The present invention is in the field of medicine, particularly in the field of
5 multispecific binding proteins, such as bispecific antibodies and trispecific binding
proteins, used in the treatment of diseases and methods of developing the same.
Multispecific binding proteins are polypeptides which comprise multiple distinct
antigen binding domains. Multiple formats of multi specific binding proteins, such as
those set forth in W02001077342, W02007110205, W02008024188, W02009089004,
10 W02012135345 and W02016118742, have been disclosed and even tested for the
treatment of various autoimmune diseases, cancers, infectious diseases and cardiovascular
disease. Although multispecific binding proteins offer the possibility for enhanced
therapeutic benefit, for example by targeting multiple antigens, potential of cost savings
and improved convenience to patients, their development as therapeutic candidates has
15 been limited.
A factor limiting the advancement of multi specific binding proteins is the
complexity of assembling, manufacturing and purifying these molecules. For example,
manufacturing ofbispecific molecules not only requires the proper assembly of distinct
antigen binding domains, but also assembly of the distinct antigen binding domains into a
20 single molecule. Often, during recombinant expression of a multi specific binding protein,
a mixture including undesired molecules (e.g., monospecific proteins, single chain pairs,
etc.) is expressed. A desired multispecific binding protein must be purified not only from
the expression medium, but also from the mixture of undesired molecules. The formation
of undesired molecules and need for additional purification steps result in reduced yield
25 of the desired multi specific binding protein and increased overall manufacturing costs.
Attempts at enhancing the development and purification of multi specific binding
proteins have been disclosed, for example, as set forth in W020 100151792,
W02013088259 and W02013136186. However, these disclosures have proven to be
limited in addressing the development issues for multispecific binding proteins. For
30 example, in some instances, these disclosures demonstrate one of more of impaired
effector function, enhanced immunogenicity concerns, altered assembly, altered affinity
and I or reduced pharmacokinetic properties such as half-life. In some instances, the
3
applicability of the disclosure is limited to a specific molecule and I or format. As such,
there remains a need for improved multispecific binding proteins and methods of
developing the same, which enhances the development of multi specific binding proteins
without altering stability or affinity and which is not attendant upon unacceptable
5 immunogenicity.
Accordingly, the present disclosure addresses one or more of the above needs by
providing improved multi specific binding proteins and methods of developing the same.
Embodiments of the multi specific binding proteins and methods of the present disclosure
provide for enhanced purification of the desired multi specific binding protein, preserve
10 and I or enhance assembly of the molecule, decreased protein aggregation, improved
physical stability and are not attendant upon increased immunogenicity risk, altered
effector function and I or altered pharmacokinetic properties. Additionally, embodiments
of the present disclosure preserve affinity of the multi specific binding protein and reduces
or eliminates undesired binding of kappa light chain to purification reagent. Furthermore,
15 embodiments of the present disclosure do not add time and I or cost to the purification
process or development process as a whole.
Accordingly, in particular embodiments, the present disclosure provides a
multispecific binding protein that binds a first antigen and a second antigen. According
to some embodiments, multispecific binding proteins are provided that bind a first
20 antigen and a second antigen, the multispecific binding protein comprising a first antigen
binding domain comprising a first light chain Fab region and a first heavy chain Fab
region, wherein the first light chain Fab region is a kappa light chain and comprises: a
lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid residue 199
(EU numbering); a lysine at amino acid residue 143 (EU numbering), a lysine at amino
25 acid residue 199 (EU numbering), and an alanine at amino acid residue 109 (EU
numbering); a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid
residue 199 (EU numbering), and an aspartic acid at amino acid residue 110 (EU
numbering); a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid
residue 199 (EU numbering), an alanine at amino acid residue 109 (EU numbering), and
30 an aspartic acid at amino acid residue 110 (EU numbering); an aspartic acid at amino
acid residue 110 (EU numbering) and a lysine at amino acid residue 143 (EU
numbering); an aspartic acid at amino acid residue 110 (EU numbering), a lysine at
4
amino acid residue 143 (EU numbering) and an alanine at amino acid residue 109 (EU
numbering); an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at
amino acid residue 199 (EU numbering); an aspartic acid at amino acid residue 110 (EU
numbering), a lysine at amino acid residue 199 (EU numbering) and an alanine at amino
5 acid residue 109 (EU numbering); an alanine at amino acid residue 109 (EU numbering)
and a lysine at amino acid residue 143 (EU numbering); an alanine at amino acid residue
109 (EU numbering) and a lysine at amino acid residue 199 (EU numbering); or an
alanine at amino acid residue 109 (EU numbering) and an aspartic acid at amino acid
residue 110 (EU numbering); and a second antigen binding domain comprising a second
10 light chain Fab region and a second heavy chain Fab region, wherein the first antigen
binding domain binds the first antigen and the second antigen binding domain binds the
second antigen. According to some such embodiments, if the first light chain Fab region
comprises a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid
residue 199 (EU numbering), then the second light chain Fab region does not comprise a
15 lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid residue 199
(EU numbering); if the first light chain Fab region comprises a lysine at amino acid
residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an
alanine at amino acid residue 109 (EU numbering), then the second light chain Fab
region does not comprise a lysine at amino acid residue 143 (EU numbering), a lysine at
20 amino acid residue 199 (EU numbering), and an alanine at amino acid residue 109 (EU
numbering); if the first light chain Fab region comprises a lysine at amino acid residue
143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an
aspartic acid at amino acid residue 110 (EU numbering), then the second light chain Fab
region does not comprise a lysine at amino acid residue 143 (EU numbering), a lysine at
25 amino acid residue 199 (EU numbering), and an aspartic acid at amino acid residue 110
(EU numbering); if the first light chain Fab region comprises a lysine at amino acid
residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), an
alanine at amino acid residue 109 (EU numbering), and an aspartic acid at amino acid
residue 110 (EU numbering), then the second light chain Fab region does not comprise a
30 lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199
(EU numbering), an alanine at amino acid residue 109 (EU numbering); if the first light
chain Fab region comprises an aspartic acid at amino acid residue 110 (EU numbering)
5
and a lysine at amino acid residue 143 (EU numbering), then the second light chain Fab
region does not comprise an aspartic acid at amino acid residue 110 (EU numbering) and
a lysine at amino acid residue 143 (EU numbering); if the first light chain Fab region
comprises an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino
5 acid residue 143 (EU numbering) and an alanine at amino acid residue 109 (EU
numbering), then the second light chain Fab region does not comprise an aspartic acid at
amino acid residue 110 (EU numbering), a lysine at amino acid residue 143 (EU
numbering) and an alanine at amino acid residue 109 (EU numbering); if the first light
chain Fab region comprises an aspartic acid at amino acid residue 110 (EU numbering)
10 and a lysine at amino acid residue 199 (EU numbering), then the second light chain Fab
region does not comprise an aspartic acid at amino acid residue 110 (EU numbering) and
a lysine at amino acid residue 199 (EU numbering); if the first light chain Fab region
comprises an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino
acid residue 199 (EU numbering) and an alanine at amino acid residue 109 (EU
15 numbering), then the second light chain Fab region does not comprise an aspartic acid at
amino acid residue 110 (EU numbering), a lysine at amino acid residue 199 (EU
numbering) and an alanine at amino acid residue 109 (EU numbering); if the first light
chain Fab region comprises an alanine at amino acid residue 109 (EU numbering) and a
lysine at amino acid residue 143 (EU numbering), then the second light chain Fab region
20 does not comprise an alanine at amino acid residue 109 (EU numbering) and a lysine at
amino acid residue 143 (EU numbering); if the first light chain Fab region comprises an
alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue
199 (EU numbering), then the second light chain Fab region does not comprise an
alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue
25 199 (EU numbering); and if the first light chain Fab region comprises an alanine at
amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110
(EU numbering), then the second light chain Fab region does not comprise an alanine at
amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110
(EU numbering).
30 According to some embodiments, the multispecific binding protein comprises a
first antigen binding domain comprising a first light chain Fab region and a first heavy
chain Fab region, wherein the first light chain Fab region is a kappa light chain and
6
comprises a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid
residue 199 (EU numbering); and a second antigen binding domain comprising a second
light chain Fab region and a second heavy chain Fab region, wherein the first antigen
binding domain binds the first antigen and the second antigen binding domain binds the
5 second antigen. In some embodiments, the first light chain Fab region further comprises
an aspartic acid at amino acid residue 110 (EU numbering). In some embodiments, the
first light chain Fab region further comprises an alanine at amino acid residue 109 (EU
numbering).
According to further embodiments of the multi specific binding proteins provided
10 herein, the multi specific binding protein comprises a first antigen binding domain
comprising a first light chain Fab region and a first heavy chain Fab region, wherein the
first light chain Fab region is a kappa light chain and comprises an aspartic acid at amino
acid residue 110 (EU numbering) and a lysine at amino acid residue 143 (EU numbering);
and a second antigen binding domain comprising a second light chain Fab region and a
15 second heavy chain Fab region, wherein the first antigen binding domain binds the first
antigen and the second antigen binding domain binds the second antigen. In some
embodiments, the first light chain Fab region further comprises an alanine at amino acid
residue 109 (EU numbering).
According to embodiments of the multi specific binding proteins provided herein,
20 the multispecific binding protein comprises a first antigen binding domain comprising a
first light chain Fab region and a first heavy chain Fab region, wherein the first light chain
Fab region is a kappa light chain and comprises an aspartic acid at amino acid residue 110
(EU numbering) and a lysine at amino acid residue 199 (EU numbering); and a second
antigen binding domain comprising a second light chain Fab region and a second heavy
25 chain F ab region, wherein the first antigen binding domain binds the first antigen and the
second antigen binding domain binds the second antigen. In some embodiments, the first
light chain Fab region further comprises an alanine at amino acid residue 109 (EU
numbering).
According to embodiments of the multi specific binding proteins provided herein,
30 the multispecific binding protein comprises a first antigen binding domain comprising a
first light chain Fab region and a first heavy chain Fab region, wherein the first light
chain Fab region is a kappa light chain and comprises an alanine at amino acid residue
7
5
109 (EU numbering) and a lysine at amino acid residue 143 (EU numbering); and a
second antigen binding domain comprising a second light chain Fab region and a second
heavy chain Fab region, wherein the first antigen binding domain binds the first antigen
and the second antigen binding domain binds the second antigen.
According to some embodiments of the multi specific binding proteins provided
herein, the multispecific binding protein comprises a first antigen binding domain
comprising a first light chain Fab region and a first heavy chain Fab region, wherein the
first light chain Fab region is a kappa light chain and comprises an alanine at amino acid
residue 109 (EU numbering) and a lysine at amino acid residue 199 (EU numbering);
10 and a second antigen binding domain comprising a second light chain Fab region and a
second heavy chain Fab region, wherein the first antigen binding domain binds the first
antigen and the second antigen binding domain binds the second antigen.
According to even further embodiments of the multi specific binding proteins
provided herein, the multispecific binding protein comprises a first antigen binding
15 domain comprising a first light chain Fab region and a first heavy chain Fab region,
wherein the first light chain Fab region is a kappa light chain and comprises an alanine at
amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110
(EU numbering); and a second antigen binding domain comprising a second light chain
Fab region and a second heavy chain Fab region, wherein the first antigen binding
20 domain binds the first antigen and the second antigen binding domain binds the second
antigen.
According to some embodiments of the multi specific binding proteins of the
present disclosure, the first antigen binding domain of the multispecific binding protein
further comprises a first heavy chain Fe region. In even further embodiments of the
25 multispecific binding proteins of the present disclosure, the first heavy chain Fe region
comprises a human IgG 1, a human IgG2 or a human IgG4 constant region. In some
embodiments of the multi specific binding proteins of the present disclosure, the second
antigen binding domain further comprises a second heavy chain Fe region. In even
further embodiments of the multi specific binding proteins of the present disclosure, the
30 second heavy chain Fe region comprises a human IgG1, a human IgG2 or a human
IgG4 constant region. In some embodiments of the multispecific binding proteins of the
present disclosure, the first heavy chain Fe region comprises an arginine at amino
8
acid residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU
numbering). According to some embodiments of the multi specific binding proteins of
the present disclosure the second heavy chain Fe region comprises an arginine at
amino acid residue 311 (EU numbering) and a glutamic acid at amino acid residue
5 317 (EU numbering). In some embodiments of the multispecific binding proteins of the
present disclosure both the first and second heavy chain Fe regions comprise a human
IgG 1 constant region; both comprise a human IgG2 constant region; or both comprise a
human IgG4 constant region. In even further embodiments of the multi specific binding
proteins of the present disclosure both the first and second heavy chain Fe regions
10 comprise an arginine at amino acid residue 311 (EU numbering) and a glutamic acid
at amino acid residue 317 (EU numbering). According to some embodiments of the
multispecific binding proteins of the present disclosure the second light chain Fab region
does not comprise an alanine at amino acid residue 109; does not comprise an
aspartic acid at amino acid residue 110; does not comprise a lysine at amino acid
15 residue 143; or does not comprise a lysine at amino acid residue 199. In some
embodiments of the multi specific binding proteins of the present disclosure the second
light chain Fab region does not comprise an alanine at amino acid residue 109; does
not comprise an aspartic acid at amino acid residue 11 0; does not comprise a lysine
at amino acid residue 143; and does not comprise a lysine at amino acid residue 199.
20 According to some embodiments of the multi specific binding proteins of the present
disclosure the second light chain Fab region is a Kappa light chain. Further, according
to some embodiments of the multi specific binding proteins of the present disclosure the
second light chain Fab region is a Lambda light chain.
According to some embodiments, the multispecific binding protein comprises
25 a bispecific binding protein. According to some such embodiments, the bispecific
binding protein is an immunoglobulin heteromab. In some more specific embodiments,
the immunoglobulin heteromab is an IgG heteromab. According to even further
embodiments, the multispecific binding protein comprises a multispecific binding
protein.
30 Furthermore, embodiments of the present disclosure also provide
pharmaceutical compositions comprising a multi specific binding protein of the present
disclosure and one or more pharmaceutically acceptable carriers, diluents or excipients.
9
According to additional embodiments of the present disclosure, methods of
purifying multispecific binding proteins of the present disclosure is provided.
According to some such embodiments, the method comprises introducing into the
first antigen binding domain a first light chain Fab region comprising a lysine at amino
5 acid residue 143 (EU numbering) and a lysine at amino acid residue 199 (EU
numbering), wherein the first light chain Fab region is a kappa light chain; expressing
the multispecific binding protein, wherein the first antigen binding domain assembles
with the second antigen binding domain; subjecting the multispecific binding protein to
an affinity chromatography column; and recovering purified multispecific binding
10 protein. According to some such embodiments, the step of introducing further
comprises introducing into the first antigen binding domain an alanine at amino acid
residue 109 (EU numbering). In some embodiments, the step of introducing further
comprises introducing into the first antigen binding domain an aspartic acid at amino
acid residue 110 (EU numbering).
15 Additional embodiments of methods of purifying multi specific binding proteins
of the present disclosure are provided which comprise introducing into the first antigen
binding domain a first light chain Fab region comprising an aspartic acid at amino acid
residue 110 (EU numbering) and a lysine at amino acid residue 143 (EU numbering),
wherein the first light chain Fab region is a kappa light chain; expressing the
20 multispecific binding protein, wherein the first antigen binding domain assembles with
the second antigen binding domain; subjecting the multi specific binding protein to an
affinity chromatography column; and recovering purified multispecific binding
protein. According to some embodiments, the step of introducing further comprises
introducing into the first antigen binding domain an alanine at amino acid residue 109
25 (EU numbering).
Additional embodiments of methods of purifying multi specific binding proteins
of the present disclosure are provided which comprise introducing into the first antigen
binding domain a first light chain Fab region comprising an aspartic acid at amino acid
residue 110 (EU numbering) and a lysine at amino acid residue 199 (EU numbering),
30 wherein the first light chain Fab region is a kappa light chain; expressing the
multispecific binding protein, wherein the first antigen binding domain assembles with
the second antigen binding domain; subjecting the multi specific binding protein to an
10
5
affinity chromatography column; and recovering purified multispecific binding
protein. According to some embodiments, the step of introducing further comprises
introducing into the first antigen binding domain an alanine at amino acid residue 109
(EU numbering).
According to additional embodiments, methods of purifying multi specific
binding proteins of the present disclosure are provided comprising introducing into the
first antigen binding domain a first light chain Fab region comprising an alanine at
amino acid residue 109 (EU numbering) and a lysine at amino acid residue 143 (EU
numbering), wherein the first light chain Fab region is a kappa light chain; expressing
10 the multispecific binding protein, wherein the first antigen binding domain assembles
with the second antigen binding domain; subjecting the multispecific binding protein to
an affinity chromatography column; and recovering purified multispecific binding
protein.
Additional embodiments of methods of purifying multi specific binding proteins
15 of the present disclosure are provided comprising introducing into the first antigen
binding domain a first light chain Fab region comprising an alanine at amino acid
residue 109 (EU numbering) and a lysine at amino acid residue 199 (EU numbering),
wherein the first light chain Fab region is a kappa light chain; expressing the
multispecific binding protein, wherein the first antigen binding domain assembles with
20 the second antigen binding domain; subjecting the multi specific binding protein to an
affinity chromatography column; and recovering purified multispecific binding
protein.
Even further embodiments of methods of purifying multi specific binding
proteins of the present disclosure are provided comprising introducing into the first
25 antigen binding domain a first light chain Fab region comprising an alanine at amino
acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110 (EU
numbering), wherein the first light chain Fab region is a kappa light chain; expressing
the multispecific binding protein, wherein the first antigen binding domain assembles
with the second antigen binding domain; subjecting the multispecific binding protein to
30 an affinity chromatography column; and recovering purified multispecific binding
protein.
11
According to some embodiments of the methods of purifying multi specific
binding proteins of the present disclosure the step of introducing further comprises
introducing into the first antigen binding domain a first heavy chain F c region. In
even further embodiments of the methods of the present disclosure, the first heavy
5 chain Fe region comprises a human IgG1, a human IgG2 or a human IgG4 constant
region. In some embodiments of the methods of the present disclosure, the step of
introducing further comprises introducing into the second antigen binding domain a
second heavy chain Fe region. In even further embodiments of the methods of the
present disclosure, the second heavy chain Fe region comprises a human IgG1, a
10 human IgG2 or a human IgG4 constant region. According to some embodiments of the
methods of the present disclosure, the step of introducing further comprises
introducing into the first heavy chain Fe region an arginine at amino acid residue 311
(EU numbering) and a glutamic acid at amino acid residue 317 (EU numbering). In
some embodiments of the methods of the present disclosure, the step of introducing
15 further comprises introducing into the second heavy chain Fe region an arginine at
amino acid residue 311 (EU numbering) and a glutamic acid at amino acid residue
317 (EU numbering). According to some embodiments of the methods of the present
disclosure, both the first heavy chain Fe region and the second heavy chain Fe region
comprise a human IgG 1 constant region; both comprise a human IgG2 constant region;
20 or both comprise a human IgG4 constant region. In some embodiments of the methods
of the present disclosure, the step of introducing further comprises introducing, into
both the first heavy chain Fe region and the second heavy chain Fe region, arginine at
amino acid residues 311 (EU numbering) and glutamic acid at amino acid residues
317 (EU numbering). In some embodiments of the methods of the present disclosure,
25 the second light chain Fab region does not comprise an alanine at amino acid residue
109; does not comprise an aspartic acid at amino acid residue 110; does not
comprise a lysine at amino acid residue 143; or does not comprise a lysine at amino
acid residue 199. In some embodiments of the methods of the present disclosure, the
second light chain Fab region does not comprise an alanine at amino acid residue 109;
30 does not comprise an aspartic acid at amino acid residue 110; does not comprise a
lysine at amino acid residue 143; and does not comprise a lysine at amino acid
residue 199. In some embodiments of the methods of the present disclosure, the
12
second light chain Fab region is a Kappa light chain. In further embodiments of the
methods of the present disclosure, the second light chain Fab region is a Lambda light
chain.
According to further embodiments of the methods of the present disclosure,
5 the affinity chromatography column comprises a kappa affinity ligand. In some
embodiments of the methods of the present disclosure the affinity chromatography
column comprises a lambda affinity ligand. According to some embodiments of the
methods ofthe present disclosure, the affinity chromatography column comprises
Protein A In some embodiments of the methods of the present disclosure, the
10 second light chain Fab region binds to the affinity chromatography column with
greater affinity than the first light chain Fab region. In even further embodiments of
the methods of the present disclosure, the first light chain Fab region does not bind
to the affinity chromatography column.
According to even further embodiments of the methods of purifying
15 multi specific binding proteins of the present disclosure, the methods further comprise
subjecting the purified multispecific binding protein to a second affinity
chromatography column after the step of recovering purified multispecific binding
protein; and recovering purified multispecific binding protein after the step of
subjecting the purified multispecific binding protein to a second affinity
20 chromatography column. According to some embodiments, the second affinity
chromatography column comprises a kappa affinity ligand. In some embodiments,
the second affinity chromatography column comprises a lambda affinity ligand.
According to some embodiments, the second affinity chromatography column
comprises Protein A In even some further embodiments, the second light chain Fab
25 region binds to the second affinity chromatography column with greater affinity than
the first light chain Fab region. Even further, in some embodiments, the first light
chain Fab region does not bind to the second affinity chromatography column.
According to even further embodiments, the present disclosure provides a method
of making a multispecific binding protein of the present disclosure. In some such
30 embodiments, such methods comprise a multispecific binding protein of the present
invention prepared according to a process, wherein said process comprises cultivating a
13
host cell comprising a polynucleotide sequence, the polynucleotide sequence encoding a
first antigen binding domain and a second antigen binding domain of the present
disclosure, under conditions such that the multispecific binding protein is expressed, and
recovering from said host cell a multi specific binding protein of the present invention.
5 According to some embodiments, the polynucleotide sequence comprises a single vector
encoding the first antigen binding domain and the second antigen binding domain.
According to further embodiments, the polynucleotide sequence comprises a first vector
encoding the first antigen binding domain and a second vector comprising the second
antigen binding domain. In some embodiments, the method of the present disclosure
10 further comprises the steps of subjecting the recovered multi specific binding protein to
an affinity chromatography column and recovering purified multispecific binding
protein. In some embodiments, the affinity chromatography column comprises Protein
A. In some embodiments, the affinity chromatography column comprises a kappa
affinity ligand. In some embodiments, the affinity chromatography column
15 comprises a lambda affinity ligand. According to some embodiments, the first
antigen binding domain comprises a first light chain Fab region and the second antigen
binding domain comprises a second light chain Fab region, the second light chain Fab
region binding to the affinity chromatography column with greater affinity than the
first light chain Fab region. In some embodiments, the first light chain Fab region
20 does not bind to the affinity chromatography column. According to even further
embodiments, the method of the present disclosure further comprises the steps of
subjecting the purified multispecific binding protein to a second affinity
chromatography column after the step of recovering purified multispecific binding
protein and recovering purified multispecific binding protein after the step of
25 subjecting the purified multispecific binding protein to a second affinity
chromatography column. In some embodiments, the second affinity chromatography
column comprises Protein A. In some embodiments, the second affinity
chromatography column comprises a kappa affinity ligand. In some embodiments,
the second affinity chromatography column comprises a lambda affinity ligand.
30 According to some embodiments, the first antigen binding domain comprises a first
light chain Fab region and the second antigen binding domain comprises a second light
14
5
chain Fab region, the second light chain Fab region binding to the second affinity
chromatography column with greater affinity than the first light chain Fab region. In
some embodiments, the first light chain Fab region does not bind to the second
affinity chromatography column
In even further embodiments, the present disclosure provides multispecific
binding proteins for use in therapy. In some embodiments, the present disclosure
provides multi specific binding proteins for use in the treatment of a medical condition. In
some such embodiments, the medical condition is one of cancer, cardiovascular disease,
autoimmune disease or a neurodegenerative disease.
10 In further embodiments, the present disclosure provides multi specific binding
proteins for use in the manufacture of a medicament. In some embodiments, the present
disclosure provides multi specific binding proteins for use in the manufacture of a
medicament for therapy. In further embodiments, the present disclosure provides
multi specific binding proteins for use in the manufacture of a medicament for the
15 treatment of a medical condition. In some such embodiments, the medical condition is
one of cancer, cardiovascular disease, autoimmune disease or a neurodegenerative
disease.
The term "multispecific binding protein", as used herein, refers to a molecule
having two or more distinct antigen-binding domains. Multispecific binding proteins of
20 the present disclosure bind two or more different antigens or two or more different
epitopes of the same antigen. Embodiments of multi specific binding proteins of the
present disclosure include bispecific antibodies, as well as trispecific or tetraspecific
binding molecules as known in the field as well as single chain multispecific binding
molecules including diabodies. Multi specific binding proteins of the instant disclosure
25 can differ in size and geometry and can comprise multiple formats as known in the art.
As referred to herein, "antigen binding domain" refers to a portion of a
multispecific binding protein that comprises amino acid residues that interact with, and
confer specificity for, the respective antigen. Antigen binding domains of multi specific
binding proteins of the present disclosure include a light chain Fab region and a heavy
30 chain Fab region. Both the heavy and light chain Fab regions include a variable portion,
at the amino-terminus, comprising CDRs interspersed with regions that are more
conserved termed framework regions. Both the heavy and light chain Fab regions also
15
include a conserved region (e.g., a CL for the light chain and CH1 for the heavy chain Fab
region, as known in the field). The light chain Fab regions are classified as kappa or
lambda, as known in the art.
Some embodiments of multi specific binding proteins of the present disclosure
5 include heavy chain Fe regions linked at the carboxy terminus of the heavy chain Fab
region (e.g., forming a heavy chain as known in the field). Heavy chain Fe regions of the
present disclosure are classified as gamma and define the isotype of heavy chain as IgG
and one of subclasses IgG 1, IgG2, IgG3 or IgG4. The heavy chain Fe region may further
comport an effector function (as known in the field) upon the multispecific binding
10 protein.
According to some particular embodiments, multispecific binding proteins of the
instant disclosure comprise an IgG heteromab molecule, or fragment thereof As known
in the art, IgG heteromab molecules comprise archetypical Fab architecture and IgG
structure (with one Fab "arm", or antigen binding domain, binding the first antigen and
15 the other Fab "arm", or antigen binding domain, binding the second antigen).
The term "EU numbering", which is recognized in the art, refers to a system of
numbering amino acid residues of immunoglobulin molecules. EU numbering is
described, for example, at Kabat et al., Sequences of Proteins oflmmunological Interest,
5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991);
20 Edelman, G.M, et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); and
http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html#refs. The
term "Kabat numbering" is recognized in the art as referring to a system of numbering
amino acid residues which are more variable (i.e., hypervariable) than other amino acid
residues in heavy and light chain variable regions (see, for example, Kabat, et al., Ann.
25 NY Acad Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication
No. 91-3242 (1991)). The term "North numbering", refers to a system of numbering
amino acid residues which are more variable (i.e., hypervariable) than other amino acid
residues in heavy and light chain variable regions and is based, at least in part, on affinity
30 propagation clustering with a large number of crystal structures, as described in (North et
al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular
Biology, 406:228-256 (2011).
16
As used herein, the term "affinity chromatography" refers to a chromatographic
method for separating biochemical mixtures (e.g., a multispecific binding protein and
undesired biomolecule species) based on specific, reversible interactions between
biomolecules. Exemplary embodiments of affinity chromatography include Protein A
5 affinity columns, kappa affinity ligand chromatograph (such as CaptureSelect™,
KappaXL™, KappaSelect™, KappaXP™) or lambda affinity ligand
chromatography.
A "parent" or "parental" molecule as referred to herein, is a molecule encoded by
an amino acid sequence which is used in the preparation of one of the exemplified
10 embodiments set forth herein, for example through amino acid substitutions and structural
alteration. A parental molecule may comprise, for example, a murine antibody, or
fragment thereof, or a binding protein derived through phage display or transgenic nonhuman
animals, for example.
A multispecific binding protein of the present disclosure can be incorporated into
15 a pharmaceutical composition which can be prepared by methods well known in the art
and which comprise a multi specific binding protein of the present disclosure and one or
more pharmaceutically acceptable carrier(s) and/or diluent(s) (e.g., Remington, The
Science and Practice of Pharmacy, 22nd Edition, Loyd V., Ed., Pharmaceutical Press,
2012, which provides a compendium of formulation techniques as are generally known to
20 practitioners). Suitable carriers for pharmaceutical compositions include any material
which, when combined with the multi specific binding protein, retains the molecule's
activity and is non-reactive with the patient's immune system.
Expression vectors capable of directing expression of genes to which they are
operably linked are well known in the art. Expression vectors can encode a signal peptide
25 that facilitates secretion of the polypeptide(s) from a host cell. The signal peptide can be
an immunoglobulin signal peptide or a heterologous signal peptide. Each of the
expressed polypeptides may be expressed independently from different promoters to
which they are operably linked in one vector or, alternatively, may be expressed
independently from different promoters to which they are operably linked in multiple
30 vectors. The expression vectors are typically replicable in the host organisms either as
episomes or as an integral part of the host chromosomal DNA Commonly, expression
17
vectors will contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate
reductase, to permit detection of those cells transformed with the desired DNA sequences.
A host cell refers to cells stably or transiently transfected, transformed, transduced
or infected with one or more expression vectors expressing one or more polypeptide chain
5 of a multi specific binding protein of the present disclosure. Creation and isolation of host
cell lines producing binding proteins of the present disclosure can be accomplished using
standard techniques known in the art. Mammalian cells are preferred host cells for
expression ofmultispecific binding proteins of the present disclosure. Particular
mammalian cells include HEK 293, NSO, DG-44, and CHO. Preferably, the binding
10 proteins are secreted into the medium in which the host cells are cultured, from which the
binding proteins can be recovered or purified by for example using conventional
techniques. For example, the medium may be applied to and eluted from a Protein A
affinity chromatography column and I or a kappa affinity ligand or lambda affinity ligand
chromatography column. Undesired biomolecule species including soluble aggregate and
15 multimers may be effectively removed by common techniques, including size exclusion,
hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product
may be immediately frozen, for example at -70°C, refrigerated, or may be lyophilized.
Various methods of protein purification may be employed and such methods are known
in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89
20 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer,
NY (1994).

CLAIMS
1. A multispecific binding protein that binds a first antigen and a second antigen, the
multispecific binding protein comprising:
a first antigen binding domain comprising a first light chain Fab region and a first
5 heavy chain Fab region, wherein the first light chain Fab region is a kappa light
10
15
20
25
30
chain and comprises:
a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid
residue 199 (EU numbering);
a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid
residue 199 (EU numbering), and an alanine at amino acid residue 109 (EU
numbering);
a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid
residue 199 (EU numbering), and an aspartic acid at amino acid residue 110 (EU
numbering);
a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid
residue 199 (EU numbering), an alanine at amino acid residue 109 (EU
numbering), and an aspartic acid at amino acid residue 110 (EU numbering);
an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino
acid residue 143 (EU numbering);
an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino
acid residue 143 (EU numbering) and an alanine at amino acid residue 109 (EU
numbering);
an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino
acid residue 199 (EU numbering);
an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino
acid residue 199 (EU numbering) and an alanine at amino acid residue 109 (EU
numbering);
an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid
residue 143 (EU numbering);
an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid
residue 199 (EU numbering); or
36
an alanine at amino acid residue 109 (EU numbering) and an aspartic acid at
amino acid residue 110 (EU numbering); and
a second antigen binding domain comprising a second light chain Fab region and
a second heavy chain Fab region,
5 wherein the first antigen binding domain binds the first antigen and the second
antigen binding domain binds the second antigen.
2. The multispecific binding protein of claim 1, wherein:
if the first light chain Fab region comprises a lysine at amino acid residue 143
10 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), then the
second light chain Fab region does not comprise a lysine at amino acid residue 143
(EU numbering) and a lysine at amino acid residue 199 (EU numbering);
if the first light chain Fab region comprises a lysine at amino acid residue 143
(EU numbering), a lysine at amino acid residue 199 (EU numbering), and an alanine
15 at amino acid residue 109 (EU numbering), then the second light chain Fab region
does not comprise a lysine at amino acid residue 143 (EU numbering), a lysine at
amino acid residue 199 (EU numbering), and an alanine at amino acid residue 109
(EU numbering);
if the first light chain Fab region comprises a lysine at amino acid residue 143
20 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an aspartic
acid at amino acid residue 110 (EU numbering), then the second light chain Fab
region does not comprise a lysine at amino acid residue 143 (EU numbering), a
lysine at amino acid residue 199 (EU numbering), and an aspartic acid at amino acid
residue 110 (EU numbering);
25 if the first light chain Fab region comprises a lysine at amino acid residue 143
(EU numbering), a lysine at amino acid residue 199 (EU numbering), an alanine at
amino acid residue 109 (EU numbering), and an aspartic acid at amino acid residue
110 (EU numbering), then the second light chain Fab region does not comprise a
lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199
30 (EU numbering), an alanine at amino acid residue 109 (EU numbering);
if the first light chain Fab region comprises an aspartic acid at amino acid residue
110 (EU numbering) and a lysine at amino acid residue 143 (EU numbering), then
37
the second light chain Fab region does not comprise an aspartic acid at amino acid
residue 110 (EU numbering) and a lysine at amino acid residue 143 (EU numbering);
if the first light chain Fab region comprises an aspartic acid at amino acid residue
110 (EU numbering), a lysine at amino acid residue 143 (EU numbering) and an
5 alanine at amino acid residue 109 (EU numbering), then the second light chain Fab
region does not comprise an aspartic acid at amino acid residue 110 (EU numbering),
a lysine at amino acid residue 143 (EU numbering) and an alanine at amino acid
residue 109 (EU numbering);
if the first light chain Fab region comprises an aspartic acid at amino acid residue
10 110 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), then
the second light chain Fab region does not comprise an aspartic acid at amino acid
residue 110 (EU numbering) and a lysine at amino acid residue 199 (EU numbering);
if the first light chain Fab region comprises an aspartic acid at amino acid residue
110 (EU numbering), a lysine at amino acid residue 199 (EU numbering) and an
15 alanine at amino acid residue 109 (EU numbering), then the second light chain Fab
region does not comprise an aspartic acid at amino acid residue 110 (EU numbering),
a lysine at amino acid residue 199 (EU numbering) and an alanine at amino acid
residue 109 (EU numbering);
if the first light chain Fab region comprises an alanine at amino acid residue 109
20 (EU numbering) and a lysine at amino acid residue 143 (EU numbering), then the
second light chain Fab region does not comprise an alanine at amino acid residue
109 (EU numbering) and a lysine at amino acid residue 143 (EU numbering);
if the first light chain Fab region comprises an alanine at amino acid residue 109
(EU numbering) and a lysine at amino acid residue 199 (EU numbering), then the
25 second light chain Fab region does not comprise an alanine at amino acid residue
109 (EU numbering) and a lysine at amino acid residue 199 (EU numbering); and
if the first light chain Fab region comprises an alanine at amino acid residue 109
(EU numbering) and an aspartic acid at amino acid residue 110 (EU numbering),
then the second light chain Fab region does not comprise an alanine at amino acid
30 residue 109 (EU numbering) and an aspartic acid at amino acid residue 110 (EU
numbering).
38
3. A multispecific binding protein that binds a first antigen and a second antigen, the
multispecific binding protein comprising:
a first antigen binding domain comprising a first light chain Fab region and a first
heavy chain Fab region, wherein the first light chain Fab region is a kappa light
5 chain and comprises a lysine at amino acid residue 143 (EU numbering) and a lysine
at amino acid residue 199 (EU numbering); and
a second antigen binding domain comprising a second light chain Fab region and
a second heavy chain Fab region,
wherein the first antigen binding domain binds the first antigen and the second
10 antigen binding domain binds the second antigen.
4. The multi specific binding protein of Claim 3, wherein the first light chain Fab region
further comprises an aspartic acid at amino acid residue 110 (EU numbering).
15 5. The multispecific binding protein of any of Claims 3 or 4, wherein the first light
chain Fab region further comprises an alanine at amino acid residue 109 (EU
numbering).
6. A multispecific binding protein that binds a first antigen and a second antigen, the
20 multispecific binding protein comprising:
25
a first antigen binding domain comprising a first light chain Fab region and a first
heavy chain Fab region, wherein the first light chain Fab region is a kappa light
chain and comprises an aspartic acid at amino acid residue 110 (EU numbering) and
a lysine at amino acid residue 143 (EU numbering); and
a second antigen binding domain comprising a second light chain Fab region and
a second heavy chain Fab region,
wherein the first antigen binding domain binds the first antigen and the second
antigen binding domain binds the second antigen.
30 7. The multi specific binding protein of Claim 6, wherein the first light chain Fab region
further comprises an alanine at amino acid residue 109 (EU numbering).
39
8. A multispecific binding protein that binds a first antigen and a second antigen, the
multispecific binding protein comprising:
a first antigen binding domain comprising a first light chain Fab region and a first
heavy chain Fab region, wherein the first light chain Fab region is a kappa light
5 chain and comprises an aspartic acid at amino acid residue 110 (EU numbering) and
a lysine at amino acid residue 199 (EU numbering); and
a second antigen binding domain comprising a second light chain Fab region and
a second heavy chain Fab region,
wherein the first antigen binding domain binds the first antigen and the second
10 antigen binding domain binds the second antigen.
9. The multi specific binding protein of Claim 8, wherein the first light chain Fab region
further comprises an alanine at amino acid residue 109 (EU numbering).
15 10. A multispecific binding protein that binds a first antigen and a second antigen, the
multispecific binding protein comprising:
a first antigen binding domain comprising a first light chain Fab region and a first
heavy chain Fab region, wherein the first light chain Fab region is a kappa light
chain and comprises an alanine at amino acid residue 109 (EU numbering) and a
20 lysine at amino acid residue 143 (EU numbering); and
25
a second antigen binding domain comprising a second light chain Fab region and
a second heavy chain Fab region,
wherein the first antigen binding domain binds the first antigen and the second
antigen binding domain binds the second antigen.
11. A multispecific binding protein that binds a first antigen and a second antigen, the
multispecific binding protein comprising:
a first antigen binding domain comprising a first light chain Fab region and a first
heavy chain Fab region, wherein the first light chain Fab region is a kappa light
30 chain and comprises an alanine at amino acid residue 109 (EU numbering) and a
lysine at amino acid residue 199 (EU numbering); and
40
5
a second antigen binding domain comprising a second light chain Fab region and
a second heavy chain Fab region,
wherein the first antigen binding domain binds the first antigen and the second
antigen binding domain binds the second antigen.
12. A multispecific binding protein that binds a first antigen and a second antigen, the
multispecific binding protein comprising:
a first antigen binding domain comprising a first light chain Fab region and a first
heavy chain Fab region, wherein the first light chain Fab region is a kappa light
10 chain and comprises an alanine at amino acid residue 109 (EU numbering) and an
aspartic acid at amino acid residue 110 (EU numbering); and
a second antigen binding domain comprising a second light chain Fab region and
a second heavy chain Fab region,
wherein the first antigen binding domain binds the first antigen and the second
15 antigen binding domain binds the second antigen.
13. The multispecific binding protein of any one of Claims 1 to 12, wherein the first
antigen binding domain further comprises a first heavy chain Fe region.
20 14. The multispecific binding protein of Claim 13, wherein the first heavy chain Fe
25
region comprises a human IgG1, a human IgG2 or a human IgG4 constant region.
15. The multispecific binding protein of any one of Claims 1 to 14, wherein the second
antigen binding domain further comprises a second heavy chain Fe region.
16. The multispecific binding protein of Claim 15, wherein the second heavy chain
Fe region comprises a human IgG1, a human IgG2 or a human IgG4 constant
region.
30 17. The multispecific binding protein of any one of Claims 13 to 16, wherein the first
heavy chain Fe region comprises an arginine at amino acid residue 311 (EU
41
numbering) and a glutamic acid at amino acid residue 317 (EU numbering).
18. The multi specific binding protein of any one of Claims 13 to 17, wherein the
second heavy chain Fe region comprises an arginine at amino acid residue 311
5 (EU numbering) and a glutamic acid at amino acid residue 317 (EU numbering).
19. The multispecific binding protein of any one of Claims 13 to 18, wherein both the
first and second heavy chain Fe regions comprise a human IgG1 constant region;
both comprise a human IgG2 constant region; or both comprise a human IgG4
10 constant region.
20. The multi specific binding protein of any one of Claims 13 to 19, wherein both the
first and second heavy chain Fe regions comprise an arginine at amino acid
residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU
15 numbering).
21. The multi specific binding protein of any one of Claims 1 to 20, wherein the
second light chain Fab region does not comprise an alanine at amino acid residue
109; does not comprise an aspartic acid at amino acid residue 110; does not
20 comprise a lysine at amino acid residue 143; or does not comprise a lysine at
amino acid residue 199.
22. The multi specific binding protein of any one of Claims 1 to 21, wherein the
25 second light chain Fab region is a Kappa light chain.
23. The multi specific binding protein of any one of Claims 1 to 22, wherein the
second light chain Fab region is a Lambda light chain.
30 24. A method of purifying a multi specific binding protein comprising a first antigen
binding domain that binds a first antigen and a second antigen binding domain that
binds a second antigen, the method comprising:
introducing into the first antigen binding domain a first light chain Fab region
42
comprising a lysine at amino acid residue 143 (EU numbering) and a lysine at amino
acid residue 199 (EU numbering), wherein the first light chain Fab region is a
kappa light chain;
expressing the multispecific binding protein, wherein the first antigen binding
5 domain assembles with the second antigen binding domain; and
subjecting the multi specific binding protein to an affinity chromatography
column; and
recovering purified multispecific binding protein.
10 25. The method of Claim 24, wherein the step of introducing further comprises
introducing into the first antigen binding domain an alanine at amino acid residue
109 (EU numbering).
26. The method of any of Claims 24 or 25, wherein the step of introducing further
15 comprises introducing into the first antigen binding domain an aspartic acid at amino
acid residue 110 (EU numbering).
27. A method of purifying a multispecific binding protein comprising a first antigen
binding domain that binds a first antigen and a second antigen binding domain that
20 binds a second antigen, the method comprising:
25
30
introducing into the first antigen binding domain a first light chain Fab region
comprising an aspartic acid at amino acid residue 110 (EU numbering) and a lysine
at amino acid residue 143 (EU numbering), wherein the first light chain Fab region
is a kappa light chain;
expressing the multispecific binding protein, wherein the first antigen binding
domain assembles with the second antigen binding domain; and
subjecting the multi specific binding protein to an affinity chromatography
column; and
recovering purified multispecific binding protein.
43
28. The method of Claim 27, wherein the step of introducing further comprises
introducing into the first antigen binding domain an alanine at amino acid residue
109 (EU numbering).
5 29. A method of purifying a multi specific binding protein comprising a first antigen
binding domain that binds a first antigen and a second antigen binding domain that
binds a second antigen, the method comprising:
introducing into the first antigen binding domain a first light chain Fab region
comprising an aspartic acid at amino acid residue 110 (EU numbering) and a lysine
10 at amino acid residue 199 (EU numbering), wherein the first light chain Fab region
is a kappa light chain;
expressing the multispecific binding protein, wherein the first antigen binding
domain assembles with the second antigen binding domain; and
subjecting the multi specific binding protein to an affinity chromatography
15 column; and
recovering purified multispecific binding protein.
30. The method of Claim 29, wherein the step of introducing further comprises
introducing into the first antigen binding domain an alanine at amino acid residue
20 109 (EU numbering).
25
31. A method of purifying a multi specific binding protein comprising a first antigen
binding domain that binds a first antigen and a second antigen binding domain that
binds a second antigen, the method comprising:
introducing into the first antigen binding domain a first light chain Fab region
comprising an alanine at amino acid residue 109 (EU numbering) and a lysine at
amino acid residue 143 (EU numbering), wherein the first light chain Fab region is
a kappa light chain;
expressing the multispecific binding protein, wherein the first antigen binding
30 domain assembles with the second antigen binding domain; and
subjecting the multi specific binding protein to an affinity chromatography
44
column; and
recovering purified multispecific binding protein.
32. A method of purifying a multi specific binding protein comprising a first antigen
5 binding domain that binds a first antigen and a second antigen binding domain that
binds a second antigen, the method comprising:
introducing into the first antigen binding domain a first light chain Fab region
comprising an alanine at amino acid residue 109 (EU numbering) and a lysine at
amino acid residue 199 (EU numbering), wherein the first light chain Fab region is
10 a kappa light chain;
15
20
expressing the multispecific binding protein, wherein the first antigen binding
domain assembles with the second antigen binding domain; and
subjecting the multi specific binding protein to an affinity chromatography
column; and
recovering purified multispecific binding protein.
33. A method of purifying a multispecific binding protein comprising a first antigen
binding domain that binds a first antigen and a second antigen binding domain that
binds a second antigen, the method comprising:
introducing into the first antigen binding domain a first light chain Fab region
comprising an alanine at amino acid residue 109 (EU numbering) and an aspartic
acid at amino acid residue 110 (EU numbering), wherein the first light chain Fab
region is a kappa light chain;
expressing the multispecific binding protein, wherein the first antigen binding
25 domain assembles with the second antigen binding domain; and
subjecting the multi specific binding protein to an affinity chromatography
column; and
recovering purified multispecific binding protein.
45
34. The method of any one of Claims 24 to 33, wherein the step of introducing further
comprises introducing into the first antigen binding domain a first heavy chain Fe
regwn.
5 35. The method of Claim 34, wherein the first heavy chain Fe region comprises a
human IgG1, a human IgG2 or a human IgG4 constant region.
36. The method of any one ofClaims 24 to 35, wherein the step ofintroducing further
comprises introducing into the second antigen binding domain a second heavy
10 chain Fe region.
37. The method of Claim 36, wherein the second heavy chain Fe region comprises a
human IgG1, a human IgG2 or a human IgG4 constant region.
15 38. The method of any one of Claims 34 to 37, wherein the step ofintroducing further
comprises introducing into the first heavy chain Fe region an arginine at amino acid
residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU
numbering).
20 39. The method of any one of Claims 34 to 38, wherein the step ofintroducing further
comprises introducing into the second heavy chain Fe region an arginine at amino
acid residue 311 (EU numbering) and a glutamic acid at amino acid residue 317
(EU numbering).
25 40. The method of any one of Claims 24 to 39, wherein both the first heavy chain Fe
region and the second heavy chain Fe region comprise a human IgG1 constant
region; both comprise a human IgG2 constant region; or both comprise a human
IgG4 constant region.
30 41. The method of any one of Claims 24 to 40, wherein the step ofintroducing further
comprises introducing, into both the first heavy chain Fe region and the second
46
heavy chain Fe region, arginine at amino acid residues 311 (EU numbering) and
glutamic acid at amino acid residues 317 (EU numbering).
42. The method of any one of Claims 24 to 41, wherein the second light chain Fab
5 region does not comprise an alanine at amino acid residue 109; does not
comprise an aspartic acid at amino acid residue 110; does not comprise a lysine
at amino acid residue 143; or does not comprise a lysine at amino acid residue
199.
10 43. The method of any one of Claims 24 to 41, wherein the second light chain Fab
15
region does not comprise an alanine at amino acid residue 109; does not
comprise an aspartic acid at amino acid residue 110; does not comprise a lysine
at amino acid residue 143; and does not comprise a lysine at amino acid residue
199.
44. The method of any one of Claims 24 to 43, wherein the second light chain Fab
region is a Kappa light chain.
45. The method of any one of Claims 24 to 43, wherein the second light chain Fab
20 region is a Lambda light chain
46. The method of any one of Claims 24 to 45, wherein the affinity chromatography
column comprises a kappa affinity ligand.
25 47. The method of any one of Claims 24 to 46, wherein the affinity chromatography
30
column comprises a lambda affinity ligand.
48. The method of any one of Claims 24 to 47, wherein the affinity chromatography
column comprises Protein A.
49. The method of any one of Claims 24 to 48, wherein the second light chain Fab
47
region binds to the affinity chromatography column with greater affinity than the
first light chain Fab region.
50. The method of any one of Claims 24 to 49, wherein the first light chain Fab
5 region does not bind to the affinity chromatography column.
51. The method of any one of Claims 24 to 50, further comprising the steps of:
subjecting the purified multispecific binding protein to a second affinity
chromatography column after the step of recovering purified multispecific
10 binding protein; and
recovering purified multispecific binding protein after the step of subjecting
the purified multispecific binding protein to a second affinity chromatography
column.
15 52. The method of 51, wherein the second affinity chromatography column
20
comprises a kappa affinity ligand.
53. The method of any one of Claims 51 or 52, wherein the second affinity
chromatography column comprises a lambda affinity ligand.
54. The method of any one of Claims 51 to 53, wherein the second affinity
chromatography column comprises Protein A
55. The method of any one of Claims 51 to 54, wherein the second light chain Fab
25 region binds to the second affinity chromatography column with greater affinity
than the first light chain Fab region.
30
56. The method of any one of Claims 51 to 55, wherein the first light chain Fab
region does not bind to the second affinity chromatography column.