U.S. patent application number 15/855258 was filed with the patent office on 2018-04-26 for recombinant production of mixtures of antibodies.
This patent application is currently assigned to Merus N.V.. The applicant listed for this patent is Merus N.V.. Invention is credited to Abraham BOUT, Ronald Hendrik BRUS, Ton LOGTENBERG, Patricius Hendrikus VAN BERKEL.
Application Number | 20180112247 15/855258 |
Document ID | / |
Family ID | 43768900 |
Filed Date | 2018-04-26 |
United States Patent
Application |
20180112247 |
Kind Code |
A1 |
VAN BERKEL; Patricius Hendrikus ;
et al. |
April 26, 2018 |
RECOMBINANT PRODUCTION OF MIXTURES OF ANTIBODIES
Abstract
Provided is methods for producing mixtures of antibodies from a
single host cell clone, wherein, a nucleic acid sequence encoding a
light chain and nucleic acid sequences encoding different heavy
chains are expressed in a recombinant host cell. The recombinantly
produced antibodies in the mixtures according to the invention
suitably comprise identical light chains paired to different heavy
chains capable of pairing to the light chain, thereby forming
functional antigen-binding domains. Mixtures of the recombinantly
produced antibodies are also provided by the invention. Such
mixtures can be used in a variety of fields.
Inventors: |
VAN BERKEL; Patricius
Hendrikus; (Berkel en Rodenrjis, NL) ; BRUS; Ronald
Hendrik; (Voorschoten, NL) ; BOUT; Abraham;
(Leiden, NL) ; LOGTENBERG; Ton; (Utrecht,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merus N.V. |
Utrecht |
|
NL |
|
|
Assignee: |
Merus N.V.
Utrecht
NL
|
Family ID: |
43768900 |
Appl. No.: |
15/855258 |
Filed: |
December 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15090505 |
Apr 4, 2016 |
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15855258 |
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12932719 |
Mar 4, 2011 |
9303081 |
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15090505 |
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12221021 |
Jul 29, 2008 |
7927834 |
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12932719 |
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11593279 |
Nov 6, 2006 |
7429486 |
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12221021 |
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11039767 |
Jan 18, 2005 |
7262028 |
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11593279 |
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PCT/EP2003/007690 |
Jul 15, 2003 |
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11039767 |
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60397066 |
Jul 18, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/73 20130101;
A61P 35/02 20180101; C07K 16/32 20130101; C07K 2317/626 20130101;
A61P 35/00 20180101; C07K 16/2851 20130101; C07K 2319/30 20130101;
C07K 2317/12 20130101; A61P 31/12 20180101; C07K 2317/21 20130101;
C07K 2317/622 20130101; C07K 2317/732 20130101; C07K 16/2833
20130101; A61P 31/04 20180101; A61P 37/00 20180101; C07K 2317/56
20130101; C07K 16/10 20130101; C07K 2317/31 20130101; C07K 2317/734
20130101; C07K 16/00 20130101; A61P 35/04 20180101; A61P 37/06
20180101; C07K 16/2803 20130101; Y02P 20/582 20151101; C12P 21/005
20130101; C07K 16/30 20130101; C07K 2317/51 20130101; A61P 43/00
20180101; C07K 2317/50 20130101; A61K 39/39558 20130101; C07K
16/2896 20130101 |
International
Class: |
C12P 21/00 20060101
C12P021/00; A61K 39/395 20060101 A61K039/395; C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; C07K 16/00 20060101
C07K016/00; C07K 16/10 20060101 C07K016/10; C07K 16/32 20060101
C07K016/32 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 18, 2002 |
EP |
02077953.4 |
Claims
1.-92. (canceled)
93. An isolated host cell comprising an exogenously introduced
nucleic acid sequence encoding an immunoglobulin light chain
variable region and three or more immunoglobulin heavy chain
variable regions.
94. The isolated host cell of claim 93, wherein the three or more
immunoglobulin heavy chain variable regions are capable of pairing
with the immunoglobulin light chain variable region to form a
multispecific immunoglobulin.
95. The isolated host cell of claim 94, wherein the multispecific
immunoglobulin comprises an antigen-binding antibody fragment.
96. The isolated host cell of claim 95, wherein the antibody
fragment is a Fv, Fab, Fab', or F(ab').sub.2 fragment.
97. The isolated host cell of claim 93, which expresses an
immunoglobulin comprising two antigen-binding domains.
98. The isolated host cell of claim 97, wherein said
antigen-binding domains are not identical.
99. The isolated host cell of claim 93, wherein the three or more
immunoglobulin heavy chain variable regions are capable of paring
with immunoglobulin light chain variable region to form three or
more non-identical antigen-binding antibody fragments.
100. The isolated host cell of claim 93, wherein the immunoglobulin
heavy chain variable regions have different specificities and/or
affinities.
101. The isolated host cell of claim 93, wherein the immunoglobulin
heavy chain variable regions bind to different epitopes on the same
antigen.
102. The isolated host cell of claim 93, wherein the immunoglobulin
heavy chain variable regions bind to different antigens.
103. The isolated host cell of claim 93, wherein the exogenously
introduced nucleic acid sequence comprises a tissue-specific
promoter.
104. The isolated host cell of claim 93, wherein the exogenously
introduced nucleic acid sequence is integrated into the host cell's
genome.
105. The isolated host cell of claim 104, wherein the exogenously
introduced nucleic acid sequence is stably integrated at a
predetermined position.
106. The isolated host cell of claim 105, wherein the integration
is performed using homologous recombination or site-specific
recombinases.
107. The isolated host cell of claim 93, wherein the nucleic acid
sequence encoding an immunoglobulin light chain variable region and
the three or more immunoglobulin heavy chain variable regions are
introduced into the host cell consecutively.
108. The isolated host cell of claim 93, wherein the nucleic acid
sequence encoding an immunoglobulin light chain variable region and
the three or more immunoglobulin heavy chain variable regions are
introduced into the host cell concomitantly.
109. A cell culture comprising the host cell of claim 93 and a
culture medium.
110. The cell culture of claim 109, which produces two or more
non-identical antibodies for more than 20 population doublings.
111. The isolated host cell of claim 93, wherein said encoded
immunoglobulin light chain variable region and three or more
immunoglobulin heavy chain variable regions are encoded by a single
nucleic acid.
112. The isolated host cell of claim 93, wherein said encoded
immunoglobulin light chain variable region and three or more
immunoglobulin heavy chain variable regions are encoded by one or
more nucleic acids.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/932,719, filed Mar. 4, 2011, pending, which application
is a continuation of U.S. patent application Ser. No. 12/221,021,
filed Jul. 29, 2008, now U.S. Pat. No. 7,927,834, issued Apr. 19,
2011, which is a divisional of U.S. patent application Ser. No.
11/593,279, filed Nov. 6, 2006, now U.S. Pat. No. 7,429,486, issued
Sep. 30, 2008, which is a divisional patent application of patent
application Ser. No. 11/039,767, filed Jan. 18, 2005, now U.S. Pat.
No. 7,262,028, issued Aug. 28, 2007, which is a continuation of PCT
International Patent Application No. PCT/EP2003/007690, filed on
Jul. 15, 2003, designating the United States of America, published
in English as International Publication No. WO 2004/009618 A2 on
Jan. 29, 2004, which itself claims the benefit of PCT International
Patent Application No. PCT/EP03/50201, filed May 27, 2003, European
Patent Application No. 02077953.4, filed Jul. 18, 2002, and U.S.
Provisional Patent Application Ser. No. 60/397,066, filed Jul. 18,
2002, the contents of the entirety of each of which are
incorporated herein by this reference.
STATEMENT ACCORDING TO 37 C.F.R. .sctn. 1.821(c) or (e)--SEQUENCE
LISTING SUBMITTED AS PDF FILE WITH A REQUEST TO TRANSFER CRF FROM
PARENT APPLICATION
[0002] Pursuant to 37 C.F.R. .sctn. 1.821(c) or (e), a file
containing a PDF version of the Sequence Listing has been submitted
concomitant with this application, the contents of which are hereby
incorporated by reference. The transmittal documents of this
application include a Request to Transfer CRF from the parent
application.
TECHNICAL FIELD
[0003] The invention relates generally to the field of
biotechnology, and more particularly, to the field of medicine and
the production of antibodies, and even more particularly, to the
production of mixtures of antibodies.
BACKGROUND
[0004] The essential function of the immune system is the defense
against infection. The humoral immune system combats molecules
recognized as non-self, such as pathogens, using immunoglobulins.
These immunoglobulins, also called antibodies, are raised
specifically against the infectious agent, which acts as an
antigen, upon first contact (Roitt, Essential Immunology, Blackwell
Scientific Publications, fifth edition, 1984; all references cited
herein are incorporated in their entirety by reference). Antibodies
are multivalent molecules comprising heavy (H) chains and light (L)
chains joined with interchain disulfide bonds. Several isotypes of
antibodies are known, including IgG1, IgG2, IgG3, IgG4, IgA, IgD,
IgE, and IgM. An IgG contains two heavy and two light chains. Each
chain contains constant (C) and variable (V) regions, which can be
broken down into domains designated C.sub.H1, C.sub.H2, C.sub.H3,
V.sub.H, and C.sub.L, V.sub.L (FIG. 1). Antibody binds to antigen
via the variable region domains contained in the Fab portion and,
after binding, can interact with molecules and cells of the immune
system through the constant domains, mostly through the Fc
portion.
[0005] B-lymphocytes can produce antibodies in response to exposure
to biological substances like bacteria, viruses and their toxic
products. Antibodies are generally epitope-specific and bind
strongly to substances carrying these epitopes. The hybridoma
technique (Kohler and Milstein, 1975) makes use of the ability of
B-cells to produce monoclonal antibodies to specific antigens and
to subsequently produce these monoclonal antibodies by fusing
B-cells from mice exposed to the antigen of interest to
immortalized murine plasma cells. This technology resulted in the
realization that monoclonal antibodies produced by hybridomas could
be used in research, diagnostics and therapies to treat different
kinds of diseases like cancer and auto-immune-related
disorders.
[0006] Because antibodies that are produced in mouse hybridomas can
induce strong immune responses in humans, it has been appreciated
in the art that antibodies required for successful treatment of
humans needed to be less immunogenic or, preferably,
non-immunogenic. For this to be done, murine antibodies were first
engineered by replacing the murine constant regions with human
constant regions (referred to as chimeric antibodies).
Subsequently, domains between the complementarity-determining
regions (CDRs) in the variable domains, the so-called framework
regions, were replaced by their human counterparts (referred to as
humanized antibodies). The final stage in this humanization process
has been the production of fully human antibodies.
[0007] In the art, bispecific antibodies, which have binding
specificities for two different antigens, have also been described.
These are generally used to target a therapeutic or diagnostic
moiety, for instance, T-cell, a cytotoxic trigger molecule, or a
chelator that binds a radionuclide, that is recognized by one
variable region of the antibody to a cell that is recognized by the
other variable region of the antibody, for instance, a tumor cell
(for bispecific antibodies, see Segal et al., 2001).
[0008] One very useful method known in the art to obtain fully
human monoclonal antibodies with desirable binding properties,
employs phage display libraries. This is an in vitro, recombinant
DNA-based, approach that mimics key features of the humoral immune
response (for phage display methods, see, e.g., C. F. Barbas III et
al., Phage Display, A laboratory manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2001). For the
construction of phage display libraries, collections of human
monoclonal antibody heavy- and light-chain variable region genes
are expressed on the surface of bacteriophage particles, usually in
single-chain Fv (scFv) or in Fab format. Large libraries of
antibody fragment-expressing phages typically contain more than
10.sup.9 antibody specificities and may be assembled from the
immunoglobulin V regions expressed in the B lymphocytes of
immunized or non-immunized individuals.
[0009] Alternatively, phage display libraries may be constructed
from immunoglobulin variable regions that have been partially
assembled or rearranged in vitro to introduce additional antibody
diversity in the library (semi-synthetic libraries) (De Kruif et
al., 1995b). For example, in vitro-assembled variable regions
contain stretches of synthetically produced, randomized or
partially randomized DNA in those regions of the molecules that are
important for antibody specificity.
[0010] The genetic information encoding the antibodies identified
by phage display can be used for cloning the antibodies in a
desired format, for instance, IgG, IgA or IgM, to produce the
antibody with recombinant DNA methods (Roel et al., 2000).
[0011] An alternative method to provide fully human antibodies uses
transgenic mice that comprise genetic material encoding a human
immunoglobulin repertoire (Fishwild et al., 1996; Mendez et al.,
1997). Such mice can be immunized with a target antigen and the
resulting immune response will produce fully human antibodies. The
sequences of these antibodies can be used in recombinant production
methods.
[0012] Production of monoclonal antibodies is routinely performed
by use of recombinant expression of the nucleic acid sequences
encoding the H and L chains of antibodies in host cells (see, e.g.,
EP0120694; EP0314161; EP0481790; U.S. Pat. No. 4,816,567; WO
00/63403, the contents of the entirety of each which are
incorporated herein by reference).
[0013] To date, many different diseases are being treated with
either humanized or fully human monoclonal antibodies. Products
based on monoclonal antibodies that are currently approved for use
in humans include HERCEPTIN.TM. (trastuzumab, anti-Her2/Neu),
REOPRO.TM. (abciximab, anti-Glycoprotein IIB/IIIA receptor),
MYLOTARG.TM. (gemtuzumab, anti-CD33), RITUXAN.TM. (Rituximab,
anti-CD20), SIMULECT.TM. (basiliximab, anti-CD25), REMICADE.TM.
(infliximab, anti-TNF), SYNAGIS.TM. (palivizumab, anti-RSV),
ZENAPAX.TM. (daclizumab, IL2-receptor), and CAMPATH.TM.
(alemtuzumab, anti-CD52). Despite these successes, there is still
room for new antibody products and for considerable improvement of
existing antibody products.
[0014] The use of monoclonal antibodies in cancer treatment has
shown that so-called "antigen-loss tumor variants" can arise,
making the treatment with the monoclonal antibody less effective.
Treatment with the very successful monoclonal antibody
RITUXIMAB.RTM. (anti-CD20) has, for instance, shown that
antigen-loss escape variants can occur, leading to relapse of the
lymphoma (Massengale et al., 2002). In the art, the potency of
monoclonal antibodies has been increased by fusing them to toxic
compounds, such as radionuclides, toxins, cytokines, and the like.
Each of these approaches, however, has its limitations, including
technological and production problems and/or high toxicity.
[0015] Furthermore, it appears that the gain in specificity of
monoclonal antibodies compared to traditional undefined polyclonal
antibodies, comes at the cost of loss of efficacy. In vivo,
antibody responses are polyclonal in nature, i.e., a mixture of
antibodies is produced because various B-cells respond to the
antigen, resulting in various specificities being present in the
polyclonal antibody mixture. Polyclonal antibodies can also be used
for therapeutic applications, for instance, for passive vaccination
or for active immunotherapy, and currently are usually derived from
pooled serum from immunized animals or from humans who recovered
from the disease. The pooled serum is purified into the
proteinaceous or gamma globulin fraction, so named because it
contains predominantly IgG molecules.
[0016] Polyclonal antibodies that are currently used for treatment
include anti-rhesus polyclonal antibodies, gamma globulin for
passive immunization, anti-snake venom polyclonal (CroFab),
THYMOGLOBULIN.TM. for allograft rejection, anti-digoxin to
neutralize the heart drug digoxin, and anti-rabies polyclonal
antibodies. In currently marketed therapeutic antibodies, an
example of the higher efficacy of polyclonal antibodies compared to
monoclonal antibodies can be found in the treatment of acute
transplant rejection with anti-T-cell antibodies. The monoclonal
antibodies on the market (anti-CD25 BASILIXIMAB.RTM.) are less
efficacious than a rabbit polyclonal antibody against thymocytes
(THYMOGLOBULIN.TM.) (press releases dated Mar. 12, Apr. 29, and
Aug. 26, 2002, on sangstat.com). The use of pooled human sera,
however, potentially bears the risk of infections with viruses such
as HIV or hepatitis, with toxins such as lipopolysaccharide, with
proteinaceous infectious agents such as prions, and with unknown
infectious agents. Furthermore, the supply that is available is
limited and insufficient for widespread human treatments. Problems
associated with the current application of polyclonal antibodies
derived from animal sera in the clinic include a strong immune
response of the human immune system against such foreign
antibodies. Therefore, such polyclonals are not suitable for
repeated treatment or for treatment of individuals that were
injected previously with other serum preparations from the same
animal species.
[0017] The art describes the idea of the generation of animals with
a human immunoglobulin repertoire, which can subsequently be used
for immunization with an antigen to obtain polyclonal antibodies
against this antigen from the transgenic animals (WO 01/19394, the
entirety of which is incorporated herein by reference). However,
many technological hurdles still will have to be overcome before
such a system is a practical reality in larger animals than mice
and it will take years of development before such systems can
provide the polyclonal antibodies in a safe and consistent manner
in sufficient quantities. Moreover, antibodies produced from pooled
sera, whether being from human or animal origin, will always
comprise a high amount of unrelated and undesired specificities, as
only a small percentage of the antibodies present in a given serum
will be directed against the antigen used for immunization. It is,
for instance, known that in normal, i.e., non-transgenic, animals,
about 1% to 10% of the circulating immunoglobulin fraction is
directed against the antigen used for hyper-immunization; hence,
the vast majority of circulating immunoglobulins is not
specific.
[0018] One approach towards expression of polyclonal antibody
libraries has been described (WO 95/20401; U.S. Pat. Nos. 5,789,208
and 6,335,163, the contents of the entirety of each of which are
incorporated herein by reference). A polyclonal library of Fab
antibody fragments is expressed using a phage display vector and
selected for reactivity towards an antigen. To obtain a sub-library
of intact polyclonal antibodies, the selected heavy and light
chain-variable region gene combinations are transferred en mass as
linked pairs to a eukaryotic-expression vector that provides
constant region genes. Upon transfection of this sub-library into
myeloma cells, stable clones produce monoclonal antibodies that can
be mixed to obtain a polyclonal antibody mixture. While in theory
it would be possible to obtain polyclonal antibodies directly from
a single recombinant production process using this method by
culturing a mixed population of transfected cells, potential
problems would occur concerning the stability of the mixed cell
population and, hence, the consistency of the produced polyclonal
antibody mixture. The control of a whole population of different
cells in a pharmaceutically acceptable large-scale process (i.e.,
industrial) is a daunting task. It would seem that characteristics,
such as growth rates of the cells and production rates of the
antibodies, should remain stable for all of the individual clones
of the non-clonal population in order to keep the ratio of
antibodies in the polyclonal antibody mixture more or less
constant.
BRIEF SUMMARY
[0019] Disclosed are means and methods for producing a mixture of
antibodies in recombinant hosts.
[0020] In one aspect, provided is a method of producing a mixture
of antibodies in a recombinant host, the method comprising
expressing in a recombinant host cell a nucleic acid sequence or
nucleic acid sequences encoding at least one light chain and at
least three different heavy chains that are capable of pairing with
at least one light chain. A further aspect is the elimination of
the production of potentially non-functional light-heavy chain
pairing by using pre-selected combinations of heavy and light
chains. It has been recognized that phage display libraries built
from a single light chain and many different heavy chains can
encode antibody fragments with very distinct binding properties.
This feature can be used to find different antibodies having the
same light chain but different heavy chains, against the same
target or different targets, wherein a target can be a whole
antigen or an epitope thereof. Such different targets may, for
instance, be on the same surface (e.g., cell or tissue). Such
antibody fragments obtained by phage display can be cloned into
vectors for the desired format, e.g., IgG, IgA or IgM, and the
nucleic acid sequences encoding these formats can be used to
transfect host cells. In one approach, H and L chains can be
encoded by different constructs that, upon transfection into a cell
wherein they are expressed, give rise to intact Ig molecules. When
different H chain constructs are transfected into a cell with a
single L chain construct, H and L chains will be assembled to form
all possible combinations. However, in contrast to approaches where
different light chains are expressed, such as for the production of
bispecific antibodies, this method will result only in functional
binding regions. It would be particularly useful when the host, for
example, a single cell line, is capable of expressing acceptable
levels of recombinant antibodies without the necessity to first
amplify in the cell the nucleic acid sequences encoding the
antibodies. The advantage is that cell lines with only a limited
copy number of the nucleic acids are expected to be genetically
more stable, because there will be less recombination between the
sequences encoding the heavy chains, than in cell lines where a
multitude of these copies is present. A cell line suitable for use
in these methods is the human cell line PER.C6.RTM. (human retina
cells that express adenovirus E1A and E1B proteins). Using this
method, a mixture of antibodies with defined specificities can be
produced from a single cell clone in a safe, controlled, and
consistent manner.
[0021] In certain embodiments, provided is a method for producing a
mixture of antibodies in a recombinant host, the method comprising
expressing a nucleic acid sequence or nucleic acid sequences
encoding at least one light chain and at least three different
heavy chains that are capable of pairing with at least one light
chain in a recombinant host cell. In certain embodiments, the
recombinant host cell comprises a nucleic acid sequence encoding a
common light chain that is capable of pairing with at least three
different heavy chains, such that the produced antibodies comprise
a common light chain. 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
the light chain exist wherein mutations (deletions, substitutions,
additions) are present that do not materially influence the
formation of functional binding regions.
[0022] Further provided is a composition comprising a mixture of
recombinantly produced antibodies, wherein at least three different
heavy chain sequences are represented in the mixture. In certain
embodiments, the light chains of such mixtures have a common
sequence. The mixture of antibodies can be produced by the method
according to the invention. Preferably, the mixture of antibodies
is more efficacious than the individual antibodies it comprises.
More preferably, the mixture acts synergistically in a functional
assay.
[0023] Further provided is a recombinant host cell for producing
mixtures of antibodies and methods for making such host cells.
[0024] Independent clones obtained from the transfection of nucleic
acid sequences encoding a light chain and more than one heavy chain
may express the different antibodies in the mixture at different
levels. It is another aspect to select a clone using a functional
assay for the most potent mixture of antibodies. Further provides a
method for identifying at least one host cell clone that produces a
mixture of antibodies, wherein the mixture of antibodies has a
desired effect according to a functional assay, the method
comprising: (i) providing a host cell with nucleic acid sequences
encoding at least one light chain and nucleic acid sequences
encoding at least two different heavy chains, wherein the heavy and
light chains are capable of pairing with each other; (ii) culturing
at least one clone of the host cell under conditions conducive to
expression of the nucleic acid sequences; (iii) screening at least
one clone of the host cell for production of a mixture of
antibodies having the desired effect by a functional assay; and
(iv) identifying at least one clone that produces a mixture of
antibodies having the desired effect. This method, as used herein,
can be performed using high-throughput procedures if desired. The
clones identified by the method can be used to produce antibody
mixtures.
[0025] In certain embodiments, further provided are transgenic
non-human animals and transgenic plants or transgenic plant cells
capable of expressing mixtures of antibodies and mixtures of
antibodies produced by these.
[0026] In certain embodiments, further provided are pharmaceutical
compositions comprising a mixture of recombinantly produced
antibodies and a suitable carrier.
[0027] In certain embodiments, further provided are mixtures of
antibodies for use in the treatment or diagnosis and for the
preparation of a medicament for use in the treatment or diagnosis
of a disease or disorder in a human or animal subject.
[0028] In certain embodiments, further provided is a method for
producing a mixture of antibodies comprising different isotypes
from a single host cell clone.
[0029] In certain embodiments, further provided is a method for
identifying a mixture of antibodies having a desired effect in a
functional assay.
[0030] In certain embodiments, further provided is a method for
producing a mixture of antibodies that are capable of binding to a
target, the method comprising: i) bringing a phage library
comprising antibodies into contact with material comprising a
target, ii) at least one step of selecting phages binding to the
target, iii) identifying at least two phages that comprise
antibodies binding to the target, wherein at least two antibodies
comprise a common light chain, iv) introducing a nucleic acid
sequence encoding the light chain and a nucleic acid sequence or
sequences encoding the heavy chains of at least two antibodies into
a host cell, v) culturing a clone of the host cell under conditions
conducive to expression of the nucleic acid sequences.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 is a schematic representation of an antibody. The
heavy and light chains are paired via interchain disulfide bonds
(dotted lines). The heavy chain can be either of the .alpha.,
.gamma., .mu., .delta. or isotype. The light chain is either
.lamda. or .kappa.. An antibody of IgG1 isotype is shown.
[0032] FIG. 2 is a schematic representation of a bispecific
monoclonal antibody. A bispecific antibody contains two different
functional F(Ab) domains, indicated by the different patterns of
the V.sub.H-V.sub.L regions.
[0033] FIGS. 3A and 3B show a sequence alignment of V.sub.L (FIG.
3A) and V.sub.H (FIG. 3B) of K53, UBS-54 and 02-237. The DNA
sequence of common V.sub.L of UBS54 and K53 is SEQ ID NO:1, while
the amino acid sequence is given as SEQ ID NO:2. DNA sequences of
V.sub.L of 02-237, V.sub.H of UBS54, K53 and 02-237 are SEQ ID
NOS:3, 5, 7 and 9, respectively, while the amino acid sequences are
given in SEQ ID NOS:4, 6, 8 and 10, respectively.
[0034] FIG. 4 is an overview of plasmids pUBS3000Neo and pCD46_3000
(Neo).
[0035] FIG. 5, Panel A, shows the isoelectric focusing (IEF) of
transiently expressed pUBS3000Neo, pCD46_3000(Neo) and a
combination of both. In Panel B, the upper part shows a schematic
representation of the expected molecules when a single light chain
and a single heavy chain are expressed in a cell, leading to
monoclonal antibodies UBS-54 or K53. The lower part under the arrow
shows a schematic representation of the combinations produced when
both heavy chains and the common light chain are co-expressed in a
host cell, with theoretical amounts when both heavy chains are
expressed at equal levels and pair to each other with equal
efficiency. The common light chain is indicated with the vertically
striped bars.
[0036] FIG. 6 is a schematic representation of a possible
embodiment of the method according to the invention (see, e.g.,
Example 9). At (1), introduction of nucleic acid sequences encoding
one light chain and three different heavy chains capable of pairing
to the common light chain to give functional antibodies into host
cells is shown; at (2), selection of stable clones; (3) shows
clones can be screened for, for instance, expression levels,
binding; at (4), clones are expanded; and at (5), production of
functional mixtures of antibodies is shown. Some or all of steps
2-5 could be performed simultaneously or in a different order.
[0037] FIGS. 7A and 7B show the sequence of V.sub.H and V.sub.L of
phages directed against CD22 (clone B28), CD72 (clone II-2) (FIG.
7A), and HLA-DR (class II; clone I-2) (FIG. 7B). DNA sequences of
V.sub.L of clones B28, II-2 and I-2 are SEQ ID NOS:11, 13 and 15,
respectively, while the amino acid sequences are SEQ ID NOS:12, 14
and 16, respectively. DNA sequence of the common light chain of
these clones is SEQ ID NO:17, while the amino acid sequence is SEQ
ID NO:18.
[0038] FIG. 8 is a map of pUBS54-IgA (pCRU-L01 encoding human IgA1
against EPCAM).
[0039] FIG. 9 shows dimeric bispecific IgA with a single light
chain (indicated by horizontally striped bar). The method of the
invention will produce a mixture of forms wherein different heavy
chains can be paired. Only the most simple form is depicted in this
schematic representation. A J-chain is shown to join the two
monomers.
[0040] FIG. 10 is a pentameric multispecific IgM with a single
light chain (indicated by horizontally striped bars). The method of
the invention will produce a mixture of many different forms,
wherein different heavy chains can be paired. Only the most simple
form is depicted in this schematic representation when five
different heavy chains are expressed with a single light chain and
all five different heavy chains are incorporated in the pentamer
and paired to the same heavy chain. Pentamers with less
specificities can also be formed by incorporation of less than five
different heavy chains. Hexamers can also be obtained, especially
when the J-chain is not expressed.
[0041] FIG. 11 depicts expression of a mixture of human IgG
isotypes consisting of a common light chain but with different
binding specificities in a single cell to avoid the formation of
bispecific antibodies. The different binding specificities are
indicated by the different colors of the V.sub.H sequences. The
common light chain is indicated with the vertically striped bars.
The IgG1 isotype is indicated with the grey Fc and the IgG3 isotype
is indicated with the black Fc part.
[0042] FIGS. 12A-12E depict DNA and protein sequences of variable
domains of heavy chains of K53 (FIG. 12A), UBS54 (FIG. 12C) and
02-237 (FIG. 12B) IgG (SEQ ID NOS:7, 9 and 5, respectively) and
light chains (SEQ ID NOS:1 and 3, respectively, for K53/UBS54 (FIG.
12D) and 02-237 IgG (FIG. 12E)).
[0043] FIG. 13 shows alignment of the variable sequences of the
heavy chains of K53, 02-237 and UBS54 (SEQ ID NOS:7, 9, and 5,
respectively). CDR1, CDR2 and CDR3 regions are indicated in
bold.
[0044] FIG. 14 is a BIACORE.TM. (surface plasmon resonance)
analysis of K53 and 02-237. Affinity-purified human CD46 from
LS174T cells was coupled (640 RU) to CM5 chips (BIACORE
BR-1000-14.TM.). Binding of 1000 (A), 500 (B), 250 (C), 125 (D), 63
(E), 31 (F), 16 (G), 8 (H) or 0 (I) nM 02-237 or K53 purified from
stable PER.C6.RTM. (human retina cells that express adenovirus E1A
and E1B proteins)-derived cell lines to the CD46 was monitored
using a BIACORE 3000.TM. system at 37.degree. C. Using this
experimental set-up, a K.sub.d of 9.1.times.10.sup.7 and
2.2.times.10.sup.8 was found for K53 and 02-237, respectively.
[0045] FIG. 15 shows binding of K53 and 02-237 to LS174T cells.
Serial dilutions of purified 02-237 (.box-solid.), K53
(.star-solid.) and the negative control GBSIII (.diamond.)
conjugated to biotin were incubated with LS147T cells pre-incubated
with normal human serum to block Fc.gamma. receptor interaction.
Binding (MFI, ordinate) was determined by FACS after incubation
with streptavidin-conjugated phycoerythrin.
[0046] FIG. 16A is an SDS-PAGE analysis of purified IgG fractions.
Three .mu.g purified IgG was analyzed on a non-reduced 4-20%
NUPAGE.RTM. gel (NOVEX) according to recommendations of the
manufacturer. Proteins were visualized by staining with colloidal
blue (NOVEX Cat. No LC6025) according to recommendations of the
manufacturer. Clone identity is indicated on top of the SDS-PAGE.
Each gel contains a control, which is either purified 02-237 or
K53. FIGS. 16B and 16C are continuations of the gel in FIG.
16A.
[0047] FIG. 16D is an SDS-PAGE analysis of purified IgG fractions.
Three .mu.g purified IgG was analyzed on a reduced 4-20%
NUPAGE.RTM. gel according to recommendations of the manufacturer.
Proteins were visualized by staining with colloidal blue (NOVEX
cat. No LC6025) according to recommendations of the manufacturer.
Clone identity is indicated on top of the SDS-PAGE. Each gel
contains a control, which is either purified 02-237 or K53. NR,
Non-reduced; R, reduced. FIGS. 16E and 16F are continuations of the
gel in FIG. 16D.
[0048] FIG. 17A shows an IEF analysis of purified IgG fractions.
Ten .mu.g purified IgG was analyzed on an Isogel 3-10 gel (BMA)
according to recommendations of the manufacturer. Proteins were
visualized by staining with colloidal blue according to
recommendations of the manufacturer. Clone identity is indicated on
top of the IEF. Each gel contains a control, consisting of a 1:1:1
mixture of 02-237, K53 and UBS54. FIGS. 17B through 17D are
continuations of the gel in FIG. 17A.
[0049] FIG. 18 is an IEF analysis of polyclonal mixtures 241, 280,
282, 361 and 402 in comparison to single K53, 02-237 and UBS54. Ten
.mu.g purified IgG was analyzed on an Isogel 3-10 gel (BMA)
according to recommendations of the manufacturer. Proteins were
visualized by staining with colloidal blue according to
recommendations of the manufacturer. IgG identity is indicated on
top of the IEF.
[0050] FIG. 19 contains mass chromatograms of CDR3 peptides of K53,
02-237, UBS54 and the two unique light chain peptides L1-K53/UBS54
and L1-237 in IgG fraction Poly1-280. On the right-hand side of
each mass chromatogram, the isotopic pattern of the peptide is
shown. The doubly charged ion at m/z 1058.98 (Mw 2115.96 Da)
results from peptide H11-K53. The doubly charged ion at m/z 1029.96
(Mw 2057.92 Da) results from peptide H11-02-237. The triply charged
ion at m/z 770.03 (Mw 2307.09 Da) results from peptide H9-UBS54.
The doubly charged ion at m/z 1291.08 (Mw 2580.16 Da) results from
peptide L1-K53/UBS54. The doubly charged ion at m/z 1278.11 (Mw
2554.22 Da) results from peptide L1-02-237.
[0051] Purified IgG was dissolved in a 0.1% RAPIGEST.TM. (Waters)
in 50 mM NH.sub.4HCO.sub.3. The disulfides were reduced using 1 M
DTT (1,4-dithio-DL-threitol), followed by incubation at 65.degree.
C. for 30 minutes. Then, for alkylation of all sulfhydryl groups, 1
M iodoacetamide was added, followed by incubation at room
temperature for 45 minutes in the dark. Alkylation was stopped by
addition of 1 M DTT. The buffer was exchanged to 25 mM
NH.sub.4HCO.sub.3, pH 7.5. Finally, the antibodies were digested
overnight at 37.degree. C. by addition of a freshly prepared
trypsin solution in 25 mM NH.sub.4HCO.sub.3. The peptide mixture
was analyzed by LC-MS. The LC-system consisted of a Vydac
reversed-phase C18 column that was eluted by applying a gradient of
solvent A (5/95/1 acetonitrile, water, glacial acetic acid v/v/v)
and solvent B (90/10/1 acetonitrile, water, glacial acetic acid
v/v/v). The LC was on-line coupled to a Q-TOF2 mass spectrometer
(Micromass), equipped with an electrospray source operated at 3 kV.
Mass spectra were recorded in a positive ion mode from m/z 50 to
1500 at a cone voltage of 35 V. The instrumental resolution of
>10,000 enabled unambiguous determination of the charge and,
therefore, the mass of most ions up to at least +7. In this way,
all peptides were identified according to their molecular weight.
The amino acid sequence of the peptide was confirmed by
MS/MS-experiments. MS/MS spectra were recorded in a positive ion
mode from m/z 50-2000 with collision energy between 20 and 35
eVolts.
[0052] FIG. 20 is a BIACORE.TM. (surface plasmon resonance)
analysis of polyclonal 280. Affinity-purified human CD46 from
LS174T cells was coupled (640 RU) to CM5 chips (BIACORE
BR-1000-14.TM.). Binding of 1000 (A), 500 (B), 250 (C), 125 (D), 63
(E), 31 (F), 16 (G), 8 (H) or 0 (I) nM Poly1-280 to CD46 was
monitored using a BIACORE 3000.TM. system at 37.degree. C.
[0053] FIG. 21 is an IEF analysis of sub-clones from clones poly
1-241, poly 1-280 and poly 1-402 producing a mixture of
antibodies.
[0054] Panel A contains clones poly 1-241 and poly 1-280. Lane 1
contains a pI marker (Amersham, Cat. No. 17-0471-01). Lane 2
contains isolated IgG from the parent clone poly 1-241 (as in FIG.
18). Lanes 3, 4 and 5, respectively, contain isolated IgG from
three independent sub-clones derived from poly 1-241 by limiting
dilution. Lane 6 contains isolated IgG from the parent clone poly
1-280 (as in FIG. 18). Lanes 7, 8 and 9, respectively, contain
isolated IgG from three independent sub-clones derived from poly
1-280 by limiting dilution.
[0055] Panel B contains clone poly 1-402. Lanes 1 and 7 contain a
pI marker. Lane 2 contains isolated IgG from the parent clone poly
1-402 (as in FIG. 18). Lanes 3, 4 and 5, respectively, contain
isolated IgG from three independent sub-clones derived from poly
1-402 by limiting dilution. Lane 6 contains a control (a 1:1:1
mixture of 02-237, K53 and UBS54).
[0056] FIG. 22 is a fluorescence activated cell sorting (FACS)
analysis of mixtures of antibodies produced from sub-clones of poly
1-241 (A), poly 1-280 (B) and poly 1-402 (C). Binding of the
mixtures of antibodies to cells transfected with cDNA of CD46,
EpCAM, or a negative control (CD38), was determined with FACS
analysis. Mean fluorescent intensity (MFI) is shown for the various
parent clones and three independent sub-clones of each. Control
antibodies GBS-III (negative control), anti-CD72 (02-004; negative
control) and the single antibodies UBS54, 02-237 and K53 are also
included.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Provided is a method for producing a mixture of antibodies
in a recombinant host, the method comprising expressing, in a
recombinant host cell, a nucleic acid sequence or nucleic acid
sequences encoding at least one light chain and at least three
different heavy chains that are capable of pairing with at least
one light chain. In certain embodiments, the light and heavy chains
form functional antigen-binding domains when paired. A functional
antigen-binding domain is capable of specifically binding to an
antigen.
[0058] In certain embodiments, the method for producing a mixture
of antibodies further comprises the step of recovering the
antibodies from the cell or the host cell culture to obtain a
mixture of antibodies suitable for further use.
[0059] In certain embodiments, a method is provided for production
of a mixture of antibodies, the method comprising expressing in a
recombinant host cell a nucleic acid sequence encoding a common
light chain and nucleic acid sequence or sequences encoding at
least three different heavy chains that are capable of pairing with
the common light chain, such that the antibodies that are produced
comprise common light chains. In one aspect, the common light chain
is identical in each light chain/heavy chain pair.
[0060] The term "antibody," as used herein, means a polypeptide
containing one or more domains that bind an epitope on an antigen,
where such domains are derived from, or have sequence identity
with, the variable region of an antibody. The structure of an
antibody is schematically represented in FIG. 1. Examples of
antibodies according to the invention include full length
antibodies, antibody fragments, bispecific antibodies,
immunoconjugates, and the like. An antibody, as used herein, may be
isotype IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM, and the
like, or a derivative of these. Antibody fragments include Fv, Fab,
Fab', F(ab').sub.2 fragments, and the like. Antibodies according to
the invention can be of any origin, including murine, of more than
one origin, e.g., chimeric, humanized, or fully human antibodies.
Immunoconjugates comprise antigen-binding domains and a
non-antibody part such as a toxin, a radiolabel, an enzyme, and the
like.
[0061] An "antigen-binding domain" preferably comprises variable
regions of a heavy and a light chain and is responsible for
specific binding to an antigen of interest. Recombinant antibodies
are prepared by expressing both a heavy and a light chain in a host
cell. Similarly, by expressing two chains with their respective
light chains (or a common light chain), wherein each heavy
chain/light chain has its own specificity, so-called "bispecific"
antibodies can be prepared. "Bispecific antibodies" comprise two
non-identical heavy-light chain combinations (FIG. 2), and both
antigen-binding regions of a bispecific antibody may recognize
different antigens or different epitopes on an antigen. "Epitope"
means a moiety of an antigen to which an antibody binds. A single
antigen may have multiple epitopes.
[0062] A "common light chain," refers to light chains which may be
identical or have amino acid sequence differences. Common light
chains may comprise mutations which do not alter the specificity of
the antibody when combined with the same heavy chain without
departing from the scope of the invention. It is, for instance,
possible within the scope of the definition of common light chains
as used herein, to prepare or find light chains that are not
identical but still functionally equivalent, e.g., by introducing
and testing conservative amino acid changes, 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. In an
exemplary embodiment, provided is the use of a common light chain,
one identical light chain, to combine with different heavy chains
to form antibodies with functional antigen-binding domains. The use
of one common light chain avoids the formation of heterodimers in
which pairing of light and heavy chains results in antigen-binding
domains that are not functional or, in other words, which are not
capable of binding to the target antigen or antigens. The use of a
common light chain and two heavy chains has been proposed (Merchant
et al., 1998; WO 98/50431, the entirety of which are incorporated
herein by reference) for a different purpose, viz., to increase the
formation of functional bispecific antibodies at the expense of
antibody mixture complexity. These publications teach a method for
preferentially producing one defined and desired bispecific
antibody, thereby minimizing the complexity of the produced
mixture. Hence, Merchant specifically teaches to prevent the
production of monospecific antibodies because these are undesired
byproducts in the process for bispecific antibody production
described in those publications. Clearly, there is no teaching in
the prior art to prepare a complex mixture of antibodies from a
recombinant host cell avoiding the formation of non-functional
binding domains or the benefits thereof, let alone how. In the
method according to the invention, at least three different heavy
chains that are capable of pairing with the common light chain are
expressed. In other embodiments, the host cell, as used herein, is
provided with nucleic acid sequences encoding for 4, 5, 6, 7, 8, 9,
10, or more, heavy chains capable of pairing with the common light
chain, to increase the complexity of the produced mixture of
antibodies.
[0063] "Different heavy chains," according to the invention, may
differ in the variable region and have the same constant region. In
other embodiments, where it is clear from the context, they may
have the same variable region and differ in the constant region,
e.g., be of a different isotype. The use of a mixture of antibodies
having different constant regions, such as the Fc-portion, may be
advantageous if different arms of the immune system are to be
mobilized in the treatment of the human or animal body. In yet
other embodiments, also to be clear from the context, both the
variable and the constant regions may differ.
[0064] A "mixture of antibodies," according to the invention,
comprises at least two non-identical antibodies, but may comprise
3, 4, 5, 6, 7, 8, 9, 10, or more, different antibodies and may
resemble a polyclonal or at least an oligoclonal antibody mixture
with regard to complexity and number of functional antigen-binding
molecules. The mixtures produced according to the invention usually
will comprise bispecific antibodies. If desired, formation of
monospecific antibodies in the mixture can be favored over the
formation of bispecific antibodies.
[0065] When n heavy chains and one common light chain are
expressed, as used herein, in a host cell at equal levels, the
theoretical percentage of bispecific antibodies produced by the
method according to the invention is (1-1/n).times.100%. The total
number of different antibodies in the mixture produced by the
method according to the invention is theoretically n+{(n.sup.2-
n)/2}, of which (n.sup.2- n/2) are bispecific antibodies.
Distortion of the ratio of expression levels of the different heavy
chains may lead to values deviating from the theoretical values.
The amount of bispecific antibodies can also be decreased, compared
to these theoretical values, if all heavy chains do not pair with
equal efficiency. It is, for instance, possible to engineer the
heavy chains, for example, by introducing specific and
complementary interaction surfaces between selected heavy chains,
to promote homodimer pairing over heterodimer pairing, contrary to
what has been proposed by Merchant, supra. Heavy chains may also be
selected so as to minimize heterodimer formation in the mixture. A
special form of this embodiment involves heavy chains of two or
more different isotypes (e.g., IgG1, IgG3, IgA). When heavy chains
of different isotype are expressed in the same host cell in
accordance with the invention and one light chain that can pair to
these heavy chains, the amount of bispecific antibodies will be
reduced, possibly to very low or even undetectable levels. Thus,
when bispecific antibodies are less desirable, it is possible to
produce a mixture of antibodies according to the invention, wherein
a nucleic acid sequence encoding a common light chain and nucleic
acid sequences encoding at least two different heavy chains with a
different variable region capable of pairing to the common light
chain are expressed in a recombinant host, and wherein the heavy
chains further differ in their constant regions sufficiently to
reduce or prevent pairing between the different heavy chains. The
mixtures of antibodies may be produced from a clone that was
derived from a single host cell, i.e., from a population of cells
containing the same recombinant nucleic acid sequences.
[0066] It will be understood that the different heavy chains can be
encoded on separate nucleic acid molecules, but may also be present
on one nucleic acid molecule comprising different regions encoding
at least three heavy chains. The nucleic acid molecules usually
encode precursors of the light and/or heavy chains, which, when
expressed, are secreted from the host cells, thereby becoming
processed to yield the mature form. These and other aspects of
expressing antibodies in a host cell are well known to those having
ordinary skill in the art.
[0067] A "recombinant host cell," as used herein, is a cell
comprising one or more so-called transgenes, i.e., recombinant
nucleic acid sequences not naturally present in the cell. These
transgenes are expressed in the host cell to produce recombinant
antibodies encoded by these nucleic acid sequences when these cells
are cultured under conditions conducive to expression of nucleic
acid sequences. The host cell, as used herein, can be present in
the form of a culture from a clone that is derived from a single
host cell wherein the transgenes have been introduced. To obtain
expression of nucleic acid sequences encoding antibodies, 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 antibodies.
[0068] "Functionally linked" is meant to describe that the nucleic
acid sequences encoding the antibody fragments or precursors
thereof is linked to the sequences capable of driving expression
such that these sequences can drive expression of the antibodies or
precursors thereof.
[0069] Useful expression vectors are available in the art, for
example, 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, for instance,
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.alpha. (EF-1.alpha.) promoter,
an 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
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, the entirety of which is
incorporated herein by reference), and the like. This may enhance
the stability and/or levels of expression.
[0070] Protein production in recombinant host cells has been
extensively described, e.g., in Current Protocols in Protein
Science, 1995, J. E. Coligan, B. M. Dunn, H. L. Ploegh, D. W.
Speicher, P. T. Wingfield, ISBN 0-471-11184-8; Bendig, 1988, the
entirety of which is incorporated herein by reference. Culturing a
cell is done to enable it to metabolize, grow, 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.
[0071] "Host cells," according to the invention, may be any host
cell capable of expressing recombinant DNA molecules, including
bacteria such as Escherichia (e.g., E. coli), Enterobocter,
Salmonella, 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, 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.RTM. (human
retina cells that express adenovirus E1A and E1B proteins), and the
like. Often, the expression system of choice will involve a
mammalian cell expression vector and host so that the antibodies
are appropriately glycosylated. A human cell line, preferably
PER.C6.RTM. (human retina cells that express adenovirus E1A and E1B
proteins), 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), the entirety of which is
incorporated herein by reference) and the conditions for expression
of the recombinant product may differ somewhat and optimization of
the process is usually performed to increase the product yields
and/or growth of the cells with respect to each other, according to
methods generally known to one of ordinary skill in the art.
[0072] 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), the entirety of which is
incorporated herein by reference. Expression of antibodies in
recombinant host cells has been extensively described in the art
(see, e.g., EP0120694; EP0314161; EP0481790; EP0523949; U.S. Pat.
No. 4,816,567; WO 00/63403, the entirety of which are incorporated
herein by reference). 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. With regard
to stability of production, the latter is preferred.
[0073] The antibodies are expressed in the cells according to the
invention and may be recovered from the cells or, preferably, from
the cell culture medium, by methods generally known to persons
skilled 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., U.S.
Pat. Nos. 4,801,687 and 5,151,504, the entirety of which are
incorporated herein by reference).
[0074] In one embodiment, at least two antibodies from the mixture
produced according to the invention comprise a heavy-light chain
dimer having different specificities and/or affinities. The
specificity determines which antigen or epitope thereof is bound by
the antibody. The affinity is a measure for the strength of binding
to a particular antigen or epitope. Specific binding is defined as
binding with an affinity (K.sub.a) of at least 5.times.10.sup.4
liter/mole, more preferably, 5.times.10.sup.5, even more
preferably, 5.times.10.sup.6, and still more preferably,
5.times.10.sup.7, or more. Typically, monoclonal antibodies may
have affinities which go up to 10.sup.10 liter per mole or even
higher. The mixture of antibodies produced according to the
invention may contain at least two antibodies that bind to
different epitopes on the same antigen molecule and/or may contain
at least two antibodies that bind to different antigen molecules
present in one antigen-comprising mixture. Such an
antigen-comprising mixture may be a mixture of partially or wholly
purified antigens, such as toxins, membrane components and
proteins, viral envelope proteins, or it may be a healthy cell, a
diseased cell, a mixture of cells, a tissue or mixture of tissues,
a tumor, an organ, a complete human or animal subject, a fungus or
yeast, a bacteria or bacterial culture, a virus or virus stock, or
combinations of these, and the like. Unlike monoclonal antibodies
that are able to bind to a single antigen or epitope only, the
mixture of antibodies according to the invention may, therefore,
have many of the advantages of a polyclonal or oligoclonal antibody
mixture.
[0075] In a preferred embodiment, the host cell according to the
method of the invention is capable of high-level expression of
human immunoglobulin, i.e., at least 1 picograms per cell per day,
preferably, at least 10 picograms per cell per day and, even more
preferably, at least 20 picograms per cell per day or more without
the need for amplification of the nucleic acid molecules encoding
the heavy and light chains in the host cell.
[0076] Preferably, host cells according to the invention contain in
their genome between one and ten copies of each recombinant nucleic
acid to be expressed. In the art, amplification of the copy number
of the nucleic acid sequences encoding a protein of interest in,
e.g., CHO cells can be used to increase expression levels of the
recombinant protein by the cells (see, e.g., Bendig, 1988; Cockett
et al., 1990; U.S. Pat. No. 4,399,216, the entirety of which are
incorporated herein by reference). This is currently a widely used
method. However, a significant time-consuming effort is required
before a clone with a desired high copy number and high expression
levels has been established and, moreover, clones harboring very
high copy numbers (up to hundreds) of the expression cassette often
are unstable (e.g., Kim et al., 1998, the entirety of which is
incorporated herein by reference). It is, therefore, a preferred
embodiment of the invention to use host cells that do not require
such amplification strategies for high-level expression of the
antibodies of interest. This allows fast generation of stable
clones of host cells that express the mixture of antibodies
according to the invention in a consistent manner. We provide
evidence that host cells according to the invention can be
obtained, sub-cloned and further propagated for at least around 30
cell divisions (population doublings) while expressing the mixture
of antibodies according to the invention in a stable manner, in the
absence of selection pressure. Therefore, in certain aspects, the
methods of the invention include culturing the cells for at least
20, preferably 25, more preferably 30, population doublings and, in
other aspects, the host cells according to the invention have
undergone at least 20, preferably 25, more preferably 30,
population doublings and are still capable of expressing a mixture
of antibodies according to the invention. Also provided is a
culture of cells producing a mixture of immunoglobulins from a
single cell, the mixture comprising at least three different heavy
chains. Also provided is a culture of cells producing at least
three different monospecific immunoglobulins from a single cell. In
certain exemplary aspects, the culture produces the mixture or at
least three different monospecific immunoglobulins in a single cell
for more than 20, preferably more than 25, more preferably, more
than 30 population doublings.
[0077] Preferably, host cells according to the method are derived
from human retina cells that have been immortalized or transformed
with adenoviral E1 sequences. A particularly preferred host cell
according to methods of the invention is PER.C6.RTM. (human retina
cells that express adenovirus E1A and E1B proteins) as deposited
under ECACC no. 96022940, or a derivative thereof.
PER.C6.RTM.-derived clones can be generated fast, usually contain a
limited number of copies (about 1-10) of the transgene, and are
capable of high-level expression of recombinant antibodies (Jones
et al., 2003, the entirety of which is incorporated herein by
reference). Therefore, such clones are expected to maintain a
stable copy number over many generations, which is an advantage in
the production of biopharmaceuticals. PER.C6.RTM. (human retina
cells that express adenovirus E1A and E1B proteins) cells have been
extensively characterized and documented, demonstrating good
process of scaling up, suspension growth and growth factor
independence. Furthermore, PER.C6.RTM. (human retina cells that
express adenovirus E1A and E1B proteins) can be incorporated into a
suspension in a highly reproducible manner, making it particularly
suitable for large-scale production. In this regard, the
PER.C6.RTM. cell line (human retina cells that express adenovirus
E1A and E1B proteins) has been characterized for bioreactor growth,
where it can grow to very high densities. The use of PER.C6.RTM.
(human retina cells that express adenovirus E1A and E1B proteins)
for recombinant production of antibodies has been described in
detail in publication WO 00/63403 and in (Jones et al., 2003, the
entirety of which is incorporated herein by reference).
[0078] Also provided is a mixture of antibodies obtainable by a
method described herein. Such mixtures of antibodies are expected
to be more effective than the sole components it comprises, in
analogy to polyclonal antibodies usually being more effective than
monoclonal antibodies to the same target. Such mixtures can be
prepared against a variety of target antigens or epitopes.
[0079] It certain embodiments, provided is a recombinant host cell
comprising a nucleic acid sequence encoding a light chain and a
nucleic acid sequence or nucleic acid sequences encoding at least
three different heavy chains of an antibody, wherein the light
chain and heavy chains are capable of pairing, preferably to form a
functional binding domain. The paired heavy and light chains form
functional antigen-binding regions against the target antigen or
target antigens. The host cells are useful in the described
methods. They can be used to produce mixtures of antibodies.
[0080] In certain embodiments, provided is a composition comprising
a mixture of recombinantly produced antibodies, wherein at least
three different heavy chain sequences are represented in the
mixture of recombinant antibodies. Monoclonal antibodies are
routinely produced by recombinant methods. Also disclosed are
mixtures of antibodies useful for diagnosis or treatment in various
fields. In certain embodiments, the compositions of the invention
comprise mixtures of at least three different heavy chains paired
to light chains in the form of antibodies. Preferably, the light
chains of the antibodies in the mixtures have a common light chain.
The mixtures may comprise bispecific antibodies. The mixtures may
be produced from a clone that was derived from a single host cell,
e.g., from a population of cells containing the same recombinant
nucleic acid sequences. The mixtures can be obtained by methods
according to the invention or be produced by host cells according
to the invention. In other embodiments, the number of heavy chains
represented in the mixture is 4, 5, 6, 7, 8, 9, 10, or more. The
optimal mixture for a certain purpose may be determined empirically
by methods known to one of ordinary skill in the art or by methods
provided by the invention. Such compositions according to the
invention may have several of the advantages of a polyclonal
antibody mixture, without the disadvantages usually inherently
associated with polyclonal antibody mixtures, because of the manner
in which they are produced. It is furthermore expected that the
mixture of antibodies is more efficacious than separate monoclonal
antibodies. Therefore, the dosage and, hence, the production
capacity required may be less for the mixtures of antibodies
according to the invention than for monoclonal antibodies.
[0081] It has, for instance, been described that although no single
monoclonal antibody to botulinum neurotoxin (BoNT/A) significantly
neutralized toxin, a combination of three such monoclonal
antibodies (oligoclonal antibody) neutralized 450,000 50% lethal
doses of BoNT/A, a potency 90 times greater than human hyperimmune
globulin (Nowakowski et al., 2002, the entirety of which is
incorporated herein by reference). This result demonstrates that
oligoclonal mixtures of antibodies comprising only two to three
different specificities may have very high potency.
[0082] Furthermore, the chances of a mixture herein losing its
activity due to target or epitope loss are reduced, when compared
to a single monoclonal antibody. In particular embodiments, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more of the antibodies present in the
mixture according to the invention have different specificities.
Different specificities may be directed to different epitopes on
the same antigen and/or may be directed to different antigens
present in one antigen-comprising mixture. A composition as
described herein may also further comprise 2, 3, 4, 5, 6, 7, 8, 9,
10, or more antibodies having different affinities for the same
epitope. Antibodies with differing affinities for the same epitope
may, for instance, be generated by methods of affinity maturation
known to one of ordinary skill in the art.
[0083] In a particularly preferred embodiment, the composition
according to the invention has an effect that is greater than the
effect of each individual monospecific antibody present in the
composition. The effect can be measured in a functional assay. A
"functional assay," as used herein, is an assay that can be used to
determine one or more desired parameters of the antibody or the
mixture of antibodies subject to the assay conditions. Suitable
functional assays may be binding assays, apoptosis assays,
antibody-dependent cellular cytotoxicity (ADCC) assays,
complement-dependent cytotoxicity (CDC) assays, inhibition of cell
growth or proliferation (cytostatic effect) assays, cell-killing
(cytotoxic effect) assays, cell-signaling assays, assays for
measuring inhibition of binding of pathogen to target cell, assays
to measure the secretion of vascular endothelial growth factor
(VEGF) or other secreted molecules, assays for bacteriostasis,
bactericidal activity, neutralization of viruses, assays to measure
the attraction of components of the immune system to the site where
antibodies are bound, including in situ hybridization methods,
labeling methods, and the like. Clearly, also in vivo assays, such
as animal models, including mouse tumor models, models of
auto-immune disease, virus-infected or bacteria-infected rodent or
primate models, and the like, can be used for this purpose. The
efficacy of a mixture of antibodies according to the invention can
be compared to individual antibodies in such models by methods
generally known to one of ordinary skill in the art.
[0084] In certain embodiments, provided is a method for identifying
at least one host cell clone that produces a mixture of antibodies,
wherein the mixture of antibodies has a desired effect according to
a functional assay, the method comprising (i) providing a host cell
comprising a nucleic acid sequence encoding at least one light
chain and nucleic acid sequence or sequences encoding at least two
different heavy chains, wherein the heavy and light chains are
capable of pairing with each other; (ii) culturing at least one
clone of the host cell under conditions conducive to expression of
nucleic acid sequences; (iii) screening at least one clone of the
host cell for production of a mixture of antibodies having the
desired effect by a functional assay; and (iv) identifying at least
one clone that produces a mixture of antibodies having the desired
effect. Preferably, the host cell comprises a nucleic acid sequence
encoding a common light chain that is capable of pairing with at
least two different heavy chains, such that produced antibodies
comprise common light chains, as described above. In specific
embodiments, culturing in step (ii) and screening in step (iii) of
the method is performed with at least two clones. The method may
optionally include an assay for measuring the expression levels of
the antibodies that are produced, which assay may be during or
after step (ii) according to the method, or later in the procedure.
Such assays are well known to one of ordinary skill in the art and
include protein concentration assays, immunoglobulin-specific
assays such as ELISA, DELFIA, and the like. In particular
embodiments of the method according to the invention, the host cell
comprises nucleic acid sequence or sequences encoding at least 3,
4, 5, 6, 7, 8, 9, 10, or more, heavy chains capable of pairing with
at least one light chain. Functional assays useful for the method
according to the invention may be assays for apoptosis, ADCC, CDC,
cell killing, inhibition of proliferation, virus neutralization,
bacterial opsonization, receptor-mediated signaling, cell
signaling, bactericidal activity, and the like. Useful screening
assays for anti-cancer antibodies have, for instance, been
described in U.S. Pat. No. 6,180,357, the entirety of which is
incorporated herein by reference. Such assays may also be used to
identify a clone according to the method of the invention. It is,
for instance, possible to use enzyme-linked immunosorbent assays
(ELISAs) for the testing of antibody binding to their target. Using
such assays, it is possible to screen for antibody mixtures that
most avidly bind the target antigen (or mixture of target antigens
against which the mixture of antibodies is to be tested). Another
possibility that can be explored is to directly screen for
cytotoxicity or cytostatic effects. It is possible that upon such a
different screen, other or the same clones producing mixtures of
antibodies will be chosen than with the ELISA mentioned above. The
screening for cell killing or cessation of growth of cancerous
cells may be suitably used according to the invention. Cell death
can be measured by various endpoints, including the absence of
metabolism or the denaturation of enzymes. In one possible
embodiment of the invention, the assay is conducted by focusing on
cytotoxic activity toward cancerous cells as an endpoint. For this
assay, a live/dead assay kit, for example, the LIVE/DEAD.RTM.
Viability/Cytotoxicity Assay Kit (L-3224) by Molecular Probes
(Eugene, Oreg.), can suitably be used. Other methods of assessing
cell viability, such as tryspan blue exclusion, .sup.51Cr release,
Calcein-AM, ALAMAR BLUE.TM., LDH activity, and similar methods, can
also be used. The assays may also include screening of the mixture
of antibodies for specificity to the desired antigen-comprising
tissue. The antibodies according to the invention may have a
limited tissue distribution. It is possible to include testing the
mixtures of antibodies against a variety of cells, cell types, or
tissues, to screen for mixtures of antibodies that preferably bind
to cells, cell types or tissues of interest.
[0085] Irrespective of a functional assay as described above, also
disclosed herein are ways to determine the identity of the
antibodies expressed by a clone, using methods such as isoelectric
focusing (IEF), mass-spectrometry (MS), and the like. In certain
embodiments, therefore, provided is use of MS and/or IEF in
selecting a clone that expresses a mixture of antibodies according
to the invention.
[0086] When monoclonal antibodies are produced by recombinant host
cells, a screening step is usually performed to assess expression
levels of the individual clones that were generated. The addition
of more heavy chains to produce mixtures adds a level of complexity
to the production of antibodies. When host cells are transfected
with nucleic acid molecules encoding the light and heavy chains
that will form the mixture of antibodies desired, independent
clones may arise containing the same genetic information but,
nevertheless, differing in expression levels, thereby producing
different ratios of the encoded antibodies, giving rise to
different mixtures of antibodies from the same genetic repertoire.
The method according to the invention is useful for identifying a
clone that produces an optimal mixture for a certain purpose.
[0087] The culturing and/or screening according to steps (ii) and
(iii), respectively, may be suitably performed using
high-throughput procedures, optionally in an automated fashion.
Clones can, for instance, be cultured in 96-well plates or other
multi-well plates, e.g., in arrayed format, and screened for
production of a desired mixture. Robotics may be suitably employed
for this purpose. Methods to implement high-throughput culturing
and assays are generally available and known to one of ordinary
skill in the art. It will also be clear that for this method
according to the invention, it is beneficial to use host cells
capable of high-level expression of proteins, without the need for
amplification of the nucleic acid encoding the proteins in the
cell. In one embodiment, the host cell is derived from a human
embryonic retinoblast cell that has been immortalized or
transformed by adenoviral E1 sequences. In a preferred embodiment,
the cell is derived from PER.C6.RTM. (human retina cells that
express adenovirus E1A and E1B proteins). This cell line has
already been shown to be amenable to high-throughput manipulations,
including culturing (WO 99/64582, the entirety of which is
incorporated herein by reference).
[0088] In specific embodiments of the invention, the mixture of
antibodies according to the method of identifying at least one host
cell according to the invention comprises at least 2, 3, 4, 5, 6,
7, 8, 9, 10, or more, antibodies having different specificities
and/or affinities.
[0089] A potential advantage of the method will be that it will
allow exploring many possible combinations simultaneously, the
combinations inherently including the presence of bispecific
antibodies in the produced mixture. Therefore, more combinations
can be tested than by just mixing purified known monoclonal
antibodies, both in number of combinations and in ratios of
presence of different antibodies in these combinations.
[0090] The clone that has been identified by the method according
to the invention can be used for producing a desired mixture of
antibodies. In certain embodiments, provided is a method of
producing a mixture of antibodies, the method comprising culturing
a host cell clone identified by the method of identifying at least
one host cell clone that produces a mixture of antibodies according
to the invention, culturing being under conditions conducive to
expression of the nucleic acid molecules encoding at least one
light chain and at least two different heavy chains. The produced
antibodies may be recovered from the host cells and/or from the
host cell culture, for example, from the culture medium. The
mixture of antibodies can be recovered according to a variety of
techniques known to one of ordinary skill in the art.
[0091] In certain embodiments, provided is a mixture of antibodies
obtainable by the method according to the invention described
above. The mixtures can be used for a variety of purposes, such as
in the treatment or diagnosis of disease, and may replace, or be
used in addition to, monoclonal or polyclonal antibodies.
[0092] The methods according to the invention may suitably use
nucleic acid molecules for encoding the antibodies, which nucleic
acid molecules have been obtained by any suitable method, including
in vivo, e.g., immunization, methods or in vitro, for instance,
antibody display methods (A. Pluckthun et al., In vitro selection
and evolution of proteins, in Adv. Prot. Chem., F. M. Richards et
al., Eds, Academic Press, San Diego, 2001, vol. 55:367-403, the
entirety of which is incorporated herein by reference), such as
phage display, ribosome display or mRNA display (C. Schaffitzel et
al., In vitro selection and evolution of protein-ligand
interactions by ribosome display, in Protein-Protein Interactions,
A Molecular Cloning Manual, E. Golemis, Ed., Cold Spring Harbor
Laboratory Press, New York, 2001, pp. 535-567, the entirety of
which is incorporated herein by reference), and yeast display
(e.g., WO 99/36569, the entirety of which is incorporated herein by
reference). Methods of identifying antibodies to a certain target,
which target may be a known antigen or an unknown antigen present
in an antigenic mixture, by phage display are known to one of
ordinary skill in the art. In general, a library of phages that
express an antigen-binding domain or derivative thereof on their
surface, the antigen-binding domain encoded by genetic material
present in the phages, is incubated with the antigen or antigen
mixture of interest, after which binding of a sub-population of the
phages that display antigen-binding sites binding to the desired
antigen is obtained whereas the non-binding phages are discarded.
Such selection steps may be repeated one, two, or more times to
obtain a population of phages that are more or less specific for
the antigen of interest. Phage display methods to obtain
antibodies, parts or derivatives thereof have been extensively
described in C. F. Barbas III et al., Phage Display, A laboratory
manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 2001, the entirety of which is incorporated herein by
reference. The library used for such screening may be generated by
using the genetic information of one or more light chains, combined
with genetic information encoding a plurality of heavy chains. The
library described by De Kruif et al. (1995b), the entirety of which
is incorporated herein by reference, comprises seven light chains,
the entirety of which is incorporated herein by reference.
Therefore, in a panel of phages binding to a target, which can,
e.g., be obtained by methods described in De Kruif et al. (supra),
and U.S. Pat. No. 6,265,150 (the entirety of which is incorporated
herein by reference), not more than seven different light chains
will be represented and, if the panel is large enough, several
phages with the same light chain coupled to unrelated heavy chains
may be found. Such phages can be used to obtain the nucleic acid
molecules useful in the methods according to the invention.
[0093] In certain embodiments, provided is a method for producing a
mixture of antibodies to a target, the method comprising i)
bringing an antibody display library comprising antibodies or
antibody fragments into contact with material comprising a target,
ii) at least one step of selecting antibodies or antibody fragments
binding to the target, iii) identifying at least two antibodies or
antibody fragments binding to the target, wherein at least two
antibodies or antibody fragments comprise a common light chain, iv)
introducing a nucleic acid sequence encoding the light chain and a
nucleic acid sequence or nucleic acid sequences encoding the heavy
chains of at least two antibodies into a host cell, v) culturing a
clone of the host cell under conditions conducive to expression of
nucleic acid sequences. The antibody display library may be a phage
display library, a ribosome display library, an mRNA display
library, or a yeast display library. Steps i) and ii) may
optionally be repeated one or more times.
[0094] The nucleic acid sequences encoding the antibodies obtained
by the phage display, ribosome display or yeast display method may
be converted to encode any desired antibody format such as IgG1,
IgG2, IgG3, IgG4, IgA, IgM, IgD, IgE, before introducing them into
a host cell, using standard molecular cloning methods and means
known to one of ordinary skill in the art (e.g., described in Boel
et al., 2000, the entirety of which is incorporated herein by
reference).
[0095] It will be clear to one of ordinary skill in the art that
libraries in which only one light chain is represented are
especially useful in light of the invention, since all antibodies
that can be obtained from such a library will have a common light
chain that is functional in binding target antigen with each of the
heavy chains. In other words, in accordance with the methods of the
invention, the formation of non-functional light chain-heavy chain
dimers is avoided. Phage antibody display libraries having
extensive H chain repertoires and unique or very few L chain
sequences have been disclosed in the art (Nissim et al., 1994;
Vaughan et al., 1996, the entirety of which are incorporated herein
by reference). In general, the specificity of an antibody appears
to be determined to a large extent by its heavy chain. It is even
possible to screen for and identify light chains that do not
contribute significantly to binding of the antibody, which light
chains also could be suitably used according to the invention. It
may also be possible to follow the teachings of the invention but
use one heavy chain and vary the light chains. However, the use of
a common light chain and different heavy chains appears preferable
and the following observations support the idea that the
specificity of an antibody appears to be dominated by its heavy
chain sequence. In the process of receptor editing, a mechanism of
B-cells to monitor if their immunoglobulin receptor encodes a
potentially harmful auto-antibody, B-cells expressing an
auto-antibody replace the expressed heavy chain with another heavy
chain while retaining the expressed light chain. Thus, a new
antibody specificity is generated that does not encode an
auto-antibody. This shows that a single light chain can
successfully dimerize with multiple heavy chains to form different
antibody specificities (Nemazee, 2000; Casellas et al., 2001, the
entirety of which are incorporated herein by reference). Series of
transfected cell lines using a single heavy chain gene with
different light chain genes have been reported, the antibodies
produced to a large extent maintaining their specificity,
regardless of the light chain (Radic et al., 1991, the entirety of
which is incorporated herein by reference).
[0096] Different antibodies have been obtained from a library that
has been constructed using a single light chain (Nissim et al.,
1994). Several antibodies have been obtained from the library
described by De Kruif et al. (1995, the entirety of which is
incorporated herein by reference), which was constructed using
seven light chains, that have the same light chain but different
specificities (see, e.g., Example 1: antibodies binding to EpCAM
and to CD46, described in WO 01/48485 and WO 02/18948,
respectively, the entirety of which are incorporated herein by
reference).
[0097] Besides screening a phage library against a target, it will
also be possible to start with an antibody that has already proven
its merits and use the light chain of this antibody in the
preparation of a library of heavy chains combined with this
particular light chain only, according to methods known to one of
ordinary skill in the art, such as phage display. Using this
strategy, a monoclonal antibody can be used to obtain a mixture of
antibodies according to the invention, functionally resembling a
polyclonal or oligoclonal antibody to the same target.
Alternatively, a method reminiscent of the method described by
Jespers et al. (1994, the entirety of which is incorporated herein
by reference) to obtain a human antibody based on a functional
rodent antibody can be used. The heavy chain of a known antibody of
non-human origin is first cloned and paired as a template chain
with a repertoire of human light chains for use in phage display,
after which the phages are selected for binding to the antigen or
mixture of antigens. The selected light chain is, in turn, paired
with a repertoire of human heavy chains displayed on a phage and
the phages are selected again to find several heavy chains that,
when paired with the light chain, are able to bind to the antigen
or mixture of antigens of interest. This enables creating a mixture
of human antibodies against a target for which thus far only a
non-human monoclonal antibody is described. It is possible that a
mixture according to the invention already has beneficial
functional effects when the individual antibodies do not have high
affinities for the target, whereas high affinities are often
required for monoclonal antibodies to be effective. This would have
the advantage that affinity maturation may be required in less
instances for methods and mixtures according to the invention than
when an approach with monoclonal antibodies is envisaged.
[0098] The heavy and light chain coding sequences can be introduced
simultaneously or consecutively into the host cell. It is also an
aspect to prepare a host cell comprising a recombinant nucleic acid
encoding a light chain of an antibody. Such a cell can, for
instance, be obtained by transfection of the nucleic acid and,
optionally, a clone can be identified that has a high expression of
the light chain. An established clone may then be used to add
genetic information encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, or more,
heavy chains of the invention by introducing the nucleic acid
molecules encoding these into cells of the clone that already
contains the light chain. The nucleic acid molecules encoding the
heavy chains may be introduced into the host cell concomitantly. It
is, of course, also possible to introduce them consecutively, for
instance, by using different selection markers, which can be
advantageous if not all heavy chains can be introduced
simultaneously because the cells do not take up enough copies of
recombinant nucleic acid molecules. Methods to introduce
recombinant nucleic acid molecules into host cells are well known
to one of ordinary skill in the art and include transfection,
electroporation, calcium phosphate precipitation, virus infection,
and the like. One of ordinary skill in the art has several
possibilities to introduce more vectors with nucleic acid sequences
of interest into the same host cell, see, e.g., Sambrook, Fritsch
and Maniatis, Molecular Cloning: A Laboratory Manual, 2.sup.nd
edition, 1989; Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds, 1987; the series Methods in Enzymology
(Academic Press, Inc.), the entirety of which are incorporated
herein by reference.
[0099] Suitable dominant selection markers for introducing nucleic
acids into eukaryotic host cells, as used herein, may be G418 or
neomycin (geneticin), hygromycin or mycophenolic acid, puromycin,
and the like, for which genes encoding resistance are available on
expression vectors. Further possibilities include, for instance,
the use of vectors containing DHFR genes or glutamate synthetase to
select in the presence of methotrexate in a DHFR.sup.- cell or the
absence of glutamine in a glutamine auxotroph, respectively. The
use of expression vectors with different selection markers enables
subsequent transfections with heavy chain sequences of interest
into the host cell, which already stably contains other heavy
chains introduced previously by use of other selection markers. It
is also possible to use selection markers that can be used more
than once, for instance, when containing mutations, introns, or
weakened promoters that render them concentration-dependent (e.g.,
EP0724639; WO 01/32901; U.S. Pat. No. 5,733,779, the entirety of
which are incorporated herein by reference). Alternatively, a
selection marker may be re-used by deleting it from the host cell
after use, for example, by site-specific recombination. A
selectable marker located between sequences recognized by a
site-specific recombinase, for example, lox-sites or FRT-sites, is
used for the generation of the first stable transfectant (for
Cre-lox site-specific recombination, see, Wilson and Kola, 2001,
the entirety of which is incorporated herein by reference).
Subsequently, the selectable marker is excised from the host cell
DNA by the matching site-specific recombinase, for example, Cre or
Flp. A subsequent transfection can suitably use the same selection
marker.
[0100] Different host cell clones each comprising the genetic
information encoding a different light chain may be prepared. If
the antibodies are identified by an antibody display method, it is
thus possible to prepare several host cells, each comprising one
light chain present in the antibody display library. After
identifying antibodies that bind to a target using antibody
display, the nucleic acid molecules encoding the heavy chains can
be introduced into the host cell containing the common light chain
that is capable of pairing to the heavy chains. It is, therefore,
an aspect to provide a method for making a host cell for production
of a mixture of antibodies, the method comprising the steps of:
introducing into the host cell a nucleic acid sequence encoding a
light chain and nucleic acid sequence or sequences encoding 3, 4,
5, 6, 7, 8, 9, 10, or more, different heavy chains that are capable
of pairing with the light chain, wherein the nucleic acid molecules
are introduced consecutively or simultaneously. It is, of course,
also possible to introduce at least two of the nucleic acid
molecules simultaneously, and introduce at least one other of the
nucleic acid molecules consecutively.
[0101] In yet another aspect, a method is provided for making a
recombinant host cell for production of a mixture of antibodies,
the method comprising the step of: introducing a nucleic acid
sequence or nucleic acid sequences encoding 2, 3, 4, 5, 6, 7, 8, 9,
10, or more, different heavy chains into a recombinant host cell
comprising a nucleic acid sequence encoding a light chain capable
of pairing with at least two of the heavy chains.
[0102] If it appears that a recombinant host cell of the invention
does not express sufficient light chain to dimerize with all of the
expressed at least two heavy chains, extra copies of the nucleic
acid molecules encoding the light chain may be transfected into the
cell.
[0103] Besides random integration after transfection, methods to
integrate the transgenes in predetermined positions of the genome
resulting in favorable expression levels can also be used according
to the invention. Such methods may, for instance, employ
site-specific integration by homologous recombination (see, e.g.,
WO 98/41645, the entirety of which is incorporated herein by
reference) or make use of site-specific recombinases (Gorman and
Bullock, 2000, the entirety of which is incorporated herein by
reference).
[0104] It is yet another aspect to provide a transgenic non-human
mammal or a transgenic plant comprising a nucleic acid sequence
encoding a light chain and a nucleic acid sequence or nucleic acid
sequences encoding at least two different heavy chains that are
capable of pairing with the light chain, wherein the nucleic acid
sequences encoding the light and heavy chains are under the control
of a tissue-specific promoter. Promoters in plants may also be
non-tissue specific and general gene-expression elements, such as
the CaMV 35S promoter and nopaline synthase polyA addition site,
can also be used. The light chain is a common light chain according
to the invention. In specific embodiments, the transgenic animal or
plant according to the invention comprises 3, 4, 5, 6, 7, 8, 9, 10,
or more, heavy chain sequences. Besides cell culture as a
production system for recombinant proteins, the art also discloses
the use of transgenic animals, transgenic plants and, for instance,
transgenic chickens to produce proteins in the eggs, and the like
to produce recombinant proteins of interest (Pollock et al., 1999;
Larrick and Thomas, 2001; WO 91/08216, the entirety of which are
incorporated herein by reference). These usually comprise the
recombinant gene or genes encoding one or more proteins of interest
in operable association with a tissue-specific promoter. It has,
for instance, been shown that recombinant antibodies can be
produced at high levels in the milk of transgenic animals that
contain the nucleic acids encoding a heavy and a light chain behind
a mammary gland-specific promoter (e.g., Pollock et al., 1999; WO
95/17085, the entirety of which are incorporated herein by
reference). Particularly useful in this respect are cows, sheep,
goats, pigs, rabbits, mice, and the like, which can be milked to
obtain antibodies. Useful promoters are the casein promoters, such
as the .beta.-casein promoter, the .alpha.S1-casein promoter, the
whey acidic protein (WAP) promoter, the .beta.-lactoglobulin
promoter, the .alpha.-lactalbumin promoter, and the like.
Production of biopharmaceutical proteins in the milk of transgenic
mammals has been extensively described (e.g., Pollock et al., 1999,
the entirety of which is incorporated herein by reference). Besides
mammary gland-specific promoters, other tissue-specific promoters
may be used, directing the expression to the blood, urine, saliva,
and the like. The generation of transgenic animals comprising
recombinant nucleic acid molecules has been extensively documented
and may include micro-injection of oocytes (see, e.g., Wilmut and
Clark, 1991, the entirety of which is incorporated herein by
reference), nuclear transfer after transfection (e.g., Schnieke et
al., 1997, the entirety of which is incorporated herein by
reference), infection by recombinant viruses (e.g., U.S. Pat. No.
6,291,740, the entirety of which is incorporated herein by
reference), and the like. Nuclear transfer and cloning methods for
mammalian cells are known to one of ordinary skill in the art, and
are, for example, described in Campbell et al., 1996; Wilmut et
al., 1997; Dinnyes et al., 2002; WO 95/17500; and WO 98/39416, the
entirety of which are incorporated herein by reference. It is
possible to clone animals and to generate lines of animals that are
genetically identical, which renders it possible for a person
skilled in the art to create such a line once an individual animal
producing the desired mixture of antibodies has been identified.
Alternatively, classical breeding methods can be used to generate
transgenic offspring. Strategies for the generation of transgenic
animals for production of recombinant proteins in milk are
described in Brink et al., 2000, the entirety of which is
incorporated herein by reference.
[0105] Transgenic plants or plant cells producing antibodies have
also been described (Hiatt et al., 1989; Peeters et al., 2001, the
entirety of which are incorporated herein by reference) and useful
plants for this purpose include corn, maize, tobacco, soybean,
alfalfa, rice, and the like. Constitutive promoters that can, for
instance, be used in plant cells are the CaMV 35S and 19S promoters
and Agrobacterium promoters nos and ocs. Other useful promoters are
light-inducible promoters such as rbcS. Tissue-specific promoters
can, for instance, be seed-specific, such as promoters from zein,
napin, beta-phaseolin, ubiquitin, or tuber-specific, leaf-specific
(e.g., useful in tobacco), root-specific, and the like. It is also
possible to transform the plastid organelle by homologous
recombination to express proteins in plants.
[0106] Methods and means for expression of proteins in recombinant
plants or parts thereof, or recombinant plant cell culture, are
known to one of ordinary skill in the art and have been, for
instance, described in Giddings et al., 2000; WO 01/64929; WO
97/42313; U.S. Pat. Nos. 5,888,789, 6,080,560 (for practical
guidelines, see Methods In Molecular Biology vol. 49 "Plant Gene
Transfer And Expression Protocols," H. Jones, 1995), the entirety
of which are incorporated herein by reference. Other transgenic
systems for producing recombinant proteins have also been
described, including the use of transgenic birds to produce
recombinant proteins in eggs (e.g., WO 97/47739, the entirety of
which is incorporated herein by reference) and the use of
transgenic fish (e.g., WO 98/15627, the entirety of which is
incorporated herein by reference), and can be used in combination
with the teachings of the invention to obtain mixtures of
antibodies. It is also possible to use an in vitro
transcription/translation or in vitro translation system for the
expression of mixtures of antibodies according to the invention. It
will be clear to one of ordinary skill in the art that the
teachings of the current invention will allow producing mixtures of
antibodies in systems where recombinant nucleic acids encoding the
light chain and heavy chains can be introduced and expressed.
Preferably, such systems are able to produce antibodies encoded by
nucleic acid sequences, without the use of amplification of nucleic
acid sequences in the systems. In another aspect, a cell from a
transgenic non-human animal or a transgenic plant according to the
invention is provided. Such cells can be used to generate the
animals or plants according to the invention, using techniques
known to one of ordinary skill in the art, such as nuclear transfer
or other known methods of cloning whole organisms from single
cells. The cells according to the invention may also be obtained by
introducing the light and at least two heavy chain sequences into
isolated cells of non-human animals or plants, which cells are
capable of becoming part of a transgenic animal or plant.
Particularly useful for such purposes are embryonic stem cells.
These can contribute to the germ line and, therefore, the genetic
information introduced into such cells can be passed to future
generations. In addition, plant cell cultures of cotton, corn,
tomato, soybean, potato, petunia, and tobacco can be utilized as
hosts when transformed with the nucleic acid molecules encoding the
light chain and the heavy chains, for instance, by use of the
plant-transforming bacterium A. tumefaciens or by particle
bombardment or by infecting with recombinant plant viruses.
[0107] In certain embodiments, provided is a pharmaceutical
composition comprising a mixture of recombinantly produced
antibodies and a suitable carrier, wherein at least two different
heavy chains are represented in the mixture of recombinantly
produced antibodies. Pharmaceutically acceptable carriers as used
herein are exemplified, but not limited to, adjuvants, solid
carriers, water, buffers, or other carriers used in the art to hold
therapeutic components, or combinations thereof. In particular
embodiments, 3, 4, 5, 6, 7, 8, 9, 10, or more, different heavy
chains are represented in the mixture. The mixture can be obtained
by mixing recombinantly produced monoclonal antibodies, but may
also be obtained by methods according to the invention. The mixture
may, therefore, comprise a common light chain for the antibodies.
The mixture may comprise bispecific antibodies. The mixture may be
produced from a clone that was derived from a single host cell,
e.g., from a population of cells containing the same recombinant
nucleic acid molecules. The term "recombinantly produced" as used
herein refers to production by host cells that produce antibodies
encoded by recombinant nucleic acids introduced in such host cells
or ancestors thereof. It does not, therefore, include the classical
method of producing polyclonal antibodies, whereby a subject is
immunized with an antigen or antigen-comprising mixture, after
which the antibodies produced by this subject are recovered from
the subject, for example, from the blood.
[0108] In certain embodiments, provided is a mixture of antibodies
wherein at least two heavy chains are represented for use in the
treatment or diagnosis of a human or animal subject. In another
aspect, provided is the use of a mixture of antibodies wherein at
least two different heavy chains are represented for the
preparation of a medicament for use in the treatment or diagnosis
of a disease or disorder in a human or animal subject. In
particular embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, heavy
chains are represented in the mixture. The mixtures of antibodies
may be mixtures of antibodies according to the invention or
obtained by methods according to the invention. Antibodies present
in the mixture may preferably comprise a common light chain. The
mixtures may comprise bispecific antibodies and may be
recombinantly produced from a clone that was derived from a single
host cell, i.e., from a population of cells containing the same
recombinant nucleic acid molecules. The targets may be used to
screen an antibody display library, as described supra, to obtain
2, 3, 4, 5, 6, 7, 8, 9, 10, or more, antibodies comprising a common
light chain that bind to the target and produce a mixture of these
according to the teachings of the invention. Virtually any area of
medicine where monoclonal antibodies can be used is amenable for
the use of the mixtures according to the invention. This can, e.g.,
include treatment of auto-immune diseases and cancer, including
solid tumors of the brain, head, neck, breast, prostate, colon,
lung, and the like, as well as hematologic tumors, such as B-cell
tumors. Neoplastic disorders which can be treated with the mixtures
according to the invention include leukemias, lymphomas, sarcomas,
carcinomas, neural cell tumors, squamous cell carcinomas, germ cell
tumors, metastases, undifferentiated tumors, seminomas, melanomas,
myelomas, neuroblastomas, mixed cell tumors, neoplasias caused by
infectious agents, and other malignancies. Targets for the antibody
mixtures may include, but are not limited to, the HER-2/Neu
receptor, other growth factor receptors (such as VEGFR1 and VEGFR2
receptors), B-cell markers (such as CD19, CD20, CD22, CD37, CD72,
etc.), T-cell markers (such as CD3, CD25, etc.), other leukocyte
cell surface markers (such as CD33 or HLA-DR, etc.), cytokines
(such as TNF), interleukins, receptors for these cytokines (such as
members of the TNF receptor family), and the like. It is
anticipated that the use of such mixtures of antibodies in the
treatment of cancerous tissues or other complex
multi-antigen-comprising cells such as microorganisms or viruses
will give rise to less occurrence of epitope-loss escape variants
than the use of single monoclonal antibodies. Several treatments
nowadays use polyclonal mixtures of antibodies, which are derived
from immunized humans or animals. These treatments may be replaced
by use of the mixtures according to the invention. Use of these
mixtures can also include use in graft-versus-host rejections known
in the art of transplantation, e.g., by use of anti-thymocyte
antibodies. It is anticipated that the mixtures of antibodies are
superior to monoclonal antibodies in the treatment of complex
antigens or antigen-comprising mixtures such as bacteria or
viruses. Therefore, use according to the invention can also include
use against strains of bacteria and fungi, e.g., in the treatment
of infectious diseases due to pathogenic bacteria such as
multidrug-resistant S. aureus and the like, fungi such as Candida
albicans and Aspergillus species, yeast and the like. The mixtures
according to the invention may also be used for post exposure
prophylaxis against viruses, such as members of the genus
Lyssavirus, e.g., rabies virus, or for therapeutic or prophylactic
use against viruses such as Varicella-Zoster Virus, Adenoviruses,
Respiratory Syncitium Virus, Human Immunodeficiency Virus, Human
Metapneumovirus, influenza virus, West Nile Virus, the virus
causing Severe Acute Respiratory Syndrome (SARS), and the like.
Mixtures according to the inventions can also be used to protect
against agents, both bacteria and viruses, and against toxic
substances that are potential threats of biological warfare.
Therefore, use according to the invention can also include use
against strains of bacteria such as Bacillus anthracis, Clostridium
botulinum toxin, Clostridium perfringens epsilon toxin Yersinia
Pestis, Francisella tulariensis, Coxiella burnetii, Brucella
species, Staphylococcus enterotoxin B, or against viruses such as
Variola major, alpha viruses causing meningoencephalitis syndromes
(EEEV, VEEV, and WEEV), viruses known to cause hemorrhagic fevers
such as Ebola, Marburg and Junin virus or against viruses such as
Nipah virus, Hantaviruses, Tick borne encephalitis virus and Yellow
fever virus or against toxins, for example, ricin toxin from
Ricinus communis and the like. Use of the mixtures according to the
invention can also include use against unicellular or multicellular
parasites. Recombinant mixtures of antibodies according to the
invention may become a safe alternative to polyclonal antibodies
obtained from pools of human sera for passive immunization or from
sera of hyper-immunized animals. The mixtures may be more
efficacious than recombinant monoclonal antibodies in various
therapeutic applications, including cancer, allergy, viral
diseases, chronic inflammation, and the like.
[0109] It has been described that homodimerization of
tumor-reactive monoclonal antibodies markedly increases their
ability to induce growth arrest or apoptosis of tumor cells (Ghetie
et al., 1997, the entirety of which is incorporated herein by
reference). Possibly, when antibodies against receptors or other
surface antigens on target cells, such as tumor cells or infectious
microorganisms, are produced according to the invention, the
bispecific antibodies present in mixtures according to the
invention may also cross-link different receptors or other antigens
on the surface of target cells and, therefore, such mixtures may be
very suitable for killing such cells. Alternatively, when
bispecific antibodies are less desirable, the invention also
provides methods to recombinantly produce mixtures of antibodies
comprising mainly monospecific antibodies. It has been described
that the efficacy of treatment with Rituximab.TM. (anti-CD20
monoclonal antibody) was increased when anti-CD59 antibodies were
added (Herjunpaa et al., 2000, the entirety of which is
incorporated herein by reference).
[0110] Therefore, it is thought that inclusion of antibodies
against CD59 in a mixture comprising anti-tumor antibodies in the
form of B-cell receptor-recognizing antibodies increases the
sensitivity of tumor cells to complement attack. It has also been
shown that a triple combination cocktail of anti-CD19, anti-CD22,
and anti-CD38-saporin immunotoxins is much more effective than the
individual components in the treatment of human B-cell lymphoma in
an immunodeficient mouse model (Flavell et al., 1997, the entirety
of which is incorporated herein by reference). Many other
combinations may also be feasible and can be designed by one of
ordinary skill in the art. In general, the use of antibody mixtures
that are capable of recognizing multiple B-cell epitopes will
likely decrease the occurrence of escape variants.
[0111] Another possible target is a transmembrane tyrosine kinase
receptor, encoded by the Her-2/Neu (ErbB2) proto-oncogene (see,
e.g., U.S. Pat. Nos. 5,772,997 and 5,783,186 for anti-Her2
antibodies, the entirety of which are incorporated herein by
reference). Her-2 is overexpressed on 30% of highly malignant
breast cancers and successful antibodies against this target
marketed under the name HERCEPTIN.TM. (Trastuzumab) have been
developed. It has been shown that targeting multiple Her-2 epitopes
with a mixture of monoclonal antibodies results in improved
antigrowth activity of a human breast cancer cell line in vitro and
in vivo (Spiridon et al., 2002, the entirety of which is
incorporated herein by reference). Her-2 may, therefore, be a good
target for antibody mixtures according to the invention. Antibodies
useful for this purpose can be obtained by methods described in the
invention, including antibody display methods.
[0112] Human antibodies are capable of eliciting effector function
via binding to immunoglobulin receptors on immune effector cells.
Human IgG and, in particular, IgG1 and IgG3, fix complement to
induce CDC and interact with Fc.gamma. receptors to induce
antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis,
endocytosis, induction of respiratory burst and release of
inflammatory mediators and cytokines. Human IgA interacts with
Fc.alpha.R, also resulting in efficient activation of ADCC and
phagocytosis of target cells. Hence, due to the differential
distribution of Fc.gamma.R and Fc.alpha.R on peripheral blood cells
(Huls et al., 1999, the entirety of which is incorporated herein by
reference), using a mixture of antibodies directed against the
target and consisting of both IgG and IgA would potentially
maximize the recruitment and activation of different immune
effector cells. Such a mixture of both IgG and IgA could be
obtained by producing the IgG and IgA monoclonal antibody in a
separate production process using two distinct production cell
lines, but could also be obtained from a single cell line producing
both the IgG and the IgA monoclonal antibody. This would have the
advantage that only a single production process has to be
developed. Thus, when different heavy chains are mentioned, heavy
chains differing in their constant regions are also encompassed in
the invention. The principle of using a common light chain can also
be used for the production of a mixture of isotypes from a host
cell. Therefore, certain embodiments of the invention provide a
method for producing a mixture of antibodies comprising different
isotypes from a host cell, the method comprising the step of:
culturing a host cell comprising a nucleic acid sequence encoding a
light chain and nucleic acid sequences encoding at least two heavy
chains of different isotype that are capable of pairing with the
light chain, under conditions conducive to expression of the
nucleic acid sequences. According to this aspect, different heavy
chains may have identical variable regions and only differ in their
constant regions (i.e., be of different isotype and have the same
specificity). In a particular embodiment, the isotypes comprise at
least an IgG and an IgA and/or IgM, preferably IgG1 or IgG3 and
IgA. Other combinations of IgG1, IgG2, IgG3 and IgG4 can also be
used. In these embodiments, bispecific antibodies will not be
produced because the variable regions are the same.
[0113] In other embodiments of this aspect, not only the constant
regions of the heavy chains may differ, but also the variable
regions, thereby giving rise to different specificities paired with
the same light chain. When bispecific antibodies are not desired
for a given purpose, for example, because the mixtures of
antibodies are less efficacious because of the presence of the
bispecific antibodies, it is possible to use at least two heavy
chains combined with the common light chain according to the
invention wherein the heavy chains differ sufficient in their
constant regions to reduce or prevent pairing between the different
heavy chains, for example, by using heavy chains of different
isotypes, such as an IgG1 and an IgG3 (see FIG. 11 for a schematic
representation). It is anticipated that the heavy chains of
different isotype will pair much less efficient, if at all,
compared to the same heavy chains. Alternatively, it is also
possible to engineer the different heavy chains in their constant
region such that homodimerization is favored over
heterodimerization, e.g., by introducing self-complementary
interactions (see, e.g., WO 98/50431 for possibilities, such as
"protuberance-into-cavity" strategies (see, WO 96/27011, the
entirety of which is incorporated herein by reference)). It is,
therefore, another aspect to provide a method for producing a
mixture of antibodies in a recombinant host, the method including
the step of: expressing in a recombinant host cell a nucleic acid
sequence encoding a common light chain and nucleic acid sequences
encoding at least two different heavy chains that differ in the
variable region and that are capable of pairing with the common
light chain, and wherein the heavy chains further differ in their
constant regions sufficiently to reduce or prevent pairing between
the different heavy chains. In one embodiment, the heavy chains are
of different isotype. In specific embodiments, 3, 4, 5, 6, 7, 8, 9,
10, or more, different heavy chains are expressed. Mixtures of
antibodies obtainable by this method are also embodied in the
invention. Such mixtures will comprise mainly monospecific
antibodies.
[0114] The teachings herein can also be used to obtain novel
multispecific antibodies or mixtures thereof. Therefore, in another
aspect, provided is a method for producing a mixture of antibodies
comprising dimeric IgA isotype {(IgA).sub.2} antibodies in a
recombinant host, wherein at least part of the dimeric IgA
antibodies have different binding regions in each of the IgA
sub-units, the method comprising the step of: expressing in a
recombinant host cell a nucleic acid sequence encoding a common
light chain and nucleic acid sequences encoding at least two
different heavy chains of IgA isotype capable of pairing to the
common light chain, wherein the different heavy chains differ in
their variable region. Dimerization of the IgA molecules can be
enhanced by co-expressing J-chain (Yoo et al., 1999, the entirety
of which is incorporated herein by reference). Dimeric IgA
antibodies have two specificities (see FIG. 9 for a schematic
representation of one possible form produced and present in the
mixture).
[0115] In certain embodiments, provided is a method for producing a
mixture of antibodies comprising an IgM antibody having at least
two different specificities, the method comprising expressing in a
recombinant host cell a nucleic acid sequence encoding a common
light chain and nucleic acid sequences encoding at least two
different heavy chains of IgM isotype, wherein the heavy chains are
capable of pairing to the common light chain and form functional
antigen-binding regions. Up to five specificities can be comprised
in an IgM pentamer in the presence of a J-chain and up to six in an
IgM hexamer in the absence of a J-chain (Yoo et al., 1999).
Therefore, in specific embodiments, 3, 4, 5, or 6 IgM heavy chains
are co-expressed with the common light chain according to this
aspect. See FIG. 10 for a schematic representation of one of the
possible forms that can be produced and present in the mixture
according to this aspect, when five different heavy chains are
expressed with a common light chain. Also provided is for IgA
dimers, IgM pentamers or hexamers having at least two different
specificities. These molecules can be produced from a clone of a
single host cell according to the invention. Such molecules
harboring antigen-binding regions with different specificities can
bind different epitopes on the same antigen, different antigens on
one cell, or different antigens on different cells, thereby
cross-linking the antigens or cells.
[0116] In certain embodiments, provided is a method for identifying
a mixture of antibodies having a desired effect in a functional
assay, the method comprising i) adding a mixture of antibodies in a
functional assay, and ii) determining the effect of the mixture in
the assay, wherein the mixture of antibodies comprises antibodies
having a common light chain. In a preferred embodiment, the mixture
is comprised in a composition of the invention.
[0117] Also provided is a method for recombinant expression of one
or more proteins in a single host cell, wherein at least four
different polypeptides are expressed in the single host cell. Each
polypeptide is independently expressed and may be under control of
a heterologous promoter. The protein or proteins may be isolated
separately or as a mixture from a culture of the host cell.
Preferably, the host cell of this embodiment is a human cell and/or
may be derived from a retina cell, more preferably a cell
comprising adenovirus E1 sequences in its genome, most preferably a
PER.C6.RTM. cell (human retina cells that express adenovirus E1A
and E1B proteins).
EXAMPLES
[0118] The following examples are provided to illustrate the
invention and are not to be construed in any way to limit the scope
of the invention. The practice of this invention will employ,
unless otherwise indicated, conventional techniques of immunology,
molecular biology, microbiology, cell biology, and recombinant DNA,
which are within the skill of the art. See, e.g., Sambrook, Fritsch
and Maniatis, Molecular Cloning: A Laboratory Manual, 2.sup.nd
edition, 1989; Current Protocols in Molecular Biology, F. M.
Ausubel, et al., eds, 1987; the series Methods in Enzymology
(Academic Press, Inc.); PCR2: A Practical Approach, M. J.
MacPherson, B. D. Hams, G. R. Taylor, eds, 1995; Antibodies: A
Laboratory Manual, Harlow and Lane, eds, 1988, the entirety of
which are incorporated herein by reference.
Example 1 Production of a Mixture of Monoclonal Antibodies with a
Common Light Chain and Two Different Heavy Chain-Variable Regions
in a Single Cell
[0119] Clone UBS-54 and Clone K53 were previously isolated by
selections on the colorectal cell line SW40 (Huls et al., 1999) and
on a heterogeneous mixture of mononuclear cells of a patient with
multiple myeloma (WO 02/18948, the entirety of which is
incorporated herein by reference), respectively, with a
semi-synthetic library (de Kruif et al., 1995b). Further studies
revealed that clone UBS-54 and K53 bound to the EP-CAM homotypic
adhesion molecule (Huls et al., 1999) and the membrane cofactor
protein CD46 (WO 02/18948), respectively. DNA sequencing of the
clones revealed that they were unique in the Heavy chain CDRs, but
that they contained an identical light chain sequence (FIG. 3). The
V.sub.H and V.sub.L of clones UBS-54 and K53 were inserted into an
expression vector containing the HAVT20 leader sequence and all the
coding sequences for the constant domains of a human IgG1 with a
Kappa light chain by a method essentially as described (Boel et
al., 2000), which resulted in plasmids pUBS3000Neo and
pCD46_3000(Neo) (FIG. 4). These plasmids were transiently
expressed, either alone or in combination in PER.C6.RTM. cells
(human retina cells that express adenovirus E1A and E1B proteins).
In brief, each 80 cm.sup.2 flask was transfected by incubation for
four hours with 140 .mu.l lipofectamine+10 .mu.g DNA (either
pUBS3000Neo, pCD46_3000(Neo) or 10 .mu.g of both) in serum-free
DMEM medium at 37.degree. C. After four hours this was replaced
with DMEM+10% FBS and the cells were grown overnight at 37.degree.
C. Cells were then washed with PBS and the medium was replaced with
Excell 525 medium (JRH Bioscience). The cells were allowed to grow
at 37.degree. C. for six days, after which the cell culture
supernatant was harvested. Human IgG-specific ELISA analysis
(described in WO 00/63403, the entirety of which is incorporated
herein by reference) indicated that IgG was present at
approximately 10 .mu.g/ml for all flasks containing expression
plasmids. No IgG1 was present in a control flask which was not
transfected with expression plasmid.
[0120] Human IgG from each supernatant was subsequently purified
using Protein A-affinity chromatography (Hightrap Protein A HP,
cat. no. 1-040203) according to standard procedures, following
recommendations of the manufacturer (Amersham Biosciences). After
elution, samples were concentrated in a Microcon YM30 concentrator
(Amicon) and buffer exchanged to 10 mM sodium phosphate, pH 6.7.
Twelve .mu.g of purified IgG was subsequently analyzed on
Isoelectric-focusing gels (Serva Pre-cast IEF gels, pH range 3-10,
cat. no. 42866). The samples were loaded on the low pH side and
after focusing, stained with colloidal blue (FIG. 5). Lane 1 shows
transiently expressed K53, Lane 2 shows transiently expressed
UBS-54 and Lane 3 shows the IgG sample of the cells in which both
antibodies were co-transfected. Clearly, K53 and UBS-54 each have a
unique pI profile and the sample from the co-transfection showed
other unique isoforms, with the major isoform having a pI in
between those of K53 and UBS-54. This is also anticipated on the
basis of the theoretic pI when calculated with the ProtParam tool
provided on the Expasy homepage (expasy.ch; Appel et al., 1994, the
entirety of which is incorporated herein by reference). K53 and
UBS-54 have a theoretic pI of 8.24 and 7.65, respectively, whereas
an isoform representing a heterodimer of one UBS-54 heavy chain and
one K53 heavy chain has a theoretical pI of 8.01. Assembly of such
a heterodimer can only occur when a single cell translates both the
heavy chain of K53 and the heavy chain of UBS-54 and assembles
these into a full length IgG molecule together with the common
light chain.
[0121] Therefore, this experiment shows that it is possible to
express two unique human IgG molecules in a single cell and that a
heterodimer consisting of these two unique binding specificities is
also efficiently formed.
Example 2 Production of a Mixture of Antibodies Against Human
B-Cell Markers in a PER.C6.RTM. Cell Line (Human Retina Cells that
Express Adenovirus E1A and E1B Proteins)-Derived Clone
[0122] A method for producing a mixture of antibodies according to
the invention, using expression in a recombinant host cell of a
single light chain and three different heavy chains capable of
pairing to the single light chain to form functional antibodies, is
exemplified herein and is schematically shown in FIG. 6. Phages
encoding antibodies capable of binding proteins present on human
B-cells, i.e., CD22, CD72 and Major Histocompatibility Complex
(MHC) class II (further referred to as HLA-DR) were previously
isolated from a semi-synthetic phage library (de Kruif et al.,
1995; van der Vuurst de Vries & Logtenberg, 1999, the entirety
of which is incorporated herein by reference). DNA sequencing of
the V.sub.H and V.sub.L sequences of the phages clone B28
(anti-CD22), clone I-2 (anti-HLA-DR) and clone II-2 (anti-CD72)
revealed that they all contain a unique V.sub.H sequence but a
common light chain sequence (V.lamda.3) with an identical CDR
region (FIG. 7).
[0123] The V.sub.H and V.sub.L sequences of clones B28, I-1 and
II-2 are cloned behind the HAVT20 leader sequences of an expression
plasmid comprising a heavy chain. An example of such a plasmid is
pCRU-K01 (contains kappa heavy chain sequences that can be easily
interchanged for lambda heavy chain sequences if desired by a
person skilled in the art), as deposited at the ECACC under number
03041601. The cloning gives rise to plasmids encoding a full length
human IgG1 with binding specificities for CD22, CD72 and HLA-DR.
These plasmids will further be referred to as pCRU-CD22, pCRU-CD72
and pCRU-HLA-DR, respectively.
[0124] Stable PER.C6.RTM. (human retina cells that express
adenovirus E1A and E1B proteins)-derived cell lines are generated,
according to methods known to one of ordinary skill in the art
(see, e.g., WO 00/63403), the cell lines expressing antibodies
encoded by genetic information on either pCRU-CD22, pCRU-CD72 or
pCRU-HLA-DR and a cell line expressing antibodies encoded by all
three plasmids. Therefore, PER.C6.RTM. cells (human retina cells
that express adenovirus E1A and E1B proteins) are seeded in DMEM
plus 10% FBS in tissue culture dishes (10 cm diameter) or T80
flasks with approximately 2.5.times.10.sup.6 cells per dish and
kept overnight under their normal culture conditions (10% CO.sub.2
concentration and 37.degree. C.). The next day, transfections are
performed in separate dishes at 37.degree. C. using Lipofectamine
(Invitrogen Life Technologies) according to standard protocols
provided by the manufacturer, with either 1-2 .mu.g pCRU-CD22, 1-2
.mu.g pCRU-CD72, 1-2 .mu.g pCRU-HLA-DR or 1 .mu.g of a mixture of
pCRU-CD22, pCRU-CD72 and pCRU-HLA-DR. As a control for transfection
efficiency, a few dishes are transfected with a LacZ control
vector, while a few dishes will not be transfected and serve as
negative controls.
[0125] After four to five hours, cells are washed twice with DMEM
and given fresh medium without selection. The next day, the medium
is replaced with fresh medium containing 500 .mu.g/ml G418. Cells
are refreshed every two or three days with medium containing the
same concentrations of G418. About 20 to 22 days after seeding, a
large number of colonies are visible and from each transfection, at
least 300 are picked and grown via 96-well plates and/or 24-well
plates via 6-well plates to T25 flasks. At this stage, cells are
frozen (at least one, but usually four vials per sub-cultured
colony) and production levels of recombinant human IgG antibody are
determined in the supernatant using an ELISA specific for human
IgG1 (described in WO 00/63403). Also, at this stage, G418 is
removed from the culture medium and never re-applied again. For a
representative number of colonies, larger volumes will be cultured
to purify the recombinant human IgG1 fraction from the conditioned
supernatant using Protein A affinity chromatography according to
standard procedures. Purified human IgG1 from the various clones is
analyzed on SDS-PAGE, Iso-electric focusing (IEF) and binding to
the targets CD22, CD72 and HLA-DR using cell transfectants
expressing these human antigens on their cell surface
(transfectants expressing CD72 and HLA-DR have been described by
van der Vuurst-de Vries and Logtenberg, 1999; a CD22 transfectant
has been prepared according to similar standard procedures in
PER.C6.RTM. (human retina cells that express adenovirus E1A and E1B
proteins)).
[0126] Colonies obtained from the co-transfection with pCRU-CD22,
pCRU-CD72 and pCRU-HLA-DR are screened by PCR on genomic DNA for
the presence or absence of each of the three constructs. The
identity of the PCR products is further confirmed by DNA
sequencing.
[0127] Next, it is demonstrated that a clonal cell line accounts
for the production of each of the three binding specificities,
i.e., proving that a single cell is able to produce a mixture of
more than two functional human IgGs. Therefore, a limited number of
colonies, which screened positive for the production of each of the
three binding specificities (both by PCR at the DNA level as well
as in the specified binding assays against CD22, CD72 and HLA-DR),
are subjected to single cell sorting using a fluorescence-activated
cell sorter (FACS) (Becton & Dickinson FACS VANTAGESE.TM.
(high-performance, high-speed cell sorter)). Alternatively,
colonies are seeded at 0.3 cells/well to guarantee clonal
outgrowth. Clonal cell populations, hereafter designated as
sub-clones, are refreshed once a week with fresh medium. Sub-clones
are grown and transferred from 96-well plates via 24- and 6-well
plates to T25 flasks. At this stage, sub-clones are frozen (at
least one, but usually four vials per sub-clone) and production
levels of recombinant human IgG1 antibody are determined in the
supernatant using a human IgG1-specific ELISA. For a representative
number of sub-clones, larger volumes are cultured to purify the
recombinant human IgG1 fraction from the conditioned supernatant
using Protein A-affinity chromatography according to standard
procedures.
[0128] Purified human IgG1 from the various sub-clones is
subsequently analyzed as described above for human IgG1 obtained
from the parental clones, i.e., by SDS-PAGE, Iso-electric focusing
(IEF) and binding to the targets CD22, CD72 and HLA-DR. Sub-clones
will also be screened by PCR on genomic DNA for the presence or
absence of each of the three constructs pCRU-CD22, pCRU-CD72 and
pCRU-HLA-DR. The identity of the PCR products is further confirmed
by DNA sequencing.
[0129] Other methods such as Southern blot and/or FISH can also be
used to determine whether each of the three constructs are present
in the clonal cell line.
[0130] Sub-clones that are proven to be transgenic for each of the
three constructs are brought into culture for an extensive period
to determine whether the presence of the transgenes is stable and
whether expression of the antibody mixture remains the same, not
only in terms of expression levels, but also for the ratio between
the various antibody isoforms that are secreted from the cell.
Therefore, the sub-clone culture is maintained for at least 25
population doubling times, either as an adherent culture or as a
suspension culture. At every four to six population doublings, a
specific production test is performed using the human IgG-specific
ELISA and larger volumes are cultured to obtain the cell pellet and
the supernatant. The cell pellet is used to assess the presence of
the three constructs in the genomic DNA, either via PCR, Southern
blot and/or FISH. The supernatant is used to purify the recombinant
human IgG1 fraction as described supra. Purified human IgG1
obtained at the various population doublings is analyzed as
described, i.e., by SDS-PAGE, Iso-electric focusing (IEF) and
binding to the targets CD22, CD72 and HLA-DR using cell
transfectants expressing these antigens.
Example 3 Screening of Clones Expressing Multiple Human IgGs for
the Most Potent Mixture of Functional Human IgGs
[0131] Functionality of the antibody mixture is analyzed in
cell-based assays to determine whether the human IgG1 mixture
inhibits proliferation and/or induces apoptosis of B-cell lines,
such as, for example, Ramos. Other cell lines can also be used. In
addition, the antibody mixtures are analyzed for their potential to
induce antibody-dependent cellular toxicity and
complement-dependent cytotoxicity of, for example, Ramos cells.
[0132] In each of the following experiments, the functionality of
the antibody mixture recognizing the targets CD22, CD72 and HLA-DR
is analyzed and can be compared to each of the individual IgG1
antibodies and to an equimolar combination of the three individual
IgG1 specificities.
[0133] To assess the ability of the antibody mixtures to inhibit
the proliferation of Ramos cells, these cells are incubated in
96-well plates (0.1-1.0.times.10.sup.5/ml) with several
concentrations (5-20 .mu.g/ml) of the antibody mixtures against
CD22, CD72 and HLA-DR for 24 hours. The proliferation of the cells
is measured by .sup.3H-thymidine incorporation during another 16
hours of culture. Inhibition of growth is determined by plotting
the percentage of .sup.3H-thymidine incorporation compared to
untreated cells (taken as 100% reference value).
[0134] To analyze apoptosis induction of Ramos cells, these cells
are stimulated in 48-well plates (0.2-1.0.times.10.sup.6/ml) with
several concentrations (5-20 .mu.g/ml) of the antibody mixtures
against the targets CD22, CD72 and HLA-DR for 24 or 48 hours. After
the incubation period, the phosphatidyl serine exposure on
apoptotic cells is analyzed (G. Koopman et al., 1994, the entirety
of which is incorporated herein by reference). Therefore, the cells
are harvested, washed twice with PBS and are incubated at RT for 10
minutes with 100 .mu.l FITC-labeled annexin V (Caltag) diluted 1:25
in annexin V-binding buffer (Caltag). Prior to the analysis of the
samples by flow cytometry (FACSCalibur, Becton Dickinson, San Jose,
Calif.), propidium iodide (PI)(Sigma) is added to a final
concentration of 5 .mu.g/ml to distinguish necrotic cells (annexin
V-/PI+) from apoptotic cells (annexin V+/PI-, early apoptotic
cells; annexin V+/PI+, late apoptotic cells).
[0135] In an alternative assay, apoptosis is induced by
cross-linking the antibody mixtures against CD22, CD72 and HLA-DR
on the cell surface of Ramos cells with 25 .mu.g/ml of F(ab)2 of
goat-anti-human (Fe-specific) polyclonal antibodies (Jackson
Immunoresearch Laboratories, West Grove, Pa.) during the incubation
period.
[0136] In another alternative assay, apoptosis is induced by
incubating the Ramos cells with several concentrations (5-20
.mu.g/ml) of the antibody mixtures against CD22, CD72 and HLA-DR
while co-incubating them with the chemosensitizing agents
doxorubicin (Calbiochem) or dexamethasone (UMCU, Utrecht, NL).
[0137] Antibody-Dependent Cellular Cytotoxicity (ADCC) of the
antibody mixtures is analyzed using peripheral blood mononuclear
cells as effector cells in a standard .sup.51Cr release assay (Huls
et al., 1999). To this purpose, 1-3.times.10.sup.6 Ramos cells are
labeled with 100 .mu.Ci (Amersham, Buckinghamshire, UK) for one
hour at 37.degree. C. After three washes with medium, the Ramos
target cells are plated in U bottom 96-well plates at
5.times.10.sup.3 cells/well. Peripheral blood mononuclear cells
that are obtained from healthy donors by Ficoll-Hypaque density
gradients are then added to each well at effector:target ratios
ranging from 80:1 to 10:1 in triplicate. The cells are incubated at
37.degree. C. in the presence of various concentrations of the
antibody mixtures (5-20 .mu.g/ml) in a final volume of 200
.mu.l.
[0138] After four hours of incubation, part of the supernatant is
harvested and .sup.51Cr release is measured. The percentage of
specific lysis is calculated using the following formula: %
specific lysis=([experimental cpm-spontaneous cpm]/[maximal
cpm-spontaneous cpm].times.100%). Maximal .sup.51Cr release is
determined by adding triton X-100 to a final concentration of 1% to
the target cells and spontaneous release is determined after
incubation of the target cells with medium alone.
[0139] Complement-dependent cytotoxicity is determined in a similar
assay. Instead of the effector cells, now 50 .mu.l human serum is
added to the target cells. Subsequently, the assay is performed in
the same manner.
[0140] Alternatively, ADCC and CDC of the antibody mixtures is
determined using a Europium release assay (Patel and Boyd, 1995,
the entirety of which is incorporated herein by reference) or using
an LDH release assay (Shields et al., 2001, the entirety of which
is incorporated herein by reference).
Example 4 Use of Phage Display to Isolate Multiple Phages with an
Identical V.sub.L Sequence Against a Predefined Target (her-2) and
Production in a Recombinant Host Cell of a Mixture of Antibodies
Capable of Binding this Target
[0141] Phages displaying scFv fragments capable of binding multiple
epitopes present on the same protein, for example, the epidermal
growth factor receptor Her-2, can be isolated from a semi-synthetic
phage library (de Kruif et al., 1995a, b). It is possible to
identify several of such phages and select the ones comprising the
same light chain sequence for further use according to the
invention. The semi-synthetic library is formed by mixing seven
sub-libraries that each contains a different light chain (de Kruif
et al., 1995a, b). It is, therefore, particularly practical to use
such a sub-library, containing only one light chain and many heavy
chains, for screening so that multiple antibodies with an identical
V.sub.L sequence are obtained and further used for expressing the
antibody mixtures according to the invention.
[0142] For the selection of phages against Her-2, several fusion
proteins are generated comprising different parts of the
extracellular domain of Her-2 that are fused to the CH2 and CH3
domains of human IgG1. For this purpose, a pCDNA3.1zeo-expression
vector (Invitrogen) has been constructed that contains in its
multiple cloning region an XhoI restriction site in the hinge
region in frame prior to the CH2 and CH3 domains of human IgG1.
Using a Her-2 cDNA clone as a template, PCR fragments are generated
using standard molecular biology techniques known to a person
skilled in the art. These fragments consist of a unique 5'
restriction site, a start codon followed by a eukaryotic leader
sequence that is linked in frame to either the total extracellular
(EC) domain of Her-2 or to a part of the EC domain of Her-2 that is
followed in frame by an XhoI restriction site. These PCR fragments
are subsequently cloned in frame with the CH2-CH3 IgG1 region into
the pCDNA3.1zeo-expression vector. In addition to the fusion
protein containing the total EC domain of Her-2, several smaller
fusion proteins are generated containing non-overlapping fragments
of the Her-2 EC domain. These constructs encoding the Her-2-Ig
fusion proteins are used for transient transfection of 293T cells
using the lipofectamine reagent (Gibco). Five days after
transfection, the supernatants of the 293T cells are harvested and
Her-2-Ig fusion proteins are purified using protein A-affinity
chromatography according to standard procedures.
[0143] Her-2-Ig fusion proteins containing non-overlapping
fragments of the Her-2 EC domain are coated for two hours at
37.degree. C. onto the surface of MAXISORP.TM. (polystyrene based
modified surface with a high affinity for polar groups) plastic
tubes (Nunc) at a saturating concentration (0.5-5 .mu.g/ml). The
tubes are blocked for one hour in 2% fat-free milk powder dissolved
in PBS (MPBS). Simultaneously, 500 .mu.l (approximately 10.sup.13
cfu) of a semi-synthetic phage display library (a sub-library
according to the terminology used above) in which only one
V.kappa.1 light chain is represented (prepared as described by De
Kruif et al. (1995a, b) and referenced therein), is added to two
volumes of 4% MPBS. In addition, human serum is added to a final
concentration of 15% and blocking is allowed to proceed for 30 to
60 minutes. The Her-2-Ig-coated tubes are emptied and the blocked
phage library is added. The tube is sealed and rotated slowly for
one hour, followed by two hours of incubation without rotation. The
tubes are emptied and washed ten times in PBS containing 0.1%
TWEEN.RTM.-20, followed by washing five times in PBS. One ml
glycine-HCL, 0.05 M, pH 2.2 is added, and the tube is rotated
slowly for ten minutes. The eluted phages are added to 500 .mu.l 1
M Tris-HCl pH 7.4. To this mixture, 3.5 ml of exponentially growing
XL-1 blue bacterial culture is added. The tubes are incubated for
30 minutes at 37.degree. C. without shaking. Subsequently, the
bacteria are plated on 2TY agar plates containing ampicillin,
tetracycline and glucose. After overnight incubation of the plates
at 37.degree. C., the colonies are scraped from the plates and used
to prepare an enriched phage library, essentially as described by
De Kruif et al. (1995a). Briefly, scraped bacteria are used to
inoculate 2TY medium containing ampicillin, tetracycline and
glucose and are grown at 37.degree. C. to an OD.sub.600nm of
.about.0.3. Helper phages are added and allowed to infect the
bacteria after which the medium is changed to 2TY containing
ampicillin, tetracycline and kanamycin. Incubation is continued
overnight at 30.degree. C. The next day, the bacteria are removed
from the 2TY medium by centrifugation, after which the phages are
precipitated using polyethylene glycol 6000/NaCl. Finally, the
phages are dissolved in a small volume of PBS-1% BSA,
filter-sterilized and used for a next round of selection. The
selection/re-infection procedure is performed twice. After the
second round of selection, individual E. coli colonies are used to
prepare monoclonal phage antibodies. Essentially, individual
colonies are grown to log phase and infected with helper phages,
after which phage antibody production is allowed to proceed
overnight. Phage antibody containing supernatants are tested in
ELISA for binding activity to Her-2-total EC-Ig coated 96-well
plates.
[0144] Selected phage antibodies that are obtained in the screen
described above are validated by ELISA for specificity. For this
purpose, Her-2-Ig fusion proteins containing non-overlapping
fragments of the Her-2 EC domain are coated to MAXISORP ELISA
plates. After coating, the plates are blocked in 2% MPBS. The
selected phage antibodies are incubated in an equal volume of 4%
MPBS. The plates are emptied, washed once in PBS, after which the
blocked phages are added. Incubation is allowed to proceed for one
hour, the plates are washed in PBS 0.1% TWEEN.RTM.-20 and bound
phages are detected using an anti-M13 antibody conjugated to
peroxidase. The procedure is performed simultaneously using a
control phage antibody directed against thyroglobulin (De Kruif et
al. 1995a, b), which serves as a negative control.
[0145] In another assay, the selected phage antibodies are analyzed
for their ability to bind BT474 human breast cancer cells that
express Her-2. For flow cytometry analysis, phage antibodies are
first blocked in an equal volume of 4% MPBS for 15 minutes at
4.degree. C. prior to the staining of the BT474 cells. The binding
of the phage antibodies to the cells is visualized using a
biotinylated anti-M13 antibody (Santa Cruz Biotechnology) followed
by streptavidin-phycoerythrin (Caltag).
[0146] Alternatively, phage antibodies recognizing multiple
epitopes on Her-2 are selected using a method based upon
competition of phage binding to Her-2 with binding of the
well-characterized murine anti-Her-2 antibodies HER50, HER66 and
HER70 (Spiridon et al., 2002, the entirety of which is incorporated
herein by reference). To this purpose, 2.times.10.sup.6 BT474 cells
are incubated at 4.degree. C. with approximately 10.sup.13 cfu (0.5
ml) of a semi-synthetic phage display library in which only one
V.kappa.1 light chain is represented, prepared as described supra,
and blocked with two volumes of medium containing 10% of FBS. The
mixture is slowly rotated at 4.degree. C. for two hours in a sealed
tube.
[0147] Subsequently, non-bound phages are removed by two washes
with 50 ml of cold medium containing 10% FBS. Hereafter, phages
recognizing multiple epitopes on Her-2 are eluted by resuspending
the BT474 cells in 1 ml of cold medium containing saturating
concentrations (5-20 .mu.g/ml) of the HER50, HER66 and HER70 murine
anti-Her-2 antibodies. The cells are left on ice for 10 minutes,
spun down and the supernatant containing the anti-Her-2 phage
antibodies is used to reinfect XL1-Blue cells as described
supra.
[0148] From the panel of Her-2-specific phage antibodies generated
by the screens described above, three phage antibodies are selected
that recognize three different non-overlapping epitopes on the
Her-2 protein.
[0149] The V.sub.H sequences and the unique V.kappa.1 light chain
sequence of these clones, provisionally designated V.kappa.1HER2-1,
V.kappa.1HER2-2 and V.kappa.1HER2-3, are cloned behind the HAVT20
leader sequences of expression plasmid pCRU-K01 (ECACC deposit
03041601), or a similar expression plasmid, to obtain plasmids
encoding a full-length human IgG1-.kappa. with binding
specificities for Her-2. These plasmids are provisionally
designated as pCRU-V.kappa.1HER2-1, pCRU-V.kappa.1HER2-2 and
pCRU-V.kappa.1HER2-3, respectively.
[0150] Stable PER.C6.RTM. (human retina cells that express
adenovirus E1A and E1B proteins)-derived cell lines are generated,
according to methods known to one of ordinary skill in the art, the
cell lines expressing antibodies encoded by genetic information on
either pCRU-V.kappa.1HER2-1, pCRU-V.kappa.1HER2-2 or
pCRU-V.kappa.1HER2-3 and a cell line expressing antibodies encoded
by all three plasmids. Therefore, PER.C6.RTM. cells are seeded in
DMEM plus 10% FBS in tissue culture dishes (10 cm diameter) or T80
flasks with approximately 2.5.times.10.sup.6 cells per dish and
kept overnight under their normal culture conditions (10% CO.sub.2
concentration and 37.degree. C.). The next day, transfections are
performed in separate dishes at 37.degree. C. using Lipofectamine
(Invitrogen Life Technologies) according to standard protocols
provided by the manufacturer, with either 1-2 .mu.g
pCRU-V.kappa.1HER2-1, 1-2 .mu.g pCRU-V.kappa.1HER2-2, 1-2 .mu.g
pCRU-V.kappa.1HER2-3 or 1 .mu.g of a mixture of
pCRU-V.kappa.1HER2-1, pCRU-V.kappa.1HER2-2 and
pCRU-V.kappa.1HER2-3. As a control for transfection efficiency, a
few dishes are transfected with a LacZ control vector, while a few
dishes are not transfected and serve as negative controls.
[0151] After five hours, cells are washed twice with DMEM and
re-fed with fresh medium without selection. The next day, medium is
replaced with fresh medium containing 500 .mu.g/ml G418. Cells are
refreshed every two or three days with medium containing the same
concentrations of G418. About 20 to 22 days after seeding, a large
number of colonies are visible and from each transfection, at least
300 are picked and grown via 96-well plates and/or 24-well plates
via 6-well plates to T25 flasks. At this stage, cells are frozen
(at least one, but usually four vials per sub-cultured colony) and
production levels of recombinant human IgG antibody are determined
in the supernatant using an ELISA specific for human IgG1. Also, at
this stage, G418 is removed from the culture medium and never
re-applied again. For a representative number of colonies, larger
volumes are cultured to purify the recombinant human IgG1 fraction
from the conditioned supernatant using Protein A-affinity
chromatography according to standard procedures. Purified human
IgG1 from the various clones is analyzed on SDS-PAGE, Iso-electric
focusing (IEF), assayed binding to Her-2-Ig fusion proteins by
ELISA, and analyzed for binding to Her-2 on the surface of BT474
cells by flow cytometry.
[0152] Clones obtained from the co-transfection of
pCRU-V.kappa.1HER2-1, pCRU-V.kappa.1HER2-2 and pCRU-V.kappa.1HER2-3
are screened by PCR on genomic DNA for the presence or absence of
each of the three constructs. The identity of the PCR products is
further confirmed by DNA sequencing.
[0153] Next, it is demonstrated that a clonal cell line accounts
for the production of each of the three binding specificities.
Therefore, a limited number of colonies, which screened positive
for the production of each of the three binding specificities (both
by PCR at the DNA level as well as in the specified binding assays
against Her-2), are subjected to single cell sorting using a
fluorescence-activated cell sorter (FACS) (Becton & Dickinson
FACS VANTAGE SE.TM.). Alternatively, colonies are seeded at 0.3
cells/well to guarantee clonal outgrowth.
[0154] Clonal cell populations, hereafter designated as sub-clones,
are refreshed once a week with fresh medium. Sub-clones are grown
and transferred from 96-well plates via 24- and 6-well plates to
T25 flasks. At this stage, sub-clones are frozen (at least one, but
usually four vials per sub-clone) and production levels of
recombinant human IgG1 antibody are determined in the supernatant
using a human IgG1-specific ELISA. For a representative number of
sub-clones, larger volumes are cultured to purify the recombinant
human IgG1 fraction from the conditioned supernatant using Protein
A-affinity chromatography according to standard procedures.
[0155] Purified human IgG1 from the various sub-clones is
subsequently analyzed as described above for human IgG1 obtained
from the parental clones, i.e., by SDS-PAGE, Iso-electric focusing
(IEF) and binding to Her-2. Sub-clones will also be screened by PCR
on genomic DNA for the presence or absence of each of the three
constructs pCRU-V.kappa.1HER2-1, pCRU-V.kappa.1HER2-2 and
pCRU-V.kappa.1HER2-3. The identity of the PCR products is further
confirmed by DNA sequencing.
[0156] Other methods such as Southern blot and/or FISH can also be
used to determine whether each of the three constructs is present
in the clonal cell line.
[0157] Sub-clones that are proven to be transgenic for each of the
three constructs are brought into culture for an extensive period
to determine whether the presence of the transgenes is stable and
whether expression of the antibody mixture remains the same, not
only in terms of expression levels, but also for the ratio between
the various antibodies that are secreted from the cell. Therefore,
the sub-clone culture is maintained for at least 25 population
doubling times, either as an adherent culture or as a suspension
culture. At every four to six population doublings, a specific
production test is performed using the human IgG-specific ELISA and
larger volumes are cultured to obtain the cell pellet and the
supernatant. The cell pellet is used to assess the presence of the
three constructs in the genomic DNA, either via PCR, Southern blot
and/or FISH. The supernatant is used to purify the recombinant
human IgG1 fraction as described supra. Purified human IgG1
obtained at the various population doublings is analyzed as
described, i.e., by SDS-PAGE, Iso-electric focusing (IEF) and
binding to Her-2 by ELISA and by flow cytometry using BT474
cells.
[0158] Functionality of the antibody mixture of anti-Her-2
antibodies is analyzed in cell-based assays to determine whether
the human IgG1 mixture inhibits proliferation and/or induces
apoptosis of BT474 cells. In addition, the antibody mixtures are
analyzed for their potential to induce antibody-dependent cellular
toxicity and complement-dependent cytotoxicity of BT474 cells.
[0159] In each of the experiments described below, the
functionality of the antibody mixture recognizing Her-2 can be
analyzed and compared to each of the individual IgG1 antibodies and
to an equimolar combination of the three individual monospecific
IgG1 molecules.
[0160] To assess the ability of the antibody mixtures to inhibit
the proliferation of BT474 cells, these cells are allowed to adhere
overnight in 96-well plates (1.5.times.10.sup.5/well) and are
subsequently incubated with several concentrations (5-20 .mu.g/ml)
of the antibody mixtures against Her-2 for 72 hours. The
proliferation of the cells is measured by .sup.3H-thymidine
incorporation during the last six hours of culture. Inhibition of
growth is determined by plotting the percentage of
.sup.3H-thymidine incorporation compared with untreated cells
(taken as 100% reference value).
[0161] To analyze apoptosis induction of BT474 cells, these cells
are allowed to adhere overnight in 48-well plates
(2.5.times.10.sup.5/well in 1 ml) and are subsequently incubated
with several concentrations (5-20 .mu.g/ml) of the antibody
mixtures against Her-2 for four hours. Hereafter, the cells are
harvested by trypsinization, washed twice with PBS and incubated at
RT for ten minutes with 100 .mu.l FITC-labeled annexin V (Caltag)
diluted 1:25 in annexin V-binding buffer (Caltag). Prior to the
analysis of the samples by flow cytometry (FACSCalibur, Becton
Dickinson, San Jose, Calif.) propidium iodide (PI)(Sigma) is added
to a final concentration of 5 .mu.g/ml to distinguish necrotic
cells (annexin V.sup.-/PI.sup.+) from apoptotic cells (annexin
V.sup.+/PI.sup.-, early apoptotic cells; annexin V.sup.+/PI.sup.+,
late apoptotic cells).
[0162] Antibody-Dependent Cellular Cytotoxicity of the antibody
mixtures is analyzed using peripheral blood mononuclear cells as
effector cells and BT474 cells as target cells in a standard
.sup.51Cr release assay as described supra (Huls et al., 1999).
Complement-dependent cytotoxicity is determined in a similar assay.
Instead of the effector cells, now 50 .mu.l human serum is added to
the target cells. Subsequently, the assay is performed as described
supra.
[0163] Alternatively, ADCC and CDC of the antibody mixtures is
determined using a Europium release assay (Patel and Boyd, 1995) or
using an LDH release assay (Shields et al., 2001).
[0164] The functionality of the antibody mixtures against Her-2 is
also tested using in vivo animal models, such as, for instance,
described in Spiridon et al., 2002.
Example 5
Expression of Different Functional Human IgGs in the Milk of
Transgenic Animals
[0165] The V.sub.H and V.sub.H sequences of phages against proteins
present on human B-cells, i.e., CD22 (clone B28), CD72 (clone II-2)
and HLA-DR (clone I-2) (FIG. 7) are cloned into expression plasmid
pBC1 (as provided in the pBC1 Mouse Milk Expression System,
Invitrogen Life Technologies) to obtain mammary gland- and
lactation-specific expression of these human IgG molecules in
transgenic animals, according to the manufacturer's instructions.
These mammary gland-specific expression vectors encoding the
antibody sequences for anti-CD22, anti-CD72 and anti-HLA-DR, are
introduced into the murine germline according to the manufacturer's
instructions. Obtained pups are screened for the presence of each
of the three constructs by PCR on DNA isolated from the tail. Pups,
either male or female, confirmed for being transgenic for each of
the three antibodies, are weaned and matured. Female transgenic
mice are fertilized at the age of 6-8 weeks and milk samples are
obtained at several time points after gestation. Male transgenic
mice are mated with non-transgenic females and female transgenic
offspring (as determined with PCR as described above) is mated and
milked as described above for the female transgenic founders.
Whenever needed, female or male transgenic founders are mated for
another generation to be able to obtain sufficient amounts of
transgenic milk for each founder line. Transgenic milk is analyzed
for the presence of human IgG with a human IgG-specific ELISA,
which does not cross-react with mouse IgG or other mouse milk
components. Human IgG is purified from transgenic mouse milk using
Protein A-affinity chromatography according to standard procedures.
Purified human IgG is analyzed on SDS-PAGE, Iso-electric focusing
and binding on the targets CD22, CD72 and HLA-DR. Functionality of
the antibody mixture is analyzed as described supra.
Example 6 Production of an IgA/IgG Mixture Against a Predefined
Target in a PER.C6.RTM. (Human Retina Cells that Express Adenovirus
E1A and E1B Proteins)-Derived Clone
[0166] The V.sub.H-V.sub.L sequences of the phage UBS-54 directed
against the homotypic adhesion molecule EP-CAM (Huls et al., 1999)
was not only cloned into a vector encoding the constant domains of
a human IgG1 with Kappa light chain (expression vector
pUBS3000Neo), but also into an expression vector encoding the
constant domains of a human IgA1 with Kappa light chain (expression
vector pUBS54-IgA, FIG. 8). Hence, antibodies derived from
pUBS3000Neo and pUBS54-IgA do bind to the same epitope on EPCAM.
The only differences antibodies derived from pUBS3000Neo and
pUBS54-IgA are in the sequences encoding the constant domains of
the heavy chain, resulting in either an IgG1 or IgA1 isotype. The
Kappa light chain sequences of these two vectors are identical.
[0167] Stable PER.C6.RTM. (human retina cells that express
adenovirus E1A and E1B proteins)-derived cell lines expressing
antibodies encoded by genetic information on pUBS3000Neo and
pUBS54-IgA are generated by procedures well known to persons
skilled in the art. Therefore, PER.C6.RTM. cells (human retina
cells that express adenovirus E1A and E1B proteins) are seeded in
DMEM plus 10% FBS in tissue culture dishes (10 cm diameter) or T80
flasks with approximately 2.5.times.10.sup.6 cells per dish and
kept overnight under their normal culture conditions (10% CO.sub.2
concentration and 37.degree. C.). The next day, transfections are
performed in separate dishes at 37.degree. C. using Lipofectamine
(Invitrogen Life Technologies) according to standard protocols
provided by the manufacturer, with either 1-2 .mu.g pUBS3000Neo and
pUBS54-IgA. As a control for transfection efficiency, a few dishes
are transfected with a LacZ control vector, while a few dishes are
not transfected and serve as negative controls.
[0168] After four to five hours, cells are washed twice with DMEM
and given fresh medium without selection. The next day, medium is
replaced with fresh medium containing 500 .mu.g/ml G418. Cells are
refreshed every two or three days with medium containing the same
concentrations of G418. About 20 to 22 days after seeding, a large
number of colonies are visible and from each transfection, at least
300 are picked and grown via 96-well plates and/or 24-well plates
via 6-well plates to T25 flasks. At this stage, cells are frozen
(at least one, but usually four vials per sub-cultured colony) and
production levels of recombinant human IgG and human IgA antibody
are determined in the supernatant using an ELISA specific for human
IgG1 as well as an ELISA specific for human IgA. Also, at this
stage, G418 is removed from the culture medium and never re-applied
again. For a representative number of colonies, larger volumes are
cultured to purify the recombinant human IgG1 and human IgA
fraction from the conditioned supernatant using, for instance, a
combination of Protein L- or LA-affinity chromatography, cation
exchange chromatography, hydrophobic interaction chromatography and
gel filtration. Purified human immunoglobulins from the various
clones are analyzed on SDS-PAGE, Iso-electric focusing (IEF) and
binding to the target EPCAM using cell lines having a high
expression of this molecule. The clones will also be screened by
PCR on genomic DNA for the presence or absence of pUBS3000Neo and
pUBS54-IgA. The identity of the PCR products is further confirmed
by DNA sequencing.
[0169] A limited number of clones, which are screened positive for
the production of both EPCAM IgG1 and EPCAM IgA, are subjected to
single cell sorting using a fluorescence-activated cell sorter
(FACS) (Becton Dickinson FACS VANTAGE SE.TM.). Alternatively,
colonies are seeded at 0.3 cells/well to guarantee clonal
outgrowth. Clonal cell populations, hereafter designated as
sub-clones, are refreshed once a week with fresh medium. Sub-clones
are grown and transferred from 96-well plates via 24- and 6-well
plates to T25 flasks. At this stage, sub-clones are frozen (at
least one, but usually four vials per sub-clone) and production
levels of recombinant human IgG1 and IgA antibody are determined in
the supernatant using a human IgG1-specific ELISA and a human
IgA-specific ELISA. For a representative number of sub-clones,
larger volumes are cultured to purify the recombinant human IgG1
and human IgA1 fraction from the conditioned supernatant using, for
instance, a combination of Protein L- or LA-affinity
chromatography, cation exchange chromatography, hydrophobic
interaction chromatography and gel filtration. Purified human
immunoglobulins from the various clones are analyzed on SDS-PAGE,
Iso-electric focusing (IEF) and binding to the target EPCAM using
cell lines having a high expression of this molecule.
[0170] Sub-clones will also be screened by PCR on genomic DNA for
the presence or absence of pUBS3000Neo and pUBS54-IgA. The identity
of the PCR products is further confirmed by DNA sequencing.
[0171] Other methods such as Southern blot and/or FISH may also be
used to determine whether both constructs are present in the clonal
cell line.
Example 7 Production of a Human IgG1/IgG3 Mixture Against Multiple
Targets in a Clonal PER.C6.RTM. Cell Line (Human Retina Cells that
Express Adenovirus E1A and E1B Proteins)
[0172] Phage clone UBS-54 and Clone K53 (FIG. 3) were obtained as
described in Example 1. The V.sub.H and V.sub.L of clone UBS-54 was
inserted into an expression vector containing the HAVT20 leader
sequence and all the coding sequences for the constant domains of a
human IgG1 with a Kappa light chain by a method essentially as
described (Boel et al., 2000). The resulting plasmid was designated
as pUBS3000Neo (FIG. 4). It will be clear that expression vectors
containing heavy chain constant domains of any desired isotype can
be constructed by routine methods of molecular biology, using the
sequences of these regions that are all available in the art. The
V.sub.H and V.sub.L sequences of Phage clone K53 are cloned into an
expression vector containing the HAVT20 leader sequence and all the
coding sequences for the constant domains of a heavy chain of a
human IgG3 with a Kappa light chain by a method essentially as
described (Boel et al., 2000). This expression vector is designated
as pK53IgG3.
[0173] These plasmids are transiently expressed, either alone or in
combination, in PER.C6.RTM. cells (human retina cells that express
adenovirus E1A and E1B proteins). In brief, each 80 cm.sup.2 flask
is transfected by incubation for four hours with 140 .mu.l
lipofectamine+10 .mu.g DNA (either pUBS3000Neo, pK53IgG3 or 10
.mu.g of both) in serum-free DMEM medium at 37.degree. C. After
four hours, this is replaced with DMEM+10% FBS and the cells are
grown overnight at 37.degree. C. Cells are then washed with PBS and
the medium is replaced with Excell 525 medium (JRH Bioscience). The
cells are allowed to grow at 37.degree. C. for six days, after
which the cell culture supernatant is harvested. Human IgG-specific
ELISA analysis, i.e., measuring all IgG sub-types, is done to
determine the IgG concentration in transfected and non-transfected
PER.C6.RTM. cells (human retina cells that express adenovirus E1A
and E1B proteins). Human IgG from each supernatant is subsequently
purified using Protein A-affinity chromatography (Hightrap Protein
A HP, cat. no. 1-040203) according to standard procedures,
following recommendations of the manufacturer (Amersham
Biosciences). After elution, samples are concentrated in a Microcon
YM30 concentrator (Amicon) and buffer exchanged to 10 mM sodium
phosphate, pH 6.7. Samples are analyzed for binding to the targets
EPCAM and CD46 using cell lines having a high expression of these
molecules such as LS174T cells. Twelve .mu.g of purified IgG,
either transiently expressed UBS-54 IgG1, K53 IgG3 or IgG from the
cells in which both antibodies were co-transfected, is subsequently
analyzed on iso-electric-focusing gels (Serva Pre-cast IEF gels, pH
range 3-10, cat. no. 42866). Samples are loaded on the low pH side
and, after focusing, stained with colloidal blue. The pI values of
the major isoforms for each sample are determined to illustrate
whether there has been expression of UBS-54 IgG1, K53 IgG3 or
bispecific heterodimers, depending on how the cells were
transfected. The identification of heterodimers would indicate that
single cells have translated both the IgG3 heavy chain of K53 and
the IgG1 heavy chain of UBS-54 and assembled these into a
full-length IgG molecule together with the common light chain.
[0174] The absence of bispecific heterodimers indicates that it is
possible to translate both the IgG3 heavy chain of K53 and the IgG1
heavy chain of UBS-54 in single cells, but that these do not
assemble into a full-length IgG molecule together with the common
light chain, i.e., there is preferential binding of IgG1 and IgG3
heavy chains. This could, however, also be explained by the lack of
co-expression of UBS-54 IgG1 and K53 IgG3. Therefore, stable clonal
cell lines expressing both pUBS3000Neo and pK53IgG3 are generated
by procedures as such well known to persons skilled in the art.
PER.C6.RTM. cells (human retina cells that express adenovirus E1A
and E1B proteins) are seeded in DMEM plus 10% FBS in tissue culture
dishes (10 cm diameter) or T80 flasks with approximately
2.5.times.10.sup.6 cells per dish and kept overnight under their
normal culture conditions (10% CO.sub.2 concentration and
37.degree. C.). The next day, transfections are performed in
separate dishes at 37.degree. C. using Lipofectamine (Invitrogen
Life Technologies) according to standard protocols provided by the
manufacturer, with either 1-2 .mu.g pUBS3000Neo, pK53IgG3 or both.
As a control for transfection efficiency, a few dishes are
transfected with a LacZ control vector, while a few dishes will be
not transfected and serve as negative controls.
[0175] After four to five hours, cells are washed twice with DMEM
and given fresh medium without selection. The next day, medium is
replaced with fresh medium containing 500 .mu.g/ml G418. Cells are
refreshed every two or three days with medium containing the same
concentrations of G418. About 20 to 22 days after seeding, a large
number of colonies are visible and from each transfection, at least
300 are picked and grown via 96-well plates and/or 24-well plates
via 6-well plates to T25 flasks. At this stage, cells are frozen
(at least one, but usually four vials per sub-cultured colony) and
production levels of recombinant human IgG antibody are determined
in the supernatant using an ELISA specific for all sub-types of
human IgG. Also, at this stage, G418 is removed from the culture
medium and never re-applied again. For a representative number of
colonies, larger volumes are cultured to purify the recombinant
human IgG from the conditioned supernatant using Protein A-affinity
chromatography (Hightrap Protein A HP, cat. no. 1-040203) according
to standard procedures, following recommendations of the
manufacturer (Amersham Biosciences). Purified human immunoglobulins
from the various clones are analyzed on SDS-PAGE, Iso-electric
focusing (IEF) and binding to the targets EPCAM and CD46 using cell
lines having a high expression of these molecules such as LS174T
cells. The clones are also screened by PCR on genomic DNA for the
presence or absence of pUBS3000Neo and pK53IgG3. The identity of
the PCR products is further confirmed by DNA sequencing.
[0176] A limited number of clones, which are screened positive for
the production of both EPCAM IgG1 and K53 IgG3, are subjected to
single cell sorting using a fluorescence-activated cell sorter
(FACS) (Becton Dickinson FACS VANTAGE SE.TM.) Alternatively,
colonies are seeded at 0.3 cells/well to guarantee clonal
outgrowth. Clonal cell populations, hereafter designated as
sub-clones, are refreshed once a week with fresh medium. Sub-clones
are grown and transferred from 96-well plates via 24- and 6-well
plates to T25 flasks. At this stage, sub-clones are frozen (at
least one, but usually four vials per sub-clone) and production
levels of recombinant human IgG antibody are determined in the
supernatant using a human IgG-specific ELISA. For a representative
number of sub-clones, larger volumes are cultured to purify the
recombinant human IgG fraction from the conditioned supernatant
using Protein A-affinity chromatography (Hightrap Protein A HP,
cat. no. 1-040203) according to standard procedures, following
recommendations of the manufacturer (Amersham Biosciences).
Purified human immunoglobulins from the various clones are analyzed
on SDS-PAGE, Iso-electric focusing (IEF) and binding to the targets
EPCAM and CD46 using cell lines having a high expression of these
molecules, such as, for instance, LS174T cells, or transfectants
expressing these molecules.
[0177] Sub-clones are also screened by PCR on genomic DNA for the
presence or absence of pUBS3000Neo and pK53IgG3. The identity of
the PCR products is further confirmed by DNA sequencing.
[0178] Other methods such as Southern blot and/or FISH may also be
used to determine whether both constructs are present in the clonal
cell line.
[0179] Once the clonal sub-clones are available and confirmed
positive for the expression of both UBS-54 IgG1 and K53 IgG3, the
presence of functional K53 and UBS-54 shows that it is possible to
generate a mixture of functional IgGs with different isotypes with
the common light chain in a single cell. Analysis of the expression
of bispecific antibodies binding both EpCAM and CD46 will reveal to
what extent the different heavy chains having a different sub-type
will pair, which will influence the amount of bispecific antibodies
produced. It is expected that no or very low levels of bispecific
antibodies will be found in this case.
Example 8. Selection of Phage Carrying Single Chain Fv Fragments
Specifically Recognizing Rabies Virus Glyco Protein (RVGP) Using
RVGP-Ig Fusion Protein, and Expression of Mixtures of Antibodies
Against the Rabies Virus
[0180] This example describes the production of mixtures of
antibodies against the rabies virus as another potential target. As
an antigen, the Rabies Virus Glycoprotein (RVGP) is chosen, but
other rabies antigens may be chosen or included as well for this
purpose. Several monoclonal antibodies recognizing RVGP have
already been described in the art, and polyclonal antibodies have
been recognized to be useful in treatment of rabies infections as
well (e.g., EP0402029; EP0445625, the entirety of which are
incorporated herein by reference).
[0181] Antibody fragments are selected using antibody phage display
libraries and MAbstract.TM. technology, essentially as described in
U.S. Pat. No. 6,265,150 and in WO 98/15833, the entirety of which
is incorporated herein by reference. All procedures are performed
at room temperature unless stated otherwise. The sequence of RVGP
is available to one of ordinary skill in the art for cloning
purposes (e.g., Yelverton et al., 1983, the entirety of which is
incorporated herein by reference). An RVGP-Ig fusion protein
consisting of whole RVGP fused genetically to the CH2 and CH3
domains of human IgG1 is produced using vector pcDNA3.1 Zeo-CH2-CH3
expressed in PER.C6.RTM. (human retina cells that express
adenovirus E1A and E1B proteins) and coated for two hours at
37.degree. C. onto the surface of MAXISORP.TM. (polystyrene based
modified surface with a high affinity for polar groups) plastic
tubes (Nunc) at a concentration of 1.25 .mu.g/ml. The tubes are
blocked for one hour in 2% fat-free milk powder dissolved in PBS
(MPBS). Simultaneously, 500 .mu.l (approximately 10.sup.13 cfu) of
a phage display library expressing single chain Fv fragments
(scFvs) essentially prepared as described by De Kruif et al.
(1995a, b) and references therein, is added to two volumes of 4%
MPBS. In this experiment, selections are performed using fractions
of the original library constructed using only one single variable
light chain gene species (e.g., a "V.kappa.1"-library). In
addition, human serum is added to a final concentration of 15% and
blocking is allowed to proceed for 30 to 60 minutes. The
RVGP-Ig-coated tubes are emptied and the blocked phage library is
added. The tube is sealed and rotated slowly for one hour, followed
by two hours of incubation without rotation. The tubes are emptied
and washed ten times in PBS containing 0.1% TWEEN.RTM.-20, followed
by washing five times in PBS. One ml glycine-HCL, 0.05 M, pH 2.2 is
added, and the tube is rotated slowly for ten minutes. The eluted
phages are added to 500 .mu.l 1 M Tris-HCl pH 7.4. To this mixture,
3.5 ml of exponentially growing XL-1 blue bacterial culture is
added. The tubes are incubated for 30 minutes at 37.degree. C.
without shaking. Then, the bacteria are plated on 2TY agar plates
containing ampicillin, tetracycline and glucose. After overnight
incubation of the plates at 37.degree. C., the colonies are scraped
from the plates and used to prepare an enriched phage library,
essentially as described by De Kruif et al. (1995a, b). Briefly,
scraped bacteria are used to inoculate 2TY medium containing
ampicillin, tetracycline and glucose and grown at a temperature of
37.degree. C. to an OD.sub.600nm of .about.0.3. Helper phages are
added and allowed to infect the bacteria, after which the medium is
changed to 2TY containing ampicillin, tetracycline and kanamycin.
Incubation is continued overnight at 30.degree. C. The next day,
the bacteria are removed from the 2TY medium by centrifugation,
after which the phages are precipitated using polyethylene glycol
6000/NaCl. Finally, the phages are dissolved in a small volume of
PBS-1% BSA, filter-sterilized and used for a next round of
selection. The selection/re-infection procedure is performed
twice.
[0182] After the second round of selection, individual E. coli
colonies are used to prepare monoclonal phage antibodies.
Essentially, individual colonies are grown to log-phase and
infected with helper phages, after which phage antibody production
is allowed to proceed overnight. Phage antibody-containing
supernatants are tested in ELISA for binding activity to human
RVGP-Ig coated 96-well plates.
[0183] Selected phage antibodies that are obtained in the screen
described above are validated in ELISA for specificity. For this
purpose, human RVGP-Ig is coated to Maxisorp ELISA plates. After
coating, the plates are blocked in 2% MPBS. The selected phage
antibodies are incubated in an equal volume of 4% MPBS. The plates
are emptied, washed once in PBS, after which the blocked phages are
added. Incubation is allowed to proceed for one hour, the plates
are washed in PBS 0.1% TWEEN.RTM.-20 and bound phages are detected
using an anti-M13 antibody conjugated to peroxidase. As a control,
the procedure is performed simultaneously using a control phage
antibody directed against thyroglobulin (De Kruif et al. 1995a, b),
which serves as a negative control.
[0184] The phage antibodies that bind to human RVGP-Ig are
subsequently tested for binding to human serum IgG to exclude the
possibility that they recognized the Fc part of the fusion
protein.
[0185] In another assay, the phage antibodies are analyzed for
their ability to bind PER.C6.RTM. cells (human retina cells that
express adenovirus E1A and E1B proteins) that express RVGP. To this
purpose, PER.C6C.RTM. cells (human retina cells that express
adenovirus E1A and E1B proteins) are transfected with a plasmid
carrying a cDNA sequence encoding RVGP or with the empty vector and
stable transfectants are selected using standard techniques known
to a person skilled in the art (e.g., J. E. Coligan et al. (2001),
Current Protocols In Protein Science, volume I, John Wiley &
Sons, Inc. New York, the entirety of which is incorporated herein
by reference). For flow cytometry analysis, phage antibodies are
first blocked in an equal volume of 4% MPBS for 15 minutes at
4.degree. C. prior to the staining of the RVGP- and
control-transfected PER.C6.RTM. cells (human retina cells that
express adenovirus E1A and E1B proteins). The blocked phages are
added to a mixture of unlabeled control-transfected PER.C6.RTM.
cells (human retina cells that express adenovirus E1A and E1B
proteins) and RGVP-transfected PER.C6.RTM. cells that have been
labeled green using a lipophylic dye (PKH67, Sigma). The binding of
the phage antibodies to the cells is visualized using a
biotinylated anti-M13 antibody (Santa Cruz Biotechnology), followed
by streptavidin-phycoerythrin (Caltag). Anti RVGP scFv selectively
stains the PER.C6.RTM. RVGP transfectant while they do not bind the
control transfectant.
[0186] An alternative way of screening for phages carrying single
chain Fv fragments specifically recognizing human RVGP, is by use
of RVGP-transfected PER.C6.RTM. cells (human retina cells that
express adenovirus E1A and E1B proteins).
[0187] PER.C6.RTM. cells (human retina cells that express
adenovirus E1A and E1B proteins) expressing membrane-bound RVGP are
produced as described supra. Phage selection experiments are
performed as described supra, using these cells as target. A
fraction of the phage library comprised of scFv phage particles
using only one single scFv species (500 .mu.l, approximately
10.sup.13 cfu) is blocked with 2 ml RPMI/10% FCS/1% NHS for 15
minutes at RT. Untransfected PER.C6.RTM. cells (human retina cells
that express adenovirus E1A and E1B proteins)
(.about.10.times.10.sup.6 cells) are added to the PER.C6.RTM.-RVGP
cells (.about.1.0.times.10.sup.6 cells). This mixture is added to
the blocked light chain restricted phage library and incubated for
2.5 hours while slowly rotating at 4.degree. C. Subsequently, the
cells are washed twice and were resuspended in 500 .mu.l RPMI/10%
FCS and incubated with a murine anti-RVGP antibody (Becton
Dickinson) followed by a phycoerythrin (PE)-conjugated
anti-mouse-IgG antibody (Caltag) for 15 minutes on ice. The cells
are washed once and transferred to a 4 ml tube. Cell sorting is
performed on a FACSvantage fluorescence-activated cell sorter
(Becton Dickinson) and RVGP (PE positive) cells are sorted. The
sorted cells are spun down, the supernatant is saved and the bound
phages are eluted from the cells by resuspending the cells in 500
.mu.l 50 mM Glycin pH2.2 followed by incubation for five minutes at
room temperature. The mixture is neutralized with 250 .mu.l 1 M
Tris-HCl pH 7.4 and added to the rescued supernatant. Collectively,
these phages are used to prepare an enriched phage library as
described above. The selection/re-infection procedure is performed
twice. After the second round of selection, monoclonal phage
antibodies are prepared and tested for binding to RVGP-PER.C6.RTM.
cells and untransfected PER.C6.RTM. cells (human retina cells that
express adenovirus E1A and E1B proteins) as described supra. Phages
that are positive on RVGP-transfected cells are subsequently tested
for binding to the RVGP-IgG fusion protein in ELISA as described
supra.
[0188] The selected scFv fragments are cloned in a human IgG1
format, according to methods known in the art (e.g., Boel et al.,
2000). To this purpose, the V.sub.L fragment shared by the selected
scFv is PCR amplified using oligos that add appropriate restriction
sites. A similar procedure is used for the V.sub.H genes. Thus,
modified genes are cloned in expression pCRU-K01 (ECACC deposit
03041601), which results in expression vectors encoding a complete
huIgG1 heavy chain and a complete human light chain gene having the
same specificity as the original phage clone. By this method, three
different heavy chains are cloned into separate expression vectors,
while only one of the vectors needs to comprise the common light
chain sequence. These expression vectors are provisionally
designated pCRU-RVGP-1, pCU-RVGP-2, and pCRU-RVGP-3. Alternatively,
these three vectors may lack DNA encoding the V.sub.L region, which
can then be encoded in a fourth, separate expression vector not
encoding a heavy chain. It is also possible to have V.sub.L
sequences present in all three or two of the three vectors
comprising the different V.sub.H sequences.
[0189] Stable PER.C6.RTM. (human retina cells that express
adenovirus E1A and E1B proteins)-derived cell lines are generated,
according to methods known to one of ordinary skill in the art
(see, e.g., WO 00/63403), the cell lines expressing antibodies
encoded by genetic information on either pCRU-RVGP-1, pCRU-RVGP-2
or pCRU-RVGP-3 and a cell line expressing antibodies encoded by all
three plasmids. Therefore, PER.C6.RTM. cells are seeded in DMEM
plus 10% FBS in tissue culture dishes (10 cm diameter) or T80
flasks with approximately 2.5.times.10.sup.6 cells per dish and
kept overnight under their normal culture conditions (10% CO.sub.2
concentration and 37.degree. C.). The next day, transfections are
performed in separate dishes at 37.degree. C. using Lipofectamine
(Invitrogen Life Technologies) according to standard protocols
provided by the manufacturer, with either 1-2 .mu.g pCRU-RVGP-1,
1-2 .mu.g pCRU-RVGP-2, 1-2 .mu.g pCRU-RVGP-3 or 1 .mu.g of a
mixture of pCRU-RVGP-1, pCRU-RVGP-2 and pCRU-RVGP-3. As a control
for transfection efficiency, a few dishes are transfected with a
LacZ control vector, while a few dishes will not be transfected and
serve as negative controls.
[0190] After four to five hours, cells are washed twice with DMEM
and given fresh medium without selection. The next day, the medium
is replaced with fresh medium containing 500 .mu.g/ml G418. Cells
are refreshed every two or three days with medium containing the
same concentrations of G418. About 20 to 22 days after seeding, a
large number of colonies are visible and from each transfection, at
least 300 are picked and grown via 96-well plates and/or 24-well
plates via 6-well plates to T25 flasks. At this stage, cells are
frozen (at least one, but usually four vials per sub-cultured
colony) and production levels of recombinant human IgG antibody are
determined in the supernatant using an ELISA specific for human
IgG1 (described in WO 00/63403). Also, at this stage, G418 is
removed from the culture medium and never re-applied again. For a
representative number of colonies, larger volumes will be cultured
to purify the recombinant human IgG1 fraction from the conditioned
supernatant using Protein A-affinity chromatography according to
standard procedures. Purified human IgG1 from the various clones is
analyzed on SDS-PAGE, Iso-electric focusing (IEF) and binding to
the target RVGP using an RVGP PER.C6.RTM.-transfectant described
above.
[0191] Colonies obtained from the co-transfection with pCRU-RVGP-1,
pCRU-RVGP-2 and pCRU-RVGP-3 are screened by PCR on genomic DNA for
the presence or absence of each of the three constructs. The
identity of the PCR products is further confirmed by DNA
sequencing.
[0192] A limited number of colonies, which screened positive for
the production of each of the three binding specificities (both by
PCR at the DNA level as well as in the specified binding assays
against RVGP), are subjected to single cell sorting using a
fluorescence-activated cell sorter (FACS) (Becton & Dickinson
FACS VANTAGE SE.TM.).
[0193] Alternatively, colonies are seeded at 0.3 cells/well to
guarantee clonal outgrowth. Clonal cell populations, hereafter
designated as sub-clones, are refreshed once a week with fresh
medium. Sub-clones are grown and transferred from 96-well plates
via 24- and 6-well plates to T25 flasks. At this stage, sub-clones
are frozen (at least one, but usually four vials per sub-clone) and
production levels of recombinant human IgG1 antibody are determined
in the supernatant using a human IgG1-specific ELISA. For a
representative number of sub-clones, larger volumes are cultured to
purify the recombinant human IgG1 fraction from the conditioned
supernatant using Protein A-affinity chromatography according to
standard procedures.
[0194] Purified human IgG1 from the various sub-clones is
subsequently analyzed as described above for human IgG1 obtained
from the parental clones, i.e., by SDS-PAGE, Iso-electric focusing
(IEF) and binding to the target RVGP.
[0195] Sub-clones are also screened by PCR on genomic DNA for the
presence or absence of each of the three constructs pCRU-RVGP-1,
pCRU-RVGP-2 and pCRU-RVGP-3. The identity of the PCR products is
further confirmed by DNA sequencing.
[0196] Other methods such as Southern blot and/or FISH can also be
used to determine whether each of the three constructs are present
in the clonal cell line.
[0197] Sub-clones that are proven to be transgenic for each of the
three constructs are brought into culture for an extensive period
to determine whether the presence of the transgenes is stable and
whether expression of the antibody mixture remains the same, not
only in terms of expression levels, but also for the ratio between
the various antibody isoforms that are secreted from the cell.
Therefore, the sub-clone culture is maintained for at least 25
population doubling times, either as an adherent culture or as a
suspension culture. At every four to six population doublings, a
specific production test is performed using the human IgG-specific
ELISA and larger volumes are cultured to obtain the cell pellet and
the supernatant. The cell pellet is used to assess the presence of
the three constructs in the genomic DNA, either via PCR, Southern
blot and/or FISH. The supernatant is used to purify the recombinant
human IgG1 fraction as described supra. Purified human IgG1
obtained at the various population doublings is analyzed as
described, i.e., by SDS-PAGE, Iso-electric focusing (IEF) and
binding to the target RVGP.
[0198] The efficacy of the antibody mixtures against rabies is
tested in in vitro cell culture assays where the decrease in spread
of rabies virus is measured, as well as in in vivo animal models
infected by rabies. Such models are known to one of ordinary skill
in the art and are, e.g., described in EP0402029.
Example 9. Production of a Mixture of Antibodies with a Common
Light Chain and Three Different Heavy Chain-Variable Regions in a
Single Cell
[0199] A method for producing a mixture of antibodies according to
the invention using expression in a recombinant host cell of a
single light chain and three different heavy chains capable of
pairing to the single light chain to form functional antibodies, is
exemplified herein and is schematically shown in FIG. 6.
[0200] Human IgGs UBS54 and K53 against the EP-CAM homotypic
adhesion molecule (Huls et al., 1999) and the membrane cofactor
protein CD46 (WO 02/18948), respectively, are described in Example
1. Another clone that was identified to bind to cofactor protein
CD46 was clone 02-237 (sequence of V.sub.H provided in FIG. 12, SEQ
ID NO:10). DNA sequencing of this clone revealed that it contained
the same light chain as UBS54 and K53 but a unique heavy
chain-variable sequence (see alignment in FIG. 3). As a result, the
CDR3 of the heavy chain of 02-237 differs at four positions from
that of K53 (see alignment in FIG. 13). The heavy and light
chain-variable sequences of phage 02-237 were cloned into the
expression plasmid pCRU-K01 (pCRU-K01 is deposited at the European
Collection of Cell Cultures (ECACC) under number 03041601), which
contains the heavy and light chain constant domains for an IgG1
antibody.
[0201] The resulting plasmid was designated pgG102-237. Due to the
cloning strategy followed, the resulting N-terminus of the light
chain of 02-237 as encoded by pgG102-237 differed slightly from the
N-terminus of UBS54 and K53 as present by pUBS3000Neo, pCD46_3000
(Neo), respectively (FIG. 3). Plasmid pgG102-237 was transiently
produced in human 293(T) cells or stably in PER.C6.RTM. cells
(human retina cells that express adenovirus E1A and E1B proteins).
It appeared that purified 02-237 IgG had a much higher affinity for
purified CD46 (FIG. 14) than K53 IgG, i.e., the affinity had
increased from 9.1.times.10.sup.-7 M to 2.2.times.10.sup.-8M for
K53 and 02-237, respectively. Also, 02-237 bound much better to
CD46 on human colon carcinoma LS174T cells than K53 (FIG. 15).
[0202] Stable PER.C6.RTM. (human retina cells that express
adenovirus E1A and E1B proteins)-derived cell lines expressing a
combination of the plasmids pUBS3000Neo, pCD46_3000 (Neo) and
pgG102-237 encoding human IgG 02-237 were generated according to
methods known as such to one of ordinary skill in the art (see,
e.g., WO 00/63403). Therefore, PER.C6.RTM. cells (human retina
cells that express adenovirus E1A and E1B proteins) were seeded in
DMEM plus 10% FBS in tissue culture dishes (10 cm diameter) with
approximately 2.5.times.10.sup.6 cells per dish and kept overnight
under their normal culture conditions (10% CO.sub.2 concentration
and 37.degree. C.). The next day, transfections were performed in
separate dishes at 37.degree. C. using Lipofectamine (Invitrogen
Life Technologies) according to standard protocols provided by the
manufacturer, with 2 .mu.g of an equimolar mixture of pUBS3000Neo,
pCD46_3000(Neo) and pgG102-237. As negative control for selection,
a few dishes were not transfected.
[0203] After four to five hours, cells were washed twice with DMEM
and given fresh medium without selection. The next day, medium was
replaced with fresh medium containing 500 .mu.g/ml G418. Cells were
refreshed every two or three days with medium containing the same
concentrations of G418. About 20 to 22 days after seeding, a large
number of colonies were visible and about 300 were picked and grown
via 96-well plates and/or 24-well plates via 6-well plates to T25
flasks. During sub-culturing, production levels of recombinant
human IgG antibody were determined in the supernatant using an
ELISA specific for human IgG1 (described in WO 00/63403). About 25%
of all colonies appeared to be positive in this highly specific
assay. The production levels measured at this stage were comparable
to the levels when a single IgG is expressed in PER.C6.RTM. cells
(human retina cells that express adenovirus E1A and E1B proteins)
(expression of a single IgG described in Jones et al., 2003). It is
important to stress that these high expression levels were obtained
without any methods for amplification of the transgene and that
they occur at a low copy number of the transgene.
[0204] The 30 best producing colonies were frozen down in vials and
the 19 highest producing clones were selected for purification of
the IgG (Table 1). They were sub-cultured in T80 flasks and human
IgG from each clone was subsequently purified using Protein
A-affinity chromatography. Therefore, 15 to 25 ml of conditioned
medium was loaded on a 5 ml Protein A FF Sepharose column (Amersham
Biosciences). The column was washed with 4 mM phosphate buffered
saline, pH 7.4 (PBS) before elution with 0.1 M citrate pH 3.0. The
eluted fraction was subsequently desalted on a Sephadex G25 Fine
HIPREP.RTM. Desalting column (Amersham Biotech) to PBS. The
concentration of the purified IgG fraction was determined by
absorbance measurement at 280 nm using a coefficient of 1.4 for a
0.1% (w/v) solution (Table 1).
[0205] The purified IgG samples were analyzed on non-reduced and
reduced SDS-PAGE and IEF. Non-reduced SDS-PAGE (FIG. 16A) showed
that all IgG samples migrated comparable to the control K53 or
02-237 as an assembled, intact IgG molecule of approximately 150
kDa. On reduced SDS-PAGE (FIG. 16B), the IgG samples migrated as
heavy and light chains of about 50 and 25 kDa, respectively,
comparable to the heavy and light chain of the control K53 or
02-237.
[0206] On IEF, the purified IgG fractions were first compared to a
mixture of equal amounts of K53, UBS54 and 02-237 (FIG. 17).
Clearly, some of the samples contained isoforms with a unique pI
profile when compared to the mixture containing purified K53, UBS54
and 02-237. Some major unique isoforms have a pI in between the pI
of K53 and 02-237 on one hand and UBS54 on the other hand. This is
also anticipated on the basis of the theoretic pI when calculated
with the ProtParam tool provided on the Expasy homepage (expasy.ch;
Appel et al., 1994). K53, 02-237 and UBS54 have a theoretic pI of
8.24, 8.36 and 7.65, respectively, whereas an isoform representing
a heterodimer of one UBS54 heavy chain and one K53 heavy chain, has
a theoretical pI of 8.01. Assembly of such a heterodimer can only
occur when a single cell translates both the heavy chain of K53 and
the heavy chain of UBS54 and assembles these into a full-length IgG
molecule together with the common light chain. Hence, these results
suggest that certain clones at least express two functional
antibodies. To confirm the unique identity of some of the isoforms,
samples of the most interesting clones were run in parallel with
K53, UBS54 and 02-237, either alone or in a mixture (FIG. 18). This
furthermore showed that some clones expressed at least two
antibodies (241, 282, 361). Moreover, it provided evidence that
some clones express all three functional antibodies (280 and
402).
[0207] To confirm that the clones expressed IgG mixtures comprising
all three heavy chains, peptide mapping (Garnick, 1992; Gelpi,
1995, the entirety of which are incorporated herein by reference)
was used to analyze the polyclonal IgG fraction. We previously
employed peptide mapping to recover 99% of the protein sequence of
K53.
[0208] Based on the protein sequence provided in FIG. 12, the mass
of the theoretical tryptic peptides of K53, UBS54 and 02-237 was
calculated (Table II and III). A few unique peptides for each IgG
could be identified, for instance, the CDR3 peptides for K53,
02-237 and UBS54 with a Mw of 2116.05, 2057.99 and 2307.15 Da,
respectively. Next, a tryptic digest of Poly1-280 was prepared and
this was analyzed using LC-MS (FIG. 19).
[0209] Peptides with Mw of 2116, 2057 and 2308 Da, representing the
unique CDR3 peptides of K53, 02-237 and UBS54, respectively, were
detected. The precise amino acid sequence of these peptides (as
listed in Table III) was confirmed by MS-MS analysis (Tables IV, V
and VI). The presence of the two unique N-terminal light chain
peptides with Mw of 2580 and 2554 Da, respectively, was also
confirmed. The peptide mapping data unequivocally showed that a
mixture of antibodies comprising a common light chain and three
different heavy chains was expressed by PER.C6.RTM. (human retina
cells that express adenovirus E1A and E1B proteins) clone
Poly1-280. Also, clones 055, 241 and 402 were screened by peptide
mapping. Clones 241 and 402 were confirmed positive for all three
heavy chain sequences, whereas clone 055 only showed expression of
the heavy chains of K53 and 02-237, and not of UBS54. This confirms
the IEF screening (FIG. 18) where no UBS54-related band was seen in
sample 055.
[0210] Poly1-280 was analyzed by BIACORE.TM. (surface plasmon
resonance) for binding to CD46 (FIG. 20). The affinity of poly1-280
for CD46 was 2.1.times.10.sup.-8 M, which shows that the IgG
mixture contains CD46-binding molecules having the same affinity as
02-237 IgG alone.
[0211] Taken together, this experiment shows that it is possible to
express a mixture of functional IgG molecules comprising three
unique heavy chains in a single cell and that next to the
homodimers, heterodimers consisting of two binding specificities
are also formed. Furthermore, the frequency of clones expressing
three different heavy chains suggests that it will also be possible
to obtain clones expressing at least 4, 5, or more, heavy chains,
using the same procedure. In the case where it would be difficult
to obtain clones expressing higher numbers of heavy chains, a clone
expressing at least three heavy chains according to the invention
can be used to introduce more heavy chains in a separate round of
transfection, for instance by using a different selection
marker.
[0212] Next, it was demonstrated that a single cell is able to
produce a mixture of more than two functional human IgGs.
Therefore, clones 241, 280 and 402, which were screened positive
for the production of each of the three IgGs, both by IEF and MS,
were subjected to limiting dilution, i.e., seeded at 0.3 cells/well
in 96-well plates to guarantee clonal outgrowth.
[0213] Clonal cell populations, hereafter designated as sub-clones,
were refreshed once a week with fresh medium. Sub-clones were grown
and transferred from 96-well plates via 24- and 6-well plates, T25,
T80 and T175 flasks. At the T80 stage, sub-clones were frozen.
Production levels of recombinant human IgG1 antibody were
determined in the supernatant using a human IgG1-specific ELISA.
For each parental clone, three sub-clones were chosen and cultured
in a few T175 flasks to obtain sufficient conditioned medium for
purification using Protein A-affinity chromatography as described
above.
[0214] Purified human IgG1 from the sub-clones was subsequently
analyzed as described above for human IgG1 obtained from the
parental clone by iso-electric focusing (IEF). The result is shown
in FIG. 21. Sub-clones from clone poly 1-241 each have the same
pattern, but differ from the parental clone in that they appear to
miss certain bands.
[0215] Sub-clones from clone poly 1-280 all appear to differ from
each other and from the parental clone. Patterns obtained by IEF
for sub-clones from parental clone poly 1-402 are identical for all
three sub-clones and the parent clone.
[0216] From these data, it can be concluded that clone 402 is
stably producing a mixture of antibodies. This demonstrates that it
is feasible to produce a mixture of antibodies according to the
invention from a single cell clone. The clones have undergone about
25 population doublings (cell divisions) from the transfection
procedure up to the first analysis (shown in FIG. 18) under
selection pressure and, from that point on, have undergone about 30
population doublings during the sub-cloning procedure in the
absence of selection pressure before the material analyzed in FIG.
21 was harvested. Therefore, the production of a mixture of
antibodies from a clone from a single cell can be stable over at
least 30 generations.
[0217] Purified IgG1 from the parental 241, 280 and 402 clones, and
sub-clones, were also analyzed for binding reactivity towards the
CD46 and EpCAM antigens. To this end, cDNA of EpCAM, CD46, and
control antigen CD38 were cloned into expression vectors pcDNA
(Invitrogen). These vectors were transfected into CHO (dhfr-) cells
using Fugene (Roche) according to the protocol supplied by the
manufacturer. Cells were cultured in Iscove's medium containing 10%
FBS and HT supplement (Gibco). After culturing for two days, cells
were harvested by trypsinization and suspended in PBS-1% BSA (PBSB)
for use in FACS analysis.
[0218] Purified IgG1 of the clones producing the mixtures of
antibodies and control IgG1 samples of anti-GBSIII, an anti-CD72
antibody (02-004), as well as antibodies from anti-EpCAM clone
UBS54 and anti-CD46 clones K53 and 02-237, were diluted in PBSB to
a concentration of 20 .mu.g IgG1/ml. Twenty .mu.l of each was added
to 200,000 transfected cells and incubated on ice for one hour.
Thereafter, cells were washed once in ice-cold PBSB. Bound IgG was
then detected using incubation with goat-anti-human IgG-biotin
followed by streptavidin-PE. After a final washing step, cells were
suspended in PBSB containing 1 .mu.g/ml propidium iodide. The
samples were analyzed on a FACS (FACSvantage, Becton Dickinson).
Live cells were gated and Mean Fluorescent Intensities (MFI) were
calculated from the FACS plots. The results are represented in FIG.
22. As expected, UBS54 bound selectively to EpCAM-transfected cells
and 02-237 and K53 bound selectively to CD46 transfectants, while
unrelated antibodies did not bind to these transfectants.
[0219] The results demonstrate that binding activities towards both
EpCAM and CD46 were present in the purified IgG1 preps of most
clones expressing a mixture of antibodies according to the
invention, demonstrating that a mixture of functional antibodies
was produced by sub-clones that have undergone more than 30 cell
divisions and that result from a single cell. In sub-clone 280-015,
binding patterns towards CD46 and EpCAM were similar as in the
parent clone poly 1-280, in contrast to the other clones.
[0220] It should be stated that the quantitative aspect of this
assay is not completely clear. Routine screening, for example, by a
functional test, can be used to find a clone with the desired
expression profile. Quantitative aspects may also be included in
such screens. Such screening allows for the identification of
desired clones, which express the mixture of antibodies with a
given functionality in a quantitatively stable manner.
[0221] All references, including publications, patents, and patent
applications, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
TABLE-US-00001 TABLE I Overview of the clones used for purification
of IgG. Screening Purification Clone ELISA Conc. in feed Purified
Poly1- (.mu.g/ml) (.mu.g/ml) (mg) 209 6.1 98 1.37 233 10.0 53 0.75
234 8.0 51 0.71 241 6.6 91 1.42 250 12.5 117 2.10 280 6.3 36 0.80
282 8.5 67 1.48 289 8.2 33 0.64 304 7.2 161 3.91 320 6.3 43 0.83
322 15.2 168 3.27 340 6.0 109 2.64 361 10.4 71 1.73 379 9.5 78 1.75
402 39.9 135 3.14 022 16.2 83 1.69 040 7.8 67 1.43 048 6.5 43 0.94
055 11 55 1.04
TABLE-US-00002 TABLE II Tryptic peptides of the variable domains of
the light chain of K53/UBS54 and 02-237. Monoiso- Monoiso- First
Last topic M.sub.W (Da) topic M.sub.W (Da) Peptide AA.sup.1) AA
K53/UBS54 02-237 L1 1 24 2580.31.sup.(2) 2554.28.sup.(2) L2 25 59
4039.02 4039.02 L3 60 66 700.35 700.35 L4 67 79 1302.61 1302.61 L5
80 82 374.23 374.23 L6 83 107 2810.29.sup.(2) 2810.29.sup.(2) L7
108 111 487.30 487.30 L8 112 112 174.11 174.11 .sup.1)AA, amino
acid .sup.(2)One Cysteine residue alkylated
TABLE-US-00003 TABLE III Tryptic peptides of variable domains of
heavy chains of K53, 02-237 and UBS54. K53 02-237 UBS54 A B C D A B
C D A B C D H1 1 12 1267.68 H1 1 12 1267.68 H1 1 12 1267.68 H2 13
19 685.41 H2 13 19 685.41 H3 20 23 492.24 H3 20 23 492.24 H3 20 23
492.24 H4 24 38 1693.81 H4 24 38 1693.81 H5 39 63 2783.28 H5 39 63
2783.28 H6 64 67 472.28 H6 64 67 472.28 H7 68 84 1906.87 H7 68 84
1906.87 H8 85 87 374.23 H8 85 87 374.23 -- -- -- -- H9 88 98
1319.55 H9 88 98 1319.55 H10 H11 -- -- -- -- Key: A: peptide B:
first amino acid C: last amino acid D: monoisotopic M.sub.W (Da)
Remarks: 1) for H1, amino acid residue 1 is a pyroglutamic acid 2)
peptides H3 and H9 from K53 and 02-237, and peptides H3 and H8 of
UBS54 contain one alkylated cysteine residue 3) Unique peptides
that can be used to confirm the presence of the respective IgGs are
indicated in bold italics
TABLE-US-00004 TABLE IV MS/MS-data of CDR3 peptide (H11) of K53,
obtained by collision induced dissociation of doubly charged m/z
1059.06. Ion m/z Ion m/z Y''.sub.1 147.12 B.sub.1 n.d. Y''.sub.2
248.18 B.sub.2 157.10 Y''.sub.3 335.21 .sup.(1) B.sub.3 304.18
Y''.sub.4 406.25 B.sub.4 419.22 Y''.sub.5 507.30 B.sub.5 582.31
Y''.sub.6 594.33 B.sub.6 768.38 Y''.sub.7 693.40 B.sub.7 825.39
Y''.sub.8 794.46 B.sub.8 953.43 Y''.sub.9 893.54 B.sub.9 n.d.
Y''.sub.10 1006.63 B.sub.10 n.d. Y''.sub.11 1107.67 B.sub.11
1224.65 Y''.sub.12 1164.68 B.sub.12 1323.68 Y''.sub.13 1292.81
B.sub.13 1424.79 Y''.sub.14 1349.77 B.sub.14 1523.86 Y''.sub.15
1535.85 B.sub.15 n.d. Y''.sub.16 1698.95 B.sub.16 n.d. Y''.sub.17
1813.95 B.sub.17 1782.96 Y''.sub.18 1960.97 B.sub.18 n.d.
Y''.sub.19 n.d. .sup.(2) B.sub.19 n.d. .sup.(1) Underlined
m/z-values are main peaks in the MS/MS-spectrum. .sup.(2) n.d. is
not detected.
TABLE-US-00005 TABLE V MS/MS-data of CDR3 peptide (H11) of 02-237,
obtained by collision induced dissociation of doubly charged m/z
1030.02. Ion m/z Ion m/z Y''.sub.1 147.12 B.sub.1 n.d. Y''.sub.2
248.18 B.sub.2 189.09 Y''.sub.3 335.20 B.sub.3 n.d. Y''.sub.4
406.24 B.sub.4 451.22 Y''.sub.5 493.30 B.sub.5 n.d. Y''.sub.6
580.32 B.sub.6 n.d. Y''.sub.7 679.40 B.sub.7 n.d. Y''.sub.8 780.44
B.sub.8 n.d. Y''.sub.9 879.53 B.sub.9 n.d. Y''.sub.10 992.60
B.sub.10 n.d. Y''.sub.11 1093.65 B.sub.11 n.d. Y''.sub.12 1150.67
B.sub.12 n.d. Y''.sub.13 1278.80 B.sub.13 n.d. Y''.sub.14 1335.80
B.sub.14 n.d. Y''.sub.15 1521.83 B.sub.15 n.d. Y''.sub.16 1608.90
B.sub.16 n.d. Y''.sub.17 1724.00 B.sub.17 n.d. Y''.sub.18 n.d.
B.sub.18 n.d. Y''.sub.19 n.d. B.sub.19 n.d. .sup.1 Underlined
m/z-values are main peaks in the MS/MS-spectrum. .sup.2 n.d. is not
detected.
TABLE-US-00006 TABLE VI MS/MS-data of CDR3 peptide (H9) of UBS54,
obtained by collision induced dissociation of triply charged m/z
770.09. Ion m/z Ion m/z Y''.sub.1 n.d. B.sub.1 n.d. Y''.sub.2
248.17 B.sub.2 213.17 Y''.sub.3 335.20 B.sub.3 360.16 Y''.sub.4
406.25 B.sub.4 473.27 Y''.sub.5 507.30 B.sub.5 610.32 Y''.sub.6
594.33 B.sub.6 773.41 Y''.sub.7 693.42 B.sub.7 959.48 Y''.sub.8
794.45 B.sub.8 1016.50 Y''.sub.9 893.53 B.sub.9 1144.57 Y''.sub.10
1006.64 B.sub.10 1201.59 Y''.sub.11 1107.67 B.sub.11 1302.68
Y''.sub.12 1164.68 B.sub.12 1415.72 Y''.sub.13 n.d. B.sub.13
1514.78 Y''.sub.14 n.d. B.sub.14 n.d. Y''.sub.15 n.d. B.sub.15 n.d.
Y''.sub.16 n.d. B.sub.16 n.d. Y''.sub.17 n.d. B.sub.17 n.d.
Y''.sub.18 n.d. B.sub.18 n.d. Y''.sub.19 n.d. B.sub.19 n.d.
Y''.sub.20 n.d. B.sub.20 n.d. .sup.1 Underlined m/z-values are main
peaks in the MS/MS-spectrum. .sup.2 n.d. is not detected.
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Sequence CWU 1
1
181333DNAArtificialVL sequence of UBS54 (anti-EpCAM) and K53
(anti-CD46)CDS(1)..(333) 1gaa att gag ctc act cag tct cca ctc tcc
ctg ccc gtc acc cct gga 48Glu Ile Glu Leu Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 gag ccg gcc tcc atc tcc tgc agg
tct agt cag agc ctc ctg cat agt 96Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25 30 aat gga tac aac tat ttg
gat tgg tac ctg cag aag cca ggg cag tct 144Asn Gly Tyr Asn Tyr Leu
Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca cag ctc ctg
atc tat ttg ggt tct aat cgg gcc tcc ggg gtc cct 192Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 gac agg
ttc agt ggc agt gga tca ggc aca gat ttt aca ctg aaa atc 240Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
agc aga gtg gag gct gag gat gtt ggg gtt tat tac tgc atg caa gct
288Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95 cta caa act ttc act ttc ggc cct ggg acc aag gtg gag atc
aaa 333Leu Gln Thr Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys
100 105 110 2111PRTArtificialVL sequence of UBS54 (anti-EpCAM) and
K53 (anti-CD46) 2Glu Ile Glu Leu Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95
Leu Gln Thr Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100 105
110 3333DNAArtificialVL sequence of 02-237 (anti-CD46)CDS(1)..(333)
3gac atc gtg atg act cag tct cca ctc tcc ctg ccc gtc acc cct gga
48Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1
5 10 15 gag ccg gcc tcc atc tcc tgc agg tct agt cag agc ctc ctg cat
agt 96Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30 aat gga tac aac tat ttg gat tgg tac ctg cag aag cca
ggg cag tct 144Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 cca cag ctc ctg atc tat ttg ggt tct aat cgg
gcc tcc ggg gtc cct 192Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg
Ala Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga tca ggc
aca gat ttt aca ctg aaa atc 240Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 agc aga gtg gag gct gag gat
gtt ggg gtt tat tac tgc atg caa gct 288Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 cta caa act ttc act
ttc ggc cct ggg acc aag gtg gag atc aaa 333Leu Gln Thr Phe Thr Phe
Gly Pro Gly Thr Lys Val Glu Ile Lys 100 105 110 4111PRTArtificialVL
sequence of 02-237 (anti-CD46) 4Asp Ile Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu
Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
Ala 85 90 95 Leu Gln Thr Phe Thr Phe Gly Pro Gly Thr Lys Val Glu
Ile Lys 100 105 110 5345DNAArtificialVH sequence of UBS54
(anti-EpCAM)CDS(1)..(345) 5cag gtg cag ctg gtg cag tct ggg gct gag
gtg aag aag cct ggg tcc 48Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser 1 5 10 15 tcg gtg agg gtc tcc tgc aag gct
tct gga ggc acc ttc agc agc tat 96Ser Val Arg Val Ser Cys Lys Ala
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 gct atc agc tgg gtg cga
cag gcc cct gga caa ggg ctt gag tgg atg 144Ala Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga ggg atc atc
cct atc ttt ggt aca gca aac tac gca cag aag ttc 192Gly Gly Ile Ile
Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 cag ggc
aga gtc acg att acc gcg gac gaa tcc acg agc aca gcc tac 240Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80
atg gag ctg agc agc ctg aga tct gag gac acg gct gtg tat tac tgt
288Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 gca aga gac ccg ttt ctt cac tat tgg ggc caa ggt acc ctg
gtc acc 336Ala Arg Asp Pro Phe Leu His Tyr Trp Gly Gln Gly Thr Leu
Val Thr 100 105 110 gtc tcg aca 345Val Ser Thr 115
6115PRTArtificialVH sequence of UBS54 (anti-EpCAM) 6Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val
Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Phe Leu His Tyr Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Thr 115
7354DNAArtificialVH sequence of K53 (anti-CD46)CDS(1)..(354) 7cag
gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg gcc 48Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 tca gtg aag gtc tcc tgc aag gct tct ggt tac acc ttt acc agc tat
96Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 ggt atc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag
tgg atg 144Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 gga tgg atc agc gct tac aat ggt aac aca aac tat
gca cag aag ctc 192Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr
Ala Gln Lys Leu 50 55 60 cag ggc aga gtc acc atg acc aca gac aca
tcc acg agc aca gcc tac 240Gln Gly Arg Val Thr Met Thr Thr Asp Thr
Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gag ctg agg agc ctg aga tct
gac gac acg gcc gtg tat tac tgt 288Met Glu Leu Arg Ser Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca agg ggc atg atg agg
ggt gtg ttt gac tac tgg ggc caa ggt acc 336Ala Arg Gly Met Met Arg
Gly Val Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 ctg gtc acc gtc
tcg aca 354Leu Val Thr Val Ser Thr 115 8118PRTArtificialVH sequence
of K53 (anti-CD46) 8Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr
Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val
Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Gly Met Met Arg Gly Val Phe Asp Tyr Trp Gly Gln Gly Thr 100
105 110 Leu Val Thr Val Ser Thr 115 9354DNAArtificialVH sequence of
02-237 (anti-CD46)CDS(1)..(354) 9cag gtg cag ctg gtg cag tct ggg
gct gag gtg aag aag cct ggg gcc 48Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtc tcc tgc
aag gct tct ggt tac acc ttt acc agc tat 96Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 ggt atc agc tgg
gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144Gly Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga tgg
atc agc gct tac aat ggt aac aca aac tat gca cag aag ctc 192Gly Trp
Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60
cag ggc aga gtc acc atg acc aca gac aca tcc acg agc aca gcc tac
240Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80 atg gag ctg agg agc ctg aga tct gac gac acg gcc gtg tat
tac tgt 288Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 gca agg ggc ttt ccg cgt acg tcg ttt gac tcc tgg
ggc cag ggc acc 336Ala Arg Gly Phe Pro Arg Thr Ser Phe Asp Ser Trp
Gly Gln Gly Thr 100 105 110 ctg gtg acc gtc tcc tca 354Leu Val Thr
Val Ser Ser 115 10118PRTArtificialVH sequence of 02-237 (anti-CD46)
10Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn
Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp
Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Pro
Arg Thr Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr
Val Ser Ser 115 11369DNAArtificialVH sequence of clone B28
(anti-CD22 phage)CDS(1)..(369) 11atg gcc gag gtg cag ctg gtg gag
tct ggg gga ggt gtg gta cgg cct 48Met Ala Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Arg Pro 1 5 10 15 gga ggg tcc ctg aga ctc
tcc tgt gca gcc tct gga ttc acc ttt gat 96Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 20 25 30 gat tat ggc atg
agc tgg gtc cgc caa gct cca ggg aag ggg ctg gag 144Asp Tyr Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 tgg gtc
tct ggt att aat tgg aat ggt ggt agc aca ggt tat gca gac 192Trp Val
Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp 50 55 60
tct gtg aag ggc cga ttc acc atc tcc aga gac aac gcc aag aac tcc
240Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
65 70 75 80 ctg tat ctg caa atg aac agt ctg aga gcc gag gac acg gcc
gtg tat 288Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr 85 90 95 tac tgt gca aga ggc ttt ctt cgt ttt gct tcc tcc
tgg ttt gac tat 336Tyr Cys Ala Arg Gly Phe Leu Arg Phe Ala Ser Ser
Trp Phe Asp Tyr 100 105 110 tgg ggc caa ggt acc ctg gtc acc gtc tcg
aga 369Trp Gly Gln Gly Thr Leu Val Thr Val Ser Arg 115 120
12123PRTArtificialVH sequence of clone B28 (anti-CD22 phage) 12Met
Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro 1 5 10
15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp
20 25 30 Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu 35 40 45 Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr
Gly Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Phe
Leu Arg Phe Ala Ser Ser Trp Phe Asp Tyr 100 105 110 Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Arg 115 120 13369DNAArtificialVH sequence
of clone II-2 (anti-CD72 phage)CDS(1)..(369) 13atg gcc cag gtg cag
ctg gtg cag tct ggg gct gag gtg aag aag cct 48Met Ala Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10 15 ggg gcc tca
gtg aag gtt tcc tgc aag gca tct gga tac acc ttc acc 96Gly Ala Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 agc
tac tat atg cac tgg gtg cga cag gcc cct gga caa ggg ctt gag 144Ser
Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40
45 tgg atg gga ata atc aac cct agt ggt ggt ggc aca agc tac gca cag
192Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Gly Thr Ser Tyr Ala Gln
50 55 60 aag ttc cag ggc aga gtc acc atg acc agg gac acg tcc acg
agc aca 240Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr 65 70 75 80 gtc tac atg gag ctg agc agc ctg aga tct gag gac
acg gcc gtg tat 288Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr 85 90 95 tac tgt gca aga gac tac tat gtt acg tat
gat tcc tgg ttt gac tcc 336Tyr Cys Ala Arg Asp Tyr Tyr Val Thr Tyr
Asp Ser Trp Phe Asp Ser 100 105 110 tgg ggc caa ggt acc ctg gtc acc
gtc tcg aga 369Trp Gly Gln Gly Thr Leu Val Thr Val Ser Arg 115 120
14123PRTArtificialVH sequence of clone II-2 (anti-CD72 phage) 14Met
Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 1 5 10
15
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20
25 30 Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu 35 40 45 Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Gly Thr Ser
Tyr Ala Gln 50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp
Thr Ser Thr Ser Thr 65 70 75 80 Val Tyr Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr Tyr
Val Thr Tyr Asp Ser Trp Phe Asp Ser 100 105 110 Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Arg 115 120 15360DNAArtificialVH sequence of
clone I-2 (anti-class II phage)CDS(1)..(360) 15atg gcc gag gtg cag
ctg gtg gag tct ggg gga ggc ttg gta cag cct 48Met Ala Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 ggc agg tcc
ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt gat 96Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 20 25 30 gat
tat gcc atg cac tgg gtc cgg caa gct cca ggg aag ggc ctg gag 144Asp
Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40
45 tgg gtc tca ggt att agt tgg aat agt ggt agc ata ggc tat gcg gac
192Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp
50 55 60 tct gtg aag ggc cga ttc acc atc tcc aga gac aac gcc aag
aac tcc 240Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser 65 70 75 80 ctg tat ctg caa atg aac agt ctg aga gct gag gac
acg gcc gtg tat 288Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95 tac tgt gca agg gac ctt tat ctt gcg cat
ttt gac tac tgg ggc caa 336Tyr Cys Ala Arg Asp Leu Tyr Leu Ala His
Phe Asp Tyr Trp Gly Gln 100 105 110 ggt acc ctg gtc acc gtc tcg aga
360Gly Thr Leu Val Thr Val Ser Arg 115 120 16120PRTArtificialVH
sequence of clone I-2 (anti-class II phage) 16Met Ala Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 20 25 30 Asp
Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40
45 Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp
50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Leu Tyr Leu Ala His
Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Arg
115 120 17336DNAArtificialcommon VL sequence of clones B28
(anti-CD22 phage), II-2 (anti-CD72 phage) and I-2 (anti-class II
phage)CDS(1)..(336) 17tcg tct gag ctg act cag gac cct gct gtg tct
gtg gcc ttg gga cag 48Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser
Val Ala Leu Gly Gln 1 5 10 15 aca gtc agg atc aca tgc caa gga gac
agc ctc aga agc tat tat gca 96Thr Val Arg Ile Thr Cys Gln Gly Asp
Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 agc tgg tac cag cag aag cca
gga cag gcc cct gta ctt gtc atc tat 144Ser Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 ggt aaa aac aac cgg
ccc tca ggg atc cca gac cga ttc tct ggc tcc 192Gly Lys Asn Asn Arg
Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 agc tca gga
aac aca gct tcc ttg acc atc act ggg gct cag gcg gaa 240Ser Ser Gly
Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 gat
gag gct gac tat tac tgt aac tcc cgg gac agc agt ggt aac cat 288Asp
Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90
95 gtg gta ttc ggc gga ggg acc aag ctg acc gtc cta ggt gcg gcc gca
336Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ala Ala Ala
100 105 110 18112PRTArtificialcommon VL sequence of clones B28
(anti-CD22 phage), II-2 (anti-CD72 phage) and I-2 (anti-class II
phage) 18Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu
Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg
Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly
Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala
Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp
Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90 95 Val Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ala Ala Ala 100 105
110
* * * * *