albumin binding as a general strategy for improving the ... · albumin binding as a general...

JBC 1 7/9/02

Albumin Binding as a General Strategy for Improving

the Pharmacokinetics of Proteins

Mark S. Dennis*§, Min Zhang§, Y. Gloria Meng‡, Miryam Kadkhodayan¶

Daniel Kirchhofer∏, Dan Combs†, Lisa A. Damico†

Department of Protein Engineering§,

Department of Assay and Automation Technology‡,

Department of Analytical Chemistry¶

Department of Physiology∏, and

Department of Clinical and Experimental Pharmacology†

Genentech, Inc.

1 DNA Way, South San Francisco, California 94080

Running title: Improved t1/2 through albumin binding

* To whom all correspondence should be sent

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1The abbreviations used are: Fv, the variable light and variable heavy domains of an

IgG; scFv, a single-chain Fv; Fab, the antigen binding fragment consisting of the light

chain and the variable and first constant domains of the heavy chain; Fab’2, two Fab

fragments joined by disulfides at the hinge region; SA06, an albumin binding peptide

with the sequence: QRLMEDICLPRWGCLWEDDF; SA08b, an albumin binding peptide

with the sequence: Ac-QGLIGDICLPRWGCLWGDSVKb-NH2 where Kb refers to lysine-

biotin; GFR, glomerular filtration rate; TF, the extracelluar domain of human tissue factor

(residues 1-219); D3H44, a humanized IgG directed against human TF; D3H44 Fab, the

Fab portion of D3H44; D3H44-L, D3H44 Fab with SA06 fused to the carboxyl terminal

of the light chain; D3H44-Ls, D3H44 Fab lacking the light-heavy chain disulfide with

SA06 fused to the carboxyl terminal of the light chain; FX, coagulation Factor X; t1/2,

half-life; PEG, polyethylene glycol; HRP, horse radish peroxidase; TCEP, Tri(2-

carboxyethyl)phosphine hydrochloride. Both the standard three letter and single letter

codes for amino acids are used.


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ABSTRACT

Plasma protein binding can be an effective means of improving the pharmacokinetic

properties of otherwise short-lived molecules. Using peptide phage display we identified

a series of peptides having the core sequence: DICLPRWGCLW that specifically bind

serum albumin from multiple species with high affinity. These peptides bind to albumin

with 1 to 1 stoichiometry at a site distinct from known small molecule binding sites.

Using surface plasmon resonance, the dissociation equilibrium constant of peptide

SA21 (Ac -RLIEDICLPRWGCLWEDD-NH2) was determined to be 266 ± 8, 320 ± 22

and 467 ± 47 nM for rat, rabbit and human albumin, respectively. SA21 has an

unusually long half-life of 2.3 h when injected by I.V. bolus into rabbits. A related

sequence, fused to the anti-tissue factor Fab of D3H44 (Presta et. al. (2001) Thromb.

Haemost. 85, 379), enabled the Fab to bind albumin with similar affinity to that of SA21

while retaining the ability of the Fab to bind tissue factor. This interaction with albumin

resulted in reduced in vivo clearance of 25- and 58-fold in mouse and rabbit,

respectively, when compared to the wild-type D3H44 Fab. The half-life was extended

37-fold to 32.4 h in rabbit and 26-fold to 10.4 h in mouse achieving 25 to 43 percent of

the albumin half-life in these animals. These half-lives exceed those of a Fab’2 and are

comparable to those seen for PEG conjugated Fab molecules, immunoadhesins, and

albumin fusions suggesting a novel and generic method for improving the

pharmacokinetic properties of rapidly cleared proteins.


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INTRODUCTION

The effectiveness of recombinant protein pharmaceuticals depends heavily on the

intrinsic pharmacokinetics of the natural protein. Because the kidney generally filters out

molecules below 60 kDa, efforts to reduce clearance have focused on increasing

molecular size through protein fusions, glycosylation or the addition of polyethylene

glycol polymers (i.e. PEG)1. For example, fusions to large long-lived proteins such as

albumin (1,2) or the Fc portion of an IgG (3), the introduction of glycosylation sites (4) or

conjugation with PEG (5-7) have been used. Through these methods, the in vivo

exposure of protein therapeutics has been extended.

Small molecule drugs have long relied on their association with various plasma

components to improve their pharmacokinetic properties in vivo; however, a drug

associated with plasma protein is usually unavailable for binding to the target even

though its half-life is extended. Since only the unbound fraction of the small molecule is

generally functionally active, a fine balance must be maintained between the

concentration of free drug required for efficacy and the frequency at which it must be

administered (8).

Albumin (molecular weight ~ 67 kDa) is the most abundant protein in plasma, present

at 50 mg/ml (600 µM), and has a half-life of 19 days in humans (9,10). Albumin serves

to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of

many metabolites and fatty acids, and serves as a major drug transport protein in

plasma. There are several major small molecule binding sites in albumin that have been

described. Warfarin is known to bind at site I, benzodiazepines and indoles at site II and


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cardenolides and biliary acids at site III. In addition, there is an important metal ion

binding site.

Non-covalent association with albumin has been shown to extend the half-life of

short-lived proteins. A recombinant fusion of the albumin binding domain from

Streptococcal protein G to human complement receptor type 1 increased its half-life 3-

fold to 5 h in rats (11). In addition, fusion to this domain has served to enhance the

immunological response directed to peptide antigens (12). In another example, when

insulin was acylated with fatty acids to promote association with albumin (13,14), a

protracted effect was observed when injected subcutaneously in rabbits or pigs.

Together, these studies demonstrate a linkage between albumin binding and prolonged

action.

In this report, peptide phage display was used to develop peptides that selectively

bind albumin with high affinity. These peptides bind to albumin from multiple species at

a novel site distinct from the known classical binding sites. To test whether association

of a short-lived protein with albumin could improve its pharmacokinetic properties, one

albumin binding peptide was added to a Fab through the use of a simple recombinant

fusion that rendered it capable of binding albumin without affecting antigen binding. We

demonstrate this approach as a viable route to increasing the half-life of potentially

important protein pharmaceuticals.


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METHODS

Phage Libraries and Selection Conditions – Eighteen phage libraries expressing

random peptide sequences fused to the major coat protein, P8 (15) were pooled into 4

groups: Pool A contained CX2GPX4C, X4CX2GPX4CX4, and XiCXjCXk where j = 8-10;

Pool B contained X20 and XiCXjCXk where j = 4-7; Pool C contained X8 and X2CXjCX2

where j =4-6; Pool D contained X2CXjCX2 where j =7-10. X represents any of the 20

naturally occurring L-amino acids and in Pools A and B, i + j + k = 18 and | i - k | < 2.

Each library has in excess of 1010

clones.

The phage library pools were suspended in Binding Buffer (PBS, 1% ovalbumin,

0.05% Tween 20) and sorted against rabbit, rat or human albumin (Sigma, St. Louis,

MO) immobilized directly on Maxisorp plates (Nunc, Roskilde, Denmark) at 10 µg/ml in

PBS, overnight at 4oC. Plates were blocked for 1 h at 25oC using PBS, containing 1%

ovalbumin except for round 4 where TBS-Casein Blocker (Pierce) was used. Phage

were allowed to bind for 2 h. Unbound phage were removed by repetitive washing with

PBS, 0.05% Tween 20 and bound phage were eluted with 500 mM KCl, 10 mM HCl, pH

2. Eluted phage were propagated in XL1-Blue cells with VCSM13 helper phage

(Stratagene, La Jolla, CA). Enrichment was monitored by titering the number of phage

that bound to an albumin coated well compared to a well coated with ovalbumin or

casein.

Phage Binding Assay—Phage clones (~1011 phage) were added to Maxisorp plates

coated with mouse, rat, rabbit, bovine, rhesus or human albumin (Sigma, St. Louis, MO)

as described above. The microtiter plate was washed with PBS, 0.05% Tween 20 and


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bound phage were detected following incubation with HRP/Anti-M13 Conjugate

(Amersham Pharmacia Biotech, Piscataway, NJ) in PBS, 0.05% Tween 20. The

amount of HRP bound was measured using ABTS/H2O2 substrate (Kirkegaard & Perry

Laboratories, Gaithersburg, MD) and monitoring the absorbance at 405 nm.

Partial and Complete Randomization on Monovalent Phage- A soft randomized

library was designed using an oligonucleotide coding for clone RB, but synthesized with

a 70-10-10-10 mixture of bases as described (16). A fully randomized library was

designed holding highly selected residues (underlined) constant: X5DXCLPXWGCLWX4;

randomized positions (X) were coded by NNS. Both the soft randomized and fully

randomized libraries were sorted against rat, rabbit and human serum albumin as

above.

Peptide Synthesis—Peptides were synthesized by either manual or automated

(Milligen 9050) Fmoc-based solid phase synthesis on a 0.25 mmol scale using a PEG-

polystyrene resin as described (17). The carboxy terminal lysine of peptide SA08 was

derivatized with NHS-LC-biotin as recommended by the manufacturer (Pierce Chemical,

Rockford, IL) and purified by reversed phase HPLC yielding SA08b (Ac-

QGLIGDICLPRWGCLWGDSVKb–NH2 where Kb refers to lysine-biotin).

Peptide Competition Assay—Rat, rabbit or mouse albumin was immobilized directly

on Maxisorp plates and blocked as above. Samples, serially diluted in Binding Buffer

were added to the plate followed immediately by the addition of 10 nM SA08b for 1 h, at

25oC. SA08b has an EC50 of 2 and 4 nM for rat and rabbit albumin, respectively. The

microtiter plate was washed with PBS, 0.05% Tween 20 and bound SA08b was


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detected with Streptavidin/HRP (Roche Molecular Biochemicals, Indianapolis, IN). The

amount of HRP bound was measured using ABTS/H2O2 substrate as above.

Affinity Measurements by Surface Plasmon Resonance—The binding affinities

between SA peptides and albumin were obtained using a BIAcore 3000 (BIAcore Inc.,

Piscataway, NJ). Human, rabbit and rat albumin were captured on a CM5 chip using

amine coupling at approximately 5000 resonance units (RU). SA peptides at 0, 0.625,

1.25, 2.5, 5, and 10 µM were injected at a flow rate of 20 µl/min for 30 seconds. The

bound peptides were allowed to dissociate for 5 min before matrix regeneration using

10 mM glycine, pH 3. The signal from an injection passing over an uncoupled cell was

subtracted from that of an immobilized cell to generate sensorgrams of the amount of

peptide bound as a function of time. The running buffer, PBS containing 0.05% Tween-

20, was used for all sample dilutions. BIAcore kinetic evaluation software (version 3.1)

was used to determine KD from the association and dissociation rates using a one-to-

one binding model.

Pharmacokinetic Study of SA21 in Rabbits – Three male New Zealand White (NZW)

rabbits were administered an intravenous (IV) bolus dose of 2 mg/kg of SA21 in PBS.

Eighteen blood samples were collected at serial time-points just prior to dosing and from

1 min to 21 days post-dosing. Samples were collected in tubes containing sodium

citrate as an anticoagulant, then centrifuged and the plasma portion frozen at -70oC

until analysis using an ESI (electrospray), LC/MS/MS method.

The mass spectrometer used was an API 4000 (Applied Biosystems / MDS Sciex,

Foster City, CA). The autosampler was a CTCPAL System (Leap Technologies, Chapel


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Hill, NC) equipped with a cooling stack. The HPLC system consisted of the Shimadzu

SCI-10A System Controller with two Shimadzu LC-10A pumps. A pre-filter was placed

in front of the analytical column (C18, 2.1 X 50 mm, Phenomenex Synergi 4µ MAX-RP

80A). The column flow was set at 500 µL per minute. Solvent systems A (100% H2O)

and B (100% acetonitrile) both contained 1% formic acid. A fast gradient (0.0-0.4 min,

90% A; 0.4-0.8 min, from 90% A to 10% A; 0.8-1.8 min, 10% A; 1.8-2.0 min, from 10%

A to 90% A; 2.0-3.0 min, 90% A) was used for all analysis. The injection volume was 10

µL.

SA 21 was initially characterized via direct infusion (1 µM in 20% acetonitrile) into the

API 4000. The transition of triply charged ion 758 to fragment 948 was optimized for

analysis in plasma matrix. Standard curves were prepared in citrated rabbit plasma in

96 well plates by adding 10 µL of diluted SA21 into 190 µL of plasma over a range of

2500 nM to 4.9 nM. TCEP (Tri(2-carboxyethyl)phosphine hydrochloride, Sigma-Aldrich,

St. Louis, MO) was used as a reducing agent to enhance peptide recovery and was

added to all samples at a final concentration of 2 mM for 20 min at 37oC. Plasma

proteins were then precipitated by addition of 160 µL of 80% acetonitrile to 40 µL of

plasma for 10 min and removed by centrifugation for 10 min at 10oC. The supernatant

was transferred to another 96 well plate and the plate was sealed with silicone sealing

mat (AxyGen, Inc., Union City, CA). Samples were placed in the autosampler at 5oC to

be analyzed by LC/MS/MS. Samples with a high concentration of SA21 (PK samples

from 1 min to 7 h) were diluted 10-fold with blank rabbit plasma prior to TCEP addition.


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Pharmacokinetic parameters were fitted to a 1-compartment elimination model using

WinNonlin software version 3.1 (Pharsight Corp, Mountain View, CA) to obtain the

clearance (CL), volume of distribution (V1), elimination half-life (t1/2), and drug exposure

(AUC).

Construction, Expression and Purification of D3H44-L and D3H44-Ls – D3H44 Fab

was produced as described (18). D3H44-L was constructed by inserting DNA encoding

a linker sequence (GGGS) followed by SA06 (QRLMEDICLPRWGCLWEDDF) onto the

carboxyl terminal end of the light chain of D3H44 using Kunkel mutagenesis (19).

D3H44-Ls was constructed in the same manner, however, additional mutations were

added to remove the disulfide between the light and heavy chains of the Fab. Each

chain was terminated one residue prior to the heavy-light inter-chain disulfide followed

by the addition of the linker sequence and peptide SA06 to the light chain. The resulting

plasmids, pD3H44-L and pD3H44-Ls, were confirmed by DNA sequencing. Expression

of D3H44-L and D3H44-Ls was carried out as described (18). Cells were harvested,

frozen, suspended in 1 mM EDTA, 10 mM Tris pH 8, 0.5 mM PMSF and disrupted using

a tissue homogenizer. D3H44-L and D3H44-Ls were rapidly purified using a Hi-Trap TF

affinity column followed by a Hi-Trap rabbit albumin affinity column (Amersham

Pharmacia Biotech, Piscataway, NJ), each generated as recommended by the

manufacturer. Both columns were washed with PBS and eluted with 50 mM HCl. Eluted

fractions were immediately neutralized using 1 M Tris pH 8. D3H44-L and D3H44-Ls

were further purified using Sephacryl S-200 gel filtration (Amersham Pharmacia

Biotech, Piscataway, NJ) in PBS followed by an extraction with Triton X-114 to remove


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traces of endotoxin (20). D3H44-L and D3H44-Ls were judged greater than 99% pure

by SDS-PAGE.

FX Activation and Prothrombin Time Assays - The FX activation assay and the

Prothrombin Time (PT) assay were performed as described previously (18).

Albumin/TF Sandwich Assay – A soluble mutant of TF (E219C) (21) was specifically

biotinylated using a 4-fold molar excess of biotin BMCC (Pierce, Rockford, IL) in 200

mM Tris pH 7.5, 20% DMSO. The reaction was desalted using a NAP5 (Amersham

Pharmacia Biotech, Piscataway, NJ) column and concentrated via Centricon YM10

(Millipore, Bedford, MA ) to 260 µM. Biotinylated-TF binds to D3H44 Fab immobilized

directly on a Maxisorp plate (10 µg/ml in PBS, overnight at 4oC and blocked for 1 h at

25oC using Casein Blocker (Pierce, Rockford, IL)) with an EC50 of 11 nM.

For the Albumin/TF sandwich assay, rabbit albumin was immobilized as described

above. Dilutions of D3H44 Fab, D3H44-L or D3H44-Ls were added in Binding Buffer for

1 h. The plate was washed with PBS, 0.05% Tween 20 and 50 nM biotinylated-TF in

Binding Buffer was added for 1 h. The microtiter plate was washed with PBS, 0.05%

Tween 20 and Streptavidin/HRP was added. After a final wash, bound HRP was

measured as above.

Pharmacokinetics of D3H44 Variants in Rabbits and Mice - Groups of 3 NZW

rabbits were given an IV bolus of 400-525 ug/kg D3H44 variants (D3H44 Fab,

D3H44-L, D3H44-Ls) into the marginal ear vein. Plasma samples were obtained

from an arterial catheter placed in the contralateral ear over a 21 day period for

analysis by TF ELISA (see below). Individual plasma concentration versus time


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curves were fitted to a 2-compartment elimination model using WinNonlin v3.0

(Pharsight, Inc., Mountain View, CA). The pharmacokinetic parameters of

clearance (CL), volume of distribution (V1), steady state volume (Vss), elimination

half-life (t1/2), drug exposure (AUC), and AUC corrected for actual dose

administered (AUC/dose) were averaged for each treatment group. Differences

between groups were determined by ANOVA, with significance at p < 0.05.

Groups of 9 BalbC mice received a 5.0 mg/kg IV bolus of D3H44 F(ab) or D3H44-L

into the tail vein. Plasma samples were obtained by eye bleed from 3 mice per time-

point over 2-9 days, and assayed for concentration of D3H44 using the TF ELISA. The

average plasma concentration was obtained for each time-point and fitted to a 2-

compartment elimination model using WinNonlin v3.0. As the analysis of the mouse

experiment produces one average concentration versus time profile for each variant, PK

parameters are presented as a single estimate for the group of 9 mice.

Quantitation of D3H44 Variants - The concentration of D3H44 Fab, D3H44-L and

D3H44-Ls in rabbit plasma was determined using a TF ELISA. Maxisorp plates were

coated overnight at 4°C with 1 µg/ml TF (Genentech, Inc., South San Francisco, CA) in

50 mM sodium carbonate buffer, pH 9.6. Plates were blocked with 0.5% ovalbumin in

PBS, pH 7.4. Diluted antibody standards (0.23-50 ng/ml) and samples (minimum

dilution 1: 100) in PBS containing 0.5% ovalbumin, 0.05% polysorbate 20, 0.35 N NaCl,

5 mM EDTA, 0.25% CHAPS, 0.2% bovine γ-globulins (Sigma, St. Louis, MO) and 1%

plasma were added to the plates for 2 h. Antibody bound to the plates was detected

with HRP conjugated goat anti-human Fab’2 antibody (Jackson ImmunoResearch, West


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Grove, PA). Bound HRP was measured using the substrate 3,3',5,5'-tetramethyl

benzidine/H2O2 (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and the change in

absorbance was monitored at 450 nm. Data falling in the linear range of the standard

curve was used to calculate D3H44 concentrations in the samples. D3H44 Fab and

D3H44-L in mouse plasma were assayed in the same ELISA except samples were

diluted in buffer without 1% plasma and the standard curve range was 0.31-40 ng/ml.


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RESULTS

Identification and Maturation of Peptides that Bind to Serum Albumin – Naïve peptide

phage library pools A through D were selected against rat, rabbit and human albumin.

Each pool, with the exception of pool D when human albumin was the target, showed

enrichment for each species of albumin. The sequences from the enriched pools

revealed in each case that a single clone had taken over the pool. The inferred peptide

sequences from these clones are shown in Table I.

Interestingly, albumin has greater than 70 percent amino acid sequence identity

between these species yet unique peptide sequences originating from within a given

phage library pool were identified for each species. The sequence similarity observed

between clones HB and HC, RA and RD, and RB and RC, despite their origins from

independent phage library pools, suggests the importance of the homologous residues

in binding to the respective species of albumin. Individual phage clones were examined

using a phage binding assay, a quick qualitative screen to assess species selectivity.

While phage clones generally bound only to the albumin for which they were selected,

clones HB and HC, selected for binding to human albumin, also bound to rat albumin

and clone RB, selected for binding to rat albumin, bound albumin from all 3 species

(Table I). None of the phage clones bound to structurally unrelated ovalbumin indicating

that the interaction with albumin was specific.

Because of its broad recognition of rat, rabbit and human albumin, clone RB was

chosen for sequence maturation on phage using ‘soft randomization’. The potential

diversity of a soft randomized library is the same as the starting naïve libraries,


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however, a soft randomized library maintains a bias towards a particular sequence, in

this case, DNA coding for the peptide sequence from clone RB. Sequences after 4

rounds of selection against rat, rabbit or human albumin are shown in Table II along

with results from the phage binding assay. All clones were specific for the albumin to

which they were selected based upon their lack of binding to immobilized ovalbumin

and casein; however, several clones also bound to albumin from other species including

bovine, rhesus and mouse albumin (Table II).

Since at any given position, the amino acid present in the parent sequence was

designed to appear approximately half the time in these libraries, only fully conserved

positions are likely to indicate important structural or contact elements that support

albumin binding. A final library that kept these highly selected residues (underlined)

constant X5DXCLPXWGCLWX4 while allowing all 20 amino acids at the 11 remaining

positions allowed a more extensive search of pertinent sequence space. The sequence

preferences at each randomized position resulting from selection against rabbit albumin

are shown in Figure 1. A similar profile was observed from sequences selected for

binding rat and human albumin (not shown). For each species of albumin, there was a

strong preference for Ile at position 7 and Arg at position 11, thus generating a core

consensus of DICLPRWGCLW. Additionally, there was a general preference for

negatively charged residues (Asp or Glu) at positions flanking this core, particularly on

the carboxy terminus.

Characterization of Albumin Binding Peptides – Several peptides patterned after the

sequences selected for albumin binding were synthesized. Their binding to human,


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rabbit and rat albumin was assessed by Biacore and a peptide competition assay was

used to assess their affinity for rabbit and mouse albumin (Tables III and IV). The IC50

values obtained for binding to rabbit albumin compared favorably to Kd values

determined by Biacore (Table III). In comparison to rabbit and rat albumin, the peptides

bind weaker to human and tighter to mouse albumin, however, the rank affinity of a

given peptide is generally maintained from species to species. Peptide SA15,

representing the consensus for binding rabbit albumin (Figure 1), had the lowest IC50

value in the peptide binding assay and highest affinity by surface plasmon resonance

for rabbit albumin (Table III). A linear peptide, identical to SA06, but with both Cys

residues changed to Ala had an IC50 greater than 50 µM demonstrating the importance

of the disulfide. In addition, the affinity of the peptides for rabbit albumin diminished with

reduction in the length of the peptides (Table IV). A core of about 10 amino acids (SA34

and SA19, Table IV) having an IC50 of ca. 25 µM, could be improved 6-fold by the

addition of 4 residues to its amino terminus (SA33) or 8.6-fold by the addition of 3

residues to its carboxy terminus (SA26). The addition of all 7 residues resulted in a 60-

fold improvement in the IC50 (SA22) indicating that these additions have an additive

effect.

Characterization of the Albumin Binding Site- When the binding of RB-B8 or RB-H1

phage to rabbit albumin was monitored over a pH range from 2.9 to 9.0, optimum

binding was observed above pH 6.0 for both clones (data not shown). Binding

decreased below pH 6.0 until no binding was observed at pH 2.9. A similar pattern was

observed for the binding of these clones to human and rat albumin. The similar amino


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acid preferences and pH profile are consistent with a similar binding environment on

each species of albumin.

Since albumin plays an important role as a carrier of many ligands and drugs, we

tested whether known albumin ligands might compete with peptide binding. Addition of

site I (indomethacin, phenylbutazone, warfarin) or site II (ibuprofen, L-tryptophan,

dansylsarcosine, diazepam) ligands, a fatty acid (myristic acid) or a metal ion (CuCl2) at

concentrations up to 100 µM had no effect on SA08b peptide binding to rat or rabbit

albumin in the peptide competition assay (not shown).

We were curious as to whether unrelated clones initially identified for binding to

albumin (Table I) might compete with our matured multi-species binding peptides. While

RD and BA phage selectively bind only to rat and rabbit albumin, respectively, these

clones were clearly blocked by the addition of SA08 (Figure 2). In contrast, binding of

clones HA and HB to human albumin was not blocked by SAO8 and thus bind to a

different site.

Pharmacokinetics of SA21 – In vivo, peptides can be rapidly metabolized or

eliminated due to glomerular filtration resulting in a short half-life. We hypothesized that

association with albumin would result in a peptide with improved pharmacokinetics and

chose to study SA21 because it was stable in citrated rabbit serum in vitro over a 24 h

period at 37oC as monitored by LC/MS/MS. With its high affinity for rabbit albumin

(Table III) and the high concentration of albumin in plasma, we calculated that SA21

should remain greater than 99.95% bound to rabbit albumin in vivo.


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The pharmacokinetic profile of SA21 in rabbit is shown in Figure 3 compared to an

unrelated control peptide of similar size, 1a (Ac-ALCDNPRIDRWYCQFVEG-NH2) and

an engineered variant which binds to albumin, 1m (an amino terminal napthalene acyl

sulfonamide derivative of 1a) (22). SA21 showed reduced clearance compared to 1a

and 1m with a significantly longer half-life of 2.3 h compared to 7.6 and 30 min,

respectively (Table V).

Characterization of Albumin Binding Fab Fusions—Compared to an IgG, Fab

fragments have relatively fast clearance of 42-72 ml/kg/h in rabbit (23). D3H44 (18) is a

humanized antibody that binds human tissue factor (TF) and acts as an anticoagulant.

To test whether association of the D3H44 Fab with albumin can increase its half-life in

vivo, the SA06 sequence was recombinantly fused through a short flexible linker to the

carboxy terminus of the light chain yielding D3H44-L. D3H44-Ls was also constructed

and lacks the disulfide linking the light and heavy chains of the Fab. D3H44-Ls was

designed to avoid potential folding problems that may be caused by the addition of a

disulfide bonded peptide. The addition of SA06 provided a simple purification scheme

utilizing a TF affinity column followed by an albumin affinity column. Although D3H44-L

and D3H44-Ls were judged to be greater than 90 percent pure following the TF affinity

column alone, only 14 percent of the D3H44-L was bound and retained on the

subsequent rabbit albumin affinity column in contrast to 54 percent of the D3H44-Ls.

The higher overall yield obtained with D3H44-Ls suggested improper folding of the

SA06 disulfide due to its proximity to the inter-chain disulfide between the heavy and

light chains.


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Following both column purification steps, D3H44-L was incubated with TF, rabbit

albumin or both, and analyzed by analytical gel filtration along with SDS-PAGE analysis.

The shift in the retention time of the eluting fractions was consistent with a 1:1

stoichiometry between D3H44-L and either TF or rabbit albumin, and a stoichiometry of

1:1:1 in the presence of both TF and rabbit albumin. For example, the retention time of

the peak that contained rabbit albumin and D3H44-L by SDS-PAGE analysis suggested

a molecular weight of 123 kDa compared to the calculated molecular weight of 119 kDa

for a 1:1 complex.

The affinity of purified Fab fusions for rabbit albumin and TF were examined in the

following assays. First, in comparison to SA06, both D3H44-L and D3H44-Ls had a

similar ability to compete for binding to immobilized rabbit albumin. In contrast, D3H44

Fab is unable to bind to rabbit albumin (Figure 4). Second, D3H44-L and D3H44-Ls not

only bind to TF, but inhibit its function to the same degree as D3H44 Fab (Figure 5a).

Third, to further investigate whether the binding of rabbit albumin to D3H44-L or D3H44-

Ls would preclude binding to TF, an albumin/TF sandwich assay was used (Figure 6). In

this assay, binding to immobilized rabbit albumin was detected with biotinylated TF. The

results demonstrate that D3H44-L and D3H44-Ls are able to simultaneously bind

albumin and TF whereas D3H44 Fab is unable to bind albumin and thus does not

generate a signal upon addition of biotinylated TF.

Consistent with these assays, D3H44-L and D3H44-Ls also prolong the prothrombin

time assay that measures TF dependent clotting in human plasma (Figure 5b). Since

D3H44-L and D3H44-Ls bind more tightly to rabbit than to human albumin, they were


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also tested in a prothrombin time assay using rabbit plasma where clotting was initiated

with human TF. Similar results were obtained (not shown). Taken together, D3H44-L

and D3H44-Ls have essentially equivalent combined functions of the D3H44 Fab and

an albumin binding peptide and these two functions do not interfere with each other.

Pharmacokinetic Analysis of D3H44 variants – The pharmacokinetics of D3H44 Fab,

D3H44-L and D3H44-Ls were compared in rabbits (Figure 7a, Table V). The clearance

of D3H44-L and D3H44-Ls decreased 58 and 43-fold and the half-life increased ca. 40-

fold to 32.4 and 38.3 h, respectively, compared to 0.8 h for D3H44 Fab (Figure 7a). In

mouse, D3H44-L had a 25-fold reduction in clearance compared to D3H44 Fab

consistent with the results obtained in the rabbits (Figure 7b and Table V). D3H44-L

had a half-life of 10.4 h, representing a 26-fold increase over the D3H44 Fab.


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DISCUSSION

A variety of albumin binding peptide phage were identified from the naïve peptide

libraries that were screened. During the affinity maturation of clone RB, which gave rise

to a series of peptides that recognize albumin from multiple species, the core sequence

DICLPRWGCLW was identified. Although a linear peptide, having both Cys substituted

with Ala indicated the importance of the disulfide, none of the matured peptides

examined by NMR, appeared to be structured in solution (N. Skelton, unpublished

result). Interestingly, SA08, a matured sequence derived from libraries patterned after

the sequence of the multi-species binding clone RB, inhibited the binding of clones RD

and BA which selectively bound rat and rabbit albumin, respectively. Although they

shared no sequence similarity they may share the same or overlapping binding site(s)

on albumin. SA08 does not appear to compete with classical site I or site II albumin

binding ligands, nor does it affect binding at fatty acid and metal binding sites. Peptide

binding to albumin is also unaffected over a broad pH range from 6 to 9. Apparently this

conserved peptide binding site on albumin is unique and remains unperturbed above pH

8 where albumin is known to undergo a conformational change (9). Whether

conformational changes in the structure of albumin at low pH or side chain titration

contributes to a loss of peptide binding remains to be determined.

Most peptides are rapidly cleared in vivo as a result of metabolism and renal filtration

(24). The 2.3 h half-life of SA21 in rabbit is relatively long when compared to other

peptides of similar size presumably owing to its association with albumin. The half-life of

the coagulation factor VIIa peptide exosite inhibitor 1a, for example, is only 7.6 min in


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rabbits but can be prolonged 4-fold by the addition of aromatic groups to its amino

terminus to induce binding to albumin (22). While both peptides were stable in citrated

rabbit serum for 24 h, differences in metabolism in vivo may occur. Alternatively,

differences in the affinities of SA21 and 1m for rabbit albumin may explain the superior

pharmacokinetic profile observed for SA21 (Figure 3); the affinity of 1m for rabbit

albumin was not reported.

In another example, the in vivo response to insulin was prolonged using insulin

derivatives acylated with fatty acids to enable association of the hormone with albumin

(14). Unlike these chemically modified peptides, however, the association of SA21 with

albumin is achieved through an amino acid peptide sequence that can simply be added

to any recombinantly expressed protein.

The ability to rapidly generate antibodies as potential therapeutics has stimulated

interest in extending their valence, binding affinity, effector functions and

pharmacokinetics through engineering. Their antigen binding domains can be readily

presented in numerous formats including Fv, scFv, diabodies, Fab and Fab’2. These

immunoglobin fragments as well as many other promising protein pharmaceuticals,

however, are rapidly cleared from the blood limiting their potential usefulness (25). To

test the possibility of extending the half-life of such molecules, an albumin binding

peptide was recombinantly fused to the carboxyl terminus of the light chain of the

D3H44 Fab directed against human tissue factor (TF) (18). The addition of SA06 to

D3H44 provided a simple purification scheme utilizing a TF affinity column followed by

an albumin affinity column. A higher overall yield was obtained with D3H44-Ls


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compared to D3H44-L likely due to the absence of the nearby disulfide between the

light and heavy chain of the Fab. The loss of this disulfide had no effect on the ability of

D3H44-Ls to bind to TF or albumin, but is associated with a 40% increase in drug

clearance relative to D3H44-L, as evidenced from the pharmacokinetic study in rabbit

(Table V). Rodrigues et al. (26) found that the pharmacokinetics of an Fab’2 lacking the

light chain–heavy chain disulfides was not affected and suggested that the stability of a

Fab’2 is not dramatically altered in the absence of this disulfide bond. In this study, we

observed a difference in the clearance as a result of the increased half-lives of the

D3H44-L and D3H44-Ls.

The utility of D3H44-L or D3H44-Ls greatly depends upon the capability of the

albumin bound fraction to bind TF. Both the prothrombin time assay and the albumin/TF

sandwich assay indicate D3H44-L and D3H44-Ls can bind to albumin and TF

simultaneously. Although not surprising given that the two binding sites are at different

ends of the Fab, the ability of the albumin bound fraction to remain functional may not

be retained when trying to enhance the half-life of other protein-peptide fusions. If an

increased half-life comes at the expense of impaired function, a higher dose may be

required to provide an efficacious concentration of free drug (8). The fraction of free

protein could be increased by using shorter peptides with reduced affinity for albumin

(Table IV), however, this is also likely to reduce the half-life.

Besides renal filtration, metabolism is an important parameter affecting half-life.

Although clearance is greatly reduced relative to the D3H44 Fab, both SA21 and

D3H44-L are cleared faster than one might expect simply based on their calculated free


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concentration in plasma and the glomerular filtration rate (GFR) in rabbit (27). The

pharmacokinetics of SA21 and D3H44-L are not influenced by binding to any in vivo

target other than albumin since D3H44 does not recognize rabbit TF. Based on these

assumptions, metabolism accounts for the vast majority of the clearance for both SA21

and D3H44-L.

Further, the faster clearance and shorter half-life of SA21 compared to D3H44-L is

seemingly at odds with their similar affinity for albumin and likely reflects an increased

metabolism of the peptide rather than differences in the rate of filtration. Susceptibility of

the peptide to metabolic proteolysis while bound to albumin may be shielded upon

fusion with the Fab or reduced as a result of the introduction of an amino terminal

fusion. As an example, stabilization of the 10 amino acid peptide hormone, GnRH,

which has a half-life of 2-8 min, was achieved by introducing carboxy terminal

modifications and other stabilizing changes to yield variants with half-lives over 4 h (28).

While association with albumin can extend the exposure of molecules in vivo, the

stability of these molecules can remain as a limiting feature that governs their half-life.

This study represents the first attempt to improve the half-life of an immunoglobulin

fragment without significantly altering the hydrodynamic size of the molecule. Through

the use of a simple recombinant fusion, selective tight binding to albumin and a

prolonged half-life are achieved. D3H44-L has a half-life of 32.4 h in rabbit or an

increase of 37-fold relative to the D3H44-Fab (Table V). This half-life is comparable to

that of a D3H44 Fab conjugated to 20k or 40k PEG and is superior to the half-life of a

D3H44 Fab’2 (half-lives of 18, 69 and 8.8 hr, respectively; L. Damico, unpublished


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result). In mouse, the 10.4 h half-life for D3H44-L represents a 26-fold improvement.

Interestingly, these half-lives correlate with the reported half-life of albumin in these

species of 5 to 6 days in rabbit (29) and 1 day in mouse (30). Despite potential

metabolism differences and the weaker affinity for human albumin (Table 3), the19-day

half-life for human albumin suggests large improvements in the half-life of a Fab in

human are possible.

Moreover, the ability to achieve an increased half-life without a dramatic increase in

size may present an advantage when trying to generate tumor targeting and imaging

molecules (25,31,32). A low molecular weight agent may have an advantage in its

ability to diffuse into tissues; however, a sufficient time of exposure is required for

adequate absorption. Generally, a small protein such as a scFv can diffuse rapidly into

tissues but the bulk of the material is lost due to extremely fast renal filtration. On the

other hand, an IgG remains circulating for several days providing ample exposure but

minimal tumor penetration due to the poor diffusion of such a large protein. A small

long-lived molecule, such as an albumin binding Fab, could be ideal as an imaging or

tumor-targeting agent.

ACKNOWLEDGMENTS

We are grateful to P. Sims for performing the TF ELISA, B. Commons for running the

LC/MS/MS, and P. Moran for prothrombin time assays. A. Arata, M. Reich and M.

Gonzales assisted in the pharmacokinetic studies. We thank C. Quan, J. Tom and K.

Zobel for synthesis of peptides, A. Zhong for DNA sequencing and R. Kelley for TF


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affinity column and TF (E219C). The support of M. Vanderlin, T. Zioncheck, and A. de

Vos is appreciated. Finally we especially thank R. Lazarus, M. Beresini and M. Koehler

for their support and many helpful discussions


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FIGURES

Figure 1. Amino acid preferences selected for binding rabbit albumin following

full randomization. Sequence preference following 4 rounds of selection on rabbit

albumin for the library: X5DXCLPXWGCLWX4, where all 20 amino acids were

substituted at X and underlined amino acids were held constant. Amino acids identified

at the indicated randomized positions are plotted as a function of their preference. The

preference for any amino acid is reported as the number of standard deviation units (σ)

above a random chance occurrence of a given residue assuming a binomial distribution

and accounts for codon bias and sampling statistics (33). Fixed amino acids in the

library are boxed. Only amino acids that occurred more frequently than 2 standard

deviations (2σ) above an expected random chance occurrence are shown.

Figure 2. Inhibition of RD and BA phage binding to rat or rabbit albumin.

RD (Ο), BA ( ), HA ( ) and HB ( ) phage binding to immobilized rat, rabbit or human

albumin, respectively, was inhibited by increasing concentrations of peptide SA08.

Relative phage binding was determined using the phage binding assay.

Figure 3. The pharmacokinetic profile of SA21 in Rabbit. The percent of initial

concentration of SA21 ( ), 1a ( ) and 1m (Ο) in plasma samples obtained from rabbits

following IV bolus administration is plotted versus time. Data obtained for SA21 is

compared to similar sized peptides reported by Koehler et al. (data replotted by

permission from author) (22). SA21 concentrations were determined by LC/MS/MS as


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described in methods. The concentration of 1a and 1m was determined using a binding

ELISA (16).

Figure 4. D3H44-L and D3H44-Ls bind to Rabbit Albumin with similar affinity to

SA06. A comparison of SA06 (Ο), D3H44-L ( ), D3H44-Ls (∆, dashed line) and D3H44

Fab ( ) in the peptide competition assay using immobilized rabbit albumin. Lines drawn

represent the data fit to a four-parameter equation from which the following IC50 values

were calculated for this representative data set: SA06 = 80 nM, D3H44-L = 130 nM,

D3H44-Ls = 50 nM and D3H44 Fab > 100 µM.

Figure 5. Effect of D3H44-L and D3H44 Fab on FX Activation and the

prothrombin time (PT). (A) Inhibition of TF•FVIIa mediated FX activation by D3H44-L

( ), D3H44-Ls (∆, dashed line) and D3H44 Fab ( ). Data from 3 independent

experiments was fit to a four-parameter equation from which the following IC50 values

were calculated: D3H44-L = 0.38 nM, D3H44-Ls = 0.33 nM and D3H44 Fab = 0.23 nM.

(B) The fold prolongation of TF-dependent clotting by D3H44-L ( ), D3H44-Ls (∆) and

D3H44 Fab ( ) in the human PT assay. Uninhibited clotting time was 9.6 s.

Figure 6. D3H44-L can bind Tissue Factor and Albumin Simultaneously. The

binding of D3H44-L ( ), D3H44-Ls (∆, dashed line) and D3H44 Fab ( ) to immobilized

rabbit serum albumin was detected using biotinylated-TF followed by streptavidin-HRP.


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Figure 7. Pharmacokinetics of D3H44-L and D3H44 Fab in Rabbit and Mouse. (A)

D3H44-L ( ), D3H44-Ls (∆, dashed line) and D3H44 Fab ( ) were dosed at 0.40-0.52

mg/kg into New Zealand white rabbits (3 rabbits/group). (B) D3H44-L ( ) and D3H44

Fab ( ) were dosed at 5 mg/kg into balbC mice (9 mice/group). Samples taken at the

indicated times were assayed in a TF ELISA.


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Table I. Sequences of phage clones selected from polyvalent naïve libraries for binding

rat, rabbit or human albumin.

The location of fixed cysteines from the library design are shaded. Sequence identity among clonesderived from different phage libraries is boxed. A qualitative assessment of the ability of phage bearingthe indicated peptide sequence to bind human (HSA), rabbit (BuSA) or rat (RSA) albumin is indicated.

Library Phage Binding

Pool Clones Selected for Binding to Human Serum Albumin HSA BuSA RSA

HA E V R S F C T D W P A E K S C K P L R G +++ - -

HB R A P E S F V C Y W E T I C F E R S E Q ++ - (+)

HC E M C Y F P G I C W M +++ - ++

Clones Selected for Binding to Rabbit Serum Albumin

BA G E N W C D S T L M A Y D L C G Q V N M - +++ -

BB M D E L A F Y C G I W E C L M H Q E Q K - +++ -

BC D L C D V D F C W F - +++ -

BD K S C S E L H W L L V E E C L F - +++ -

Clones Selected for Binding to Rat Serum Albumin

RA R N E D P C V V L L E M G L E C W E G V - - +++

RD D T C V D L V R L G L E C W G - - +++

RB Q R Q M V D F C L P Q W G C L W G D G F + ++ +++

RC D L C L R D W G C L W - - +++


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Table II. Sequences of phage clones selected for binding rat, rabbit or human albumin following soft randomization of clone RB.

Amino acids positions identical in clone RB, the starting sequence used for soft randomization are shaded. A qualitative assessment of the ability of phage bearing the indicated peptide sequence to bind different species of albumin is indicated (nd indicates not determined). Abbreviations indicate human (HSA), rabbit (BuSA), rat (RSA), bovine (BSA), rhesus (RhSA ) and mouse (MSA) albumin.

Phage BindingClone RB Q R Q M V D F C L P Q W G C L W G D G F HSA BuSA RSA BSA RhSA MSA

Clones Selected for Binding to Human Serum AlbuminRB-H1 Q R H P E D I C L P R W G C L W G D D D ++ +++ +++ ++ +++ +++RB-H6 N R Q M E D I C L P Q W G C L W G D D F ++ +++ +++ ++ +++ +++

Clones Selected for Binding to Rabbit Serum AlbuminRB-B2 Q R L M E D I C L P R W G C L W G D R F ++ +++ +++ nd nd ndRB-B5 Q W H M E D I C L P Q W G C L W G D V L ++ +++ +++ - +++ +++RB-B6 Q W Q M E N V C L P K W G C L W E E L D ++ +++ +++ ++ +++ +++RB-B4 L W A M E D I C L P K W G C L W E D D F ++ +++ +++ ++ +++ +++RB-B7 L R L M D N I C L P R W G C L W D D G F ++ +++ +++ ++ +++ +++RB-B8 H S Q M E D I C L P R W G C L W G D E L ++ +++ +++ +++ +++ +++RB-B11 Q W Q V M D I C L P R W G C L W A D E Y ++ +++ +++ ++ +++ +++RB-B12 Q G L I G D I C L P R W G C L W G D S V ++ +++ +++ +++ +++ +++RB-B16 H R L V E D I C L P R W G C L W G N D F ++ +++ +++ nd nd ndRB-B9 Q M H M M D I C L P K W G C L W G D T S + +++ +++ nd nd ndRB-B14 L R I F E D I C L P K W G C L W G E G F + +++ +++ nd nd ndRB-B3 Q S Y M E D I C L P R W G C L S D D A S + +++ +++ nd nd ndRB-B10 Q G D F W D I C L P R W G C L S G E G Y - +++ +++ nd nd ndRB-B1 R W Q T E D V C L P K W G C L F G D G V - +++ +++ nd nd nd

Clones Selected for Binding to Rat Serum AlbuminRB-R8 Q G L I G D I C L P R W G C L W G D S V ++ +++ +++ +++ +++ +++RB-R16 L I F M E D V C L P Q W G C L W E D G V + +++ +++ + +++ +++RB-R10 Q R D M G D I C L P R W G C L W E D G V ++ +++ +++ ++ +++ +++RB-R4 Q R H M M D F C L P K W G C L W G D G Y - + +++ nd nd ndRB-R7 Q R P I M D F C L P K W G C L W E D G F - + +++ nd nd ndRB-R11 E R Q M V D F C L P K W G C L W G D G F - + +++ nd nd ndRB-R12 Q G Y M V D F C L P R W G C L W G D A N - + +++ nd nd ndRB-R13 K M G R V D F C L P K W G C L W G D E L - + +++ nd nd ndRB-R15 Q S Q L E D F C L P K W G C L W G D G F - + +++ nd nd ndRB-R17 Q G G M G D F C L P Q W G C L W G E D L - + +++ nd nd ndRB-R5 Q R L M W E I C L P L W G C L W G D G L - - +++ nd nd ndRB-R18 Q R Q I M D F C L P H W G C L W G D G F - - +++ nd nd ndRB-R2 G R Q V V D F C L P K W G C L W E E G L - - +++ nd nd ndRB-R3 Q M Q M S D F C L P Q W G C L W G D G Y - - +++ nd nd ndRB-R9 K S R M G D F C L P E W G C L W G D E L - - +++ nd nd ndRB-R1 E R Q M E D F C L P Q W G C L W G D G V - - +++ nd nd ndRB-R14 Q R Q V V D F C L P Q W G C L W G D G S - - +++ nd nd nd

Peptide Competition Assay Surface Plasmon Resonance

PEPTIDE Sequence BuSA MSA HuSA BuSA RSA

IC50 (nM) Kd (nM)SA21 Ac-R L I E D I C L P R W G C L W E D D -NH2 270 ± 110 7 ± 2 467 ± 47 320 ± 22 266 ± 8

SA06 Q R L M E D I C L P R W G C L W E D D F -NH2 130 ± 50 6 ± 2 803 ± 62 143 ± 5 229 ± 9

SA08 Ac-Q G L I G D I C L P R W G C L W G D S V K -NH2 51 ± 11 12 ± 2 858 ±59 108 ± 5 158 ± 3

SA15 G E WW E D I C L P R W G C L W E E E D -NH2 13 ± 12 5 ± 1 878 ± 58 65 ± 3 150 ± 5

Table III. Comparison of the peptide competition assay with Biacore for selected peptides.

Selected peptides were tested for binding human (HSA), rabbit (BuSA), rat (RSA) and mouse (MSA) albumin using the peptide competition assay or surface plasmon resonance as indicated and described in the methods.


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PEPTIDE Sequence IC50 (nM)

SA20 Ac- Q R L I E D I C L P R W G C L W E D D F 260SA21 Ac- R L I E D I C L P R W G C L W E D D 270 ± 110SA22 Ac- R L I E D I C L P R W G C L W E D 430 ± 170SA29 Ac- R L I E D I C L P R W G C L W E 400 ± 90SA31 Ac- R L I E D I C L P R W G C L W 200SA33 Ac- R L I E D I C L P R W G C L 4310 ± 2770SA35 Ac- R L I E D I C L P R W G C >250000

SA23 Ac- L I E D I C L P R W G C L W E D 360 ± 140SA24 Ac- I E D I C L P R W G C L W E D 1380 ± 410SA25 Ac- E D I C L P R W G C L W E D 2730 ± 1300SA26 Ac- D I C L P R W G C L W E D 3120 ± 660SA27 Ac- I C L P R W G C L W E D 86700 ± 21800SA28 Ac- C L P R W G C L W E D >400000

SA30 Ac- I E D I C L P R W G C L W E 1800 ± 590SA32 Ac- E D I C L P R W G C L W 2170 ± 520SA04 D I C L P R W G C L W 8540 ± 4620SA34 Ac- D I C L P R W G C L 28210 ± 6500SA19 D I C L P R W G C L 24510 ± 2100SA18 I C L P R W G C L W 124900SA36 Ac- I C L P R W G C >250000

-NH2-NH2

-NH2-NH2

-NH2

-NH2-NH2-NH2

-NH2-NH2-NH2-NH2

-NH2-NH2

-NH2

-NH2

-NH2-NH2

-NH2-NH2

Table IV. The effect of peptide length on binding rabbit albumin in the peptide competition assay.

SA21 D3H44 Fab D3H44-L D3H44-Ls

Parameter Units Average Stdev Average Stdev Average Stdev Average Stdev

Dose mg/kg 2.00 0.40 0.42 0.52

AUC/Dose (hr*ug/mL)/(mg/kg) 77.7 4.1 14.80 3.1 840 78 633 157

CL mL/hr/kg 12.9 0.7 69.9 16.2 1.20 0.11 1.64 0.37t1/2 hr 2.31 0.24 0.876 0.213 32.4 3.2 38.3 8.8V1 mL/kg 42.8 2.9 90.6 38.4 56.2 10.1 87.6 1.7Vss mL/kg 221 95 113 7 176 11

PK Parameters in Mouse

PK Parameters in Rabbit

Fab D3H44-L

Parameter Units Estimate Estimate

Dose mg/kg 5.0 5.0

AUC/Dose (hr*ug/mL)/(mg/kg) 8.49 214

CL mL/hr/kg 118 4.68t1/2 hr 0.398 10.4V1 ml/kg 67.6 70.4Vss ml/kg 151 138

Table V. Pharmacokinetic Parameters from Rabbit and Mouse Studies.


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Figure 1.

0

5

10

15

20

AG EG L W LW D G LW D EG D I C L P KR WGC LW D EG D E D EG AD EN

1 2 3 4 5 7 11 17 18 19 20

Pre

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(∆ σ

)

Figure 2.

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0.2

0.4

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102

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Concentration SA08 (nM)

Rel

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Figure 3.

0.1

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10

100

0 1 2 3 4 5 6 7Time (hr)

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n (m

g/m

l)

Figure 4.

0

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Concentration (nM)

Rel

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Rab

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Figure 5a.

0

0.2

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0.001 0.01 0.1 1 10 100 1000

D3H44 (nM)

FX

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Figure 5b.

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D3H44 (µM )


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Figure 6.

0

0.1

0.2

0.3

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0.5

10 -4 10 -2 10 0 10 2 10 4

D3H44 (nM)

Abs

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(405

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Figure 7a & b.

0.01

0.1

1

10

0 7 14 21Time (day)

D3H

44 (

µg/

ml)

0.1

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100

0 24 48 72 96 120 144 168

Time (h)

D3H

44 (

mg/

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Dan Combs and Lisa A. DamicoMark S. Dennis, Min Zhang, Y. Gloria Meng, Miryam Kadkhodayan, Daniel Kirchhofer,

Albumin binding as a general strategy for improving the pharmacokinetics of proteins

published online July 15, 2002J. Biol. Chem.

10.1074/jbc.M205854200Access the most updated version of this article at doi:

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