NZ728228B2 - Methods and means for the production of ig-like molecules - Google Patents
Methods and means for the production of ig-like molecules Download PDFInfo
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- NZ728228B2 NZ728228B2 NZ728228A NZ72822813A NZ728228B2 NZ 728228 B2 NZ728228 B2 NZ 728228B2 NZ 728228 A NZ728228 A NZ 728228A NZ 72822813 A NZ72822813 A NZ 72822813A NZ 728228 B2 NZ728228 B2 NZ 728228B2
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
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- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/526—CH3 domain
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Abstract
Discloses a method of producing at least two different monospecific CH3 domain-comprising molecules from a single host cell, wherein each of said two CH3 domain-comprising molecules comprises two CH3 domains that are capable of forming an interface, said method comprising providing in said cell: a. A first nucleic acid molecule encoding a 1st CH3 domain-comprising polypeptide chain, b. A second nucleic acid molecule encoding a 2nd CH3 domain-comprising polypeptide chain, wherein said 1st CH3 domain-comprising polypeptide chain comprises negatively charged amino acid residues at amino acid positions 392 and 409 and a positively charged amino acid residue at amino acid position 399, and wherein said 2nd CH3 domain-comprising polypeptide chain comprises: - either a wildtype CH3 domain, or - positively charged amino acid residues at amino acid positions 356 and 357 and negatively charged amino acid residues at amino acid positions 370 and 439, said method further comprising the step of culturing said host cell and allowing for expression of said nucleic acid molecules and harvesting said at least two different CH3 domain-comprising molecules from the culture. A first nucleic acid molecule encoding a 1st CH3 domain-comprising polypeptide chain, b. A second nucleic acid molecule encoding a 2nd CH3 domain-comprising polypeptide chain, wherein said 1st CH3 domain-comprising polypeptide chain comprises negatively charged amino acid residues at amino acid positions 392 and 409 and a positively charged amino acid residue at amino acid position 399, and wherein said 2nd CH3 domain-comprising polypeptide chain comprises: - either a wildtype CH3 domain, or - positively charged amino acid residues at amino acid positions 356 and 357 and negatively charged amino acid residues at amino acid positions 370 and 439, said method further comprising the step of culturing said host cell and allowing for expression of said nucleic acid molecules and harvesting said at least two different CH3 domain-comprising molecules from the culture.
Description
METHODS AND MEANS FOR THE PRODUCTION OF IG-LIKE MOLECULES
This application is a divisional of New Zealand patent application 630551, which is the
national phase entry in New Zealand of PCT international application
PCT/NL2013/050293 (published as WO2013/157953), dated 19 April 2013, and claims the
benefit of priority to U.S. provisional application no. 61/635935, filed 20 April 2012, all of
which are incorporated herein by reference. The reader’s attention is also directed to our
related divisional, New Zealand patent application 772318.
FIELD
The invention relates to the fields of molecular biology, medicine and biological
therapeutics. It particularly relates to the field of therapeutic antibodies for the treatment of
various diseases.
BACKGROUND
Many currently used biological therapeutics are isolated recombinant, human or
humanized monoclonal antibodies that enhance the ability of the body's immune system to
neutralize or eliminate cells and/or molecules involved in disease processes or to eradicate
invading pathogens or infectious agents. Monoclonal antibodies bind to a single specific
area, or epitope, of an antigen and, for use in therapy, are often selected for a desirable
functional property such as for example killing of tumor cells, blocking of receptor-ligand
interactions or virus neutralization. Nowadays, there are about 30 FDA approved
monoclonal antibodies, which are typically produced at large quantities and their
biophysical and biochemical characteristics can be analyzed in great detail to ensure batch-
to-batch consistency, which facilitates regulatory acceptability. Despite these favorable
characteristics, monoclonal antibodies have several disadvantages, some of which relate to
their monospecific nature and the complexity of diseases. Diseases processes are often
multifactorial in nature, and involve redundant or synergistic action of disease mediators or
up-regulation of different receptors, including crosstalk between their signaling networks.
Consequently, blockade of multiple, different factors and pathways involved in pathology
2
may result in improved therapeutic efficacy. By nature of their monospecificity,
monoclonal antibodies can only interfere with a single step within the complex disease
processes which often does not have an optimal effect. In addition to not fully addressing
multiple aspects of a disease process, it has become clear that targeting a single epitope on
a single cellular or soluble protein or pathogen often will not suffice to efficiently treat
disease because the target epitope may no longer be available for the monoclonal antibody
to bind to and exert the desired effect. As an example, tumor cells often escape from
monoclonal antibody therapy by down-regulation, mutation or shielding of the target
epitope present on a growth factor receptor. By activating alternative receptors and/or their
ligands, tumor cells than may exploit a different path leading to continued growth and
metastasis. Similarly, viruses and other pathogens frequently mutate and lose or shield the
target epitope, thereby escaping monoclonal antibody treatment. Monoclonal antibodies
that bind to a single epitope often do not recruit the full spectrum of effector mechanisms
evoked by polyclonal antibodies, including, amongst other things, opsonization (enhancing
phagocytosis of antigens), steric hindrance (antigens coated with antibodies are prevented
from attaching to host cells or mucosal surfaces), toxin neutralization, agglutination or
precipitation (antibodies binding several soluble antigens cause aggregation and
subsequent clearance), activation of complement and antibody-dependent cellular
cytotoxicity (antibodies enable the killing of target cells by natural killer cells and
neutrophils).
Polyclonal antibodies for therapeutic applications may be obtained from pooled human
serum. Such serum-derived therapeutic polyclonal antibodies may for example be used to
treat or prevent infections caused by viruses such as the rabies virus, cytomegalovirus and
respiratory syncytial virus, to neutralize toxins such as tetanus toxin and botulinum toxin or
to prevent Rhesus D allograft immunization. A more widespread use of serum-derived
polyclonal antibody preparations has been prevented by the fact that source plasma is only
available for a limited range of targets such as infectious diseases and toxins. Moreover,
the products are highly dependent on donor blood availability, both in terms of quantity
and suitability, resulting in considerable variation between batches. In addition, screening
technologies fail to keep up with constantly evolving viruses, thus, immunoglobulin
3
products carry a potential risk of infectious disease transmission. Finally, the long process
of blood collection, screening and immunoglobulin purification means plasma-derived
immunoglobulins are expensive to produce.
Mixtures of monoclonal antibodies may improve the efficacy of monoclonal antibodies
while avoiding the limitations associated with serum-derived polyclonal antibodies. In the
art, combinations of two human or humanized monoclonal antibodies have been tested in
preclinical models and in clinical trials (for example mixtures of 2 monoclonal antibodies
against the HER2 receptor, mixtures of 2 antibodies against the EGFR receptor and , 2
monoclonal antibodies against the rabies virus).
In the art, it has been shown that combinations of 2 monoclonal antibodies may have
additive or synergistic effects and recruit effector mechanisms that are not associated with
either antibody alone. For example, mixtures of 2 monoclonal antibodies against the EGFR
or HER2 were shown to more potently kill tumor cells based on a combination of activities
including enhanced receptor internalization, improved blockade of signalling pathways
downstream of the receptors as well as enhanced immune effector-mediated cytotoxicity.
For combination therapies based on 2 monoclonal antibodies, the component antibodies
may be produced separately and combined at the protein level. A drawback of this
approach is the staggering cost of developing the 2 antibodies individually in clinical trials
and (partially) repeating that process with the combination. This would lead to
unacceptable cost of treatments based on antibody combinations. Alternatively, the 2
recombinant cell lines producing the component monoclonal antibodies may be mixed in a
fermentor and the resultant mixture of antibodies may be purified as a single preparation
(WO 2004/061104). A drawback of this approach is the poor control over the composition
and hence reproducibility of the resulting recombinant polyclonal antibody preparation,
especially when considering that such compositions may change over time as the cells are
being cultured.
During the past decade, bispecific antibodies have emerged as an alternative to the use of
combinations of 2 antibodies. Whereas a combination of 2 antibodies represents a mixture
of 2 different immunoglobulin molecules that bind to different epitopes on the same or
4
different targets, in a bispecific antibody this is achieved through a single immunoglobulin
molecule. By binding to 2 epitopes on the same or different targets, bispecific antibodies
may have similar effects as compared to a combination of 2 antibodies binding to the same
epitopes. Furthermore, since bispecific antibodies of the IgG format combine 2 different
monovalent binding regions in a single molecule and mixtures of 2 IgG antibodies
combine 2 different bivalent binding molecules in a single preparation, different effects of
these formats have been observed as well. From a technological and regulatory
perspective, this makes development of a single bispecific antibody less complex because
manufacturing, preclinical and clinical testing involve a single, molecule. Thus, therapies
based on a single bispecific antibody are facilitated by a less complicated and cost-
effective drug development process while providing more efficacious antibody therapies.
Bispecific antibodies based on the IgG format, consisting of 2 heavy and two light chains
have been produced by a variety of methods. For instance, bispecific antibodies may be
produced by fusing two antibody-secreting cell lines to create a new cell line or by
expressing two antibodies in a single cell using recombinant DNA technology. These
approaches yield multiple antibody species as the respective heavy chains from each
antibody may form monospecific dimers (also called homodimers), which contain two
identical paired heavy chains with the same specificity, and bispecific dimers (also called
heterodimers) which contain two different paired heavy chains with different specificity. In
addition, light chains and heavy chains from each antibody may randomly pair to form
inappropriate, non-functional combinations. This problem, known as heavy and light chain
miss-pairings, can be solved by choosing antibodies that share a common light chain for
expression as bispecific. But even when a common light chain is used, expression of two
heavy chains and one common light chain in a single cell will result in 3 different antibody
species, i.e. two monospecific 'parental' antibodies and the bispecific antibody so that the
bispecific antibody of interest needs to be purified from the resulting antibody mixture.
Although technologies have been employed to further increase the percentage of bispecific
antibodies in the mixtures of parental and bispecific antibodies and to decrease the
percentage of miss-paired heavy and light chains, there remains a need for bispecific
formats that eliminate or minimize some of the disadvantages mentioned above.
Taken together, the art provides a variety of technologies and methods for generating
monoclonal antibodies, bispecific antibodies, mixtures of monoclonal antibodies, or
mixtures of monospecific and bispecific antibodies that can subsequently be used for
therapeutic application in patients. However, as discussed above, each of these existing
technologies and methods have their drawbacks and limitations.
There is thus a need for improved and/or alternative technologies for producing biological
therapeutics in the form of mixtures or bispecific approaches for targeting multiple disease-
modifying molecules.
SUMMARY OF INVENTION
In a first aspect, the present invention provides an in vitro method for producing at least
two different monospecific CH3 domain-comprising molecules from a single host cell,
wherein each of said two CH3 domain-comprising molecules comprises two CH3 domains
that are capable of forming an interface, said method comprising providing in said cell
st
a. a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide
chain, and
nd
b. a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
st
chain, wherein said 1 CH3 domain-comprising polypeptide chain comprises negatively
charged amino acid residues at amino acid positions 392 and 409 and a positively charged
amino acid residue at amino acid position 399,
nd
and wherein said 2 CH3 domain-comprising polypeptide chain comprises:
- either a wildtype CH3 domain, or
- positively charged amino acid residues at amino acid positions 356 and 357 and
negatively charged amino acid residues at amino acid positions 370 and 439, said method
further comprising the step of culturing said host cell and allowing for expression of said
nucleic acid molecules and harvesting said at least two different CH3 domain-comprising
molecules from the culture.
6
In a second aspect, the present invention provides an in vitro method for producing at least
two different monospecific CH3 domain-comprising molecules from a single host cell,
wherein each of said two CH3 domain-comprising molecules comprises two CH3 domains
that are capable of forming an interface, said method comprising providing in said cell
a) a first nucleic acid molecule encoding a 1st CH3 domain-comprising polypeptide
chain, and
b) a second nucleic acid molecule encoding a 2nd CH3 domain-comprising
polypeptide chain,
wherein said first CH3 domain-comprising polypeptide chain comprises:
- either a wildtype CH3 domain, or
- negatively charged amino acid residues at amino acid positions 392 and 409 and a
positively charged amino acid residue at amino acid position 399,
and wherein said second CH3 domain-comprising polypeptide chain comprises positively
charged amino acid residues at amino acid positions 356 and 357 and negatively charged
amino acid residues at amino acid positions 370 and 439,
said method further comprising the step of culturing said host cell and allowing for
expression of said nucleic acid molecules and harvesting said at least two different CH3
domain-comprising molecules from the culture.
In a third aspect, the present invention provides a mixture of at least two different
monospecific CH3 domain-comprising molecules obtained by the methods according to
the first and second aspects of the invention.
In a fourth aspect, the present invention provides an in vitro recombinant host cell
comprising nucleic acid sequences encoding at least a first and a second CH3 domain-
st
comprising polypeptide chain, wherein said 1 CH3 domain-comprising polypeptide chain
comprises negatively charged amino acid residues at amino acid positions 392 and 409 and
a positively charged amino acid residue at amino acid position 399,
nd
and wherein said 2 CH3 domain-comprising polypeptide chain comprises:
- either a wildtype CH3 domain, or
7
- positively charged amino acid residues at amino acid positions 356 and 357 and
negatively charged amino acid residues at amino acid positions 370 and 439.
In a fifth aspect, the present invention provides an in vitro recombinant host cell
comprising nucleic acid sequences encoding at least a first and a second CH3 domain-
st
comprising polypeptide chain, wherein said 1 CH3 domain-comprising polypeptide chain
comprises:
- either a wildtype CH3 domain, or
- negatively charged amino acid residues at amino acid positions 392 and 409 and a
positively charged amino acid residue at amino acid position 399,
and wherein said second CH3 domain-comprising polypeptide chain comprises positively
charged amino acid residues at amino acid positions 356 and 357 and negatively charged
amino acid residues at amino acid positions 370 and 439.
In a sixth aspect, the present invention provides a pharmaceutical composition comprising
the at least two different monospecific CH3 domain-comprising molecules according to the
third aspect of the invention, and a pharmaceutically acceptable carrier.
In a seventh aspect, the present invention provides an in vitro method for making a host
cell for production of at least two different monospecific CH3 domain-comprising
molecules, the method comprising the step of introducing into said host cell nucleic acid
sequences encoding at least a first and a second CH3 domain-comprising polypeptide
chain,
st
wherein said 1 CH3 domain-comprising polypeptide chain comprises negatively charged
amino acid residues at amino acid positions 392 and 409 and a positively charged amino
acid residue at amino acid position 399,
nd
and wherein said 2 CH3 domain-comprising polypeptide chain comprises:
- either a wildtype CH3 domain, or
- positively charged amino acid residues at amino acid positions 356 and 357 and
negatively charged amino acid residues at amino acid positions 370 and 439, wherein said
nucleic acid sequences are introduced consecutively or concomitantly.
8
In an eighth aspect, the present invention provides an in vitro method for making a host
cell for production of at least two different monospecific CH3 domain-comprising
molecules, the method comprising the step of introducing into said host cell nucleic acid
sequences encoding at least a first and a second CH3 domain-comprising polypeptide
chain,
st
wherein said 1 CH3 domain-comprising polypeptide chain comprises:
- either a wildtype CH3 domain, or
- negatively charged amino acid residues at amino acid positions 392 and 409 and a
positively charged amino acid residue at amino acid position 399,
and wherein said second CH3 domain-comprising polypeptide chain comprises positively
charged amino acid residues at amino acid positions 356 and 357 and negatively charged
amino acid residues at amino acid positions 370 and 439, wherein said nucleic acid
sequences are introduced consecutively or concomitantly.
In a ninth aspect, the present invention provides a culture of recombinant host cells of the
invention, or of recombinant host cells obtained by the method according to the seventh or
eighth aspects of the invention, producing at least two different monospecific CH3
domain-comprising molecules
Certain statements that appear below are broader than what appears in the statements of the
invention above. These statements are provided in the interests of providing the reader
with a better understanding of the invention and its practice. The reader is directed to the
accompanying claim set which defines the scope of the invention.
DESCRIPTION OF THE INVENTION
The invention generally provides methods and means for improved and/or alternative
technologies for producing biological therapeutics in the form of mixtures or bispecific
approaches for targeting multiple disease-modifying molecules. Products and uses
resulting from these methods and means are also described.
9
Various approaches are described in the art in order to promote the formation of a certain
bispecific antibody of interest, thereby reducing the content of undesired antibodies in the
resulting mixture.
For antibodies, it is well-known that the CH3-CH3 interaction is the primary driver for Fc
dimerization (Ellerson JR., et al., J. Immunol 1976 (116) 510-517; Deisenhofer J.
biochemistry 1981 (20) 2361-2370). It is furthermore well-known that when two CH3
domains interact with each other they meet in a protein-protein interface which comprises
"contact" residues (also called contact amino acids, interface residues or interface amino
acids). Contact amino acids of a first CH3 domain interact with one or more contact amino
acids of a second CH3 domain. Contact amino acids are typically within 5.5 Å (preferably
within 4.5 Å) of each other in the three-dimensional structure of an antibody. The
interaction between contact residues from one CH3 domain and contact residues from a
different CH3 domain may for instance be via Van der Waals forces, hydrogen bonds,
water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive
interactions between aromatic side chains, disulfide bonds, or other forces known to one
skilled in the art. It was previously shown that approximately one-third of the contact
amino acid side chains at the human IgG1 CH3 domain interface can account for the
majority of contributions to domain folding and association. It can further be envisaged
that other (neighbouring) amino acid residues may affect the interactions in the protein-
protein interface.
Approaches to interfere with the dimerization of antibody heavy chains have been
employed in the art. Specific engineering in the CH3 domains was applied in order to
favour heterodimerization over homodimerization. Examples of such engineering of the
CH3-CH3 interface include the introduction of complementary protuberance and cavity
mutations, also known as ‘knob-into-hole’ approaches as described for instance in
WO1998/050431, Ridgeway et al., 1996 and Merchant et al. 1998.
Generally, the method involves introducing a protuberance at the interface of a first
polypeptide and a corresponding cavity in the interface of a second polypeptide, such that
the protuberance can be positioned in the cavity so as to promote heteromultimer formation
and hinder homomultimer formation. ''Protuberances'' or “knobs” are constructed by
replacing small amino acid side chains from the interface of the first polypeptide with
larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" or “holes” of
identical or similar size to the protuberances are created in the interface of the second
polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or
threonine). The protuberance and cavity can be made by synthetic means such as altering
the nucleic acid encoding the polypeptides or by peptide synthesis.
Using the knob-into-hole technology alone, the proportion of a bispecific antibody of
interest is at best 87% of the mixture of the 2 parental and bispecific antibodies. Merchant
et al., succeeded in raising the proportion of bispecific antibodies to 95% of the mixture by
introduction of an additional disulfide bond between the two CH3 domains in the CH3-
CH3 interface. Still, in order to use such bispecific antibody as a medicament, the
bispecific antibody has to be purified (separated) from the homodimers and formulated into
a pharmaceutically acceptable diluent or excipient. Purification of heterodimers from such
mixtures poses a major challenge because of the similarity in physico-chemical properties
of the homodimers and heterodimers. Described herein are methods for producing a
bispecific antibody in a single cell clone with a further improved proportion of the
bispecific antibody in the mixture. Accordingly, knob-into-hole technology can thus be
used as one of the means, alone or together with other means, to achieve said further
improved bispecific proportion in a mixture.
Another example of such engineering of the CH3-CH3 interface is provided by a
heterodimeric Fc technology that supports the design of bispecific and asymmetric fusion
proteins by devising strand-exchange engineered domain (SEED) CH3 heterodimers.
These SEED CH3 heterodimers are derivatives of human IgG and IgA CH3 domains that
are composed of alternating segments of human IgA and IgG
CH3 sequences which results in pairs of complementary human SEED CH3 heterodimers,
the so-called SEED-bodies (Davis JH. Et al., Protein Engineering, Design & Selection
2010(23)195-202; WO2007/110205).
Yet another approach for the production of a given bispecific antibody of interest is based
on electrostatic engineering of contact residues within the CH3-CH3 interface that are
naturally charged, as for example described in EP01870459 or US2010/0015133,
WO2007/147901, WO2010/129304, Gunasekaran et al (2010) and WO 2009/089004.
11
These publications describe mutations in the CH3 domains of heavy chains wherein
naturally occurring charged amino acid contact residues are replaced by amino acid
residues of opposite charge (i.e. a charge reversal strategy). This creates an altered charge
polarity across the Fc dimer interface such that co-expression of electrostatically matched
Fc chains support favorable attractive interactions thereby promoting desired Fc
heterodimer formation, whereas unfavorable repulsive charge interactions suppress
unwanted Fc homodimer formation.
It was described that within the CH3-CH3 interface four unique charges residue pairs are
involved in the domain-domain interaction. These are D356/K439’, E357/K370’,
K392/D399’ and D399/K409’ (numbering according to Kabat (1991) where residues in the
first chain are separated from residues in the second chain by ‘/’ and where the prime ( ’ )
indicates the residue numbering in the second chain). As the CH3-CH3 interface displays a
2-fold symmetry, each unique charge pair is represented twice in intact IgG (i.e., also
K439/D356’, K370/E357’, D399/K392’ and K409/D399’ charge interactions are present in
the interface). Taking advantage of this two-fold symmetry, it was demonstrated that a
single charge reversion, e.g. K409D in the first chain, or D399’K in the second chain
resulted in diminished homodimer formation due to repulsion of identical charges.
Combining different charge reversions further enhanced this repulsive effect. It was
demonstrated that expression of different CH3 domains comprising different,
complementary charge reversions, could drive heterodimerization, resulting in an increased
proportion of the bispecific species in the mixture.
Using the approach described above, it is possible to produce a bispecific antibody in a
single cell with proportions ranging between about 76% and about 96%. Described are
methods for producing a bispecific antibody in a single cell with a further improved
percentage of desired bispecific antibodies. As described herein, electrostatic engineering
technology can be used as one of the means, alone or together with other means, e.g knob-
into-hole approaches, to achieve said further improved percentages of desired (bispecific)
antibodies.
12
Described herein is a method for producing at least two different Ig-like molecules from a
single host cell, wherein each of said two Ig-like molecules comprises two CH3 domains
that are capable of forming an interface, said method comprising providing in said cell
st
a) a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain,
nd
b) a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain,
rd
c) a third nucleic acid molecule encoding a 3 CH3 domain-comprising polypeptide chain,
and
th
d) a fourth nucleic acid molecule encoding a 4 CH3 domain-comprising polypeptide
chain,
wherein at least two of said nucleic acid molecules are provided with means for
st nd rd
preferential pairing of said 1 and 2 CH3 domain-comprising polypeptides and said 3
th
and 4 CH3-domain comprising polypeptides, said method further comprising the step of
culturing said host cell and allowing for expression of said at least four nucleic acid
molecules and harvesting said at least two different Ig-like molecules from the culture.
It is often desired to produce more than one (bispecific) antibody, for instance in order to
more efficiently interfere with multiple biological pathways involved in a disease process
or with the invasion, replication and/or spreading of a pathogen.
A mixture of more than one bispecific antibody is also particularly useful for the treatment
of certain diseases. For example, tumor cells use many different strategies to develop
resistance during treatment with antibodies or small molecule drugs. Resistance may
involve multiple cell surface receptors and soluble molecules and it is considered
beneficial to develop antibody-based treatments for cancers that address multiple such
disease- and escape-associated molecules simultaneously. In case more than 2 such
disease- and escape-related target molecules or epitopes are involved, a mixture of
bispecific antibodies provides an innovative and attractive therapeutic format. Preferably,
such mixtures of bispecific antibodies are produced by a single cell to facilitate a drug
development process that is less complicated from a regulatory point of view and cost-
effective and feasible from a drug manufacturing and clinical development point of view.
13
In a single cell-based approach, it is desirable to use methods that allow controlled and
efficient production of the bispecific antibodies, thus reducing or even completely
abrogating the need of separating the desired mixture of bispecific IgG molecules from
non-desired monospecific IgG molecules. In the prior art, mixtures of monospecific and
bispecific antibodies have been produced by a single cell (WO2004/009618), but these
mixtures represent complex concoctions of several different bispecific and monospecific
antibody species. Also described are means and methods for producing defined mixtures of
bispecific antibodies in single cells. Preferably, methods are described which result in
mixtures of (bispecific) antibodies with a proportion of at least 95%, at least 97% or even
more than 99% of dimeric IgG molecules, irrespective of the amount of monomeric by-
products, see herein below. Typically, in a cell where multiple intact IgG molecules are
produced, half molecules (monomeric by-products) may be present that can be simply
removed by size exclusion chromatography known in the art.
In one embodiment described are methods for producing a defined mixture of at least two
different Ig-like molecules in single cells, instead of a single (bispecific) antibody of
interest, wherein the formation of other, undesired dimeric antibody species is diminished
or even absent. The resulting mixture is well defined and its composition is controlled by
the design of CH3 domain mutants. Furthermore, regulation of expression levels and/or
different transfection ratios used for expression affects the composition of the mixture. In a
method as described herein, a first nucleic acid molecule encodes a CH3 domain which
preferentially pairs with a CH3 domain encoded by a second nucleic acid molecule, and a
third nucleic acid molecules encodes a CH3 domain which preferentially pairs with a CH3
domain encoded by a fourth nucleic acid molecule. Also described are mixtures of at least
two different Ig-like molecules obtainable by the methods of the invention.
st nd
As used herein, the term “preferential pairing of said 1 and 2 CH3 domain-comprising
st
polypeptides” means that essentially all the resulting dimers comprising the 1 CH3
nd
domain-comprising polypeptide and/or the 2 CH3 domain-comprising polypeptide will
st nd
be dimers consisting of one 1 CH3 domain-comprising polypeptide paired with one 2
rd
CH3 domain-comprising polypeptide. Likewise, the term “preferential pairing of said 3
14
th
and 4 CH3 domain-comprising polypeptides” means that essentially all of the resulting
rd th
dimers comprising the 3 CH3 domain-comprising polypeptide and/or the 4 CH3
rd
domain-comprising polypeptide will be dimers consisting of one 3 CH3 domain-
th
comprising polypeptide paired with one 4 CH3 domain-comprising polypeptide. As a
result, when nucleic acid molecules encoding four different (A, B, C, D) CH3 domain-
comprising polypeptides are introduced in a single cell, instead of a mixture of 10 different
Ig-like dimers (AA, AB, AC, AD, BB, BC, BD, CC, CD and DD), a mixture of
predominantly two specific Ig-like molecules is produced.
As explained herein below in more detail, in a preferred embodiment said first CH3-
domain comprising polypeptide chain comprises the amino acid substitution T366K, and
said second CH3-domain comprising polypeptide chain comprises the amino acid
substitution L351D. These amino acid changes are preferred means for preferential pairing
of said first and second CH3-domain comprising polypeptide chains. Said first CH3-
domain comprising polypeptide chain preferably further comprises the amino acid
substitution L351K. Moreover, said second CH3-domain comprising polypeptide chain
preferably further comprises an amino acid substitution selected from the group consisting
of Y349E, Y349D and L368E, most preferably L368E. In yet another preferred
embodiment, said third CH3-domain comprising polypeptide chain comprises the amino
acid substitutions E356K and D399K, and said fourth CH3-domain comprising
polypeptide chain comprises the amino acid substitutions K392D and K409D.
In a method as described herein, each of the CH3-domain comprising polypeptide chains
preferably further comprises a variable region recognizing a target epitope. The variable
regions that are part of the CH3-domain comprising polypeptide chains preferably share a
common light chain. In that case only the VHs of the variable regions differ whereas the
VL in all variable regions is essentially the same. Hence, in a preferred embodiment,
described is a method which further comprises providing said host cell with a nucleic acid
molecule encoding a common light chain. In one particularly preferred embodiment, each
of said 4 variable regions of the 4 CH3-domain comprising polypeptide chains recognizes
a different target epitope. For instance, if the first nucleic acid molecule encodes a heavy
chain that further contains a variable domain with specificity for antigen A, the second
nucleic acid molecule encodes a heavy chain that further contains a variable domain with
specificity for antigen B, the third nucleic acid molecule encodes a heavy chain that further
contains a variable domain with specificity for antigen C, and the fourth nucleic acid
molecule encodes a heavy chain that further contains a variable domain with specificity for
antigen D, a mixture will then be produced containing bispecific Ig-like molecules that are
specific for AB and bispecific Ig-like molecules that are specific for CD. The formation of
monospecific antibodies (with AA, BB, CC or DD specificity) or bispecific antibodies with
specificity for AC, AD, BC or BD is lowered or even absent due to the means for
st nd rd
preferential pairing of said 1 and 2 CH3 domain-comprising polypeptides and said 3
th
and 4 CH3 domain-comprising polypeptides. It is, of course, possible to use further
th th
nucleic acid molecules, for instance encoding a 5 and a 6 CH3 domain-comprising
polypeptide, in order to produce defined mixtures comprising more than two different Ig-
like molecules.
Of note, the ratio of the nucleic acids used in a method as described herein does not need to
be 1:1:1:1 and the ratio of the resulting Ig-like molecules that are expressed does not need
to be 1:1. It is possible to use means known in the art to produce mixtures of antibodies
with optimized ratios. For instance, expression levels of nucleic acid molecules and hence
the ratios of the resulting Ig-like molecules produced may be regulated by using different
genetic elements such as promoters, enhancers and repressors or by controlling the
genomic integration site of copy number of the DNA constructs encoding antibodies.
Said means for preferential pairing preferably may comprise engineered complementary
knob-into-hole mutations, disulfide bridges, charge mutations including charge reversal
mutations, or combinations thereof. The skilled person will appreciate that said means for
preferential pairing may be chosen within a certain type of mutations, i.e. all at least 4
nucleic acid molecules encoding CH3-domain comprising polypeptide chains may for
example comprise charge mutations as means for preferential pairing. Additionally, also
non-engineered wildtype CH3 may in certain instances be used for preferential pairing of
two wildtype CH3-domain comprising polypeptide chains. In a particularly preferred
embodiment, said means for preferential pairing comprise at least one CH3 mutation
selected from Table B, as explained elsewhere in this application. One preferred
embodiment describes a method wherein all 4 of said nucleic acid molecules are provided
st nd
with means for preferential pairing of said 1 and 2 CH3 domain-comprising
16
rd th
polypeptides and said 3 and 4 CH3-domain comprising polypeptides, wherein said
st nd
means for preferential pairing of said 1 and 2 CH3 domain-comprising polypeptides are
rd th
different from those means for preferential pairing of said 3 and 4 CH3-domain
comprising polypeptides.
st nd
Also described is a method wherein said means for preferential pairing of said 1 and 2
CH3 domain-comprising polypeptides are different from said means for preferential
rd th
pairing of said 3 and 4 CH3-domain comprising polypeptides. By ‘different’ it is meant
st nd
that the means for preferential pairing of said 1 and 2 CH3 domain comprising
st nd
polypeptides are designed such that preferential pairing of the 1 and 2 chain is
st rd
favoured. The design is such that essentially no interaction between the 1 and the 3
th
and/or 4 CH3 domain comprising polypeptide chain will take place. In other words,
st rd th
dimerization between said 1 CH3 domain comprising polypeptide and said 3 or 4
rd th
polypeptide is reduced to essentially zero and so forth. The 3 and the 4 CH3 domain-
comprising polypeptides may either be wildtype or may comprise means for preferential
st nd
pairing that are different from the means for preferential pairing of the 1 and 2 CH3
domains. Current studies have focused on the production of a single bispecific antibody,
using for instance the knob-into-hole technology or mutations (reversions) of charged
contact amino acids present in CH3 domains. Production of defined mixtures of at least
two (bispecific) Ig-like molecules, without significant co-production of other dimeric by-
products, has, however, not been realized prior to the present invention.
Also described are methods for the efficient and controlled production of a well-defined
mixture of Ig-like molecules, with a high proportion of bispecifics in the mixture. Even a
proportion of (two) bispecifics of at least 95%, at least 97% or more is obtained in a system
where two bispecifics are desired. This means that only at most 5%, at most 3% or less
monospecific bivalent by-products are obtained. Of note, the amount of monomeric by-
products, i.e. half molecules, is less important since these half-molecules are easily
separated from dimers using their size difference.
st nd
In another preferred embodiment, the variable regions of the 1 and the 2 CH3-domain
comprising polypeptide chains recognize different target epitopes, whereas the variable
17
rd th
regions of the 3 and the 4 CH3-domain comprising polypeptide chains recognize the
same target epitopes. This will result in the predominant production of one kind of
bispecific Ig-like molecule and one kind of monospecific Ig-like molecule. For instance, if
st nd
the variable regions of the 1 and the 2 CH3-domain comprising polypeptide chains
rd th
recognize different target epitopes and if the variable regions of the 3 and the 4 CH3-
domain comprising polypeptide chains both recognize the same target epitope which is
st nd
different from the target epitopes recognized by the 1 and the 2 CH3-domains, a mixture
of Ig-like molecules having specificity for AB or CC will be formed. Also described is a
th
method wherein the target epitope recognized by the variable regions of the 3rd and 4
CH3 domain comprising polypeptide chain is the same, but different from the target
st nd
epitope recognized by the variable region of the 1 or the 2 CH3-domain comprising
polypeptide chain.
st nd
Alternatively, when the variable regions of the 1 and the 2 CH3-domain comprising
polypeptide chains recognize different target epitopes and when the variable regions of the
rd th
3 and the 4 CH3-domain comprising polypeptide chains both recognize the same
st nd
epitope as the 1 or the 2 CH3-domain comprising polypeptide chains, a mixture of Ig-
like molecules having specificity for AB and AA, or AB and BB will be formed. A method
th
wherein the target epitope recognized by the variable regions of the 3rd and 4 CH3
domain comprising polypeptide chain is the same as the target epitope recognized by the
st nd
variable region of the 1 or the 2 CH3-domain comprising polypeptide chain is therefore
also described herein.
Also described are means and methods for producing defined mixtures of bispecific
antibodies and monospecific antibodies in a single cell culture. A non-limiting example of
such well-defined mixture is a mixture of bispecific antibodies with specificity AB and
monospecific antibodies with specificity AA. Another example is a mixture of bispecific
antibodies with specificity AB and monospecific antibodies with specificity BB. Yet
another example is a mixture of bispecific antibodies with specificity AB and monospecific
antibodies with specificity CC. Again, preferably means and methods are described which
yield mixtures of antibodies of interest with at least 90%, more preferably at least 95% and
most preferably at least 97% or even more than 99% of desired antibodies.
18
st
In yet another embodiment, described is a method wherein the variable regions of the 1
nd
and the 2 CH3-domain comprising polypeptide chains recognize the same target epitope,
rd th
whereas the variable regions of the 3 and the 4 CH3-domain comprising polypeptide
chains recognize a second target epitope which differs from the target epitope recognized
st nd
by said 1 and 2 variable regions. This will result in the predominant production of
monospecific Ig-like molecules having either specificity for AA or specificity for BB. The
formation of bispecific Ig-like molecules is diminished or even avoided. In several
embodiments it is preferred to produce mixtures of monospecific antibodies in a single
cell, rather than mixtures of bispecific antibodies. For instance when cross-linking of two
identical target molecules is desired, or when two targets are located too far away from
each other so that they cannot be bound by a single bispecific antibody. It can also be
advantageous to produce mixtures of monospecific antibodies in a single cell as the
mixture can be regarded as a single therapeutic product. In the art, the therapeutic efficacy
and safety of various monospecific antibodies has already been proven and market
authorisation has been obtained. Production of mixtures of monospecific antibodies in a
single cell will thus facilitate the testing for efficacy and safety of several of such mixtures
and will reduce the efforts and costs for regulatory approval and manufacturing. There are,
however, currently no methods available for producing specific mixtures of monospecific
antibodies in a single cell wherein the formation of bispecific by-products is reduced to
below 5%. Also described are means and methods for producing such well-defined
homodimeric antibody mixtures in single cells wherein the formation of bispecific
antibodies is reduced to below 5%.
Hence, a method as described herein is suitable for the production of any desired mixture
of bispecific and/or monospecific Ig-like molecules. Again, it is possible to use further
th th th th
nucleic acid molecules, for instance encoding a 5 and a 6 (and 7 and 8 and so forth)
CH3 domain-comprising polypeptide, in order to produce defined mixtures comprising
more than two different Ig-like molecules.
19
Preferably, in a method according to the present invention at least two CH3 domains are
used that comprise at least one combination of mutations as described herein. Through
these mutations novel specific interactions are formed between two CH3 domains. These
mutations useful in the present invention are discussed below in more detail.
The term ‘Ig-like molecule’ as used herein means a proteinaceous molecule that possesses
at least one immunoglobulin (Ig) domain. Said Ig-like molecule comprises a sequence
comprising the function of at least an immunoglobulin CH3 domain, preferably the
sequence comprises an IgG1 CH3 domain. Proteinaceous molecules that possess at least a
CH3 domain can be further equipped with specific binding moieties. The CH3 domains
useful in the present invention, containing means for preferential pairing, can thus be used
for preferential pairing of two CH3-domain comprising proteinaceous molecules to design
desired heterodimeric binding molecules or mixtures of binding molecules. Binding
moieties that can be engineered to the CH3-domain comprising proteinaceous molecules
can be any binding agent, including, but not limited to, single chain Fvs, single chain or
Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, a BiTE®, a Fab,
ankyrin repeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody,
Adnectins®, Affilins®, Trans-bodies®, Affibodies®, a TrimerX®, MicroProteins,
Fynomers®, Centyrins® or a KALBITOR®. In a preferred embodiment, the binding
moieties are antibody variable regions (i.e. VH/VL combinations). Variable regions that
are part of the CH3-domain comprising polypeptide chains preferably share a common
light chain. In that case, only the VHs of the variable regions differ whereas the VL in all
variable regions is essentially the same.
Alternatively, or in addition, other molecules can be engineered to the CH3 domains useful
in the present invention, including cytokines, hormones, soluble ligands, receptors and/or
peptides.
In a more preferred embodiment, said Ig-like molecule comprises a full length Fc
backbone. In a most preferred embodiment, the Ig-like molecules are antibodies. The
variable regions of these antibodies preferably share a common light chain, but they may
differ in their VH regions. The term ‘antibody’ as used herein means a proteinaceous
molecule belonging to the immunoglobulin class of proteins, containing one or more
domains that bind an epitope on an antigen, where such domains are derived from or share
sequence homology with the variable region of an antibody. Antibodies are known in the
art and include several isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM.
An antibody as described herein may be any of these isotypes, or a functional derivative
and/or fragment of these. In a preferred embodiment, Ig-like molecules are produced that
are antibodies of the IgG isotype because IgG antibodies e.g. have a longer half life as
compared to antibodies of other isotypes.
Antibodies produced with methods according to the present invention can have sequences
of any origin, including murine and human sequences. Antibodies can consist of sequences
from one origin only, such as fully human antibodies, or they can have sequences of more
than one origin, resulting for instance in chimeric or humanized antibodies. Antibodies for
therapeutic use are preferably as close to natural antibodies of the subject to be treated as
possible (for instance human antibodies for human subjects). Antibody binding can be
expressed in terms of specificity and affinity. The specificity determines which antigen or
epitope thereof is bound by the binding domain. The affinity is a measure for the strength
of binding to a particular antigen or epitope. Specific binding is defined as binding with
-5 -7
affinities (K ) of at least 1x10 M, more preferably 1x10 M, more preferably higher than
D
-9
1x10 M. Typically, monoclonal antibodies for therapeutic applications have affinities of
-10
up to 1x10 M or even higher. The term ‘antigen’ as used herein means a substance or
molecule that, when introduced into the body, triggers the production of an antibody by the
immune system. An antigen, among others, may be derived from pathogenic organisms,
tumor cells or other aberrant cells, from haptens, or even from self structures. At the
molecular level, an antigen is characterized by its ability to be bound by the antigen-
binding site of an antibody. Also mixtures of antigens can be regarded as ‘antigen’, i.e. the
skilled person would appreciate that sometimes a lysate of tumor cells, or viral particles
may be indicated as ‘antigen’ whereas such tumor cell lysate or viral particle preparation
exists of many antigenic determinants. An antigen comprises at least one, but often more,
epitopes. The term ‘epitope’ as used herein means a part of an antigen that is recognized by
the immune system, specifically by antibodies, B cells, or T cells. Although epitopes are
usually thought to be derived from non-self proteins, sequences derived from the host that
can be recognized are also classified as epitopes.
21
The term ‘CH3 domain’ is well known in the art. The IgG structure has four chains, two
light and two heavy chains; each light chain has two domains, the variable and the constant
light chain (VL and CL) and each heavy chain has four domains, the variable heavy chain
(VH) and three constant heavy chain domains (CH1, CH2, CH3). The CH2 and CH3
domain region of the heavy chain is called Fc (Fragment crystallizable) portion, Fc
fragment, Fc backbone or simply Fc. The IgG molecule is a heterotetramer having two
heavy chains that are held together by disulfide bonds (-S-S-) at the hinge region and two
light chains. The heavy chains dimerize through interactions at the CH3-CH3 domain
interface and through interactions at the hinge region. The number of hinge disulfide bonds
varies among the immunoglobulin subclasses (Papadea and Check 1989). The Fc fragment
of an immunoglobulin molecule is a dimer of the two C-terminal constant regions, i.e. CH2
and CH3 domains, of the heavy chain. Among its physiological functions are interactions
with the complement system and with specific receptors on the surface of a variety of cells.
Interactions between the CH3 domains of two individual heavy chains are known to play
an important role in driving heavy chain dimerization. Thus, CH3 domains direct the
association of antibody heavy chains, and it is known that the interface between CH3
domains contains more than 20 contact residues from each chain that play a role in the
CH3-CH3 interaction (Deisenhofer J., Biochemistry 1981(20)2361-2370; Miller S., J. Mol.
Biol. 1990(216)965-973; Padlan, Advances in Protein Chemistry 1996 (49) 57-133). The
CH3 variants useful in the present invention can thus be used in association with other
antibody domains to generate full length antibodies that are either bispecific or
monospecific. The specificity of the antibody as defined by the VH/VL combinations
typically does not affect the heavy chain dimerization behaviour that is driven by the CH3
domains.
The terms ‘contact residue’, ‘contact amino acid’, ‘interface residue’ and ‘interface amino
acid’ as used herein typically refers to any amino acid residue present in the CH3 domain
that can be involved in interdomain contacts, as can be calculated by technologies known
in the art, including calculating solvent accessible surface area (ASA) of the CH3 domain
residues in the presence and absence of the second chain (Lee and Richards J. Mol.
2
Biol.1971(55)379) where residues that show difference (> lÅ ) in ASA between the two
22
calculations are identified as contact residues. Contact residues that have been identified
include residues at positions 347, 349, 350, 351, 352, 353, 354, 355, 356, 357, 360, 364,
366, 368, 370, 390, 392, 394, 395, 397, 399, 400, 405, 407, 409, 439 according to the EU
numbering system (Table A).
23
Table A: List of CH3 domain interface residues
Interface residue Contacting residues in chain
in chain A B
Q347 K360
Y349 S354, D356, E357, K360
T350 S354, R355
L351 L351, P352, P353, S354,
T366
S354 Y349, T350, L351
R355 T350
D356 Y349, K439
E357 Y349, K370
K360 Q347, Y349
S364 L368, K370
T366 L351, Y407
L368 S364, K409
K370 E357, S364
N390 S400
K392 L398, D399, S400, F405
T394 T394, V397, F405, Y407
P395 V397
V397 T394, P395
D399 K392, K409
S400 N390, K392
F405 K392, T394, K409
Y407 T366, T394, Y407, K409
K409 L368, D399, F405, Y407
K439 D356
Contact residues within the CH3-CH3 interface can either be amino acids that are charged,
or amino acid residues that are neutral. The term ‘charged amino acid residue’ or ‘charged
residue’ as used herein means amino acid residues with electrically charged side chains.
These can either be positively charged side chains, such as present in arginine (Arg, R),
histidine (His, H) and lysine (Lys, K) or can be negatively charged side chains, such as
present in aspartic acid (Asp, D) and glutamic acid (Glu, E). The term ‘neutral amino acid
residue’ or neutral residue as used herein refers to all other amino acids that do not carry
electrically charged side chains. These neutral residues include serine (Ser, S), threonine
(Thr, T), asparagine (Asn, N), glutamine (GLu, Q), Cysteine (Cys, C), glycine (Gly, G),
proline (Pro, P), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L),
methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, T).
24
The term ‘CH3-CH3 domain interface’, or ‘CH3 interface’, ‘CH3-CH3 pairing’, ‘domain
interface’ or simply ‘interface’, as used herein, refers to the association between two CH3
domains of separate CH3-domain comprising polypeptides that is a result of interacting
amino acid residues, i.e. at least one interaction between an amino acid of a first CH3
domain and an amino acid of a second CH3 domain. Such interaction is for instance via
Van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or
other electrostatic forces, attractive interactions between aromatic side chains, the
formation of disulfide bonds, or other forces known to one skilled in the art.
As used herein, said means for preferential pairing of the first and second CH3 domain-
comprising polypeptides and said third and fourth CH3 domain-comprising polypeptide
can be any means known in the art. In one embodiment, at least one nucleic acid molecule
encodes a CH3 domain which contains at a contact residue position a large amino acid
residue (i.e. a “knob” or “protuberance”) such as for instance R, F, Y, W, I or L, whereas at
least one other nucleic acid molecule encodes a CH3 domain which contains at a
complementary contact residue position a small amino acid residue (i.e. a “hole” or
“cavity”) such as for instance G, A, S, T or V. The resulting CH3 domains will
preferentially pair with each other due to the steric conformation of said contact amino
acids. The knob-into-hole technology is described herein before in more detail. In a further
embodiment, at least one nucleic acid molecule encodes a CH3 domain which contains at a
contact residue position that is naturally charged, i.e. a naturally occurring K, H, R, D or E,
an amino acid that now carries the opposite charge as compared to wildtype, whereas at
least one other nucleic acid molecule encodes a CH3 domain which contains at a
complementary contact residue position that is naturally charged, an amino acid that now
carries the opposite charge as compared to wildtype. The resulting engineered CH3
domains will preferentially pair with each other due to the opposite charges of said contact
amino acids, whereas pairing of identical CH3 domains will be diminished due to
electrostatic repulsion. In one embodiment, CH3 mutations as described in EP01870459,
WO 2009/089004, Gunasekaran et al (2010), are used. In one embodiment, the means for
st nd
preferential pairing of said 1 and 2 CH3 domain-comprising polypeptides are “knob”
th th
and 4
and “hole” amino acid residues and the means for preferential pairing of said 3
CH3 domain-comprising polypeptides are charge-engineered amino acids. Preferably, both
st nd
said means for preferential pairing of said 1 and 2 CH3 domain-comprising polypeptides
th th
and said 3 and 4 CH3 domain-comprising polypeptides are charge-engineered amino
acids. In one embodiment, different amino acid residues are engineered for preferential
st nd
pairing of said 1 and 2 CH3 domain-comprising polypeptides as compared to the amino
th th
acid residues that are engineered for preferential pairing of said 3 and 4 CH3 domain-
comprising polypeptides. In a particularly preferred embodiment at least a first and a
second nucleic acid molecule encode CH3 domains with novel mutations as described
herein. As described herein below in more detail, described are novel CH3 mutations
which enable the production of certain bispecific Ig-like molecules of interest without a
significant amount of undesired (dimeric) by-products. Also described are novel CH3
mutations which enable the production of certain monospecific Ig-like molecules of
interest without a significant amount of undesired (dimeric) by-products. The use of at
least one of these CH3 mutations is, therefore, preferred.
The term ‘polypeptide’, ‘polypeptide molecule’ or ‘polypeptide chain’ as used herein
refers to a chain of amino acids that are covalently joined together through peptide bonds.
Proteins are typically made up of one or more polypeptide molecules. One end of every
polypeptide, called the amino terminal or N-terminal, has a free amino group. The other
end, with its free carboxyl group, is called the carboxyl terminal or C-terminal.
Polypeptides as described herein may have gone through post-translational modification
processes and may e.g. be glycosylated. The CH3 domain-comprising polypeptide chains
as described herein thus refer to polypeptide chains that at least encompass an Ig CH3
domain and that may have gone through post-translational modification processes.
The term “nucleic acid molecule” as used herein is defined as a molecule comprising a
chain of nucleotides, more preferably DNA and/or RNA. In one embodiment, double-
stranded RNA is used. In other embodiments a nucleic acid molecule comprises other
kinds of nucleic acid structures such as for instance a DNA/RNA helix, peptide nucleic
acid (PNA), locked nucleic acid (LNA) and/or a ribozyme. Hence, the term “nucleic acid
molecule” also encompasses a chain comprising non-natural nucleotides, modified
26
nucleotides and/or non-nucleotide building blocks which exhibit the same function as
natural nucleotides.
Also described is a method for making a host cell for production of at least two different
Ig-like molecules, the method comprising the step of introducing into said host cell nucleic
acid sequences encoding at least a first, a second, a third and a fourth CH3-domain
comprising polypeptide chain, wherein at least two of said nucleic acid sequences are
provided with means for preferential pairing of said first and second CH3-domain
comprising polypeptides and said third and fourth CH3-domain comprising polypeptides,
wherein said nucleic acid sequences are introduced consecutively or concomitantly.
Also described is a method for making a host cell for production of a heterodimeric Ig-like
molecule, the method comprising the step of introducing into said host cell nucleic acid
sequences encoding at least a first and a second CH3-domain comprising polypeptide
chain, wherein said first CH3 domain-comprising polypeptide chain comprises at least one
substitution of a neutral amino acid residue by a positively charged amino acid residue and
wherein said second CH3 domain-comprising polypeptide chain comprises at least one
substitution of a neutral amino acid residue by a negatively charged amino acid residue,
wherein said nucleic acid sequences are introduced consecutively or concomitantly. Said
methods for making said host cells preferably further comprise the step of introducing into
said host cell a nucleic acid sequence encoding a common light chain.
Also described herein is a recombinant host cell comprising nucleic acid sequences
encoding at least a first, a second, a third and a fourth CH3-domain comprising polypeptide
chain, wherein at least two of said nucleic acid molecules are provided with means for
preferential pairing of said first and second CH3-domain comprising polypeptides and said
third and fourth CH3-domain comprising polypeptides.
Also described is a recombinant host cell comprising nucleic acid sequences encoding at
least a first and a second CH3-domain comprising polypeptide chain, wherein said first
CH3 domain-comprising polypeptide chain comprises at least one substitution of a neutral
amino acid residue by a positively charged amino acid residue and wherein said second
27
CH3 domain-comprising polypeptide chain comprises at least one substitution of a neutral
amino acid residue by a negatively charged amino acid residue.
A recombinant host cell according to the invention preferably further comprises a nucleic
acid sequence encoding a common light chain.
A "host cell" according to the invention may be any host cell capable of expressing
recombinant DNA molecules, including bacteria such as for instance Escherichia (e.g. E.
coli), Enterobacter, Salmonalla, Bacillus, Pseudomonas, Streptomyces, yeasts such as S.
cerevisiae, K. lactis, P. pastoris, Candida, or Yarrowia, filamentous fungi such as
Neurospora, Aspergillus oryzae, Aspergillus nidulans and Aspergillus niger, insect cells
such as Spodoptera frugiperda SF-9 or SF-21 cells, and preferably mammalian cells such
as Chinese hamster ovary (CHO) cells, BHK cells, mouse cells including SP2/0 cells and
NS-0 myeloma cells, primate cells such as COS and Vero cells, MDCK cells, BRL 3A
cells, hybridomas, tumor-cells, immortalized primary cells, human cells such as W138,
HepG2, HeLa, HEK293, HT1080 or embryonic retina cells such as PER. C6, and the like.
Often, the expression system of choice will involve a mammalian cell expression vector
and host so that the antibodies can be appropriately glycosylated. A human cell line,
preferably PER.C6, can advantageously be used to obtain antibodies with a completely
human glycosylation pattern. The conditions for growing or multiplying cells (see e. g.
Tissue Culture, Academic Press, Kruse and Paterson, editors (1973)) and the conditions for
expression of the recombinant product may differ somewhat, and optimization of the
process is usually performed to increase the product proportions and/or growth of the cells
with respect to each other, according to methods generally known to the person skilled in
the art. In general, principles, protocols, and practical techniques for maximizing the
productivity of mammalian cell cultures can be found in Mammalian Cell Biotechnology: a
Practical Approach (M. Butler, ed., IRL Press, 1991). Expression of antibodies in
recombinant host cells has been extensively described in the art (see e.g. EP0120694;
EP0314161; EP0481790; EP0523949; US patent 4,816,567; WO 00/63403). The nucleic
acid molecules encoding the light and heavy chains may be present as extrachromosomal
copies and/or stably integrated into the chromosome of the host cell, the latter is preferred.
28
Also described is a culture of recombinant host cells according to the invention or as
described herein, or a culture of recombinant host cells obtainable or obtained by a method
according to the invention or as described herein, said culture either producing at least two
different Ig-like molecules or a heterodimeric Ig-like molecule.
To obtain expression of nucleic acid sequences encoding the CH3 domain-comprising
polypeptides, it is well known to those skilled in the art that sequences capable of driving
such expression can be functionally linked to the nucleic acid sequences encoding the CH3
domain-comprising polypeptides. Functionally linked is meant to describe that the nucleic
acid sequences encoding the CH3 domain-comprising polypeptides or precursors thereof is
linked to the sequences capable of driving expression such that these sequences can drive
expression of the CH3 domain-comprising polypeptides or precursors thereof. Useful
expression vectors are available in the art, e.g. the pcDNA vector series of Invitrogen.
Where the sequence encoding the polypeptide of interest is properly inserted with
reference to sequences governing the transcription and translation of the encoded
polypeptide, the resulting expression cassette is useful to produce the polypeptide of
interest, referred to as expression. Sequences driving expression may include promoters,
enhancers and the like, and combinations thereof. These should be capable of functioning
in the host cell, thereby driving expression of the nucleic acid sequences that are
functionally linked to them. Promoters can be constitutive or regulated, and can be
obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or
artificially designed. Expression of nucleic acids of interest may be from the natural
promoter or derivative thereof or from an entirely heterologous promoter. Some well-
known and much used promoters for expression in eukaryotic cells comprise promoters
derived from viruses, such as adenovirus, e.g. the E1A promoter, promoters derived from
cytomegalovirus (CMV), such as the CMV immediate early (IE) promoter, promoters
derived from Simian Virus 40 (SV40), and the like. Suitable promoters can also be derived
from eukaryotic cells, such as methallothionein (MT) promoters, elongation factor 1 α (EF-
1 α) promoter, actin promoter, an immunoglobulin promoter, heat shock promoters, and the
like. Any promoter or enhancer/promoter capable of driving expression of the sequence of
interest in the host cell is suitable in the invention. In one embodiment the sequence
29
capable of driving expression comprises a region from a CMV promoter, preferably the
region comprising nucleotides –735 to +95 of the CMV immediate early gene
enhancer/promoter. The skilled artisan will be aware that the expression sequences used in
the invention may suitably be combined with elements that can stabilize or enhance
expression, such as insulators, matrix attachment regions, STAR elements (WO
03/004704), and the like. This may enhance the stability and/or levels of expression.
Protein production in recombinant host cells has been extensively described, e.g. in
Current Protocols in Protein Science, 1995, Coligan JE, Dunn BM, Ploegh HL, Speicher
DW, Wingfield PT, ISBN 011184-8; Bendig, 1988. Culturing a cell is done to enable
it to metabolize, and/or grow and/or divide and/or produce recombinant proteins of
interest. This can be accomplished by methods well known to persons skilled in the art,
and includes but is not limited to providing nutrients for the cell. The methods comprise
growth adhering to surfaces, growth in suspension, or combinations thereof. Several
culturing conditions can be optimized by methods well known in the art to optimize protein
production yields. Culturing can be done for instance in dishes, roller bottles or in
bioreactors, using batch, fed-batch, continuous systems, hollow fiber, and the like. In order
to achieve large scale (continuous) production of recombinant proteins through cell culture
it is preferred in the art to have cells capable of growing in suspension, and it is preferred
to have cells capable of being cultured in the absence of animal- or human-derived serum
or animal- or human-derived serum components. Thus purification is easier and safety is
enhanced due to the absence of additional animal or human proteins derived from the
culture medium, while the system is also very reliable as synthetic media are the best in
reproducibility.
Ig-like molecules are expressed in host cells and are harvested from the cells or, preferably,
from the cell culture medium by methods that are generally known to the person skilled in
the art. After harvesting, these Ig-like molecules may be purified by using methods known
in the art. Such methods may include precipitation, centrifugation, filtration, size-exclusion
chromatography, affinity chromatography, cation- and/or anion-exchange chromatography,
hydrophobic interaction chromatography, and the like. For a mixture of antibodies
comprising IgG molecules, protein A or protein G affinity chromatography can be suitably
used (see e.g. US patents 4,801,687 and 5,151,504).
Ig-like molecules, and/or mixtures thereof, produced with methods according to the present
invention preferably have a common light chain. Also described is a method further
comprising providing said host cell with a nucleic acid molecule encoding a common light
chain. This is a light chain that is capable of pairing with at least two different heavy
chains, thereby forming functional antigen binding domains. A functional antigen binding
domain is capable of specifically binding to an antigen. Preferably, a common light chain
is used that is capable of pairing with all heavy chains produced with a method according
to the invention, thereby forming functional antigen binding domains, so that mispairing of
unmatched heavy and light chains is avoided. In one aspect, only common light chains
with one identical amino acid sequence are used. Alternatively, those of skill in the art will
recognize that “common” also refers to functional equivalents of the light chain of which
the amino acid sequence is not identical. Many variants of said light chain exist wherein
mutations (deletions, substitutions, additions) are present that do not materially influence
the formation of functional binding regions. Such variants are thus also capable of binding
different heavy chains and forming functional antigen binding domains. The term
‘common light chain’ as used herein thus refers to light chains which may be identical or
have some amino acid sequence differences while retaining the binding specificity of the
resulting antibody after pairing with a heavy chain. It is for instance possible to prepare or
find light chains that are not identical but still functionally equivalent, e.g. by introducing
and testing conservative amino acid changes, and/or changes of amino acids in regions that
do not or only partly contribute to binding specificity when paired with the heavy chain,
and the like. A combination of a certain common light chain and such functionally
equivalent variants is encompassed within the term “common light chain”. Reference is
made to WO 2004/009618 for a detailed description of the use of common light chains.
Preferably, a common light chain is used in the present invention which is a germline-like
light chain, more preferably a germline light chain, preferably a rearranged germline
human kappa light chain, most preferably either the rearranged germline human kappa
light chain IgVκ1-39/Jκ or IGVκ3-20/Jκ.
31
Alternatively, the skilled person may select, as an alternative to using a common light
chain and to avoid mispairing of unmatched heavy and light chains, means for forced
pairing of the heavy and light chain, such as for example described in WO2009/080251,
WO2009/080252 and/or WO2009/080253.
Described herein are novel engineered CH3 domains as well as novel combinations of
CH3 mutations. Before the present invention, charged contact amino acids of CH3
domains that were known to be involved in CH3-CH3 pairing were substituted by amino
acids of opposite charge (charge reversal), thereby influencing the CH3-CH3 pairing. The
mutations useful according to the present invention are an inventive alternative to this
approach, because now CH3 amino acids that are non-charged or neutral in wildtype CH3
are substituted with charged residues. The present description in this embodiment does not
exchange charged contact amino acids by amino acids of opposite charge but substitutes
non-charged CH3 amino acids for charged ones. The approach of the present invention
provides not only a method for efficiently steering the dimerization of CH3 domains but
also has the advantage that at least one additional charge-charge interaction in the CH3
interface is created. In view of this additional charge-charge interaction on top of the
existing charge-pairs in the CH3-CH3 interface, the dimers according to the invention are
generally more stable as compared to the wild type dimers (the wild type dimer is defined
as a bispecific IgG (AB) without CH3 engineering in contrast to its parental homodimers
(AA or BB)). Moreover, it has surprisingly become possible to increase the proportion of
one or more Ig-like molecules of interest in a mixture even further. As described herein
before, methods known in the art for preferential production of a bispecific antibody
typically involves the production of some undesired dimeric side products. For instance,
the proportion of a bispecific antibody of interest using the knob-into-hole technology is at
best 87%, whereas the electrostatic engineering approach wherein charged contact amino
acids are substituted by amino acids of opposite charge, also results in proportions of up to
96% (see for instance Example 11). Quite surprisingly, the present inventors have
succeeded in introducing mutations that further enhance the proportion of an Ig-like
molecule of interest in a mixture. For instance, Example 17 discloses a method using
mutations according to the present description, wherein the proportion of a bispecific
32
antibody of interest was raised to such extent that no dimeric by-product was detectable in
the resulting mixture at all. Unpaired half-molecules consisting of only a single heavy
chain paired with a common light chain were present to some extent in the mixtures, but
these are the result of unbalanced expression of the heavy chains and can be easily
separated from the mixture by size exclusion chromatography. Hence, with such mutations,
a bispecific Ig-like molecule can be produced in a single cell with a high proportion with
essentially no contaminating dimeric by-products being present, which is particularly
suitable for the production of a pharmaceutical composition.
One preferred embodiment describes a method for producing a heterodimeric Ig-like
molecule from a single cell, wherein said Ig-like molecule comprises two CH3 domains
that are capable of forming an interface, said method comprising providing in said cell
st
a. A first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide
chain,
nd
b. A second nucleic acid molecule encoding a 2 CH3 domain-comprising
polypeptide chain,
wherein said first CH3 domain-comprising polypeptide chain comprises at least one
substitution of a neutral amino acid residue by a positively charged amino acid residue and
wherein said second CH3 domain-comprising polypeptide chain comprises at least one
substitution of a neutral amino acid residue by a negatively charged amino acid residue,
said method further comprising the step of culturing said host cell and allowing for
expression of said two nucleic acid molecules and harvesting said heterodimeric Ig-like
molecule from the culture.
Said method preferably further comprises the step of providing said host cell with a
nucleic acid molecule encoding a common light chain, which has advantages as outlined
herein before.
The amino acids at position 366 of one CH3 domain and position 351 of a second CH3
domain have been reported to be a pair of contact residues in the CH3-CH3 interface,
meaning that they are located sufficiently close to each other in the three-dimensional
conformation of the resulting Ig-like molecule in order to be capable of interacting with
33
each other. Hence, the first CH3 domain will preferentially pair with the second CH3
domain.
In one embodiment, threonine (T) at position 366 of a first CH3 domain is replaced by a
first charged amino acid and leucine (L) at position 351 of a second CH3 domain is
replaced by a second charged amino acid, wherein said first and second charged amino
acids are of opposite charge. If the first CH3 domain-comprising polypeptide, that carries a
charged residue at position 366, further comprises a variable domain which has specificity
for antigen A, and if the second CH3 domain-comprising polypeptide, that carries an
oppositely charged residue at position 351, further comprises a variable domain which has
specificity for antigen B, bispecific Ig-like molecules with an AB specificity will be
predominantly formed. Also described is a method, wherein said means for preferential
st nd
pairing of said 1 and 2 CH3 domain-comprising polypeptides or said means for
rd th
preferential pairing of said 3 and 4 CH3 domain-comprising polypeptides are a
st rd
substitution of threonine at position 366 of said 1 or 3 CH3 domain by a first charged
nd th
amino acid and substitution of leucine at position 351 of said 2 or 4 CH3 domain by a
second charged amino acid, wherein said first and second charged amino acids are of
opposite charge.
One preferred combination of mutations is the substitution of threonine (T) by lysine (K) at
position 366 of a first CH3 domain-comprising polypeptide which further comprises a
variable domain (for instance with specificity A) and the substitution of leucine (L) by
aspartic acid (D) at position 351 of a second CH3 domain-comprising polypeptide which
further comprises a variable domain (for instance with specificity B). This is denoted as a
T366K/L351’D pair mutation. As explained before, the amino acids at position 366 of one
CH3 domain and position 351 of a second CH3 domain have been reported to be a pair of
contact residues in the CH3-CH3 interface. The lysine that is introduced at position 366
and the aspartic acid introduced at position 351 have opposite charges, so that these amino
acids will electrostatically attract each other. Hence, the first CH3 domain will
preferentially attract the second CH3 domain and Ig-like molecules comprising a first CH3
domain containing lysine at position 366 paired with a second CH3 domain containing
aspartic acid at position 351 will be predominantly formed. If the first CH3 domain-
34
comprising polypeptide has specificity for antigen A, and if the second CH3 domain-
comprising polypeptide has specificity for antigen B, bispecific Ig-like molecules with
‘AB’ specificity will be predominantly formed. Nota bene, in some embodiments the
specificity of the variable domains of both said first and second CH3-domain comprising
polypeptide chains may be the same, which will result in the formation of monospecific Ig-
like molecules (for instance with ‘AA’ specificity) . As mentioned above, one of the
advantages of the mutations as described herein is the fact that a novel interaction between
a newly introduced pair of charged amino acids is created, instead of replacing existing
charged amino acid interactions. This was not previously disclosed or suggested. Also
described is a method for producing at least two different Ig-like molecules from a single
st
host cell, wherein said 1 CH3 domain-comprising polypeptide chain comprises the amino
nd
acid substitution T366K, and said 2 CH3 domain-comprising polypeptide chain
comprises the amino acid substitution L351D. One embodiment describes a method for
producing a heterodimeric Ig-like molecule from a single cell, wherein said Ig-like
molecule comprises two CH3 domains that are capable of forming an interface, said
method comprising providing in said cell:
st
- a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain,
and
nd
- a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain,
wherein said first CH3 domain-comprising polypeptide chain comprises the amino acid
substitution T366K and wherein said second CH3 domain comprising polypeptide chain
comprises the amino acid substitution L351D, said method further comprising the step of
culturing said host cell and allowing for expression of said two nucleic acid molecules and
harvesting said heterodimeric Ig-like molecule from the culture.
Using the above mentioned amino acid substitutions, it has become possible to produce a
heterodimeric Ig-like molecule from a single cell, whereby the presence of contaminating
homodimers is less than 5%, preferably less than 2%, more preferably less than 1%, or,
most preferably, whereby contaminating homodimers are essentially absent. One
embodiment therefore describes a method for producing a heterodimeric Ig-like molecule
from a single cell, wherein said Ig-like molecule comprises two CH3 domains that are
capable of forming an interface and wherein the presence of contaminating homodimers is
less than 5%, preferably less than 2%, more preferably less than 1%, and most preferably
contaminating homodimers are essentially absent, said method comprising providing in
said cell:
st
- a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain,
and
nd
- a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain,
wherein said first CH3 domain-comprising polypeptide chain comprises the amino acid
substitution T366K and wherein said second CH3 domain comprising polypeptide chain
comprises the amino acid substitution L351D, said method further comprising the step of
culturing said host cell and allowing for expression of said at two nucleic acid molecules
and harvesting said heterodimeric Ig-like molecule from the culture.
Also described is a method for producing at least two different Ig-like molecules, or a
method for producing a heterodimeric Ig-like molecule, wherein said first CH3-domain
comprising polypeptide chain further comprises the amino acid substitution L351K. It is
further preferred that said second CH3-domain comprising polypeptide chain further
comprises an amino acid substitution selected from the group consisting of Y349E, Y349D
and L368E. Most preferably said second CH3-domain comprising polypeptide chain
further comprises the amino acid substitution L368E.
Thus, in a preferred embodiment the above mentioned T366K/L351’D mutations are
further combined with the substitution of leucine (L) by glutamic acid (E) at position 368
of the second CH3 domain. This is, for example, denoted as a T366K/L351’D,L368’E
mutation (but alternative ways of denoting are also possible, such as T336K/L351D-L368E
or T366K/L351D,L368E or T366K - L351D,L368E). As shown in Example 17,
introduction of this mutation into a first CH3 domain-comprising polypeptide with
specificity for antigen A, and a second CH3 domain-comprising polypeptide with
specificity for antigen B results in a particular good proportion of bispecific Ig-like
36
molecules with dual AB specificity. With this mutational pair it has even become possible
to obtain bispecific antibody without any detectable amount of homodimers formed. A
particularly preferred embodiment therefore describes a method for producing a
heterodimeric Ig-like molecule from a single cell, wherein said Ig-like molecule comprises
two CH3 domains that are capable of forming an interface and wherein the presence of
contaminating homodimers is less than 5%, preferably less than 2%, more preferably less
than 1%, and most preferably contaminating homodimers are essentially absent, said
method comprising providing in said cell:
st
- a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain,
and
nd
- a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain,
wherein said first CH3 domain-comprising polypeptide chain comprises the amino acid
substitution T366K and wherein said second CH3 domain comprising polypeptide chain
comprises the amino acid substitutions L351D and L368E, said method further comprising
the step of culturing said host cell and allowing for expression of said at two nucleic acid
molecules and harvesting said heterodimeric Ig-like molecule from the culture.
In yet another preferred embodiment, threonine (T) is substituted by lysine (K) at position
366 of a first CH3 domain and leucine (L) is substituted by aspartic acid (D) at position
351 of a second CH3 domain and tyrosine (Y) is substituted by glutamic acid (E) at
position 349 of said second CH3 domain. This is for example denoted as a
T366K/L351’D,Y349’E mutation but other ways of denoting these mutations may include
for example T366K – L351D:Y349E, or T366K/L351D,Y349E or simply
T366K/L351DY349E. Residue Y349 is a neighboring residue of the residue at position
351 that may contribute to dimer interactions. According to in silico data, Y349E adds to
the stability of the heterodimer (lower in silico scores) as well as to the destabilization of
the monodimer (higher in silico scores) and glutamic acid (E) on position 349 is more
favorable than aspartic acid (D). Thus, introduction of a second amino acid substitution in
the second CH3 domain comprising polypeptide, comprising already the amino acid
substitution at position 351, favors heterodimerization further.
37
A particularly preferred embodiment therefore describes a method for producing a
heterodimeric Ig-like molecule from a single cell, wherein said Ig-like molecule comprises
two CH3 domains that are capable of forming an interface and wherein contaminating
homodimers are less than 5%, more preferably less than 2%, even more preferably less
than 1%, and most preferably essentially absent, said method comprising providing in said
cell:
st
- a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain,
and
nd
- a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain,
wherein said first CH3 domain-comprising polypeptide chain comprises the amino acid
substitution T366K and wherein said second CH3 domain comprising polypeptide chain
comprises the amino acid substitutions L351D and Y349E, said method further comprising
the step of culturing said host cell and allowing for expression of said at two nucleic acid
molecules and harvesting said heterodimeric Ig-like molecule from the culture.
In yet another preferred embodiment, threonine (T) is substituted by lysine (K) at position
366 of a first CH3 domain and leucine (L) is substituted by aspartic acid (D) at position
351 of a second CH3 domain and tyrosine (Y) is substituted by glutamic acid (E) at
position 349 of said second CH3 domain and leucine (L) is substituted by glutamic acid
(E) at position 368 of said second CH3 domain. This is denoted as a
T366K/L351’D,Y349’E,L368’E mutation. The two residues Y349 and L368 are residues
that may contribute to dimer interactions. According to the in silico data, Y349E and
L368E add to the stability of the heterodimer (lower in silico scores) as well as to the
destabilization of the BB dimer (higher in silico scores) and glutamic acids (E) on positions
349 and 368 are more favorable than aspartic acids (D). Thus, introduction of a second and
third amino acid substitution in the B-chain, which already comprises the amino acid
substitution at position 351, favors heterodimerization further. A particularly preferred
embodiment therefore describes a method for producing a heterodimeric Ig-like molecule
from a single cell, wherein said Ig-like molecule comprises two CH3 domains that are
capable of forming an interface and wherein contaminating homodimers are less than 5%,
38
more preferably less than 2%, even more preferably less than 1%, and most preferably
essentially absent, said method comprising providing in said cell:
st
- a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain,
and
nd
- a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain,
wherein said first CH3 domain-comprising polypeptide chain comprises the amino acid
substitution T366K and wherein said second CH3 domain comprising polypeptide chain
comprises the amino acid substitutions L351D and Y349E and L368E, said method further
comprising the step of culturing said host cell and allowing for expression of said at two
nucleic acid molecules and harvesting said heterodimeric Ig-like molecule from the
culture.
In yet another preferred embodiment, threonine (T) is substituted by lysine (K) at position
366 of a first CH3 domain and leucine (L) is substituted by lysine (K) at position 351 of
said first CH3 domain and leucine (L) is substituted by aspartic acid (D) at position 351 of
a second CH3 domain and leucine (L) is substituted by glutamic acid (E) at position 368 of
said second CH3 domain. This is denoted as a T366K,L351K/L351’D,L368’E mutation.
This mutation also enhances the proportion of the (bispecific) antibody of interest, as
shown in the Examples. Also with this mutation it has become possible to obtain bispecific
antibody without any detectable amount of homodimers formed. Further described is
therefore a method for producing a heterodimeric Ig-like molecule from a single cell,
wherein said Ig-like molecule comprises two CH3 domains that are capable of forming an
interface and wherein contaminating homodimers are less than 5%, preferably less than
2%, more preferably less than 1%, and most preferably essentially absent, said method
comprising providing in said cell:
st
- a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain,
and
nd
- a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain,
39
wherein said first CH3 domain-comprising polypeptide chain comprises the amino acid
substitutions T366K and L351K, and wherein said second CH3 domain comprising
polypeptide chain comprises the amino acid substitutions L351D and L368E, said method
further comprising the step of culturing said host cell and allowing for expression of said at
two nucleic acid molecules and harvesting said heterodimeric Ig-like molecule from the
culture.
In yet another preferred embodiment, threonine (T) is substituted by lysine (K) at position
366 of a first CH3 domain and leucine (L) is substituted by lysine (K) at position 351 of
said first CH3 domain and leucine (L) is substituted by aspartic acid (D) at position 351 of
a second CH3 domain and tyrosine (Y) is substituted by aspartic acid (D) at position 349 of
said second CH3 domain and arginine (R) is substituted by aspartic acid (D) at position
355 of said second CH3 domain. This is denoted as a
T366K,L351K/L351’D,Y349’D,R355’D mutation. The T366K-L351K/L351’D-Y349’D
pair may be further improved by the R355’D mutation in the B-chain, which results in a
higher BB-in silico score, but also the AB in silico score is slightly higher. Further
described is therefore a method for producing a heterodimeric Ig-like molecule from a
single cell, wherein said Ig-like molecule comprises two CH3 domains that are capable of
forming an interface and wherein contaminating homodimers are less than 5%, more
preferably less than 2%, even more preferably less than 1%, and most preferably
essentially absent, said method comprising providing in said cell:
st
- a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain,
and
nd
- a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain,
wherein said first CH3 domain-comprising polypeptide chain comprises the amino acid
substitutions T366K and L351K, and wherein said second CH3 domain comprising
polypeptide chain comprises the amino acid substitutions L351D and Y349D and R355D,
said method further comprising the step of culturing said host cell and allowing for
expression of said at two nucleic acid molecules and harvesting said heterodimeric Ig-like
molecule from the culture.
40
Table B provides an overview of mutations that can be introduced in CH3 domains as
preferred means for preferential pairing to create either heterodimers or homodimers.
Table B:
AA substitutions in CH3 Construct # Preferentially pairs with
- (wildtype) - Wildtype
E356K, D399K 1 Construct 2 or 3
K392D, K409D 2 Construct 1
K392D, K409D, K439D 3 Construct 1
K392D, D399K, K409D 4 Construct 4
E356K, E357K, K439D, K370D 5 Construct 5
T366W 6 Construct 7
T366S, L368A, Y407V 7 Construct 6
T366K 43 Construct 63, 69, 70, 71, 73
L351D 63 Construct 43, 68
T366K, L351K 68 Construct 63, 69, 70, 71, 72, 75
L351D, L368E 69 Construct 43, 68
L351E, Y349E 70 Construct 43, 68
L351D, Y349E 71 Construct 43, 68
L351D, R355D 72 Construct 43, 68
L351D, Y349E, L368E 73 Construct 43
L351D, Y349D, R355D 75 Construct 68
A method as described herein for producing at least two different Ig-like molecules, or a
method as described herein for producing a heterodimeric Ig-like molecule, wherein said
st nd
means for preferential pairing of said 1 and 2 CH3 domain-comprising polypeptides
rd th
and/or said means for preferential pairing of said 3 and 4 CH3 domain-comprising
polypeptides comprise at least one combination of mutations as depicted in Table B is
41
st nd
therefore also described. Preferably, said means for preferential pairing of said 1 and 2
rd
CH3 domain-comprising polypeptides and said means for preferential pairing of said 3
th
and 4 CH3 domain-comprising polypeptides comprise at least two combinations of
mutations as depicted in Table B.
Also described are novel combinations of CH3 mutations with which it has become
possible to produce a mixture of at least two monospecific Ig-like molecules in a single
cell, wherein contaminating bispecific Ig-like molecules are less than 5%, preferably more
than 2%, even more preferably less than 1%, and most preferably even essentially absent.
These mutations are, therefore, particularly suitable for the production of a mixture of
monospecific antibodies, which is for instance advantageous when a high level of
crosslinking of two identical target molecules is desired, when the density of antibodies on
a target cells needs to be high enough to recruit certain effector functions such as
complement-mediated lysis of a tumor cell, or when two targets are located too far away
from each order so that they cannot be bound by as single bispecific antibody, or in order
to simplify regulatory approval procedures. In such cases, it is often desired to optimize the
production platform for such monospecific antibodies. As shown in Example 10, the
present invention provides the insight that when lysine (K) at position 392 of a first CH3
domain-comprising polypeptide (for instance having specificity A) is substituted by
aspartic acid (D) and when aspartic acid (D) at position 399 of said first CH3 domain-
comprising polypeptide is substituted by lysine (K) and when lysine (K) at position 409 of
said first CH3 domain-comprising polypeptide is substituted by aspartic acid (D), it has
become possible to produce a mixture of at least two different monospecific Ig-like
molecules in a single cell, including monospecific Ig-like molecules with specificity AA,
wherein the formation of bispecific by-products (bispecific Ig-like molecules) is reduced to
below 5%, or even to below 3%, or even essentially not detectable at all. Hence, the above
mentioned combination of mutations (denoted herein as K392D, D399K, K409D) is
particularly preferred for the production of a mixture of monospecific Ig-like molecules.
The skilled person will appreciate that functional variants thereof, i.e., K392E, D399R,
K409E, may result in similar effects. Additionally, double mutants comprising D399K and
42
K409D substitutions, or other functional variants such as e.g. K392D and K409D, D399R
and K409E and so forth, may also result in similar effects.
The same holds true for a combination of mutations wherein glutamic acid (E) at position
356 of a first CH3 domain-comprising polypeptide is substituted by lysine (K) and wherein
glutamic acid (E) at position 357 of said first CH3 domain-comprising polypeptide is
substituted by lysine (K) and wherein lysine (K) at position 439 of said first CH3 domain-
comprising polypeptide is substituted by aspartic acid (D) and wherein lysine (K) at
position 370 of said first CH3 domain-comprising polypeptide is substituted by aspartic
acid (D). This combination of mutations (denoted herein as E356K, E357K, K439D,
K370D) is also particularly preferred for the production of a mixture of monospecific Ig-
like molecules. The skilled person will appreciate that functional variants thereof, i.e.,
E356R, E357R, K439E, K370E, may result in similar effects. Additionally, triple or double
mutants comprising E356K and K439D, and E357K and K370D substitutions, or other
functional variants may also result in similar effects. A further embodiment therefore
describes a method for producing at least two different monospecific Ig-like molecules
from a single host cell, wherein each of said two Ig-like molecules comprises two CH3
domains that are capable of forming an interface, said method comprising providing in said
cell
st
a) a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain
having a specificity A,
nd
b) a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain having a specificity B,
wherein said first CH3 domain-comprising polypeptide chain comprises a K392D, D399K,
K409D mutation and said second CH3 domain-comprising polypeptide chain comprises
either a wildtype CH3 domain or comprises a E356K, E357K, K439D, K370D mutation,
said method further comprising the step of culturing said host cell and allowing for
expression of said nucleic acid molecules and harvesting said at least two different Ig-like
molecules from the culture.
An alternative embodiment describes a method for producing at least two different
monospecific Ig-like molecules from a single host cell, wherein each of said two Ig-like
43
molecules comprises two CH3 domains that are capable of forming an interface, said
method comprising providing in said cell
st
a) a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain
having a specificity A,
nd
b) a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide
chain having a specificity B,
wherein said first CH3 domain-comprising polypeptide chain comprises either a wildtype
CH3 domain or comprises a K392D, D399K, K409D mutation and said second CH3
domain-comprising polypeptide chain comprises a E356K, E357K, K439D, K370D
mutation, said method further comprising the step of culturing said host cell and allowing
for expression of said nucleic acid molecules and harvesting said at least two different Ig-
like molecules from the culture.
As shown in Example 10, two monospecific Ig-like molecules can be produced in a single
cell, wherein the formation of bispecific Ig-like molecules is essentially undetectable. The
rd
skilled person may select a 3 nucleic acid molecule encoding a wildtype or engineered
CH3 domain-comprising polypeptide chain to provide to said host cell such that a mixture
of 3 monospecific antibodies is produced, and so forth.
Also described is a method for producing at least two different Ig-like molecules or for
producing a heterodimeric Ig-like molecule wherein each of the CH3-domain comprising
polypeptide chains further comprises a variable region recognizing a different target
epitope, wherein the target epitopes are located on the same molecule. This often allows
for more efficient counteraction of the (biological) function of said target molecule as
compared to a situation wherein only one epitope is targeted. For example, a heterodimeric
Ig-like molecule may simultaneously bind to 2 epitopes present on, e.g., growth factor
receptors or soluble molecules critical for tumors cells to proliferate, thereby effectively
blocking several independent signalling pathways leading to uncontrolled proliferation,
and any combination of at least two Ig-like molecules may simultaneously bind to 2, or
even 3 or 4 epitopes present on such growth factor receptors or soluble molecules.
44
In a preferred embodiment, the target molecule is a soluble molecule. In another preferred
embodiment, the target molecule is a membrane-bound molecule.
Also described is a method for producing at least two different Ig-like molecules or for
producing a heterodimeric Ig-like molecule wherein each of the CH3-domain comprising
polypeptide chains further comprises a variable region recognizing a target epitope,
wherein the target epitopes are located on different molecules. In this case, each of the
different target molecules may either be a soluble molecule or a membrane-bound
molecule. In one embodiment, the different target molecules are soluble molecules.
Alternatively, one target molecule is a soluble molecule whereas the second target
molecule is a membrane bound molecule. In yet another alternative, both target molecules
are membrane bound molecules. In one embodiment the different target molecules are
expressed on the same cells, whereas in other embodiments the different target molecules
are expressed on different cells. As a non-limiting example, any heterodimeric Ig-like
molecule or any combination of at least two Ig-like molecules may be suitable for
simultaneously blocking multiple membrane-bound receptors, neutralizing multiple soluble
molecules such as cytokines or growth factors for tumor cells or for neutralizing different
viral serotypes or viral strains.
One preferred embodiment describes a method for producing at least two different Ig-like
molecules or for producing a heterodimeric Ig-like molecule, wherein at least one of said
target epitopes is located on a tumor cell. Alternatively, or additionally, at least one of said
target epitopes is located on the surface of an effector cell. This is for instance suitable for
recruitment of T cells or NK cells for tumor cell killing. For instance, at least one Ig-like
molecule is produced with a method as described herein that is capable of recruiting
immune effector cells, preferably human immune effector cells, by specifically binding to
a target molecule located on immune effector cells. In a further embodiment, said immune
effector cell is activated upon binding of the Ig-like molecule to the target molecule.
Recruitment of effector mechanisms may for instance encompass the redirection of
immune modulated cytotoxicity by administering an Ig-like molecule produced by a
method as described herein that is capable of binding to a cytotoxic trigger molecule such
45
as the T cell receptor or an Fc gamma receptor, thereby activating downstream immune
effector pathways. The term ‘immune effector cell’ or ‘effector cell’ as used herein refers
to a cell within the natural repertoire of cells in the mammalian immune system which can
be activated to affect the viability of a target cell. Immune effector cells include cells of the
lymphoid lineage such as natural killer (NK) cells, T cells including cytotoxic T cells, or B
cells, but also cells of the myeloid lineage can be regarded as immune effector cells, such
as monocytes or macrophages, dendritic cells and neutrophilic granulocytes. Hence, said
effector cell is preferably an NK cell, a T cell, a B cell, a monocyte, a macrophage, a
dendritic cell or a neutrophilic granulocyte.
Target antigens present on immune effector cells may include CD3, CD16, CD25, CD28,
CD64, CD89, NKG2D and NKp46. Also described is a method for producing at least two
different Ig-like molecules or for producing a heterodimeric Ig-like molecule, wherein said
target epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or a
NKp46 molecule.
The viability of a target cell may include cell survival, proliferation and/or ability to
interact with other cells.
Also described are methods for producing a heterodimeric Ig-like molecule, wherein each
of the CH3-domain comprising polypeptide chains further comprises a variable region
recognizing a target epitope. In one embodiment, each of the 2 variable regions of the
CH3-domain comprising polypeptide chains recognizes the same target epitope but with
different affinities. In another embodiment, each of the 2 variable regions of the CH3-
domain comprising polypeptide chains recognizes a different target epitope. In another
embodiment, the different target epitopes are located on the same target molecule, which
can be either a membrane-bound molecule or a soluble molecule. In another embodiment,
the different target epitopes are located on different target molecules, which can be either
expressed on the same cells or on different cells. Alternatively, the different target
molecules can be soluble molecules, or one target molecule can be a soluble molecule
whereas the second target molecule is a membrane bound molecule. In a preferred
embodiment, at least one of the target molecules of the heterodimeric Ig-like molecule is
located on a tumor cell. In yet another preferred embodiment, at least one of the target
46
molecules of the heterodimeric Ig-like molecule is located on an effector cell (i.e. an NK
cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell or a neutrophilic
granulocyte, and said target epitope may be located on a CD3, CD16, CD25, CD28, CD64,
CD89, NKG2D or a NKp46 molecule).
In a preferred embodiment, a method for producing at least two different Ig-like molecules
or for producing a heterodimeric Ig-like molecule is described, wherein said at least two
different Ig-like molecules are antibodies, most preferably antibodies of the IgG isotype,
even more preferably the IgG1 isotype, as described herein above.
Further described is an Ig-like molecule, a heterodimeric Ig-like molecule, or a mixture of
at least two Ig-like molecules, obtainable by a method as described herein. Said
(heterodimeric) Ig-like molecule or mixture of Ig-like molecules preferably comprises at
least one CH3 mutation as depicted in Table B. An (heterodimeric) Ig-like molecule or a
mixture of at least two Ig-like molecules, comprising at least one mutation as depicted in
Table B is therefore also herewith described, as well as a pharmaceutical composition
comprising at least one Ig-like molecule, or a mixture of at least two Ig-like molecules, as
described herein. In one embodiment said Ig-like molecule is a bispecific Ig-like molecule,
such as a bispecific antibody. In another embodiment said Ig-like molecule is a
monospecific Ig-like molecule, such as a monospecific antibody. One preferred
embodiment describes a mixture of at least two different Ig-like molecules obtainable by a
method as described herein, wherein said at least two different Ig-like molecules bind to
different epitopes on the same antigen and/or to different epitopes on different antigens.
Further described is a heterodimeric Ig-like molecule obtainable by a method as described
herein, wherein said heterodimeric Ig-like molecule binds to different epitopes on the same
antigen and/or to different epitopes on different antigens. Advantages and preferred uses of
such mixtures and antibodies are described herein before. Also described is a mixture of at
least two different Ig-like molecules obtainable by a method as described herein, wherein
said at least two different Ig-like molecules comprise at least one heterodimeric Ig-like
molecule. In one embodiment, two of said at least two different Ig-like molecules are
heterodimeric Ig-like molecules. Yet another preferred embodiment describes a
47
heterodimeric antibody comprising two CH3 domains, wherein one of said two CH3
domains comprises the amino acid substitutions L351D and L368E and wherein the other
of said two CH3 domains comprises the amino acid substitutions T366K and L351K.
These amino acid substitutions are preferred means for preferential pairing of said two
CH3 domains, as explained before. The amino acid substitutions L351D and L368E in one
of said two CH3 domains and the amino acid substitutions T366K and L351K in the other
of said two CH3 domains are together dubbed the ‘DEKK combination of mutations’,
‘DEKK variant’, ‘DEKK pair’, ‘DEKK engineered CH3 domains’, ‘DEKK’ or alternative
names referring to DEKK are used. The CH3 domain that carries the amino acid
substitutions L351D and L368E is also dubbed ‘the DE-side’ and the CH3 domain that
carries the amino acid substitutions T366K and L351K is also dubbed ‘the KK-side’.
Also described is a pharmaceutical composition comprising a (heterodimeric) Ig-like
molecule, or a mixture of at least two Ig-like molecules obtainable by any method as
described herein. Said (heterodimeric) Ig-like molecule, or said at least two Ig-like
molecules as described herein is/are preferably (an) antibody/antibodies. Said
pharmaceutical composition may comprise said (heterodimeric) Ig-like molecule, a
mixture comprising monospecific or bispecific Ig-like molecules, or a combination of
monospecific and bispecific Ig-like molecules. In addition, a pharmaceutical composition
according to the invention comprises a pharmaceutically acceptable carrier. As used
herein, such ‘pharmaceutically acceptable carrier’ includes any and all solvents, salts,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible. Depending on the route
of administration (e.g., intravenously, subcutaneously, intra-articularly and the like) the Ig-
like molecules may be coated in a material to protect the Ig-like molecules from the action
of acids and other natural conditions that may inactivate the Ig-like molecules. In one
embodiment, a pharmaceutical composition comprising a mixture of at least two Ig-like
molecules obtainable by any method as described herein is described, wherein said at least
two different Ig-like molecules have been produced by recombinant host cells according to
the present invention. Furthermore, a pharmaceutical composition is described comprising
a heterodimeric Ig-like molecule obtainable by any method as described herein, wherein
48
said heterodimeric Ig-like molecule has been produced by recombinant host cells as
described herein.
A nucleic acid molecule encoding a CH3 domain-comprising polypeptide chain that
comprises at least one mutation as depicted in Table B is also provided herewith, as well as
a recombinant host cell comprising at least one nucleic acid molecule encoding a CH3
domain-comprising polypeptide chain that comprises at least one mutation as depicted in
Table B.
The term “comprising” as used in this specification and claims means “consisting at least
in part of”. When interpreting statements in this specification, and claims which include
the term “comprising”, it is to be understood that other features that are additional to the
features prefaced by this term in each statement or claim may also be present. Related
terms such as “comprise” and “comprised” are to be interpreted in similar manner.
The invention is further illustrated by the following examples. These examples are not
limiting the invention in any way, but merely serve to clarify the invention.
Brief description of the drawings
Figure 1: A) schematic representation of construct vector MV1057. The stuffer region is
the region into which an antibody VH region is cloned. B) schematic representation of
phage display vector MV1043.
Figure 2: amino acid sequence of wildtype IgG1 Fc, as present in construct vector
MV1057 (EU numbering scheme applied).
Figure 3: nucleotide and amino acid sequences of VH regions used for cloning into the
various constructs.
Figure 4: mass spec data of transfections A, G and H.
Figure 5: mass spec data of transfections M and U.
Figure 6: mass spec data of transfection O.
Figure 7: prevention of homodimerisation by substitution of neutral amino acids for
charged amino acids.
49
Figure 8: Native MS spectrum of transfection sample ZO (T366K/L351’D) (A) and
Convoluted MS spectrum of transfection sample ZO (T366K/L351’D). The second/main
peak represents the bispecific molecule (B).
Figure 9: HADDOCK scores on experimentally verified mutation pairs
Figure 10: Cartoons of interactions in the CH3-CH3 interface; A)
K409D:K392D/D399’K:E356’K, B) D399K:E356K/D399’K:E356’K, C) K409D:K392D/
K409’D:K392’D
Figure 11: HADDOCK scores for various 366/351’ charge mutants
Figure 12: Cartoons of interactions in the CH3-CH3 interface ; A) L351D/L351’D, B)
L351D:S354A:R355D/ L351’D:S354’A:R355’D
Figure 13: HADDOCK scores for additional charge mutations around position L351
Figure 14: HADDOCK scores for additional charge mutations around position T366 in
chain A and position L351 in chain B.
Figure 15: Cartoons of interactions in the CH3-CH3 interface
Figure 16: HADDOCK scores for variants around T366/L351
Figure 17: HADDOCK scores for additional variants around T366/L351
Figure 18: Examples of nMS spectra for bispecific IgG obtained after the co-expression of
construct T366K,L351K with either construct L351D (left hand panel) or L351D,Y349E
(right hand panel), zoomed in on a single charge state of the full IgG (half bodies not
shown)
Figure 19: A) Results of native MS showing relative abundances of AA, AB, BB, A and B
(total of all species is 100%); B) idem but now without AB to have a better overview on
the undesired species AA, BB, A and B
Figure 20: Results of thermostability assay. Squares: wildtype; triangles: charge reversal
pair E356K:D399K/K392D:K409D; circles: mutant CH3 combinations as indicated above
each graph.
st
Figure 21: Results of 10x freeze-thaw experiment. 1122= 1 parental antibody BB; 1337=
nd
2 parental antibody AA; wildtype=AA, AB, BB; CR= bispecific of charge reversal pair
E356K:D399K/K392D:K409D; 3-6 and 9-12=bispecific molecules from combinations 3-6
and 9-12 from Table 15.
50
Figure 22: Results in serum stability, measured by ELISA using fibrinogen as coated
antigen. A) ELISA data with IgG samples diluted to 0.5 μg/ml; B) ELISA data with IgG
samples diluted to 0.05 μg/ml;. Results are normalized to the T=0 days time point (100%).
nd
1337= 2 parental antibody AA; wildtype=AA, AB, BB; CR= bispecific of charge
reversal pair E356K:D399K/K392D:K409D; 3-6 and 9-12=bispecific molecules from
combinations 3-6 and 9-12 from Table 15.
Figure 23: nMS results of ratio experiments with transfection ratios from 1:5 to 5:1. A)
DEKK combination of mutations, with specificity ‘A’ on the DE-side and ‘B’ on the KK-
side; B) DEKK combination of mutations, with specificity ‘C’ on the DE-side and ‘B’ on
the KK-side; C) charge reversal combination of mutations, with specificity ‘A’ on the
E356K:D399K-side and ‘B’ on the K392D:K409D-side
Figure 24: nMS results of transfections # 1-11 from Table 20.
Figure 25: HADDOCK scores for dimers with different CH3 engineered vectors. Grey
bars: Desired species AB and CD; black bars: undesired species AA, BB, CC, DD, AC,
BC, AD, BD.
Figure 26: SDS-PAGE of transfections # 1-11 from Table 20. Control samples DE/KK,
DE/DE and KK/KK are also included.
Figure 27: nMS of transfections # 9 (A) and # 11 (B).
Figure 28: nMS of gel filtrated samples 1516:1516 (A), 1337:1337 (B) and 1516:1337 (C).
Figure 29: serum levels of samples of DEKK engineered antibody and its two parental
antibodies (pK study).
Examples
Example 1: amino acid substitutions to create various different CH3-domains
In order to have a wide variety of Ig-like molecules that differ in their CH3 domains such
that pairing of CH3-domain comprising Ig-like molecules is preferentially promoted or
inhibited, a number of amino acid substitutions that were known to promote heterodimer
formation, as well as a number of alternative amino acid substitutions that were not
previously reported nor tested but that were chosen to promote homodimer formation, were
introduced into a construct vector (construct vector MV1057; Figure 1A). The construct
vector MV1057 comprises nucleic acid sequences encoding the normal wildtype IgG1 Fc
part, as depicted in figure 2. Table 1 lists the amino acid substitutions that were introduced
51
in this wildtype Fc, resulting in a series of seven constructs. All constructs were made at
Geneart. Constructs 1, 2 and 3, or alternatives thereof, have previously been described to
drive heterodimerization (EP01870459, WO2009/089004) as have constructs 6 and 7
(WO98/50431). Constructs 4 and 5 are new and are designed to promote
homodimerization.
Table 1
AA substitutions in CH3 construct Will pair with % bispecific
# product reported
- (wildtype) - - (wildtype) ~50%
E356K, D399K 1 Construct 2 or 3 ~100%
K392D, K409D 2 Construct 1 ~100%
K392D, K409D, K439D 3 Construct 1 ~100%
K392D, D399K, K409D 4 Construct 4
E356K, E357K, K439D, K370D 5 Construct 5
T366W 6 Construct 7 ~86,7%
T366S, L368A, Y407V 7 Construct 6 ~86,7%
52
Example 2: cloning of VH into constructs with CH3 mutations
Several antibody VH regions with known specificities and known ability to pair with the
human IGKV1-39 light chain were used for cloning into these constructs.
As indicated earlier, all CH3 variants can be used in association with other antibody
domains to generate full length antibodies that are either bispecific or monospecific. The
specificity of the antibody as defined by the VH/VL combinations will not affect the heavy
chain dimerization behaviour that is driven by the CH3 domains. Model VH/VL
combinations were used throughout the studies, wherein all VLs are based on the germline
human IGKV1-39 and VHs vary. Figure 3 provides full sequences and specificities of the
antibody VH regions used throughout the studies. The MF coding refers to internal Merus
designation for various VHs, e.g. VH MF1337 has specificity for tetanus toxoid, MF1025
for porcine thyroglobulin, MF1122 for bovine fibrinogen.
VH regions present in phage display vector MV1043 (Figure 1B) are digested with
restriction enzymes SfiI and BstEII (New England Biolabs/ cat# R0123L and R0162L/
according to manufacturer’s instructions) that release the VH fragment from this vector.
Vector MV1057 is digested with SfiI and BstEII according to standard procedures
(according to manufacturer’s instructions). Fragments and vector are purified over gel
(Promega/ cat# V3125/ according to manufacturer’s instructions) to isolate the cut vector
and VH gene inserts. Both are combined by ligation after which the ligation is transformed
into E. coli DH5α (Invitrogen/ cat# 12297-016/ according to manufacturer’s instructions).
After overnight selection single colonies are picked and vectors with a correct insert
identified by sequencing.
Example 3: transfection and expression of full IgG in HEK293T cells
Transfection of the various plasmids encoding the recloned VH variants, and further
encoding the common light chain huIGKV1-39, in HEK293T cells was performed
according to standard procedures such that IgG could express (de Kruif etal Biotech
Bioeng. 2010). After transfection, IgG expression levels in supernatants were measured
using the ForteBIO Octet-QK system, which is based on Bio-Layer Interferometry (BLI)
and which enables real-time quantitation and kinetic characterization of biomolecular
53
interactions; for details see www.fortebio.com. When expression levels exceeding 5 μg/ml
were measured, the IgG was purified using Protein A affinity purification.
Example 4: purification of IgG
Culture supernatants were purified using protein A columns (GE Healthcare/ cat# 11
95/ according to manufacturer’s instructions) and eluted in 0,1 M citrate buffer pH 3.0 and
immediately neutralized in an equal volume of 1,0 M Tris-HCL pH 8.0 or directly
rebuffered to PBS using a desalting column. Alternatively one could purify IgG using
protein A beads (sepharose beads CL-4B, GE healthcare cat #170780-01)
Example 5: Ag-specific ELISA’s
Antigen specific ELISAs were performed to establish binding activity against the antigens
and capture ELISAs were carried out to demonstrate binding activity of the bispecific
antibodies. Biotinylated second antigen was used for detection of the complex. (de Kruif
etal Biotech Bioeng. 2010)
Example 6: SDS-PAGE
The purified IgG mixtures were analysed by SDS-PAGE (NuPAGE® 4-12% bis-tris gel/
Invitrogen/ cat# NP0323BOX) under reduced and non-reducing conditions according to
standard procedures, and staining of proteins in gel was carried out with colloidal blue
(PageBlue™ protein staining solution/ Fermentas/ cat# RO571).
Example 7: Enzymatic deglycosylation of IgG1
As there is heterogeneity in the glycosylation of the IgGs, the proteins were deglycosylated
in order to create a single product with a distinct mass, suitable for mass spectrometric
analysis. One unit of N-glycosidase F (PNGase F; Roche Diagnostics, Mannheim,
o
Germany) was incubated per 10 µg of IgG1, overnight at 37 C. Buffer exchange using 10
kDa MWCO centrifugal filter columns (Millipore) was performed to remove the original
purification buffer (0,1 M citrate buffer pH 3.0 / 1,0 M Tris-HCL pH 8.0) and to rebuffer
to PBS. Similar buffer exchange procedures were performed to remove the detached
glycan chains, and to change the buffer to 150 mM ammonium acetate pH 7.5. Filters were
54
o
washed with 200 µl 150 mM ammonium acetate pH 7.5, for 12 min 11,000 rpm and 4 C.
After washing 50 µl deglycosylated IgG was loaded on the filter and 450 µl of 150 mM
ammonium acetate pH 7.5 was added, subsequently followed by another centrifugation
o
round of 12 min at 11,000 rpm at 4 C. In total the centrifugation was repeated 5 times,
each time fresh 150 mM ammonium acetate pH 7.5 buffer was added to a total volume of
500 µl. After the last centrifugation step the remaining buffer exchanged deglycosylated
IgG1, approximately 25 µl, was collected and transferred to an eppendorf tube, ready for
mass spectrometric analysis.
Example 8: Native mass spectrometric analysis
Mass Spectrometry was used to identify the different IgG species in the purified IgG
mixtures and to establish in what ratios these IgG species are present. Briefly, 2-3 µl at a 1
µM concentration in 150 mM ammonium acetate pH 7.5 of IgG’s were loaded into gold-
plated borosilicate capillaries made in-house (using a Sutter P-97 puller [Sutter Instruments
Co., Novato, CA, USA] and an Edwards Scancoat six sputter-coater [Edwards
Laboratories, Milpitas, CA, USA]) for analysis on a LCT 1 mass spectrometer (Waters
Corp., Milford, MA, USA), adjusted for optimal performance in high mass detection
(Tahallah et al., RCM 2001). A capillary voltage of 1300 V was used and a sampling cone
voltage of 200 V; however, these settings were adjusted when a higher resolution of the
‘signal-to-noise’ ratio was required. The source backing pressure was elevated in order to
promote collisional cooling to approximately 7.5 mbar. To measure the IgG1’s under
denaturing conditions the proteins were sprayed at a 1 µM concentration in 5% formic
acid.
Example 9: Data processing and quantification
Processing of the acquired spectra was performed using MassLynx 4.1 software (Waters
Corp., Milford, MA, USA). Minimal smoothing was used, after which the spectra were
centered. The mass of the species was calculated using each charge state in a series. The
corresponding intensities of each charge state were assigned by MassLynx and summed.
This approach allowed the relative quantification of all species in a sample. Alternatively,
quantification of the peaks can be performed using area-under-the-curve (AUC) methods,
55
known in the art. All analyses were repeated three times to calculate standard deviations of
both the masses of the IgG’s as well as their relative abundance.
Example 10: mixtures of 2 or 3 monospecific antibodies from a single cell
Several antibody VH regions with known specificities and known ability to pair with the
human IGKV1-39 light chain (Figure 3) were used for recloning into the wildtype
construct vector MV1057, or in construct 4 or construct 5 of Table 1, resulting in vectors I-
III (Table 2). The resulting vectors I, II and III, each containing nucleic acid sequences
encoding for the common human light chain as well as an Ig heavy chain with different
CH3 region and different VH specificity, were subsequently transfected into cells, either
alone to demonstrate formation of intact monospecific antibodies only, or in combination
with one or two other construct vectors to obtain mixtures of two monospecific or three
monospecific antibodies. Table 3 depicts the transfection schedule and results.
Table 2: VH specificity inserted in different constructs
Vector VH Antigen VH mass Merus Cloned in
gene specificity (Da) designation construct #
I IGHV Tetanus (A) 13703 MF1337 wildtype
1.08
II IGHV Thyroglobulin 12472 MF1025 4
3.23 (B)
III IGHV Fibrinogen 12794 MF1122 5
3.30 (C)
56
Table 3: transfection schedule and results
# Transfecti Transfecti Expect Calcula Experim AA BB CC Othe
differen on of on code ed ted ental foun foun foun r
t mono- and ratio species mass - mass d d d mole
specific 2LYS (%) (%) (%) cules
s (%)
produce
d
1 Only A AA 146521 146503 100
vector I
1 Only G BB 144032 144087 100
vector II
1 Only H CC 144647 144656 100
vector III
2 Vector I M AA 146521 146518 51 45 4
and II (I:II=1:1) BB 144032 144030
2 Vector I N AA 146521 146509 88 9 3
and III (I:III=1:1) CC 144647 144633
U (I:III= AA 146521 146522 47 48 5
1:5) CC 144647 144643
2 Vector II nd BB
and III CC
3 Vector I, II O AA 146521 146525 66 4 30
and III (I:II:III=1:1 BB 144032
144032
:1) CC 144647 144650
V AA 146521 146531 8 81 9 2
(I:II:III=1:1 BB 144032 144043
:10) CC 144647 144654
nd= not done.
57
It was observed that transfections A, G and H resulted in formation of homodimers only,
and 100% of bivalent monospecific AA, BB or CC was retrieved from cells transfected
with any one of vectors I, II or III (Fig. 4). Although this was to be expected and
previously demonstrated for transfection A, it is actually now shown for the first time that
homodimerisation of CH3-engineered Ig heavy chains containing either the triple amino
acid substitution of construct 4 (i.e., K392D, D399K, K409D) or the quadruple amino acid
substitution of construct 5 (i.e., E356K, E357K, K439D, K370D) is reported (transfections
G and H).
Next, co-expression experiments of two vectors in a single cell were performed.
Interestingly, transfections M and N show that wildtype and CH3 engineered Ig heavy
chains can be co-expressed in a single cell together with a common light chain resulting in
mixtures of two species of monospecific antibodies without the presence of undesired
bispecific antibodies and with as little as 4-5% contaminating ‘other molecules’ present in
the mixture. ‘Other molecules’ is defined as all molecules that do not have the mass of an
intact IgG, and includes half molecules consisting of a single heavy and light chain pair.
Importantly, the fraction ‘other’ does not include bispecific product. In transfection M, the
ratio of AA:BB was close to 1:1 upon transfection of equal ratios of vector DNA.
However, transfection N resulted in an almost 10:1 ratio of AA:CC. Therefore, this
transfection was repeated with adjusted ratios of DNA (transfection U). Indeed, a 1:5 ratio
of vector DNA I:III equalized the ratio of AA:CC antibody product in the mixture towards
an almost 1:1 ratio. Thus, transfections M and U show that it is possible to express two
different, essentially pure, monospecific antibodies in a single cell, without undesired by
products (i.e., no abundant presence of AC or half molecules A or C) (fig. 5). The novel
CH3 modifications of constructs 4 and 5 differ substantially from wildtype CH3 such that
heterodimerization between wildtype and 4, or wildtype and 5, does not occur, which is
advantageous for application in large scale production of mixtures of monospecific
antibodies from single cells.
Analogous to these results, also transfection of two different CH3 engineered Ig heavy
chains (constructs 4 and 5) are expected to result in mixtures of two different monospecific
antibodies only, without further undesired species present. It is reasoned that the CH3
modifications of construct 4 differ substantially from the CH3 modifications of constructs
58
such that heterodimerization does not occur. In that case, co-expression of CH3-
engineered heavy chains of constructs 4 and 5, together with wildtype CH3 heavy chains in
a single cell would results in 3 monospecific antibodies only.
Indeed, this was observed to be the case as it was found that also a mixture of three pure
monospecific antibodies could be obtained by expression of three different Ig heavy
chains, designed to form homodimers over heterodimers, together with a common light
chain in a single cell, with no contaminations present in the mixture (transfection O) (Fig.
6). As is clear from Table 3, with equal ratios of vector DNA used during transfection O,
no 1:1:1 ratio of AA:BB:CC antibodies was obtained. Transfections with altered vector
DNA ratios (1:1:10, transfection V) demonstrated that ratios of AA:BB:CC in the mixtures
can be steered towards desired ratios.
Taken together, these experiments show that two or three essentially pure monospecific
antibodies can be expressed in a single cell without undesired by products, offering
advantages for large scale production of mixtures of therapeutic monospecific antibodies.
Example 11: mixtures of 2 bispecific antibodies from a single cell
Whereas use of CH3-engineered heavy chains for production of single bispecific
antibodies has been reported elsewhere, this experiment was designed to investigate
whether it is feasible to produce mixtures of 2 different bispecific antibodies from a single
cell.
Antibody VH regions with known specificities and known ability to pair with the human
IGKV1-39 light chain (fig. 3) were used for recloning into vectors containing constructs 1-
3 or 6-7 of Table 1 resulting in vectors IV-X (Table 4). Vectors IV-X, each containing
nucleic acid sequences encoding the common human light chain as well as an Ig heavy
chain with different CH3 region and different VH specificity, were subsequently
transfected into cells, either alone to demonstrate that formation of intact monospecific
antibodies was hampered, or in combination with another construct vector to obtain
bispecific antibodies or mixtures of two bispecific antibodies. Table 5 depicts the
transfection schedule and results.
59
Table 4: VH specificity inserted in different constructs
Vector VH gene Antigen VH Cloned in
specificity mass construct
(Da) #
IV IGHV 3.23 Thyroglobulin 12472 1
(B)
V IGHV 3.30 Fibrinogen (C) 12794 2
VI IGHV 1.08 Tetanus (A) 13703 2
VII IGHV 3.30 Fibrinogen (C) 12794 3
VIII IGHV 1.08 Tetanus (A) 13703 3
IX IGHV 1.08 Tetanus (A) 13703 6
X IGHV 3.23 Thyroglobulin 12472 7
(B)
Table 5:
# differ- Trans- Transf Expect Calculated Experim Half Full Bispec Other
rent fection ection ed mass - ental molecul IgG ific molec
bispe- of code spe- 2LYS mass es foun found ules
cifics and cies found d (%) (%)
produ- ratio (%) (%)
ced
0 vector B Half B 144082 144066 40 60
IV
0 vector V C Half C 144651 144622 77 23
0 vector D Half A 146469 146459 23 77
VI
0 vector E Half C 144625 144643 76 24
VII
0 vector F Half A 146443 146468 64 36
VIII
60
0 vector P Half A 146691 146677 82 18
IX
0 vector X Q Half B 143818 143844 58 42
1 Vector I (1:1) BC 144367 144352 96 4
IV and
V
1 Vector J (1:1) BC 144354 144382 96 4
IV and
VII
2 Vector K(1:1: BC + 144367 + 144351+ 38 + 15 (A
IV, V 1) AB 145276 145260 47 + C)
and VI
S(2:1:1 BC + 144367 + 144371 + 42 + 3 (BB)
) AB 145276 145277 55
2 Vector L BC + 144354 + 144346 + 16 + 24 (A
IV, VII (1:1:1) AB 145263 145255 60 + C)
and VIII T BC + 144354 + 144385 + 58 + 3 (BB)
(2:1:1) AB 145263 145292 39
It was previously demonstrated that CH3-engineered Ig heavy chains encoded by
constructs 1 and 2 are still able to form homodimers when expressed alone in single cells
(WO2009/089004). However, WO2009/089004 further reports that CH3 domains that are
engineered to comprise triple charge pair mutations, such as present in construct 3, are no
longer capable of forming homodimers when expressed alone.
In the present study, these findings were only partly confirmed. Indeed, the results of
transfections B, C and D demonstrated the presence of full IgGs, in addition to a high
proportion of unpaired half molecules, demonstrating some homodimerization of CH3
domains encoded by constructs 1 and 2. Transfections E and F also resulted in production
of full IgGs in addition to unpaired half molecules, demonstrating that the triple charge
mutations of construct 3 do not fully impair homodimerisation.
61
It was furthermore demonstrated that also the ‘knob’ and ‘hole’ CH3 variants of constructs
6 and 7 form homodimers (18% homodimers for ‘knob-knob’ and 42% homodimers for
‘hole-hole’).
CH3 variants that fully prevent homodimerisation when expressed alone are preferred, to
prevent or minimize undesired byproducts (homodimers) upon co-expression with a
second CH3 variant for heterodimerization.
Interestingly, the present experiments demonstrate for the first time that also mixtures of
bispecific antibodies can be expressed in single cells with virtually no homodimers in the
mixture. Transfections K and L clearly show that the expected bispecific species BC + AB
are indeed obtained (38% + 47% in transfection K, and 16% + 60% in transfection L). In
both transfections a relatively high percentage of undesired half molecules was observed
(15% half molecule A + half molecule C in transfection K, and 24% half molecule A + half
molecule C in transfection L). The relatively high percentage of half molecules still present
was attributed to low amounts of matching heavy chains of vector IV due to unbalanced
expression of heavy chains in a matched pair. Therefore, transfections were repeated with
an adjusted ratio of vector DNA, 2:1:1, in transfections S and T. This resulted in equal
amounts of IgG heavy chains constituting a matched pair and pure mixtures of bispecific
IgG without the presence of half IgG molecules and with as little as 3% homodimeric BB
present. Ideally, this low proportion of contaminating monospecific product should be
reduced to essentially zero. It is therefore desired to find additional CH3-mutants that
would result in mixtures of bispecific antibodies with minimal contaminating monospecific
antibodies present.
The present study demonstrates for the first time that essentially pure mixtures of two
bispecific antibodies recognizing 3 different target epitopes can be produced in a single
cell, with minimal presence of monospecific antibodies in the mixture.
Example 12: varieties of mixtures
As it was demonstrated that production of mixtures of 2 bispecific antibodies recognizing 3
epitopes from a single cell, or production of mixtures of 2 or 3 monospecific antibodies
from a single cell is technically feasible, we next explored the feasibility of controlled
production of a variety of other mixtures. A fourth antibody VH region with known
62
specificity and known ability to pair with the human IGKV1-39 light chain will be used for
recloning into vectors containing constructs 1-3 or 7 of Table 1, resulting in vectors I’, II’,
III’ or X’ (the ’ indicating a different specificity as compared to corresponding vector
numbers). The resulting vectors I’-III’, X’ and IV-IX, each containing nucleic acid
sequences encoding for the common human light chain as well as an Ig heavy chain with
different CH3 region and different VH specificity, will subsequently be transfected into
cells, in combination with other construct vectors to obtain a variety of mixtures of
bispecific and/or monospecific antibodies. The variety of mixtures that will be obtained
include mixtures of 2 bispecific antibodies recognizing 4 epitopes, 2 bispecific antibodies
and one monospecific antibody, or mixtures of 1 bispecific and one monospecific antibody
from a single cell. Table 6 depicts the transfection schedule and expected results.
Table 6
Variety of Transfect- Transfectio Expected Expected % Expected %
mixture ion of n code and species monospecific Bispecific
ratio IgG
2 BsAbs, 4 IV+V+IX+ ZA (1:1:1:1) BC + AD 0 50 + 50
epitopes X’
2 BsAbs, 4 IV+VII+IX ZB (1:1:1:1) BC + AD 0 50 + 50
epitopes +X’
2 bsAbs + 1 IV+V+VI+ ZC (2:1:1:2) BC + AB 33 33 + 33
mAb wt’ + DD
2 bsAbs + 1 IV+V+VI+ ZD (2:1:1:2) BC + AB 33 33 + 33
mAb II’ + DD
2 bsAbs + 1 IV+V+VI+ ZE (2:1:1:2) BC + AB 33 33 + 33
mAb III’ + DD
1 bsAb + 1 IV+V+wt’ ZF (1:1:2) BC + DD 50 50
mAb
1 bsAb + 1 IV+V+II’ ZG(1:1:2) BC + DD 50 50
mAb
63
1 bsAb + 1 IV+V+III’ ZH(1:1:2) BC + DD 50 50
mAb
1 bsAb + 1 IV+VII+wt ZI (1:1:2) BC + DD 50 50
mAb ’
1 bsAb + 1 IV+VII+II’ ZJ (1:1:2) BC + DD 50 50
mAb
1 bsAb + 1 IV+VII+III ZK (1:1:2) BC + DD 50 50
mAb ’
1 bsAb + 1 IX+X+wt’ ZL (1:1:2) AB + DD 50 50
mAb
1 bsAb + 1 IX+X+II’ ZM (1:1:2) AB + DD 50 50
mAb
1 bsAb + 1 IX+X+III’ ZN (1:1:2) AB + DD 50 50
mAb
Although, theoretically, production of all mixtures should be feasible, it is known from
previous work by others that large scale production of classical knob-into-hole variants is
hampered by instability issues. Mixtures resulting from transfections ZA, ZB, ZL, ZM and
ZN are thus expected to become problematic when transferred to larger scale production.
Thus, the current set of constructs present in Table 1 would not allow production of all
theoretical mixtures from single cells at a larger scale, as knob-into-hole variants are
reported to be unstable, and it cannot be excluded that CH3 domains comprising a ‘knob’
or a ‘hole’ will dimerize with either charge variants or wildtype CH3 domains. It is thus
desired to design new CH3-variants that are engineered to preferentially form homodimers
or heterodimers only and which will not homo- or heterodimerize with constructs 1-5 of
Table 1 as to allow for co-expression in single cells.
Example 13: identification of novel charge pair mutants
The objective of this study was to engineer the IgG CH3 region to result in the production
of only heterodimers or only homodimers upon mixed expression of different IgG heavy
chains in a single cell, wherein the novel engineered CH3 domains will not homo- or
64
heterodimerize with known engineered CH3 domains, or with wildtype CH3 domains.
Therefore, as a first step in identifying novel engineered CH3 domains that would meet the
criteria, many interface contact residues in the IgG CH3 domain were scanned one by one
or in groups for substitutions that would result in repulsion of identical heavy chains – i.e.,
reduced homodimer formation - via electrostatic interactions. The objective was to obtain a
list of residues that, when substituted by a charged residue, would result in repulsion of
identical chains such that these mutations may be used to drive homo- and/or heterodimer
formation upon mixed expression of different IgG heavy chains, whereby the obtained full
length IgGs are stable and are produced with high proportions. In a follow up, the
identified substitutions will be used to generate bispecific antibodies or mixtures of
bispecific or monospecific antibodies by engineering matched pairs of CH3 residues in one
or more IgG heavy chains - CH3 regions. Additionally, newly identified charge mutant
pairs may be combined with existing pairs, such that multiple nucleic acid molecules
encoding different heavy chains, all carrying different and complementing CH3 mutations,
can be used for expression in cells such that mixtures of monospecific antibodies only, or
bispecific antibodies only, or mixtures of defined monospecific and bispecific antibodies
can preferentially be obtained. The residues to be tested in the present study are contact
residues as previously identified (Deisenhofer J., 1981; Miller S., 1990; Padlan, 1996,
Gunasekaran, 2010). The rationale for this approach is that repulsive charges are
engineered into each available pair of contacting residues. Samples are subsequently
analyzed on non-reducing SDS-PAGE to identify pairs in which dimer formation is
reduced, as visualized by the presence of bands of approximately 72 kD. All available pairs
will be screened as single mutations or in combination with a single other mutation as the
repulsive electrostatic interaction between one non-matching pair may or may not be
sufficient to result in sufficient amounts of half-molecules for detection by this method, the
mutations are also combined.
Amino acid substitutions were introduced in construct vector MV1057 by Geneart
according to the table 7 and expression of constructs was performed by transfection in
HEK293T cells, according to standard procedures. IgG expression levels were measured in
Octet. When production failed twice, the mutation was considered to be detrimental to
expression and the mutation was not pursued further.
65
Table 7: list of amino acid substitutions in the various constructs that were made (EU
numbering)
AA substitutions in construct # Effect on homodimer
CH3 formation
(- = no effect; +++ = max.
inhibition; NT= not tested
on gel)
Q347K 8 -
Y349D 9 +-
Y349K 10 +-
T350K 11 -
T350K, S354K 12 +-
L351K, S354K 13 +-
L351K, T366K 14 ++
L351K, P352K 15 +-
L351K, P353K 16 ++
S354K, Y349K 17 ++
D356K 18 -
E357K 19 -
S364K 20 ++
T366K, L351K 21 ++
T366K, Y407K 22 +++
L368K 23 NT
L368K, S364K 24 ++
N390K, S400K 25 +-
T394K, V397K 26 +
T394K, F405K 27 +++
T394K, Y407K 28 +++
P395K, V397K 29 +-
66
AA substitutions in construct # Effect on homodimer
CH3 formation
(- = no effect; +++ = max.
inhibition; NT= not tested
on gel)
S400K 30 -
F405K 31 +++
Y407K 32 ++
Q347K, V397K, 33 +
T394K
Y349D, P395K, 34 +
V397K
T350K, T394K, 35 NT
V397K
L351K, S354K, S400K 36 +
S354K, Y349K, 37 +-
Y407K
T350K, N390K, 38 +-
S400K
L368K, F405K 39 ++
D356K, T366K, 40 +++
L351K
Q347K, S364K 41 +++
L368D, Y407F 42 +
T366K 43 +
L351K, S354K, T366K 44 +
Y349D, Y407D 45 +
Y349D, S364K, 46 +
Y407D
Y349D, S364K, 47 +
S400K, T407D
67
AA substitutions in construct # Effect on homodimer
CH3 formation
(- = no effect; +++ = max.
inhibition; NT= not tested
on gel)
D399K 48 +-
D399R 49 +-
D399H 50 +-
K392D 51 +-
K392E 52 +-
K409D 53 +
Supernatants containing ≥5 μg/ml IgG were analyzed in SDS-PAGE and IgG was purified
using protein A. The proteins were stained using colloidal blue. Homodimers were visible
as a band of approximately 150 kD. Smaller bands of approx 75 kD represented the
presence of half molecules (see negative control: K392D, K409D). Blots are shown in
Figure 7.
The results of SDS-PAGE gels were analyzed and scored as presented in table 7, right
hand column. A number of residues were considered promising for further testing in
combination, including residues Q347, S354, Y349, L351, K360, T366, T394, and V397.
The choice was based on high scores in the inhibition of formation of homodimers
combined with the availability of contacting residues that can be modified without running
into issues such as other non-complementary charges. For example, it is known that
residues F405 and Y407 have multiple interactions at the CH3-CH3 interface, including
interactions with residues that are already charged, which may be problematic after
introduction of multiple charge mutations among these interacting residues (see Table A).
New constructs were made in vector MV1057 (Table 8), and antibody VH regions with
known specificities and known ability to pair with the human IGKV1-39 light chain were
used for recloning into vectors containing these new constructs (see Table 9) such that
combinations could further be tested. Table 10 depicts the transfection schedules and
results.
68
Table 8:
AA substitutions construct #
in CH3
L351K 61
T394K 62
L351D 63
T366D 64
S354D, Y349D 65
V397D 66
K360D 67
Table 9: VH specificity inserted in different constructs
Vector VH gene Antigen specificity VH mass Cloned in
(Da) construct
#
XI IGHV 1.08 Tetanus (A) 13703 8
XII IGHV 1.08 Tetanus (A) 13703 17
XIII IGHV 1.08 Tetanus (A) 13703 43
XIV IGHV 1.08 Tetanus (A) 13703 61
XV IGHV 1.08 Tetanus (A) 13703 62
XVI IGHV 3.30 Fibrinogen (C) 12794 63
XVII IGHV 3.30 Fibrinogen (C) 12794 64
XVIII IGHV 3.30 Fibrinogen (C) 12794 65
XIX IGHV 3.30 Fibrinogen (C) 12794 66
XX IGHV 3.30 Fibrinogen (C) 12794 67
69
Table 10:
Transfectio Transfectio Expecte AA AC CC Half A Half C other
n of n code d species foun found found found found (%)
(ratio) d (%) (%) (%) (%)
(%)
XIII + XVI ZO (1:1) AC 0 69 7 24 0 0
ZT (3:1) AC 10 45 16 27 0 0
ZU (1:1) AC 5 61 10 13 0 0
ZV (1:3) AC 3 61 23 13 0 0
ZW (1:1) AC 0 88.3 2.4 7 0 2.3
XIV + XVII ZP AC 30 52 13 0 0 5
XII + XVIII ZQ AC 4 51 33 2 1 8
XV + XIX ZR AC 20 42 11 0 1 26
XI + XX ZS AC 34 41 15 0 0 10
Combinations of CH3 variants were expressed, and analyzed in SDS-PAGE (data not
shown) and in native mass spectrometry (MS). Results are summarized in Table 10. The
ZO transfection resulted in the highest proportion of heterodimers in the mixtures (69%
AC). Interestingly, in the ZO transfection, the AA homodimer was not present whereas the
CC homodimer comprised a small proportion (7%). Mass spectrometric analysis unveiled
that the remaining protein in the mixture consisted of half A molecules, probably resulting
from unequal expression of the A and C heavy chains. The raw MS data from transfection
sample ZO are shown in Figure 8.
Surprisingly, whereas transfection ZO resulted in fair amounts of bispecific product, the
reverse charge pair of transfection ZP (L351K/T366’D versus T366K/L351’D of ZO) did
not result in similar results, and only 52% of bispecific product was observed, with
considerable amounts of the two homodimers being present (30% AA and 13% CC). An
explanation for this may be that the negatively charged D structurally closely resembles T,
hence the T366D may not be potent enough to repulse itself and T366D will thus still form
homodimers, as was indeed observed.
70
It can be envisaged that subtle variants of the newly found T366K/L351’D pair (e.g. by
testing all permutations including new constructs T366R and L351E) may result in similar
percentages of BsAbs.
Example 14: HADDOCK for design of new CH3 mutants to drive efficient
heterodimerization.
As described in example 13, the newly found charge pair T366K/L351’D increases the
proportion of heterodimers in the mixture (69 %) with a small fraction of undesired CC
homodimers (7%) (L351D/L351’D) and a substantial fraction of half A molecules (24%)
‘contaminating’ the mixture. In this example, an in silico approach was used to generate
further insight in amino acid residues involved CH3 interface interactions, to test
complementary substitutions in opposing CH3 regions and to find novel CH3 pairs
containing complementary substitutions that further increase efficient heterodimerization
while preventing efficient formation of homodimers of the two heavy chains.
HADDOCK (High Ambiguity Driven protein-protein DOCKing) is an information-driven
flexible docking approach for the modeling of biomolecular complexes. HADDOCK
distinguishes itself from ab-initio docking methods in the fact that it encodes information
from identified or predicted protein interfaces in ambiguous interaction restraints (AIRs) to
drive the docking process. (de Vries et al., 2010).
The input for the HADDOCK web server consists of a protein structure file, which can be
a crystal structure, NMR structure cluster or a modeled structure. After the docking or
refinement, HADDOCK returns a so-called HADDOCK score, which is a weighted
average of VanderWaals energy, electrostatic energy, buried surface area and desolvation
energy. The HADDOCK score can be interpreted as an indication of binding energy or
affinity, even though a direct translation to experimental data is often hard to achieve. In
addition to this, HADDOCK provides structure files for the ‘top four’ structures that
resulted from the docking run. These structure files can be downloaded and visualized,
enabling the detailed analysis of the interactions of the individual residues.
In this example, the interactions between the CH3-domains of the IgG1 heavy chains were
studied. A high-resolution crystal structure of the Fc part of the IgG (structure 1L6X) was
71
used as starting structure (http://www.rcsb.org/pdb/explore/explore.do?structureId=1l6x ;
Idusogie, E.E. et al., J.I. 2000(164)4178-4184).
In example 13, it was found that co-transfection of vectors XIII and XVI resulted in the
formation of the CC homodimeric contaminant (Table 10). HADDOCK was used to search
for additional mutations to the T366K/L351’D pair that prevent homodimerization.
The HADDOCK output consists of a set of calculated energies, a HADDOCK score
(which is a weighted average of the energies) and four structure files corresponding to the
four lowest-energy structures found by the program. The HADDOCK-scores are used to
compare different structures; the other energies are merely used to get an indication about
what is happening in the structures (e.g. good electrostatic interactions, smaller buried
surface, high Van der Waals energy). The lower the HADDOCK score, the better. For each
mutation pair, the scores were calculated for the AA, AB and BB dimers.
Sets of mutation pairs from example 12 were run in HADDOCK to see whether the
calculated energies would correlate to the experimental data. Table 11 presents all
theoretical energies, which are visualized in Figure 9.
Table 11:
Construct combinations HADDOCK VdW Electrostatic Desolvation Buried
Score energy energy energy surface
area
Wildtype-wildtype -208.2 -62.8 -773 9.2 2505.8
1-2 (E356KD399K – -225.8 -56.4 -862 3 2458.3
K392DK409D)
2-2 (K392DK409D – -180.3 -67.9 -562.1 0.1 2312.5
K392DK409D)
1-1 (E356KD399K – -176.7 -75.5 -469.3 -7.3 2349.6
E356KD399K)
1-3 (E356KD399K – -220.6 -67.9 -793.8 6.1 2499.8
K392DK409DK439D)
3-3 (K392DK409DK439D -150.1 -76.6 -387.6 4.1 2261.2
- K392DK409DK439D)
72
6-7 (T366W – -221.3 -65.8 -735.5 -8.3 2509.0
T366SL368AY407V)
6-6 (T366W – T366W) 1916.9* 2072.3 -681.3 -19.2 2499.9
7-7 (T366SL368AY407V -191.9 -55.0 -683.2 -0.2 2427.2
– T366SL368AY407V)
43-63 (T366K – L351D) -210.6 -64 -758.4 5.1 2456.5
43-43 (T366K – T366K) -191.7 -71.2 -634.1 6.3 2533.5
63-63 (L351D – L351D) -212.5 -60.4 -774 2.6 2445.6
*this value is unusually high due to high VanderWaals energy score, probably due to steric
clash of T366W/T366’W
With 2 wildtype CH3 domains, the HADDOCK scores are the same for AA, AB and BB
because the A and B CH3 regions are identical. In most other cases, the AB pair has the
lowest score, which is as expected. For the T366K/L351D pair the BB score is slightly
better than the AB score (-210.6 vs. -212.5), but this difference is within the error of the
calculations. Using HADDOCK, the structures of the heterodimers of these pairs were
visualized. For example, the construct combinations 1-2, 1-1 and 2-2 are presented in
Figure 10. From these visualizations it is apparent that salt bridges are formed in the
heterodimer (Figure 10A left hand panel) whereas electrostatic repulsion occurs between
residues of identical chains (Figure 10B and C, middle and right hand panel). The higher
HADDOCK scores for the homodimers can thus be explained by the electrostatic repulsion
of the mutated interface residues. These residues have to bend away from each other and
don’t have interaction with residues on the other chain, causing a drop in the affinity.
Table 11 and Figure 9 confirm what was observed in example 13. The T366K/L351’D AC
heterodimer and the L351D/L351’D CC homodimer form with a similar energy, explaining
the presence of both the heterodimer and homodimer in the mixture. The T366K/T366’K
AA homodimer, on the other hand, is barely detectable in the mixture although T366K half
A molecules are present. Table 11 and Figure 9 indeed show that the HADDOCK score for
the T366K/T366’K AA homodimer is higher than the score for the AC heterodimer; hence
formation of this homodimer is energetically less favorable.
73
Example 15: 366/351 variations
In example 13, it is hypothesized that alternatives for the T366K/L351’D mutant charge
pair can be designed that may have similar results in terms of percentage of bispecific
antibodies in the mixture. Alternatives may include substitutions T366R, T366D, T366E,
L351E, L351K and L351R. The proportion of CC homodimers of L351D/L351’D may be
diminished by creating variants of the 366/351 pair. All possible mutation pairs were run in
HADDOCK and the resulting scores are presented in Table 12 and visualized in Figure 11.
Table 12
Construct HADDOCK VdW Electrostatic Desolvation Buried
combinations Score energy energy energy surface
area
T366K – L351D -210.6 -64 -758.4 5.1 2456.5
T366K – T366K -191.7 -71.2 -634.1 6.3 2533.5
L351D – L351D -212.5 -60.4 -774 2.6 2445.6
T366K – L351E -216.9 -55.7 -854.7 9.8 2532.7
L351E – L351E -217.9 -65.5 -802.2 8 2532
T366R – L351D -210.5 -68.8 -760.8 10.4 2514.5
T366R – T366R -201.8 -77.4 -626.4 0.9 2608
T366R – L351E -225.8 -56.2 -874.8 5.4 2579.2
T366D – L351R -211.2 -71.3 -723.6 4.8 2455.6
T366D – T366D -198.1 -58.1 -713.4 2.1 2477
L351R – L351R -220.7 -75.5 -806.5 16.1 2552.2
T366D – L351K -223.9 -62.1 -810.1 0.3 2487.8
L351K – L351K -224.4 -75.6 -812.1 13.6 204.5
T366E – L351R -222.3 -69 -783 3.4 2557.2
T366E – T366E -201.9 -57.6 -741 4 2487.5
T366E – L351K -215.9 -58.4 -808.9 4.3 2486
74
When looking at the HADDOCK scores, it was observed that some of the mutations have a
similar ‘pattern’ when compared to T366K/L351’D. For most permutations the AA
homodimer was found to have a higher HADDOCK-score than the AB heterodimer, but
the BB homodimer appeared as favorable as the AB heterodimer. Even though the 351
residue is known to be a ‘neighbor’ to itself on the other chain, i.e. residue 351 of chain A
pairs with residue 351 of chain B at the CH3-CH3 interface, there is barely a negative
influence of the identical charges when the BB dimer is formed. Looking at the
L351D/L351’D structure this is explained by the aspartic acids bending away from each
other and the stabilizing influence of at least the naturally occurring Arginine at position
355 and also some stabilization of negative charge by the naturally occurring Serine at
position 354 (see Figure 12A). Mutation of these residues (S354A and R355D) provides
only little improvement. From figure 12B it is clear that the backbone-hydrogen of A354
causes stabilization of the homodimer. From this series, the T366R/L351’E pair seems to
be the most favorable, with the lowest HADDOCK score for the bispecific molecule.
Example 16: mutations around T366K/L351’D
In the series of HADDOCK analyses in this example, the T366K/L351’D or
T366K/L351’E pair were taken as a starting structure. In order to identify additional
mutations that would further increase the predicted percentage of bispecifics of these A
and B chains, additional mutations on the B-chain were used to calculate the HADDOCK-
scores and energies. When the structure of the CH3 domain is studied using a viewer for
visualization of protein structures at a molecular level (YASARA, www.yasara.org), one
can calculate the distances between individual residues. While doing so, it was observed
that the two residues Y349 and L368 are neighboring residues that may contribute
positively or negatively to dimer interactions and these have been mutated in this example
–in addition to the L351D mutation– to study the result on dimer formation of the homo-
and heterodimers (see figure 13). Both residues seem to add to the stability of the
heterodimer (lower HADDOCK scores) as well as to the destabilization of the BB dimer
(higher HADDOCK scores). Glutamic acids (E) on positions 349 and 368 seem to be more
favorable than aspartic acids (D). Thus, introduction of a second amino acid substitution in
75
the B-chain, comprising already the amino acid substitution at position 351, seems to favor
heterodimerization further.
In a next set of HADDOCK analyses, the T366K/L351’D pair was again taken as starting
structure. In addition to the substitutions in the B chain that further increased
heterodimerization (i.e. Y349D/E and L368E), additional mutations were added to the A-
chain which already comprises the T366K substitution. As shown in Figure 14, there are
several mutation pairs that seem favorable towards the formation of bispecific
heterodimers. In the T366K-L351K/L351’D-Y349’D pair, all four mutated residues are
involved in the heterodimeric pairing, which is not de case for T366K-L351K/L351’E-
L368’E in which K351 is not directly involved in the binding. However, the HADDOCK-
score for this latter heterodimer is -228.9; significantly lower than the -214.2 for the
T366K/ L351’E-L368’E, which can be explained by hydrogen bonding interactions of the
K at position 351 (see Figure 15). The T366K-L351K/L351’D-Y349’D pair may be further
improved by the R355’D mutation in the B-chain, which results in a higher BB-
HADDOCK score, but also the AB HADDOCK score is slightly higher. Overall the
additional L351K results in lower AB scores and similar AA and BB scores when
compared to the sole T366K mutation in the A chain. Theoretically this would result in
higher amounts of bispecific heterodimers in the samples.
As is apparent from figure 11, having an R rather than a K at position 366 may be more
potent in driving heterodimerization. Therefore, some of the HADDOCK analyses shown
in figure 13 were repeated but now with T366R rather than T366K in the A-chain. It was
demonstrated that it is not favourable to combine an R366 in chain A with double
mutations in chain B (figure 16). This may be due to the large size of this residue,
interfering with other interface interactions, even though all the expected salt-bridges with
R366 are present in the structures. Also, the HADDOCK score for the AA homodimer is
lower for R366 than for K366, which also doesn’t contribute favorably to heterodimer
formation. Therefore no further HADDOCK analyses were performed using R366 in the
interface.
A total of 14 best performing pairs, according to HADDOCK predictions, have been
selected (see Table 13 and Figure 17). In some pairs, an R355D substitution is included to
76
remove the stabilizing influence of the naturally occurring R355 on the L351/L351’D
interaction.
Table 13:
Construct combinations HADDOCK HADDOCK HADDOCK
Score AB Score AA Score BB
Wildtype-wildtype -208.2 -208.2 -208.2
T366K – L351D -210.6 -191.7 -212.5
T366K – L351E -216.9 -191.7 -217.9
T366R – L351E -225.8 -201.8 -217.9
T366E – L351R -222.3 -201.9 -220.3
T366K – L351DY349E -215.9 -191.7 -190
T366K – L351DL368E -223.3 -191.7 -198.9
T366K – L351EY349E -214.5 -191.7 -187.5
T366KL351K – L351D -233.2 -205 -212.5
T366K – -207.5 -191.7 -179.5
L351DY349EL368E
T366KL351K – -255.2 -205 -204.3
L351DY349D
T366KL351K – -227.2 -205 -190
L351DY349E
T366KL351K – -243.9 -205 -198.9
L351DL368E
T366KL351K – -233.6 -205 -211.9
L351DR355D
T366KL351K – -242.8 -205 -183.5
L351DY349DR355D
T366D – L351KY349K -237.9 -198.1 -228.4
77
Example 17: in vitro expression of bispecifics using CH3 mutants based on
HADDOCK predictions
The analysis in example 16 suggested that some CH3 variants with additional mutations
around the T366K/L351’D pair would yield mixtures with higher proportions of the
bispecific component and potentially lower proportions of the homodimeric component.
These best performing pairs were selected for production and further analysis. In addition,
the constructs T366R and L351E were also generated. Table 14 lists the constructs that
were made and which were used for recloning antibody VH regions with known
specificities and known ability to pair with the human IGKV1-39 light chain. Expression
of the IgGs that contain the individual constructs was previously reported in example 13,
and was repeated for the constructs as listed in Table 14. Aim was to assess which of the
constructs homodimerize in the absence of a matching heterodimerization partner. Ideally,
high percentages of half bodies would be formed and low percentages of homodimers. As
a control, constructs containing previously reported charge mutations and constructs
containing the previously reported knob-in-hole mutations were also used for expression as
whole IgG by recombinant cells. Protein A purified supernatants were analyzed in SDS-
PAGE; results were analyzed and scored as presented in Table 14
78
Table 14:
AA substitutions in CH3 Construct % IgG % half molecule
#
E356K, D399K 1 64.2 35.8
K392D, K409D 2 30.9 69.1
K392D, K409D, K439D 3 24.5 75.5
T366W 6 27.6 72.4
T366S, L368A, Y407V 7 58.6 41.4
T366K 43 32.9 67.1
L351D 63 89.8 10.2
T366D 64 89.6 10.4
T366K, L351K 68 34.7 65.3
L351D, L368E 69 83.7 16.3
L351E, Y349E 70 67.8 32.2
L351D, Y349E 71 79.7 20.3
L351D, R355D 72 100 -
L351D, Y349E, L368E 73 79.3 20.7
L351D, Y349D 74 88.6 11.4
L351D, Y349D, R355D 75 89.9 10.1
L351K, L368K 76 56.6 43.4
L351R 77 100 -
T366E 78 44.4 55.6
T366R 79 29.6 70.4
L351E 80 100 -
The results of co-expression of a common light chain and two different heavy chains
carrying the amino acid substitutions of constructs shown in Table 14 or heavy chains
carrying the amino acid substitutions of previous constructs are presented in Table 15.
Expression of two different heavy chains comprising the amino acid substitutions T366K
79
and L351’D:L368’E respectively resulted in approximately 87% of the bispecific AB
heterodimer in the mixture with no AA or BB homodimers present (combination nr. 3 of
Table 15). About 12% half molecules (half A) comprising the T366K substitution was
observed. Furthermore, it was found that the percentage of bispecific AB heterodimer
increased when the additional amino acid substitution L351K was introduced in the first
heavy chain. For example, co-expression of two different heavy chains comprising the
amino acid substitutions T366K:L351K and L351’D:L368’E respectively resulted in
approximately 92% of bispecific AB heterodimer whereas AA and BB homodimers are
essentially absent in the mixture (combination nr. 12 of Table 15). Combinations 10 and 11
also resulted in favorable distributions of high percentages heterodimers and virtually
absence of homodimers. The absence of homodimers is advantageous, because the fraction
containing the intact IgG molecules is composed of AB heterodimer only. For purification
and subsequent therapeutic application, the half molecules can be removed by standard
approaches such as size exclusion chromatography. Hence, applying these newly identified
charge mutants in the production process for generating bispecific antibodies provides
advantages over known charge mutants and knobs-into-holes mutants where the presence
of 'contaminating' homodimeric antibodies is not excluded. In addition, the
T366K/L351’D:L368’E and T366K:L351K/ L351’D:L368’E charge pairs have an
additional advantage over the previously described E356K:D399K/K392’D:K409’D and
E356K:D399K/K392’D:K409’D:K439’D charge reversal pairs, in that the previously
described charge variants are based on the reversal of existing charges within the CH3-
CH3 interface whereas the newly identified charge variants are adding additional charge
pairs (charge-charge interactions) to the CH3-CH3 interface. The introduction of additional
charge pairs in the CH3-CH3 interface may further increase the stability of the interface
and thereby of the intact antibody. The same holds true for the mutations used in
combinations nrs. 4, 5, 6, 9, 10, and 11, which also resulted in favorable proportions of
bispecific heterodimer with exceedingly low proportions of AA and BB homodimers
present in the mixtures.
80
Table 15:
Combinatio % %
chain A* / % % %
n of 2 chain B**/ half half
mutations AA AB BB
different mutations A B
(construct foun foun foun
heavy (construct #) foun foun
#) d d d
chains d d
1 T366E (78) L351R (77) 3 81 2 13 0
2 T366K (43) L351D (63) 0 88 3 9 0
3 T366K (43) L351D,L368E (69) 0 87 0 12 0
4 T366K (43) L351E,Y349E (70) 2 85 0 11 0
T366K (43) L351D,Y349E (71) 2 92 1 5 0
L351D,Y349E,L368
6 T366K (43) 0 96 1 4 0
E (73)
T366K,L351
7 L351D (63) 0 77 12 10 1
K (68)
T366K,L351
8 L351D,R355D (72) 0 79 8 10 1
K (68)
T366K,L351 L351D,Y349D,R35
9 1 93 2 4 1
K (68) 5D (75)
T366K,L351
L351D,Y349D (74) 1 95 1 3 0
K (68)
T366K,L351
11 L351D,Y349E (71) 1 95 0 3 1
K (68)
T366K,L351
12 L351D,L368E (69) 0 92 0 8 0
K (68)
13 T366K (43) L351E (80) 0 70 10 18 2
14 T366R (79) L351E (80) 4 38 36 21 1
T366D (64) L351K, L368K (76) 3 92 2,5 2,5 0
16 T366D (64) L351R (77) 30 69 1 0 0
* chain A carries specificity of MF1337 (=tetanus toxoid); ** chain B carries specificity of
MF1122 (=fibrinogen)
81
Native MS
Native MS was performed on all bispecific samples. The obtained graphs were analyzed to
determine the relative ratio's of the present species in two ways: by peak height and by
peak area. Peak area is the more scientifically correct way of analysis, but since all
previous analyses for other studies were done based on peak height, both methods were
included in the analysis, for comparison purposes. The differences between the methods
were within the error of measurement, and therefore only the peak area values were used
for future measurements. Two typical spectra are shown in Figure 18. An overview of the
results is shown graphically in Figure 19, the numerical values can be found in Table 15. In
about half of the samples the total contamination of monospecific lgG is less than 5%, and
only in three cases it is > 10% while for wt lgG it is expected to find about 50% of
monospecific lgG in the mixture.
A panel of ten combinations of 2 different heavy chains was selected from Table 15 for
further analyses. These ten combinations included combinations 1, 2, 3, 4, 5, 6, 9, 10, 11
and 12 (Table 15). Selection of these ten was based on low percentages of homodimers
present in the mixtures as determined by nMS, but also based on their overall physico-
chemical properties, including production yields, SDS-PAGE, as well as the number of
mutations present in the CH3 domain.
Example 18: IgG stability analyses
In this study, a series of CH3 mutation pairs that resulted in high proportions of bispecific
heterodimers in the intact IgG fraction and very low amounts (<5%) of parental IgGs will
be further analyzed for stability of the Fc part of the IgG molecule. The mutated CH3
domains that are used to promote the heterodimerization of the heavy chains may have
unexpected destabilizing effects on the Fc region of the IgG, that may result in undesirable
properties such as a reduction of in vivo half life, reduction in effector function and/or an
increase in immunogenicity. The newly identified charge pairs will be compared to
wildtype bispecifics and a bispecific containing previously identified charge mutations
(chain A comprising construct 1 and chain B comprising construct 2). All bispecifics in
82
this study will contain the same heavy and light chain variable regions, ensuring that the
observed effects are caused by mutations in the Fc-part of the molecule and not by
variation in the variable regions.
A series of stability studies will be performed on these bispecifics. These studies include
spectroscopic (UV-Vis absorbance, fluorescence and light-scatter) and microscopic (light
and fluorescence microscopy with Nile Red staining) analyses that provide information on
the aggregation state of the CH3 variants.
The UV-Vis absorbance spectra will be recorded with a double beam, two
monochromators Cary 300 Bio spectrophotometer at 25°C. The spectra will be monitored
between 250 and 400 nm using a path length of 1 cm. The absorbance at wavelengths of
320 nm and longer provides information on the aggregation state of the IgG.
Intrinsic fluorescence spectra will be monitored at 25°C using a FluoroMax
spectrofluorimeter. The fluorescence method will be optimized. The fluorescence
emission will provide information on conformation and aggregation properties.
90° light-scattering spectra will be monitored at 25°C using a FluoroMax
spectrofluorimeter by running a synchronous scan ( λ = λ ) between 400 nm and 750 nm
em ex
with an integration time of 0.01s. Excitation and emission slits will be optimized. For
example, right angle light-scattering can distinguish between IgG samples that have no and
% dimers.
For fluorescence microscopy with Nile Red staining, just prior to measurements, Nile Red
in ethanol will be added to the sample. The samples will be filled in a microscopy slide and
analyzed by fluorescence microscopy. Particles will be counted. The lower size limit of the
particles that can be observed by fluorescence microscopy is approximately 0.5 µm.
Application of stress such as temperature, pH, mechanical stress or denaturants on proteins
might result in a conformation change (e.g. unfolding) and/or aggregation. As it was
previously reported that charge-engineered bispecific antibodies have reduced melting
temperature of the modified CH3 (Gunasekaran 2010), these studies aim to discriminate
between the novel charge mutants useful in the present invention and existing known
charge mutants.
83
Thermo-stability studies using the Octet are explored, both with Protein A biosensors and
by using FcRn binding to IgG. To examine the thermal stability of CH3-engineered IgGs,
the samples will be incubated at a concentration of 100 ug/ml (in PBS) at 4, 50, 55, 60, 65,
70 and 75˚C for 1 hour using a PCR machine. Following this the samples will be cooled
down slowly during a period of 15 minutes to 25˚C and kept at this temperature for 2
hours, after which they will be stored overnight at 4˚C. Precipitated antibodies will be
removed by centrifugation, after which the total IgG concentration of soluble antibodies
will be determined by Octet using the protein A Biosensor (1/10 dilution in PBS). Assays
that measure binding of the CH3 engineered IgG to FcRn using the Octet are being
explored. Either protein L biosensors are used to bind the light chain of IgG to the sensor,
followed by incubation with FcRn in solution, or anti-penta-HIS biosensors are used to
bind His-tagged FcRn protein, followed by incubation with the IgG of interest. These
methods may be more sensitive than using the protein A Biosensor and can also be used
for thermal stability studies.
All samples will also be analyzed for serum stability. Briefly, (engineered) IgG samples
will be incubated at 37˚C in human serum, control samples will be kept at 4°C. After 1, 2,
3 and 4 weeks, samples are centrifuged to remove precipitated IgG. Subsequently the
sample is titrated in antigen-specific ELISA to determine the relative amounts of functional
IgG. Purified control antibody freshly spiked in human serum will be used as a reference.
Example 19: stability analyses
In previous experiments, high percentages of bispecific antibodies were obtained by co-
expression of two different heavy chains comprising CH3 mutations, and a common light
chain (example 17).
A panel of eight combinations of 2 different heavy chains was selected from Table 15 for
further analyses. These eight combinations included combinations 3, 4, 5, 6, 9, 10, 11 and
12 (Table 15). In this study, these eight combinations were analyzed, with a strong focus
on stability of the Fc part of the IgG. As controls, wildtype bispecifics (i.e. without CH3
mutations) and/or bispecifics based on previously reported CH3 charge mutations were
included. Note that for wildtype bispecifics, 2 heavy chains and the common light chain
are co-expressed without means for preferential steering towards heterodimers. These
84
‘wildtype bispecifics’ thus represent a mixture of AA, AB and BB. All bispecifics in this
study were designed to carry the same VH/VL-combinations, ensuring that the observed
effects are caused by mutations in the Fc-part of the molecule and not by variation(s) in the
Fab parts.
It was hypothesized that the mutational pairs that were used to promote the heterodimeric
pairing of the two different heavy chains could be associated with unexpected structural or
otherwise destabilizing effects on the Fc region of the IgG. This could subsequently result
in undesired issues that would hamper further clinical development, such as a reduction of
in vivo half life, a reduced effector function and/or increased immunogenicity due to the
presence of these mutations.
Thermo stability
Application of stress such as increases or decreases in temperature might result in a
conformation change (e.g. unfolding) and/or aggregation of proteins. To examine the
thermal stability of CH3-engineered IgGs, the bispecific molecules from combinations 3-6
and 9-12 (Table 15), as well as wildtype bispecifics and bispecific molecules obtained
when using constructs 1 and 2 (E356K:D399K/ K392D’:K409D’ combination, also
dubbed ‘charge reversal’ pair) were incubated at a concentration of 100 μg/ml (in PBS) at
4, 60, 62.5, 65, 67.5, 70 and 72.5˚C for 1 hour using a PCR machine. Following this the
samples were cooled down slowly during a period of 15 minutes to 25˚C and kept at this
temperature for 2 hours, after which they were stored overnight at 4˚C. The next day,
precipitated antibodies were removed by centrifugation (18,000 rpm; 4˚C, 20 min), after
which the total IgG concentration of soluble antibodies was determined by Octet using the
protein A Biosensor (1/10 dilution in PBS). Results are shown in figure 20. It was
observed that the control CH3 engineered bispecific antibody (the charge reversal
E356K:D399K/ K392D’:K409D’ combination (triangles)) has a reduced thermal stability
as compared to the wildtype bispecific (squares). The bispecific molecules from
combinations 3-6 and 9-12 (diamonds) also demonstrated a reduced thermal stability as
compared to wildtype. Remarkably, three combinations, however, demonstrated an
improved stability as compared to the control CH3 engineered bispecific antibody.
Bispecifics of combinations 9, 10 and 11 are significantly more stable than the other CH3
85
engineered (charge reversal) bispecifics and are as stable as wildtype bispecifics at the
highest temperature measured.
Freeze-thaw stability
To examine the stability of CH3-engineered IgGs upon repetitive freezing and thawing, the
bispecific molecules from combinations 3-6 and 9-12 (Table 15), as well as wildtype
bispecifics and bispecific molecules obtained when using constructs 1 and 2
(E356K:D399K/ K392D’:K409D’ combination (charge reversal pair)) were exposed to ten
subsequent freeze-thaw cycles by putting the samples at -80 ⁰C for at least 15 minutes until
they were completely frozen. Thereafter, samples were thawed at room temperature. When
they were completely thawed, the freeze-thaw cycle was repeated. After 10 freeze-thaw
cycles, precipitated antibodies were removed by centrifugation (18,000 rpm; 4˚C, 20 min),
after which the total IgG concentration of soluble antibodies was determined by Octet
using the protein A Biosensor (1/10 dilution in PBS). The freeze-thaw stability test was
repeated three times. Results are shown in figure 21. It was observed that the control
charge reversal CH3 engineered bispecific antibody seemed to have a slightly reduced
stability as compared to the wildtype bispecific. In contrast, the bispecific molecules from
combinations 3, 4 and 9 seemed to have a slightly improved stability as compared to the
wildtype bispecific. Overall, it can be concluded that the stringent conditions of freeze-
thaw cycles do not cause major stability issues for the CH3 engineered variants.
In vitro serum stability
To examine the stability of CH3-engineered IgGs in serum kept at 37 ⁰C, the bispecific
molecules from combinations 3-6 and 9-12 (Table 15), as well as wildtype bispecifics and
the charge reversal bispecific molecules were incubated at 37 ⁰C in 10% human serum.
Control samples were kept in human serum at 4 ⁰C. After 1, 2 or 5 days, precipitated
antibodies were removed by centrifugation. Thereafter, the samples were titrated in a
fibrinogen-specific ELISA, to determine the relative amounts of functional IgG. Purified
control antibody freshly spiked in human serum was used as reference.
Data of the fibrinogen ELISA show that all samples were quite stable in 10% human serum
at 37°C for 5 days. At the lower IgG concentration bispecific molecules from combinations
86
4 and 5 seem to be slightly less stable, especially at T=1 and T=2, but the difference is only
minimal at the end-point of this experiment (see Figure 22).
Example 20: Further stability tests
A further series of analytical methods was used to assess the stability of the variant IgGs.
Bispecific molecules from combinations 3-6 and 9-12 (Table 15), as well as wildtype
bispecifics (AA, AB, BB), the individual parental antibodies (AA and BB) and bispecific
molecules obtained when using constructs 1 and 2 (E356K:D399K / K392D’:K409D’
combination (charge reversal pair)) were used as samples in these stability assays. All IgGs
were diluted to 0.2 mg/ml and several stress conditions (2 days at 50 ⁰C, 2 weeks at 40 ⁰C,
5x freeze-thawing) were applied, aiming to be able to discriminate between the different
samples. Of note, these high stress levels resulted in conditions in which one of the
parental antibodies (the BB parental, carrying two 1122 Fabs) as used in all bispecifics
became unstable. At 2 days at 50°C, aggregation of this protein was detected by UV
absorbance. This suggested that this stress condition may not differentiate between
instability of the Fab and the CH3 in the bispecific and data resulting from the 50°C
incubation should be used cautiously.
The results are summarized in Table 16. Analytical methods that were used included:
- Fluorescence microscopy with Nile Red (‘Nile Red particles’ in Table 16); to
observe the amount of particles > 0.5 µm after addition of Nile Red dye.
- UV spectrometry at 350 nm (‘UV 350 nm’); a change in absorption at wavelengths
> 320 nm gives information about the aggregation state of the protein.
- 90° Light scatter at 400 nm (‘LS 400 nm’); a sensitive technique to observe
changes in protein aggregation, e.g. the difference between monomers and dimers
of IgG.
- Intrinsic fluorescence; the fluorescence wavelength maximum and intensity of the
aromatic residues in a protein change upon changes in the environment (e.g.
unfolding)
87
- 1,8-ANS fluorescence spectroscopy; 1,8-ANS binds through electrostatic
interactions to cationic groups through ion pair formation and changes in protein
structure and/or conformation can be detected
UV-VIS spectroscopy
UV-Vis absorbance spectra were measured at 25°C with a double beam, two
monochromators Cary 300 Bio spectrophotometer from Varian in different quartz cuvettes
(such as black low volume Hellma cuvettes with a pathlength of 1.0 cm and clear Hellma
cuvettes of 0.2 cm x 1.0 cm). The spectra were monitored between 220 and 450 nm using a
pathlength of 1.0 cm. The absorbance around 280 nm provides information on the protein
concentration. The region between 320 nm and 450 nm can provide information on the
aggregation state of the samples.
90° light-scattering
The 90° light-scattering spectral method was developed to study protein aggregation and
was performed as described in Capelle, 2005; Demeule, 2007a. 90° light-scattering spectra
were monitored at 25°C using a FluoroMax spectrofluorimeter (Spex, Instruments S.A.,
Inc. U.K.) by running a synchronous scan ( λem = λex) between 400 nm and 750 nm with an
integration time of 0.01 s. Different slits settings were tested in order to find the optimal
conditions. After optimization, the same slit settings were used for all measurements.
Steady-state fluorescence emission
The fluorescence emission of tryptophan, tyrosine and phenylalanine residues gives
information on the local environment of these fluorophores. Changes or differences in
hydrophobicity and/or rigidity are measured. Typically, a more hydrophobic and rigid
environment leads to an increase in the fluorescence intensity and a blue shift of the
emission maximum. Intrinsic fluorescence spectroscopy can provide information on the
current state of the protein and monitor changes in the physical and chemical properties.
More information on the fluorescence of tyrosine and tryptophan can be found in the book
of Lakowicz [Lakowicz, 2006].
88
The fluorescence emission and excitation spectra were recorded at 25°C in different quartz
cuvettes. The samples were excited at different wavelengths. Integration times and slit
settings were optimized. After optimization, the same integration times and slit settings
were applied for all samples.
Fluorescence microscopy with Nile Red staining
The Nile Red staining method was developed to visualize protein aggregates and was
performed as described in Demeule et al., 2007b.
The microscopy observations were performed on a Leica DM RXE microscope (Leica
Microsystems GmbH, Wetzlar, Germany) equipped with a mercury lamp. The images were
acquired with a Sony NEX-5 camera and its firmware. The objectives were 10x, 20x and
40x. For microscopy investigations slides with a fixed distance of 0.1 mm between the
slide and the cover glass were used. The size of the 4x4 grids is 1 mm x 1 mm and
corresponds to 0.1 µl.
1,8-ANS fluorescence spectroscopy
1-anilinonaphthalenesulfonic acid (1,8-ANS) is an uncharged small hydrophobic
fluorescent probe (Mw 299.34 Da) used to study both membrane surfaces and proteins.
1,8-ANS is essentially non-fluorescent in water and only becomes appreciably fluorescent
when bound to membranes (quantum yields ~0.25) or proteins (quantum yields ~0.7). This
property of 1,8-ANS makes it a sensitive indicator of protein folding, conformational
changes and other processes that modify the exposure of the probe to water. References on
1,8-ANS can be found on the Internet home page of Molecular Probes, www.probes.com.
The fluorescence emission spectra of 1,8-ANS were recorded using a FluoroMax
spectrometer. A direct comparison of the 1,8-ANS fluorescence between IgGs will not be
performed. Each IgG can have different number of 1,8-ANS binding sites and can
therefore not be compared. In principle, the lower the 1,8-ANS fluorescence, the less 1,8-
ANS molecules are bound to the antibody. The changes in the 1,8-ANS fluorescence
intensity and emission wavelength due to stress will be evaluated.
89
Table 16: Overview of the different forced degradation results on various IgG samples
after dilution to 0.2 mg/ml. The colour of the cells indicate the variations between T= 0 and
after stress: dark grey = large change, light grey = small change and no colour = no change
(=stable).
* ‘combi. #’ refers to the combination of mutations as listed in Table 15; ** very small
particles by fluorescence microscopy, relevance of these particles unknown; 2d4 ⁰C= 2
days at 4 ⁰C; 2d50 ⁰C= 2 days at 50 ⁰C; 2w4 ⁰C= 2 weeks at 4 ⁰C; 2w40 ⁰C= 2 weeks at 40 ⁰C;
T0= start of experiment; 5FT=5 freeze thaw cycles
Protein Stress Nile red UV 350 LS 400 Intrinsic 1,8-ANS fluorescence
sample particles nm nm fluorescence
7
(10 cps)
fluo.int λ 1,8- λ Shif
. Max. ANS Max t
6
(10 cps (nm) int. . (nm
6
) (10 cps (nm )
) )
BB 2d4 ⁰C 0-10 0.001 0.7 4.2 335
2d50 ⁰C 0-10 0.013 0.8 4.2 335
AA 10-20 0 1.2 5.7 338
2d4 ⁰C
2d50 ⁰C 10-20 0.002 1.0 5.5 338
Wildtype 2d4 ⁰C 30-50 0.003 0.9 5.1 336 7.1 507
bispecific
2d50 ⁰C >10000* 0.007 0.9 5.0 336 7.1 507
(AA AB BB)
*
2w4 ⁰C 0 0.9 5.0 336
2w40 ⁰ >2000** 0 0.8 5.0 336
C
T0 0.001 0.8 5.0 336
5FT >2000** 0.009 1.2 4.8 336
Charge 2d4 ⁰C 10-20 0.001 1.3 5.9 336 7.0 507
reversal
2d50 ⁰C 10-20 0.002 1.2 5.7 336 7.0 507
90
bispecific >2000** 0 1.1 5.5 336
2w4 ⁰C
(E356K,D39
2w40 ⁰ >2000** 0.002 1.1 5.5 336
9K/
C
K392D,K409
T0 0.001 1.3 5.7 336
D)
5FT 30-50 0.007 1.8 5.5 336
Combi. # 3* 2d4 ⁰C 30-50 0 0.9 5.0 337
2d50 ⁰C 30-50 0.001 0.8 4.9 337
Combi. # 4 2d4 ⁰C 20-30 0 1.0 6.2 337 7.5 505
>3000** 0.001 1.0 6.2 337 7.5 505
2d50 ⁰C
2w4 ⁰C 0.001 1.0 6.3 337
2w40 ⁰ >2000** 0.003 0.9 6.3 337
C
T0 0.002 1.1 6.3 337
5FT >2000** 0.003 1.2 6.0 337
Combi. # 5 2d4 ⁰C >2000** 0.001 1.1 4.9 337
2d50 ⁰C >10000* 0.001 0.9 5.0 337
*
Combi. # 6 2d4 ⁰C 10-20 0 0.7 4.3 337
2d50 ⁰C 20-30 0.001 0.7 4.3 337
Combi. # 9 30-50 0 1.0 5.5 337 7.5 507
2d4 ⁰C
2d50 ⁰C 50-100 0 1.0 5.5 337 8.1 500 -7
2w4 ⁰C >2000** 0 0.9 5.1 337
2w40 ⁰ >2000** 0 0.9 5.2 337
C
T0 0.002 0.8 5.1 337
5FT >2000** 0.007 1.4 4.9 337
Combi. # 10 2d4 ⁰C 30-50 0.002 1.0 5.6 337 7.0 505
150-200 0.001 1.1 5.9 337 8.7 499 -6
2d50 ⁰C
2w4 ⁰C >2000** 0 0.9 5.2 337
91
>2000** 0 0.9 5.4 337
2w40 ⁰
C
T0 0.005 1.0 5.3 337
5FT 20-30 0.004 1.1 5.4 337
Combi. # 11 2d4 ⁰C 20-30 0 0.9 4.9 337
2d50 ⁰C 30-50 0.002 0.9 5.1 337
2w4 ⁰C >2000** 0 0.8 5.0 337
2w40 ⁰ >2000** 0 0.8 5.1 337
C
T0 0.004 1.1 5.0 337
5FT >2000** 0.002 1.2 5.0 337
Combi. # 12 2d4 ⁰C 10-20 0.001 0.8 3.8 337 6.2 511
-20 0.002 0.7 3.8 337 6.5 508 -3
2d50 ⁰C
2w4 ⁰C >2000** 0.003 0.6 3.6 337
2w40 ⁰ >2000** 0.001 0.5 3.5 337
C
T0 0.005 0.6 3.7 337
5FT 0.004 0.7 3.6 337
Taken together, these data indicate that the various IgG samples are remarkably stable.
Severe stress conditions (e.g. 2 days at 50 ⁰C) were needed to generate measurable
differences between the tested samples. Under these conditions, samples of combinations #
9 and # 10 seem to aggregate more than other samples.
The most discriminating factors for stability between the proteins are the freeze-thaw
cycles and increased temperature. Taking into account the very stringent stress factor of
incubating at 50°C, the T366K/L351E,Y349E (combi.#4) and
T366K,L351K/L351D,Y349E (combi.#11) variants are the two most stable proteins within
panel, closely followed by T366K,L351K/L351D,Y349D (combi.#10) and
T366K,L351K/L351D,L368E (combi.#12).
92
Example 21: Native MS on ratio experiments; transfection ratio’s from 1:5 to 5:1
To become more knowledgeable about the behavior of the CH3 mutated IgGs in skewed
transfection mixtures, in particular about the T366K:L351K/L351D’:L368E’ combination
(from now on dubbed KK/DE or DEKK), a more elaborate ratio experiment was
conducted.
Previously used antibody VH regions with known ability to pair with the common light
chain IGKV1-39 were used for recloning into constructs 1, 2, 68 and 69, resulting in
vectors I-V of Table 17. Vectors I-V, each containing nucleic acid sequences encoding the
common human light chain as well as an Ig heavy chain with different CH3 region and
different antigen specificity, were subsequently transfected into cells with different
transfection ratios as indicated in Table 18. Results are shown in figure 23.
Table 17:
Vector VH gene Antigen VH mass Merus Cloned in construct
specificity (Da) designation #
I IGHV Fibrinogen 12794 MF1122 69 (L351D, L368E)
3.30 (A)
II IGHV RSV (C) 13941 MF2729 69 (L351D, L368E)
3.23
III IGHV Tetanus (B) 13703 MF1337 68 (T366K, L351K)
1.08
IV IGHV Fibrinogen 12794 MF1122 1 (E356K, D399K)
3.30 (A)
V IGHV Tetanus (B) 13703 MF1337 2 (K392D, K409D)
1.08
93
Table 18:
Transfection nr vectors ratio
1 I and III 5:1
2 I and III 3:1
3 I and III 1:1
4 I and III 1:3
I and III 1:5
6 II and III 5:1
7 II and III 3:1*
8 II and III 1:1
9 II and III 1:3
II and III 1:5
11 IV and V 5:1
12 IV and V 3:1
13 IV and V 1:1
14 IV and V 1:3
IV and V 1:5
*due to a technical error, this sample has not been measured.
Figures 23A and B show that for the DEKK combination of mutations, when an excess of
A or C is present (A or C are on the ‘DE side’ and B is on the ‘KK side’), AB or BC is
formed but the surplus of A or C is present as a mixture of both homodimers and half
bodies in all cases. However, when an excess of B is present (B is on the ‘KK side’ and A
or C are on the ‘DE side’), there is a clear difference. AB or BC is still formed but the
surplus of B is essentially absent as homodimer and only half bodies are formed.
Percentages were again measured by peak height Nota bene: peaks detected in the range of
2% or lower are below the threshold of what the nMS technology as applied can accurately
measure. Measurements of <2% are therefore regarded to be within the noise level of
analysis and therefore ignored. It is striking that the excess of B results in high percentages
of half body B only. Especially at the 1:3 and 1:5 ratios of A:B, high percentages of half
94
body B were observed (Fig. 23A and 23B) in the absence of homodimer BB, indicating
that the CH3 mutations of the KK-side disfavour homodimerization. The absence of
homodimers offers a crucial advantage, as this ‘KK side’ of the DEKK combination can be
chosen to incorporate a specificity that may have known adverse effects when present as a
homodimer (for example cMET or CD3 antibodies are known to have undesired adverse
side effects when present as bivalent homodimers in therapeutic compositions).
The observed findings for the different ratio’s of DE:KK are in contrast to the control
charge reversal CH3 mutations in vectors IV and V. Figure 23C shows that for the
E356K:D399K/K392D’:K409D’ combination of mutations when an excess of A is present
(A is on the ‘K392D:K409D side’), the surplus of A is present as a mixture of both
homodimers and half bodies in all cases, but also when an excess of B is present (B is on
the ‘E356K:D399K side’), the surplus of B is present as a mixture of both homodimers and
half bodies in all cases. Even at the higher ratios 1:3 and 1:5 no half bodies B are observed
although homodimers are present, indicating that the E356K:D399K side does not
disfavour homodimerization as much as the KK-side of the DEKK combination.
Taken together, the DEKK combination of mutations offers a clear benefit over the charge
reversal CH3 mutations, in that one of the chains of the heterodimer does not form
homodimers.
Example 22: varieties of mixtures using the DEKK combination
As it was demonstrated that the DEKK combination of mutations drives the formation of
bispecific IgG molecules (‘AB’) with high purity, we next explored the feasibility of
controlled production of more complex antibody mixtures from one cell, such as ‘AB and
AA’ or ‘AB and AC’ mixtures. Previously used model Fabs were incorporated in vectors
that contain either the ‘DE construct’ or the ‘KK construct’ and various combinations of
these vectors were co-expressed to create mixtures, to demonstrate the versatility of the
technology. Model Fabs MF1337 (tetanus toxoid), MF1122 (fibrinogen) and MF1025
(thyroglobulin) were chosen based on their overall stable behaviour, good expression
levels and mass differences between the IgGs containing these Fabs (see Table 19)
95
Table 19:
Specificity Fab name IgG mass Δ-mass MF1122
Tetanus (A) (MF)*1337 146747.03 +1842.05
Fibrinogen (B) (MF)1122 144904.98 0
Thyroglobulin (C) (MF)1025 144259.87 -645.11
* MF=Merus Fab, designations such as MF1337 and 1337 are both used interchangeably.
Table 20: Transfection schedule:
Tr Heavy Heavy Heavy Tr. Expected species Observed species
. # chain 1 chain 2 chain 3 ratio (%) (%)
1 1337-KK 1122-DE 1025-DE 2:1:1 AB (50%) AC (50%) AB (43%) AC (57%)
2 1337-DE 1122-KK 1025-KK 2:1:1 AB (50%) AC (50%) AB (40%) AC (54%)
AA (6%)
3 1337-KK 1122-DE 1025-KK 1:2:1 AB (50%) BC (50%) AB (54%) BC (46%)
4 1337-KK 1122-KK 1025-DE 1:1:2 AC (50%) BC (50%) AC (66%) BC (33%)
CC (1%)
1337-KK 1337-DE 1122-DE 2:1:1 AA (50%) AB AA (57%) AB
(50%) (43%)
6 1337-KK 1122-KK 1122-DE 1:1:2 AB (50%) BB (50%) AB (75%) BB (25%)
7 1337-KK 1337-DE 1025-DE 2:1:1 AA (50%) AC AA (46%) AC
(50%) (54%)
8 1337-KK 1025-KK 1025-DE 1:1:2 AC (50%) CC (50%) AC (60%) CC (40%)
9 1337-KK 1122-DE 1:1 AB (100%) AB (>98%)
1337-KK 1025-DE 1:1 AC (100%) AC (>98%)
11 1122-KK 1025-DE 1:1 BC (100%) AC (>98%)
SDS-PAGE analysis demonstrated that most samples consisted of predominantly full IgGs
and in some cases half bodies were present at small percentages. Furthermore, many of the
samples showed two bands at ca. 150 kDa on non-reduced gels, reflecting the presence of
96
two distinct IgG species in the sample. Also on the reduced gels, two heavy chain bands
were visible in some samples (data not shown).
Native MS was performed on all samples and the percentages of observed species were
calculated based on peak height (% of observed species in Table 20). Results are presented
in Figure 24. In all eight samples where three heavy chains were co-expressed, two main
peaks were observed which corresponded to the expected species. In two of these samples
(transfections 2 and 4), and in transfection 11, a small amount of contaminating DE-DE
homodimer was observed. Half bodies were detected in very small amounts in most of the
samples (less than 2%), which are not problematic as they can be easily separated from the
full length IgG fraction as discussed previously. After nMS it was discovered that the
observed mass of the IgG in sample 11 corresponded to a different species than expected,
and it was concluded that this was due to an transfection error, i.e. in sample 11 apparently
1025-DE was co-transfected with 1337-KK instead of 1122-KK.
The IgG samples were further tested in a sandwich ELISA to confirm the functional
presence of the desired specificities. Coating of ELISA plates was done with fibrinogen or
thyroglobulin and detection was performed with fluorescein-labelled thyroglobulin or -
tetanus toxoid. The detection antigens were labelled with fluorescein (Pierce NHS-
fluorescin Antibody Labeling kit, cat. # 53029) according to the manufacturer’s
instructions. Fluorescein-labeled antigens could subsequently be detected by a FITC-
conjugated anti-fluorescein antibody (Roche diagnostics, cat. # 11426346910).
Results of the bispecific ELISA (OD450 values) are summarized in Table 21. The greyed
cells indicate the expected species for each transfection. Generally, the results meet the
expected outcome with view exceptions as indicated in italic or bold. In transfections 1-3,
the supposed ‘negative’ well for species BC (tr. #1 and 2) or AC (tr.# 3) demonstrated a
significant background signal. It is known from previous studies that bispecific ELISAs
may suffer from high background levels. These background levels may also be caused by
the potential presence of half-bodies in the sample. Of note is that the results of bispecific
ELISA indeed confirmed that an error had occurred in transfection #11, as the species AC
(bold value) was detected rather than BC.
97
Table 21: OD450 values from bispecific ELISA
Detected IgG Species
Tr. # AB (Tet-Fib) AC (Tet-Thyr) BC (Fib-thyr)
1 0.989 1.792 0.438
2 1.085 1.852 0.418
3 1.419 0.775 1.547
4 0.205 1.795 1.22
1.367 0.047 0.057
6 1.359 0.043 0.06
7 0.054 1.779 0054
8 0.04 1.338 0.052
9 1.588 0.048 0.051
0.044 1.805 0.055
11 0.043 1.821 0.056
Example 23: improved mixtures of two bispecific antibodies recognizing 4 different
epitopes (AB and CD) from a single cell
In example 12 it was hypothesized that mixtures resulting from transfections ZA or ZB are
expected to become problematic when transferred to larger scale production, as knob-into-
hole variants are reported to be unstable and it cannot be excluded that CH3 domains
comprising a ‘knob’ or a ‘hole’ will dimerize with charge-engineered CH3 domains. As it
was demonstrated in the above examples that novel charge pair mutants have been found
that preferentially drive heterodimerization with virtually no formation of homodimers,
CH3 domain-comprising polypeptide chains comprising these novel charge pair mutants
can be expressed in cells together with previously known charge-engineered CH3 domain-
comprising polypeptide chains or potentially with SEED bodies, and are likely to result in
the preferential formation of two bispecific molecules only.
98
From the above examples it was clear that the DEKK combination of mutations is
excellent for the production of one bispecific (AB) or two bispecifics (AB plus AC) by
clonal cells where dimerization of the heavy chains is driven by the CH3 domains.
However, using only one vector set of complementary CH3 mutations limits the number of
possibilities of mixture-varieties that can be produced. It would be possible to produce
more complex mixtures of IgGs and/or bispecifics, such as ‘AB and CD’ or ‘AB and CC’
mixtures if a second ‘orthogonal’ vector set could be used in combination with DEKK.
When combining two vector sets, an important requirement is that the heavy chains
expressed from the two different sets of CH3 engineered vectors cannot make ‘crossed’
dimers, which is that the heavy chains produced by one of the vector sets dimerize into full
IgG with heavy chains expressed by the other vector set.
To test for such potential formation of ‘crossed’ dimers, an in silico analysis was
performed using HADDOCK to obtain further insights whether possible pairing between
wildtype CH3 domains and CH3 domains containing DE- or KK-mutations would occur.
Similarly, potential pairings between wildtype CH3 domains and CH3 domains containing
E356K,D399K or K392D,K409D mutations were analyzed, as well as potential pairings
between wildtype CH3 domains and CH3 domains containing knob-into-hole mutations
and any combination of the above. Combinations of CH3-mutants that were analyzed in
HADDOCK are listed in Table 22 and the resulting HADDOCK scores are summarized in
Figure 25.
99
Table 22: CH3 variants analyzed in HADDOCK, with one letter codes for assigned for
each CH3-variant carrying heavy chain. *Wildtype chains are designated ‘C’ and ‘D’ for
matters of consistency; **The charge reversal variants are designated ‘A and B’ when
combined with knob-into-hole variants, and are designated ‘C and D’ when combined with
DE/KK variants.
CH3 combination Mutations One letter code in
HADDOCK
DEKK Chain 1: T366K,L351K A
Chain 2: L351D,L368E B
Wildtype (WT) Chain 1: none C*
Chain 2: none D*
Charge reversal (CR) Chain 1: K392D,K409D A/C**
Chain 2: E356K,D399K B/D**
Knob-into-hole Chain 1: T366W C
(KIH)
Chain 2: T366S,L368A,Y407V D
Figure 25 shows that, based on these HADDOCK predictions, combining the CH3
combinations of DEKK with charge reversal CH3 combinations is most likely to be
successful in forming the desired combination of two bispecifics (AB and CD) without
contaminating by-products (especially AC, AD, BC, BD) when co-transfected in a single
cell. As can be seen from figure 25, these undesired bispecific species AC, AD, BC, and
BD have relatively high HADDOCK scores, whereas the desired AB and CD species have
the lowest HADDOCK scores. Of course, when either the CH3 combinations of DEKK or
charge reversal will be put into a construct carrying the same specificity (e.g. ‘C’ on the
DE-side, ‘C’ on the KK-side, ‘A’ on the E356K,D399K-side and ‘B’ on the
E356K,D399K-side, or ‘A’ on the DE-side, ‘B’ on the KK-side, ‘C’ on the E356K,D399K-
side and ‘C’ on the E356K,D399K-side) this will result in the production of predominantly
CC and AB upon co-expression in a cell.
100
In contrast, when looking at the predictions for co-expressing DEKK with wildtype, it can
be seen that the HADDOCK scores for AC and AD are lower than the HADDOCK score
for CD, which indicates that AC and AD are very likely contaminants when trying to
produce a mixture of AB and CD by co-expression of vectors encoding for CH3
combinations of DEKK together with vectors encoding wildtype CH3. Lastly, the
predictions for co-expressing either DEKK or charge reversal variants together with the
knob-into-hole variants results in undesired bispecific variants with relatively low
HADDOCK scores, i.e. a high likelihood that these undesired species will be produced
upon co-expression.
It is thus concluded that combining the CH3 combinations of DEKK with charge reversal
CH3 combinations (E356K,D399K/K392’D,K409D’) is ideally suited for obtaining
essentially pure ‘AB and CD’ and/or ‘AB and CC’ mixtures of antibodies.
Next, mixtures of 2 bispecifics recognizing 4 targets/epitopes (AB and CD) and mixtures
of one bispecific and 1 monospecific antibody recognizing 3 targets/epitopes (AB and CC)
were created by putting the above into practice. These mixtures were created using 4
different VHs that are all capable of pairing with the common light chain IGVK1-39, but
the individual VH/VL combinations all have different specificities. To enable native MS
analysis, the mass difference between the (expected) species has to be sufficient, i.e. > 190
Da. Four individual VHs have been selected and the masses of these were such that the
expected species upon co-transfection could be identified and separated by nMS.
Furthermore, the mass differences between the 4 selected VHs are also large enough to
identify most of the possible contaminants in the mixtures, in addition to the two desired
species. Selected VHs are listed in Table 23.
101
Table 23:
VH (target) Mass as wt IgG
A (RTK1) 146736.78
B (Tetanus toxoid) 146106.20
C (Fibrinogen) 144904.98
D (RTK2) 145421.37
The 4 different VHs were cloned into vectors containing the ‘DE’ or ‘KK’ constructs or
the charge reversal constructs, and several co-transfections were performed as indicated in
Table 24. NB: as always, all vectors also contained the nucleic acid encoding the common
light chain IGKV1-39. As previously indicated, when combining two vector sets, an
important requirement is that the heavy chains expressed from the two different sets of
CH3 engineered vectors cannot make ‘crossed’ dimers, which is that the heavy chains
produced by one of the vector sets dimerize into full IgG with heavy chains expressed by
the other vector set. To test for such potential formation of ‘crossed’ dimers between heavy
chains containing charge reversal mutations and heavy chains containing DE or KK
mutations, control transfections were performed.
102
Table 24:
st nd
Tr. # 1 VH / 2 VH / Expected species
construct # construct #
1 D / 68 A / 68 mismatch ‘KK’ with ‘KK’; Mostly half-bodies
expected
2 D / 68 A / 69 match ‘KK’ with ‘DE’; AD product expected
3 D / 68 A / 1 Expected mismatch ‘KK’ with ‘E356K:D399K’
4 D / 68 A / 2 Expected mismatch ‘KK’ with ‘K392D:K409D’
D / 69 A / 68 match ‘DE’ with ‘KK’; AD product expected
6 D / 69 A / 69 mismatch ‘DE’ with ‘DE’; mixture of half-
bodies, AA, AD and DD expected
7 D / 69 A / 1 Expected mismatch ‘DE’ with ‘E356K:D399K’
8 D / 69 A / 2 Expected mismatch ‘DE’ with ‘K392D:K409D’
st nd rd th
Tr. # 1 VH / 2 VH / 3 VH / 4 VH / Expected
construct # construct # construct # construct # species
9 A / 68 B / 69 C / 1 D / 2 AB and CD
A / 68 A / 69 C / 1 D / 2 AA and CD
11 A / 68 B / 69 C / 1 C / 2 AB and CC
Table 25 provides a further overview of masses of the expected species, and the possible
contaminants, of transfections # 9-11 of Table 24.
Table 25: For each of transfections # 9-11, the species are sorted by mass, mass difference
is calculated with the mass above. Grey cells: expected (and desired) species; italics: mass
difference too small to separate in nMS analysis. *Species: single letters represent half-
bodies; two-letter code intact IgG.
Transfection # 9
Species* Mass Mass difference
C 72464,62
D 72684,53 219,90
B 73070,99 386,47
A 73410,46 339,47
CC 144929,2 71518,78
CD 145149,2 219,90
103
DD 145369,1 219,90
BC 145535,6 166,56
BD 145755,5 219,90
AC 145875,1 119,57
AD 146095 219,90
BB 146142 47,00
AB 146481,5 339,47
AA 146820,9 339,47
Transfection # 10
Species Mass Mass difference
C 72464,62
D 72684,53 219,90
A 73410,46 725,94
CC 144929,2 71518,78
CD 145149,2 219,90
DD 145369,1 219,90
AC 145875,1 506,03
AD 146095 219,90
AA 146820,9 725,94
Transfection # 11
Species Mass Mass difference
C 72464,62
B 73070,99 606,37
A 73410,46 339,47
CC 144890,95 71480,49
BC 145535,61 644,66
AC 145875,08 339,47
BB 146141,98 266,90
AB 146481,45 339,47
AA 146820,92 339,47
All purified protein samples obtained from transfections # 1-11 were analyzed on SDS-
PAGE, and three control samples were included (Figure 26). In addition, nMS analysis was
performed on protein samples from transfections # 9-11 to identify all species in the
samples. As can be seen from Figure 26, transfections # 3 and # 4 resulted in the expected
mismatch between ‘KK’ constructs and either ‘E356K:D399K’ or ‘K392D:K409D’ and
the amount of half bodies in protein samples from these transfections exceeded the amount
of full IgG molecules. Transfections # 7 and # 8 resulted in protein samples wherein both
half bodies and full IgG is present in about equal amounts. However, from SDS-PAGE it
104
cannot be deduced whether the full IgG represents a DE/DE dimer, a DE/E356K:D399K
dimer or a DE/K392D:K409D dimer. Remarkably, virtually no half bodies were observed
in samples from transfections # 9-11.
In Figure 27, the nMS analysis of transfections # 9 and # 11 are presented. Percentages of
expected species and contaminating species were calculated by peak height. It was
demonstrated that, for transfection # 9, the expected species ‘AB and CD’ are represented
for 97% in the mixture (30% AB and 67% CD) whereas only as little of about 3% of
contaminating BD is present (Figure 27A). For transfection # 11, the expected species ‘AB
and CC’ are represented for 94% in the mixture (33% AB and 61% CC) whereas only as
little of about 6% of contaminating BC (4.1%) and AC (1.8%) is present (Figure 27B).
These data show that it is indeed possible to produce more complex mixtures of IgGs
and/or bispecifics, such as ‘AB and CD’ or ‘AB and CC’ mixtures when a second
‘orthogonal’ vector set is used in combination with DEKK. Combination of the charge
reversal constructs together with the DEKK constructs results in only very limited
formation of ‘crossed’ dimers. By adjusting the transfection ratio’s it is expected that the
low percentages of these contaminating by-products can be even further reduced.
Example 24: single dose pharmacokinetic study in mice
To study the pharmacokinetic (pK) behavior of bispecific antibodies carrying the DEKK
combination of mutations in their CH3 regions, in this study the pK parameters for three
different IgG batches were determined and compared.
The three IgG batches included 1) wildtype anti-tetanus toxoid parental antibody
1337:1337 (two MF1337 Fabs on a wildtype Fc backbone); 2) wildtype anti-tetanus toxoid
parental antibody 1516:1516 (two MF1516 Fabs on a wildtype Fc backbone); 3) CH3
engineered bispecific anti-tetanus toxoid antibody 1516:1337 that carries the DEKK
combination of mutations in its Fc region (MF1516 Fab on DE-side, MF1337 Fab on KK-
side).
The parental antibodies 1337:1337 and 1516:1516 were chosen as specificities to be
included in the DEKK-bispecific product, as it was known based on previous studies that
no pre-dose serum response against these antibodies was present in several mice strains.
105
NB: the presence of a pre-dose serum response would of course invalidate the study. In
addition, there is sufficient mass difference between the parental antibodies to enable the
identification of 1337:1337 (wt Fc), 1516:1337 (DEKK Fc) and 1516:1516 (wt Fc) species
by nMS. The three IgG batches were prepared as previously described, but the DNA used
for transfection was made using an endo-free maxiprep kit to ensure that the amount of
endotoxins is as low as possible. The batches were subsequently tested for protein
concentration, aggregate levels, endotoxin levels and percentage bispecific product. It was
demonstrated that the acceptance criteria for subsequent use of the IgG batches in a pK
study were met, i.e. the IgG concentration after gel filtration was >0.3 mg/ml, aggregate
levels were <5%, endotoxin levels were <3 EU/mg protein and the DEKK batch contained
> 90% bispecific IgG.
Native mass spectrometry of the gel filtrated samples showed that the expected species
were present in high percentages. In sample 1516:1337 a small amount of the DE:DE
homodimer is detected, which is estimated to be ca. 2% (Figure 28). It was concluded that
the 3 IgG batches are qualified to be used in the pK study.
For comparison of pK parameters between the three batches, 3 groups of female C57BL/6J
mice (Harlan, The Netherlands) were dosed at 1 mg/kg human lgG (5 ml/kg
immunoglobulin solution/kg body weight). At dosing time, the animals were between 7-8
weeks of age and had a body weight of about 18-20 grams. Blood samples were collected
pre-dose and at 15, 60 minutes, and 2, 4, 8, 24, 48, 96, 168, 268 and 336 h after dosing.
Serum samples were prepared and stored at < -20 ⁰C until analysis. Each group consisted
of 3 subgroups of 4 mice, i.e. 12 mice/group. From each mice 6 time points were sampled.
The welfare of the animals was maintained in accordance with the general principles
governing the use of animals in experiments of the European Communities (Directive
86/609/EEC) and Dutch legislation (The Experiments on Animals Act, 1997). This study
was also performed in compliance with the Standards for Humane Care and Use of
Laboratory Animals, as issued by the Office of Laboratory Animal Welfare of the U.S.
National lnstitutes of Health under identification number 45859-01 (expiration date: 30
April 2015).
106
Mice of Group 1 received the full length monospecific IgG 1516:1516 antibody (triangles);
Mice of Group 2 received the full length monospecific IgG 1337:1337 antibody (squares);
Mice of Group 3 received the full length bispecific IgG 1516:1337 antibody (diamonds),
with DEKK engineered CH3 regions (1516 on the DE-side and 1337 on the KK-side).
An ELISA assay was applied for the quantitative analysis of monoclonal human antibodies
in mouse serum using a quantitative human lgG ELISA (ZeptoMetrix, NY USA; ELISA
kit nr. 0801182). Briefly, the ELISA assay is based on the principle that the human
monoclonal antibody binds to anti-human lgG coated in a 96-wells ELISA plate. Bound
antibody was subsequently visualized using a polyclonal antihuman lgG antibody
conjugated with horseradish peroxidase (HRP). The optical density (OD) of each well is
directly proportional to the amount of antibody in the serum sample. Results are shown in
Figure 29, and it was observed that serum levels of both the bispecific full length IgG
antibody carrying the DEKK combination of mutations and its parental monospecific
antibodies are strikingly similar. It is concluded that the CH3 mutations as present in the
DEKK-bispecific antibody does not alter stability nor half life, and the DEKK variant is
behaving as wildtype IgG.
In this specification where reference has been made to patent specifications, other external
documents, or other sources of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless specifically stated otherwise,
reference to such external documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction, are prior art, or form part of
the common general knowledge in the art.
107
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ISBN 046093-9.
Claims (35)
1. An in vitro method for producing at least two different monospecific CH3 domain- comprising molecules from a single host cell, wherein each of said two CH3 domain- comprising molecules comprises two CH3 domains that are capable of forming an interface, said method comprising providing in said cell st a. a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain, and nd b. a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide st chain, wherein said 1 CH3 domain-comprising polypeptide chain comprises negatively charged amino acid residues at amino acid positions 392 and 409 and a positively charged amino acid residue at amino acid position 399, nd and wherein said 2 CH3 domain-comprising polypeptide chain comprises: - either a wildtype CH3 domain, or - positively charged amino acid residues at amino acid positions 356 and 357 and negatively charged amino acid residues at amino acid positions 370 and 439, said method further comprising the step of culturing said host cell and allowing for expression of said nucleic acid molecules and harvesting said at least two different CH3 domain-comprising molecules from the culture.
2. An in vitro method for producing at least two different monospecific CH3 domain- comprising molecules from a single host cell, wherein each of said two CH3 domain- comprising molecules comprises two CH3 domains that are capable of forming an interface, said method comprising providing in said cell st a) a first nucleic acid molecule encoding a 1 CH3 domain-comprising polypeptide chain, and nd b) a second nucleic acid molecule encoding a 2 CH3 domain-comprising polypeptide chain, wherein said first CH3 domain-comprising polypeptide chain comprises: - either a wildtype CH3 domain, or - negatively charged amino acid residues at amino acid positions 392 and 409 and a positively charged amino acid residue at amino acid position 399, 109 and wherein said second CH3 domain-comprising polypeptide chain comprises positively charged amino acid residues at amino acid positions 356 and 357 and negatively charged amino acid residues at amino acid positions 370 and 439, said method further comprising the step of culturing said host cell and allowing for expression of said nucleic acid molecules and harvesting said at least two different CH3 domain-comprising molecules from the culture.
3. The method of claim 1 or 2, further comprising providing said host cell with a nucleic acid molecule encoding a common light chain.
4. The method of any one of claims 1-3, wherein said first CH3 domain-comprising polypeptide chain comprises an aspartic acid (D) residue at amino acid position 392 and a lysine (K) residue at amino acid position 399 and an aspartic acid (D) residue at amino acid position 409.
5. The method of any one of claims 1-4, wherein said second CH3 domain-comprising polypeptide chain comprises a lysine (K) residue at amino acid position 356 and a lysine (K) residue at amino acid position 357 and an aspartic acid (D) residue at amino acid position 439 and an aspartic acid (D) residue at amino acid position 370.
6. The method of any one of claims 1-5, wherein the formation of bispecific CH3 domain-comprising molecules is reduced to below 5%.
7. The method of any one of claims 1-6, wherein the formation of bispecific CH3 domain-comprising molecules is reduced to below 3%.
8. The method of any one of claims 1-7, wherein each of the CH3 domain-comprising polypeptide chains further comprises a variable region recognizing a target epitope.
9. The method of claim 8, wherein the target epitope recognized by the variable nd regions of the 2 CH3 domain-comprising polypeptide chain is different from the target 110 st epitope recognized by the variable region of the 1 CH3 domain-comprising polypeptide chain.
10. The method of claim 8 or 9, wherein the target epitopes are located on the same target molecule.
11. The method of claim 10, wherein the target molecule is a soluble molecule.
12. The method of claim 10, wherein the target molecule is a membrane-bound molecule.
13. The method of claim 8 or 9, wherein the target epitopes are located on different target molecules.
14. The method of claim 13, wherein the different target molecules are expressed on the same cells.
15. The method of claim 13, wherein the different target molecules are expressed on different cells.
16. The method of claim 13, wherein the different target molecules are soluble molecules.
17. The method of claim 13, wherein one target molecule is a soluble molecule whereas the second target molecule is a membrane-bound molecule.
18. The method according to any one of claims 1-17, wherein said at least two different CH3 domain-comprising molecules are antibodies.
19. The method of any one of claims 8-9, 13-15 or 17, wherein at least one of said target epitopes is located on a tumor cell. 111
20. The method of any one of claims 8-9, 13-15 or 17, wherein at least one of said target epitopes is located on an effector cell.
21. The method of claim 20, wherein said effector cell is an NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell or a neutrophilic granulocyte.
22. The method of claims 20 or 21, wherein said target epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or a NKp46 molecule.
23. A mixture of at least two different monospecific CH3 domain-comprising molecules obtained by the methods according to any one of claims 1-22.
24. A mixture according to claim 23, wherein said at least two different monospecific CH3 domain-comprising molecules bind to different epitopes on the same antigen and/or to different epitopes on different antigens.
25. An in vitro recombinant host cell comprising nucleic acid sequences encoding at st least a first and a second CH3 domain-comprising polypeptide chain, wherein said 1 CH3 domain-comprising polypeptide chain comprises negatively charged amino acid residues at amino acid positions 392 and 409 and a positively charged amino acid residue at amino acid position 399, nd and wherein said 2 CH3 domain-comprising polypeptide chain comprises: - either a wildtype CH3 domain, or - positively charged amino acid residues at amino acid positions 356 and 357 and negatively charged amino acid residues at amino acid positions 370 and 439.
26. An in vitro recombinant host cell comprising nucleic acid sequences encoding at st least a first and a second CH3 domain-comprising polypeptide chain, wherein said 1 CH3 domain-comprising polypeptide chain comprises: - either a wildtype CH3 domain, or 112 - negatively charged amino acid residues at amino acid positions 392 and 409 and a positively charged amino acid residue at amino acid position 399, and wherein said second CH3 domain-comprising polypeptide chain comprises positively charged amino acid residues at amino acid positions 356 and 357 and negatively charged amino acid residues at amino acid positions 370 and 439.
27. A recombinant host cell according to claim 25 or 26, wherein said host cell further comprises a nucleic acid sequence encoding a common light chain.
28. A pharmaceutical composition comprising the at least two different monospecific CH3 domain-comprising molecules of any one of claims 23-24, and a pharmaceutically acceptable carrier.
29. A pharmaceutical composition according to claim 28, wherein said at least two different monospecific CH3 domain-comprising molecules have been produced by recombinant host cells according to any one of claims 25-27.
30. An in vitro method for making a host cell for production of at least two different monospecific CH3 domain-comprising molecules, the method comprising the step of introducing into said host cell nucleic acid sequences encoding at least a first and a second CH3 domain-comprising polypeptide chain, st wherein said 1 CH3 domain-comprising polypeptide chain comprises negatively charged amino acid residues at amino acid positions 392 and 409 and a positively charged amino acid residue at amino acid position 399, nd and wherein said 2 CH3 domain-comprising polypeptide chain comprises: - either a wildtype CH3 domain, or - positively charged amino acid residues at amino acid positions 356 and 357 and negatively charged amino acid residues at amino acid positions 370 and 439, wherein said nucleic acid sequences are introduced consecutively or concomitantly. 113
31. An in vitro method for making a host cell for production of at least two different monospecific CH3 domain-comprising molecules, the method comprising the step of introducing into said host cell nucleic acid sequences encoding at least a first and a second CH3 domain-comprising polypeptide chain, st wherein said 1 CH3 domain-comprising polypeptide chain comprises: - either a wildtype CH3 domain, or - negatively charged amino acid residues at amino acid positions 392 and 409 and a positively charged amino acid residue at amino acid position 399, and wherein said second CH3 domain-comprising polypeptide chain comprises positively charged amino acid residues at amino acid positions 356 and 357 and negatively charged amino acid residues at amino acid positions 370 and 439, wherein said nucleic acid sequences are introduced consecutively or concomitantly.
32. A method according to claim 30 or 31, further comprising the step of introducing into said host cell a nucleic acid sequence encoding a common light chain.
33. A recombinant host cell according to any one of claims 25-27, or a method according to any one of claims 30-32, wherein said first CH3 domain-comprising polypeptide chain comprises an aspartic acid (D) residue at amino acid position 392 and a lysine (K) residue at amino acid position 399 and an aspartic acid (D) residue at amino acid position 409.
34. A recombinant host cell according to any one of claims 25-27 or 33, or a method according to any one of claims 30-33, wherein said second CH3 domain-comprising polypeptide chain comprises a lysine (K) residue at amino acid position 356 and a lysine (K) residue at amino acid position 357 and an aspartic acid (D) residue at amino acid position 439 and an aspartic acid (D) residue at amino acid position 370.
35. A culture of recombinant host cells of any of claims 25-27 or 33-34, or of recombinant host cells obtained by the method according to any one of claims 30-34, producing at least two different monospecific CH3 domain-comprising molecules.
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NZ772318A NZ772318A (en) | 2012-04-20 | 2013-04-19 | Methods and means for the production of ig-like molecules |
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US201261635935P | 2012-04-20 | 2012-04-20 | |
US61/635,935 | 2012-04-20 | ||
NZ630551A NZ630551A (en) | 2012-04-20 | 2013-04-19 | Methods and means for the production of ig-like molecules |
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