`
`The opinion in support of the decision being entered today (1) was not written for
`publication in a law journal and (2) is not binding precedent of the Board.
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`Paper No. 57 V
`
`MAILED
`
`AUG I 3 1993
`
`PAT.&T.M. OFFICE
`BOARD OF PATENT APPEALS
`AND INTERFEPIENCES
`
`BEFORE THE BOARD OF PATENT APPEALS
`AND INTERFERENCES
`
`SHMUEL CABILLY, HERBERT L. HEYNEKER,
`‘WILLIAM E. HOLMES, ARTHUR D. RIGGS
`and RONALD B. WETZEL
`Junior party‘
`
`V.
`
`MICHAEL A. BOSS, JOHN H. KENTEN,
`JOHN S. EMTAGE and CLIVE R. WOOD
`Senior party?
`
`Patent Interference No. 102,572
`
`
`
`‘ Application 08/205,419, filed June 10, 1988. According benefit of Application
`06/483,457, filed April 8, 1983, now patent No. 4,816,567, issued March 28, 1989.
`Assignee for Genentech, Inc., South San Francisco, CA, A California Corporation.
`
`2 Application 06/672,265, filed November 14, 1984, now Patent No. 4,816,397,
`issued March 28, 1989. Accorded benefit of PCT application, PCT/GB84/00094, filed
`March 23, 1984 and UK application No. 83/08235, filed March 25, 1983. Assignee for
`Celltech Limited, Berkshire SL1 4DY, U.K., A British Company.
`
`1
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 959
`
`
`
`Interference No. 102,572
`
`Before RONALD H. SMITH, DOWNEY and SCHAFER,3 Administrative Patent Judges.
`
`DOWNEY, Administrative Patent Judge.
`
`
`FINAL DECISION
`
`The interference concerns a two step process for producing either an
`
`immunoglobulin (lg) molecule or an immunologically functional lg fragment comprising
`
`at least the variable domains of the lg heavy and light chains in a single host cell.
`
`The subject matter at issue is defined by a single count, which count is identical
`
`to claim 1 of the Boss et al. patent. The count reads as follows:
`
`Qount 1
`
`A process for producing an lg molecule or an immunologically functional
`lg fragment comprising at least the variable domains of the lg heavy and
`light chains, in a single host cell, comprising the steps of:
`
`(i) transforming said single host cell with a first DNA sequence encoding
`at least the variable domain of the lg heavy chain and a second DNA
`sequence encoding at least the variable domain of the lg light chain, and
`
`(ii) independently expressing said first DNA sequence and said second
`DNA sequence so that said lg heavy and light chains are produced as
`separate molecules in said transformed single host cell.
`
`Boss et al. claims 1-18 and Cabilly et al. claims 101-120 correspond to the count.
`
`During the preliminary motion stage of this proceeding, the administrative patent
`.___._________._____
`
`3 APJ Schafer has been substituted for APJ Pellman who has retired.
`Bose, 772 F.2d 866, 868-869, 227 USPQ 1, 2-4 (Fed. Cir. 1985).
`2
`
`In re
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 960
`
`
`
`Interference No. 102,572
`
`judge (APJ), granted the Boss et al. motion for benefit of the March 25, 1983 and March
`
`23, 1984, filing dates of their United Kingdom application, No. 83/08235 and PCT
`
`application, PCT/GB84/00094, respectively. With the granting of the motion for benefit,
`
`party Boss et al. became senior party in this interference.
`
`Boss et al. took no testimony and thus stand on their March 25, 1983,
`
`filing date
`
`accorded them during the motion period.
`
`Junior party Cabiily et al. raise the following issues in their brief (Brief, page 3):
`
`(1) does the record establish that Cabiily et al. actually reduced to practice the
`
`invention of the count prior to the March 25, 1983, effective filing date accorded Boss et
`
`al., and if not, then,
`
`(2) does the record establish that Cabiily et al. conceived of the invention of the
`
`count prior to the March 25, 1983, filing date accorded Boss et al. and proceeded with
`
`reasonable diligence to either an actual or constructive reduction to practice (April 8,
`
`1983) from a time prior to conception of Boss et al. (March 25, 1983).
`
`In addition, we have before us, a Cabiily et al. motion, pursuant to 37 CFR §
`
`1.635,
`
`to have certain Cabiily et al. pages, 224-231 attached to exhibit 8 and page 993
`
`attached to Exhibit 20, entered into the record (Paper No. 49). The motion stands
`opposed (Paper No. 50); and a reply was filed (Paper No. 51).
`
`The following issues have ggt been raised by the parties:
`
`(1) a question of no interference-in-fact;
`
`3
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 961
`
`
`
`Interference No. 102,572
`
`(2) a question of separate patentability of any clalm(s);
`
`(3)a question of whether Cabilly et al. claims are unpatentable. Boss et al. filed
`
`a motion forjudgment against Cabilly et al. claims during the motion stage, which
`
`motion was denied; Boss et al. do not seek review of this motion at final hearing; and
`
`(4) a question of whether Cabilly et al. rely upon attorney diligence for their
`
`priority case. Cabilly et al. allege priority based on conception coupled with reasonable
`
`diligence to filing of their application. Cabilly et al. could have but did not offer any
`
`evidence relating to attorney diligence in preparing and filing the Cabilly et al. patent
`
`application during the critical period.
`
`Cabilly et al. filed a record (CR) consisting of exhibits 1—20 (CX) 4 and the
`
`declarationsof coinventors: Arthur D. Riggs, Ph.D, (Riggs) and Shmuel Cabilly
`
`(Cabilly), employees of City of Hope; William E. Holmes (Holmes) and Ronald B.
`
`Wetzel, Ph.D., (Wetzel), employees of Genentech, |nc.; and corroborators Paul J.
`
`“ The record and exhibits will be referred to as CR and CX followed by the
`appropriate number.
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 962
`
`
`
`interference No. 102,572
`
`Carter, Ph.D (Carter)5, Michael B. Mumford (Mumford), L. Jeanne Perry (Perry), Michael
`
`W. Rey (Rey), all employees of Genentech and John E. Shively, Ph.D., (Shively), an
`
`employee of City of Hope. Boss et al. did not cross examine any of the witnesses.
`
`Both parties filed briefs and appeared through counsel at final hearing.
`
`Cabilly et al. motion to correct the record
`
`I.
`
`With their reply brief, Cabilly et al. filed a motion to have entered into the record
`
`certain pages which were referred to and relied upon in various declarations but were
`
`omitted from the record when it was filed and served upon Boss et al. The omission
`
`was first realized when Boss et al. noted, in their brief, that the pages were not in the
`
`Cabilly et al. record.
`
`The motion is gm. in view of the fact that Cabilly et al. referred to CX-8,
`
`pages 224-231 in the Wetzel and Perry declarations and CX-20, page 993 in the
`
`declaration, we find the failure to file these pages with their respective exhibits an
`
`oversight on the part of Cabilly et al.
`
`5 The Carter testimony was submitted in response to the Boss et al.
`motion for judgment; as noted the motion is not being reviewed at final hearing and thus
`the Carter testimony is not relevant to the issues before us and has not
`been considered in rendering this decision.
`
`5
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 963
`
`
`
`interference No. 102,572
`
`The Boss et al. arguments are without merit.
`
`It is true that the present rules do
`
`not require Boss et al. to notify Cabilly et al. of the oversight. As a matter of courtesy,
`
`Boss et al. could have notified Cabilly et al. of the omission when the exhibits were
`
`served. We find no prejudice to Boss et al. by entering the omitted exhibits into the
`
`record.
`
`Background:
`
`lmmunoglobulins (lg), also called antibodies, are protein molecules produced in
`
`vertebrates by B cells in response to foreign antigenic agents. (Boss et al. patent,
`
`column 1, lines 60-64, Cabilly et al. specification, page 1). The basic structure of all
`immunoglobulin molecules is a unit consisting of two identical light (L) polypeptide
`
`chains of molecular weight of approximately 23,000 daltons and two identical heavy (H)
`
`polypeptide chains of molecular weight 53,000-70,000 daltons, shaped to form a Y:
`
`
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 964
`
`
`
`interference No. 102,572
`
`Each H and L chain is held together by disulfide bonds to form a monomer, and the two ‘
`
`monomers are linked by disulfide bonds to form the basic dimeric structure of the
`
`molecule (Cabilly et al. specification, page 5). There are five classes or types of H
`
`chains, gamma, mu, alpha, delta, or epsilon which characterize an individual lg as an
`
`lgG, lgM, lgA, lgD or lgE, respectively; and two classes of chains, kappa (K) and
`
`lambda (A) (Cabilly et al. specification, page 5). Each antibody chain contains a
`
`variable region (V) and a constant region. The variable region is about 100 amino acids
`
`in length and is specific for the antigen which elicited it.
`
`(Cabilly et al. specification,
`
`page 6). The constant region does not take part in the binding of any antigenic
`
`determinant but does serve to link the antibody to other participants in the immune
`
`defenses, e.g. to fix complement, and thus makes an antibody bifunctional. The
`
`variable region of the H and L chain interact closely and when correctly folded form a
`
`three dimensional site at each branch of the Y for binding to a particular portion or
`
`epitope of the specific antigen which elicited the antibody(Cabi|ly et al. specification,
`
`page 5-6, and Boss et al. patent, column 1, line 60—column 2, line 47).
`
`Kohler and Milstein developed a technique that made it possible to produce
`
`monoclonal antibodies, i.e., homogenous antibodies of a single class and single,
`
`specificity, by the use of hybridoma technology. Monoclonal antibodies are produced in
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 965
`
`
`
`interference No. 102,572
`
`a laboratory by a hybridoma cell line, created by injecting the mouse with antigen,
`
`harvesting its spleen cells and fusing the same with cells from an immortal cancer cell
`line. Monoclonal antibodies are specific to one antigen which may have multiple I
`
`determinants or epitopes. Antibodies have the ability to detect and bind to antigens.
`The strength of the antibody-antigen binding is referred to as specificity and is
`
`quantatively measured by an affinity value.
`
`III.
`
`THE COUNT
`
`It is well-settled that, absent ambiguity, a count in an interference is to be given
`
`the broadest reasonable interpretation that the language of the count permits without
`
`resort to either party's disclosure. DeGeorge v. Bernier, 768 F.2d 1318, 1322, 226
`
`USPQ 758, 761 (Fed. Cir. 1985); Fontiin v. Okamoto, 518 F.2d 610, 618, 186 USPQ
`
`97, 103-104 (CCPA 1975); Lamont v. Berguer, 7 USPQ2d 1580, 1582 (Bd. Pat. App.
`
`& Int. 1988). We find the count is clear and unambiguous.
`
`Accordingly, we construe the count as being directed to a two step process for
`
`the production of either an lg molecule or an immunologically functional lg fragment
`
`encoding at least the variable domains of the lg heavy and light chains. The firststep
`
`comprises transforming a single host cell (e.g., E.COIi) with first and second DNA
`
`sequences encoding at least the variable regions of both the heavy chain and light
`
`chain and the second step comprises expressing, in the transformed host cell, the
`
`8
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 966
`
`
`
`Interference No. 102,572
`
`respective heavy and light chains as separate molecules.
`
`The count is broad enough to include adding a single plasmid or vector
`
`containing both the DNA sequences encoding for at least the variable regions of the
`
`heavy and light chains to the host cell or by adding two plasmids to the host cell each
`
`containing said DNA sequences individually. The count does not require that the
`
`expression step directly result in the production in the host cell of an lg molecule or an
`
`immunological functional fragment of lg containing at least the variable regions of the
`heavy and light chains.
`if the process results in the production of inclusion bodies,‘‘
`
`then in order to show that a useful product was produced it would be necessary to
`
`reassociate or refold after extraction of the inclusion bodies and denaturing its contents.
`
`Hence, while the count does not set forth any additional steps it is clear that the count
`
`does not exclude other process steps because the count utilizes the open-ended term
`
`A “comprising “. In re Baxter, 656 F.2d 679, 686-687, 210 USPQ 795, 802-803 (CCPA
`
`1931).
`
`IV.
`
`THE PARTIES BRIEFS
`
`The requirements for the parties briefs are set forth in 37 CFR § 1.656(b).
`
`in
`
`.__.___:___:g_—____.
`
`6 inclusion bodies are also known as refractile bodies. They are insoluble
`particles which require cell lysis and solubilization in denaturant to permit recovery.
`(Cabilly et al. specification, page 23, lines 18-21).
`9
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 967
`
`
`
`Interference No. 102,572
`
`particular, 37 CFR § 1.656(b)(4) requires:
`
`
`|a|n argument, which may be preceded by a summary, which shall contain
`the contentions of the party with respect to the issues to be decided, and
`the reasons therefor, with citations to the cases, statutes, other
`authorities, and part of the record relied on. (Emphasis added)
`
`Conclusions of fact and law made without appropriate citation to the record or citation of
`
`authority will be taken as attorney argument. Compare Ex parte McCullough, 7
`
`USPQ2d 1889, 1891 (Bd. Pat. App. & Int. 1988); Ex parte Myer, 6 USPQ2d 1966, 1968
`
`(Bd. Pat. App. & Int. 1988); In re Mehta, 347 F.2d 859, 863-864, 146 USPQ 284, 286
`
`(CCPA 1965). Attorney argument cannot take the place of evidence lacking in the
`
`record. Meitzner v. Mindick, 549 F.2d 775, 782, 193 USPQ2d 17, 22 (CCPA), cert.
`
`denied, 434 U.S. 854 (1977).
`
`V.
`
`The Cabilly et al. Case for Priority (as set forth in their brief):
`
`(1) Coinventor Riggs testified that during the period of July 1980 through
`
`September 1980 he was at Genentech on sabbatical from City of Hope.
`
`It was his
`
`intention to explore the possibility of producing antibodies in bacteria. According to
`
`Riggs, after returning to City of Hope, he submitted a proposal (CX—2)
`
`to Genentech
`
`on October 5, 1981, which was based in part on conversations he had with Dr.
`
`10
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 968
`
`
`
`Interference No. 102,572
`
`Heyneker7 and Cabilly. Riggs contended that he proposed the use of a single bacterial
`
`strain for coexpression of the heavy and light chain genes. Riggs, 11 3. Riggs stated
`
`that he discussed this project with Shively who was involved in the study of human anti-
`
`CEA3 antibodies and that he requested and received from Shively, on
`
`or about February, 1981, a mouse hybridoma cell line, CEA.66-E3, said to express
`
`anti—CEA antibodies. Riggs,
`
`1111 3-5 (CR-15-16).
`
`(2) Shively testified that he had conversations with Riggs regarding the project
`
`and that upon request from Riggs he supplied Riggs with the cells requested on or
`
`about February, 1981. Shively, 11 5 (CR—17).
`
`(3) Cabilly, a post doctoral fellow in Riggs‘ laboratory at City of Hope, testified
`that in September 1981, he received CEA.66-E3 cells from Shively and that he used
`
`these cells to extract total RNA. The polyA mRNA was purified from the total RNA by
`
`using an oligo-dT cellulose column. Following the isolation of the mRNA, Cabilly
`
`testified that he gave a sample of it to Holmes at Genentech for the preparation of an E.
`
`coli colony cDNA library. Cabilly 11 4~(CR-39).
`
`(4) Coinventor Holmes testified that on July 12, 1982 he began working on the
`
`7 Dr. Herbert Heyneker. a coinventor of the Cabilly et al. application, is said to be
`a senior scientist from Genentech; he did not testify in this proceeding.
`
`8 Carcinoembryonic antigen (CEA) is an antigen unique to humans and is found
`mainly in intestinal tumors. (CX-2, page 2).
`
`11
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 969
`
`
`
`interference No. 102,572
`
`project to express antibodies directed against human CEA in E. coli. Holmes, ‘[1 3.
`Holmes testified that he received a sample of polyA mRNA from City of Hope and
`
`prepared cDNA which was incorporated into plasmids to make a E. coli colony library.
`
`Holmes, 11 4. He testified that he inoculatedicolonies into microliter plates and the
`
`cultures therefrom were stamped onto agar plates and allowed to grow. Holmes, 1l 5
`
`(CR-29).
`
`(5) Rey, a research assistant at Genentech reporting to Heyneker, testified that
`
`he began work on the project in July 1982 when he received microliter dishes with
`
`cultures containing cDNA from the hybridoma cell line CEA.66-E3. He transferred
`
`these cultures to agar plates and allowed them to grow and later transferred the
`
`colonies to nitrocellulose filters, layered them onto agar plates and allowed them to
`
`grow. Once grown. he lysed the colonies on the filters and treated them for subsequent
`
`probing. Rey, 11 3 (CR-33).
`
`(6) Holmes used oligonucleotides from the Organic Chemistry Department at
`
`Genentech to prepare light and heavy chain oligonucleotide probes to hybridize with the
`
`filters. After exposure to X-ray film, he picked several colonies which hybridized to the
`
`light or heavy chain oligonucleotide probes, characterized the colonies by Pst
`
`restriction endonuclease digestion9 and fractionation by [sodium dodecylsulfate(S‘DS)]
` _:_:__.:
`
`9 Restriction digestion is a process involving the use of enzymes which recognize
`different DNA sequences and cleave the DNA backbone at the site recognized forming
`either blunt or stick ends.
`
`12
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 970
`
`
`
`Interference No. 102,572
`
`polyacrylamide [gel] electrophoresis (PAGE)‘°. Several colonies which hybridized to
`
`the heavy chain probe were also digested with both Pstl and Ncol and analyzed by
`PAGE. Holmes subcloned these DNA sequences into M13 vectors. Holmes, 11 6,11 7
`
`(CR—30).
`
`(7) Rey testified that he assisted in the sequencing of the heavy chain cDNA by
`
`subcloning DNA into M13 vectors, preparing single-stranded template and carrying out
`
`the sequencing reactions. Rey also testified that he assisted in the sequencing of the
`
`heavy and light chain cDNA's. Rey, ‘II 4 (CR-33).
`
`(8) Holmes testified that he and Heyneker analyzed the sequences and found
`
`that the entire coding region of the light chain was found in the cDNA insert of pK17G4
`
`and that portions of the nucleotide sequence of the heavy chain were found in two
`
`isolated plasmids: pGamma298 and pGamma11. Holmes, ‘[1 7.
`
`(9) Holmes indicated that the plasmid, pKCEA|nt 2, for direct expression of the
`
`anti-CEA light chain gene was prepared from five DNA fragments, 1-5. According to
`
`Holmes, fragment 1 was prepared by digesting pHGH207-1* with EcoRl, filling in“ and
`
`digesting with BamHI. Following purification of this fragment, Holmes stated that he
`
`‘O ASDS-PAGE stands for sodium dodecylsuIfate-polyacrylamide gel
`electrophoresis which is a techniquewhich separates various species of proteins or
`polynucleotides of different sizes in an electric field.
`
`" The Cabilly et al. brief (page 11)al|eges that the “filling in" was done with DNA
`polymerase I large fragment . No one testified as to how the filling in was done.
`‘Meitzner, 549 F.2d at 782, 193 USPQ at 22.
`
`13
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 971
`
`
`
`Interference No. 102,572
`
`treated the DNA with bacterial alkaline phosphatase (BAP). The large fragment
`
`(fragment 1) was purified by PAGE. Holmes, 11 8 (CR-30). For fragment 2, Holmes
`
`testified that he digested pK17G4 DNA with Pstl, purified the fragment by PAGE, 1
`
`digested with Avall and isolated the 333 bp‘Pst|-Avall fragment by PAGE; he used the
`
`Pstl-Avall fragment and an oligonucleotide primer in a primer repair reaction to
`
`introduce the initiation codon to the light chain gene. Following the primer repair, Rey
`
`sequenced a Pstl to Avall DNA fragment of the light chain. Rey, 11 5 (CR-34). Holmes
`
`indicated that he and Heyneker analyzed the sequencing results . Holmes, 11 9 (CR-30).
`
`The fragment was purified by PAGE, cleaved with Sau3A and the 182 bp fragment
`
`isolated by PAGE (fragment 2). Holmes, 11 9 . Thereafter, fragments 1 and 2 were
`
`ligated together and the ligation reaction transformed into E. coli. The resultant
`
`transformants were analyzed by restriction digestion and sequencing to confirm the
`
`construction of pKCEAlnt1.” Holmes, 11 10. To prepare fragment 3, Holmes testified
`
`that he digested pK17G4 DNA with Pstl and purified the fragment by PAGE. This
`
`fragment was partially digested with Avall, filled in and purified by PAGE. This
`
`fragment was subsequently digested with Hpall and the 497 bp fragment isolated by
`
`PAGE (fragment3). Holmes, 11 11 (CR-31). For fragment 4, Holmes testified that he
`
`digested‘ the plasmid pKCEAlnt1 with Aval, filled in and digested with Xbal. The large
`
`
`'2 The Cabilly et al. brief (page 11, first paragraph) alleges that this analysis and
`isolation of pKCEAlnt1 was done on or about October 30, 1982. No one testified as to
`this date.
`i_d.
`
`14
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 972
`
`
`
`Interference No. 102,572
`
`fragment was treated with BAP and isolated by PAGE (fragment 4). Holmes, 11 12. The
`small fragment was digested with Hpall and the 169 bp fragment isolated by PAGE
`(fragment 5). Ho|mes,‘11 12 (CR-31). Fragments 3, 4 and 5 were ligated and the
`
`ligation reaction transformed into E.coIi. Resultant transformants were analyzed by
`
`restriction digestion to confirm the construction of pKCEAlnt2. Holmes, 11 13 (CR-31).
`
`(10) Cabilly testified that he modified plasmid pKCEAtrp207—1* by cleaving out
`
`the Pstl-Pvul fragment from the ampicillin resistance gene, filling it in and relegating the
`
`blunt ends to yield plasmid pKCEAtrp207-1*delta which is resistant to tetracycline but
`
`sensitive to ampicillin. Cabilly 11 5 (CR-39).
`
`(11) According to Holmes, six fragments, A-F, had to be isolated to make the
`
`heavy chain expression plasmid, pGammaCEAlnt2. To make the first fragment,
`
`Holmes digested pHGH207-1* with Aval, filled in, digested with BamHI, treated with
`
`BAP and purified the large fragment by PAGE (fragment A). Holmes, 11 14. He digested
`
`pGamma11 with Pstl, the fragment was purified by PAGE, digested with Avall, filled in,
`
`and digested with Taql. The 375 bp fragment was isolated by PAGE (fragment B).
`Holmes, 11 15. He digested pGamma298 with Taql, BamHI, and isolated the 496 bp
`
`fragment by PAGE (fragment C). Holmes, 11 16. Holmes ligated fragments A, B and C
`
`and transformed the ligation reaction into E. coli. The resultant transformants were
`
`analyzed by restriction digestion to confirm the construction of pGammaCEAlnt1.
`
`Holmes, 1117 (CR-31). Holmes used a 15 nucleotide DNA primer in a primer repair
`
`15
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 973
`
`
`
`lnterference No. 102,572
`
`reaction to introduce the initiation codon into an Alul to Rsal fragment of pGamma 298
`
`(fragment D). Holmes, 1) 18 (CR—31). He digested pGamma298 with Pstl, BamHl,
`
`Hpall and purified the fragment by PAGE (Fragment E). Holmes, 1] 18 (CR-31). He
`
`digested pGammaCEA|nt1 with EcoRl, filled in, and digested with BamHl. The then
`
`treated this fragment with BAP and purified the fragment by PAGE (Fragment F).
`
`Holmes, 1] 18. He then ligated fragments D, E and F and transformed the ligation
`
`reaction into E. coli. The plasmid, pGammaCEA|nt2 was said to be confirmed by
`
`restriction analysis and sequencing. Holmes, ‘ll 18 (CR-31).
`
`(12) Holmes stated that he prepared the expression plasmid
`
`pGammaCEAtrp207—1* when he digested plasmid pBR322(Xap) with EcoR|, filled in,
`
`digested with Pstl, and purified by PAGE. He isolated a 1543 bp fragment by treating
`
`pGammaCEA|nt2 with Pstl followed by BamHl and purification by PAGE. He also
`
`isolated a 869 bp fragment from pGammaCEA|nt2 by digestion with Aval, filling in,
`
`cleaving with BamHl and subsequent PAGE purification. He then ligated these
`
`fragments, transformed the ligation reaction into E. coli and analyzed the resultant
`
`colonies by restriction analysis to confirm pGammaCEAtrp207—1*.” Holmes, 1] 19
`
`(CR-31-32).
`
`(13) Accordingly to Cabilly hetransformed competent E. coli cells with
`
`13 The Cabilly et al. brief (page 13) argues that this work was done by December
`8, 1982. This argument is also not supported by any testimony. Q.
`
`16
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 974
`
`
`
`Interference No. 102,572
`
`pK§)EAtrp207—1*de|ta and retransformed the successful E.coIi cells with
`
`pGammaCEAlnt2 which confers resistance to ampicillin but not to tetracycline.
`Cabilly,1[6 (CR—39-40). V He grew the cotransformed cells in minimal media containing
`
`ampicillin and tetracycline and induced the cultures with indoleacrylic acid (IAA) to
`
`make refractile body preparations. Cabilly testified that he gave a sample to Jeanne
`
`Perry for SDS-PAGE analysis. Cabilly indicated that he analyzed several samples by
`
`SDS-PAGE; subsequently these were silver stained or subjected to Western blot using
`
`anti—mouse IgG for the identification of light and heavy chain protein.” Cabilly, ‘ll 7 (CR-
`
`40).
`
`(14) Perry indicated that the first sample she analyzed from a "cotransformed
`
`refractile body preparation" was supplied to her by Cabilly. She analyzed this sample
`
`by PAGE. Perry, 11 12 (CR-26).
`
`(15) Cabilly testified that he also constructed pGammaCEAFABtrp207-1*, a
`
`plasmid vector for the direct expression of the FAB fragment of the heavy chain gene.
`
`(CR-40). Accordingly to Cabilly, he digested pBR322 with Hindlll, filled in, digested
`
`with Pstl and treated with BAP. He isolated the vector fragment by PAGE (fragment l).
`
`Cabilly, ‘ii 9. Cabilly indicates that he received a sample of pGammaCEAtrp207-1* from
`
`Ho|mes,‘he digested this plasmid with BamHI and Pstl and isolated the fragmentby
`
`“‘ The Cabilly et al. brief (page 14, sec. 4) alleges that the Western Blot used
`rabbit anti—mouse primary antibodies and ‘—2§|-labeled protein A. This is attorney
`argument. id.
`
`17
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 975
`
`
`
`Interference No. 102,572
`
`P'AGE (fragment ll). Another sample of this plasmid was digested with Ncol and Ndel.
`
`he isolated the 260 bpbDNA by PAGE. He used a 13 bp oligonucleotide primer in a
`
`primer repair reaction" in order to introduce a termination codon. The fragment was then
`
`digested with BamHI, the 179 bp fragment isolated by PAGE, filled in (fragment Ill).
`
`Fragments I,
`
`II and ill were ligated and transformed into E. coli. Cabilly, 1] 9. These
`
`transformants were said to be analyzed by Rey, Holmes and Cabilly by restriction
`
`cleavage analysis and sequencing.” Cabilly, 11 9, Holmes, 11 20 and Rey, 11 7 (CR—40-
`
`41).
`
`(16) Cabilly testified that he conducted a refolding experiment with the material
`
`from the cotransformed heavy and light chain E. coli cells. He grew the
`
`cotransformants, lysed them by sonication, solubilized the pellet with guanidine
`
`hydrochloride and incubated this material overnight at room temperature. He then,
`
`dialyzed the reaction mixture against urea buffer at room temperature followed by
`
`dialysis into phosphate buffered saline (PBS). Cabilly testified that he performed an
`
`assay to detect active anti-CEA antibody. He indicated that he found the heavy chain
`
`and light chain protein had recombined to yield antigen binding activity significantly
`
`higher than background. Cabilly, 11
`
`(CR-40).
`
`(17) Mumford, an employee of Genentech, was responsible for microbial
`
`‘5 The Cabilly et al. brief (page 14) alleges that this analysis was performed on
`or about January 22, 1983. No one testified as to this date for the analysis. id.
`
`18
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 976
`
`
`
`interference No. 102,572
`
`fermentation optimization of gene products. Mumford, 11 2 (CR-35) . The record shows
`
`that on three occasions between December 13, 1982 to March 25, 1983, he or
`someone in his lab received and recorded receipt of labeled microbial samples.
`Mumford, 1111 5-7 (CFt—36). Of specific interest is the receipt on February 2, 1983 of
`
`W3110/p102 and W311O/p62 from Heyneker's laboratory. Mumford, 1] 7. Mumford
`recorded:
`
`[T]hese two organisms are E. coli strains, which had been co-transformed
`with two plasmids for the co-expression of heavy and light chain of an
`anti—CEA antibody. These samples were used to prepare the DMSO
`stocks 1246-31 and 1246-32, respectively.
`(11 7) (CR—36)
`
`(18) Fermentations were run on these two stock solutions.” Thereafter, on
`
`February 14, 1983, Mumford recorded the fermentations in CX-18. Mumford, ‘ll 13
`
`(CR-38).
`
`(19) Coinventor Wetzel, a senior scientist at Genentech, testified that he had
`
`some success in folding other recombinant proteins and his help was enlisted on the
`
`project. Wetzel, 11 5 (CR 19-20). He and Perry, a research associate in his lab, began
`
`working on the project in January, 1983. Wetzel, 1] 6 (CR-20) , and Perry, 11 2
`
`(CR-21-22 ).
`
`Initially they attempted to isolate and purify the heavy and light chains
`
`producedpln two different E. coli strains from cell pastes received by Mumford. Wetzel,
`
`$17 (CR-20).
`
`
`‘6 The Cabilly et al. brief (page 16) alleges that the fermentations occurred on
`Feb. 8, 1983. Rey did not give any specific run dates for these fermentations.
`lg.
`19
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 977
`
`
`
`Interference No. 102,572
`
`(20) Perry” testified that their strategy to refold the heavy and light chains from
`
`singly transformed bacteria was to first purify the refractile bodies from the bacteria,
`
`soiubiiize the protein in denaturant, followed by sulfitolysis. The chains would then be
`further purified by S3(lO gel filtration chromatography and possibly DEAE ion—exchange
`
`chromatography. The plan was to then reconstitute the antibodies by folding the heavy
`
`chain first, adding it to the light chain, allowing both chains to fold together and then
`
`oxidize the disulfide bonds. Perry, 1[ 3 (CR-22). She testified that they tried this
`
`strategy and found a loss of heavy chain protein after the removal of the denaturant by
`
`dialysis into native buffer.
`
`In order to alleviate proteolysis, they tried adding PMSF,
`
`EDTA, EGTA, and altering pH and temperature. Protease was found to be inactivated
`
`by addition of PMSF, Perry, 11 7 (CR-23-24), and protease could be removed by DEAE
`
`ion exchange chromatography. Perry, 11 8 (CR-24).
`
`(21) The strategy to reconstitute immunoglobulin chains also included the
`
`comparison of the refolding results of heavy and light chains from the anti—CEA
`
`antibodies produced from hybridoma cells. Perry, 1] 10. The antibodies were said to be
`
`supplied by Cabilly. Optimal conditions were determined and used.” Perry, 1] 10.
`
`(22) Perry stated that later they found that they could isolate the heavy and light
`
`‘7 Other than referring to January, 1983, Perry, in her testimony, does not set
`forth any date for the work she did or observed.
`
`‘8 The Cabilly et al. brief (page 19, last 5 lines-page 20, line 1) lists a number of
`conditions not identified by Perry in her testimony. Q.
`
`20
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 978
`
`
`
`interference No. 102,572
`
`chains from both single and cotransformed bacteria by guanidine solubilization of
`refractile body preparations without any further purification by column chromatography.
`
`These samples could then be used directly in the refolding reactions. The final
`
`conditions involved a mixture of sulfitolized‘ heavy chain and an extract of light chain
`
`cells. The chains were reconstituted using the same conditions as the optimal
`
`conditions for the reconstitution of antibody chains produced in hybridoma cells. Perry,
`
`‘ll 11 (CR-26).
`
`(23) Wetzel testified that he recorded in his notebook the results of a Western
`
`blot of SDS-PAGE run by Perry. He testified that from the blot they noted production of
`
`heavy and light chain protein product in the co-transformed E.coIi cells and that they
`
`were able to estimate the level of expression (%) from cell paste from Mumford. Wetzel
`
`testified that the results were used to calculate the theoretical maximum possible yield
`
`which were in turn used to calculate % yield. Wetzel, 1] 9 (CR-25).
`
`(24) Perry testified that she performed a refolding experiment on the
`
`cotransformed cell paste received from Mumford. The paste was sonicated and
`
`centrifuged to isolate the refractile bodies. Perry analyzed the refractile body
`
`preparations by SDS-PAGE. The sample was resuspended in urea, dialyzed, and then
`
`assayed-. Later Perry analyzed another reconstitution experiment from denaturant
`
`solubilized refractile body preparations of cotransformed cells. Perry found that the
`
`heavy and light chains were insoluble, and that dialysis into urea was necessary to
`
`21
`
`Genzyme Ex. 1044, p
`
`Genzyme Ex. 1044, pg 979
`
`
`
`Interference No. 102,572
`
`obtain activity. She stated “[T]he reconstitution of cotransformed cell extracts utilizing
`
`the optimal conditions for the reconstitution of hybridoma cell produced antibody chains
`was significantly higher than the background". Perry, 11 13 (CR26-27).
`
`(25) Wetzel testified that he and Perry , between March 18, and March 24, 1983,
`
`performed an experiment in which C