Board of Patent Appeals and Interferences
Patent and Trademark Office (P.T.O.)
*1 EX PARTE HERBERT JUNGFER, HEINRICH BARCHET AND WINFRIED ALBERT
Appeal No. 88-3337
August 27, 1990
HEARD: June 17, 1990
Application for Patent filed October 3, 1985, Serial No. 06/784,696, which is a continuation of Serial No. 06/490,094 filed April 29, 1983. Process For The Production Of Permanently Culturable Animal And Human Cell Lines And The Use Thereof.
Norman D. Hanson et al. for appellants
Primary Examiner--John E. Tarcza
Before Pellman, W. Smith and Metz
This is an appeal from the final rejection of claims 2 through 4, 15, 17, 19 and 20, all the claims remaining in the application. Claims 15 and 20 are illustrative of the subject matter on appeal and read as follows:
15. A process for obtaining animal and human cells which are permanently culturable in vitro, comprising fusing normal animal or human cells with karyoplasts or cytoplasts which are incapable of multiplication which karyoplasts or cytoplasts are obtained from transformed cells to obtain a fused product of normal cells and karyoplasts or cytoplasts and cultivating said fused product in non-selective medium to obtain permanently culturable cells separated from said cytoplasts or karyoplasts.
20. A permanently culturable cell line comprising a normal human or animal cell line which has been fused with a caryoplast or cytoplast from a transformed cell which is incapable of multiplication.
The references relied upon by the examiner are:
Szoka et al. (Szoka) 4,393,338 July 19, 1983
Olsson et al. (Olsson), "Human-human Hybridomas Producing Monoclonal Antibodies Of Predefined Antigenic Specificity"D', Proc. Natl. Acad. Sci. USA, Vol. 77, No. 10, pages 5429-5431 (Sept. 1980).
Prujansky-Jakobovits et al. (Prujansky-Jakobovits), "Alteration Of Lymphocyte Surface Properties By Insertion Of Foreign Functional Components Of Plasma Membrane"D', Proc. Natl. Acad. Sci. USA, Vol. 77, No. 12, pages 7247-7251 (Dec. 1980).
Veomett et al. (Veomett), "Reconstruction Of Mammalian Cells From Nuclear And Cytoplasmic Components Separated By Treatment With Cytochalasin B"D', Proc. Natl. Acad. Sci. USA, Vol. 71, No. 5, pages 1999-2002 (May 1974).
Shih et al. (Shin), "Passage Of Phenotypes Of Chemically Transformed Cells Via Transfection Of DNA And Chromatin"D', Proc. Natl. Acad. Sci. USA, Vol. 76, No. 11, pages 5714-5718 (Nov. 1979).
Claims 2 through 4, 15, 17, 19 and 20 stand rejected under 35 USC § 103 as unpatentable over either Szoka or Prujansky-Jakobovits in view of Veomett and Shih. Claim 20 stands separately rejected under 35 USC § 102(b) as anticipated by, or in the alternative, under 35 USC § 103 as unpatentable over either Sugimoto or Olsson. We affirm.
*2 The claims on appeal are directed to a process for obtaining animal and human cells which are permanently culturable in vitro and the permanently culturable cell lines obtained from this process. The claimed method comprises fusing normal animal or human cells with karyoplasts or cytoplasts which are themselves incapable of multiplication which have been obtained from transformed cells. The fused product is cultivated in a non-selective medium whereby the permanently culturable cells are separated from cytoplasts or karyoplasts.
The present invention is an improvement over the hybridoma technology of Kohler and Milstein. Appellants set forth on pages 7-8 of the present specification that a disadvantage in the hybridoma technology arises from the fact that, while hybridomas require a certain starting up time before they are able to commence proliferation, the non-fused myeloma cells used to form the hybridomas immediately grow further. Thus, the selective suppression of the non-fused myeloma cells is essential for the production of hybridoma clones. The standard selection process in hybridoma technology is based upon the so-called HAT selection medium. The myeloma cells used in hybridoma technology are selected on the basis of their non-viability in HAT medium. Hybridoma technology and its attendent problems is summarized by appellants at page 13, lines 1-13 of the specification as follows:
B-lymphocytes of normal donors can be artificially "immortalised"D'. The hybridoma technique uses living myeloma cells which, in a culture, having an unlimited ability to multiply, which are fused with antigen-stimulated B-lymphocytes. The hybrid cells obtained by cell-cell fusion are isolated by HAT selection and cloned by application of individual cell cultures. Hybridoma clones which form antibodies with the desired specificity are multiplied for the mass production of monoclonal antibodies. However, considerable disadvantages arise from the HAT selection, by chromosome losses and hybrid immunoglobulins.
The present invention overcomes these problems by using isolated karyoplasts or cytoplasts from transformed cells, e.g., myeloma cells. Appellants have found that such karyoplasts and cytoplasts are capable of imparting so-called immorality upon normal cells when fused as in conventional hybridoma technology. Since the normal cells which are fused to the karyoplast or cytoplast are incapable of significant further growth and the karyoplast or cytoplast are themselves incapable of further growth, there is no need to use a selective medium in order to isolate the resulting hybrid cells.
Turning to the examiner's rejection under 35 USC § 103 first, we find that Szoka and Veomett are the most relevant references relied upon with the remaining references being, at best, cumulative. Szoka discloses methods for inserting DNA into a living cell which comprise encapsulating the DNA in a lipid vesicle and bringing the vesicle in contact with the cell so that insertion occurs. Of particular interest is Example 2 of Szoka where chromosomes extracted from HELA cells, which are transformed cells, were encapsulated and used to transform A9 mouse cells. [FN1] The A9 cells lack the enyzme hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT), which means that these cells are not capable of surviving in HAT medium. See page 8 of the present specification. A vesicle suspension containing the encapsulated HELA chromosomes is mixed with the A9 mouse cells and incubated, after which the mouse cells are exposed to HAT medium and clonal growth is monitored. Example 2 reports the presence of HGPRT which indicates that the encapsulated HELA chromosomes were effective for transferring genetic information to the A9 cells which lacked the necessary information to form HGPRT.
*3 Veomett discloses reconstruction of mammalian cells from nuclear and cytoplasmic components (karyoplasts and cytoplasts) separated by treatment with Cytochalasin B. Cytoplasts and karyoplasts derived from mouse L929 cells with Cytochalasin B were fused using Sendai virus, a conventional fusing agent. As set forth in the last full paragraph of column 2 of page 2000 of veomett, some of the reconstructed cells entered mitosis from which Veomett concludes that the reconstructed cells were capable of the resumption and completion of the cell cycle and are capable of proliferation. Veomett also concludes in the closing paragraph of the document that in view of the experiments which show that whole cells can be reconstructed from separated karyoplasts and cytoplasts, the feasibility of constructing mixed cell types by combining karyoplasts and cytoplasts derived from parental cells of different types has been established.
Assuming that the A9 cells of Example 2 of Szoka are normal per counsel's statement, Szoka provides evidence that normal cells may be transformed with genetic information from less than a completely transformed cell such as the HELA cells used in this example. Again, assuming the A9 cells are normal cells, the monitoring of clonal growth of the fused cell products in Example 2 of Szoka indicates that the immortality of the HELA cells was transferred into the A9 cells along with the HGPRT gene. Inasmuch as the vesicle containing the HELA DNA used in Example 2 of Szoka is similar to the karyoplasts used in the present invention, we find Example 2 of Szoka would have rendered the subject matter on appeal prima facie obvious.
In any event, Veomett discloses that cells may be separated into karyoplasts and cytoplasts and subsequently reconstructed into a whole cell which is capable of proliferation and that use of the disclosed technique makes the construction of mixed cell types by combining karyoplasts and cytoplasts derived from parental cells of different types feasible.
Keeping in mind that the claimed invention is directed to an improvement over the known hybridoma technology of Kohler and Milstein where normal cells are fused with immortal myeloma cells for the purpose of imparting immortality to the normal cells, we agree with the examiner's conclusion that one of ordinary skill in the art would have found it prima facie obvious to fuse normal cells with karyoplasts or cytoplasts of transformed cells, such as myeloma cells, for the purpose of imparting immortality upon the normal cells. Szoka and Veomett disclose that the desired genetic information may be transferred into a given cell by using less than a complete cell; Szoka using a synthetically produced vesicle containing the genetic information and Veomett using karyoplasts produced from the desired cell itself.
Turning to the dependent claims, we note that Veomett discloses Sendai virus as a fusion agent and the use of Cytochalasin B in obtaining karyoplasts or cytoplasts as required by claims 2 and 3. Inasmuch as Veomett discloses the obtention of karyoplasts and cytoplasts and their subsequent fusion to obtain a reconstructed cell, the alternative use of lysis or mechanical digestion of transformed cells as set forth in claim 4 on appeal to form karyoplasts or cytoplasts would have been prima facie obvious. Claim 17 requires that the karyoplasts or cytoplasts are obtained from myeloma cells, ascites tumor cells or Epstein-Barr virus infected cells. Conventional hybridoma technology confers immortality upon normal cells through use of myeloma cells. Therefore, myeloma cells would have been an obvious source of karyoplasts or cytoplasts in implementing the teachings of Szoka and Veomett. Inasmuch as the object of conventional hybridoma technology is to culture the obtained immortal cell line to produce a cell product, the requirements of claim 19 in this regard are similarly prima facie obvious from a consideration of this prior art.
*4 Appellants argue that Example 2 of Szoka uses HAT selective medium and therefore is not relevant to the claims on appeal which specify that a non-selective medium is used to obtain the hybrid cells. The examiner argues that HAT medium is used in Example 2 of Szoka as an assay to determine the presence of HGPRT and is not used to select hybrid cells.
The resolution of this issue depends upon whether the A9 cells used in Example 2 of Szoka are normal. If the cells are normal, then clearly the HAT medium is not being used as a selective medium as there would be nothing to select. If the A9 cells are transformed cells similar to the myeloma cells used in conventional hybridoma technology, then appellants' argument has some credence in that non-fused A9 cells would apparently proliferate as myleoma cells do in conventional hybridoma technology and selection for the fused cells in Example 2 of Szoka would be needed. However, as set forth above, appellants have stated that we are to assume that the A9 cells of Example 2 of Szoka are normal cells. Accordingly, appellants' arguments in this regard are not relevant [FN2].
Appellants argue on pages 15-17 of the Appeal Brief that Veomett does not fuse a whole cell with a fragment or use fusion material from two different sources. While the experiments performed in Veomett are as described by appellants, Veomett is not applied against the subject matter on appeal by itself. Szoka discloses that whole cells may be transformed by genetic information which is contained in a vehicle which is less than the whole natural cell. The two references considered together would suggest to one of ordinary skill in the art the use of a karyoplast or cytoplast to transfer desired genetic information into a whole cell via fusion. Such a hypothetical person would have recognized that normal cells and karyoplasts or cytoplasts are themselves not immortal so that the use of a selection medium would not be needed. Again, Veomett does state that the experimental work set forth in that reference establishes the feasibility of constructing mixed cells derived from parental cells of different types. Absolute predictability is not needed in order to reach a conclusion of obviousness under § 103. Rather, only a reasonable expectation of success is needed. In re O'Farrell, 853 F.2d 894, 7 USPQ2d 1673 (Fed.Cir.1988).
Turning to the examiner's alternative rejection of claim 20, we note that this claim is a product-by-process claim. Therefore, the patentability of claim 20 is to be determined based upon the product formed and not by the method by which it was produced. In re Thorpe, 777 F.2d 695, 227 USPQ 964 (Fed.Cir.1985).
The basis of the examiner's rejection is that the product encompassed by claim 20 on appeal when formed via a fusion of a whole normal cell and a karyoplast would resemble the cell-cell hybridomas formed in Sugimoto or Olsson after a few cell generations since the cytoplasm portion of the tumor cell used in forming the hybrids of the two references would be rapidly diluted and replaced over a period of time.
*5 The arguments made by appellants in the various Briefs concerning this rejection are directed to the purported lack of anticipation of claim 20 by the references. These arguments misapprehend the basis of the examiner's rejection. As set forth by the court in In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA1977):
Where, as here, the claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes, the PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his claimed product. ... whether the rejection is based on 'inherency' under 35 USC 102, on 'prima facie obviousness' under 35 USC 103, jointly or alternatively, the burden of proof is the same, and its fairness is evidenced by the PTO's inability to manufacture products or to obtain and compare prior art products [footnote omitted].
Here, the examiner's position is that the cell-cell hybrids of the references would resemble the products of the present invention when karyoplasts are fused with whole cells after the hybrids of the references have passed through a few cell generations due to loss of cytoplasm. We find that the examiner has properly shifted the burden of proof to appellants to establish through objective evidence that the claimed products do, in fact, differ from the hybrids of the references. Appellants have not established on this record through objective evidence that the examiner's position is incorrect. The reference relied upon by appellants in the Reply Brief in rebuttal of this rejection, Reid, is not directed to this issue.
The decision of the examiner is affirmed.
No time period for taking any subsequent action in connection with this appeal may be extended under 37 CFR 1.136(a). See the final rule notice, 54 F.R. 29548 (July 13, 1989), 1105 O.G. 5 (August 1, 1989).
BOARD OF PATENT APPEALS AND INTERFERENCES
Irving R. Pellman
William F. Smith
Andrew H. Metz
FN1. While appellants argue on page 10 of the Appeal Brief that the examiner has not established on this record that the A9 cells of Szoka are normal cells as required by the present invention, counsel stated at oral argument that for the purposes of this appeal it may be assumed that the A9 cells of Szoka are normal cells.
FN2. If prosecution is continued on this subject matter in a continuing application, appellants and the examiner should determine the specifics of the A9 cells used in Example 2 of Szoka so that such further prosecution need not be based upon assumptions.