elements of the claims, which it does not, the claimed invention would still be nonobvious due to the well-documented high level of unpredictability in the art as of the filing date” (App. Br. 12- 13). In view of these conflicting positions, we frame the obviousness issue before us as follows: Does the evidence of record support the Examiner’s conclusion that the prior art rendered obvious the method of cryopreservation of human embryonic cells of Claim 17? Findings of Fact (FF) 1. Ure teaches “freezing ES cells in situ on 24-well plates. We have developed a simplified method in which the ES cells are exposed to the minimum volume of freezing mix, which is frozen uniformly and thawed very rapidly” (Ure 6). 2. Ure teaches that “clones were picked and expanded in gelatine- coated 24-well plates. Individual clones were trypsinized when near Appeal 2010-001157 Application 10/993,468 6 confluent and one-third of the cell suspension was passaged into a fresh well” (Ure 6). 3. Ure teaches that the “minimum volume of freezing mix required to coat the wells completely (0.25 ml of complete media containing 10% dimethyl sulphoxide, DMSO) was then added” (Ure 6). 4. Ure teaches that “[p]lates were placed in polystyrene boxes packed with sytrofoam and immediately transferred to a -80°C freezer” (Ure 6). 5. The Examiner acknowledges that “Ure et al. do not teach cryopreserving human embryonic stem cells” (Ans. 4). The Examiner also acknowledges that “Ure et al. taken with Reubinoff, and Funk et al. do not teach adding a carbohydrate-based cryopreservation medium over the matrix adherent cells” (Ans. 5). 6. Reubinoff teaches that “to exploit this remarkable potential of human ES cells . . . an efficient cryopreservation method would be highly valuable for the development and widespread use of these cell lines” (Reubinoff 2187, col. 1-2). 7. Reubinoff teaches that: Slow-rate freezing and rapid thawing methods are most commonly used for the cryopreservation of cell lines (Freshney 1994). While these standard methods are efficient for the cryopreservation of mouse ES cells (Robertson, 1987), it has been observed that the survival of undifferentiated human ES cells following slow-rate freezing is very poor, and most of the cells either differentiate or die. (Reubinoff 2187, col. 2). Appeal 2010-001157 Application 10/993,468 7 8. Reubinoff teaches that “ES cells were cryopreserved in clumps of about 100-200 cells by using either the conventional slow-freezing method . . . or the open pulled straw (OPS) vitrification method” (Reubinoff 2188, col. 10). 9. Reubinoff teaches that “[h]uman pluripotent ES cells from both cell lines could be successfully recovered and propagated after cryopreservation with conventional slow-rate freezing and rapid thawing methods . . . and the cells retained their fundamental characteristics. Nevertheless, the efficiency of these standard methods with human ES cells was relatively low” (Reubinoff 2190, col. 1-2). 10. Funk teaches “a system for obtaining expression libraries from primate pluripotent stem (pPS) cells. pPS cells can be maintained in vitro without requiring a layer of feeder cells to inhibit differentiation” (Funk, col. 5, ll. 39-42). 11. Funk teaches that “the role of feeder cells can be replaced by a combination of one or more features in the culture environment that support proliferation of the cells without differentiation. One such feature is a suitable substrate on the culture surface, such as extracellular matrix exemplified by Matrigel® and laminin” (Funk, col. 5, ll. 43-48). 12. Wolkers teaches “[s]mall carbohydrates, such as trehalose . . . have an exceptional ability to stabilize cells during freezing, freeze-drying, and air-drying” (Wolkers 79, col. 1). 13. Wolkers teaches that “[t]rehalose . . . is particularly effective at stabilizing dry cells. . . . Its remarkable effectiveness is apparently due to its ability to replace the water shell of macromolecules, thus preventing Appeal 2010-001157 Application 10/993,468 8 damaging effects during drying, and to its high glass transition temperature, resulting in the formation of stable glasses at room temperature that preserve cells in the dried state” (Wolkers 79, col. 1-2). 14. The Specification teaches that “trehalose is believed to maintain thermodynamic stability of membranes by preserving phospholipid head group spacing and inhibiting lipid phase transitions and separation during freezing and drying” (Spec. 1-2 ¶ 0004). 15. The Specification teaches that “[i]t has been proposed that freezing destruction from extracellular ice is essentially a plasma membrane injury resulting from osmotic dehydration of the cell” (Spec. 9 ¶ 0052). Principles of Law “Often, it will be necessary . . . to look to interrelated teachings of multiple [references] . . . and the background knowledge possessed by a person having ordinary skill in the art, all in order to determine whether there was an apparent reason to combine the known elements in the fashion claimed.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 418 (2007). “[T]his analysis should be made explicit,” and it “can be important to identify a reason that would have prompted a person of ordinary skill in the relevant field to combine the elements in the way the claimed new invention does.” Id. An obviousness “analysis need not seek out precise teachings directed to the specific subject matter of the challenged claim, for a court can take account of the inferences and creative steps that a person of ordinary skill in the art would employ.” Id. Appeal 2010-001157 Application 10/993,468 9 In assessing a claim’s obviousness, “a court must ask whether the improvement is more than the predictable use of prior art elements according to their established functions.” Id. at 417. “The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 416. A reference may be said to teach away when a person of ordinary skill, upon reading the reference, would be discouraged from following the path set out in the reference, or would be led in a direction divergent from the path that was taken by the applicant. The degree of teaching away will of course depend on the particular facts; in general, a reference will teach away if it suggests that the line of development flowing from the reference’s disclosure is unlikely to be productive of the result sought by the applicant. In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994). “[A] reference may teach away from a use when that use would render the result inoperable.” In re ICON Health and Fitness Inc., 496 F.3d 1374, 1381 (Fed. Cir. 2007). Analysis Ure teaches a method of cryopreserving embryonic stem cells by growing the stem cells on gelatine coated plates, adding a freezing medium to the cells, and cooling the matrix to cryopreserve the cells (FF 1-4). There is no dispute that Ure does not teach freezing of human embryonic stem cells or adding trehalose to the cells (FF 5). Applying the KSR standard of obviousness to the findings of fact, we conclude that the person of ordinary creativity would have predictably applied the cryopreservation method of Ure to human embryonic stem cells Appeal 2010-001157 Application 10/993,468 10 for the reasons of Reubinoff that cryopreservation is “highly valuable” and expected to work to at least some degree (FF 6-7) and incorporated trehalose since Wolker teaches that trehalose has “an exceptional ability to stabilize cells during freezing” (Wolkers 79, col. 1; FF 12). Such a combination is merely a “predictable use of prior art elements according to their established functions.” KSR, 550 U.S. at 417. Appellants argue that: Although the Examiner alleged that one of skill in the art would have been motivated to use the Ure method with human ESCs (office action dated October 30, 2007, page 3, lines 17-18), Reubinoff explicitly discourages such combination by explaining that while “slow-rate freezing and rapid thawing methods ... are efficient for the cryopreservation of mouse ES cells, it has been observed that the survival of undifferentiated human ES cells following slow-rate freezing is very poor, and most often the cells either differentiate or die” (Reubinoff, page 2187, right column, second paragraph (emphasis added)) (App. Br. 9). Appellants argue that “Funk teaches away from freezing ESCs on a Matrigel matrix by separating the cells from the matrix before cryopreservation” (App. Br. 10). We are not persuaded. While Reubinoff teaches that “the efficiency of these standard methods with human ES cells was relatively low,” Reubinoff also teaches that “[h]uman pluripotent ES cells from both cell lines could be successfully recovered and propagated after cryopreservation with conventional slow-rate freezing and rapid thawing methods . . . and the cells retained their fundamental characteristics” (Reubinoff 2190, col. 1-2; FF 9). The claim does not require any degree of efficiency or survival rate. Appeal 2010-001157 Application 10/993,468 11 Reubinoff does not teach or suggest that cryopreservation of human embryonic stem cells by the method of Ure would be unlikely to work (see FF 6-9). Rather, Reubinoff simply does not prefer slow-rate freezing, but Reubinoff clearly shows that the conventional methods are operable (FF 6- 9). Regarding Funk, Appellants argue that Funk teaches to separate the cells into solution prior to cryopreservation (App. Br. 10). However, Funk teaches that “[c]ells maintained in Matrigel®/conditioned medium and laminin/conditioned medium were cryopreserved as follows: The cells were frozen in standard hES medium (not conditioned medium) supplemented with 10% DMSO and additional 10% SR(total 30%). The cells were thawed onto Matrigel® or laminin in conditioned medium” (Funk, col. 29, ll. 42- 47). This teaching of Funk does not indicate that the cells should be separated and certainly does not teach away from freezing cells on the matrix. Like our appellate reviewing court, “[w]e will not read into a reference a teaching away from a process where no such language exists.” DyStar Textilfarben GmbH v. C.H. Patrick Co., 464 F.3d 1356, 1364 (Fed. Cir. 2006). Appellants argue that “Wolkers teaches . . . that human cell lines require genetic engineering to express a bacterial pore protein to permit trehalose penetration into the cells . . . Therefore, a skilled artisan would . . . have no reasonable expectation of success using trehalose on cells not provided with such structures” (App. Br. 10). We are not persuaded. In fact, Wolkers teaches that “[s]mall carbohydrates, such as trehalose . . . have an exceptional ability to stabilize Appeal 2010-001157 Application 10/993,468 12 cells during freezing, freeze-drying, and air-drying” (Wolkers 79, col. 1; FF 12). While Wolkers gives one example where the use of genetically engineered pores enhanced the freeze tolerance of human cell lines, Wolkers’ own experiments do not use such cells with genetically engineered pores, but rather use normal platelets (see Wolkers 79, col. 1 and 80, col. 1- 2). The Specification recognizes, consistent with Wolkers, that “trehalose is believed to maintain thermodynamic stability of membranes by preserving phospholipid head group spacing and inhibiting lipid phase transitions and separation during freezing and drying” (Spec. 1-2 ¶ 0004). The Specification teaches that “[i]t has been proposed that freezing destruction from extracellular ice is essentially a plasma membrane injury resulting from osmotic dehydration of the cell” (Spec. 9 ¶ 0052; FF 15). Thus, the ordinary artisan would have expected that trehalose would enhance the stability of cells during freezing, whether the trehalose penetrated the cells or not (FF 12-15). Appellants argue that “there is no absolute Matrigel requirement in human ESC cultures. Rather, the cells can be cultured on feeder layers or other matrices, which is explicitly stated by Funk . . .Therefore, the Examiner failed to show a motivation to combine Ure with Reubinoff and Funk” (App. Br. 11). We are not persuaded. First, the use of Matrigel is not a requirement of Claim 17 (or indeed of any of independent claims 1, 16, 28, or 32). “[L]imitations are not to be read into the claims from the specification.” In re Van Geuns, 988 F.2d 1181, 1184 (Fed. Cir. 1993) (citing In re Zletz, 893 F.2d 319, 321 (Fed. Cir. 1989)). Appeal 2010-001157 Application 10/993,468 13 Second, Funk teaches that in growing embryonic stem cells “the role of the feeder cells can be replaced by a combination of one or more features in the culture environment that support proliferation of the cells without differentiation. One such feature is a suitable substrate on the culture surface, such as extracellular matrix exemplified by Matrigel® and laminin” (Funk, col. 5, ll. 43-48; FF 11). The Supreme Court has clearly stated that the “combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results,” KSR, 550 U.S. at 401. This reasoning is applicable here, since Funk teaches an alternative matrix for growing human embryonic stem cells which would predictably support the cell growth (FF 10-11). Appellants have provided no evidence that the use of Matrigel® would have been unpredictable. Appellants argue that even “if the prior art could be combined as suggested by the Examiner, and even if such combination of four or more documents would teach the elements of the claims, which it does not, the claimed invention would still be nonobvious due to the well-documented high level of unpredictability in the art as of the filing date” (App. Br. 12- 13). We are not persuaded. Appellants argument that there is a “well- documented high level of unpredictability” requires evidentiary support. Attorney argument is not evidence. In re Pearson, 494 F.2d 1399, 1405 (CCPA 1974). Nor can it take the place of evidence lacking in the record. Meitzner v. Mindick, 549 F.2d 775, 782 (CCPA 1977). For example, Appellants did not provide evidence that it would be unpredictable to apply the method of Ure to human embryonic stem cells with the incorporation of Appeal 2010-001157 Application 10/993,468 14 trehalose which has “an exceptional ability to stabilize cells during freezing, freeze-drying, and air-drying” (Wolkers 79, col. 1; FF 12). Conclusion of Law The evidence of record supports the Examiner’s conclusion that the prior art rendered obvious the method of cryopreservation of human embryonic cells of Claim 17. C. 35 U.S.C. § 103(a) Rejection over Ure, Reubinoff, Funk, Wolkers and Reid Appellants argue that “Reid does not teach adding of a top layer of a solid support matrix.” (App. Br. 16). Appellants argue that “it would not have been obvious to the skilled artisan reading Reid to add a layer of matrix over adherent human ESCs for cryopreservation because the Reid cells are in suspension” (App. Br. 16). Appellants also argue that “Funk does not teach growing cells to about 1000-10,000 cell colonies” (App. Br. 16). The Examiner finds that “Reid et al. teach that cryopreservation of the human liver stem cells embedded in extracellular matrix results in better cell viability and function” (Ans. 29). The Examiner finds that the “skilled Artisan would have known and would have been motivated to increase the efficiency of the cryopreservation method . . . by further covering their Matrigel-adherent cells with a layer of Matrigel (i.e., to embed the HES cell in Matrigel)” (Ans. 29). The Examiner also finds that “since Funk et al. teach that only colonies comprising 10-2,000 cells are optimal for growth and propagation . . . one of skill in the art would have known and been motivated to freeze colonies of this size” (Ans. 30). Appeal 2010-001157 Application 10/993,468 15 We find that the Examiner’s position is supported by a preponderance of the evidence. Reid teaches “optionally embedding the cells in forms of extracellular matrix” (Reid 10 ¶ 0109). We agree with the Examiner that Reid’s teaching to embed cells in the extracellular matrix reasonably suggests coating Matrigel-adherent cells with a layer of Matrigel to the ordinary artisan (see Ans. 29). We also agree with the Examiner that Funk’s teaching of a particular size colony as optimal would suggest the use of these sized colonies. B., D., and E. 35 U.S.C. § 103(a) Rejections Appellants do not separately argue the additional rejections under 35 U.S.C. § 103(a) which depend upon the primary rejection over Ure, Reubinoff, Funk, and Wolkers. Instead, Appellants state with regard to each of these rejections that for “the reasons discussed above, Ure, Reubinoff, Funk, and Wolkers do not teach cryopreservation of adherent human ESCs as recited by the claims” (see, e.g., App. Br. 14). The Examiner has found specific evidence and reasoning to support the rejections (see Ans. 7-11). Appellants have not provided substantial reasons to doubt the correctness of the Examiner’s rejections. On appeal to this Board, Appellant must show that the Examiner has not sustained the required burden. See Ex parte Yamaguchi, 88 USPQ2d 1606, 1608 and 1614 (BPAI 2008) (precedential); Ex parte Fu, 89 USPQ2d 1115, 1118 and 1123 (BPAI 2008) (precedential). Therefore, we affirm the Examiner’s rejections. SUMMARY In summary, we affirm the rejections of: Appeal 2010-001157 Application 10/993,468 16 claims 17-20, 22, 24-27, 32, 33, 36, 38, 42, 43, and 45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, and Wolkers. claims 17-22, 24-27, 32-34, 36, 38, 42, 43, and 45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, and Voigt. claims 1, 2, 5-9, 12-14, 16-22, 24-33, 35-38, and 40-45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, and Reid. claims 1, 2, 5-10, 12-14, 16-22, 24-38, and 40-45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, Reid, and Voigt. claims 1, 2, 5-9, 12-22, 24-33, 35-38, and 40-45 under 35 U.S.C. § 103(a) as obvious over Ure, Reubinoff, Funk, Wolkers, Reid, and Limaye. No time period for taking any subsequent action in connection with this appeal may be extended under 37 C.F.R. § 1.136(a)(1)(iv)(2006). AFFIRMED Appeal 2010-001157 Application 10/993,468 17 dm QUARLES & BRADY LLP 33 E. 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