Regeneron Pharmaceuticals, Inc. v Kymab Ltd

Hearing dates: 17–20 October 2017

The claimant and appellant (“Regeneron”) appeals from the decision of Henry Carr J that European Patent (UK) No 1 360 287 and its divisional European Patent (UK) No 2 264 163 (“the 287 patent” and “the 163 patent” respectively) are invalid. The first respondent (“Kymab”) cross-appeals against the judge's finding that its various strains of transgenic mice would infringe claims 5 and 6 of the 287 patent and claim 1 of the 163 patent if those patents had not been invalid. The second respondent, Novo Nordisk, although formally a party to Regeneron's appeal, has not taken any active part because Regeneron discontinued its infringement claim against it shortly before the trial. Its counterclaim for revocation survives, and for that purpose it adopts Kymab's submissions. This is the judgment of the court on the appeal.

The disclosure of the two patents is substantially the same, the material differences lying in the claims. For that reason we will refer to paragraphs in the description of the 287 patent, as the judge did.

The technical background in outline

The patents are concerned with biotechnology, and in particular the production of human antibodies using transgenic mice. This is a field of great technical complexity. The judge began his judgment by describing some of the basic concepts in a passage which was not the subject of any dispute either before him or before us: see [2016] EWHC 87 (Pat) at [8]–[79] to which the reader can refer for the fine detail. In the summary which follows we draw heavily on that description. All of it formed part of the common general knowledge of the relevant skilled team at the priority date of the patents, which was 16 February 2001.

Antibodies

By the priority date, the potential uses of antibodies (also known as immunoglobulins) for use in treating human disease had been well recognised, and a number of different antibodies had been developed and approved for such use. These included mouse antibodies, chimeric antibodies and humanised antibodies.

Antibodies all share a characteristic unit structure consisting of four polypeptide chains: two identical “heavy” chains and two identical “light” chains. The structure of an antibody (or “Ig”) is traditionally depicted as having a Y-formation as shown below. The light and heavy chains are so called because the former are made up of only two immunoglobulin domains, while heavy chains are made up of four (or five). One end of both the heavy and light chains is variable in sequence and is known as the variable region, whilst the other end is constant in sequence (for a given class or isotype) and is known as the constant region.

The light chains are found in two isotypes, kappa (“κ” or “K”) and lambda (“λ” or “L”). There is no known functional difference between antibodies having K and L chains but they are encoded by genes on different chromosomes.

The immunoglobulin genes, which are responsible for encoding the heavy and light chains, are not present in germline B cells in a form which is transcribed as a functional unit that encodes an antibody. Instead the relevant loci contain a series of segments which recombine during the B cell maturation process to form unique immunoglobulin heavy and light chain loci. These segments are known as variable (V), diversity (D), joining (J) and constant (C) gene segments. The heavy chain of an antibody has V, D, J and C segments. The light chains have only V, J and C segments.

During B cell development the V, D, and J segments (in the case of the heavy chain) and the V and J segments (in the case of the light chain) are joined together at the DNA level in a process known as VDJ recombination or somatic gene rearrangement. The eventual result is the production, after transcription and translation, of a huge array of different antibodies. The process of rearrangement, and then transcription and translation of the heavy chain of an antibody is shown schematically below:

After VDJ rearrangement, if a B cell is engaged by a fully mature and activated T cell of appropriate specificity, the B cell turns on activation induced cytidine deaminase which results in a wide spectrum of mutations in the antibody genes. Any given V segment may undergo anything from zero to dozens of mutations leading to numerous amino acid substitutions in the V regions. Favourable mutations confer an increase in affinity for the antigen, and B cells harbouring such mutations have a selective advantage, whereas deleterious mutations eventually ensure that the irrelevant B cells are eliminated. This process of mutation is known as somatic hypermutation and it improves still further the strength of the antibody response.

Antibodies in therapy

Antibodies incorporating mouse regions can create an immune response in humans (known as the HAMA response or human anti-mouse antibody response). To avoid the HAMA response it was known to be preferable to use an antibody that was fully human, as opposed to a murine antibody or a chimeric antibody (which has mouse variable regions and human constant regions) or a humanised antibody (which has the complementarity determining regions of a murine antibody grafted onto a human antibody).

The antibodies used for therapy at the priority date were monoclonal antibodies. Monoclonal antibodies were first described in Köhler and Milstein's 1975 paper in the journal Nature where they proposed a technique whereby a transformed (cancerous) B cell (i.e. myeloma) was fused with a normal antibody producing B cell to create a “hybridoma” that grows freely in vitro while continuing to produce an antibody. The hybridoma thus carried a single rearranged immunoglobulin heavy (“IgH”) gene, and a single rearranged immunoglobulin kappa (“IgK”) or lambda (“IgL”) gene, and secreted antibody molecules of a single specificity into the culture supernatant. The ability to produce monoclonal antibodies specific to particular human receptors opened up the possibility of major advances in the treatment of disease.

Immunoglobulin locus size

The mouse and human Ig loci are of different sizes and contain different numbers of V, (D) and J gene segments. The human IgH locus is approximately 1,250 kb long. The human IgK locus is approximately 1,820 kb long, and the IgL locus is approximately 1,050 kb long and is located on chromosome 22.

The murine IgH locus is approximately 3 Mb in length. The murine IgK locus is approximately 3.2 Mb in length, and the murine IgL locus is smaller than the other loci at just 240 kb in length.

Transgenics

Transgenics is the term used to describe the introduction of a DNA fragment encoding a functional gene product into the germ line of a different species. In transgenics, one can take a human antibody gene and insert it into the mouse genome. If one challenges the mouse with a target antigen of interest the mouse will produce antibodies with its B cells. One can then screen for the B cell which is producing the antibody of interest.

Transgenics can be carried out broadly by two techniques. The first is random integration into the target genome, and the second is by targeted integration. Random integration was a technique practised by groups including those of Brüggeman, Lonberg and Abgenix before the priority date. Targeted integrations can be achieved by two methods, namely homologous recombination and site-specific recombination (“SSR”).

Most methods of gene targeting involve rare events so it is very desirable to be able to select or screen for the desired gene alteration and to discriminate against the others. Most methods of gene targeting use embryonic stem (“ES”) cells. Selectable markers can be used to select for ES cells in which the targeting construct has integrated.

Vectors to carry the DNA fragment to be incorporated into the host genome differ depending on whether they are to be used for homologous recombination or SSR. A targeting vector for homologous recombination includes flanking homology arms which are selected so that they are highly related and preferably identical in DNA sequence to the sequences that flank the target sequence being modified or replaced in the genome. The length and degree of sequence homology of the homology arms is an important factor in determining the efficiency of incorporation of the targeting construct. The process is illustrated in the following diagram:

A targeting vector for SSR uses a site-specific recombinase enzyme and its target sequence. The enzyme cleaves the DNA at a distinct target sequence and ligates it to the cleaved DNA of a second target of the same nature to generate a specific recombination event. The SSR sequence is first introduced into the host (by homologous recombination), followed by the use of the site for site-specific recombination. SSR can be used for targeted insertion or replacement of an endogenous gene in whole or in part. This system was particularly efficient at making deletions from the host genome.

Site directed integration...