(Received for publication, May 24, 1996, and in revised form, September 13, 1996)
From the Departments of Analytical Chemistry and
Formulation, ¶ Protein Chemistry,
Immunobiology, ** Product
Recovery, and
Cellular Immunology, Immunex
Corporation, Seattle, Washington 98101
Interleukin (IL)-15 is a multifunctional cytokine
that shares many biological activities with IL-2. This functional
overlap, as well as receptor binding subunits shared by IL-15 and IL-2, suggests tertiary structural similarities between these two cytokines. In this study, recombinant human IL-15 was PEGylated via
lysine-specific conjugation chemistry in order to extend the
circulation half-life of this cytokine. Although PEGylation did extend
the -elimination circulation half-life of IL-15 by greater than
50-fold, the biological activity of polyethylene glycol (PEG)-IL-15 was
significantly altered. Specifically, PEG-IL-15 lost its ability to
stimulate the proliferation of CTLL but took on the properties of a
specific IL-15 antagonist in vitro. In comparing sequence
alignments and molecular models for IL-2 and IL-15, it was noted that
lysine residues resided in regions of IL-15 that may have selectively disrupted receptor subunit binding. We hypothesized that PEGylation of
IL-15 interferes with
but not
receptor subunit binding, resulting in the IL-15 antagonist activity observed in
vitro. The validity of this hypothesis was tested by engineering
site-specific mutants of human IL-15 as suggested by the IL-15 model
(IL-15D8S and IL-15Q108S block
and
receptor subunit binding,
respectively). As with PEG-IL-15, these mutants were unable to
stimulate CTLL proliferation but were able to specifically inhibit the
proliferation of CTLL in response to unmodified IL-15. These results
supported our model of IL-15 and confirmed that interference of
receptor subunit binding by adjacent PEGylation could be responsible
for the altered biological activity observed for PEG-IL-15.
IL-151 and IL-2 share many biological
activities, including the ability to stimulate T cells, NK cells, and
activated B cells (1-5). The overlapping biological activities of
IL-15 and IL-2 can be explained, at least in part, by the composition
of the multisubunit receptor complexes for these two cytokines. The
high affinity IL-2R complex consists of at least three chains,
designated ,
, and
, and interaction of IL-2 with both the
and
chains is necessary for initiation of signaling events (6). The
IL-15 receptor complex also consists of at least three subunits. It shares use of the common
and
chains of the IL-2R complex but contains an
subunit that is distinct from IL-2R
(3, 7, 8).
Similar to the IL-2R complex, interaction of IL-15 with both the
and
subunits is necessary for initiation of signaling. The IL-2R
and IL-15R
subunits share significant structural homology (8);
however, there is at least one distinct difference in the interaction
of these subunits with their respective ligands. The IL-2R
subunit
binds IL-2 with very low affinity (Ka = 108), whereas the IL-15R
subunit binds IL-15 with
unusually high affinity (Ka = 1011).
This difference suggests that IL-15 may have in vivo
functions distinct from those it shares with IL-2.
Mutagenesis studies with recombinant human IL-2 have indicated several
regions and key amino acid residues involved in ,
, and
receptor subunit binding. In particular, mutation of mature IL-2
residue Lys35, Arg38, Phe42, or
Lys43 reduces or blocks IL-2R
subunit binding (9-11),
mutation of residue Asp20 inhibits
receptor subunit
binding and signal transduction (10, 12, 13), and mutation of residue
Gln126 blocks
receptor subunit binding and signaling
(12-14). Mutagenesis studies with murine IL-2 have demonstrated
analogous regions of amino acids involved in
,
, and
receptor
subunit binding (15).
Whereas protein mutagenesis allows for specific investigation of protein structure to function relationships, other protein modification techniques may also influence specific protein-ligand interactions. The process of conjugating chains of polyethylene glycol (PEG) onto proteins such as IL-2, granulocyte-macrophage colony-stimulating factor, asparaginase, immunoglobulins, hemoglobin, and others has traditionally been used to prolong circulation half-lives in vivo, enhance solubility, and reduce immunogenicity (16-18). However, PEGylation has also been found to reduce or alter biological activities, the extent of which depends on the degree of conjugation, the type of conjugation chemistry used, and the specific location on the protein where the conjugation has occurred. The independent variation of two biological activities has also been demonstrated with immunoglobulin and granulocyte-macrophage colony-stimulating factor by selectively PEGylating regions of these proteins involved with one biological function while not interfering with the other (19, 20). These examples likely demonstrate the importance of the location of lysine residues within the tertiary protein structure when using lysine-specific PEG conjugation chemistry.
In this paper we describe the PEGylation of IL-15 and the in
vivo and in vitro characterization of this molecule.
Whereas the pharmacokinetics were enhanced following PEGylation of
IL-15, the biological activity of this molecule was significantly
altered. Specifically, PEG conjugation eliminated the CTLL
proliferative activity of IL-15 and generated a molecule that
functioned as a specific antagonist in vitro. We hypothesize
that lysine-specific PEGylation of IL-15 resulted in steric
interference of the region of IL-15 that interacts with the receptor subunit, while not affecting the region of IL-15 that
interacts with the
receptor subunit. This hypothesis is based on
sequence alignment of IL-15 with IL-2 and the construction of a
molecular model of IL-15. Two mutants of IL-15, designed to disrupt
either
or
receptor subunit binding, were prepared to verify the
accuracy of these regions of the IL-15 model. The in vitro
characterization of these IL-15 mutants was consistent with the
predictions of the molecular model and supported the hypothesis
concerning PEGylated IL-15. Furthermore, when taken together, the
PEGylation and mutagenesis data support the model of IL-15 in which
each of the receptor subunits,
,
, and
bind to IL-15 in a
spatial orientation similar to that established for IL-2.
The cloning of simian IL-15 has been described (1) and the sequences for both human and simian IL-15 have been recently reported (21). All of the PEGylation studies described below were carried out with simian IL-15, and all of the mutagenesis studies were carried out with human IL-15. Human and simian IL-15 are 96% identical at the amino acid level, and the number and position of lysine residues are identical. Also, the binding characteristics and biological activity of human and simian IL-15 as measured by CTLL-2 cell proliferation are identical.2 Human IL-15 was used in all studies as the control material.
Briefly, for the expression of either human or simian IL-15, cDNA encoding IL-15 from either species was ligated into a yeast expression vector that directs secretion of the recombinant protein into the yeast medium (22). Supernatants were collected following ultrafiltration of the yeast broth, and IL-15 was purified as described previously (21).
Site-specific mutants of huIL-15 (IL-15D8S and IL-15Q108S) were
generated by two separate PCR amplifications. In the primary PCR
reaction, amplification was with primer pairs that either introduced
the appropriate mutation or amplified the wild-type sequence. In the
secondary PCR reaction, material from the first round was reamplified
with a primer set that introduced restriction sites necessary for
cloning into the yeast expression vector. The mutagenic
oligonucleotides had the sequence 5-AATGTAATAAGTTCTTTGAAAAAAATT-3
(Asp8
Ser, 5
sense oligo), and
5
-GTTGATGAACATAGAGACAATATG-3
(Gln108
Ser, 3
antisense oligo). PCR amplification was performed under standard
conditions using Taq polymerase (Boehringer Mannheim) and
cycling conditions of denaturation at 94 °C for 45 s,
annealing at 45 °C for 45 s, and extension at 72 °C for 1 min for a total of 30 cycles, using approximately 10 ng of wild-type
human IL-15 cDNA as template.
Approximately 20 ng of gel purified product from the primary amplification was used as the template for the secondary PCR amplification, which used nonmutagenic, flanking oligonucleotides that appended the necessary restriction enzyme sites. Cycling conditions were denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, and extension at 72 °C for 1 min for a total of 20 cycles.
Amplification products were gel purified and digested with
Asp718 (Boehringer Mannheim) and NcoI (New
England Biolabs) and ligated into the 2-µm yeast expression vector
pIXY456, which contains the ADH2 promoter and the -factor leader.
Ligations were transformed into Escherichia coli DH10b cells
by electroporation. Plasmid DNA from single transformants was sequenced
to confirm sequence integrity and used to transform S. cerevisiae XV2181. Purification of the mutant proteins was carried
out by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B
(Pharmacia Biotech, Inc.).
Recombinant human IL-2 (des-alanyl-1-, Ser125 human interleukin-2, Aldesleukin, Proleukin®) was obtained from Chiron Corp. (Emeryville, CA).
PEG conjugation reactions were carried out with 5-kDa PEG, which was obtained in the activated form of succinimidyl carbonate PEG from Shearwater Polymers (Huntsville, AL). PEGylation of simian IL-15 was carried out in 50 mM NaH2PO4, pH 8.5. For PEGylation of IL-2, the addition of 0.1% SDS improved the solubility of IL-2 and the extent of PEGylation. Reactions with both IL-15 and IL-2 were carried out in 0.5-ml volumes at 100 µg/ml for in vitro characterization studies. Larger volumes were prepared for in vivo pharmacokinetic studies. Succinimidyl carbonate PEG was added to the reaction mixtures at molar ratios of PEG to lysine of 1:1, 3:1, 10:1, and 100:1 (7 lysine residues + 1 N terminus per IL-15 molecule and 11 lysine residues + 1 N terminus per IL-2 molecule). All reactions were allowed to proceed overnight at 4 °C. For subsequent in vivo pharmacokinetics experiments, PEG-IL-15 was dialyzed against PBS to remove residual N-hydroxysuccinimide, a byproduct of succinimidyl carbonate PEG hydrolysis.
Protein concentrations of IL-15, PEG-IL-15, and PEG-IL-2 species were determined by amino acid analysis.
Physical Characterization of PEG-IL-15 and PEG-IL-2PEG-IL-15 and PEG-IL-2 were characterized by SDS-polyacrylamide gel electrophoresis, SE-HPLC, and MALDI-TOF mass spectroscopy. In the chromatography experiments, a Waters HPLC system (Millipore Corp., Milford, MA) was equipped with two 300 × 8 mm SEC-250 Biosil columns (Bio-Rad), which were run in series in order to resolve the various PEG-modified species. For the mass spectroscopy experiments, PEG-IL-15 (PEG to lysine ratio of 100 to 1) was first purified by SE-HPLC in order to reduce residual levels of unconjugated PEG and other reaction byproducts. PEG-IL-15 samples were further prepared by buffer exchange with water and concentrated to approximately 1 mg/ml. PEG-IL-15 (0.5 µl) was applied to targets along with a sinapinic acid matrix (0.5 µl of 10 mg/ml solution in 50:50 acetonitrile/water), allowed to air dry, and then analyzed with a Finnigan Mat LaserMat time-of-flight laser desorption mass spectrometer (San Jose, CA).
PharmacokineticsExperiments were performed to compare the
circulation half-lives of IL-15 and PEG-IL-15 (PEG:lysine ratio of
10:1). Groups (3 mice per group) of 10-12-week-old female BALB/c mice
(The Jackson Laboratory, Bar Harbor, ME) were injected intravenously
with 10 µg of protein in a total volume of 150 µl. Following the
injections, mice were sacrificed and blood samples were collected at 3, 5, 15, and 30 min and 1, 2, 4, 8, 16, 20, 40, and 48 h via cardiac puncture, and the plasma was analyzed by CTLL-2 competitive inhibition assay, CTLL-2 proliferation assay, and IL-15 ELISA. The ELISA used a
murine monoclonal capture antibody (M111, Immunex designation) and a
murine monoclonal antibody for detection (M112, Immunex designation).
These antibodies were found to bind to both the PEGylated and
unmodified forms of IL-15, but with different affinities. Therefore,
amino acid-analyzed standards of both IL-15 and PEG-IL-15 were used as
controls in the ELISA experiments. Pharmacokinetic parameters of the
PEGylated and unmodified forms of IL-15 in the blood were determined
and the apparent elimination rate constant (K) and half-life
(t1/2) for each form of the molecule were calculated using a pharmacokinetics half-life program on a RS/1 system.
The log linear portion of the blood concentration/time curve was used
to calculate K with t1/2
determined as t1/2 = ln2K. Half-life values are presented as
t1/2 ± S.E., where S.E. indicates the
standard error in fitting the log linear line to the data points in
calculating the K value. The distribution (t1/2) and elimination
(t1/2
) half-lives were calculated
using a biphasic pharmacokinetics program that related the respective
log linear concentration/time curves to specific K and
t1/2 values.
Bioactivity of IL-15, IL-2, PEG-IL-15, PEG-IL-2, IL-15D8S, and IL-15Q108S were assessed using a modified CTLL-2 cell [3H]thymidine incorporation assay (23). CTLL-2 competitive inhibition assays were performed by adding suboptimal amounts of IL-15 (final concentration, 50 pg/ml) or IL-2 (20 pg/ml) to all assay wells after serial dilution of samples and prior to addition of cells.
Sequence Alignment and Molecular ModelingThe amino acid
sequences for huIL-15 and huIL-2 have been previously reported (21,
24). In spite of the overwhelming clues to the structural similarities
between these cytokines based on their functional similarities and
common receptor usage, the amino acid sequences show only weak sequence
identities. This is not uncommon among members of the helical cytokine
family, of which IL-2 is a member (25, 26). Those amino acid sequences
that are conserved between IL-2 and IL-15 were used for alignment. In
particular, amino acid residues of each cytokine presumed to define the
helical structure were aligned first. Secondary consideration was
given to the conserved cysteine and other conserved residues found in
the loop regions that connect each
helix. This alignment strategy
required the placement of gaps in either the IL-2 or IL-15 sequence in
the loop regions that connect the
helices.
A three-dimensional computer model of IL-15 was generated using FOLDER,
a distance geometry based homology modeling package (27, 28). For the
model, a "template" sequence was defined that included the
structurally conserved amino acid residues within the helices as
defined by other cytokines within the four-helix bundle cytokine family
(25). Other amino acids included in the template sequence were
short loop regions between or flanking the
helices. The IL-15 model
was then generated using the template sequence and the established
coordinates for IL-2 (PDB code 3ink) (29, 30). Amino acids in the
template sequence of IL-2 were the only residues used to derive
structural constraints between atoms of the corresponding amino acids
in the IL-15 sequence. For all the other amino acids of IL-15, for
which there are no corresponding amino acids in the template sequence,
only chemical, steric and loop closure constraints were used to define
spatial locations (27).
SDS-polyacrylamide gel electrophoresis analysis and
SE-HPLC were used to characterize and separate PEG-IL-15 and PEG-IL-2 species, respectively. Heterogeneous mixtures of PEG-IL-15 and PEG-IL-2
were produced, depending on the PEG:lysine ratio used in the
conjugation reaction (Fig. 1). At high PEG:lysine
ratios, significant smearing of the bands on the gels was observed.
SE-HPLC was used to separate PEGylated species for further analysis
(Fig. 2), and MALDI-TOF mass spectroscopy of PEG-IL-15
(PEG:lysine ratio of 100:1 in reaction mixture) confirmed at least
seven distinct species ranging in size from 14,000 to approximately
50,000 kDa in intervals of about 5,000 kDa (Fig. 3).
Pharmacokinetics
A comparison of the circulating serum
concentrations of IL-15 and PEG-IL-15 as measured by ELISA is shown in
Fig. 4. Based on these data, pharmacokinetic circulation
half-lives were measured for IL-15
(t1/2 = 0.9 ± 0.3 min;
t1/2
= 16.0 ± 1.7 min) and
PEG-IL-15 (t1/2
= 87.6 ± 8.2 min; t1/2
= 2445 ± 167 min).
Also, the circulation half-life of IL-15 was estimated by CTLL-2
proliferation assay (t1/2
= 1.0 ± 0.7 min; t1/2
= 16.5 ± 4.7 min), and the circulation half-life of PEG-IL-15 was estimated by
CTLL-2 inhibition assay (t1/2
= 107 ± 41 min; t1/2
= 859 ± 133 min). PEG-IL-15 clearly was eliminated more slowly than
un-PEGylated IL-15; t1/2
was slower by
a factor of 52 (by CTLL-2 analysis) or 153 (by ELISA).
In Vitro Bioassay Characterization
CTLL-2 bioassays were used
to measure the bioactivity of each of the molecules investigated in
this study (Fig. 5). Biological activity was measurable
for IL-2, IL-15, and PEG-IL-2 (PEG:lysine ratio of 100:1). Similar
specific activities were measured for IL-15 and IL-2, whereas the
bioactivity of PEG-IL-2 was lower than these by 2 orders of magnitude.
No bioactivity could be measured for PEG-IL-15 (PEG:lysine ratio of
100:1), IL-15D8S, or IL-15Q108S. Nonconjugated PEG did not inhibit
CTLL-2 proliferation at concentrations used in the PEGylated cytokine
assays (data not shown). Intermediate bioactivities were measured for
PEG-IL-15 and PEG-IL-2 with PEG:lysine ratios of 1:1, 3:1, and 10:1
(data not shown). The ability of PEGylated forms of IL-15 and IL-2 and
mutated forms of IL-15 to specifically inhibit the bioactivity of
unmodified IL-15 or IL-2 was measured. The specific inhibitory effects
of PEG-IL-15, IL-15D8S, and IL-15Q108S on the biological activity of a
standard solution of IL-15 (50 pg/ml) are clearly demonstrated (Fig.
6). No such inhibitory effect was found for PEG-IL-2 in
this assay (Fig. 7). It should be noted that the mutated
forms of IL-15 were able to inhibit IL-15 binding at concentrations of
approximately three orders of magnitude lower than those required to
inhibit IL-15 binding with PEG-IL-15. A 2000-5000-fold molar excess of
PEG-IL-15 (based on protein weight) was required to obtain 100%
inhibition of IL-15 induced bioactivity, whereas only a 10-20-fold
molar excess of IL-15D8S or IL-15Q108S was required to obtain 100%
inhibition of IL-15 induced bioactivity. None of the cytokines
investigated in this study were able to inhibit the biological activity
of a standard solution of IL-2 (20 pg/ml) (Fig. 7).
Sequence Alignment of IL-15 and IL-2
The sequence alignment
for huIL-15 and huIL-2 is shown in Fig. 8. A disulfide
bridge is conserved between IL-15 (Cys42-Cys88)
and IL-2 (Cys58-Cys105), and one disulfide
bridge unique to IL-15 (Cys35-Cys85) has been
proposed (1). The structural significance of the sequence alignment can
be evaluated by the number and site of identities within the four
blocks defining the four helices. The conserved
EFLXXXXXXXQXXI (Xs are nonidentical
amino acids) in the D-helix, the conserved CXXXEL in
B-helix, the conserved cysteine within the loop that connects the
C-helix and D-helix, and conserved DLXXI in the A-helix
further support the expected structural similarities between the two
cytokines. Conservation of XDX in the A-helix and
XQX in the D-helix also suggests similar receptor
binding modes between these two cytokines because
XDX and XQX of IL-2 are
known to be involved in
and
receptor binding, respectively.
Furthermore, the level of identities found within the four helices are
no lower than those between other helical cytokines that are known to
belong to helical cytokines family using crystal structure analysis
(25).
Molecular Modeling
Ribbon models of IL-2 and IL-15 are
illustrated in Fig. 9, A-D. The side chains
of amino acid residues of IL-2 that are known to influence binding to
the ,
, and
receptor subunits are highlighted in Fig.
9A. As a result of sequence alignment and spatial comparison
between IL-2 and IL-15, residues on IL-15 hypothesized to interact
with
and
receptor subunits were identified (Asp8
and Gln108, respectively). The positions of single point
mutations made for IL-15D8S and IL-15Q108S are illustrated in Fig.
9B. Potential PEGylation sites (lysine residues and the N
terminus) are highlighted for IL-2 and IL-15 in Fig. 9, C
and D, respectively. In comparing these figures, it is
evident that potential PEGylation sites are absent in the region of
IL-15 that, by comparison with IL-2, may bind IL-15R
. Also,
potential PEGylation sites in the region of IL-15 which may bind
IL-15R
were noted. On the basis of these observations, we
hypothesized that PEG-IL-15 (as well as IL-15D8S and IL-15Q108S) could
still bind to IL-15R
but would not signal through the
receptor complex.
Our initial goal for PEGylation of IL-15 was to extend its circulation half-life in a manner similar to that reported for IL-2 (31, 32). Toward this end, both IL-15 and IL-2 were PEGylated with lysine-specific, succinimidyl carbonate-activated PEG. The physical characteristics of PEG-IL-15 were found to be very similar to those of PEG-IL-2. Specifically, SDS-polyacrylamide gel electrophoresis and SE-HPLC demonstrated similar distinct banding patterns that were attributable to the number of PEG conjugates on each protein molecule (Figs. 1 and 2). Also, pharmacokinetic studies carried out in a murine system demonstrated a significant enhancement of the circulation half-life of PEG-IL-15 in comparison with unmodified IL-15 (Fig. 4), an observation similar to that reported in the literature for PEG-IL-2 (31).
However, despite the physical similarities of PEG-IL-15 and PEG-IL-2, the biological activity of IL-15 was significantly altered by PEGylation, whereas the activity of IL-2 was largely unaffected by PEGylation. Specifically, PEG-IL-15 was inactive in the CTLL-2 bioassay (Fig. 5) but was able to competitively inhibit the biological activity of unmodified IL-15 (Fig. 6). By comparison, the biological activity of PEG-IL-2 was only slightly reduced relative to unmodified IL-2 (Fig. 5).
In order to account for the discrepancies in the biological activity of
PEG-IL-15 and PEG-IL-2, a critical analysis of the spatial orientation
of the binding domains of each cytokine was carried out. The sequences
of IL-15 and IL-2 were aligned (Fig. 8), and a molecular model of IL-15
was constructed (Fig. 9, B and D). On the basis
of this model, we hypothesized that IL-15 and IL-2 could bind to their
respective subunits and to their and common
and
receptor
subunits through a similar spatial orientation of each cytokine. To
test this hypothesis, muteins were generated at positions
Asp8 or Gln108, which by analogy with
mutagenesis studies carried out on IL-2, would interfere with
or
receptor subunit binding, respectively.
The resulting muteins, IL-15D8S and IL-15Q108S, were tested for their
biological activity in vitro. As with PEG-IL-15, each of the
muteins was inactive in the CTLL-2 bioassay (Fig. 5) but was able to
competitively inhibit the biological activity of unmodified IL-15 (Fig.
6). These results supported the model of IL-15 and confirmed our
rationale for mutating IL-15 at positions Asp8 and
Gln108. The antagonistic activity of IL-15D8S and
IL-15Q108S, as well as that of PEG-IL-15, could best be explained by
the model. Disruption of binding to either the or the
receptor
subunit would prevent signal transduction and cell proliferation, while
still allowing competitive binding to the
receptor complex
through IL-15R
. In further support of this conclusion, neither
IL-15D8S nor IL-15Q108S was able to inhibit the bioactivity of IL-2,
which binds to a unique IL-2R
subunit but signals through the common
receptor subunit complex (Fig. 7).
That PEGylation of IL-15 could disrupt or
receptor subunit
binding is seen by the presence of two lysine residues at positions Lys10 and Lys11 (Fig. 9D). PEG
conjugation to either of these residues could sterically interfere with
receptor binding to the region of IL-15 containing
Asp8. Equally important in the IL-15 model is the
observation that no lysine residues are present in the region
hypothesized to bind to the
receptor subunit (approximately between
residues 21 and 33). The closest lysine residues to the hypothesized
receptor subunit binding domain are Lys11 in helix A
and Lys35 near the carboxyl end of the strand separating
helix A and B.
In contrast to IL-15, no lysine residues exist on IL-2 in regions that
would interfere with either or
receptor subunit binding, and
two lysine residues (Lys35 and Lys43) are known
to interact with the IL-15R
subunit (Fig. 9C) (9-11). These observations are also consistent with the in vitro
biology results in which PEG-IL-2 retains biological activity (Fig. 5) but is unable to act as a specific antagonist in vitro in
the presence of unmodified IL-2 (Fig. 7).
For the bioassay and pharmacokinetic studies, we chose to use forms of PEGylated molecules that were heavily PEG-modified in order to avoid heterogeneous mixtures of non-PEG and PEG-modified proteins. However, even in mixtures that were highly PEGylated, characterization by MALDI-TOF mass spectroscopy identified a heterogeneous population of PEGylated species (Fig. 3). Because multiple species of PEG-IL-15 conjugates were present in these samples, the specific activity could be expected to be reduced (i.e. some species of IL-15 may exist that have not been PEG conjugated at either position Lys10 or position Lys11). It should also be considered that a single chain of PEG may be extended in solution such that a chain attached at one site may interfere with the protein-receptor interactions at another distant site. Each of these possibilities is consistent with our CTLL-2 bioassay data, in which the concentration of PEG-IL-15 required to inhibit the activity of unmodified IL-15 was approximately 100-fold higher than that required for similar activities of IL-15D8S and IL-15Q108S (Fig. 6).
The technique of protein PEGylation is usually considered in the context of extending the half-life of circulating proteins, reducing protein antigenicity, or improving the resistance of a given protein to proteolysis. In this study, we have demonstrated the potential use of PEG as a probe for protein structure and function. The observation that PEG can selectively interfere with a specific biological function has been reported previously in the literature. For example, in the process of PEGylating IgG, the complement-fixing activity of this molecule can be reduced, whereas its ability to bind antigen remains unaltered (19). Also, in the process of PEGylating granulocyte-macrophage colony-stimulating factor, neutrophil priming can be maintained or even enhanced, whereas the colony-stimulating activity of this molecule is reduced (20). In fact, the knowledge that PEGylation of proteins can interfere with protein-ligand interactions in general has led investigators to introduce cysteine residues in recombinant proteins to enable site-specific PEGylation and reduce the possibility of steric interference from PEG (33, 34). Site-directed mutagenesis has also been used to eliminate lysine residues in locations where lysine-specific PEGylation could interfere with specific protein function (35).
We suggest that protein PEGylation may also be used as a complementary technique to protein engineering in order to sterically control protein-ligand interactions. Protein-ligand interactions typically involve many amino acids over the surfaces of proteins: e.g. lysozyme/antibody (36), trypsin/trypsin inhibitor (37), and growth hormone/receptor (38). By traditional protein engineering techniques, amino acid residues must be systematically mutated over a wide target region in order to uncover key residues or combinations of residues that are critical to protein function. In the case of protein PEGylation, the extended polymer chain may serve to sterically interfere with protein-protein interactions over a fairly large region on the surface of a protein molecule. In the particular example of IL-15 described in this paper, the likely PEGylation of residues Lys10 and/or Lys11 were sufficient to block the action of the nonadjacent critical residue, Asp8.
The potential exists (by introducing specific amino acid residues in critical regions) to PEG modify other ligands that bind multisubunit receptors in order to make specific antagonists (e.g. growth hormone, TNF, IL-2, IL-4, etc.). These molecules should have the added attributes conferred by PEG conjugation, including extended serum half-life. This approach could be particularly useful when single point mutations are insufficient to block the binding of specific receptor subunits.
The authors thank Dr. Linda Park for critical reading and Anne Bannister and Alison House for assistance in preparation of the manuscript.