B e f o r e :
| Regeneron Pharmaceuticals Inc
|- and -
Novo Nordisk A/S
Michael Tappin QC & James Whyte (instructed by Powell Gilbert LLP) for the First Defendant
Piers Acland QC (instructed by DLA Piper UK LLP) for the Second Defendant
Hearing dates: 18-20, 23- 27, 30 November and 7 & 8 December 2015
|VDJ recombination / somatic hypermutation||11|
|Immunoglobulin Locus Size||19|
|Screening for Integration||30|
|Making Targeting Vectors||33|
|BACs and BAC Libraries||37|
|The Expert Witnesses||39|
|Sir Martin Evans||39|
|The Witnesses of Fact||52|
|Dr Andrew Murphy||52|
|Dr Yancopoulos, Professor DeFranco and Professor Ishida||53|
|The Skilled Team||55|
|Common General Knowledge||60|
|An undiscovered 5' enhancer (construction and infringement)||61|
|Size of insertions and deletions by homologous recombination (sufficiency)||66|
|Homology arms (sufficiency)||69|
|Site-specific recombination (sufficiency)||70|
|BACs and BAC libraries (sufficiency)||76|
|Modifying the endogenous mouse Ig loci (inventive step)||80|
|Inactivating the endogenous murine locus (construction)||100|
|LTVECs and the MOA assay||102|
|Materials and Methods of Example 3||127|
|The first proposed approach||130|
|The second proposed approach||137|
|The light chains||140|
|The Claims in Issue||141|
|In situ replacement||148|
|Scope of Claim 1 of 287||174|
|Claims 5 and 6 of the 287 Patent||177|
|Claim 1 of the 163 Patent||184|
|Inversion and displacement upstream of mouse sequences||193|
|Infringement of the claims in issue||195|
|Ordinary methods of trial and error/undue burden||203|
|Excessive claim breadth||208|
|Insufficiency: the facts||215|
|Claim 1 of 287 – breadth of claim||216|
|The second proposed approach of Example 3||229|
|The importance of Example 3||230|
|The Macdonald paper||233|
|Multiple insertions without deletion||239|
|Repetition of the homologous recombination process||244|
|Assessment of insufficiency||257|
|Further insufficiency objections||259|
|Effect of long homology arms||260|
|The reduced amount of DNA||262|
|Identifying the 5' end||273|
|Submissions following circulation of the draft judgment||274|
|The Prior Art||290|
|The 287 Patent||312|
|The 163 Patent||320|
Mr Justice Henry Carr:
(a) Muromonab-CD3 (anti CD3) approved in 1986, which was one of the earliest examples of a mouse monoclonal antibody;
(b) Abciximab (glycoprotein IIb/IIIa) approved in 1994, a chimeric antibody;
(c) Daclizumab (anti CD25) approved in 1997, a humanised antibody;
(d) Rituximab (anti CD20) approved in 1997, which was one of the best known examples of a chimeric antibody;
(e) Palivizumab (anti respiratory syncytial virus glycoprotein) approved in 1998, a humanised antibody;
(f) Infliximab (anti TNF) approved in 1998, a chimeric antibody;
(g) Basiliximab (anti CD25) approved in 1998, a chimeric antibody; and
(h) Alemtuzumab (anti CD52) approved in 2001, which was one of the best known examples of a humanised antibody.
VDJ recombination / somatic hypermutation
The typical organisation of an immunoglobulin molecule composed of two identical heavy chains and two identical light chains. The lines that connect the two heavy chains and that connect the light chain to the heavy chain are disulphide bonds.
Immunoglobulin Locus Size
General scheme for gene targeting by homologous recombination: (a) the targeting vector consists of a left and right homology arm that flanks the payload to be inserted; (b) the locus to be targeted; and (c) homologous recombination may occur anywhere within the arm of homology resulting in insertion of the payload as well as portions of the homology arms. The position of recombination is indicated by the hatched vertical line.
Cre/Lox recombination. Left: The green triangles denote the sequence elements that constitute the LoxP site. If two LoxP sites in the same orientation flank a stretch of DNA (insert), then expression of active Cre recombinase will result in excision of that DNA insert. This reaction is in principle reversible, as indicated, however the intramolecular reactions are favoured over intermolecular reactions (left half of diagram). For inversions (right) the reactions in either direction are intramolecular and thus have more or less equal probability. Right: If the LoxP sites are in opposite orientation, then the insert is inverted upon expression of active Cre recombinase, as indicated by the blue arrowhead.
Screening for Integration
a) PCR involves amplification of a DNA fragment spanning the integration site, using a primer specific to a sequence in the exogenous DNA and a primer specific to a sequence in the endogenous chromosome, adjacent to the homology arm or site-specific recombination site.
b) Southern blotting involves using restriction enzymes to cut the chromosome at two sites, one within the exogenous DNA and one within the endogenous chromosome, outside of the homology arm or site-specific recombination site. The fragments are separated by size using gel electrophoresis, transferred to a membrane, and sequences of interest are identified by hybridization with a labelled homologous DNA probe. The restriction enzyme(s) and probe(s) are chosen so that a change in fragment size will be detected when there is a correct integration event.
Making Targeting Vectors
BACs and BAC Libraries
The Expert Witnesses
Sir Martin Evans
"For the development of a powerful technology for manipulating the mouse genome with exquisite precision, which allows the creation of animal models of human disease."
In 2007 he shared the Nobel Prize for Physiology and Medicine (with Mario Capecchi and Oliver Smithies) for the "Discovery of principles for introducing specific gene modifications in mice by the use of embryonic stem cells". He is a pre-eminent mouse embryologist, and a leader in his field.
The Witnesses of Fact
Dr Andrew Murphy
Dr Yancopoulos, Professor DeFranco and Professor Ishida
The Skilled Team
"Q. If you hit a problem in this field, it is often unclear what the cause of the problem is.
Q. Let alone how to fix it.
A. Absolutely, and the standard protocol would be to try not to change more than one parameter at a time. Things really go wrong when you have got two faults at the same time. So you try to get back to the point where there is one fault which you can then test, and that would be the standard scientific technique in this sort of field."
Common General Knowledge
An undiscovered 5' enhancer (construction and infringement)
"An attractive alternative would be to replace the mouse Ig loci with the human Ig loci; in this way it would be possible to retain and exploit any possible regulatory sequences in the mouse loci that are located distal to protein coding regions."
"Although the human genes [in existing transgenic mice] can utilise expression, selection and mutation mechanisms provided by the mouse B-cells, the efficiency with which they do so is clearly reduced. It will be interesting to find out whether the provision of V or C region genes or as yet unidentified regulatory sequences - in short, driving the generation of antibody repertoires from large almost locus size regions - provides a transgenic construct with authentic expression capabilities."
Size of insertions and deletions by homologous recombination (sufficiency)
Homology arms (sufficiency)
Site-specific recombination (sufficiency)
BACs and BAC libraries (sufficiency)
"I agree with Prof. Stewart at paragraph 64 of his report that DNA purification and sequencing from BACs was considered simple and straightforward at the Priority Date. However I disagree with him at paragraph 66 that the incomplete knowledge of the mouse genome did not allow ready identification of BACs. Whilst it is true that the entire nucleotide sequence of the mouse genome had not been published at the Priority Date, extensive work had already been carried out to characterise the mouse genome. By the Priority Date unique sequence markers had been identified along the entirety of the mouse gene at an interval of around 300 kb… In addition many gene specific markers have also been identified… Using the information available at Priority Date the Skilled Genetic Engineer would have been able to design a probe from almost any region of the mouse gene and this would have been standard laboratory work."
"A. I do agree with that. I have indeed done these sorts of exercises myself and when I approached this case, when in the first time I was thinking about this I thought, okay, it is fine, doing chromosomal walking, fishing out BAC clones, that is obviously straightforward and I was very surprised to then find out about the troubles that Regeneron had to isolate the V regions from the mouse genome.
Q. So let us leave Regeneron on one side for a moment. When you say "the troubles", did you get that from the VelocImmune History document or did you go to the notebooks to find this?
A. No, in the course of discussions about the case I became aware of ----
Q. Discussions with?
A. With my legal colleagues.
Q. Right, but you have not been through the notebooks yourself.
A. No, I have not."
Modifying the endogenous mouse Ig loci (inventive step)
"From the work described in the Rajewsky publications, the Skilled Immunologist was aware that one of the implications of engineering the endogenous locus (as opposed to using random insertions) was that the introduced DNA need not provide all the sequences necessary for antibody production. This applied both to regulatory sequences and protein coding sequences. … This is in contrast to random insertions, for which the introduced DNA has to provide all of the regulatory and protein coding sequences necessary for antibody rearrangement and expression. The Skilled Immunologist therefore knew that targeting to the endogenous locus brought with it a flexibility to retain certain mouse sequences instead of having to provide them all."
"At no time during my involvement with Abgenix did it occur to me that deficiencies in B cell development and antibody responses could be addressed by creating a hybrid locus at the endogenous immunoglobulin heavy locus with human variable regions and mouse constant regions. Such an approach was not obvious to me."
"Nor at any time before 16 February 2001 did anyone associated with Abgenix or its SAB (or anyone else) suggest in my presence that this was a strategy of interest or raise the potential benefits of such an approach as disclosed in paragraphs  and  of the Patent."
"I have a recollection of discussing this matter with Nils Lonberg during our collaboration and his suggesting it could perhaps be addressed by altering the cytokine environment in the mouse by, for example, administering interleukin 6. Neither he, nor anyone else, suggested this had anything to do with the nature of the human constant region and its interaction with the mouse B cell, and this did not occur to me either."
"Having experienced the issues associated with the Medarex mice, I became very excited about an idea that I had which I thought had a very significant chance of fixing all of the problems of the Kirin-Medarex mice, without requiring new adjuvant solutions, or the breeding of excessive numbers of animals. My idea was that the problem could be addressed by retaining the mouse constant regions and creating a hybrid locus, as we have described in the Patent. Such a mouse would theoretically not only be much more efficient and optimised in its immune function but also simultaneously avoid all the inherent breeding problems seen in the Medarex and Kirin mice, in terms of having to account for multiple transgenic loci at multiple non-endogenous locations, not to mention the instability problems with the Kirin mini-chromosome.
The solution seemed to me to be simple and elegant. Until I introduced the possibility to them under confidentiality (see paragraph 45 below), at no time did any Medarex scientist even raise the possibility of any solution remotely akin to what we now refer to as VelocImmune®. They never raised the possibility that putting variable segments into the endogenous mouse locus would have any advantage. Nor did they raise the possibility that retaining mouse constant regions would have any utility or advantages."
"Q. Now, these are leaders in the field and nowhere do they mention the potential benefits of the reverse chimeric locus, and by that I mean retaining mouse constant genes in their transgenic loci."
A. No, that is correct."
"In most of the published mouse strains currently under use, segments of the human IgH and IgL loci comprising differing numbers of human V, D, and J segments linked to human C regions have been integrated into the mouse germline (16,17) with the endogenous mouse Ig loci having been rendered non-functional through targeted gene disruption (18).
…Because the transgenic mice can essentially be viewed as a source of Ag-specific IgV genes (with the desired IgC region provided at a later stage during the creation of cell lines for bulk Ab production), we wondered whether the transgenic approach could be improved if the germline configuration human IgVH-D-JH segments were linked to endogenous (rather than human) IgCH regions."
"the early Zou papers were following on very shortly on the discovery of gene targeting by homologous recombination. The Rajewsky lab was one of the very first to adapt Cre-lox based strategies to engineer the genome in a specific manner. I view these papers as demonstrating proof of concept. Yes, one can do these things. As we discussed yesterday, the number of academic laboratories that were engaged in producing fully human antibodies can be counted on the fingers of one hand. Brüggemann, Neuberger, I could not think of many others, they were narrowly focused on producing fully human immunoglobulins. As I pointed out, the Rajewsky lab is focused on studying B-cell physiology, the regulation of a normal B-cell response, elucidating mechanisms of hypermutation class switching and so forth."
"Furthermore technologies for direct gene replacement (e.g. using the Cre-LoxP system) might allow the generation of animals in which much of the DNA of the mouse Ig loci is substituted by human Ig-gene DNA."
The focus of the publication is on producing human antibodies by substituting as much as possible of the mouse Ig loci with human Ig-gene DNA. In the conclusions (page 6) the authors note that:
"Human Ig genes introduced in germline configuaration can be re-arranged, expressed and hypermutated in the mouse background. While it is not clear that all these processes operate with optimal efficiency, they certainly operate at a level sufficient to ensure that the transgenic mice can be used to obtain hybridomas secreting high-affinity human antibodies against a variety of antigens. "
They explain that it now technically feasible to contemplate making a mouse having the whole human Ig loci but that it might be better to be judicious in the selection of certain human V segments.
"the various strategies that had been used to introduce and express human Ig heavy and light chain genes in transgenic animals and show that a diverse human antibody repertoire can be obtained in a mouse background with silenced endogenous IgH and IgL chain loci…I also draw attention to present shortcomings and speculate what the future may provide."
"several new technologies will be used to produce high-affinity fully human antibodies. Whole Ig loci will be routinely transferred into different species of animals."
"was unidirectional, narrowly focussed, myopically obsessed, one might say, with the production of human antibodies."
By the priority date, the art had moved on from early, tentative suggestions by the Rajewsky group, and it was thought that progress would be made by the introduction of more human Vs into the mouse locus. There was no suggestion to use a reverse chimeric locus to produce chimeric antibodies, or that such a locus would offer any benefits over the "fully-human" approach.
Inactivating the endogenous murine locus (construction)
"We have produced mice that carry the human Ig heavy (IgH) and both κ and λ light chain transloci in a background in which the endogenous IgH and κ loci have been inactivated."
This is elaborated on page 2 which refers to the insertion of a stop codon and Neo cassette and explains that:
"Mice with their endogenous H chain or λ L chain loci rendered non-functional have been described previously."
LTVECs and the MOA assay
"target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells."
(i) bacterial homologous recombination (i.e. recombineering) to engineer a desired genetic modification within a large cloned genomic fragment to create large targeting vectors ("LTVECs") for use in eukaryotic cells;
(ii) introducing LTVECs into eukaryotic cells to modify the endogenous chromosomal locus of interest; and
(iii) using an assay for modification of allele ("MOA") of the parental allele "that does not require sequence information outside of the targeting sequence, such as, for example, quantitative PCR" to determine those eukaryotic cells in which the targeted locus has been modified as desired.
"The field of this invention is a method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. The field of the invention further provides for a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci). The field also encompasses the use of these cells to generate organisms bearing the genetic modification, the organisms, themselves, and methods of use thereof."
"The use of LTVECs provides substantial advantages over current methods. For example, since these are derived from DNA fragments larger than those currently used to generate targeting vectors, LTVECs can be more rapidly and conveniently generated from available libraries of large genomic DNA fragments (such as BAC and PAC libraries) than targeting vectors made using current technologies. In addition, larger modifications as well as modifications spanning larger genomic regions can be more conveniently generated than using current technologies. Furthermore, the present invention takes advantage of long regions of homology to increase the targeting frequency of "hard to target" loci, and also diminishes the benefit, if any, of using isogenic DNA in these targeting vectors."
"…Assays for successful targeting involve standard Southern blotting or long PCR …from sequences outside the targeting vector and spanning an entire homology arm (see Definitions); thus, because of size considerations that limit these methods, the size of the homology arms are restricted to less than 10-20 kb in total."
"The ability to utilize targeting vectors with homology arms larger than those used in current methods would be extremely valuable. For example, such targeting vectors could be more rapidly and conveniently generated from available libraries containing large genomic inserts (e.g. BAC or PAC libraries) than targeting vectors made using current technologies, in which such genomic inserts have to be extensively characterized and trimmed prior to use."
" It should be emphasized that previous methods to detect successful homologous recombination in eukaryotic cells cannot be utilized in conjunction with the LTVECs of Applicants' invention because of the long homology arms present in the LTVECs. Utilizing a LTVEC to deliberately modify endogenous genes or chromosomal loci in eukaryotic cells via homologous recombination is made possible by the novel application of an assay to determine the rare eukaryotic cells in which the targeted allele has been modified as desired, such assay involving a quantitative assay for modification of allele (MOA) of a parental allele, by employing, for example, quantitative PCR or other suitable quantitative assays for MOA."
""Modification of allele" (MOA) refers to the modification of the exact DNA sequence of one allele of a gene(s) or chromosomal locus (loci) in a genome. This modification of allele (MOA) includes, but is not limited to, deletions, substitutions, or insertions of as little as a single nucleotide or deletions of many kilobases spanning a gene(s) or chromosomal locus (loci) of interest, as well as any and all possible modifications between these two extremes."
"In contrast to traditional methods, in which a difference in restriction fragment length spanning the entire homology arm or arms indicates the modification of one of two alleles, the quantitative TaqMan® method [an example of the MOA assay] will detect the modification of one allele by measuring the reduction in copy number (by half) of the unmodified allele. Specifically, the probe detects the unmodified allele and not the modified allele."
"Taking these passages together, and the document as a whole, it would be clear to the person skilled in the art that the MOA assay requires the detection by an appropriate probe (using various techniques) to detect the unmodified parental allele. If the gene has been correctly targeted there will only be one modified allele and one unmodified allele. Modifying both alleles in a single manipulation would be unexpected. The assay measures this reduction in the frequency of the unmodified allele from two to one.
The proposal in the '287 Patent is therefore not to detect the targeted ES cell clone by detailed analysis of the targeted allele but just its alteration or loss. The detection of a targeted ES cell clone is seen by concentrating on the intact endogenous alleles and seeing a change from two copies to one in the correctly targeted cell clone (MOA), whereas an incorrectly targeted cell clone retains two copies. This is an elegant and clever approach that was not generally known or used by those skilled in the art. It obviates interference in the analysis from a randomly integrated targeting vector. It also facilitates using larger targeting vectors because the size of arms is immaterial and detailed restriction maps (or sequence information) are no longer required. It particularly lends itself to replacement and/or deletion events. It may perhaps be particularly powerful coupled with the use of bacterial recombination to construct the targeting vectors because here, too, detailed knowledge of the restriction maps is not needed.
"Let us just refresh our minds a little bit of what this very clever assay is. You established in our first meeting, I am of the old school. We used to try very hard to use homologous recombination to make a deletion knock-out change in a desired locus and we always had to find that not only by things, as we have discussed, like PCR ending or Southern blot but also looking to make sure we have actually made the change we want, so we are always looking at our changed locus. One is always completely forgetting about the fact that there is an unchanged locus in the background except that we have to discriminate against that when we are doing our tests. What the MOA has done is to invert that logic."
"While many of the techniques used in practicing the individual steps of the methods of the invention are familiar to the skilled artisan, Applicants contend that the novelty of the method of the invention lies in the unique combination of those steps and techniques coupled with the never-before-described method of introducing an LTVEC directly into eukaryotic cells to modify a chromosomal locus, and the use of quantitative MOA assays to identify eukaryotic cells which have been appropriately modified. This novel combination represents a significant improvement over previous technologies for creating organisms possessing modifications of endogenous genes or chromosomal loci."
As can be seen from this figure, this LTVEC, when introduced into ES cells, is intended to lead to the replacement of 20 kb of mOCR10 sequence with the modification cassette of about 6 kb in length. The removal of 20 kb of sequence is described in  as "a very large deletion". Profs. Evans and Stewart agreed that this reflects the view of the skilled person in 2001/2. The experts also agreed that the skilled genetic engineer, given Example 1, would have been able to reproduce it.
" Q. Just looking back at Table 1 again if you could with me, these are all cases in which targeting constructs of a similar nature to that in Example 1 have been used, in that they are LTVECs containing fairly short modification cassettes, are they not? For example, if you look at the data in the first line, we see that the LTVEC is 147 kb, the two arms are together 140 kb and so the cassette is 7 kb -- cassette plus vector backbone is 7 kb.
A. Yes, and they also have a figure for the deletion there.
Q. That is right, but in terms of what is going into the genome they are all fairly short cassettes. The largest deletion here is 30 kb. Professor, there is no evidence here that replacements can be made which consists of more than replacing 30 kb with a few kbs?
A. That is what this table showed."
"More recently, endogenous genes have been knocked out of mice, and the genes replaced with their human counterparts to produce entirely human antibodies. Unfortunately, the use of these constructs has highlighted the importance of an endogenous constant region in the development and optimization of antibodies in B cells. Mice producing fully human antibodies have reduced immune responses. This may because human antibodies produced by transgenic mice with entirely human constructs have reduced affinity as compared to their mouse counterparts. Reduced affinity could effect B-cell maturation and survival. Accordingly, the much acclaimed methods of producing humanized antibodies in mice and other organisms, wherein endogenous variable and constant regions of the mice are knocked out and replaced with their human counterparts, has not resulted in optimal antibodies."
"A transgenic mouse is created that produces hybrid antibodies containing human variable regions (VDJ/VJ) and mouse constant regions. This is accomplished by a direct, in situ replacement of the mouse variable region genes with their human counterparts. The resultant hybrid immunoglobulin loci will undergo the natural process of rearrangements during B-cell development to produce the hybrid antibodies."
"…Antibodies generated by the new mouse will retain murine Fc regions which will interact more efficiently with the other components of the mouse B cell receptor complex, including the signaling components required for appropriate B cell differentiation (such as Iga and Igb). Additionally, the murine Fc regions will be more specific than human Fc regions in their interactions with Fc receptors on mouse cells, complement molecules, etc. These interactions are important for a strong and specific immune response, for the proliferation and maturation of B cells, and for the affinity maturation of antibodies."
"Because there is a direct substitution of the human V-D-J/V-J regions for the equivalent regions of the mouse loci all of the sequences necessary for proper transcription, recombination, and/or class switching will remain intact. For example, the murine immunoglobulin heavy chain intronic enhancer, Em, has been shown to be critical for V-D-J recombination as well as heavy chain gene expression during the early stages of B cell development … whereas the immunoglobulin heavy chain 3' enhancer region appears to be critical for class switching … as well as heavy chain gene expression at later stages of B cell differentiation…Given these various, yet crucial, functions of the transcriptional control elements, it is desirable to maintain these sequences intact."
Materials and Methods of Example 3
"Precise replacement of the mouse heavy chain locus variable region (VDJ) with its human counterpart is exemplified using a combination of homologous and site-specific recombination in the following example, which utilizes a two-step process. One skilled in the art will recognize that replacement of the mouse locus with the homologous or orthologous human locus may be accomplished in one or more steps. Accordingly, the invention contemplates replacement of the murine locus, in whole or in part, with each integration via homologous recombination."
The first proposed approach
" In the first step, LTVEC1 (Figure 4D) is constructed by bacterial homologous recombination in E. coli. LTVEC1contains, in order: a large mouse homology arm derived from the region upstream from the mouse DJ region but whose absolute endpoints are not important; a cassette encoding a selectable marker functional in ES cells (PGK-neomycinR in this example); a loxP site; a large human insert spanning from several V gene segments through the entire DJ region;and a mouse homology arm containing the region immediately adjacent to, but not including, the mouse J segments. The 5' end of the downstream arm and the placement of the loxP sites define the 3' end of the region to be replaced in the locus. Mouse ES cells will be transformed by standard techniques, for example, electroporation, with linearized LTVEC1. Because direct introduction of LTVEC1 results in a modification of the endogenous variable gene locus, neomycin resistant colonies can be screened for correct targeting using a MOA assay. These targeted ES cells can give rise to mice that produce antibodies with hybrid heavy chains. However, it will be preferable to proceed with subsequent steps that will eliminate the remainder of the mouse variable segments."
The second proposed approach
The light chains
"The final steps in creating the human variable/mouse constant monoclonal antibody producing mouse will be performing the equivalent variable region substitutions on the lambda and kappa light chain loci and breeding all three hybrid loci to homozygo[s]ity in the same mouse. The resultant transgenic mouse will have a genome comprising entirely human heavy and light chain variable gene loci operably linked to entirely endogenous mouse constant region such that the mouse produces a serum containing an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. …"
The Claims in Issue
"A method of modifying an endogenous immunoglobulin heavy chain variable region gene locus in an isolated mouse embryonic stem (ES) by an in situ replacement of V, D, and J gene segments of the endogenous locus with orthologous human V, D and J gene segments, to create a modified immunoglobulin locus that produces hybrid antibodies containing human variable regions and mouse constant regions, said method comprising:
a) obtaining a large cloned genomic fragment greater than 20 kb containing orthologous human V, D, and J gene segments;
b) using bacterial homologous recombination to genetically modify the cloned genomic fragment of (a) to create a large targeting vector for use in a mouse ES cell (LTVEC);
c) introducing the LTVEC of (b) into a mouse ES cell to replace said V, D, and J segments in situ with the orthologous human V, D and J gene segments; and
d) using a quantitative assay to detect modification of allele (MOA) in the mouse ES cell of (c) to identify a mouse ES cell in which said V, D and J segments have been replaced in situ with the orthologous human V, D and J gene segments."
In situ replacement
"Although it has often been said that the question of construction does not depend on the alleged infringement ('as if we had to construe it before the defendant was born'…) questions of construction seldom arise in the abstract. That is why most sensible discussion of the meaning of language runs on the general lines 'does it mean this, or that, or the other?' rather than the open-ended 'what does it mean?'"
"It must be remembered, however, that the specification and claims of the patent serve different purposes. The specification describes and illustrates the invention, the claims set out the limits of the monopoly which the patentee claims. As with the interpretation of any document, it is conceivable that a certain, limited, meaning may be implicit in the language of a claim, if that is the meaning that it would convey to a skilled person, even if that meaning is not spelled out expressly in the language. However it is not appropriate to read limitations into the claim solely on the ground that examples in the body of the specification have this or that feature. The reason is that the patentee may have deliberately chosen to claim more broadly than the specific examples, as he is fully entitled to do."
"Before the court gets to the examination room it has to do some swotting: to get into its mind the relevant knowledge of the skilled man. For how a document will be understood depends on the reader."
"Another embodiment of the invention is a method wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element; alteration of a coding sequence, gene segment, or regulatory element; insertion of a new coding sequence, gene segment, or regulatory element; creation of a conditional allele; or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from a different species."
"Accordingly, the much acclaimed methods of producing humanized antibodies in mice and other organisms, wherein endogenous variable and constant regions of the mice are knocked out and replaced with their human counterparts, has not resulted in optimal antibodies."
 defines a "gene knockout" as "a genetic modification resulting from the disruption of the genetic information encoded in a chromosome locus." So in knockout mice, the endogenous locus is disrupted and not removed. I have found that the skilled person would be aware from his common general that the endogenous loci in the transgenic mice produced by Brüggemann, Abgenix and Medarex, were inactivated. This is reflected in the text of  which refers to the methods of producing humanised antibodies in such mice as "much acclaimed".
"Q. …the person skilled in the art would have no doubt that this was a reference to the mice made by Brüggemann, Lonberg, Abgenix. Yes?
A. I believe that is what it is referred to, yes."
So, in my judgment, the 287 Patent refers to the variable and constant regions of mice as having been "replaced" with their human counterparts, when the corresponding mouse segments have been inactivated but not removed. Kymab accepts this, but submits that this is a reference to the prior art and not the invention. However, Kymab's construction requires that the word "replacement" be interpreted in a more narrow sense in the claims than it is used in paragraph  of the description. Whilst this conclusion could conceivably be right, it would need a powerful reason to support it.
"These targeted ES cells can give rise to mice that produce antibodies with hybrid heavy chains. However, it will be preferable to proceed with subsequent steps that will eliminate the remainder of the mouse variable segments."
"Because there is a direct substitution of the human V-D-J/V-J regions for the equivalent regions of the mouse loci all of the sequences necessary for proper transcription, recombination and/or class switching will remain intact."
"The substitutions of the human V-D-J or V-J regions into the genuine murine chromosomal immunoglobulin loci should be substantially more stable, with increased transmission rates to progeny and decreased mosaicism of B cell genotypes compared with the currently available mice (Tomizuka, K., Shinohara, T., Yoshida, H., Uejima, H., Ohguma, A., Tanaka, S., Sato, K., Oshimura, M., and Ishida, I. Proc Natl Acad Sci (USA) 97:722-727 (2000)). Furthermore, introduction of the human variable regions (VDJ/VJ) at the genuine murine loci in vivo will maintain the appropriate global regulation of chromatin accessibility previously shown to be important for appropriately timed recombination events (Haines, B. B., and Brodeur, P. H. Eur J Immunol. 28:4228-4235 (1998))."
"In conclusion, our data and others' indicate that D-distal VH gene segments exist in a distinct regulatory environment relative to the D-proximal segments. It has been suggested that conserved regulatory elements such as an enhancer or locus control region (LCR) may reside upstream of the Igh locus [1, 15]. These elements could function in the propagation of chromatin changes as well as mediate transcriptional and recombinational regulation at D-distal V regions."
Therefore, Kymab submits that the skilled person is expressly referred to a paper which suggests that a 5' regulatory sequence could be significant and would appreciate that there might be a benefit, contemplated by the patentee, to keeping it in place, rather than displacing it upstream, away from the human variable genes.
"A. Yes, that is absolutely fine.
Q. This is perfectly straightforward, is it not? There is nothing at all inventive or unusual or surprising about this which would not be plain as a pikestaff in the light of the patent in suit, the '287 patent?
A. It is fine. However, I think the skilled genetic engineer would prefer to do a junction PCR."
Scope of Claim 1 of 287
"Q. Now, we have looked at what  is about, what it is describing. Now, we have talked about the amount of mouse sequence that is being deleted in the replacement step. It is 100 kb of mouse sequence at least and I think you would agree that what  is envisaging is putting in its place about 200-300 kb of human sequence?
A. I believe it is, yes.
Q. I think, and we looked at these numbers earlier, you say, well, if you wanted to replace at least one mouse V, as well as the Ds and Js, you would have to replace 150 kb of mouse sequence.
A. Yes, I think that is probably about right.
Q. And if you wanted to replace it with one human V, as well as the human Ds and Js, that is about 75 kb.
Claims 5 and 6 of the 287 Patent
"5. A genetically modified eukaryotic cell or a mouse comprising a genetically modified immunoglobulin heavy chain variable region locus obtainable by the method of any one of the preceding claims in situ in place of the endogenous immunoglobulin heavy chain variable region gene locus."
"6. A mouse embryonic stem (ES) cell containing a genetically modified immunoglobulin heavy chain variable region gene locus obtainable by the method of any one of claims 1 to 4 in situ in place of the endogenous immunoglobulin heavy chain variable region gene locus."
i) their purpose is to claim a product irrespective of how it was made but with a shared characteristic which results from using a given process ;
ii) the claim has to specify the characteristic being referred to ;
iii) "obtainable by" claims present clarity problems and should only be permitted if there is no alternative way of defining the product in question ; and
iv) for a product to be "obtainable by" a process it must have every characteristic which is the inevitable consequence of that process .
i) As with claim 1, they include products in which 100 kb of endogenous sequence has been deleted and 200-300 kb of orthologous sequence has been inserted and products in which 150 kb of endogenous sequence has been deleted and 75 kb of orthologous sequence has been inserted.
ii) Unlike claim 1, they contain no limitation as to the use of LTVECs or the MOA assay. Provided that the product is obtainable by the process of claim 1, it does not matter how it is produced.
iii) They contain no limitation as to the amount of endogenous sequence which may have been deleted and include products in which the entire endogenous heavy chain sequence has been deleted.
iv) They contain no limitation as to the amount of orthologous sequence which may have been inserted and include products in which the entire orthologous heavy chain sequence has been inserted.
Accordingly, claims 5 and 6 extend to cells and mice in which the entire mouse variable IgH locus has been replaced by the entire human variable IgH locus.
Claim 1 of the 163 Patent
"A transgenic mouse that produces hybrid antibodies containing human variable regions and mouse constant regions, wherein said mouse comprises an in situ replacement of mouse VDJ regions with human VDJ regions at a murine chromosomal immunoglobulin heavy chain locus and an in situ replacement of mouse VJ regions with human VJ regions at a murine chromosomal immunoglobulin light chain locus."
i) The claim is not confined to a single product. It includes mice in which different amounts of VDJ and VJ regions have been replaced with human VDJ and VJ regions, for example a mouse in which one VDJ and one VJ region have been replaced, and mice in which several such regions have been replaced.
ii) As with claim 1 of 287 Patent, it includes products in which 100 kb of endogenous sequence has been deleted and 200-300 kb of orthologous sequence has been inserted; and products in which 150 kb of endogenous sequence has been deleted and 75 kb of orthologous sequence has been inserted.
iii) Unlike claim 1 of the 287 Patent, it contains no limitation as to the use of LTVECs or the MOA assay.
iv) The terms "VDJ regions" and "VJ regions" as used in claim 1 of the 163 Patent, encompass the whole mouse and human variable gene loci. Accordingly the claim extends to a mouse in which the entire murine variable gene loci have been replaced with the entire human variable gene loci.
i) Product claims 5 and 6 of the 287 Patent. Regeneron claims that every strain of Kymouse is and was generated from a product within the scope of those claims;
ii) Product claim 1 of the 163 Patent. Regeneron's case is that Kymouse strains having both modified IgH and IgK loci (namely, the HK and HKL strains) are mice within the scope of claim 1; and
iii) Process claims 2 and 3 of the 163 Patent. However, these claims stand or fall with claim 1 of 163, both in respect of infringement and validity. Neither party addressed them separately in their closing speeches, and no separate issue arises in respect of these claims which I am required to determine.
Inversion and displacement upstream of mouse sequences
Infringement of the claims in issue
"We analyzed the species origin of V, D and J segment usage in mRNA transcripts of antibody genes in mice with human BACs inserted via S-RMCE. No mouse D or J segments were found in heavy-chain or kappa light-chain transcripts. However, we detected both human and mouse V-segment sequences in IGH and IGK transcripts. The frequency of mouse V-segment usage diminished as more BACs were inserted (Fig. 2a). Nevertheless, to prevent mouse V-segments from being used to generate antibodies in the transgenic mice, we created inversions in the heavy-chain and kappa light-chain loci. The inversions moved the entire mouse variable gene clusters several megabases away on the same chromosome and inverted them relative to the mouse constant regions."
Ordinary methods of trial and error/undue burden
"The section requires the skilled man to be able to perform the invention, but does not lay down the limits as to the time and energy that the skilled man must spend seeking to perform the invention before it is insufficient. Clearly there must be a limit. The sub-section, by using the words clearly enough and completely enough, contemplates that patent specifications need not set out every detail necessary for performance, but can leave the skilled man to use his skill to perform the invention. In so doing he must seek success. He should not be required to carry out any prolonged research, enquiry or experiment. He may need to carry out the ordinary methods of trial and error, which involve no inventive step and generally are necessary in applying the particular discovery to produce a practical result. In each case, it is a question of fact, depending on the nature of the invention, as to whether the steps needed to perform the invention are ordinary steps of trial and error which a skilled man would realise would be necessary and normal to produce a practical result."
"Even though a reasonable amount of trial and error is permissible when it comes to the sufficiency of disclosure in an unexplored field or – as in this case – where there are many technical difficulties, there must then be available adequate instructions in the specification or on the basis of common general knowledge which would lead the skilled person necessarily and directly towards success through the evaluation of initial failures or through an acceptable statistical expectation rate in case of random experiments."
i) The patent need not set out every detail necessary for performance of the invention, as the skilled person applies his common general knowledge.
ii) The skilled person is seeking success when trying to perform the invention.
iii) There is a balance to be struck between ordinary steps of trial and error on the one hand, and prolonged research, enquiry or experiment on the other.
iv) Ordinary methods of trial and error involve no inventive step.
v) The enquiry is fact-sensitive and depends on the nature of the invention.
"All the same, one must be on one's guard against formulations that gloss the statutory requirement as there is always a risk that they will end up being substituted for it. This is a particular risk where the subject of the specification is very complex and its development would anyway be expected to be accompanied by a great amount of work. What is 'prolonged' in this context? It is always necessary to keep a balance between the interests of the public and the interests of the patentees in the sense that it is necessary to guard against imposing too high a standard of disclosure merely because the subject matter is inherently complex."
Excessive claim breadth
"122. The main points which I drew from Lord Hoffmann's opinion in Biogen v Medeva were as follows:
i) A claim will be invalid for insufficiency if the breadth of the claim exceeds the technical contribution to the art made by the invention. It follows that it is not necessarily enough to disclose one way of performing the invention in the specification.
ii) The breadth of the claim will exceed the technical contribution if the claim covers ways of achieving the desired result which owe nothing to the patent or any principle it discloses. Two classes of this are where the patent claims results which it does not enable, such as making a wider class of products when it enables only one and discloses no principle to enable the others to be made, and where the patent claims every way of achieving a result when it enables only one way and it is possible to envisage other ways of achieving that result which make no use of the invention.
iii) The patent in Biogen v Medeva was invalid because it was an example of the second class of objectionable claim.
123. The key point which emerges from Lord Hoffmann's opinion in Kirin-Amgen v Hoechst is his explanation at  of what he had meant by "a principle of general application" in Biogen v Medeva:
"In my opinion there is nothing difficult or mysterious about it. It simply means an element of the claim which is stated in general terms. Such a claim is sufficiently enabled if one can reasonably expect the invention to work with anything which falls within the general term."
124. I summarised the reasoning of the House in Generics v Lundbeck as follows:
i) The House agreed with Lord Hoffmann in Biogen v Medeva that it was important for United Kingdom patent law to be aligned, so far as possible, with the jurisprudence of the EPO. Furthermore, the House also agreed with Lord Hoffmann that the statement of principle which he quoted from Exxon/Fuel oils correctly stated the law.
ii) The House considered that the instant case was to be distinguished from Biogen v Medeva because it was concerned with a claim to a single chemical compound whereas Biogen v Medeva concerned a product-by-process claim of broad scope.
iii) It was a mistake to equate the technical contribution of the claim with its inventive concept. In the instant case, the technical contribution made by claims 1 and 3 was the product, and not the process by which it was made, even though the inventive step lay in finding a way to make the product. It followed that the breadth of the claim did not exceed the technical contribution which the invention made to the art."
"It is therefore now settled law that the disclosure must be sufficient to enable the whole width of the claimed invention to be performed … The EPO applies the same principle that all members of a claimed class must be enabled …
The principle was elegantly illustrated in Nokia v IPCom where the claim required "coarse frequency synchronization at least if the accuracy of the carrier frequencies is not adequate"; the patentee contended that if the former feature could not be made to work it did not matter because adequate accurate oscillators were available. The contention was rejected, because:
"It is not permissible in law expressly to claim product features or process steps which are not enabled. … The claim can be thought of as split notionally into two parts: one where the coarse frequency step is not needed and the other where it is. If the part where coarse frequency synchronisation is needed is not enabled, the claim will be insufficient."
Accordingly, in order to be sufficient, the description must enable the invention to be performed across the full width of the claim."
"…the requirement of sufficient disclosure can only mean that the whole subject-matter that is defined in the claims, and not only a part of it, must be capable of being carried out by the skilled person without the burden of an undue amount of experimentation or the application of inventive ingenuity." (T 435/91 Unilever / detergents at [2.2])
"…claims may not be considered allowable if they encompass subject-matter which in the light of the disclosure provided by the description can only be performed with undue burden or with application of inventive skill." (T 694/92 Mycogen / modifying plant cells at )
"Even though a reasonable amount of trial and error is permissible when it comes to assessing sufficiency of disclosure, there must still be adequate instructions in the specification, or on the basis of common general knowledge, leading the skilled person necessarily and directly towards success, through evaluation of initial failures. … the principle underlying Article 83 [is] that the skilled person should be given sufficient guidance for performing the invention without undue burden over the whole range claimed." (T 1743/06 Ineos / amorphous silica at [1.9]-[1.10])
"The heart of the test is: "Can the skilled person readily perform the invention over the whole area claimed without undue burden and without needing inventive skill?""
"It must therefore be possible to make a reasonable prediction the invention will work with substantially everything falling within the scope of the claim or, put another way, the assertion that the invention will work across the scope of the claim must be plausible or credible. The products and methods within the claim are then tied together by a unifying characteristic or a common principle. If it is possible to make such a prediction then it cannot be said the claim is insufficient simply because the patentee has not demonstrated the invention works in every case.
On the other hand, if it is not possible to make such a prediction or if it is shown the prediction is wrong and the invention does not work with substantially all the products or methods falling within the scope of the claim then the scope of the monopoly will exceed the technical contribution the patentee has made to the art and the claim will be insufficient. It may also be invalid for obviousness, there being no invention in simply providing a class of products or methods which have no technically useful properties or purpose."
"It is now well established that the scope of the monopoly, as defined in the claims, must correspond to the technical contribution the patentee has made to the art. An aspect of this requirement is that the specification must enable the invention to be performed to the full extent of the monopoly claimed."
Insufficiency: the facts
Claim 1 of 287 – breadth of claim
i) the minimum replacement by LTVEC1 that is described in  is a deletion of 100 kb of mouse sequence (being the DJ region, which is flanked by the inner ends of the arms as described in ) and an insertion of 200-300 kb of human sequence (as shown in Fig.4);
ii) the minimum replacement that is required by claim 1 of the 287 patent involves a larger deletion (150 kb) but a smaller insertion (75 kb); and
iii) The claim also includes the case where the mouse sequence is displaced and deactivated, but it is not limited to exclude deletion of the mouse sequence, which is specifically described in the specification.
"Construct BACs with human inserts, selection cassettes and mouse homology boxes. The initial idea … was to do three steps of recombination in ES cells to sequentially replace the proximal, distal and middle third of the IgH VDJ region using very large hybrid BACs. This method required the capability to stitch together BACs to add the long mouse homology arms onto human BAC segments and to combine multiple human inserts – typically BACs contain 100-200 kb of sequence with ends "randomly" located. This capability had not been established at the time. In addition, we were not sure that even with long (>20 kb, but up to >100 kb) mouse homology arms we could insert long sequences (100s of kb) delete long sequences (100s of kb) in ES cells."
The second proposed approach of Example 3
The importance of Example 3
The Macdonald paper
"we eventually were able to target the distal end of the mouse IgH locus and delete the 3 megabases of mouse sequence in between as we had predicted. At around this point (I think), I had the idea of doing sequential targeting using overlapping human sequences (from overlapping BACs) and repeatedly using the same distal mouse arm, but alternating selection (neoR hygS to neoS hygR etc)."
"an original, and elegant approach…one that I had not previously come across before reading Macdonald" (Stewart 2 (d));"
" a beautiful piece of science…an outstanding solution to the problem" [T5/7156-12]"
" I think you are doing Drew Murphy a great disservice by saying that is pretty all obvious. The solution he came up with I think is elegant. It was a really outstanding piece of thinking and very creative." [T6/91314-18]"
Multiple insertions without deletion
"Well, that was my personal take. That was using my inventiveness, if you like. I do not want to say that the person skilled in the art would have chosen any other way of doing this."
Mr. Tappin: "As I understood it, what this diagram [X2/35] was being suggested for was the idea of doing sequential insertions.
A. I think it may have been, but it is a very useful diagram for this ----
Q. And of course sequential insertions were what you were being asked about at the outset and which you did not put in your report because you said that was not what the skilled person would have done.
A. That is right. So shunting it up, as I think we have heard."
Repetition of the homologous recombination process
"Yet another preferred embodiment is a method of replacing, in whole or in part, in a mouse embryonic stem cell, an endogenous immunoglobulin variable region gene locus with its homologous or orthologous human gene locus comprising:
a) obtaining a large cloned genomic fragment containing, in whole or in part, the homologous or orthologous human gene locus;
b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the embryonic stem cells;
c) introducing the large targeting vector of (b) into mouse embryonic stem cells to replace, in whole or in part, the endogenous immunoglobulin variable gene locus in the cells; and
d) using a quantitative PCR assay to detect modification of allele (MOA) in the mouse embryonic stem cells of (d) to identify those mouse embryonic stem cells in which the endogenous variable gene locus has been replaced, in whole or in part, with the homologous or orthologous human gene locus."
"In another embodiment, the method further comprises:
e) obtaining a large cloned genomic fragment containing a part of the homologous or orthologous human gene locus that differs from the fragment of (a);
f) using bacterial homologous recombination to genetically modify the cloned genomic fragment of (e) to create a large targeting vector for use in the embryonic stem cells;
g) introducing the large targeting vector of (f) into the mouse embryonic stem cells identified in step (d) to replace, in whole or in part, the endogenous immunoglobulin variable gene locus; and
h) using a quantitative assay to detect modification of allele (MOA) in the mouse embryonic stem cells of (g) to identify those mouse embryonic stem cells in which the endogenous immunoglobulin variable region gene locus has been replaced, in whole or in part, with the homologous or orthologous human gene locus."
"Still another preferred embodiment is a method of wherein steps (e) through (h) above are repeated until the endogenous immunoglobulin variable region gene locus is replaced in whole with an homologous or orthologous human gene locus."
"introducing the LTVEC of (b) into a mouse ES cell to replace said V, D and J segments in situ with the orthologous human V, D and J gene segments."
Assessment of insufficiency
Further insufficiency objections
Effect of long homology arms
"Q. But you accept that there are benefits to having long homology arms. There is nothing between us on that.
The reduced amount of DNA
"Standard methods for targeted modification of genes in mouse embryonic stem (ES) cells typically employ 20 to 40 µg of targeting vector in the electroporation procedure. Applicants have discovered that with LTVECs, electroporation with much lower amounts of DNA—in the range of about 1 to 5 µg per 1x107 cells—doubles the frequency of correctly targeted homologous recombination events while greatly reducing the number of secondary, non-homologous insertion events. This clear improvement in targeting efficiency is important because it significantly reduces the number of ES cells clones that need to be screened to find several positive clones with a correctly targeted, single-copy modification."
"Q. All right, but what they are saying here is that, despite the increased molecular weight of the LTVECs, they are using a very low amount of DNA.
A. Yes, they do.
Q. And they [say] that has two benefits. "[It] doubles the frequency of correctly targeted homologous recombination events while greatly reducing the number of secondary, non-homologous insertion events", and that is clear improvement of targeting efficiency. They do not tell us exactly what greatly reducing means, but obviously the overall benefit is more than double. Yes? So they are doubling the frequency of the correct event and greatly using [reducing] the number of others.
A. Yes, it is more than double.
Q. More than double. The targeting frequency is more than double. We do not know quite how much. What we know, professor, is that when Regeneron managed to get their 3VH targeting vector to target, they used a level of DNA, which of course the patent does not suggest you should use this special low level of DNA, does it?
Q. And if they had used standard levels of DNA, we have no way of knowing that they would have been successful?
A. No, we do not, but I think we should comment on this, my Lord, that this is a very interesting claim here. Once again I do not think this is a claim that would have been known about or available to the standard person skilled in the art."
Identifying the 5' end
Submissions following circulation of the draft judgment
"Such a replacement involving a deletion of 17 kb and an insertion of 75 kb was put to Prof. Stewart (Stewart XX [T6/92417-9263]), on the assumption that the targeting construct could be made and everything else was working perfectly. He said that in 2001 he would have considered that it was very ambitious and would have had no idea if it would work. Naturally he agreed that today, it could be made to work – a fact that we only know from the Regeneron work and the Macdonald 2014 publication, achieved only with the application of VelociGene advances not described in the patents. The evidence does not support the making of such a replacement without undue burden, or indeed making it at all without the VelociGene advances – indeed, even with them, the targeting efficiency was only 0.2% (Macdonald Table 1)."
General observations about submissions on draft judgments
"In a complex case, it might well be prudent, and certainly not out of place, for the judge, having handed down or delivered judgment, to ask the advocates whether there are any matters which he has not covered. Even if he does not do this, an advocate ought immediately, as a matter of courtesy at least, to draw the judge's attention to any material omission of which he is then aware or then believes exists. It is well-established that it is open to a judge to amend his judgment, if he thinks fit, at any time up to the drawing of the order. In many cases, the advocate ought to raise the matter with the judge in pursuance of his duty to assist the court to achieve the overriding objective (CPR 1.3, which does not as such apply to these proceedings); and in some cases, it may follow from the advocate's duty not to mislead the court that he should raise the matter rather than allow the order to be drawn. It would be unsatisfactory to use an omission by a judge to deal with a point in a judgment as grounds for an application for appeal if the matter has not been brought to the judge's attention when there was a ready opportunity so to do. Unnecessary costs and delay may result. I should make it clear that there are general observations for assistance in future cases, and that I make no criticisms of Counsel in this case."
"3…Just because any draft is a draft judgment the opportunity for correction is available, and from time to time it is taken, not only on the application of one of the parties, but also on the judge's personal initiative if, on re-reading his draft, he thinks it appropriate to do so. In short, the judge is not bound by the terms of the draft judgment which has been circulated in confidence…
4 The primary purpose of this practice is to enable any typographical or similar errors in the judgments to be notified to the court. The circulation of the draft judgment in this way is not intended to provide an opportunity to any party (and in particular the unsuccessful party) to reopen or re-argue the case, or to repeat submissions made at the hearing, or to deploy fresh ones. However, on rare occasions, and in exceptional circumstances, the court may properly be invited to reconsider part of the terms of its draft…"
The Prior Art
"In order for a functional heavy chain Ig polypeptide to be produced, three discontinuous DNA segments, from the VH, D and JH regions must be joined."
"As already indicated, the target locus may be substituted with the analogous human locus. In this way, the human locus will be placed substantially in the same region as the analogous host locus, so that any regulation associated with the position of the locus will be substantially the same for the human immunoglobulin locus. For example, by isolating the entire VH gene locus (including V, D, and J sequences), or portion thereof, and flanking the human locus with sequences from the mouse locus, preferably sequences separated by at least about 5 kbp, in the host locus, preferably at least about 10 kbp in the host locus, one may insert the human fragment into this region in a recombinational event(s), substituting the human immunoglobulin locus for the variable region of the host immunoglobulin locus. In this manner, one may disrupt the ability of the host to produce an endogenous immunoglobulin subunit, while allowing for the promoter of the human immunoglobulin locus to be activated by the host enhancer and regulated by the regulatory system of the host."
"Q. You would agree that Kucherlapati is teaching nothing about the benefits of the reverse chimeric locus in terms of B-cell development. Yes?
A. Nothing about B-cell development at all.
Q. It provides no motivation to the person reading the document in 2001 to engineer a mouse with a reverse chimeric locus at the endogenous position. It provides no motivation to do that.
A. The motivation of course has more to do with the position effects -- the preservation of the intronic enhancer, which is explicit that that should be an advantage of a construction of this kind."
"Once the human loci have been introduced into the host genome, either by homologous recombination or random integration, and host animals have been produced with the endogenous immunoglobulin loci inactivated by appropriate breeding of the various transgenic or mutated animals, one can produce a host which lacks the native capability to produce endogenous immunoglobulin subunits, but has the capacity to produce human immunoglobulins with at least a significant portion of the human repertoire."
"The functional inactivation of the two copies of each of the three host Ig loci, where the host contains the human Ig kappa and/or lambda loci would allow for the production of purely human antibody molecules without the production of host or host/human chimeric antibodies."
"Q. Now, having looked at at least parts of this article, it is correct that it is not teaching the reverse chimeric locus or the benefits to be achieved by retaining mouse constant regions, is it?
Q. And the skilled team reading this in 2001, it provides no motivation to them to make the reverse chimeric locus or to retain the mouse constant regions, does it?
A. No, I do not think I would wish to say that; no. I believe there is a subliminal motivation throughout the entire story, but I agree that this paper does not stress it.
Q. And it offers no practical teaching on how to make a reverse chimeric locus of the endogenous position, does it?
Q. Can I suggest to you that in 2001 the person skilled in the art who was interested in using transgenes to produce human antibodies and who was aware of how this was being done in the prior art with the non-targeted transgenes and the fully human locus who picked up Brüggemann would not see any reason for making a reverse chimeric locus. It is just not there in Brüggemann, is it?
A. No, you are right from that point of view, but on the other hand I believe that the person to whom you refer would also pick up quite a lot of other documentation about this problem."
(i) An intermediate generalisation occurs when "a feature is taken from a specific embodiment, stripped of its context and then introduced into the claim in circumstances where it would not be apparent to the skilled person that it has any general applicability to the invention" (Nokia v IPCom at ).
(ii) The question is whether the feature in question would be seen by the skilled person as being generally applicable or only of significance in the context in which it was specifically disclosed (Nokia v IPCom at -).
(iii) An amendment does not require literal support in the application: The test is whether the skilled person is presented in the patent with new information relating to the invention in the patent as compared to the application. This depends on whether the combination of claimed features in the patent derives directly and unambiguously from the application, read as a whole (T 667/08 of 20 April 2012, and the EPO Guidelines for Examination Part H, Chapter IV, §2.2).
(iv) There is a distinction between what a claim covers and what it discloses. Not everything falling within the scope of a claim is necessarily disclosed (Nokia v IPCom at ).
The 287 Patent
"One embodiment of the invention is a method of replacing, in whole or in part, in a non-human eukaryotic cell, an endogenous immunoglobulin variable region gene locus with a homologous or orthologous human gene locus comprising:
a) obtaining a large cloned genomic fragment containing, in whole or in part, the homologous or orthologous human gene locus;
b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the eukaryotic cells (LTVEC);
c) introducing the LTVEC of (b) into the eukaryotic cells to replace, in whole or in part, the endogenous immunoglobulin variable gene locus; and
d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous immunoglobulin variable region gene locus has been replaced, in whole or in part, with the homologous or orthologous human gene locus."
"… 'in situ' replacement is not specifically linked to a method of replacement exemplified in the application but is a general feature of the claimed invention, i.e. modifying an endogenous locus in place. The term 'in situ' is used in the first paragraph of the section "brief description" of example 3. Said section is not associated with specific features of the example but is a general introduction to the application of the method of the invention to the replacement of mouse VDJ/VJ genes with their human counterparts. Said method is generally disclosed on pages 9-11 of the application as originally filed. Therefore the term 'in situ' is not inextricably linked to the specific methods used to replace the murine immunoglobulin locus in example 3 but refers to the general method subject-matter of claims 1 and 9 and the products of claims 14-16 obtained by said method. Thus the OD comes to the conclusion that the introduction of the terms 'in situ replacement', 'in situ in place' or 'replaced in situ' in claims 1, 9, 14, 15 and 16 does not violate Article 123(2) EPC."
"As indicated with regard to the Main Request, the OD finds on pages 46-48 a basis for the replacement of V and J or V, D and J gene segments. Moreover if page 43 refers to VDJ/VJ genes, the person skilled in the art knows that the variable region is formed by V(D)J gene segments as shown in Figure 4 and disclosed for example on page 46, line 26 or page 48, line 9. Therefore the person skilled in the art would directly and unambiguously derive from the content of the application as filed the replacement of V and J or V, D and J gene segments. Claim 1 is directed to the generic replacement of V and J or V, D and J gene segments and not to the specific replacement of one V and J, i.e. of a specific part of the V(D)J region. Therefore claim 1, and as a consequence the AR 5 fulfills the requirements of Article 123(2) EPC."
"The OD notes that the background and summary of the invention refer extensively to the use of large vectors and in particular to vectors that can accommodate large DNA fragments. On page 6, lines 4-6, the application specifically discloses that the LTVEC vectors of the invention are capable of accommodating large DNA fragments greater than 20 kb. Even though the term "capable of" does not require that the vectors indeed accommodate fragments greater than 20 kb, the person skilled in the art will find on page 8 of the description a specific embodiment disclosing the cloning of a fragment greater than 20 kb. Moreover all the cloned DNA fragments of example 3 fall within the range of greater than 20 kb (see for example figure 4 disclosing human fragments of 200 to 300 kb). The application as filed discloses the lower end of the range greater than 20 kb in the context of the LTVEC vector as well as examples comprising cloned fragments falling within the range of greater than 20 kb. Therefore the OD considers that the feature of claims 1 and 2 "cloned DNA fragment greater than 20 kb" does not add subject-matter."
The 163 Patent
i) All strains of Kymouse mice addressed in the PPDs were or would be generated from a product within the scope of claims 5 and 6 of the 287 Patent; and Kymouse strains having both modified IgH and IgK loci (namely, the HK and HKL strains) are mice within the scope of claim 1 of the 163 Patent
ii) None of the claims which I have been asked to consider, namely claims 1, 5 and 6 of the 287 Patent and claim 1 of the 163 Patent is anticipated or rendered obvious by the prior art.
iii) There is no added matter in respect of the 287 or 163 Patents..
iv) However, all of the claims of the 287 and 163 Patents which I have been asked to consider are invalid for insufficiency, on the basis that, at the priority date, the skilled person would not have been able to perform the invention over the whole area claimed without undue burden and without needing inventive skill.