Board of Patent Appeals and Interferences
Patent and Trademark Office (P.T.O.)
*1 EX PARTE DAVID F. MARK, LEO S. LIN, SHI-DA YU LU AND ALICE M. WANG
Appeal No. 88-2811
July 24, 1989
HEARD: May 11, 1989
Application for Patent filed February 7, 1985, Serial No. 06/698,939, which is a continuation-in-part of Serial No. 06/564,224 filed December 20, 1983, now Patent No. 4,518,584; which is a continuation-in-part of Serial No. 06/486,162 filed April 15, 1983, now abandoned; which is a continuation-in-part of Serial No. 06/435,154 filed October 19, 1982. It is also a continuation-in-part of Serial No. 06/698,936 filed February 7, 1985, which is a continuation-in-part of Serial No. 06/670,360 filed November 9, 1984, which is a continuation-in-part of Serial No. 06/661,026 filed October 15, 1984. Cysteine-Depleted Muteins of Biologically Active Proteins.
Supervisory Patent Examiner--Thomas G. Wiseman
Before Goldstein, Pellman and W. Smith
This is an appeal from the final rejection of claims 1 through 5 and 45 through 69. The appeal as to claims 54 and 56 was withdrawn by appellants' counsel at oral argument. [FN1] Thus, claims 1 through 5, 45 through 53, 55 and 57 through 69 remain for our consideration, which are all of the claims remaining in the application.
Claims 1, 45, 54, 55, 56, 57 and 64 are illustrative of the subject matter on appeal and read as follows:
1. A synthetic mutein of a biologically active native protein in which native protein has at least one cysteine residue that is free to form a disulfide link and is nonessential to said biological activity, said mutein having at least one of said cysteine residues substituted by another amino acid and said mutein exhibiting the biological activity of said native protein.
45. A structural gene having a DNA sequence that encodes a synthetic mutein of a biologically active native protein which native protein has at least one cysteine residue that is free to form a disulfide link and is nonessential to said biological activity, said mutein having at least one of said cysteine residues substituted by another amino acid and said mutein exhibiting the biological activity of said native protein.
54. A method of preventing a protein having at least one cysteine residue that is free to form a disulfide link from forming said link comprising mutationally altering the protein by deleting the cysteine residue or replacing the cysteine residue with another amino acid.
55. The method of claim 54 wherein the protein is biologically active and the cysteine is not essential to said biological activity.
56. The method of claim 54 wherein the cysteine residue is replaced with serine or threonine.
57. A method for making a gene having a DNA sequence that encodes a synthetic mutein of a biologically active native protein which native protein has at least one cysteine residue that is free to form a disulfide link and is nonessential to said biological activity, said mutein having at least one of said cysteine residues substituted by another amino acid and said mutein exhibiting the biological activity of said native protein comprising:
*2 (a) hybridizing single-stranded DNA comprising a strand of a structural gene that encodes said protein with a mutant oligonucleotide primer that is complimentary to a region of said strand that includes the codon for said cysteine residue or the antisense triplet paired with said codon, as the case may be, except for a mismatch with said codon or said antisense triplet which mismatch defines a triplet that codes for said other amino acid;
(b) extending the primer with DNA polymerase to form a mutational heteroduplex; and
(c) replicating said mutational heteroduplex.
64. An oligonucleotide for use in making a structural gene, said gene having a DNA sequence that encodes a synthetic mutein of a biologically active native protein which native protein has at least one cysteine residue that is free to form a disulfide link and is nonessential to said biological activity, and said mutein having at least one of said cysteine residues substituted by another amino acid and said mutein exhibiting the biological activity of said native protein, by oligonucleotide-directed mutagenesis, said oligonucleotide having a nucleotide sequence that is complimentary to a region of the strand of the structural gene that includes the codon for the cysteine residue or the anti-sense triple paired with said codon, as the case may be, except for a mismatch of said codon that defines a triplet that codes for said other amino acid.
The reference relied upon by the examiner is:
Mark et al. (Mark '584) 4,518,584 May 21, 1985
A reference relied upon by the Board is:
Mark et al. (Mark '585) 4,588,585 May 13, 1986
The sole rejection of the claims remaining on appeal is under 35 USC § 112, first paragraph, as being nonenabled. In support of the rejection, the examiner relies upon a statement of prior art which appears at page 3, lines 22-line 34 of the present specification and at column 1, lines 55-56 of U.S. Patent No. 4,518,584 to Mark et al., one of the present parent applications, which reads as follows:
In this regard Shepard, H.M., et al, Nature (1981) 294:563-565 describe a mutein of IFN-B in which the cysteine at position 141 of its amino acid sequence (there are three cysteines in native human IFN-B at positions 17, 31, and 141, Gene (1980) 10:11-15 and Nature (1980) 285:542-547) is replaced by tyrosine. This mutein was made by bacterial expression of a hybrid gene constructed from a partial IFN-B cDNA clone having a G ?? A transition at nucleotide 485 of the IFN-B gene. The mutein lacked the biological activity of native IFN-B leading the authors to conclude that the replaced cysteine was essential to activity.
The review of the newly allowed claims with the inventors in light of the presently available data concerning the claimed species revealed that the seven Cys to Ser substitutions possible within the mature CSF-1 sequence shown in Figure 5 each result in a substantial reduction in the in vitro colony stimulation assay specified in claim 53. Thus, the ser@90CSF-1 species claimed in claim 20 (and also in claims 22 and 29) does not meet the requirement specified by claim 53. Nevertheless, applicants are of the view that the DNA encoding the ser@90CSF-1 species as well as the other Cys substitution species may have other uses, as experimental probes for example. Accordingly, claim 20 which specifically claims ser90CSF-1 has been made independent. For the above described reasons, the ser@90CSF-1 species has been deleted from claims 22 and 29.
*3 The examiner's rejection is summarized at page 3 of the Examiner's Answer as follows:
Essentially, the position taken in the rejection is that it would require undue further experimentation to construct by recombinant methods (site specific mutagenesis) the innumerable muteins encompassed by the instant claims (claims encompass modification of any protein which comprises a "non-essential" cysteine residue) and to screen the muteins produced for any of those which exhibit biological activity after modification.
The examiner further reasons that it is generally known in the art that cysteine residues facilitate the proper disulfide bonds and consequently the proper folding of a protein. The examiner concludes that it is likely that most of the muteins prepared by appellants' claimed methodology "would be inoperative simply because the removal of the cysteine would disturb proper folding of the molecule, thereby potentially blocking the active site or sites of the resulting mutein." (Examiner's Answer, page 4)
The examiner points out on page 6 of her Answer that the claims on appeal encompass any protein, even those which have not been characterized or cloned and that the mere sequencing of all possible proteins encompassed by the claims on appeal, would entail an undue amount of experimentation.
As set forth on page 7 of the Appeal Brief:
Appellants' position is that given the disclosure of the present invention substituting a nonessential cysteine with a neutral amino acid, the nonessential cysteine residues of any candidate protein could be identified and substituted in ten days employing the methods disclosed in the instant disclosure and the general knowledge of the art at the time the application was filed. Such limited amount of experimentation based on the disclosure in the application and the success shown by three proteins certainly does not constitute undue experimentation.
These arguments are supported by the declaration of co-appellant Alice M. Wang filed under 37 CFR 1.132 on August 10, 1987. In her declaration Ms. Wang sets forth what she terms a reasonable scheme for determining which cysteine residues in a generic biologically active protein would be available for substitution without destroying the biological activity. The declaration sets forth a step-by-step scheme for implementing the claimed invention which parallels the disclosure of the present application.
The examiner sets forth on page 5 of her Answer that Ms. Wang's declaration does not refute the determination that undue experimentation is needed for implementation of the claimed invention because of the "limited succcessful embodiments shown and the established unpredictability associated with such modifications as to how many such site-specific mutageneses would need to be undergone to obtain even one alternative biologically active mutein."
We have carefully considered the respective positions of the examiner and the appellants and find that we agree with appellants that the claims remaining on appeal are enabled by the present disclosure. The working examples of the present specification set forth experiments which establish that three proteins, IFN-B, IL-2 and TNF, have nonessential cysteine residues which may be deleted or replaced with the resulting mutein retaining the biological activity of the native protein. When it is considered that the claims remaining on appeal all require that the mutein produced retain the biological activity of the native protein, we consider the disclosure of this application to be enabling. The passages relied upon by the examiner from Mark '584 and copending Serial No. 06/876,819 are merely examples of work which is outside the claims on appeal. The record before us establishes that for a given protein having cysteine residues, one skilled in the art would be able to routinely determine whether deletion or replacement of the cysteine residues would result in a mutein which is within the claims on appeal.
*4 To the extent that the examiner is concerned that undue experimentation would be required to determine other proteins suitable for use in the present invention, we find Ms. Wang's declaration to be persuasive that only routine experimentation would be needed for one skilled in the art to practice the claimed invention for a given protein. The fact that a given protein may not be amenable for use in the present invention in that the cysteine residues are needed for the biological activity of the protein does not militate against a conclusion of enablement. One skilled in the art is clearly enabled to perform such work as needed to determine whether the cysteine residues of a given protein are needed for retention of biological activity.
The examiner's rejection under 35 USC § 112, first paragraph, is reversed.
NEW GROUND OF REJECTION
The present application lists four co-inventors, Mark, Lin, Lu and Wang. Appellants state on page 1 of the present specification that this application has two lines of parent applications. It is the first line of parent applications, i.e., Serial Nos. 06/564,224, 06/486,162 and 06/435,154 which is of present interest.
This application is stated to be a continuation-in-part of Serial No. 06/564,224 which is a continuation-in-part of Serial No. 06/486,162 which is a continuation-in-part of Serial No. 06/435,154. Each of these parent applications lists only three inventors, Mark, Lin and Lu. Wang, who is a co-inventor of the present application, is not a co-inventor in the parent applications. Mark '584 issued from Serial No. 06/564,224. Mark '585 is a division of Serial No. 06/564,224, and shares common parentage with Mark '584 of Serial Nos. 06/486,162 and 06/435,154.
In order for the present claims to be entitled under 35 USC § 120 to the benefit of the earlier filing date of any of the parent applications, their subject matter must be disclosed in the parent applications in the manner provided by 35 USC § 112, first paragraph, including the description requirement of this section of the statute. In re van Langenhoven, 458 F.2d 132, 173 USPQ 426 (CCPA 1972).
Here, our review leads us to the conclusion that the earliest filing date the present generic claims are entitled to is the December 20, 1983 filing date of parent application Serial No. 06/564,224 since this appears to be the first application in this chain which sets forth a generic description of the synthetic muteins of the present invention. Parent application Serial No. 06/486,162 describes only a synthetic mutein of IFN-B. The entire original disclosure of Serial No. 06/486,162 describes and is strictly limited to synthetic muteins of IFN-B except for original claim 20 of that application which was directed to "a nucleotide primer for mutagenesis, comprising an oligonucleotide of about 12 to about 24 bases." The specification of this application contains a corresponding disclosure of this generic nucleotide primer. However, comparing this generic disclosure of a nucleotide primer with that of the present application, i.e., claim 64, it is apparent that claim 20 of this parent application does not provide descriptive support for the broader oligonucleotide disclosed and claimed in this application. Thus, none of the present claims are entitled to the benefit of the earlier filing date of Serial No. 06/486,162, at best, only Serial No. 06/564,224.
*5 Having made this determination, we find that Mark '584 or Mark '585 is available as prior art against the appealed claims under 35 USC § 102(e) as these patents are by "others" having the effective filing date required by this section of the statute. The effective filing date of these two references, to the extent they disclose synthetic muteins of IFN-B, is October 19, 1982, the filing date of common parent application Serial No. 06/435,154. They are anticipatory of the claims included in this rejection in that these references describe the IFN-B synthetic mutein species of the present generic claims. In re May, 574 F.2d 1082, 1089, 197 USPQ 601, 607 (CCPA 1978).
Claims 68 and 69 are rejected under 35 USC § 103 as being unpatentable over Mark '584 or Mark '585.
These claims are directed to a therapeutic formulation which comprises an effective amount of the mutein of the present invention and at least one other anti-cancer or anti-viral compound, e.g., gamma-interferon. While Mark '584 and Mark '858 describe such a therapeutic formulation, this description does not appear in common parent Serial No. 06/486,162. This parent application only indicates that the synthetic mutein of IFN-B is useful for treatment of viral infections, and various types of cancer where interferon therapy is indicated.
However, in view of the disclosed utility of the synthetic mutein of IFN-B as an anti-cancer or anti-viral compound in Serial No. 06/486,162, it would have been prima facie obvious to one of ordinary skill in the art to use the disclosed synthetic muteins of IFN-B in combination with other known anti-cancer or anti-viral compounds such as gamma-interferon. In re Kerkhoven, 626 F.2d 846, 205 USPQ 1069 (CCPA 1980).
Any request for reconsideration or modification of this decision by the Board of Patent Appeals and Interferences based upon the same record must be filed within one month from the date of the decision (37 CFR 1.197). Should appellants elect to have further prosecution before the examiner in response to the new rejection under 37 CFR 1.196(b) by way of amendment or showing of facts, or both, not previously of record, a shortened statutory period for making such response is hereby set to expire two months from the date of this decision.
37 CFR 1.136(a) does not apply to the times for taking any subsequent action in connection with this appeal.
REVERSED, 37 CFR 1.196(b).
BOARD OF PATENT APPEALS AND INTERFERENCES
Irving R. Pellman
William F. Smith
FN1. It became apparent at oral argument that appellants' invention revolves around the present synthetic muteins retaining the biological activity of the native protein. The method of claims 54 and 56 is not so limited. When this was brought to counsel's attention during oral argument, counsel orally withdrew claims 54 and 56 from appeal.