Patent Date
January 26, 2023
Court Intellectual Property High
Right
Court, Fourth Division
Case number 2021 (Gyo-Ke) 10094- With regard to an invention having an invention-specifying matter of "which canneutralize binding between PCSK9 and LDLR protein" and which "competes" "forbinding to PCSK9" "with an antibody which comprises: a heavy chain comprising aheavy chain variable region consisting of an amino acid sequence of SEQ ID NO:
67; and a light chain comprising a light chain variable region consisting of an aminoacid sequence of SEQ ID NO: 12" (hereinafter referred to as a "reference
antibody"), a technical significance of the present invention should be deemed toexist in that it has been identified that an antibody which competes with anreference antibody for binding to PCSK9 has a functional property as a binding
neutralizing antibody. However, it cannot be deemed that this point was disclosed
in the present description. Therefore, the present invention does not comply withthe support requirement.
- Even if it is interpreted that the present invention is directed to only an antibody
which "can neutralize binding between PCSK9 and LDLR protein" among
antibodies which compete for binding to PCSK9 with a reference antibody, thepresent invention also comprises an antibody which competes in a manner that binds
to a position for steric hindrance to the binding between PCSK9 and LDLR protein
to occur. However, the present description does not state anything about amechanism, etc. by which these antibodies neutralize the binding. From this point
as well, the present invention does not comply with the support requirement.
Case type: Rescission of Trial Decision
Result: Appeal granted (Granted)
References: Article 36, paragraph (6), item (i) of the Patent Act
Related rights, etc.: Invalidation Trial No. 2020-800012, Patent No. 5906333
Summary of the Judgment
No. 1

Background

1. The Defendant filed a patent application (hereinafter referred to as "the present
application") with regard to an invention titled "ANTIGEN BINDING PROTEIN TO
PROPROTEIN CONVERTASE SUBTILISIN KEXIN TYPE 9 (PCSK9)" on February
23, 2015 by dividing a part of a patent application (Patent Application No. 2010522084) of which the international filing date is August 22, 2008 (Priority date:August 23, 2007, December 21 of the same year, January 9, 2008, and August 4 of thesame year (hereinafter referred to as "the priority date of the present case"), Priority
country: United States of America).

The Defendant obtained a registration of

establishment of a patent right (Patent No. 5906333, Number of claims: 5, this patentis hereinafter referred to as "the present patent") on March 25, 2016.
For the present patent, in invalidation trial procedures of a trial for invalidation(Invalidation Trial No. 2016-800066) by Sanofi (a French corporation), among aigroup of claims consisting of Claims 1, 2, and 5 in the scope of claims, the following
corrections were made: Claims 1 and 5 were corrected; Claim 2 was deleted; and agroup of claims consisting of Claims 3 and 4 were deleted (hereinafter referred to as"the correction of the present case") (Note that the JPO made a trial decisiondismissing the request for a trial for invalidation of the present patent with regard tocorrected Claims 1 and 5, then Sanofi instituted a suit against the above trial decision
made by the JPO, but Sanofi's claim was dismissed in the suit, and the judgment
became final).
2. The Plaintiff filed a request for a trial for invalidation of the present patent withregard to a portion according to Claims 1 and 5 (Invalidation Trial No. 2020-800012)
on February 12, 2020, but received a decision that "The request for a trial isdismissed." (hereinafter referred to as "the trial decision of the present case") on April
7, 2021.

Accordingly, the Plaintiff instituted a suit of the present case to seek

rescission of the trial decision of the present case.
3. The recitation of Claims 1 and 5 after the correction of the present case is asfollows (hereinafter, an invention according to Claim 1 is referred to as "Present
Invention 1", an invention according to Claim 5 is referred to as "Present Invention 5",
and Present Invention 1 and Present Invention 5 are collectively referred to as "thepresent invention," and in addition, "an antibody which comprises: a heavy chain
comprising a heavy chain variable region consisting of an amino acid sequence of
SEQ ID NO: 67; and a light chain comprising a light chain variable region consistingof an amino acid sequence of SEQ ID NO: 12" is referred to as "31H4 antibody" andis also referred to as a "reference antibody.").
[Claim 1] An isolated monoclonal antibody, which can neutralize binding between
PCSK9 and LDLR protein and which competes for binding to PCSK9 with anantibody which comprises: a heavy chain comprising a heavy chain variable region
consisting of an amino acid sequence of SEQ ID NO: 67; and a light chain comprisinga light chain variable region consisting of an amino acid sequence of SEQ ID NO:
12."[Claim 5] A pharmaceutical composition, comprising the isolated monoclonal
antibody as claimed in Claim 1.
No. 2

Summary of the Court decision

1. The term "neutralize" in the present invention includes an embodiment ofaltering a binding ability of PCSK9 to LDLR protein through indirect means (such as
structural or energetic alterations in a ligand) in addition to interfering with, blocking,reducing, or modulating an interaction between PCSK9 and LDLR protein by directlyiiblocking a protein binding site.

However, a reference antibody itself can be

acknowledged as a neutralizing antibody which sterically interferes with binding
between PCSK9 and LDLR protein and which strongly blocks the binding at a
position which partially overlaps with a position of EGFa domain of LD LR (thisEGFa domain binds to a catalytic domain of PCSK9, and an antibody which interacts
with or blocks any of PCSK9 residues present within the region can be useful as an
antibody which inhibits the interaction between PCSK9 and LDLR) in the crystal
structure.

Based on this fact, the invention-specifying matter of "which competes forbinding to PCSK9 with 31H4 antibody" in the present invention also has a technicalsignificance in that it is revealed that an antibody which competes with the 31H4
antibody interferes with, blocks, reduces, or modulates the interaction between
PCSK9 and LDLR protein by directly blocking a binding site of LDLR protein(specifically, by the antibody binding to PCSK9 at a position which overlaps with aposition of EGFa domain of LDLR in the crystal structure) by a mechanism similar tothat of the 31H4 antibody.
2. Regarding an antibody stated as having a neutralizing activity among antibodies
identified as antibodies which compete with 31H4 antibody, the present description
does not specifically state a position where the above antibody stated as having aneutralizing activity binds on PCSK9.

In this regard, it can be deemed to be highly

probable that a group of antibodies having an amino acid sequence with high identity
to the 31H4 antibody bind to PCSK9 in a position similar to the 31H4 antibody.
However, regarding several groups of antibodies having an amino acid sequence otherthan the above, a knowledge that a position where such an antibody binds on PCSK9
is revealed by the fact that the antibody was evaluated as competing in an assay suchas epitope binning cannot be acknowledged to be common general technicalknowledge.

Thus, the position where the above antibody binds to PCSK9 cannot be

deemed to be apparent.
Further, it is obvious that an antibody having a property "which competes forbinding to PCSK9 with a reference antibody" of the present invention encompasses avery wide variety of antibodies in addition to several groups of antibodies specificallystated in the Detailed Description of the Invention in the present description.
Furthermore, the antibody of the present invention encompasses not only an antibody
which prevents or inhibits (e.g., reduces) specific binding of the reference antibody bybinding to a site which overlaps with a site on PCSK9 where 31H4 antibody binds to
PCSK9, but also an antibody which prevents or inhibits (e.g., reduces) specificbinding of the reference antibody to PCSK9 in various degrees by binding to PCSK9iii

in a manner that sterically interferes with the binding between the reference antibody
and PCSK9.

Then, the antibodies mentioned above can include, for example, anantibody which prevents or inhibits (e.g., reduces) specific binding of the 31H4
antibody to PCSK9 by binding to a site which differs from a site where the 31H4
antibody binds to PCSK9 and which differs from a position of EGFa domain of LDLR
in the crystal structure and bringing minor steric hindrance to the 31H4 antibody.
However, a site where such an antibody binds to PCSK9 is not a position where theantibody overlaps with a position of EGFa domain of LDLR in the crystal structure.
Thus, such an antibody cannot be deemed to interfere with, block, reduce, or modulate
the interaction between PCSK9 and LDLR protein by directly blocking a binding siteof LDLR protein.
As mentioned above, it cannot be deemed that the "antibody which competes forbinding to PCSK9 with 31H4 antibody" interferes with, blocks, reduces, or modulates
the interaction between PCSK9 and LDLR protein by directly blocking a binding siteof LDLR protein (specifically, by the antibody binding to PCSK9 at a position which
overlaps with a position of EGFa domain of LDLR in the crystal structure) in amanner similar to 31H4 antibody.

Thus, it also cannot be acknowledged that the"antibody which competes for binding to PCSK9 with 31H4 antibody" has afunctional property as a binding-neutralizing antibody.
3. The Defendant asserts that there is no reason why the present invention violates
the support requirement, on the grounds that even if there exists an antibody which
competes with 31H4 antibody (a reference antibody) but which cannot neutralize thebinding between PCSK9 and LDLR protein, such an antibody is literally excluded
from the technical scope of Present Invention 1.
However, a technical significance of the present invention should be deemed toexist in that it has been identified that an antibody which competes with 31H4
antibody has a functional property as an antibody which neutralizes the binding
between PCSK9 and LDLR protein by the mechanism similar to that of 31H4
antibody.

If an antibody which competes with 31H4 antibody includes one which

does not have a binding neutralizing activity, it is apparent that the assumption of its
technical significance will collapse.
Note that even if it is interpreted that the scope of claims of Present Invention 1 is,as asserted by the Defendant, directed to only an antibody which "can neutralize
binding between PCSK9 and LDLR protein" among antibodies which compete forbinding to PCSK9 with a reference antibody, the invention-specifying matter of thatwhich competes for binding to PCSK9 with a reference antibody according to theivpresent invention is not limited to an antibody which binds to a position that is thesame as or overlaps with a position where the reference antibody binds as asserted by
the Defendant, but also includes an antibody which competes in a manner that binds
to a position for steric hindrance to the binding between PCSK9 and LDLR protein tooccur.

Thus, it must be supported that such an antibody is also a binding

neutralizing antibody.

In this regard, unlike the case of an antibody which binds to a

position that is the same as or overlaps with a position where a reference antibody
binds, the present description does not state anything about a mechanism by which an
antibody neutralizes the binding between PCSK9 and LDLR protein in which theantibody competes in a manner that binds to a position for steric hindrance to thebinding to occur.

In addition, binding neutralizing antibodies based on experimental

results by binning are all likely to be antibodies which bind to a position that is thesame as or overlaps with a position where a reference antibody binds, of which themechanism pertaining to binding and neutralizing is disclosed.

Even if this point is

excluded, at least, the present description does not state anything to suggest that theseare sterically interfering antibodies.
Thus, it must be deemed that the Detailed Description of the Invention in thepresent description does not disclose anything about the fact that among antibodies
which compete with a reference antibody, when an antibody competes in a mannerthat binds to a position for steric hindrance to the binding between PCSK9 and LDLR
protein to occur, the antibody has a binding neutralizing activity.

From this point as

well, the present invention does not comply with the support requirement.

v

Judgment rendered on January 26, 2023
2021 (Gyo-Ke) 10094 Case of seeking rescission of trial decision
Date of Conclusion of Oral Argument: November 7, 2022
Judgment
Plaintiff: Regeneron Pharmaceuticals Incorporated
Defendant: Amgen Incorporated
Main text
1. The court shall rescind the decision made by the Japan Patent Office (JPO) on April
7, 2021 with regard to the case of Invalidation Trial No. 2020-800012.
2. The court costs shall be borne by the Defendant.
3. The additional period for filing a final appeal and a petition for acceptance of final
appeal against this judgment shall be 30 days.
Facts and reasonsNo. 1 Claim
The same gist as the main text, the first paragraph.
No. 2 Outline of the case
1. History, etc. of Procedures at the JPO(1)

The Defendant filed a patent application (hereinafter referred to as "the Present

Application") with regard to an invention titled "ANTIGEN BINDING PROTEIN TO
PROPROTEIN CONVERTASE SUBTILISIN KEXIN TYPE 9 (PCSK9)" on February
23, 2015 by dividing a part of a patent application (Patent Application No. 2010 522084) whose international filing date is August 22, 2008 (Priority date: August 23,
2007, December 21 of the same year, January 9, 2008, and August 4 of the same year
(hereinafter referred to as "the priority date of the present case"), Priority country:
United States of America).

The Defendant obtained a registration of establishmentof a patent right (Patent No. 5906333, Number of claims: 5, this patent is hereinafterreferred to as "the Present Patent") on March 25, 2016.
(2)

Sanofi (a French corporation) filed a request for a patent invalidation trial withregard to the present patent on May 31, 2016 (the case of Invalidation Trial No. 2016 800066, hereinafter referred to as "the invalidation trial of the separate case").
The Defendant received an advance notice of a trial decision dated March 9,2017 and thus, on May 8 of the same year, filed a request for correction to the effectthat in the scope of claims, Claims 1 and 5 be corrected and Claim 2 be deleted among1

a group of claims consisting of Claims 1, 2 and 5 and that a group of claims consistingof Claims 3 and 4 be deleted (hereinafter referred to as "the correction of the present
case").
Thereafter, on August 2, 2017, the Japan Patent Office allowed the correctionof the present case and rendered a trial decision that "The request for the trial withregard to the inventions according to Claims 1 and 5 of the present patent isgroundless.

The request for the trial with regard to the inventions according to

Claims 2 to 4 shall be dismissed" (hereinafter referred to as "the trial decision of theseparate case").
Sanofi instituted a suit for seeking rescission of the portion pertaining to
Claims 1 and 5 of the Present Patent in the trial decision of the separate case(hereinafter referred to as "the suit against the trial decision of the separate case") on
December 8, 2017 (Intellectual Property High Court, 2017 (Gyo-Ke) 10226).
However, the Intellectual Property High Court dismissed Sanofi's claim on December
27, 2018 (the Intellectual Property High Court denied the presence of all grounds forrescission (error in the determination of an inventive step, error in the determinationof the support requirement, and error in the determination of the enablement
requirement) asserted by Sanofi).

Sanofi filed a petition for acceptance of a final

appeal against the same judgment.

However, the Supreme Court ruled not to accept

the final appeal on April 24, 2020, and the same judgment became final and binding.
(3)

The Plaintiff filed a request for a patent invalidation trial with regard to the

portion pertaining to Claims 1 and 5 of the Present Patent on February 12, 2020 (thecase of Invalidation Trial No. 2020-800012).
However, the Japan Patent Office rendered a trial decision that "The request for
the trial of the present case is groundless." (hereinafter referred to as "the trial
decision of the present case") on April 7, 2021, a certified copy of which w as servedon the Plaintiff on the 16th of the same month (additional time frame: 90 days).
(4)

The Plaintiff instituted a suit of the present case for seeking rescission of thetrial decision of the present case on August 13, 2021.
2. Statement of the Scope of Claims
The statement of Claims 1 and 5 after the correction of the present case is asfollows (Hereinafter, an invention according to Claim 1 is referred to as "Present
Invention 1," an invention according to Claim 5 is referred to as "Present Inven tion
5," and Present Invention 1 and Present Invention 5 are collectively referred to as "thepresent invention."

In addition, "an antibody which comprises: a heavy chain

comprising a heavy-chain variable region consisting of an amino acid sequence of2

SEQ ID NO: 67; and a light chain comprising a light-chain variable region consistingof an amino acid sequence of SEQ ID NO: 12" is referred to as "31H4 antibody" andis also referred to as a "reference antibody.").
[Claim 1] An isolated monoclonal antibody, which can neutralize binding between
PCSK9 and LDLR protein and which competes for binding to PCSK9 with anantibody which comprises: a heavy chain comprising a heavy-chain variable region
consisting of an amino acid sequence of SEQ ID NO: 67; and a light chain comprisinga light-chain variable region consisting of an amino acid sequence of SEQ ID NO:
12."[Claim 5] A pharmaceutical composition, comprising the isolated monoclonal
antibody as claimed in Claim 1.
3. Summary of the Trial Decision of the Present Case
(1) Reason 1 for Invalidation (Violation of the Support Requirement)
The present invention is "an isolated monoclonal antibody" that has both of aproperty "which can neutralize binding between PCSK9 and LDLR protein" and aproperty "which competes for binding to PCSK9 with 31H4 antibody" and is "apharmaceutical composition comprising" this.

In this regard, according to eachstatement of [0002], [0003], [0066], [0071], [0155], [0270], [0271], and [0276] of thedescription attached to the written application of the present application (hereinafterreferred to as "the present description," including the Drawings), it can be understoodthat a problem of the present invention is to provide such a novel antibody and to
produce a pharmaceutical composition comprising this, which neutralizes binding
between PCSK9 and LDLR and increases the amount of LDLR, thereby achieving aneffect of causing a decrease in serum cholesterol in a subject, treating or preventing
diseases related to elevated cholesterol levels such as hypercholesterolemia, andreducing the risk of such diseases.
In addition, the present description specifically states a method of producing ananti-PCSK9 monoclonal antibody (preparation of immunized mice and production of
hybridomas (antibody-producing cells) using immunized mice), a method of screening
for an antibody which neutralizes binding between PCSK9 and LDLR, and a methodof screening for an antibody which competes with 31H4 antibody ([0138], [0312],[0313], [0320], [0322] to [0328], [0332] to [0334], [0336], [0377], [0378], and Table
1 to Table 3).

Further, the working examples of the present description show resultsof two independent experiments in which hybridomas that produced antibodies whichstrongly neutralized binding between PCSK9 and LDLR were selected fromhybridomas obtained by injecting human PCSK9 antigen into two groups of mice3containing human immunoglobulin genes, and epitope binning of these antibodies was
performed (Example 10, Example 37), which specifically demonstrates that a numberof antibodies of the present invention can be repeatedly identified with sufficiently
high probability by performing two screenings of anti-PCSK9 monoclonal antibodies;that is, one screening is to select antibodies "which can neutralize binding between
PCSK9 and LDLR" and the other screening is to select antibodies "which competewith 31H4 antibody."

Furthermore, the present description states a mechanism ofaction in which the binding between PCSK9 and LDLR is neutralized the reby toincrease the amount of LDLR, causing a decrease in serum cholesterol in a subject([0066], [0155], [0270], [0271], [0276]).

Thus, it can be reasonably acknowledgedthat an antibody of the present invention having a property "which can neutralize
binding between PCSK9 and LDLR protein" achieves an effect of causing a decrease
in serum cholesterol in a subject and can solve the problem of treating or preventing
diseases related to elevated cholesterol levels such as hypercholesterolemia, andreducing the risk of such diseases.
Hence, a person ordinarily skilled in the art can acknowledge from thestatement of the present description that the antibody of the present invention cansolve the above problem, and the present invention can be deemed to be stated in thedescription.

Therefore, the present patent complies with the support requirement.

(2) Reason 2 for Invalidation (Violation of the Enablement Requirement)
As mentioned in (1) above, the present description specifically states a methodof producing an antibody of the present invention, such as a method of producing ananti-PCSK9 monoclonal antibody (preparation of immunized mice and production of
hybridomas using immunized mice), a method of screening for an antibody which
neutralizes binding between PCSK9 and LDLR, and a method of screening for an
antibody which competes with 31H4 antibody.

In addition, the present description

shows through specific data that a number of antibodies of the present invention canbe repeatedly identified with sufficiently high probability by performing two
screenings of anti-PCSK9 monoclonal antibodies; that is, the screening for being able
to neutralize binding between PCSK9 and LDLR and the screening for competingwith 31H4 antibody.

Thus, it can be deemed that a person ordinarily skilled in theart can produce an antibody of the present invention with sufficiently high probabilityon the basis of these specific statements of the present description.
Further, a structure (amino acid sequence) of a binding region in an antibody isa resulting product from reconstituting antibody genes in immune cells of an
immunized animal.

Thus, it can be acknowledged that a person ordinarily skilled in4the art will reasonably understand that a wide variety of antibodies other than theworking examples encompassed in the present invention can be produced countlessly
by changing the types of animals to be immunized and the immunization programusing the statement of the present description as a hint.
Hence, the present description can be deemed to be stated to the extent that aperson ordinarily skilled in the art can obtain an antibody of the present invention
without undue burden.

Therefore, the present patent complies with the enablement

requirement.
(3) Reason 3 for Invalidation (Lack of Inventive Step)
A. Invention Disclosed in Nature Structural & Molecular Biology, vol. 14(5), pp.
413-419 (2007) (Exhibit Ko 1, hereinafter referred to as "Exhibit Ko 1 document," seeAttachment 2) as Found by the Trial Decision of the Present Case (hereinafter referred
to as "Exhibit Ko 1 invention")"PCSK9 which binds to LDLR, i.e., gain-of-function PCSK9 mutants F216L,
S127R, D374Y, or wild-type PCSK9."
B. Common Feature and Difference as Found by the Trial Decision of the Present
Case
Both of the "monoclonal antibody" of Present Invention 1 and "PCSK9" of the
Exhibit Ko 1 invention are proteins.

Thus, the two are common in that they areproteins and differ in the following point.
(Difference)
Present Invention 1 is "an isolated monoclonal antibody, which can neutralize
binding between PCSK9 and LDLR protein and which competes for binding to
PCSK9 with 31H4 antibody."

In contrast, the Exhibit Ko 1 invention is "PCSK9

which binds to LDLR; i.e., gain-of-function PCSK9 mutants F216L, S127R, D374Y,
or wild-type PCSK9."
C. Determination on Difference
From the disclosures of Exhibit Ko 1-11 in Attachment 2 (hereinafter referred
to simply as "Exhibit Ko 1-11" or the like) and Exhibit Ko 1-12, by obtaining antiPCSK9 antibodies using full-length PCSK9 as an antigen through a method of
obtaining monoclonal antibodies, such as an animal immunization method and aphage display method, and then screening the obtained anti-PCSK9 antibodies forneutralizing binding between PCSK9 and LDLR, it is possible to acknowledge up to
the extent that there is possibility of obtaining some anti-PCSK9 monoclonal
antibodies which neutralize binding between PCSK9 and LDLR.

However, whenanti-PCSK9 monoclonal antibodies which neutralize binding between PCSK9 an d5

LDLR are obtained by obtaining anti-PCSK9 monoclonal antibodies using full-length
PCSK9 as an antigen and then screening the obtained anti-PCSK9 monoclonal
antibodies through a binding-neutralizing assay, it can be considered that such antiPCSK9 monoclonal antibodies encompass a considerable variety of monoclonal
antibodies which bind to various epitopes present on or around binding surfaces.
Thus, in order to obtain an anti-PCSK9 monoclonal antibody which can compete with
21B12 antibody among anti-PCSK9 monoclonal antibodies which can neutralize
binding between PCSK9 and LDLR, it is essential to select anti-PCSK9 monoclonal
antibodies by performing a competition assay with 31H4 antibody, and in order to doso, it is a prerequisite that 31H4 antibody has been obtained.
However, the Exhibit Ko 1 document neither discloses nor suggests 31H4
antibody and does not disclose any information that would provide a hint to obtaining
31H4 antibody among anti-PCSK9 monoclonal antibodies which can inhibit binding
between PCSK9 and LDLR antibody and to obtain a monoclonal antibody which
competes for binding to PCSK9 with 31H4 antibody.

Further, it cannot be

acknowledged that 31H4 antibody had been widely known prior to the priority date of
the present case.
Thus, even a person ordinarily skilled in the art cannot arrive at obtaining a
monoclonal antibody which competes with 31H4 antibody.

Therefore, it cannot be

deemed that Present Invention 1 that comprises the invention-specifying matter of"which competes with 31H4 antibody" could have easily been made by a person
ordinarily skilled in the art on the basis of the Exhibit Ko 1 invention and well -known
art.

The same applies to Present Invention 5 that is a pharmaceutical composition

comprising the antibody of Present Invention 1.
(4) Reason 4 for Invalidation (Violation of the Clarity Requirement)
In the present invention, it should be interpreted that the term "compete" means
a common-general knowledge degree of competing to a person ordinarily skilled in
the art.

In the present description as well, as in [0140], the term "compete" is used in

the meaning of common general knowledge, testing methods therefor are stated
together with specific documents, and it is stated that the term "compete" means acommon-general knowledge degree of inhibiting binding.

Therefore, it cannot be

deemed that the outer limit of "which competes with 31H4 antibody" is unclear.
(5) Reason 5 for Invalidation (Violation of the Requirement that the Invention
Should Fall within Its Definition)
Both Present Inventions 1 and 5 provide specific means for solving theirproblem and can be deemed to be creation of technical ideas utilizing the laws of6

nature.

Therefore, both Present Inventions 1 and 9 fall within the definition of an"invention" under the Patent Act.
4. Ground for Rescission(1) Error in Determination on Inventive Step Based on Exhibit Ko 1 Invention(Ground 1 for Rescission)
A. Error in Finding Common Feature and Difference Between Present Invention 1
and the Exhibit Ko 1 Invention (Ground 1-1 for Rescission)
B. Error in Determination on Whether Present Invention could have been Easily
Conceived of (Ground 1-2 for Rescission)(2) Error in Determination on Violation of the Support Requirement (Ground 2 for
Rescission)(3) Error in Determination on Violation of the Enablement Requirement (Ground 3
for Rescission)(4) Error in Determination on Violation of the Clarity Requirement (Ground 4 for
Rescission)(5) Error in Determination on Violation of the Requirement that the Invention should
Fall within Its Definition (Ground 5 for Rescission)(omitted)
No. 4 Judgment of this court
1. Statements of the Present Description (Exhibit Ko 201), etc.
A. In the [Detailed Description of the Invention] of the present description, there
are statements as shown in Attachment 1.

According to these statements, thefollowing matters are disclosed with regard to the present invention.
(A) PCSK9 (proprotein convertase subtilisin kexin type 9) is a serine proteaseinvolved in regulating the levels of low-density lipoprotein receptor (LDLR) protein,
directly interacts with LDLR protein, and is endocytosed into liver cells along with
LDLR, decreasing the levels of LDLR protein in the liver and further decreasing theamount of LDLR protein available to bind to LDL on cell surfaces (extracellularly),
thereby increasing the amount of LDL in a subject ([0002], [0003], [0071]).
(B)

The antibody which comprises: a heavy chain comprising a heavy-chain

variable region consisting of an amino acid sequence of SEQ ID NO: 67 and a lightchain comprising a light-chain variable region consisting of an amino acid sequenceof SEQ ID NO: 12 ("31H4 antibody") ("reference antibody") is a neutralizing
antibody which strongly blocks binding between PCSK9 and LDLR protein (Example
711, [0377] to [0379], [0138], Table 2).

The reference antibody partially overlaps

with a position of EGFa domain of LDLR in the crystal structure and stericallyinterferes with its binding to PCSK9 (Example 31, [0444], Figure 20A).
The EGFa domain of LDLR binds to the catalytic domain of PCSK9.

PCSK9amino acid residues that are present within 5 angstroms of the EGFa domain in the
crystal structure are specific core PCSK9 amino acid residues of the interaction
interface with the EGFa domain of LDLR (core residues).

PCSK9 residues that arepresent at 5 angstroms to 8 angstroms of the EGFa domain are boundary PCSK9amino acid residues of the interaction interface with the EGFa domain of LDLR.

An

antibody which interacts with or blocks any of these amino acid residues can be usefulas an antibody which inhibits the interaction between PCSK9 and the EGFa domain of
LDLR (and/or LDLR in general) (Example 28, [0428] to [0432]).
(C)

The monoclonal antibody which "competes" with the reference antibody isan antibody which prevents or inhibits (e.g., reduces) binding of the reference
antibody to PCSK9 ([0138], [0140], [0262], [0269]).
(D) A neutralizing ABP (antibody) to PCSK9 neutralizes binding between
PCSK9 and LDLR to increase the amount of LDLR, thereby achieving an effect of
causing a decrease in the amount of LDL in a subject and a decrease in serum
cholesterol in the subject.

In addition, owing to this effect, diseases related toelevated cholesterol levels, such as hypercholesterolemia, can be treated or prevented,
and the risk of such diseases can be reduced.

Thus, the neutralizing ABP to PCSK9

can be therapeutically useful ([0155], [0270], [0271], [0276]).
B. According to the disclosures of the present description as summarily excerpted
in A above, the present invention increases the amount of LDLR protein, thereby
achieving an effect of causing a decrease in the amount of LDL in a subject and adecrease in serum cholesterol in the subject, and owing to this effect, diseases related
to elevated cholesterol levels, such as hypercholesterolemia, can be treated or
prevented, and the risk of such diseases can be reduced.

For this purpose, thepresent invention made its problem to provide an antibody which neutralizes binding
between PCSK9 and LDLR protein, in which the PCSK9 binds to the LDLR protein
to thereby decrease the amount of LDLR protein in a subject and increase the amountof LDL in the subject, or to provide a pharmaceutical composition comprising this.
On this basis, the present invention reveals that PCSK9 binds to the EGFa domain of
LDLR, and that the reference antibody is a neutralizing antibody which stericallyinterferes with binding between PCSK9 and LDLR protein at a position that partiallyoverlaps with a position of the EGFa domain of the LDLR in the crystal structure and8which strongly blocks the binding, and that the monoclonal antibody which"competes" with the reference antibody is an antibody which prevents or inhibits (e.g.,
reduces) binding of the reference antibody to PCSK9.
(2) Statements of Exhibit Ko 1 Document
A. In the Exhibit Ko 1 document that is a publication distributed prior to thepriority date of the present case, there are statements as shown in Attachment 2 (notethat the statements are shown as translations).

According to these statements, theExhibit Ko 1 document can be found to disclose the following matters.
(A) Familial hypercholesterolemia results from elevated LDL cholesterol levels
in plasma or the like.

Proprotein convertase subtilisin kexin type 9 (PCSK9) reduces

the abundance of low-density lipoprotein receptor (LDLR) present on cell surfaces,
and gain-of-function mutations of PCSK9 result in more severe decrease in thenumber of LDLR with consequent hypercholesterolemia.

(The disclosure up to hereis from Exhibit Ko 1-2.)
Secreted PCSK9 interacts with human liver cell surfaces and can be co immunoprecipitated with LDLR, and study results have shown that PCSK9 binds to
LDLR with more greatly increased affinity by as much as 170-fold at the endosomal
pH (Exhibit Ko 1-5).
(B)

Gain-of-function mutations in human PCSK9 are associated with familial

hypercholesterolemia.

In particular, the D374Y mutant is about 10 times more

active than wild-type PCSK9 in lowering the number of LDLR.

The increased effectof the D374Y mutant in lowering LDLR is likely to result from enhanced binding tocell-surface LDLR (Exhibits Ko 1-6, 1-9).
(C)

Genetic evidence suggests that PCSK9 is an attractive target for thetreatment of cardiovascular disease.

Because binding to LDLR protein in plasma

and receptor-dependent intracellular uptake are mostly the rate-determining step for
PCSK9 function, antibodies or small molecules which bind to PCSK9 in plasma andinhibit its binding to LDLR can be effective inhibitors of PCSK9 function.

In

particular, the structure of PCSK9-LDLR complex is useful for designing novel
therapies (Exhibit Ko 1-12).
B. According to each statement as summarily excerpted in the above, it can befound that the Exhibit Ko 1 document discloses the following matters: familial
hypercholesterolemia results from elevated LDL cholesterol levels in plasma; in thisregard, proprotein convertase subtilisin kexin type 9 (PCSK9) reduces the abundanceof low-density lipoprotein receptor (LDLR) present on cell surfaces; thus, PCSK9 is
an attractive target for the treatment, and antibodies or the like which bind to PCSK99

in plasma and inhibit its binding to LDLR can be effective inhibitors.
(3) Results of the Demonstration Experiment and Its Evaluation as Stated in
Affidavit (1) by Dr. [A] (Exhibit Ko 2-1)
A. (A) According to the abridged translation of Affidavit (1) by Dr. [A] (Exhibit Ko2-1), experimental results as shown in Attachment 3 are stated.
(B)

According to the abridged translation of Affidavit (1) by Dr. [B] (Exhibit Ko2-2), there are the following statements.
a.

"4. I have also been provided with details of experiments performed by

Yumab GmbH (hereinafter "Yumab") on a panel of antibodies to hPCSK9 (a group of
antibodies) and with a copy of the "Affidavit (1) by Dr. [A]([A])" (hereinafter "D1")from Yumab.

The Affidavit shows the overall explanation of these experiments.

I

have been requested to review and comment on these results, ..." (Page 2, lines 19 to
24 (The relevant part refers to the original text.
b.
14.

The same applies hereinafter.))"C. Analysis of Competition and Neutralization

Data on the Regeneron antibodies are summarized in Table 1, using thecriterion for competition; i.e., "a reduction of greater than or equal to 50% in thebinding of 21B12 or 31H4 antibody."
Table 1: Neutralizing Property of Regeneron Antibody which Competes with
21B12/31H4 Using 50% Threshold Value for Competition
Competing with 21B12
Competing with 31H4
Neutralizing
3
6
Non-neutralizing
10
28
Total
13
34
Non-neutralizing
76.90%
82.40%(%)
15.

As Table 1 shows, about 80% of Regeneron antibodies classified as competingwith 21B12 antibody cannot neutralize the hPCSK9-LDLR interaction.

Further, as

Table 1 shows, more than 80% of Regeneron antibodies classified as competing with
31H4 antibody cannot neutralize the hPCSK9-LDLR interaction.
16.

....Similarly, in summary, these results demonstrate that most of the antibodies

which compete for binding with 31H4 cannot neutralize binding between hPCSK9 and
LDLR....

According to the present patent, this is because the binding site of 31H4

antibody only partially overlaps with the binding site of LDLR on hPCSK9.

Thus, abinding site of another antibody can overlap with the 31H4 binding site without
overlapping with the binding site of LDLR, and in this manner, another antibody can

10compete with 31H4 without neutralizing the hPCSK9-LDLR interaction.

Further,

two antibodies can compete with each other without necessarily binding to the samesite (e.g., in a case where one antibody sterically prevents another antibody frombinding to a close binding site).

Therefore, in order for competition to be seen,

overlap of binding sites is not required." (Page 5, lines 8 to page 6, line 11)c.
19.

"D. Conclusion....Similarly, taking these results into consideration, it is scientificallyerroneous to state that an antibody which competes with 31H4 antibody would"neutralize binding to LDLR."... The fact that antibodies which compete with 31H4

antibody do not have the effect of strongly neutralizing binding of hPCSK9 -LDLR
obviously indicates that such antibodies do not have any effects on this interaction.
20.

I also disagree that an antibody which competes with 21B12 antibody would

necessarily have affinity and/or a binding site similar to that of the 21B12 antibody....
21.

Therefore, the idea that an antibody which competes with 21B12 must have an

activity similar to said specific 21B12 antibody cannot be accepted as my scientificopinion.

Therefore, the idea that an antibody which competes with 31H4 must

similarly have an activity similar to that of said specific 31H4 antibody cannot be
accepted as my scientific opinion.

It is certainly erroneous to state that all of suchantibodies would have an effect of neutralizing binding.

The fact that so many ofsuch antibodies cannot actually inhibit binding of hPCSK9-LDLR demonstrates that
this idea is unrealistic.

Whatever the case may be, any biological effect of these

non-neutralizing antibodies can be produced through a mechanism different fromthose of 21B12 and 31H4 and does not fall within the definition of neutralization in
the claims of the present patent (i.e., inhibition by preventing hPCSK9 and LDLR
from interacting with each other as verified in an in vitro competition assay)." (Page 6,line 19 to page 7, line 15)
B. (A) As mentioned in No. 3, 2(2)C(A) above, with regard to the demonstration
experiment stated in Affidavit (1) by Dr. [A] (Exhibit Ko 2-1), the Defendant relies on
the Expert Opinion by Professor [C] (Exhibit Otsu 24) and points out the following
matters: [i] In Example 10, the "premix" method is used, but Dr. [A] did not use thismethod, and in the method used by Dr. [A] in which PCSK9 was first added to atested antibody on a plate to allow to bind and the reference antibody was then added,
the binding site of the reference antibody on PCSK9 was occluded depending on the
direction in which the tested antibody was immobilized on the plate, which caused thereference antibody to be unable to bind to the PCSK9 + tested antibody complex,
showing the result as if they had been apparently competing; [ii] PCSK9 at a much11

higher concentration than in Example 10 and the tested antibody at a higher
concentration than in Example 10 were used, and nevertheless a blocking buffer at aslightly lower concentration than in Example 10 was used, which caused PCSK9 tononspecifically bind to the tested antibody in such a manner as to occlude the bindingsite of the reference antibody, making it impossible to prevent the phenomenon suchthat the reference antibody became unable to bind to PCSK9, leading to the results as
if they had been apparently competing; [iii] Experiments were performed using astrong blocking buffer, which showed that 081006A, 081006B, and 190515-41
antibodies, which Dr. [A] concluded to be "competing," did not compete with thereference antibody.

On the above basis, the Defendant asserts that the results of thedemonstration experiment by Dr. [A] contain false positives and are thus erroneous.
However, the above assertion compares the above demonstration experiment by
Dr. [A] with the experimental conditions in Example 10 of the present description.
For example, Example 37 of the present description does not use the premix method,
but the blocking buffer is phosphate buffered saline plus 1% BSA that has an evenlower concentration than 2% BSA (containing 0.05% Tween 20) used in the abov edemonstration experiment.

Thus, on the basis of the comparison of the experimental

conditions with Example 10, it cannot be deemed that the conditions of the above
demonstration experiment are inappropriate.

In addition, needless to say, the present

invention does not define the measurement method and measurement conditions forcompetition and neutralization of monoclonal antibodies for neutralizing binding
between PCSK9 and LDLR.
Further, in the above demonstration experiment, among 63 Regeneron
antibodies, it has been confirmed that five antibodies (081211B, 190515-35, 190515-7,
190515-8, 190515-9) do not bind to PCSK9 (see Material B1 of Attachment 3).
From this point as well, it cannot be deemed that the above demonstration experiment
was performed under conditions in which nonspecific binding of PCSK9 produced
false-positive results.

In addition, the above demonstration experiment is intended toverify binding specificity, compatibility, and neutralizing property using theantibodies disclosed in the present description (9C9, 3B6, and 27B2) as controls.

Asshown in Material B1 of Attachment 3, it has been confirmed that 9C9 and 3B6 bindto PCSK9, compete with 21B12 antibody, and have neutralizing property.

It hasbeen confirmed that the results of this demonstration experiment are the same as theresults disclosed in the present description (see [0374], [0493]).
Thus, it cannot be deemed that the above results of the demonstration
experiment are inappropriate experimental results, (If the results of the experiment12

were inappropriate, the Defendant should have presented experiment results
performed under the same conditions as in Example 10, as pointed out by the Plaintiff.
However, the Defendant failed to do so and instead entirely focused on imp eaching
the above results of the experiment.)

Further, it also cannot be deemed that Affidavit(1) by Dr. [B] (Exhibit Ko 2-2) based on this demonstration experiment is an
inappropriate opinion.
(B) Incidentally, after the date for preparatory proceedings (both parties gave
technical explanations concerning the present invention under the involvement of
three technical advisors) had concluded, it was agreed that both parties may submit
any written arguments to the extent that they would supplement their explanations
given on the same date (see the record of the first preparatory proceedings).
Nevertheless, immediately before the date for the first oral argument, the Defendant
newly submitted the Expert Opinion by Professor [C] (Exhibit Otsu 48).

Then, theDefendant points out the following matters about the demonstration experiment by Dr.
[A]: [i] 25 of the 63 Regeneron antibodies used are disclosed in the Plaintiff's U.S.
provisional application (No. 61/122,482), among which 081211B and 190515-35 are
identical to antibodies H1H314P and H1H317P disclosed in the Plaintiff's provisional
application, respectively, and the Plaintiff's provisional application states that both ofthese antibodies bind to PCSK9, whereas the above demonstration experimentconcludes that these antibodies do not bind to PCSK9 at all; [ii] 190515-36 and
190515-37 are identical to antibodies H1H320P and H1H321P disclosed in the
Plaintiff's provisional application (Exhibit Otsu 48), respectively, and in theprovisional application, the blocking activity of binding between PCSK9 and LDLR isconfirmed, whereas in the results of the above demonstration experiment, the activity
is described as non-neutralizing; [iii] 081008B and 190515-43 have the same aminoacid sequence, and thus competition test results should be the same, and in spite ofthis, the above results of the demonstration experiment conclude that 190515 -43
competes with 31H4 antibody, whereas 081008B does not compete with 31H4
antibody.

On the above basis, the Defendant asserts that these points diminish thereliability of the results of the above demonstration experiment.
The above assertion goes beyond the limit of supplementing the Defendant's
explanation concerning the reliability of the results of the above demonstration
experiment on the date of preparatory proceedings, and falls under delayed allegationand evidence, the dismissal of which is inescapable.

However, since the Plaintiff

submitted the ninth brief as a counter-assertion against this on the date of conclusionof the oral argument, the following can be pointed out as additional remarks just to13

make sure.

Regarding point [i], it is apparent that both H1H314P and H1H317P

disclosed in the provisional application have lower binding activity than otherantibodies (see Table 4 in Exhibit Ko 222).

Thus, the fact that these are evaluated as

not binding in the experimental assay different from that in the provisional application
does not necessarily impair the reliability of the results of the above demonstration
experiment.

Regarding point [ii], the provisional application (Exhibit Ko 222)

discloses in paragraph [0076] that H1H320P (190515-36) and H1H321P (190515-37)
block binding between hPCSK9 and hLDLR-EGF-A domain.

However, H1H320P

and H1H321P are not stated in Table 6 prepared as examples of antibodies having
clear binding blocking property to PCSK9 (paragraph [0077]).

Further, similarly topoint [i], the fact that different evaluations are made in the experimental assay
different from that in the provisional application does not necessarily impair theoverall reliability of the results of the above demonstration experiment.

Regardingpoint [iii], in the Plaintiff's provisional application, it can be found from the sequencelistings in Exhibit Ko 222 and Exhibit Otsu 10 that 190515-43 (H1M505 (SEQ ID
NOs: 266/274) in the provisional application) and 081008B (H1HM504 (SEQ ID
NOs: 242/250) in the provisional application) are stated as different antibodies, andTable 7 differently states the binding specificity to the chimeric protein and D374Y
mutant of PCSK9.

Thus, the fact that different evaluations are made in thedemonstration experiment by Dr. [A] does not necessarily affect the reliability of theresults of the same demonstration experiment.
(C) According to the foregoing, the results of the demonstration experiment statedin Affidavit (1) by Dr. [A] (Exhibit Ko 2-1) should be deemed to have reliability.
2. Ground 2 for Rescission (Error in Determination on Violation of the Support
Requirement)
In accordance with the case, first, a determination will be made on Ground 2
for Rescission (error in determination on violation of the support requirement).
(1)

The Defendant asserts, as mentioned in No. 3, 3(2)A above, that it violates

Article 167 of the Patent Act that the Plaintiff asserts Ground 2 for Rescission on thepremise that the Plaintiff and Sanofi substantially share common interests and on the
grounds that the trial decision of the separate case has become final and binding withregard to the invalidation trial of the separate case which was requested for reasonsthat the present patent violates the support requirement, etc.
However, Article 167 of the Patent Act provides that "Once the trial decision ina trial for patent invalidation ... has become final and binding, neither the parties nor
intervenors may file a request for either such kind of trial on the basis of the same14

facts or evidence."

In this regard, although the Plaintiff and Sanofi or Sanofi K.K.

have a relationship in which they jointly commercialize a formulation, etc. concerning
the dispute on the present patent, the Plaintiff is a corporation separate from Sanofi,
and it cannot be acknowledged that there are special circumstances in which thePlaintiff and Sanofi or Sanofi K.K. should be regarded as the same party from apractical point of view, such as a relationship between parent company and subsidiarycompany, or a relationship between a Japanese corporation and a foreign corporation(needless to say, the Plaintiff is not also an intervenor in the invalidation trial of theseparate case).

Therefore, in the first place, it should be deemed that the same

Article is not applied.
(2)

Article 36, paragraph (6), item (i) of the Patent Act provides that the statementof the scope of claims shall not be made beyond the scope of the invention stated in
the Detailed Description of the Invention.

The purpose of this provision can beinterpreted as follows: if an invention that is not stated in the Detailed Description of
the Invention were to be claimed in the scope of claims, it would be improper to claim
a monopolistic and exclusive right for the unpublished invention; and thus, thepurpose of this provision is to prevent this.
Then, it is reasonable to interpret that whether or not the statement of the scopeof claims complies with the requirement provided in the same item (support
requirement) should be determined by comparing between the statement of the scopeof claims and the statement of the Detailed Description of the Invention, thenexamining: whether or not the invention stated in the scope of claims is the invention
stated in the Detailed Description of the Invention, and falls within the scope where aperson ordinarily skilled in the art can recognize that the problem of the inven tion canbe solved by the statement of the Detailed Description of the Invention; or whether ornot, even if it is neither stated nor suggested in the Detailed Description of theInvention, the invention stated in the scope of claims falls within the scope where aperson ordinarily skilled in the art can recognize that the problem of the invention canbe solved in light of the common general technical knowledge at the time of filing theapplication.
(3)

Under the circumstances, the statement of the scope of claims according to thepresent invention will be examined.

It can be interpreted that Claim 1 of the present

invention has the invention-specifying matters: [i] "which can neutralize binding
between PCSK9 and LDLR protein"; [ii] "which competes" for binding to PCSK9with "an antibody which comprises: a heavy chain comprising a heavy-chain variable
region consisting of an amino acid sequence of SEQ ID NO: 67; and a light chain
15

comprising a light-chain variable region consisting of an amino acid sequence of SEQ
ID NO: 12" (31H4 antibody) (reference antibody); and [iii] "An isolated monoclonal
antibody," and that the invention-specifying matters [i] and [ii] determine propertiesof [iii] the isolated monoclonal antibody.
A. When the statement of the present description is examined in order to interpretthe technical significance of "neutralize" in the invention-specifying matter [i] "which
can neutralize binding between PCSK9 and LDLR protein," there are the following
statements: "The term 'neutralizing antigen binding protein' or 'neutralizing antibody'
represents an antigen binding protein or antibody, respectively, which binds to aligand and prevents or reduces a biological effect of that ligand.

This can be done,for example, by directly blocking a binding site on the ligand or by binding to theligand to alter the binding ability of the ligand through indirect means (such as
structural or energetic alterations in the ligand)." ([0138]); and "the antigen binding
protein provided in the present description can interfere with, block, reduce, or
modulate the interaction between PCSK9 and LDLR.
protein is denoted as 'neutralizing'....

Such an antigen binding

the neutralizing ABP binds to PCSK9 in aposition and/or manner that prevents PCSK9 from binding to LDLR.

Such an ABP

can be specifically described as a 'competitively neutralizing' ABP." ([0155]).
According to these statements, the term "neutralize" in the present invention meansinterfering with, blocking, reducing, or modulating the interaction between PCSK9and LDLR protein and includes not only an aspect of directly blocking a binding site
between PCSK9 and LDLR protein, but also an aspect of altering the binding abilityof PCSK9 to LDLR protein through indirect means (such as structural or energ etic
alterations in the ligand).
B. Next, when the statement of the present description is examined in order tointerpret the technical significance of the invention-specifying matter [ii] "which'competes' for binding to PCSK9 with a reference antibody," there are the following
statements in the present description: "Antigen binding proteins identified by thecompetition assay (competing antigen binding proteins) include an antigen binding
protein which binds to the same epitope as a reference antigen binding protein and an
antigen binding protein which binds to an adjacent epitope sufficiently close to an
epitope bound by the reference antigen binding protein for steric interference tooccur...." ([0140]); "Competing Antigen Binding Proteins ... for specific binding to
PCSK9, there are provided antigen binding proteins which compete with one of the
exemplified antibodies or functional fragments binding to the epitope described in thepresent description.

Such antigen binding proteins can also bind to the epitope that16

is the same as or overlaps with one of the antigen binding proteins exemplified in thepresent description" ([0269]); "The term 'compete' when used in the context of anantigen binding protein (e.g., a ... neutralizing antibody) which competes for the same
epitope means competition between antigen binding proteins as measured by an assay
in which the antigen binding protein (e.g., an antibody or immunologically functional
fragment thereof) being tested prevents or inhibits (e.g., reduces) spec ific binding of areference antigen binding protein (e.g., a ligand or reference antibody) to a common
antigen (e.g., PCSK9 or a fragment thereof)." ([0140]).

According to thesestatements, it can be interpreted that "competing" with a reference antibody in thepresent invention means preventing or inhibiting (e.g., reducing) specific binding of
the reference antibody by binding to a site on PCSK9 that is the same as or overlaps
with a site where the reference antibody binds to PCSK9 and also means prevent ing or
inhibiting (e.g., reducing) specific binding of the reference antibody by stericallyinterfering with binding between the reference antibody and PCSK9, which isevaluated as "competing" between antibodies if the fact that an antibody prevents orinhibits (e.g., reduces) specific binding of the reference antibody to PCSK9 is
measured by an assay.

In the present invention, the degree of "competing" is not

specified.
Thus, it can be acknowledged that the monoclonal antibody of the present
invention, which competes with the reference antibody, prevents or inhibits (e.g.,
reduces) specific binding of the reference antibody in various degrees and is notnecessarily limited to a monoclonal antibody having a property of preventing or
inhibiting (e.g., reducing) specific binding of the reference antibody by binding to thesame site on PCSK9 as a site where the reference antibody binds to PCSK9, but also
includes a monoclonal antibody having a property of preventing or inhibiting (e.g.,
reducing) specific binding of the reference antibody by binding to a site that overlaps
with a site on PCSK9 where the reference antibody binds to PCSK9, and also includes
a monoclonal antibody having a property of preventing or inhibiting (e.g., reducing)
specific binding of the reference antibody to PCSK9 by binding to PCSK9 in amanner that sterically interferes with binding between the reference antibody and
PCSK9.
(4) A. Next, when the statement of the present description is further specificallyexamined, there are the following statements in addition to the statements mentionedin 1(1)A above:(A) In order to produce antibodies to PCSK9, two groups of XenoMouse (R)mice were used to prepare immunized mice according to the schedule of the17

immunization program in Table 3.

Then, mice (10 mice) that produced antibodies

specific to PCSK9 were selected, and splenocytes and lymphocytes were isolated
from the spleens and lymph nodes (Example 1, [0312] to [0314], [0320], [0321]).
(B)

B cells were dissociated from the lymphoid tissue of the mice selected in (A)and mixed with non-secretory myeloma P3X63Ag8.653 cells.

Then, throughprocedures such as centrifugal sedimentation of the fused cells, hybridomas that
produce antigen binding proteins to PCSK9 were produced (Example 2, [0322] to[0324]).
(C)

The primary screening by ELISA using biotinylated-PCSK9 without a V5

tag bound to a NeutrAvidin-coated plate as a capture sample was performed, thereby
obtaining a total of 3104 antigen (wild-type PCSK9) specific hybridomas (Example 3,[0325] to [0328]).
In order to confirm that stable hybridomas were established, a total of 3000
positives mentioned above were rescreened for binding to wild-type PCSK9, and a
total of 2441 positives were repeated in the second screening (confirmato ry screening).
Then, in order to confirm that the antibodies can bind to both human and mouse,"mouse cross-reaction screening" was performed to confirm that 579 antibodies cross reacted with mouse PCSK9 ([0329], [0330]).
(D) The hybridoma exhaust supernatant and biotinylated D374Y PCSK9 mutant
having a high binding affinity to LDLR were transferred to a plate bound to LDLR asa capture sample.

Screening to detect the LDLR-bound biotinylated D374Y mutant

using streptavidin HRP (large-scale receptor ligand blocking screening) was
performed to identify 384 antibodies which blocked the interaction between PCSK9
and the LDLR wells, among which it was confirmed that 100 antibodies inhibited thebinding interaction between PCSK9 and LDLR by more than 90% ([0332]).
Then, the receptor ligand assay was repeated using the D374Y mutant on 384
neutralizing substances identified in the first large-scale receptor ligand inhibition
assay, which identified 85 antibodies that blocked the interaction between the D374Y
mutant and LDLR by more than 90% ([0333], [0334]).
(E)

31H4 antibody (reference antibody) produced from the hybridoma that

produces an antibody having the desired interaction with PCSK9 as identified on thebasis of these assays (screenings) is a neutralizing antibody which strongly blocks
binding between PCSK9 and LDLR (Example 11, [0377] to [0379], [0138], Table 2).
(F)

The EGFa domain of LDLR binds to the catalytic domain of PCSK9.

PCSK9 amino acid residues that are present within 5 angstroms of t he EGFa domain
in the crystal structure are specific core PCSK9 amino acid residues of the interaction18

interface with the EGFa domain of LDLR (core residues).

PCSK9 residues that arepresent at 5 angstroms to 8 angstroms of the EGFa domain are boundary PCSK9amino acid residues of the interaction interface with the EGFa domain of LDLR.

An

antibody which interacts with or blocks any of these amino acid residues can be usefulas an antibody which inhibits the interaction between PCSK9 and the EGFa domain of
LDLR (and/or LDLR in general) (Example 28, [0428] to [0432], Figure 17).
(G) As illustrated in Figure 19B and Figure 19A, 31H4 antibody binds to thecatalytic domain of PCSK9, has a binding site different from that of 21B12 antibody,and binds to PCSK9 simultaneously with 21B12 antibody.

PCSK9 amino acidresidues that are present within 5 angstroms of 31H4 antibody in the crystal structure
are specific core PCSK9 amino acid residues of the interaction interface with 31H4
antibody (core residues).

An antibody which interacts with or blocks any residue ofthe specific core PCSK9 amino acid residues of the interaction interface with the
EGFa domain of LDLR (core residues) or boundary PCSK9 amino acid residues of
the interaction interface with the EGFa domain of LDLR can be useful for inhibitionof the PCSK9/LDLR interaction (Example 29, [0433], [0434], [0437], Figure 18B,
Example 30, [0438] to [0440], [0443], Figure 19A).
The

resultby superimposing the structure of the ternary complex(PCSK9/31H4/21B12) (Figure 19A) obtained from Example 30 on the PCSK9/EGFa
domain structure (Example 28, Figure 17A) is Figure 20A, in which both 21B12
antibody and 31H4 antibody partially overlap with the position of the EGFa domain
of LDLR and sterically interfere with its binding to PCSK9 (Example 31, [0444],
Figure 20A).

Fifteen specific core PCSK9 amino acid residues of the interaction

interface with the EGFa domain of LDLR (core residues) and 32 specific core PCSK9amino acid residues of the interaction interface with 31H4 antibody (core residues)
have three amino acid residues in common.

Further, the residues that are involved inbinding to the EGFa domain and are close to the regions to which the antigen binding
protein (21B12 antibody or 31H4 antibody) binds can be useful for manipulating thebinding of PCSK9 to LDLR ([0446], [0447], Table 12).

19

Catalytic domain(H) An ELISA plate was coated with antibodies containing a first antibody (each2 μg/mL) and blocked with 3% bovine serum albumin (BSA), and biotinylated human
PCSK9 (30 ng/mL) together with a second antibody was applied on the ELISA plate,which was washed.

Then, antibodies having the same binding property weregrouped into the same epitope bin.

The results are shown in Table 8.3, in which

21B12 antibody was grouped into bin 1 and 31H4 antibody was grouped into bin 3(Example 10, [0373], [0374], Table 8.3).
Then, bead codes coated with human IgG capture antibody were washed threetimes with phosphate buffered saline plus 1% BSA (PBSA), and 2 μg/mL anti-PCSK9
antibody was added, which was washed three times with PBSA.

Then, 2 μg/mL

PCSK9 was added and 2 μg/mL anti-PCSK9 antibody was further added to performbinning and identify competing antibodies, the results of which are as shown in Table
37.1.

Bin 1 (competing with 21B12 antibody) and bin 3 (competing with 31H4

antibody) are exclusive of each other, bin 2 competes with bin 1 and bin 3, and bin 4
does not compete with bin 1 and bin 3.

The antibodies in each of the bins are

representative of different types of epitope positions on PCSK9, some of which
overlap with each other (Example 37, [0489] to [0495], Table 37.1, Figures 23A to D).

20Bin 1

Table 37.1
Bin 2
Bin 3 Bin 4

Bin 5(I) Figure 27D illustrates the 12H11 epitope hits mapped onto the crystal structureof PCSK9 with 31H4 and 21B12 antibodies.

12H11 competes with 21B12 antibody

and 31H4 antibody in the binning assay ([0523], [0526], Figure 27D).
(J) Single bolus injections of either 10 mg/kg or 30 mg/kg of 21B12 antibody and
31H4 antibody were performed to gene recombinant mice that express human PCSK9.
As a result, both 31H4 and 21B12 showed significant LDL-cholesterol lowering up to
48 hours (including 48 hours) after administration, as compared to cont rol mice
(Example 26, [0422], [0423], Figure 14A, Figure 14B).
B. (A) According to the disclosures of the present description as summarily
excerpted in A above, the following matters are disclosed: [i] 3104 hybridomas that
produce antibodies specific to PCSK9 were obtained, and the assay (screening) ofantibodies produced by these hybridomas was performed for the binding interaction
between PCSK9 and LDLR protein, which identified antibodies having activity toneutralize binding between PCSK9 and LDLR protein; [ii] From the identified
hybridomas, 31H4 antibody was produced, and this antibody is a neutralizing
antibody which strongly blocks binding between PCSK9 and LDLR protein; [iii] Thebinding region of the 21B12 antibody on PCSK9 was identified in the crystal structure,
and a mechanism has been solved in which the 31H4 antibody partially overlaps the
position of the EGFa domain of LDLR to sterically interfere with its binding to
PCSK9; [iv] Amino acid residues that are involved in binding to the EGFa do main
and that are present at a position on PCSK9 close to the regions to which the antigen
binding protein (21B12 antibody or 31H4 antibody) binds can be useful for21

manipulating the binding of PCSK9 to LDLR protein; [v] In the working examples,
epitope binning was performed on antibodies confirmed to have high bindingneutralizing activity, and antibodies that belong to the group which competes with
31H4 antibody were grouped.
According to these disclosures, it can be deemed that a person ordinarily
skilled in the art can understand that among antibodies having high neutralizing
binding for binding between PCSK9 and LDLR protein, antibodies which competewith 31H4 antibody (reference antibody) are selected, and that 31H4 antibody
partially overlaps with a position of EGFa domain of LDLR in the crystal structure tosterically interfere with its binding to PCSK9.
(B)

Next, Table 37.1 of the present description is a table grouped on the basis of

the data of Figures 23A to D.

(It can be found that some of the clones stated inTable 8.3 are contained, but there are no statements of the original data for groupingin Table 8.3 in the Detailed Description of the Invention and the Drawings in thepresent description.

A few unclear points on the relationship between the two can befound; that is, in relation to 27B2 designated as ND in Table 8.3, Table 37.1 showsthat 27B2.6 is included in bin 1, and 27B2.1 and 27B2.5 are included in bin 2; inrelation to 30A4 included in bin 1 in Table 8.3, Table 37.1 shows that 30A4.1 isincluded in bin 5; and other points.).

Regarding 19 antibodies other than 31H4

antibody included in bin 3 of Table 37.1 that is stated as competing with 31H4
antibody, and for three antibodies in bin 2 that can be deemed to include an antibody
which competes with 31H4 antibody in view of their competing with bin 3, thefollowing matters are confirmed.
That is, each of 16F12.1, 22E2.1, 27A6.1, 28B12.1, 28D6.1, and 31G1.1
antibodies has high identity of amino acid sequence, but does not have high i dentityof amino acid sequence with 31H4 antibody.

Each of 08A1.2, 08A.3.1, and 11F1.1

antibodies has high identity of amino acid sequence, but it is not stated that these
antibodies are neutralizing antibodies.

Regarding 12A11.1 antibody as well, it is notstated that this is a neutralizing antibody.

Further, each of 27B2.1 and 27B2.5

antibodies is a non-neutralizing antibody ([0138]).
On the basis of these disclosures, as an antibody which competes with 31H4
antibody and which neutralizes binding between PCSK9 and LDLR protein, theDetailed Description of the Invention in the present description can be found to
disclose a group of antibodies that differ from 31H4 antibody in the amino acidsequence and have high identity of amino acid sequences with each other.
(5) A. Examination based on the premises above will be conducted.
22

As explained in(2) above, it is reasonable to interpret that whether or not the statement of the scope ofclaims complies with the support requirement should be determined by comparing
between the statement of the scope of claims and the statement of the Detailed
Description of the Invention, then examining: whether or not the invention stated in
the scope of claims is the invention stated in the Detailed Description of the Invent ion,and falls within the scope where a person ordinarily skilled in the art can recognizethat the problem of the invention can be solved by the statement of the Detailed
Description of the Invention; or whether or not, even if it is neither stated nor
suggested in the Detailed Description of the Invention, the invention stated in thescope of claims falls within the scope where a person ordinarily skilled in the art canrecognize that the problem of the invention can be solved in light of the common
general technical knowledge at the time of filing the application.

In this regard, as

shown in 1(1) above, the present invention can be understood as follows: the present
invention increases the amount of LDLR protein, thereby achieving an effect of
causing a decrease in the amount of LDL in a subject and a decrease in serum
cholesterol in the subject, and owing to this effect, diseases related to elevatedcholesterol levels, such as hypercholesterolemia, can be treated or prevented, and therisk of such diseases can be reduced; and for this purpose, the present invention madeits problem to provide an antibody which neutralizes binding between PCSK9 and
LDLR protein, in which the PCSK9 binds to the LDLR protein to thereby decrease theamount of LDLR protein in a subject and increase the amount of LDL in the subject,or to provide a pharmaceutical composition comprising this; and the present invention
reveals that an antibody which competes with the reference antibody that is aneutralizing antibody which strongly blocks binding between PCSK9 and LDLR
protein is an isolated monoclonal antibody which prevents or inhibits binding of thereference antibody to PCSK9.
In addition, according to (3) above, the term "neutralize" in the present
invention includes an aspect of altering a binding ability of PCSK9 to LDLR protein
through indirect means (such as structural or energetic alterations in the ligand) in
addition to interfering with, blocking, reducing, or modulating the interaction between
PCSK9 and LDLR protein by directly blocking the protein binding site.

However, as

mentioned in 1(1) above, the reference antibody itself can be acknowledged as aneutralizing antibody which sterically interferes with binding between PCSK9 and
LDLR protein and which strongly blocks the binding at a position which partiallyoverlaps with a position of the EGFa domain of LDLR (this EGFa domain binds to acatalytic domain of PCSK9, and an antibody which interacts with or blocks any of
23

PCSK9 residues present within the region can be useful as an antibody which inhibits
the interaction between PCSK9 and LDLR) in the crystal structure.

On this basis, it

should be deemed that the invention-specifying matter of "which competes forbinding to PCSK9 with 31H4 antibody" in the present invention also has a technicalsignificance in that it is revealed that an antibody which competes with the 31H4
antibody interferes with, blocks, reduces, or modulates the interaction between
PCSK9 and LDLR protein by directly blocking the binding site of LDLR protein(specifically, by the antibody binding to PCSK9 at a position which overlaps with aposition of the EGFa domain of LDLR in the crystal structure) by a mechanism
similar to that of the 31H4 antibody.

Conversely, it can also be deemed thatprecisely because an antibody which competes with the reference antibody binds atsuch a position, it makes neutralization possible.

This point is also supported by thefact that the Defendant itself asserts in No. 3, 3(2)C above that according to the
Detailed Description of the Invention in the present description, in light of thecommon general technical knowledge at the time of filing the application, it can bedeemed that a person ordinarily skilled in the art understands that an antibody whichbinds to a specific position within a specific region among multiple binding surfaces
on PCSK9 (a position that overlaps with the site which binds to the EGFa domain of
LDLR (or a similar position)) by competing with the reference antibody can neutralize
binding between PCSK9 and LDLR protein, and the person has been able to recognizethat the problem of the invention can be solved throughout the entire technical scopeof the invention.
In addition, according to the disclosures of the Exhibit Ko 1 document as foundin 1(2) above, familial hypercholesterolemia results from elevated LDL cholesterollevels in plasma.

In this regard, since PCSK9 reduces the abundance of LDLR

protein present on cell surfaces, it can be found to have already been shown thatPCSK9 is an attractive target for the treatment and that antibodies or the like whichbind to PCSK9 in plasma and which inhibit its binding to LDLR protein can beeffective inhibitors.

Thus, from these points of view as well, the technicalsignificance of the present invention can also be deemed to lie in the point that it has
been identified that an antibody which competes with 31H4 antibody has a functional
property as an antibody which inhibits the binding to LDLR protein as mentioned
above by a mechanism similar to that of the 31H4 antibody; i.e., a bindingneutralizing antibody.

Originally, as found in 1(1)B above, the problem of thepresent invention is to provide an antibody which neutralizes binding between PCSK9and LDLR protein, in which the PCSK9 binds to the LDLR protein to thereby24decrease the amount of LDLR protein in a subject and increase the amount of LDL in
the subject, or to provide a pharmaceutical composition comprising this.

In arelationship between such a problem and its solution, competing itself with thereference antibody cannot be found to have any unique meaning.

Thus, from thesepoints of view as well, as mentioned above, the technical significance of the present
invention should be deemed to lie in the point that it has been identified that an
antibody which competes with 31H4 antibody has a functional property as a binding neutralizing antibody by the mechanism similar to that of the 31H4 antibody.
B. Further examination will be made.

As mentioned in (4)B(B) above, theDetailed Description of the Invention in the present description discloses that epitope
binning was performed and as a result, a group of antibodies with amino acidsequences that cannot be deemed to have high identity with 31H4 antibody was
identified as competing with the 31H4 antibody.

Regarding an antibody stated ashaving a neutralizing activity among antibodies identified as an antibody which
competes as mentioned above, the present description does not specifically state aposition where the above antibody stated as having a neutralizing activity binds on
PCSK9.

In addition, regarding a group of antibodies that differs from 31H4

antibody in the amino acid sequence, knowledge that a position where such an
antibody binds on PCSK9 is revealed by the fact that the antibody was evaluated ascompeting in an assay such as epitope binning cannot be acknowledged to be common
general technical knowledge.

Thus, the position where the above antibody binds on

PCSK9 cannot be deemed to be apparent.
Further, it is obvious that an antibody having a property "which competes forbinding to PCSK9 with a reference antibody" of the present invention encompasses avery wide variety of antibodies in addition to several groups of antibodies specificallystated in the Detailed Description of the Invention in the present description
mentioned above.

Furthermore, as mentioned in 2(3)B above, the antibody of thepresent invention encompasses not only an antibody which prevents or inhibits (e.g.,
reduces) specific binding of the reference antibody by binding to a site which overlaps
with a site on PCSK9 where 31H4 antibody binds to PCSK9, but also an antibody
which prevents or inhibits (e.g., reduces) specific binding of the reference antibody to
PCSK9 in various degrees by binding to PCSK9 in a manner that sterically interfereswith binding between the reference antibody and PCSK9, as the Defendant asserts.
Then, the antibodies mentioned above can include, for example, an antibody which
prevents or inhibits (e.g., reduces) specific binding of the 31H4 antibody to PCSK9 bybinding to a site which differs from a site where the 31H4 antibody binds to PCSK925

and which differs from a position of EGFa domain of LDLR in the crystal structure
and bringing minor steric hindrance to the 31H4 antibody.

However, a site wheresuch an antibody binds to PCSK9 is not a position where the antibody overlaps with aposition of EGFa domain of LDLR in the crystal structure.

Thus, such an antibody

cannot be deemed to interfere with, block, reduce, or modulate the interactio nbetween PCSK9 and LDLR protein by directly blocking the binding site of LDLR
protein.
Incidentally, the present description states that "An antigen binding protein andfragment which compete with or bind to the same epitope as the exemplified antigen
binding proteins are expected to show similar functional properties." ([0269])
However, as mentioned above, since the matter "which competes for binding to
PCSK9 with 31H4 antibody" does not specify binding to PCSK9 at the same position
as the 31H4 antibody, it cannot be deemed that an antibody which competes with
31H4 antibody is an antigen binding protein (antibody) which competes with or binds
to the same epitope as the 31H4 antibody.

Further, no specific explanations can befound on a mechanism supporting that such antibodies in general show functional
properties similar to those of the 31H4 antibody.

Therefore, it cannot be deemedthat the "antibody which competes for binding to PCSK9 with 31H4 antibody" of thepresent invention "shows functional properties similar" to the 31H4 antibody.
As mentioned above, the technical significance of the present invention should
be deemed to lie in the point that the present invention identifies that an antibody
which competes with 31H4 antibody has a property as an antibody which neutralizes
binding between PCSK9 and LDLR protein by the mechanism similar to that of the
31H4 antibody.

In this regard, as mentioned above, it cannot be deemed that an

antibody which competes with 31H4 antibody would not directly block the bindingsite between PCSK9 and LDLR protein by binding to the site where the antibody
interacts with the EGFa domain of LDLR (from the statement of the present
description, it can be understood as specific core PCSK9 amino acid residues of theinteraction interface with the EGFa domain of LDLR (core residues), which aredefined as PCSK9 residues present within 5 angstroms of the EGFa domain, andboundary PCSK9 amino acid residues of the interaction interface with the EGFa
domain of LDLR, which are defined as PCSK9 residues present at 5 angstroms to 8
angstroms of the EGFa domain).

Other than the above, there is no disclosure on themechanism by which any antibody which competes with 31H4 antibody will be an
antibody which inhibits the interaction (binding) between PCSK9 and the EGFa
domain of LDLR (and/or LDLR in general).
26

Therefore, it can only be deemed to bedifficult for a person ordinarily skilled in the art to arrive at the understanding that an
antibody which competes with 31H4 antibody is a binding-neutralizing antibody.
C. As mentioned in the foregoing, it cannot be deemed that an "antibody which
competes for binding to PCSK9 with 31H4 antibody" interferes with, blocks, reduces,or modulates the interaction between PCSK9 and LDLR protein by directly blocking
the binding site of LDLR protein (specifically, by the antibody binding to PCSK9 at a
position which overlaps with a position of EGFa domain of LDLR in the crystal
structure) in a manner similar to 31H4 antibody.

Thus, it also cannot be

acknowledged that the "antibody which competes for binding to PCSK9 with 31H4
antibody" has a functional property as a binding-neutralizing antibody.

Incidentally,

as mentioned in (3)A above, the term "neutralize" in the present invention includes
not only an aspect of directly blocking the binding site between PCSK9 and LDLR
protein, but also an aspect of altering the binding ability of PCSK9 to LDLR protein
through indirect means (such as structural or energetic alterations in the ligand).
However, it cannot be deemed that it was the common general technical knowledge at
the time of filing the present application that an "antibody which competes for binding
to PCSK9 with 31H4 antibody" will be an antibody which alters the binding ability of
PCSK9 to LDLR protein through the above indirect means, nor can it be deemed that
it was disclosed in the Detailed Description of the Invention in the present description.
D. These points are supported by the results of the demonstration experiment by
Dr. [A], the reliability of which has been acknowledged in 1(3) above, and Affidavit(1) by Dr. [B] based on the same demonstration experiment.

That is, in thisdemonstration experiment, 63 antibodies of Regeneron were tested for competition
with the reference antibody and their binding-neutralizing activity.

As a result ofusing a threshold value of 50% for competition, it was confirmed that 34 antibodies
competed with the reference antibody, among which 28 antibodies (more than 80%)
had no binding-neutralizing activity (Material B1 of Attachment 3 and 1(3)A(B)babove).

Thus, the specific experimental result demonstrates that it cannot be deemedthat an antibody which competes with the reference antibody has a bindingneutralizing activity.

Further, in addition to this experimental result, Dr. [B] statesthat "According to the present patent, this is because the binding site of 31H4
antibody only partially overlaps with the binding site of LDLR on hPCSK9 ... abinding site of another antibody can overlap with the 31H4 binding site without
overlapping with the binding site of LDLR, and in this manner, another antibody canoverlap with the 31H4 binding site without overlapping with the hPCSK9 -LDLR
binding site" (1(3)A(B)b above) and provides an opinion that "it is certainly erroneous27

to state that 'all of ... antibodies which compete with 31H4 antibody would have aneffect of neutralizing binding.'" (1(3)A(B)c above).
E. The Defendant asserts in No. 3, 3(2)C above that there is no reason why thepresent invention violates the support requirement, on the grounds that even if there
exists an antibody which competes with 31H4 antibody (a reference antibody) but
which cannot neutralize binding between PCSK9 and LDLR protein, such an antibody
is literally excluded from the technical scope of Present Invention 1.

However, as

already explained, a technical significance of the present invention should be deemed
to lie in the point that it has been identified that an antibody which competes with
31H4 antibody has a functional property as an antibody which neutralizes binding
between PCSK9 and LDLR protein by a mechanism similar to that of the 31H4
antibody.

If an antibody which competes with 31H4 antibody includes one which

does not have a binding-neutralizing activity, it is apparent that the assumption of its
technical significance will collapse. (In an instance like the present case, if it wereinterpreted to be sufficient to literally exclude an antibody that does not have abinding-neutralizing activity, it would be allowed to make a very broad definition of aposition where the antibody binds to PCSK9, such as the most part of PCSK9, which
would allow the scope of claims to be made broad without a justifiable basis.
Therefore, such an interpretation is not reasonable.)

In addition, even if it isinterpreted that the scope of claims of Present Invention 1 is, as asserted by the
Defendant, directed to only an antibody which "can neutralize binding between
PCSK9 and LDLR protein" among antibodies which compete for binding to PCSK9with a reference antibody, the invention-specifying matter of that which competes forbinding to PCSK9 with a reference antibody according to the present invention is not
limited to an antibody which binds to a position that is the same as or overlaps with aposition where the reference antibody binds as asserted by the Defendant, but also
includes an antibody which competes in a manner that binds to a position for stericinterference with the binding between PCSK9 and LDLR protein to occur, as
explained above.

Thus, it must be supported that such an antibody is also a binding-neutralizing antibody.

In this regard, unlike the case of an antibody which binds to a

position that is the same as or overlaps with a position where the reference antibody
binds, the present description does not state anything about a mechanism by which an
antibody neutralizes the binding between PCSK9 and LDLR protein in which theantibody competes in a manner that binds to a position for steric interference with thebinding to occur.

In addition, binding-neutralizing antibodies based on experimental

results by binning ((4)B(B) above) are all likely to be antibodies which bind to a28

position that is the same as or overlaps with a position where the reference antibody
binds, whose mechanism on binding-neutralizing is disclosed.

Even if this point is

excluded, at least, the present description does not state anything to suggest that theseare sterically interfering antibodies.

Thus, it must be deemed that the Detailed

Description of the Invention in the present description does not disclose anything
about the fact that among antibodies which compete with a reference antibody, when
an antibody competes in a manner that binds to a position for steric interference withthe binding between PCSK9 and LDLR protein to occur, the antibody has a bindingneutralizing activity.

From this point as well, the present invention does not comply

with the support requirement.
Further, as mentioned in No. 2, 3(1) above, the trial decision of the present casedetermines that the present description specifically demonstrates that a number ofantibodies of the present invention are repeatedly identified with sufficiently highprobability by performing the preparation and selection of immunized mice accordin gto the procedure and schedule of the immunization program as stated in the present
description, the production of hybridomas using the selected immunized mice, and thescreening and epitope binning assay for identifying an antibody which strongly blocks
the binding interaction between PCSK9 and LDLR as stated in the present description
from the beginning, repeatedly.

However, as the second Expert Opinion byProfessor [F]([F]) (referred to as Professor [F]) (Exhibit Ko 230) states that "It is
impossible to generate and screen all possible candidate antibodies, because whether aparticular mouse generates a particular antibody is controlled by luck," even if the
production process of antibodies stated in the present description has been undergone,
it is "controlled by luck" what position on PCSK9 an antibody obtained in an
immunized mouse will bind to.

Also, it cannot be deemed that a method of

producing an antibody which binds to an antigen protein in a manner that stericallyinterferes with binding of the antibody to the antigen protein was common generaltechnical knowledge at the time of filing the present application.

Therefore, on thebasis of the statement on a method of producing an antibody as stated in the present
description, it cannot be deemed that various antibodies encompassed in the present
invention were stated in the Detailed Description of the Invention in the present
description.
F. Furthermore, Present Invention 5, which is an invention relating to apharmaceutical composition comprising the monoclonal antibody of Present Invention
1, also does not comply with the support requirement, for the same reasons asmentioned above.
29

(6)

According to the foregoing, since neither of Present Inventions 1 and 5 can be

acknowledged to comply with the support requirement, the determination of the trial
decision of the present case which differs from this is erroneous.

(Note that among

the Plaintiff's assertions, it is considered that the point concerning "EGFa mimic
antibodies" mentioned in No. 3, 3(1)B(C) above contains something worth affirming
and raises doubts about the Defendant's assertion that the support requirement iscomplied with.

However, without making a determination on this point, neither ofPresent Inventions 1 and 5 can be acknowledged to comply with the support
requirement, as mentioned above.

Therefore, the court shall refrain from adding

further determinations.)(7)

Hereinafter, additional remarks will be made just to be sure.

A. With regard to the international situation surrounding the present invention, thePlaintiff asserts that in Europe, the corresponding European patent, which issubstantially the same as the present invention, was judged to be invalid for lack of an
inventive step in 2020 in the court of appeal against opposition, and that in the U.S.,
the corresponding U.S. patent, which is more limited than the present invention, wasjudged to be invalid for lack of the enablement requirement on February 11, 2021 in
the Court of Appeals for the Federal Circuit of the United States, and that Japan iscurrently the only country in the world where the validity of the present patent has
been maintained by the court.

On the other hand, with regard to the above judgmentby the Court of Appeals for the Federal Circuit, the Defendant asserts that since theFederal Supreme Court granted the petition for acceptance of discretionary appeal on
November 4, 2022, the above judgment is extremely likely to be overturned.
However, needless to say, it is apparent that the judgments in other countries do not
immediately affect the judgment in the present case (Note that with regard to the U.S.,
even if the judgment of invalidation by the Court of Appeals for the Federal Circuit isoverturned, the corresponding U.S. patent is not directly relevant to the judgment on
the present invention in either case, because it can be found that the matter"competition" with the reference antibody is not defined as an invention -specifying
matter (For example, Claim 1 of the U.S. Patent No. 8829165 recites the inventionspecifying matter that is "An isolated monoclonal antibody, which, when binding to
PCSK9, 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 inhibits PCSK9 from binding to LDLR" (Exhibit Ko 19).).).
B. In the suit against the trial decision of the separate case concerning the present
invention, as mentioned in No. 2, 1(2) above, Sanofi's assertion on violation of the30

support requirement has been rejected.

However, this can also be understood to be

due to the fact that, in view of the circumstances of the assertions and proof at thetime, it was naturally premised that an antibody which competes with 31H4 antibody
would bind to almost the same position on PCSK9 as the 31H4 antibody and have afunction similar to that of the 31H4 antibody.

In contrast thereto, in the present suit,although doubts about the above premise have been raised by new assertions based on
new evidence such as the structural analysis according to each of the Affidavits by Dr.
[A] and Dr. [B], the Expert Opinion by Professor [F], etc. (Exhibits Ko 18, 230), andthe relevant documentary evidence concerning "EGFa mimic antibodies" (Exhibits Ko
4-1 and 4-2), no information for determination to support this premise can be found.
Therefore, there should be a reasonable reason why the conclusion of the judgment in
the separate case differs from the determination in the present case.
3. Conclusion
According to the foregoing, Ground 2 for Rescission asserted by the Plaintiff iswell founded.

Therefore, without going so far as to determine other grounds forrescission, the trial decision of the present case should be rescinded.
For the foregoing reasons, the judgment is rendered as mentioned in the main
text.
Intellectual Property High Court, Fourth Division
Presiding Judge

KANNO Masayuki

Judge

NAKAMURA Kyo

Judge

OKAYAMA Tadahiro

31

(Attachment 1)[Detailed Description of the Invention][Technical Field][0002]
Field of the Invention
The present invention relates to an antigen binding protein which binds toproprotein convertase subtilisin kexin type 9 (PCSK9) and to a method of using andproducing said antigen binding protein.
[Background Art]
[0003]
Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serine proteaseinvolved in regulating the levels of low-density lipoprotein receptor (LDLR) protein(Horton et al., 2007; Seidah and Prat, 2007).

In vitro experiments have shown that

adding PCSK9 to HepG2 cells lowers the levels of cell-surface LDLR (Benjannet etal., 2004; Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004).

Experimentswith mice have shown that increasing PCSK9 protein levels lowers the levels of the
LDLR protein in the liver (Benjannet et al., 2004; Lagace et al., 2006; Maxwell et al.,2005; Park et al., 2004), while PCSK9 knockout mice have increased levels of LDLR
in the liver (Rashid et al., 2005).

Additionally, various human PCSK9 mutations that

result in either increased or decreased levels of plasma LDL have been identified(Kotowski et al., 2006; Zhao et al., 2006).

PCSK9 has been shown to directlyinteract with the LDLR protein, be endocytosed into cells along with the LDLR, andemit immunofluorescence simultaneously with the LDLR throughout the endosomal
pathway (Lagace et al., 2006).

Degradation of LDLR by PCSK9 has not been

observed, and the mechanism through which extracellular LDLR protein levels arelowered is uncertain.
[Description of Embodiments][0066]
As will be appreciated by a person ordinarily skilled in the art, in light of thepresent disclosure, altering the interaction between PCSK9 and LDLR increases theamount of LDLR available for binding to LDL, which in turn decreases the amount ofserum LDL in a subject, resulting in a reduction in the subject's serum cholesterollevel.

Hence, antigen binding proteins to PCSK9 can be used in various methods

and compositions for treating a subject with elevated serum cholesterol levels, a
subject at risk for elevated serum cholesterol levels, or a subject that could benefit
from a reduction in serum cholesterol levels.
32

Thus, various methods and techniques

for lowering, maintaining, or preventing an increase in serum cholesterol are also
described in the present description.

In some embodiments, the antigen binding

protein allows for binding between PCSK9 and LDLR, but the antigen binding protein
prevents or reduces the adverse activity of PCSK9 on LDLR.

In some embodiments,

the antigen binding protein prevents or reduces the binding of PCSK9 to LDLR.
[0071]The term "PCSK9 activity" includes any biological effect of PCSK9.

In

certain embodiments, PCSK9 activity includes the ability of PCSK9 to interact with asubstrate or receptor or to bind to a substrate or receptor.

In some embodiments,

PCSK9 activity is represented by the ability of PCSK9 to bind to an LDL receptor(LDLR).

In some embodiments, PCSK9 binds to and catalyzes a reaction including

LDLR.

In some embodiments, PCSK9 activity includes the ability of PCSK9 to alter(e.g., reduce) the availability of LDLR.

In some embodiments, PCSK9 activity

includes the ability of PCSK9 to increase the amount of LDL in a subject.

In someembodiments, PCSK9 activity includes the ability of PCSK9 to decrease the amountof LDLR available for binding to LDL.

In some embodiments, "PCSK9 activity"

includes any biological activity resulting from PCSK9 signaling.

Exemplary

activities include, but are not limited to, binding of PCSK9 to LDLR, PCSK9 enzyme
activity which cleaves LDLR or other proteins....
[0109]
An "antigen binding protein" ("ABP") as used in the present description means
any protein which binds a specified target antigen.

In the present application, thespecified target antigen is the PCSK9 protein or a fragment thereof.

The "antigen

binding protein" includes, but is not limited to, an antibody and a binding part thereof(such as an immunologically functional fragment).
of antigen binding proteins.

A peptibody is another example

The term "immunologically functional fragment" (orsimply "fragment") of an antibody or immunoglobulin chain (heavy chain or lightchain) antigen binding protein as used in the present description is a species ofantigen binding protein comprising a portion (regardless of how said portion is
obtained or synthesized) of an antibody that lacks at least some of the amino acids
present in a full-length chain but which can still bind specifically to the antigen.
Such a fragment is biologically active, in that the fragment binds to the target antigen
and can compete with other antigen binding proteins, including intact antibodies, forbinding to a certain epitope.
fragment.

In some embodiments, the fragment is a neutralizing

In some embodiments, the fragment can block or reduce the possibility of

the interaction between LDLR and PCSK9.
33

In one aspect, such a fragment retains at

least one CDR present in the full-length light chain or heavy chain and, in someembodiments, comprises a single heavy chain and/or light chain or a p ortion thereof....
[0123]
An "antigen binding region" means a protein or a portion of a protein which
specifically binds to a particular antigen (e.g., a paratope).

For example, that portionof an antigen binding protein comprising amino acid residues which interact with an
antigen and give the antigen binding protein its specificity and affinity for the antigen
is referred to as the "antigen binding region."

The antigen binding region generally

includes one or more "complementary binding regions" ("CDRs").

A certain antigen

binding region also includes one or more "framework" regions.

The "CDR" is anamino acid sequence that contributes to antigen binding specificity and affinity.

The"framework" region can aid in maintaining the proper conformation of the CDR topromote binding between an antigen binding region and an antigen.

Structurally, theframework region can be located between CDRs in an antibody.

Examples offramework and CDR regions are shown in Figures 2A to 3D, 3CCC to JJJ, and 15A to
15D....
[0127]The variable region typically exhibits the same general structure of relatively
conserved framework regions (FR) joined by three hyper variable regions (alsoreferred to as complementarity determining regions or CDRs).

The CDR obtained

from the two chains of each pair is generally aligned by the framework regions, which
can enable binding to a specific epitope.

From N-terminal to C-terminal, both light-

chain and heavy-chain variable regions generally include domains FR1, CDR1, FR2,
CDR2, FR3, CDR3, and FR4.

The assignment of amino acids to each domain is

generally in accordance with the definition of Kabat sequences of proteins of
immunological interest (National Institutes of Health, Bethesda, Md. (1987 and1991)), or "Chothia & Lesk, J. Mol. Biol., 196: 901-917 (1987); Chothia et al., Nature,
342: 878-883 (1989)".
[0132]The term "light chain" includes a full-length light chain and a fragment thereof
having a variable region sequence sufficient to give binding specificity.

The full -length light chain includes a variable region domain V L and a constant region domain
C L.

The variable region domain of the light chain is located at the amino-terminus ofa polypeptide.

The light chain includes a κ chain and a λ chain.

[0133]The term "heavy chain" includes a full-length heavy chain and a fragment
34

thereof having a variable region sequence sufficient to give binding specificity.

The

full-length heavy chain includes a variable region domain V H and three constantregion domains C H1, C H2, and C H3.

The VH domain is located at the amino-terminusof a polypeptide, and the C H domains are located at the carboxyl-terminus, in which
the CH3 is closest to the carboxy-terminus of the polypeptide.

The heavy chain canbe any isotype, such as IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA(including IgA1 and IgA2 subtypes), IgM, and IgE.
[0138]The term "neutralizing antigen binding protein" or "neutralizing antibody"
represents an antigen binding protein or antibody, respectively, which binds to aligand and prevents or reduces a biological effect of that ligand.

This can be done,for example, by directly blocking a binding site on the ligand or by binding to theligand to alter the binding ability of the ligand through indirect means (such as
structural or energetic alterations in the ligand).

In some embodiments, this term canalso represent an antigen binding protein that prevents a protein to which it binds
from exerting a biological function.

In assessing the binding and/or specificity of anantigen binding protein (e.g., an antibody or immunologically functional fragment
thereof), when an excess of antibody reduces the amount of binding partner bound toa ligand by at least about 1 to 20%, 20 to 30%, 30 to 40%, 40 to 50%, 50 to 60%, 60
to 70%, 70 to 80%, 80 to 85%, 85 to 90%, 90 to 95%, 95 to 97%, 97 to 98%, 98 to
99%, or more (when used in an in vitro competitive binding assay), an antibody or
fragment can substantially inhibit binding of the ligand to its binding partner....

Insome embodiments, in the case of PCSK9 antigen binding protein, such neutralizing
molecules can reduce the ability of PCSK9 to bind LDLR.

In some embodiments,

the neutralizing ability is characterized and/or described via a competition assay....
In some embodiments, ABP27B2, 13H1, 13B5, and 3C4 are non-neutralizing ABPs,
3B6, 9C9, and 31A4 are weak neutralizing substances, and the remaining ABPs in
Table 2 are strong neutralizing substances.

In some embodiments, the antibody or

antigen binding protein neutralizes by binding to PCSK9 and preventing PCSK9 frombinding to LDLR (or reducing the ability of PCSK9 to bind to LDLR).

In someembodiments, the antibody or ABP neutralizes by binding to PCSK9 and whileallowing the PCSK9 to bind to LDLR, preventing or reducing PCSK9-mediateddegradation of the LDLR....
[0140]The term "compete" when used in the context of an antigen binding protein(e.g., a neutralizing antigen binding protein or neutralizing antibody) which competes
35

for the same epitope means competition between antigen binding proteins as measured
by an assay in which the antigen binding protein (e.g., an antibody or
immunologically functional fragment thereof) being tested prevents or inhibits (e.g.,
reduces) specific binding of a reference antigen binding protein (e.g., a ligand orreference antibody) to a common antigen (e.g., PCSK9 or a fragment thereof).

Inorder to determine if one antigen binding protein competes with another binding
protein, there can be used numerous types of competitive binding assays, e.g., solid
phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect
enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983,
Methods in Enzymology 9: 242-253); solid phase direct biotin-avidin EIA (see, e.g.,
Kirkland et al., 1986, J. Immunol. 137: 3614-3619) ... solid phase direct biotin-avidin
EIA (see, e.g., Cheung, et al., 1990, Virology 176: 546-552).

Typically, such anassay includes using a purified antigen bound to a solid surface or cell havi ng any ofthese, an unlabeled test antigen binding protein, and a labeled reference antigen
binding protein.

Competitive inhibition is determined by measuring the amount oflabel bound to the solid surface or cell in the presence of the test antigen bindi ng
protein.

Usually, the test antigen binding protein is present in excess.

Antigenbinding proteins identified by the competition assay (competing antigen binding
proteins) include an antigen binding protein which binds to the same epitope as areference antigen binding protein, and an antigen binding protein which binds to an
adjacent epitope sufficiently close to an epitope bound by the reference antigen
binding protein for steric interference to occur....
[0142]The term "epitope" includes any determinant which can be bound by an antigen
binding protein such as an antibody or T-cell receptor.

An epitope is a region of anantigen bound by an antigen binding protein which targets the antigen, and when theantigen is a protein, the epitope includes a particular amino acid that directly contacts
the antigen binding protein.

Most often, the epitope is present on a protein.

However, in some instances, the epitope can be present on other kinds of molecules
such as nucleic acids.

Epitope determinants can include chemically active surface

groups of molecules such as amino acids, sugar side chains, a phosphoryl or sulfonylgroup, and can have specific three-dimensional structural characteristics and/or
specific charge characteristics.

Generally, an antibody specific to a particular target

antigen will preferentially recognize an epitope on a target antigen in a complex
mixture of proteins and/or macromolecules.
[0154]
36

Antigen Binding Proteins to PCSK9Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serine proteaseinvolved in regulating the levels of low-density lipoprotein receptor (LDLR) protein(Horton et al., 2007; Seidah and Prat, 2007).

PCSK9 is a prohormone-proprotein

convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003)....
The structure of the PCSK9 protein has recently been solved by two groups....
PCSK9 comprises a signal sequence, an N-terminal prodomain, a subtilisin-likecatalytic domain, and a C-terminal domain.
[0155]
Antigen binding proteins (ABPs) which bind PCSK9, including human PCSK9,
are described in the present description.

In some embodiments, the antigen binding

protein provided is a polypeptide comprising one or more complementary determining
regions (CDRs), as described in the present description.

In the same antigen binding

protein, the CDR is embedded into a "framework" region that orients the CDR suchthat the proper antigen binding property of the CDR is achieved.

In someembodiments, the antigen binding protein provided in the present description caninterfere with, block, reduce, or modulate the interaction between PCSK9 and LDLR.
Such an antigen binding protein is denoted as "neutralizing."

In some embodiments,

binding between PCSK9 and LDLR can still occur even though the antigen binding
protein is neutralizing and bound to PCSK9.

For example, in some embodiments,

the ABP prevents or reduces the adverse influence of PCSK9 on LDLR without
blocking the LDLR binding site on PCSK9.

Thus, in some embodiments, the ABP

modulates or alters the ability of PCSK9 to cause degradation of LDLR without
having to prevent the binding interaction between PCSK9 and LDLR.

Such an ABP

can be specifically denoted as a "non-competitively neutralizing" ABP.

In someembodiments, the neutralizing ABP binds to PCSK9 in a position and/or manner thatprevents PCSK9 from binding to LDLR.

Such an ABP can be specifically described

as a "competitively neutralizing" ABP.

Both of the above neutralizing substances

can result in a greater amount of free LDLR present in a subject, which results inmore LDLR bound to LDL (thereby reducing the amount of LDL in a subject).

Inturn, this results in a reduction in the amount of serum cholesterol present in a subject.
[0170]
Some specific examples of the light-chain and heavy-chain variable regions of
the antibodies provided and their corresponding amino acid sequences are
summarized in Table 2.
[0171]
37

[Table 2]
Table 2
Exemplary Heavy-Chain andLight-Chain Variable Regions
Antibody

Light/Heavy
SEQ ID NO[0172]
Likewise, 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 pairs of light chains and heavy chains found insome of the antibodies disclosed in the present description....
[0262]... In some embodiments, the ABP competes with ABP 31H4.
[0268]
In some embodiments, ABP 21B12 binds to an epitope comprising residues 162
to 167 (e.g., residues D162 to E167 of SEQ ID NO: 1)....
[0269]
Competing Antigen Binding Proteins
In another aspect, for specific binding to PCSK9, there are provided antigen
binding proteins which compete with one of the exemplified antibodies or functional
fragments binding to the epitope described in the present description.

Such antigen

binding proteins can also bind to the epitope that is the same as or overlaps with one
38

of the antigen binding proteins exemplified in the present description.

An antigen

binding protein and fragment which competes with or binds to the same epitope as the
exemplified antigen binding proteins are expected to show similar functional
properties.

The exemplified antigen binding proteins and fragments include thosedescribed above, such as those having heavy-chain and light-chain variable region
domains, and CDRs included in Table 2 and/or Figures 2 to 3 and 15.

Thus, asspecific examples, the antigen binding proteins provided include those which competewith an antibody or antigen binding protein having:(a) all 6 of the CDRs listed for antibodies listed in Figures 2 to 3 and 15;(b) VH and VL listed for antibodies listed in Table 2; or(c) two light chains and two heavy chains as specified for antibodies listed in Table 2.
[0270]
Certain Therapeutic Uses and Pharmaceutical Compositions
In certain instances, PCSK9 activity correlates with a number of human disease
conditions.

For example, in certain instances, too high or too low PCSK9 activity

correlates with certain conditions such as hypercholesterolemia.

Thus, in certain

instances, modulating PCSK9 activity can be therapeutically useful.

In certain

embodiments, a neutralizing antigen binding protein to PCSK9 is used to modulate atleast one PCSK9 activity (e.g., binding to LDLR).

Such a method can treat and/or

prevent diseases that relate to elevated serum cholesterol levels or to which elevatedcholesterol levels relate, and/or reduce the risk of these diseases.
[0271]
As will be appreciated by a person ordinarily skilled in the art, in light of thepresent disclosure, diseases that relate to varied cholesterol, LDL, or LDLR levels,
involve varied cholesterol, LDL, or LDLR levels, or can be influenced by variedcholesterol, LDL, or LDLR levels can be addressed by various embodiments of theantigen binding proteins.

In some embodiments, "cholesterol related diseases"(including "serum cholesterol related diseases") include any one or more of those thatcan be manifested, for example, by elevated total serum cholesterol, elevated LDL,
elevated

triglycerides,

elevated

VLDL,

and/or

low

HDL,

as

follows:

hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heartdisease, stroke, cardiovascular disease, Alzheimer's disease, and dyslipidemia ingeneral....
[0276]
In some embodiments, an antigen binding protein to PCSK9 is used to decrease
the amount of PCSK9 activity from an abnormally high level or a normal level.
39

Insome embodiments, an antigen binding protein to PCSK9 is used to treat or prevent
hypercholesterolemia

and/orinthepreparationof

medicamentsforhypercholesterolemia and/or for other cholesterol related diseases (such as thosedescribed in the present description).

In certain embodiments, an antigen binding

protein to PCSK9 is used to treat or prevent conditions such as hypercholesterolemia
in which PCSK9 activity is normal.

In such conditions, for example, a reduction in

PCSK9 activity to below normal can provide a therapeutic effect.
[0312](Example 1)
Immunization and Titer Measurement
Production of Anti-PCSK9 Antibodies and Hybridomas
Antibodies to the mature form of PCSK9 (illustrated as the sequence in Figure
1A, where the prodomain is underlined) were produced in XenoMouse (R) mice
(Abgenix, Fremont, CA) that are mice containing human immunoglobulin genes.
Two groups of XenoMouse (R) mice (Groups 1 and 2) were used to produce antibodies
to PCSK9.

Group 1 included mice of XenoMouse (R) strain XMG2-KL that produce

fully human IgG2 κ and IgG2λ antibodies.
human PCSK9.

Group 1 mice were immunized withPCSK9 was prepared by use of standard recombinant techniques

using the GenBank sequence as reference (NM_174936).
XenoMouse(R)

Group 2 included mice ofstrain XMG4-KL that produce fully human IgG4 κ and IgG4 λ antibodies.

Group 2 mice were also immunized with human PCSK9.
[0313]The mice of both groups were injected with the antigen 11 times according to
the schedule in Table 3.

In the first immunization, each mouse was injected with a

total of 10 μg of the antigen delivered intraperitoneally into the abdomen.
Subsequent boost immunizations were doses of 5 μg, and the injection method wasstaggered between intraperitoneal injections into the abdomen and subcutaneous
injections at the base of the tail.

For intraperitoneal injections, the antigen was

prepared as an emulsion with TiterMax (R) Gold (Sigma, Cat #T2684) added.

Forsubcutaneous injections, the antigen was mixed with Alum (aluminum phosphate) and
CpG oligos.

In injections 2 to 8 and 10, each mouse was injected with a total of 5 μgof the antigen in the adjuvant alum gel.

A final injection of 5 μg of the antigen permouse was delivered in phosphonate-buffered saline and delivered into two sites(50% intraperitoneal into the abdomen and 50% subcutaneous at the base of the tail).
The immunization program is summarized in Table 3 shown below.
[0314]
40

[Table 3]
Table 3
Mouse strain
Number of animals
ImmunogenFirst boost immunization

Secondimmunization

boost

Third boost immunization

Fourthimmunization

boost

Fifth boost immunization

Sixth boost immunization

Seventhimmunization

boost

Eighthimmunization

boost

Blood collection
Ninth boost immunization

10th boost immunization

11th boost immunization

XMG2/kl
10
PCSK9-V5/His
Intraperitoneal injection
10 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODNIntraperitoneal injection
5 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODNIntraperitoneal injection
5 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODNIntraperitoneal injection
5 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODN

XMG4/kl
10
PCSK9-V5/His
Intraperitoneal injection
10 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODNIntraperitoneal injection
5 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODNIntraperitoneal injection
5 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODNIntraperitoneal injection
5 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODNIntraperitoneal injection
5 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODN
BIP
5 μg each
PBS

Intraperitoneal injection
5 μg each
Titermax Gold
Tail injection
5 μg each
Alum/CpG ODN
BIP
5 μg each
PBS

Harvest[0320]
Titers of antibodies to human PCSK9 were tested by ELISA assay for mice41

immunized with the soluble antigen as described.

Table 4 summarizes the ELISA

data and indicates that there were some mice that appeared to be specific to PCSK9.
See, for example, Table 4.

Therefore, at the end of the immunization program, 10

mice (in bold type in Table 4) were selected for harvest, and splenocytes andlymphocytes were isolated from the spleens and lymph nodes, respectively, as
described in the present description.
[0321][Table 4]
Table 4
Summary
results

of

ELISA
Titer

Titer

AnimalID
Group 1

Group 2

Naive
Naive

[0322](Example 2)

N
a
N
i
av
ie
v
e

Recovery of Lymphocytes, Isolation of B-cells, Fusions, and Production ofHybridomas
This example outlines how the immune cells were recovered and how
42hybridomas were produced.
dislocation.

Selected immunized mice were sacrificed by cervical

From each cohort, draining lymph nodes were harvested and pooled.

In order to release cells from the tissue, B cells were dissociated from the lymphoidtissue by grinding in DMEM, and the cells were suspended in DMEM.
were counted.

The cells

In order to resuspend the cells gently but completely, 0.9 mL of

DMEM per 100 million lymphocytes was added to the cell sediment.
[0323]
Lymphocytes were mixed with non-secretory myeloma P3X63Ag8.653 cells
purchased from ATCC, cat. #CR11580 (Kearney et al., (1979) J. Immunol. 123, 1548 1550) at a ratio of 1:4.

The cell mixture was gently sedimented by centrifugation at

400 × g for 4 minutes.

After removing the supernatant by tipping the container, thecells were gently mixed using a 1 mL pipette.

Preheated PEG/DMSO solution

obtained from Sigma (cat #P7306) (1 mL per million of B-cells) was slowly addedwith gentle agitation over 1 minute and then mixed for 1 minute.

Subsequently,

preheated IDMEM (2 mL per million of B cells) (DMEM without glutamine, Lglutamine, penicillin/streptomycin, MEM non-essential amino acids) (all obtained
from Invitrogen) was added over 2 minutes with gentle agitation.

Finally, preheated

6

IDMEM (8 mL per 10 B-cells) was added over 3 minutes.
[0324]
The fused cells were spun down at 400 × g for 6 minutes and resuspended in 20
mL of selection medium (DMEM (Invitrogen), 15% FBS (Hyclone), supplemented
with L-glutamine, penicillin/streptomycin, MEM non-essential amino acids, sodium
pyruvate,

2-mercaptoethanol(allobtainedfrom

Invitrogen),

HA-azaserine

hypoxanthine and OPI (oxaloacetate, pyruvate, bovine insulin) (both obtained from
Sigma) and IL-6 (Boeringer Mannheim)) per million B cells.

The cells wereincubated at 37°C for 20 to 30 minutes, and then resuspended in 200 mL of selection
medium and cultured in T175 flasks for 3 to 4 days prior to seeding into a 96 -well.
Thus, hybridomas that produce antigen binding proteins to PCSK9 were produced.
[0325](Example 3)
Selection of PCSK9 Antibodies
The present example outlines how the various PCSK9 antigen binding proteins
were characterized and selected.

The binding of secreted antibodies (produced fromthe hybridomas produced in Examples 1 and 2) to PCSK9 was assessed.

Selection of

antibodies was based on binding data and inhibition of binding of PCSK9 to LDLR
and affinity.

As described below, binding to soluble PCSK9 was analyzed by ELISA.
43

BIAcore (R) (surface plasmon resonance) was used to quantify binding affinity.
[0326]
Primary Screening
Primary screening for antibodies which bind to wild-type PCSK9 was
performed.

The primary screening was performed on two harvests.

The primary

screening comprised an ELISA assay and was performed using the following protocol.
[0327]
Costar 37-2 medium-binding 384-well plates (Corning Life Sciences) wereused.

The plates were coated with NeutrAvidin at a concentration of 4 μg/mL in

1×PBS/0.05% azide at a volume of 40 μL/well.
overnight.

The plates were incubated at 4°C

Then, the plates were washed using a Titertek plate washer (Titertek,

Huntsville, AL).

Three cycles of washing were performed.

The plates were

blocked with 90 μL of 1×PBS/1% milk and incubated at room temperature for about
30 minutes.

Then, the plates were washed.

Again, three cycles of washing were

performed.

The capture sample was biotinylated-PCSK9 without a V5 tag and added

at 0.9 μg/mL in 1×PBS/1% milk/10 mM Ca 2+ at a volume of 40 μL/well.
plates were incubated at room temperature for 1 hour.

Then, theNext, the plates were washedusing the Titertek plate washer operated using three-cycle washing.

Ten microlitersof the supernatant was transferred into 40 μL of 1×PBS/1% milk/10 mM Ca 2+ andincubated at room temperature for 1.5 hours.

Again, the plates were washed using

the Titertek plate washer operated using three-cycle washing.

Forty microliters/wellof goat anti-human IgG Fc POD at a concentration of 100 ng/mL (1:4000) in
1×PBS/1% milk/10 mM Ca 2+ was added to the plates and incubated at room
temperature for 1 hour.
washing.

The plates were washed once again using three-cycle

Finally, 40 μL/well of One-step TMB (Neogen, Lexington, Kentucky) was

added to the plates, and quenching was performed with 40 μL/well of 1N hydrochloric
acid at room temperature after 30 minutes.

OD was read immediately at 450 nm

using a Titertek plate reader.
[0328]The primary screening resulted in a total of 3104 antigen specific hybridomas
identified from the two harvests.

Based on the highest ELISA OD, 1500 hybridomas

per harvest were used for further manipulation for a total of 3000 positives.
[0329]
Confirmatory Screening
Then, in order to confirm that stable hybridomas were established, 3000
positives were rescreened for binding to wild-type PCSK9....
44

A total of 2441

positives were repeated in the second screening.

Then, these antibodies were used in

the subsequent screenings.
[0330]
Mouse Cross-Reaction Screening
In order to confirm that the antibodies can bind to both human and mouse
PCSK9, the panel of hybridomas was then screened for cross-reactivity to mouse
PCSK9....

It was observed that 579 antibodies cross-reacted with mouse PCSK9.

Then, these antibodies were used in the subsequent screenings.
[0331]
D374Y Mutant Binding Screening
The D374Y mutation in PCSK9 in human populations has been stated in
documents (e.g., Timms KM et al., "A mutation in PCSK9 causing autosomaldominant hypercholesterolemia in a Utah pedigree", Hum. Genet. 114: 349-353,
2004).

In order to determine if the antibodies were specific to the wild type or also

bound to the D374Y form of PCSK9, the samples were then screened for binding to
the mutant PCSK9 sequence comprising the mutation D374Y....

More than or equal

to 96% of the positive hits on the wild-type PCSK9 also bound the mutant PCSK9.
[0332]
Large-Scale Receptor Ligand Blocking Screening
An assay was developed using the D374Y PCSK9 mutant in order to screen for
antibodies which block PCSK9 binding to LDLR.

The mutant was used for thisassay because the mutant has a higher binding affinity to LDLR, enabling thedevelopment of a more sensitive receptor ligand blocking assay.
ligand blocking screening, the following protocol was used.

In the receptor

In the screening, Costar

3702 medium-binding 384-well plates (Corning Life Sciences) were used.

The

plates were coated with 2 μg/mL goat anti-LDLR (R&D Cat #AF2148) in
1×PBS/0.05% azide at a volume of 40 μL/well.
overnight.

The plates were incubated at 4°C

Then, the plates were washed using a Titertek plate washer (Titertek,

Huntsville, AL).

Three cycles of washing were performed.

The plates were

blocked with 90 μL of 1×PBS/1% milk and incubated at room temperature for about
30 minutes.

Then, the plates were washed using the Titertek plate washer.

cycles of washing were performed.

Three

The capture sample was LDLR (R&D, Cat#2148LD/CF), and was added at 0.4 μg/mL in 1×PBS/1% milk/10 mM Ca 2+ at avolume of 40 μL/well.

Then, the plates were incubated at room temperature for 1

hour and 10 minutes.

Simultaneously, 20 ng/mL of biotinylated human D374Y

PCSK9 was incubated together with 15 μL of hybridoma exhaust supernatant in Nunc
45

polypropylene plates and the exhaust supernatant concentration was diluted at 1:5.
Then, the plates were preincubated at room temperature for about 1 hour and 30
minutes.

Next, the plates were washed using the Titertek plate washer operatedusing three-cycle washing.

Fifty microliters/well of the preincubated mixture wastransferred onto the LDLR-coated ELISA plates and incubated at room temperaturefor 1 hour.

To detect LDLR-bound b-PCSK9, 40 μL/well of 500 ng/mL streptavidin

HRP in assay diluent was added to the plates.
temperature for 1 hour.
washer.

The plates were incubated at room

Again, the plates were washed using the Titertek plateThree cycles of washing were performed.

Finally, 40 μL/well of One-step

TMB (Neogen, Lexington, Kentucky) was added to the plates, and quenching was
performed with 40 μL/well of 1N hydrochloric acid at room temperature after 30
minutes.

OD was read immediately at 450 nm using a Titertek plate reader.

The

screening identified 384 antibodies which blocked the interaction between PCSK9 andthe LDLR wells, among which 100 antibodies blocked the interaction strongly (OD <0.3).

These antibodies inhibited the binding interaction between PCSK9 and LDLR

by more than 90% (more than 90% inhibition).
[0333]
Receptor Ligand Binding Assay on Subset of Blocking Substances
The receptor ligand assay was then repeated using the mutant enzyme on thesubset of 384 neutralizing substances identified in the first large-scale receptor ligand
inhibition assay.

The screening of the 384 blocking substance subset assays used thesame protocol as that performed in the large-scale receptor ligand blocking screening.
This repeated screening confirmed the first screening data.
[0334]
This screening of the 384-member subset identified 85 antibodies whichblocked the interaction between the PCSK9 mutant enzyme and LDLR by more than
90%.
[0335]
Receptor Ligand Binding Assay of Blocking Substances Which Bind WildType PCSK9 but Does Not Bind the D374Y Mutant

In the initial panel of 3000

supernatants, there were 86 antibodies shown to specifically bind to the wild -type
PCSK9 and not to the huPCSK9 (D374Y) mutant.

These 86 supernatants were tested

for the ability to block the wild-type PCSK9 from binding to the LDLR receptor....
[0336]
Screening Results
Based on the results of the assays described, several hybridoma lines were46

identified as producing antibodies having the desired interaction with PCSK9.
Limiting dilution was used to isolate a manageable number of clones from each line.
The clones were designated by hybridoma line number (e.g., 21B12) and clone
number (e.g., 21B12.1).

Generally, differences among different clones of aparticular line were detected by the functional assays described in the present
description.

In a few instances, clones were identified from a particular line thatbehaved differently in the functional assays.

For example, it was found that 25A7.1

does not block PCSK9/LDLR, but 25A7.3 (referred to as 25A7 in the present
description) has a neutralizing property.

Each of the isolated clones was allowed to

grow in 50 to 100 mL of hybridoma solvent until exhausted (i.e., cell viability of lessthan about 10%).

The concentration and potency of the antibodies to PCSK9 in the

supernatants of these cultures were measured by ELISA and by in vitro functional
testing, as described in the present description.

As a result of the screening

described in the present description, the hybridoma having the highest titer ofantibodies to PCSK9 was identified.

The selected hybridomas are shown in Figures

2A to 3D and Table 2.
[0373](Example 10)
Epitope Binning
Competition ELISA was used for anti-PCSK9 antibody binning.

In summary,in order to determine if two antibodies belong to the same epitope bin, one of theantibodies (mAb1) was first coated onto an ELISA plate (NUNC) at 2 μg/mL byovernight incubation.

Then, the plate was washed and blocked with 3% BSA.

On

the other hand, 30 ng/mL of biotinylated hPCSK9 was incubated with the second
antibody (mAb2) at room temperature for 2 hours.

The mixture was applied tocoated mAb1 and incubated at room temperature for 1 hour.

Then, the ELISA plate

was washed and incubated with Neutravidin-HRP (Pierce) at a dilution of 1:5000 for
1 hour.

After further washing, the plate was incubated with TMB substrate and thesignal was detected at 650 nm using a Titertek plate reader.

Antibodies having thesame binding property were grouped into the same epitope bin.
antibody binning studies are shown in Table 8.3.
[0374][Table 11]

47

The results of theTable 8.3
Clone

Clone

Bin

Bin

[0377](Example 11)
Effects of 31H4 and 21B12 on Blocking D374Y PCSK9/LDLR Binding
The present example provides IC50 values for two of the antibodies in blocking
the ability of PCSK9 D374Y to bind to LDLR.

Clear 384-well plates (Costar) were

coated with 2 μg/mL of goat anti-LDL receptor antibody (R&D Systems) diluted inbuffer A (100 mM sodium cacodylate, pH 7.4).

The plates were washed thoroughlywith buffer A and then blocked for 2 hours with buffer B (1% milk in buffer A).
After washing, the plates were incubated with 0.4 μg/mL of LDL receptor (R&D
Systems) diluted in buffer C (buffer B supplemented with 10 mM CaCl 2) for 1.5 hours.
Simultaneously with this incubation, 20 ng/mL of biotinylated D374Y PCSK9 wasincubated with various concentrations of the 31H4 IgG2, 31H4 IgG4, 21B12 IgG2, or
21B12 IgG4 antibody that was diluted in buffer A, or buffer A alone (control).
48

The

LDL receptor-containing plates were washed, and the biotinylated D374Y
PCSK9/antibody mixture was transferred to the plates and incubated at room
temperature for 1 hour.

Binding of the biotinylated D374Y to the LDL receptor was

detected by incubation with 500 ng/mL of streptavidin-HRP (Biosource) in buffer C,
followed by incubation with TMB substrate (KPL).

The signal was quenched with

1N HCl and the absorbance was read at 450 nm.
[0378]The results of this binding study are shown in Figures 6A to 6D.

In summary,

IC50 values were measured for each antibody and found to be 199 pM for 31H4 IgG2
(Figure 6A), 156 pM for 31H4 IgG4 (Figure 6B), 170 pM for 21B12 IgG2 (Figure 6C),
and 169 pM for 21B12 IgG4 (Figure 6D).
[0379]
The antibodies also blocked the binding of wild-type PCSK9 to LDLR in thisassay.
[0380](Example 12)
Cell LDL Uptake Assay
The present example demonstrates that various antigen binding proteins can
reduce LDL uptake by cells....
[0381]The results of the cell uptake assay are shown in Figures 7A to 7D.

Insummary, IC 50 values were measured for each antibody and found to be 16.7 nM for
31H4 IgG2 (Figure 7A), 13.3 nM for 31H4 IgG4 (Figure 7B), 13.3 nM for 21B12
IgG2 (Figure 7C), and 18 nM for 21B12 IgG4 (Figure 7D).

These results

demonstrate that the applied antigen binding proteins can reduce the effect of PCSK9(D374Y) to block LDL uptake by cells.

The antibodies also blocked the effect ofwild-type PCSK9 in this assay.
[0382](Example 13)
Serum Cholesterol Lowering Effect of 31H4 Antibody in a 6-Day Study
In order to assess total serum cholesterol (TC) lowering in wild type (WT)
mice via antibody therapy against PCSK9 protein, the following procedure was
performed.
[0383]
Male WT mice (C57BL/6 strain, 9 to 10 weeks old, 17 to 27 g) obtained from
Jackson Laboratory (Bar Harbor, ME) were fed a normal diet (Harland-Teklad, Diet49

2918) throughout the duration of the experiment.

Mice were administered either

anti-PCSK9 antibody 31H4 (2 mg/mL in PBS) or control IgG (2 mg/mL in PBS) at alevel of 10 mg/kg through the mouse's tail vein at t = 0.
aside as a naive control group.

Naive mice were also set

Dosing groups and time of sacrifice are shown in

Table 9.
[0384][Table 12]
Table 9Group

Treatment

1
2
3
4
5
6
7
8
9

IgG
31H4
IgG
31H4
IgG
31H4
IgG
31H4
Naive

Time point
dosing8 hours
8 hours
24 hours
24 hours
72 hours
72 hours
144 hours
144 hours
n/a

after

Number
7
7
7
7
7
7
7
7
7

[0385]
Mice were sacrificed with CO 2 asphyxiation at the predetermined time points
shown in Table 9.

Blood was collected via vena cava into Eppendorf tubes and wasallowed to clot at room temperature for 30 minutes.

Then, in order to separate theserum, the samples were spun down in a table-top centrifuge at 12,000 × g for 10
minutes.

Serum total cholesterol and HDL-C were measured using Hitachi 912

clinical analyzer and Roche/Hitachi TC and HDL-C kits.
[0386]The results of the experiment are shown in Figures 8A to 8D.

In summary,mice to which antibody 31H4 was administered showed decreased serum cholesterollevels over the duration of the experiment (Figure 8A and Figure 8B).

In addition, it

is noted that the mice also showed decreased HDL levels (Figure 8C and Figure 8D).
For Figure 8A and Figure 8C, the change in % is relative to the control IgG at thesame time point (*P < 0.01, #P < 0.05).

For Figure 8B and Figure 8D, the change

in % is relative to total serum cholesterol and HDL levels measured in naive animals
at t = 0 hours (*P < 0.01, #P < 0.05).
[0387]For the lowered HDL levels, it is noted that it will be appreciated by a person

50

ordinarily skilled in the art that the decrease in HDL in mice does not suggest that an
HDL decrease will occur in humans, and merely further reflects that the serum
cholesterol in this organism decreased.

It is noted that mice transport most of serum

cholesterol into high-density lipoprotein (HDL) particles, which differs from humans
having most serum cholesterol on LDL particles.

In mice, the measurement of total

serum cholesterol most closely resembles the serum HDL-C levels.

Mouse HDL

contains apolipoprotein E (apoE) that is a ligand for the LDL receptor (LDLR) andallows HDL to be cleared by the LDLR.

Thus, examining HDL is an appropriate

indicator for the present example in mice (it is appreciated that a decrease in HDL isnot expected for humans).

In contrast thereto, for example, human HDL does notcontain apoE and is not a ligand for LDLR.

PCSK9 antibodies increase LDLR

expression in mouse, allowing the liver to clear more HDL, thus lowering serum
HDL-C levels.
[0388](Example 14)
Effect of Antibody 31H4 on LDLR Levels in a 6-Day Study
The present example demonstrates that an antigen binding protein alters LDLR
levels in a subject over time, as expected.

A Western blot analysis was performed inorder to confirm the effect of antibody 31H4 on LDLR levels.

Fifty to 100 mg ofliver tissue obtained from the sacrificed mice described in Example 13 was
homogenized in 0.3 mL of RIPA buffer (Santa Cruz Biotechnology Inc.) containing acomplete protease inhibitor (Roche).

The homogenate was incubated on ice for 30

minutes and centrifuged to sediment the cellular debris.

Protein concentration in the

supernatant was measured using BioRad protein assay reagent (Bio Rad laboratories).
One hundred micrograms of protein was denatured at 70°C for 10 minutes andseparated on 4 to 12% Bis-Tris SDS gradient gel (Invitrogen).

The protein wastransferred to a 0.45 μm PVDF membrane (Invitrogen) and blocked in washing buffer(50 mM Tris PH7.5, 150 mM NaCl, 2 mM CaCl 2, and 0.05% Tween 20) containing
5% non-fat milk at room temperature for 1 hour.

Then, the blot was probed withgoat anti-mouse LDLR antibody (R&D system) 1:2000 or anti-β actin (sigma) 1:2000
at room temperature for 1 hour.

The blot was washed for a short time and incubatedwith bovine anti-goat IgG-HRP (Santa Cruz Biotechnology Inc.) 1:2000 or goat antimouse IgG-HRP (Upstate) 1:2000.

After incubation at room temperature for 1 hour,

the blot was washed thoroughly, and immunoreactive bands were detected using ECL
plus kit (Amersham biosciences).

The Western blot showed an increase in LDLR

protein levels in the presence of antibody 31H4, as illustrated in Figure 9.
51

[0389](Example 15)
Serum Cholesterol Lowering Effect of Antibody 31H4 in a 13-Day Study
In order to assess total serum cholesterol (TC) lowering in wild type (WT)
mice via antibody therapy against PCSK9 protein in a 13-day study, the following
procedure was performed.
[0390]
Male WT mice (C57BL/6 strain, 9 to 10 weeks old, 17 to 27 g) obtained from
Jackson Laboratory (Bar Harbor, ME) were fed a normal diet (Harland-Teklad, Diet
2918) throughout the duration of the experiment.

Mice were administered either

anti-PCSK9 antibody 31H4 (2 mg/mL in PBS) or control IgG (2 mg/mL in PBS) at alevel of 10 mg/kg through the mouse's tail vein at t = 0.

Naive mice were also set

aside as a naive control group.
[0391]
Dosing groups and time of sacrifice are shown in Table 10.

Animals wer esacrificed, and livers were extracted and prepared as in Example 13.
[0392][Table 13]
Table 10Group

Treatment

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

IgG
31H4
31H4
IgG
31H4
31H4
IgG
31H4
31H4
IgG
31H4
31H4
IgG
31H4
31H4
Naive

Time point
dosing72 hours
72 hours
72 hours
144 hours
144 hours
144 hours
192 hours
192 hours
192 hours
240 hours
240 hours
240 hours
312 hours
312 hours
312 hours
n/aafter Number Dose
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6

10 mg/kg
10 mg/kg
1 mg/kg
10 mg/kg
10 mg/kg
1 mg/kg
10 mg/kg
10 mg/kg
1 mg/kg
10 mg/kg
10 mg/kg
1 mg/kg
10 mg/kg
10 mg/kg
1 mg/kgn/a[0393]
When the 6-day experiment was extended to a 13-day study, the same serum52

cholesterol lowering effect observed in the 6-day study was also observed in the 13day study.

More specifically, animals dosed at 10 mg/kg showed a 31% decrease inserum cholesterol on day 3, which gradually returned to pre-dosing levels by day 13.
Figure 10A illustrates the results of this experiment.

Figure 10C illustrates theresults of repeating the above procedure using 31H4 at a dose of 10 mg/kg andanother antibody 16F12 also at a dose of 10 mg/kg.

Dosing groups and time ofsacrifice are shown in Table 11.
[0395]As shown in Figure 10C, both 16F12 and 31H4 resulted in significant andsubstantial decreases in total serum cholesterol after only a single dose, and were
beneficial over a week or more (10 days or more).

The results of the repeated 13 -day study are consistent with those of the first 13-day study, and a decrease in serum
cholesterol levels of 26% on day 3 is observed.

For Figure 10A and Figure 10B, thechange in % is relative to the control IgG at the same time point (*P < 0.01).

For

Figure 10C, the change in % is relative to the control IgG at the same time poin t (*P <0.05).
[0422](Example 26)
Mouse Model for the Ability of PCSK9 and ABP to Lower LDL in Vivo
In order to prepare mice which over-express human PCSK9, three-week-old
WT C57B1/6 mice were injected via tail vein administration with various
concentrations of adeno-associated virus (AAV) recombinantly modified to express
human PCSK9 so as to measure the correct titer that would give a measurable increaseof LDL-cholesterol in the mice.

By using this virus which expresses human PCSK9,

it was determined that 4.5 × 10E12 pfu of virus resulted in LDL-cholesterol levels of
about 40 mg/dL in the circulating blood (normal levels of LDL in WT mice are about
10 mg/dL).
μg/mL.

The human PCSK9 levels in these animals were found to be about 13

A colony of mice was prepared using this injection criteria.

[0423]
One week after injection, mice were assessed for LDL-cholesterol levels andrandomly assigned to different treatment groups.

Then, a single bolus injection ofeither 10 mg/kg or 30 mg/kg of 16F12, 21B12, or 31H4 antigen binding protein was
administered to the animals via tail vein injection.
separate group of animals as a dosing control.

IgG2 ABP was administered to a

Then, subgroups of the animals (n = 6

to 7) were euthanized at 24 and 48 hours after ABP animals.

After IgG2

administration, there were no effects on LDL-cholesterol levels at either dose.
53

Both

31H4 and 21B12 showed significant LDL-cholesterol lowering up to 48 hours(including 48 hours) after administration, as compared to IgG2 control (shown in
Figures 14A and 14B at two different doses).

By the time point of 48 hours, 16F12

showed an intermediary LDL-cholesterol lowering response at levels returning tobaseline of about 40 mg/dL.

These data are consistent with in vitro binding data

(Biacore and Kinexa) that show a nearly equivalent binding affinity between 31H4
and 21B12 for human PCSK9 and a lesser affinity of 16F12 for PCSK9.
[0426](Example 27)
31H4 and 21B12 Bind to ProCat Region of PCSK9The present example describes one method for determining where various
antibodies bind to PCSK9.
[0427]The ProCat (31 to 449 of SEQ ID NO: 3) or V domain (450 to 692 of SEQ ID
NO: 3) of the PCSK9 protein was combined with either antibody 31H4 or 21B12.
The samples were analyzed by non-denaturing PAGE for complex formation.

As isapparent from Figure 16A and Figure 16B, gel shifts were present for the samples of
ProCat/31H4 and ProCat/21B12, demonstrating that the antibodies bound to the
ProCat domain.
[0428](Example 28)
LDLR EGFa Domain Binds to Catalytic Domain of PCSK9The present

example presents the solved crystal structure of PCSK9 ProCat (31 to 454 of SEQ ID
NO: 3) bound to the LDLR EGFa domain (293 to 334) at 2.9 angstrom resolution (thecondition described in the following examples).
[0429]
An illustrated explanation of the structure of PCSK9 bound to EGFa is shownin Figure 17.

The crystal structure (and its illustrated explanation in Figure 17)

reveals that the EGFa domain of LDLR binds to the catalytic domain of PCSK9.

In

addition, the interaction between PCSK9 and EGFa appears to occur across a surfaceof PCSK9 that is present between residues D374 and S153 in the structure illustrated
in Figure 17.
[0430]
Specific core PCSK9 amino acid residues of the interaction interface with the
LDLR EGFa domain were defined as PCSK9 residues that are present within 5
angstroms of the EGFa domain.

The core residues are as follows: S153, I154, P155,
54

R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380, and S381.
[0431]
Boundary PCSK9 amino acid residues of the interaction interface with the
LDLR EGFa domain were defined as PCSK9 residues that are present at 5 angstroms
to 8 angstroms of the EGFa domain.

The boundary residues are as follows: W156,

N157, L158, E159, H193, E195, H229, R237, G240, K243, D367, I368, G370, A371,
S373, S376, and Q382.

The underlined residues are mostly or completely buried

within PCSK9.
[0432]
As will be appreciated by a person ordinarily skilled in the art, the results fromthe present example demonstrate that PCSK9 and EGFa interact with each other.
Thus, an antibody which interacts with or blocks any of these residues can be usefulas an antibody which inhibits the interaction between PCSK9 and the EGFa domain of
LDLR (and/or LDLR in general).

In some embodiments, when bound to PCSK9,

antibodies which interact with or block any of the above residues or are present at 15
to 8, 8, 8 to 5, or 5 angstroms of the above residues are contemplated to provide
useful inhibition of PCSK9 binding to LDLR.
[0438](Example 30)
21B12 binds to the catalytic domain of PCSK9, has a binding site different
from 31H4, and can bind to PCSK9 simultaneously with 31H4.
[0439]The present example presents the crystal structure of PCSK9 ProCat (31 to 449
of SEQ ID NO: 3) bound to the Fab fragments of 31H4 and 21B12, which wasmeasured at 2.8 angstrom resolution (the conditions described in the following
examples).

This crystal structure illustrated in Figures 19A and 19B shows that

31H4 and 21B12 have different binding sites on PCSK9 and that both antigen binding
proteins can bind to PCSK9 simultaneously.

The structure shows that 21B12

interacts with amino acid residues derived from the catalytic domain of PCSK9.

In

this structure, the interaction between PCSK9 and 31H4 is similar to what wasobserved above.
[0440]
Specific core PCSK9 amino acid residues of the interaction interface with
21B12 were defined as PCSK9 residues that are present within 5 angstroms of the
21B12 protein.

The core residues are as follows: S153, S188, I189, Q190, S191,

D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376,
55

T377, and F379.
[0443]
As will be appreciated by a person ordinarily skilled in the art, the results
obtained from Example 30 demonstrate where the antigen binding protein to PCSK9
can interact with PCSK9, and that the antigen binding protein to PCSK9 can still
block PCSK9 from interacting with EGFa (and thus with LDLR).

Thus, an antigen

binding protein which interacts with any of these PCSK9 residues or which blocks
any of these residues can be useful as an antibody which inhibits the interaction
between PCSK9 and EGFa (and thus LDLR).

Therefore, in some embodiments, anantibody which interacts with any of the above residues or which interacts withresidues that are present within 5 angstroms of the above residues is contemplated toprovide useful inhibition of PCSK9 binding to LDLR.

Similarly, an antigen binding

protein which blocks any of the above residues (which can be determined, e.g., via acompetition assay) can also be useful for inhibition of the PCSK9/LDLR interaction.
[0444](Example 31)
Interaction between EGFa, PCSK9, and Antibodies
The structure of the ternary complex (PCSK9/31H4/21B12) obtained from theabove example was superimposed on the PCSK9/EGFa structure (determined as
described in Example 28) and the result of this combination is illustrated in Figure
20A.

This figure shows regions on PCSK9 which can be usefully targeted so as toinhibit PCSK9 interaction with EGFa.

The figure shows that both 31H4 and 21B12

partially overlap with the position of the EGFa domain of LDLR and stericallyinterfere with its binding to PCSK9.

In addition, as is apparent from the structures,

21B12 directly interacts with a subset of amino acid residues specifically involved inbinding to the LDLR EGFa domain.
[0445]As described above, analysis of the crystal structures identified specific aminoacids involved in the interaction between PCSK9 and the counter-proteins (the core
and the interface boundary regions on the PCSK9 surface) and the spatial
requirements of these counter-proteins to interact with PCSK9.

These structures

suggest methods to inhibit the interaction between PCSK9 and LDLR.

First, asdescribed above, binding of a factor to PCSK9 that shares residues in common withthe binding site of the LDLR EGFa domain inhibits the interaction between PCSK9and LDLR.

Second, a factor which binds outside of the residues in common cansterically interfere with the EGFa domain or regions of the LDLR that are either N56

terminal or C-terminal to the EGFa domain to interfere with the interaction between
PCSK9 and LDLR.
[0446]
In some embodiments, the residues that are involved in EGFa binding and areclose to the regions to which the above antigen binding proteins bind are especially
useful for manipulating the binding of PCSK9 to LDLR.

For example, amino acidresidues derived from common interfaces in both the core region and boundary region
for different binding partners are listed in Table 12 below.

Amino acid residues

completely buried within the PCSK9 protein are underlined.
[0447][Table 15]
Table 12
Parameter
31H4/EGFa, both less than 5 angstroms
31H4, less than 5 angstroms / EGFa, 5 to
8 angstroms
31H4, 5 to 8 angstroms / EGFa, less than
5 angstroms
31H4/EGFa, both 5 to 8 angstroms
21B12/EGFa, both less than 5 angstroms
21B12, less than 5 angstroms / EGFa, 5
to 8 angstroms
21B12, 5 to 8 angstroms / EGFa, lessthan 5 angstroms
21B12/EGFa, both 5 to 8 angstroms

Amino acid position
D374, V380, S381
D367, Q382
I369, S372, C378, F379
H229, S373
S153, R194, D238, D374, T377, F379
R237, K243, S373, S376
I154, A239, I369, S372, C375, C378
H193, E195

[0448]
As will be appreciated by a person ordinarily skilled in the art, in someembodiments, the antigen binding protein binds to and/or blocks at least one of theabove residues.
[0489](Example 37)
Epitope Mapping - Binning
Another set of binning experiments was performed in addition to the set in
Example 10.

As in Example 10, ABPs which compete with each other can beconsidered to bind to the same site on the target and, in common parlance, are said toform "bin" with each other.
[0490]A modification of the multiplexed binning method stated by Jia et al. (J.
57

Immunological Methods, 288 (2004) 91-98) was used.

Each bead cord of

streptavidin-coated Luminex beads was incubated in 100 μL of 0.5 μg/mL
biotinylated monovalent mouse anti-human IgG capture antibody (BD Pharmingen,#555785) at room temperature for 1 hour in the dark, and then washed three times
with PBSA (phosphate buffered saline (PBS) plus 1% bovine serum albumin (BSA)).
Each bead cord was separately incubated with 100 μL of 2 μg/mL anti-PCSK9
antibody (Coating Antibody) for 1 hour, and then washed three times with PBSA.
The beads were pooled and then dispensed to a 96-well filter plate (Millipore,#MSBVN1250).

One hundred microliters of 2 μg/mL purified PCSK9 protein wasadded to half the wells.

Buffer was added to the other half as control.

was incubated for 1 hour and then washed.

The reaction

One hundred microliters of 2 μg/mL

anti-PCSK9 antibody (Detection Ab) was added to all the wells, and the solution was
incubated for 1 hour, and then washed.

As another subject, irrelevant human IgG(Jackson, #009-000-003) was allowed to run.

To each well, 20 μL of PE-conjugatedmonovalent mouse anti-human IgG (BD Pharmingen, #555787) was added, thesolution was incubated for 1 hour, and then washed.

Beads were resuspended in 100

μL of PBSA, and a minimum of 100 events/bead cord were collected on the BioPlex
instrument (BioRad).
[0491]The median fluorescent intensity (MFI) of the antibody pair without PCSK9
was subtracted from the signal of the corresponding reaction containing PCSK9.

For

the antibody pair to be considered to bind simultaneously (and thus in different bins),
the subtracted signal had to be three times greater than the signal of the antibody
competing with itself and three times greater than the signal of the antibody
competing with the irrelevant antibody.
[0492]The data obtained from the above is illustrated in Figures 23A to 23D.
ABPs belonged to five bins.

The

The shaded boxes indicate ABPs which can bindsimultaneously to PCSK9.

The non-shaded boxes indicate ABPs which competewith each other for binding.

A summary of the results is shown in Table 37.1.

[0493][Table 17]58

Table 37.1
Bin 1

Bin 2

Bin 3

Bin 4

Bin 5

[0494]
Bins 1 (competing with ABP 21B12) and 3 (competing with 31H4) areexclusive of each other, bin 2 competes with bins 1 and 3, and bin 4 does not competewith bins 1 and 3.

In this example, bin 5 is represented as a "catch all" bin todescribe ABPs that fit into other bins.

Thus, the above ABPs in each of the bins are

representative of different types of epitope positions on PCSK9, some of which
overlap with each other.
[0495]
As will be appreciated by a person ordinarily skilled in the art, if the reference
ABP prevents the binding of the probe ABP, the antibodies are referred to be in thesame bin.

The order in which the ABPs are used can be important.

If ABP A is

used as the reference ABP and blocks the binding of ABP B, the converse is notalways true.

ABP B used as the reference ABP does not necessarily block ABP A.

There are a number of factors that play a role here.

The binding of an ABP cancause conformational changes in the target, which prevents the binding of the second
ABP, or epitopes which overlap but does not completely block each other can allow
the second ABP to still have enough high-affinity interaction with the target to allow
binding.

ABPs with a much higher affinity can have a greater ability to push out

blocking ABPs.

In general, if competition is observed in any order, the ABPs are
59referred to as bins with each other, and if both ABPs can block each other, it is likelythat the epitopes overlap more completely.
[0521]
Table 39.5 displays a summary of all of the hits for the various antibodies.
[0522][Table 22]
Table 39.5
EC50 shift hits

Bmax shift hits

* Decreases EC50
[0523]
To further examine how these residues form some or all of the relevant
epitopes, the above positions were mapped onto various crystal structure models, ofwhich the results are shown in Figures 27A to 27E....
[0526]
Figure 27D illustrates the 12H11 epitope hits mapped onto the crystal structureof PCSK9 with 31H4 and 21B12 antibodies.
residues as follows.

The structure identifies PCSK9Light gray indicates unmutated residues (except for residues

explicitly indicated on the structure) and darker gray indicates mutated residues (someof which could not express).

Residues explicitly indicated (regardless of the shading

indicated on the figure) were tested, and a significant change in EC50 and/or Bmax
were obtained.

12H11 competes with 21B12 and 31H4 in the binning assay

described above.

60

% Of inhibition% Of inhibition

SEQ ID NO: 1

61

Total cholesterol (mg/dlL)

Total cholesterol

Time (hr)62% Of inhibition% Of inhibitionTotal cholesterol (mg/dlL)

Total cholesterol

Time (hr)Time (hr)Time (hr)63Total cholesterol (mg/dlL)Time (days)

Time (hr)Time (hr)64Prodomain

Heavy chain

Light
chain

Catalytic
domaindomainHeavy chain

Light chain
Catalytic
domain65

Catalytic
domainCatalytic
domain66

β-propeller

Catalytic domain

V domainCatalytic
domainProdomain

V domain67

Blue: Bmax shift
Green: EC50 shift

68

(Attachment 2)* When the relevant portion of Exhibit Ko 1 below (page number and line
number are based on the original text) is summarily excerpted, it is abbreviated as, forexample, "Exhibit Ko 1-1".
1. Page 413, Abstract"Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the number ofsurface low-density lipoprotein (LDL) receptors through an undefined mechanism....
Biosensor studies show that PCSK9 binds to the extracellular domain of LDL receptor
with Kd = 170 nM at the neutral pH of plasma and with a K d as low as 1 nM at theacidic pH of endosomes.

The D374Y gain-of-function mutant is associated withhypercholesterolemia and early-onset cardiovascular disease, binds to the receptor 25
times more strongly than wild-type PCSK9 at neutral pH, and remains exclusively ahigh-affinity complex at acidic pH.

PCSK9 can decrease LDL receptors by amechanism that requires direct binding but not necessarily receptor proteolysis."
2. Page 413, Left Column, Lines 1 to 21"Familial hypercholesterolemia produces results of mutations that cause defects
in hepatic cholesterol clearance, elevated levels of plasma LDL cholesterol (LDL-C),
and an early onset of cardiovascular disease....

Recently, a third locus related tofamilial hypercholesterolemia has been found: it was a gene encoding a serine
protease PCSK9.

The protein is predominantly expressed in the liver and lowers thenumber of cell-surface LDLR by an unknown mechanism.

Gain-of-function

mutations of PCSK9 are characterized through clinical studies in multiple populations
and result in more severe decrease in the number of LDLR with consequent
hypercholesterolemia.

Loss-of-function mutations lead to increased number ofreceptors, increasing clearance of LDL-C from the circulating blood and reducing therisk of cardiovascular disease.

Thus, PCSK9 is viewed as an attractive new target

for therapeutic intervention in dyslipidemia.
heightened by the following evidence.

In addition, this attraction is further

That is, lipid-lowering effects provided by

PCSK9 have synergistic effects with those provided by statins widely used ascholesterol biosynthesis inhibitor.

This suggests that a PCSK9 inhibitor has benefits

that improve those existing therapies."
3. Page 413, Left Column, Lines 22 to 25"PCSK9 (or NARC-1, Document 11) is the ninth known member of the
proprotein convertase family.

Convertase zymogens invariably have an N -terminal

prodomain, a subtilisin-like catalytic domain, and a C-terminal domain."69

4. Page 416, Figure 4"Figure 4 Structure and orientation of the C-terminal domain....
view of PCSK9 and furin superimposed with catalytic domains.

(d) Stereo

Gray, furin; red,

PCSK9 P' peptide; yellow, PCSK9 catalytic domain (ends at 449); cyan, PCSK9 C terminal domain (starts at 453); magenta, space-fill, serine 386.

Some other PCSK9

residues are displayed."
5. Page 417, Left Column, Lines 1 to 20"Biophysical Analysis of PCSK9 Binding to LDLR
Secreted PCSK9 interacts with human liver cell surfaces and can be
immunoprecipitated with LDLR.

Ligand blotting of the recombinant extracellulardomain (ECD) of LDLR subjected to SDS-PAGE under non-denaturing conditions
has also indicated that the LDLR ECD directly binds to PCSK9....

Most importantly,

these studies show that PCSK9 binds to LDLR with more greatly increased affinity(by as much as 170-fold) at the endosomal pH."6. Page 417, Left Column, Lines 28 to 41"Gain-of-function Mutations
Gain-of-function mutations in human PCSK9 are associated with familial
hypercholesterolemia.

In particular, the D374Y mutant is about 10 times more

active than wild-type PCSK9 in lowering the number of LDLR.

We overexpressedand purified D374Y and two other gain-of-function mutants and tested their binding
to LDLR ECD by SPR (Table 1).

At pH 7.5, the D374Y showed 25 times greateraffinity (Kd = 6 nM) for LDLR extracellular domain (ECD) than that of wild-type
PCSK9.

Moreover, at pH 5.4, D374Y had a single binding mode with a K d of 1.6

nM (Table 1).

Because 50% of the wild-type PCSK9 had a K d of 42 nM at acidic pH,

70this portion of the binding is strengthened by 25-fold in the case of the D374Y mutant.
The results strongly suggest that the increased effect of the D374Y mutant in lowering
LDLR results from its enhanced binding to LDLR ECD."
7. Page 417, Table 1"Table 1

Affinities of PCSK9 and Mutants Binding to Immobilized LDLR

ECD
Each value represents the average of three to four determinations and is shownwith standard deviations.

a

Two values indicate that the data fit a model with two

populations of binding sites or conformations; the fractions of each population are
shown in parentheses."
8. Page 418, Left Column, Lines 1 to 17"LDLR Lowering by PCSK9The extracellular domain of LDLR contains seven LDLR type A modules (LA1
to LA7), followed by two epidermal growth factor (EGF) repeats, a YWTD βpropeller domain, another EGF repeat, and a highly glycosylated 58-residue segment.
At neutral pH, LDL particles primarily bind to the LA3 to LA5 modules.

The LA

modules often use three conserved, Ca 2+-binding acidic residues for protein-protein
interactions.

Binding of LDL to the cell-surface LDLR is followed by intracellular

uptake of the LDL-bound receptor into the endosomal compartment, where the acidic
pH promotes a conformational change, which leads to self-association of LDLR
between the LA3 to LA5 modules and the β-prodomain.

This rearrangement aids inthe release of bound LDL and recycling of the ligand-free LDLR to the cell surface,where the LDL liberated in the endosome is degraded in the lysosome.

Usually,

LDLRs undergo a rapid process of intracellular uptake and recycling, but some LDLR
mutants which do not release the bound ligand in endosomes are not recycled to thecell surface."
9. Page 418, Left Column, Lines 18 to 28"Ligand-blotting experiments have shown that PCSK9 binds directly to LDLR
71

extracellular domain....

Thus, the increased effect of D374Y in LDLR lowering canindeed arise from enhanced binding to cell-surface LDLR."
10. Page 418, Left Column, Line 29 to Right Column, Line 6"PCSK9 moves with LDLR into endosomes, suggesting that LDLR-bound
PCSK9, like LDL, moves the receptor together into this acidic compartment; however,in contrast to the weakened binding between LDL and LDLR, the affinity between
PCSK9 and LDLR can be increased in the endosome.

Failure to release PCSK9 canprevent recycling and reduce the number of LDLR on the cell-surface.

The D374Y

mutant binds to LDLR even more strongly than wild-type PCSK9 does, which canaccount for its increased activity in LDLR lowering."
11. Page 418, Right column, Lines 10 to 17"Whether PCSK9 interacts with an unidentified partner to facilitate the releaseof the prodomain and activate PCSK9 as a protease for LDLR lowering is an open
question.

The retention of the prodomain in recombinant and plasma PCSK9,

together with enhanced LDLR binding of the prodomain S127R mutant, suggests thatthe prodomain contributes to LDLR binding.

The prodomain and catalytic domain

are insufficient for activity, suggesting that all three domains are involved in forming
an extensive binding surface for LDLR."
12. Page 418, Right column, Lines 23 to 32"Genetic evidence suggests that PCSK9 is an attractive target for the treatmentof cardiovascular disease.

In theory, PCSK9 could be targeted by a cell -permeable

protease inhibitor which inhibits self-processing and secretion of PCSK9 and achieves
effects similar to those of PCSK9 loss-of-function mutations.

Because binding to

LDLR in plasma and receptor-dependent intracellular uptake are mostly the ratedetermining step for PCSK9 function, antibodies or small molecules which bind to
PCSK9 in plasma and inhibit its binding to LDLR can also be effective inhibitors of
PCSK9 function.

Our structure reported here, particularly the structure of thePCSK9-LDLR complex, will be useful for designing novel therapies."72

(Attachment 3)
1.

"3. By a representative of Regeneron, I was requested to test an ability ofvarious antibodies which bind specifically to hPCSK9 to compete with two reference
antibodies (including the variable heavy chain (VH) and variable light chain (VL)
regions of 21B12 antibody and 31H4 antibody, respectively)." (Page 1, lines 28 to 32)
2.

"4.1 Regeneron Antibody
From Dr. [G]([G]) of Regeneron, we have received 63 different monoclonal

antibodies named by the following designations....
4.2 Amgen Antibody
From Dr. [H]([H]) of InSCREENeX, I have received five monoclonal
antibodies named 9C9, 3B6, 27B2, 21B12, and 31H4...." (Page 2, line 1 to page 3,line 19)3.

"5.1 Binding of MAb (translator's note: monoclonal antibody) to PCSK9...
Antibodies from Regeneron and Amgen were diluted with PBS and coated on a384-well ELISA plate at room temperature for 1 hour (Greiner #781061, > 5 μg/ml,
30 μl/well).
controls.

Uncoated (antibody-free) wells on the plate were used as negative

After uncoated antibodies were removed by washing the plate with H 2O

(containing 0.05% Tween 20), blocking of the ELISA plate was performed using 2%
BSA solution (containing 0.05% Tween 20) at room temperature for 30 minutes (80μl/well).

At the time when the blocking solution was removed, PCSK9 (received

from InSCREENeX) was added to each immobilized antibody in duplicate (30 μl/well,
5 μg/ml in 2% BSA solution containing 0.05% Tween 20).

As negative controls, 2%

BSA solution (containing 0.05% Tween 20) was added to each immobilized antibody
in duplicate.

The solution was incubated at room temperature for 45 minutes.

After the plate was washed with H 2O (containing 0.05% Tween 20), the captured
antigen (Myc-tagged-PCSK9) was detected with HRP-bound anti-Myc antibody (2%BSA solution (containing 0.05% Tween 20) in 1:100,000 dilution, 30 μl/well) at room
temperature for 1.5 hours.

After a further washing step, TMB (30 μl/well) was

added and incubated at room temperature for 20 minutes before stopping the HRP
reaction with 30 μl of H 2SO4 (0.5 M).

Absorbance was measured at 450 nm(reference wavelength 620 nm).
Based on the absorbance value, the arithmetic mean value and standard
deviation for the same measurement value were calculated.
The results of this experiment are shown in column 2 of the table attached as
Material B1.

If a signal-to-noise ratio of < 10 is detected when comparing binding73

signals with/without PCSK9, it is considered that the MAb (translator's note:monoclonal antibody) concerned "does not bind to soluble PCSK9", which has beenhighlighted in red.

If a signal-to-noise ratio of > 10 is detected when comparing

binding signals with/without PCSK9, it is considered that the MAb concerned "binds
to soluble PCSK9", which has been highlighted by shading." (Page 4, line 8 to page 5,line 10)4.

"5.2 Competition with 31H4 or 21B12 for Binding to PCSK9"...
Antibodies from Regeneron and Amgen were diluted with PBS and coated on a384-well ELISA plate at room temperature for 1 hour (Greiner #781061, > 5 μg/ml,
30 μl/well).
controls.

Uncoated (antibody-free) wells on the plate were used as negative

After uncoated antibodies were removed by washing the plate with H 2O

(containing 0.05% Tween 20), blocking of the ELISA plate was performed using 2%
BSA solution (containing 0.05% Tween 20) at room temperature for 30 minutes (80μl/well).

At the time when the blocking solution was removed, PCSK9 (received

from InSCREENeX) was added to each immobilized antibody in duplicate (30 μl/well,
5 μg/ml in 2% BSA solution containing 0.05% Tween 20).

As negative controls, 2%

BSA solution (containing 0.05% Tween 20) was added to each immobilized antibody
in duplicate.

The solution was incubated at room temperature for 45 minutes.

After the plate was washed with H2O (containing 0.05% Tween 20), the captured
antigen was detected at room temperature for 45 minutes by adding biotinylated
21B12 or biotinylated 31H4 antibody (30 μl/well, 1.25 μg/ml in 2% BSA solution
containing 0.05% Tween 20).

After a further washing step, a streptavidin-HRP

reagent was added (30 μl/well, 1:5000 dilution in 2% BSA solution containing 0.05%
Tween 20) and the solution was incubated at room temperature for 45 minutes.

The

plate was washed again, and TMB was added (30 μl/well) and incubated at room
temperature for 20 minutes before stopping the HRP reaction with 30 μl of H 2SO4 (0.5M).

Absorbance was measured at 450 nm (reference wavelength 620 nm).
Based on the absorbance value, the arithmetic mean value and standard

deviation for the same measurement value were calculated.

The compatibility of the

21B12 antibody and the immobilized antibody was quantified by standardizing eachindividual binding signal to the binding signal of the 21B12 antibody and theimmobilized 31H4 antibody (100%).

The compatibility of the 31H4 antibody andthe immobilized antibody was quantified by standardizing each individual binding
signal to the binding signal of the 31H4 antibody and the immobilized 21B12
antibody (100%).
74

The results of this experiment are shown in column 3 from the left (for 21B12
antibody) and column 4 from the left (for 31H4 antibody) in the table attached as
Material B1.
I think that it is necessary to note that Claim 1 of Present Patent 1 and Claim 1of Present Patent 2 do not indicate a specific value for inhibition of the binding; i.e.,competition, between the claimed antibody and the reference antibodies to beachieved in order that other antibodies can be considered to be "competing
antibodies" with one of the reference antibodies.

As mentioned above, thecompatibility of the 31H4 antibody and the immobilized antibody was quantified bystandardizing each individual binding signal to the binding signal of the 31H4
antibody and the immobilized 21B12 antibody (100%).

However, as is apparent

from Material B1 attached, when BSA was immobilized instead of the PCSK9
antibody, the remaining fluorescence (background control) resulted in a signal of 3%
and 4% for 21B12-binding compatibility or 31H4-binding compatibility, respectively.
Thus, full competition is achieved when the signal is reduced to about 3%.

On suchgrounds, I thought that it was reasonable to use an MAb (translator's note: monoclonal
antibody) having a binding compatibility of < 50% with either 21B12 or 31H4 as acompeting antibody.

The competing antibodies are highlighted by shading incolumn 3 from the left (for 21B12 antibody) and column 4 from the left (for 31H4
antibody) in the table attached as Material B1." (Page 5, line 11 to page 7, line 2).
5.

"5.3 Neutralization of LDLR Binding to PCSK9...
LDL-R was diluted with PBS and coated on a 384-well ELISA plate at 4°C for
16 hours (Greiner #781061, 1 μg/ml, 30 μl/well).
the plate were used as negative controls.

Uncoated (antibody-free) wells on

After uncoated antigens were removed,

blocking of the ELISA plate was performed using 2% BSA solution (containing
0.05% Tween 20) at room temperature for 2 hours (80 μl/well).

Then, the plate waswashed with 2% BSA solution (containing 0.05% Tween 20).
In the meantime, dilution series of Regeneron and Amgen antibodies wereprepared (20 μg/ml, 2 μg/ml, 0.2 μg/ml, and 0 μg/ml in 2% BSA solution (containing
0.05% Tween 20)).

Each 50 μl of the antibody dilutions was added to 50 μl of

PCSK9 (from Regeneron) in a 96-well plate (1 μg/ml in 2% BSA solution (containing
0.05% Tween 20)) and preincubated at room temperature for 1 hour.

Afterpreincubation, each antibody:antigen mixture was transferred to the washed ELISA
plate (30 μl/well) and incubated at room temperature for 1 hour.

After the plate waswashed with H2O (containing 0.05% Tween 20), PCSK9 was detected using anti-Myc
75

antibody (30 μl/received, diluted 1:1000 in 2% BSA solution (containing 0.05%
Tween 20)) at room temperature for 1 hour.

The plate was washed again, and TMB

was added (30 μl/well) and incubated at room temperature for 20 minutes before
stopping the HRP reaction with 30 μl of H 2SO4 (0.5 M).

Absorbance was measured

at 450 nm (reference wavelength 620 nm).
The PCSK9 binding signals in the absence of antibodies were standardized to100%.

The remaining PCSK9 binding signals in the presence of the antibody were

calculated accordingly.

If the PCSK9 binding activity at the highest antibody

concentration was ≤ 60%, the antibody was considered to neutralize.
The values for "PCSK9 activity on LDL-R" observed in these experiments areshown in columns 6 to 9 of the table in Material B1 for 10 μg/ml Mab, 1 μg/ml Mab,
0.1 μg/ml Mab, and 0 μg/ml Mab, respectively.

I think that it should be noted that

Claim 1 of Present Patent 1 and Claim 1 of Present Patent 2 do not indicate a specificvalue that needs to be observed for reduced binding of LDLR to PCSK9.

However,in order to classify the results, it was necessary to select a threshold value for defining
the antibody as neutralizing or non-neutralizing.

I thought that it was reasonable torefer to an MAb which reduces binding of PCSK9 to LDL-R by at least 40% at aconcentration of 10 μg/ml (i.e., a high MAb with the remaining PCSK9 binding to
LDLR being toward 60%) as "neutralizing".

Similarly, at 10 μg/ml MAb, if theremaining PCSK9 which binds to LDLR is higher than 60% of the maximum level(i.e., the reduction in PCSK9-LDLR binding is less than 40%), the MAb was referred
to as non-neutralizing.

In column 5 from the left of the table in Material B1, theneutralizing MAbs are highlighted by shading." (Page 7, line 3 to page 8, line 20)(Material B1)Material B1
Antibo Antibo 21B12
31H4
Neutralizi
dydybindingbinding
ng
binding compatibili compatibili property
propert ty [%]ty [%]y190515 Bind to 76-34
solublePCSK947

Nonneutralizi
ng
76

PCSK PCSK PCSK PCSK9999activit activit activit activit
y on y on y on y on
LDL- LDL- LDL- LDLR [%] R [%] R [%] R [%](10 μg (1 μg (0.1(0 μg
IgG/m IgG/m μg
IgG/m
l)
l)
IgG/m l)l)-

081211 Not
Bbind tosolublePCSK9081203 Bind to 3
AsolublePCSK9081205 Bind to 3
BsolublePCSK9190515 Not-35bind tosolublePCSK9081212 Bind to 96
AsolublePCSK9190515 Bind to 100-36solublePCSK9190515 Bind to 4
-37
solublePCSK9190515 Bind to 97-48
solublePCSK9190515 Bind to 6
-49
solublePCSK9190515 Bind to 4
-38
solublePCSK9190515 Bind to 3
-1
solublePCSK9190515 Bind to 53-50
solublePCSK9190515 Bind to 54-51
solublePCSK9190515 Bind to 62-2solublePCSK9190515 Bind to 80-3soluble
PCSK9

-

-

-

-

-

-

26

Neutralizi 29
ng

31

101

100

6

Neutralizi 44
ng

40

89

100

-

-

-

-

-

-

11

Neutralizi 46
ng

45

108

100

107

NonneutralizingNonneutralizingNonneutralizingNonneutralizing
Neutralizi
ng

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

55

43

96

100

NonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizi
ng

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

126

74

6

5

102

35

34

39

48

77

190515 Bind to 94-4solublePCSK9190515 Bind to 67-5
solublePCSK9190515 Bind to 52-6solublePCSK9190515 Not-7bind tosolublePCSK9190515 Bind to 3
-39
solublePCSK9190515 Not-8bind tosolublePCSK9190515 Not-9bind tosolublePCSK9190515 Bind to 89-10
solublePCSK9190515 Bind to 63-11
solublePCSK9190515 Bind to 66-12solublePCSK9190515 Bind to 64-13solublePCSK9190515 Bind to 97-14
solublePCSK9190515 Bind to 84-15
solublePCSK9190515 Bind to 70-16
solublePCSK9190515 Bind to 69-17
soluble73

NonneutralizingNonneutralizingNonneutralizi
ng
-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Nonneutralizi
ng
-

-

-

-

-

-

-

-

-

-

-

76

NonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizi

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

41

31

-

116

42

43

46

33

57

46

45

78

190515-18
190515-19190515-20
190515-21
190515-22
190515-23
190515-24
190515-25
190515-26
190515-27
190515-28
190515-29
190515-30
190515-31
190515-33
190515PCSK9Bind to 98
solublePCSK9Bind to 59
solublePCSK9Bind to 45
solublePCSK9Bind to 94
solublePCSK9Bind to 83
solublePCSK9Bind to 98
solublePCSK9Bind to 66
solublePCSK9Bind to 80
solublePCSK9Bind to 86
solublePCSK9Bind to 50
solublePCSK9Bind to 89
solublePCSK9Bind to 53
solublePCSK9Bind to 61
solublePCSK9Bind to 101
solublePCSK9Bind to 85
solublePCSK9Bind to 101

84

35

28

76

64

90

48

68

84

31

74

35

44

102

75

3

ng
NonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizing
Neutralizi
79

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

40

49

101

100

-46
080924
A
080924
D
190515-40080924
C
080927A
190515-41080930A
190515-42081006A
081006B
081013
C
190515-45
190515-44081008B
190515-43

solublePCSK9Bind to 82
solublePCSK9Bind to 63
solublePCSK9Bind to 3
solublePCSK9Bind to 62
solublePCSK9Bind to 33
solublePCSK9Bind to 53
solublePCSK9Bind to 86
solublePCSK9Bind to 101
solublePCSK9Bind to 4
solublePCSK9Bind to 62
solublePCSK9Bind to 3
solublePCSK9Bind to 89
solublePCSK9Bind to 99
solublePCSK9Bind to 101
solublePCSK9Bind to 101
soluble
PCSK9

ng
45

38

3

41

23

32

63

101

7

19

64

63

102

127

4

NonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizing
Neutralizi
ng

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

42

111

110

100

NonneutralizingNonneutralizingNonneutralizingNonneutralizingNonneutralizing
Neutralizi
ng

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

42

56

98

100

Neutralizi 46
ng

55

94

100

80

081017 Bind to 81
CsolublePCSK9
9C9
Bind to 3
solublePCSK9
3B6
Bind to 3
solublePCSK927B2
Bind to 9
solublePCSK956

Nonneutralizing
Neutralizi 37
ng

-

-

-

58

84

100

105

Neutralizi 43
ng

66

96

100

5

Nonneutralizi
ng

-

-

-

70

81

-