Note: Descriptions are shown in the official language in which they were submitted.
- 1 -
STABLE N-TERMINALLY LINKED DTPA:PROTEIN COMPOSITIONS AND
METHODS
The present invention broadly relates to the
field of protein modification, more specifically,
to diethlyenetriaminepentaacetic acid (DTPA) :protein
composition wherein the chelating agent DTPA has been
conjugated site-specifically to the N-terminus of the
protein, thereby providing a homogenous and well-defined
product capable of forming complexes with a variety of
metallic radionuclides. In another aspect, the
invention relates methods of conjugating DTPA to
granulocyte colony stimulating factor (G-CSF) or
interleukin-2 (IL-2), thereby providing a useful
procedure of radio-labeling such proteins and related
proteins including cytokines, while maintaining the
structural and functional integrity of the protein.
Background of the Invention
Radioactive labeling of the proteins and other
biological compounds is commonly achieved by iodination.
Proteins may be successfully labeled with radioisotopes
of iodine by a number of methods; Reogoeczi, E., Iodine-
Labeled Plasma Proteins, 1, 53, (CRC Press, Boca Raton,
Fla 1982), and antibodies so labeled have been used in
radioimmunodetection studies in which tumor localization
is determined by external imaging; Keenan et al., J.
Nucl. Med., 26, 531 (1985). However, in the course of
these investigations and others involving the use of
radioisotopes of iodine, certain limitations to the use
of radioiodine imaging procedures became apparent (e.g.,
the poor imaging characteristics of many of the
radioisotopes of iodine, the involved labeling
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procedures, and the high degree of instability of the
label in vivo). In addition, the most common iodination
methods involve oxidizing conditions in the reaction
mixtures which can modify other sensitive groups and
cause alteration of protein structure, and possible
biological inactivation.
To avoid the difficulties encountered when
trying to iodinate these proteins and other compounds,
alternative methods were employed. One such method is
the "bifunctional chelate" method, in which strong
chelating groups are covalently attached to proteins so
that the protein bound chelate can then form complexes
with a variety of metallic radionuclides; Meares &
Goodwin, ~. Prot. Chem., 3, 215-228, (1984),
paramagnetic metal ions; Lauffer & Brady, Magn. Reson.
Imag., 3, 11-16, (1985); Ogan et al., Invest. Radiol.,
~, 665-671, (1987), and flourescent metals; Mukkala et
al., Anal. Biochem., 176, 319-325, (1989).
The reagent most commonly used for the
covalent modification of proteins with a chelating agent
is the cyclic dianhydride of DTPA. The cyclic
dianhydride of DTPA generally forms stronger chelates
than the analogs of ethylenetriaminetetraacetic acid
(EDTA); Perrin et al., Organic Ligands, IUPAC Chemical
Data Series No. 22,(New York, Pergamon Press 1982), and
involves less complicated synthesis procedures than
those involved when using analogs of EDTA. In addition,
the cyclic dianhydride of DTPA is stable indefinitely at
room temperature, thereby providing for greater control
on the conditions of coupling. Hnatowich and McGann,
Int. ~. Rad. Appl. Instrum., [B], 14, 563-568 (1987).
Coupling of DTPA to proteins is routinely performed at
pH > 7.0, where the dianhydride reacts primarily with
free amine groups (e.g., available lysine residues) to
35 form amide bonds; Hnatowich et al., Science, 220, 613-
615, (1983a).
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A primary concern for one performing these
covalent modifications is that there can be many
possible sites on each protein where chelators can be
attached. The currently existing methods provide for
non-selective attachment at any reactive group, whether
located within the protein, such as a lysine side group,
or at the N-terminus. This results in a heterogenous
population. For example, reaction of DTPA dianhydride
with insulin yielded a complex mixture of several
products, including cross-linked protein and acylated
tyrosine residues; Maisano et al., Bioconj. Chem., 3,
212-217 (1992), while reaction of albumin with DTPA
dianhydride produced protein molecules with multiple
chelating groups attached; Lauffer & Brady, Magn. Reson.
Imag., 3, 11-16, (1985).
The number of DTPA groups conjugated to the
protein is often given as an average number, as sample
preparations are heterogenous, each having protein with
both more and less chelating groups than the average
number; Hnatowich and McGann, Int. J. Rad. Appl.
Instrum., [B], 1~, 563-568 (1987). It is well known
that proteins may be degraded by covalent attachment of
chelating groups, with the extent of degradation
increasing with increasing substitution; Sakahara et
al., J. Nucl. Med., 26, 750, (1985). Those protein
molecules containing several chelating groups are least
likely to retain their native biological properties;
Meares and Goodwin, Jour. of Prot. Chem., 3, 215-228
(1984). From a producer's point of view, garnering
30 regulatory approval for sale of these heterogenous
therapeutic proteins may have added complexities.
The properties in vivo of chelate-tagged
proteins have been reviewed; Meares et al., Adv. Chem.,
198, 369-387 (1982). The most general observations are
3S that in vivo stability depends critically on the
chemical nature of the protein-chelator conjugate, that
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the more lightly labeled proteins have longer biological
half-lives, and that retention of activity is made more
likely by procedures (e.g. specific labeling) that
~;ni~i ze the labeling of residues involved in the active
site(s). For example, horse serum albumin (HSA) was
conjugated with chelating agent, labeled with lllIn, and
when injected in vivo was rapidly cleared by the liver
(as compared to results following l2sI label on iodinated
HSA); Leung and Meares, Biochem. Biophys . Res . Commun .,
75, 149-155 (1977). The chelate-conjugated HSA, at
least the population with the most numerous chelating
groups and thus representing a large percentage of the
followed radioactivity, may have been recognized in vivo
as foreign protein; Meares and Goodwin, ~our. of Prot.
Chem., 3, 215-228 (1984). The advantage of avoiding a
random and numerous distribution of products by
specifically labeling a single (nonessential) site on a
protein is evident.
Covalent coupling of DTPA to proteins using
DTPA dianhydride has been described by several
investigators. For example, Khaw et al., Science, ~Q~,
295, (1980) coupled DTPA to immunoglobulin G (IgG)
fragments active against myosin and investigated the
localization of the labeled protein in canine myocardial
infarcts. Using the same method, Scheinberg et al.,
Science, 215, 1511, (1982) prepared labeled monoclonal
antibody specific for erythroleukemic cells in mice.
Although these methods and others provide coupled
proteins, they are invariably characterized by
complicated syntheses and by low coupling efficiencies.
Hnatowich et al., Science, 220, 613-615, (1983a).
U.S. Patent No. 4,479,930 (Hnatowich)
discloses compositions comprising a dicyclic dianhydride
coupled to an amine, and chelated with a radioisotope
metallic cation. The compositions are reported to be
stable in vivo. Methods of preparing the compositions
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are also disclosed. It is reported that the initial and
final pH of the coupling reaction mixture is pH 7.0 in
~ all instances, and that coupling efficiency (defined as
the percentage of anhydride molecules which covalently
attach to the polypeptide or protein) is high when
anhydride to antibody molar ratios are held at 1:1, but
decrease at pH values above or below neutrality. There
is no teaching as to the distribution of the DTPA moiety
on the proteins or polypeptides of the various reaction
products.
Nothing can be drawn from the literature
concerning the preparation of DTPA:protein conjugates
which are advantageous over those previously described
due to the fact that the conjugation is site-specific to
the N-terminus of the protein, thereby yielding a more
well-defined, homogenous composition. The compositions
can be produced in large quantities and retain full in
vivo bioactivity, either with or without chelated
metallic radionuclide. The synthesis described in the
present invention is a simple one step reaction wherein
a single reactive site is created, providing a useful
method for labeling proteins. The DTPA:protein
conjugates of the present invention may have potential
use in diagnosis, imaging, and/or treatment of leukemia
and related diseases.
SUMMARY OF THE INV~NTION
The present invention relates to substantially
homogenous preparations of N-terminally chemically
modified proteins, and methods therefor. Unexpectedly,
the conjugation of the chelating agent DTPA is site-
- specific to the N-terminus of the protein, thereby
providing a more homogenous and well-defined product as
compared to other chelating agent:protein compositions.
Also unexpectedly, the preferred DTPA:G-CSF conjugates
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can chelate a number of metallic radionuclides yielding
a radio-labeled product, while maintaining the
structural and functional integrity of the protein, and
this method is broadly applicable to other proteins (or
analogs thereof), as well as G-CSF.
In one aspect, the present invention relates
to a substantially homogenous preparation of DTPA:G-CSF
(or analog thereof) and related methods. One working
example below demonstrates that the chelating agent DTPA
is conjugated site-specifically to the N-terminus of
rhG-CSF, and that such compostion is capable of forming
complexes with a variety of metallic radionuclides.
Since the conjugation is specific to the N-terminus of
the G-CSF molecule, the resulting product is a more
homgenous and well-defined product than those previously
described.
The present invention also relates to a method
for preparing a labeled protein, said method comprising:
(a) reacting a chelating agent with said protein at a
pH sufficiently acidic to selectively activate the a-
amino group at the amino terminus of said protein; (b)
separating the conjugated protein from non-conjugated
protein; (c) adding a metallic cation to said
conjugate; and (d) obtaining the labeled protein. This
method is described below for rhG-CSF and IL-2, and
these provide for additional aspects of the present
invention.
BRIFF DESCRIPTION OF TH~ DRAWINGS
FIGURE l shows the effects of initial pH and
DTPA:protein molar ratio on the coupling of rhG-CSF.
SDS-PAGE analysis of the following samples was
performed: lane l- MW markers; lanes 2-4, DTPA:rhG-CSF
at 5:l, 50:l, and 500:l, respectively, at initial pH
6.0; lanes 5-7, DTPA:rhG-CSF at 5:1, 50:1, and 500:1,
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WO96/1~816 PCT~S95/1~072
respectively, at initial pH 7.0; lanes 8-10, DTPA:rhG-
CSF at 5:1, 50:1, and 500:1, respectively, at initial pH
~ 8.0; lane 11, rhG-CSF at pH 6.0; lane 12, rhG-CSF at pH
8Ø
., 5
FIGURE 2 shows size-exclusion HPLC elution
plots for rhG-CSF starting material (line 1), DTPA:rhG-
CSF conjugation reaction mixture before passage through
a G50 spin column (line 2), and DTPA:rhG-CSF conjugation
reaction mixture after passage through a G50 spin column
(line 3). Elution was monitored for absorbance at 280
nm.
FIGURE 3 shows preparative cation-exchange
FPLC elution plots for rhG-CSF starting material (dashed
line) and DTPA:rhG-CSF reaction mixture (solid line).
Elution was monitored for absorbance at 280 nm.
FIGURE 4 shows analytical cation-exchange HPLC
analysis of rhG-CSF (lines 2 and 3) and DTPA:rhG-CSF
conjugate (lines 1 and 4) preincubated with lllIn.
Elution was monitored for absorbance at 220 nm (lines 3
and 4) and for radioactivity ~lines 1 and 2, inverted).
FIGURE 5 shows analytical cation-exchange HPLC
analysis of rhG-CSF (line 1), DTPA:rhG-CSF conjugate
(line 2), and DTPA:rhG-CSF conjugate treated with excess
InCl3 (line 3). Elution was monitored for absorbance at
220 nm. EDTA (lmM) was added to Buffer A.
- FIGURE 6 shows silica gel TLC plate analysis
of the following samples: lane 1, 0.1 nmol 1l1In (indium
- with a trace of 111In); lane 2, 10 nmol 111In added to 20
nmol DTPA; lane 3, 10 nmol 111In incubated with 2 nmol
rhG-CSF, followed by addition of 20 nmol DTPA; and lane
4, 10 nmol lllIn incubated with 2 nmol DTPA:rhG-CSF
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WO96/lS816 PCT~S95/lS072
conjugate, followed by addition of 20 nmol DTPA. For
lanes 2-4, aliquots containing O.l nmol 1l1In were taken
from the mixtures and loaded onto the plate.
FIGURE 7 shows the MALDI-MS spectrum of the
DTPA:rhG-CSF conjugate. The spectrum shows the multiply
protonated species (l, 2, 3 and 4 protons attached).
FIGURE 8 shows the ion-spray mass spectrum of
the DTPA:rhG-CSF conjugate with chelated indium. The
conjugate was preincubated with saturating InCl3
(In:conjugate, lO:l, mol/mol) before analysis.
FIGURE 9 shows the ion-spray mass spectrum of
rhG-CSF.
FIGURE lO shows peptide mapping of the
DTPA:rhG-CSF conjugate. Peptide fragments generated
from the DTPA-rhG-CSF conjugate (solid line) and rhG-CSF
(dashed line) by proteolysis were reduced, alkylated,
and then resolved by reversed-phase HPLC. Elution was
monitored for absorbance at 215 nm. The arrow indicates
elution of the N-termi n~l peptide from the digested rhG-
CSF sample.
FIGURE ll shows an isoelectric focusing gel
(pH 3-lO) containing the following samples: lane l, rhG-
CSF; lane 2, DTPA:rhG-CSF conjugate preincubated with
excess InCl3 (In:conju~ate, lO:l, mol/mol); lane 3,
DTPA:rhG-CSF conjugate; and lane 4, isoelectric point
markers.
FIGURE 12 shows circular dichroism (CD)
spectra of the DTPA:rhG-CSF conjugate without (----) and
with (_ _) chelated indium, and of unmodified rhG-CSF
( ) and DTPA (_._.). Samples (0.078 mg/ml protein,
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and 4.07 ~M DTPA) were analyzed at 10~C in 20mM sodium
acetate, pH 5.4. The unmodified rhG-CSF sample and
DTPA:rhG-CSF sample (without indium) are identical.
FIGURE 13 shows the effects of DTPA
conjugation on the in vivo activity of rhG-CSF. Activity
(WBC count) was measured after subcutaneous injection of
hamsters. The rhG-CSF dose was 100 ~g/kg. Bars
represent standard deviation (n = 8-10 for protein
samples, and n = 5-6 for baseline).
FIGURE 14 shows analytical cation-exchange
HPLC analysis of IL-2 (lines 2 and 3) and DTPA:IL-2
conjugate (lines 1 and 4) preincubated with 1llIn.
Elution was monitored for absorbance at 220 nm (lines 3
and 4) and for radioactivity (lines 1 and 2, inverted).
FIGURE 15 shows analytical cation-exchange
HPLC analysis of IL-2 (line 1), DTPA:IL-2 conjugate
(line 2), and DTPA:IL-2 conjugate treated with excess
InCl3 (line 3). Elution was monitored for absorbance at
220 nm. EDTA (lmM) was added to Buffer A.
FIGURE 16 shows silica gel TLC plate analysis
of the following samples: lane 1, 0.1 nmol 1l1In (indium
with a trace of ll1In); lane 2, 10 nmol 11lIn added to 20
nmol DTPA; lane 3, 10 nmol 111In incubated with 2 nmol
IL-2, followed by addition of 20 nmol DTPA; and lane 4,
10 nmol 111In incubated with 2 nmol DTPA:IL-2 conjugate,
followed by addition of 20 nmol DTPA. For lanes 2-4,
~ aliquots containing 0.1 nmol l11In were taken from the
mixtures and loaded onto the plate.
.
FIGURE 17 shows peptide mapping of the
DTPA:IL-2 conjugate. Peptide fragments generated from
the DTPA-IL-2 conjugate (solid line) and IL-2 (dashed
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WO96/lS816 PCT~S95/15072
-- 10 --
line) by proteolysis were reduced, alkylated, and then
resolved by reversed-phase HPLC. Elution was monitored
for absorbance at 215 nm. The arrow indicates elution
of the N-terminal peptide from the digested IL-2 sample.
D~TATTl~n DE~CRIPTION
The DTPA:protein conjugates of the present
invention are described in more detail in the discussion
that follows and are illustrated by the examples
provided below. The examples show various aspects of
the invention and include results of biological activity
testing of various DTPA:protein conjugates.
Surprisingly, using the methods of the present
invention, a single reactive site was created such that
the resulting DTPA conjugation is site-specific to the
N-terminus of the protein, yielding a well-defined and
homogeneous composition capable of forming complexes
with a variety of metallic radionuclides, while
maintaining the structural and functional integrity of
the protein.
Contemplated for use in the practice of the
present invention are a variety of cytokines and related
proteins. Exemplary proteins contemplated include
various hematopoietic factors such as the aforementioned
G-CSF, GM-CSF, M-CSF, the interferons (alpha, beta, and
gamma), the interleukins (1-14), erythropoietin (EPO),
fibroblast growth factor, stem cell factor (SCF),
megakaryocyte growth and development factor (MGDF),
platelet-derived growth factor (PDGF), and tumor growth
factor (alpha, beta).
Granulocyte colony stimulating factor
(G-CSF) is a glycoprotein which induces differentiation
of hemapoietic precursor cells to neutrophils, and
stimulates the activity of mature neutrophils.
Recombinant human G-CSF (rhG-CSF), expressed in E. coli,
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WO9611S816 r~~ 3~15~72
contains 175 amino acids, has a molecular weight of
18,798 Da, and is biologically active. Currently,
Filgrastim, a recombinant G-CSF, is available for
therapeutic use.
The structure of G-CSF under various
conditions has been extensively studied; Lu et al.,
J. Biol. Chem. Vol. 267, 8770-8777 (1992), and the
three-~;m~n~ional structure of rhG-CSF has recently
been determined by x-ray crystallography. G-CSF is a
member of a class of growth factors sharing a common
structural motif of a four a-helix bundle with two long
crossover connections; Hill et al., P.N.A. S. USA, Vol.
90, 5167-5171 (1993). This family includes GM-CSF,
growth hormone, interleukin-2, interleukin-4, and
interferon $. The extent of secondary structure is
sensitive to the solvent pH, where the protein acquires
an even higher degree of alpha helical content at acidic
pH; Lu et al., Arch. Biochem. Biophys., ~, 81-92
(1989).
In general, G-CSF useful in the practice of
this invention may be a form isolated from m~mm~lian
organisms or, alternatively, a product of chemical
synthetic procedures or of prokaryotic or eukaryotic
host expression of exogenous DNA sequences obtained by
genomic or cDNA cloning or by DNA synthesis. Suitable
prokaryotic hosts include various bacteria (e.g.,
E. coli); suitable eukaryotic hosts include yeast (e.g.,
S. cerevisiae) and m~mm~lian cells (e.g., Chinese
hamster ovary cells, monkey cells). Depending upon the
host employed, the G-CSF expression product may be
glycosylated with mammalian or other eukaryotic
carbohydrates, or it may be non-glycosylated. The G-CSF
expression product may also include an initial
methionine amino acid residue (at position -1). The
present invention contemplates the use of any and all
such forms of G-CSF, although recombinant G-CSF,
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WO g6/1S816 PCI'IUS9S/lS072
especially E. coli derived, is preferred, for, among
other things, greatest commercial practicality.
Certain G-CSF analogs have been reported to be
biologically functional, and these may also be
chemically modified. G-CSF analogs are reported in U.S.
Patent No. 4,810,643. Examples of other G-CSF analogs
which have been reported to have biological activity are
those set forth in AU-A-76380/91, EP O 459 630, EP O 272
703, EP O 473 268 and EP O 335 423, although no
representation is made with regard to the activity of
each analog reportedly disclosed. See also
AU-A-10948/92, PCT US94/00913 and EP 0 243 153.
Generally, the G-CSFs and analogs thereof
useful in the present invention may be ascertained by
practicing the chemical modification procedures as
provided herein and testing the resultant product for
the desired biological characteristic, such as the
biological activity assays provided herein. Of course,
if one so desires when treating non-human m~mmAls, one
may use recombinant non-human G-CSF's, such as
recombinant murine, bovine, canine, etc. See PCT WO
9105798 and PCT WO 8910932, for example.
Interleukin-2, a glycoprotein with a molecular
weight of approximately 15,000 daltons, is a member of
the group called lymphokines that mediate immune
responses in the body. This protein is produced by
activated T-cells and is known to possess various
activities in vivo. For instance, IL-2 has been
reported to enhance thymocyte mitogenesis, induce T-cell
reactivity, regulate gamma interferon, and augment the
recovery of the immune function of lymphocytes in
selected immunodeficient states. It has potential
application in research and the treatment of neoplastic
and immunodeficiency diseases and has been employed in
therapies for the treatment of cancer.
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IL-2 useful in the practice of this invention
may be a form isolated from m~mm~l ian organisms or,
~ alternatively, and especially if an IL-2 analog, a
product of chemical synthetic procedures or of
< 5 prokaryotic or eukarYotic host expression of exogenous
DNA se~uences obtained by genomic or cDNA cloning or by
DNA synthesis. Suitable prokaryotic hosts include
various bacteria (e.g., E. coli); suitable eukaryotic
hosts include yeast (e.g., S. cerevisiae) and mammalian
10 cells (e.g., Chinese hamster ovary cells, monkey cells).
Depending upon the host employed, the IL-2 expression
product may be glycosylated with m~mmAlian or other
eukaryotic carbohydrates, or it may be non-glycosylated.
The IL-2 expression product may also include an initial
15 methionine amino acid residue (at position -1). The
present invention contemplates the use of any and all
such ~orms of IL-2 and its analogs, although recombinant
IL-2 and analogs, especially E. coli derived, are
preferred, for, among other things, greatest commercial
20 practicality.
Methods for the preparation of IL-2 by
isolation and purification of the naturally occurring
form or by genetically engineered means are known. See,
for instance, U.S. Patent Nos. 4,778,879; 4,908,434; and
25 4,925,919 (Mertelsman et al.); U.S. Patent No. 4,490,289
(Stein); U.S. Patent No. 4,738,927 (Taniguchi et al.);
U.S. Patent No. 4,569,790 (Koths et al.); U.S. Patent
No. 4,518,584 (Mark et al.); U.S. Patent No. 4,902,502
(Nitecki et al.) and European patent 0136489 (Souza
et al.).
- The IL-2 receptor (IL-2R) is constitutively
overexpressed in various hematologic malignancies
- including adult T-cell leukemia (Uchiyama, et al.,
1985), hairy cell leukemia (Trentin, et al., 1992),
chronic lymphocyte leukemia (Rosolen, et al., 1989),
Hodgkin's disease (Strauchen and Breakstone, 1987), and
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- 14 -
non-Hodgkin's lymphoma (Grant, et al., 1986).
Lymphocytes involved in several autoimmune diseases,
including rheumatoid arthritis (Lemm and Warnatz, 1986)
and allograft rejection (Waldmann, 1989) also
overexpress the IL-2R. This receptor has therefore been
actively pursued as a target for cytotoxic therapy.
Recombinant fusion toxins have been produced in which
the cell-binding domain of Pseudomonas exotoxin
(Lorberboum-Galski, et al., 1988a) or the receptor-
binding domain of diphtheria toxin (Williams, et al.,1987) have been replaced with IL-2. These fusion
proteins are specifically cytotoxic to cells that
express the high affinity IL-2R (Lorberboum-Galski, et
al., 1988b; Williams, et al., 1990). A recently
described Pseudomonas exotoxin/IL-4 ch;meric protein may
also prove useful for the treatment of autoimmune
diseases, allograft rejections, and many hematologic
malignancies where cells express elevated levels of IL-4
receptor (Puri, et al., 1994). A diphtheria toxin-
related human G-CSF fusion protein has also recently
been constructed which may have usefulness in the study
and treatment of leukemia (Chadwick, et al., 1993).
Conjugation of DTPA to IL-2, IL-4, as well as rhG-CSF
may also have potential use in diagnosis, imaging and/or
treatment of leukemia and related diseases. Chelatable
radiometals for cytotoxic therapy may include 2l2Bi,
21lAt, and 90Y. Indeed, antibodies to the IL-2R a chain,
and bearing radioisotopes 2l2Bi and 90Y via conjugated
bifunctional chelates, are being examined by several
investigators (Junghans, et al., 1993; Parenteau, et
al., 1992; Kozak, et al., 1990) for cytotoxicity towards
alloreactive T-cell lines, and for potential
radiotherapy.
The DTPA useful in the conjugations of the
present invention is technical grade DTPA dianhydride.
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-- 15 --
In a preferred embodiment involving E. coli
derived rhG-CSF, the DTPA:rhG-CSF conjugation occurs at
an initial pH of 6.0, and a 50:1 DTPA:rhG-CSF molar
ratio.
In a preferred embodiment involving E. coli
derived IL-2, the DTPA:IL-2 conjugation occurs at an
initial pH of 6.0, and a 50:1 DTPA:IL-2 molar ratio.
Although the invention has been described and
illustrated with respect to specific DTPA:protein
conjugates and treatment methods, it will be apparent to
one of ordinary skill that a variety of related
conjugates, and treatment methods may exist without
departing from the scope of the invention.
The following examples will illustrate in more
detail the various aspects of the present invention.
F~XZ~,MpT.F. 1
The DTPA used for conjugation is initially the
dianhydride form, and therefore there exists the
potential for undesirable side-reactions such as
protein:protein crosslinking; Hnatowich et al.,J.
Immuno. Methods, 65, 147-157, (1983b). Reaction
conditions such as initial pH and DTPA dianhydride:rhG-
CSF molar ratio were therefore investigated in order to
min;mize the formation of such products. The rhG-CSF
was produced using recombinant DNA technology in which
E. coli cells were transfected with a DNA se~uence
encoding human G-CSF as described in U.S. Patent No.
- 4,810,643 to Souza. The rhG-CSF was prepared as a 2.75-
4 mg/ml solution in 100 mM sodium phosphate buffer, pH
- 6Ø DTPA dianhydride and tributylphosphine (TBP,
technical grade) were obtained from Aldrich (Milwaukee,
WI).
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WO g6/1S816 P~ 9SI1SO72
- 16 -
Pre~aration of DTPA:rhG-CSF Coniuaate
Various amounts of DTPA dianhydride were
placed in dry acid-washed (Meares, et al., J. Prot.
Chem., 3, 215-228, (1984)) test tubes. Two milliliters
of anhydrous chloroform (Aldrich Chemicals, Milwaukee,
WI) was then added, and the tube vortexed under a light
stream of nitrogen gas to evaporate the chloroform and
form a thin film of DTPA dianhydride at the bottom of
the tube. rhG-CSF at a concentration of 2.75-4.0 mg/ml
in lOOmM sodium phosphate buffer, pH 6.0, pH 7.0, or pH
8.0 was added to the DTPA dianhydride-coated tubes to a
final molar ratio of 5:1, 50:1, or 500:1 (DTPA:rhG-CSF)
while gently swirling. Aliauots of each sample were
maintained and the bulk of the sample passed through a
G50 spin column as described; Penefske, H.S., Methods
Enzymol., 56, 527-530, (1979), in order to remove
unconjugated DTPA.
An~lvsis of the DTPA:rhG-CSF Coniuaate
1. SDS-PAGE Analysis.
SDS-PAGE was performed on the G50 spin column
treated samples using 17-27% ISS MiniPlus gels (Nattick,
MA). Samples were diluted with nonreducing buffer, and
5~g of protein was loaded into each well. The gels were
run on a discontinuous buffer system and stained with
Coomassie Blue R-250 (Laemmli, U.K., Nature, 227, 680-
685, (1970). SDS-PAGE analysis of the samples prepared
above is shown in Figure 1.
The reactions conducted with an initial pH of
7.0 (lanes 5-7) or 8.0 (lanes 8-10) yielded significant
amounts of higher molecular weight species in comparison
to the apparent molecular weight observed for untreated
rhG-CSF (lanes 11 and 12). However, the reactions with
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-- 17 -
an initial pH of 6.0 (lanes 2-4) yielded a single
detectable higher molecular weight species (which
corresponded to one of the species detected in the pH
7 . 0 and 8.0 samples). The apparent molecular weight of
this species suggests it may be a DTPA-cross-linked
protein dimer. With the pH 6.0 samples, this higher
molecular weight band became less intense with
increasing DTPA:rhG-CSF molar ratio, and was virtually
absent for the 500:1 sample (lane 4).
2. Size Exclusion HPLC.
The reaction mixture with an initial pH of
6.0, and DTPA:rhG-CSF ratio of 50:1 was further analyzed
by size-exclusion HPLC. A pre-G50 spin column and post-
G50 spin column sample was analyzed. HPLC was performed
on a Waters Li~uid Chromatograph (Milliford, MA)
equipped with a WISP 717 plus auto sampler refrigerated
at 5~C, and a 490E multiwavelength W /Vis detector in
line with a Raytest Ramona LS radioisotope detector
(Pittsburgh, PA). The void volume between the W /Vis
detector and the radioisotope detector was 50 ~l. For
size-exclusion HPLC, samples were analyzed with an
isocratic mobile phase of O.lM sodium phosphate buffer,
0.5M NaC1, pH 6.9, on a Phenomenex BioSep S2000 column
(Torrance, CA) eluted at 1.0 ml/min at 25~C. Elution
was monitored for absorbance at 280 nm and recorded by
Waters Millennium software on a PC computer.
As shown in Figure 2, the pre-G50 spin column
sample revealed two major peaks with elution times of
8.65 and 9.53 minutes (Figure 2, line 2). The second
major peak coelutes with free DTPA and was nearly
eliminated in the post-G50 spin column sample (Figure 2,
line 3), indicating successful removal of unbound DTPA
from the reaction mixture. The elution time of the
r~m~;n;ng major peak was unchanged by the G50 spin
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- 18 -
column and eluted slightly before unreacted rhG-CSF
(Figure 2, line 1). This peak represents monomeric
DTPA-conjugated rhG-CSF. Thus, the behavior of the
DPTA-conjugated rhG-CSF on this size-exclusion column
allows it to be resolved from unmodified rhG-CSF.
The analysis above shows that initial pH and
DTPA:rhG-CSF molar ratio can affect formation of
undesirable side reactions. By initiating the reaction
in buffer with an initial pH of 6.0, the formation of
products resulting from such side reactions (e.g.
protein crosslinking) is greatly reduced.
~AMPr,~ 2
In this example, the ability of the
DTPA:rhG-CSF conjugate to chelate l1lIn was determined
using analytical cation-exchange HPLC and thin layer
chromatography. The conjugate was then further analyzed
to determine the mass of the conjugate (with and without
chelated indium), the stoichiometic molar ratio of DTPA
to rhG-CSF, and the location of the conjugated DTPA
moiety on the rhG-CSF.
Pre~aration of DTPA:~hG-CSF Coniuaate
In this example, analysis was conducted on a
DTPA:rhG-CSF conjugate prepared using an initial pH of
6.0, and a 50:1 DTPA dianhydride:rhG-CSF molar ratio as
described in Example 1. However, instead of passing the
sample through a G50 spin column, preparative cation-
exchange chromatography was performed using a Pharmacia
Hi-Load SP-Sepharose High Performance, 16/10, strong
cation-exchange column (Pharmacia, Sweden). Separation
was accomplished at 5~C by a Pharmacia FPLC system
e~uipped with a 50 ml injection loop. The column was
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-- 19 --
equilibrated in Buffer A (20mM sodium acetate, pH 5.4)
and elution was carried out with a 0-40% Buffer B (20mM
sodium acetate, 0.5M NaCl, pH 5.4) gradient over 180
minutes at 1.0 ml/minute. Elution was monitored for
absorbance at 280 nm and recorded.
A reaction mixture (initial pH ~.0, 50:1 DTPA
dianhydride:rhG-C~F mola~ ra~io) originally containing
20 mg of rhG-CSF was diluted to 50 ml with Milli-Q water
and directly applied to the Hi-Load SP-Sepharose column.
A peak representing approximately 13% of the integrated
peak areas (Figure 3, peak 2) coeluted with control
unreacted rhG-CSF (Figure 3, dashed line). This
indicates that approximately 13% of the rhG-CSF remained
unmodified. A peak eluted between 120 and 130 minutes
contained approximately 84% of the total eluted protein
(Figure 3, peak 1). The shift in this material to elute
at a lower salt concentration is in agreement with an
increase in negative charge on the protein via
conjugation with DTPA.
This simple, efficient chromatography step
yields a homogeneous and well-defined product with both
unbound DTPA and unconjugated rhG-CSF separated from the
purified DTPA:rhG-CSF conjugate.
~n~lvsis of the DTPA:rhG-CSF Coniua~te
1. Analytical Cation-Exchange HPLC.
Analytical cation-exchange HPLC was performed
with mobile phases of Buffer A (20mM sodium acetate, pH
5.4), and Buffer B (20mM sodium acetate, 0.5M NaCl, pH
5.4) on a Tosohaas SP-5PW, 7.5 X 7.5 mm column
(Montgomery, PA) using the Waters HPLC system. The
column was equilibrated with mobile phase A, and
separation was performed at 25~C with a 1% B/min linear
gradient over 30 minutes at 1.0 ml/minute. Separation
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-- 20 --
was detected by monitoring absorbance at 22 0 nm, and
where applicable, with the radioisotope detector. For
samples cont~;n;ng indium, lmM EDTA was added to Buffer
A before adjusting the pH to 5.4.
rhG-CSF and DTPA:rhGCSF conjugate were
preincubated with 111In for 15 minutes. An excess of cold
indium was then added (In:protein, 2:1, mol/mol), and
the samples analyzed by analytical cation-exchange HPLC
as described above. For the DTPA:rhG-CSF conjugate
(Figure 4, lines 1 and 4, solid), 99.5% of the l1lIn
radioactivity coeluted from the cation-exchange column
with the protein, whereas no detectable radioactivity
co-eluted with unmodified rhG-CSF (Figure 4, lines 2 and
3, dashed), indicating chelation of lllIn by the
DTPA:rhG-CSF conjugate, and absence of lllIn binding by
unmodified rhG-CSF.
The effect of metal chelation on analytical
cation-exchange HPLC analysis of the DTPA:rhG-CSF
conjugate is depicted in Figure 5. Elution of rhG-CSF
(line 1), the DTPA:rhG-CSF conjugate (line 2), and the
conjugate preincubated with excess InCl3 (In:conjugate,
10:1, mol/mol, line 3) was monitored for absorbance at
220 nm. The DTPA:rhG-CSF conjugate eluted at a lower
salt concentration than unmodified rhG-CSF. The
chelated conjugate then elutes at a slightly higher salt
concentration than the non-chelated conjugate, but still
at a salt concentration lower than that of unmodified
rhG-CSF. The characteristic retention times of the
DTPA:rhG-CSF conjugate with and without chelated metal
may be used to monitor metal contamination of the
conjugate preparation. Furthermore, this analysis may
be used to monitor metal labeling of the conjugate.
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WO96115816 PCT~S9~lS072
2. Thin Layer Chromatography (TLC)
TLC was performed as previously described
(Meares et al., ~. Prot. Chem., 3, 215-228, (1984)) with
slight modification. An indium stock solution
containing InCl3 with a trace of lllIn was prepared in
lOmM HCl, and was used to prepare the following samples:
(1) indium added to l00mM sodium phosphate, pH 6.0; (2)
10 nmol indium added to 20 nmol DTPA in 20mM sodium
acetate, pH 5.4; (3) l0 nmol indium incubated with 2
nmol rhG-CSF at room temperature for l0 minutes,
followed by addition of 20 nmol DTPA in 20mM sodium
acetate, pH 5.4, and (4) l0 nmol indium incubated with 2
nmol DTPA-conjugated rhG-CSF at room temperature for l0
minutes, followed by addition of 20 nmol DTPA in 20mM
sodium acetate, pH 5.4. One ~l of each sample
(containing 0.l nmol indium) was spotted onto 250 ~m
thick silica gel (60 A) on glass backing (Whatman,
Clifton, NJ). The TLC plate was developed using 10%
(w/v) ~mm~;um acetate in distilled H2O:methanol (l:l,
v/v) as the solvent. The developed plate was then
analyzed using a Molecular Dynamics PhosphorImager
(Sunnyvale, CA).
The stoichiometric molar ratio of DTPA to
rhG-CSF was determined as described above. Chelation of
In by DTPA results in migration of all radioactivity
from near the solvent front (Figure 6, compare lanes l
and 2). Incubation of lllIn (l0 nmol) with the DTPA:rhG-
CSF conjugate (2 nmol), followed by addition of DTPA,
resulted in retention of a portion o~ the radioactivity
~ at the origin ~Figure 6, lane 4). Line graphs of the
individual lanes were generated and integration of the
- peak areas from lane 4 revealed 18% of the radioactivity
remained at the origin. The r~m~i n; ng unbound lllIn was
scavenged by the added DTPA and migrated near the
solvent front. Thus, approximately l.8 nmol of lllIn was
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-- 22 --
bound by 2 nmol of the DTPA:rhG-CSF conjugate,
indicating a DTPA to rhG-CSF molar ratio of 0.9.
Unmodified rhG-CSF did not retain radioactivity at the
origin, indicating absence of 111In binding (Figure 6,
lane 3).
3. Mass Spectrometry
Matrix-assisted laser desorption/ionization
mass specrometry (MALDI-MS) was performed with a Kompact
MALDI III mass spectrometer (Kratos Analytical, Ramsey,
NJ) fitted with a standard 337 nm nitrogen laser. The
spectra were recorded with the analyzer in linear mode
at an accelerating voltage of 20 kV. A sample aliquot
15 containing 15 pmol of protein and 1.0 ~l alpha-cyano-4-
hydroxyc;nnAm;c acid were mixed in the sample wells of
the probe slide and allowed to air dry. The laser
fluence of the instrument was set at 30 (adjustable over
a relative scale of 0-lOOj.
The mass of the DTPA:rhG-CSF conjugate was
determined by MALDI-MS (Figure 7~. The acquired
spectrum revealed multiply charged ions in addition to
the monoprotonated species. The mass obtained by
averaging the peak series was 19,171.7 (+ 7.3) Da. The
calculated MW for a single DTPA conjugated to rhG-CSF is
19,170.8 Da. Therefore, in general agreement with the
TLC analysis, the observed mass indicated a DTPA to
rhG-CSF molar ratio of l:1 for the DTPA:rhG-CSF
conjugate.
4. Ion-spray mass spectrometry.
Ion-spray mass spectrometry was performed with
a Perkin-Elmer Sciex API III mass spectrometer (Norwalk,
CT) equipped with an ion-spray interface by method of
flow injection. Samples were diluted in
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water/acetonitrile/formic acid (50:50:0.1, v/v) and flow
injected into the same solvent flowing at 25 ~l/minute.
The orifice was set at 70 V, and the mass spectrometer
operated in Q1 mode.
~ 5 The mass of the DTPA:rhG-CSF conjugate with
chelated indium was determined as described above.
Analysis of the conjugate, preincubated with saturating
indium (In:conjugate, 10:1, mol/mol), yielded a series
of peaks with differing m/z values. This multiply
charged ion series, arising from multiple protonation of
the protein, was deconvoluted to produce the MW spectrum
shown in Figure 8. The measured mass of the conjugate
with chelated indium was 19,286 (+ 1.7) Da, which is in
agreement with the calculated mass of 19,285.6 Da.
For rhG-CSF, the measured mass was 18,798 (+ 1.8) Da
(Figure 9), in agreement with the calculated mass of
18,798.5 Da.
5. Peptide Mapping
For peptide analysis, approximately 0.5 mg of
rhG-C~F or DTPA:rhG-CSF was dried in a speed vacuum,
reconstituted in 100 ~l of 8M Urea and sonicated for 10
minutes. After sonication, 10 ~l of lM Tris-HCl, pH 8.5
and 2.5 ~g of EndoLys-C (Wako Chemicals, Richmond, VA)
from a 1 mg/ml stock solution in lOmM Tris HCl, pH 8.5,
was added. The total volume was adjusted to 200 ~l with
distilled water, and the proteolytic digestion was
carried out for 7 hours at room temperature. Following
the hydrolysis with EndoLys-C, the disulfide bonds were
- simultaneously reduced with 5 ~l of 80mM TBP and
alkylated with 10 ~l of 40mM ABD-F ~2mM final
- concentrationJ as described; Kirley, T.K., Anal.
Blochem., 180, 231-236, (1989). Immediately after
reduction and alkylation, the generated peptides (200
~l) were injected directly onto a 300 A pore size C4
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reversed-phase HPLC column (Separations Group, Vydac,
Hesperia, CA) equilibrated with Solvent A (0.1~ TFA in
distilled water). Peptide analysis was performed using
a Waters HPLC system consisting of two 510 pumps, a WISP
712 autoinjector, and a 481LC spectrophotometer all
controlled through a system interface module by the
system software, Maxima. The generated peptides were
eluted with a linear gradient of 3-76% Solvent B (0.1%
TFA, 95% acetonitrile) over 115 minutes. Elution was
monitored for absorbance at 215 nm. Individual peptides
from the rhG-CSF standard peptide map were collected and
identified by amino acid composition analysis and N-
terminal sequencing as describedi Souza et al., Science,
232, 61-65, (1986).
Peptide fragments of unmodified rhG-CSF and
DTPA:rhG-CSF were prepared and analyzed as described
above. A peak eluting from the unmodified rhG-CSF
sample at 60 minutes was absent from the DTPA:rhG-CSF
conjugate sample (Figure 10). The material eluting in
this peak was determined by amino acid composition
analysis and N-terminal sequencing to be the N-term; n~l
17 residues of rhG-CSF. Thus, the corresponding N-
terminal peptide fragment from the DTPA:rhG-CSF
conjugate was modified, yielding a new partially split
double peak eluting at 62 minutes. Analysis of peptide
from each of these partially separated peaks by mass
spectrometry revealed the first peak to have the
expected mass of the N-terminal peptide with conjugated
DTPA, while the mass of the second peak material
suggested conjugated peptide contaminated with iron.
Peptide mapping therefore indicated that the conjugated
DTPA group is localized to the N-terminal 17 amino
acids. This N-terminal peptide contains the N-terminus,
one threonine, three serine residues and a lysine
residue. Cleavage of the peptide by EndoLys-C indicates
that the lysine is unmodified. Acylation of threonine
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-- 25 --
or serine residues is highly unlikely at pH 6Ø
Undigested DTPA:rhG-CSF conjugate subjected to
N-terminal sequencing revealed >99% blocked N-terminus,
indicating the single DTPA moiety on the protein is
conjugated to the N-terminus.
6. Isoelectric Focusing.
Isoelectric focusing was performed using Novex
pH 3-10 gels (San Diego, CA) with a pI 3.5 - 8.5
performance range. Samples were diluted 1:1 with sample
buffer, and 5 ~g of protein was loaded into each lane.
The gels were run at constant voltages of 100 V for 1
hour, 200 V for 2 hours, and then 500 V for 0.5 hour.
All fixing, staining and destaining procedures were done
to the manufacturer's specifications.
The DTPA:rhG-CSF conjugate revealed a single
major band of pI 4.9 following isoelectric focusing
(Figure 11, lane 3). Preincubation of the conjugate
with excess InCl3 (In:conjugate, 10:1, mol/mol) shifted
the band to pI 5.3 (Figure 11, lane 2). The pI values
of the conjugate, both with and without indium, were
lower than that of rhG-CSF, pI 6.0 (Figure 11, lane 1).
The conjugation of DTPA, concomitant with the loss of
the N-terminal free amino group, substantially decreased
the pI of the rhG-CSF. Furthermore, chelated indium
slightly increased the pI of the conjugate. The
characteristic isoelectric points of the DTPA:rhG-CSF
conjugate with and without chelated metal may also be
used to monitor metal contamination and metal labeling
- of the conjugate preparation.
The data above demonstrate that: (1) the
DTPA:rhG-CSF conjugate is able to chelate lllIn; (2) the
stoichiometric molar ratio of DTPA to rhG-CSF is
approximately 1.0 for the DTPA:rhG-CSF conjugate;
(3) the DTPA conjugation is specific to the N-terminus
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of the rhG-CSF; and (4) the conjugation of DTPA to
rhG-CSF decreases the pI of rhG-CSF.
.MPT,I;~ 3
In this example, circular dichroism analysis
was used to study the effects on rhG-CSF secondary
structure resulting from conjugation of a chelating
group to the N-terminus of rhG-CSF.
Circular Dichroism (CD) spectra were obtained
with a Jasco J-720 spectropolarimeter (Japan
Spectroscopic Co., LTD., Tokyo, Japan). Samples (0.078
mg/ml protein) were analyzed at 10~C in 20mM sodium
acetate, pH 5.4. The CD spectra of the DTPA:rhG-CSF
lS conjugate overlays that of unmodified rhG-CSF (Figure
12), each revealing ellipticity minima at 208nm and
222nm. Addition of excess indium to saturate all
chelating sites on the conjugate (In:conjugate, 10:1,
mol/mol) did not change the overall shape of the
spectra, yet caused a slight (approx. 5%) reduction in
alpha helicity. Therefore, the secondary structure (at
pH 5.4) is shown here not to be influenced by the
conjugation of a chelating group to the N-terminus.
~PJ,~ 4
In this example, the effects of DTPA
conjugation and subsequent chelation of indium on the
biological activity of rhG-CSF was determined.
Peripheral WBC counts in hamsters by rhG-CSF,
the DTPA:rhG-CSF conjugate, and the conjugate with
chelated indium were evaluated after subcutaneous
injection of 100 ~g/kg rhG-CSF in hamsters (Figure 13).
The animals were sacrificed at the indicated time
intervals, and collected blood samples were analyzed
using a Sysmex F800 microcell counter.
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- 27 -
Injection of the conjugate (Figure 13, (~))
induced the level of peripheral WBC counts in a manner
similar to unmodified rhG-CSF (Figure 13, (o)).
Injection of the conjugate preincubated with excess
~ 5 indium (In:conjugate, 10:1, molJmol)(Figure 13, (0))
also induced a similar response, with m~X~ mA 1 WBC levels
reached at 24 to 36 hours post injection. Thus, the
conjugation of DTPA and rhG-CSF did not significantly
alter the observed in vivo activity of the rhG-CSF, and
furthermore the activity of the conjugate is unchanged
after chelation of indium.
~AMPT~ 5
In this example, the conjugation reaction
described above was carried out on the related growth
factor, interleukin-2 (IL-2). The ability of the
DTPA:IL-2 conjugate to chelate lllIn was evaluated using
cation-exchange HPLC as described above. In addition,
the stoichiometric molar ratio of DTPA:IL-2 was
determined as well as the distribution of the DTPA
moiety on the IL-2.
The IL-2 was produced using recombinant DNA
technology in which E. coli cells were transfected with
a DNA sequence encoding IL-2 as described in European
patent 0136489 (Souza e~ al.). The IL-2 was prepared as
a 1.82 mg/ml solution in 100 mM sodium phosphate buffer,
pH 6Ø DTPA dianhydride and tributylphosphine (TBP,
technical grade) were obtained from Aldrich (Milwaukee,
WI). The conjugates were prepared as described in
- Example 2 above.
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An~lvsis of the DTPA:IT-2 Coniuaate
1. Analytical Cation-Exchange HPLC.
Analytical cation-exchange HPLC was performed
as described above. For the DTPA:IL-2 conjugate (Figure
14, lines 1 and 4, solid), 99.5% of the lllIn
radioactivity coeluted from the cation-exchange column
with the protein, whereas no detectable radioactivity
co-eluted with unmodified IL-2 (Figure 14, lines 2 and
3, dashed), indicating chelation of lllIn by the DTPA:IL-
2 conjugate, and absence of lllIn binding by unmodified
IL-2.
The effect of metal chelation on analytical
cation-exchange HPLC analysis of the DTPA:IL-2 conjugate
is depicted in Figure 15. Elution of IL-2 (line 1), the
DTPA:IL-2 conjugate (line 2), and the conjugate
preincubated with excess InCl3 (In:conjugate, 10:1,
mol/mol, line 3) was monitored for absorbance at 220 nm.
The DTPA:IL-2 conjugate eluted at a lower salt
concentration than unmodified IL-2. The chelated
conjugate then elutes at a slightly higher salt
concentration than the non-chelated conjugate, but still
at a salt concentration lower than that of unmodified
IL-2. As was the case with rhG-CSF analysis above, the
characteristic retention times of the DTPA:IL-2
conjugate provide a useful method of monitoring metal
contamination and metal labeling of the conjugate.
2. Thin Layer Chromatography (TLC).
TLC was performed as described above in order
to determine the ability of the DTPA:IL-2 conjugate to
chelate lllIn, and to determine the stoichiometric molar
ratio of DTPA to IL-2.
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WO 9611!;816 PCI/US95~1~S072
- 29 -
As shown in Figure 16, chelation of l1lIn by
DTPA results in migration of all radioactivity from near
the solvent front (Figure 16, compare lanes 1 and 2).
Incubation of ll1In (10 nmol) with the DTPA:IL-2
S conjugate (2 nmol), followed by addition of DTPA,
resulted in retention of a portion of the radioactivity
at the origin (Figure 16, lane 4). Line graphs of the
individual lanes were generated and integration of the
peak areas from lane g revealed 18% of the radioactivity
remained at the origin. The rPm~in;ng unbound lllIn was
scavenged by the added DTPA and migrated near the
solvent front. Thus, approximately 1.8 nmol of 1l1In was
bound by 2 nmol of the DTPA:IL-2 conjugate, indicating a
DTPA to IL-2 molar ratio of 0.9:1. Unmodified IL-2 did
not retain radioactivity at the origin, indicating
absence of 11lIn binding (Figure 16, lane 3).
3. Peptide Mapping.
Peptide analysis was performed on the
DTPA:IL-2 conjugate in order to determine the location
of the conjugated DTPA moiety on the IL-2. Peptide
fragments were prepared as described above.
As shown in Figure 17 (arrow), a peak eluting
from the unmodified IL-2 sample at approximately 36
minutes was absent from the DTPA:IL-2 conjugate sample.
The material eluting in this peak was determined by
amino acid composition analysis and N-terminal
sequencing to be the N-term' n~ 1 peptide of IL-2.
Therefore, the corresponding N-terminal peptide fragment
from the DTPA:IL-2 conjugate was modified, yielding a
new partially split peak at 40 minutes. As was the case
with rhG-CSF, peptide mapping indicated that the
conjugated DTPA group is localized to the N-terminus.
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These data demonstrate that the DTPA: IL-2
conjugate is able to chelate 111In, that the
stoichiometric molar ratio of DTPA to IL-2 is
approximately 1.0, and that DTPA is conjugated site-
specifically to the N-terminus of IL-2.
