Sanofi v Amgen Inc. (No 3)
[2025] FCA 387
•23 April 2025
FEDERAL COURT OF AUSTRALIA
Sanofi v Amgen Inc. (No 3) [2025] FCA 387
File number: NSD 876 of 2022 Judgment of: NICHOLAS J Date of judgment: 23 April 2025 Catchwords: PATENTS – appeal from decision of Delegate of Commissioner of Patents holding oppositions to patent applications unsuccessful – applications for patents including claims to isolated monoclonal antibodies that bind to epitopes on PCSK9 that include one or more specified amino acid residues and which block binding of PCSK9 to LDLR and claims to isolated monoclonal antibodies that bind to at least one specified amino acid on PCSK9 and which block binding of PCSK9 to LDLR – where opposition based on (inter alia) alleged failure to meet requirements of s 40 of the Patents Act 1990 (Cth) in the form it took before amendment by the Intellectual Property Laws Amendment (Raising the Bar) Act 2012 (Cth) – whether claims would be clearly invalid if Applications proceeded to grant – whether claims fairly based on the matter described in the specifications – whether claims fail to define the invention – whether claims fail to describe the invention fully –whether claims to a single invention comprising a class of antibodies or a multiplicity of different inventions not fully described – whether claims for a manner of manufacture – whether claims entitled to priority date of 23 August 2007 based on provisional application filed on that date – whether claims involve an inventive step – whether notional skilled team would have been directly led to try to generate antibodies in the expectation that they may well block binding between PCSK9 and LDLR – consideration of state of the art at the priority date
PATENTS – whether claims to isolated monoclonal antibodies that compete with specified reference antibodies for binding to PCSK9 lack clarity by failing to specify a numerical value or otherwise failing to provide a workable standard with respect to such competition
Held: Appeal dismissed
Legislation: Evidence Act 1995 (Cth) s 57
Patents Act 1990 (Cth) ss 18(1)(a), 40, 60
Patents Act 1952 (Cth) s 40
Intellectual Property Laws Amendment (Raising the Bar) Act 2012 (Cth)
Patents Act 1949 (UK) s 4(3)(c)
Patents Act 1977 (UK) s 14
Cases cited: Aktiebolaget Hässle v Alphapharm Pty Ltd (2002) 212 CLR 411
Albany Molecular Research Inc v Alphapharm Pty Ltd (2011) 90 IPR 457
AMP Inc v Utilux Pty Ltd (1971) 45 ALJR 123
Anaesthetic Supplies Pty Ltd v Rescare Ltd (1994) 50 FCR 1
Ariosa Diagnostics, Inc v Sequenom, Inc (2021) 391 ALR 473
Aristocrat Technologies Australia Pty Ltd v Commissioner of Patents (2022) 274 CLR 115
AstraZeneca AB v Apotex Pty Ltd (2014) 226 FCR 324
Atlantis Corporation Pty Ltd v Schindler (1997) 39 IPR 29
Austal Ships Pty Ltd v Stena Rederi Aktiebolag (2005) 66 IPR 420
British Thomson-Houston Company Ld. v Corona Lamp Works Ld. (1922) 39 RPC 49
Burroughs Corp (Perkins’) Application [1974] RPC 147
CCOM Pty Ltd v Jiejing Pty Ltd (1994) 51 FCR 260
Commissioner of Patents v Microcell Ltd (1958) 102 CLR 232
Coopers Animal Health Australia Ltd v Western Stock Distributors Pty Ltd (1987) 15 FCR 382
D’Arcy v Myriad Genetics Inc (2015) 258 CLR 334
Electric & Musical Industries Ltd v Lissen Ltd (1938) 56 RPC 23
Eli Lilly and Co v Pfizer Overseas Pharmaceuticals (2005) 218 ALR 408
Encompass Corporation Pty Ltd v InfoTrack Pty Ltd (2019) 372 ALR 646
Evans Medical Ltd’s Patent [1998] RPC 517
F Hoffman-La Roche AG v New England Biolabs Inc (2000) 99 FCR 56
Genentech Inc.’s Patent [1987] RPC 553
General Tire & Rubber Company v Firestone Tyre & Rubber Company Limited [1972] RPC 457
Gilead Sciences Pty Ltd v Idenix Pharmaceuticals LLC (2016) 117 IPR 252
GlaxoSmithKline Consumer Healthcare Investments (Ireland) (No 2) Ltd v Generic Partners Pty Ltd (2018) 264 FCR 474
Grant v Commissioner of Patents (2006) 154 FCR 62
ICI Chemicals and Polymers Ltd v Lubrizol Corporation Inc (1999) 45 IPR 577
Interlego AG v Toltoys Pty Ltd (1973) 130 CLR 461
Jupiters Ltd v Neurizon Pty Ltd (2005) 222 ALR 155
Kimberly-Clark Australia Pty Ltd v Arico Trading International Pty Ltd (2001) 207 CLR 1
Kirin-Amgen Inc v Hoechst Marion Roussel Ltd) [2005] RPC 169
Leonardis v Sartas No 1 Pty Ltd (1996) 67 FCR 126
Lockwood Security Products Pty Limited v Doric Products Pty Ltd (2004) 217 CLR 274
Meat & Livestock Australia Ltd v Cargill, Inc (2018) 354 ALR 95
Merck & Co Inc v Arrow Pharmaceuticals Ltd (2006) 154 FCR 31
Merck Sharp & Dohme Corporation v Wyeth LLC (No 3) (2020) 155 IPR 1
Minnesota Mining and Manufacturing Company v Beiersdorf (Australia) Limited (1980) 144 CLR 253
Monsanto Co v Commissioner of Patents (1974) 48 ALJR 59
Mullard Radio Valve Co Ltd v British Belmont Radio Ltd (1938) 56 RPC 1
National Resource Development Corporation v Commissioner of Patents (1959) 102 CLR 252
Neurim Pharmaceuticals (1991) Ltd v Generic Partners Pty Ltd (No 5) [2024] FCA 360
NV Philips Gloeilampenfabrieken v Mirabella International Pty Ltd (1995) 183 CLR 655
Olin Corporation v Super Cartridge Co Pty Ltd (1977) 180 CLR 236
Pfizer Overseas Pharmaceuticals v Eli Lilly & Co (2005) 225 ALR 416
Rehm Pty Ltd v Websters Security Systems (International) Pty Ltd (1988) 81 ALR 79
Research Affiliates and Commissioner of Patents v RPL Central Pty Ltd (2015) 238 FCR 27
Research Affiliates LLC v Commissioner of Patents (2014) 227 FCR 378
Reynolds v Herbert Smith & Co Ltd (1902) 20 RPC 123
Sequenom, Inc v Ariosa Diagnostics, Inc (2019) 143 IPR 24
Shave v H V McKay Massey Harris Pty Ltd (1935) 52 CLR 701
Stauffer Chemical Co’s Application [1977] RPC 33
ToolGen Incorporated v Fisher (No 2) [2023] FCA 794
Tramanco Pty Ltd v BPW Transpec Pty Ltd (2014) 105 IPR 18
Welch Perrin & Co Pty Ltd v Worrel (1961) 106 CLR 588 at 610
Bodkin C, Patent Law in Australia (4th ed, Law Book Co, 2024)
Terrell T, Terrell on the Law of Patents (13th ed, Sweet & Maxwell, 1982)
Division: General Division Registry: New South Wales National Practice Area: Intellectual Property Sub-area: Patents and associated Statutes Number of paragraphs: 470 Date of last submissions: 16 February 2024 Date of hearing: 6-17 November and 11-13 December 2023 Counsel for the Appellant: Mr D Shavin KC with Ms K Beattie Solicitor for the Appellant: Jones Day Counsel for the Respondent: Mr T Cordiner KC with Ms C Cunliffe and Ms M McGrath Solicitor for the Respondent: Wrays Lawyers Pty Ltd ORDERS
NSD 876 of 2022 BETWEEN: SANOFI
Appellant
AND: AMGEN INC.
Respondent
ORDER MADE BY:
NICHOLAS J
DATE OF ORDER:
23 APRIL 2025
THE COURT ORDERS THAT:
1.The appeal be dismissed.
2.The decision of the Delegate of the Commissioner of Patents made on 26 September 2022 be affirmed.
3.Each of Australian Patent Applications AU 2013203677, AU 2013203748, AU 2013203685, AU 2013203689 and AU 2013203751 proceed to grant.
4.The time within which any application for leave to appeal must be made is extended to 21 May 2025.
5.Order 3 is stayed subject to any further order:
(a)until 21 May 2025; or
(b)if any application for leave to appeal is filed by that date, until the determination of that application or the determination of any appeal if leave to appeal is granted.
6.The appellant pay the respondent’s costs of the appeal.
7.Exhibits GAP-A7 and GAP-A8 be returned to the respondent’s solicitors.
Note: Entry of orders is dealt with in Rule 39.32 of the Federal Court Rules 2011.
REASONS FOR JUDGMENT
INTRODUCTION
[1]
GENERAL PRINCIPLES
[23]
WITNESSES
[28]
SCIENTIFIC BACKGROUND
[54]
THE COMMON SPECIFICATION
[119]
SECTION 40
[176]
PRIORITY DATE
[344]
THE NOTIONAL PERSON SKILLED IN THE ART
[368]
COMMON GENERAL KNOWLEDGE
[373]
INVENTIVE STEP
[391]
LACK OF CLARITY
[428]
SUFFICIENCY
[455]
ADDITIONAL MATTERS
[467]
DISPOSITION
[470]
NICHOLAS J:
INTRODUCTION
The appellant (“Sanofi”) appeals from a decision of the Delegate of the Commissioner of Patents made on 26 September 2022 dismissing Sanofi’s opposition of the grant of five patent applications in the name of the respondent (“Amgen”): Sanofi v Amgen Inc. [2022] APO 67. The appeal is brought under s 60 of the Patents Act 1990 (Cth) (“the Act”).
The patent applications (collectively, “the Applications”) are:
(1)AU 2013203677 (“677”);
(2)AU 2013203748 (“748”);
(3)AU 2013203685 (“685”);
(4)AU 2013203689 (“689”); and
(5)AU 2013203751 (“751”).
Each of the Applications is entitled “Antigen Binding Proteins to Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9)”.
The Applications were filed on 11 April 2013. The earliest asserted priority date for each of the relevant claims is 23 August 2007 (“the priority date”).
Each of the Applications claims divisional status from Application No. 2008288791 (“the Parent Application”) filed on 22 August 2008 under the provisions of the Patent Cooperation Treaty as PCT/US2008/074097. The Parent Application was first published on 26 February 2009.
Low density lipoprotein receptor (“LDLR”) on the surface of the liver takes up low density lipoprotein (“LDL”) which is then transported to lysosomes for degradation. The LDLR is then returned to the liver surface to take up more LDL. High levels of LDL cholesterol are associated with increased risk of cardiovascular and related diseases. Reduction in LDL cholesterol concentrations may therefore lead to improved health outcomes.
PCSK9 refers to proprotein convertase subtilisin kexin Type 9. PCSK9 is a protein involved in the degradation of LDLR. The Applications are concerned with isolated monoclonal antibodies (“isolated MAbs”) that target PCSK9 (the antigen). The MAbs block or inhibit the activity of PCSK9 by binding to it, thereby increasing the level of LDLR available to clear LDL and reduce LDL cholesterol levels.
The binding of an antibody to its antigen occurs via non-covalent interactions between amino acid residues of the antibody and the antigen.
The Applications have a Common Specification (“CS”), which differs as between the Applications in the consistory-like clauses and some figures. Each of the independent claims of the Applications is to an isolated MAb. The term “isolated” means the MAb exists separate from the other components with which it would usually be found, and the term monoclonal describes uniform antibodies of the same specificity and comprising the same heavy and light chains, with some minor variations to their amino acid sequences to be expected. Most relevantly, MAbs will bind to the same epitope, a term which is defined immediately below.
The CS and the claims of some of the Applications refer to amino acid residues on PCSK9. The claims identify residues by reference to (inter alia) “SEQ ID NO: 1” and SEQ ID NO: 3”, both being amino acid sequences of PCSK9 set out in Figures 1A and 1B to the CS, respectively. The term “epitope” refers to the specific region on the antigen to which the antibody binds. The amino acid sequences in Figures 1A and 1B are relevantly the same, except that Figure 1B includes the signal sequence, whereas Figure 1A does not. The specification teaches that “any amino acid position in SEQ ID NO: 1, will correspond to an amino acid position 30 amino acids further into the protein in SEQ ID NO: 3”.
The parties have agreed upon a set of exemplary claims in each of the Applications (“the Exemplary Claims”). Those claims can be divided into the “Epitope Claims” and the “Residue Claims” of 677, 689 and 751, and the “Competition Claims” of 677, 685 and 748.
677 includes independent claims 1 and 5. Claim 1, an Epitope Claim, is for:
1.An isolated monoclonal antibody that binds an epitope on hPCSK9 comprising one or more of amino acid residues 207, 208, 162, 164, 167, or 123 of SEQ ID NO: 1, and wherein the monoclonal antibody blocks binding of PCSK9 to LDLR.
Claim 5, a Residue Claim, is for:
5.An isolated monoclonal antibody that binds to at least one of amino acid residues 207, 208, 162, 164, 167, or 123 of SEQ ID NO: 1, and wherein the monoclonal antibody blocks binding of PCSK9 to LDLR.
677 also includes a Competition Claim:
23.The isolated monoclonal antibody according to any one of claims 1-20, wherein the isolated human monoclonal antibody competes for binding to PCSK9 with an antibody comprising a heavy chain amino acid sequence comprising an amino acid sequence of SEQ ID NO: 49 and a light chain amino acid sequence comprising an amino acid sequence of SEQ ID NO: 23.
689 includes independent claim 1, a Residue Claim for:
An isolated monoclonal antibody, wherein, when bound to PCSK9, the monoclonal antibody binds to at least one of the following residues: S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380, or S381 of SEQ ID NO: 3, and wherein the monoclonal antibody blocks binding of PCSK9 to LDLR.
751 includes independent claims 1, 2, 10, and 13. Claims 1 and 10 are Epitope Claims. Claims 2 and 13 are Residue Claims. These claims provide:
1.An isolated monoclonal antibody that recognizes an epitope on human PCSK9 comprising amino acid residues: S153, R194, D238, D374, T377, and F379 of SEQ ID NO: 3, wherein the monoclonal antibody reduces binding between PCSK9 and EGFa domain of LDLR.
2.An isolated monoclonal antibody that binds to amino acids in human PCSK9 of SEQ ID NO: 1, wherein the amino acids comprise S153, R194, D238, D374, T377, and F379 of SEQ ID NO: 3, and wherein the monoclonal antibody reduces binding between PCSK9 and an EGFa domain of LDLR.
…
10.An isolated monoclonal antibody that recognizes an epitope on human PCSK9, wherein the epitope comprises at least ten of the following residues: S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380, or S381 of SEQ ID NO: 3, wherein the monoclonal antibody reduces binding between PCSK9 and an EGFa domain of LDLR.
…
13.An isolated monoclonal antibody that binds to amino acids in human PCSK9, wherein the amino acids comprise ten or more of the following amino acids: S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380, or S381 of SEQ ID NO: 3, and wherein the monoclonal antibody reduces binding between PCSK9 and an EGFa domain of LDLR.
Claims 2 and 13 do not use the word “residue”, however, as with claims 1 and 10, they identify particular amino acid residues by reference to their place in the sequence.
Claim 1 of 677 refers to a MAb that binds to an epitope of PCSK9 so as to block binding of PSK9 to LDLR. Claims 1 and 10 of 751 adopt the same approach as claim 1 of 677, although using slightly different terminology. Claims 5 of 677, 1 of 689 and 2 and 13 of 751 refer to MAbs that bind to particular residues of PCSK9, rather than epitopes. The “EGFa domain” of LDLR refers to a section of LDLR, which is the region of LDLR that binds to PCSK9.
Claim 23 of 677, and claims 1 of 685 and 748, introduce the concept of competition in referring to a MAb that competes for binding to PCSK9 with another antibody with a particular amino acid sequence.
685 includes claim 1, a Competition Claim for:
An isolated monoclonal antibody that binds to human PCSK9 and reduces binding between human PCSK9 and an EGFa domain of LDLR, wherein said monoclonal antibody competes for binding to PCSK9 with an antibody that comprises a heavy chain variable region of the amino acid sequence in SEQ ID NO: 67; and a light chain variable region of the amino acid sequence in SEQ ID NO: 12.
748 includes claim 1, another Competition Claim, which provides:
An isolated monoclonal antibody that binds to human PCSK9 and is neutralizing in that an excess of said antibody reduces the quantity of human PCSK9 bound to LDLR in an in vitro competitive binding assay, wherein said monoclonal antibody competes for binding to PCSK9 with an antibody that comprises: a heavy chain variable region of the amino acid sequence in SEQ ID NO: 49; and a light chain variable region of the amino acid sequence in SEQ ID NO: 23.
The antibodies identified in claims 23 of 677, 1 of 685 and 1 of 748 by reference to their heavy and light chain regions are identified in the CS as either of antibodies 31H4 and 21B12, which are proffered as benchmarks against which other MAbs may be assessed for the purpose of determining whether they have the relevant binding effect. It is convenient to refer to these two antibodies as the “reference antibodies”.
CS [0607] includes the following definition of “comprise” and “comprising”
Throughout the specification and claims, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Applying this definition to the Epitope and Residue Claims, the MAb need only bind to, or bind to an epitope that includes, one or more (or in the case of claims 10 and 13 of 751, at least 10) of the specified amino acid residues. For the Epitope Claims, there does not need to be a non-covalent interaction between the MAb and any particular residue, provided that the specified residue is part of the epitope on PCSK9 bound by the MAb.
Sanofi contended that any patent granted on any of the Applications would be invalid on various grounds. Sanofi summarised its opposition to the Applications as follows:
(a)Failure to define the invention – The claims of each of the Applications fail to define the invention in that they pertain to any “isolated monoclonal antibody” having some claimed functional features only. The “blocking” function of an antibody, knowledge of the epitope or a residue or residues on an antigen to which an antibody binds, or reference to another antibody with which an antibody competes, does not define the structure of, or characterise the construction of, an antibody as claimed.
(b)Not a manner of manufacture – The claims of each of the Applications fail to define a manner of manufacture because they are for no more than a mere desideratum, namely, any MAb providing particular functional outcomes.
(c)Lack of fair basis – The claims travel well beyond any invention described in the specification and omit essential features of that invention and are therefore not fairly based.
(d)Lack of clarity – The Competition Claims lack clarity because it is not clear whether “competes” means any degree of competition or some particular numerical degree of competition and, in any event, there is no workable standard to determine the exact boundaries of the claims. Various alternative competitive binding assays described in the specification can produce different results.
(e)Failure to describe the invention fully – The claims of each of the Applications are properly characterised as claims to a multiplicity of inventions, each a different antibody with a unique amino acid sequence and binding to a different epitope on PCSK9. The Applications are therefore insufficient because the person skilled in the art (“PSA”) would need to engage in the research project described in Examples 1 to 3 of the Applications in an attempt to produce one embodiment of each such invention.
(f)Lack of inventive step – The use of an antibody to inhibit PCSK9 had been postulated in well-known, peer-reviewed literature. LDLR residues in the PCSK9 and LDLR EGFa domain interface (“the PCSK9/EGFa interface”) were known. It was “logical” that an antibody that blocks the binding of PCSK9 to LDLR (to any degree) would do so by binding to an epitope that included any one of the residues in the PCSK9/EGFa interface. It follows that the inventions do not involve any inventive step.
GENERAL PRINCIPLES
Standard of Proof
It is common ground that the Applications should proceed to grant unless the Court is satisfied that any such grant would be clearly invalid. Sanofi, the opponent, bears the onus of showing that the grant of a standard patent based on the Applications would be clearly invalid.
Although contested issues of fact are to be decided on the balance of probabilities, the ultimate question raised under each of the relevant grounds of opposition, typically involving mixed issues of fact and law, is to be decided by reference to the higher standard. This has important implications for the present case in which the relevant grounds of opposition are the subject of significant differences of opinion between expert witnesses. As Bennett J observed when deciding an opposition based on an alleged lack of inventive step in Austal Ships Pty Ltd v Stena Rederi Aktiebolag (2005) 66 IPR 420 (“Stena”) at [12]:
I can accept that a lower standard may apply to proof of evidence such as whether a document has been published or, indeed, whether a prior art vessel was well-known. I do not accept that it properly applies to the factual question that itself is the test for obviousness or lack of inventive step. Where the factual question is itself the legal test, as set out in s 7(3) of the Act, it seems to me that it should be determined at the higher standard. That means that where there are two opposing expert views that are conclusive on obviousness, both presented bona fide by witnesses of accepted expertise, unless one set of views can be rejected on proper grounds, the legal burden to establish a ground of opposition is not discharged; the court cannot be practically certain that obviousness or lack of inventive step is established.
Her Honour’s approach to the expert evidence was followed by Beach J in Meat & Livestock Australia Ltd v Cargill, Inc (2018) 354 ALR 95 (“Meat & Livestock v Cargill”) at [11], although his Honour favoured the language of “clearly invalid” used by Emmett J in F Hoffman-La Roche AG v New England Biolabs Inc (2000) 99 FCR 56 at [67], rather than that of “practically certain”. I have adopted the same approach as Beach J.
Principles of Construction
There was no dispute between the parties as to the principles governing the construction of a patent specification or, in this case, a patent application. These principles were summarised by the Full Court in Jupiters Ltd v Neurizon Pty Ltd (2005) 222 ALR 155 (Hill, Finn and Gyles JJ) as follows at [67]:
…
(i)the proper construction of a specification is a matter of law: Décor Corporation Pty Ltd v Dart Industries Inc (1988) 13 IPR 385 at 400 [(Décor Corporation Pty Ltd)];
(ii)a patent specification should be given a purposive, not a purely literal, construction: Flexible Steel Lacing Co v Beltreco Ltd (2000) 49 IPR 331; [2000] FCA 890 at [81] (Flexible Steel Lacing); and it is not to be read in the abstract but is to be construed in the light of the common general knowledge and the art before the priority date: [Kimberly]-Clark Australia Pty Ltd v Arico Trading International Pty Ltd (2001) 207 CLR 1; 177 ALR 460; 50 IPR 513; [2001] HCA 8 [(Kimberly-Clark v Arico)] at [24];
(iii)the words used in a specification are to be given the meaning which the normal person skilled in the art would attach to them, having regard to his or her own general knowledge and to what is disclosed in the body of the specification: Décor Corporation Pty Ltd at 391;
(iv)while the claims are to be construed in the context of the specification as a whole, it is not legitimate to narrow or expand the boundaries of monopoly as fixed by the words of a claim by adding to those words glosses drawn from other parts of the specification, although terms in the claim which are unclear may be defined by reference to the body of the specification: [Kimberly]-Clark v Arico at [15]; Welch Perrin & Co Pty Ltd v Worrel (1961) 106 CLR 588 at 610; Interlego AG v Toltoys Pty Ltd (1973) 130 CLR 461 at 478; the body of a specification cannot be used to change a clear claim for one subject matter into a claim for another and different subject matter: Electric & Musical Industries Ltd v Lissen Ltd [1938] 4 All ER 221 at 224–5; (1938) 56 RPC 23 at 39;
(v)experts can give evidence on the meaning which those skilled in the art would give to technical or scientific terms and phrases and on unusual or special meanings to be given by skilled addressees to words which might otherwise bear their ordinary meaning: Sartas No 1 Pty Ltd v Koukourou & Partners Pty Ltd (1994) 30 IPR 479 at 485–6 (Sartas No 1 Pty Ltd); the court is to place itself in the position of some person acquainted with the surrounding circumstances as to the state of the art and manufacture at the time ([Kimberly]-Clark v Arico at [24]); and
(vi)it is for the court, not for any witness however expert, to construe the specification; Sartas No 1 Pty Ltd at 485–6.
The notion of purposive construction requires that the specification be read through the eyes of the PSA with practical knowledge and experience in the field of work in which the invention was intended to be used, and with the proper purpose of the invention in mind: GlaxoSmithKline Consumer Healthcare Investments (Ireland) (No 2) Ltd v Generic Partners Pty Ltd (2018) 264 FCR 474 (“GSK v Generic”) at [106].
WITNESSES
Sanofi’s Witnesses
Professor Jay D. Horton
Professor Jay Horton is a Professor in the Department of Internal Medicine and the Department of Molecular Genetics, University of Texas Southwestern Medical Center (“UT Southwestern”), in Dallas. He holds an undergraduate degree in Zoology and a Doctor of Medicine (“MD”) from the University of Iowa. After obtaining his MD, he completed an internal medicine residency and gastroenterology fellowship at UT Southwestern. He then completed a post-doctoral fellowship in the Department of Molecular Genetics at UT Southwestern. The work he undertook subsequent to his fellowship, as a junior member of the Department of Molecular Genetics, led to the identification of novel proteins regulated by sterol regulatory element binding proteins, one of which was PCSK9. Following that, his group undertook research into the function and biology of PCSK9.
Professor Horton gave evidence about PCSK9 and the therapeutic modalities used to inhibit it. Professor Horton provided consultancy services to a number of pharmaceutical companies before the priority date in relation to PCSK9. This work, which commenced early 2005, involved consulting on projects investigating PCSK9 as a possible drug target for the treatment of high cholesterol. He provided services to Merck, Pfizer, Amgen, Eli Lily, Alnylam, Bristol-Myers Squibb (“BMS”), Novartis, Regeneron and Schering-Plough in relation to PCSK9. There appeared to be no dispute that he was, at the relevant times, one of the world’s leading experts on PCSK9. Professor Horton and his team were not directly involved in drug discovery or drug development.
As I will later explain, I found that some of Professor Horton’s evidence as to the state of the art at the priority date was most likely affected by hindsight. That said, Professor Horton impressed me as a highly knowledgeable expert on PCSK9 as is evident from his enormous contribution to the scientific literature on that topic.
Professor Stephen Michael Mahler
Professor Stephen Mahler is an Emeritus Professor at the Australian Institute for Biotechnology at the University of Queensland, and the Global Chief, Science and Technology at the Aegros Group. He holds a Bachelor of Science (Honours) from the University of Sydney and a Doctor of Philosophy in Biochemistry from the University of Queensland.
Professor Mahler primarily gave evidence about monoclonal antibodies, including antibody production and analysis of binding activity. He has more than 35 years of experience in the antibody field, including the production, discovery, and development of monoclonal antibodies for therapeutic and diagnostic purposes. He has an extensive academic career focused on monoclonal antibody technology.
Professor Mahler was criticised by Amgen on the ground that he considered it his role as an expert witness for Sanofi to find fault with the CS wherever possible. Examples of this tendency identified by Amgen included evidence given by him concerning “antibody excess” which I refer to in more detail later in these reasons. Apart from that example, the other matters upon which Amgen relied in support of its attack on Professor Mahler’s reliability as an expert witness were to my mind relatively inconsequential. I considered Professor Mahler an impressive witness and I found much of his evidence helpful.
Professor Michael Parker
Professor Michael Parker is the Head of the Structural Biology Unit at St Vincent’s Institute of Medical Research, in Melbourne, and a Professorial Fellow and Professor at the Department of Biochemistry and Molecular Biology and Director of the Bio21 Institute at the University of Melbourne. He holds a Bachelor of Science in Chemistry with Honours from the Australian National University, and a Doctor of Philosophy from the University of Oxford.
Professor M Parker’s evidence was directed to crystallography and analysis of antibody-antigen interactions. He is a structural biologist, and expert in X-ray crystallography including the use of X-ray crystallography to solve the atomic structure of proteins. He has more than 40 years of experience in this field.
Amgen was critical of Professor M Parker’s evidence as “overly pedantic” and as expecting the CS to meet standards of disclosure that were not always adhered to in his own publications. Professor M Parker asserted in his written evidence that certain data files (the PDB files used in the PyMOL analysis) were not available to him, but Amgen submits that he did not request that they be provided to him. Amgen described his failure to request this data so that he could conduct his own analysis of it (rather than merely relying on Professor Petsko’s analysis) as “inexplicable”.
There is considerable force in Amgen’s submission, though ultimately the validity of Professor M Parker’s criticisms of the PyMOL analysis relied on by Amgen to establish certain non-covalent interactions need not be addressed, since my conclusions with respect to the fair basis challenge do not depend on it.
Other witnesses
Sanofi also relied on affidavit evidence of Anthony Muratore, Samin Raihan, Lisa Taliadoros and Maria Garcia of Jones Day. They were not cross-examined and their affidavit evidence was uncontroversial.
Amgen’s Witnesses
Dr Rex Arnold Parker
Dr Rex Parker was a Director and Senior Research Fellow of the Hopewell Biology Discovery Department at BMS. He holds undergraduate degrees in chemistry and biology, and a PhD in biochemistry, from Indiana University. After completing his doctoral research, he joined BMS's cardiovascular and metabolic diseases research department looking at ways to regulate LDL cholesterol and sterol metabolism.
Dr R Parker gave evidence about PCSK9 and the therapeutic modalities to inhibit it. Between 2002 and 2014, he was responsible for BMS’s lipid-lowering and anti-atherosclerotic drug discovery, and was co-leader of BMS's PCSK9 inhibitor discovery research program. In 2009, he was promoted to the position of Senior Research Fellow for Cardiovascular Drug Discovery at BMS, which was the position he held until he retired in 2014. He has many years’ experience in the field of drug discovery and drug development.
Sanofi was critical of Dr R Parker’s evidence on the basis that he was a “contrarian”, and that he was excessively critical of all scientific literature supporting the view that PCSK9 had an extracellular mode of action. I do not accept Sanofi’s criticisms of Dr R Parker as a witness, which I deal with in further detail below when considering the parties’ submissions on the topic of inventive step. Generally speaking, I considered Dr R Parker an impressive witness whose evidence was of considerable assistance to me on that topic.
Professor Angel Francisco Lopez
Professor Lopez holds a degree in medicine from the School of Medicine at the University of Rosario, Argentina, and a PhD from the National Institute for Medical Research, University of London. He has over 40 years of experience working in the field of antibodies and has created antibodies within his laboratory for the treatment of acute myeloid leukaemia. He is currently the Head of the Cytokine Receptor Laboratory and head of the Division of Human Immunology at SA Pathology in Adelaide, an Adjunct Clinical Professor of the Faculty of Medicine at the University of Adelaide and an Adjunct Professor at the University of South Australia. Professor Lopez gave evidence about antibody generation using hybridoma technology, immunisation and screening protocols.
Professor Lopez was criticised in Sanofi’s submissions on the grounds that his evidence was often unresponsive and that, particularly in relation to crystallography, he volunteered opinions that travelled outside his area of expertise. Sanofi also referred to what it said was “the tendentious approach he took when he perceived his evidence to be unhelpful to Amgen’s case”. I took that to mean that Professor Lopez was biased against Sanofi. There is nothing in Sanofi’s submissions which justifies that very serious attack upon Professor Lopez’s credit. I found Professor Lopez’s evidence concerning antibody development helpful in understanding the technology and in assessing the difficulties that may be experienced by the PSA in generating an anti-PCSK9 antibody without the benefit of the CS. I reject Sanofi’s submission that he was biased or that he sought to argue Amgen’s case in a manner that was inconsistent with his duty as an independent expert.
Professor Peter John Hudson
Professor Peter Hudson has expertise and extensive experience in the field of antibody structure, function and development, and in the area of X-ray crystallography. He currently provides scientific consultancy services in the development of antibodies as clinical products as the Managing Director of Diantron Biomed Pty Ltd, and also serves as the Chief Scientific Officer of AviPep Pty Ltd and as Senior Scientific Advisor to CarTherics Pty Ltd (for the development of T-cell therapies). He holds a Bachelor of Science (Honours) from the Department of Biochemistry, University of Adelaide and a PhD from the MRC Laboratory of Molecular Biology, University of Cambridge. Professor Hudson gave evidence as to antibody technology and structural biology (including crystallography).
As with Professor Lopez, Sanofi submitted that Professor Hudson gave evidence which displayed what it said was a “disturbing tendency” to argue Amgen’s case. The evidence to which Sanofi points in support of this submission does not support it. For example, it criticised Professor Hudson for having by his own admission speculated about the inventors’ reasons for having followed an immunisation protocol involving more than 10 immunisations of mice in the process of generating antibodies. But he freely admitted that this was his own interpretation of why the inventors may have proceeded as they did. Later in his evidence he agreed that he was mistaken in suggesting in earlier evidence that mice were given a “massive double boost” of 10mg of antigen as opposed to 5mg split across two different injections.
I do not consider this or any other evidence given by Professor Hudson as reflecting a tendentious approach to the giving of evidence. Nor do I think that any other inconsistency to which Sanofi pointed in his evidence diminishes the quality of his evidence in areas where it most matters. I generally found Professor Hudson to be an impressive witness whose evidence was quite helpful.
Professor Gregory Anthony Petsko
Professor Petsko is a Professor of Neurology at Harvard Medical School and Brigham Women’s Hospital, Adjunct Professor of Biomedical Engineering at Cornell University and Emeritus Professor in Biochemistry and Chemistry at Brandeis University. He holds a Bachelor of Chemistry from Princeton University and a PhD in Molecular Biophysics from Oxford University. He is an elected member of the National Academy of Sciences (1995) and the American Academy of Arts and Sciences (2002). He has been recognised by the latter for his pioneering work in the field of X-ray crystallography. In 2023 he was awarded a National Medal of Science by the President of the USA.
Professor Petsko has expertise in various related fields including, most relevantly, structural biochemistry, and X-ray crystallography. He has authored or co-authored over 300 peer reviewed articles mostly detailing the relationship between protein structure and function. Much of his work has included the use of X-ray crystallography to solve the atomic structure of proteins either alone or in complex with other molecules. He is a co-author (with Professor Dagmar Ringe) of a textbook first published in 2003 concerning basic principles of protein structure and function entitled “Primers in Biology: Protein Structure and Function”.
Sanofi submitted that Professor Petsko, while highly qualified, was often loquacious in his answers and “passionately committed to Amgen’s case”. Sanofi was also critical of Professor Petsko for not disclosing in his written evidence that he prepared or relied on an electron density map in respect of residue S153 on PCSK9.
Professor Petsko made clear in his oral evidence that his analysis of S153 relied only on the structural information that would be available to Professor M Parker and did not rely on the electron density map for S153. Professor Petsko’s cross examination revealed he had seen the electron density map for S153 “a number of years ago” and that he could not “unsee” this information. When Professor Petsko was pressed on the topic of S153, he made clear he did not use the electron density map as part of his analysis, but instead used the B-Factors provided in Table 35.3 and the rotamer for S153 recorded in the coordinates in Table 35.3.
There are a few points I would make with regard to the suggestion that Professor Petsko was “loquacious” and “passionately committed to Amgen’s case”. First, much of Professor Petkso’s oral evidence was directed to explaining the relevant science and the CS, often in answer to questions from the Court. In this regard, the impression I formed of Professor Petsko was not that he was loquacious, but that he is an excellent communicator with an impressive ability to explain the relevant science pellucidly. I reject Sanofi’s submission that Professor Petsko’s evidence was tendentious or affected by bias. I regard him as a very impressive witness whose evidence I found helpful.
Other witnesses
Amgen also relied on affidavit evidence of Louisa King, Bindhu Holavanahalli, and Mark O’Leavey, none of whom were cross-examined. Some of this evidence was contentious in that it related to 3D models created by Mr O’Leavey, an engineer with expertise in production of 3D models of molecules of interest using crystallographic data. These 3D models produced by Mr O’Leavey formed part of the “Modelling Evidence” the subject of objection by Sanofi and a ruling on evidence recorded in MFI-10. I note that it was not necessary for me to refer to the Modelling Evidence for the purpose of deciding any issue in the case.
Joint Expert Reports
Several expert conclaves were conducted, and four separate joint expert reports (“JERs”) were produced. Dr R Parker and Professor Horton produced a JER regarding knowledge as to the mechanism of action of PCSK9 reducing LDLR levels and approaches to the inhibition of PCSK9. Professors Petsko, Hudson, Lopez, M Parker and Mahler (“the Antibody and Crystallography Experts”) jointly produced a JER documenting the results of a conclave dealing with both antibody and crystallography related issues. Professors Mahler, Lopez and Hudson (“the Antibody Experts”) also produced a JER dealing with further antibody issues, and Professors M Parker, Petsko and Hudson (“the Crystallography Experts”) produced a JER dealing with crystallography issues.
SCIENTIFIC BACKGROUND
The parties have filed an “Agreed Primer”, which contains some scientific background relevant to the invention and the CS. The following background information has been extracted from the Agreed Primer, the CS, or evidence otherwise agreed by the relevant experts.
Antibody structure
An antibody (also known as an immunoglobulin) is a type of protein. Proteins are complex molecules made up of amino acids (often referred to as “amino acid residues” or “residues”).
The sequence of amino acids is referred to as the “primary structure” of the protein. There are 20 naturally occurring amino acids, and in sequences they are represented by either a one-letter or a three-letter abbreviation. For example, aspartic acid is represented by the three-letter abbreviation “asp”, and the one-letter abbreviation “D”. A specific amino acid residue on a protein can be referred to using the abbreviated name and its position number in the protein. For example, D238 means the aspartic acid residue at position number 238 of the protein.
Stretches of protein will typically arrange into distinct local conformations, referred to as “secondary structures”. The secondary structure of a protein is determined by the primary structure. The secondary structure of the protein will assemble into a 3D structure, the overall shape of which is referred to as the “tertiary structure” of the protein.
Antibodies can be represented two dimensionally as having a Y shaped structure, comprised of heavy and light chains of amino acids, as shown below:
In reality, antibodies have extremely complex three-dimensional (“3D”) shapes. The biological function of a protein is dependent on its 3D shape, because the shape of a protein affects how it interacts with other molecules.
As depicted above, there are five different classes of antibodies in mammals (IgA, IgD, IgE, IgG and IgM). Each of the classes is made up of various sub-classes (or isotypes). For example, the IgG class has four subclasses (IgG1, IgG2, IgG3 and IgG4 antibodies).
The light and heavy chains are made up of different regions, also referred to as “domains”. Each light chain consists of one variable domain, and one constant domain. Each heavy chain consists of one variable domain and three constant domains. The constant domains are referred to as such because the amino acid sequence in these domains is highly similar in all antibodies of the same class. The light and heavy chains are held together by disulfide (-S-S-) bonds, as shown below:
The “constant end” of an antibody (in the antibody depicted above, the two heavy chain CH2-CH3 regions) is called the Fc region.
The variable domains of the light and heavy chains contain the amino acid residues that contribute to the binding of the antibody to the antigen. Variable domains of the antibody comprise conserved regions referred to as “framework regions”, and hypervariable regions referred to as “complementarity determining regions” (“CDRs”). CDRs are typically around four to 20 amino acids in length. CDRs interact with the target protein (i.e. the antigen) and make up the functional binding site of the antibody. The antigen-binding site on the antibody is referred to as the paratope.
Antibody-antigen interactions
Each antibody typically binds to a particular antigen. The site on the antigen to which the antibody binds is referred to as the epitope. The CS refers to an epitope at [0233] as:
… a region of an antigen that is bound by an antigen binding protein [i.e., antibody] that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding protein.
The CS distinguishes between structural epitopes and functional epitopes as follows at CS [0571]:
Epitopes can be further defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction (e.g. hydrogen bonds, ionic interactions). Structural epitopes can be thought of as the patch of the target which is covered by the antibody.
The Antibody and Crystallography Experts agreed that the term “structural epitope” refers to the region of the target antigen covered by the antibody that binds it. They also agreed that the term “functional epitope” refers to the region of the target antigen that is directly involved in non-covalent interactions with the antibody that contribute to the affinity the two proteins have for each other.
The CS also distinguishes between linear and conformational epitopes. Amgen defines a conformational epitope as one “composed of discontinuous amino acid sequences that are brought together by protein folding in the antigen”. Sanofi’s definition, which varies only slightly, draws a distinction between conformational epitopes which “[conform] with the native three-dimensional structure of the antigen” and linear epitopes, composed of residues which are contiguous. I do not consider that there is a meaningful distinction between the two proposed definitions, but in any event, it is sufficient to refer to Amgen’s definition.
The measure of the strength of binding between the antibody and antigen is referred to as affinity. The affinity of the binding between an antibody and its antigen is the sum of all the non-covalent interactions between the antibody and the antigen.
The specificity of an antibody is the ability of the antibody to distinguish between a particular molecular structure and all other structures. Specific binding occurs when an antibody binds preferentially to its antigen over other proteins. Non-specific binding occurs when an antibody binds to an epitope that is common across a range of proteins, including its antigen and unrelated proteins. Specificity is measured through assays that may examine, for example, whether an antibody binds to proteins other than the target protein.
Protein-protein (including antibody-antigen) binding occurs when the 3D shape and contours of one protein allow some of its amino acid residues to align with that of another protein, such that one or (typically) multiple individual non-covalent interactions between those residues can take place. The three major types of non-covalent interactions that may contribute to binding of an antibody to its antigen are:
(a)Salt bridge (ionic bond) – an interaction between the positive charge of an atom of one amino acid side chain in the first protein with the negative charge of an atom of another amino acid side chain in the second protein. This is the strongest of the non-covalent interactions;
(b)Hydrogen bond – an interaction between the hydrogen atom attached to a highly electronegative atom in an amino acid residue of one protein, and a highly electronegative atom in an amino acid residue of another protein; and
(c)Van der Waals interactions – these include a range of different types of typically very weak interactions that arise due to a temporary redistribution of electrons of atoms.
Protein-protein interactions may also include other interactions involving ligands (“ligand mediated interactions”) such as water, where, for example, an atom in an amino acid residue in a first protein forms a hydrogen bond with a water molecule, and the same water molecule also forms a hydrogen bond with an atom in an amino acid residue in a second protein.
Typically, approximately 15 to 20 amino acid residues are involved in the binding of an antibody to its antigen. Although each individual interaction may be weak, the combination of interactions can result in strong binding. Put another way, although a relatively small amount of energy is needed to overcome (or “break”) an individual non-covalent interaction, a relatively large amount of energy may be needed to “break” all the non-covalent interactions and separate the antibody from its antigen.
The binding of an antibody to an antigen is a dynamic process in which the antibody rapidly binds to and dissociates from the antigen over time. In general, the higher the affinity that the two proteins have for one another, the more time the proteins will spend in their bound state. The lower the affinity that two proteins have for one another, the more time they will spend in their unbound state.
The binding of an antibody to its antigen is a dynamic process in which the antibody rapidly binds to and dissociates from the antigen over time. The antigen-antibody complex is in equilibrium with free antigen and free antibody, which can be represented by the following equation:
In general, the higher the affinity two proteins have for one another, the more time the proteins will spend in the “bound” state (i.e. the right-hand side of the equation: “AB”), and the lower the affinity two proteins have for one another, the more time the proteins will spend in the “unbound” state (i.e. the left hand side of the equation: “A + B”).
The dissociation constant (“KD”) is a quantitative estimation of the strength of binding of two proteins (e.g., antigen-antibody). For example, in an antibody-antigen interaction, a KD of 10-9 to 10-10 moles (“M”) is considered a strong interaction, whereas a KD of between 10-6 and 10-7 M is considered a relatively weak interaction. The higher the affinity, the smaller the dissociation constant.
The binding of an antibody to its antigen does not necessarily result in a biological outcome. Whether or not a biological outcome is produced may depend on the strength of the binding (the affinity), which is particularly important in the case of therapeutic antibodies. Typically, though not necessarily, a therapeutic antibody has a KD of 10-9 M.
Antibody production
In the human body, hematopoietic stem cells give rise to B cells, which produce antibodies.
Two methodologies were developed in the early 1990s for creating new fully human antibodies against a particular antigen, namely, transgenic mice and phage display. With the advent of these technologies, it was routine by the priority date, to produce fully human antibodies in high yield cell lines. Further, by the priority date, the methods, and processes for the manufacture of antibodies were mature and well established.
Transgenic mice
Transgenic mice are mice that have been genetically modified, in this context to produce fully human antibodies. When a transgenic mouse is immunised (i.e. injected with) the antigen of interest, B cells are created, some of which produce antibodies that bind to the antigen. These B cells are “remembered” by the immune system and undergo random mutations, which may lead to antibodies having increased or reduced binding affinity.
After the spleens of the mice are removed, the B cells are isolated. Isolation of a single B cell from this sample and clonal expansion results in a colony of cells arising from that single “parent” cell. Antibodies produced from a unique parent cell (and its cloned colony) are termed “monoclonal antibodies” (i.e., MAbs).
B cells must be “immortalised” to survive outside the body. To do so, a B cell is fused with a myeloma cell to create a “hybridoma”, which has the characteristics of both the B cell (in that it expresses and secretes a MAb) and the myeloma cell (in that it can grow and divide indefinitely ex vivo).
The hybridoma thus provides a cell “factory” that can be used in the large-scale production of MAbs. The hybridoma is cultured and MAbs are secreted to the outside of the cell, accumulating in the surrounding media. After an appropriate time in the cell culture, the media is harvested, and the antibody is purified from the media. The panel of antibodies produced by these hybridomas can then be screened to identify those with the desired attributes including, for example, their ability to bind to a target antigen.
The recovery of B cells from mice and their immortalisation, and the techniques for screening the binding of antibodies to a target antigen, as a general matter, were known procedures as at the priority date.
The Antibody Experts agreed that, once a mouse has been injected with the selected protein or part thereof which is the intended target antigen, the researcher has no control over how the mouse’s immune system will react. As Professor Mahler said, once injection has occurred, “[t]he mouse immune system then takes over, and whatever antibodies are raised are raised…and you get what you get”. I accept that evidence.
Phage Display
Phage display is a technique involving the use of certain types of viruses (phage) that use the cells they infect as a “factory” to produce viral particles.
Phage display also utilises an immunoglobulin gene library, which is a large collection of DNA sequences that encode the variable regions of antibodies. These libraries were available for purchase by the priority date. This DNA is inserted into ‘phagemids’ and cultured in bacteria, resulting in the production of a library of phage with the antibody variable regions ‘displayed’ on their surface.
The phage library is then screened against an antigen of interest, in a process called “bio-panning”. This results in a sub-library of phage antibodies which bind to the antigen, as well as some non-specific binders. The process is then repeated using this sub-library of phage antibodies (and subsequent sub-libraries) to increase the ratio of phage antibodies that bind specifically to the antigen.
The resultant sub-library of phage antibodies can then be screened to identify those with desired attributes. Preparation of MAbs by screening phage display libraries was well known by the priority date.
Techniques to analyse antibodies and antibody-antigen interactions
The following information is relevant to the techniques employed by the PSA in their analysis of individual antibodies and the interactions as between antibodies and antigens.
X-ray Crystallography
Atoms within chemical molecules exist in a 3D conformation. X-ray crystallography (or simply, crystallography) enables determination of that 3D arrangement (x, y and z coordinates). From well before 2007/2008, crystallography has been universally accepted as the ‘gold standard’ technique for studying the precise manner of binding between two proteins.
The technique involves shining an X-ray beam onto a sample of the protein or protein complex. Some of the X-rays interact with electrons of the atoms in the sample, resulting in the production of new X-rays that are emitted in many directions (“diffracted”).
However, when a beam of X-rays is directed at a single protein molecule, the scatter of the X‑ray beam will be too small to detect. Instead, a sample of the protein (or protein complex) in the form of a 3D crystal lattice consisting of a regular, repeating arrangement of the protein molecule (or protein complex) is required. A typical crystal has tens of thousands of identical, repeating “unit cells” which amplify the X-ray beam, creating a signal that has sufficient intensity to be detected. Obtaining crystals of a protein or protein complex relies on various factors (such as the stability of the protein complex) and is usually the most rate-limiting step of this process.
Using a protein (or protein complex) crystal, the ‘diffraction pattern’ of the scattered X-rays can be collected and then analysed mathematically to construct an initial 3D image of the electron clouds of the protein, referred to as an “electron density map”. The electron density map is then compared to the amino acid sequence of the protein, and, based on knowledge of the chemical structure of each amino acid, the electron clouds observed can be assigned to particular amino acid residues. This process is called ‘refinement’ and involves considering all possibilities until the best consensus fit with the amino acid sequence is reached. It is done using a computer program. The overall percentage disagreement between the model’s calculated diffraction pattern and the observed diffraction data is referred to as the “R factor”.
The process of fitting amino acid residues in a protein to the electron density map makes it possible to identify 3D atomic coordinates for atoms in a protein (or protein complex), represented as x, y and z values.
Subject to the resolution of the crystallography experiment, the 3D coordinates of ligands, such as water, will be identified separately. Resolution can be thought of as the extent to which it is possible to identify atoms or groups of atoms. The higher the resolution, the greater the level of detail of the map and the greater the extent to which it is possible to distinguish one atom from another in the electron density map. Resolution is typically measured in angstroms (“Å”), which is 1×10-10 m (or 0.0000001 mm). Even at a high resolution, it may still be difficult to detect the position of certain side chains, if they are too mobile (or not well ordered enough).
Two relevant crystallography parameters are temperature (or ‘B’) factor, which is a measure of the spread of electron density, and occupancy, which is the fraction of an atom present within a particular site.
Computer programs can be used to generate depictions of the structure of a protein or a protein-protein complex from the atomic coordinates. These can be viewed in 3D on a computer. One such computer program, which was widely available as at 23 August 2007, is known as PyMOL.
Mutagenesis
Mutagenesis studies are another means by which the PSA may assess whether a particular residue is involved in a protein-protein interaction. In such studies, one or more mutated versions of the antigen are prepared whereby, in each mutated form of the antigen, one residue is mutated from the ‘wild-type’ residue to a different residue. The degree of binding of the antibody to the mutated antigen is then compared to the degree of binding of the antibody to the wild-type antigen.
Assays which measure binding, competition and blocking
The Agreed Primer sets out the details of various assays that the PSA can use to measure the binding of an antibody to an antigen, competition for binding as between two antibodies and whether an antibody blocks another molecule from binding with the target antigen.
ELISA
ELISA is a technique designed to detect and quantify a protein of interest (e.g., an antigen or antibody). The technique was first designed in the early 1970s.
A sample of one protein is immobilised onto the surface of the well in a micro-plate, and then a sample containing another protein is added to the well. Detection of any binding between the two proteins is achieved in a number of different ways. The immobilisation of the first protein can also be achieved a number of different ways, in order to orient the antigen differently.
ELISA can be used to measure the binding of an antibody to an antigen or to detect competition between two or more antibodies binding the same antigen. For example, ELISA can be used to determine if an antibody blocks the binding of an antigen (e.g., PCSK9) to its target (e.g., LDLR).
There are different forms of ELISA, depending on the purpose of the assay to be undertaken. Direct ELISA involves the immobilisation of an antigen to the surface of a well, and the addition of an antibody which has been conjugated with a detection label. The antibody and antigen are incubated for a period of time to allow for binding, after which point, the well is washed to remove any unbound antibody. A substrate is added, which reacts with the detection label. If any antibody (now bound to the antigen) is present, the substrate reaction with the detection label results in a detectable colour. Typically, the detection methodology involves a colorimetric reaction that can be read by a spectrophotometer. The spectrophotometer detects the intensity of the colour, and the readout is measured in optical density (“OD”) units. A higher OD indicates more antibodies are bound to the antigen in the well. Generally, an antibody that has a stronger affinity for the antigen will have a higher OD than an antibody with a weaker affinity.
Indirect Detection ELISA similarly involves the immobilisation of an antigen to a well, the addition of an antibody (“the primary antibody”) and incubation to allow for binding. The well is then washed to remove the unbound primary antibody, and a “secondary antibody” is added and incubated for a period of time to allow the secondary antibody to bind to the primary antibody. The secondary antibody typically carries a label, which generates a colour, typically following a chemical reaction. The colour can be detected and measured, and the binding between the antigen and primary antibody can be calculated from the OD.
Sandwich ELISA is an alternative format of Indirect Detection ELISA. It can be used in competition assays, to detect whether an antibody can compete with the binding of another antibody to the same antigen. When used in this way, the surface of the well is coated with the “capture antibody”, and the antigen is added to the well and captured. A “detection antibody”, which carries a label that generates a colour, is then added. According to the Agreed Primer, the detection antibody can bind the antigen:
if the epitope on the antigen to which it binds does not overlap with the epitope which binds to the …capture antibody. If the epitopes overlap, the antibodies compete for binding and the …detection antibody will not bind.
Sandwich ELISAs can also be performed using a “premix” protocol, where the capture antibody is immobilised to the well, but the detection antibody is premixed or prebound with the antigen before being added to the well. Again, the primer explains:
Binding of the…detection antibody indicates that the…capture antibody and…detection antibody do not compete because they can bind to the…antigen at the same time. However, if the…detection antibody is not bound, this indicates that the two antibodies are not capable of binding to the…antigen at the same time.
Surface Plasmon Resonance (“SPR”)
SPR is used to monitor the interaction between two proteins without the use of labels and colour reactions. Biacore is the trade name for a bench top instrument that measures SPR.
In this assay, an automatic sampler injects samples through flow cells on a sensor chip. The sensor chip provides the site where the interaction between an antibody and antigen takes place, measures the mass change on the surface over time and converts it into an SPR signal.
The method is used to detect the binding of a soluble antigen to an antibody. The antibody is immobilised onto the sensor chip surface. Next, the antigen is added to the surface of the sensor chip. The binding interaction that takes place between the antibody and the antigen (if there is one) is represented as an increase in response units (“RU”) on a graph (referred to as a “sensorgram”).
SPR can also be used to test whether two antibodies compete for binding to a given antigen. One way of detecting competition involves immobilising a first antibody to the sensor chip. The antigen is then flowed over the surface of the chip, and the sensorgram shows an increase in RU that corresponds to the mass of the antigen which is now bound. Then, a second antibody is flowed over the sensor chip. If the first and second antibodies do not compete for binding to the antigen, the sensorgram will show another increase in RU that corresponds to the mass of the second antibody, reflecting that the two antibodies are capable of binding to the antigen at the same time. However, if the first and second antibodies compete for binding to the antigen, there will be no increase in RU when the second antibody is flowed over the sensor chip.
Kinetic Exclusion Assay (“KinExA”)
KinExA is a technique for measuring the binding affinity between two proteins, for example, an antigen and antibody. The antigen and antibody are mixed together to form an equilibrium solution, which is then passed through a column to which the antigen has previously been immobilised. Any free antibody that binds to the column is measured by a detection antibody, which carries a fluorescent label. Fluorescence is continuously monitored and represented graphically.
Luminex
Luminex is a form of multiplex bead array assay designed to rapidly test a large number of antibody candidates for their binding to a protein target of interest. In one variation of this method, target competition between a capture antibody and a detection antibody is measured. In this assay, target proteins are captured onto spherical beads in suspension. Different coloured beads are premixed with different antibody candidates, such that each coloured bead is coated with a different antibody. The beads are then mixed together with a protein target (e.g., antigen). The ability of the individual antibody candidate to bind to the protein target is measured by a detection antibody carrying a label, using a flow cell to separate the beads. As with the ELISA method, the amount of binding of an antibody to its target can be measured on each bead by fluorescence.
In an inverted format, mutated target proteins (e.g., different mutated forms of the antigen) can be bound to the coloured beads, and the assay measures the ability of an antibody to bind to the mutant target protein.
The cholesterol pathway and PCSK9
Serum cholesterol levels in humans predict the risk of atherosclerotic cardiovascular disease (“ASCVD”), coronary heart disease, cerebral ischemia and peripheral arterial disease.
Serum cholesterol is insoluble in water and cannot be transported in isolation. It is transported in blood as a component of water-soluble lipoproteins. Serum cholesterol is found in all the lipoproteins in the circulatory system. These lipoproteins are termed very low density lipoprotein, low density lipoprotein (referred to above as “LDL”) and high density lipoprotein (“HDL”). LDL cholesterol is responsible for the epidemiological association between ASCVD and serum cholesterol.
The uptake of LDL takes place primarily via coated pit regions of the plasma membrane of hepatocytes (liver cells), by the pathway of receptor-mediated endocytosis (internalisation). In the 1980s, Brown and Goldstein identified LDL receptor (referred to above as “LDLR”) and showed it is responsible for removing LDL from the blood.
The binding between LDLR and LDL takes place on the outer surface of the plasma membrane of the cell. With or without LDL bound to it, cell surface LDLR is reinternalized by endocytosis. It can either be recycled back to the cell surface or routed to the lysosome, where the LDL is degraded. After LDL degradation, LDLR remains intact and is trafficked back to the cell surface where the cycle is repeated.
As I have explained above, PCSK9 is a protein involved in the degradation of LDLR. The antibodies the subject of the Applications target PCSK9, blocking or inhibiting its activity degrading LDLR, thereby increasing the level of LDLR available to degrade LDL.
PCSK9 consists of three regions or domains; starting from the N-terminus of the protein there is a “prodomain” followed by a “catalytic domain”, followed by a C-terminal domain (also referred to as the V domain). The two-dimensional primary structure of PCSK9 can be represented as shown below.
THE COMMON SPECIFICATION
Introduction
According to the CS, the field of the invention relates to antigen binding proteins (“ABPs”, which include antibodies) that bind to PCSK9 and methods of making and using them. The CS provides some background to PCSK9 including its role in regulating the levels of LDLR.
The CS includes a section entitled “Summary of Various Embodiments” which includes a large number of consistory statements that mirror many of the claims, including the Exemplary Claims. These statements include that appearing at CS [0009] which states that “the invention comprises an isolated antigen binding protein that specifically binds to an epitope that is bound by any of the ABPs disclosed herein”. The consistory clauses vary amongst the Applications in accordance with the claims.
The various figures included in the CS are identified at CS [0063]-[0159]. These figures include various figures depicting:
(a)the amino acid sequence of PCSK9 (Figures 1A and 1B);
(b)the amino acid sequences of parts of various antibodies (Figures 2A – 3JJJ); and
(c)structural models of PCSK9 and the reference antibodies.
The section of the CS entitled “Detailed Description of Certain Exemplary Embodiments” includes definitions of many terms used throughout the CS. I have previously referred to the definition of “epitope” at CS [0233]. I will refer to other definitions in the course of these reasons when addressing the specific topics.
The CS refers at [0245] to the amino acid sequence for PCSK9 presented in Figures 1A and 1B, noting that the structure of the protein had recently been solved by two groups. The CS states at [0246] that the ABPs that bind PCSK9 “are provided herein”. According to CS [0250], these ABPs can be used in a variety of therapeutic applications including for treating cholesterol related disorders including hypercholesterolemia, and also in the diagnosis of such conditions.
The CS at [0259] describes the general structure of the variable regions of light and heavy chains which include “conserved framework regions (FR)” and CDRs. The CS relevantly states at [0259]:
The CDRs from the two chains of each heavy chain/light chain pair mentioned above typically are aligned by the framework regions to form a structure that binds specifically with a specific epitope on the target protein (e.g., PCSK9).
Production and selection of Table 2 Antibodies
CS [0260]-[0262] describes by reference to various figures 32 different examples of antibodies and the amino acid sequences for the variable regions of their light and heavy chains. The MAbs referred to in Table 2 (“the Table 2 Antibodies”), at CS [0261], are a selection of those generated in XenoMouse mice which can produce fully human antibodies (Example 1). The mice were injected with PCSK9 eleven times. Selected mice were then sacrificed and their lymph nodes were used to create hybridomas to produce MAbs capable of binding to PCSK9 (Example 2).
Following the production of the hybridomas, a screening process was undertaken (Example 3). The first “primary screen” identified 3104 PCSK9 specific hybridomas of interest. This number was reduced to 2441 following a “confirmatory screen”. Further screening identified 579 MAbs that bound to both mouse and human PCSK9. These MAbs were then screened for binding to a mutant variant of PCSK9 (“D374Y mutant”). This mutant was used because it has a higher binding affinity to LDLR. Over 96% of those antibodies identified as binding to wild-type PCSK9 (i.e., non-mutated) were found to bind to the D374Y mutant. A further assay then tested whether the MAbs could block the D374Y mutant from binding to LDLR, The D374Y screen identified a subset of 384 MAbs that blocked binding between D374Y and LDLR “well”, and 100 which blocked the interaction “strongly”.
This assay was repeated and 85 antibodies were found to block the interaction between the D374Y mutant and LDLR greater than 90%. A screen was also performed on those antibodies found to bind to wild-type PCSK9 but not the D374Y mutant, for their ability to block binding between wild-type PCSK9 and LDLR.
CS [0422] explains that based on the results of the assays described, “several hybridoma lines were identified as producing antibodies with desired interactions with PCSK9”, and “[l]imiting dilution was used to isolate a manageable number of clones from each line” which were given line numbers (e.g., 21B12) and clone numbers (e.g., 21B12.1). The clones of each line generally behaved the same. The isolated clones were then expanded in hybridoma media, and the concentration and potency of the antibodies to PCSK9 in the supernatants of those cultures were tested. Those hybridomas with the highest titer of antibodies (i.e., highest detected amount) were identified. The selected hybridomas are listed in Table 2. Relevantly, CS [0260]-[0262] sets out:
[0260] Various heavy chain and light chain variable regions are provided herein and are depicted in FIGs. 2A-3JJ and 3LL-3BBB. In some embodiments, each of these variable regions can be attached to the above heavy and light chain constant regions to form a complete antibody heavy and light chain, respectively. Further, each of the so generated heavy and light chain sequences can be combined to form a complete antibody structure.
[0261] Specific examples of some of the variable regions of the light and heavy chains of the antibodies that are provided and their corresponding amino acid sequences are summarized in TABLE 2.
[0262] Again, each of the exemplary variable heavy chains listed in Table 2 can be combined with any of the exemplary variable light chains shown in Table 2 to form an antibody. Table 2 shows exemplary light and heavy chain pairings found in several of the antibodies….
As I have alluded to above, the amino acid sequences for the variable regions of the light and heavy chains of the Table 2 Antibodies are included in the figures to the specification. The nucleic acid sequences for the light and heavy chains were first determined by nucleotide sequencing, from which their amino acid sequences were then deduced (Example 9, “Sequence Analysis of Antibody Heavy and Light Chains”). Each of the numbers in Table 2 corresponds to a particular sequenced variable region. For example, the sequence for the light and heavy chain variable regions for 30A4 are found in SEQ ID NO: 5 and SEQ ID NO: 74, respectively, both of which are reproduced as Figure 3EE.
The CS also explains that, in addition to combining the light and heavy chains identified in Table 2, other pairings and combinations can be made. For example, CS [0262] continues:
In some instances, the antibodies include at least one variable heavy chain and one variable light chain from those listed in Table 2. In other instances, the antibodies contain two identical light chains and two identical heavy chains. As an example, an antibody or antigen binding protein can include a heavy chain and a light chain, two heavy chains, or two light chains. In some embodiments the antigen binding protein comprises (and/or consists) of 1, 2, and/or 3 heavy and/or light CDRs from at least one of the sequences listed in Table 2 (CDRs for the sequences are outlined in FIGs. 2A‑3D, and other embodiments in FIGs. 3CCC-3JJJ and 15A-15D). In some embodiments, all 6 CDRs (CDR1-3 from the light (CDRL1, CDRL2, CDRL3) and CDR1-3 from the heavy (CDRH1, CDRH2, and CDRH3)) are part of the ABP. In some embodiments, 1, 2, 3, 4, 5, or more CDRs are included in the ABP. In some embodiments, one heavy and one light CDR from the CDRs in the sequences in Table 2 is included in the ABP (CDRs for the sequences in table 2 are outlined in FIGs. 2A‑3D). In some embodiments, additional sections (e.g., as depicted in FIG. 2A-2D, 3A-3D, and other embodiments in 3CCC-3JJJ and 15A-15D) are also included in the ABP. Examples of CDRs and FRs for the heavy and light chains noted in Table 2 are outlined in FIGs. 2A-3D (and other embodiments in FIGs. 3CCC-3JJJ and 15A-15D). Optional light chain variable sequences (including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4) can be selected from the following: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. Optional heavy chain variable sequences (including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4) can be selected from the following: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60.
The antibodies described in these paragraphs of the CS (including Table 2) are not said to be embodiments of the invention as claimed. Table 2 identifies what are called “Exemplary Heavy and Light Chain Variable Regions” of 32 antibodies. Which of those antibodies falls within the claims depends on, in the case of claim 1 of 677, whether they bind to an epitope on PCSK9 that includes one or more of the relevant residues and also block binding of PCSK9 to LDLR. This must also be true of the various antibodies generally described in CS [0262] with different arrangements of the exemplary heavy and light chain variable regions.
Residues bound on PCSK9
The CS then shifts to a discussion of antibody binding to PCSK9 and the site at which it may bind. It does so by reference to (inter alia) the amino sequence for PCSK9 disclosed in SEQ ID NO: 1 (Figure 1A) and the three domains of the antigen. The CS states at [0264]:
In some embodiments, the ABP binds selectively to the form of PCSK9 that binds to LDLR (e.g., the autocatalyzed form of the molecule). In some embodiments, the antigen binding protein does not bind to the c-terminus of the cataylytic [sic] domain (e.g., the 5. 5-10, 10-15, 15-20, 20-25, 25-30, 30-40 most [sic] amino acids in the c-terminus). In some embodiments, the antigen binding protein does not bind to the n-terminus of the catalytic domain (e.g., the 5. 5-10, 10-15, 15-20, 20-25, 25-30, 30-40 most [sic] amino acids in the n-terminus). In some embodiments, the ABP binds to amino acids within amino acids 1-100 of the mature form of PCSK9. In some embodiments, the ABP binds to amino acids within (and/or amino acid sequences consisting of) amino acids 31-100, 100-200, 31-152, 153-692, 200-300, 300-400, 452-683, 400-500, 500-600, 31-692, 31-449, and/or 600-692. In some embodiments, the ABP binds to the catalytic domain. In some embodiments, the neutralizing and/or non-neutralizing ABP binds to the prodomain. In some embodiments, the ABP binds to both the catalytic and pro domains. In some embodiments, the ABP binds to the catalytic domain so as to obstruct an area on the catalytic domain that interacts with the pro domain. In some embodiments, the ABP binds to the catalytic domain at a location or surface that the pro-domain interacts with as outlined in Piper et al … In some embodiments, the ABP binds to the catalytic domain and restricts the mobility of the prodomain. In some embodiments, the ABP binds to the catalytic domain without binding to the pro-domain. In some embodiments, the ABP binds to the catalytic domain, without binding to the pro-domain, while preventing the pro domain from reorienting to allow PCSK9 to bind to LDLR. In some embodiments, the ABP binds in the same epitope as those surrounding residues 149-152 of the pro-domain in Piper et al. In some embodiments, the ABPs bind to the groove (as outlined in Piper et al.) on the V domain. In some embodiments, the ABPs bind to the histidine-rich patch proximal to the groove on the V domain. In some embodiments, such antibodies (that bind to the V domain) are not neutralizing. In some embodiments, antibodies that bind to the V domain are neutralizing. In some embodiments, the neutralizing ABPs prevent the binding of PCSK9 to LDLR. In some embodiments, the neturalizing [sic] ABPs, while preventing the PCSK9 degradation of LDLR, do not prevent the binding of PCSK9 to LDLR (for example ABP3 1A4). In some embodiments, the ABP binds to or blocks at least one of the histidines depicted in Figure 4 of the Piper et al. paper. In some embodiments, the ABP blocks the catalytic triad in PCSK9.
The various antibodies (or ABPs) are described by reference to the domain of PCSK9 and the amino acids on PCSK9 to which the antibody binds or does not bind. Looking at the various ranges of residues specified in CS [0264], it is apparent that every residue of PCSK9 is mentioned, not merely those referred to in the claims.
Antibody variants
The CS describes further variants of ABPs at CS [0280]-[0295]. These paragraphs describe the ABPs by reference to their amino acid sequences when compared to those of the Table 2 Antibodies and sequences identified in various figures in the CS. The CS includes the following disclosures at [0280]-[0294]:
[0280] Other antibodies that are provided are variants of the ABPs listed above formed by combination or subparts of the variable heavy and variable light chains shown in Table 2 and comprise variable light and/or variable heavy chains that each have at least 50%, 50-60, 60-70, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or above 99% identity to the amino acid sequences of the sequences in Table 2 (either the entire sequence or a subpart of the sequence, e.g., one or more CDR). In some instances, such antibodies include at least one heavy chain and one light chain, whereas in other instances the variant forms contain two identical light chains and two identical heavy chains (or subparts thereof). In some embodiments, the sequence comparison in FIG. 2A-3D (and 13A-13J and other embodiments in 15A-15D) can be used in order to identify sections of the antibodies that can be modified by observing those variations that impact binding and those variations that do not appear to impact binding. For example, by comparing similar sequences, one can identify those sections (e.g., particular amino acids) that can be modified and how they can be modified while still retaining (or improving) the functionality of the ABP. In some embodiments, variants of ABPs include those consensus groups and sequences depicted in FIGs. 13A, 13C, 13F, 13G, 13H, 131 and/or 13J and variations are allowed in the positions identified as variable in the figures …
[0281] In certain embodiments, an antigen binding protein comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In certain embodiments, an antigen binding protein comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from at least one of the sequences of [the previously mentioned SEQ IDs]. In certain embodiments, an antigen binding protein comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 99% identical to an amino acid sequence selected from at least one of the sequences of [the previously mentioned SEQ IDs].
[0282] In some embodiments, the antigen binding protein comprises a sequence that is at least 90%, 90-95%, and/or 95-99% identical to one or more CDRs from the CDRs in at least one of sequences of [the previously mentioned SEQ IDs]. In some embodiments, 1, 2, 3, 4, 5, or 6 CDR (each being at least 90%, 90-95%, and/or 95-99% identical to the above sequences) is present.
[0283] In some embodiments, the antigen binding protein comprises a sequence that is at least 90%, 90-95%, and/or 95-99% identical to one or more FRs from the FRs in at least one of sequences of [the previously mentioned SEQ IDs]. In some embodiments, 1, 2, 3, or 4 FR (each being at least 90%, 90-95%, and/or 95-99% identical to the above sequences) is present.
[0284] In certain embodiments, an antigen binding protein comprises a light chain comprising a variable region comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In certain embodiments, an antigen binding protein comprises a light chain comprising a variable region comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from at least one of the sequences of [the previously mentioned SEQ IDs]. In certain embodiments, an antigen binding protein comprises a light chain comprising a variable region comprising an amino acid sequence at least 99% identical to an amino acid sequence selected from at least one of the sequences of [the previously mentioned SEQ IDs].
[0285] In some embodiments, the antigen binding protein comprises a sequence that is at least 90%, 90-95%, and/or 95-99% identical to one or more CDRs from the CDRs in at least one of sequences of [the previously mentioned SEQ IDs]. In some embodiments, 1, 2, 3, 4, 5, or 6 CDR (each being at least 90%, 90-95%, and/or 95-99% identical to the above sequences) is present.
[0286] In some embodiments, the antigen binding protein comprises a sequence that is at least 90%, 90-95%, and/or 95-99% identical to one or more FRs from the FRs in at least one of sequences of [the previously mentioned SEQ IDs]. In some embodiments, 1, 2, 3, or 4 FR (each being at least 90%, 90-95%, and/or 95-99% identical to the above sequences) is present.
[0287] In light of the present disclosure, a skilled artisan will be able to determine suitable variants of the ABPs as set forth herein using well-known techniques. In certain embodiments, one skilled in the art can identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that can be important for biological activity or for structure can be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
[0288] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art can opt for chemically similar amino acid substitutions for such predicted important amino acid residues.
[0289] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar ABPs. In view of such information, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues can be involved in important interactions with other molecules. Moreover, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.
It was submitted by Sanofi that CS [0231] defines “compete”. I agree that it defines that term, in its first sentence, in the context of antibodies competing for the same epitope, to mean competition determined by an assay in which a “test” ABP prevents or inhibits a “reference” ABP from binding to a “common antigen”. That definition does not exclude the possibility that competition may occur between antibodies for other reasons. The skilled addressee reading the CS and the Competition Claims would understand the reference ABP to relevantly include MAbs 31H4 and 21B12 and the reference to a common antigen to refer to PCSK9.
CS [0231] refers (non-exhaustively) to the types of competition which might occur (e.g., the test ABP competes with the reference ABP to bind to the same epitope or an adjacent epitope “sufficiently proximal” so that steric hindrance occurs). Later, in CS [0565], reference is made to competition observed due to “conformational changes”, meaning that the ABPs need not compete for the same or adjacent epitopes, but the binding of the test ABP causes a conformational change (i.e., change in shape) to the antigen which impedes binding of the reference ABP. This is discussed in more detail below. CS [0229] also contemplates the concept of competition by means other than competition for the same or adjacent epitopes:
The term "neutralizing antigen binding protein" or "neutralizing antibody" refers to an antigen binding protein or antibody, respectively, that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be done, for example, by directly blocking a binding site on the ligand or by binding to the ligand and altering the ligand's ability to bind through indirect means (such as structural or energetic alterations in the ligand)…
(Emphasis added)
In my view, “competes” as used in the CS and the Competition Claims can refer to any scenario in which the test ABP prevents or inhibits the reference ABP from binding to the common antigen. While Sanofi’s closing written submissions suggested that Amgen attempted to confine “competes” to ABPs competing for the same epitope, Amgen’s submissions are agnostic to the mechanism of competition. It explained:
Sanofi addresses the various causes of competition that are posited in the [CS]… For the purposes of construction, these matters are irrelevant. Competition is determined by an assay, and it does not matter what causes the competition.
I agree with that interpretation of the CS and the Competition Claims.
CS [0231] identifies various types of assays that may be used to determine whether the test ABP competes with a reference ABP, but it does not require that they compete to any particular degree. The range of percentages provided in the paragraph is not exhaustive, and is prefaced with the word “usually”. The claims require that the MAb competes with a reference antibody so as to prevent or inhibit the latter binding to PCSK9. Some MAbs will compete strongly, while others will compete less strongly. Either way, on what I regard as the proper construction of the Competition Claims, if there is any competition between the relevant MAb and the reference antibody, this requirement of the claim will be met.
My construction of the Competition Claims broadly accords with Professor Hudson’s and Professor Lopez’s evidence of their understanding of the term. Although Professor Hudson did, at one point, suggest that for antibodies to be competing within the meaning of the specification there had to be “significant competition”, his response indicates that he was focussed on CS [0231] and was qualified by his clarification, “[a]nd I think you’re asking me is 45 per cent significant. Yes. If that was the question.” His responses to other questions during cross-examination support the view that he understood competition to be a binary concept, in particular his statement that “competition is competition” and “[i]t’s a sliding scale from 100 per cent down to zero”. His evidence, and Professor Lopez’s evidence, indicates that competition which was less than “complete” would still be understood to fall within the Competition Claims. Contrary to Sanofi’s submission, I do not accept that the Competition Claims lack clarity because they fail to impose a numerical threshold for the purpose of determining whether there is competition.
CS [0231] does not mandate the use of any particular type of competitive binding assay but merely describes a number of different types that can be used. It follows that there is nothing in the CS which would preclude the skilled addressee from ascertaining whether a MAb competes with a reference antibody for binding to PCSK9 using a different type of competition assay to those referred to in CS [0231].
Professor Hudson stated that it was not unusual to get different results from different competitive binding assays. In this regard, Professor Hudson said:
I think, whenever you do experiments using different technologies or analytical techniques, you can end up with different results. I mean, not – they’re usually in the same order, if I’m making myself clear … from a high binding antibody to a weak antibody or a weak blocker to a high blocker, but the actual numerical value may differ.
I take from this evidence that while the numerical values produced by different assays may vary, they will usually produce consistent results in terms of identifying which antibodies compete and whether they do so strongly or weakly.
Professor Mahler gave evidence that the CS did not teach the PSA how to produce antibodies falling within the Competition Claims. In the course of giving that evidence he did not suggest that he would have any difficulty determining whether a particular antibody competed with either of the reference antibodies. Asked whether the CS assists in generating antibodies within the claims, Professor Mahler said:
PROF MAHLER: So what it means is that – what it means is that when you – when you go through immunisation protocol and raise antibodies, you don’t have any control over what you get. You can’t – it’s not as though you have a preconceived idea that, “I’m going to make antibodies that compete and therefore I’m going to do this” … You don’t know what you’re going to get and so you’re going to immunise with PCSK9, you’re going to generate a panel of antibodies, and then you will test them, and then you will know whether they compete or not.
But you can’t have a preconceived idea about – about what you want and translate that into some protocol that gives you something that – that the animal does…You have no control over which antibodies are going to be raised. Everything happens downstream with the testing of what you have got so – so what I meant by that is that you – you – you can’t have a preconceived idea that the application is going to assist me to make antibodies that compete, because it – it doesn’t. That – that’s the way I read the question.
HIS HONOUR: Well, it assists you to determine whether or not they compete at the very end of your program.
PROF MAHLER: It’s after you – it’s after you let the animal do the work. You – you screen the antibodies, you end up with a panel, then you can say whether they compete. But the fact that you want – you want antibodies that compete – well, there’s nothing you can do. The application doesn’t assist or give you any – it can’t. It – it’s not possible. It can’t.
HIS HONOUR: Well - - -
PROF MAHLER: It can’t tell you how to make antibodies that compete.
HIS HONOUR: What about the assistance it gives at that very last step … though?
PROF MAHLER: Which last step is that, sorry?
HIS HONOUR: Determining whether or not they do compete.
PROF MAHLER: Of course, because you have got – right at the end you have got the antibodies that they compete with so then you can test them. But that’s totally independent of do these antibodies assist me in generating antibodies that compete. They – they don’t.
In fairness to Professor Mahler, in this part of his evidence, he was not addressing the reliability of competition assays specifically. In his extensive written evidence he asserted that:
… the different methodologies in the different types of assays discussed in paragraph [0231] are likely to yield different results. Therefore, it is typical in the industry when reporting results, to identify the assay by which the results were obtained.
However, I understood this evidence to be referring to potential numerical differences in assay results. He did not say that any of the assays identified in CS [0231] would be likely to produce either a false positive or false negative.
Sanofi drew attention to the treatment of MAb 30A4 in the CS which was allocated to bin 1 in the competition assay used in Example 10 and bin 5 in the different competition assay used in Example 37. As I have explained above, antibodies that compete with each other were grouped together in the same bin in each example. Table 8.3 shows 30A4 as binning with 21B12, whereas in Table 37.1, antibody 30A4 is shown in bin 5 when 21B12 is in bin 1. This indicates that in the competition assay used in Example 10, 30A4 competed with 21B12, but the same could not be said of the competition assay used in Example 37. CS [0564] describes bin 5 as “a catch all” bin for antibodies that do not fit into other bins. It is not clear from CS [0564] what results were observed to warrant placement in this bin, as opposed to bin 4, which was for antibodies that did not compete with either 21B12 or 31H4.
Importantly, the Antibody and Crystallography Experts agreed in the JER that “the results from Examples 10 and 37 were generally consistent and complementary”. In the JER Professor Mahler also specifically observed that the Example 37 data was quite consistent with that of Example 10 and that there was no added teaching from Example 37. He said:
Examples 10 and 37: There were some discrepancies in the binning for antibodies listed in Tables 2 and 8.3 of the Application, from Examples 10 and 37, respectively, as pointed out in Paragraph 167 of my Declaration dated 28th [February] 2018. Overall there was consistency in the binning of antibodies with a few differences. It is my experience that different methods can yield different results in some circumstances.
In the declaration to which Professor Mahler refers he noted that in Example 37 antibody 30A4 was listed in bin 5. Although he noted that antibodies 31D1, 25A7 and 20D10 were not listed in Example 37, he did not identify any other inconsistency in the binning results. It is not apparent from Professor Mahler’s evidence whether he had regard to the binning data for Example 37 in the tables reproduced in Figures 23A-23D to the CS.
In the JER, Professor Hudson said that 30A4 had been incorrectly allocated to bin 5 rather than bin 1. When cross-examined about the binning of 30A4 in Table 37.1, he referred to the data in Figures 23A-23D, which he said showed that the binding of 30A4 is particularly weak, but that it did compete with some antibodies in bin 1 and there was a “slight overlap” with bin 3 antibodies. He suggested that the inventors may have put 30A4 in bin 5 because they were uncertain as to whether it should be put in bin 1 (i.e., competing with 21B2) and bin 2 (i.e., competing with both 21B2 and 31H4) and therefore put it in the “catch-all” bin. That is a reasonable hypothesis, given that it was not put into bin 4 (i.e., the bin for antibodies that did not compete with bins 1 and 3). Professor Hudson was not referred to any other example of what Sanofi submitted are inconsistent test results based on the use of different competition assays.
Professor Hudson described 30A4 as “an unusual antibody” based on its binning data and the different results as “anomalous”. Professor Petsko said in the JER:
There are a few minor discrepancies between the results … All antibodies that were tested in both Example 10 and 37 and were found in Example 10 to co-bin with 31H4 were still found to do so in Example 37. All but one of the antibodies that co-binned with 21B12 in Example 10 were found to do so in Example 37; the exception was 30A4, which co-binned with 21B12 in Example 10 but was put in the “catch-all” bin in Example 37. However, when I examine the Example 37 data in Figures 23A-23C of the 677 Application, I conclude that 30A4 in fact DOES co-bin with 21B12. The apparent discrepancy may therefore be a clerical error.
I accept that there is good reason to believe that 30A4 has been incorrectly assigned to bin 5 rather than bin 1. I am not persuaded that the different binnings for 30A4 in Example 10 and Example 37 demonstrate that different competition assays will give different answers to the binary question of whether two antibodies compete for binding to PCSK9.
As to the order in which the antibodies are tested, the CS makes clear that the order in which the ABPs are employed can be important (see CS [0565] reproduced below). In this regard, Professor Hudson gave this evidence:
MR SHAVIN: The order in which the ABPs are employed can be important - - -
PROF HUDSON: Yes.
MR SHAVIN: - - - and you accept, don’t you, that if you’re doing a competitive binding assay, you may get different results depending upon the sequence that you use.
PROF HUDSON: Correct.
MR SHAVIN: Yes. And if ABP(a) is employed as the reference antibody and blocks the binding of ABP(b), the converse is not always true, and you accept that.
PROF HUDSON: Correct.
Although the order in which the competition assay is run (i.e. which antibody is added first) may affect the results, this is something that would be known to the skilled addressee. CS [0565] states:
As will be appreciated by one of skill in the art, if the reference ABP prevents the binding of the probe ABP then the antibodies are said to be in the same bin. The order in which the ABPs are employed can be important. If ABP A is employed as the reference ABP and blocks the binding of ABP B the converse is not always true: ABP B used as the reference ABP will not necessarily block ABP A. There are a number of factors in play here: the binding of an ABP can cause conformational changes in the target which prevent the binding of the second ABP, or epitopes which overlap but do not completely occlude each other may allow for the second ABP to still have enough high-affinity interactions with the target to allow binding. ABPs with a much higher affinity may have a greater ability to bump a blocking ABP out of the way. In general, if competition is observed in either order the ABPs are said to bin together, and if both ABPs can block each other then it is likely that the epitopes overlap more completely.
This paragraph of the CS teaches the skilled addressee that if competition is observed in either order then the test antibody and the reference antibody will bin together, thereby implying that they will compete for binding to PCSK9.
Sanofi also submitted that the Competition Claims lacked clarity because the CS did not disclose how much excess antibody should be used in a competition assay. Claim 1 of 685 does not require that any competitive assay utilise an “excess” of an antibody. However, claim 1 of 748 is for a MAb that:
… binds to human PCSK9 and is neutralizing in that an excess of said antibody reduces the quantity of human PCSK9 bound to LDLR in an in vitro competitive binding assay, wherein said monoclonal antibody competes for binding to PCSK9 with [21B12].
I note, though, that in this claim the requirement for an “excess” of antibody in an in vitro competitive binding assay appears to relate to the assay used to determine if the MAb competes with PCSK9, rather than the assay used to determine if the MAb competes with 21B12.
In any event, Sanofi’s submission on this point appears to relate to both 748 and 685. It submitted that the amount of “excess” antibody affects the results of a competitive binding assay, and too little excess (and, I infer, the absence of an excess) can result in a false negative. The concept of an “excess” is referred to in CS [0231], which explains that when conducting a competitive binding assay, “[u]sually the test antigen binding protein is present in excess”.
Professor Mahler gave evidence concerning the term “excess” and suggested, at least at some points in his evidence, that it was unclear to him what amount of excess needed to be used. However, I ultimately understood him to accept that he could determine how much antibody would be required to give “an excess of said antibody”, something which his evidence does not suggest would be beyond the capacity of the PSA. Professor Mahler agreed that, if necessary, the amount of antibody to be used in the competition assay could be determined by carrying out a separate assay for that purpose.
Professor Hudson said that “excess means excess” and that an assay could be used to identify a relevant excess. Professor Lopez said that “when we talk about excess antibody … [we are] trying to make sure we have got enough antibody … to be able to detect an inhibitory effect”. Both Professors Hudson and Lopez agreed that the results of a competitive binding assay would be the same regardless of whether a small or large excess was used, because in each case the excess would by definition be more than the amount required. It was not put to either of Professors Hudson Lopez that it would be beyond the skill of the PSA to determine an appropriate concentration of antibody to provide an excess in a competition assay.
Professor Mahler is the co-inventor of a patent published in 2014 entitled “Anti-CD83 antibodies and use thereof”. Claim 15 of that patent is for a CD83 binding protein which (inter alia) competitively inhibits binding of a reference antibody or which binds to the same epitope in CD83 as that antibody. The specification includes a statement that “[a]ssays for determining a CD83 binding protein that competitively inhibits binding of an antibody will be apparent to the skilled artisan”. Professor Mahler accepted that the specification did not include any description of any competitive binding assay. In particular, he agreed that there was no description in the specification of the level of excess antibody required to perform the competition assay. That evidence strongly suggests to me that Professor Mahler would accept that ascertaining how much antibody to use when conducting a competition assay is something that the PSA could do using standard and well-known techniques.
I am not persuaded that the Competition Claims do not provide a workable standard due to a failure to identify a particular competition assay. In particular, I am not satisfied that the use of different assays, to determine whether two antibodies compete for binding to the same antigen, would be likely to produce materially different results. I am also satisfied that the skilled addressee could determine how much antibody to use in a competition assay to ensure that there was an excess of the relevant antibody. In the result, I am not persuaded that any of the Competition Claims lack clarity.
SUFFICIENCY
A complete specification must “describe the invention fully”: s 40(2)(a) of the Act pre-amendment by the RTB Act. In Kimberly-Clark, the High Court identified the relevant question under s 40(2)(a) as follows at [25]:
… [W]ill the disclosure enable the addressee of the specification to produce something within each claim without new inventions or additions or prolonged study of matters presenting initial difficulty?”
(Footnote omitted)
Sanofi accepted that if the invention in each claim of the Applications is properly characterised as a single invention, namely a class of monoclonal antibodies, then on the standard of proof required in this appeal, the claims are fully described under the principles in Kimberly-Clark. That is because Sanofi accepted that the PSA could generate at least one antibody within each such claim.
Sanofi submitted that where a claim is in effect a claim to more than one invention, the specification must describe how to perform an embodiment of each invention within the claim. It says that the claims of the 677, 689 and 751 are not claims to a class of antibodies, but are claims to a very large number of individual, monoclonal antibodies, each binding to its own particular epitope on PCSK9. In support of its submission Sanofi relied on Tramanco Pty Ltd v BPW Transpec Pty Ltd (2014) 105 IPR 18 (“Tramanco”).
Tramanco was concerned with a method for logging the performance of a vehicle suspension system, by which method one or more of three different performance parameters was determined. It was conceded by the patentee in that case that the claim would be invalid for lack of sufficiency if the complete specification did not enable the PSA to determine all three parameters using the method. The Full Court accepted (obiter) that the concession was properly made.
Sanofi relies on the following statement in Tramanco at [207] (per Nicholas J, Allsop CJ at [53] and Greenwood J at [164]) agreeing):
In Kimberly-Clark and [Lockwood No 1] the High Court was concerned with various product claims each of which consisted of a number of features all of which were essential (in the sense of non-optional) integers of the claim. However, there are some types of claim that may need to be approached slightly differently including, in particular, claims to methods for producing one or more specified results. For example, a claim for a method of producing one or more of outcomes A, B or C might be infringed if the alleged infringer uses the method to produce outcome A, but not outcome B or C…it would seem to me to be wrong in principle to hold that the description of the invention is sufficient if the specification enables the use of the method to achieve outcome A, but not outcomes B or C. It would be inconsistent with the purposes of the Act to confer a monopoly on a patentee for a method of producing any of outcomes A, B or C, if the patentee’s disclosure only enabled the use of the method to produce some of those outcomes.
The question arising from the cited passage of Tramanco is whether there is “more than one invention around which the claims are drawn”: Ariosa at [198]. In Ariosa the Full Court observed at [201]:
The Court in Tramanco did not eschew the requirement set out in Kimberly-Clark at [25] that the disclosure enable the addressee to produce something within each claim. Rather it considered that if a claim properly construed had the effect of claiming three distinct alternative outcomes, the position would not be any different to a case where each outcome was expressed in a separate claim. If the disclosure of the specification did not enable at least one method falling within each of the alternatives, then it would not conform with the requirement of s 40(2)(a) …
In the same proceeding at first instance, the trial judge, Beach J, referred to Tramanco in some detail: Sequenom, Inc v Ariosa Diagnostics, Inc (2019) 143 IPR 24 at [827]-[856]. In particular, his Honour said at [854]-[855]:
[854]… in my view the peculiar claims at issue in Tramanco were to a method for logging the performance of a vehicle suspension and determining one or more parameters selected from the group consisting of A, B, and C (ie the claim included methods for determining only one parameter out of A, B, C, combinations of two parameters A+B, A+C, B+C and all three parameters A+B+C).
[855]But leaving aside special classes of claims to methods producing one or more specified optional outcomes, it is well settled that only one embodiment within each claim need be enabled for sufficiency purposes. An invention is sufficiently disclosed if a skilled person could make a single embodiment of the invention which falls within the scope of the claims …
In Meat & Livestock v Cargill, Beach J at [906] also observed that the claim under consideration in Tramanco at [207] “was in form and in substance a claim to a method of producing alternative results or outcomes”. His Honour distinguished the claims in the case before him on that basis, finding that the claims were sufficiently enabled because the PSA could readily perform an embodiment of the invention within the scope of each claim.
The claims in the present case do not claim distinct alternative outcomes. The outcome postulated in claims 1 and 5 of 677 and claim 1 of 689 is one in which “the monoclonal antibody blocks binding of PCSK9 to LDLR”. Claim 23 of 677 necessarily describes that same outcome, as it is dependent on earlier claims, but through an antibody which also competes for binding to PCSK9 with 21B12. In the case of claims 1, 2, 10 and 13 of 751, the outcome is that “the monoclonal antibody reduces binding between PCSK9 and an EGFa domain of LDLR”. It follows that Tramanco is distinguishable.
Nor am I persuaded that each of the claims is for a number of different inventions. When considering the issue of fair basis I concluded that the each of the specifications for 677, 689 and 751, which include various consistory clauses and mutual examples, describe a class of antibodies which bind either directly to certain nominated residues, or to epitopes containing certain nominated residues. That conclusion was based not merely on matters of form, but also the substance of the relevant disclosures. That characterisation of the invention is at odds with Sanofi’s argument (which I reject) that each of the claims of 677, 689 and 751 include many different inventions.
In the result, I am not persuaded that any of the claims of 677, 689 or 751 would be clearly invalid for non-compliance with s 40(2)(a) of the Act.
Although Sanofi’s submissions on this point focussed on the Epitope and Residue Claims, it also submitted that the Competition Claims in 685 and 748 had a “similar vice” and were also properly understood to be claims to a multiplicity of inventions. This submission can be rejected for the same reasons given in relation to the Epitope and Residue Claims. The Competition Claims are to a class of antibodies defined by competition and to one singular outcome (i.e., reduction of binding between PCSK9 and the EGFa domain of LDLR in the case of 685, and neutralisation of PCSK9 as observed in a competitive binding assay in the case of 751).
ADDITIONAL MATTERS
The evidence of the Later Crystallised Antibodies (i.e., the crystal structures for antibodies 8A3, 11F1, 1A12 and 25G4) was admitted by me under s 57 of the Evidence Act 1995 (Cth). Evidence regarding the crystal structures of the Later Crystallised Antibodies that is not included in the CS is not relevant to fair basis. Given the limited way in which Sanofi argued the insufficiency ground, evidence of the Later Crystallised Antibodies is also not relevant to that ground. I therefore give that evidence no weight.
The parties disagreed as to whether the Applications as filed included colour versions of the crystal structures reproduced in Figures 27A-27E. Amgen informed the Court that for the purposes of this proceeding only it did not rely on the colour versions of the Figures to address any of the grounds of invalidity asserted by Sanofi. I have only had regard to the black and white reproductions of the relevant figures as they appear in the Court Book.
The parties also disagreed as to whether a revised written closing submission filed by Sanofi was outside the scope of the leave given to the parties, in that it did more than make corrections to the original version of that document. I have had regard to the revised document filed by Sanofi even though some material included in it was outside the leave granted. I do not consider that this will have caused Amgen any real prejudice.
DISPOSITION
For Sanofi to succeed in this appeal it is necessary that it satisfy me that the Applications include one or more claims which would be clearly invalid if the relevant application proceeded to grant. I am not so satisfied. In those circumstances there will be an order dismissing the appeal together with orders affirming the decision of the Delegate and directing that each of the Applications proceed to grant. There will also be an order that Sanofi pay Amgen’s costs of the appeal.
I certify that the preceding four hundred and seventy (470) numbered paragraphs are a true copy of the Reasons for Judgment of the Honourable Justice Nicholas. Associate:
Dated: 23 April 2025
Annexure A
Bibliography
Benjannet 2004: Suzanne Benjannet et al, (2004) “NARC-1/PCSK9 and Its Natural Mutants” The Journal of Biological Chemistry 279(47):48865-48875.
Cameron 2006: Jamie Cameron et al, (2006) “Effect of mutations in the PCSK9 gene on the cell surface LDL receptors” Human Molecular Genetics 15(9):1551-1558.
Cohen 2006: Jonathan C Cohen et al, (2006) “Sequence Variations in PCSK9, Low LDL, and Protection against Coronary Heart Disease” The New England Journal of Medicine 354(12): 1264-1272.
Cunningham 2007: David Cunningham et al, “Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia” Nature Structural Molecular Biology 2007 May;14(5):413-9.
Fisher 2007: Timothy S Fisher et al, “Effects of pH and Low Density Lipoprotein (LDL) on PCSK9-dependant LDL Receptor Regulation” The Journal of Biological Chemistry 282(28):20502-20512.
Graham 2007: Mark J Graham et al, (2007) “Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice” Journal of Lipid Research 48:763-767.
Holla 2007: Øystein L Holla et al, (2007) “Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly” BMC Cell Biology 8:9.
Homer 2008: Vivienne M Homer et al, (2008) “Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa” Atherosclerosis 196:659-666.
Horton 2007: Jay D Horton et al, (2007) “Molecular biology of PCSK9: its role in LDL metabolism” TRENDS in Biochemical Sciences 32(2):71-77.
Horton 2009: Jay D Horton et al, (2009) “PCSK9: a convertase that coordinates LDL catabolism” Journal of Lipid Research April Supplement S172-S177.
Kwon 2008: Hyock Joo Kwon et al, “Molecular basis for LDL receptor recognition by PCSK9” PNAS 105(6):1820-1825.
Lagace 2006: Thomas A Lagace et al, (2006) “Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice” The Journal of Clinical Investigation 116(11): 2995-3005 and Supplementary data.
Li 2007: Jun Li et al, (2007) “Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity” Biochem. J 406:203-207
Maxwell 2005: Kara N Maxwell et al, (2005) “Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment” PNAS 102(6):2069-2074.
McNutt 2007: Markey C McNutt et al, (2007) “Catalytic Activity Is Not Required for Secreted PCSK9 to Reduce Low Density Lipoprotein Receptors in HepG2 Cells” The Journal of Biological Chemistry 282(29):20799-20803.
Nassoury 2007: Nasha Nassoury et al, (2007) “The Cellular Trafficking of the Secretory Proprotein Convertase PCSK9 and Its Dependence on the LDLR” Traffic 8:718-732.
Piper 2007: Derek E Piper et al, (2007) “The Crystal Structure of PCSK9: A Regulator of Plasma LDL-Cholesterol” Structure 75:545-552.
Qian 2007: Yue-Wei Qian et al, (2007) “Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis” Journal of Lipid Research 48:1488-1498.
Rashid 2005: Shirya Rashid et al, (2005) “Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9” PNAS 102(15):537.
Seidah 2007: Nabil G Seidah and Annik Prat (2007) “The proprotein convertases are potential targets in the treatment of dyslipidemia” J Mol Med 85:6.
Zhang 2007: Da-Wei Zhang et al, (2007) “Binding of Proprotein Convertase Subtilisin/Kexin Type 9 to Epidermal Growth Factor like Repeat A of Low Density Lipoprotein Receptor Decreases Receptor Recycling and Increases Degradation” The Journal of Biological Chemistry 282(25):18602-18612.
0
13
6