Note: Descriptions are shown in the official language in which they were submitted.
W094/26772 2 1 6 2 6 7 1 PCT~S94/0~78
SEPARATION OF ANTI-METAL CHELATE ANTIBODIES
BachyLo~,d of the Invention
This invention relates to methods of separating
antibodies and, more specifically, to a method of separating
anti-metal chelate antibodies from other antibodies and
proteins.
The last twenty-five years have witnessed a revolution
in the field of immunology. During the 1970's methods were
developed of producing in quantity single species of
antibodies, the proteins which the immune system uses to
recognize, bind and eventually eliminate substances which
are recognized as foreign. In order to specifically bind to
the enormous range of potential antigens, each of the some
105 to I08 lymphocyte lineages which an individual possesses
produces different antibodies with specificity for a
different foreign substance, or antigen. By fusing a single
antibody-producing lymphocyte with an immortalized cell,
such as a cancer cell, it is now possible to make a single
lymphocyte clone, each cell producing the same monoclonal
antibody. The development of such hybridomas to produce
monoclonal antibodies has had an enormous impact on the
ability to both diagnose and treat a vast array of diseases.
Antibodies comprise two identical pairs of a heavy and
light peptide chain arranged in the shape of a "Y". Each of
the arms contains a site which binds to the antigen through
various non-covalent interactions, including ionic
interactions, hydrogen bonding and van der Waals forces,
which result in an affinity between the antibody and its
cognate antigen. This antigen-binding site is encompassed
within an antigen reactive region which corresponds to the
so called variable region of the antibody. In native
antibodies, both antigen reactive binding regions will be
identical. "Bifunctional antibodies" have been produced
which express two different variable regions. These
bifunctional antibodies can be produced chemically or by
W094/26772 2 21626 71 PCT~S94/0~78
engineered cells which simultaneously express two different
sets of genes encoding the antibody proteins. These gene
products then assemble into a variety of species of
antibodies exhibiting different combinations of antibody
gene products including two which exhibit identical antigen-
binding sites (~monofunctional antibodies") and one
exhibiting dissimilar antigen-binding sites.
Bifunctional antibodies have great utility in allowing
simultaneous binding to more than one antigen. As one
example of such use, a bifunctional antibody having one arm
specific for an antigen expressed on the surface of a tumor
cell and one arm specific for an imaging or therapeutic
moiety may be effectively used to target such a moiety to
the site of a tumor. Among the moieties so used for such
therapeutic and diagnostic purposes are metals in metal
chelates. Antibodies having binding affinity for metal
chelates are termed '~anti-metal chelate antibodies.~
The recent developments of antibody technology have
generated a need for methods to purify antibodies from
proteins and other cont~m;n~nts and to isolate specific
species of antibodies from other antibodies.
Conventionally, two methods have been used to separate
antibodies: ion exchange chromatography and affinity
chromatography. Ion exchange chromatography utilizes a
solid support to which charged functional groups are
covalently attached. The ionic interaction of these charged
groups with charges available on the surface of various
proteins provides a means of separating many types or
families of protein. A protein whose surface is negatively
charged will likely bind to an anion exchanger which has
positively charged functional groups, while a protein whose
surface exposes predominately positive charges will likely
bind to a cation exchanger. The binding of these proteins
is influenced by pH, salt composition and concentration and
as such these parameters can be utilized to isolate
antibodies as a family from other types of proteins. While
ion exchange chromatography has proved quite useful in
W094/26772 3 ~l 6 2 6 7 I PCT~S94/0~78
certain applications, it has the critical limitation that
antibodies having similar physical characteristics but
distinct functional characteristics, such as antigen
binding, are usually not differentiated.
More specific purification can be achieved using an
affinity column containing a resin to which is bound the
cognate antigen or hapten of the antibody to be isolated. A
hapten is a small molecule which is antigenic when attached
to a carrier. The antibody preparation is passed over the
column, with antibodies specific to the antigen binding to
and being retained on the column. Because of the strength
of the antigen-antibody binding, a solution containing the
cognate is required to elute the antibody. For instance,
as disclosed in U.S. Patent Number 5,112,951, anti-metal
chelate antibodies show extended retention times on a
sulfopropyl column, as compared with non-specific
antibodies, in the absence of hapten. Moreover, retention
times were similar for murine antibodies and their chimeric
derivatives despite the fact that the murine antibodies have
a much lower pI than do the chimerics. However, the
retention time of anti-metal chelate antibodies in the
presence of the hapten analog, lmM Co/EDTA, was dramatically
reduced as compared to only slight changes with the non-
specific antibodies. This longer retention time of anti-
metal chelate antibodies and its abrogation in the presence
of metal chelate hapten indicate that the binding of these
antibodies to oxo acid resin reflects interaction in the
antigen reactive binding region.
Despite its potential for separating antibodies with
similar physical characteristics, affinity purification has
certain serious drawbacks. For example, it may be difficult
or impossible to separate the antibodies from the antigen or
analog hapten with which they elute. In addition, the
cognate and cognate-resin may be unavailable or costly.
Even more importantly, the antibody may be denatured by the
severe elution conditions, such as extreme pH or the
presence of chaotropic agents.
W094l26772 4 2 1 6 2 6 7 ¦ PCT~4/0~78
There thus exists a need for an inexpensive and
effective method for specifically isolating anti-metal
chelate antibodies from non-specific antibodies or proteins
which does not result in their being denatured during the
process. Preferably, such a method should be effective in
isolating polyclonal fractions enriched in anti-metal
chelate antibodies as well as monoclonal antibodies and
their bifunctional derivatives. The present invention
satisfies these needs and provides related advantages as
well.
Summary of the In~ention
The present invention provides a method for the
separation of anti-metal chelate antibodies from non-
specific proteins, including antibodies, by applying apreparation containing the anti-metal chelate antibodies to
a carboxylic acid derivatized solid support and eluting
first with an elution buffer containing sufficient salt
concentration to elute non-specific proteins but not
sufficient to elute the anti-metal chelate antibodies and
then increasing the salt concentration of the elution
solution so as to elute the anti-metal chelate antibodies,
wherein the pH of the elution buffer is 7.5 or below. In
one embodiment, the carboxylic acid derivatized solid
support is a carboxymethyl resin. Appropriate salts include
sodium phosphate, sodium chloride, sodium sulphate and
sodium acetate. Alternatively, the anti-metal chelate
antibody can be selectively eluted with a solution
containing a second non-cognate carboxylic acid having less
affinity for the anti-metal chelate antibody than does the
cognate hapten.
The method can be used to separate monoclonal or
polyclonal anti-metal chelate antibodies from non-specific
proteins as well as to separate bifunctional anti-metal
chelate antibodies from monoclonal anti-metal chelate
antibodies and other non-specific proteins without
precipitating out the eluted antibodies. The method is also
W094/26772 5 2 1 6 2 6 7 1 PCT~Sg4/0~78
useful for separating anti-metal chelate antibody fragments
bearing antigen reactive regions from non-specific proteins.
Brief Description of the Figures
Figure 1 is a graph illustrating reactivity with a
series of non-cognate carboxylic acids of an antibody raised
against a metal chelate, Indium-benzyl EDTA.
Figure 2 is a graph of ultraviolet absorbance at 28Onm
over time showing the elution scan of a bifunctional anti-
metal chelate antibody (BxBFA) chromatographed on a
sulfopropyl derivatized column as described in Example I
using sodium phosphate as the elutant salt.
Figure 3 is a graph of ultraviolet absorbance at 280nm
over time showing the elution scan of a bifunctional anti-
metal chelate antibody (BxBFA) chromatographed on a
carboxymethyl derivatized column as described in Example II
using sodium phosphate as the elutant salt.
Figure 4 is a graph of ultraviolet absorbance at 280nm
over time showing the elution scan of a bifunctional anti-
metal chelate antibody (ECA 001) chromatographed on a
carboxymethyl derivatized column as described in Example III
using sodium phosphate as the elutant salt.
Figure 5 is a graph of ultraviolet absorbance at 28Onm
over time showing the elution scan of a bifunctional anti-
metal chelate antibody (BxBFA) chromatographed on a
iminodiacetic acid derivatized column as described in
Example IV using sodium phosphate as the elutant salt.
Figures 6a and 6b are graphs of ultraviolet absorbance
at 280nm over time showing the elution scan of a
bifunctional anti-metal chelate antibody (BxBFA)
chromatographed on an iminodiacetic acid derivatized column
as described in Example V using glutamic acid and/or glycine
as the elutant.
Figure 7 is a graph of ultraviolet absorbance at 28Onm
over time showing the elution scan of a bifunctional anti-
metal chelate antibody (ECA 001) chromatographed on a
iminodiacetic acid derivatized column as described in
W094/26772 PCT~S9410~78
6 - 2162671
Example VI using sodium sulfate as the elutant salt.
Figure 8 is a graph of ultraviolet absorbance at 280nm
over time showing the elution scan of a bifunctional anti-
metal chelate antibody (BxBFA) chromatographed on a glutamic
acid derivatized column as described in Example VII using
sodium phosphate as the elutant salt.
Detailed Description of the Invention
The present invention provides an effective method for
separating anti-metal chelate antibodies wherein the hapten
chelate is an carboxylic acid or acid derivative from non-
specific proteins, which may include other non-specific
antibodies. The method exploits the unexpected ability of
anti-metal chelate antibodies to bind to a non-cognate
antigen, a carboxylic acid moiety with at least some
structural similarity to the cognate hapten chelate, but
with reduced affinity for their antigen reactive regions,
thus differentiating them from other non-specific antibodies
and proteins. While antigens and haptens, or their analogs,
such as derivatives and fragments, have been used on the
solid phase of affinity columns, the present method is based
on a binding between anti-metal chelate antibodies and a
non-cognate hapten or analog through their antigen-binding
site. It is, moreover, an advantage of the present
invention that an elevated salt concentration is sufficient
to elute the anti-metal chelate antibodies. As an
additional advantage of the present invention, the pH of the
elevated salt concentration can be 6.5 or above.
Alternatively, anti-metal chelate antibodies can be
selectively eluted with mono-, di- or tri-carboxylic acids
having less affinity for the anti-metal chelate antibody
than does the cognate hapten (for example In-benyzl-EDTA).
Further, no harsh or denaturing elution conditions, common
to conventional affinity purification systems, are required.
The invention is premised on the unexpected ability of
anti-metal chelate antibodies to bind to negatively charged
multi-oxygen resonance structures in a manner which reflects
WOg4/26772 7 2 1 6 2 6 7 1 PCT~S94/0~78
their immunological specificity, indicating that the binding
is with their antigen reactive region, at or near the
antigen-binding site. In particular, anti-metal chelate
antibodies raised against oxo acid hapten chelates, such as
carboxylic acids and their derivatives, exhibit attraction
for non-cognate hapten carboxylic acids, such as those
having one or two carboxylic acid functionalities situated
so as to cooperate in bonding with the antigen reactive
region of the antibody. For example, non-cognate haptens,
including EDTA with non-Indium metals, and non-cognate
carboxylic acids, having lesser affinity for the binding
site of an antibody raised against metal benzyl-EDTA than
does the cognate, including acetic, iminodiacetic, benzyl-
EDTA, as shown in Figure 1 of the drawings, as well as
glycine or other acids such as citric aspartic or glutamic
acid.
Such non-cognate carboxylic acids can be attached to a
solid support, to form a carboxylic acid derivatized solid
support. When such a non-cognate carboxylic acid solid
support is used as the solid phase in a chromatographic
method, monoclonal anti-metal chelate antibodies can be
separated from non-specific proteins and similarly
polyclonal anti-metal chelate antibody enriched fractions
can be obtained from antiserum.
In addition to distinguishing monoclonal and polyclonal
anti-metal chelate antibodies from non-specific proteins,
the invention permits the separation of bifunctional anti-
metal chelate antibodies from monofunctional anti-metal
chelate antibodies, such as would be found together in the
culture fluid from a polydoma. The various species of
antibodies in this culture fluid exhibit discrete retention
times when eluted with a gradient having an increasing salt
concentration; the active bifunctional anti-metal chelate
antibodies elute from the non-cognate carboxylic acid
derivatized solid support at a lower salt concentration than
the active monofunctional anti-metal chelate antibodies.
This separation reflects the difference in avidity between a
W094/26772 2 ~ ~ 2 PCT~S94/0~78
bifunctional antibody having a single anti-metal chelate
binding site (~monovalent") and a monofunctional anti-metal
chelate antibody having two antigen-binding sites
("bivalent"). Thus, the bifunctional anti-metal chelate
antibodies can be effectively separated from the other, non-
desired, species, as disclosed in U.S. Patent Number
5,112,951.
As used herein, the term "anti-metal chelate
antibodies" refers to antibodies and antibody constructs
well known in the art, such as chimeric, CDR-grafted, and
humanized antibodies which have a variable region with a
high affinity for at least one metal chelate or metal
chelate analog wherein the hapten chelate is a carboxylic
acid such as benzyl-EDTA. Generally the affinity of the
anti-metal chelate antibody is greater than about 106 L/M,
preferably greater than 107 L/M, most preferably greater
than lO~L/M. However, a particular anti-metal chelate
antibody will, of course, exhibit differing affinities for
different metal chelates. Antibodies not exhibiting such an
affinity for metal chelate haptens wherein the hapten is
carboxylic acid or acid derivative are referred to as "non-
specific antibodies." Non-specific antibodies together with
non-antibody proteins are termed "non-specific proteins."
For a description of anti-metal chelate antibodies see U.S.
Patent Number 4,722,892 and Reardan, et al., Nature,
316:265-268 (1985). Metal chelates include any metal ion in
the (II) or (III) oxidation state, including radioactive
isotopes, complexed with a polycarboxylate chelating agent,
including but not limited to EDTA, DTPA, and DOTA, termed
herein the '~cognate acid" chelator. For a list of such
metals, see Reardan, suPra. For a discussion of chelating
agents see U.S. Patent Number 4,678,667.
The method of the invention is also suited to the
separation of anti-metal chelate antibody fragments which
contain the antigen reactive region from non-specific
proteins, including non-specific antibodies and their
fragments. As used herein, the term anti-metal chelate
W094/26772 PCT~S94/0~78
9 2162~1
antibodies includes fragments thereof which bear antigen
reactive regions (Fab fragments including Fab; F(ab' )2 and
Fab').
The method of the present invention has several
advantages over more conventional affinity chromatography
methods for the purification of anti-metal chelate
antibodies. As indicated, not only does the method permit
separation of anti-metal chelate antibodies from non-
specific antibodies, but the method also permits the
differentiation of antibodies having different avidity, by
virtue of their valence, for the carboxylic acid derivatized
solid phase support. In addition, it is more cost
effective, as carboxylic acid derivatized solid phase
supports, buffers and salts are less expensive and more
readily available than metal chelate resins and metal
chelate haptens. Also, the column can be easily sanitized,
depyrogenated, cleaned of accumulated protein and
regenerated, as by treatment with 0.2 N sodium hydroxide.
Anti-metal chelate monoclonal antibodiec bind much more
tightly to such a non-cognate carboxylic acid derivatized
solid support than do other proteins commonly found in
tissue culture supernatants and, unexpectedly, bind even
tighter than do non-specific monoclonal antibodies with
higher pI's. The pI or "isoelectric point" of an individual
protein or antibody is determined primarily by amino acid
composition and is defined as the pH at which the net charge
of that protein is zero. Above its isoelectric point, the
protein has a net negative charge and below its isoelectric
point, the protein has a net positive charge. At any given
pH, a protein with a higher pI would be more positively
charged, or conversely be less negatively charged, than a
protein with a lower pI. Particularly among proteins with
similar structure and physical characteristics, such as
antibodies, a higher pI would be expected to predict
stronger interaction with the negatively charged functional
groups on a carboxylic acid derivatized solid support, as
ionic forces generally account for the interaction of
W094l26772 1 PCT~S94/0~78
~ 21 6267~
proteins with negatively charged functional groups. Yet,
even among antibodies genetically engineered to have
identical constant regions, the anti-metal chelate antibody
will be retained longer on the non-cognate carboxylic acid
derivatized solid support than a non-metal chelate specific
antibody having a higher pI. By the same token at a higher
pH the same antibody has a less positive charge and will be
held less securely by the negatively charged functional
groups on a carboxylic acid derivatized solid support.
Despite this, it has been discovered that metal chelate
antibody separations can be performed using non-cognate
carboxylic acid supports at a pH of 7.5 and below.
As an indication that this behavior of anti-metal
chelate antibodies on non-cognate derivatized solid supports
exhibits immunologically specific binding, U.S. Patent
Number 5,112,951 discloses that anti-metal chelate
antibodies bearing the same antigen reactive binding region
sequences, but having different constant regions and pI's,
as in the case of chimeric antibodies and the native murine
antibodies from which they were derived, exhibit the same
extended retention time on the non-cognate oxo acid
derivatized solid support. These unexpected observations
indicate that pI does not, in this case, explain the primary
behavior of antibodies having a metal chelate specificity.
Even more conclusively, the presence of a metal chelate
hapten in solution-phase will effectively eliminate the
anti-metal chelate antibodies' unexpectedly strong binding
to the non-cognate derivatized solid phase by shortening
their retention times so as to be comparable to those of
non-specific antibodies. Considering the small size of
-these competing solution phase metal chelate haptens, the
site of interaction which accounts for this unexpected
binding behavior of anti-metal chelate antibodies must be
near or identical with the antigen-binding site of these
anti-metal chelate antibodies.
The unexpectedly strong non-cognate oxo acid binding
reaction and its abrogation in the presence of metal chelate
W094/26772 2 ~ 6 2 6 7 1 PCT~S94/0~78
haptens indicates that anti-metal chelate antibodies bind to
non-cognate carboxylic acid derivatized solid supports by a
mechanism which is distinct from normal mechanisms of cation
exchange chromatography and that this new mechanism of
interaction is related to the immunological specificity of
these antibodies. Moreover, this unexpected behavior
extends to bifunctional antibodies derived from anti-metal
chelate antibodies. Such bifunctional antibodies have only
a single metal chelate binding site, and it is this
monovalence which accounts for their being measurably less
well retained on a non-cognate carboxylic acid derivatized
column than is the monofunctional anti-metal chelate
antibody from which they were derived, which has two metal
chelate hapten-binding sites.
The interaction between anti-metal chelate antibodies
and the non-cognate carboxylic acid derivatized solid
support exhibits a behavior characteristic of
immunologically specific binding and reflects an interaction
at or near the antigen-specific binding site. However, the
fact that only increasing salt concentration is required for
their elution indicates that any affinity that these
antibodies have for the non-cognate carboxylic acid
derivatized solid phase support is low in comparison to
their affinity for the metal chelate. It is believed that
non-cognate carboxylic acid groups on the solid support may
mimic the three dimensional spatial configuration or charge
density pattern of a portion of the metal chelate hapten,
thereby accounting for the antibody's affinity for the
matrix, albeit lower than for the metal chelate itself.
Such conformational similarity is plausible as the non-
cognate carboxylic acid derivatized solid support does
possess oxygen atoms with negative charges like the metal
chelate hapten. This characteristic indicates that other
negatively charged multi-oxygen resonance structures would
also be suitable derivatives for use in the method of the
invention. Whatever the basis of this interaction, the
behavior of anti-metal chelate antibodies on the non-cognate
~`--
W094/26772 l2 2 1 6 2 6 7 1 PCT~Sg4/04878
,
carboxylic acid derivatized column can best be described as
an interaction reflecting the antigen reactivity of the
anti-metal chelate antibody. As illustrated in Figure 1,
reactivity with a series of non-cognate carboxylic acids of
an antibody raised against a metal chelate, Indium-benzyl
EDTA, increases as the structure of the non-cognate acid
increases in similarity to that of the acid in the metal
chelate, benzyl-EDTA.
It will be noted that the relative affinities of a
subject antibody for various candidate non-cognate acids can
be determined empirically using elution from solid support
columns and methods well known in the art. These methods
are further illustrated in the Examples herein. One skilled
in the art will appreciate that candidate non-cognate acids
can also be selected by inspection simply by comparing the
degree of similarity in the chemical structure of the
candidates to that of the cognate hapten. As a general
rule, it will be expected that the non-cognate acid having
the greatest degree of structural similarity to the cognate
hapten will have an affinity for the anti-metal chelate
antibody which is closer to that of the cognate metal
chelate.
Various anti-metal chelate antibodies are known or
available. Examples include CHA255 and CHB235, which are
monoclonal antibodies of murine origin which have particular
affinity for an Indium-EDTA complex. See U.S. Patent No.
4,722,892. Anti-metal chelate antibodies and non-specific
antibodies referred to herein are listed with their
characteristics in Table I.
W094/26772 l3 2 1 6 2 6 ~ I PCT~S94/04878
TABLE I
Antibody pI Specificity Characteristics
s
CHA255 7.5 l1lIn metal murine monoclonal
chelate monofunctional
CHB235 6.6- lllIn metal murine monoclonal
7.2 chelate monofunctional
XCHA351 8.5 lllIn metal chimeric monoclonal
chelate monofunctional
XCEM449.08 8.8 tumor CEA chimeric monoclonal
monofunctional
CYA339 90Y metal murine monoclonal
chelate monofunctional
CEVMSC 7.0- tumor CEA murine monoclonal
7.2 monofunctional
ECH037 lllIn metal murine
chelate/tumor polydoma produced
CEA bifunctional
BxBFA 8.6 lllIn metal chimeric monoclonal
chelate/tumor bifunctional
CEA
pCYA Y-metal murine polyclonal
chelate monofunctional
ECA 001 6.6 lllIn metal murine polydoma
chelate/tumor produced
CEA bifunctional
Both monoclonal and polyclonal anti-metal chelate
antibodies can be made by methods known to those skilled in
the art. For example, an antigen can be prepared by
W094/26772 l4 2 1 6 2 6 7 1 PCT~S94/0~78
conjugating a chelating agent to a carrier in solution. The
resulting solution is then mixed with a metal salt, such as
indium citrate, and dialyzed. Alternatively, one could use
gel filtration in place of dialysis. The amount of attached
chelate can be determined from the absorbance or by
radioactive titration. Hybridoma cells producing anti-metal
chelate antibodies can be prepared by methods well known in
the art. See, for example, Antibodies, a Laboratory Manual,
(Harlow and Lane, eds.) Cold Spring Harbor, New York (1988).
In the preparation of CHA255 and CHB235, for example,
an indium-EDTA antigen was prepared. Keyhole limpet
haemocyanin (9.3mg) was allowed to react in 265~L aqueous
solution, pH 6.0, with (L)-SCN-C6H4-CH2-EDTA (isothiocyanate
benzyl-EDTA; ITCBE) for eight hours at 36C. The resulting
solution was mixed with 90~L of O.lM indium citrate and
dialyzed against lmM EDTA, 0.15M NaCl. From the absorbance
of the thiourea group at 310nm, it was determined that there
was approximately O.lmg of attached chelate per mg of
protein.
Spleen cells from BALB/c mice, multiply immunized with
the antigen described above, were fused with a variant of
the P3.653 myeloma cell line using the technique of Gerhard,
Monoclonal Antibodies, (Kennett, et al., eds.) Plenum Press
New York (1980). The resulting hybridomas were screened,
using a solid phase second antibody radioimmunoassay, for
their ability to bind In(III) aminobenzyl-EDTA according to
the method of Wang, et al., J. Immunol. Meth., 18:157
(1977). Those hybridomas exhibiting high titer and
relatively high affinity antibodies as determined by
inhibition of binding by unlabelled antigen, were selected
and injected intraperitoneally into BALB/c mice for ascites
production. In the case of murine antibodies, the
monoclonal anti-metal chelate antibodies were purified from
mouse ascites by ion-exchange chromatography on DEAE-
cellulose as described by Parham, et al., J. Immunol. Meth.,
53:133 (1982).
The binding constants of the antibodies for the
W094/26772 l5 2 1 6 2 6 7 1 PCT~S9410~78
chelates were determined by the method of Eisen, Metn. Med.
Res. 10:106 (1964). Briefly, the antibody and metal
chelates were dialyzed to near equilibrium for 24 hours at
37C in 0.05M 2-hydroyethyl-piperazine-ethanesulfonate
(HEPES), O.lM NaCl, 0.1~ NaN3 and 0.1~ bovine serum albumin
at pH 7. The concentration of antibody-binding sites inside
the dialysis bag was 10-7 M and the concentration of free
In(III)-(L)-aminobenzyl EDTA complex was in the same range.
CHA255 and CHB235 have affinities (binding constants) for
In(III) EDTA complex on the order of 109 L/M and 108L/M,
respectively.
Chimeric anti-metal metal chelate antibodies can be
produced expressing, for example, a variable region of
murine origin and constant regions of human origin. CDR-
grafted anti-metal chelate antibodies can be produced
expressing, for example, CDR's of murine origin and
framework and constant regions of human origin. To prepare
such chimeric or CDR-grafted antibodies, DNA sequences of
the variable and constant regions can be obtained from
genomic DNA. Genomic DNA may be prepared and cloned by a
variety of conventional techniques such as those described
in Basic Methods in Molecular Bioloqy, (L.G. Davis, M.D.
Dibner and J.F. Battey, eds.), Elsevier, New York (1986);
Feder, J., et al., Am. J. Hum. Genetics, 37:635-649 (1985);
and Steffer, D. and Weinberg, R.A., Cell 15:1003-1010
(1978); Beidler, et al., J. Immunol., 141:4053-4060 (1988).
For example, the DNA sequences encoding the desired variable
light and heavy chain regions may be obtained from cellular
DNA of a murine hybridoma expressing a desired anti-metal
chelate antibody, while the DNA sequence encoding for the
constant region may be derived from human lymphocytes,
preferably human peripheral blood lymphocytes. Cellular DNA
may be isolated by standard procedures, the genomic DNA
fragmented into restriction fragments by restriction
endonucleases, and the resulting fragments cloned into
suitable recombinant DNA cloning vectors and screened with
radiolabeled or enzymatically labeled probes for the
W094/26772 l6 2 1 6 2 6 7 PCT~S94/0~78
presence of the desired DNA sequences. Methods for
incorporating DNA constructs containing the desired
sequences into cloning vectors and expression vectors are
now well known in the art and described by numerous
references such as Eukaryotic Viral Vectors, (Y. Gluzman,
ed.) Cold Spring Harbor Laboratories publications, Cold
Spring Harbor, New York (1982); Eukaryotic Transcription,
(Y. Gluzman, ed.) Cold Spring Harbor, New York (1985);
Sequence S~ecificity in Transcription & Translation, (R.
Calendar and L. Gold, eds.) Allan R. Liss, Inc., New York
(1985); Maximizinq Gene Expression, (W. Reznikoff and L.
Gold, eds.) Butterworths, New York (1986); M~mm~lian Cell
Technology, (W. G. Thilly, ed.) Butterworths, New York
(1986); J. Sambrook and M.J. Gething, Focus, (Bethesda
Research Laboratories/Life Technologies, Inc.) 10 #3, pp.
41-48 (1988).
Appropriate host cells, preferably eukaryotic cells,
may be transformed to incorporate the expression vectors by
any one of several standard transfection procedures well
known in the art, including, for example, electroporation
techniques, protoplast fusion and calcium phosphate
precipitation techniques. Such techniques are generally
described by Toneguzzo, F., et al., Mol. and Cell Biol.,
6:703-706 (1986); Chu, G., et al., Nucleic Acid Res.,
15:1311-1325 (1987); Rice, D., et al., Proc. Natl. Acad.
Sci. USA, 79:7862-7865 (1979); and Oi, V., et al., Proc.
Natl. Acad. Sci. USA, 80:825-829 (1983). Preferably, the
recombinant expression vectors comprising the chimeric
constructs are transfected sequentially into host cells.
For example, the expression vectors comprising the chimeric
light chain DNA constructs are first transfected into the
host cells. Transformed host cells expressing the chimeric
light chain polypeptides are then selected by standard
procedures known in the art as described, for example, in
Engvall, E. and Perlmann, P., Immunochemistry, 8:871-874
(1971). The expression vectors comprising the chimeric
heavy chain DNA constructs are thereafter transfected into
W094~6772 ~7 ' PCT~S94/0~78
21 62671
the selected host cells. Alternatively, both the chimeric
light and heavy chain expression vectors can be introduced
simultaneously into the host cells or both chimeric gene
constructs can be combined on a single expression vector for
transfection into cells. Following transfection and
selection, standard assays are performed for the detection
of chimeric antibodies directed against desired metal
chelates.
The method of the present invention finds particular
utility in separating bifunctional anti-metal chelate
antibodies from the monofunctional anti-metal chelate
antibodies. Bifunctional antibodies exhibiting one
specificity against metal chelates and the other against a
different antigen can be obtained. See, for example, U.S.
Patent Number 4, 722,892, U.S. Patent Number 4,475,893 and
Martinis, et al., in Protides of the Bioloqical Fluids, (H.
Peters, ed.) pp. 311-316, Pergamon Press, Oxford (1983).
For example, polydomas able to express bifunctional
antibodies can be formed by fusing a cell secreting
antibodies of the one specificity with a cell secreting
antibodies of a different specificity. The heavy and light
chains of the two antibodies then assemble to form a variety
of antibody species including two active monofunctional,
bivalent antibodies (corresponding to those of the parental
cells), an active bifunctional antibody having one antigen-
binding site comprising the light and heavy chain of one
parent and the other antigen-binding site comprising the
light and heavy chain of the other parent, and various other
inactive species. The term "active" refers to constructs in
which each antigen-binding site is composed of a light and a
heavy chain from the same parent, thus giving it the
parental specificity. "Inactive" refers to those constructs
which lack the binding specificity of either parent because
one or both antigen-binding sites comprise a heavy chain
from one parent and a light chain from the other parent.
The culture fluid from these polydomas contains various
antibody species, including both the active monofunctional
W094/26772 1~ 2 1 62 PCT~S94/0~78
antibodies as well as active bifunctional antibodies.
Polyclonal anti-metal chelate antibodies can be
obtained by means known to those skilled in the art. See,
for example, Ghose, et al., Methods in Enzymoloqy,
93:326-327 (1983). Because such antiserum will contain a
plurality of both anti-metal chelate antibodies and non-
specific antibodies, the method of the present invention is
of particular utility as it permits separation of anti-metal
chelate antibodies from non-specific antibodies and proteins
and allows identification of fractions enriched in anti-
metal chelate antibodies. Such antiserum can contain anti-
metal chelate antibodies having only low affinity for metal
chelates whose retention time non-cognate carboxylic acid
derivatized solid supports can overlap the retention time of
other proteins found in the supernatant. The invention,
therefore, is particularly suited to separate those
antibodies having affinities for metal chelates of greater
than 108 L/M although it can also be used to separate those
antibodies having affinities of 107 L/M or even as low as 106
L/M, from non-specific antibodies, although the resulting
preparations may be accordingly less pure. The method is
particularly well suited to preparing fractions having a
high concentration of anti-metal chelate antibodies.
In accordance with the separation method of the present
invention, a solution containing the anti-metal chelate
antibodies, such as a cell culture supernatant, is dialyzed
into a buffer solution, such as 50mM sodium phosphate,
pH 7.5 and below, preferably between 5.6 and 6.8, most
preferably 6.8, and applied to a column of a non-cognate
carboxylic acid derivatized resin which has been
equilibrated in a starting solution, usually in the same
buffer solution or one having the same conductivity as that
in which the antibody is applied. Preferably, the resin is
a carboxymethyl ion exchange resin. Examples of
commercially available carboxymethyl (CM) resins include TSK
CM 5/PW (TosoHaas, Philadelphia, Pennsylvania) and (Bio-Rad
Laboratories, Richmond, California) and Pharmacia CM
W094/26772 l9 2 1 6 2 6 7 1 PCT~Sg4/0~78
Sepharose Fast Flow (Pharmacia Biotech Inc., Piscataway, New
Jersey). Examples of commercially available iminodiacetic
acid (IDA) resins include Chelate Column (Poros, Cambridge,
Massachusetts and TosoHaas). Other non-cognate carboxylic
acid derivatized solid supports can be used. Appropriate
support materials include polymeric resins, such as
polystyrene and polyester, glass and glass matrices, dextran
and cellulose, and polymer-coated supports although others
will be known to those skilled in the art. The non-cognate
carboxylic acid can be conjugated to the solid support
through means known to those skilled in the art. These
carboxylic acids can be attached to resin through aliphatic,
aromatic, or branched alkyls of varying length, provided
that a carboxylic acid group is available.
In one embodiment of the invention, such as is
appropriate for separating monoclonal anti-metal chelate
antibodies from non-specific antibodies and other non-
specific proteins, bound material is eluted from the column
using an elution solution with a linear gradient of
increasing salt concentration, by means well known in the
art. Various buffer salts can be used for this purpose
including, but not limited to, sodium phosphate, sodium
chloride sodium acetate and sodium sulfate. Preferably, a
linear gradient to 300 mM sodium phosphate, pH 7.5 or below,
is used. Alternatively, sodium phosphate buffer, pH 7.5 or
below in conjunction with a sodium sulfate gradient can be
employed. Collected eluate fractions can be assayed for
protein by means well known in the art, such as, for
example, ultraviolet absorbance. Anti-metal chelate
antibodies elute from the column at a later retention time
than do non-specific antibodies, thereby permitting their
separation.
An alternative embodiment as is appropriate for
separating active bifunctional anti-metal chelate antibodies
from other antibodies and proteins such as would be found in
the culture of a polydoma. When the culture fluid is
applied to the non-cognate carboxylic acid derivatized solid
W094/2C772 20 2 1 6 2 ~ 7 1 PCT~Sg4/0~78
phase support as described above and the eluants assayed for
protein concentration, various peaks are evident. The
bifunctional antibody will normally elute between the two
parental types. The identity of the peak which corresponds
to the active bifunctional can be determined by, for
example, a modified ELISA requiring both activities to be
present in a single moiety. The elution conditions can then
be selected so as to permit separation of the active
bifunctional anti-metal chelate antibodies from the other
- 10 antibodies species.
In a further embodiment, the invention provides a
method for obtaining elution fractions enriched in anti-
metal chelate antibodies from a polyclonal antiserum. When
the antiserum is run on a non-cognate carboxylic acid
derivatized solid phase support, as described above, protein
concentration approaches a Gaussian distribution. Anti-
metal chelate activity, as determined by, for example, a
quantitative ELISA determination, increases in the later
eluting fractions. Thus, by running the antiserum on a non-
cognate carboxylic acid derivatized solid phase support andselecting later eluting material, anti-metal chelate
antibody-enriched material will be obtained.
Although the invention is described in the following
examples under particular conditions, including the identity
of the solid support, the composition of the buffer, nature
of the gradient, the affinity of the particular antibodies
and so forth, it will be clear to one skilled in the art
that the particular eluant conditions to be used to separate
the desired antibody can be determined empirically. For
example, using the teaching herein, an eluent condition may
be found empirically in which non-specific antibodies will
not be retained on non-cognate carboxylic acid derivatized
solid supports, yet at which anti-metal chelate antibodies
will be retained. The anti-metal chelate antibody can be
removed from the non-cognate carboxylic acid derivatized
solid support by this same eluent having a salt
concentration selected to discriminate between the two types
wog4n6772 2l 2 1 6 2 6 7 ~ PCT~S94/0~78
of antibodies. The exact eluent conditions will depend on
the type of derivatized support, eluent composition, and
physical characteristics of both the anti-metal chelate
antibodies and non-metal chelate antibodies being separated
The determination of appropriate elution conditions can then
be applied to batch-mode purification, where the starting
solution is chosen so as to prevent binding of non-specific
proteins and the elution buffer is chosen to elute the
desired anti-metal chelate antibody.
The following examples are intended to illustrate but
not limit the invention.
EXAMPLE I
HP~C Chromatography of Anti-metal Chelate Antibodies
On a Sulfopropyl Column
A sulfopropyl (SP) column (75 x 7.5mm), packed with 10
micron TSK SP 5PW resin beads, was purchased from BioRad,
prepared according to the manufacturer's instructions and
equilibrated in 50mM sodium phosphate buffer, at the desired
pH for each run. The anti-metal chelate antibody, BxBFA, a
murine/human chimeric monoclonal bispecific antibody which
has a pI of 8.6 and specificities for In-benzyl EDTA metal
chelate and tumor CEA, was diluted 1:3 with the
equilibration buffer. lOOug of antibody was loaded onto the
column and allowed to bind to the matrix. In each trial the
antibody was eluted from the column using three or, in some
cases four pumps to mix mono and dibasic phosphate solutions
with water to obtain the desired combinations of pH and
phosphate concentration. A series of linear gradients of
increasing salt from 50mM to 300mM sodium phosphate over a
period of 100 minutes, were run at a pH of 8.0, 7.3, 6.7,
6.2 or 5.5 and a flow rate of lml/ minute. The presence of
antibody was determined by absorbance at 28Onm using a
Waters 490E (Milford, Massachusetts) variable wavelength
ultraviolet detector.
As can be seen from Figure 2, the retention time of the
antibody on the column increased as the pH was decreased.
W094/26772 22 2 1 6 2 6 7 ~ PCT~Sg4/0~78
This occurs due to the increasingly positive charge held by
the antibody at these lower pH values and their attraction
for the negative charges on the sulfopropyl column. The
retention time of BxBFA at pH 5.5 was about 50 minutes.
EXAMP~E II
Separation of Anti-metal Chelate Antibodies
On A Carboxymethyl Coll-~n
A carboxymethyl (CM) column (75 x 7.5mm) packed with 10
micron TSK CM 5/PW resin beads was purchased from Bio-Rad
Laboratories, prepared according to the manufacturer's
instructions, equilibrated, and antibody was prepared and
loaded onto the column as in Example 1. Also as in Example
I a series of elutions were conducted at pH 8.0, 7.3, 6.7,
6.2 and 5.5. The elution times of the antibody from the
carboxymethyl column are shown in Figure 3. Relative to the
sulfopropyl column, the BxBFA never eluted from the
carboxymethyl column at a pH of 5.5. Instead of pH 5.5, the
BxBFA can be eluted from the carboxymethyl column at pH 6. 2
with a retention of about 61 minutes, a longer retention
time than that seen with the sulfopropyl column at pH 5.5
under the same gradient conditions. This stroner retention
of the BxBFA by the carboxylmethyl column in comparison with
the sulfopropyl column occurs despite the fact that the
antibody becomes less positively charged at the higher pH.
This reduction in ionic attraction means that forces other
than the attraction of opposite cnarge are responsible for
the longer retention times on the carboxymethyl column.
Generally longer retention times on the carboxymethyl column
also mean that a purification can be done at a higher pH,
above pH 6 or even closer to the physiological pH of the
starting cell culture material. In the case of antibodies
produced through in vitro cell culture procedures, adjusting
the pH of the starting material to pH about 5.5 or below can
cause protein precipitates that include the antibody
therein.
W094/26772 23 2 1 6 2 6 7 I PCT~Sg4/0~78
EXAMPLE III
Effect of Hapten on Anti-metal
A carboxymethyl (CM) column (75 x 7.5mm) packed with 10
micron TSK CM 5/PW resin beads was purchased from Bio-Rad
Laboratories, and prepared according to the manufacturer~s
instructions. The column was equilibrated, a purified
sample of ECA 001, a murine polydoma produced bispecific
antibody with a pI of 6.5 and with specificities against In-
benzyl EDTA metal chelate and tumor CEA, was prepared and
loaded onto the column as in Example I. Elution proceeded
as in Example I except that the pH of the solutions were
9.4, 8.3, 7.4, 6.8, 6.2 and 5.5. The elution times were as
shown in Figure 4. The retention time for ECA 001 was about
5 minutes less at pH 6.2 than for the BxBFA in Example 2
above. The ECA 001 antibody has an attraction for the
carboxymethyl column similar in principle to that of the
BxBFA antibody, but since this antibody has more negative
charges relative to the BxBFA, as can be seen by comparing
their respective pI's, it is to some degree repelled by the
negatively charged carboxymethyl column. Although the two
antibodies have greatly different pI's, their common
specificity against In-benzyl EDTA metal chelate accounts
for similar retention on the column. As in Example 2 above,
at pH 5.5 the phosphate elution was unable to remove the
ECA 001 antibody from the column.
EXAMPLE IV
A iminodiacetic acid (IDA) column (75 x 7.5 mm) packed
with 10 micron resin beads was purchased as a Chelate Column
from TosoHaus and prepared according to the manufacturer/s
instructions. The column was equilibrated without metal
loading, and anti-metal chelate antibody BxBFA was prepared
and loaded onto the column as in Example I. A series of
phosphate buffer elutions were conducted as in Example I
except that the pH values were 9.0, 8.0, 7.3, 6.7, 6.2 and
5.5 and were monitored over time as shown in Figure 5. It
was impossible to elute the antibody from the column even
W094/26772 24 2 1 6 2 6 7 1 PCT~S94/0~78
with a buffer solution of 500mM sodium phosphate having a pH
of 9.4. It is presumed that the structure of iminodiacetic
acid is too similar to that of benzyl-EDTA, the cognate acid
chelator, to elute the metal chelate with a simple phosphate
salt solution.
EXAMPLE V
A iminodiacetic acid (IDA) column (75 x 7.5 mm) packed
with 10 micron resin beads was purchased as a Chelate Column
- from TosoHaus, and prepared according to the manufacturer~s
instructions. The column was equilibrated without metal
loading, and metal chelate antibody BxBFA was prepared and
loaded onto the column as in Example I. Elutions were
conducted following the procedure of Example I except that a
linear gradient from zero to 240mM of glutamic acid was used
at pH values of 8.2, 7.2, 6.7, 6.2 and 5.6. The same
procedure was followed using a glycine elution at pH values
of 8.1, 7.3, 6.7, 6.2 and 5.6 The elution times were as
shown in Figures 6a and 6b. The pH was maintained
throughout the gradient with sodium phosphate buffers
adjusted to a final concentration of 60mM. It is believed
that the structures of glutamic acid and glycine were
similar enough to that of the cognate metal/benzyl-EDTA to
free the antibody from the column whereas a phosphate salt
solution could not.
EXAMPLE VI
A iminodiacetic acid (IDA) column was prepared,
equilibrated and loaded as in Example V, except that the
antibody was a purified sample of ECA 001. Elutions were
conducted following the procedure of Example I, but using a
linear gradient of sodium sulfate to elute the column.
Again the pH of the elution solution was maintained using 60
mM phosphate at a value of 9.4, 8.1, 7.4, 6.8, 6.2 or 5.2.
The elution times were as shown in Figure 7. This example
shows that sulfate acts as a non-cognate hapten in a manner
like that of glutamic acid.
W094/26772 25 2 1 6 2 6 7 I PCT~S94,04878
-
EXAMPLE VII
A glutamic acid (GLU) column was prepared with 1o
micron resin beads according to the manufacturer's
instructions using a Tresyl 5/PW column (TosoHaus). The
column was equilibrated and loaded as in Example V, except
that the antibody was BxBFA. Elutions were conducted
following the procedure of Example I, but using a linear
gradient of 50 to 300 mM sodium phosphate to elute the
column at pH values of 8.1, 7.4, 6.9, 6.4 and 5.7. The
- 10 elution times were as shown in Figure 8.
Although the invention has been described in terms of
the presently preferred embodiments, it will be apparent to
one skilled in the art that modifications can be made
without departing from the spirit of the invention. Thus,
the invention is limited only by the following claims.
