CONTROL NOS. 90/007,542 AND 90/007,859
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`DOCKET NOS. 22338~10230 AND -1 0231
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`CONTROL NOS. 90/007,542 AND 90/007,859
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`ATTORNEY DOCKET NOS. 22338-10230, »l023l
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`Control Nos.:
`
`Confirrnation Nos.:
`
`90/007,542
`'90/007,859
`
`7585 (’542)
`6447 (’859)
`
`Filed:
`'
`
`Patent Owner:
`
`13 May 2005
`23 December 2005
`
`(’542)
`C859)
`
`Genentech, Inc. and
`City of Hope
`
`Group Art Unit:
`
`3991
`
`Examiner:
`
`B.M. Celsa
`
`For:
`
`Merged Reexaminations of U.S. Patent No. 6,331,415 (Cabilly et al.)
`
`DECLARATION OF MICHAEL BOTCHAN UNDER 37 C.F.R. § 1.132
`\
`
`1, Michael Botchan, do hereby declare and state:
`
`1.
`
`.
`
`I am a citizen of the United States, and reside in Kensington, California. My CV. is
`
`I
`
`attached as Exhibit A.
`
`2.
`
`I have been retained by Genentech and City of Hope to provide my opinion on certain
`
`issues in the patent reexamination proceedings involving U.S. Patent No. 6,331,415.
`
`I
`
`am being compensated for my time at a rate of $550 per hour.
`
`3.
`
`H
`
`I am not now affiliated with either Genentech or City of Hope.
`
`I served as an expert for
`
`Genentech in City ofHope Nat 7 Med. Center v. Genentech, Inc., Case No. BC215 1 52
`
`(Los Angeles Co. (Cal.) Super. Ct.), and provided deposition testimony in that litigation.
`
`4.
`
`I have reviewed the following documents in the course of preparing this declaration:
`
`Cabilly eta1., U.S. Patent No. 6,331,415 (the ’415 patent)
`
`Cabilly et al., U.S. Patent No. 4,816,567 (the ’567 patent)
`
`Moore et al., U.S. Patent No. 5,840,545 (the ’545 patent)
`
`Moore et al., U.S. Patent No. 4,642,334;
`Moore et al., U.S. application no. 06/358,414 (the ’414 application)
`Boss et al., U.S. Patent~.No. 4,816,397
`
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`GENENTECH 2018
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`GENENTECH 2018
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`CONTROL NOS. 90/007,542 AND 90/007,859
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`DOCKET NOS. 22338-10230 AND -10231
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`CONTROL NOS. 90/007,542 AND 90/007,859
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`ATTORNEY DOCKET NOS. 22338-10230, -10231
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`-
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`-
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`-
`
`—
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`Axel et al., U.S. Patent No. 4,399,216
`
`Rice et al., Proc. Nat ’l Acad. Sci. USA 79: 7862-65 (1982)
`
`Kaplan et al., EP 0044722
`
`Builder et al., U.S. Patent No. 4,511,502
`
`Accolla er al., Proc. Nat ’l Acad. Sci. USA 77: 563-66 (1980)
`
`Dallas, W0 82/03088
`
`Deacon et al., Biochem. Soc. Trans. 4: 818-20 (1976)
`
`Valle et al., Nature 291: 338-40 (1981)
`
`Valle et al., Nature 300: 71-74 (1982)
`
`Ochi et al., Nature 302: 340-42 (1981)
`
`Oi et al., Proc. Nat ’lAcad. Sci. USA 80: 825-29 (1983)
`
`5.
`
`V I have also reviewed the documents associated with the two reexamination proceedings,
`
`including the PTO communication dated February 16, 2007 (the Office Action).
`
`6.
`
`I understand that patentability is to "be evaluated using the perspective of a person of
`
`ordinary skill in the technical field of the invention just prior to the filing date of the
`
`patent (i.e., in this case, early April of 1983). A person of ordinary skill in the field of the
`
`’4l 5 patent would have had a Ph.D. in molecular biology or a comparable scientific
`
`discipline and two to three years of postdoctoral experience.
`
`I believe I am well-qualified
`
`to express an opinion on what a person of ordinary skill in the art of the ’4l5 patent
`
`would have believed or expected in early April of 1983 because I worked with many
`
`people at that time with these qualifications.
`
`7.
`
`I understand that the ’545 patent issued in 1998 from an application filed on June 5, 1995.
`
`I also understand that there were several earlier applications filed between 1982 and
`
`1995, and that the first of these was the ’414 application, which was filed in March of
`
`1982.
`
`I understand that the question of what is described in the ’4l4 application (the
`
`1982 application) relative to what is described in the ’545 patent is an issue in this
`
`reexamination proceeding.
`
`8.
`
`I have been asked to explain the techniques described in the_ ’414 application and whether
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`there is a description of a host cell that produces two different polypeptide chains/or a '
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`I
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`_ process which produces two different polypeptide chains in a single host cell in that
`application.
`I have also been asked to address what a person of ordinary skill in the art in
`early April of 1983 would have taken away from the infonnation in a variety of patents
`
`and publications, and whether that information would have made the coexpression
`
`procedures in the ’415 patent claims obvious at that time.
`
`Analysis ofthe '414 Application and the '545 Patent
`
`9.
`
`I
`
`The ’4l4 application describes procedures for cloning DNA that were conventional in
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`early April of 1983. At that time, it was known that to “clone” a DNA sequence, you
`would:
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`-
`
`-
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`»
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`-
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`isolate or prepare desired DNA;
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`insert the DNA into a vector;
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`insert the vector into a host cell, and grow the host cell;
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`isolate the copies of the DNA (within the vector) from the host cell culture
`(which now contains multiple progeny of the cells, and therefore multiple
`copies of the vector containing the desired DNA).
`
`10. A
`
`The ’4l4 application describes cloning procedures having these steps at pages 5, line 16,
`
`I
`
`to page 9, line 20.
`
`1].
`
`The process for isolating DNA encoding the individual immunoglobulin chains is
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`described at page 6, line 14 to page 8, line 7 of the ’414 application. First, an mRNA
`
`extract is produced from a hybridoma that is making a desired antibody. This niRNA
`
`extract will contain many different mRNA “transcripts” corresponding to the messengers
`
`of the genes being expressed in the cell. Each of the mRNA transcripts is a discrete
`molecule containing a sequence corresponding to the amino acid sequence of a single
`polypeptide encoded by the DNA in the cell. The mixture of mRNA transcripts isolated I
`
`from the hybridoma in the ’4] 4 application will contain mRNA transcripts produced
`
`during transcription of the immunoglobulin light chain gene, and different mRNA
`transcripts produced during transcription of the immunoglobulin heavy chain gene.
`
`There will be no mRNA transcripts in the extract that contain sequences from both heavy
`
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`and light chain genes, because the mRNAs for the chains are encoded by different genes
`
`expressed from separate promoters at different chromosomal positions.
`
`12.
`
`The mRNA extract is then purified and used to prepare a cDNA library. The process as
`
`described is standard for the time, as described at page 7, line 37 to page 8, line 7. It
`
`involves using the “reverse transcriptase” enzyme that ‘produces a complementary DNA
`
`(cDNA) molecule corresponding to each mRNA transcript in the purified mRNA extract.
`
`Again, because no mRNA transcript will contain sequences for both heavy and light
`
`chains, no individual cDNA in this cDNA library willcontain heavy and light chain
`
`sequences.
`
`13.
`
`The next step described in the application is amplification of the cDNA library.
`
`Amplification involves incorporating all of the cDNA molecules in the cDNA library into
`
`individual plasmids, and then inserting the plasmids into cells in culture by a
`
`transformation process. This procedure is specified at 8, line 12, to page 9, line 19. The
`
`procedures being described make it absolutely clear that each plasmid incorporates a
`
`single cDNA encoding a light or heavy immunoglobulin chain, and that each bacterial
`
`cell transformed will contain one plasmid.
`
`-
`
`At page 8, lines 15-18, the application states that “the ds cDNA obtained from the
`
`reverse transcription of the mRNA” is being used. As I explained above, each
`
`discrete ds cDNA molecule in the cDNA library encodes only one
`
`immunoglobulin polypeptide sequence because it is produced from individual
`
`mRNA transcripts in the mRNA extract.
`
`-
`
`Thedesign of the plasmid indicates that one cDNA insert will be incorporated
`
`into each plasmid. See page 8, lines 20~24 i(“. .. the vector will have a unique
`
`restriction site in one of multiple markers so that transformants may be selected
`
`by the expression of one marker and the absence of expression of the other
`
`marker”). Certainly, this is the desired outcome.
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`ATTORNEY DOCKET NOS. 22338-10230, -10231
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`-
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`The selection of screening techniques for bacterial clones indicates that each
`
`clone has one plasmid with one cDNA in it. The descriptions of these techniques
`
`could not be clearer in stating that each clone contains one plasmid with one
`
`cDNA encoding only one of the two immunoglobulin chains. Specifically, at
`
`page 9, lines 10-13, the ’414 application states:
`
`The host colonies, usually bacterial, which have DNA which
`hybridizes to either the light or heavy chain probes are picked and
`then grown in culture under selective pressure.
`
`14.
`
`After each clone has been propagated in culture, the bacterial cells‘ are lysed_, and the
`copies of the plasmid are isolated, sequenced, and subjected to restriction mapping. The
`
`sites for specific restriction enzyme hydrolysis are mapped on the genome of the plasmid.
`
`The sequencing and restriction mapping techniques in the application indicate that
`
`individual cDNA sequences encoding the light or heavy immunoglobulin chain are being
`
`used in the process. For example, at page 9, lines 22-31, the application states:
`
`These analyses insure that the isolated cDNA clones completely encode the
`variable region and, optionally, the leader sequences for the light or heavy
`_c_l;a_ip_ of the desired immunoglobulin. Furthermore, by having a restriction
`map of the variable regions and leader sequences, as well as the flanking
`sequences, one can determine the appropriate restriction sites for excising a
`DNA fragment which will allow for appropriate modification of the DNA
`sequence for insertion into a vector and expression of the polypeptide of
`interest. (emphasis added)
`
`15.
`
`Someone who was familiar with basic molecular biology principles would know that
`
`unless special steps were taken to culture the bacterial cells under “selective pressure,”
`
`those cell cultures will become uniform with respect to plasmid content within each cell.
`
`Specifically, if a bacterial cell is transformed with a plasmid that contains an antibiotic
`
`resistance gene, copies (clones) of that bacterial cell can be selectively cultivated by
`
`adding the relevant antibiotic to the cell culture (i.e., the antibiotic kills the cells that have
`
`not incorporated the plasmid). This concept of selective pressure is central to the design
`
`of genetic engineering experiments. In the case of the ’4l4 application, the procedures
`
`employ cell culture techniques that use only a single source of selective pressure (i.e., a
`
`single antibiotic is used to exert selective pressure on transformed cells).
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`16.
`
`In my opinion, a person of ordinary skill also would have known that if a bacterial cell
`
`was transformed to contain two plasmids that contain the same marker and regulatory
`
`elements, within an overnight period of growth, in the absence of appropriate selective
`
`pressure, the bacterial culture would be devoid of “double transformants.” This is the
`
`consequence of several aspects of cell biology.
`
`—
`
`-
`
`First, propagation of bacterial cells is geometric (one cell divides into two, two
`
`divide into four, etc.) and the final number of cells in the culture is limited by
`
`nutrient resources and other competitive forces in the culture medium.
`
`Second, transformation efficiency using procedures prevalent in the early 1980’s
`
`were low — approximately one in 10,000 bacterial cells would incorporate a
`
`foreign plasmid. See S. N. Cohen et al., Proc. Nat’1Acad. Sci, USA 69:2110-
`
`2114, 21 12V( 1972).
`
`In the absence of some strategy to increase the odds of
`
`incorporation of two different plasmids, the probability of one cell incorporating
`
`two different plasmids during a single transformation step is roughly the square of
`
`the rate of transformation with a single plasmid (ie, one in 108). This means that
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`a “double transformant,” if it were produced at all, would be vastly outnumbered
`
`in the culture medium by “single” transformants.
`
`-
`
`Finally, and very significantly, bacterial cells exhibit “plasmid incompatibility”
`
`when plasmids with the same regulatory elements but different neutral genetic
`
`elements (e.g., cDNA inserts which have no selective influence on the plasmid
`
`replication or survival) are inserted into a cell. B. Polisky, Cell 552929-932, 929
`
`(1988). This incompatibility results from the mechanisms of plasmid DNA
`
`replication as follows.
`
`1.
`
`Individual plasmids within a cell are chosen randomly from the pool to be
`
`replicated.
`
`2.
`
`Thetotal number of plasmids within a cell are under strict copy number
`
`control, and once a copy number is achieved, repression of plasmid
`
`replication occurs.
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`ATFORNEY DOCKET NOS. 22338-10230, -1023!
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`3.
`
`Thus the progeny of any given cell will contain one or the other of the
`
`original _rare “double transformants” but not both. This is because a bias is
`
`introduced toward one or the other of the plasmids in the first cell
`
`doubling (see points 1 and 2 above). This bias is amplified in each
`
`successive doubling until all copies of the other plasmid are lost to the
`
`progeny of the first transformed cell.
`
`17. When all these factors are considered together, it would be very clear to a person skilled
`in this field that a bacterial cell culture, left alone, will eventually be dominated by the
`
`“most successful” bacterial clones. Given the natural forces exerted on these cells during
`
`propagation, and in the absence of multiple sources of selective pressure, a bacterial cell
`
`culture that contained one or more double transformants would, soon would contain
`
`effectively no progeny of that double transforrnant that maintained the two different
`
`plasmids.
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`.
`
`18.
`
`Accordingly, I do not believe a person skilled in the field of molecular biology would
`
`have read any of the sections of the ’414 application as describing transformation
`
`procedures where bacterial cells are being transformed with two different plasmids.
`
`None of the steps listed in the application indicate that two different plasmids should be
`inserted into a single host cell, and there is no description of any strategy for exerting
`
`selective pressure to cause a cell culture to maintain “doubly transformed” clones. There
`
`are simply no suggestions of these types of techniques or approaches anywhere in the
`
`’414 application.
`
`19.
`
`Someone who was familiar with molecular biology techniques also would immediately
`
`recognize that the amplification steps described in the ’4l4 application involve
`
`manipulations of individual plasmids. For example, the restriction mapping procedures
`described in the application are procedures where arrays of fragments of a nucleotide
`
`sequence are produced by enzymatic digestion of a particular nucleotide sequence.
`
`Restriction maps may be used to confirm the presence or absence of a particular DNA
`
`sequence (here the sequence encoding either the immunoglobulin heavy or light chain) in
`
`a particular transformed cell. Ordinarily, each map is produced from a single sequence,
`
`because of the complexity of mapping multiple sequences from a single test medium. If
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`these steps were being performed on mixtures of plasmids or different cDNA sequences,
`
`the application would have clearly indicated this.
`
`20. i
`i
`
`The next step in the process is preparation of the tailored cDNA sequences encoding the
`A variable region sequence of each immunoglobulin chain. To do this, a short oligomer ‘is
`
`synthesized which will hybridize to a region in the light or ‘heavy chain variable region
`
`being produced. The oligomer incorporates a “stop codon” which will temiinate
`
`translation of the cDNA at the end of the variable region. The oligomer is combined with
`
`a restriction fragment encoding the light or heavy chain variable region from the
`
`amplified cDNA step (see paragraph 12 above). After the oligomer hybridizes to the V
`
`restriction fragment, it is enzymatically elongated to produce a DNA strand
`
`complementary to the original source heavy or light chain sequence — except that it has
`
`the incorporated stop codon at the end of the variable region. This produces a double
`
`stranded (ds) cDNA sequence encoding the variable region and upstream flanking
`
`regions of the immunoglobulin chain sequence being manipulated.
`
`21.
`
`This ds cDNA is referred to as a “heteroduplexed” ds cDNA in the application, meaning
`
`that the two DNA strands are not 100% complementary. The application also refers to
`
`the plasmid into which this ds cDNA is incorporated as a “hybrid” plasmid because it
`
`,
`
`contains “mismatched” sequences.
`
`22.
`
`I note that these hybridization techniques described in the ’414 application depend on the
`
`use of specific reaction conditions (eg, temperature, salt concentration, etc.) appropriate
`
`for forming each heteroduplex. Because the “melting temperature” of a heteroduplex
`
`will be sensitive to a particular mix of these conditions, it would not be possible to
`
`manipulate both a heavy chain cDNA and a light chain cDNA to introduce stop codons in
`
`the same reaction mixture.
`
`23.
`
`As the application points out, when the resulting plasmid is incorporated into a bacterial
`
`cell, and the cell divides; one of the daughter cells will contain a ds cDNA corresponding
`
`to the original or “native” sequence (i.e., produced from the mRNA extract); and the
`
`other daughter cell will contain a ds cDNA having the sequence of the “tailored” cDNA.
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`24.
`
`The clones containing the tailored sequence are then amplified. I note that at page 12,
`
`where this amplification step is described, all of the references to the tailored cDNA
`indicate that it encodes only one polypeptide. See, e.g., page 12, lines 15~18 (“... to
`
`provide‘ individual clones replicating me tailored sequence”) (emphasis added). After
`
`amplification, the tailored cDNA is treated to introduce a start codon at the other end (the
`
`5' terminus) of the sequence. See, page 12, line 26 to page 14, line 15. Once that is
`
`completed, the tailored cDNA is incorporated into a plasmid for expression of the tailored
`
`gene and production of the desired polypeptide.
`
`25.’
`
`At this point, the application is crystal clear that individual heavy chain and light chain
`
`polypeptides are being produced in separate cell cultures. At page 14, the process for
`
`preparing an expression vector is described. As it explains, the vector (plasmid) contains
`
`the transcriptional and translational regulatory signals required for successful expression
`
`of an introduced cDNA sequence. This indicates that each vector will be instructing the
`
`cell to express only a single cDNA inserted into the vector. Even in the very general
`
`guidance provided at page 15, lines 6 to 19, the application indicates that the host cells
`
`, are being engineered to produce a single polypeptide. See, e.g., page 15, lines 9 to 11
`(“the availability of vectors which allow for insertion of the ds cDNA sequence into the
`
`vector and expression of the variable region polypeptide"). See also page 16, lines 33 to
`37 (“The ribosome binding site and variable-region initiation codon may be properly
`
`spaced to optimize expression of the variable region polypeptide”).
`
`26.
`
`If there were any doubt that the procedures described in the ’4l4 application are designed
`
`to produce one polypeptide in each host cell, that doubt is erased by the explanation of
`
`the procedures for isolation and purification of the expression product, and preparation of
`the rFv. For example, at page 15, lines 27~30, the application states that the polypeptides
`
`3
`
`made by this procedure “are prepared as a homogeneous composition containing identical
`
`sequences and chain lengths.” If each cell were producing a mixture of heavy and light
`
`chain variable region polypeptides, the composition would not be “a homogeneous
`
`composition containing identical sequences.”
`
`27.
`
`Then, the application outlines the procedure for assembling the rFv. In very clear terms,
`
`the application indicates that each‘ transformed host cell will produce one of the two
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`variable region polypeptides, and then, after each is isolated, the polypeptides will be
`
`combined outside of the cell to form the rFv. As the application explains at page 16,
`
`lines 24 to 29:
`
`'
`
`The resulting construct is then introduced into an appropriate host to provide
`expression of the heavy or light polypeptide members of the rFv and the
`polypeptides isolated. The heavy and light polypeptide members of the rFv
`are then combined in an appropriate medium to form the rFv.
`
`28.
`
`This one protein-one host cell concept is reiterated throughout the section of the
`
`application describing polypeptide isolation procedures. See, for example, page 17, line
`
`35, to page 18, line 7:
`
`Where the light or heavy chain is not secreted, the transformed
`microorganisms containing the appropriate ds cDNA for either light or
`heavy chains are grown in liquid cultures and cleared lysates prepared.
`The bound variable regions are eluted from the column with an appropriate
`denaturing solvent. The eluates from each of the heavy and light chain
`isolations are pooled, followed by treatment to renature the polypeptides to
`form L-rFv and H—rFv respectively.
`
`29.
`
`Again, this makes it absolutely clear that each variable region polypeptide is produced in
`
`a separate host cell.
`
`30.
`
`The application contains an example that illustrates use of the general procedure outlined
`
`earlier. See Example 1, starting at page 19, line 10 and continuing to page 42, line 13. I
`
`note that several details in this example clearly demonstrate that only one variable region
`
`polypeptide will be produced in each host cell.
`
`31.
`
`At pages 40~4l, the example describes a procedure where each tailored cDNA is
`
`incorporated into a separate plasmid. As the application states at page 41, lines 6 to 15,
`
`“the ‘tailored’ pGMl is isolated, partially restricted with PstI and the DNA sequences
`
`coding for the light and heavy chain variable regions prepared above inserted individually
`into the tailored site to provide two plasmids having DNA sequences coding for the light
`
`(pGM1L) and heavy (pGMll-I) chains
`
`. The resulting plasmids are used to transform
`
`E. coli HB 101 and clones having the light and heavy variable region sequences in the
`
`desired orientation identified by restriction mapping and purified.’’_
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`32.
`
`0 Reading this, a person familiar with basic molecular biology techniques would
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`immediately see several points that would erase any doubt about the procedures being
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`described.
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`-
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`-
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`First, because separate plasmids are being produced, the “genetic constructs" used
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`for expression will contain only one cDNA encoding either the light or heavy
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`chain variable region polypeptide, not both.
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`Second, because the same plasmid (pGMl) having the same regulatory elements
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`and antibiotic resistance gene is used to prepare the two plasmids (ie., the ‘
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`pGMlH plasmid containing the heavy chain sequence, and the pGMlL plasmid
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`containing the light chain sequence), the two plasmids are clearly not being
`incorporated into the same host cell. The PstI site used in the pGM1 plasmids lies
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`within the ampicillin resistance gene. See F. Bolivar et al., Gene 2295-113, 95
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`(1977). Inserting a cDNA at this site will render the ampicillin resistance gene
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`non-functional. Host cells transformed with this plasmid thus will be resistant to
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`tetracycline, but not ampicillin. Transforming a single cell culture with both
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`plasmids would make little sense in this process as it is described. One cannot use
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`tetracycline to select “double transformants” using the methods described because
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`of the plasmid incompatability mechanism discussed above. Furthermore, using
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`only tetracycline, it would not have been possible to differentiate the host cells
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`that had been transfonned with the first plasmid, the second plasmid, or both.
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`Thus, any reading of the ’4l4 application reveals a strategy where only a single
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`plasmid is to be propagated in an individual clone.
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`-
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`Third, as I explained above, the few bacterial clones in the culture that might have
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`incorporated two different plasmids would quickly be outnumbered by clones
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`containing only one of the plasmids. The culture would then become uniform and
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`not contain any of these “double transformants" —— especially since no selective
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`pressure for double transformants could be exerted by the scheme that has been
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`described.
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`EvIDENc€‘Z\Tr9l5t§Ni3Ii32
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`PAGE ‘PAGE B595
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`

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`CONTROL NOS. 90/007,542 AND 90/007,859
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`DOCKET NOS. 22338-10230 AND -1 0231
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`CONTROL NOS. 90/007,542 AND 90/007,359
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`ATTORNEY DOCKET NOS. 22338-10230, -10231
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`~
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`Finally, the use of restriction mapping as described to confirm successful
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`transfonnation indicates to me that each host cell is being transformed with, and
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`will contain, only one plasmid containing one cDNA encoding a single
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`polypeptide sequence.
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`33.
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`34.
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`3
`
`1
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`The example also follows the procedures outlined earlier in the application for isolating
`the individual light chain polypeptides and combining them in vitro to fon'n the rFv.
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`Specifically, at page 41, lines 29-35, the application refers to plural column extracts (“the
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`supematants are passed over the immunoabsorbant columns”), and at page 52, lines l~3,
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`it refers to the process of mixing these separately prepared eluates to form the rFv (“the
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`renatured heavy and light chains of the rFv are further purified by combining the eluates
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`containing the rFv components”).
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`As I indicated earlier, I do not believe a person familiar with basic molecular biology
`techniques and concepts in early April of 1983 could have read these various sections of
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`the application and in any way conclude the application is describing a procedure for
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`coexpression of heavy and light chain variable region sequences in a single transformed
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`host cell.
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`35.
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`The PTO indicates that it interprets portions of the application as stating that individual
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`bacterial clones containing DNA encoding both the heavy and the light immunoglobulin
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`chains will be produced. For example, in the Office Action at page 20, the PTO states
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`that there is a disclosure of “a ‘host cell’ transformed with a single genetic construct
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`or
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`two separate constructs
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`comprising DNA encoding variable light and heavy chains.”
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`The PTO indicates specific passages in the ’545 patent support this reading.
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`I do not
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`agree.
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`36.
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`As I explained earlier, the procedures outlined in the application will not produce any
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`single cDNA that contains heavy and light chain sequences. The cDNA being amplified
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`during the initial steps is produced via reverse transcription of an mRNA extract. None
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`of the mRNA transcripts will contain sequences corresponding to both immunoglobulin
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`chains because the immunoglobulin chains are encoded by separate genes.
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`Consequently, none of the cDNA molecules will contain heavy and light chain
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`ATTORNEY DOCKET NOS. 2233 8-l0230, -10231
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`sequences. Similarly, the procedures for producing the tailored cDNA make it clear that
`the cDNA will not contain heavy and light chain sequences. Also, the application
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`contains no description of a procedure where two different cDNA sequences are
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`incorporated into a single genetic construct (e.g., a single plasmid): Instead, both in the
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`general description and in the example, each cDNA is incorporated into a separate
`plasmid. Thus, there is no description in the ’414 application of a procedure for
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`producing two different polypeptides in a single transformed host cell. I could find
`nothing that supports "the PTO’s view that the ’4l4 application describes a host cell that
`contains a single genetic construct or two separate constructs containing cDNAs
`V
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`encoding variable light and heavy chains (pages 20-21 of the Office Action). The
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`sections of the patent the PTO identifies certainly do not describe what the PTO states.
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`-
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`Col. 5, lines 32-35, is simply indicating that many vector choices were available
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`for amplification and expression of cDNA sequences. This is the opening
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`sentence of a section of the patent that is explaining a process where cDNAs in a
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`‘library are amplified. The “host cells” made during this process each incorporate
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`a single plasmid, into which has been inserted a single cDNA from the cDNA
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`library. None of these cells will contain a single plasmid with two cDNA inserts,
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`or {two plasmids each with a different cDNA.
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`-
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`Col. 7, lines 39-50, is describing the steps where the “hybrid” plasmid containing
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`the “mismatched” DNA strands is being replicated. The mismatched double
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`stranded sequence separates during division of the transformed cell, and forms
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`two sets of double stranded cDNA, one with the native sequence, and one with the
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`tailored sequence. Each new plasmid is contained in one daughter cell;‘this is
`basic cell biology. As I explained above (see paragraph 31), the sections
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`preceding this passage clearly indicate that a single cDNA encoding either the
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`light or the heavy chain variable region is used to prepare the “mismatched”
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`double-stranded cDNA. There is no indication here that two different cDNAs are
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`to be inserted into a single host cell for amplification.
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`EvIDENcI';Bi2l‘l9l‘-‘lé’IlIl3‘ri<”
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`DOCKET NOS. 22338-10230 AND ~10231‘
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`CONTROL Nos. 90/007,542 AND 90/007.359
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`.
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`-
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`Col. 10, lines 1~5, is describing the process of inserting start and stop codons at
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`either end of the coding sequence for the variable region polypeptide. This is not
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`describing a host cell transformed with a single genetic construct containing
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`cDNA encoding light and heavy chain variable region polypeptides, or two
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`plasmids that encode, respectively, light and heavy chain variable region
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`polypeptides.
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`-
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`Col. 23, lines 35-45, is describing an example corresponding to the “hybrid
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`plasmid” replication step outlined at col. 7, lines 39-50. This section concerns
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`cDNA cloning and amplification, not protein expression. As I explained above
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`(see paragraph 19), the process of generating “tailored” sequences involves
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`insertion of a plasmid containing a mismatched double—stranded cDNA sequence
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`into a host cell. When the cell replicates, it produces daughter cells that contain
`double stranded copies of the two mismatched /cDNA sequences. In addition, it is
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`absolutely clear that each of the tailored cDNA sequences is inserted into a
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`separate plasmid, and only one plasmid is incorporated into each transformed cell.
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`Each of these cDNA inserts (and thus each plasmid) encodes only one of the two
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`immunoglobulin polypeptides. There is no description at this point of the patent
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`of a host cell with one plasmid that has incorporated two different cDNA ins