Patent
Right

Date
Case number

June 26, 20192018 (Gyo-Ke) 10043

CourtIntellectual
PropertyHigh
Court,
Third
Division- A case in which, with regard to the invention titled "ANTIGEN-BINDING
MOLECULE CAPABLE OF BINDING TO TWO OR MORE ANTIGEN
MOLECULES REPEATEDLY", the Court has determined that the Detailed
Description of the Invention does not conform to the enablement requirement of
Article 36, paragraph (4), item (i) of the Patent Act, stating that it cannot be said
that the Detailed Description of the Invention provides a description to the extent
that allows a person ordinarily skilled in the art to implement the whole range ofpharmaceutical composition encompassed by the invention.
Case type: Rescission of Trial Decision to Maintain
Result: Granted
References: Article 36, paragraph (4), item (i) of the Patent Act
Number of related rights, etc.: Patent No. 4954326
Summary of the Judgment
1

The case is a suit against a trial decision that dismissed a request for aninvalidation trial of the patent according to the invention titled "antigen-binding
molecule capable of binding to two or more antigen molecules repeatedly".
The issues of the case are: [i] conformity to the clarity requirement; and [ii]conformity to the enablement requirement and the support requirement.
2

The court decision has rescinded the JPO decision, ruling as in the following

with regard to [ii] conformity to the enablement requirement.
(1) The scope of the claims of Invention 1 fails to specify an original antibody and theposition and number of histidine substitution or insertion.

Thus it can also be seenthat neither an original antibody nor the position and number of histidine substi tution
or insertion is limited for an antibody included in a pharmaceutical composition of
Invention 1.

Therefore, the technical scope of Invention 1 encompasses apharmaceutical composition comprising every antibody having one or more ofhistidine substitutions and/or insertions, a certain pH-dependent binding property, anda long half-life in plasma.
Consequently, in order that Invention 1 conforms to the enablement requirement,
the Detailed Description of the Invention must provide a description to the extent thatallows a person ordinarily skilled in the art to implement the whole range ofpharmaceutical compositions encompassed by Invention 1.
(2) The CDR sequence described in [Description of Embodiment] of the descriptionⅰas a site to be substituted with histidine is only an example, and a desired antibody
may likely be obtained by modification of the other site.

Thus it does not apply to

the whole range of the pharmaceutical composition encompassed by Invention 1.
(3) Example 2 of the description describes a method using homology modeling andconformation model ([0285]).
However, homology modeling is a method that predicts a conformation of aprotein with an unknown structure by a computer on the basis of a conformation of aprotein with a known structure having amino acid sequence homology.

It is atechnique that is based on the premise of information of a conformation of a protein
homologous in amino acid sequence to a protein to be subjected to structural
prediction.
Consequently, the method of Example 2 using homology modeling may notalways be utilized when a position of histidine substitution is investigated for generalantibodies with unknown structures.
Example 3 of the description describes a method of preliminarily selecting sites
where the binding ability was not greatly changed even when a residue of CDR was
substituted with histidine by a histidine scanning method to prepare an antibody
where any one of the sites is substituted with histidine ([0288] to [0290]).

Differingfrom the method of the above Example 2, this method is applicable to antibodies withunknown structures.
It is indefinite from the description, however, as to whether "sites where thebinding ability was not greatly changed even when a residue of CDR was substituted
with histidine" of Example 3 ([0289]) always includes a site of an antibody ofInvention 1 substituted with histidine.

Further, there is no evidence sufficient to findthe common technical knowledge that a site of an antibody of Invention 1 substituted
with histidine is always encompassed by "sites where the binding ability was not
greatly changed even when a residue of CDR was substituted with histidine " asmentioned in the description.
Therefore, the methods of Example 2 and Example 3 are not applicable to thewhole range of pharmaceutical composition encompassed by Invention 1.
As described above, it cannot be said that the Detailed Description of theInvention provides a description to the extent that allows a person ordinarily skilled in
the art to implement Invention 1 without undue trials and errors on the basis of thedescription of the Detailed Description of the Invention and the common generalknowledge as of the filing.
(4) Paragraph [0029] of the description describes alanine scanning.
ⅱ

It is recognizedthat alanine scanning was a matter of common technical knowledge as of the filingdate as a means for replacing each residue of amino acid sequences one by one withalanine and analyzing the role of each residue (Exhibits Otsu 19 to 23).

Therefore, it

can be said that a person ordinarily skilled in the art who read the description could
have produced an antibody in which each of 220 amino acid residues of variable siteof an antibody was exhaustively substituted with histidine one by one on the basis of
the common technical knowledge.
Defendant alleges that the identification of a position substituted with histidine
after preparing an antibody should be made with the criterion of "a site showing adesired pH dependency (found to be promising or cause pH-dependent binding
properties)"; however, the description fails to describe as such, and the criterion for
identifying a position substituted with histidine is indefinite from the description orcommon technical knowledge appeared as evidence to implement the whole range ofpharmaceutical composition encompassed into Invention 1.
As seen above, the description has insufficient information on how to identify aposition to be substituted with histidine by histidine scanning as Defendant alleges,
and thus it cannot be said that the Detailed Description of the Invention provides a
description to the extent that allows a person ordinarily skilled in the art to implement
Invention 1 without undue trials and errors on the basis of the description of the
Detailed Description of the Invention and the common general knowledge as of thefiling.

ⅲ

Judgment rendered on June 26, 20192018 (Gyo-Ke) 10043

A case of seeking rescission of the JPO decision

Date of conclusion of oral argument: April 15, 2019
Judgment

Plaintiff Alexion pharmaceuticals, incorporated
Defendant CHUGAI PHARMACEUTICAL CO., LTD.
Main text
1

The decision on Invalidation Trial No. 2016-800136 that JPO has made on

November 22, 2017 shall be rescinded.
2

The court costs shall be borne by Defendant.
Facts and reasonsNo. 1 Claim
The same as the main text
No. 2 Summary of the case (Facts to be found from the evidences listed below andthe overall import of oral argument)
1

History of the procedures, etc. in Japan Patent Office(1) Defendant is a patentee of a patent right according to the invention titled"antigen-binding molecule capable of binding to two or more antigen molecules
repeatedly" (Patent No. 4954326, Filing date: April 10, 2009 (claiming priority datesof April 11, 2008, September 26, 2008, March 19, 2009) (hereinafter the filing date isreferred to as "the filing date"), Registered Date: March 23, 2012, Number of claims:
6, hereinafter referred to as "the patent right", and the patent according to the patentright is referred to as "the Patent") (Exhibit Ko 16).
(2) Plaintiff requested a trial for patent invalidation with respect to the Patent on
December 19, 2016, which was assigned to a collegial body as a case of Invalidation
Trial No. 2016-800136 in JPO.
(3) JPO made a trial decision that dismissed the request for a patent invalidation
trial on November 22, 2017 (hereinafter referred to as "the trial decision") and its
certified copies were served for Plaintiff on November 30.
as a period for filing a suit.
1

Ninety days are offered(4) Plaintiff filed a suit for the case, seeking for the rescission of the trial decision,
on March 29, 2018.
2

The recitation of the Claims
The recitation of Claims 1 to 6 of the scope of the claims of the Patent is set

forth as below: Hereinafter, the inventions according to the claims are referred to as"Invention 1", "Invention 2", etc. according to the number of the claim, and arecollectively referred to as "the Invention".

The specification of the Patent (Exhibit

Ko 17) is referred to as "the specification", including the drawings.
[Claim 1] A pharmaceutical composition comprising an antibody having aKD(pH5.8)/KD(pH7.4) value, defined as the ratio of KD for the antigen at pH 5.8 andKD for the antigen at pH 7.4, of 2 or higher to 10000 or less, as well as a longer halflife in plasma, wherein at least one amino acid of a variable region is substituted withhistidine, or one histidine is inserted into at least variable region.
[Claim2]

The

pharmaceuticalcomposition

of

Claim

1,

whereina

KD(pH5.8)/KD(pH7.4) value, defined as the ratio of KD for the antigen at pH 5.8 andKD for the antigen at pH 7.4, is 10 or higher.
[Claim3]

The

pharmaceuticalcomposition

of

Claim

1,

whereina

KD(pH5.8)/KD(pH7.4) value, defined as the ratio of KD for the antigen at pH 5.8 andKD for the antigen at pH 7.4, is 40 or higher.
[Claim 4] The pharmaceutical composition of any one of Claims 1 to 3, wherein said
antibody has an antagonist activity.
[Claim 5] The pharmaceutical composition of any one of Claims 1 to 4, wherein said
antibody binds to a membrane antigen or a soluble antigen.
[Claim 6] The pharmaceutical composition of any one of Claims 1 to 5, wherein said
antibody is an antibody binding an antigen selected from the group con sisting of IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-31, IL-23,
IL-2 receptor, IL-6 receptor, OSM receptor, gp130, IL-5 receptor, CD40, CD4, Fas,
osteopontin, CRTH2, CD26, PDGF-D, CD20, monocyte chemotactic factor, CD23,
TNF-α, HMGB-1, α4 integrin, ICAM-1, CCR2, CD11a, CD3, IFNγ, BLyS, HLA-DR,
TGF-β, CD52, and IL-31 receptor.
3

Summary of reasons of trial decision
Plaintiff alleges that [i] nonconformance to the enablement requirement andsupport requirement with respect to Inventions 1 to 6 (Reason 1 for invalidation), [ii]violation of the provision concerning prior art effect on the grounds of an application
corresponding to the following Exhibit Ko 6 (Japanese Patent Application No. 2009531851, hereinafter referred to as "Prior application 1", which translation relies on
2

Exhibit Ko 7 document) with respect to the Inventions 1 to 5 (Reason 2 forinvalidation), [iii] lack of inventive step on the basis of the following Exhibits Ko 8 to
11 documents and the common general knowledge with respect to Inventions 1, 2, and
4 to 6 (Reason 3 for invalidation), and [iv] nonconformance to the clarity requirementwith respect to Inventions 1 to 6 (Reason 4 for invalidation).
The reasons of the trial decision are as per described in the written trial
decision (copy) of the attachment.

In summary, [i] regarding Reason 1 forinvalidation, Inventions 1 to 6 conform to the enablement requirement and support
requirement, [ii] regarding Reason 2 for invalidation, it cannot be said that Inventions
1 to 5 are identical to the inventions described in the description of Prior application 1,
nor is the prior art effect recognized, [iii] regarding Reason 3 for invalidation, it
cannot be said that Inventions 1, 2, and 4 to 6 were easily conceivable on the basis of
the following Exhibits Ko 8 to 11 documents and the common general knowledge, nor
can it be said that these inventions do not involve inventive step, [iv] regarding
Reason 4 for invalidation, Inventions 1 to 6 conform to the clarity requirement.
Exhibit Ko 6: International Publication No. WO2008-043822
Exhibit Ko 7: National Publication of International Patent Application No. 2010505436
Exhibit Ko 8: Ito et al., FEBS Lett., 1992, 309(1), pp. 85-88
Exhibit Ko 9: U.S. patent publication No. 2006/0141456
Exhibit Ko 10: Junghans et al., Proc.

Natl.

Acad.

Sci.

USA (1996) Vol. 93,

pp. 5512-5516
Exhibit Ko 11: Sarkar et. al., Nature Biotechnology (2002) Vol 20, pp. 908-913
4

Reasons for rescission

Reason 1 for rescission: Reason 4 for invalidation (Nonconformance to the clarity
requirement)
Reason 2 for rescission: Erroneous Determination of Reason 1 for invalidation(Nonconformance to the Enablement requirement and Support requirement)
Reason 3 for rescission: Erroneous Determination of Reason 3 for invalidation (Lackof inventive step)
Reason 4 for rescission: Erroneous Determination of Reason 2 for invalidation ( Priorart effect)(omitted)
No. 5 Judgment of this court
3

1

The Invention(1) The recitation of the Claims
The scope description of the claims of the Invention is as per described in the

aforesaid No. 2-2.
(2) The description of the specification
The specification has the following descriptions (Exhibit Ko 17):
A. [Technical Field][0001]

The

present

invention

relatestomethodsforimprovingthepharmacokinetics of antigen-binding molecules and methods for increasing thenumber of times of antigen-binding of antigen-binding molecules, as well as
antigen-binding molecules having improved pharmacokinetics, antigen-binding
molecules having increased number of times of antigen-binding, and methods for
producing such molecules.
B. [Background Art]
[0002] Antibodies are drawing attention as pharmaceuticals as they are highly
stable in plasma and have few adverse effects.

At present, a number of IgG-

type antibody pharmaceuticals are available on the market and many more
antibody pharmaceuticals are currently under development In general, therequisite dose of an antibody pharmaceutical is very high.

In theory, the doseof an antibody pharmaceutical may be reduced by improving antibody
pharmacokinetics or improving the affinity between antibodies and ant igens.
[0003]

The

literaturehas

reportedmethodsforimproving

antibody

pharmacokinetics using artificial substitution of amino acids in constant regions.
Similarly, affinity maturation has been reported as a technology for enhancing
antigen-binding ability or antigen-neutralizing activity.

This technology

enables enhancement of antigen-binding activity by introduction of amino acidmutations into the CDR region of a variable region or such.

The enhancementof antigen-binding ability enables the reduction of dosage, and further enables
improvement of in vivo efficacy.
[0004] Meanwhile, the antigen-neutralizing capacity of a single antibody
molecule depends on its affinity.

By increasing the affinity, an antigen can be

neutralized by smaller amount of an antibody.
enhance the antibody affinity.

Various methods can be used toFurthermore, if the affinity could be madeinfinite by covalently binding the antibody to the antigen, a single antibody
molecule could neutralize one antigen molecule (a divalent antibody canneutralize two antigen molecules).

However, the stoichiometric neutralization4

of one antibody against one antigen (one divalent antibody against two antigens)is the limit of pre-existing methods, and thus it is impossible to completely
neutralize antigen with an amount of antibody smaller than that of antigen.

Inother words, the affinity enhancing effect has a limit (Non-Patent Document 9).
To prolong the neutralization effect of a neutralizing antibody for a certain
period, the antibody must be administered at a dose higher than the amount of
antigen produced in the body during the same period.

With the improvement ofantibody pharmacokinetics or affinity maturation technology alone describedabove, there is thus a limitation in the reduction of the required antibody dose.
[0005] Accordingly, in order to sustain antibody's antigen-neutralizing effect fora target period with an amount of the antibody smaller than that of antigen, asingle antibody must neutralize multiple antigens.

Methods for neutralizing

multiple antigens with a single antibody include antigen inactivation using
catalytic antibodies, which are antibodies conferred with a catalytic function .
When the antigen is a protein, it can be inactivated by hydrolyzing its peptide
bonds.

It is supposed that an antibody can repeatedly neutralize antigens by

catalyzing such hydrolysis (Non-Patent Document 8).

There are many previous

reports published on catalytic antibodies and technologies for producing them .
However, there have been no reports of catalytic antibodies having sufficient
catalytic activity as a pharmaceutical agent.

Specifically, in an antibody in vivo

study for a certain antigen, there has been no publication of catalytic antibodies
which can produce a comparable or stronger effect even at low doses or produce
a more prolonged effect even at a same dose as compared to an ordinary non catalytic neutralizing antibody.
[0006] As described above, there have been no reports of antibodies that can
produce a superior in vivo effect than ordinary neutralizing antibodies through asingle antibody neutralizing multiple antigen molecules.

Thus, from theviewpoint of dose reduction and sustained effect, there is a need for new
technology to produce an antibody having a stronger in vivo effect than ordinary
neutralizing antibodies by individually neutralizing multiple antigens with one
antibody.
C. [Means for solving problem][0010] The present inventors have intensively investigated a method of binding
multiple times to an antigen of a polypeptide having an antigen binding property
such as an antigen binding molecule, and a method of improving (improving
pharmacokinetics) a half-life in plasma (half-life in blood).
5

As a result, thepresent inventors have found that an antigen binding molecule with a weak
antigen-binding activity at a pH in an earlier stage endosome compared to an
antigen-binding activity at a pH in plasma (blood) binds to an antigen multiple
times and has a longer half-life in plasma.
D. [Advantageous Effects of Invention][0012] The present invention provides a method of repeatedly binding one
antigen binding molecule to a plurality of antigens.

Binding one antigen

binding molecule to a plurality of antigens may improve pharmacokinetics ofantigen binding molecule and cause more excellent effects compared to ordinary
antigen binding molecule in vivo.
E. [Description of Embodiment][0025] In the present invention, the difference in the antigen-binding activity
between acidic and neutral pHs is not particularly limited so long as the antigenbinding activity at acidic pH is lower than that at neutral pH.

However, thevalue of KD(pH5.8)/KD(pH7.4), which is a ratio of dissociation constant (KD)against an antigen at pH 5.8 and that at pH 7.4, is preferably 2 or greater, more
preferably the value of KD(pH5.8)/KD(pH7.4) is 10 or greater, and still more
preferably the value of KD(pH5.8)/KD(pH7.4) is 40 or greater.

The upper limitof KD(pH5.8)/KD(pH7.4) value is not particularly limited, and may be any value,
for example, 400, 1,000, or 10,000, as long as the molecule can be produced bytechnologies of a person ordinarily skilled in the art. ...
[0029] The methods for impairing the antigen-binding activity of an antigenbinding molecule at pH 5.8 as compared to that at pH 7.4 (methods forconferring the pH-dependent binding ability) are not particularly limited and may
be any methods.

Such methods include, for example, methods for impairing theantigen-binding activity at pH 5.8 as compared to that at pH 7.4 by substituting
histidine for amino acids in the antigen-binding molecule or inserting histidine
into the antigen-binding molecule.

It is already known that an antibody can beconferred with a pH-dependent antigen-binding activity by substituting histidine
for amino acids in the antibody (FEBS Letter, 309(1), 85-88 (1992)).

Such

histidine mutation (substitution) or insertion sites are not particularly limited,
and any site is acceptable so long as the antigen-binding activity at pH 5.8 islower than that at pH 7.4 (the value of KD(pH5.8)/KD(pH7.4) gets greater) as
compared to before mutation or insertion.

When the antigen-binding molecule

is an antibody, such sites include, for example, sites within an antibody variable
region.

The appropriate number of histidine mutation or insertion sites can be6

appropriately determined by a person ordinarily skilled in the art.

Histidine

may be substituted or inserted at a single site, or two or more sites .

It is also

possible to introduce non-histidine mutation (mutation with amino acids otherthan histidine) at the same time.

Furthermore, histidine mutation may beintroduced simultaneously with histidine insertion.

It is possible to substitute orinsert histidine at random using a method such as histidine scanning, which uses
histidine instead of alanine in alanine scanning known to a person ordinarily
skilled

in

the

art.

Alternatively,

antigen-binding

molecules

whose

KD(pH5.8)/KD(pH7.4) is increased as compared to before mutation can beselected from an antigen-binding molecule library with random histidine
mutation or insertion.
[0030] When histidine is substituted for amino acids of an antigen -binding
molecule or inserted between amino acids of the molecule, it is preferred, but notrequired, that the antigen-binding activity of the antigen-binding molecule at pH
7.4 after histidine substitution or insertion is comparable to that at pH 7.4 before
histidine substitution or insertion. ...

When the antigen-binding activity of theantigen-binding molecule has been impaired due to histidine substitution or
insertion, the antigen-binding activity may be adjusted by introducing
substitution, deletion, addition, and/or insertion of one or more amino acids into
the antigen-binding molecule so that the antigen-binding activity becomescomparable to that before histidine substitution or insertion.

The present

invention also includes such antigen-binding molecules having a comparable
binding activity as a result of substitution, deletion, addition, and/or insertion of
one or more amino acids after histidine substitution or insertion.
[0070]In the present invention, when the antigen-binding molecule is anantibody, possible sites of histidine or non-natural amino acid substitution
include, for example, sites within the CDR sequence or sequence responsible for
the CDR structure of an antibody.
listed below.

Such sites include, for example, the sites

The amino acid positions are numbered based on Kabat

numbering (Kabat EA et al., (1991) Sequences of Proteins of ImmunologicalInterest, NIH).
[0071] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63,
H64, H65, H99, H100b, and H102

Light chain: L24, L27, L28, L32, L53, L54,

L56, L90, L92, and L94[0072] Among the above sites, H32, H61, L53, L90, and L94 could be universal
modification sites.
7

[0073] When the antigen is the IL-6 receptor (e.g., human IL-6 receptor),
preferable modification sites include, but are not particularly limited to, thefollowing.
[0074] Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65,
H100b, and H102

Light chain: L24, L27, L28, L32, L53, L56, L90, L92, andL94[0075] When histidine or non-natural amino acid is substituted at multiple sites,
preferred combinations of substitution sites include,for example, thecombination of H27, H31, and H35; combination of H27, H31, H32, H35, H58,
H62, and H102; combination of L32 and L53; and combination of L28, L32, and
L53.

In addition, preferred combinations of substitution sites of heavy and lightchains include the combination of H27, H31, L32, and L53.
[0076] When the antigen is IL-6 (e.g., human IL-6), preferable modification sites
include, but are not particularly limited to, the following.
[0077] Heavy chain: H32, H59, H61, and H99

Light chain: L53, L54, L90, andL94[0078] When the antigen is the IL-31 receptor (e.g., human IL-31 receptor),
preferable modification sites include, but are not particularly limited to, H33.
[0106] In particular, the present inventors noted that the pH in the plasma wasdifferent from the pH in the endosomes, and thus discovered that antibodies thatstrongly bind to antigens under plasma pH condition but that weakly bind to
antigens under endosomal pH condition were superior in retention in the plasma,
because one antibody molecule could bind to multiple antigens.
[0108] Accordingly, an antigen-binding molecule whose antigen-binding activity
at acidic pH is weaker than the antigen-binding activity at neutral pH binds to theantigen in the plasma which have a neutral pH, is taken up into cells, and thendissociates from the antigen in the endosomes which have an acidic pH.

Theantigen-binding molecule that dissociated from the antigen binds to FcRn,translocates to the cell surface, and returns again in the plasma in a state notbound to antigens.

As a result, the antigen-binding molecule can bind to

antigens multiple times, thereby improving pharmacokinetics.
[0183] Antigen-binding substances that are screened by the screening methods ofthe present invention may be prepared by any method.

For example, it ispossible to use pre-existing antibodies, pre-existing libraries (phage libraries andthe like), antibodies and libraries that are prepared from hybridomas obtained byimmunizing animals or from B cells of immunized animals, antibodies and8libraries prepared by introducing histidine mutations or non-natural amino acidmutations into the above-described antibodies and libraries (libraries with highcontent of histidine or non-natural amino acid, libraries introduced with histidine
mutations or non-natural amino acid mutations at specific sites, and the like), andso on.
[0191] In addition, the present invention provides libraries in which the histidine
content is increased as compared to the original libraries.

Libraries containing

antigen-binding molecules with increased histidine content can be used in thescreening methods described above and the production methods describedhereinafter.
[0192] Libraries with increased histidine content can be prepared by methods
known to those skilled in the art, which include the following method. 20 typesof triplet codons (trinucleotides) encoding 20 types of amino acids can beincorporated at equal frequency when synthesizing nucleic acids to prepare alibrary by the trinucleotide-method (J Mol Biol. 2008 Feb. 29; 376(4): 1182-200).
As a result, the position mutated for the library can be made to contain 20 typesof amino acids at equal probability.

The frequency of histidine in the position

mutated for the library can be increased by increasing the proportion of ahistidine-encoding trinucleotide as compared to the remaining amino acids
among the 20 types in the synthesis.
[0254]<Pharmaceutical Compositions>
The present invention also relates to pharmaceutical compositions that include
antigen-binding molecules of the present invention, antigen-binding molecules
isolated by the screening methods of the present invention, or antigen -binding
molecules produced by the production methods of the present invention.

The

antigen-binding molecules of the present invention and antigen-binding
molecules produced by the production methods of the present invention aresuperior in retention in plasma, and thus, are expected to reduce theadministration frequency of the antigen-binding molecules, and are therefore
useful as pharmaceutical compositions.

The pharmaceutical composition of thepresent invention may include pharmaceutically acceptable carriers.
F. [Example(s)][0264] Herein below, the present invention will be specifically described withreference to Examples, but is not to be construed as being limited thereto.
[0265]
9

[Example 1] Preparation of modified humanized PM1 antibody ...
[0272]
Preparation of modified humanized IL-6 receptor antibody
In this context, mutations were introduced into the framework sequence andCDR sequence of humanized mouse PM1 antibody (the term "wild type" isabbreviated as WT, the term "wild type H chain" is abbreviated as H(WT)(amino acid sequence of SEQ ID NO: 9), and the term "wild type L chain isabbreviated as L(WT) (amino acid sequence: SEQ ID NO. 10)) described inCancer Res. 1993, Feb. 15; 53(4): 851-6, to produce modified H chains H53(amino acid sequence: SEQ ID NO: 1) and PF1H (amino acid sequence: SEQ ID
NO: 11), and modified L chains L28 (amino acid sequence: SEQ ID NO: 12) andPF1L (amino acid sequence: SEQ ID NO: 2). ...
[0274][Example 2] Production of pH-Dependently-Binding Antibody H3pI/L73
Method for Creating Antibody Capable of Neutralizing Antigen Multiple Times
Since IgG molecules are divalent, a single IgG molecule can neutralize up to
two antigen molecules when the two sites bind to the antigens; however, itcannot neutralize three or more antigen molecules.

Therefore, to maintain theneutralizing effect of a neutralizing antibody over a certain period, it is necessary
to administer an amount of the antibody equal to or greater than the amount of
antigen produced during the period.

Thus, there is a limitation on the extent towhich the required dose of antibody can be reduced by improving thepharmacokinetics or affinity of antibody.

Therefore, if it were possible toneutralize two or more antigen molecules with a single IgG molecule, the same
dose could improve the duration of neutralizing effect, or alternatively the doseof antibody required to achieve the same duration could be reduced.
[0275] For neutralizing antibodies, there are two types of target antigens:soluble-type antigens, which are present in plasma, and membrane-boundantigens, which are expressed on the surfaces of cells.
[0276] When the antigen is a membrane-bound antigen, an administered antibody
binds to the membrane antigen on the cellular surface, and the antibody issubsequently taken up into endosomes within the cell by internalization together
with the membrane antigen bound to the antibody.

Then, the antibody which is

kept bound to the antigen moves to a lysosome where it is degraded by lysosome
together with the antigen.

The elimination of antibody from the plasma

mediated by internalization by membrane antigen is referred to as antigen10

dependent elimination, and this has been reported for numerous antibody
molecules (Drug Discov. Today, 2006 January; 11(1-2): 81-8).

Since a single

IgG antibody molecule binds to two antigen molecules when it divalently binds
to antigens, and is then internalized and directly degraded by lysosome, a single
ordinary IgG antibody cannot neutralize two or more antigen molecules (FIG. 1).
[0277] The reason for the long retention (slow elimination) of IgG molecules inplasma is that FcRn, known as an IgG molecule salvage receptor, functions (Nat.
Rev. Immunol. 2007 September; 7(9): 715-25).

IgG molecules that have beentaken up into endosomes by pinocytosis bind to FcRn expressed in endosomes
under intraendosomal acidic conditions.

IgG molecules bound to FcRn move tothe cell surface where they dissociate from FcRn under neutral conditions inplasma and return to plasma, while IgG molecules unable to bind to FcRnproceed into lysosomes where they are degraded (FIG. 2).
[0278] IgG molecules bound to a membrane antigen are taken up into
intracellular endosomes by internalization, move into lysosomes while bound to
the antigen, and undergo degradation.

When an IgG antibody divalently binds

to antigens, it neutralizes two antigen molecules and undergoes degradation
together with the antigens.
endosomes

If the IgG antibody, when taken up into intracellular

by internalization,candissociate

from

the

antigenunder

intraendosomal acidic conditions, the dissociated antibody may be able to bind t o
FcRn expressed in the endosomes.

The IgG molecule dissociated from theantigen and bound to FcRn is transferred to the cell surface and then dissociatedfrom FcRn under neutral conditions in the plasma, to thereby return to theplasma again.

The IgG molecule that has returned to the plasma is able to bindto a new membrane antigen again.

The repetition of this process allows a single

IgG molecule to repeatedly bind to membrane antigens, thereby enablingneutralization of multiple antigens with a single IgG molecule (FIG. 3).
[0279] In the case of a soluble antigen, an antibody administered binds to theantigen in the plasma, and remains in the plasma in the form of an antigen antibody complex.

Normally, while the retention of antibody in plasma is very

long (elimination rate is very slow) due to the function of FcRn as describedabove, the retention of antigen in plasma is short (elimination rate is fast).

Thus,

antibody-bound antigens show retention in plasma comparable to that ofantibody (elimination rate is very slow).

Antigens are produced in the body at aconstant rate and, in the absence of antibody, remain present in plasma at aconcentration at which the antigen production rate and the antigen elimination11

rate are under equilibrium.

In the presence of antibody, most of the antigens arebound to antibodies, resulting in the very slow elimination of antigens.

Thus,

the antigen concentration in plasma increases as compared with that in theabsence of antibody (Kidney Int. 2003, 64, 697-703; J. National Cancer Institute2002, 94(19), 1484-1493; J. Allergy and Clinical Immunology 1997, 100(1),110-121; Eur. J. Immunol. 1993, 23; 2026-2029).

Even if the affinity ofantibody for antigen is infinite, antigen concentration elevates as antibody isslowly eliminated from the plasma, and the neutralizing effect of antibody
terminates after the concentrations of antibody and antigen become equal.
Although antibodies with a stronger dissociation constant (KD) can neutralize
soluble antigens at a lower antibody concentration, antibodies at a concentration
half or less than the concentration of antigen present are unable to neutralize
antigens regardless of how strong the affinity of antibody is (Biochem. Biophys.
Res. Commun. 2005 Sep. 9; 334(4): 1004-13).

As is the case with IgG

molecules not bound to antigens, IgG molecules bound to antigens in the plasma
are also taken up into endosomes by pinocytosis, and bind to FcRn expressed inendosomes under intraendosomal acidic conditions.

The IgG molecules boundto FcRn move to the cell surface while the antibody is kept bound to the antigen ,and then dissociate from the FcRn under neutral conditions in the plasma.
Since the IgG molecules return to the plasma while bound to the antigen, theycannot bind to new antigens in the plasma.

In this case, if IgG molecules candissociate from the antigen under intraendosomal acidic conditions, thedissociated antigen will not be able to bind to FcRn and thereby may be degradedby lysosomes.

On the other hand, the IgG molecules can return to the plasma

again by binding to FcRn.

Since the IgG molecules that have returned to theplasma have already dissociated from the antigen in endosomes, they are able tobind to a new antigen again in the plasma.

The repetition of this process allows

a single IgG molecule to repeatedly bind to soluble antigens.

This enables asingle IgG molecule to neutralize multiple antigens (FIG. 4).
[0280] Thus, regardless of whether the antigen is a membrane antigen or asoluble antigen, if the dissociation of IgG antibody from the antigen is possible
under intraendosomal acidic conditions, a single IgG molecule would be able torepeatedly neutralize antigens.

In order for IgG antibodies to dissociate fromantigens under intraendosomal acidic conditions, it is necessary that antigenantibody binding be considerably weaker under acidic conditions than under
neutral conditions.

Since membrane antigens on the cell surface need to be
12neutralized, antibodies have to strongly bind to antigens at the cell surface pH;
namely, pH 7.4.

Since the intraendosomal pH has been reported to be typically

pH 5.5 to 6.0 (Nat. Rev. Mol. Cell. Biol. 2004 February; 5(2): 121-32), an
antibody that weakly binds to an antigen at pH 5.5 to 6.0 is considered todissociate from the antigen under intraendosomal acidic conditions.

Morespecifically, a single IgG molecule that strongly binds to an antibody at the cellsurface pH of 7.4 and weakly binds to the antigen at the intraendosomal pH of
5.5 to 6.0 may be able to neutralize multiple antigens and thereby improve thepharmacokinetics.
[0281] In general, protein-protein interactions consist of hydrophobic interaction,
electrostatic interaction, and hydrogen bonding, and the binding strength istypically expressed as a binding constant (affinity) or apparent binding constant(avidity). pH-dependent binding, whose binding strength varies between neutral
conditions (pH 7.4) and acidic conditions (pH 5.5 to 6.0), is present in naturallyoccurring protein-protein interactions.

For example, the above-mentioned

binding between IgG molecules and FcRn known as a salvage receptor for IgG
molecules is strong under acidic conditions (pH 5.5 to 6.0) but remarkably weakunder neutral conditions (pH 7.4).

Most of such pH-dependently changing

protein-protein interactions are associated with histidine residues.

Since thepKa of histidine residue is in the vicinity of pH 6.0 to 6.5, the proton dissociation
state of histidine residues varies between neutral conditions (pH 7.4) and acidic
conditions (pH 5.5 to 6.0).

Specifically, histidine residues are not charged andfunction as hydrogen atom acceptors under neutral conditions (pH 7.4), while
they become positively charged and function as hydrogen atom donors underacidic conditions (pH 5.5 to 6.0).

It has been reported that the pH-dependent

binding of the above-described IgG-FcRn interaction is also associated withhistidine residues present in IgG (Mol. Cell. 2001 April; 7(4): 867-77).
[0282] Therefore, pH-dependence can be imparted to protein-protein interactions
by substituting an amino acid residue involved in protein-protein interactions
with a histidine residue, or by introducing a histidine into an interaction site .
Such attempts have also been made in protein-protein interactions between
antibodies and antigens, and a mutant antibody with antigen-binding ability
decreased under acidic conditions has been successfully acquired by introducing
histidine into the CDR sequence of an anti-egg white lysozyme antibody (FEBS
Letter (vol. 309, No. 1, 85-88, 1992)).

In addition, an antibody that is preparedby introducing histidine into its CDR sequence and specifically binds to an13

antigen at the low pH of cancer tissues but weakly binds under neutral conditions
has been reported (WO 2003-105757).
[0283] Although methods for introducing pH dependency into antigen-antibody
reactions have been reported as described above, an IgG molecule thatneutralizes multiple antigens by strongly binding to antigens at the body fluid pHof 7.4 but weakly binding to antigens at the intraendosomal pH of pH 5.5 to 6.0
has not been reported.

In other words, there have been no reports relating tomodifications that significantly reduce the binding under acidic conditions while
maintaining the binding under neutral conditions such that, as compared to an
unmodified antibody, a modified antibody binds to antigens multiple times invivo and thereby shows improved pharmacokinetics as well as improved durationof the neutralizing effect at the same dose.
[0284] The IL-6 receptor is present in the body in the form of either soluble IL-6
receptor or membrane IL-6 receptor (Nat. Clin. Pract. Rheumatol. 2006
November; 2(11): 619-26).

Anti-IL-6 receptor antibodies bind to both thesoluble IL-6 receptor and membrane IL-6 receptor, and neutralize their biologicalaction.

It is considered that, after binding to the membrane IL-6 receptor, anti-

IL-6 receptor antibodies are taken up into intracellular endosomes byinternalization while bound to the membrane IL-6 receptor, then move intolysosomes while the antibodies are kept bound to the membrane IL-6 receptor,
and undergo degradation by lysosomes together with the membrane IL-6 receptor.
In fact, it has been reported that a humanized anti-IL-6 receptor antibody exhibits
non-linear clearance, and its antigen-dependent elimination greatly contributes tothe elimination of the humanized anti-IL-6 receptor antibody (The Journal of
Rheumatology, 2003, 30; 71426-1435).

Thus, one humanized anti-IL-6

receptor antibody binds to one or two membrane IL-6 receptors (monovalently or
divalently), and is then internalized and degraded in lysosomes.

Therefore, if it

is possible to produce modified antibodies that exhibit greatly reduced binding
ability under acidic conditions but retain the same binding ability as the wild
type humanized anti-IL-6 receptor antibody under neutral conditions (pHdependent binding anti-IL-6 receptor antibody), multiple IL-6 receptors can be
neutralized with a single humanized anti-IL-6 receptor antibody.

Thus, incomparison with wild type humanized anti-IL-6 receptor antibodies, pHdependent binding anti-IL-6 receptor antibodies may improve the duration of theneutralizing effect in vivo at the same dosage.
[0285]
14

Production of pH-Dependently Binding Humanized Anti-IL-6 Receptor Antibody
H3pI/L73:
Introduction of histidine into a CDR has been reported as a method for
introducing pH-dependent binding to antigen-antibody reaction (FEBS Letter(vol. 309, No. 1, 85-88, 1992)).

In order to find amino acid residues exposed on

the surface of the variable region of the H53/PF1L produced in Example 1 andpossible residues interacting with the antigen, an Fv region model of H53/PF1L
was created by homology modeling using MOE software (Chemical Computing
Group Inc.).

A three-dimensional model constructed on the basis of thesequence information of H53/PF1L was used to select H27, H31, H35, L28, L32 ,and L53 (Kabat numbering, Kabat, E. A. et al., 1991, Sequences of Proteins of
Immunological Interest, NIH) as mutation sites that may introduce pH-dependent
antigen-binding by histidine introduction.

The product in which the residues at

H27, H31, and H35 in H53 produced in Example 1 were substituted withhistidines was designated as H3pI (amino acid sequence: SEQ ID NO: 3), and the
product in which the residues at L28, L32, and L53 in PF1L produced in
Example 1 were substituted with histidines was designated as L73 (amino acidsequence: SEQ ID NO: 6).
[0286] Production, Expression and Purification of H3pI/L73 Expression Vector
Amino acid modification was carried out to produce antibodies modified at the
selected sites.

Mutations were introduced into H53 (nucleotide sequence: SEQ

ID NO: 13) and PF1L (nucleotide sequence: SEQ ID NO: 14) produced in
Example 1 to produce H3pI (amino acid sequence: SEQ ID NO: 3) and L73(amino acid sequence: SEQ ID NO: 6).

More specifically, the QuikChange

Site-Directed Mutagenesis Kit (Stratagene) was used according to the method
described in the instructions provided, and the resulting plasmid fragments wereinserted into a mammalian cell expression vector to produce the desired H chain
expression vector and L chain expression vector.

The nucleotide sequences of

the resulting expression vectors were determined using a method known topersons skilled in the art.

H3pI/L73 which uses H3pI for the H chain and L73

for the L chain was expressed and purified by the method described in Example 1.
[0287][Example 3] Conferring pH-Dependent Antigen Binding Ability by His
Modification of CDR Using Phage Display Technology
Production of scFv Molecule of Humanized PM1 Antibody
The humanized PM1 antibody, which is a humanized anti-IL-6R antibody
15

(Cancer Res. 1993 Feb. 15; 53(4): 851-6), was converted into scFv.

The VHand VL regions were amplified by PCR, and humanized PM1 HL scFv having
the linker sequence GGGGSGGGGSGGGGS (SEQ ID NO. 15) between VH and
VL was produced.
[0288]
Selection of Histidine-Introducible Positions by Histidine Scanning
PCR was performed using the produced humanized PM1 HL scFv DNA as atemplate to produce a histidine library in which any one of the CDR amino acids
is replaced with histidine.

The library portions were constructed by PCR using

primers in which the codon of an amino acid desired to be mutated for the library
was replaced with CAT, a codon corresponding to histidine, and other portions
were constructed by normal PCR.
PCR.

These portions were then linked by assemble

The constructed library was digested with SfiI, inserted into a phagemide

vector pELBG lacI vector that was also digested with SfiI, and then used totransform XL1-Blue (Stratagene).

The resulting colonies were used to evaluate

antigen binding by phage ELISA and analyze the sequence of HL scFv.

Phage

ELISA was carried out using a plate coated with SR344 at 1μg/mL in accordancewith J. Mol. Biol. 1992; 227: 381-388.

Clones that were found to bind toSR344 were subjected to sequence analysis using specific primers.
[0289] Phage titer was determined by ELISA with an anti-Etag antibody (GEHealthcare) and anti-M13 antibody (GE Healthcare).

This value was then used

to select sites where the binding ability was not greatly changed even when aresidue of CDR was substituted with histidine as compared to humanized PM1
HL scFv, based on the results of phage ELISA for SR344.
positions are shown in Table 2.

The selected

Numbering of each residue was in accordancewith Kabat numbering (Kabat, et al., 1991, Sequences of Proteins of
Immunological Interest, NIH).
[0290][TABLE 2] Positions of Histidine Substitution Not Significantly Affecting
Binding Ability
H31, H50, H54, H56, H57, H58, H59, H60, H61, H62, H63, H64, H65, H100a,
H100b, H102
L24, L26, L27, L28, L30, L31, L32, L52, L53, L54, L56, L90, L92, L93, L94[0291]
Construction of Histidine-Modified CDR Library
A library was designed in which the amino acids of CDR residues that did not16

significantly alter the binding ability when substituted with histidine as shown in
Table 2 (histidine-introducible positions) are their original sequence (wild typesequence) or histidine.

The library was constructed based on the sequences ofthe H chain PF1H and the L chain PF1L produced in Example 1 such that the
mutated positions for the library have the original sequences or histidines (either
the original sequence or histidines).
[0292] The library portions were constructed by PCR using primers that were
designed such that a position desired to be mutated for the library has theoriginal amino acid codon or histidine codon, and other portions were produced
by normal PCR, or by PCR using synthetic primers as in the library portions.
These portions were then linked by assemble PCR (J. Mol. Biol. 1996; 256: 7788).
[0293] This library was used to construct a ribosome display library inaccordance with J. Immunological Methods 1999; 231: 119-135.

In order tocarry out Escherichia coli cell-free in vitro translation, an SDA sequence(ribosome binding site) and T7 promoter were added to the 5′ side, and a gene3
partial sequence serving as a linker for ribosome display was ligated to the 3′
side using SfiI.
[0294] Acquisition of pH-Dependent Binding scFv from Library by Bead
Panning
In order to concentrate only scFv having the ability to bind to SR344, panningwas carried out twice by the ribosome display method in accordance with Nature
Biotechnology 2000 December; 18: 1287-1292.

The prepared SR344 wasbiotinylated using NHS-PEO4-Biotin (Pierce) to obtain an antigen.

Panning

was carried out using 40 nM of the biotinylated antigen.
[0299] However, no clones exhibiting potent pH-dependent binding ability wereobtained by this panning using the antigen immobilized on the magnetic beads .
Clones that were found to show weak pH-dependent binding ability were
subjected to sequence analysis using specific primers.

The positions in theseclones where histidine was present at a high rate are shown in Table 3.
[0300][TABLE 3] Positions of Histidine Substitution Detected Using Phage Library(Magnetic Bead Panning)
H50, H58, H61, H62, H63, H64, H65, H102
L24, L27, L28, L32, L53, L56, L90, L92, L94[0301]
17

Acquisition of pH-Dependently Binding scFv from Library by Column Panning
No clones having strong pH-dependent binding ability were obtained by typicalpanning using the magnetic bead-immobilized antigen.
following reasons.

This may be due to the

In the panning using an antigen immobilized on magnetic

beads or a plate, all phages dissociated from the magnetic beads or plate underacidic conditions are collected.

Thus, phage clones having weak pH

dependency recovered together reduce the likelihood that clones having strong
pH dependency are included in the finally concentrated clones.
[0302] Therefore, panning using a column immobilized with an antigen was
examined as a more stringent panning method (FIG. 5). ...
[0305]
Evaluation by Phage ELISA
The resulting phages were evaluated by phage ELISA.

Clones that were

found to have strong pH dependency were subjected to sequence analysis using
specific primers.

As a result, several clones showing strong pH-dependent

binding as compared to WT were obtained.

As shown in FIG. 6, clone CL5 (Hchain: CLH5, L chain: CLL5) (CLH5: amino acid sequence of SEQ ID NO: 5,
CLL5: amino acid sequence of SEQ ID NO: 8) was found to exhibit particularly
strong pH-dependent binding as compared to WT.

It was thus confirmed that

antibodies exhibiting strong pH-dependent binding, while being unable to beobtained by typical panning using the antigen immobilized onto magnetic beads,
can be obtained by panning using a column immobilized with the antigen.
Therefore, panning using an antigen-immobilized column was found to be ahighly effective method for obtaining pH-dependently binding antibodies from alibrary.

The amino acid sequences of the clones showing pH-dependent binding

were analyzed, and the positions where histidine was present at a highprobability in the concentrated clones are shown in Table 4.
[0306][TABLE 4] Positions of Histidine Substitution found by Phage Library (Column
Panning)
H31, H50, H58, H62, H63, H65, H100b, H102
L24, L27, L28, L32, L53, L56, L90, L92, L94[0307][Example 4] Expression and Purification of Histidine-Modified Humanized IL-6
Receptor Antibody
Production, Expression, and Purification of Expression Vector of Histidine18

Modified Humanized IL-6 Receptor Antibody
In order to convert clones showing strong pH dependency in phage ELISA to
IgG, VH, and VL were respectively amplified by PCR, digested with XhoI/NheI
and EcoRI, and inserted to a mammalian cell expression vector.

The nucleotide

sequence of each DNA fragment was determined by a method known to persons
skilled in the art.

CLH5/L73, in which CLH5 was used for the H chain and L73

obtained in Example 2 was used for the L chain, was expressed and purified as
IgG.

Expression and purification were carried out by the method described in

Example 1.
[0308] Antibody having even higher pH dependency was produced by combining
mutation sites.

Based on the locations where His was concentrated in the phagelibrary as well as the structural information and the like, H32, H58, H62 , andH102 in H3pI which was obtained as an H chain in Example 2 were substituted
with histidine, and H95 and H99 were further substituted with valine andisoleucine, respectively, to produce H170 (SEQ ID NO: 4).

The variant

production was carried out using the method described in Example 1.

In

addition, L82 (SEQ ID NO: 7) was produced by substituting the 28th histidine of
L73, which was produced as an L chain in Example 2, with aspartic acid.

The

variant production was carried out using the method described in Example 1.
H170/L82, in which H170 was used for the H chain and L82 was used for the L
chain, was expressed and purified as IgG using the method described in Example
1.
[0311][Example 6] Biacore Analysis of pH-Dependently Binding Antibody
Analysis of Binding of pH-Dependently Binding Clones to Soluble IL-6
Receptor
Kinetic analyses of antigen-antibody reactions at pH 5.8 and pH 7.4 were carriedout using Biacore T100 (GE Healthcare) on the four antibodies: humanized PM1
antibody (WT) and H3pI/L73, CLH5/L73, and H170/L82 produced in Examples
2 and 4 (buffer: 10 mM MES (pH 7.4 or pH 5.8), 150 mM NaCl, and 0.05%
Tween 20). ...
[0312] As a result of calculating the affinity ratio between pH 5.8 and pH 7.4 foreach clone, the pH-dependent binding (affinity) of H3pI/L73, H170/L82, andCLH5/L73 to SR344 was 41-fold, 394-fold, and 66-fold, respectively, eachshowing the pH-dependent binding more than 15 times higher than that of WT.
[0313] Anti-IL-6 receptor antibodies that strongly bind to the antigen at the19plasma pH of 7.4 but weakly bind to the antigen at the intraendosomal pH of 5.5
to 6.0 have not yet been reported.

In this study, antibodies were obtained thatretain the biological neutralization activity equivalent to the WT humanized IL-6
receptor antibody and the affinity at pH 7.4, but exhibit the affinity at pH 5.8 thathas been specifically lowered by a factor of more than 10 times.
[0314][TABLE 5] Comparison of Binding of pH-Dependently Binding Clones Directed
Against SR344 to Soluble IL-6 Receptor[0335][Example 10] Improvement of pH-Dependent Binding to Membrane-Type IL-6
Receptor by Optimization of Variable Region
Optimization of Variable Regions H3pI/L73 and CLH5/L82
Antibodies having pH-dependent binding abilities were shown to demonstratesuperior effects in Example 9.

Therefore, to further improve the pH-dependent

binding abilities, mutations were introduced into the CDR sequence of CLH5
obtained in Example 3 to construct VH1-IgG1 (SEQ ID NO: 21) and VH2-IgG1(SEQ ID NO: 22).

In addition, mutations were introduced into the framework

sequence and CDR sequence of H3pI to construct the modified H chains VH3IgG1 (SEQ ID NO: 23) and VH4-IgG1 (SEQ ID NO: 24).

Mutations wereintroduced into the CDR sequences of L73 and L82 to construct the modified L
chains VL1-CK (SEQ ID NO: 25), VL2-CK (SEQ ID NO: 26), and VL3-CK(SEQ ID NO: 27). ...
[0336] The antibody having VH2-IgG1 (SEQ ID NO: 22) as H chain and VL2CK (SEQ ID NO: 26) as L chain was denoted as Fv1-IgG1, the antibody having
VH1-IgG1 (SEQ ID NO: 21) as H chain and L82 as L chain was denoted as Fv2IgG1, the antibody having VH4-IgG1 (SEQ ID NO: 24) as H chain and VL1-CK(SEQ ID NO: 25) as L chain was denoted as Fv3-IgG1, and the antibody having
VH3-IgG1 (SEQ ID NO: 23) as H chain and VL3-CK (SEQ ID NO: 27) as L
chain was denoted as Fv4-IgG1.
expressed and purified.

Of these, Fv2-IgG1 and Fv4-IgG1 were

The expression and purification were carried out by themethod described in Example 1. ...
20

[0391][Example 18] Repetitive Binding to Antigen by pH-Dependently Binding
Antibody
Expression and Purification of Antibody Administered to Mice
The four types of humanized IL-6 receptor antibodies described below wereprepared.

As the antibodies that do not pH-dependently bind to the IL-6

receptor, WT-IgG1 including H (WT) (amino acid sequence: SEQ ID NO: 9) and
L (WT) (amino acid sequence: SEQ ID NO: 10), and H54/L28-IgG1 including
H54 (amino acid sequence: SEQ ID NO: 70) and L28 (amino acid sequence: SEQ
ID NO: 12) were expressed and purified using the method indicated in Example
1.

As the antibodies that pH-dependently bind to the IL-6 receptor, H170/L82-

IgG1of Example 3 including H170 (amino acid sequence: SEQ ID NO: 4) andL82 (amino acid sequence: SEQ ID NO: 7), and Fv4-IgG1of Example 10
including VH3-IgG1 (SEQ ID NO: 23) and VL3-CK (SEQ ID NO: 27) wereexpressed and purified using the method indicated in Example 1.
[0392]
Analysis of Binding of Each Type of Antibody to Soluble IL-6 Receptor
Kinetic analysis of antigen-antibody reactions at pH 7.4 and pH 5.8 was carriedout using Biacore T100 (GE Healthcare) for the four types of antibodies
prepared: WT-IgG1, H54/L28-IgG1, H170/L82-IgG1, and Fv4-IgG1 (buffer: 10
mM MES (pH 7.4 or pH 5.8), 150 mM NaCl, 0.05% Surfactant-P20).

The

antibodies were bound to a sensor chip on which recomb-protein A/G (Pierce)
was immobilized by amine coupling, and SR344 adjusted to an appropriate
concentration was injected therein as an analyte.

The association with anddissociation from SR344 of each type of antibody were observed on a real-timebasis.

All the measurements were carried out at 37°C.

Association rate

constants ka (1/Ms) and dissociation rate constants kd (1/s) were calculated using
Biacore T100 Evaluation Software (GE Healthcare), and dissociation constants
KD (M) were calculated based on those values (Table 17).
[0393][TABLE 17] Comparison of Association Rates (ka), Dissociation Rates (kd), andDissociation Constants (KD) of Each Type of Antibody Against Soluble IL-6
Receptor (SR344)

21

pH dependency

Sample[0394] The ratio of affinity (KD) at pH 5.8 and pH 7.4 for each antibody wascalculated.

The KD ratio, which indicates the pH-dependent binding to SR344,

was 1.6, 0.7, 61.9, and 27.3 for WT-IgG1, H54/L28-IgG1, H170/L82-IgG1, andFv4-IgG1, respectively.

In addition, the ratio of dissociation rate (kd) at pH 5.8

and pH 7.4 for each antibody was calculated.

The kd ratio, which indicates thepH-dependent dissociation rate for SR344, was 2.9, 2.0, 11.4, and 38.8 for WTIgG1, H54/L28-IgG1, H170/L82-IgG1, and Fv4-IgG1, respectively.

Thus, itwas confirmed that H170/L82-IgG1 and Fv4-IgG1 demonstrate the pH-dependent
binding, while the conventional antibodies WT-IgG1 and H54/L28-IgG1 hardly
exhibit the ability.

In addition, since the affinity (KD) of these antibodies at pH

7.4 was nearly equal, their ability to bind to SR344 in the plasma was thought tobe equivalent.
(3) Feature of the Invention
According to the above (1) and (2), the following matters are recognized withregard to the Invention: The present invention relates to methods for improving thepharmacokinetics of antigen-binding molecules and methods for increasing thenumber of times of antigen-binding of antigen-binding molecules, as well as antigenbinding molecules having improved pharmacokinetics, antigen-binding molecules
having increased number of times of antigen-binding, and methods for producing suchmolecules. ([0001])
In general, the requisite dose of an antibody pharmaceutical is very high.

The

literature has reported methods for improving antibody pharmacokinetics using
artificial substitution of amino acids in constant regions.

Similarly, affinity

maturation has been reported as a technology for enhancing antigen-binding ability or
antigen-neutralizing activity.

The enhancement of antigen-binding ability enables

the reduction of dosage, and further enables improvement of in vivo efficacy. ([0002],[0003])The stoichiometric neutralization of one antibody against one antigen (onedivalent antibody against two antigens) is the limit of pre-existing methods; in a caseof neutralizing antibody, to prolong the neutralization effect of a neutralizing antibody
22

for a certain period, the antibody must be administered at a dose higher than theamount of antigen produced in the body during the same period.

With theimprovement of antibody pharmacokinetics or affinity maturation technology alone
described above, there is thus a limitation on the reduction of the required antibody
dose. ([0004])
From the viewpoint of dose reduction and sustained effect, there is a need for
new technology to produce an antibody having a stronger in vivo effect than ordinary
neutralizing antibodies by individually neutralizing multiple antigens with one
antibody. ([0006])
The present inventors have found that an antigen binding molecule with a weak
antigen binding property at a pH in an earlier stage endosome as compared to an
antigen binding property at a pH in plasma (blood) binds to an antigen multiple times
and has a longer half-life in plasma. ([0010])
Binding one antigen binding molecule to a plurality of antigens may improvepharmacokinetics of antigen binding molecule and cause more excellent effects ascompared to ordinary antigen binding molecule in vivo. ([0012])
2

Reasons 2 for rescission (Erroneous Determination of Reason 1 for invalidation(Enablement requirement and Support requirement))
In view of the nature of the case, Reason 2 for rescission is firstly considered in
the following.
(1) Enablement requirement
A. Article 36(4)(i) of the Patent Act specifies that the Detailed Description of
the Invention is required to disclose matters necessary for those who have an
ordinary knowledge in a technical field to which the invention pertains tounderstand a technical significance of the invention, including a problem to besolved by the invention and a means for solving the problem, definitely andsufficiently to the extent that allowed those who have expert knowledge in thetechnical field to implement the invention.

In order that the description of the

Detailed Description of the Invention conforms to the enablement requirement, theDetailed Description of the Invention is required to provide a description to theextent that allows a person ordinarily skilled in the art to implement the invention
without undue trials and errors on the basis of the description of the Detailed
Description of the Invention and the common general knowledge as of the filing.
B. The scope of the claims of Invention 1 neither specifies an original antibody
nor the position and number of histidine substitution or insertion.

Thus it can beseen that an antibody included in a pharmaceutical composition of Invention 1
23

specifies neither an original antibody nor the position and number of histidine
substitution or insertion.

Therefore, the technical scope of the invention 1

encompasses every pharmaceutical composition comprising every antibody ha ving
one or more of histidine substitutions and/or insertions, a certain pH-dependent
binding property, and a long half-life in plasma.
Consequently, in order that Invention 1 conforms to the enablement
requirement, the Detailed Description of the Invention must provide a description
to the extent that allows a person ordinarily skilled in the art to implement thewhole range of pharmaceutical compositions encompassed by Invention 1.
(2) The descriptions of the Detailed Description of the Invention
A. Description of [Description of Embodiment](A) Regarding a method of weakening an antigen binding activity of anantigen binging molecule at a pH of 7.4 as compared with that at a pH of 5.8 (amethod of imparting a pH-dependent binding ability), paragraph [0029] of thedescription discloses that [i] the position of histidine substitution or insertion is
not particularly limited, [ii] the position may include a variable region of an
antibody in a case of an antigen binding molecule being an antibody, [iii] thenumber of histidine substitutions or insertions may be determined as necessary
by a person ordinarily skilled in the art, [iv] mutation other than histidine
mutation (mutation to amino acid other than histidine) may be introduced
simultaneously,[v]

histidine

substitution

andinsertion

may

be

done

simultaneously.

Furthermore, it is possible to substitute or insert histidine at

random using a method such as histidine scanning, which uses histidine insteadof alanine in alanine scanning known to those skilled in the art.
itisdescribed

that

antigen-binding

molecules

Alternatively,

withincreased

KD(pH5.8)/KD(pH7.4) compared to before substitution can be selected from anantigen-binding molecule library with random histidine substitution or insertion.
Further, with respect to the site substituted with histidine, paragraphs [0070] to[0078] disclose that CDR sequence of antibody and a sequence for determinationof CDR structure are assumed in a case of an antigen binding molecule being anantibody, including, for example, 16 sites for heavy chain and 10 sites for lightchain, and furthermore, 4 sites of these are supposed to be universal modifying
sites, and some examples of preferable combinations may be given in a casewhere a plurality of sites are substituted with histidine in combination.
(B) However, the above CDR sequence is only an example, and a desired
antibody may be obtained by the modification of another site.
24

Thus it does notapply to the whole range of the pharmaceutical composition encompassed by
Invention 1.
B. Description of [Example](A) According to the description of H3pI/L73 in [0285], the description of
CLH5/L73 in [0287] to [0291], [0294], [0305], and [0307], the description of
HL170/L82 in [0308], the description of H170/L82-IgG1 in [0308] and [0391],
and the description of Fv4-IgG1 in [0335], [0336], and [0391], it can be said as
the methods of Example 2 and Example 3 for which a method of identifying theposition of histidine substitution or insertion with regard to the "antibody having
a KD(pH5.8)/KD(pH7.4) value, defined as the ratio of KD for the antigen at pH
5.8 and KD for the antigen at pH 7.4, of 2 or higher to 10000 or less, wherein atleast one amino acid of a variable region is substituted with histidine, or one
histidine is inserted into at least a variable region" of Invention 1; i.e., an
antibody with a determined pH-dependent binding property that is substituted
with or inserted by histidine.
(B) Example 2
Example 2 describes a method using homology modeling and conformation
model ([0285]).
However, homology modeling is a method that predicts a conformation of aprotein with unknown structure by a computer on the basis of a conformation ofa protein with known structure having amino acid sequence homology.

It is atechnique that is based on the premise of information of a conformation of aprotein homologous in amino acid sequence to a protein to be subjected tostructural prediction (no dispute between parties).
Consequently, the method of Example 2 using homology modeling may notalways be utilized when a position of histidine substitution is investigated forgeneral antibodies with unknown structures.
Therefore, the method of Example 2 is not applicable to the whole range ofpharmaceutical composition encompassed by Invention 1.
(C) Example 3
Example 3 describes a method of preliminarily selecting sites where thebinding ability was not greatly changed even when a residue of CDR was
substituted with histidine by a histidine scanning method to prepare an antibody
where any one of the sites is substituted with histidine ([0288] to [0290]).
Differing from the method of Example 2 of the above (B), this method isapplicable to antibodies with unknown structures.
25

It is indefinite from the description, however, as to whether "sites where thebinding ability was not greatly changed even when a residue of CDR was
substituted with histidine" of Example 3 ([0289]) always includes a site of an
antibody of Invention 1 substituted with histidine.

Further, there is no evidence

sufficient to find the common technical knowledge that a site of an antibody ofInvention 1 substituted with histidine is always encompassed by "sites where thebinding ability was not greatly changed even when a residue of CDR was
substituted with histidine" as mentioned in the description.
Therefore, the method of Example 3 is not applicable to the whole range ofpharmaceutical composition encompassed by Invention 1.
C. As described above, it cannot be said that the Detailed Description of theInvention of the description is described to the extent that allows a person
ordinarily skilled in the art to implement Invention 1 without undue trials and errors
on the basis of the description of the Detailed Description of the Invention and thecommon general knowledge as of the filing.
(3) Defendant's allegation
A. Defendant alleges that a person ordinarily skilled in the art could implement
Invention 1 in view of the description including paragraph [0029] and [0116] andthe common general knowledge by [i] a test for preparing an antibody, in which
each of 220 amino acid residues of a variable site of an antibody is substituted withhistidine, measuring the KD value and identifying the substituted position for
identifying a substituted site of histidine (hereinafter referred to as "the first half oftest") and [ii] a test of blood kinetics in a case where a desired pH dependency isshown by the above [i] (found to be promising and cause pH-dependent binding
properties) (hereinafter referred to as "the second half of test") (histidine scanning
as Defendant alleges).
A consideration is given hereinafter.
describes alanine scanning.

Paragraph [0029] of the description

It is recognized that the alanine scanning was a matterof common technical knowledge as of the filing date as a means for replacing eachresidue of amino acid sequences one by one with alanine and analyzing the role ofeach residue (Exhibits Otsu 19 to 23).

Therefore, it can be said that a person

ordinarily skilled in the art who read the description could have produced an
antibody in which each of 220 amino acid residues of variable sites of an antibody
was exhaustively substituted with histidine one by one on the basis of the common
technical knowledge.
Defendant alleges that the identification of a position substituted with histidine
26

after preparing an antibody is made with the criteria of "a site showing a desired pH
dependency (found to be promising or cause pH-dependent binding properties)";
however, the description fails to describe as such, and the criteria for identifying a
position substituted with histidine is indefinite from the description or common
technical knowledge appearing in evidence to implement the whole range ofpharmaceutical composition encompassed by Invention 1.
As seen above, the description has insufficient information on how to identify
the position to be substituted with histidine by histidine scanning as alleged by
Defendant, and thus it cannot be said that the Detailed Description of the Inventionof the description is described to the extent that allows a person ordinarily skilledin the art to implement Invention 1 without undue trials and errors on the basis of
the description of the Detailed Description of the Invention and the common
general knowledge as of the filing.
B. When it comes to the identification of the position of histidine substitution
in a first half test of histidine scanning as Defendant alleges, it can be seen as [i] asite "where KD(pH5.8)/KD(pH7.4) is increased as compared to before mutation " asdescribed in [0029] of the description, or [ii] a site "having certain pH-dependent
binding properties" as recited in the scope of claims, it cannot be said that suchhistidine scanning allows for the implementation of the whole range ofpharmaceutical compositions of Invention 1 for the following reasons:(A) "Antibody" "in which at least one amino acid of a variable region issubstituted with histidine, or one histidine is inserted into at least variable
region" of Invention 1 includes an antibody substituted with a plurality ofhistidines.

Defendant alleges that it is sufficient to combine the positions ofhistidine substitution identified by a first half test for identifying the positions ofa single histidine substitution of antibodies substituted with a plurality ofhistidines.
(B) Accordingly, a consideration is given as to whether the position ofhistidine substitution can be always identified in an antibody of Invention 1
substituted with a plurality of histidines by a method of combining the positionsof a single substitution as Defendant alleges.
a. The description fails to show that a site of histidine substitution in an
antibody having a certain pH-dependent binding property can always be
identified by a first half test of histidine scanning as Defendant alleges,
wherein the antibody is substituted with a plurality of histidines.

Further,

there is no proper evidence showing the common general knowledge as of the27

filing for this fact.
Consequently, the Detailed Description of the Invention fails to describe theconstitution of the invention to the extent that allows a person ordinarily
skilled in the art to implement the case where a plurality of sites aresubstituted with histidines.
b. In addition, while it is a document after filing date, the description of
Exhibit Ko 43 of anti-C5 antibody where a plurality of sites are substituted
with histidines (Exhibit Ko 43, [0276], [0281], [0282], Table 3) also supports
the above determination.
(a) Specifically, an antibody in which a reference antibody is substituted
with histidine at any one site of E62, D66, or S104 for heavy chain and N28,
I29, or A55 for light chain has a lower KD(pH5.5)/KD(pH7.4) compared toa value of the reference antibody (2.6).

The KD(pH5.5)/KD(pH7.4) is 6.73

to 19.4 for an antibody substituted with histidine at three sites of any one ofthese sites, heavy chain F100 and light chain S26.
Further, KD(pH5.5)/KD(pH7.4) is 1.83 and 1.8 respectively in acase where a reference antibody is substituted with histidine solely at heavy
chain N63 or light chain A51.

These values are lower than 2.

In contrast,

an antibody in which a reference antibody is substituted with histidine atthree sites of the above heavy chain N63, heavy chain F100, and light chain
S26 has a KD(pH5.5)/KD(pH7.4) of 10.03.

An antibody in which areference antibody is substituted with histidine at three sites of the above
light chain A51, heavy chain F100, and light chain S26 has aKD(pH5.5)/KD(pH7.4) of 4.02.
(b) According to this, it is presumed that a site of histidine substitution of an
antibody substituted with a plurality of histidines included in Invention 1
may include [i] a site in which a single histidine substitution provides aKD(pH5.8)/KD(pH7.4) value lower than a value of an antibody before
substitution, and [ii] a site in which a single histidine substitution may notprovide a certain pH-dependent binding property (In addition, thedescription of the above (a) relates to KD(pH5.5)/KD(pH7.4).

This might

possibly be a little bit higher than a KD(pH5.8)/KD(pH7.4) value of
Invention 1.

Thus the above presumption could have been made (Exhibit

Otsu 34 and the overall import of oral argument)).
Further, the above sites [i] and [ii] do not apply to the specificcriteria of the position of histidine substitution in a first half test ([i] a site28

"where KD(pH5.8)/KD(pH7.4) is increased as compared to before mutation ",or [ii] a site "having a certain pH-dependent binding properties").

Thus

these sites would not be identified as the position of histidine substitution by
a first half test.
Therefore, the combination of single substitution positions as
Defendant alleges may not implement the pharmaceutical composition
according to Invention 1 comprising antibody including these sites ofsubstitution.
(c) Regarding the credibility of Exhibit Ko 43, Defendant alleges that it is
only N28H and I29H of light chain that decrease KD(pH5.5)/KD(pH7.4) ina reference antibody and increases KD(pH5.5)/KD(pH7.4) in an antibody
where heavy chain F100 and light chain S26 are substituted with histidine ;however, a plurality of the other positions can be found as a similar
substitution position as in the above (a).
Further, Defendant alleges thata degree of decrease in

KD(pH5.5)/KD(pH7.4) by substitution falls within an error range; however,as in the above (a), a reference antibody has a KD(pH5.5)/KD(pH7.4) of 2.6,
whereas KD(pH5.5)/KD(pH7.4) is about 1.8 for the substitution of lightchain A51H and heavy chain F100H.

Thus it is doubtful whether this canbe said as falling within an error range.
C. Defendant alleges that single substitution or insertion only has an added
effect in most cases for an antibody where a plurality of histidine substitution s orinsertions are introduced, and thus is not necessary to reconsider the value ofKD(pH5.8)/KD(pH7.4) for a combination of individual substitutions or insertions
found to be promising by histidine scanning as Defendant alleges.
However, as explained in the above B, it cannot be said that a plurality ofpositions of histidine substitution can be always identified by histidine scanning as
Defendant alleges even if single substitution or insertion only has an added effect inmany cases for an antibody where a plurality of histidine substitutions or insertions
are introduced.

Thus the Defendant's allegation does not affect the determinationof the above (2).
D. Defendant refers to the use of libraries ([0183], [0191], [0192]) andconformation model (Example 2); however, information for identifying histidine
substitution position still remains insufficient.

Thus such allegation does notaffect the determination of the above (2).
(4) As aforementioned, Invention 1 does not conform to the enablement
29

requirement.

Further, Inventions 2 to 6 are inventions depending from Invention 1.

Thus the above explanation holds true with respect to the conformity to Inventions 2
to 6 to the enablement requirement.

Therefore, the Inventions do not conform to theenablement requirement.
3.

As aforementioned, the Invention should be invalidated for the Reason 1 forinvalidation.

The determination of the trial decision that negates this is erroneous.

Thus Reason 2 for rescission has a point, and the trial decision should be rescindedwithout the determination of the remaining reasons for rescission.
Therefore, the judgment shall be made as described in the main text.
Intellectual Property High Court, Third Division
Presiding Judge

TSURUOKA ToshihikoJudge YAMAKADO Masaru
Judge TAKAHASHI Aya

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