1. Site-Specific Modification of a Mutant Cyanovirin-N with
a Large Polyethylene Glycol Moiety Yields a Potent Anti-HIV Agent
with Reduced Immunogenicity and Toxicity in Mice
M. Elizabeth Green1, Michael Roberts1, Richard Goodin1, Miroslav Novak1, Michael Boyd2,
Toshiyuki Mori2, and Barry O’Keefe2
1Nektar Therapeutics, Huntsville, AL; 2 Molecular Targets Discovery Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD
! PEG-CV-N was separated from free PEG and unmodified The cysteine mutant version of CV-N retained significant
ABSTRACT SUMMARY CV-N using gel filtration followed by anion exchange activity when conjugated to different molecular weight mPEG
chromatography. maleimide reagents.
Cyanovirin-N (CV-N) is a potent anti-HIV protein.
Mutagenesis has been used to replace an amino acid in ! Protein purity and yield were monitored via reverse phase
native CV-N with a cysteine residue, which is then site- and gel permeation HPLC. Figure 3: Relative Activity of CV-N and mPEG-MAL-CV-N
specifically modified with a single 30K methoxypolyethylene conjugates in an in vitro anti-HIV assay. Activity is measured as
glycol maleimide to form a CV-N cysteine mutant conjugate.
! Protein concentration was monitored using the BCA protein the ability of the compound to protect 50% of cells from infection with
assay. HIV (IC50). The IC50 is depicted relative to AZT, with AZT activity
In contrast to the CV-N-PEG moieties obtained using
assigned as 1.0. The cell-based assay showed significant variability
conventional random PEGylation, the cysteine mutant from one assay to the next; therefore all sample IC50 values were
conjugate retains significant activity against HIV in vitro. Activity, Acute Toxicity and Immunogenicity
normalized against AZT for each run.
! The in vitro activity of all proteins and protein conjugates
OBJECTIVES was monitored using an XTT-based cytprotection assay 4.5
using CEM-SS cells and the HIV-1 RF strain (SRI, 4.0
! Generate a cysteine mutant of cyanovirin-N for site-specific
EC50 Relative to AZT
Frederick, MD). 3.5
PEGylation with sulfhydryl reactive mPEG-maleimides of ! Acute toxicity of the native CV-N and CV-N(Q62C)-MAL 3.0
varying molecular weight. 30K conjugate were compared using an in vivo dose 2.5
! Demonstrate activity of the PEG-CV-N conjugates in vitro. escalation study in which mice were administered 2.0
modified or unmodified CV-N i.v. on three consecutive 1.5
! Demonstrate reduced toxicity of the PEG-CV-N conjugates days (BAS Evansville, Mt. Vernon, IN). 1.0
in vivo in mice. 0.5
! The immune response elicited in mice by unmodified CV-
! Demonstrate reduced immunogenicity of the PEG-CV-N N(Q62C), CV-N(Q62C)-MAL 20K and CV-N(Q62C)-MAL
0.0
conjugates in vivo in mice.
ZT
)
N
K
K
C
30K was studied.
V-
20
30
62
A
C
L
L
(Q
A
A
e
N
iv
)-M
)-M
! Three groups of mice were injected three times with 50
V-
at
C
C
N
C
62
62
INTRODUCTION
(Q
(Q
mg of one of the above compounds mixed with
N
N
V-
V-
incomplete Freund’s adjuvant on days 0, 21 and 35.
C
C
The rationale for attaching PEG to proteins has been covered
extensively in the literature. Compared to their unmodified
! Blood was drawn from all mice on indicated days.
counterparts, PEG-proteins may have some or all of the ! Anti-CV-N antibody levels were determined using an In a preliminary acute toxicity study, administration of 50
following characteristics: ELISA format. mg/kg of native CV-N resulted in the death of all mice while
mice given an equivalent dose of CV-N(Q62C)-MAL 30K
! Reduced toxicity exhibited only photosensitivity.
! Reduced immunogenicity RESULTS AND DISCUSSION Unmodified CV-N(Q62C) elicited a large immune response in
! Improved pharmacokinetics mice having a 50% endpoint titer of 16384. In comparison,
The initial, nonspecific PEGylation of lysines and/or the N- CV-N(Q62C)-MAL 20K had a 50% endpoint titer of 1825 and
! Greater solubility terminus of CV-N resulted in either very low yields or CV-N(Q62C)-MAL 30K a titer of only 512, merely twice that of
Cyanovirin-N is a suitable candidate for PEGylation because: conjugates with insignificant levels of activity (data not the baseline reading.
shown). Theorizing that the loss of activity of the CV-N-PEG
! It is a bacterial protein (originally isolated from Nostoc conjugates resulted from interference with one or more of the
ellipsosporum and produced recombinantly in Escherichia active sites, we sought to generate a modifiable mutation Figure 4: Immunologic Response in Mice to Dosing with mPEG-
coli)1,2 and therefore likely to cause toxicity and MAL-Conjugated or Unconjugated CV-N(Q62C).
located as far as possible from these sites.
immunogenicity when used in vivo.
16000
! It is a small protein (eleven kilodaltons) likely to be cleared CV-N(Q62C)
Figure 1: Proximity of mutation site to high mannose binding mPEG-MAL 20K CV-N(Q62C)
rapidly from the body.
Reciprocal of End-point Titer
14000
sites on CV-N. CV-N structure from Bewley, et al.3 mPEG-MAL 30K CV-N(Q62C)
Additionally, CV-N is an ideal candidate for site-directed 12000
PEGylation, which aims to attach a PEG moiety to a specific High-mannose oligosaccharide 10000
amino acid residue within a protein. binding sites
8000
! Traditional PEGylation targeting amine groups may result
in protein inactivation when PEGylation occurs in the 6000
proximity of the active site. 4000
! Since it is produced in E. coli, CV-N can be subjected to 2000
site-specific mutagenesis resulting in a protein with only
one site available for PEGylation. 0
0 21 35 49
! As CV-N’s active sites are known3, the introduced Day of Bleed
PEGylation site can be placed as remotely as possible from
the high-mannose binding sites that make CV-N active
against HIV. Q62C mutation site CONCLUSIONS
! Native CV-N randomly PEGylated at amine groups is
EXPERIMENTAL METHODS The substituted cysteine was then modified with 5, 20 or 30 essentially inactive.
kDa mPEG-maleimide with a yield approaching 70% following
Mutagenesis, Expression and Purification of CV- purification. ! Cyanovirin-N mutant Q62C retains activity when
N(Q62C) conjugated to 20K or 30K mPEG-maleimide at the
substituted cysteine residue.
! Glutamine 62 was replaced by a cysteine using a PCR- Figure 2: Reverse phase analysis of PEG-CV-N conjugation
based method. reaction. A) unreacted PEG; B) unreacted CV-N(Q62C); ! CV-N(Q62)-MAL 30K is less toxic in mice than its native
C) unknown; D) PEG-CV-N(Q62C) conjugate. Samples run on a counterpart.
! CV-N(Q62C) was expressed in E. coli according to the Vydac C-18 column using a 0.1M ammonium acetate pH
method of Mori, et al.2 7.4/methanol gradient. ! CV-N(Q62C)-MAL 20K and 30K generate a significantly
lower immune response than unconjugated CV-N(Q62C).
! The protein was purified using acid precipitation followed
by cation exchange chromatography.
PEGylation and Purification of CV-N REFERENCES
! Initially, mPEG-succinimidyl propionic acid or mPEG-
propionaldehyde, ranging in size from 2 kDa to 30 kDa, 1. Boyd MR, Gustafson KR, McMahon JB, Shoemaker RH, O’Keefe
BR, Mori T, Gulakowski RJ, Wu L, Rivera MI, Laurencot CM,
was attached to lysine residues and/or the N-terminus of
Currens MJ, Cardellina JH 2nd, Buckheit RW Jr., Nara PL, Pannell
native CV-N. LK, Sowder RC 2nd, Henderson LE. (1997) Antimicrob Agents
! After mutagenesis, 5 kDa, 20 kDa, or 30 kDa mPEG- Chemother. 41(7):1521-30.
maleimide was attached at the introduced cysteine. 2. Mori T, Gustafson KR, Pannell LK, Shoemaker RH, Wu L,
McMahon JB, Boyd MR. (1998). Protein Exp and Purif.
! The reaction was carried out at neutral pH using a two-fold 12(2):151-8.
molar excess of mPEG-maleimide. 3. Bewley CA. (2001). Structure (Camb). 9(10):931-40.
4. Shenoy SR, O’Keefe BR, Bolmstedt AJ, Cartner LK, Boyd MR.
(2001). J Pharmacol Exp Ther. 297(2):704-710.
30th Annual Meeting and Exposition of the Controlled Release Society, Glasgow, Scotland. July 19-23, 2003.
